Multidrug resistant

Am J Trop Med Hyg, 1998 Jan, 58(1), 11 - 6
Comparative clinical trial of artesunate suppositories and oral artesunate in combination with mefloquine in the treatment of children with acute falciparum malaria; Sabchareon A et al.; A randomized pilot study to compare the safety and efficacy of artesunate suppositories (15 mg/kg/day for three days) versus oral artesunate (6 mg/kg/day for three days), both in combination with mefloquine (25 mg/kg), was conducted in 52 Thai children with uncomplicated multidrug-resistant falciparum malaria . Forty-five patients (87%) had a full 28-day follow-up in the hospital to assess efficacy and exclude reinfection . Mean {range} times to fever clearance of the two groups were similar (42 hr {15-104} versus 42 hr {6-119}) . Artesunate suppositories resulted in significantly longer times to achieve 50% and 90% reductions of the initial parasite counts (17 and 26 hr versus 9 and 15 hr; P < 0.05 and P < 0.001) . Time {range} to parasite clearance was longer in the artesunate suppositories group (42 hr {14-93} versus 35 hr {16-69}), but the difference was not significant . The cure rates by days 28 were not significantly different, 92% for artesunate suppository-treated patients and 100% for oral artesunate-treated patients . Both drug regimens are safe and effective . Further studies are needed to characterize the pharmacokinetic properties and the optimum regimen of artesunate suppositories for the treatment of severe malaria.

Br J Haematol, 1998 Jan, 100(1), 194 - 7
Functional multidrug resistance in acute myeloblastic leukaemia: a standardized flow cytometric assay for intracellular daunorubicin accumulation; Pallis M et al.; We have developed a standardized flow cytometric method for the measurement of in vitro multidrug resistance in acute myeloblastic leukaemia (AML) blasts, using carboxylate microspheres which bind the fluorescent drug daunorubicin . Cells and beads were incubated concurrently with the drug . Fluorescence was expressed as a cell:bead ratio . Bead fluorescence at a fixed cytometer voltage was consistent over at least a 3-month period (CV 5.47%), and repeat assays up to 8 months later correlated well (R = 0.86) . Bead to drug binding provides a valuable measure of quality assurance as well as a standard for cellular drug accumulation assays and would therefore be suitable for reproducibly reporting the results of multidrug resistance analysis in a clinical setting.

J Chromatogr B Biomed Sci Appl, 1997 Nov 21, 702(1-2), 203 - 10
High-performance liquid chromatographic determination of pyrazoloacridine, a nitro-9-methoxyacridine anticancer agent, in human plasma; Jayewardene AL et al.; Pyrazoloacridine (PZA) is a 9-methoxy substituted acridine with a reducible nitro group . PZA has shown selective solid tumor cytotoxicity with activity against hypoxic cells, non-cycling cells and cells expressing the multidrug resistant phenotype . A high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of PZA in human plasma to support phase II clinical trials . PZA and ethyl orange, the internal standard, were isolated from human plasma by precipitating plasma proteins with methanol, and centrifuging to pellet the proteins . The resulting supernatant was injected onto a cyanopropyl HPLC column eluted isocratically with a mobile phase consisting of 125 mM ammonium acetate buffer pH 4.75-acetonitrile (76:24, v/v) . A single wavelength at 460 nm was used for detection . Relative standard deviations for the assay ranged from 5.0% to 12.2% for four different drug concentrations and the limit of quantitation was 100 ng/ml . During the validation short term stability of the drug in plasma and stability of PZA on repeated freezing and thawing of plasma was evaluated . Overall recovery of PZA was 88% . This simple assay was found suitable for studying the clinical pharmacokinetics of PZA.

J Chromatogr B Biomed Sci Appl, 1997 Nov 21, 702(1-2), 181 - 91
On the use of ion-pair chromatography to elucidate doxorubicin release mechanism from polyalkylcyanoacrylate nanoparticles at the cellular level; Pepin X et al.; The major hypothesis underlying the remarkable efficiency of polyalkylcyanoacrylate particles loaded with doxorubicin against multidrug resistant tumor cells in vitro, is based on the ion-pair association of doxorubicin with soluble hydrolysis products of polyalkylcyanoacrylate . In an attempt to demonstrate the validity of this hypothesis, we have used ion-pair reversed-phase high-performance liquid chromatography and a laboratory-synthetized compound, i.e., the 2-cyano-2-butylhexanoic acid, as a model for polyalkylcyanoacrylate highly polydispersed degradation products . It is shown that, compared to a counter-ion, like heptane sulfonic acid, 2-cyano-2-butylhexanoic acid exhibits an effective ion-pairing effect at different pH values and organic mobile phase conditions . Moreover, at pH close to physiological conditions and at low mobile phase organic modifier percentage, this effect is experimentally observed, which strongly supports the initial hypothesis.

Antimicrob Agents Chemother, 1998 Jan, 42(1), 135 - 9
Randomized comparison of artemether-benflumetol and artesunate-mefloquine in treatment of multidrug-resistant falciparum malaria; van Vugt M et al.; An open, randomized comparison of artemether-benflumetol (CGP 56 697; Novartis) with artesunate-mefloquine was conducted in 617 patients with acute uncomplicated multidrug-resistant falciparum malaria on the western border of Thailand . Both treatments rapidly and reliably cleared fever and parasitemia, and there was no significant difference in the initial therapeutic response parameters . Parasite genotyping was used to distinguish recrudescences from new infections . The 63-day cure rate for artesunate-mefloquine (94%) was significantly higher than the cure rate for artemether-benflumetol (81%) (P < 0.001) . Both regimens were well tolerated . Nausea, vomiting, dizziness, sleep disorders, and other neurological side effects were between two and four times more common in the artesunate-mefloquine group than in the artemether-benflumetol group (P < 0.001) . Artemether-benflumetol is effective and very well tolerated in the treatment of multidrug-resistant falciparum malaria . A higher dose than that used in the present study may improve efficacy.

Antimicrob Agents Chemother, 1998 Jan, 42(1), 88 - 93
Characterization of mutations contributing to sulfathiazole resistance in Escherichia coli; Vedantam G et al.; A sulfathiazole-resistant dihydropteroate synthase (DHPS) present in two different laboratory strains of Escherichia coli repeatedly selected for sulfathiazole resistance was mapped to folP by P1 transduction . The folP mutation in each of the strains was shown to be identical by nucleotide sequence analysis . A single C-->T transition resulted in a Pro-->Ser substitution at amino acid position 64 . Replacement of the mutant folP alleles with wild-type folP significantly reduced the level of resistance to sulfathiazole but did not abolish it, indicating the presence of an additional mutation(s) that contributes to sulfathiazole resistance in the two strains . Transfer of the mutant folP allele to a wild-type background resulted in a strain with only a low level of resistance to sulfathiazole, suggesting that the presence of the resistant DHPS was not in itself sufficient to account for the overall sulfathiazole resistance in these strains of E . coli . Additional characterization of an amplified secondary resistance determinant, sur, present in one of the strains, identified it as the previously identified bicyclomycin resistance determinant bcr, a member of a family of membrane-bound multidrug resistance antiporters . An additional mutation contributing to sulfathiazole resistance, sux, has also been identified and has been shown to affect the histidine response to adenine sensitivity displayed by these purU strains.

Biochem Pharmacol, 1998 Jan 15, 55(2), 131 - 9
Altered drug membrane permeability in a multidrug-resistant Leishmania tropica line; Chiquero MJ et al.; We selected a Leishmania tropica cell line resistant to daunomycin (DNM) that presents a multidrug-resistant (MDR) phenotype characterized by overexpression of a P-glycoprotein of 150 kDa . The resistant line overexpressed an MDR-like gene, called ltrmdr1, located in an extrachromosomal circular DNA . DNM uptake experiments using laser flow cytometry showed a significant reduction in drug accumulation in the resistant parasites . The initial stages of the interaction of DNM with membranes from wild-type and DNM-resistant parasites were defined by a rapid kinetic stopped-flow procedure which can be described by two kinetic components . On the basis of a previous similar kinetic study with tumor cells, we ascribed the fast component to rapid interaction of DNM with membrane surface components and the slow component to passive diffusion of the drug across the membranes . The results reported here indicate that entrance of DNM into wild-type parasites was facilitated in respect to the resistant ones . We propose that resistance to DNM in L . tropica is a multifactorial event involving at least two complementary mechanisms . an altered drug membrane permeability and the overexpression of a protein related to P-glycoprotein that regulates drug efflux.

Biol Pharm Bull, 1997 Dec, 20(12), 1303 - 6
Potentiation of pirarubicin activity in multidrug resistant cells by rifampicin; Furusawa S et al.; The effect of the anti-tuberculosis drug rifampicin on pirarubicin activity was investigated in multidrug-resistant cells overexpressing P-glycoprotein . Rifampicin increased the sensitivity of pirarubicin to anthracycline-resistant mouse leukemic P388 cells and significantly enhanced the cytotoxicity and intracellular accumulation of pirarubicin in resistant cells, but had no effect in parent cells . By contrast, two other rifamycins, rifamycin B and SV, had no effect on pirarubicin accumulation in resistant cells . Rifampicin also enhanced pirarubicin-induced apoptosis and G2/M blockade on the cell cycle in resistant cells . These results show that rifampicin enhances the cytotoxic action of pirarubicin in resistant cells, at least partly via the inhibition of cellular pirarubicin efflux.

Biol Pharm Bull, 1997 Dec, 20(12), 1257 - 60
Anti-proliferative effect of toremifene and tamoxifen on estrogen receptor-lacking anaplastic thyroid carcinoma cell lines; Kishino T et al.; The antitumor effects of toremifene were studied in vitro using 10 anaplastic thyroid carcinoma cell lines and were compared with those of tamoxifen . The antitumor effect of toremifene was dose-dependent and the IC50 values for these cell lines ranged 20.1-58.5 microM . Although the cell lines expressed multidrug resistance gene mRNA, multidrug resistance-associated protein gene mRNA and insulin-like growth factor-1 (IGF-1) receptor mRNA to various degrees, nine of them lacked estrogen receptor expression as evaluated by immunocytochemistry . These data indicate that toremifene, as well as tamoxifen, is cytolytic at relatively high doses for multidrug-resistant anaplastic thyroid carcinoma cell lines.

Bull World Health Organ, 1997, 75(5), 477 - 89
Practical and affordable measures for the protection of health care workers from tuberculosis in low-income countries; Harries AD et al.; With the global upsurge in tuberculosis (TB), fueled by the human immunodeficiency virus (HIV) pandemic, and the increase in multidrug-resistant TB, the condition has become a serious occupational hazard for health care workers worldwide . Much of the current understanding about nosocomial TB transmission stems from the USA; however, little is known about the risk of such transmission in low-income countries . The focus of this review is on sub-Saharan Africa, since this is the region with the highest TB incidence, the highest HIV incidence, the worst epidemic of HIV-related TB, and where the risk to health care workers is probably greatest . Measures used in industralized countries to control nosocomial TB transmission (ventilation systems, isolation rooms, personal protective equipment) are beyond the resources of low-income countries . Protecting health care workers in these settings involves practical measures relating to diagnosis and treatment of infectious cases; appropriate environmental control; and relevant personal protection and surveillance of health care workers . Research needs to be carried out to examine the feasibility and cost-effectiveness of measures such as voluntary HIV-testing of health care workers (to enable known HIV-positive health care workers to avoid high-risk settings) and isoniazid preventive therapy for workers in high-risk settings . More resources are also needed to ensure full implementation of currently recommended measures to decrease the risk of nosocomial and laboratory-acquired TBPIP: This review considers the occupational hazard to health care workers posed by the global increase in tuberculosis (TB), especially in multidrug-resistant TB . The review opens by focusing on the following aspects of TB in industrialized countries: the risk of TB among health care workers, reasons for the increase in nosocomial TB, which health care workers are most at risk, and effective interventions to reduce nosocomial TB transmission . Next, the review turns to developing countries, using Africa South of the Sahara as an example and considering the impact of AIDS and TB and the risk of nosocomial transmission to health workers . The review then notes that the measures used to control nosocomial TB in developed countries are not generally affordable in developing countries and discusses the following 1993 guidelines offered by the World Health Organization to address this problem as well as practical measures introduced in some African hospitals that 1) improve the diagnosis and treatment of TB patients, 2) involve appropriate environmental control, and 3) institutionalize the means of personally protecting health care workers . It is concluded that operations research is needed to test and evaluate inexpensive, sustainable, and cost-effective control measures in low-resource settings and that health care workers are a vital resource and must be protected against this occupational risk .

Blood, 1998 Feb 1, 91(3), 1001 - 7
Cyclosporin A induces apoptosis in childhood acute lymphoblastic leukemia cells; Ito C et al.; In an effort to identify novel antileukemic agents that can bypass the mechanisms of multidrug resistance, we found that cyclosporin A ({CyA} 5 mumol/L) produced a median cell kill of 69% (range, 47% to 85%) in seven B-lineage acute lymphoblastic leukemia (ALL) cell lines (OP-1, SUP-B15, KOPN-55bi, RS4;11, NALM6, REH, and 380) and three T-lineage ALL cell lines (MOLT4, CCRF-CEM, and CEM-C7) after 4 days of culture . At 10 mumol/L, median CyA toxicity was 99% (range, 88% to > 99%) . CyA was equally toxic to both a multidrug-resistant cell line, CEM-VLB100, which overexpresses gp-170 P-glycoprotein, and one resistant to topoisomerase II inhibitors, CEM-VM1-5, which has a mutation in the topoisomerase II gene . CyA was also toxic to primary leukemic cells maintained in stroma-based culture, a system that substantially prolongs in vitro cell survival . Against lymphoblasts from 21 patients with B-lineage ALL, the compound (at 5 mumol/L) reduced the leukemic cell number by a median of 87% (range, 27% to > 99%) compared with results for parallel control cultures lacking CyA . Seven of these samples were from cases with unfavorable genetic features (e.g., Philadelphia-chromosome or MLL gene rearrangements); three were obtained at relapse . Against T lymphoblasts (from six patients), the median reduction in cell number was 79% (range, 30% to > 99%) . At 10 mumol/L, the cell kill exceeded 97% in all cases studied . The mechanism of CyA cytotoxicity was found to be the activation of apoptosis, which was suppressed by phorbol myristate acetate but not by inhibitors of ceramide-mediated apoptosis, phosphatidyl inositol-3 kinase activity, or tyrosine kinase activity . These findings demonstrate high levels of CyA-induced toxicity against ALL cells at concentrations achievable in vivo, thus providing a strong rationale for clinical testing of this agent in patients with ALL.

Tumori, 1997 Sep-Oct, 83(5 Suppl), S21 - 4
{SDZ PSC 833: a novel modulator of MDR}; Covelli A; SDZ PSC 833 is a novel compound able to reverse the resistance to chemotherapy of cancer cells with the multidrug resistance (MDR) phenotype by inhibiting the 170 kd P-glyco-protein (P-gp) . In vitro studies show that SDZ PSC 833 directly interacts with, but is not transported by P-gp, although the exact mechanism of action has not yet been defined . In cells with the MDR phenotype, intracellular concentration of various P-gp-transported anticancer drugs is restored to the same level as in sensitive cells by SDZ PSC 833 concentrations of 0.8 microM to 3.0 microM . In vivo SDZ PSC 833 was highly active in potentiating the anti-tumour activity of all tested anticancer drugs (ACs) in both sensitive and MDR tumours . Sensitivity of non-MDR tumours was increased by SDZ PSC 833 through pharmacokinetic interactions, that result in enhanced area-under-the-curve (AUC) of P-gp-transported ACs . However, an increased AC bioavailability is not sufficient to explain the therapeutic benefit of SDZ PSC 833 co-treatment in MDR tumour-bearing mice: in these animals, no survival increase could be achieved with the AC alone by simply increasing the cytotoxin dosage up to doses that were severely toxic for the non-tumour-bearing mice . In a series of phase I/II studies, the recommended doses of SDZ PSC 833 were established at: 10 mg/kg/day i.v . as a 24-hour continuous infusion after a 2 mg/kg loading dose as a 2-hour infusion; 20 mg/kg orally divided four times daily in solid tumours or 16 mg/kg orally divided four times daily in multiple myeloma . The dose limiting toxicity of SDZ PSC 833 is ataxia, which appears to be reversible and dose-related . Moreover, a predictable change in pharmacokinetic parameters of concomitantly administered P-gp-transported AC(s) which usually necessitate a 30-60% reduction from the standard dose of the AC in order to maintain the same time-exposure and dose-related toxicity of the cytotoxic drug alone . The results of experiments both in vitro and in vivo suggested that adequate blood levels (i.e . > or = 1.0 microM) of SDZ PSC 833 must be reached before and maintained during the administration of concomitant AC(s), in order to maximally reverse MDR . At the recommended doses, blood concentrations exceeding 1000 ng/mL (1.0 microM) can be achieved after both i.v . and oral administration . Indeed, SDZ PSC 833 concentrations that fully reverse MDR in vitro are achievable in vivo, plasma samples from patients treated with SDZ PSC 833 restored the sensitivity of MDR human sarcoma cells to paclitaxel, etoposide and doxorubicin . Clinical studies completed so far aimed first to determine the dose of both SDZ PSC 833 and the concomitant AC(s) to be used in ongoing pivotal trials . These studies accrued advanced stage cancer patients, however, tumour responses have been observed in both solid and hematological tumours . The in vitro finding that treatment with SDZ PSC 833 may suppress the activation of the MDR1 gene and prevent the emergence of resistant cancer cell clones with the MDR phenotype might support the use of this MDR modulator in earlier stages of disease.

Tumori, 1997 Sep-Oct, 83(5 Suppl), S17 - 20
{Treatment of multidrug resistance in oncology and hematology}; Petrini M et al.; BACKGROUND: Drug resistance, which often occurs also during chemotherapy, is yet a great obstacle to the success of the human malignancies treatments . Among many possible mechanisms of drug resistance (biological, biochemical, kinetic or pharmacological), both typical and atypical multidrug-resistance (MDR) have been extensively studied . Multidrug resistance is the phenomenon whereby the development of resistance to one drug is accompanied by the simultaneous development of resistance to a variety of structurally unrelated drugs . Typical MDR seems to depend on the expression of an 170 KD protein, the P-170 or P-glycoprotein, that acts as an energy-dependent pump removing possible toxic agents, including antiblastic drugs, from the cell . High levels of the mdr1 expression have been found in normal adrenal gland, kidney, colon, jejunum and liver, whereas low levels were detected in skin, muscle, brain, nervous system and bone marrow, where CD34+ stem cells are P-170 positive . Moreover, heat shock proteins or oncogene transfection, cell differentiation or proliferation could modulate the P-glycoprotein expression: low protein levels were described in resting B lymphocytes and monoblast cells, whereas plasma cells and monocytes express higher P-170 levels . On the contrary, more differentiated myeloid cells show a low MDR1 expression . METHODS: Since normal kidney and haematopoietic cells physiologically express P-170 protein, 20 renal cell carcinomas at the surgery and many hematological patients were evaluated in this study, including acute leukemias, chronic myeloid, chronic lymphocytic leukemias and multiple myeloma . Moreover we evaluated the MDR expression in 23 non small cell lung cancers . The MDR1 gene expression was showed by RT-PCR assays, whereas the P-170 protein was detected by immunological and cytofluorometric reactions employing the C-219 and JSB1 monoclonal antibodies . RESULTS AND CONCLUSIONS: Of the 32 acute leukemia patients, 15 (47%) resulted P-170 positive; 14 out of the 15 positive, but 12 out of the 17 negative cases were resistant to the chemotherapy, with an higher positivity in monoblastic types; on the contrary, all lymphoblastic leukemias resulted P-170 negative and only 2 of 13 patients resulted drug resistant . A very high MDR expression was showed in chronic disorders: in the lymphocytic ones, only samples CD5/CD19 negative (3 of 42) resulted P-170 negative, confirming the basal MDR phenotype expression . In these cases, nevertheless, the protein expression was not related to the treatment response . Also in the multiple myeloma patients, no relations between clinic parameters and P-170 expression were found; however a significant relationship between treatment response and P170 expression was found . In kidney and lung cancer samples examined, a low percentage of positive samples was detected without any relationship to the evaluated clinical parameters.

Leuk Res, 1997 Nov-Dec, 21(11-12), 1077 - 86
The MDR1 (P-glycoprotein) and MRP (P-190) transporters do not play a major role in the intrinsic multiple drug resistance of Jurkat T lymphocytes; Martel J et al.; The response of T cells in relation to the cell cycle has not been extensively studied . We have attempted to address this question using Jurkat T cells treated with cytostatic drugs known to arrest cells at various transition points of their cycle . We tested several concentrations of drugs that act at G1/S (hydroxyurea, lovastatin, thymidine), early S (aphidicolin, cyclosporin A, rapamycin) or G2+M (colchicine, nocodazole) in 24 h cultures . Cytofluorimetric analyses showed that cycling Jurkat cells were equally distributed between the G1 (44.9 +/- 6.5%) and S (42.3 +/- 8.0%) phases . Cell distribution in G2+M was 12.7 +/- 2.8% . Hydroxyurea but not lovastatin increased the percentage of cells in S phase to approximately 60-70% and both drugs decreased it to approximately 30% in G1 . Thymidine had no effects . Aphidicolin increased the distribution in S phase to approximately 70% with a decrease in G1 to approximately 30% . Cyclosporin A and rapamycin increased the percentage of the cells in G1 to approximately 70% and decreased it to approximately 25% in S phase . Nocodazole increased cell distribution in G2+M to approximately 60% and induced a decrease in G1 to approximately 10% . The effects of the drugs were not related to their toxicity and their limited efficiency raised the possibility that Jurkat cells possessed an intrinsic resistance to these xenobiotics . Time-course analysis showed (scanning electron microscopy) that the early morphological changes induced by colchicine were reversible . Drug efflux experiments (vinblastine) suggested that an ATP-dependent process could be involved . However, Northern blot analyses showed a weak signal for MDR1 (P-glycoprotein) . In contrast, a probe for MRP (P-190) showed a strong signal in Jurkat and peripheral lymphocytes . The presence of drugs (cyclosporin A, nocodazole, thymidine) (24 h) did not upregulate its message and cell treatment with DL-butathione (S,R)-sulfoximine only moderately affected the efficiency of the glutathione S-conjugate MRP transporter . Our data suggest that the intrinsic multidrug resistance of leukemic Jurkat T cells does not appear to involve the MDR1 and MRP members of the ABC family of reverse drug transporters and these observations raise the possibility of the involvement of multifaceted mechanisms.

Southeast Asian J Trop Med Public Health, 1997 Jun, 28(2), 387 - 90
Kat G mutations in isoniazid resistant Mycobacterium tuberculosis isolates from Thai patients; Paca-uccaralertkun S et al.; Isoniazid resistant mechanisms in Mycobacterium tuberculosis have been shown to involve at least two genes, kat G and inh A . Alteration in the kat G gene has been found in a great number of resistant isolates . Percentage of resistant isolates harboring alteration in this gene varied among laboratories suggesting that different mutations were presented in different geographic areas . Fourteen isoniazid resistant and five multidrug resistant isolates from the Central Chest Hospital, Thailand, were examined for the kat G gene mutations in the region between base position 17 to 299 . No different pattern of mutations were found between these two groups . Among nineteen isolates, there were nine isolates which showed point mutations and five isolates with base insertions of the kat G gene . The remaining five isolates revealed gene deletion . Heteroduplex formation technique also confirmed base alterations in these nine mutants.

Mol Pharmacol, 1998 Jan, 53(1), 141 - 7
Kinetics of anthracycline efflux from multidrug resistance protein-expressing cancer cells compared with P-glycoprotein-expressing cancer cells; Marbeuf-Gueye C et al.; The multidrug resistance protein (MRP) has been shown to mediate ATP-dependent efflux of anticancer agents of diverse structure, such as daunorubicin (DNR), vincristine and etoposide . Thus, this protein does confer a multidrug resistant phenotype to cancer cells, similar to P-glycoprotein (Pgp) . The substrate specificity of both transporter proteins is partly overlapping but is otherwise very distinct; because MRP is a multiple organic anion transporter, it transports certain glutathione conjugates and may be partly dependent on intracellular glutathione levels for the transport of anthracyclines . We have studied the transport kinetics of a series of anthracyclines in MRP and Pgp that overexpress tumor cell lines to obtain information on the substrate specificity of these proteins . The anthracyclines have modifications in the sugar moiety . The mean active efflux coefficient Ka, used to characterize the efficiency of the active efflux, was very similar for DNR and one of its 4'-deoxy-derivatives (eso-DNR) for MRP and Pgp {10-20 x 10(-10)/sec/(cells/ml)} . The permanently neutral derivatives 3'-deamino-3'-hydroxy-doxorubicin (OH-DOX) and 3'-deamino-3'-hydroxy-daunorubicin (OH-DNR) were effluxed by both proteins but had a lower Ka {2 x 10(-10) and 6 x 10(-10)/sec/(cells/ml) (OH-DOX)} and 2 x 10(-10) and 5 x 10(-10)/sec/(cells/ml) (OH-DNR)} for MRP and Pgp . Two anthracyclines, the doxorubicin derivative pirarubicin and 2'-bromo-4'-epi-DNR seemed to have a slightly higher Ka value for Pgp than for MRP . The apparent Michaelis-Menten constants (K(m)) and maximal efflux rates (VM) for the active transport were within a narrow range for both transporters, except for OH-DOX and OH-DNR, which had a lower VM in the case of MRP-mediated transport, suggesting a role of the amino group in the interaction with glutathione . Determination of the Hill coefficient (nH) of the MRP-mediated efflux gave most values close to 2, which suggests cooperativity of the transport of anthracyclines as reported before for Pgp . In conclusion, the transport kinetics of anthracyclines by MRP and Pgp are very similar.

J Nucl Med, 1998 Jan, 39(1), 91 - 4
Technetium-99m-MIBI uptake in small cell lung cancer; Bom HS et al.; Patients with small cell lung cancer (SCLC) often fail to respond to chemotherapy due to multidrug resistance (MDR) . Technetium-99m-MIBI was reported to be a suitable transport substrate of P-glycoprotein, which is a cytoplasmic membrane protein encoded by the MDR gene . The purpose of this study was to evaluate whether or not the degree of MIBI uptake in SCLC or its retention on delayed imaging correlated with response to chemotherapy . METHODS: Twenty-five patients (19 men, 6 women; mean age 59 +/- 10 yr) with biopsy-proven SCLC had MIBI SPECT 3-7 days before starting chemotherapy . Imaging was acquired 1 and 4 hr after injection of 740 MBq MIBI using a single-head rotating gamma camera . Tumor-to-normal lung uptake ratio (T/NL) was measured . Percent retention (%R) was measured as: %R = 100 x (T/NL at 4 hr)/(T/NL at 1 hr) . All patients received VAP chemotherapy (VP-16 100 mg/m2, adriamycin 40 mg/m2, cisplatin 25 mg/m2) every 4 wk for at least three times . Response to chemotherapy was grouped as complete remission, partial remission and no remission according to the change of tumor size on chest radiograph and CT images . Differences in T/NL and %R among the three groups were analyzed using ANOVA . RESULTS: T/NL of patients with complete remission (n = 7) and partial remission (n = 10) were significantly higher than that of no remission (n = 8) in 1 hr and 4 hr . T/NL at 1 hr in three groups were 2.75 +/- 0.78, 2.35 +/- 0.31 and 1.65 +/- 0.36, respectively . T/NL at 4 hr in three groups was 2.61 +/- 0.94, 2.48 +/- 0.50 and 1.66 +/- 0.42, respectively . However, %R was not different among three groups . Percent retention in three groups was 109.40 +/- 22.10, 96.71 +/- 14.25 and 103.59 +/- 28.43, respectively . CONCLUSION: SCLC with a higher MIBI uptake was more likely to respond to chemotherapy than that with a lower uptake . However, there was a considerable overlap of MIBI uptake among subjects . No significant correlation between the MIBI retention between 1 hr and 4 hr, and the response to chemotherapy was noted.

J Nucl Med, 1998 Jan, 39(1), 77 - 86
Novel technetium (III)-Q complexes for functional imaging of multidrug resistance (MDR1) P-glycoprotein; Crankshaw CL et al.; Overexpression of the multidrug resistance (MDR1) P-glycoprotein (Pgp) correlates with cancer chemotherapeutic failure . Lipophilic cationic radiopharmaceuticals such as 99mTc-sestamibi, 99mTc-tetrofosmin and 99Tc-furifosmin (Tc-Q12) have been validated as transport substrates for the MDR1 Pgp and may enable functional imaging of the MDR phenotype in cancer by observing enhanced washout rates of the tracers in those tumor areas expressing Pgp . To further explore and optimize the Pgp recognition properties of Schiff base phosphine mixed-ligand complexes of the Tc-Q series of nonreducible (Tc(III) cations, a variety of Tc-Q complexes were synthesized and tested in vitro for recognition as transport substrates by the human MDR1 Pgp . METHODS: Tracer assays with human drug-sensitive KB-3-1 epidermal carcinoma and MDR KB-8-5 cells expressing nonimmunodetectable and modest levels of MDR1 Pgp, respectively, were used to screen and pharmacologically characterize 37 novel 99mTc-Q analogs . RESULTS: The ideal agent should have low nonspecific binding, high distinction in net uptake between drug-sensitive cells and MDR tumor cells, and high enhancement of uptake in resistant cells after treatment with an MDR modulator, indicating selective blockade of Pgp-mediated efflux of the radiotracer . Three analogs, trans-{5,5'-(1,2-ethanediyldiimino)bis(2-OEt-2-Me-4-penten-3 -one)}bis{dimethyl(3-OMe-1-propyl)phosphine}99mTc(III) (99mTc-Q63) and two trans-{bis(methyl-bis(3-OMe-1-propyl)phosphine)} analogs (99mTc-Q57 and 99mTc-Q58) displayed transport distinctions between drug-sensitive and MDR cell lines that were equal to or greater than all previously available agents . Cyclosporin A, an MDR modulator, had no significant effect in KB-3-1 cells for these 99mTc-complexes but enhanced tracer accumulations in KB-8-5 cells with IC50 values of approximately 1 microM . In contrast, the non-MDR agents methotrexate and cisplatin had no effect on accumulation of 99mTc-Q complexes and 99mTc-sestamibi in KB-8-5 cells . CONCLUSION: Technetium-99m-Q57, 99mTc-Q58 and 99mTc-Q63 are avid transport substrates recognized by the human MDR1 Pgp, and have enhanced in vitro properties that may enable functional imaging of Pgp in vivo with improved signal-to-noise ratios and tissue contrast compared to currently available agents.

Urol Res, 1997, 25(6), 407 - 12
Correlation of expression levels of P-glycoprotein with resistance to adriamycin in a renal adenocarcinoma cell line; Kawamoto S et al.; We have demonstrated that low-level expression of P-glycoprotein (PGP), detectable by reverse transcriptase polymerase chain reaction (RT-PCR) assay and flow cytometric assay, is an important factor in multidrug resistance (MDR) in ACHN cancer cells . In this study, we established a subline highly resistant to adriamycin (ACHN/ADM) from ACHN cells, and determined the correlation between PGP levels and MDR levels using ACHN/ADM cells and their parent ACHN cells . The ACHN/ADM cells showed overexpression of PGP, and sensitivity to antitumor agents was lower than that found in ACHN cells . Intracellular accumulation of ADM in ACHN/ADM cells was approximately half the amount of its accumulation in ACHN cells . Sensitivity to ADM in ACHN/ADM cells was enhanced by chemosensitizers with an increase in intracellular ADM accumulation . These results indicate that PGP levels correlate with MDR levels and suggest that chemotherapy using chemosensitizers might be effective in the treatment of renal cancers with overexpression of PGP.

Cancer Chemother Pharmacol, 1997, 41(1), 48 - 52
Pharmacokinetics of the multidrug-resistance-converting drug dexniguldipine and its pyridine metabolite M-1 in the plasma, tumor, and renal tissue of tumor-bearing Wag/Rij rats; Schellens JH et al.; The pharmacokinetics of oral dexniguldipine, a new multidrug-resistance-modifying agent under clinical evaluation, and its pyridine metabolite M-1 were determined in plasma, tumor, and renal tissue in Wag/Rij rats bearing a multidrug-resistant CC531 colon adenocarcinoma tumor under the renal capsule . The pharmacokinetics were studied in four experiments . After a single administration of dexniguldipine (30 mg/kg), tumors and kidneys were collected after 5 (experiment 1), 24 (experiment 2), and 48 h (experiment 3) . In the fourth experiment, dexniguldipine was given once daily for 3 consecutive days at a dose of 30 mg/kg . In all experiments, plasma samples were collected at regular intervals . The concentrations of dexniguldipine and M-1 could be determined in plasma in most of the rats at up to 32 h after drug administration . The area under the curve (AUC) of dexniguldipine and M-1 varied by a factor of 2-6 in the four experiments . High tumor-tissue concentrations of dexniguldipine were observed . The concentrations were highest in the multiple-dose experiment (2014 +/- 1005 ng/g tissue) . High degrees of correlation (> 0.8) were established between the concentrations of dexniguldipine measured in plasma and tumor as well as renal tissue . Overall, tumor-tissue concentrations of M-1 comprised one-third of the dexniguldipine concentrations measured.

Cancer Res, 1998 Jan 15, 58(2), 256 - 62
BCL-X expression in multiple myeloma: possible indicator of chemoresistance; Tu Y et al.; Because murine myeloma plasma cells and normal human lymph node plasma cells express BCL-X, we evaluated BCL-X expression in malignant human plasma cells . BCL-X expression was detected in several human myeloma cell lines, as well as in CD38-sorted bone marrow cells obtained from some patients . Only the antiapoptotic long form of BCL-X (BCL-X-L), was detected . Because BCL-X-L expression can protect tumor cells from apoptotic death induced by chemotherapeutic agents, we tested the clinical relevance of expression in 55 archival bone marrow biopsies . The biopsies were stained by immunohistochemistry, and BCL-X expression was correlated with the subsequent response to treatment . BCL-X expression in malignant plasma cells strongly correlated with decreased response rates in patient groups treated with either melphalan and prednisone or vincristine, Adriamycin, and dexamethasone . Response rates were 83-87% in non-BCL-X-expressing cases and 20-31% in BCL-X-expressing cases . In addition, BCL-X expression was more frequent in specimens taken from patients at relapse (77%), when compared to those at initial diagnosis (29%) . Further support for the association of drug resistance with BCL-X-L expression came from studies of the 8226 dox-40 cell line . This line, which expresses p-glycoprotein and serves as a model of multidrug resistance in multiple myeloma cells, demonstrated an up-regulated expression of BCL-X-L, which was relatively specific, in that BCL-2 or BAX expression was not altered . In addition, dox-40 cells demonstrated a generalized resistance to apoptosis that was induced by several different agents . These results indicate that malignant plasma cells can express BCL-X-L and that such expression may be a marker of chemoresistant disease.

J Biol Chem, 1998 Jan 23, 273(4), 2098 - 104
Divergent transcriptional control of multidrug resistance genes in Saccharomyces cerevisiae; Hallstrom TC et al.; Improper control of expression of ATP binding cassette transporter-encoding genes is an important contributor to acquisition of multidrug resistance in human tumor cells . In this study, we have analyzed the function of the promoter region of the Saccharomyces cerevisiae YOR1 gene, which encodes an ATP binding cassette transporter protein that is required for multidrug tolerance in S . cerevisiae . Deletion analysis of a YOR1-lacZ fusion gene defines three important transcriptional regulatory elements . Two of these elements serve to positively regulate expression of YOR1, and the third element is a negative regulatory site . One positive element corresponds to a Pdr1p/Pdr3p response element, a site required for transcriptional control by the homologous zinc finger transcription factors Pdr1p and Pdr3p in other promoters . The second positive element is located between nucleotides -535 and -299 and is referred to as UASYOR1 (where UAS is upstream activation sequence) . Interestingly, function of UASYOR1 is inhibited by the downstream negative regulatory site . Promoter fusions constructed between UASYOR1 and the PDR5 promoter, another gene under Pdr1p/Pdr3p control, are active, whereas analogous promoter fusions constructed with the CYC1 promoter are not . This suggests the possibility that UASYOR1 has promoter-specific sequence requirements that are satisfied by another Pdr1p/Pdr3p-regulated gene but not by a heterologous promoter.

Semin Cancer Biol, 1997 Jun, 8(3), 205 - 13
Do cMOAT (MRP2), other MRP homologues, and LRP play a role in MDR?
Borst P, Kool M, Evers R.
The discovery of the Multidrug Resistance-associated Protein (MRP or MRP1) as a GS-X pump able to transport both anionic drug conjugates and unmodified anti-cancer drugs out of the cell, has raised the question whether other members of the MRP family might contribute to drug resistance of human tumours . The most extensively studied member of this family is cMOAT, the canalicular Multispecific Organic Anion Transporter . The substrate specificity of this pump was originally defined by an inborn error in rats, lacking this protein . These rats are mildly hyperbilirubinemic, because of their inability to secrete bilirubin glucuronides into their bile . In addition, they have diminished capacity to secrete a variety of other organic anions . Absence of cMOAT in humans results in an analogous inborn error of metabolism, the Dubin-Johnson syndrome . Attempts to determine the effect of cMOAT on the sensitivity of cells to anti-cancer drugs have run into technical problems . Most cells transfected with a cMOAT cDNA construct and overproducing cMOAT seem unable to transport the protein to the cell surface and are not MDR . However, in polarized kidney cell monolayers cMOAT is correctly routed to the apical cell surface and able to transport vinblastine . Hence, overexpression of cMOAT in cancer cells could potentially lead to drug resistance . In studies of cells selected for drug resistance no correlation was found thus far between cMOAT overexpression and MDR, but there was a positive association with cisplatin resistance, raising the possibility that cMOAT might contribute to cisplatin resistance by mediating excretion of cisplatin-glutathione complexes . This remains to be verified by more direct experiments and clinical studies, however . Database searches have yielded four additional MRP family members, MRP3-6 . The physiological functions of these putative transporters are not yet known and whether they can contribute to drug resistance needs to be determined . Another putative transporter found in many MDR cells not overproducing P-glycoprotein is the Lung Resistance Protein (LRP), which is the major vault protein . Scheper et al have detected LRP in many MDR cell lines and they have shown that elevated LRP values are a strong and independent predictor of unfavourable outcome for several types of drug-treated human tumours . LRP is a cytoplasmic protein and attempts to demonstrate its involvement in drug transport have failed thus far . The possibility that this protein is only an indicator of resistance caused by upregulation of other proteins, rather than a drug transporter, remains open.

Semin Cancer Biol, 1997 Jun, 8(3), 193 - 204
Function, evolution and structure of multidrug resistance protein (MRP); Deeley RG et al.; Multidrug Resistance Protein (MRP) confers resistance to natural product drugs when overexpressed in cultured cells . It has also been detected in human tumors and in some cases, expression has been correlated with a poor response to chemotherapy . MRP is present in normal tissues where it probably functions as an active transporter of amphiphilic anions . It is also presumed to transport the drugs to which it confers resistance, but how and in what form has not been resolved . Unlike other members of the ATP Binding Cassette superfamily, MRP and several related proteins have three potential membrane spanning domains . The additional NH2-proximal domain in MRP contains five membrane spanning helices with an extracytosolic NH2-terminus and is essential for transport . Conserved features of gene organization and protein structure suggest that MRP and its related proteins share their ancestry with the cystic fibrosis conductance regulator.

Semin Cancer Biol, 1997 Jun, 8(3), 143 - 50
ATP hydrolysis cycles and mechanism in P-glycoprotein and CFTR; Senior AE et al.; P-glycoprotein and CFTR are prominent members of the ABC Transporter family . Both use ATP, the former to drive extrusion of drugs from cells and confer multidrug-resistance, the latter to drive opening and closing of anion channels . We compare current working models of catalytic cycle and mechanism of the two proteins . In Pgp the NBDs appear functionally equivalent, in CFTR they appear functionally distinct . In both proteins, ATP hydrolysis occurs in both NBDs, and it is proposed that the two NBDs alternate in catalysis . Other differences and similarities are noted, fostering ideas for future developments.

Semin Cancer Biol, 1997 Jun, 8(3), 135 - 42
Structure of the multidrug resistance P-glycoprotein; Higgins CF et al.; In order to elucidate the mechanism by which the multidrug resistance P-glycoprotein extrudes cytotoxic drugs from the cell, and particularly the number and nature of the drug binding site(s), knowledge of the structure of P-gp is essential . A considerable body of genetic and biochemical data has accrued which gives insights into P-gp structure and function . These data are critically reviewed, particularly in relation to the low resolution structure of P-gp which has recently been determined by electron microscopy . P-gp is one of the best characterised of the ABC transporters and these structure-function studies may have more general implications.

Int J Tuberc Lung Dis, 1997 Oct, 1(5), 405 - 10
Anti-tuberculosis drug resistance in Madagascar in 1994-1995; Chanteau S et al.; SETTING: A new tuberculosis control programme has been implemented in Madagascar since 1991 . A survey on Mycobacterium tuberculosis resistance to the major drugs was conducted between August 1994 and December 1995 . OBJECTIVE: To determine primary and acquired resistance in pulmonary tuberculosis patients in four main cities . DESIGN: Were included 401 randomly sampled new smear positive patients (36.2% of declared new patients) and 137 recurrent cases (72.9% of declared cases) from 8 centres . Drug susceptibility testing was performed on Lowenstein Jensen medium according to the proportion method . RESULTS: The male to female ratio was 1.35:1 in new patients (age range 11-74 years) and 1.98:1 in recurrent patients (age range 16-76 years) . The primary resistance rate to any drug was 20% (95% Confidence Interval {CI} 16-23) and the acquired resistance rate 40% (95% CI 32-48, P < 2.10(-7) . Primary resistance to one drug was 18% (95% CI 15-22), mainly attributable to streptomycin resistance (14.5%) . Multidrug resistance (MDR) to isoniazid and rifampicin was 0.25% (95% CI 0-0.7) for primary resistance and 5% (95% CI 2.6-10.6) for secondary resistance . No difference was observed between sexes or ages . CONCLUSION: This survey conducted in big cities gives a very negative picture of resistance in Madagascar.

Int J Tuberc Lung Dis, 1997 Apr, 1(2), 187 - 90
Cycloserine-induced Stevens-Johnson syndrome in an AIDS patient with multidrug-resistant tuberculosis; Akula SK et al.; We report a case of cycloserine-induced Stevens-Johnson syndrome (SJS) in a 38-year-old male with the acquired immune deficiency syndrome (AIDS) and multidrug resistant tuberculosis (MDR-TB) . The patient developed a cutaneous reaction after 60 days of therapy with ofloxacin, streptomycin (SM), pyrazinamide (PZA), ethambutol (EMB), and cycloserine (CSN) . All drugs were stopped and the rash improved . Due to the severity of his disease, anti-tuberculosis drugs were resumed, one at a time . The patient developed a recurrent rash consistent with SJS, which began when CSN was restarted . CSN was stopped and the SJS began to gradually resolve with palliative treatment despite continuation of the other anti-tuberculosis drugs . However, the patient's overall condition gradually deteriorated and he died . To our knowledge, this is the first case of probable CSN-related SJS.

Int J Tuberc Lung Dis, 1997 Feb, 1(1), 59 - 63
Nature of drug resistance and predictors of multidrug-resistant tuberculosis among patients seen at the Philippine General Hospital, Manila, Philippines; Mendoza MT et al.; SETTING: This study was conducted at the University of the Philippines--Philippine General Hospital (UP-PGH), Manila, Philippines . OBJECTIVES: To determine the nature of drug resistance among patients with pulmonary tuberculosis (PTB), and to establish clinical predictors of drug-resistant tuberculosis . DESIGN: Descriptive, prospective study . METHODS: Patients with positive culture for Mycobacterium tuberculosis were interviewed regarding past history of anti-tuberculosis treatment, BCG vaccination, chest X-ray and family contact . M . tuberculosis isolates from 299 patients were tested for susceptibility to rifampicin, isoniazid (INH), ethambutol and streptomycin using the submerged disc proportion method . Pyrazinamide (PZA) susceptibility test was done with standard laboratory powder in 7H10 media . RESULTS: Of the 299 M . tuberculosis isolates, 17% were fully susceptible to the 5 primary drugs and 54% were resistant to 2 or more drugs (multidrug resistant TB {MDR-TB}) . Initial drug resistance rate was high with ethambutol (39%) and INH (17%) . Previous history of anti-tuberculosis treatment was significantly associated with MDR-TB (Odds Ratio {OR} 2.44, 95% confidence interval) . Incomplete anti-TB treatment taken for longer than 3 months increased the likelihood of MDR-TB (OR 4.6, P < 0.0001) . CONCLUSION: The high rate of MDR-TB was associated with previous anti-tuberculosis treatment . The chance of developing MDR-TB was significantly increased when inadequate prior treatment was given for more than 3 months.

J Thorac Imaging, 1998 Jan, 13(1), 65 - 71
Radiographic findings and patterns in multidrug-resistant tuberculosis; Fishman JE et al.; Multidrug-resistant tuberculosis (MDR TB) is prevalent in urban areas with large HIV-positive populations . We retrospectively evaluated the chest radiographs of MDR TB patients at presentation and compared them to patients with drug-sensitive tuberculosis (DS TB) . Although the overall radiographic findings and patterns of MDR TB and DS TB were similar, there were significant differences among the MDR TB patients depending on how MDR TB was acquired . Patients who developed MDR TB during an outbreak showed noncavitary consolidations, pleural effusions, and a primary radiographic pattern (70%) . On the other hand, patients who acquired MDR TB due to noncompliance with antituberculous therapy often had cavitary consolidations (50%) and generally demonstrated a postprimary radiographic pattern . Cavitation occurred equally in patients with MDR TB who are HIV positive regardless of CD4 cell count . Chest radiographic findings and patterns in MDR TB are most accurately interpreted in conjunction with clinical history, specifically prior TB treatment . Nevertheless, approximately one-third of patients did not show the "expected" radiographic pattern.

J Photochem Photobiol B, 1997 Nov, 41(1-2), 136 - 44
A hydroxypyridinone (CP94) enhances protoporphyrin IX formation in 5-aminolaevulinic acid treated cells; Bech O et al.; Different cell lines were given photodynamic treatment with 5-aminolaevulinic acid (ALA) and light . In addition, the iron chelator 1,2-diethyl-3-hydroxypyridin-4-one (CP94) was used . The porphyrin species produced was spectrofluorimetrically identified as protoporphyrin IX . All the cell lines responded to treatment, including a multidrug resistance gene expressing bladder cancer line and, to a lesser degree, cells derived from untransformed human skin fibroblasts . CP94 enhanced both porphyrin fluorescence, total porphyrin content and photosensitivity of the cells . CCD fluorescence microscopy showed a granular extranuclear porphyrin fluorescence distribution for all the cell lines involved, but the untransformed cells showed a distribution pattern different from the ones seen in the other cells.

Jpn J Cancer Res, 1997 Nov, 88(11), 1100 - 7
Combination therapy with antibody and interleukin-2 gene transfer against multidrug-resistant cancer cells; Shinohara T et al.; In the present study, we examined the effect of interleukin-2 (IL-2) gene transfer into multidrug resistance (MDR) cancer cells on the therapeutic efficacy of MRK16 . Human MDR ovarian cancer cells, AD10, were transduced with a bicistronic IL-2 retrovirus, Ha-IL2-IRES-Neo . The G418-resistant population, IL2-AD10, secreted IL-2 into the culture supernatant and did not form a tumor mass in nude mice . The IL2-AD10 cells were more susceptible to the cytotoxicity of murine spleen cells than AD10 cells in vitro . For examination of the effect of IL-2 gene transfer on the antitumor activity of MRK16 against P-glycoprotein-positive tumors, IL2-AD10 cells were co-transplanted s.c . with AD10 cells into nude mice in a ratio of 1:3, and the mice were treated with MRK16 on days 2 and 7 . MRK16 markedly inhibited the growth of AD10 cells mixed with IL2-AD10 cells under conditions (0.3-1 microgram/body) where it showed only marginal effects on the growth of AD10 tumors . These findings suggest that IL-2 gene transfer potentiates the antitumor activity of MRK16 against MDR tumors.

Arch Biochem Biophys, 1998 Jan 1, 349(1), 113 - 21
Uncouplers of mitochondrial oxidative phosphorylation are not substrates of the erythrocyte glutathione-S-conjugate pump; Sokal A et al.; Uncouplers of mitochondrial oxidative phosphorylation, dinitrophenol (DNP) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), were found to stimulate Mg(2+)-ATPase activity of human erythrocyte membranes in a manner competitive with respect to 2,4-dinitrophenyl-S-glutathione (DNP-SG) which suggested that these compounds may also be substrates of the glutathione-S-conjugate pump . We confirm that the stimulation of erythrocyte membrane ATPase activity by DNP and by another uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), is competitive with respect to DNP-SG . However, we found no evidence for active transport of DNP and CCCP out of erythrocytes and demonstrate that they inhibit the low-affinity component of DNP-SG transport noncompetitively while stimulating the high-affinity DNP-SG transport (mediated by multidrug resistance-associated protein, MRP1) . Implications of these findings may indicate the electrogenic nature of MRP1-mediated transport of glutathione-S conjugates and stimulation of aminophospholipid translocase (flippase) rather than the glutathione-S-conjugate pump by the uncouplers.

Zhonghua Yi Xue Za Zhi (Taipei), 1997 Oct, 60(4), 184 - 90
Expression of DNA topoisomerase II alpha and multidrug resistance p-glycoprotein in acute leukemia; Chiu CF et al.; BACKGROUND: Drug resistance is a major cause of treatment failure in acute leukemia . Overexpression of multidrug resistance gene and decreased activity of topoisomerase II alpha are suggested as two important mechanisms for this resistance . METHODS: We used immunohistochemical method to determine the expressions of both topoisomerase II alpha (topo II alpha) and p-glycoprotein (gp-170) in bone marrow biopsy specimens from 68 cases of acute leukemia . Patients were divided into four groups: (1) leukemia cells with high score for topo II alpha and negative for gp-170; (2) leukemia cells with high score for topo II alpha and positive for gp-170; (3) leukemia cells with low score for topo II alpha and negative for gp-170; and (4) leukemia cells with low score for topo II alpha and positive for gp-170 . The clinical responses were then followed as routine, and the clinical correlation was evaluated by analysis of variance and Pearson Chi-Square test . RESULTS: The measure of the single parameter (either topo II alpha or gp-170 alone) did not show a significant difference in the overall survival . However, the complete response rate was much higher in the first group patients whose bone marrow reading score was high in topo II alpha and negative for gp-170 expression . Survival duration increased with the increase in the complete response rate . CONCLUSIONS: Combined parameters of topo II alpha and gp-170 are more useful than any individual parameter for the prognosis of acute leukemia.

Exp Cell Res, 1997 Dec 15, 237(2), 410 - 8
Prolonged weightlessness affects promyelocytic multidrug resistance; Piepmeier EH et al.; An immortalized promyelocytic cell line was studied to detect how doxorubicin uptake is affected by microgravity . The purpose of this experiment was to identify the effect that microgravity may have on multidrug resistance in leukocytes . HL60 cells and HL60 cells resistant to anthracycline (HL60/AR) were grown in RPMI and 10% FBS . Upon reaching orbit in the Space Shuttle Endeavour, the cells were robotically mixed with doxorubicin . Three days after mixing, cells were fixed with paraformaldehyde/glutaraldehyde . Ground control experiments were conducted concurrently using a robot identical to the one used on the Shuttle . Fixed cells were analyzed within 2 weeks of launch . Confocal micrographs identified changes in cell structure (transmittance), drug distribution (fluorescence), and microtubule polymerization (fluorescence) . Flight cells showed a lack of cytoskeletal polymerization resulting in an overall amorphic globular shape . Doxorubicin distribution in ground cells included a large numbers of vesicles relative to flight cells . There was a greater amount of doxorubicin present in flight cells (85% +/- 9.7) than in ground control cells (43% +/- 26) as determined by image analysis . Differences in microtubule formation between flight cells and ground cells could be partially responsible for the differences in drug distribution . Cytoskeletal interactions are critical to the function of P-glycoprotein as a drug efflux pump responsible for multidrug resistance.

Exp Cell Res, 1997 Dec 15, 237(2), 307 - 17
Effects of multidrug resistance-related ATP-binding-cassette transporter proteins on the cytoskeletal activity of cytochalasins; Berger W et al.; Cytochalasins are microfilament-active mould metabolites, widely utilized to study the involvement of the actin cytoskeleton in cellular processes as well as in genotoxicity and cell kinetic research . In this study we have investigated whether multidrug-resistance phenotypes, caused by overexpression of the ATP-binding-cassette transporter proteins P-glycoprotein (P-gp) or multidrug-resistance-associated protein (MRP), influence the microfilament-depolymerizing effect of cytochalasins . Using four well-characterized multidrug-resistance cell models, we have shown that both the microfilament-disrupting (phalloidine staining) and the cytotoxic (MTT-assay) activity of cytochalasins are reduced in parallel with increased P-gp expression and restorable by P-gp-modulating agents . This also applied to the cytochalasin D-mediated induction of polykaryons (microscopic evaluation) which arise as a consequence of impaired cytokinesis but unaffected karyokinesis . The reduced cellular activity of cytochalasins in P-gp-positive cell lines was correlated with decreased intracellular accumulation ({3H}cytochalasin B accumulation) which was also restorable by P-gp modulators . Moreover, the dose-dependent inhibition of P-gp photoaffinity labeling ({3H}-azidopine) suggested cytochalasins as P-gp-binding agents . In contrast, MRP overexpression had no effect on either cytochalasin microfilament activity or cytotoxicity . In conclusion, data indicate that the microfilament-destructive effects of cytochalasins are impaired due to a reduction of the intracellular cytochalasin accumulation by P-gp but not by MRP . Results are discussed with regard to P-gp as a resistance factor when cytochalasins are utilized to study microfilament dynamics, cell cycle kinetics or chromosomal damage . Moreover, the polykaryon-inducing activity of cytochalasin D is suggested as a specific indicator for a P-gp-mediated multidrug-resistance phenotype and the reversing potency of chemosensitizers.

Invest Ophthalmol Vis Sci, 1998 Jan, 39(1), 164 - 70
Intravitreal daunomycin induces multidrug resistance in proliferative vitreoretinopathy; Esser P et al.; PURPOSE: Adjuvant intravitreal daunomycin is frequently used for the management of proliferative vitreoretinopathy (PVR) . In this study the authors examined whether daunomycin could induce multidrug resistance (MDR), mediated by the mdr-1 gene product P-glycoprotein, in the cells responsible for reproliferation in vivo and in human retinal pigment epithelial (RPE) cells in vitro . METHODS: Expression of P-glycoprotein was examined by immunohistochemistry in surgically removed epiretinal membranes . The cellular source of P-glycoprotein was examined by colabeling for cytokeratin, glial fibrillary acidic protein, and the macrophage marker EBM-11 . P-glycoprotein expression by cultured RPE cells was assessed by reverse transcription-polymerase chain reaction and immunoblot analysis . Daunomycin toxicity was quantified by crystal violet assay . RESULTS: P-glycoprotein expression was detected in 10 of 10 patients pre-exposed to intravitreal daunomycin . In contrast, epiretinal membranes from only 2 of 13 patients never exposed to daunomycin showed faint P-glycoprotein expression . P-glycoprotein expression was strong within 8 months after daunomycin treatment and faded thereafter . Colocalization studies demonstrated predominant expression of P-glycoprotein by RPE cells . Pre-exposure of cultured human RPE cells to subtoxic concentrations of daunomycin induced resistance to daunomycin that was sensitive to the MDR inhibitor, verapamil . Induction of the MDR phenotype in RPE cells by daunomycin was associated with a minor increase in the mdr-1 mRNA level but a prominent increase in P-glycoprotein expression, thus suggesting a primarily translational mechanism of MDR development in human RPE cells . CONCLUSIONS: Intravitreal daunomycin induced P-glycoprotein expression in PVR . Reproliferation in daunomycin-pretreated patients probably necessitates cotreatment with daunomycin and inhibitors of multidrug resistance such as verapamil or administration of antiproliferative drugs such as 5-fluorouracil, which act in a MDR-independent fashion.

J Nucl Med, 1997 Dec, 38(12), 1915 - 9
Technetium-99m-furifosmin as an agent for functional imaging of multidrug resistance in tumors; Ballinger JR et al.; There has been a preliminary report that furifosmin, like the other lipophilic 99mTc cations sestamibi and tetrofosmin, is a substrate for P-glycoprotein, the membrane transporter that is a mechanism of multidrug resistance (MDR) in tumors . This has been further investigated in the rat mammary carcinoma cell line MatB/WT and its doxorubicin-selected resistant variant MatB/AdrR . METHODS: In vitro studies were performed by adding furifosmin to stirred single-cell suspensions of MatB/WT and MatB/AdrR in the presence or absence of the Pgp-modulating drug PSC833 . Dynamic imaging studies over 30 min were performed in rats bearing MatB/WT or MatB/AdrR tumors growing in the leg . RESULTS: Accumulation of furifosmin in MatB/AdrR cells in vitro was much lower than that in MatB/WT cells . The addition of 1 microM PSC833 increased the plateau accumulation in MatB/AdrR cells 2.4-fold, but did not affect accumulation in MatB/WT cells . In rats, furifosmin accumulated rapidly in MatB/WT tumors and washed out with a mean t3 of 78 min . Washout from MAtB/AdrR tumors was more rapid, with a t3 of 46 min (p < 0.025) . Following dissection of animals at 30 min, mean tumor-to-muscle ratios were 1.57 and 1.05 in MatB/WT and MatB/ AdrR tumors, respectively (p < 0.025) . CONCLUSION: Furifosmin is suitable for functional imaging of multidrug resistance in tumors.

FEBS Lett, 1997 Dec 8, 419(1), 112 - 6
Transport of glutathione prostaglandin A conjugates by the multidrug resistance protein 1; Evers R et al.; The human multidrug resistance protein MRP1 mediates transport of organic substrates conjugated to glutathione, glucuronide, or sulfate . The naturally occurring prostaglandins A1 and A2 can form two diastereomeric glutathione S-conjugates, and it has been speculated that these might be substrates for MRP1 . Here we present evidence that polarized MDCKII cells expressing MRP1 cDNA transport PGA1-GS to the basolateral side of a cell monolayer, in accordance with the lateral localization of human MRP1 in these cells . Furthermore, we show that vesicles made from yeast cells expressing MRP1 cDNA and from mouse erythrocytes (known to contain mrpl) actively accumulate both diastereomers of PGA2-GS with a similar efficiency . Recently, we generated mice with a homozygous mutant mrp1 allele . Uptake of PGA2-GS in vesicles made from erythrocytes of these mice was 3.2 times lower than in wild-type vesicles, but was still significantly above background . This residual transport activity was partly inhibited by methotrexate and cAMP, whereas mrp1-mediated activity was unaffected by these compounds . We conclude that mouse erythrocytes contain at least two transport systems for PGA2-GS . One of these is mrp1; the other one has not been identified yet, but can be inhibited by methotrexate and cAMP.

Biochem Biophys Res Commun, 1997 Dec 18, 241(2), 548 - 52
Another link between phospholipid transmembrane migration and ABC transporter gene family, inferred from a rare inherited disorder of phosphatidylserine externalization; Toti F et al.; The mechanisms involved in the maintenance or loss of the asymmetric distribution of phospholipids in the cell plasma membrane remain mysterious . In the yeast Saccharomyces cerevisiae, the transmembrane migration of certain phospholipids is controlled by transcription regulators of various ATP-binding cassette (ABC) transporters . The P-glycoprotein membrane transporters encoded by the multidrug resistance (MDR) genes, members of the ABC protein family, act as lipid translocases in mammalian cells . We report here the lack of expression of MDR genes in lymphoblasts derived from the B cells of a patient with an inherited Scott syndrome, characterized by impaired transmembrane migration of procoagulant phosphatidylserine and hemorrhagic complications . From microsatellite analysis of 7q21.1 and functional assessment, the most likely explanation accounting for Scott phenotype is a mutation in an unlinked gene coding for a regulatory protein necessary for the expression of MDR genes . Because phosphatidylserine externalization is also one of the hallmarks of cells undergoing apoptosis, these observations are suggestive of a relationship between basic processes such as multidrug transport, apoptosis and procoagulant phospholipid exposure.

Mol Pharmacol, 1997 Dec, 52(6), 948 - 57
Characterization of a novel bisacridone and comparison with PSC 833 as a potent and poorly reversible modulator of P-glycoprotein; Horton JK et al.; Novel compounds, composed of two acridone moieties connected by a propyl or butyl spacer, were synthesized and tested as potential modulators of P-glycoprotein (P-gp)-mediated multidrug resistance . The propyl derivative 1,3-bis(9-oxoacridin-10-yl)-propane (PBA) was extremely potent and, at a concentration of 1 microM, increased steady state accumulation of vinblastine (VLB) approximately 9-fold in the multidrug-resistant cell line KB8-5 . In contrast to the readily reversible effects of VRP and cyclosporin A on VLB uptake and similar to the effects of the cyclosporin analog PSC 833, this modulation by PBA was not fully reversed 6-8 hr after transfer of cells to PBA-free medium . Continuous exposure to 3 microM PBA was nontoxic and could completely reverse VLB resistance in KB8-5 cells . Consistent with its effects on VLB transport, the drug resistance-modulating effect of PSC 833 was significantly more persistent than that of VRP . However, the effect of PBA was, like that of VRP, rapidly reversed once the modulator was removed from the extracellular environment . PBA was able to compete with radiolabeled azidopine for binding to P-gp and to stimulate P-gp ATPase activity . However, both the steady state accumulation of PBA and the rate of efflux of PBA were similar in drug-sensitive KB3-1 and drug-resistant KB8-5 cells, suggesting that this compound is not efficiently transported by P-gp . These results indicate that PBA represents a new class of potent and poorly reversible synthetic modulators of P-gp-mediated VLB transport.

Biochemistry, 1997 Dec 9, 36(49), 15208 - 15
Binding of steroid modulators to recombinant cytosolic domain from mouse P-glycoprotein in close proximity to the ATP site; Dayan G et al.; We recently found that recombinant NBD1 cytosolic domain corresponding to segment 395-581 of mouse mdr1 P-glycoprotein bound fluorescent 2'(3')-N-methylanthraniloyl-ATP (MANT-ATP) with high affinity {Dayan, G., Baubichon-Cortay, H., Jault, J.-M., Cortay, J . -C., Deleage, G., & Di Pietro, A . (1996) J . Biol . Chem . 271, 11652-11658} . The present work shows that a longer 371-705 domain (extended-NBD1), including tryptophan-696 as an intrinsic probe, which bound MANT-ATP with identical affinity, also interacted with steroids known to modulate anticancer drug efflux from P-glycoprotein-positive multidrug-resistant cells . Progesterone, which is not transported, its hydrophobic derivatives medroxyprogesterone acetate and megestrol acetate, and Delta6-progesterone produced nearly a 50% saturating quenching of the domain intrinsic fluorescence, with dissociation constants ranging from 53 to 18 microM . The even more hydrophobic antiprogestin RU 486 produced a complete quenching of tryptophan-696 fluorescence, in contrast to more hydrophilic derivatives of progesterone containing hydroxyl groups at positions 11, 16, 17, and 21 and known to be transported, which produced very little quenching . A similar differential interaction was observed with full-length purified P-glycoprotein . The steroid-binding region within extended-NBD1 appeared distinct from the nucleotide-binding site as the RU 486-induced quenching was neither prevented nor reversed by high ATP concentrations . In contrast, MANT-ATP binding was efficiently prevented or displaced by RU 486, suggesting that the hydrophobic MANT group of the bound nucleotide analogue overlaps, at least partially, the adjacent steroid-binding region revealed by RU 486.

J Biol Chem, 1997 Dec 5, 272(49), 30962 - 8
ATPase activity of purified multidrug resistance-associated protein; Chang XB et al.; Human multidrug resistance protein (MRP) was expressed at high levels in stably transfected baby hamster kidney (BHK-21) cells . These cells exhibited a pattern of cross-resistance to several different drugs typical of an MRP-mediated phenotype despite the addition of 10 histidine residues at the C terminus to facilitate purification . Consistent with this functional evidence of the presence of MRP at the surface of these transfectants, strong signals were detected by immunoblotting and immunofluorescence using a specific monoclonal antibody to MRP . There was intense uniform staining of the cell surface as well as weaker staining of intracellular membranes . MRP-containing membranes were solubilized in 1% N-dodecyl-beta-D-maltoside in the presence of 0.4% sheep brain phospholipids . Two sequential affinity purification steps on Ni-NTA agarose and wheat germ agglutinin agarose provided substantial enrichment, and contaminating bands were not detected . ATPase activity of the purified protein was assayed in the presence of the phospholipids, which had been maintained throughout all purification steps . ATP was hydrolyzed in proportion to the amount of purified protein assayed, and typical Michaelis-Menten behavior was exhibited, yielding estimations of Km of approximately 3.0 mM and Vmax of 0.46 micromol mg-1 min-1 . This activity was moderately stimulated by the drugs that others have shown to be transported by MRP-containing membrane vesicles . This stimulation was enhanced by reduced glutathione as is its drug transport, and oxidized glutathione, itself a substrate for transport, caused a strong stimulation . These data describe the first purification of MRP and provide the first direct evidence that the molecule possesses drug-stimulated ATPase activity.

J Gen Physiol, 1997 Dec, 110(6), 655 - 64
Octameric stoichiometry of the KATP channel complex; Shyng S et al.; ATP-sensitive potassium (KATP) channels link cellular metabolism to electrical activity in nerve, muscle, and endocrine tissues . They are formed as a functional complex of two unrelated subunits-a member of the Kir inward rectifier potassium channel family, and a sulfonylurea receptor (SUR), a member of the ATP-binding cassette transporter family, which includes cystic fibrosis transmembrane conductance regulators and multidrug resistance protein, regulators of chloride channel activity . This recent discovery has brought together proteins from two very distinct superfamilies in a novel functional complex . The pancreatic KATP channel is probably formed specifically of Kir6.2 and SUR1 isoforms . The relationship between SUR1 and Kir6.2 must be determined to understand how SUR1 and Kir6.2 interact to form this unique channel . We have used mutant Kir6.2 subunits and dimeric (SUR1-Kir6.2) constructs to examine the functional stoichiometry of the KATP channel . The data indicate that the KATP channel pore is lined by four Kir6.2 subunits, and that each Kir6.2 subunit requires one SUR1 subunit to generate a functional channel in an octameric or tetradimeric structure.

Br J Pharmacol, 1997 Oct, 122(4), 765 - 71
The multi-drug resistance reversal agent SR33557 and modulation of vinca alkaloid binding to P-glycoprotein by an allosteric interaction; Martin C et al.; 1 . The interaction of the indolizin sulfone SR33557 with the multidrug resistance P-glycoprotein (P-gp), was used to explore the nature of drug binding site(s) on this transporter . The steady-state accumulation of {3H}-vinblastine in P-gp expressing CHrB30 cells was increased by SR33557 with greater potency than verapamil . Furthermore, SR33557 potentiated the affinity of verapamil to modulate vinblastine transport when added simultaneously . 2 . Verapamil elicited a 1.5 to 2.5 fold stimulation of basal ATPase activity in CHrB30 membranes, whereas SR33557 and vinblastine inhibited activity, but only at relatively high concentrations . However, SR33557 and vinblastine decreased the Vmax but not the Km for verapamil stimulation of ATPase activity . This is indicative of a non-competitive interaction, most likely at distinct sites . 3 . The specific {3H}-vinblastine binding to P-gp in CHrB30 cell membranes was displaced by SR33557 with an IC50 of 8.3 +/- 4.5 nM . Moreover, SR33557 caused a 3 fold increase in the dissociation rate of vinblastine binding to P-gp indicating a negative allosteric effect on the vinca alkaloid acceptor site . 4 . These results demonstrate that SR33557 interacts with a site on P-gp which is distinct from, but allosterically linked to the vinca alkaloid site . The apparent broad substrate specificity displayed by P-gp may be explained by a multiple drug binding site model.

Tumour Biol, 1998, 19(1), 41 - 51
Comparison between anthracyclines and rhodamine-123 accumulation in chronic lymphoid leukemia: effect of cyclosporin A and verapamil; Maia RC et al.; Multidrug resistance in leukemic cells is associated with decreased drug accumulation . A resistant cell line and cells from 11 patients with chronic lymphoid leukemia B were used for the evaluation of intracellular accumulation of daunorubicin (DNR), idarubicin (IDA), epirubicin (EPI) and rhodamine-123 (Rh-123) . Cyclosporin A (CSA) and verapamil were used to test their modulatory effects on anthracyclines and the fluorescent dye . In leukemic samples there was a tendency for a lower accumulation index in samples tested with Rh-123 as compared to anthracyclines . IDA was a poorer substrate to P-glycoprotein (Pgp) than two of its analogues, e.g . DNR and EPI . A good correlation (80%) was found between Rh-123 accumulation and Pgp expression by phosphatase-anti-alkaline phosphatase . A strict correlation (100%) was found between modulation by CSA of Rh-123 accumulation and immunoreactivity to Pgp . Two discordant results were seen suggesting that other mechanisms of resistance could be present . The Rh-123 accumulation test seems to give a better indication than anthracyclines, however, it is not selective and may allow the detection of other drug-transport pumps.

Gan To Kagaku Ryoho, 1997 Dec, 24(15), 2190 - 5
{Structures and functions of xenobiotic efflux pump P-glycoprotein and MRP--important molecular targets for cancer chemotherapy}; Ueda K; This paper deals with the basic features of the xenobiotic efflux pump (P-glycoprotein and MRP) and the clinical significance of the search for specific modulators of these proteins . P-glycoprotein and MRP function as ATP-dependent efflux pumps that extrude cytotoxic drugs from the cells before the drugs reach their intracellular targets, thus conferring resistance to many structurally dissimilar anti-cancer drugs . These proteins are responsible for multidrug resistance of tumor cells, a major obstacle to cancer chemotherapy . To develop well-designed modulators, structural information regarding the specific drug binding sites is important . We recently found that mutations in the putative transmembrane domain (TM) 1 of human P-glycoprotein alter the drug resistance pattern . Some amino acid residues in TM1 together with TM5-6 and TM11-12 may help to govern substrate specificity . The features common to substrates for P-glycoprotein and MRP are also discussed.

Cell Growth Differ, 1997 Dec, 8(12), 1243 - 7
Regulation of P-glycoprotein expression in cyclic AMP-dependent protein kinase mutants; Cvijic ME et al.; Multidrug resistance (MDR) in cancer poses a major obstacle to the success of chemotherapy . We previously reported that cyclic AMP (cAMP)-resistant mutants of the Chinese hamster ovary and the mouse adrenal cortical carcinoma cells harboring defective regulatory (RI alpha) subunits of the cAMP-dependent protein kinase (PKA) are more sensitive than wild-type cells to chemotherapeutic agents that are substrates for P-glycoprotein . In addition, a transfectant overexpressing a mutant RI alpha cDNA showed similar increased sensitivity to these drugs . The altered drug sensitivity in the RI alpha mutants results from reduced expression of the mdr gene, suggesting that PKA may regulate its expression . In this study, we evaluated the sensitivity of several Chinese hamster ovary catalytic (C) subunit mutants to various anticancer drugs . Like the RI alpha subunit mutant, the C subunit mutants also exhibit decreased kinase activity and unresponsiveness to growth inhibition by cAMP . However, in contrast to the RI alpha subunit mutant, the C subunit mutants are not multidrug sensitive and maintain P-glycoprotein expression levels comparable to those of wild-type cells . Furthermore, the C subunit mutants display the same resistance patterns as wild-type cells to P-glycoprotein substrates, including Adriamycin, Taxol, and colchicine . No significant difference was observed in their sensitivity to non-MDR drugs, such as 5-fluorodeoxyuridine, between wild-type, RI alpha, and C subunit mutant cells . These results suggest that the increased multidrug sensitivity in the PKA mutant cells results from alteration of the RI alpha subunit and not the kinase activity, thus implying novel functions for the RI alpha subunit . Therefore, genetic alteration of the RI alpha subunit of PKA may modulate drug resistance in cancer.

Cancer Res, 1997 Dec 15, 57(24), 5460 - 4
The 95-kilodalton membrane glycoprotein overexpressed in novel multidrug-resistant breast cancer cells is NCA, the nonspecific cross-reacting antigen of carcinoembryonic antigen; Ross DD et al.; Human breast carcinoma MCF-7/AdrVp cells display a novel multidrug resistance phenotype that is characterized by the overexpression of a 95-kDa membrane glycoprotein (p95) and by marked reduction in intracellular anthracycline accumulation, without overexpression of P-glycoprotein or the multidrug resistance protein MRP . p95 is also highly expressed in multidrug-resistant NCI-H1688 cells derived from a human small cell lung carcinoma . Deglycoslyated p95 from NCI-H1688 cells was isolated by two-dimensional gel electrophoresis and then digested with trypsin . The tryptic peptides were analyzed by mass spectrometry and microsequencing . These analyses identified p95 to be identical to NCA-90, the nonspecific cross-reacting antigen related to the carcinoembryonic antigen (CEA) . Further confirmation that p95 is indeed NCA-90 was obtained by Northern and Western blot studies using probes or antibodies specific for p95, NCA-90, or CEA family members . Western blot studies also revealed that CEA itself is overexpressed in MCF-7/AdrVp cells compared to parental MCF-7/W cells . The enforced expression of NCA-90 protein in HeLa cells stably transfected with NCA-90 cDNA did not result in increased resistance of the transfected cells to daunorubicin or a decrease in daunorubicin accumulation in the transfected cells compared to cells transfected only with the expression vector . However, a recent report by H . Kawaharata et al . (Int . J . Cancer, 72: 377-382, 1997) of diminished accumulation, retention, and cytotoxicity of doxorubicin in EJNIH3T3 cells in which enforced expression of CEA was accomplished leaves open the possibility that the overexpression of CEA, possibly in combination with that of NCA-90, could account at least in part for the drug resistant phenotype displayed by MCF-7/AdrVp cells.

J Exp Ther Oncol, 1996 Sep, 1(5), 286 - 95
In vitro evaluation of new anticancer drugs, exemplified by vinorelbine, using the fluorometric microculture cytotoxicity assay on human tumor cell lines and patient biopsy cells; Fridborg H et al.; The feasibility of combined studies on a cell-line panel and primary cultures of patient tumor cells in the preclinical evaluation of new anticancer drugs was evaluated in a study of the activity and cross-resistance pattern in vitro of the new semi-synthetic vinca alkaloid vinorelbine (Vrb) . The activity of Vrb was investigated in ten cell lines representing different resistance mechanisms and in a total of 256 fresh human tumor samples, using the fluorometric microculture cytotoxicity assay (FMCA) . Resistance to Vrb in the cell lines was associated with expression of the multidrug resistance-mediating P-glycoprotein and the multidrug resistance-associated protein (MRP) and by a recently described tubulin-associated mechanism, while the cell lines with topoisomerase II- and glutathion-associated resistance did not show decreased sensitivity to the drug . Cross-resistance to vincristine (Vcr) and other tubulin-active agents was high in cell lines as well as in patient cells . As with most commonly used anti-cancer drugs, Vrb was more active in hematological than in solid tumor samples . Among the solid tumors investigated, the highest in vitro response rates were observed in ovarian cancer (27%), sarcoma (25%), non-small cell lung cancer (21%) and bladder cancer (20%), while no response was observed in renal or colorectal cancer . Compared to Vcr, Vrb appeared to be slightly more active in solid tumors and slightly less active in hematological tumors . The results show that although Vrb displays a high degree of cross-resistance to Vcr and other tubulin-active drugs, some difference in the activity spectrum could be detected and that the drug is sensitive to multiple mechanisms of resistance . The results also suggest that leukemias, ovarian cancer, sarcoma and bladder cancer are possible further targets for Vrb . The combination of studies on a cell-line panel and patient tumor cells from a broad spectrum of diagnoses to evaluate a new drug seems feasible and may give information on the mechanism of action and target diagnoses for phase II trials.

J Exp Ther Oncol, 1996 Sep, 1(5), 278 - 85
Biochemical consequences of resistance to a recently discovered IMP dehydrogenase inhibitor, benzamide riboside, in human myelogenous leukemia K562 cells; Jayaram HN et al.; Benzamide riboside (BR) exhibits potent antitumor activity in a variety of cultured human tumor cells . The drug is metabolized to benzamide adenine dinucleotide (BAD), which in turn functions as a selective inhibitor of IMP dehydrogenase (IMPDH) activity with a Ki of 0.118 microM . In vitro, BR is a more potent antitumor inhibitor of IMPDH than tiazofurin, another IMPDH inhibitor which has shown significant oncolytic activity in adult patients with end-stage leukemia . To elucidate the mechanism of resistance, a variant of human myelogenous leukemia K562 cells was developed by subculturing sensitive cells in sublethal concentrations of BR over 60 generations . The BR resistant line that emerged exhibited an IC50 (a concentration producing 50% reduction in cell proliferation) of 148 microM, compared to the sensitive line which had an IC50 of 1.6 microM . The activity of the target enzyme, IMPDH, was increased 3-fold in the resistant variant . Studies on BR metabolism revealed that resistant cells formed only 18% of the active metabolite, BAD, compared to sensitive cells . This finding, in turn, correlated with the specific activity of NAD pyrophosphorylase (the enzyme responsible for the synthesis of BAD) which was reduced to undetectable levels in the resistant variant . The basal levels of NAD and guanylates were also significantly decreased to 41% and 48%, respectively, in the resistant line compared to the parent line . Additionally, after treatment with BR a decrease in guanylate level was observed only in the sensitive cells . Sensitive and resistant cells exhibit comparable cytotoxicity to agents outside the tiazofurin family, suggesting that a multidrug resistance was unlikely to explain the resistance to BR . Moreover, BR resistant cells exhibit collatoral sensitivity to 6-aminopurine, cytarabine and 5-fluorouracil, which have different mechanisms of action . In conclusion, these studies establish that the primary mechanism of resistance to BR involves an increase in IMPDH (target enzyme) activity with a concurrent decrease in NAD pyrophosphorylase (BAD synthetic enzyme) activity.

J Exp Ther Oncol, 1996 Jul, 1(4), 260 - 70
Behavior of a novel monoclonal antibody for investigation of ovarian cancer and with possible involvement in multiple drug resistance; McGarry Y et al.; Monoclonal antibodies (MAbs) were raised to an ovarian cancer cell line, OAW42, derived from a patient with a histology of serous cystadenocarcinoma of the ovary, in an attempt to identify novel antigens with a possible role in cancer medicine . One antibody P1H10, subclass IgG1, with a high titer was isolated and shown to recognize an antigen of 48 kDa . Enzyme-linked immunosorbent assay and immunocytochemical studies showed the presence of the target antigen in a number of carcinoma cell lines, including lung and breast, and in two out of three frozen breast tissue specimens . The antigen was not detected in normal human lymphocytes and there was minimal binding of the antibody to normal buccal cells . The antigen was not secreted by the OAW42 or the HepG2 cell lines and was not detected in the sera of a number of ovarian cancer patients . Indirect immunofluorescence studies confirmed the localization of the antigen to be intracellular . The binding of the antibody P1H10 to a number of multidrug resistant variants of the OAW42 cell line showed that the presence and the localization of the antigen in the drug-sensitive parental line and resistant variant cell lines was distinctly different and varied with the degree of drug resistance . The relative specificity of the antibody suggests it may be a possible diagnostic agent in human cancer . A possible role of the antigen in multiple drug resistance (MDR) is also suggested.

J Exp Ther Oncol, 1996 Jul, 1(4), 251 - 9
Metastatic potential and multidrug resistance correlation in the B16 melanoma system; Staroselsky AN et al.; The question of whether metastatic potential and drug resistance are related phenotypes was addressed by comparing the biological behavior of the parental B16 melanoma and a multidrug resistant variant derived from it, the B16/Col/R . A more pronounced metastatic spread to lungs was observed in mice inoculated i.v . with the B16/Col/R variant than in those bearing the parental line . In addition, in the mice injected with the drug resistant melanoma, unusual tumor masses were observed . Large abdominal and spinal cord growths were seen with the MDR variant but not encountered in mice inoculated with the original B16 melanoma . We further attempted to test the capacity of the two cell types to perform several cellular functions relevant to the metastatic process . The B16/Col/R cells displayed a higher aggregability and cell motility than did the B16 cells . Adherence to endothelial cells was higher in the parental line than in the B16/Col/R, possibly supporting a more efficient extravasation of the variant cells . The drug resistant variant displayed a higher capacity to grow locally in kidney, spleen, cecum and peritoneum, as compared to the parental melanoma, indicating a higher ability of homing and growth in these potential target organs for metastasis . A correlation between metastatic potential and multidrug resistance appears therefore to exist in the system examined.

J Exp Ther Oncol, 1996 Jul, 1(4), 226 - 30
Modulation of chemosensitivity by alpha interferon in multiple myeloma and non-Hodgkin's lymphoma; Iaffaioli RV et al.; Low-grade non-Hodgkin's lymphoma and multiple myeloma are chemosensitive malignancies, but are rarely curable because of primary or acquired drug resistance . Interferon has been shown to modulate the multidrug resistance phenotype and to reinduce chemosensitivity in patients with chemoresistant tumors . Fifteen patients with multiple myeloma and 64 patients with low/intermediate grade non-Hodgkin's lymphoma unresponsive to initial chemotherapy were treated with alpha 2b interferon for 2 months . In case of an objective response, treatment was continued until disease progression; non-responding patients received the same chemotherapy to which they were resistant, preceded by a 5 day course of interferon . Interferon salvage monotherapy induced an objective response in 1/15 patients with multiple myeloma and in 7/64 patients with non-Hodgkin's lymphoma . An objective response was achieved after retreatment with first-line chemotherapy preceded by interferon in 4/14 patients (28.6%) with multiple myeloma and in 20/56 evaluable patients (35.7%) with non-Hodgkin's lymphoma . Toxicity was moderate, predictable, manageable, and never caused interruption of the treatment . Interferon appears to be able to modulate chemosensitivity of tumors refractory to chemotherapy with several potential mechanisms, including an effect on drug accumulation; its utilization in this setting warrants further evaluation.

J Exp Ther Oncol, 1996 Jan, 1(1), 49 - 61
Ultrastructural changes related to multidrug resistance in CEM cells: role of cytoplasmic vesicles in drug exclusion; Bobichon H et al.; The multidrug resistance phenotype is found to be frequently associated with the overexpression of proteins which lead to a decrease of drug accumulation within human tumor cells . A 170 kDa membrane glycoprotein which is related to the overexpression of the mdr1 gene is inserted in the plasma membrane and pumps the cytotoxic drugs out of the cells . The aim of this work was to study the morphological modifications of resistant CEM/VLB 100 cells relative to their parental drug-sensitive ones and the detection of the ultrastructural localization of P-glycoprotein at the cytoplasmic level . Using a scanning electron microscope, CEM resistant cells showed wide smooth protrusions while CEM sensitive cells showed microvilli and fine folds . With transmission electron microscopy, an enhanced secretory system was observed in CEM resistant cells: both electron transparent and electron opaque vesicles were associated with the Golgi system, revealed by wheat germ agglutinin-colloidal gold labelling . These vesicles were the binding site of C 219 and MRK 16 antimembrane glycoprotein antibodies, and some of them were determined to belong to the lysosomal system after PTA staining . These vesicles may be an additional way to decrease the cellular uptake of drugs in multidrug resistant cells . Moreover, some nuclear and nucleolar modifications were also observed . These observations show that MDR has wide morphological features which concern several organelles.

J Exp Ther Oncol, 1996 Jan, 1(1), 39 - 48
Messenger RNA expression of resistance factors in human tumor cell lines after single exposure to radiation; Stammler G et al.; Resistance of tumor cells to chemotherapeutic drugs can not only be caused by treatment with antineoplastic agents but also by radiotherapy . The aim of this study was to analyze whether ionizing radiation can influence the mRNA expression of proteins which have been found to be involved in drug resistance of tumor cells . Human tumor cell lines (MCF-7, LXF and Sk-Mel) were treated with single doses of irradiation (5, 10 and 20 Gy) . The expression of the resistance related proteins glutathione S-transferase-pi (GST-pi), topoisomerase II alpha (Topo II), thymidylate synthase (TS), O6-methylguanine-DNA-methyltransferase (MGMT), P-glycoprotein (Pgp), glutathione peroxidase (GPX) multidrug resistance-associated protein (MRP) and also of the heat-shock protein 70 (HSP 70) were determined at the mRNA level during the time interval from 1.5 to 72 h post-irradiation and compared with their corresponding controls . We also examined whether a relationship exists between these proteins and the proliferative activity (histone 3, Ki-67, statin) of the cells . We found that exposure of MCF-7, LXF and Sk-Mel cells to ionizing radiation increases the expression of the mRNA of GST-pi . Topo II, TS, HSP 70 and proliferation markers were also altered by exposure to ionizing radiation, but there was no common response of the three cell lines . No significant changes were observed in the expression of MGMT, Pgp, GPX and MRP after radiation treatment . Drug resistance tests revealed that irradiated MCF 7 cells were less sensitive to doxorubicin than non-irradiated control cells . Our results indicate that ionizing irradiation modifies the expression of some proteins involved in drug resistance and the response of MCF 7 cells to doxorubicin and may, therefore, play a role in clinical drug response.

J Exp Ther Oncol, 1996 Jan, 1(1), 23 - 9
In vivo reversibility of multidrug resistance by the MDR-modulator dexniguldipine (niguldipine derivative B859-35) and by verapamil; Dietel M et al.; The newly synthesized dihydropyridine derivative B859-35 was previously shown in vitro to be highly effective in reversing multidrug resistance (MDR) of P-glycoprotein positive tumor cell lines, such as the adriamycin (ADR) resistant erythroleukemia F4-6RADR cells . In the current study B859-35 was investigated for its efficiency in reversing MDR in an in vivo tumor model for preclinical testing of MDR-modulators . F4-6RADR cells were injected into the right flank of nude mice while the parent cells were injected into the left flank . The animals were treated i.p . with ADR (9.0 mg/kg body weight) combined with B859-35 (5, 10, or 25 mg/kg) or, for comparison and validation, with verapamil (VRP) (75 mg/kg) . The effects of ADR and the MDR-modulator combination were evaluated by histological morphometry of the tumors . While ADR alone was shown to be ineffective in resistant cells, the combinations of ADR + B859-35 as well as of ADR + VRP were highly active in reducing the number of viable cells in the resistant tumor nodule by 67 +/- 9% or by 53 +/- 11% of controls . This model provides evidence that even in vivo, MDR modulators can be effective in reversing drug resistance . In addition, it presents a potentially useful and rapid preclinical system for in vivo studies on the modification of drug resistance.

J Exp Ther Oncol, 1996 Jan, 1(1), 13 - 22
Multidrug resistance-modifying components in human plasma with potential clinical significance; Mulder HS et al.; P-Glycoprotein (P-gp) and multidrug resistance protein (MRP) are plasma membrane associated proteins which can confer multidrug resistance (MDR) to cancer cells by lowering the intracellular amount of drug . Although clinical trials with MDR-reverting agents have been initiated, not much attention has been paid to blood components which may modulate the activity of P-gp or MRP . The present investigation was performed to identify and characterize blood components which may influence the drug content and the drug cytotoxicity of MDR cells . Human plasma, from healthy volunteers, was tested for its effects on the daunorubicin (DNR) accumulation and cytotoxicity in the MDR cell lines SW-1573/2R160 (2R160) and GLC4/ADR containing P-gp and MRP, respectively . The data were compared to the effects observed in wild-type cells . MDR-modifying plasma components were isolated by extraction procedures and characterized using ultrafiltration, high-performance liquid chromatography (HPLC) and mass spectrometry . An increase in the proportion of plasma in the culture medium led to a reduction of the ratio between the DNR content of wild-type and corresponding MDR cells . At 100% plasma we observed an increase in the cellular DNR content of 2R160 cells, which was 10-30% (median 18%) of the maximum possible increase induced by well-known MDR-reverting agents, such as verapamil (for GLC4/ADR cells: 10-20%, median 15%) . The DNR cytotoxicity in MDR cells also increased with an increasing amount of plasma included in the culture media . There was neither an increase in the cellular DNR content nor an effect on the DNR cytotoxicity in wild-type cells . Plasma extract analysis by HPLC showed a major peak which increased the DNR content of MDR cells . The HPLC column retention time of this fraction was identical to that of a standard of cortisol and it was further confirmed to be cortisol using mass spectrometry . Moreover, inclusion of a standard of cortisol in culture media induced a similar effect . We analyzed the data for one of the plasma pools and found that blood cortisol was responsible for the MDR-modulating effect only for 35% of the effect of 100% plasma . Other plasma components were responsible for the remaining modulation effect on MDR cells . In conclusion, the DNR pumping activity of P-gp and MRP is inhibited by human plasma, resulting in 10-30% of the maximum possible increase in cellular drug content . Based on cellular pharmacokinetic calculations this percentage will most likely increase at clinical levels of drug resistance (reaching 40-50%) . In one sample blood cortisol accounted for 35% of the effect of plasma on the DNR content in MDR 2R160 cells . These data show the need for additional studies to test plasma samples for their MDR modulating effects before the administration of MDR-reverting agents in chemotherapy . The data suggest that the effectiveness of chemotherapeutic drugs may be enhanced when administered in accordance with the circadian peak of endogenous corticoids.

Lancet, 1997 Dec 13, 350(9093), 1738 - 42
Nosocomial transmission of Mycobacterium bovis resistant to 11 drugs in people with advanced HIV-1 infection; Guerrero A et al.; BACKGROUND: Since 1990, several nosocomial outbreaks of multidrug-resistant (MDR) tuberculosis have occurred, none of which have involved Mycobacterium bovis . We describe an epidemic of nosocomial and primary MDR M bovis tuberculosis from December, 1993, to February, 1995, among HIV-1-infected patients in a district of Madrid . METHODS: We undertook genetic characterisation of the M bovis strain and investigated its presence in a tuberculosis epidemic in a Madrid hospital in a case-controlled study . We assessed 19 cases diagnosed with MDR tuberculosis due to M bovis during the study period . For the control group, we randomly selected 33 patients with HIV-1 infection and isolation of a strain of M tuberculosis susceptible to isoniazid, rifampicin, or both, who were treated in Ramon y Cajal Hospital . Infection-control policies and practices were implemented . FINDINGS: We detected 19 cases in HIV-1-infected patients of primary MDR tuberculosis produced by M bovis resistant to 11 antituberculosis drugs . We found phenotypic and genotypic similarities in the strains of M bovis . In the case group, the index case and two other cases had had previous contact with another hospital that had had an MDR tuberculosis outbreak . All patients died after a mean of 44 days (range 2-116), despite multidrug treatment with first-line and second-line antituberculosis drugs . The cases with M bovis MDR tuberculosis were significantly more likely than controls to have been admitted to a hospital ward at the same time as patients already infected with MDR tuberculosis during the 10 months before their diagnosis (adjusted odds ratio 94.6 {95% CI 9.4-956.3}, p < 0.0001) . Advanced HIV-1 immunosuppression was associated with the development of MDR tuberculosis . Implementation of control measures stopped the epidemic . INTERPRETATION: An M bovis primary MDR tuberculosis epidemic that cannot be treated effectively and with high mortality has emerged in Europe and has been transmitted between hospitals.

Anticancer Res, 1997 Sep-Oct, 17(5A), 3707 - 11
Evaluation of topoisomerase I catalytic activity as determinant of drug response in human cancer cell lines; Voigt W et al.; The prognostic value of topoisomerase I (Topo I) catalytic activity and expression of the multidrug resistance (MDR) marker P-glycoprotein (Pgp) and multidrug resistance protein (MRP) for in vitro sensitivity to Topo I interactive agents were evaluated . The efficacy of short term (2 h) and long term (24 h) exposures of camptothecin (CPT), two CPT derivatives (SN-22, SN-38) and the indolocarbazole compound NB-506, was determined against human ovarian carcinoma (A2780 and A2780 DX5), human fibrosarcoma (HT1080 and IIT1080/DR4) and human ileocecal carcinoma (HCT-8) . For each cell line the Topo I protein levels and catalytic activity were determined and correlated with drug-induced cytotoxicity . In general, the Topo I protein levels correlated with Topo I catalytic activity . Drug-induced cytotoxicity increased significantly with prolongation of the exposure time . With the 2 h exposure, the multidrug resistant A2780 DX5 cell line (Pgp+, MRP-) was moderately resistant to all four drugs compared to its parental cell line . In case of CPT and SN-22 but not for SN-38 and NB-506, this resistance was no longer detectable following 24 h drug exposure . No resistance was detectable for the multidrug resistant HT1080/DR4 (Pgp-, MRP+) cell line when compared to its parental cell line . With short term exposures a strong trend was observed toward increased cytotoxicity with increased Topo I catalytic activity, especially if this correlation was studied between derivative cell lines (A2780 vs . A2780 DX5 and HT1080 vs . HT1080/DR4) . This correlation weakened when all 5 cell lines and both exposure conditions were considered . Thus, although overall the correlation between Topo I catalytic activity and sensitivity to Topo I interactive drugs between different cell lines is weak, this correlation may be stronger when comparing derivative cell lines . For CPT and SN-22 but not for SN-38 and NB-506, the moderate resistance levels observed in the Pgp-expressing cell line could be negated by prolongation of exposure duration . MRP expression did not effect drug efficacy . The data demonstrate that the importance of Topo I catalytic activity as single prognostic factor for drug response to Topo I interactive agents is weak and that additional mechanisms affecting drug response have to be taken into consideration.

Anticancer Res, 1997 Sep-Oct, 17(5A), 3633 - 45
Expression of CD44 standard and isoforms in human breast cancer xenografts and shedding of soluble forms into serum of nude mice; Fichtner I et al.; Standard CD44 (CD44s) and variant isoforms (CD44v) are expressed on different malignant cells and tissues . Their upregulation has been implicated, in the progression and metastasis of malignomas . In this work we addressed the question of whether these molecules are also expressed on xenografted human breast carcinomas and if certain expression patterns are correlated with biological parameters like tumour size, hormone receptor status, histology, growth rate, chemoresistance and microenvironment . Additionally, we were interested in the shedding of soluble CD44 (sCD44) into the blood circulation of tumour bearing nude mice . The human breast carcinomas MCF-7, MCF-7/ADR, 4296 and MDA-MB435, 4134 and 4151 were transplanted subcutaneously (sc.) or into the mammary fat pad (mfp.) of nude mice . The expression of the CD44s, -v6, and -v9 isoforms was determined at different time points on tissue samples by immunohistochemistry or RT-PCR employing human-specific antibodies or primers, respectively . The serum concentration of CD44s and -v6 was measured by human specific ELISAs . All tumours expressed CD44s . The lowest level was observed in the MCF-7 cancer . The CD44v6 and -v9 sequences and epitopes were distinctly expressed in MCF-7/ADR . MDA-MB435, 4134, 4151 and 4296, while MCF-7 lacked these isoforms . The highest serum concentration of the v6 isoform was detected in mice bearing the tumour 4296 with a high tendency for lymphogenic metastasis . The serum levels of sCD44 were in 5/6 xenografts linearly correlated with the tumour size . Interestingly, there was a remarkable difference between the two sublines MCF-7 and MCF-7/ADR: Both the tissue and serum levels of CD44 isoforms indicated that the development of multidrug resistance is accompanied by an alteration in the expression of membrane proteins discussed to be involved in metastasis . There was no relation of tissue expression with the transplantation site and the hormone receptor status of the tumour lines . CD44s and its variant isoforms are expressed in human xenotransplanted breast cancers in very different levels and patterns . The highest expression in the tumour 4296 is related to lymphogenic metastasis while the absence of isoforms in the model MCF-7 is related to non-metastatic behaviour . CD44 is shed into the serum and can be used for monitoring of tumour growth.

Anticancer Res, 1997 Sep-Oct, 17(5A), 3531 - 6
Characteristics of human gastric carcinoma cell lines with induced multidrug resistance; Kang MS et al.; In contrast to intrinsic drug resistance, induced multidrug resistance in gastric cancer cells has not been well studied . Therefore, two doxorubicin-resistant cell lines, (SNU-1DOX, SNU-16DOX), were derived in vitro from gastric carcinoma cell lines (SNU-1, SNU-16) respectively, and their characteristics were investigated . These resistances were not associated with overexpression of mdrl, multidrug resistance associated protein 1 (MRP1), pi or liver class of glutathione S transferase (GST pi, GSTL), heat shock protein 70 (HSP70), p53 or transglutaminase C (TGC) . Levels of p21WAF1 RNA and topoisomerase II protein were decreased in the SNU-16DOX, but not in SNU-1DOX . However, the subsequent enzyme activity of topoisomerase II in SNU-16DOX was not decreased, but rather increased in SNU-16DOX . Furthermore, both resistant cell lines showed lower uptake and higher efflux of doxorubicin and induced cross-resistance to etoposide and vincristine in addition to doxorubicin, indicating a multi-drug resistance phenotype . In summary, we report two gastric carcinoma cell lines exhibiting induced multidrug resistance phenotype and suggest that mdrl, MRP1, GST, TGC, HSP70 and p53 do not play important roles in induced drug resistance in these cell lines . The role of changes in topoisomerase II activity and/or protein is still inconclusive, and p21WAF1 is associated with induced multidrug resistance in the SNU-16DOX gastric carcinoma cell line.

Anticancer Res, 1997 Sep-Oct, 17(5A), 3493 - 7
Multidrug resistance-associated protein gene expression and drug sensitivity in human lung cancer cells; Narasaki F et al.; Multidrug resistance-associated protein (MRP) mRNA expression and drug sensitivity in lung cancer cells were examined, and the effects of verapamil, a modulating agent for MRP, on drug sensitivity were also tested . Nine cell lines expressed various levels of MRP gene expression but not the MDR1 gene . The levels were higher in non-small cell carcinoma cells (NSCLC) than in small cell carcinoma cells (SCLC) . Clear correlations between the MRP gene level and the sensitivity to etoposide (VP-16) and doxorubicin (Dox) were observed except for one cell line which highly expressed DNA topoisomerase II . Positive correlations between the MRP gene levels in three cell lines and the modulation effects of verapamil in VP-16, Dox, and vincristine were observed . The present results indicate that MRP probably confers intrinsic multidrug resistance in NSCLC rather than in SCLC.

Anticancer Res, 1997 Sep-Oct, 17(5A), 3409 - 23
The primary in vitro antitumor screening of "half-mustard type" phenothiazines; Wuonola MA et al.; The antitumor effects of "half-mustard type" phenothiazines were studied on 57 different tumor cell lines, including leukemias, non-small lung cancer, colon, central nervous system, ovarian, renal, breast, and prostate cancer, as well as melanoma cell cultures . Alkyl-urea derivatives of phenothiazines displayed in vitro antitumor activity . The phenothiazine phthalimido derivatives (1-6) were not active on the majority of cancer cell cultures . In contrast, propylureas (9, 11) were active against some leukemia cell types . Only two compounds with the butylene {(CH2)4} linker (10, 12) were active against non-small lung cancer cells . Compounds containing the propylene linker were less effective . On colon cancer lines, tumor cells from the central nervous system and on melanoma cells the same compounds were effective, however, having substituents at the 2-position of phenothiazine seems to be important . Surprisingly, the majority of ovarian cancer cell lines (except one type, IGROVI) and five of eight renal cancer lines were not sensitive to these phenothiazine derivatives . The two butylene linked phenothiazine ureas (10, 12) had moderate antiproliferative action on two renal cancer cell lines . The prostate cancer and some breast cancer cell lines were not sensitive . Nevertheless some breast cancer cell lines were apparently sensitive to CF3-substituted phenothiazine alkylureas . On the basis of these experiments one may postulate that in the case of insensitive cells an mdr-gene encoded multidrug resistance efflux pump is responsible for the resistance . The selectivity or organ cell specificity of the effective phenothiazines will be targeted for improvement in further studies, in order to avoid the general cytotoxic effects of "half mustard type" phenothiazines.

Anticancer Res, 1997 Sep-Oct, 17(5A), 3393 - 401
Cytoskeleton alteration in MCF7R cells, a multidrug resistant human breast cancer cell line; Bichat F et al.; Various cytoskeleton modifications are associated with malignant cell transformation and have been used as prognostic factors . A human breast cancer cell line (MCF7S) and its multidrug resistant (MDR) subline (MCF7R) were characterized here for their intermediate filaments (IFs) expression (cytokeratin 8, 18, 19 and vimentin) as a function of their resistance phenotype . Modifications of these cytoskeleton molecules were analyzed by flow cytometry, immunofluorescence, electrophoresis and immunoblotting techniques . Cytokeratins 8 and 18 were similarly expressed in the cell lines . Cytokeratin 19 was expressed in the MCF7S cell line and not in the MCF7R variant, while vimentin was highly expressed in MCF7R and slightly in MCF7S . Analysis of IFs after the addition of doxorubicin (Dox) in the culture medium of MCF7S, showed an increase in cytokeratin 8 filaments . Vimentin expression in MCF7R was not modified in the presence of these different MDR modulators . Acquisition of MDR was associated with an increase and a redistribution of vimentin filaments characterized by a perinuclear polarization . These drug resistance associated changes might derive from different biological processes triggered by chemotherapy . In conclusion, this suggests that this intermediate filament could be a marker associated with chemoresistance or a marker of malignancy in certain epithelial cancers.

Anticancer Res, 1997 Sep-Oct, 17(5A), 3329 - 34
Pulsed exposure of SDZ PSC 833 to multidrug resistant P388/ADR and MCF7/ADR cells in the absence of anticancer drugs can fully restore sensitivity to doxorubicin; Krishna R et al.; AIM: An in vitro cellular pharmacological study was undertaken to characterize the latency of modulating activity by the chemosensitizer, PSC 833 . METHODS: PSC 833 was evaluated for cytotoxicity in the MDR P388/ADR and MCF7/ADR cell lines . Cellular uptake levels and intracellular DOX distribution characteristics were assessed by flow cytometry and fluorescence microscopy, respectively . These parameters were correlated with the chemosensitivity of the MDR cells under varying conditions of exposure to PSC 833 and DOX . RESULTS: PSC 833 (1 microM) provided near complete MDR reversal and exhibited significant latent modulating activity indicative of a sustained inhibition of the PGP pump in P388/ADR and MCF7/ADR cells after removal of the MDR modulator . Interestingly, complete latent chemosensitizing activity could be achieved by pulsing cells with PSC 833 prior to DOX exposure . Increases in cellular DOX uptake by individual P388/ADR and MCF7/ADR cells induced by MDR modulators correlated well with their ability to chemosensitize the resistant tumor cells where intracellular DOX levels approaching those obtained for sensitive wild type cells was observed for PSC 833 . CONCLUSION: The MDR modulator PSC 833 exhibits latent MDR modulating activity that appears well suited for modulating PGP mediated MDR in vivo where chemosensitization is complicated by difficulties in synchronizing therapeutic levels of modulator and anticancer drug at the tumor site.

Am J Respir Crit Care Med, 1997 Dec, 156(6), 1993 - 8
Development of a suicide gene as a novel approach to killing Mycobacterium tuberculosis; Rom WN et al.; The increase in multidrug-resistant tuberculosis and high mortality among those co-infected with HIV-1 necessitates new therapeutic approaches directed at Mycobacterium tuberculosis . We hypothesized that a dominant-negative mutation in the DNA-dependent RNA polymerase gene would inhibit transcription of all genes by blocking access of the wild-type enzyme to promoters . An evolutionarily invariant lysine was substituted with arginine by site-directed mutagenesis in the rpoB gene . The dominant-negative rpoB gene product inhibited a transposon-derived kanamycin-resistance gene in both M . smegmatis and M . tuberculosis H37Rv, leading to growth inhibition of the mycobacteria on solid media containing kanamycin . The dominant-negative mutant rpoB gene is a potential suicide gene especially for the treatment of multidrug-resistant tuberculosis once a delivery strategy is also developed.

Indian Pediatr, 1997 Jun, 34(6), 530 - 4
Medical practitioners and their practices in acute diarrhea; Buch NA et al.; PIP: A study conducted in the Pediatric Outpatient Department of the Institute of Medical Sciences in Srinagar, India, assessed the acute diarrhea management strategies of various categories of health care practitioners . Of the total of 1030 infants enrolled at private clinics or other health units, 71.7% were treated by general practitioners and chemists, 11.6% saw hospital residents, and 16.7% were treated by pediatricians . Antidiarrheal and antispasmodic preparations were given to most infants, either alone (46.8%) or in conjunction with oral rehydration therapy (ORT) (45.4%) . Only 4.1% of infants received ORT alone . 87.2% of pediatricians selected a combination of drugs and ORT . Both qualified and unqualified practitioners provided drugs such as lopermaide (57.9%), pipenzolate (2.1%), metoclopramide (13.7%), and steroids (2.5%) . All categories of health workers prescribed marketed ORT preparations containing insufficient sodium; only 23.7% of cases received home-made sugar-salt solution . Parents in India express a preference for the use of drugs and parenteral fluids in acute diarrhea, and most practitioners appear to gear their practices to this preference . Education of both parents and health professionals about the adequacy of ORT in most cases of acute diarrhea is needed to prevent electrolyte imbalances, iatrogenic hazards, and the emergence of multidrug-resistant strains of microorganisms .

J Formos Med Assoc, 1997 Nov, 96(11), 890 - 4
Initial drug resistance of Mycobacterium tuberculosis in Taiwan; Yu MC et al.; The prevalence and mortality rate of pulmonary tuberculosis in adults are high in Taiwan . Because the emergence of drug-resistant tuberculosis is one of the major causes of this sustained high tuberculosis mortality, surveillance of initial drug resistance is important . We tested Mycobacterium tuberculosis isolates from 1,935 newly diagnosed tuberculosis patients from January 1990 through December 1995 at the Taiwan Provincial Chronic Disease Control Bureau . The overall initial drug resistance rate was 12.3%; 8.7% of isolates were resistant to only one drug, 2.6% to two drugs, 0.7% to three drugs, and 0.3% to four drugs . The resistance rates to individual drugs were: streptomycin, 5.7%; isoniazid, 9.2%; ethambutol, 0.7%; and rifampin, 1.5% . The frequency of multidrug-resistant M . tuberculosis (resistant to at least isoniazid and rifampin) was 1.2% . In view of the high initial isoniazid resistance rate and low initial ethambutol resistance rate, ethambutol should be added to the regimen for the initial treatment of tuberculosis in Taiwan . The emergence of multidrug-resistant M . tuberculosis is ominous and should be considered when treating patients in Taiwan.

Semin Hematol, 1997 Oct, 34(4 Suppl 5), 63 - 71
Measuring multidrug resistance expression in human malignancies: elaboration of consensus recommendations; Marie JP et al.; Before the prognostic significance of P-glycoprotein (P-gp) expression can be properly evaluated in prospective clinical trials of P-gp modulators, standard techniques for the measurement of P-gp must be widely accepted . Several multicenter trials have demonstrated large discrepancies in the observed levels of P-gp expression in the same clinical samples evaluated at different centers . The greatest discrepancies occurred with samples that expressed low levels of P-gp . Although standardized procedures have dramatically increased the interlaboratory reproducibility of flow cytometry and polymerase chain reaction assays, data from immunocytochemistry remain difficult to interpret . Consensus recommendations are presented for improving data reproducibility . These recommendations emphasize the importance of using calibrated batches of antibodies and two different antibodies for immunocytochemistry, the need for an internal standard for reverse transcriptase-polymerase chain reaction (RT-PCR) assays, the need for the presentation of data as a continuous variable, and the need for setting standard parameters for flow cytometry . It is also extremely important for the success of clinical trials that multiple techniques be employed to insure accurate measurement of P-gp expression.

Semin Hematol, 1997 Oct, 34(4 Suppl 5), 40 - 7
Pharmacologic approaches to reversing multidrug resistance; Sikic BI; The rationale for modulation of multidrug resistance (MDR) by inhibitors of the multidrug transporter, P-glycoprotein (P-gp) includes the following: (1) P-gp is expressed by human cancers, either at diagnosis or after failure of chemotherapy; (2) P-gp expression at diagnosis has been associated with a poor prognosis in some types of cancer; (3) MDR related to P-gp expression can be reversed by modulators, resulting in enhanced therapeutic efficacy in cellular and animal models of drug resistance; and (4) the emergence of MDR related to P-gp expression can be prevented in cellular models by co-administration of MDR-related cytotoxins and modulators . Clinical trials of modulation of MDR have been limited by two major factors: inability to achieve adequate levels of the modulators to reverse drug resistance in patients and the presence of other mechanisms of resistance in tumor cells in addition to P-gp . The former limitation will hopefully be overcome by new, more potent and specific inhibitors of P-gp such as PSC 833 . The latter will require further understanding of various alternative cellular mechanisms of resistance and the development of approaches to overcome or circumvent these mechanisms . PSC 833 is associated with significant drug interactions with MDR-related cytotoxic agents, which require dose reduction of the cytotoxins to achieve a dose exposure and toxicity similar to the chemotherapy agents without a modulator . These drug interactions are predictable and are at least in part due to inhibition of P-gp in normal tissues such as the liver and kidneys, where P-gp is known to play a role in drug excretion . Data from knockout mice, which lack P-gp expression, support the concept that P-gp is an important factor in MDR-related drug disposition . Early data from phase I and II trials with PSC 833 indicate that substantial inhibition of P-gp can be achieved in patients at clinically tolerable doses of both modulator and cytotoxins . The ultimate therapeutic benefit of MDR modulation with PSC 833 is currently being tested in phase III clinical trials in acute myeloid leukemias (AMLs) and multiple myeloma.

Semin Hematol, 1997 Oct, 34(4 Suppl 5), 34 - 9
Drug resistance in multiple myeloma; Sonneveld P et al.; The development of multidrug resistance (MDR) is a major obstacle to improving treatment outcomes in multiple myeloma . Recent studies have indicated that several specific mechanisms of MDR may be involved in clinically refractory multiple myeloma patients, such as expression of P-glycoprotein (P-gp), expression of the lung-resistance protein (LRP) and suppression of apoptosis via expression of Bcl-2 . The emergence of these mechanisms of MDR in multiple myeloma is enhanced by exposure to chemotherapeutic agents . Recently, clinical reversal of MDR by noncytotoxic P-gp modulators such as verapamil, cyclosporin A (CsA), and PSC 833 was explored in acute leukemia and multiple myeloma . Preliminary results from clinical phase I/II trials indicate that reversal of MDR via modulation of P-gp is possible and that coadministration of these MDR modulators with chemotherapeutic agents alters the plasma pharmacokinetics of chemotherapeutic agents . Phase II and III clinical trials investigating the efficacy of these and other agents in the reversal of MDR in hematologic malignancies are ongoing.

Semin Hematol, 1997 Oct, 34(4 Suppl 5), 25 - 33
The prognostic significance of the expression and function of multidrug resistance transporter proteins in acute myeloid leukemia: studies of the Southwest Oncology Group Leukemia Research Program; Willman CL; Resistance to chemotherapy is a major obstacle in the treatment of patients with acute myeloid leukemia (AML) . The majority of AML patients are intrinsically resistant to chemotherapy at initial diagnosis before chemotherapeutic exposure; such intrinsic resistance frequently results from expression of the multidrug resistance gene (MDR-1), which encodes a membrane transporter protein, P-glycoprotein (P-gp), that mediates drug efflux in leukemic cells . Expression of novel transporter proteins that confer alternative forms of multidrug resistance (MDR), such as the lung-resistance protein (LRP) and the MDR-associated protein (MRP), occurs more frequently in leukemic patients at relapse . Preliminary studies indicate that these proteins may also confer therapeutic resistance in leukemia . In patients older than 55 years of age, AML is characterized by a high frequency of unfavorable cytogenetics, P-gp expression, and functional drug efflux, which contribute to poor clinical outcome . In a multivariate analysis, secondary AML, unfavorable cytogenetics, and P-gp expression/function were each significantly and independently associated with a lower complete remission (CR) rate . Resistant disease was associated with P-gp expression and unfavorable cytogenetics . Strikingly, elderly patients with P-gp- de novo AML and favorable or intermediate cytogenetics had a CR rate of 81% . Patients with P-gp+ secondary AML with unfavorable cytogenetics had a CR rate of only 12% . Therefore, characterization of elderly AML patients at diagnosis using biologic parameters may help identify those patients who are likely to achieve a CR with conventional regimens, as well as those patients who require alternate treatment designed to overcome MDR . In contrast, expression of P-gp and functional drug efflux is detected in only 25% to 30% of AML patients less than 50 years of age . While P-gp expression is less strongly associated with a poor outcome in younger versus older AML patients, it remains strongly associated with resistant disease . Studies are ongoing to determine the prognostic significance of LRP and MRP in various forms of leukemia.

Semin Hematol, 1997 Oct, 34(4 Suppl 5), 20 - 4
Non-P-glycoprotein drug export mechanisms of multidrug resistance; List AF; A variety of cellular mechanisms of multidrug resistance (MDR) have been identified in human drug-resistant cell lines, and may play an important role in the clinical response of hematologic malignancies to chemotherapy . P-glycoprotein (P-gp)-mediated drug efflux is the most well-characterized cellular mechanism of MDR; however, several other non-P-gp membrane transporter proteins have also been implicated in the development of an MDR phenotype in hematologic malignancies . These include the MDR-related protein (MRP), the lung-resistance protein (LRP), and the transporter of antigenic peptides (TAP) . The transporter proteins MRP and TAP are both members of the adenosine triphosphate (ATP)-binding cassette (ABC) family of transmembrane transporters, but each has distinct differences in substrate specificity . Despite effective modulation of P-gp, one or more of these alternate mechanisms of drug resistance may contribute to an MDR phenotype in tumor cell lines . Development of multifunctional MDR modulators or novel therapeutics may be necessary to effectively circumvent MDR in hematologic malignancies.

Cytokines Mol Ther, 1996 Mar, 2(1), 47 - 57
Construction and characterization of a selectable multidrug resistance-glucocerebrosidase fusion gene; Aran JM et al.; Gene fusions can be employed to ensure concomitant expression of two different proteins under the same transcriptional control elements . We have synthesized a retroviral expression vector (pHaMG1) containing a human multidrug resistance (MDR1)-glucocerebrosidase (GC) chimeric gene inserted between the long terminal repeats of the Harvey murine sarcoma virus . When introduced into psi-CRE mouse fibroblasts, pHaMG1 conferred the drug-selectable multidrug resistance phenotype, and drug-resistant clones produced active human GC of about 60 kDa . Percoll gradient fractionation of homogenates prepared from transfectants confirmed correct targeting of P-glycoprotein to the plasma membrane and of GC to lysosomes . Although this construction was designed as a translational fusion of the MDR1 gene product, P-glycoprotein, and human GC, no evidence for a fusion protein was found in transfected cells, and an analysis of the RNAs transcribed from the integrated pHaMG1 retroviral vector suggests that either P-glycoprotein and GC are translated from one mRNA and rapidly processed into two proteins or they are translated separately from different mRNAs . These results reveal the feasibility of using fusion genes, which are smaller than alternative constructions with two promoters or with an internal ribosome entry site, for coexpression of selectable and nonselectable cDNAs in retroviral vectors.

Cytokines Mol Ther, 1995 Dec, 1(4), 301 - 7
Multidrug resistance expression and proliferative studies in poor risk acute myeloid leukemia treated with the FLAG (G-CSF plus fludarabine and Ara-C) regimen; Tafuri A et al.; Fourteen poor risk acute myeloid leukemia (AML) patients were treated with G-CSF prior (from day 0) and during chemotherapy with fludarabine and Ara-C from day 1 to day 5 (the FLAG regimen) . Several biological parameters were monitored on the blast population: multidrug resistance (MDR) functional expression by rhodamine-123 efflux (Rhd-E), cell cycle changes, induction of apoptosis and leukemic clonogenic cell growth (CFU-L) . The mean basal Rhd-E value was 14.4% (range 0-51.2), and 12/14 patients exhibited a dye efflux > 4%, efficiently blocked by the MDR-reversal agent cyclosporin A . After 24 h of G-CSF administration, cell cycle studies showed in bone marrow (BM) samples a significant mean increase in S phase (p = 0.04) and in RNA content of G1 cells (p = 0.01), coupled to a significant increase in apoptosis (p = 0.02) . Clonogenic cell growth analysis showed a twofold increase in BM CFU-L in 6 of the 14 cases tested . When G-CSF activity was assessed without the addition of exogenous growth factors (autonomous proliferation), a significant increase (p = 0.02) in CFU-L was found only in patients who achieved a complete remission (CR); these patients were also characterized by lower S-phase values at diagnosis . Eight of the 14 patients treated achieved CR, but the median response duration was three months, and only two cases are still in CR . The FLAG regimen can thus induce remission in poor risk AML patients . The responses, however, are short, suggesting that resistant cells are not efficiently affected by either the use of agents not involved in the MDR-efflux mechanism or by the G-CSF priming strategy . Other post-induction therapies need to be considered in further approaches.

Cytokines Mol Ther, 1995 Jun, 1(2), 123 - 32
Modulation of vindesine and doxorubicin resistance in multidrug-resistant pleural mesothelioma cells by tumor necrosis factor-alpha; Licht T et al.; Tumor necrosis factor-alpha (TNF-alpha) has been shown to enhance the cytotoxicity of a variety of antineoplastic agents . To examine whether multidrug-resistant cells are targets of TNF-alpha, and whether TNF-alpha is capable of modulating chemoresistance of these cells, a pleural mesothelioma cell line (PXF1118L) and two multidrug-resistant sublines thereof were used as experimental models . Drug resistance of these cells was due to P-glycoprotein expression, as confirmed by (1) staining with a monoclonal antibody (MRK16) specific for human P-glycoprotein, (2) decreased accumulation of {3H}vinblastine that was reversed by verapamil, and (3) enhanced cytotoxicity of vindesine in the presence of verapamil . Parental and multidrug-resistant cells exhibited little but comparable sensitivity to TNF-alpha alone . Combining TNF-alpha with vindesine or, to a lesser extent, with doxorubicin, but not with cisplatin, resulted in greater cytotoxicity towards multidrug-resistant cells than seen for each compound alone, indicating a synergism . In contrast, TNF-alpha failed to modulate vindesine or doxorubicin cytotoxicity in parental cells . {3H}Vinblastine accumulation was unaffected by TNF-alpha, and chemoresistance was reduced by TNF-alpha also in the presence of verapamil (10 microM), indicating that TNF-alpha was acting in a way different from calcium-channel blockers . Though the molecular mechanism by which TNF-alpha was enhancing vindesine and doxorubicin cytotoxicity remained undefined in this study, the numbers of TNF-alpha binding sites on parental and on multidrug-resistant cells were similar, and P-glycoprotein expression was unmodulated during the entire 48 h incubation period . In conclusion, we show that TNF-alpha increases the cytotoxicity of anticancer drugs in multidrug-resistant tumor cells by a mechanism that differs from most chemosensitizing agents, including verapamil . Further studies will be needed to clarify the mechanism by which TNF-alpha synergizes with anticancer drugs.

Cytokines Mol Ther, 1995 Mar, 1(1), 11 - 20
Transfer of the MDR1 (multidrug resistance) gene: protection of hematopoietic cells from cytotoxic chemotherapy, and selection of transduced cells in vivo; Licht T et al.; Expression of the drug efflux pump P-glycoprotein, encoded by the multidrug resistance (MDR1) gene, has been identified as an impediment to successful chemotherapy of neoplastic diseases . More recently, its potential use for gene therapy has been analyzed . Expression of a full-length MDR1 cDNA in hematopoietic cells renders them resistant to various anticancer drugs, as first shown in a transgenic mouse model . Similarly, mouse hematopoietic progenitor cells in bone marrow or peripheral blood are protected from the toxicity of anticancer chemotherapy by retroviral transduction of the MDR1 gene . Furthermore, cells engineered to express P-glycoprotein survived after the administration of cytotoxic drugs, indicating that the gene could function as a selectable marker in vivo . Recently, MDR1 transduction into isolated pluripotent hematopoietic stem cells has been demonstrated . Clinical studies on MDR1 gene transfer into hematopoietic cells of cancer patients are being planned . Transfer of the MDR1 gene into hematopoietic precursor cells may allow the introduction and selection of otherwise non-selectable genes in bone marrow . The ability to select transduced cells can circumvent the low transduction efficiency that has hampered efficient gene therapy . Recently, fusion genes in which the MDR1 cDNA is fused to genes that correct genetic disorders have been constructed to facilitate gene therapy of inherited metabolic disorders.

Nephron, 1997, 77(3), 284 - 9
Expression of MDR1 (multidrug resistance) gene and its protein in normal human kidney; Ernest S et al.; P-glycoprotein (Pgp), the product of the multidrug resistance (MDR) gene overexpressed in cancer cells, is present also in normal tissues . In the kidney, MDR1 Pgp has been found in the proximal tubule and in cultured mesangial cells . In situ hybridization and immunohistochemistry were used to determine the complete nephronal localization of MDR mRNA and its product, Pgp, in the human kidney . MDR mRNA expression was studied with the use of nonradioactive in situ MDR RNA probes . MDR1 Pgp was immunolocalized using the specific monoclonal antibody MRK16 . The presence of MDR mRNA was confirmed in proximal tubules and mesangium, and demonstrated as well in thick limb of Henle's loops and in collecting ducts . MDR1 Pgp colocalized in the same nephronal segments . This suggests that, in addition to secreting xenobiotics, Pgp may play a role in the transport of endogenous substrates or in the regulation of Cl- channels.

Invest Ophthalmol Vis Sci, 1997 Nov, 38(12), 2523 - 30
Multidrug resistance-related proteins in primary choroidal melanomas and in vitro cell lines; van der Pol JP et al.; PURPOSE: Metastatic uveal melanoma is strongly resistant to chemotherapy, and multidrug resistance (MDR) may be involved . To investigate the role of MDR, the presence of the MDR-associated proteins P-glycoprotein (Pgp), MRP, and lung resistance protein (LRP) was determined on primary choroidal melanomas and cell lines . METHODS: A panel of primary choroidal melanomas was examined for the presence of MDR-associated proteins by immunohistochemical analysis . In cell lines established from four primary choroidal melanomas and one metastatic choroidal melanoma, the expression of MDR-associated proteins was determined with monoclonal antibodies in cytospin preparations and flow cytometry . In addition, the functional capacities of transporter proteins Pgp and MRP as adenosine triphosphate-driven efflux pumps were determined by measuring the cellular accumulation and efflux of the fluorescent dyes rhodamine 123 and calcein-AM, with and without the presence of specific pump inhibitors PSC833 and probenecid . RESULTS: Low levels of Pgp and MRP were detected in most primary tumors and in some cell lines . Measurable transporter function of Pgp could be determined in cell line OCM-1 . Lung-resistance protein was present in all primary tumors and cell lines and showed high expression levels . CONCLUSIONS: This study revealed the involvement of LRP and at least a minor role of Pgp and MRP in chemoresistance of choroidal melanoma . Compared with cutaneous melanomas, uveal melanomas appear to express slightly higher levels of Pgp . These findings provide insights into the drug-resistant phenotype of this disease and can aid in the design of therapeutic protocols.

Chem Biol, 1997 Oct, 4(10), 711 - 5
Can prenylcysteines be exploited as ligands for mammalian multidrug-resistance transporters?
Hurwitz LM, Casey PJ.
The overexpression of specific transport proteins in the membrane of many cancer cells renders these cells resistant to many therapeutic drugs . Some lipid-modified cysteine compounds inhibit one drug-transporting protein, indicating the potential of developing such compounds as therapeutic agents.

J Biol Chem, 1997 Nov 28, 272(48), 30088 - 95
Expression cloning and characterization of ROAT1 . The basolateral organic anion transporter in rat kidney; Sweet DH et al.; Expression cloning in Xenopus laevis oocytes was used to isolate an organic anion transport protein from rat kidney . A cDNA library was constructed from size-fractionated poly(A)+ RNA and screened for probenecid-sensitive transport of p-aminohippurate (PAH) . A 2, 227-base pair cDNA clone containing a 1,656-base pair open reading frame coding for a peptide 551 amino acids long was isolated and named ROAT1 . ROAT1-mediated transport of 50 mu M {3H}PAH was independent of imposed changes in membrane potential . Transport was significantly inhibited at 4 degrees C, or upon incubation with other organic anions, but not by the organic cation tetraethylammonium, by the multidrug resistance ATPase inhibitor cyclosporin A, or by urate . External glutarate and alpha-ketoglutarate (1 mM), both counterions for basolateral PAH exchange, also inhibited transport, suggesting that ROAT1 is functionally similar to the basolateral PAH carrier . Consistent with this conclusion, PAH uptake was trans-stimulated in oocytes preloaded with glutarate, whereas the dicarboxylate methylsuccinate, which is not accepted by the basolateral exchanger, did not trans-stimulate . Finally, ROAT1-mediated PAH transport was saturable, with an estimated Km of 70 mu M . Each of these properties is identical to those previously described for the basolateral alpha-ketoglutarate/PAH exchanger in isolated membrane vesicles or intact renal tubules.

Leuk Lymphoma, 1997 Sep, 27(1-2), 119 - 25
Simultaneous expression of P-glycoprotein and BCL-2 in acute myeloid leukemia blast cells; Campos L et al.; High expression of the multidrug resistance gene product P170, and of the oncoprotein bcl-2 have been associated with in vitro resistance to chemotherapeutic agents and with poor clinical outcome in acute myeloid leukemia (AML) . More recently, it has been shown that autonomous proliferation of blast cells in liquid culture was also predictive of poor prognosis . In a series of 72 adult AML cases at diagnosis, we studied by flow cytometry the expression of P170 and bcl-2 proteins, together with autonomous growth of leukemic cells in liquid culture . Cases were classified as exhibiting no proliferation (N = 29), intermediate proliferation (N = 25) and high proliferation (N = 18) . We observed a significant correlation between the percentage of cells in each sample expressing P170 and bcl-2 . This was confirmed by double staining techniques showing that both antigens were present in the same cells . We also observed a significant association between growth pattern and P170 or bcl-2 expression . All patients were treated by intensive chemotherapy including an anthracycline drug and cytarabine . The blasts of patients achieving complete remission (N = 47) were less frequently positive for CD34, P170 and bcl-2 than those from patients who did not . Growth pattern also influenced significantly CR . In univariate analysis, CD34, P170 and bcl-2 expression, as well as growth pattern, significantly influenced survival . However, in multivariate analysis P170 expression remained the only significant factor, bcl-2 (or proliferation) having no independent value . Our study confirms the prognostic value of P170 and bcl-2 expression as well as the value of spontaneous proliferation and suggests that several drug-resistance mechanisms are implicated concomitantly in AML.

Gene, 1997 Oct 24, 200(1-2), 35 - 43
Identification and sequence of human PKY, a putative kinase with increased expression in multidrug-resistant cells, with homology to yeast protein kinase Yak1; Begley DA et al.; We have previously shown that several protein kinases are present in higher activity levels in multidrug resistant cell lines, such as KB-V1 . We have now isolated a gene that codes for a putative protein kinase, PKY, of over 130 kDa that is expressed at higher levels in multidrug-resistant cells . RNA from KB-V1 multidrug-resistant cells was reverse-transcribed and amplified by using primers derived from consensus regions of serine threonine kinases and amplified fragments were used to recover overlapping clones from a KB-V1 cDNA library . An open reading frame of 3648 bp of DNA sequence predicting 1215 aa, has been identified . This cDNA hybridizes to a mRNA of about 7 kb which is expressed at high levels in human heart and muscle tissue and overexpressed in drug-resistant KB-V1 and HL60/ADR cells . Because its closest homolog is the yeast serine/threonine kinase, Yak1, we have called this gene PKY . PKY is also related to the protein kinase family that includes Cdks, Gsk-3, and MAPK proline-directed protein kinases . This protein represents the first of its type known in mammals and may be involved in growth control pathways similar to those described for Yak1, as well as possibly playing a role in multidrug resistance.

Neoplasma, 1997, 44(3), 172 - 7
Taxol-enhanced cytotoxic effect of radiation in human promyelocytic leukemia cells: relative resistance of multidrug-resistant HL-60 cells in vitro; Sedlak J et al.; Cytotoxic effects of sequential taxol (paclitaxel) and X-irradiation on drug-sensitive human cultured promyelocytic leukemia (HL-60) cell line and its multidrug-resistant sublines were examined using photometric MTT test and flow cytometry . Paclitaxel (at concentrations 1-10 nmol) stimulated the cytotoxic effect of irradiation in HL-60 and to a lesser extent also in HL-60/ADR, but not in HL-60/VCR cells . The stimulation of radiation-induced cytotoxic effect by paclitaxel correlated with its potential to induce cell cycle and viability alterations identified with flow cytometric analysis (i.e . increased propidium iodide staining, increased side scatter, decreased forward angle scatter, accumulation of necrotic cell detritus, apoptotic pre-G0 cells and cells in the G2/M phase of the cell cycle).

Curr Opin Hematol, 1996 Jul, 3(4), 329 - 34
Prognostic markers in acute leukemia; Leith CP et al.; The identification of biologic markers for disease outcome in hematopoietic malignancies is essential for the development of "risk-adapted" therapies . Although new prognostic factors are frequently described, their real clinical and biologic impact is often difficult to determine . Factors that influence a marker's true prognostic value include several variables of study design: study size, uniform versus nonuniform patient treatment, methodologic variations, and correlations with other variables . Despite these concerns, several important prognostic factors have emerged in acute leukemias . For example, in acute myeloid leukemia, the multidrug resistant phenotype, whether conferred by the classic P-glycoprotein (multidrug resistance protein 1) or by other mechanisms, and cytogenetics are major prognostic indicators for outcome . In acute lymphoblastic leukemia, markers associated with loss of growth control (loss of tumor suppressor genes, increased proliferative fraction) likewise identify a group of poor-prognosis patients . The delineation of prognostic factors such as these allows for the better identification of patients who may benefit from risk-adapted therapies.

Biochemistry, 1997 Nov 18, 36(46), 14218 - 27
Characterization of phosphine complexes of technetium(III) as transport substrates of the multidrug resistance P-glycoprotein and functional markers of P-glycoprotein at the blood-brain barrier; Luker GD et al.; The multidrug resistance (MDR1) P-glycoprotein functions as a broad specificity efflux transporter of structurally diverse natural product and xenobiotic compounds . P-glycoprotein also is an important component of the functional blood-brain barrier . To enable further studies of function and modulation of MDR1 P-glycoprotein in vitro and in vivo, two novel phosphine technetium(III) complexes were designed and characterized: trans-{2,2'-(1, 2-ethanediyldiimino)bis(1, 5-methoxy-5-methyl-4-oxo-hexenyl)}bis{methylbis(3-methoxy-1- propyl)ph osphine}Tc(III) (Tc-Q58) and trans-{5,5'-(1,2-ethanediyl diimino)bis(2-ethoxy-2-methyl-3-oxo-4-pentenyl)}bis{dimethyl(3- methox y-1-propyl)phosphine)}Tc(III) (Tc-Q63) . In human drug-sensitive KB 3-1 cells and multidrug-resistant KB 8-5 and 8-5-11 derivative cell lines, expressing nonimmunodetectable, low, and high levels of MDR1 P-glycoprotein, respectively, accumulation of Tc-Q58 and Tc-Q63 was inverse to expression of the transporter . Differences between drug-sensitive and multidrug-resistant cells, while detectable at picomolar concentrations of each radiopharmaceutical, were independent of tracer concentration . Ratios of tracer accumulation in KB 3-1 and 8-5 cells were 62.3 and 48.1 for Tc-Q58 and Tc-Q63, respectively . Cell contents of Tc-Q58 and Tc-Q63 were enhanced up to 60-fold in MDR cells by known modulators of MDR1 P-glycoprotein, while drugs not in the multidrug-resistant phenotype had no effect on their accumulation . In KB 8-5 cells, potency of modulators was GF120918 >> cyclosporin A > verapamil . Accumulation of Tc-Q58 and Tc-Q63 in Sf9 insect cells infected with a recombinant baculovirus containing human MDR1 P-glycoprotein was reduced in a GF120918-reversible manner (EC50 </= 70 nM) compared with cells infected with a wild-type baculovirus . By contrast, cell contents of Tc-Q58 or Tc-Q63 in Sf9 cells expressing the homologous MDR3 P-glycoprotein did not differ from wild-type virus . Demonstrating molecular targeting of these complexes in vivo, distribution and retention of Tc-Q58 in brain tissue of FVB mice treated with a saturating dose of GF120918 and mice deficient in the mdr1a gene {mdr1a (-/-)} were enhanced 180% and 520% over control, respectively . Exploiting the gamma-emission spectrum of 99mTc, increased uptake of Tc-Q58 in brain tissue of mdr1a (-/-) mice was readily detected noninvasively by scintigraphic imaging . Thus, both Tc-Q58 and Tc-Q63 are demonstrated to be substrates for transport by MDR1 P-glycoprotein, broadening the specificity of this transporter to include phosphine-containing metal complexes . As shown with Tc-Q58, these Q complexes can be used to detect transport activity and modulation of MDR1 P-glycoprotein in vitro and to directly monitor the functional status of P-glycoprotein at the blood-brain barrier in vivo.

J Biol Chem, 1997 Nov 21, 272(47), 29784 - 9
A single chain Fv fragment of P-glycoprotein-specific monoclonal antibody C219 . Design, expression, and crystal structure at 2.4 A resolution; Hoedemaeker FJ et al.; A construct encoding a single chain variable fragment of the anti-P-glycoprotein monoclonal antibody C219 was made by combining the coding sequences for the heavy and light chain variable domains with a sequence encoding the flexible linker (GGGGS)3, an OmpA signal sequence, a c-myc identification tag, and a five-histidine purification tag . The construct was expressed in Escherichia coli and purified from the periplasmic fraction using a nickel chelate column and ion exchange chromatography . Three-step Western blot analysis showed that the construct retains binding affinity for P-glycoprotein . Crystals of 1.0 x 0.2 x 0.2 mm were grown in 100 mM citrate, pH 4.5, 21% polyethylene glycol 6000 in the presence of low concentrations of subtilisin, resulting in proteolytic removal of the linker and purification tags . The structure was solved to a resolution of 2.4 A with an R factor of 20.6, an Rfree of 28.5, and good stereochemistry . This result could lead to a clinically useful product based on antibody C219 for the diagnosis of P-glycoprotein-mediated multidrug resistance . The molecule will also be useful in biophysical studies of functional domains of P-glycoprotein, as well as studies of the intact molecule.

Oncol Res, 1997, 9(6-7), 295 - 302
Importance of glutathione and associated enzymes in drug response; Shen H et al.; Maintenance of cellular homeostasis is a critical survival trait in tumors when exposed to anticancer drugs . Because conjugation and elimination of drugs and their metabolites is dependent upon sequential and coordinated pathways, acquired drug resistance through a gradual adaptive response would rarely be expected to be the consequence of changes in the expression of one gene product . We have used a number of drug-resistant human cell lines to characterize those genes that are implicated in maintaining a resistant phenotype . Human HT29 colon cancer cells chronically exposed to ethacrynic acid (EA) {a glutathione (GSH) and glutathione S-transferase (GST) modulator} have acquired resistance to the drug . Commensurate with resistance, EA is more effectively conjugated to GSH and effluxed from the resistant cells . Using directed and random (differential display) approaches, a number of detoxification and/or protective gene products have been shown to be expressed at elevated levels . These include: gamma-glutamyl cysteine synthetase (gamma-GCS, the rate-limiting enzyme in GSH biosynthesis); GST pi (the enzyme catalyzing the conjugation reaction); multidrug resistance associated protein (MRP) (the membrane pump responsible for effluxing the conjugate from the cell interior) . In addition, other gene products not directly linked with EA metabolism were induced, including dihydrodiol dehydrogenase (an alpha-ketoreductase) (30-fold), DT-diaphorase (threefold), and a transcriptional regulator SSP 3521 (threefold) . HL60 cells resistant to a GSH paralog Ter199 also show increased expression of some of these gene products . Furthermore, an adriamycin-resistant human HL60 cell line also shows overexpression of GST pi, gamma-GCS, and MRP, but in addition has approximately 20-fold more DNA-dependent protein kinase catalytic subunit (DNA-PKcs) . This enzyme is an early stress response gene that can phosphorylate and activate downstream transcription factors . Such overexpression could impact on the transcriptional control of the other detoxification gene products . Both adriamycin and a typical drug-GSH conjugate (APA-SG) are inhibitors of DNA-PK . Because cellular levels of these conjugates would presumably be a good indicator of stress, it would seem reasonable to speculate that DNA-PK may act as a receiver and transmitter of signals that are crucial to the drug-resistant phenotype . Additionally, this enzyme may prove to be a potentially important target for drug design based upon the inhibitory activity of GSH conjugates.

Neurosci Lett, 1997 Oct 31, 236(2), 107 - 11
Modulation of P-glycoprotein activity by glial factors and retinoic acid in an immortalized rat brain microvessel endothelial cell line; El Hafny B et al.; P-glycoprotein (P-gp), a product of the multidrug-resistant (mdr) genes, is expressed in the endothelial cells of the blood-brain barrier (BBB) . Effects of glial factors and retinoic acid (RA) on P-gp activity and level were investigated in the immortalized rat brain endothelial cell line RBE4, which expressed immunodetectable P-gp associated with a decrease in accumulation of the P-gp substrates, vinblastine and colchicine . When RBE4 cells were cultured either in the presence of C6-conditioned medium or on C6- or astrocyte-extracellular matrix, intracellular vinblastine and colchicine concentrations were decreased . When the cells were treated with RA, increases in P-gp activities were correlated with increases in P-gp levels . Effects of simultaneous treatments with glial factors and RA were studied in RBE4 cells cultured on astrocyte-extracellular matrix and were shown to be additive on P-gp activity and level . RBE4 cells may serve as a useful in vitro model for basic research on P-gp regulation at the level of the BBB.

Stem Cells, 1997, 15(6), 420 - 9
Induction of c-kit molecules on human CD34+/c-kit < low cells: evidence for CD34+/c-kit < low cells as primitive hematopoietic stem cells; Sogo S et al.; c-kit, a receptor for stem cell factor, has been widely accepted as a distinctive marker for hematopoietic stem cells . However, the level of c-kit expression on pluripotent hematopoietic stem cells is still controversial in mice and humans . We purified CD34+/c-kit < low cells (phenotypically c-kit-negative but only detectable at the message level) from human cord blood and examined their maturational steps in relation to the expression of c-kit molecules . When the CD34+/c-kit < low cells were cultured with cytokines (flt 3 ligand, interleukin 6 and interleukin 7) plus immobilized anti-CD34 monoclonal antibody (to crosslink CD34 molecules), c-kit molecules were clearly induced within 24 h . The c-kit expression gradually increased until day 8 . When CD34+/c-kit(low) or CD34+/c-kit+ cells that had been induced from CD34+/c-kit < low cells were resorted and recultured using a methylcellulose culture system, they showed the same colony-forming ability as the freshly isolated CD34+/c-kit(low) or CD34+/c-kit+ cells, respectively . Furthermore, CD34+/c-kit < low cells have a similar hematopoietic potential to CD34+/c-kit(low) cells in assays for long-term culture initiating cell and colony-forming unit culture generated from long-term cultures . These findings suggest that CD34+/c-kit < low cells mature into CD34+/c-kit(low) and CD34+/c-kit+ cells, and acquire the reactivity to various humoral hematopoietic stimuli . Moreover, CD34+/c-kit < low cells showed a low level of rhodamine 123 retention, suggesting that CD34+/c-kit < low cells have multidrug resistance . Therefore, the CD34+/c-kit < low cells without colony-forming unit-granulocyte-erythroid-macrophage-megakaryocyte activity are also a pluripotent hematopoietic stem cell population, and the expression of c-kit on c-kit < low cells is the first maturational step of hematopoiesis.

Leuk Lymphoma, 1997 Oct, 27(3-4), 257 - 74
P-glycoprotein expression in de novo acute myeloid leukemia; Del Poeta G et al.; Detection of the multidrug resistance P-glycoprotein (PGP) phenotype was performed at the time of diagnosis in 223 patients with acute myeloid leukemia (AML) by flow cytometry using C219 Monoclonal Antibody (MoAb) . On the other hand, JSB1 MoAb was tested in 173 of these samples . At onset, PGP was detected in 57.4% of cases with C219 and 75.9% of cases with JSB1 . There was no correlation between PGP expression and sex, age, marrow blast percentage or extramedullary disease . On the contrary, strict correlations were noted either between C219 negativity and FAB M3 subtype or between C219 positivity and FAB M5 group (P = 0.003) . Significant correlation was found between PGP phenotype and CD7, as 143 of 223 samples had similar patterns of staining with C219 (P < 0.0001) . Finally, there was a close relationship between C219 and JSB1 positivity: all the C219+ cases were positive for JSB1 (P < 0.0001) . Concerning the karyotype, most patients with monosomy or del (7) were MDR positive; on the other hand, most patients with t(8;21) or t(15;17) were MDR negative . Rh123 accumulation studies showed a significant decrease of mean fluorescence intensities both in C219 and in JSB1 positive cases in comparison with PGP negative ones (P < 0.001) . A significant decrease of remission induction rates (CR) was highlighted both between C219+ and C219- and between JSB1+ and JSB1- cases (32.1% v 62.1% and 32.6% v 73.8%, respectively, with P < 0.0001) . The overall survival and the remission duration (CCR) were significantly shorter both in C219+ and in JSB1+ patients with no relationship to age . Furthermore, a higher rate of early relapses was noted among MDR+ when compared with MDR- patients both for C219+ and JSB1+ cases . The combination (C219- JSB1+) identified a subset of patients with an intermediate prognosis . On multivariate analysis, C219 and JSB1 were confirmed to be independent prognostic factors for achievement of CR, overall survival and CCR . In conclusion, the assessment of MDR phenotype by flow cytometry is a crucial prognostic factor of treatment outcome in AML.

Anticancer Drugs, 1997 Oct, 8(9), 869 - 75
Comparative effect of verapamil, cyclosporin A and SDZ PSC 833 on rhodamine 123 transport and cell cycle in vinblastine-resistant Chinese hamster ovary cells overexpressing P-glycoprotein; Petriz J et al.; The product of the mdr1 gene, P-glycoprotein (P-gp), represents a common mechanism of cellular resistance to a wide variety of structurally and functionally unrelated drugs . A range of structurally different P-gp inhibitors, such as verapamil, cyclosporin A and SDZ PSC 833, have been shown to modify multidrug resistance (MDR) . We used flow cytometry to investigate in vitro modulation of P-gp-dependent efflux of rhodamine 123 (Rh123) . The capacity to modulate the MDR phenotype of vinblastine-resistant Chinese hamster ovary (CHO) cells was assessed by analyzing the concentration of modulator needed to decrease the Rh123 mean fluorescence intensity by 50% . We found that the cyclosporin derivative SDZ PSC 833 was significantly more effective than cyclosporin A and verapamil, either in the presence or absence of fetal calf serum-supplemented media . This study indicates that analysis of Rh123 efflux modulation can be used to determine the optimal doses of MDR inhibitors in vitro and suggests that more than one modulator is needed to measure P-gp function, since verapamil had no effect on Rh123 modulation when MDR cells were used.

Br J Haematol, 1997 Dec, 99(3), 618 - 24
Activity of TNF-related apoptosis-inducing ligand (TRAIL) in haematological malignancies; Snell V et al.; T-cell cytotoxicity is primarily mediated by two cell surface proteins, Fas ligand (FasL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), and intracellular perforin and granzyme granules . FasL-deficient and perforin-deficient T lymphocytes maintain cytotoxicity but fail to induce graft-versus-host disease (GVHD) when transplanted into mice . suggesting that GVHD and graft-versus-tumour (GVT) effects can be dissociated, and that TRAIL is not involved in the pathogenesis of GVHD . Because TRAIL could mediate a favourable GVT effect it became important to study the spectrum of its activity and to investigate factors that can dissociate its expression from FasL . TRAIL induced apoptosis in 11/41 (27%) tumour specimens of haematological origin compared to 16/41 (39%) induced by FasL . Although eight specimens were sensitive to both FasL and TRAIL, no synergism was observed between these two ligands . TRAIL induced apoptosis in a dose and time dependent manner with an ED50 of 0.5 microg/ml and EDmax of 1 microg/ml . TRAIL activity was not reduced by the over-expression of the multidrug resistant (MDR) protein, and was not enhanced by 9-cis retinoic acid (RA), which can down-regulate bcl-2 protein . Both ligands were simultaneously up-regulated in normal peripheral blood lymphocytes in response to IL-2, IL-15 and anti-CD3 antibody, whereas IL-10 had no effect . Together, our data show that (1) TRAIL can mediate cell death in a variety of human haematological malignancies, (2) resistance to TRAIL is not mediated by MDR protein, (3) the lack of synergy between TRAIL and FasL suggests that either one is sufficient to mediate T-cell cytotoxicity, and (4) within the panel of cytokines tested, the expression of TRAIL and FasL could not be dissociated.

J Clin Microbiol, 1997 Dec, 35(12), 3015 - 20
IS6110 fingerprinting of drug-resistant Mycobacterium tuberculosis strains isolated in Germany during 1995; Niemann S et al.; The epidemiological relatedness of drug-resistant Mycobacterium tuberculosis strains isolated in Germany in 1995 was evaluated by the standardized IS6110 fingerprinting method . Altogether, 196 M . tuberculosis isolates from 167 patients were analyzed . A large degree of IS6110 polymorphism was found, ranging from 1 to 20 copies . Multiple isolates from one patient generally remained stable over a period of up to 1 year . However, one strain showed an additional fragment 7 months after the first isolate was obtained . Isolates from 55 patients (33%) showed identical fingerprint patterns or fingerprint patterns that differed only in one band, and thus they were clustered in 22 fingerprint groups . Specific transmission links could be established between members of four groups, e.g., transmission by family contacts . In one case, transmission of a multidrug-resistant strain to a patient initially infected with a drug-susceptible strain could be shown . Besides these fingerprint groups, 30 of the 167 isolates (approximately 18%) could be grouped in two fingerprint clusters with a similarity of at least 78% . Approximately 60% of the patients of these two clusters were known to be immigrants from the former Soviet Union, and one patient is still living in Belarus . In conclusion, our results indicate that (i) transmission of drug-resistant strains contributes substantially to the emergence of drug-resistant tuberculosis in Germany and (ii) drug-resistant M . tuberculosis strains were presumably carried over from the former Soviet Union to Germany by immigrants.

Cancer Detect Prev, 1997, 21(6), 532 - 9
Immunohistochemical assessment of proliferation markers and altered gene expression in archival specimens of ovarian epithelial tumors; Khalifa MA et al.; Recently reported morphologic and molecular genetic evidence suggests that some ovarian carcinomas arise from their benign and low malignant potential (LMP) counterparts . In order to help reach a better understanding of ovarian tumorigenesis, we studied a wide range of gene products involved in cellular growth regulation in archival material obtained from three groups of tumors with graduated malignant potential . Immunohistochemical staining was performed for Ki-67, proliferating cell nuclear antigen (PCNA), epidermal growth factor receptor (EGFR), HER-2/neu-encoded receptor protein, p53 gene product, and multidrug resistance gene product (P-glycoprotein) . The expression of EGFR, HER-2/neu-encoded receptor protein, and mutant p53 product was significantly lower in LMP tumors than in carcinomas (p < 0.05) . HER-2/neu immunopositivity was more prevalent in adenocarcinomas than in LMP tumors, and the proportion of HER-2/neu-positive adenocarcinomas increased with the progression of the disease . The staining differences between LMP tumors and adenocarcinomas with antibodies against Ki-67, PCNA, and P-glycoprotein were not statistically significant . Immunohistochemical detection of EGFR, HER-2/neu, and p53 in ovarian epithelial tumor is relevant to ovarian tumorigenesis . It could serve as a powerful tool for the pursuit of retrospective studies focused on these important biologic markers.

Biochem Biophys Res Commun, 1997 Nov 26, 240(3), 606 - 11
Human canalicular multispecific organic anion transporter (cMOAT) is expressed in human lung, gastric, and colorectal cancer cells; Narasaki F et al.; Human canalicular multispecific organic anion transporter (cMOAT), a glutathione conjugate membrane transporter, has been isolated from cisplatin-resistant cancer cells and is distributed mainly in normal liver . We analyzed the expression of human cMOAT in 14 lung, 11 gastric, and 9 colorectal non-drug-selected human cancer cells, two multidrug-resistant cells, and one cisplatin-resistant cells, using quantitative RT-PCR and newly developed anti-human cMOAT antibody . All cell lines analyzed here expressed human cMOAT at the level of mRNA and protein, and some of them expressed higher levels of human cMOAT than the cisplatin-resistant cells . The two multidrug-resistant cell lines co-expressed human cMOAT gene and both or either of MRP and MDR1 genes . Immunostaining showed that human cMOAT was predominantly localized to the cytoplasm of these single cells . Our results indicate that human cMOAT is expressed in various human cancer cells including drug-resistant cells.

Int J Cancer, 1997 Nov 27, 73(5), 763 - 8
Characterisation of novel human lung carcinoma cell lines selected for resistance to anti-neoplastic analogues of staurosporine; Courage C et al.; The staurosporine analogues CGP 41251, UCN-01 and Ro 31-8220 are specific inhibitors of protein kinase C (PKC) . CGP 41251 and UCN-01 exert anti-neoplastic activity against human tumours grown in rodents, and CGP 41251 reverses multidrug resistance . The hypothesis was tested that these agents can induce drug resistance and alter cellular levels of target kinases . Human-derived A549 lung carcinoma cells were exposed for 6 months to CGP 41251, UCN-01 or Ro 31-8220 at gradually increasing concentrations . Cells acquired resistance against these agents, 4.3-fold against CGP 41251 (A549/CGP cells), 4.0-fold against UCN-01 (A549/UCN cells) and 14-fold against Ro 31-8220 (A549/Ro cells) . Cells were neither collaterally cross-resistant towards the PKC inhibitors nor resistant against the growth-inhibitory properties of 12-O-tetradecanoylphorbol-13-acetate . However, cross-resistance was observed in A549/CGP cells against staurosporine (13-fold) and in A549/Ro cells against doxorubicin (26-fold) . All 3 cell types expressed multidrug resistance-associated protein, and A549/Ro cells expressed P-glycoprotein, as adjudged by Western blot analysis . Phorbol ester-stimulated PKC activity in these cells was decreased by between 57% and 96% compared to wild-type A549 cells . Levels of the PKC isoenzymes alpha and theta in all 3 resistant cell types and of PKC-epsilon in A549/UCN cells were concomitantly reduced . Cells regained drug sensitivity after culture in the absence of drug for 6 (A549/Ro cells), 5 (A549/CGP cells) and 1 (A549/UCN cells) months . Our results suggest the following features of this type of anti-signalling drug: (i) they can induce drug resistance, (ii) they may be potentially useful in combination because of the lack of cross-resistance between them and (iii) they can down-regulate PKC, which may have pharmacological or toxicological consequences.

Hepatology, 1997 Dec, 26(6), 1467 - 76
Modulation of liver canalicular transport processes by the tyrosine-kinase inhibitor genistein: implications of genistein metabolism in the rat; Jager W et al.; Rat liver cells express the multispecific organic anion transporter (cmoat, cmrp, mrp2) and P-glycoprotein (Pgp) in their canalicular membranes, proteins that are homologous to the multidrug-resistance related protein (MRP) and multidrug resistance (MDR) gene products in multidrug resistant tumor cells . We tested whether genistein, a modulator of drug resistance in tumor cells, affects biliary secretion of substrates of canalicular multispecific organic anion transporter (cmoat) (glucuronides of bilirubin and rhodamine, glutathione conjugate of bromsulphthalein) and of P-glycoprotein (Pgp) (rhodamine), respectively . Using the isolated perfused rat liver of control Wistar rats (TR+) and of a mutant strain (TR-) that expresses Pgp but not cmoat, we show that genistein effectively inhibits the secretion of anionic substrates of cmoat in Wistar rats but stimulates secretion of cationic rhodamine in TR- rats . Genistein is subject to glucuronidation and sulfatation and secretion of genistein and its metabolites stimulates bile flow in Wistar rats, but secretion is nearly absent in TR- rats . Because genistein and its metabolites are substrates for cmoat, inhibition of anion secretion by genistein is partially explained by competition for this transporter . Genistein is also a substrate of uridindiphosphate (UDP)-glucuronyltransferase isoenzyme(s) . Inhibition of glucuronidation reduces the availability of bilirubin and rhodamine glucuronates for transport via cmoat, but unconjugated cationic rhodamine becomes available for transport via Pgp at an increased cellular concentration . Daidzein, a genistein analogue with no effect on protein tyrosine kinase (PTK) shows Similar effects on secretion of organic anions and cations supporting the conclusion that genistein affects transport in liver mainly through competition with other substrates at the sites of glucuronidation and transport via cmoat.

Carcinogenesis, 1997 Nov, 18(11), 2255 - 63
Functional characterization of the rat mdr1b encoded P-glycoprotein: not all inducing agents are substrates; Santoni-Rugiu E et al.; The multidrug resistance (mdr) genes encode P-glycoproteins, integral membrane proteins which function as drug efflux transporters . Exposure of animals in vivo and cells in vitro to a variety of xenobiotics leads to increased mdr1 gene expression and higher levels of P-glycoprotein . This response may protect cells from the cytotoxic effects of these compounds . In this investigation we functionally expressed the rat mdr1b gene in NIH 3T3 cells and assessed the ability of the encoded P-glycoprotein to protect these cells from the cytotoxicity of xenobiotics known to induce mdr1b expression . In long-term colony survival assays, stably expressed mdr1b conferred resistance to cytotoxic drugs such as colchicine, vinblastine and doxorubicin, but not to 5-fluorouracil nor to the carcinogens aflatoxin B1 and N-hydroxy-acetylaminofluorene . The mdr reversal agent verapamil restored cytotoxicity of colchicine, doxorubicin, actinomycin D, vinblastine and taxol, but had no effect on the sensitivity of these cells to 5-fluorouracil, aflatoxin B1 or N-hydroxy-acetylaminofluorene . In a competitive transport assay, verapamil and, to a lesser extent, colchicine blocked the increased efflux of the fluorescent dye rhodamine 123 from mdr1b-transfected cells, whereas aflatoxin B1 did not compete for this export . These data demonstrate that expression of the rat mdr1b encoded P-glycoprotein can protect cells from a diverse group of compounds previously identified to be mdr substrates, however, other effective inducers of mdr expression, such as aflatoxin B1 and N-hydroxy-acetylaminofluorene, remain potent cytotoxins despite high levels of P-glycoprotein . The fact that compounds which are not themselves substrates can induce P-glycoprotein expression may have implications for pharmacokinetic interactions and chemotherapy.

Lancet, 1997 Nov 29, 350(9091), 1624 - 5
Malarone-donation programme in Africa; Bloland PB et al.; PIP: Glaxo Wellcome announced in November 1996 its intent to donate up to 1 million treatment courses per year of its new antimalarial drug, Malarone, to countries in Africa, Southeast Asia, and South America, where malaria is endemic . Because the effectiveness of the small number of available antimalarial drugs is threatened by the emergence of drug resistance, the advantages of introduction of this new drug to a given area should be given careful consideration . Chloroquine, for example, is nearing the end of its effectiveness as a first-line drug for the treatment of uncomplicated falciparum malaria in many areas of East and Central Africa . The lifespan of its replacement, sulfadoxine-pyrimethamine, is likely to be even shorter given its long half-life and the ease with which resistance-conferring mutations occur . In Southeast Asia and the Amazon basin of South America, where multidrug-resistant Plasmodium falciparum malaria is a serious problem, the advantages of Malarone introduction clearly outweigh any disadvantages . In sub-Saharan Africa, the premature distribution and increasing use of artemisinins may jeopardize their long-term effectiveness, however . Another factor complicating decisions to introduce Malarone is its required 3-day course of treatment, necessitating hospitalization if compliance is to be ensured . The donation project gives patients in developing countries access to an expensive drug that would otherwise be unavailable . Time must be taken, however, to fully debate the project's pros and cons, resolve inherent logistic problems, and establish guidelines for Malarone use in sub-Saharan Africa .

Cancer Res, 1997 Dec 1, 57(23), 5292 - 9
Transient induction of the MRP/GS-X pump and gamma-glutamylcysteine synthetase by 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3- nitrosourea in human glioma cells; Gomi A et al.; Treatment of human glioma A172 cells with 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), an alkylating antitumor agent the primary target of which has been thought to be DNA, resulted in elevated expression of mRNA for multidrug resistance-associated protein (MRP) within the first 2 h and then a decrease in expression 24 h after the treatment . Western blot analyses revealed that levels of MRP in these ACNU-treated cells paralleled mRNA levels . Membrane vesicles prepared from ACNU-treated cells also displayed elevated transport activities for leukotriene C4, a known substrate for MRP . Gamma-glutamylcysteine synthetase (gamma-GCS) mRNA expression was coinduced with MRP by ACNU . Because gamma-GCS is the rate-limiting enzyme involved in the de novo biosynthesis of glutathione, increases in glutathione were also transiently induced by ACNU . These results demonstrate for the first time that the expression of functional MRP and gamma-GCS can be transiently coinduced by ACNU . Multiple short exposures (1 h) of ACNU following a long duration (1 week) of drug-free conditions resulted in the development of an ACNU-resistant population (designated A172R) that overexpressed MRP/gamma-GCS mRNA and had elevated transport activities for leukotriene C4 . A172R exhibited cross-resistance to the antitumor drug doxorubicin and heavy metal sodium arsenate but not to cisplatin . Our results also demonstrate that intermittent treatments of human glioma cells with ACNU can lead to the development of MRP-related multidrug resistance . These results, taken together, reveal a possible new mechanism of the development of drug resistance for the antitumor nitrosoureas.

Cancer Res, 1997 Dec 1, 57(23), 5246 - 53
Liposomal doxorubicin circumvents PSC 833-free drug interactions, resulting in effective therapy of multidrug-resistant solid tumors; Krishna R et al.; Conventional methods that are used to overcome multidrug resistance (MDR) often involve the coadministration of chemosensitizers and anticancer drugs . The cyclosporin analogue SDZ PSC 833 {(3'-keto-Bmt1)-(Val2)-cyclosporin} (PSC 833) has been shown to possess powerful chemosensitization properties in vitro, in addition to being intrinsically nontoxic . However, coadministration of PSC 833 with anticancer drugs, such as daunorubicin, doxorubicin (DOX), and Taxol, have resulted in the exacerbation of anticancer drug toxicity, which is due to altered anticancer drug pharmacokinetics . Here, we hypothesized that optimization of the anticancer drug delivery, using liposomal carriers, may, by avoiding these adverse interactions, offer a significant advantage over nonencapsulated drugs . Toxicity studies were conducted in normal BDF1 mice, with i.v . DOX (free or liposome encapsulated) administration and p.o . PSC 833 in single and multiple dosage regimens over a 15-day study period . p.o . administration of PSC 833, at a dose of 100 mg/kg, reduced the maximum tolerated dose (MTD) of i.v administered free drug by 2.5-3-fold, in single- and multiple-dose regimens . In contrast, PSC 833 administration resulted in only a 20% reduction of the MTD for DOX encapsulated in 100-nm 1,2 distearoyl-sn-glycero-3-phosphocholine/cholesterol liposomes (55:45 molar lipid ratio) in a single-dose regimen and had no effect on the liposomal DOX MTD for the day 1, 5, and 9 treatment schedule . Modest modulation of P-glycoprotein-mediated MDR was observed in the murine P388/ADR solid tumor model when PSC 833 was administered with free DOX at the MTD . In contrast, liposomal DOX combined with PSC 833 resulted in tumor growth inhibition that was comparable to that observed for drug-sensitive P388/WT tumors . This efficacy of P388/ADR tumors treatment was dependent on PSC 833 because treatment with liposomal DOX alone provided significantly less antitumor activity . Pharmacokinetic and tissue distribution data demonstrated that DOX encapsulated in 1,2 distearoyl-sn-glycero-3-phosphocholine/cholesterol liposomes exhibited comparable plasma elimination and tissue distribution properties in the presence and absence of PSC 833, whereas free DOX displayed reduced plasma elimination rates and altered tissue distribution in the presence of PSC 833 . These results provide evidence that PSC 833 can induce P-glycoprotein modulation and chemosensitize MDR tumors in the absence of altered DOX pharmacokinetics when liposomal carriers are used . This suggests that the improved tumor selectivity of anticancer drugs that are administered in liposomal formulations may avoid the complications that are associated with free drug-MDR-reversing agent combinations and enhance the therapy of multidrug-resistant tumors.

Cancer Res, 1997 Dec 1, 57(23), 5238 - 42
Disruption of the murine MRP (multidrug resistance protein) gene leads to increased sensitivity to etoposide (VP-16) and increased levels of glutathione; Lorico A et al.; The mrp (multidrug resistance protein) gene has been associated with the multidrug resistance of cancer cells in vitro and in vivo . To gain information on its physiological role, embryonic stem cells were used to generate mice homozygous for a disruption of the mrp gene, resulting in complete abrogation of mrp expression . No physiological abnormalities were observed, at least up to 4 months of age . Viability, fertility, and a range of histological, hematological, and serum-chemical parameters were similar in mrp(+/+) and mrp(-/-) mice . mrp(-/-) mice displayed an increased sensitivity to etoposide phosphate (2-fold) accompanied by greater bone marrow toxicity, whereas the acute toxicity of sodium arsenite was equivalent in mrp(+/+) and mrp(-/-) mice . Tissue levels of glutathione (GSH) were elevated in breast, lung, heart, kidney, muscle, colon, testes, bone marrow cells, blood mononuclear leukocytes, and blood erythrocytes of mrp(-/-) mice and were unchanged in organs known to express little if any mrp, such as the liver and small intestine . The increase in GSH was not due to an increase in the activity of gamma-glutamylcysteine synthetase, the rate-limiting enzyme for GSH synthesis . The findings demonstrate that mrp is dispensable for development and growth but exerts a role in drug detoxification and GSH metabolism.

Cancer Res, 1997 Dec 1, 57(23), 5232 - 7
Evidence that the multidrug resistance protein (MRP) functions as a co-transporter of glutathione and natural product toxins; Rappa G et al.; The MRP (multidrug resistance protein) gene, a member of the ubiquitous superfamily of ATP-binding cassette transporters, is associated with the multidrug resistance of mammalian cells to natural product anticancer agents . We have previously shown that abrogation of MRP expression by gene targeting leads to hypersensitivity to several drugs . In two independently produced MRP double knockout clones, the baseline export of glutathione (GSH) was one-half that of wild-type embryonic stem (ES) cells . The export of GSH from wild-type ES cells, but not from the MRP double knockout clones, increased in the presence of etoposide (VP-16) and sodium arsenite, accompanied by equivalent decreases in intracellular levels of GSH . In the two MRP double knockout clones, the intracellular steady-state concentration of etoposide was twofold greater than that in wild-type cells . Depletion of intracellular GSH by D,L-buthionine sulfoximine increased the intracellular accumulation of radiolabeled etoposide in parental ES cells up to the level present in the two MRP knockout clones but did not change etoposide levels in the MRP knockout clones . These observations provide evidence that: (a) MRP exports GSH physiologically, presumably in association with an endogenous compound(s); (b) baseline MRP expression protects cells from the toxic effects of xenobiotics by effluxing the xenobiotics and GSH from the intracellular compartment into the extracellular medium by a co-transport mechanism; and (c) disruption of the gene encoding MRP abrogates the cotransport of xenobiotics and GSH.

Biochem Pharmacol, 1997 Dec 15, 54(12), 1297 - 306
Emergence of different mechanisms of resistance in the evolution of multidrug resistance in murine erythroleukemia cell lines; Modrak DE et al.; We examined the genetic and biochemical bases for drug resistance and the order of appearance of different mechanisms underlying the increasingly more resistant murine erythroleukemia cell lines established in Adriamycin (ADR) . In the first-step low-level resistant cell line PC4-A5 (able to grow in 5 ng/mL ADR), there was a 2-fold reduction in topoisomerase IIalpha and topoisomerase IIbeta mRNA levels, as well as topoisomerase IIalpha protein and activity levels as compared with the parental cell line . The topoisomerase IIalpha activity levels remained reduced as the cells became increasingly more resistant . In contrast, the topoisomerase II mRNA and protein levels returned to approximately the parental levels in resistant cells growing in higher drug concentrations (40-160 ng/mL) . Parental cells expressed the multidrug resistance protein (MRP), but beginning with PC4-A5 MRP expression decreased and remained reduced in increasingly resistant cell lines . At high levels of ADR resistance, the cells expressed the mdr3 gene concomitant with the appearance of vincristine resistance and energy-dependent daunomycin and vincristine efflux . Glutathione levels, internal pH, and expression of the major vault protein (MVP) remained unchanged in all cell lines . Fluorescence microscopy revealed no alterations in daunomycin distribution or vesicle numbers between the parental and resistant cell lines . Different resistance mechanisms emerge sequentially as cells become more resistant to ADR; the mechanisms are retained during the development of multidrug resistance (MDR) . In intermediate-level MDR cell lines (PC4-A10 and PC4-A20), resistance involves an as yet undetermined mechanism(s).

Leuk Res, 1997 Sep, 21(9), 867 - 74
Effect on cell kill of addition of multidrug resistance modifiers cyclosporin A and PSC 833 to cytotoxic agents in acute myeloid leukaemia; Grey M et al.; Multidrug resistance (MDR) mediated by the drug efflux pump P-glycoprotein (Pgp), may cause remission failure and relapse in patients with acute myeloid leukaemia (AML) by extruding cytotoxic agents such as anthracyclines from leukaemic cells thus allowing them to survive . Cell line data suggest that reversal of MDR is possible using modifying drugs such as cyclosporin A (CSA) and its analogue PSC 833 . We have investigated the effects on cell kill of the addition of CSA and PSC 833 to daunorubicin, idarubicin, mitozantrone, etoposide and cytarabine in 52 fresh cell samples from AML patients using an MTT assay . Pgp status was determined by using monoclonal antibodies JSB-1 and MRK-16 and by assessment of rhodamine efflux . Although overall each cytotoxic-modifier combination produced significant improvements in cell kill compared to cytotoxic alone (P values ranged from P < 0.001 to P = 0.017), modifiers also produced significant cytotoxicity in their own right, and no consistent difference was seen between responses in Pgp-positive and negative groups . Up to one in three Pgp-positive samples failed to show any improvement in cell kill with the addition of CSA or PSC 833, possibly owing to co-expression of alternative resistance mechanisms not affected by the MDR modifiers . The best responses were seen when PSC 833 was added to idarubicin, with 7 out of 22 Pgp-positive cases (32%) showing five-fold improvements in cell kill or better compared to idarubicin alone . Comparison of equimolar concentrations of the two modifiers in the Pgp positive group failed to show a significant difference in cell kill, though PSC 833 was markedly superior to CSA in a minority of highly responsive samples which demonstrated clear evidence of MDR reversal . Our in vitro data suggest that MDR modifiers such as CSA and PSC 833 could play an important role in the therapy of AML and indicate the need for prospective randomised trials to assess their clinical efficacy.

Mol Gen Genet, 1997 Oct, 256(4), 406 - 15
Molecular and phenotypic characterization of yeast PDR1 mutants that show hyperactive transcription of various ABC multidrug transporter genes; Carvajal E et al.; Mutations at the yeast PDR1 transcriptional regulator locus are responsible for overexpression of the three ABC transporter genes PDR5, SNQ2 and YOR1, associated with the appearance of multiple drug resistance . The nucleotide sequences of 13 alleles of PDR1, comprising 6 multidrug resistance mutants, 1 intragenic suppressor and 6 wild types, have been determined . Single amino acid substitutions were shown to result from the mutations pdr1-2 (M308I), pdr1-3 (F815S), pdr1-6 (K302Q), pdr1-7 (P298A) and pdr1-8 (L1036 W), whereas the intragenic suppressor mutant pdr1-100 is deleted for the two amino acids L537 and A538 . An isogenic series of strains was constructed containing the mutant alleles pdr1-3, pdr1-6 and pdr1-8 integrated into the genome . We found that the levels of resistance to cycloheximide, oligomycin, 4-nitroquinoline-N-oxide and ketoconazole were increased in all three mutants . The increase was more pronounced in the pdr1-3 than in the pdr1-6 and pdr1-8 mutants . Studies of the activity of the promoters of the ABC genes PDR5, SNQ2 and YOR1 demonstrated that the combination of the PDR5 promoter and the pdr1-3 mutation resulted in the highest level of promoter induction . Concomitantly, the level of PDR5 mRNA, of Pdr5p protein, and of its associated nucleoside triphosphatase activity, was strongly increased in the plasma membranes of the PDR1 mutants . Again, the pdr1-3 allele was associated with a stronger effect than the pdr1-8 and pdr1-6 alleles . The locations of the mutations in the PDR1 gene indicate that at least three different regions distributed throughout the Pdr1p transcription factor may be mutated to generate a Pdr1p with considerably increased transcriptional activation potency . These gain-of-function mutations support the concept, recently proposed, that in members of the large family of yeast Zn2Cys6 transcription factors a central inhibitory domain exists (delineated by the pdr1-7, pdr1-6 and pdr1-2 mutations) . This domain may interact in a locked conformation with a putative, more C-terminally located inhibitory domain (mutated in pdr1-3), and with the putative activation domain (mutated in pdr1-8).

Mol Gen Genet, 1997 Oct, 256(4), 397 - 405
Clustered amino acid substitutions in the yeast transcription regulator Pdr3p increase pleiotropic drug resistance and identify a new central regulatory domain; Nourani A et al.; In the yeast Saccharomyces cerevisiae mutations in the genes encoding the transcription factors Pdr1p and Pdr3p are known to be associated with pleiotropic drug resistance mediated by the overexpression of the efflux pumps Pdr5p, Snq2p, and Yor1p . Mutagenesis of PDR3 was used to induce multidrug resistance phenotypes and independent pdr3 mutants were isolated and characterized . DNA sequence analysis revealed seven different pdr3 alleles with mutations in the N-terminal region of PDR3 . The pdr3 mutants were semidominant and conferred different drug resistance patterns on host strains deleted either for PDR3 or for PDR3 and PDR1 . Transactivation experiments proved that the mutated forms of Pdr3p induced increased activation of the PDR3, PDR5, and SNQ2 promoters . The amino acid changes encoded by five pdr3 mutant alleles were found to occur in a short protein segment (amino acids 252-280), thus revealing a regulatory domain . This region may play an important role in protein-DNA or protein-protein interactions during activation by Pdr3p . Moreover, this hot spot for gain-of-function mutations overlaps two structural motifs, MI and MII, recently proposed to be conserved in the large family of Zn2Cys6 transcription factors.

J Nat Prod, 1997 Nov, 60(11), 1193 - 5
Multidrug-resistance modulators from Stephania japonica; Hall AM et al.; An alkaloidal extract of the vines of Stephania japonica showed multidrug-resistance-reversing activity as demonstrated by the bicinchoninic acid assay . Two known bisbenzylisoquinoline alkaloids, isotrilobine (1) and trilobine (2), were isolated by bioassay-directed fractionation and separation . Isotrilobine (1) was shown to be as active as verapamil (3) in reversing doxorubicin resistance in human breast cancer cells.

J Natl Cancer Inst, 1997 Nov 19, 89(22), 1706 - 15
P-glycoprotein expression: critical determinant in the response to osteosarcoma chemotherapy; Chan HS et al.; BACKGROUND: Fewer than 20% of patients with bone cancer who are treated with surgery alone are cured . Even with the best current treatment, surgery combined with chemotherapy, only 60%-80% of patients with nonmetastatic bone cancer and 10% of patients with metastatic bone cancer are cured . Thus far, the reason for treatment failure in the nonresponding subset has not been identified . It has been hypothesized that P-glycoprotein, which confers multidrug resistance, might be the cause . We sought to determine whether the expression of P-glycoprotein is associated with poor treatment outcome in osteosarcoma . METHODS: In a retrospective study, we correlated P-glycoprotein expression with the outcome of conventional chemotherapy in 62 consecutive, clinically staged patients diagnosed as having osteosarcoma between 1980 and 1989 . RESULTS: P-glycoprotein was overexpressed in 27 patients but not in another 34 patients, and expression was ambiguous in the sample from one patient . At a median follow-up of 8.9 years, the 34 patients whose tumors did not express P-glycoprotein had significantly better relapse-free rates than the 27 subjects whose tumors expressed the protein (87% versus 0%; P<.00001) and had improved survival rates (94% versus 35%; P<.00001) . Among the 46 patients who received chemotherapy before surgery, the 23 whose tumors were negative for P-glycoprotein showed significantly better long-term outcomes (P<.00002), although differences in tumor necrosis in response to therapy were only of borderline significance (P = .057) . CONCLUSIONS: P-glycoprotein expression does correlate with treatment failure in patients with osteosarcoma . This correlation raises the possibility that inhibiting the action of P-glycoprotein as part of therapy for this disease would improve outcome.

Int J Cancer, 1997 Nov 14, 73(4), 600 - 6
Evidence for reversal of multidrug resistance by quinine in LR73 cells without alteration of nuclear pirarubicin uptake and down-regulation of mdr1 gene expression; Belhoussine R et al.; Confocal laser microspectrofluorometry was used to investigate restoration of nuclear pirarubicin (THP-DOX) accumulation and sensitivity by verapamil, quinine and S9788 in 2 variants of the Chinese hamster ovary cell lines LR73, selected for resistance to doxorubicin (LR73D) or transfected with the mdr1 gene (LR73R) . The 2 resistant cell lines present a multidrug-resistance phenotype (MDR) . Verapamil and S9788, which interact with P-glycoprotein (P-gp), were able to restore nuclear THP-DOX accumulation in LR73R and LR73D cells to a level equivalent to that in sensitive cells . On the other hand, quinine was unable to increase nuclear THP-DOX accumulation significantly even at a concentration of 50 microM . All modulators completely restored THP-DOX sensitivity in resistant cell lines . Our results also show that verapamil and S9788 allow high nuclear drug accumulation, whereas quinine did not affect nuclear accumulation . The effect of quinine on the mdr1 gene expression was determined by the use of reverse transcription coupled with polymerase chain reaction . After a 2 hr treatment with 20 microM of quinine, mdr1 gene expression increased slightly.

Zhonghua Fu Chan Ke Za Zhi, 1996 Nov, 31(11), 652 - 5
{Clinical correlations of multidrug resistance gene expression in ovarian carcinoma}; Chen X et al.; OBJECTIVES: To measure the multidrug resistance gene (MDR1) messenger RNA (mRNA) content of ovarian carcinoma and to investigate the clinical significance of MDR1 expression in ovarian carcinoma . METHODS: A sensitive assay based on the polymerase chain reaction (PCR) was used to measure MDR1 mRNA in biopsy samples of 35 ovarian carcinoma, 20 ovarian benign tumors and 17 normal controls . Pathological diagnosis was made from these samples . RESULTS: (1) The expression rate of MDR1 in ovarian carcinomas was 57.1%, but there was no expression in benign ovarian tumors and in the normal controls . (2) 40.9% of patients given primary treatments and 84.6% of patients previously treated but with relapses had over expression of MDR1 gene . (3) The positive expression rate of MDR1 in cancer lesion, its adjacent sites and metastasis was 52.9%, 11.8% and 70.6% respectively . (4) The response rate to chemotherapy was lower in patients with overexpression of MDR1 gene than in patients without such expression . (5) No significant correlation was observed when the positive rate of expression of MDR1 in cancer tissues was analyzed against clinical stages and pathological types . CONCLUSIONS: The overexpression of MDR1 was correlated with acquired drug-resistance in patients with ovarian caracinoma . Measurement of the expression of MDR1 in a patient's tumor may be of value clinically in detecting drug-sensitivity and in predicting prognosis.

Zhonghua Zhong Liu Za Zhi, 1996 Jul, 18(4), 263 - 5
{Detection of expression of multidrug resistance gene in breast cancer by RT-PCR}; Liu X et al.; Using RT-PCR technique, we established a method for measuring expression of mdr-1 gene in breast cancer and the conditions affecting PCR results . Expression of mdr-1 gene was detected in 74 breast cancer samples . The results showed that 34.3%(12/35) untreated primary lesions and 59.0%(23/39) relapsed metastatic lesions had positive expression of mdr-1 gene (P < 0.05) . In 32 patients, the overall correlation of mdr-1 gene expression with clinical drug sensitivity was 78.1% . The results validate that RT-PCR assay is highly sensitive, relatively quantitative and reliable for measuring levels of mdr-1 gene expression in clinical samples.

J Bioenerg Biomembr, 1997 Aug, 29(4), 401 - 13
Biochemical, genetic, and metabolic adaptations of tumor cells that express the typical multidrug-resistance phenotype . Reversion by new therapies; Baggetto LG; Among the genetic and metabolic alterations that cancer cells undergo, several allow their survival under extreme environmental conditions . The resulting aberrant metabolism is compatible with tumor progression at the expenses of high energy needs, especially for maintaining high division rate . When treated with chemotherapeutic drugs many cancer cells take advantage of their ability to develop a resistance phenotype, as part of an adaptative mechanism . Two main actors of this multidrug phenotype (MDR) are represented by the P-glycoprotein and by the more recently discovered multidrug-resistance associated protein (MRP), two membrane proteins of the ABC superfamily of transporters that can extrude chemotherapeutic drugs under an ATP-dependent mechanism . We will briefly review the major metabolic aberrations that several cancers develop, followed by the molecular, genetic, structural, and functional aspects related mainly to P-glycoprotein, with a concern for the regulation of mdr gene expression . We will point out the role that membrane cholesterol may play in the MDR phenotype, relate this phenotype to bioenergetic considerations, and review the ways of modulating it by the use of new therapeutic approaches.

Br J Cancer, 1997, 76(10), 1376 - 81
Phase IB study of doxorubicin in combination with the multidrug resistance reversing agent S9788 in advanced colorectal and renal cell cancer; Punt CJ et al.; S9788 is a new triazineaminopiperidine derivate capable of reversing multidrug resistance (MDR) in cells resistant to chemotherapeutic agents such as doxorubicin . It does not belong to a known class of MDR revertants, but its action involves the binding of P-glycoprotein . Thirty-eight evaluable patients with advanced colorectal or renal cell cancer were treated with doxorubicin alone (16 patients) followed after disease progression with combination treatment of doxorubicin plus S9788 (12 patients) or upfront with the combination of doxorubicin plus S9788 (22 patients) . S9788 was given i.v . as a loading dose of 56 mg m-2 over 30 min followed by doxorubicin given at 50 mg m-2 as a bolus infusion . Thereafter, a 2-h infusion of S9788 was administered at escalating doses ranging from 24 to 120 mg m-2 in subsequent cohorts of 4-10 patients . Pharmacokinetic analysis demonstrated that concentrations of S9788 that are known to reverse MDR in vitro were achieved in patients at non-toxic doses . Compared with treatment with doxorubicin alone, treatment with the combination of doxorubicin and S9788 produced a significant increase in the occurrence of WHO grade 3-4 granulocytopenia . Treatment with S9788 was cardiotoxic as it caused a dose-dependent and reversible increase in corrected QT intervals as well as clinically non-significant arrhythmias on 24- or 48-h Holter recordings . Although clinically relevant cardiac toxicities did not occur, the study was terminated as higher doses of S9788 may increase the risk of severe cardiac arrhythmias . Twenty-nine patients treated with S9788 plus doxorubicin were evaluable for response, and one patient, who progressed after treatment with doxorubicin alone, achieved a partial response . We conclude that S9788 administered at the doses and schedule used in this study results in relevant plasma concentrations in humans and can safely be administered in combination with doxorubicin.

J Nucl Med, 1997 Nov, 38(11), 1674 - 7
Comparison between technetium-99m-sestamibi and hydrogen-3-daunomycin myocardial cellular retention in vitro; Cayre A et al.; This study was undertaken to verify whether 99mTc-sestamibi uptake parallels that of 3H-daunomycin in cells treated with multidrug resistance (MDR) reversing agents . Since we have detected in a previous work a moderate typical MDR phenotype in rat cardiac cells, a model of cultured myocardial cells was used . METHODS: Newborn-rat cultured myocardial cells were incubated 120 min with the MDR-reversing agent verapamil 50 microM, PSC833 1 microM or S9788 10 microM alone or in combination, and the cellular retention of 3H-daunomycin and 99mTc-sestamibi was counted . RESULTS: Hydrogen-3-daunomycin cellular accumulation was never modified by more than 15% when compared to control values, while 99mTc-sestamibi decreased to 75% +/- 32% (m +/- s.d.) of controls in the presence of S9788 and to 44% +/- 19% when S9788 was associated with verapamil . CONCLUSION: The variations of 99mTc-sestamibi and 3H-daunomycin cellular accumulation induced by MDR-reversing agents in cultured myocardial cells can be dramatically different . While some MDR-reversing agents can significantly increase the 3H-daunomycin retention in cardiac cells, they have unexpected effects on that of 99mTc-sestamibi.

Brain Res, 1997 Sep 26, 769(2), 340 - 6
Differential toxic effects of methamphetamine (METH) and methylenedioxymethamphetamine (MDMA) in multidrug-resistant (mdr1a) knockout mice; Mann H et al.; The toxic effects of methamphetamine (METH) (2.5, 5.0 and 10.0 mg/kg) and methylenedioxymethamphetamine (MDMA) (5.0, 10.0 and 20.0 mg/kg) on dopaminergic systems were assessed in the striatum and of the nucleus accumbens in mdr1a wild-type and knockout mice . METH caused significant dose-dependent decreases of dopamine (DA) and DA transporters (DAT) in the striatum and the nucleus accumbens (NAc) of both wild-type and knockout mice . The lowest doses of METH (2.5 mg/kg) caused only small changes in the wild-type, but marked . decreases in the mdr1a knockout mice . The two higher doses (5 mg/kg and 10 mg/kg) caused similar changes in both strains of mice . In contrast to METH, MDMA caused greater percentage decreases in DAT in the wild-type mice . For example, the lowest dose (5 mg/kg) caused significant decreases in DAT in the NAc of wild-type but not of mdr1a knockout mice . The highest dose (20 mg/kg) caused similar changes in both the strains . These results suggest that METH and MDMA interact differentially with P-glycoproteins . These observations document, for the first time, a role for these proteins in the entry of METH and MDMA into the brain via the blood-brain barrier, with P-glycoprotein possibly facilitating the entry of MDMA but interfering with that of METH into the brain.

Curr Opin Hematol, 1994 Jul, 1(4), 248 - 55
Acute leukemia; Ganser A et al.; Within the past year substantial progress has been made in understanding the molecular changes underlying the development of acute leukemia . Development of molecular probes allowed substantial improvements in the detection of minimal residual disease . Pilot studies underlined the correlation between the expression of the multidrug resistance gene and the likelihood of response to chemotherapy . The expression of mdr-1 apparently correlates with the expression of certain surface antigens like CD34 and CD7, which by themselves are associated with the expression of bcl-2 and poor treatment response . The value of hematopoietic growth factors to overcome drug resistance has not yet been clearly demonstrated in clinical trials, but they seem to reduce early mortality in elderly patients with acute myeloid leukemia when given after chemotherapy . Substantial progress has been made in the understanding of molecular changes in acute promyelocytic leukemia . Combination of all-trans retinoic acid and chemotherapy is the treatment of choice in acute promyelocytic leukemia . Allogeneic bone marrow transplantation proved to be superior in certain high-risk groups of patients with acute leukemia, including those with acute lymphoblastic leukemia with the bcr-abl configuration . The value of allogeneic bone marrow transplantation from related and unrelated donors, as well as the value of autologous bone marrow transplantation, is increasingly defined in randomized trials.

J Med Chem, 1997 Nov 7, 40(23), 3734 - 8
1,4-disubstituted anthracene antitumor agents; Iyengar BS et al.; Three different types of 1,4-disubstituted anthracenes were synthesized, and their cytotoxicity in a panel of tumor cells was compared with that of the corresponding 9,10-disubstituted anthracenes . The panel contained human myeloma, melanoma, colon, and lung cancer cells and sensitive and multidrug-resistant murine L1210 leukemia cells . These compounds had {{(dimethylamino)ethyl}amino}methyl, N-{(dimethylamino)ethyl}carbamoyl, and carboxaldehyde (4,5-dihydro-1H-imidazol-2-yl)hydrazone side chains . The 1,4-diamide was more potent across the tumor panel than the corresponding 9,10-isomer, but the 1,4-diamine and the 1,4-hydrazone were less potent than their 9,10-isomers . Although the 1,4-hydrazone was active against P388 leukemia in mice, it was inactive against L1210 leukemia . Within each pair of compounds, the one with greater average potency against tumor cells gave a greater increase in the transition melt temperature of DNA.

Curr Opin Oncol, 1997 Nov, 9(6), 543 - 8
Inhibitors of multidrug resistance; Sonneveld P et al.; P-glycoprotein expression on tumor cells is a frequent cause of pleiotropic drug resistance in cell lines and tumor specimens . Besides the multidrug resistance gene (MDR1), other mechanisms of increased drug extrusion have been described, such as the MDR-related protein and the lung resistance protein . In addition, other gene-regulated processes may lead to cell survival after exposure to cytostatic agents . It has been shown that p-glycoprotein can be circumvented in vitro by noncytotoxic agents such as verapamil and cyclosporin A, which interact pharmacologically with p-glycoprotein-mediated efflux . More recently, molecular approaches to downregulate p-glycoprotein expression or function have been studied . These approaches and the clinical results obtained so far will be discussed.

Leukemia, 1997 Nov, 11(11), 1850 - 7
Expression of c-Kit and functional drug efflux are correlated in de novo acute myeloid leukaemia; Sincock PM et al.; P-glycoprotein (Pgp), the major mediator of multidrug resistance (MDR) has often been implicated as a poor prognostic indicator in acute myeloid leukaemia (AML) . We have previously reported that high expression of the receptor tyrosine kinase c-Kit in AML is associated with poor prognosis . To determine whether the MDR phenotype is associated with high c-Kit expression, the monoclonal antibodies UIC-2 and YB5.B8, which identify Pgp and c-Kit, respectively, were used for indirect immunofluorescence labelling of 50 de novo AML specimens . Quantitative dye efflux studies using Rhodamine123 were also carried out to assess the functional drug efflux capability of these samples . Pgp expression by the majority of primary AML was comparable to that seen in subsets of cells from normal bone marrow and Spearman rank analysis showed no relationship with c-Kit expression (rs = 0.20, P = 0.16) . However, c-Kit expression did show a significant correlation with Rhodamine123 efflux (rs = 0.57, P = 0.0001), suggesting that the MDR phenotype, Pgp mediated or other, may contribute to the prognostic significance of high c-Kit expression . The monoclonal antibody UIC-2 was used specifically to block Pgp activity of a limited number of leukaemic specimens and cell lines, and evidence of non-Pgp-mediated efflux was found . The existence of alternative mechanisms may explain the relatively low correlation of Pgp expression with dye efflux within the leukaemic samples (rs = 0.47, P = 0.0006) and has implications for prognosis in AML . The c-Kit ligand, stem cell factor, did not influence drug efflux activity of the nine c-Kit-positive AML specimens tested . Thus the correlation between c-Kit and the MDR phenotype in AML is likely to be a consequence of co-expression at a similar stage of differentiation, and may account for the previously observed association of high c-Kit expression with poor outcome.

Ann Hematol, 1997 Sep, 75(3), 81 - 6
Functional expression of MDR-1 in acute myeloid leukemia: correlation with the clinical-biological, immunophenotypical, and prognostic disease characteristics; Martinez A et al.; Up till now, clinical data on the possible prognostic influence of multidrug resistance (MDR) in hematological malignancies have been inconsistent, probably due to technical pitfalls . Moreover, in most studies qualitative information on the presence/absence of MDR-1 expression has been used instead of quantitative results . In addition, results usually refer to the total BM population and not specifically to blast cells . In the present study we analyzed the expression of MDR-1 in a series of 50 newly diagnosed de novo AML using a double-staining technique: (a) monoclonal antibodies for the specific identification of blast cells and (b) the rhodamine-123 efflux assay, which allows a quantitative and calibrated measurement of MDR-1 function . Expression of MDR-1 was correlated with clinical, biological, and immunophenotypical disease characteristics . All patients were uniformly treated according to the AML 87/91 protocols of the Spanish Pethema Cooperative Group; the median age was 51 +/- 19 years and the FAB distribution was as follows: 2 M0, 9 M1, 9 M2, 12 M3, 11 M4, 5 M5, and 2 M6 cases . Upon grouping the 50 AML patients analyzed according to the level of rh123 elimination, it was observed that those cases with > or = 30% decrease in rh123 fluorescence displayed higher WBC counts (9 +/- 12 vs 37 +/- 73 x 10(9)/l, p = 0.02) and platelet numbers (94 +/- 11 vs 35 +/- 25 x 10(9)/l, p = 0.02), together with a higher incidence of extramedullary involvement (35% vs 24%, p = 0.02) . Half of the patients (47%) displaying a low rh123 elimination (< 30%) showed M3 morphology, while among the 33 patients with a higher rate of rh123 elimination (> or = 30%), only four (12%) corresponded to the M3 morphological subtype (p = 0.0006) . From the immunophenotypic point of view, a low rate of rh123 elimination was associated with a lower expression of HLADR antigen (p = 0.003) and a higher expression of CD117 (p = 0.01) . Regarding the possible prognostic influence of MDR1 expression, we found that a high rate of rh123 elimination (> 30%) was associated with a tendency towards poor disease outcome, illustrated by both a lower complete remission rate with the first cycle of chemotherapy (36% vs 56%) and a lower median disease-free survival (22 months vs median DFS not reached), although differences did not reach statistical significance (p = 0.1 in both comparisons) . This data shows that although MDR-1 can be a relevant parameter in the evaluation of AML patients, larger series of patients using appropriate techniques for specifically analyzing the MDR of blast cells will be necessary in order to establish the final clinical value of this parameter.

Stem Cells, 1997, 15 Suppl 2, 243 - 9
Approaches for studying radiation-induced leukemia; Gluzman DF; According to the conclusion of the International Programme on the Health Effects of the Chernobyl Accident (IPHECA) Haematology Pilot Project (1991-1995), there was no increase in the incidence of malignant disease in hematopoietic and lymphoid tissues after the Chernobyl accident . Nevertheless, since studies of A-bomb survivors indicate that the peak in morbidity may occur more than 10 years after radiation exposure, long-term studies of hemoblastoses and myelodysplastic syndromes are needed today . Study of these leukemias and lymphomas that are potentially induced by ionizing radiation must include both fundamental and applied approaches, i.e., A) epidemiological design; B) utilization of modern methods of diagnosis (cytomorphology, immunocytochemistry, cytogenetics); C) studies of gene mutations, mechanisms of apoptosis, and G1 delay; D) monitoring of oncogene and multidrug resistance gene expression, and E) tracking changes in cell-cell signaling in the bone marrow microenvironment.

N Z Med J, 1997 Sep 26, 110(1052), 352 - 4
Tuberculosis in those with HIV infection; Thomas MG et al.; AIMS: To determine the incidence, demography, clinical features, treatments and outcome for patients with tuberculosis and human immunodeficiency virus (HIV) infection in Auckland . METHODS: We reviewed the notes of all patients with HIV infection and tuberculosis seen by the Infectious Disease Unit, at Auckland Hospital since the onset of the HIV epidemic in New Zealand in 1984 until 31 December 1995 . RESULTS: Eleven patients have had HIV infection and tuberculosis, 2.4% of all those with HIV infection cared for by this unit . Ten were male and eight homosexual . The median age was 30 years (range 24-57) . The incidence in Pakeha was 1.2% (3 of 234), in Maori 20% (5 of 25) and in African 27% (3 of 11) . Until 1990 we saw one case every two years and since then one or two cases per year . Six patients had normal chest x-rays and five had abnormal chest x-rays; of the latter, three were typical of tuberculosis and two atypical . Ten of the eleven strains of Mycobacterium tuberculosis cultured were fully sensitive but one was resistant to both rifampicin and isoniazid . Conventional treatment regimens were used . Seven patients have died of HIV infection, three continue treatment and one returned to Africa . One patient relapsed with fully sensitive tuberculosis . Three patients had major side effects to rifampicin necessitating alternative treatment . CONCLUSIONS: Tuberculosis is uncommon amongst those with HIV infection in Auckland but the incidence has risen in recent years . The risks amongst Maori and Africans are high . Multidrug resistant tuberculosis is uncommon . Those caring for patients with tuberculosis need to be mindful of HIV infection: those caring for patients with HIV infection need to be increasingly alert for tuberculosis.

Curr Opin Pulm Med, 1996 May, 2(3), 236 - 45
Tuberculosis reemerges: the captain remains aboard; Efferen LS et al.; The resurgence of tuberculosis and the emergence of multidrug resistant tuberculosis have led to renewed interest in this ancient disease . Advances in the field of molecular biology have increased our understanding of the epidemiology and transmission of infection . This has had a particular impact on the documentation of, and the subsequent development of guidelines to prevent, the nosocomial transmission of tuberculosis . Molecular techniques have dominated the efforts of investigators to improve diagnostic methods and therapeutic options . Recent information regarding the mechanism of developing protective immunity to tuberculosis may lead to the development of more effective vaccines and a role for immunotherapy in treatment . National and international organizations have formulated guidelines for the diagnosis and treatment of disease and infection . The development of a global response to the problem of tuberculosis in order to ensure the establishment of long-lasting control is needed.

Curr Opin Pulm Med, 1995 May, 1(3), 171 - 6
Unusual pathogens for respiratory infections; Marrie TJ; There are a large number of unusual pathogens for respiratory tract infections . The list of such pathogens is continuously changing because of changes in our environment, changes in the host (especially immunosuppression), and advances in medical technology, which allow minimally or otherwise nonpathogenic microorganisms to cause respiratory tract infections . Changes may also occur in common microorganisms such as penicillin-resistant pneumococci or multidrug-resistant Mycobacterium tuberculosis . Finally, usual pathogens may result in unusual manifestations.

Hepatology, 1997 Nov, 26(5), 1195 - 202
Effect of a xanthine analog on human hepatocellular carcinoma cells (Alexander) in culture and in xenografts in SCID mice; Marucci L et al.; Hepatocellular carcinoma (HCC) frequently overexpresses the MDR1 gene and is resistant to drugs transported by the multidrug-resistance efflux pump . A xanthine analog, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine (CT-2584,CTI), is cytotoxic to many tumors in culture and was four times more effective than verapamil in inhibiting Rhodamine 123 secretion in MDR1-overexpressing Chinese hamster ovary cells . However, studies using PRF/PLC/5 (Alexander) cells revealed that CT-2584 is cytotoxic by another mechanism not involving inhibition of MDR1 function . Alexander cells have integrated the hepatitis B surface antigen (HBsAg) gene and quantitatively secrete HBsAg . The parent cell line, Alex 0, has low MDR1 expression and is drug-sensitive, whereas a derived line, Alex 0.5, is drug-resistant and overexpresses MDR1 100 times . Both cell lines were similarly killed within 24 or 48 hours by CT-2584 . Freshly isolated rat and human hepatocytes were considerably more resistant to killing by CT-2584 . In vivo, CT-2584 significantly reduced tumor growth in SCID mice bearing Alex 0 or 0.5 xenografts as determined by serial measurements of HBsAg . Hepatic parenchyma was normal, whereas apoptosis and cellular loss were observed in xenografts . The xenograft model is useful for testing pharmacological therapy of HCC.

Antisense Nucleic Acid Drug Dev, 1997 Oct, 7(5), 511 - 22
Efficient long-term coexpression of a hammerhead ribozyme targeted to the U5 region of HIV-1 LTR by linkage to the multidrug-resistance gene; Lee CG et al.; Ribozymes as anti-HIV-1 agents hold promise for the treatment of AIDS . They can be delivered into cells either exogenously or through an expression system . For effective protection against HIV-1, sufficient and sustained amounts of the antiviral ribozymes must be delivered into target cells . The coexpression of a dominant selectable marker with ribozymes would serve to enrich for cells containing the molecular antiviral and facilitate prolonged expression of these ribozymes . The multidrug resistance gene (MDR1) is a potential clinically relevant selectable marker and offers many advantages over other known dominant selectable markers, including the use of diverse pharmacologically characterized drug or drug combinations for selection . Harvey sarcoma-based retroviral vectors encoding the MDR1 multidrug transporter with a hammerhead ribozyme targeted to highly conserved sequences within the HIV-1 U5 LTR segment have been constructed in a bicistronic format . The internal ribosome entry site (IRES) from encephalomyocarditis virus was used to initiate translation of the MDR1 mRNA . The ribozyme remained functional despite being tethered to MDR1 . Long-term, high-level expression of both the ribozyme and MDR1, as evident by RT-PCR and FACS analysis, was observed in a human T cell line containing the construct selected with vincristine, a cytotoxic substrate for the multidrug transporter.

Int J Cancer, 1997 Nov 4, 73(3), 362 - 6
Mechanisms for high methoxymorpholino doxorubicin cytotoxicity in doxorubicin-resistant tumor cell lines; Bakker M et al.; Methoxymorpholino doxorubicin (MMRDX) is an anthracycline analogue that is able to overcome tumor cell resistance to classical anthracyclines . Mechanisms for increased MMRDX cytotoxicity were analyzed in a small cell lung carcinoma cell line (GLC4), its 300-fold doxorubicin-resistant and multidrug resistance-associated protein (MRP)-over-expressing subline (GLC4/ADR), an ovarian carcinoma cell line (A2780) and its 100-fold doxorubicin resistant and P-glycoprotein (P-gp)-overexpressing subline A2780AD . Cross-resistance, measured with the MTT assay at MMRDX concentration resulting in 50% growth inhibition, was 1.8-fold in GLC4/ADR and 4.5-fold in A2780AD compared to their respective parental cell lines . Cellular MMRDX accumulation was equal in GLC4 and GLC4/ADR and 2-fold lower in A2780AD compared to A2780 . Doxorubicin fluorescence was analyzed with confocal laser scan microscopy . Fluorescence was nuclear in sensitive, and cytoplasmic in resistant, cell lines, while MMRDX fluorescence was found in the nucleus in all cell lines . Pre-incubation with the MRP blocker MK 571 restored in GLC4/ADR cells the nuclear doxorubicin fluorescence pattern, as observed in GLC4 cells . MMRDX, thus, can largely overcome cross-resistance in these P-gp- and MRP-overexpressing doxorubicin-resistant cell lines . Our results suggest that MMRDX is not a substrate for MRP-mediated resistance.

Leuk Res, 1997 Aug, 21(8), 743 - 52
The MRD1 (P-glycoprotein) and MRP (P-190) transporters do not play a major role in the intrinsic multiple drug resistance of Jurkat T lymphocytes; Martel J et al.; The response of T cells in relation to the cell cycle has not been extensively studied . We have attempted to address this question using Jurkat T cells treated with cytostatic drugs known to arrest cells at various transition points of their cycle . We tested various concentrations of drugs that act at G1/S (hydroxyurea, lovastatin, thymidine), early S {aphidicolin, cyclosporin A (CsA), rapamycin} or G2 + M (colchicine, nocodazole) in 24 h cultures . Cytofluorimetric analyses showed that cycling Jurkat cells were equally distributed between the G1 (44.9 +/- 6.5%) and S (42.3 +/- 8.0%) phases . Cell distribution in G2 + M was 12.7 +/- 2.8% . Hydroxyurea but not lovastatin increased the percentage of cells in S phase to ca 60-70% and both drugs decreased it to ca 30% in G1 . Thymidine had no effects . Aphidicolin increased the distribution in S phase to ca 70% with a decrease in G1 to ca 30% . CsA and rapamycin increased the percentage of the cells in G1 to ca 70% and decreased it to ca 25% in S phase . Nocodazole increased cell distribution in G2 + M to ca 60% and induced a decrease in G1 to ca 10% . The effects of the drugs were not related to their toxicity and their limited efficiency raised the possibility that Jurkat cells possessed an intrinsic resistance to these xenobiotics . Time-course analysis showed (scanning electron microscopy) that the early morphological changes induced by colchicine were reversible . Drug efflux experiments (vinblastine) suggested that an ATP-dependent process could be involved . However, Northern blot analyses showed a weak signal for MDR1 (MDR, multiple drug resistance) . In contrast, a probe for multidrug resistance-associated protein (P-190; MRP) showed a strong signal in Jurkat and peripheral lymphocytes . The presence of drugs (CsA, nocodazole, thymidine) (24 h) did not up-regulate its message and cell treatment with BSO only moderately affected the efficiency of the glutathione S-conjugate MRP transporter . Our data suggest that the intrinsic multidrug resistance of leukemic Jurkat T cells does not appear to involve the MDR1 and MRP members of the ABC family of reverse drug transporters and these observations raise the possibility of the involvement of multi-faceted mechanisms.

Nat Med, 1997 Nov, 3(11), 1275 - 9
Increased sensitivity to anticancer drugs and decreased inflammatory response in mice lacking the multidrug resistance-associated protein; Wijnholds J et al.; The multidrug resistance-associated protein (MRP) mediates the cellular excretion of many drugs, glutathione S-conjugates (GS-X) of lipophilic xenobiotics and endogenous cysteinyl leukotrienes . Increased MRP levels in tumor cells can cause multidrug resistance (MDR) by decreasing the intracellular drug concentration . The physiological role or roles of MRP remain ill-defined, however . We have generated MRP-deficient mice by using embryonic stem cell technology . Mice homozygous for the mrp mutant allele, mrp-/-, are viable and fertile, but their response to an inflammatory stimulus is impaired . We attribute this defect to a decreased secretion of leukotriene C4 (LTC4) from leukotriene-synthesizing cells . Moreover, the mrp-/- mice are hypersensitive to the anticancer drug etoposide . The phenotype of mrp-/- mice is consistent with a role for MRP as the main LTC4-exporter in leukotriene-synthesizing cells, and as an important drug exporter in drug-sensitive cells . Our results suggest that this ubiquitous GS-X pump is dispensable in mice, making treatment of MDR with MRP-specific reversal agents potentially feasible.

Br J Haematol, 1997 Oct, 99(1), 76 - 83
MDR 1 expression is an independent prognostic factor for response and survival in de novo acute myeloid leukaemia; van den Heuvel-Eibrink MM et al.; The Multidrug Resistance gene (MDR 1) is frequently expressed in acute myeloid leukaemia (AML) . MDR 1 is associated with resistance to chemotherapy in vitro and with a poor response rate in AML . We have investigated the prognostic value of MDR 1 expression in relation to other patient characteristics with respect to response and survival . One hundred and thirty patients aged 0-88 years were treated for de novo AML with standard induction and consolidation chemotherapy . MDR 1 expression was determined by immunocytochemistry . Univariate and multivariate analyses were conducted to identify prognostic factors for reaching complete remission (CR) and for overall survival from diagnosis, in order to compare MDR 1 with known prognostic factors . Univariate analysis showed that higher MDR 1 expression was an adverse prognostic factor for CR (P<0.001), as was higher age (P<0.001) and unfavourable karyotype (P<0.01) . These factors were also negative prognostic factors for overall survival (P<0.001, P<0.05 and P<0.005, respectively) . In the multivariate analysis MDR 1 (P<0.001), higher age (P<0.001) and karyotype (P<0.01) were independent adverse prognostic factors for CR as well as for overall survival (P<0.001, P<0.005, P<0.001, respectively) . Our data indicate that MDR 1 expression is a disease-related unfavourable prognostic factor which has a significant impact on complete remission and overall survival in AML . Analysis of MDR 1 may be used to determine prognosis in individual patients.

Lipids, 1997 Oct, 32(10), 1045 - 54
Growth inhibitory effects of liposome-associated 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine; Peters AC et al.; The growth inhibitory effects of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3) and various liposome compositions of ET-18-OCH3 were compared in a standardized growth inhibition assay utilizing a diverse tumor cell line panel including cell lines expressing multidrug resistance . ET-18-OCH3 and ELL-12 (4:3:1:2, dioleoylphosphatidylcholine/ cholesterol/dioleoylphosphatidylethanolamine-glutaric acid/ET-18-OCH3), an optimal liposomal ET-18-OCH3 formulation, inhibited growth in the micromolar range in drug-sensitive and -resistant cells . In general, ET-18-OCH3-liposomes were about twofold less growth inhibitory than ET-18-OCH3 . However, the known hemolytic effects of ET-18-OCH3 were greatly reduced, up to 20 or more times, by liposome association . The effects of ET-18-OCH3 and ELL-12 were compared in intracellular {Ca2+} modulation and DNA fragmentation assays . ET-18-OCH3 elicited both concentration- and serum-dependent transient and permanent increases in intracellular {Ca2+} . In contrast, ELL-12 did not modulate intracellular {Ca2+} . ET-18-OCH3 and ELL-12 similarly affected DNA fragmentation, which may be indicative of apoptosis . The results suggest that, although the specific growth inhibitory effects of ET-18-OCH3 and ELL-12 are similar, associating ET-18-OCH3 with stable well-characterized liposomes eliminates nonspecific cell membrane-associated lytic effects.

Hum Gene Ther, 1997 Oct 10, 8(15), 1773 - 83
A bicistronic retroviral vector for protecting hematopoietic cells against antifolates and P-glycoprotein effluxed drugs; Galipeau J et al.; Chemoresistance gene transfer is an experimental method to protect hematopoietic cells from the toxicity of anticancer drugs . Because multiple drugs are usually given together in cancer therapy, this strategy will ultimately require vectors expressing multiple chemoresistance genes . For this reason, we designed a bicistronic retroviral vector (HaMID) containing a modified human multidrug resistance-1 cDNA and a mutant human dihydrofolate reductase cDNA bearing a leucine to tyrosine substitution at codon 22 (L22Y) . To determine if this vector would confer dual drug resistance to hematopoietic cells, recombinant retrovirus was used to transduce the human CEM T lymphoblastic cell line as well as primary murine myeloid progenitors . Growth suppression assays, using polyclonal transduced CEM cells, demonstrated increased resistance to taxol (13-fold), trimetrexate (8.9-fold), vinblastine (5.6-fold), methotrexate (2.5-fold), and etoposide (1.5-fold) when used as single agents . HaMID-transduced cells also grew at a logarithmic rate in the simultaneous presence of 25 nM taxol and 100 nM trimetrexate while control cells were entirely growth inhibited by this drug combination . Similarly, HaMID-transduced murine myeloid progenitors acquired increased resistance to taxol (2.9-fold) and trimetrexate (140-fold), and were able to form colonies in the simultaneous presence of both drugs . Our results suggest that retroviral transfer of HaMID into primary hematopoietic cells should reduce the myelosuppression associated with the combined use of antifolates and P-glycoprotein-effluxed drugs.

Hum Gene Ther, 1997 Oct 10, 8(15), 1745 - 51
Retrovirus-mediated gene transfer of the multidrug resistance-associated protein (MRP) cDNA protects cells from chemotherapeutic agents; D'Hondt V et al.; Transduction of hematopoietic progenitors with a multidrug resistance gene like mdr-1 or mrp aims to protect bone marrow from toxicity of chemotherapeutic agents . The interest in the use of mrp as an alternative to mdr-1 gene transfer for bone marrow protection lies in its different modulation . Indeed, classical P-gp reversal agents, tested in the clinic to decrease mdr-1 tumor resistance, have little or no effect on MRP function . This would allow, in the same patient, the use of reversal agents to decrease P-gp tumor resistance without reversing bone marrow protection of the transduced hematopoietic cells provided by multidrug resistance-associated protein (MRP) . As a first step, we have constructed and tested two different mrp-containing vectors with either the Harvey retroviral long terminal repeat (LTR) or PGK as promoters and generated ecotropic producer cells . We have shown by Southern blot analysis that retroviral supernatant from these producer cells can efficiently transmit the mrp gene to target cells . Mrp expression could be detected by fluorescence-activated cell sorting (FACS) analysis in the producer cells . The transduced cells have increased resistance to doxorubicin, vincristine, and etoposide . Furthermore, chemoprotection of the transduced cells was increased after selection with chemotherapeutic agents in the presence of glutathione, a co-factor for MRP function . These data indicate that mrp retroviral vectors may be useful for chemoprotection and selection.

Exp Hematol, 1997 Nov, 25(12), 1227 - 32
Expression of the human major vault protein LRP in acute myeloid leukemia; Hart SM et al.; Overexpression of a 110-kD protein (lung resistance-related protein {LRP}) may predict a poor response to chemotherapy in patients with acute myeloid leukemia (AML) and ovarian carcinoma . The LRP gene has recently been mapped to chromosome 16, close to the multidrug resistance-associated protein (MRP) gene . Seventy-seven samples from 67 patients with AML were examined for expression of LRP, MRP, and multidrug resistance (MDR1) mRNA using a semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay . Results were compared with 29 normal samples (11 normal peripheral blood and 18 normal bone marrow) . Thirty-three patients with untreated AML were evaluable for response to chemotherapy . Levels of LRP, but not of MRP or MDR1 mRNA, were significantly higher in eight patients who failed to achieve complete remission (CR) compared with 25 patients who achieved CR (p = 0.033) . A positive correlation was demonstrated between LRP and MRP (R = 0.368, p = 0.001) and between MRP and MDR1 mRNA levels (R = 0.301, p = 0.01) in the 77 clinical samples analyzed . In AML samples, a significant difference in MDR1 mRNA levels was found between presentation (47 samples) and relapse (30 samples) (p = 0.031) . No significant difference was seen in LRP mRNA levels between these two groups or in eight patients studied sequentially at both presentation and relapse . Thirteen samples (10 at presentation, 3 at relapse) were analyzed for LRP protein expression by flow cytometry . Eight (5 at presentation, 3 at relapse) displayed greater than 10% positive cells (range 15-86%) . These data suggest that LRP gene overexpression may constitute a novel mechanism of multidrug resistance.

Lancet . 1997 Nov 1;350(9087):1298.
High mortality rates among patients with tuberculosis in Bangui, Central African Republic; Garin B et al.; PIP: Survival time until death was investigated in a prospective cohort of 224 tuberculosis patients from Bangui, Central African Republic, who were randomly selected from among 1492 such patients registered in 1993 and 1994 . 6 patients (2.7%) presented with extrapulmonary tuberculosis, 186 (83%) were smear-positive, and 139 (62%) were infected with HIV-1 . 23 (10.3%) had multidrug-resistant tuberculosis strains . The treatment regimen (isoniazid, rifampicin, ethambutol, and pyrazinamide for 2 months, followed by isoniazid and ethambutol for another 6 months) was successful in 46.4% of HIV-infected patients compared with 67.1% of HIV-negative patients . At the end of 8 months, 39.1% of HIV-infected patients but only 8.2% of HIV-negative patients had died . 24 months after the start of tuberculosis treatment, the cumulative death rate was 58% in HIV-seropositive patients compared with 20% in seronegative patients . Median life expectancy to death among HIV-infected tuberculosis patients was 15 months . Decreased survival was significantly associated with HIV-seropositivity, older age, failure to complete the full treatment regimen, and a low CD4 cell count . Multidrug-resistant tuberculosis was not linked to increased mortality .

Biochem J, 1997 Oct 1, 327 ( Pt 1), 305 - 10
ATP-dependent transport of bilirubin glucuronides by the multidrug resistance protein MRP1 and its hepatocyte canalicular isoform MRP2; Jedlitschky G et al.; Bilirubin is secreted from the liver into bile mainly as monoglucuronosyl and bisglucuronosyl conjugates . We demonstrate for the first time that ATP-dependent transport of both bilirubin glucuronides is mediated by the multidrug resistance protein (MRP1) as well as by the distinct canalicular (apical) isoform MRP2, also termed cMRP or cMOAT (canalicular multispecific organic anion transporter) . In membrane vesicles from MRP1-transfected HeLa cells mono{3H}glucuronosylbilirubin and bis{3H}glucuronosylbilirubin (each at 0.5 microM) were transported with rates of 5.3 and 3.1 pmol/min per mg of protein respectively . Rat hepatocyte canalicular membrane vesicles, which contain Mrp2 (the rat equivalent of MRP2), transported mono{3H}glucuronosylbilirubin and bis{3H}glucuronosylbilirubin at rates of 8.9 and 8.5 pmol/min per mg of protein, whereas membrane vesicles from mutant liver lacking Mrp2 showed no transport of the conjugates . In membrane vesicles from human hepatoma Hep G2 cells, which predominantly expressed MRP2, transport rates were 8.3 and 4.4 pmol/min per mg of protein for monoglucuronosylbilirubin and bisglucuronosylbilirubin respectively . ATP-dependent transport of the glutathione S-conjugate -3H-leukotriene C4, an established high-affinity substrate for MRP1 and MRP2, was inhibited by both bilirubin glucuronides with IC50 values between 0.10 and 0.75 microM . The ratios of leukotriene C4 transport and bilirubin glucuronide transport, determined in the same membrane vesicle preparation, indicated substrate specificity differences between MRP1 and MRP2 with a preference of MRP2 for the glucuronides.

J Infect, 1997 Sep, 35(2), 129 - 33
Investigation of an outbreak of multidrug resistant tuberculosis among renal patients using rpo B gene sequencing and IS6110 inverse PCR; Saunders NA et al.; A cluster of cases of tuberculosis among five patients receiving treatment for renal failure was investigated . Insertion sequence (IS6110) fingerprinting and antibiotic resistance profiling of the Mycobacterium tuberculosis isolates from four of the patients (A-D), who had been on the same ward, showed that three of these cases (A-C) were related, but that the fourth (D) was distinct . An isolate from the fifth patient (E), who had been on a separate ward, was indistinguishable from the outbreak strain by IS6110 profile . However, the isolate from patient E and a second isolate from patient A differed from the previous strains in being rifampicin resistant . Sequence analysis of the rpo B genes of the two rifampicin-resistant strains demonstrated the presence of different mutations, showing that they had evolved independently from the same source strain . IS6110 and rpo B gene analyses are invaluable for the accurate investigation of outbreaks of multidrug resistant tuberculosis.

Anal Cell Pathol, 1997, 14(3), 129 - 40
Is rhodamine 123 an appropriate fluorescent probe to assess P-glycoprotein mediated multidrug resistance in vinblastine-resistant CHO cells?
Petriz J, O'Connor JE, Carmona M, Garcia-Lopez J.
Cellular drug resistance, which involves several mechanisms such as P-glycoprotein (P-gp) overexpression, kinetic and metabolic quiescence, or the increase in the intracellular levels of glutathione, limits the effectiveness of cancer treatment . It has been reported that functional assessment of the cationic dye rhodamin 123 (Rho123) efflux reveals accurately the drug-resistant phenotype . To study cellular drug resistance, we have obtained a CHO-K1 derived cell line resistant to vinblastine by means of multistep selection . This cell line (CHOVBR) displays high reactivity with a monoclonal antibody (MAb) (C219) directed against an internal domain of P-gp, and an active Rho123 efflux, as shown by parallel flow cytometric and fluorometric assays . However, under similar experimental conditions, the drug-sensitive parental cell line CHO-K1 (as well as the myeloblastic KG1 and KG1a cell lines), was also able to pump Rho123 out . These parental CHO-K1 cells had a very low reactivity against the C219 Mab, as confirmed by Western blot analysis . Both vinblastine and verapamil inhibited Rho123 efflux in CHO-K1 cells, but had no effect on CHOVBR cultures . Also, deprivation of vinblastine for one month did not affect Rho123 efflux in these cells . Our results suggest that the activity of P-gp appears to be essential, but not sufficient to confer drug resistance, and that Rho123-based functional assays of drug resistance should be evaluated for each cellular experimental model.

J Pharmacol Exp Ther, 1997 Nov, 283(2), 574 - 80
P-Glycoprotein mediates the efflux of quinidine across the blood-brain barrier; Kusuhara H et al.; Recent studies suggest that P-glycoprotein located on the blood-brain barrier restricts the brain uptake of its substrates . We examined the role of P-glycoprotein on the restricted entry of quinidine to the brain . Quinidine is a well known inhibitor of P-glycoprotein, although it is not yet clarified whether quinidine is the substrate for P-glycoprotein . Kinetic analysis of the uptake of quinidine into the rat brain after intravenous bolus administration revealed that the net uptake clearance is 25.5 microl/min/g brain . Intravenous administration of SDZ PSC 833, a multidrug resistance modifier, enhanced the net uptake clearance of quinidine by 15.7-fold . In contrast, no enhancement by SDZ PSC 833 was observed for the brain uptake of mannitol, a marker for the passive diffusion across the blood-brain barrier . The elimination of {3H} quinidine from the rat brain after microinjection into the cerebral cortex was inhibited by preadministered unlabeled quinidine and verapamil . In addition, the brain-to-plasma concentration ratio of quinidine at 10 min after intravenous administration was 27 . 6-fold higher in mdr1a knock-out mice than in control mice . These results suggest that P-glycoprotein mediates the efflux of quinidine across the blood-brain barrier, resulting in its restricted entry to the brain.

Biochem Pharmacol, 1997 Oct 1, 54(7), 791 - 9
P-glycoprotein-independent decrease in drug accumulation by phorbol ester treatment of tumor cells; Wielinga PR et al.; The effect of a change in the phosphorylation state of the drug transporter P-glycoprotein (P-gp) on its drug transport activity was studied for the substrates daunorubicin (DNR), etoposide (VP-16), and calcein acetoxymethyl ester (Cal-AM) . Phorbol ester (PMA), added to stimulate phosphorylation of P-gp by protein kinase C (PKC), caused a decrease in the cellular accumulation of DNR and VP-16, both in multidrug-resistant (MDR) P-gp-overexpressing cells and in wild-type cells . Since treatment of cells with kinase inhibitor staurosporine (ST) reversed this effect of PMA and the non-PKC-stimulating phorbol ester 4alpha-phorbol, 12,13-didecanoate (4alphaPDD) did not result in a decreased DNR accumulation, we conclude that this effect is the result of kinase activity . The concentration dependence of the inhibition of P-gp by verapamil (Vp) was not influenced by PMA . Accumulation of the P-gp substrate Cal-AM was not influenced by PMA in wild-type cells . Therefore, Cal-AM was used to study the effect of PMA-induced phosphorylation of P-gp on its transport activity . Activation of PKC with PMA or inhibition of protein phosphatase 1/2A (PP1/PP2A) with okadaic acid (OA) did not affect the accumulation of Cal-AM in the MDR cells or wild-type cells . The kinase inhibitor ST increased the Cal-AM accumulation only in the MDR cells . Neither stimulating PKC with PMA nor inhibiting PP1/PP2A with OA led to a decreased inhibition of P-gp by ST, indicating that ST inhibits P-gp directly . From these experiments, we conclude that PKC and PP1/PP2A activity do not regulate the drug transport activity of P-gp . However, these studies provide evidence that PMA-induced PKC activity decreases cellular drug accumulation in a P-gp-independent manner.

Gastroenterology, 1997 Nov, 113(5), 1438 - 42
Osmodependent dynamic localization of the multidrug resistance protein 2 in the rat hepatocyte canalicular membrane; Kubitz R et al.; BACKGROUND & AIMS: Circumstantial evidence suggests a regulation of biliary secretion by transporter insertion and retrieval into and from the canalicular membrane . This study was undertaken to provide direct evidence for such a process . METHODS: Osmosensitivity of the subcellular localization of the mrp2 gene-encoded conjugate export pump (MRP2) was studied by immunofluorescence and confocal laser scanning microscopy of isolated hepatocyte aggregates and in perfused rat liver . RESULTS: MRP2 was localized largely in membranes of the pseudocanaliculi formed by isolated hepatocyte aggregates during hypo-osmotic exposure, whereas after hyperosmotic exposure MRP2 was also detectable in intracellular vesicles . In perfused liver, the EAG15 antibody specific for rat MRP2 and the ZO-1 antibody specific for tight junctions produced immunostaining of the canalicular membrane . However, the relative amount of MRP2 increased significantly in the pericanalicular region with increasing perfusate osmolarity, as shown by confocal microscopy of intracellular vesicles containing MRP2 (but not ZO-1) and by computed densitometry . The osmodependent distribution of MRP2 between the canalicular membrane and intracellular, pericanalicular vesicles occurred within 30 minutes and was fully reversible . CONCLUSIONS: The findings provide direct evidence for an osmosensitive dynamic insertion and retrieval of the canalicular MRP2 transporter into and out of the canalicular membrane.

Gan To Kagaku Ryoho, 1997 Oct, 24(13), 1941 - 6
{Expression of ATP binding cassette superfamily (multidrug resistance-1, multidrug resistance-associated protein, human canalicular multispecific organ anion transporter) mRNA in etoposide and m-AMSA resistant cell lines}; Matsumoto Y et al.; The efficacy of all chemotherapeutic agents is limited by the occurrence of drug resistance . To further understand resistance to topoisomerase (topo) II inhibitors, 50 sublines were isolated as single clones from parental cells by exposure to etoposide or m-AMSA . Subsequently, a population of cells from each sublines was exposed to three-fold higher drug concentrations allowing 16 stable sublines to be established at higher extracellular drug concentration . Quantitative aspects of MRP and C-MOAT were studied by Northern blotting in 66 resistant cell lines . Increased MRP mRNA was observed in 48.5% of resistant cell lines (64.7% of etoposide resistant cells and 31.3% of m-AMSA resistant cell lines) . Increased C-MOAT mRNA was also observed in 39.4% of resistant cell lines (41.2% in etoposide resistant cell lines and 37.5% in m-AMSA resistant cell lines) . To characterize the function of C-MOAT, cellular accumulation assay for 3H-etoposide was performed in three resistant cell lines which overexpress C-MOAT but do not express MRP . Accumulation of etoposide was reduced in the cell lines . Our findings suggest that increased MRP and O-MOAT mRNA seems to be an important mechanism of resistance to topo II inhibitors.

J Intern Med Suppl, 1997, 740, 147 - 51
Transport proteins in drug resistance: detection and prognostic significance in acute myeloid leukemia; Broxterman HJ et al.; Resistance to natural product-derived anti-cancer drugs, such as the anthracyclines and etoposide, contributes to the failure of chemotherapeutic treatment of leukaemia . One biological resistance mechanism of potential importance is the overexpression of the plasma membrane drug transporter proteins P-glycoprotein (Pgp) and multidrug resistance protein (MRP) . Many studies have reported evidence for a correlation of Pgp/MDR1 expression with unfavourable prognostic features in acute myeloid leukaemia (AML) . Failure to achieve complete remission (CR) is correlated with Pgp and the CD34+ phenotype . For MRP fewer data are available, which suggest a basal expression level in most AMLs . Another protein reported to correlate with treatment failure in AML is the lung resistance protein or major vault protein (LRP), a protein with a still unknown function . Co-expression of Pgp and LRP especially seems to define an adverse prognostic population . Further progress towards the understanding of the clinical importance of these proteins is hampered by the lack of validation of methods to determine their expression . A reliable way to measure Pgp seems to be the assessment of the active transport of fluorescent Pgp substrates, such as rhodamine 123 out of AML cells . Such functional Pgp assays can be used to validate mRNA or protein measurements and to quantify the effect of Pgp or the magnitude of the effect of a blocker of the Pgp-mediated drug efflux on the intracellular drug concentration . The prognostic value of such methods has still to be shown.

J Intern Med Suppl, 1997, 740, 133 - 7
Transport proteins in drug resistance: biology and approaches to circumvention; Twentyman PR; At least two transport proteins, P-glycoprotein (Pgp) and the multidrug resistance associated protein (MRP), are believed to play a significant role in clinical resistance to cytotoxic therapy . These proteins are expressed at relatively high levels in a number of malignant diseases including various types of leukaemias . They are variably expressed on both the plasma membrane and intracellular vesicular membranes resulting in cellular drug efflux or vesicular drug sequestration, respectively . The action of MRP as a drug transporter depends on intracellular levels of glutathione . A number of strategies for circumvention of these drug resistance mechanisms have been developed and some of these are now in clinical trial.

Am J Ind Med, 1997 Nov, 32(5), 528 - 34
Occupational risk of Mycobacterium tuberculosis infection in hospital workers; Boudreau AY et al.; We conducted a 4-year (1/89-12/92) retrospective cohort study among employees at a large metropolitan hospital where a nosocomial outbreak of multidrug-resistant tuberculosis (TB) had occurred . We compared the risk of tuberculin skin test (TST) conversion among employees who worked on wards where patients with culture-confirmed TB were cared for ("exposed") with the risk among employees who worked on wards with no such patients ("unexposed") . Exposed employees had a higher 4-year risk of TST conversion (14.5%) than unexposed employees (1.4%) (adjusted relative risk 13.4; 95 percent confidence interval 5.1-35.2) . Exposed employees had significantly higher risks of conversion than unexposed employees during 1989-91, but not for 1992 . Among the exposed, ward clerks had a risk of conversion (15.6%) only slightly lower than nurses (18.2%) . We conclude that employees who worked in areas where patients with active M . tuberculosis infection were cared for, including workers who did not provide direct patient care, had a higher risk of TST conversion than employees who did not work in these areas . Reasons for the decline in risk over time include outbreak termination, fewer admissions of patients with TB, implementation of effective infection control measures, and possible resistance to infection in some members of the study population.

Int J Cancer, 1997 Sep 17, 72(6), 1021 - 6
Increased LRP mRNA expression is associated with the MDR phenotype in intrinsically resistant human cancer cell lines; Laurencot CM et al.; Multidrug resistance (MDR) in human cancer cells is multifactorial . Previously, we reported on the association between expression of P-glycoprotein (Pgp), the multidrug resistance-associated protein (MRP), and the lung resistance protein (LRP) with the MDR phenotype in the NCI panel of 60 human cancer cell lines used for in vitro anticancer drug screening . Eight cell lines from this panel, manifesting widely divergent levels of in vitro drug resistance were chosen to investigate the role of MRP and LRP expression at the molecular level . LRP mRNA levels, as determined by ribonuclease protection assay, varied significantly among the 8 cell lines, and correlated closely with in vitro drug resistance to both MDR and non-MDR related drugs . LRP mRNA expression was determined to be a stronger correlate of drug sensitivity than protein expression . In contrast, MRP mRNA levels were not significantly correlated with drug sensitivity . The rates of newly transcribed LRP or MRP mRNA did not correlate with mRNA levels, indicating that mRNA stability or other features of processing may be important in regulation of LRP and MRP mRNA levels . Using Southern blot analysis, LRP gene amplification was shown not to be associated with LRP overexpression . These data suggest that LRP expression may be an important determinant of the MDR phenotype in cell lines intrinsically resistant to cancer chemotherapeutic agents.

J Med Assoc Thai, 1997 Sep, 80 Suppl 1, S162 - 73
Association of mdr1 gene expression with other prognostic factors and clinical outcome in human breast cancer; Punyammalee B et al.; Multidrug resistance of cancer (CA) is one of a major problems in CA chemotherapy that is frequently associated with the expression of P-glycoprotein (P-gp) encoded by mdr1 genes . However, the controversial results exist regarding to the significance of mdr1 gene expression on clinical drug resistance to chemotherapy of breast CA cells . Recent evidence reported a strong correlation between the increased P-gp levels and the prognosis in advanced breast CA . The current study investigated whether mdr1 gene expression has any impact on prognosis and response to chemotherapy in breast CA patients . We determined mdr1 expression in 127 primary and 8 locally relapsed breast CA using a sensitive, specific and quantitative technique based on a RT-PCR and Southern blot hybridization detection by non-radioactive labelled-probe . In patients with primary breast CA, mdr1 expression were negative (mdr1-ve), low (< 10 units), high (> or = 10 units) in 63.8, 8.7 and 27.5 per cent of the patients, respectively . No differences in age, menopause status, tumor size, stage, lymph node involvement, estrogen receptor level and p53 level were observed between mdr1-ve and mdr1+ve expression patients . However, mdr1 gene expression is often associated with number of positive lymph nodes and negative estrogen receptors (p = 0.008 and 0.0007, respectively) . In locally relapsed cases, mdr1-ve was 62.5 per cent whereas 37.5 per cent were mdr1+ve with high level of mdr1 RNA . No differences in other prognostic factors: lymph nodal involvement, estrogen receptor level and p53 level, were detected in both groups . Response to chemotherapy in primary and recurrent breast CA was not different in mdr1-ve and mdr1+ve patients . Finally, our results show that mdr1 gene expression is frequently present in breast CA both before and after chemotherapy . Association of mdr1 gene overexpression with other two prognostic factors suggests that they may confer a more aggressive nature of the tumor, drug resistance and poor prognosis . Evaluation of these factors may improve the ability to identify and select breast CA patients at high risk for poor prognosis for aggressive treatment . However, in this series response to CMF chemotherapy of primary and locally recurrent breast CA were not affected by the presence or absence of mdr1 gene product.

Pharmazie, 1997 Sep, 52(9), 679 - 85
Molecular modeling study of the multidrug resistance modifiers cis- and trans-flupentixol; Wiese M et al.; Recent drug-membrane interaction and quantitative structure-activity relationship studies of thioxanthenes and related compounds acting as multidrug resistance (MDR) modifiers pointed to the importance of the stereoisomery for their MDR reversing activity . Therefore a molecular modeling study of trans-(T) and cis-flupentixol (C) was performed in order to elucidate the observed discrepancy between equal binding potency to P-glycoprotein and different MDR reversing activity of the two stereoisomers . The results show that the 2 to 3-fold difference in MDR reversing activity of T compared to C might be related to a different orientation of the molecules in the membrane lipid environment . From the conformations generated by the SYBYL systematic search procedure those comprising local energy minima were selected and further optimized with semiempirical quantum chemistry methods . From the optimized conformations those that corresponded to 1H NMR results on drug conformations in lipid environment were selected for further molecular modeling studies . The electrostatic and lipophilic fields of T and C were compared in order to identify molecular properties related to the activity difference . The results show that the electrostatic fields of the drugs when similar in shape are dissimilar and that the lipophilic and hydrophilic regions are clearer separated in T in comparison with C . This imposes a better fitting of T compared to C to membrane lipid environment in accordance with the observed higher interaction strength of T with phospholipids.

Biochem Biophys Res Commun, 1997 Oct 9, 239(1), 345 - 8
Inhibition growth of multidrug resistant KBV200 cells by MDR1 antisense RNA; Li Y et al.; Acquisition of resistance to multiple drugs of tumor cell caused by overexpression of the MDR1 gene is one of major obstacles in cancer chemotherapy . We have attempted to reverse the multidrug resistance (MDR) phenotype by treating vincristine (VCR) and adriamycin (ADM) resistant KBV200 cells with MDR1 antisense RNA . Retroviral vector expressing the antisense RNA was transfected into KBV200 . In the transfected cells, a stable expression of antisense RNA and a reduction of cellular MDR1 mRNA could be detected by RT-PCR, and a reduction of MDR1 specific P-glycoprotein (P-gp) was also detected by Western blot, whereas an increase of the drug concentration in the cells was detected by FACS . The IC50 of transfected cells to VCR and ADM was reduced by 65 and 47% . This study demonstrates that antisense RNA can increase the sensitivity of tumor cells to anticancer drug by decreasing the expression of the MDR1 gene . This strategy may be applicable to cure cancer patients with P-gp mediated MDR phenotype.

Biochem Biophys Res Commun, 1997 Oct 9, 239(1), 51 - 6
Posttranscriptional regulation of MRP/GS-X pump and gamma-glutamylcysteine synthetase expression by 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea and by cycloheximide in human glioma cells; Gomi A et al.; Treatment of human glioma A172 cells with 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethy-3-nitrosourea (ACNU) for 2 to 4 hr resulted in a 2- to 3-fold increase in steady-state levels of multidrug resistance-associated protein (MRP) and gamma-glutamylcysteine synthetase (gamma-GCS) mRNA . Nuclear run-on assays revealed a less than 0.5-fold increase in transcription rates of these genes under the same treatment conditions, suggesting that posttranscriptional regulation plays an important role for the increased mRNA levels . In the absence of ACNU, rates of MRP and gamma-GCS mRNA degradation were similar in A172 cells as determined by incubating cells with the RNase inhibitor, Actinomycin D . ACNU treatments resulted in increased MRP mRNA stability . Induction of MRP and gamma-GCS mRNA by ACNU apparently did not require de novo protein synthesis as determined by the use of protein synthesis inhibitor cycloheximide (CHX) . However, CHX alone could induce accumulation of gamma-GCS mRNA, also by posttranscriptional mechanism . Taken together, these results demonstrate that (i) posttranscriptional regulation is primarily involved in the induction of MRP and gamma-GCS expression by ACNU and CHX in human glioma cells; and (ii) despite the fact that these two genes have been reported to be frequently co-expressed, their responses to the treatments of RNA and protein synthesis inhibitors are not the same.

Biochem Biophys Res Commun, 1997 Oct 20, 239(2), 606 - 11
Polymorphic expression of multidrug resistance mRNA in lung parenchyma of nonpregnant and pregnant rats: a comparison to cystic fibrosis mRNA expression; Johannesson M et al.; Multidrug resistance (MDR1b) and cystic fibrosis transmembrane conductance regulator (CFTR) proteins are members of the "ATP-binding cassette" superfamily of transporters . They are associated with chloride channel activities and ATP secretion and have complementary patterns of expression in several organs . In the rat uterus, CFTR expression is replaced by MDR1b expression during pregnancy . We have studied whether expression of MDR1b and CFTR also vary in the lung during pregnancy . No variations in MDR1b or CFTR mRNA levels during pregnancy were detected . However, there was an unusual degree of variation in MDR1b mRNA expression in lung parenchyma between animals in both the control group and the pregnant group . If present among humans, polymorphic expression of MDR1 in lung parenchyma may explain part of the differences in lung symptomatology observed in the CF patients carrying the same mutation .

Genomics, 1997 Oct 15, 45(2), 368 - 78
Analysis of the intron-exon organization of the human multidrug-resistance protein gene (MRP) and alternative splicing of its mRNA; Grant CE et al.; Overexpression of multidrug-resistance protein (MRP) and P-glycoprotein confers similar but not identical multidrug-resistance phenotypes . However, unlike P-glycoprotein, which comprises two membrane-spanning domains (MSDs) and two nucleotide-binding domains, MRP contains a third NH2-proximal MSD, a feature now identified in several other ATP-binding cassette transmembrane transporters . MRP is located on chromosome 16 at band 13.1 close to the short-arm breakpoint of the pericentric inversion associated with the M4Eo subclass of acute myeloid leukemia . We have defined the intron-exon structure of MRP and characterized a number of splicing variants of MRP mRNA . The gene spans at least 200 kb . It contains 31 exons and a high proportion of class 0 introns, alternative splicing of which results in significant levels of variant transcripts that maintain the original open reading frame of MRP mRNA . Analyses of the conservation of intron-exon organization and protein primary structure suggest that the MRP-related transporters evolved from a common ancestor shared with the cystic fibrosis transmembrane conductance regulator, by fusion with one or more genes encoding polytopic membrane proteins .

Arch Biochem Biophys, 1997 Nov 1, 347(1), 37 - 44
Expression of protein kinase C-beta promotes the stimulatory effect of phorbol ester on phosphatidylethanolamine synthesis; Kiss Z; Stimulation of phosphatidylethanolamine (PtdEtn) synthesis by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) has reportedly been found only in hepatocytes expressing the alpha-, betaII-, epsilon-, and zeta-PKC isozymes . In contrast, stimulation of phosphatidylcholine synthesis by PKC activators, known to be mediated by PKC-alpha, is widespread in mammalian cells . In this work, various cell lines exhibiting characteristic differences in their PKC systems were used to determine the role of specific PKC isozymes in the mediation of PMA effect on PtdEtn synthesis . In NIH 3T3 fibroblasts, which express high levels of PKC-alpha but none of the beta (betaI or betaII) isoforms, PMA did not stimulate PtEtn synthesis . In contrast, in Rat-6 fibroblasts overexpressing PKC-betaI, 10-100 nM PMA considerably (1.7- to 2.6-fold) enhanced PtdEtn synthesis . In wild-type or multidrug resistant MCF-7 human breast carcinoma cells, which express PKC-alpha and PKC-betaII (to varying extents) but not PKC-betaI, PMA had only small or no effects on PtdEtn synthesis . In contrast, in MCF-7 cells overexpressing PKC-alpha, and as a consequence also expressing the betaI- and betaII-PKC isoforms, PMA effectively stimulated the synthesis of PtdEtn . Finally, in HL60 human leukemia cells, which contains PKC-betaII as the major PKC isoform, PMA again stimulated PtdEtn synthesis . The results establish that while stimulation of PtdEtn synthesis by PMA occurs only in selected cell lines, this phenomenon is not restricted to hepatocytes . Furthermore, the data indicate that expression of either PKC-betaI or PKC-betaII, but not PKC-alpha, correlates with the effect of PMA on PtdEtn synthesis . Overall, these observations strongly suggest that regulation of PtdEtn and PtdCho synthesis by PMA involves separate PKC isozymes, i.e., PKC-beta and PKC-alpha, respectively .

Kidney Int, 1997 Oct, 52(4), 953 - 61
Overexpression of ecto-5'-nucleotidase promotes P-glycoprotein expression in renal epithelial cells; Ledoux S et al.; P-glycoprotein (P-gp), responsible for multidrug resistance (MDR) of tumoral cells, is also expressed in apical membranes of normal epithelial cells, among which are proximal tubular cells . Ecto-5'-nucleotidase (5'Nu), co-located with P-gp in renal brush border membranes, could be instrumental in the expression of MDR phenotype . P-gp activity {assessed by rhodamine 123 (R123) and {3H}vinblastine (3H-VBL) accumulation} was evaluated in MDCK cell lines in which human 5'Nu was expressed at different levels after retroviral infection: MDCK-5'NU/- cells with a low 5'Nu activity (Vmax < 2 pmol/mg protein/min) and MDCK-5'NU/+ cells, which expressed a high level of 5'Nu (Vmax 150 +/- 18.5 pmol/mg protein/min) . MDCK-5'NU/- cells did not display functional expression of MDR . In MDCK-5'NU/+ cells, R123 and 3H-VBL accumulation was significantly lower than in MDCK-5'NU/- cells and was dramatically enhanced by P-gp inhibitors . This high P-gp activity in MDCK-5'NU/+ cells was confirmed by their resistance to colchicine (measured by LDH release and MTT assay) as compared to MDCK-5'NU/- and was accounted for by increased membrane expression of P-gp assessed by Western blot . Neither AMP nor adenosine, the substrate and the product of 5'Nu, respectively, affected P-gp activity . Inhibition of 5'Nu with alpha beta-methylene-adenosine-diphosphate (alpha beta MADP) or with a blocking anti-5'Nu antibody (1E9) did not blunt MDR expression in MDCK-5'NU/+ cells . Conversely, the anti-5'Nu antibody 5F/F9, which did not block the enzymatic site, induced a decrease of P-gp activity . Further, incubation of MDCK-5'NU/- cells with conditioned medium from MDCK-5'NU/+ cells, which contained significant amounts of released 5'Nu, induced MDR phenotype . In conclusion: (i) expression of ecto-5'Nu promotes multidrug resistance (MDR) activity in renal epithelial cells by enhancement of P-gp expression; (ii) this effect does not involve enzymatic activity of 5'Nu; (iii) supernatants of cells that express 5'Nu conferred P-gp activity to 5'Nu negative cells.

Yakugaku Zasshi, 1997 Aug, 117(8), 455 - 67
{Mechanism of multidrug resistant tumors and chemotherapeutic approaches against the resistant tumors}; Tsuruo T; Research on multidrug resistance (MDR) has spread widely, with the emphasis on the development of therapeutic approaches . This line of research began in the early 1970s . In 1981 and 1982, calcium channel blockers such as verapamil and calmodulin inhibitors were found to enhance the intracellular levels of vincristine (VCR) and adriamycin (ADM) in resistant tumor cells by inhibiting their outward transport and to circumvent MDR in animal experiments . Since these results were noted for verapamil, various other compounds have been investigated to overcome drug resistance . Among these compounds, two compounds were evaluated in our laboratory . The non-immunosuppressive cyclosporin derivative SDZ PSC833 (PSC) has been shown to reverse MDR completely in vitro and in vivo . The second compound is MS-209, a novel quinoline derivative . MS209 completely reversed the resistance against VCR and ADM in vitro . MS209 enhanced the chemotherapeutic effects of VCR and ADM in P388/VCR- and P388/ADM-bearing mice . MS-209 has now started clinical trials in Japan . In addition to these chemical agents, monoclonal antibodies (moAb) against P-glycoprotein such as MRK16 could be useful tools for selective killing of MDR tumor cells . Furthermore another moAb MRK17 can be used against human MDR cells transfected with macrophage-colony stimulating factor (M-CSF) gene . M-CSF can act as an enhancer of antibody dependent cellular cytotoxicity (ADCC) in therapy of human MDR cancer with the anti-P-glycoprotein antibody.

Semin Oncol, 1997 Oct, 24(5), 580 - 91
Drug resistance in the treatment of sarcomas; Colvin OM; The antineoplastic action and development of drug resistance are reviewed for chemotherapeutic agents used in the treatment of sarcoma, including alkylating agents (cyclophosphamide, ifosphamide, dacarbazine), platinum compounds (cisplatin, carboplatin), the anthracycline compound doxorubicin, the topoisomerase II inhibitor etoposide, and the taxanes (paclitaxel, taxotere) . Drug resistance mechanisms discussed include changes in intracellular glutathione and metallothione levels, increased aldehyde dehydrogenase levels, enhanced DNA repair and protection from apoptosis (for alkylating agents); increased 0-6 alkyltranferase levels (for dacarbazine); multidrug resistance 1- and multidrug resistance associated protein-mediated drug export from cells (anthracyclines, taxanes); and structural alteration of microtubules (taxanes).

Biotechniques, 1997 Oct, 23(4), 722 - 6
Calibration and storage of DNA competitors used for contamination-protected competitive PCR; Kohler T et al.; DNA fragments used as standards in competitive PCR were precisely calibrated using HPLC and commercially available DNA molecular mass markers . The accuracy of calibration was reflected by data that differed by only 2% from the mean when two independently purified and calibrated competitor preparations were compared . Highly dilute competitor solutions were stable at -20 degrees C for up to 1 year in the presence of carrier HindIII-digested lambda DNA, but progressive loss of competitor DNA with increasing storage time was observed when carrier DNA was omitted from the solution . Applying 0.2 U uracil-DNA glycosylase (UDG) per assay of remaining temperature-stable activity did not effect the ratios of synthesized products . This study describes quality management in PCR quantitation that is useful for the measurement of multidrug resistance-associated protein (MRP) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene transcripts.

J Lab Clin Med, 1997 Sep, 130(3), 297 - 306
Standardization of a single-cell assay for sensitive detection of multidrug resistance protein expression in normal and malignant cells in archival clinical samples; Chan HS et al.; Multidrug resistance protein (MRP), like P170, confers multidrug resistance, but its clinical relevance is uncertain, whereas P170 is an accepted cause of chemotherapy failure for which ongoing reversal trials are being conducted . Because such trials have been only modestly successful, we must investigate alternative drug resistance mechanisms such as MRP, which is poorly blocked by P170 inhibitors . The significance of MRP has remained undefined because MRP mRNA is difficult to assay in archival material, does not necessarily reflect MRP levels, and is widely expressed in normal or hematopoietic cells within tumors and bone marrow . Because conventional immunoblot or immunocytochemistry may not be sensitive enough to detect low or heterogeneous MRP expression in clinical samples, we elected to score MRP in single tumor cells by modifying our P170 assays that have proven valuable for correlating P170 expression with the outcome of pediatric cancer chemotherapy . We enhanced the signal-to-noise ratio with several peroxidase-tagged secondary antibody layers and staining refinements, standardizing the assay with MRP-negative and MRP-positive but P170-negative transfected or drug-selected controls in which MRP was quantified by immunoblot . We confirmed sensitivity by staining a very low MRP-expressing revertant line and "mixed" samples containing small numbers of positive cells; we confirmed specificity by applying two antibodies directed against separate MRP epitopes . We examined neuroblastoma, osteosarcoma, rhabdomyosarcoma, and retinoblastoma samples, identifying MRP-positive malignant cells, which were distinguishable from MRP-positive normal cells . This assay may be valuable for early diagnosis of low but potentially important MRP expression, which would allow timely application of alternative therapy, perhaps with MRP-specific blockers.

Eur J Immunol, 1997 Sep, 27(9), 2204 - 11
Enhanced sensitivity of P-glycoprotein-positive multidrug resistant tumor cells to complement-mediated lysis; Bomstein Y et al.; The interaction of KB-V1, a multidrug resistant (MDR) variant of the KB-3-1 human oral carcinoma, with human complement was investigated . KB-V1 cells were found to be more sensitive than KB-3-1 cells to complement-mediated lysis . Detailed analysis of the capacity of KB cells to activate human complement demonstrated that both C3b deposition and formation of the membrane attack complex (MAC) are higher on KB-V1 than on KB-3-1 cells . Furthermore, the MAC formed on KB-V1 cells, but not on KB-3-1 cells, was found to be resistant to trypsin treatment, i.e . more stably inserted into the plasma membrane . Immunofluorescence analysis by flow cytometry showed that KB-V1 cells express less decay-accelerating factor (DAF, CD55) than KB-3-1 cells . Two other complement regulatory proteins, membrane cofactor protein (MCP, CD46) and CD59 are expressed to a similar extent on both KB-V1 and KB-3-1 cells . Treatment of KB-V1 cells with neutralizing anti-P-glycoprotein (P-gp) monoclonal antibodies reduced their sensitivity to complement . In addition, KB-V1 revertants which cease to express P-gp become more resistant to complement . These results indicate that multiple factors, such as reduced expression of DAF, enhanced deposition of C3b and increased binding and stability of the MAC may contribute to the increased complement sensitivity of KB-V1 cells . It is suggested that P-gp is responsible for the complement-sensitive phenotype of KB-V1 cells.

Keio J Med, 1997 Sep, 46(3), 142 - 7
Drug resistance studies using fresh human ovarian carcinoma and soft tissue sarcoma samples; Coley HM; The relevance of continuous cell line cultures to the problem of clinical anticancer drug resistance is unclear . There is also mounting scepticism regarding the use of tumour cell lines with in vitro acquired drug resistance, possessing high levels of resistance unlikely to be seen in the clinical setting . To overcome some of these problems we have initiated a study of drug resistance using fresh tumour material obtained from patients suffering from ovarian cancer and soft tissue sarcoma (STS) . Studies involving ovarian cancer have involved over 30 specimens of stage III-IV disease . For these samples we have specifically focused on the multidrug-resistant (MDR) phenotype, examining the role of proteins P-glycoprotein (Pgp), multidrug resistance-protein (MRP) and lung-resistance-associated protein (LRP) . Techniques have involved chemosensitivity testing, immunocytochemistry and flow cytometry, to measure Pgp function (drug efflux capacity with modulator reversal) . Pgp was the most commonly expressed marker and its expression correlated with survival . MDR modulation using cyclosporins was shown to chemosensitise a proportion of the samples . Hence, in vitro screening can help to identify patients likely to benefit from resistance reversal strategies . Studies involving STS have looked at a combination of MDR and p53 disruption (commonly seen in this disease) . Data have been examined alongside clinical data and the course of disease has been closely monitored . Although our studies are ongoing, we have identified a group of patients with aggressive disease showing marked drug resistance in vitro . All patients have relapsed with persistent disease following chemotherapy or radiotherapy . A number of chemoresistant patients showed a combination of p53 disruption in the presence of an MDR phenotype . Feedback from these translational studies should be used to guide the selection of patients for clinical trials using resistance reversal strategies and may suggest new targets for drug development.

Comput Biomed Res, 1997 Aug, 30(4), 307 - 22
A computer simulation model for the spread of nosocomial infections caused by multidrug-resistant pathogens; Sebille V et al.; A Monte Carlo simulation model was developed for the spread of antibiotic-resistant bacteria in hospital units . The model allows for the representation of every patient and staff member . Staff-patient interactions, staff handwashing compliance, admission of colonized patients, and antibiotic use are included in the model . The simulation model provides colonization curves for patients and staff and offers the possibility of simulating different kinds of hospital units . Simulation of the spread of an antibiotic-resistant pathogen in an intensive care unit was performed . We studied the impact of handwashing compliance on colonization . The importance of handwashing in preventing colonization and the influence of admission of colonized patients in perpetuating an epidemic were confirmed by the model . The model offers a new approach to modeling the spread of nosocomial pathogens in hospital units . It allows one to study the impact of infection control measures and represents a valuable educational tool for staff.

Hepatology, 1997 Oct, 26(4), 980 - 5
Induction of cMrp/cMoat gene expression by cisplatin, 2-acetylaminofluorene, or cycloheximide in rat hepatocytes; Kauffmann HM et al.; The human multidrug-resistance-associated protein (MRP), a member of the adenosine triphosphate (ATP)-binding cassette transporter superfamily, is frequently overexpressed in tumor cells resistant to antineoplastic drugs . In the rat, two Mrp isoforms have been identified, Mrp and cMrp . cMrp, also called Mrp2 or cMoat (canalicular multispecific organic anion transporter), is expressed in the canalicular membrane of rat hepatocytes and mediates the excretion of glucuronate, sulfate, and glutathione conjugates into bile . We investigated the expression of cMrp and Mrp in rat hepatocytes in primary culture . Treatment with the chemical carcinogen 2-acetylaminofluorene (2-AAF), the antineoplastic drug cisplatin, and the protein-synthesis inhibitor cycloheximide led to a dose-dependent and time-dependent increase in cmrp gene expression . A 347-base pair cmrp complementary DNA (cDNA) probe served to demonstrate the induction of cmrp messenger RNA (mRNA) with 40 micromol/L 2-AAF, 5 micromol/L cisplatin, or 5 micromol/L cycloheximide . An analogous response was obtained for the increase in cMrp protein . Mrp mRNA was below the detection limit in Northern blots of RNA from liver and hepatocyte cultures, in contrast to rat testis mRNA which served as a positive control . Immunofluorescence microscopy of cultured hepatocytes was used to visualize cMrp in the plasma membrane . Treatment with 2-AAF led to a marked increase in the immunofluorescence signal confirming the cMrp-inducing potency of 2-AAF . In conclusion, the inducing effect of the compounds studied may reflect a general inducibility of hepatic cMrp by a variety of cytotoxic, carcinogenic, and chemotherapeutic agents which is likely to be of relevance for the acquisition of multidrug resistance during chemotherapy and in the process of chemical carcinogenesis in the liver.

Br J Cancer, 1997, 76(7), 862 - 9
Resistance to the new anti-cancer phospholipid ilmofosine (BM 41 440); Hofmann J et al.; The thioether phospholipid ilmofosine (BM 41 440) is a new anti-cancer drug presently undergoing phase II clinical trials . Because resistance to anti-tumour drugs is a major problem in cancer treatment, we investigated the resistance of different cell lines to this compound . Here we report that the multidrug-resistant cell lines MCF7/ADR, CCRFNCR1000, CCRF/ADR500, CEM/VLB100 and HeLa cell lines transfected with a wild-type and mutated (gly/val185) multidrug resistance 1 gene (MDR1) are cross-resistant to ilmofosine compared with the sensitive parental cell lines . In CEMNM-1 cells, in which the resistance is associated with an altered topoisomerase II gene, no cross-resistance to ilmofosine was observed . Ilmofosine is not capable of modulating multidrug resistance and neither does it reduce the labelling of the P-glycoprotein (P-gp) by azidopine nor alter ATPase activity significantly . The resistance to ilmofosine in multidrug-resistant CCRF/VCR1000 cells cannot be reversed by the potent multidrug resistance modifier dexniguldipine-HCI (B8509-035) . A tenfold excess of ilmofosine does not prevent the MDR-modulating effect of dexniguldipine-HCl . Treatment of cells with ilmofosine does not alter the levels of MDR1 mRNA . Long-term treatment of an ilmofosine-resistant Meth A subline with the drug does not induce multidrug resistance, indicating that ilmofosine does not increase the level of P-gp . Determination of the MDR2 mRNA levels in the cells revealed that the resistance pattern to ilmofosine is not correlated with the expression of this gene . It is concluded, therefore, that multidrug-resistant cells are cross-resistant to ilmofosine and that the compound is not a substrate of Pgp . No association between the expression of the MDR2-encoded P-gp and resistance to ilmofosine was observed . It is supposed that MDR1-associated alterations in membrane lipids cause resistance to ilmofosine.

Oncol Res, 1997, 9(5), 229 - 36
Functional analysis of the nucleotide binding domains of the multidrug resistance protein (MRP); Zhu Q et al.; HeLa cells were transfected with full-length multidrug resistance protein (MRP) cDNA and with MRP cDNAs that had been mutated at certain nucleotide binding domains . Stable transfectants were isolated and those producing equivalent amounts of P190 were tested in cytotoxicity assays using a variety of chemotherapeutic agents . The results demonstrate that deletions in the C-motif of NBD1 or the A-motif of NBD2 have a pronounced effect in reducing resistance levels to adriamycin, vincristine, or etoposide (VP-16) . Single-site mutations of lysine in these same motifs reduce IC50 values but less than that observed with the deletion mutants . Additional studies have demonstrated an increase in drug accumulation and reduction in drug efflux in NBD deletion and single-site mutants . The results of this study therefore identify two lysines of the NBD A- and C-motifs that are critical for MRP-mediated multidrug resistance . The results also provide definitive evidence that resistance occurring as a result of MRP overexpression is related to enhanced levels of an ATP-dependent efflux pump.

Blood, 1997 Oct 15, 90(8), 3027 - 36
Multilineage long-term engraftment potential of drug-resistant hematopoietic progenitors; Bertolini F et al.; Peripheral blood progenitor cells (PBPCs) are increasingly used instead of bone marrow for autologous or allogeneic transplantation . In this study PBPCs mobilized in cancer patients by chemotherapy and granulocyte-colony stimulating factor were collected by apheresis and first enriched by immunoaffinity removal of lineage positive cells . When these cells were exposed to both cyclophosphamide and taxol or cultured for 7 days in the presence of 5-fluorouracil, stem cell factor, and interleukin-3, 88% to 93% of the enriched PBPCs were killed and short-term clonogenic capacity in methylcellulose assays was lost, but week-5 cobblestone area-forming cell (CAFC) enrichment was higher than 10-fold in comparison to enriched PBPCs and higher than 700-fold in comparison to unmanipulated apheresis cells . After drug exposure, most of the progenitors displayed a CD34+, CD38-, multidrug-resistance (MDR+), Rhodamine 123 low, Hoechst 33342 low phenotype, and as few as 180 of these drug-resistant cells were able to generate a stable multilineage human hematopoiesis in sublethally irradiated immunodeficient mice . In these animals, the level of human hematopoietic engraftment was significantly increased by cotransplantation of irradiated cells from the human L87/4 stromal cell line . These observations are consistent with the functional isolation of a population of very early hematopoietic progenitors and might help to design new protocols for the removal of neoplastic cells from autografts.

Eur J Haematol, 1997 Oct, 59(4), 206 - 15
Five putative drug resistance parameters (MDR1/P-glycoprotein, MDR-associated protein, glutathione-S-transferase, bcl-2 and topoisomerase IIalpha) in 57 newly diagnosed acute myeloid leukaemias . Swiss Group for Clinical Cancer Research (SAKK); Lohri A et al.; Using a modified quantitative reverse transcriptase (RT) PCR assay in 57 patients with acute myeloid leukaemia (AML) from a Swiss Phase III multicentre study (SAKK 30/85), we measured the m-RNA expression of the genes from the multidrug resistance gene 1 (MDR1), the multidrug resistance associated protein (MRP), glutathione-S-transferase (GST) pi, bcl-2 and topoisomerase (topo) IIalpha . P-glycoprotein (p-gp) was measured by Western blot, and GST activity by functional assays . To analyse progression-free (PFS) and overall survival (OS), parameters were prospectively divided into "low" and "high" groups, according to their median values (exceptions: MDR1 and p-gp) . Median follow-up was 60 months . RESULTS: MDR1- and MRP mRNA levels correlated with each other (r=0.54, Spearman), FABM4/M5 and extramedullary disease . "Low" bcl-2-mRNA predicted longer PFS: 22 months vs . 7 months (median,p=0.02, log rank), and longer OS: 64 months vs . 14 months (p=0.06) . "Low" topo IIalpha predicted poorer outcome: median PFS 9 vs . 19 months (p=0.03); median survival 12 months vs . "not reached" (p=0.03) . An improved outcome tendency, albeit nonsignificant, was seen in p-gp-negative patients . In a Cox model adjusted for age, performance status, presence of Auer rods, FAB type and clinical response, bcl-2 and topo IIalpha mRNA levels retained their predictive values.

Int J Cancer, 1997 Oct 9, 73(2), 249 - 57
Identification of novel drug resistance-associated proteins by a panel of rat monoclonal antibodies; Flens MJ et al.; Since some multidrug-resistant (MDR) tumor cell lines show drug accumulation defects but do not over-express Pgp or MDR protein (MRP), a search was made for novel MDR-related transporter proteins by immunizing rats with non-small cell lung cancer SW- 1573/2R120 cells to produce monoclonal antibodies (MAbs) . Five rat MAbs (LMR-4, -12, -42, -44 and -94) were generated, showing strong membranous staining of non-Pgp MDR SW- 1573/2R120 tumor cells and minimal reactivity to the corresponding parental and revertant cell lines . In addition, a 6th MAb (LMR-5) was isolated, recognizing the MDR-related lung resistance protein (LRP), previously identified as the major vault protein . The first 5 LMR MAbs show predominantly membranous staining of several non-Pgp MDR tumor cell lines of different histogenetic origins, except for LMR-4, which recognizes only MDR sublines of the SW- 1573 cell line . Flow-cytometric analysis revealed that all MAbs, except LMR-4 and -5, detect outside epitopes . Functional studies showed that these MAbs did not restore the daunorubicin accumulation defect . All but one of the MAbs (LMR-42) showed staining of distinct normal human tissues, notably epithelial cells lining the airways and digestive tract . In addition, staining of vascular endothelial cells was found with all MAbs except LMR-4 . Three MAbs (LMR-12, -44 and -94) showed remarkable immunoreactivity with vincristine-selected SW- 1573 sublines . By immunoblotting and precipitation, the LMR antigens were found to be in the 42-69 kDa range.

Int J Cancer, 1997 Sep 26, 73(1), 164 - 7
Increased MRP expression is associated with resistance to radiation, anthracyclines and etoposide in cells treated with fractionated gamma-radiation; Harvie RM et al.; The failure of chemotherapy is often associated with the failure of radiotherapy in the treatment of cancer . To investigate this relationship, the CCRF-CEM (CEM) human T-cell leukaemia cell line was treated with fractionated gamma-radiation totalling 75 Gy (10 cycles of 1.5 Gy daily for 5 days) . This produced the CEMRR subline which was 1.5-fold resistant to radiation compared with the parental CEM cells . The CEMRR subline was also resistant to daunorbicin, idarubicin and etoposide but not to paclitaxel, cis-platinum or chlorambucil . Treatment with 50 microM buthionine sulphoximine, an inhibitor of glutathione synthesis, reversed the daunorubicin resistance in the CEMRR subline . Multidrug resistance-associated protein (MRP) mRNA was 6-fold higher in the CEMRR subline than in the CEM cells, and there was no detectable expression of P-glycoprotein in either the CEM cells or the CEMRR subline . Treatment of the CEM cells with 2 Gy of gamma-radiation caused an increase in MRP-mRNA within 4 hr which, by 24 hr, was greater than 5-fold that of the untreated CEM cells . No change in MRP mRNA was observed in the CEMRR subline with similar treatment . We conclude that MRP is involved in the immediate response to radiation and it may account for the drug resistance that often develops following radiation treatment.

Int J Cancer, 1997 Sep 26, 73(1), 84 - 93
Expression of the multidrug resistance-associated protein (MRP) and chemoresistance of human non-small-cell lung cancer cells; Berger W et al.; Human non-small-cell lung cancer (NSCLC) is considered to be a chemotherapy-refractory malignancy . The underlying mechanisms remain rather obscure . The multidrug resistance-associated protein (MRP), mediating a multidrug resistance (MDR) phenotype, has been reported to be overexpressed in several drug-selected lung cancer cell lines . A few previous studies have described intrinsic MRP expression in both NSCLC and normal lung tissues . However, the drug-transporting activity as well as the correlation with chemoresistance is unclear . Using 15 unselected cell lines, we show that MRP (mRNA and protein as detected by reverse transcriptase polymerase chain reaction and immunoblot) is frequently expressed intrinsically, with markedly varying intensity, in NSCLC . Two cell lines expressed high MRP levels, one comparable to the drug-selected controls (GLC4/ADR, HL-60/AR) without, however, amplification of the MRP gene (Southern hybridization) . Using 3H-daunomycin (3H-DM) and calcein as MRP substrates and probenecid (PRO), genistein (GEN), benzbromarone (BB), N-ethylmaleimide (NEM) and verapamil (VP) as MRP modulators, drug accumulation studies revealed a transporting activity of MRP that correlated significantly with the gene expression data . Moreover, a significant correlation between MRP expression and chemoresistance against daunomycin (DM), doxorubicin (DOX), etoposide (VP-16) and vinblastine (VBL), but not cisplatin (CDDP) and bleomycin (Bleo) (MTT-based survival assay), was detected . Correlations mainly rested on the pronounced chemoresistance of 2 highly MRP-expressing cell lines and did not reach significance when these cell lines were excluded.

Cancer Res, 1997 Oct 15, 57(20), 4451 - 4
Loss of amino acids 1490Lys-Ser-Lys1492 in the COOH-terminal region of topoisomerase IIalpha in human small cell lung cancer cells selected for resistance to etoposide results in an extranuclear enzyme localization; Wessel I et al.; The human small cell lung cancer NCI-H69 cell line selected for resistance to etoposide (H69/VP) has been reported previously to sequentially overexpress both the MRP and MDR1 multidrug resistance-conferring genes . In addition, immunocytochemistry of H69/VP cells demonstrated a distinct extranuclear localization of the nuclear enzyme topoisomerase IIalpha, the target of etoposide . Immunoblots showed a decrease in Mr 170,000 topoisomerase IIalpha in nuclear extracts in H69/VP but equal amounts of the enzyme in whole-cell extracts . Topoisomerase II catalytic activities in H69 and H69/VP whole-cell extracts were equal, as were their inhibition by etoposide . Sequencing of the entire H69/VP topoisomerase IIalpha cDNA showed a homozygous 9-nucleotide deletion encompassing nucleotides 4468-76, coding for Lys-Ser-Lys, overlapping two potential bipartite nuclear localization signals . The deletion occurred at the initial nine nucleotides of an exon, suggesting alternative splicing of topoisomerase IIalpha mRNA . Subsequent sequencing of H69/VP genomic DNA revealed a G-->T point mutation in the 3' acceptor splice site consensus sequence, resulting in the use of an alternate splice site . Comparison with previous reports on three drug-resistant cell lines with large truncations/deletions in the COOH-terminal region of topoisomerase IIalpha and extranuclear localization point to a pivotal role for the basic cluster 1490Lys-Ser-Lys1492 in the nuclear import of this enzyme.

J Natl Cancer Inst, 1997 Oct 15, 89(20), 1524 - 9
Cross-reactivity of C219 anti-p170(mdr-1) antibody with p185(c-erbB2) in breast cancer cells: cautions on evaluating p170(mdr-1)
Liu B, Sun D, Xia W, Hung MC, Yu D.
BACKGROUND: Increased expression of the multidrug resistance gene (MDR-1)-encoded P-glycoprotein (p170{mdr-1}) is a major cause of tumor cell multidrug resistance . p170(mdr-1) functions as a drug-efflux pump to reduce the cellular accumulation of specific drugs . MDA-MB-435 human breast cancer cells that have been transfected with oncogene c-erbB2 complementary DNA (435.eb cells) express high levels of the transmembrane glycoprotein p185(c-erbB2) and exhibit increased resistance to the chemotherapeutic agent paclitaxel via p170(mdr-1)-independent mechanisms . We have recently discovered that the widely used monoclonal antibody C219, which is specific for p170(mdr-1), may cross-react with p185(c-erbB2) in 435.eb cells . In this study, we have investigated the nature of this cross-reactivity . METHODS: Immunoprecipitation experiments involving the use of breast cancer cells that express different levels of p185(c-erbB2) were performed, and C219 was used for western blot analysis of immunoprecipitated proteins . Immunohistochemical analyses were performed on acetone-fixed slides of human breast cancer cells . Peptide sequence comparisons and enzyme-linked immunosorbent assays were performed to determine the molecular basis of C219 cross-reactivity with p185(c-erbB2) . RESULTS: The cross-reactivity of C219 with p185(c-erbB2) was demonstrated by both western blot and immunohistochemical analyses . Peptide sequence comparisons revealed that C219 recognizes an epitope in p170(mdr-1) (C219 epitope) that shares sequence homology with p185(c-erbB2) . Enzyme-linked immunosorbent assays demonstrated that C219 recognizes synthetic peptides derived from both the C219 epitope in p170(mdr-1) and the C219 epitope-homologous region in p185(c-erbB2) . CONCLUSIONS: The anti-p170(mdr-1) monoclonal antibody C219 cross-reacts with p185(c-erbB2) through a peptide sequence in p185(c-erbB2) that is homologous to the C219 epitope in p170(mdr-1).

J Pharmacol Exp Ther, 1997 Oct, 283(1), 108 - 15
Nonlinear intestinal absorption of 5-hydroxytryptamine receptor antagonist caused by absorptive and secretory transporters; Tamai I et al.; The mechanism of the nonlinear concentration dependence of intestinal absorption of the 5-hydroxytryptamine receptor antagonist azasetron was studied by use of rat in situ intestinal perfusion, as well as an in vitro Ussing-type chamber method mounted with rat intestinal tissue and cultured monolayers of human adenocarcinoma Caco-2 cells . The intestinal absorption rate constant of azasetron evaluated by the Doluisio method increased significantly with increasing concentration of azasetron up to 10 mM in a nonlinear fashion and tended to decrease at higher concentrations . Mucosal-to-serosal directed permeation of {14C}azasetron across rat ileal sheets evaluated by the in vitro Ussing-type chamber method also increased in a nonlinear fashion in a low concentration range, followed by a decrease as the concentration was further increased, whereas serosal-to-mucosal directed permeation decreased in a concentration-dependent manner . Vectorial transport of {14C}azasetron across a Caco-2 cell monolayer was observed, with higher transport in the basolateral-to-apical direction at a trace concentration of azasetron . When the initial uptake rate of azasetron by Caco-2 cells was measured, it was saturable with an apparent half-saturation concentration of 15 mM and was reduced in the presence of several cationic compounds . These observations suggest that azasetron is taken up by a carrier-mediated transport mechanism across the intestinal epithelial cells . When the steady-state uptake of {14C}azasetron was measured, it was increased in the presence of unlabeled azasetron and ondansetron . In addition, the steady-state uptake was enhanced in the presence of a P-glycoprotein inhibitor, cyclosporin A, and by ATP-depletion of the cells, although these treatments had no effect on the initial uptake of {14C}azasetron . Furthermore, the multidrug-resistant cancer cell line K562/ADM that overexpresses P-glycoprotein accumulated azasetron less extensively than did the parental drug-sensitive K562 cells . These results strongly suggest that azasetron is secreted into the intestinal lumen predominantly by P-glycoprotein . We conclude that intestinal transport of azasetron involves specialized transporters in both the absorptive and secretory directions, and the complex nonlinear intestinal absorption characteristics can be ascribed to the participation of multiple transport mechanisms.

J Biol Chem, 1997 Oct 17, 272(42), 26479 - 87
Topology mapping of the amino-terminal half of multidrug resistance-associated protein by epitope insertion and immunofluorescence; Kast C et al.; The multidrug resistance-associated protein (MRP) is an integral membrane protein that causes multidrug resistance when overexpressed in mammalian cells . Within the ATP-binding cassette superfamily, MRP belongs to a subgroup of structurally and functionally related proteins that includes the yeast cadmium factor 1 and yeast oligomycin resistance I proteins, and the mammalian sulfonylurea receptors SUR1 and SUR2 . Hydropathy analysis of these proteins predicts a unique membrane-associated region at the amino terminus followed by a structural unit composed of 12 transmembrane (TM) domains and two nucleotide-binding domains that is characteristic of eukaryotic ATP-binding cassette transporters . The topology of the membrane-associated regions of MRP remains largely unknown and was investigated . Small hemagglutinin epitopes (YPYDVPDYAS) were inserted in predicted hydrophilic segments of the membrane-associated regions from the amino-terminal half of MRP and these proteins were expressed in HeLa cells, and tested for their capacity to confer etoposide resistance . The polarity of the inserted tags with respect to plasma membrane was then deduced by immunofluorescence in intact and permeabilized cells . Insertion of epitopes at positions 4, 163, 271, 574, and 653 produced functional proteins while insertions at positions 127, 417, 461, and 529 abrogated the capacity of MRP to confer drug resistance . Epitopes inserted at positions 4, 163, and 574 were localized extracellularly, whereas those inserted at positions 271 and 653 revealed an intracellular location . Although a single epitope inserted at position 461 was compatible with MRP function, it was inaccessible to the anti-epitope antibody and two copies of the tag at that site abrogated MRP function . These results indicate that the amino terminus of MRP is extracellular, while the linker segment joining the first and second membrane-associated regions is intracellular as is the first nucleotide-binding domain . Our findings are therefore consistent with a topological model of MRP that contains 5 TM segments in the first membrane-associated region and 6 TM segments in the second membrane region.

Antimicrob Agents Chemother, 1997 Oct, 41(10), 2300 - 1
In vitro activities of the biguanide PS-15 and its metabolite, WR99210, against cycloguanil-resistant Plasmodium falciparum isolates from Thailand; Edstein MD et al.; The in vitro activities of the new biguanide PS-15 and its putative active metabolite, WR99210, were determined against seven different isolates or clones of Plasmodium falciparum . The mean 50% inhibitory concentrations of PS-15 and WR99210 were 1,015 and 0.06 ng/ml, respectively . WR99210 was up to 363 times more potent than cycloguanil, the active metabolite of proguanil, against cycloguanil-resistant parasites . The pronounced activity of WR99210 against multidrug-resistant P . falciparum indicates that further studies are required to determine the value of the prodrug, PS-15, as an antimalarial agent.

Cancer Chemother Pharmacol, 1997, 40(6), 489 - 94
Methyl-beta-cyclodextrin in HL-60 parental and multidrug-resistant cancer cell lines: effect on the cytotoxic activity and intracellular accumulation of doxorubicin; Grosse PY et al.; The purpose of this work was to determine the role of methyl-beta-cyclodextrin (MEBCD) in combination with doxorubicin (DOX) on the cellular proliferation of a sensitive parental and a multidrug-resistant human cancer cell line (HL-60 S and HL-60 R) and to study the effect of MEBCD on DOX intracellular accumulation . The cytotoxicity of DOX at five concentrations (50-50,000 nM) was evaluated with or without the coadministration of four fixed noncytotoxic concentrations of MEBCD (100, 200, 500, and 1,000 microM) . Intracellular DOX concentrations were determined by a high-performance liquid chromatography (HPLC) method with fluorescence detection . MEBCD applied at 500 and 1000 microM in combination with doxorubicin (DOX) significantly potentiated the activity of DOX used alone on both sensitive and multidrug-resistant cell lines; 50% growth-inhibitory (IC50) ratios (IC50 MEBCD-DOX/IC50 DOX) were about 3:4 and 1.6:4 for HL-60 S and HL-60 R, respectively . Moreover, intracellular DOX accumulation, determined by HPLC during 6 h of drug exposure, was about 2-4 times higher for cells treated with MEBCD in combination with DOX than in those treated with DOX alone . Similar results were obtained using other paired MCF 7 sensitive and resistant cell lines . Correlation between these results and an MEBCD-cell membrane interaction was discussed . These initial data provide a basis for the potential therapeutic application of MEMBCD in cancer therapy.

Biochem Biophys Res Commun, 1997 Sep 29, 238(3), 790 - 4
cDNA cloning of a short type of multidrug resistance protein homologue, SMRP, from a human lung cancer cell line; Suzuki T et al.; Members of the ATP binding cassette (ABC) superfamily are involved in the energy-dependent transport of a wide variety of substrates including anticancer agents across the membranes . We have cloned a cDNA fragment including a novel ABC sequence from a cisplatin-resistant human lung adenocarcinoma cell line, PC-14/CDDP, by reverse transcription polymerase chain reaction (RT-PCR) using degenerate primers and screened a cDNA library using the cDNA fragment as a probe . A full-length cDNA clone, BM4.8, was obtained . Sequence analysis showed that the cDNA encoded a short type of multidrug resistance protein homologue, SMRP, by computed homology search . SMRP was composed of 946 amino acids and had two ABCs with walker A and B motifs . This gene was mapped on chromosome 3 at band q27 by fluorescence in situ hybridization (FISH) analysis and was found to be expressed in various tissues by Northern blot analysis.

Mol Pharmacol, 1997 Oct, 52(4), 692 - 700
Lipopolysaccharide and the glycoside ring of staurosporine induce CD14 expression on bone marrow granulocytes by different mechanisms; Pedron T et al.; We established previously that lipopolysaccharide (LPS) can induce the expression of LPS-binding sites on bone marrow cells (BMC) . We now report that staurosporine (STP), a glycosylated indolocarbazole alkaloid with potent inhibitory activity for various protein kinases, can induce the same effect . With both agents, the newly expressed LPS receptor was found to be CD14 . The STP-induced effect was independent of its protein kinase inhibitory activity because several other protein kinase inhibitors, such as the indolocarbazole K-252a, the bisindolylmaleimide RO-31-8220, the perylenequinone calphostin C, and the isoquinolinesulfonamide H7, did not induce CD14 expression . The observation that the STP analog K-252a with an identical polyaromatic aglycon moiety was inactive yet the analog UCN-01 with an identical glycoside ring was active suggests that the induction of CD14 expression is triggered by the sugar moiety of STP . Three lines of evidence show that the mechanism of CD14 expression induced by STP differs from that induced by LPS: (i) unlike LPS, STP can stimulate BMC from LPS-unresponsive C3H/HeJ mice, (ii) LPS and STP effects are additive at a saturating dose of LPS, and (iii) the protein kinase inhibitor K-252a inhibits the LPS-induced but not STP-induced stimulation . Therefore, our findings show that both a protein kinase-dependent (LPS-induced) and a protein kinase-independent (STP-induced) mechanism can lead to the expression of the LPS receptor CD14 on BMC . We also found that the STP-induced stimulation of BMC is modulated by cyclosporin A, vinblastine, and verapamil . This observation may suggest that the inducible effect of STP could be initiated by its interaction with P-glycoprotein, a membrane pump with drug efflux function that plays a critical role in the multidrug resistance of cancer cells.

Mol Pharmacol, 1997 Oct, 52(4), 613 - 22
The microtubule-stabilizing agent discodermolide competitively inhibits the binding of paclitaxel (Taxol) to tubulin polymers, enhances tubulin nucleation reactions more potently than paclitaxel, and inhibits the growth of paclitaxel-resistant cells; Kowalski RJ et al.; The lactone-bearing polyhydroxylated alkatetraene (+)-discodermolide, which was isolated from the sponge Discodermia dissoluta, induces the polymerization of purified tubulin with and without microtubule-associated proteins or GTP, and the polymers formed are stable to cold and calcium . These effects are similar to those of paclitaxel (Taxol), but discodermolide is more potent . We confirmed that these properties represent hypernucleation phenomena; we obtained lower tubulin critical concentrations and shorter polymers with discodermolide than paclitaxel under a variety of reaction conditions . Furthermore, we demonstrated that discodermolide is a competitive inhibitor with {3H}paclitaxel in binding to tubulin polymer, with an apparent Ki value of 0.4 microM . Multidrug-resistant human colon and ovarian carcinoma cells overexpressing P-glycoprotein, which are 900- and 2800-fold resistant to paclitaxel, respectively, relative to the parental lines, retained significant sensitivity to discodermolide (25- and 89-fold more resistant relative to the parental lines) . Ovarian carcinoma cells that are 20-30-fold more resistant to paclitaxel than the parental line on the basis of expression of altered beta-tubulin polypeptides retained nearly complete sensitivity to discodermolide . The effects of discodermolide on the reorganization of the microtubules of Potorous tridactylis kidney epithelial cells were examined at different times . Intracellular microtubules were reorganized into bundles in interphase cells much more rapidly after discodermolide treatment compared with paclitaxel treatment . A variety of spindle aberrations were observed after treatment with both drugs . The proportions of the different types of aberration were different for the two drugs and changed with the length of drug treatment.

Cancer, 1997 Oct 1, 80(7), 1250 - 7
Expression of the MDR1 gene product P-glycoprotein in childhood neuroblastoma; Dhooge CR et al.; BACKGROUND: In cancer treated with chemotherapy, multidrug resistance is characterized by increased genetic expression of P-glycoprotein (P-gp), which acts as an ATP-dependent drug-efflux pump . However, the clinical significance of the expression of the multidrug resistance gene (MDR1) product P-gp in neuroblastoma (NB) is still a matter of debate . In this study, the role of the expression of P-gp in NB was evaluated . METHODS: NB tumor imprints and NB positive bone marrow smears from 23 children before and after multidrug chemotherapy were examined for P-gp expression by antialkaline phosphatase immunocytochemical analysis . RESULTS: Before chemotherapy, only 10% of the NB samples showed positivity for P-gp . At diagnosis, no difference in P-gp expression was found between primary tumor cells and NB cells from metastases to bone marrow . P-gp positivity was only observed in patients with nonlocalized disease . P-gp positivity was never found in tumor cells that were histologically well differentiated . No clear correlation of P-gp positivity with poor prognostic parameters, such as chromosome 1p deletion or MYCN amplification, were found . Multidrug chemotherapy did not induce enhanced expression of P-gp in the neuroblasts . However, at clinical recurrence, P-gp expression was found in the metastatic NB cells of five of seven bone marrow samples examined . CONCLUSIONS: The prognostic relevance of P-gp expression in NB was not clear from the results of this study . To resolve the uncertainties, a standardization of the methodology and more prospective studies are needed to determine whether routine analysis of P-gp is worth adding to the other prognostic parameters that are evaluated in NB patients . The finding that metastatic cells are capable of expressing MDR1, in contrast to the NB cells of the primary tumor, would certainly be an interesting topic for further study as work directed at understanding the progression to metastasis continues.

J Clin Microbiol, 1997 Oct, 35(10), 2521 - 5
Rapid method for testing susceptibility of Mycobacterium tuberculosis by using DNA probes; Martin-Casabona N et al.; The increasing number of multidrug-resistant Mycobacterium tuberculosis strains has stimulated the interest of investigators in finding a rapid method for susceptibility testing . We used commercially available rRNA DNA-bioluminescence-labelled probes (Accu-Probe, Gen Probe, Inc . San Diego, Calif.) for this purpose . The study was performed in three chronological steps . (i) We studied the correlation between the photometric light units (PLUs) given by the hybridization method, the numbers of CFU per milliliter, and turbidity as nephelometric units for six different inocula of an M . tuberculosis strain over 14 days . A good correlation (c > 0.9; P < 0.05) was found from the third day for all concentrations used . (ii) Over a period of 14 days we studied the evolution of the PLUs for 20 strains growing in medium with 0.2 microl of isoniazid (H) per ml and 18 strains in medium with 1 microl of rifampin (R) per ml to standardize the method . Susceptible and resistant strains were used according to the reference proportions method in Middlebrook 7H10, and the MICs were determined in solid and liquid media . The final inoculum of a 10(-2) dilution from a McFarland no . 1 standard and reading at 3 and 5 days provided the best results . A quotient was established to find a cutoff point between resistant and susceptible strains . (iii) We used the standardized parameters in 117 tests with H and R . On day 3, the sensitivity, specificity, positive predictive value, and negative predictive value for detecting resistant strains were 86.8, 100, 100, and 90.1%, respectively, and on day 5 they were 96.2, 100, 100, and 94%, respectively . We concluded that the method is readily available, is easy to perform, and could be useful for screening resistant M . tuberculosis strains.

JAMA, 1997 Oct 1, 278(13), 1073 - 7
Transmission of a highly drug-resistant strain (strain W1) of Mycobacterium tuberculosis . Community outbreak and nosocomial transmission via a contaminated bronchoscope; Agerton T et al.; CONTEXT: Nosocomial transmission of multidrug-resistant tuberculosis (MDR TB) has been reported primarily in New York State and has generally been presumed to occur via respiratory aerosols . OBJECTIVE: To assess nosocomial transmission of MDR TB . In 1995, 8 patients with MDR TB were identified in South Carolina; all were resistant to 7 drugs and had matching DNA fingerprints (strain W1) . Community linkswere identified for 5 patients (Patients 1-5) . However, no links were identified forthe other 3 patients (Patients 6-8) except being hospitalized at the same hospital as 1 community patient . DESIGN: Outbreak investigation . SETTING: Community and hospital . PATIENTS: Eight patients whose MDR TB isolates had DNA fingerprint patterns matching strain W1 . MAIN OUTCOME MEASURES: Clinical characteristics of patients with strain W1 Mycobacterium tuberculosis isolates . RESULTS: Patient 5 (community patient) and Patient 8, diagnosed April 1995 and November 1995, respectively, had clinical courses consistent with MDR TB, with smear-positive and culture-positive specimens and cavitary lesions on chest radiograph; both died of MDR TB less than 1 month after diagnosis . Patients 6 and 7 (diagnosed May 1995) each had 1 positive culture for MDR TB; specimens were collected during bronchoscopy . Patient 6 had a skin test conversion after bronchoscopy . Neither Patient 6 nor Patient 7 had a clinical course consistent with MDR TB, neither was treated for MDR TB, and both are alive and well . No evidence of laboratory contamination of specimens, transmission on inpatient wards, or contact among patients was found . All 4 received bronchoscopies in May 1995; Patients 6, 7, and 8 had bronchoscopies 1, 12, and 17 days, respectively, after Patient 5 . Observations revealed that bronchoscope cleaning was inadequate, and the bronchoscope was never immersed in disinfectant . CONCLUSIONS: Inadequate cleaning and disinfection of the bronchoscope after the procedure performed on Patient 5 led to subsequent false-positive cultures in Patients 6 and 7 and transmission of infection to Patient 6 and active MDR TB to Patient 8.

Adv Enzyme Regul, 1997, 37, 335 - 47
Pharmacological characterization of LY335979: a potent cyclopropyldibenzosuberane modulator of P-glycoprotein; Starling JJ et al.; The above data indicate that LY335979 displays the following characteristics of an 'ideal modulator' of Pgp-mediated multidrug resistance: high affinity binding to Pgp, high potency for in vitro reversal of drug resistance, high therapeutic index (activity was demonstrated at doses ranging from 1-30 mg/kg) observed in in vivo antitumor efficacy experiments, and a lack of pharmacokinetic interactions that alter the plasma concentration of coadministered oncolytic agents . These desirable features strongly suggest that LY335979 is an exciting new clinical agent to test the hypothesis that inhibition of P-glycoprotein activity will result in reversal of multidrug resistance in human tumors.

Adv Enzyme Regul, 1997, 37, 321 - 33
The canalicular multidrug resistance protein, cMRP/MRP2, a novel conjugate export pump expressed in the apical membrane of hepatocytes; Keppler D et al.; The conjugate export pump in the hepatocyte canalicular membrane is, together with the ATP-dependent bile salt export pump, one of the two major pumps determining canalicular anion secretion and bile flow . The so-called bile salt-independent bile flow is largely driven by the cmrp/cmoat gene-encoded conjugate export pump, as indicated by the markedly reduced bile flow in the GY/TR- (11, 13-16) and the EHBR mutant rats (18-20) . The importance of conjugation with glutathione (52, 53), glucuronate (11, 21), and sulfate (11, 16) for transfer of endogenous and xenobiotic substances from blood into bile has long been known . The molecular identification (7, 26, 54) and cloning (9, 10, 30) of the ATP-dependent export pump for these conjugates in the canalicular membrane was, at least in part, a consequence of the elucidation of the substrate specificity of the multidrug resistance protein (MRP) which is very similar to that of its canalicular isoform (3-6, 49) . The broad substrate specificity of the conjugate export pump enables the terminal excretion of a multitude of conjugates and amphiphilic anions which are formed by a large number of relatively specific monooxygenases and transferases in phase I and phase II metabolism of endogenous and xenobiotic substances in the hepatocyte . The predominant expression of the conjugate export pump encoded by the cmrp/cmoat gene in the canalicular membrane does not exclude overexpression of this transporter in other cells and tissues when exposed to drugs and toxins that can be excreted by this pump . The apical conjugate export pump (8-10) may thus confer multidrug resistance to tumor cells in a similar manner as MRP1 (55) . The observation that mRNA encoding rat cMrp/cMoat (10, 12) and its rabbit homolog (35) is not only detected in hepatocytes but also in small intestine and the kidneys suggests that the cmrp/cmoat gene-encoded conjugate export pump may function in the apical membrane domain of various epithelial cells.

Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 11037 - 42
p53-dependent regulation of MDR1 gene expression causes selective resistance to chemotherapeutic agents; Thottassery JV et al.; Loss of functional p53 paradoxically results in either increased or decreased resistance to chemotherapeutic drugs . The inconsistent relationship between p53 status and drug sensitivity may reflect p53's selective regulation of genes important to cytotoxic response of chemotherapeutic agents . We reasoned that the discrepant effects of p53 on chemotherapeutic cytotoxicity is due to p53-dependent regulation of the multidrug resistance gene (MDR1) expression in tumors that normally express MDR1 . To test the hypothesis that wild-type p53 regulates the endogenous mdr1 gene we stably introduced a trans-dominant negative (TDN) p53 into rodent H35 hepatoma cells that express P-glycoprotein (Pgp) and have wild-type p53 . Levels of Pgp and mdr1a mRNA were markedly elevated in cells expressing TDN p53 and were linked to impaired p53 function (both transactivation and transrepression) in these cells . Enhanced mdr1a gene expression in the TDN p53 cells was not secondary to mdr1 gene amplification and Pgp was functional as demonstrated by the decreased uptake of vinblastine . Cytotoxicity assays revealed that the TDN p53 cell lines were selectively insensitive to Pgp substrates . Sensitivity was restored by the Pgp inhibitor reserpine, demonstrating that only drug retention was the basis for loss of drug sensitivity . Similar findings were evident in human LS180 colon carcinoma cells engineered to overexpress TDN p53 . Therefore, the p53 inactivation seen in cancers likely leads to selective resistance to chemotherapeutic agents because of up-regulation of MDR1 expression.

Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 10594 - 9
Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein; Dey S et al.; Human P-glycoprotein (Pgp) confers multidrug resistance to cancer cells by ATP-dependent extrusion of a great many structurally dissimilar hydrophobic compounds . The manner in which Pgp recognizes these different substrates is unknown . The protein shows internal homology between its N- and C-terminal halves, each comprised of six putative transmembrane helices and a consensus ATP binding/utilization site . Photoactive derivatives of certain Pgp substrates specifically label two regions, one on each half of the protein . In this study, using {125I}iodoarylazidoprazosin ({125I}IAAP), a photoactive analog of prazosin, we have demonstrated the presence of two nonidentical drug-interaction sites within Pgp . Taking advantage of a highly susceptible trypsin cleavage site in the linker region of Pgp, we characterized the {125I}IAAP binding to the N- and C-terminal halves . cis(Z)-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of {125I}IAAP for the C-terminal half of the protein (C-site) by reducing the Kd from 20 to 6 nM without changing the labeling or affinity (Kd = 42-46 nM) of the N-terminal half (N-site) . Also, the concentration of vinblastine (Pgp substrate) and cyclosporin A (Pgp modulator) required for 50% inhibition of {125I}IAAP binding to the C-site was increased 5- to 6-fold by cis(Z)-flupentixol without any effect on the N-site . In addition, {125I}IAAP binding to the N-site was less susceptible than to C-site to inhibition by vanadate which blocks ATP hydrolysis and drug transport . These data demonstrate the presence of at least two nonidentical substrate interaction sites in Pgp.

Mol Microbiol, 1997 Aug, 25(4), 683 - 94
Mutational analysis of the Saccharomyces cerevisiae ATP-binding cassette transporter protein Ycf1p; Wemmie JA et al.; Ycf1p is a member of the ATP-binding cassette transporter family of membrane proteins . Strong sequence similarity has been observed between Ycf1p, the cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance protein (MRP) . In this work, we have examined the functional significance of several of the conserved amino acid residues and the genetic requirements for Ycf1p subcellular localization . Biochemical fractionation experiments have established that Ycf1p, expressed at single-copy gene levels, co-fractionates with the vacuolar membrane and that this co-fractionation is independent of vps15, vps34 or end3 gene function . Several cystic fibrosis-associated alleles of the CFTR were introduced into Ycf1p and found to elicit defects analogous to those seen in the CFTR . An amino-terminal extension shared between Ycf1p and MRP, but absent from CFTR, was found to be required for Ycf1p function, but not its subcellular localization . Mutant forms of Ycf1p were also identified that exhibited enhanced biological function relative to the wild-type protein . These studies indicate that Ycf1p will provide a simple, genetically tractable model system for the study of the trafficking and function of ATP-binding cassette transporter proteins, such as the CFTR and MRP.

J Biochem Biophys Methods, 1997 Jun 9, 34(3), 177 - 87
An homogeneous assay for measuring the uptake and efflux of radiolabelled drugs in adherent cells; Graves R et al.; We have developed an homogeneous assay to measure the uptake and efflux of {14C}adriamycin (doxorubicin hydrochloride) in human squamous lung carcinoma cells (SKMES-1), using 96 well scintillating microplates . The assay was also used to examine the effect of inhibitors of multidrug resistance in adriamycin resistant cells (SKMES-1/ADR) . The effect of adriamycin on cell growth and viability was examined by continuous monitoring of the uptake of {14C}thymidine . The non-invasive nature of these assays, and the ease of use of the microplates, suggests a role in screens for, and characterisation of, novel chemotherapeutic or chemosensitizing agents.

Br J Pharmacol, 1997 Sep, 122(2), 241 - 8
Interaction of cyclosporin derivatives with the ATPase activity of human P-glycoprotein; Watanabe T et al.; 1 . P-glycoprotein, a 170-180 kDa membrane glycoprotein that mediates multidrug resistance, hydrolyses ATP to efflux a broad spectrum of hydrophobic agents . In this study, we analysed the effects of three MDR reversing agents, verapamil, cyclosporin A and {3'-keto-Bmt1}-{Val2}-cyclosporin (PSC 833), on the adenosine triphosphatase (ATPase) activity of human P-glycoprotein . 2 . P-glycoprotein was immunoprecipitated with a monoclonal antibody (MRK-16) and the P-glycoprotein-MRK-16-Protein A-Sepharose complexes obtained were subjected to a coupled enzyme ATPase assay . 3 . While verapamil activated the ATPase, the cyclosporin derivatives inhibited both the substrate-stimulated and the basal P-glycoprotein ATPase . No significant difference was observed between PSC 833 and cyclosporin A on the inhibition of basal P-glycoprotein ATPase activity . PSC 833 was more potent than cyclosporin A for the substrate-stimulated activity . 4 . Kinetic analysis indicated a competitive inhibition of verapamil-stimulated ATPase by PSC 833 . 5 . The binding of 8-azido-{alpha-32P}-ATP to P-glycoprotein was not altered by the cyclosporin derivatives, verapamil, vinblastine and doxorubicin, suggesting that the modulation by these agents of P-glycoprotein ATPase cannot be attributed to an effect on ATP binding to P-glycoprotein . 6 . The interaction of the cyclosporin derivatives with ATPase of P-glycoprotein might present an alternative and/or additional mechanism of action for the modulation of P-glycoprotein function.

Bioorg Med Chem, 1997 Aug, 5(8), 1481 - 8
Antitumor agents--CLXXIII . Synthesis and evaluation of camptothecin-4 beta-amino-4'-O-demethyl epipodophyllotoxin conjugates as inhibitors of mammalian DNA topoisomerases and as cytotoxic agents; Bastow KF et al.; Two conjugates composed of a camptothecin and a 4'-O-demethyl epipodophyllotoxin derivative joined by an imine linkage were prepared and evaluated as inhibitors of mammalian DNA topoisomerases I and II . Target compounds stimulated cleavable complex formation with both types of enzyme in vitro although activities were reduced at least twofold relative to the activity of unconjugated constituents . The behavior of the most active conjugate as an inhibitor of cell growth closely resembled both topoisomerase I- and II- inhibitory components in that the compound displayed a combined spectrum of activity against various drug-resistant KB sublines . Cytotoxic activity and selectivity were largely retained through conjugation, the exception being a lower than expected activity against a pleiotrophic multidrug-resistant subline . The induced levels and the properties of cellular protein-associated DNA complexes were consistent with topoisomerase involvement and with the in vitro cleavage assay results . Based on the present findings, conjugation afforded cleavable complex-forming topoisomerase inhibitors which display dual target specificity and a broad spectrum of cytotoxic activity against drug-resistant cells.

J Biol Chem, 1997 Oct 3, 272(40), 25333 - 8
Modulation of cardiac ryanodine receptors by sorcin; Lokuta AJ et al.; Sorcin is a widely expressed, 22-kDa Ca2+-binding protein initially identified in multidrug-resistant cells . In the heart, sorcin localizes to the dyadic junctions of transverse tubules and sarcoplasmic reticulum and coimmunoprecipitates with the Ca2+ release channel/ryanodine receptor (RyR) (Meyers, M . B., Pickel, V . M., Sheu, S.-S., Sharma, V . K., Scotto, K . W., and Fishman, G . I . (1995) J . Biol . Chem . 270, 26411-26418) . We have investigated a possible functional interaction between sorcin and cardiac RyR using purified recombinant sorcin in {3H}ryanodine binding experiments and single channel recordings of RyR . The open probability of single RyR was decreased significantly by the addition of sorcin to the cytoplasmic side of the channel (IC50 approximately 480 nM) . In addition, sorcin completely inhibited {3H}ryanodine binding with an IC50 approximately 700 nM . Inhibition occurred over a wide range of {Ca2+}, and sorcin-modulated RyR remained Ca2+-dependent . Furthermore, caffeine-activated RyRs were also inhibited by sorcin at low {Ca2+} (pCa 7), suggesting that Ca2+ is not an obligatory factor for sorcin inhibition of RyR . Comparisons of these inhibitory effects with those of calmodulin and calpain, proteins structurally related to sorcin, suggested that the interaction of sorcin with cardiac RyR was distinct from and independent of either of these modulatory proteins . Phosphorylation of sorcin with the catalytic subunit of protein kinase A significantly decreased the ability of sorcin to modulate RyR . These results suggest that sorcin may modulate RyR function in a normal cell environment and that the level of modulation is in turn influenced by signaling pathways that increase protein kinase A activity.

Anticancer Drugs, 1997 Aug, 8(7), 708 - 13
Novel tetramethylpiperidine-substituted phenazines are potent inhibitors of P-glycoprotein activity in a multidrug resistant cancer cell line; Van Rensburg CE et al.; The multidrug resistance (MDR)-neutralizing and cytotoxic properties of 16 novel tetramethylpiperidine (TMP)-substituted phenazines were compared with those of clofazimine and B669 using a P-glycoprotein (P-gp)-expressing undifferentiated, human leukemia cell line (K562/MMB) . Unchlorinated TMP-substituted phenazine molecules were more cytotoxic than their chlorinated counterparts, while the halogenated molecules, especially those with chlorine atoms at position 3 on the aniline and phenyl rings, were less cytotoxic but more effective as chemosensitizing, P-gp-neutralizing agents . One of the TMP-substituted phenazines, B4121, increased the sensitivity of K562/MMB cells to vinblastine by 100-fold . TMP-substituted phenazines are a novel class of pharmacologic anti-cancer agents with both direct cytotoxic, as well as MDR-neutralizing anti-tumor properties.

Anticancer Drugs, 1997 Aug, 8(7), 671 - 6
Down-regulation of topoisomerase II by camptothecin does not prevent additive activity of the topoisomerase II inhibitor etoposide in vitro; Stahl M et al.; Topoisomerases (Topo) I and II are cellular enzymes that catalyze the relaxation of topologically strained DNA and that are involved in a number of DNA-related processes . Their complete inhibition by Topo I and II inhibitors gives promise for improvements in the treatment of malignant diseases . However, preclinical studies showed down-regulation of Topo II protein expression by Topo I inhibitors, which may preclude the useful application of combined topoisomerase inhibition in the clinic . We investigated the efficacy of the combination of etoposide (ETP) and camptothecin (CPT) in human gastric and lung cancer cell lines with different sensitivity towards ETP . The cytotoxicity of different drugs was assessed by the sulforhodamine B assay . Drug interactions were evaluated by isobologram analysis . The polymerase chain reaction and flow cytometry were employed for examination of the mdr1 (multidrug resistance type 1) phenotype . As reported by others, incubation of the P glycoprotein (P-gp)-negative tumor cell lines with the Topo I inhibitor CPT resulted in a significant down-regulation of Topo II protein expression . This was obviously due to changes in the cell cycle distribution of the cells induced by the treatment, with a marked increase of cells in G2/M phase and a consecutive decrease of S phase cells . Despite these biochemical changes, isobologram analysis showed additive cytotoxic activity of CPT and ETP in all the cell lines, independent of whether the drug incubation was performed simultaneously or sequentially . These data indicate that down-regulation of Topo II protein by CPT does not prevent additive activity of CPT and ETP in vitro, and therefore combined Topo I and II inhibition may be useful for investigation in clinical trials.

Rinsho Byori, 1997 Sep, 45(9), 891 - 8
{Flow cytometric analysis of P-glycoprotein function by rhodamine 123 dye-efflux assay in human leukemia cells}; Kawabata M et al.; Cells with multidrug resistance(MDR) phenotype express P-glycoprotein(P-gp) on cell membrane, which works as a drug-efflux pump with low selectivity . P-gp function can be determined microfluorometrically using the fluorescent dye rhodamine 123(Rh123), which is an artificial substrate for P-gp . In this study, we assessed P-gp function in human leukemia sublines of MOLT-3 with various magnitude of MDR phenotype using the Rh123-efflux assay . The MDR cells efficiently pumped out Rh123 outside cells in parallel with the magnitude of resistance to vincristine, while the parent MOLT-3 cells scarcely showed dye efflux . The P-gp function determined by the dye efflux assay was correlated with the degree of cell surface expression of P-gp measured by indirect flow cytometric analysis using MRK16 anti-P-gp antibody and with the amount of MDR1 mRNA (encoding P-gp) quantified by Northern blot analysis and by competitive reverse transcription-polymerase chain reaction (RT-PCR) assay . In the evaluation of 28 clinical samples obtained from patients with leukemias, 9 cases exhibited positive results Rh123-efflux . A good correlation of Rh123-efflux with MDR1 expression measured by competitive RT-PCR was observed in these samples . Since subpopulations of normal lymphocytes show low degree of P-gp function, the strict gating of leukemia cells was mandatory in the dye-efflux assay in clinical samples . Although leukemia cells could not be distinguished from normal lymphocytes in the conventional scattergram in some cases, additional staining of the former cells with specific monoclonal antibody such as CD34(labelled with PE-Cy5, a dye without interference with Rh123 fluorescence emission) enabled a selective analysis of a particular subpopulation . The Rh123 dye-efflux assay is a simple and sensitive method for the determination of P-gp expression and its function, and is particularly suitable for the analyses in the clinical laboratory.

Blood, 1997 Sep 15, 90(6), 2465 - 70
Immunophenotyping investigation of minimal residual disease is a useful approach for predicting relapse in acute myeloid leukemia patients; San Miguel JF et al.; A high complete remission rate is currently achieved in patients with acute myeloid leukemia (AML) . However, many patients eventually relapse due to the persistence of low numbers of residual leukemic cells that are undetectable by conventional cytomorphologic criteria (minimal residual disease {MRD}) . Using immunophenotypic multiparametric flow cytometry, we have investigated in sequential studies (diagnosis and follow-up) the impact of MRD detection on the outcome of 53 AML patients that had achieved morphologic remission with standard AML protocols and displayed at diagnosis an aberrant phenotype . Patients were studied at diagnosis with a panel of 35 monoclonal antibodies in triple staining combinations for detection of aberrant or uncommon phenotypic features . According to these features, a patient's probe was custom-built at diagnosis for the identification of possible residual leukemic cells during follow-up . The level of MRD at the end of induction and intensification therapy correlated with the number of relapses and relapse-free survival (RFS) . Thus, patients with more than 5 x 10(-3) residual cells (5 residual cells among 1,000 normal bone marrow {BM} cells) identified as leukemic by immunophenotyping in the first remission BM showed a significant higher rate of relapse (67% v 20% for patients with less than 5 x 10(-3) residual cells; P = .002) and a lower median RFS (17 months v not reached; P = .01) . At the end of intensification, with a cut-off value of 2 x 10(-3) leukemic cells, AML patients also separated into two distinct groups with relapse rates of 69% versus 32% (P = .02), respectively, and median RFS of 16 months versus not reached (P = .04) . In addition, overall survival was also significantly related to the level of residual cells in the marrow obtained at the end of induction and particularly after intensification therapy (P = .008) . Furthermore, we have explored whether residual disease was related with the functional expression of multidrug resistance (MDR-1) at diagnosis as assessed by the rhodamine123 assay . Patients with > or =5 x 10(-3) residual leukemic cells at the end of induction therapy had a significantly higher rhodamine-123 efflux (mean, 56% +/- 24%) than those with less than 5 x 10(-3) residual cells (mean, 32% +/- 31%; P = .04) . Finally, multivariate analysis showed that the number of residual cells at the end of induction or intensification therapy was the most important prognostic factor for prediction of RFS . Overall, our results show that immunophenotypical investigation of MRD strongly predicts outcome in patients with AML and that the number of residual leukemic cells correlates with multidrug resistance.

Eur J Biochem, 1997 Aug 15, 248(1), 104 - 12
Efficiency of P-glycoprotein-mediated exclusion of rhodamine dyes from multidrug-resistant cells is determined by their passive transmembrane movement rate; Eytan GD et al.; The aim of the present study was to examine the relationship between the rate of the passive transmembrane movement of multidrug resistance (MDR)-type substrates and the ability of P-glycoprotein to extrude them from MDR cells . For this purpose, seven rhodamine dyes were examined for their P-glycoprotein-mediated exclusion from MDR cells, their localization in wild-type drug-sensitive cells, their capacity to stimulate the ATPase activity of P-glycoprotein reconstituted in proteoliposomes, and their transmembrane movement rate in artificial liposomes . All these rhodamine dyes were accumulated in wild-type drug-sensitive cells and were localized mainly in the mitochondria . All the dyes tested were substrates of reconstituted P-glycoprotein and cellular P-glycoprotein and were excluded to a variable degree from MDR cells . The transmembrane movement rate proved the major factor determining the efficacy of the P-glycoprotein-mediated exclusion of rhodamine dyes from MDR cells . Thus, rhodamine B, the poorest cellular P-glycoprotein substrate, exhibited a high affinity toward reconstituted P-glycoprotein, but was the fastest membrane-traversing dye . In contrast, tetramethylrosamine, the best cellular MDR probe, exhibited high affinity toward reconstituted P-glycoprotein and slow transmembrane movement rate . Therefore, an anticancer drug with a fast transmembrane movement rate is expected to overcome the MDR phenomenon . Furthermore, the widely used MDR marker, rhodamine 123, was a poor cellular MDR substrate compared with other rhodamine dyes, especially tetramethylrosamine, which was a superior cellular MDR substrate for functional dye-exclusion studies.

Biochem Pharmacol, 1997 Sep 15, 54(6), 649 - 55
Overexpression of the multidrug resistance genes mdr1, mdr3, and mrp in L1210 leukemia cells resistant to inhibitors of ribonucleotide reductase; Rappa G et al.; L1210 MQ-580 is a murine leukemia cell line resistant to the cytotoxic activity of the alpha-(N)-heterocyclic carboxaldehyde thiosemicarbazone class of inhibitors of ribonucleotide reductase . The line is cross-resistant to etoposide, daunomycin, and vinblastine . L1210 MQ-580 cells expressed 8-fold resistance to 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP), a relatively newly developed inhibitor of ribonucleotide reductase . The accumulation of {14C}3-AP by L1210 MQ-580 cells was 5- to 6-fold less than by parental L1210 cells . An increased rate of efflux of 3-AP was responsible for the lower steady-state concentration of 3-AP in resistant cells . In reverse transcription-polymerase chain reaction assays, L1210 MQ-580 cells were found to overexpress the multidrug resistance genes mdr1, mdr3, and mrp, but not the mdr2 gene, compared with parental L1210 cells . Measurement of the steady-state concentration of doxorubicin, a potential substrate for both the mdr and mrp gene products, demonstrated that L1210 MQ-580 cells accumulated 4-fold less anthracycline than parental cells . These findings indicate that drug efflux is a major determinant of the pattern of cross-resistance of L1210 MQ-580 cells . To extrapolate these observations to the human homologues of the mdr1, mdr3, and mrp murine genes, the effects of 3-AP were measured in L1210/VMDRC0.06 and NIH3T3 36-8-32 cells transfected with human MDR1 and MRP cDNAs, respectively . The transfectants were 2- to 3-fold resistant to the cytotoxic effects of 3-AP and accumulated less {14C}3-AP than their parental mock-transfected counterparts . Moreover, the cytotoxic activity of 3-AP was significantly greater in two double mrp gene knockout cell lines than in parental W 9.5 embryonic stem cells . Thus, the results suggest that 3-AP is a substrate for both the P-glycoprotein and MRP and that baseline MRP expression has the capacity to exert a protective role against the toxicity of this agent.

Biomed Pharmacother, 1997, 51(6-7), 276 - 83
Treatment of multidrug-resistant murine leukemia with antisense mdr1 oligodeoxynucleotides; Hiratake S et al.; To overcome multidrug resistance in a P-glycoprotein-overexpressing P388/ADR murine leukemia cell line, antisense mdr1 phosphorothioate-oligodeoxynucleotide (AS-oligomer) was constructed . AS-oligomer inhibited P-glycoprotein expression and mdr1 mRNA in vitro in a dose-dependent manner, whereas sense mdr1 oligomer (SE-oligomer) had no effect at the doses used . When P388/ADR was treated in vitro with AS-oligomer and doxorubicin (ADR), ADR-resistance was reduced by approximately 2 logs . Furthermore, a single injection of AS-oligomer plus ADR intraperitoneally into B6D2F1 mice with P388/ADR significantly prolonged mean survival time in a dose-dependent fashion . Again, sense mdr1 oligomer had no effect in vivo . No side effects, either acute or chronic, were found with this treatment during the observation period . These results show that antisense mdr1 oligomer could be a useful tool to overcome multidrug resistance.

Recent Results Cancer Res, 1998, 144, 93 - 115
Protection of hematopoietic stem cells from chemotherapy-induced toxicity by multidrug-resistance 1 gene transfer; Fruehauf S et al.; An increased chemotherapeutic dose intensity is believed to translate into higher survival rates among cancer patients . Pancytopenia is the dose-limiting toxic result of most anticancer agents . Overexpression of the human multidrug resistance 1 (MDR1) gene in transgenic animals resulted in complete myeloprotection against high doses of cytostatic drugs . Stem cell research, vector development, and experimental pharmacology are uniting their efforts in an attempt to achieve a similar effect in human hematopoietic stem cells . This article gives an overview of the crucial steps involved, from retroviral vector design and optimization of viral titers to vector uptake, gene integration, and expression . The authors' own results are presented with special regard in vitro and in vivo assays for the detection of hematopoietic stem cell transduction.

J Gastroenterol Hepatol, 1997 Aug, 12(8), 569 - 75
Expression of P-glycoprotein and p53 in advanced hepatocellular carcinoma treated by single agent chemotherapy: clinical correlation; Chou YY et al.; Hepatocellular carcinoma (HCC), a chemoresistant tumour, is the most common fatal cancer in Taiwan . Hepatocellular carcinoma frequently expresses a high level of P-glycoprotein (P-gp), which is a specific phenotype of a multidrug-resistance gene, and harbours mutations of the tumour suppressor gene p53 . A modulatory relationship between p53 and P-gp has been reported . In this study, we analysed the expression of P-gp in relation to chemotherapeutic response and p5353 protein expression in advanced HCC . Prechemotherapeutic tumour samples were obtained from 25 patients with HCC which had been treated with either etoposide (VP-16) or doxorubicin . P-glycoprotein and p53 in HCC were visualized by immunohistochemical staining using the monoclonal antibodies JSB-1 and DO1, respectively . We investigated the correlation of P-gp expression with chemotherapeutic responses, clinicopathological features and p53 protein expression . In our study, seven cases achieved partial remission, and the remaining 18 cases had a poor response to chemotherapy . Expression of P-gp was observed in 13 tumours (52%) . Positive P-gp protein expression was significantly associated with non-responders (8% or 1/13 vs 50% or 6/12, P = 0.03) . Thus, P-gp expression inversely correlated with chemotherapeutic response . Expression of p53 protein was seen in 12 cases and did not correlate with chemosensitivity or P-gp expression . In summary, P-gp expression correlates with the chemosensitivity of HCC that has been treated with VP-16 or doxorubicin and p 53 mutations do not appear to be a major determinant of P-gp expression in advanced HCC.

Gen Pharmacol, 1996 Dec, 27(8), 1283 - 91
The P-glycoprotein multidrug transporter; Fardel O et al.; 1 . P-glycoprotein (P-gp) is a transmembrane protein involved in ATP-dependent efflux of various structurally unrelated anticancer drugs . Its overexpression in cancer cells decreases intracellular drug concentrations and, thus, confers a multidrug resistance phenotype . 2 . P-gp is encoded by MDR genes, which constitute a small gene family comprising two genes in humans and three genes in rodents . Only the MDR1 gene in humans and mdr1 and mdr3 genes in rodents have been demonstrated to be involved in drug resistance . 3 . P-gp encoded by the human MDR1 gene is a phosphorylated and glycosylated protein 1289 amino acids long, and consists of 2 halves that share a high degree of similarity . 4 . A wide variety of cancers have been shown to express P-gp, including solid tumors and hematological malignancies . This P-gp positivity can be evidenced at the time of diagnosis prior to chemotherapy or at relapse after treatment, and has been correlated with treatment failure and poor prognosis in several types of cancer . In addition, P-gp is also expressed by some normal tissues, such as liver and kidney . 5 . P-gp expression is regulated by various factors, including xenobiotics and hormones . 6 . P-gp-mediated multidrug resistance can be reversed by various unrelated compounds called chemosensitizers or reversing agents . These drugs act through inhibition of P-gp function and have entered clinical trials.

Antimicrob Agents Chemother, 1997 Sep, 41(9), 1898 - 903
In vitro life cycle of pentamidine-resistant amastigotes: stability of the chemoresistant phenotypes is dependent on the level of resistance induced; Sereno D et al.; Using a continuous drug pressure protocol, we induced pentamidine resistance in an active and dividing population of amastigote forms of Leishmania mexicana . We selected in vitro two clones with different levels of resistance to pentamidine, with clone LmPENT5 being resistant to 5 microM pentamidine, while clone LmPENT20 was resistant to 20 microM pentamidine . Resistance indexes (50% inhibitory concentration {IC50} after drug presure/IC50 before drug pressure) of 2 (LmPENT5) and 6 (LmPENT20) were determined after drug selection . Both resistant clones expressed significant cross-resistance to diminazene aceturate and primaquine . Pentamidine resistance was not reversed by verapamil, a calcium channel blocker known to reverse multidrug resistance (A . J . Bitonti, et al., Science 242:1301-1303, 1988; A . R . C . Safa et al., J . Biol . Chem . 262:7884-7888, 1987) . No difference in the in vitro infectivity for resident mouse macrophages was observed between the wild-type clone (clone LmWT) and pentamidine-resistant clones . During in vitro infectivity experiments, when the life cycle was performed starting from the intramacrophagic amastigote stage, the drug resistance of the resulting LmPENT20 amastigotes was preserved even if the intermediate promastigote stage could not be considered resistant to 20 microM pentamidine . In the same way, when a complete developmental sequence of L . mexicana was achieved axenically by manipulation of appropriate culture conditions, the resulting axenically grown LmPENT20 amastigotes remained pentamidine resistant, whereas LmPENT5 amastigotes lost their ability to resist pentamidine, with IC50s and index of resistance values close to those for the LmWT clone . These results strongly indicate that the level of pentamidine tolerated by resistant amastigotes after the life cycle was dependent on the induced level of resistance . This fact could be significant in the in vivo transmission of drug-resistant parasites by Phlebotominae . Particular attention should be given to the finding that the emergence of parasite resistance is a potential risk of the use of inadequate doses as therapy in humans.

Br J Cancer, 1997, 76(5), 571 - 81
Anti-tumour activities of a new benzo{c}phenanthridine agent, 2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo{c}phena nthridini um hydrogensulphate dihydrate (NK109), against several drug-resistant human tumour cell lines; Kanzawa F et al.; Drug resistance is one of the problems severely limiting chemotherapy in cancer patients . Thus, it is very important to develop new drugs that are effective against drug-resistant tumour cells . The novel anti-tumour agent NK109 has been developed from benzo{c}phenanthridine derivatives by Nippon Kayaku (Tokyo, Japan) . We have confirmed that NK109 shows anti-tumour effects against a number of human tumour cell lines by inhibiting DNA topoisomerase II activity through the stabilization of the cleavable complex . Further, its efficacy against several drug-resistant tumour cell lines was also shown . NK109 showed potent anti-tumour activity against doxorubicin-resistant human tumour cell lines that have a typical multidrug resistance phenotype caused by P-glycoprotein . NK109 was not pumped extracellularly by P-glycoprotein and, consequently, NK109 accumulated in resistant cells . Cisplatin-resistant human tumour cell lines, which demonstrated decreased cisplatin accumulation, were sensitive to NK109 . NK109 non-cross-resistance was confirmed using xenografts of tumour cells that were resistant to cisplatin in SCID mice . Furthermore, etoposide-resistant cells, with decreased topoisomerase II activity, were markedly sensitive to NK109 when compared with their parent cells, suggesting the possibility that the cytotoxic mechanism of NK109 differs from that of etoposide . In conclusion, NK109 is a very promising new anti-tumour drug for clinical use, because the efficacy of NK109 is not susceptible to several known molecular alterations that are associated with drug resistance . A clinical study of this compound is now in progress in Japan.

Behring Inst Mitt, 1997 Mar, (99), 51 - 7
Nonspecific resistance to Mycobacteria: the role of the Nramp1 gene; Buschman E et al.; The genus Mycobacteria consists of over 50 species that include two of the best-known human pathogens, M . tuberculosis and M . leprae, the causes of tuberculosis (TB) and leprosy, respectively . Whereas the spread of leprosy currently appears to be under control, there are presently about 30 million active cases of TB worldwide, with an alarming increase in the number of multidrug resistant case of M . tuberculosis . As strategies for antibiotic intervention against TB become more limited, it is imperative to develop new therapeutic approaches against this oppressive disease . One promising avenue is to characterize the host genes and gene products which regulate resistance to mycobacterial infections . In the mouse, resistance and susceptibility to intracellular growth of Mycobacteria in macrophages is controlled by the Bcg (Nramp1) gene, which has now been cloned and shown to encode a macrophage transmembrane protein with a putative transporter function . Sequencing of Nramp1 revealed that susceptibility to infection is associates with a single, nonconservative glycine to aspartic acid substitution at position 169 (G169D) . Although the intracellular location of the Nramp1 protein in macrophages has not yet been determined, a phagosomal site has been postulated . Consistent with the proposed role of Nramp1 in macrophage activation, recent studies of the Nramp1 promoter region have revealed consensus sequences associated with responsiveness to IFN-gamma and LPS . Finally, a total of 11 polymorphisms have been identified within the human NRAMP1 gene which are being used to test for linkage of NRAMP1 alleles with human susceptibility to TB and leprosy.

J Natl Med Assoc, 1997 Sep, 89(9), 634 - 5
Multidrug-resistant Pneumococcus causing vertebral osteomyelitis; Antony SJ; Primary vertebral osteomyelitis in a rare presentation of pneumococcal infection especially in an asymptomatic patient with no primary focus of infection . This report describes a patient who presented with lower back pain in which the magnetic resonance imaging showed little evidence of L1 and L2 vertebral body destruction . Cultures from these vertebral bodies grew penicillin and third-generation resistant pneumococcus . The patient was treated successfully with 6 weeks of vancomycin and rifampin.

Int J Cancer, 1997 Sep 4, 72(5), 885 - 91
Development and characterisation of novel human multidrug resistant mammary carcinoma lines in vitro and in vivo; Stein U et al.; Clinical chemotherapy of breast carcinomas must be considered insufficient, mainly due to the appearance of drug resistance . The multidrug resistance (MDR) phenotype, either intrinsically occurring or acquired, e.g., against a panel of different antineoplastic drugs, is discussed in relation to several MDR-associated genes such as the MDR-gene mdr1 encoding the P-glycoprotein (PGP), the MRP gene (multidrug resistance protein) encoding an MDR-related protein or the LRP gene encoding the lung resistance protein . Numerous experimental and clinical approaches aiming at reversing resistance require well-characterised in vitro and in vivo models . The aim of our work was to develop multidrug resistant sublines from human xenotransplanted breast carcinomas, in addition to the broadly used line MCF-7 and its multidrug resistant subline MCF-7/AdrR . MDR was induced in vitro with increasing concentrations of Adriablastin (ADR) for several weeks, resulting in a 3.5- to 35-fold increase in IC50 values using the MTT-test . Cell lines were cross-resistant toward another MDR-related drug, vincristine, but remained sensitive to non-MDR-related compounds such as cisplatin and methotrexate . The resistance toward Adriamycin and vincristine was confirmed in vivo by a lack of tumour growth inhibition in the nude mouse system . Gene expression data for the mdr1/PGP, MRP/MRP and LRP/LRP on both the mRNA (RT-PCR) and the protein levels (immunoflow cytometry) demonstrated that induction of mdr1 gene expression was responsible for the acquired MDR phenotype . Rhodamine efflux data, indicated by PGP overexpression, underlined the development of this MDR mechanism in the newly established breast carcinoma lines MT-1/ADR, MT-3/ADR and MaTu/ADR.

Int J Cancer, 1997 Sep 4, 72(5), 844 - 50
Anti-proliferative activity of a new class of taxanes (14beta-hydroxy-10-deacetylbaccatin III derivatives) on multidrug-resistance-positive human cancer cells; Distefano M et al.; Paclitaxel, docetaxel and a series of new analogs synthesized from 14beta-hydroxy-10-deacetylbaccatin III (14-OH-DAB), a natural diterpene closely related to the core synthon of the 2 above prototypes, were tested in vitro for their growth-inhibitory activity on different human cancer cell lines, including some expressing the classic multidrug-resistant (MDR) phenotype (MCF-7 ADRr and CEM VBLr) . The 14-OH-DAB derivatives showed enhanced anti-proliferative activity as compared to the parent compounds on the MDR-positive cancer cell lines . Particularly, IDN 5109 showed a 25- to 30-fold higher activity than paclitaxel . The fold change in activity between paclitaxel and analogs (IC50 paclitaxel/IC50 analogs) on the MDR-positive cell lines was calculated and a significant correlation observed . As far as the MDR-negative MDA-MB 231 cells are concerned, docetaxel and IDN 5109 exhibited a more potent activity than paclitaxel . On the basis of the data obtained on cell growth inhibition, we selected the most active compounds to study their effect on the cell cycle . Cell cycle analysis showed that all of the compounds tested were able to induce cell cycle block at G2/M in a concentration-dependent manner . The amount of cell block, measured as a G1/G2 ratio, was correlated significantly (p < 0.001) with apoptosis, as evaluated in the sub-G1 region (% of DNA fragmentation), thereby suggesting that the G2/M-blocked cells underwent apoptosis . To confirm the occurrence of apoptosis in this system, DNA gel agarose electrophoresis was performed and showed the typical ladder pattern.

Int J Cancer, 1997 Sep 4, 72(5), 728 - 34
Evaluation of P-glycoprotein expression in human oral oncogenesis: correlation with clinicopathological features; Ralhan R et al.; To determine whether the multidrug-resistance-gene product phospho-glycoprotein (P-gp) is implicated in progression of oral tumours and/or drug resistance, the expression of P-gp was examined in different stages of oral oncogenesis using monoclonal antibody C-219 . Cryosections from normal (41 cases), dysplastic lesions (32 cases), untreated primary SCCs (50 cases) and recurrent tumours (31 cases) were used for immunostaining, and the results were corroborated by immunoblotting . Chi-square test for trend analysis showed a significant increase in P-gp immunopositivity across the normal, leukoplakia, primary oral SCC and recurrent SCC groups (p < 0.01) . Expression of P-gp in dysplastic lesions showed significant association with severity of dysplasia, the level of P-gp protein being higher in severe and moderate dysplasia . Among the primary tumours, significant correlation was observed between P-gp positivity as well as level of P-gp expression and tumour stage . The recurrent tumours showed significant increase in P-gp expression as compared with untreated primary oral tumours . We conclude that differential expression of P-gp may be an index of the disease prognosis in oral-cancer patients in the context of the Indian population.

Leuk Res, 1997 Jul, 21(7), 681 - 90
Ricin fusion toxin targeted to the human granulocyte-macrophage colony stimulating factor receptor is selectively toxic to acute myeloid leukemia cells; Burbage C et al.; Treatment failure of patients with acute myelogenous leukemia (AML) is frequently due to the development of multidrug resistance phenotype blasts . We have expressed a fusion protein consisting of human granulocyte-macrophage colony stimulating factor (GMCSF) fused to the N-terminus of a lectin-deficient ricin toxin B chain (RTB) in Spodoptera frugiperda insect cells . The fusion protein was purified by immunoaffinity chromatography and reassociated with chemically deglycosylated ricin toxin A chain (RTA) . The resulting fusion toxin was found to react with antibodies to GMCSF, RTB and RTA and had the predicted molecular mass of 80 kDa . GMCSF-ricin bound poorly to asialofetuin (Kd = 10(6) M-1) and receptor negative cells indicating loss of lectin activity, but bound strongly to GMCSF receptor positive HL60 cells . Ligand displacement assays showed fusion toxin affinity 2.6-fold less than native GMCSF . Selective inhibition of protein synthesis was observed on receptor positive cells . Induction of apoptosis was also observed on receptor positive cells . Cells expressing multidrug resistance gene products (P-gp, Bcl2 and BclXL) were also sensitive to fusion toxin . These results suggest that GMCSF-ricin deserves further preclinical development.

Biol Chem, 1997 Aug, 378(8), 787 - 91
Transport of glutathione conjugates and glucuronides by the multidrug resistance proteins MRP1 and MRP2; Keppler D et al.; The search for the membrane proteins mediating the ATP-dependent transport of conjugates with glutathione, glucuronate, or sulfate has led to the identification of the multidrug resistance proteins MRP1 and MRP2 . Both 190-kDa membrane glycoproteins were cloned in the recent years and shown to be unidirectional ATP-driven export pumps with an amino acid identity of 49% in human . MRP1 is detected in the plasma membrane of many cell types, including erythrocytes, whereas MRP2, also termed canalicular MRP (cMRP) or canalicular multispecific organic anion transporter (cMOAT), has been localized to the apical domain of polarized epithelia, particularly to the hepatocyte canalicular membrane . Physiologically important substrates of both transporters include glutathione S-conjugates such as leukotriene C4, bilirubin glucuronides, 17 beta-glucuronosyl estradiol, dianionic bile salts such as 6 alpha-glucuronosyl hyodeoxycholate, and glutathione disulfide . Both transporters have been associated with multiple drug resistance of malignant tumors because of their capacity to pump drug conjugates and drug complexes across the plasma membrane into the extracellular space . The substrate specificity of MRP1 and MRP2 is very different from MDR1 P-glycoprotein . MRP1 and MRP2 may be termed conjugate transporting ATPases functioning in detoxification and, because of their role in glutathione disulfide export, in the defense against oxidative stress.

Anticancer Drugs, 1997 Jul, 8(6), 610 - 7
Cellular pharmacology of lipophilic anthracyclines in human tumor cells in culture selected for resistance to doxorubicin; Bennis S et al.; We have studied the cytotoxicity and intracellular accumulation of two lipophilic anthracyclines, pirarubicin and idarubicin, as compared to doxorubicin, in two human tumor cell lines, MCF7 and K562, and in their doxorubicin-resistant counterparts, presenting the multidrug-resistant (MDR) phenotype . The new lipophilic anthracyclines were found to present a higher cytotoxicity and accumulation than the reference anthracycline, doxorubicin, and there was a significant inverse correlation between drug accumulation and IC50 in both cell types . With the aim of identifying the reasons for the higher cytotoxicity and accumulation of lipophilic anthracyclines, we used and compared the efficiency of three MDR modulators, verapamil, quinine and S-9788 . We showed that all three were able to sensitize the resistant cells to the three anthracyclines, but with different efficiencies, S-9788 being the most active reverter and quinine the least active at equimolar doses . We also observed that there was no correlation between the abilities of a modulator to reverse resistance and to restore drug accumulation . In view of the sustained activity of the modulators to increase pirarubicin and idarubicin cytotoxicity and accumulation, as they do for doxorubicin, we conclude that the better efficiency of lipophilic anthracyclines is likely to be due to their high uptake rate rather than to a decreased activity of P-glycoprotein on these drug substrates.

FEMS Microbiol Rev, 1997 Aug, 21(1), 55 - 84
Mechanisms of multidrug transporters; Bolhuis H et al.; Drug resistance, mediated by various mechanisms, plays a crucial role in the failure of the drug-based treatment of various infectious diseases . As a result, these infectious diseases re-emerge rapidly and cause many victims every year . Another serious threat is imposed by the development of multidrug resistance (MDR) in eukaryotic (tumor) cells, where many different drugs fail to perform their therapeutic function . One of the causes of the occurrence of MDR in these cells is the action of transmembrane transport proteins that catalyze the active extrusion of a large number of structurally and functionally unrelated compounds out of the cell . The mode of action of these MDR transporters and their apparent lack of substrate specificity is poorly understood and has been subject to many speculations . In this review we will summarize our current knowledge about the occurrence, mechanism and molecular basis of (multi-)drug resistance especially as found in bacteria.

Cytometry, 1997 Sep 1, 29(1), 65 - 75
Sensitive immunofluorescence detection of the expression of P-glycoprotein in malignant cells; Chan HS et al.; Because reversal of multidrug resistance increases chemotoxicity, early detection of low P-glycoprotein expression is clinically relevant for justifying early treatment of those patients that might benefit most from reversal therapy . We elected to score P-glycoprotein in single tumor cells, because the gene is rarely amplified, mRNA levels do not necessarily correlate with protein levels, and many normal hematopoietic or stroma cells within tumors and leukemic marrows also express P-glycoprotein . We enhanced the "signal-to-noise" ratio for detecting low P-glycoprotein levels by a novel complex made by pre-incubating mouse peroxidase-antiperoxidase, used solely to provide a stable framework for attaching multiple DTAF-labeled F(ab')2 fragments of rabbit antimouse IgG . We improved specificity by using both C219 and C494, which are directed against separate internal P-glycoprotein epitopes . We standardized staining with two series of negative and positive controls, in which P-glycoprotein was quantified by immunoblot, and confirmed sensitivity by staining a low-expression cell line and "mixed" samples containing small numbers of positive cells . We measured P-glycoprotein by flow cytometry, examining aliquots by differential interference contrast microscopy to identify malignant cells, in which we confirmed P-glycoprotein staining by fluorescence microscopy . We detected low P-glycoprotein expression in clinical samples of leukemic blasts, distinguishing them from normal P-glycoprotein-expressing hematopoietic cells . This assay may be valuable for early diagnosis of low, but potentially important expression of P-glycoprotein, thereby allowing early application of reversal therapy.

Clin Nucl Med, 1997 Sep, 22(9), 610 - 4
Sarcomatous transformation of neurofibromas . Comparative imaging with Ga-67, Tl-201, Tc-99m pentavalent DMSA and Tc-99m MIBI; Lee J et al.; A 29-year-old man, with a history of von Recklinghausen's disease, presented with progressive dyspnea associated with a rapidly growing mass on the right chest wall . Plain radiograph and CT of the chest revealed a huge soft-tissue mass with central low-density area involving the right upper lung and chest wall . SPECT imaging with Ga-67 citrate, Tl-201 chloride, Tc-99m pentavalent DMSA (V-DMSA), and Tc-99m MIBI were performed to characterize the mass . The tumor concentrated Ga-67, Tl-201, and Tc-99m (V) DMSA, but not Tc-99m MIBI . Punch biopsy of the lesion revealed malignant transformation of a thoracic neuroma (neurofibrosarcoma) . Subsequently, findings compatible with the presence of a multidrug resistance-1 (MDR1) gene in the tumor was documented, which may explain the poor uptake of Tc-99m MIBI . The patient did not respond to intensive chemotherapeutic regimens, and died 3 months later . This case demonstrates the potential use of combined radionuclide imaging for the detection of malignant transformation of neurofibroma, as well as for predicting tumor response to chemotherapy.

Cell Biol Toxicol, 1997 Jul, 13(4-5), 375 - 86
Immortalized human hepatocytes as a tool for the study of hepatocytic (de-)differentiation; Schippers IJ et al.; Primary human hepatocytes were immortalized by stable transfection with a recombinant plasmid containing the early region of simian virus (SV) 40 . The cells were cultured in serum-free, hormonally defined medium during the immortalization procedure . Foci of dividing cells were seen after 3 months . Albumin- and fibrinogen-secreting cells were selected and cloned by limiting dilution to obtain homologous cell populations . The established IHH (immortalized human hepatocyte) cell lines were evaluated for their usefulness in studying the regulation of cell growth and of certain differentiated hepatocyte functions . IHH cells retain several differentiated features of normal hepatocytes . They display albumin secretion at a level comparable to cultured primary human hepatocytes (30 micrograms albumin/ml per day) . A portion of the IHH cells are polarized, forming bile canaliculi-like vacuoles where exogeneous organic anions accumulate . The multidrug resistance (MDR) P-glycoprotein, known to be localized at the canalicular membrane, is also present in these vacuoles . The polarized features allowed the use of IHH cells for the study of localization of the newly characterized multidrug resistance protein MRP1 . The homologues of MRP were found in hepatocytes, MRP1 and MRP2 (cMOAT), both functioning in ATP-dependent excretion of anionic conjugates . In differentiated hepatocytes, MRP1 expression is extremely low . In contrast, MRP1 is highly expressed in proliferating IHH cells, where it is localized in lateral membranes . A highly differentiated feature of short-term cultured primary hepatocytes which is not detectable in IHH cells is active uptake of the bile salt taurocholate . Furthermore, IHH cells secrete triglyceride (TG)-rich lipoproteins, apolipoprotein B (0.6 microgram/ml per day), and apolipoprotein A-I (1 microgram/ml per day) . However, they secrete apoB-containing TG-rich lipoproteins mainly in the LDL density range, while short-term cultured primary hepatocytes mainly secrete TG-rich lipoproteins in the VLDL density range . In conclusion, functions that are rapidly lost in short-term hepatocyte cultures are, in general, not displayed by IHH cells . Immortalized human hepatocytes provide a valuable tool for studying the regulation of hepatocyte proliferation-related phenomena.

Fed Regist, 1997 Oct 17, 62(201), 54160 - 308
Occupational exposure to tuberculosis--OSHA . Proposed rule and notice of public hearing; Membrane topology of the multidrug resistance protein (MRP) . A study of glycosylation-site mutants reveals an extracytosolic NH2 terminus; Department of Pathology, Queen's University, Kingston, Ontario, Canada K7L 3N6Multidrug resistance protein, MRP, is a 190-kDa integral membrane phosphoglycoprotein that belongs to the ATP-binding cassette superfamily of transport proteins and is capable of conferring resistance to multiple chemotherapeutic agents . Previous studies have indicated that MRP consists of two membrane spanning domains (MSD) each followed by a nucleotide binding domain, plus an additional extremely hydrophobic NH2-terminal MSD . Computer-assisted hydropathy analyses and multiple sequence alignments suggest several topological models for MRP . To aid in determining the topology most likely to be correct, we have identified which of the 14 N-glycosylation sequons in this protein are utilized . Limited proteolysis of MRP-enriched membranes and deglycosylation of intact MRP and its tryptic fragments with PNGase F was carried out followed by immunoblotting with antibodies known to react with specific regions of MRP . The results obtained indicated that the sequon at Asn354 in the middle MSD is not utilized and suggested approximate sites of N-glycosylation . Subsequent site-directed mutagenesis studies established that Asn19 and Asn23 in the NH2-terminal MSD and Asn1006 in the COOH-terminal MSD are the only sites in MRP that are modified with N-linked oligosaccharides . N-Glycosylation of Asn19 and Asn23 provides the first direct experimental evidence that MRP has an extracytosolic NH2 terminus . This finding, together with those of previous studies, strongly suggests that the NH2-terminal MSD of MRP contains an odd number of transmembrane helices . These results may have important implications for the further understanding of the interaction of drugs with MRP.

Diagn Microbiol Infect Dis, 1997 Jul, 28(3), 119 - 22
Synergistic antimycobacterial activity between ethambutol and the beta-lactam drug cefepime; Abate G et al.; The activity of cefepime alone and in combination with ethambutol was assessed, using the BACTEC radiometric system, on 33 mycobacterial strains, 14 Mycobacterium avium complex (MAC), 6 isolates of M . malmoense, and 13 multidrug-resistant (MDR) isolates of M . tuberculosis . All tested mycobacteria were resistant to 8 mg/l cefepime . However, at a concentration of 32 mg/l cefepime was active on 7/13 (54%) MDR isolates of M . tuberculosis and 2/6 (33%) M . malmoense strains . All MAC strains were also resistant to this concentration . Synergistic antimycobacterial effects were seen for the combination of cefepime and ethambutol against all isolates of MAC and M . malmoense and on 4/13 (31%) MDR M . tuberculosis isolates . Moreover, this drug combination rendered 17/24 (71%) initially resistant mycobacterial strains susceptible . These promising results suggest that the antimycobacterial activity of combinations of beta-lactam drugs and ethambutol should be studied further.

JAMA, 1997 Sep 10, 278(10), 838 - 42
Long-term hospitalization for tuberculosis control . Experience with a medical-psychosocial inpatient unit; Singleton L et al.; CONTEXT: Patients with tuberculosis (TB) who are nonadherent to therapy or have complicated medical or social problems pose a threat to public health . In some cases, hospitalization may be a necessary component of a comprehensive TB control program . OBJECTIVE: To describe experience with a new inpatient program for TB control . DESIGN: Retrospective review . SETTING: Eighteen-bed, secure, TB treatment unit in a state public health hospital providing a spectrum of acute and chronic care services . PATIENTS: Patients with known or suspected TB who were unable to be treated as outpatients and were hospitalized from 1990 through 1995 . INTERVENTIONS: Voluntary or involuntary hospitalization, with medical, psychosocial, and legal services . MAIN OUTCOME MEASURES: Admissions, treatment completion, and disposition . RESULTS: A total of 166 patients with a confirmed diagnosis of TB accounted for 214 hospitalizations for TB . The mean age was 42 years, 132 (79.5%) were men, 84 (50.6%) were nonwhite, and 45 (27.1%) were foreign born . At the time of admission, 58 patients (34.5%) were homeless, 116 (69.9%) had a history of abuse of alcohol or other drugs, and 46 (31.7%) were positive for human immunodeficiency virus . The mean length of stay was 119.7 days (median, 70 days; range, 7-656 days), and was higher among homeless patients than nonhomeless patients (168.8 vs 93.4 days) . Of 48 patients (28.9%) who were admitted involuntarily, 21 required long-term confinement under court order . Admission indications (not mutually exclusive) changed over 5 years: nonadherence decreased (95% to 34%), medical complexity increased (14% to 77%), short-term isolation increased (19% to 39%), and involuntary admission decreased (54% to 13%) . Of 157 patients with positive cultures for Mycobacterium tuberculosis, 36 (23.1%) were resistant to at least 1 drug, including 16 who were multidrug resistant . A total of 123 patients (74.7%) were discharged to an outpatient setting to complete therapy, 40 (24.1%) required inpatient care to complete therapy, and 3 died (1 from TB) before discharge . CONCLUSIONS: A high proportion of patients with TB who failed outpatient therapy completed treatment in a combined medical and psychosocial inpatient unit . During the 5-year study period, involuntary admissions decreased and most patients completed therapy as outpatients . In Massachusetts, this program plays an important role in protecting public health and in providing specialized medical management for patients to complete therapy in a safe and supportive environment.

JAMA, 1997 Sep 10, 278(10), 833 - 7
Trends in drug-resistant tuberculosis in the United States, 1993-1996; Moore M et al.; CONTEXT: With the resurgence of tuberculosis (TB) disease in the late 1980s and early 1990s in the United States, multidrug-resistant (MDR) TB emerged as a serious challenge to TB control . In response, the Centers for Disease Control and Prevention in 1993 added drug susceptibility test results to the information collected for the national surveillance system to monitor trends in drug resistance . OBJECTIVE: To determine the extent of drug-resistant tuberculosis (TB) in the United States . DESIGN: Descriptive analysis of TB surveillance data . STUDY POPULATION: Patients reported to the national TB surveillance system as confirmed TB cases with culture-positive disease from 1993 through 1996 by the 50 states, New York City, and the District of Columbia (DC) . MAIN OUTCOME MEASURE: Percentage of case patients with culture-positive disease whose isolates are resistant to specific anti-TB drugs . RESULTS: Overall resistance to at least isoniazid was 8.4%; rifampin, 3.0%; both isoniazid and rifampin (ie, MDR TB), 2.2%; pyrazinamide, 3.0%; streptomycin, 6.2%; and ethambutol hydrochloride, 2.2% . Rates of resistance were significantly higher for case patients with a prior TB episode . Among those without prior TB, isoniazid resistance of 4% or more was found in 41 states, New York City, and DC . A total of 1457 MDR TB cases were reported from 42 states, New York City, and DC; however, 38% were reported from New York City . Rates of isoniazid and streptomycin resistance were higher for cases among foreign-born compared with US-born patients {corrected} but rates of rifampin resistance and MDR TB were similar . Among US-born patients, resistance to first-line drugs, particularly rifampin monoresistance, was significantly higher among those with human immunodeficiency virus (HIV) infection . CONCLUSIONS: Compared with recent US surveys in 1991 and 1992, isoniazid resistance has remained relatively stable . In addition, the percentage of MDR TB has decreased, although the national trend was significantly influenced by the marked decrease in New York City . Foreign-born and HIV-positive patients and those with prior TB have higher rates of resistance . The widespread extent of isoniazid resistance confirms the need for drug susceptibility testing to guide optimal treatment of patients with culture-positive disease.

J Nucl Med, 1997 Sep, 38(9), 1348 - 51
Fractional retention of technetium-99m-sestamibi as an index of P-glycoprotein expression in untreated breast cancer patients; Del Vecchio S et al.; The multidrug-resistant phenotype is characterized by the reduced intracellular retention of several structurally and functionally unrelated cytotoxic compounds due to the energy-dependent pump activity of P-glycoprotein (Pgp) . Because 99mTc-sestamibi is a suitable transport substrate of Pgp, we tested whether the time-dependent fractional retention of this tracer could be used as an index of Pgp expression in untreated breast carcinomas . METHODS: Twenty-seven patients with histologically confirmed breast carcinoma were intravenously injected with 740 MBq (20 mCi) of 99mTc-sestamibi, and static planar images of the breast were obtained at 10, 60 and 240 min . The fractional retention of 99mTc-sestamibi was then calculated as the ratios between 60 and 10 min (R60/10) and between 240 and 10 min (R240/10) of decay-corrected counts/pixel registered in the region of interest drawn around the tumor . Surgically excised tumors were then obtained from each patient, and Pgp levels were determined using 125I-labeled MRK16 monoclonal antibody and in vitro quantitative autoradiography . RESULTS: The fractional retention of 99mTc-sestamibi at 60 and 240 min was significantly higher in tumors with low Pgp levels (Group I, n = 18) as compared to that measured in tumors with high Pgp expression (Group II, n = 9) (p < 0.001) . In particular, R60/10 values were 0.86 and 0.59 in breast carcinomas of Groups I and II, respectively, whereas the values of R240/10 were 0.56 and 0.25 in low- and high-Pgp-expressing tumors, respectively . CONCLUSION: The determination of fractional retention of 99mTc-sestamibi may be used as a simple functional test for Pgp expression in untreated breast cancer . A preliminary estimate of the sensitivity and the specificity of the test indicates its potential use in clinical practice to identify patients with a high probability of developing multidrug resistance.

J Cancer Res Clin Oncol, 1997, 123(8), 456 - 60
Immunocytochemical detection of P-glycoprotein in the management of malignant effusions; Athanassiadou P et al.; P-glycoprotein (P-gp), a cell membrane protein, has been found in multidrug-resistant cancer cells . A total of 104 smears from patients with breast-cancer-associated pleural effusions and ovarian-cancer-related peritoneal effusions were studied for P-gp with the antibody C-219 and the avidin-biotin-immunoperoxidase method . Samples were taken before and 3 and 7 days after intracavitary bleomycin therapy and reaccumulation of effusion was assessed at 30 days . Smears that were P-gp-negative by the 7th day were associated with a good 30-day response to bleomycin in the majority of cases, while P-gp-positive smears were associated with a significant reaccumulation of fluid at 30 days . P-gp status is a valuable prognostic indicator of response to intracavitary bleomycin treatment in effusions from breast or ovarian cancer.

J Cancer Res Clin Oncol, 1997, 123(8), 452 - 5
Treatment of advanced colorectal cancer with doxorubicin combined with two potential multidrug-resistance-reversing agents: high-dose oral tamoxifen and dexverapamil; Weinlander G et al.; On the basis of the overexpression of the MDR1 gene in human colorectal cancer, which may constitute a molecular basis for intrinsic drug resistance that can be reversed, and because of the limited therapeutic value of conventional cytotoxic treatment in this common disease, the present phase II study of P-glycoprotein-directed double modulation was initiated . Fifteen patients with measurable metastatic colorectal cancer, all of whom were refractory to first-line chemotherapy with 5-fluorouracil/leukovorin, were entered in this trial . Treatment consisted of 80 mg tamoxifen twice daily on days 1-9, oral dexverapamil every day on days 7-9, and 60 mg/m2 doxorubicin given by intravenous bolus injection on day 8 . Courses were repeated every 4 weeks . After a median of three (between one and six) courses, none of the 14 evaluable patients had objective response, and 4 had stable disease . Adverse reactions consisted mainly of myelosuppression (WHO grade IV granulocytopenia was noted in 40%), and mild and reversible dexverapamil-related cardiovascular side-effects, specifically hypotension (47%) . Our results suggest that, despite the histological demonstration of high levels of P-glycoprotein in colorectal cancer and administration of two potentially synergistic chemosensitizers, we were unsuccessful in circumventing its primary resistance to chemotherapy.

J Clin Lab Anal, 1997, 11(5), 258 - 66
Quantitative analysis of human multidrug resistance 1 (MDR1) gene expression by nonisotopic competitive reverse transcriptase polymerase chain reaction assay; Kobayashi H et al.; We have established competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay for the quantification of MDR1 mRNA encoding P-glycoprotein (P-gp) by analyzing leukemia sublines of MOLT-3 with various expression of MDR1 . The expression was quantified by simultaneous RT-PCR of cellular RNA with decreasing amounts of heterologous competitor RNA, which shares the MDR1 primer sequences with the cellular MDR1 mRNA, but yields a different-sized PCR product . This allows resolution of the amplified cDNA fragments . The amounts of MDR1 mRNA measured by the assay were accurate and reproducible over wide range, and were determined as 31.6, 100, and 316 amol/microgram total RNA in MOLT-3/TMQ70, MOLT-3/ TMQ800, and MOLT-3/VCR1,000, respectively . The relative ratio of MDR1 mRNA measured by the competitive RT-PCR among three sublines was similar to that of MDR1 transcript determined by Northern analysis (1:4:12) and to that of P-gp measured by flow cytometry (FCM) analysis . In mononuclear cells from patients with leukemia, MDR1 mRNA could be sufficiently quantified by the competitive RT-PCR established, while FCM assay could scarcely detet P-gp . This study demonstrated that the competitive RT-PCR assay using heterologous competitor RNA is a rapid, reliable, and non-radioactive procedure and is acceptable for the evaluation of MDR1 expression in clinical samples.

Am J Rhinol, 1997 Jul-Aug, 11(4), 317 - 21
Expression of P-glycoprotein 170 in nasal mucosa may be increased with topical steroids; Henriksson G et al.; The synthesis of P-glycoprotein 170 (P-gp), a "multidrug resistance" protein capable of extruding various drugs including 11-OH steroids from human cells, can be upregulated by certain glucocorticosteroids . This study demonstrates the presence of P-gp in the columnar surface epithelium and in glandular acini of healthy nasal mucosa with immunohistochemical technique . Furthermore, nasal polyps from 5 of 17 patients treated with clinical doses of a topical nasal steroid, budesonide, appear to show a stronger staining intensity for P-gp than polyps from 13 untreated patients . This suggests the possibility of local P-gp gene induction by topical glucocorticoid treatment . Upregulation of P-gp synthesis appears as a new possible cause of relative resistance to topical steroid medication in patients with nasal inflammatory disease.

J Infect Dis, 1997 Sep, 176(3), 786 - 9
Evolution of mutations conferring multidrug resistance during prophylaxis and therapy for cytomegalovirus disease; Chou S et al.; In a human immunodeficiency virus-infected subject, cytomegalovirus (CMV) isolated 9 months after the patient began oral ganciclovir prophylaxis was resistant to ganciclovir and cidofovir and contained mutations in both UL97 and Pol coding regions . At 1 year, retinitis developed, which progressed despite intravenous ganciclovir followed by foscarnet and then cidofovir . A subsequent buffy coat virus isolate was resistant to all three drugs and contained new mutations in UL97 and Pol . By individually transferring the observed mutations to laboratory strain AD169, it was shown that a mutation at codon 603 of UL97 conferred resistance to ganciclovir, a mutation at codon 412 of Pol conferred resistance to both ganciclovir and cidofovir, and a mutation at codon 802 of Pol conferred resistance to ganciclovir and foscarnet . This case illustrates the development of multidrug resistance during prolonged exposure to antiviral therapy for CMV and cross-resistance arising from point mutations in the CMV Pol gene.

J Infect Dis, 1997 Sep, 176(3), 637 - 42
Nosocomial spread of human immunodeficiency virus-related multidrug-resistant tuberculosis in Buenos Aires; Ritacco V et al.; A steep upsurge of human immunodeficiency virus (HIV)-associated multidrug-resistant tuberculosis (MDR-TB) was recently observed at a referral treatment center in Buenos Aires City . Between January 1994 and June 1995, TB isolates resistant to at least five drugs were recovered from 101 of 272 HIV-infected inpatients . Highly resistant isolates from 77 patients underwent restriction fragment length polymorphism study with IS6110 . After cross-contamination was eliminated, a single TB strain was found to have caused disease in 68 patients with a history of on-site exposure . The frequency of smear-positive pulmonary disease was higher among these patients than among non-MDR-TB HIV-infected patients (50/68 vs . 60/148, P < .001), and the 1-year survival was dramatically reduced (5/68 vs . 92/148) . The strain involved in the outbreak was traced back to patients hospitalized in 1992 . Institutional infection control policies were and may still be inadequate to contain the spread of TB among immunodepressed subjects, as is the case in other large urban hospitals in Argentina.

Biochemistry, 1997 Sep 16, 36(37), 11169 - 78
Human MDR 1 protein overexpression delays the apoptotic cascade in Chinese hamster ovary fibroblasts; Robinson LJ et al.; Several laboratories have reported that overexpression of the multidrug resistance (MDR) protein is associated with intracellular alkalinization, and several investigators have reported that cells induced to undergo programmed cell death (apoptosis) acidify quite significantly . Because it is difficult to fully explain the resistance to apoptosis-inducing chemotherapeutic drugs that is exhibited by MDR tumor cells solely via altered drug transport alone {Hoffman et al . (1996) J . Gen . Physiol . 108, 295-313}, we have investigated whether overexpression of the hu MDR 1 protein alters progression of the apoptotic cascade . LR73 fibroblasts induced to undergo apoptosis either via treatment with the chemotherapeutic drug colchicine or by serum withdrawal exhibit cellular volume changes, intracellular acidification, nuclear condensation, and chromosomal digestion ("ladder formation"), characteristic of apoptosis, in a temporally well-defined pattern . However, multidrug resistant LR73/20E or LR73/27 hu MDR 1 transfectants recently created in our laboratory without selection on chemotherapeutic drug are significantly delayed in the onset of apoptosis as defined by the above criteria, regardless of whether apoptosis is induced by colchicine treatment or by serum withdrawal . Thus, the delay cannot simply be due to the well-known ability of MDR protein overexpression to lower chemotherapeutic drug accumulation in MDR cells . LR73/27V500 "selectants", exhibiting similar levels of MDR protein overexpression but higher multidrug resistance due to selection with the chemotherapeutic drug vincristine, exhibit a slightly longer delay in the progression of apoptosis . Normal apoptotic cascade kinetics are partially restored by pre-treatment of the MDR cells with the MDR protein inhibitor verapamil . Untransfected LR73 cells not expressing MDR protein but elevated in pHi via manipulation of CO2/HCO3- as described {Hoffman et al . (1996) J . Gen . Physiol . 108, 295-313} are inhibited in DNA ladder formation, similar to LR73/hu MDR 1 transfectants . These results uncover an additional mechanism whereby MDR protein overexpression may promote the survival of tumor cells and further support the notion that in some systems intracellular acidification may be either causal or permissive for proper progression of the apoptotic cascade.

Biochemistry, 1997 Sep 2, 36(35), 10777 - 83
The dihydropyridine dexniguldipine hydrochloride inhibits cleavage and religation reactions of eukaryotic DNA topoisomerase I; Straub T et al.; Dexniguldipine hydrochloride (B859-35, a dihydropyridine with antitumor and multidrug resistance-reverting activity) inhibits both the DNA cleavage and religation reactions of purified human DNA topoisomerase I at concentrations >1 microM, whereas at concentrations <1 microM it inhibits selectively the religation step and stabilizes the covalent topoisomerase I-DNA intermediate in a similar fashion as camptothecin . Inhibition of religation by camptothecin can be overcome by increasing the concentration of the DNA substrate in the religation reaction, indicating a competitive type of inhibition . In contrast, dexniguldipine hydrochloride decreases rate constants of topoisomerase I-mediated DNA religation independently of the concentration of the DNA substrate, suggesting a noncompetitive mechanism of inhibition, which is different from that of camptothecin.

Cancer Res, 1997 Sep 1, 57(17), 3751 - 8
Microfilament depletion and circumvention of multiple drug resistance by sphinxolides; Zhang X et al.; Sphinxolides, a newly described family of cytotoxins from the New Caledonian sponge Neosiphonia superstes, bear structural resemblance to scytophycins . We now demonstrate that the cytotoxicity of sphinxolides is associated with cell cycle arrest in G2-M and induction of apoptosis . Like scytophycins and cytochalasins, sphinxolides caused rapid loss of microfilaments in cultured cells, without affecting microtubule organization . Microfilament reassembly was very slow after removal of the sphinxolide, consistent with the slow recovery of cellular proliferation . Sphinxolides potently inhibited actin polymerization in vitro and the microfilament-dependent ATPase activity of purified actomyosin, indicating a direct effect on actin . Importantly, sphinxolides were equally cytotoxic toward MCF-7 human breast carcinoma cells and a subline which overexpresses P-glycoprotein (MCF-7/ADR) . Similarly, overexpression of the multidrug resistance-associated protein MRP by HL-60 cells did not confer resistance to the sphinxolides . These studies demonstrate that sphinxolides are potent new antimicrofilament compounds that circumvent multidrug resistance mediated by overexpression of either P-glycoprotein or MRP . Therefore, these agents may be useful in the treatment of drug-resistant tumors.

J Histochem Cytochem, 1997 Sep, 45(9), 1255 - 64
Characterization of acidic vesicles in multidrug-resistant and sensitive cancer cells by acridine orange staining and confocal microspectrofluorometry; Millot C et al.; To study the pH gradient status through membranes of acidic vesicles, either in sensitive or in multidrug-resistant living cancer cells, we monitored the fluorescence-emission spectra of acridine orange . Successive stainings with a pH-sensitive dye and AO showed that low-pH organelles were stained red by AO . In these compartments, high AO concentrations are driven by the pH gradient through membrane vesicles . The resulting rise in the dye's oligomeric/monomeric ratio induced an increase in the red/green (655-nm/530-nm) emission intensity ratio . Therefore, the accumulation of AO in acidic organelles was appraised by determination of the contribution of the red emission intensity (R%) in each emission spectrum, using laser scanning confocal microspectrofluorometry . In vesicles of multidrug-resistant K562-R cells, R% is significantly higher (72 +/- 10%) than the value (48 +/- 8%) from K562-sensitive cells (p < 0.001) . This result is interpreted as a more important accumulation of AO in acidic cytoplasmic structures of resistant cells, which induces a shift from AO monomers (green emission) to self-associated structures (red emission) . Equilibration of the pH gradient through acidic organelles was performed by addition of weak bases and carboxylic ionophores . Ammonium chloride (0.1 mM), methylamine (0.1 mM), monensine (10 microM), or nigericine (0.3 microM) all suppressed the initial difference of local AO accumulation between both cell lines . These agents decreased the red emission intensity for the resistant cell line but not for the sensitive one . The same effects were induced by 50 microM verapamil, a pleiotropic drug-resistance modulator . Our data allow the hypothesis of a higher pH gradient through membranes of acidic organelles, which would be a potential mechanism of multidrug resistance via the sequestration of weak bases inside these organelles.

Mol Pharmacol, 1997 Sep, 52(3), 344 - 53
Pharmacological characterization of the murine and human orthologs of multidrug-resistance protein in transfected human embryonic kidney cells; Stride BD et al.; Overexpression of the human multidrug-resistance protein (MRP) causes a form of multidrug resistance similar to that conferred by P-glycoprotein, although the two proteins are only distantly related . In contrast to P-glycoprotein, human MRP has also been shown to be a primary active transporter of a structurally diverse range of organic anionic conjugates, some of which may be physiological substrates . At present, the mechanism by which MRP transports these compounds and mediates multidrug resistance is not understood . With the objective of developing an animal model for studies on the normal functions of MRP and its ability to confer multidrug resistance in vivo, we recently cloned the murine ortholog of MRP (mrp) . To assess the degree of functional conservation between mrp and MRP, we directly compared the drug cross-resistance profiles they confer when transfected into human embryonic kidney cells, as well as their ability to actively transport leukotriene C4, 17beta-Estradiol 17beta-(D-glucuronide), and vincristine; mrp and MRP conferred similar drug resistance profiles, with the exception that only MRP conferred resistance to the anthracyclines tested . Consistent with these findings, accumulation of {3H}vincristine and {3H}VP-16 was decreased, and efflux of {3H}vincristine was increased in both murine and human MRP-transfected cell populations, whereas only human MRP-transfected cells displayed decreased accumulation and increased efflux of {3H}daunorubicin . Membrane vesicles derived from both transfected cell populations transported leukotriene C4 in an ATP-dependent manner with comparable efficiency, although the efficiency of 17beta-estradiol 17beta-(D-glucuronide) transport was somewhat higher with MRP transfectants . ATP-dependent transport of vincristine was also observed with vesicles from mrp and MRP transfectants but only in the presence of glutathione . These studies reveal intrinsic differences between the murine and human MRP orthologs with respect to their ability to confer resistance to a major class of chemotherapeutic drugs.

Q J Nucl Med, 1997 Sep, 41(3), 239 - 50
Diagnostic and prognostic role of 99mTc-Tetrofosmin in breast cancer; Mansi L et al.; 99mTc-Tetrofosmin (TF) is a lipophilic diphosphine compound routinely used for myocardial scintigraphy . Extracardiac utilization has occurred in evaluation of patients with malignant neoplasms and in parathyroid adenomas . Although its uptake mechanisms are not completely understood, they appear similar to those of 99mTc Setamibi (MIBI) . The importance of flow and the metabolic status of cells with an intracellular uptake depending on mitochondria and the Na+/K+ pump have been hypothesized . It has also been demonstrated that Tetrofosmin shares with MIBI the property of being a substrate for P-glycoprotein (P-gp), a multidrug resistance transporter . In this review the possible clinical role in breast cancer is analysed . First experiences suggest that scintimammography with TF is useful in patients with indeterminate Mammography and to obtain complementary data to avoid surgery and/or biopsy . TF is a reliable tracer for diagnosis of primary cancer, of local recurrence of axillary lymph node metastases . Preliminary data stimulate a possible role in functional imaging of chemoresistance and in differential diagnosis of distant metastases with main reference to the evaluation of single hot lesions at bone scan.

FEBS Lett, 1997 Aug 18, 413(2), 344 - 8
Modulation by (iso)flavonoids of the ATPase activity of the multidrug resistance protein; Hooijberg JH et al.; The multidrug resistance protein (MRP) is an ATP-dependent transport protein for organic anions, as well as neutral or positively charged anticancer agents . In this study we report that dinitrophenyl-S-glutathione increases ATPase activity in plasma membrane vesicles prepared from the MRP-overexpressing cell line GLC4/ADR . This ATPase stimulation parallels the uptake of DNP-SG in these vesicles . We also show that the (iso)flavonoids genistein, kaempferol and flavopiridol stimulate the ATPase activity of GLC4/ADR membranes, whereas genistin has no effect . The present data are consistent with the hypothesis that certain (iso)flavonoids affect MRP-mediated transport of anticancer drugs by a direct interaction with MRP.

Biochem Biophys Res Commun, 1997 Aug 18, 237(2), 407 - 12
Functional incorporation of P-glycoprotein into Xenopus oocyte plasma membrane fails to elicit a swelling-evoked conductance; Aleu J et al.; Microinjecton of Xenopus oocytes with P-glycoprotein-containing membranes from multidrug resistant cells following a recently published procedure resulted in the transplantation of the protein to the plasma membrane of the oocytes and was confirmed by Western blot analysis . These oocytes showed a reduced intracellular accumulation of daunomycin, when compared to uninjected oocytes or to those injected with membrane vesicles lacking P-glycoprotein, thus indicating that the protein had been incorporated in a transport-competent form . On the other hand, transplantation of P-glycoprotein to the oocyte membrane did not significantly change either the appearance or the properties of swelling-elicited membrane conductance with respect to those determined in oocytes either uninjected or injected with membranes lacking P-glycoprotein . These results do not support a role for P-glycoprotein as a swelling-activated chloride channel.

Cancer Res, 1997 Aug 15, 57(16), 3537 - 47
Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines; Kool M et al.; By screening databases of human expressed sequence tags, we have identified three new homologues of MRP1, the gene encoding the multidrug resistance-associated protein, and cMOAT (or MRP2), the canalicular multispecific organic anion transporter gene . We call these new genes MRP3, MRP4, and MRP5 . MRP3, like cMOAT, is mainly expressed in the liver . MRP4 is expressed only at very low levels in a few tissues, and MRP5, like MRP1, is expressed in almost every tissue tested . To assess a possible role of these new MRP homologues in multidrug or cisplatin resistance, a large set of resistant cell lines was examined for the (over)expression of MRP1, cMOAT, MRP3, MRP4, and MRP5 . We find that even in cells selected for a low level of resistance, several MRP-related genes can be up-regulated simultaneously . However, MRP4 is not overexpressed in any of the cell lines we analyzed; MRP3 and MRP5 are only overexpressed in a few cell lines, and the RNA levels do not seem to correlate with resistance to either doxorubicin or cisplatin . cMOAT is substantially overexpressed in several cell lines, and cMOAT RNA levels correlate with cisplatin but not doxorubicin resistance in a subset of resistant cell lines . Our results emphasize the need for gene-specific blocks in gene expression to define which transporter contributes to resistance in each resistant cell line.

Blood, 1997 Aug 15, 90(4), 1643 - 8
Expression of the neural cell adhesion molecule CD56 is associated with short remission duration and survival in acute myeloid leukemia with t(8;21)(q22;q22); Baer MR et al.; Although acute myeloid leukemia (AML) with t(8;21) (q22;q22) is associated with a high complete remission (CR) rate and prolonged disease-free survival, treatment outcome is not universally favorable . Identifying factors that predict for treatment outcome might allow therapy to be optimized based on risk . AML with t(8;21) has a distinctive immunophenotype, characterized by expression of the myeloid and stem cell antigens CD13, CD15, CD34, and HLADr, and frequent expression of the B-cell antigen CD19 and the neural cell adhesion molecule CD56, a natural killer cell/stem cell antigen . Because CD56 expression has been associated with both extramedullary leukemia and multidrug resistance, we sought to correlate CD56 expression with treatment outcome in AML with t(8;21) . Pretreatment leukemia cells from 29 adult de novo AML patients with t(8;21) treated on Cancer and Leukemia Group B (CALGB) protocols were immunophenotyped by multiparameter flow cytometry as part of a prospective immunophenotyping study of adult AML (CALGB 8361) . CD56 was expressed in 16 cases (55%) . There was no correlation between CD56 expression and age, sex, white blood cell count, granulocyte count, the presence of additional cytogenetic abnormalities, or the presence of extramedullary disease at diagnosis . The CR rate to standard-dose cytarabine and daunorubicin was similar for cases with and without CD56 expression (88% v 92%; P = 1.0) . Post-CR therapy included at least one course of high-dose cytarabine in 24 of 26 patients who achieved CR; numbers of courses administered were similar in cases with and without CD56 expression . Although post-CR therapy did not differ, CR duration was significantly shorter in cases with CD56 expression compared with those without (median, 8.7 months v not reached; P = .01), as was survival (median, 16.5 months v not reached; P = .008) . We conclude that CD56 expression in AML with t(8;21) is associated with significantly shorter CR duration and survival . Our results suggest that CD56 expression may be useful in stratifying therapy for this subtype of AML.

J Biol Chem, 1997 Aug 15, 272(33), 20913 - 9
Localization of the iodomycin binding site in hamster P-glycoprotein; Demmer A et al.; P-glycoprotein, the overexpression of which is a major cause for the failure of cancer chemotherapy in man, recognizes and transports a broad range of structurally unrelated amphiphilic compounds . This study reports on the localization of the binding site of P-glycoprotein for iodomycin, the Bolton-Hunter derivative of the anthracycline daunomycin . Plasma membrane vesicles isolated from multidrug-resistant Chinese hamster ovary B30 cells were photolabeled with {125I}iodomycin . After chemical cleavage behind the tryptophan residues, 125I-labeled peptides were separated by electrophoresis and high performance liquid chromatography . Edman sequencing revealed that {125I}iodomycin had been predominantly incorporated into the fragment 230-312 of isoform I of hamster P-glycoprotein . According to models based on hydropathy plots, the amino acid sequence 230-312 forms the distal part of transmembrane segment 4, the second cytoplasmic loop, and the proximal part of transmembrane segment 5 in the N-terminal half of P-glycoprotein . The binding site for iodomycin is recognized with high affinity by vinblastine and cyclosporin A.

Biochemistry, 1997 Aug 12, 36(32), 9807 - 15
Interaction of P-glycoprotein with defined phospholipid bilayers: a differential scanning calorimetric study; Romsicki Y et al.; One of the major causes of multidrug resistance in human cancers is expression of the P-glycoprotein multidrug transporter, which acts as a drug efflux pump . P-Glycoprotein is a member of the ABC superfamily of membrane proteins, and is composed of 12 hydrophobic membrane-spanning segments and 2 cytoplasmic nucleotide binding domains . Membrane lipids are known to play an important role in the function of P-glycoprotein . In the present study, purified P-glycoprotein of high specific ATPase activity was reconstituted into defined bilayers of dimyristoylphosphatidylcholine (DMPC), and its effects on lipid thermodynamic properties were then investigated using differential scanning calorimetry . P-Glycoprotein had a large perturbing effect on DMPC bilayers, even at relatively high lipid:protein ratios . The gel to liquid-crystalline phase transition temperature, Tm, was lowered on inclusion of P-glycoprotein in the bilayer, and the cooperativity of the transition was markedly reduced . The phase transition enthalpy, DeltaH, declined in a linear fashion with increasing P-glycoprotein content for lipid:protein ratios between 63:1 and 16:1 (w/w) . Evaluation of these data using two different analytical methods indicated that P-glycoprotein perturbed either 375 or 485 phospholipids, withdrawing them from the phase transition . The DeltaH value for those lipids undergoing melting was similar to that of pure DMPC, which implies that their thermodynamic properties are essentially unchanged in the presence of P-glycoprotein . At lipid:protein ratios below 16:1 (w/w), transition enthalpy increased with higher P-glycoprotein content, until the DeltaH value reached that of pure DMPC . However, the lipid remained highly perturbed, as indicated by a very broad phase transition peak . This behavior may arise from either aggregation/oligomerization of P-glycoprotein within the bilayer or changes in the interaction of the transporter with the membrane at high density.

Int J Cancer, 1997 Aug 7, 72(4), 649 - 56
Molecular targeting of mitomycin C chemotherapy; Nishiyama M et al.; In 10 human cancer cell lines, the activity of mitomycin C (MMC) was found to be determined by an interplay between activation by DT-diaphorase (DTD) and inactivation by glutathione S-transferase (GST) . NADPH/cytochrome P-450 reductase was not responsible for MMC activation and expression of MDRI (Mr 170,000 P-glycoprotein), and MRP (multidrug resistance-associated protein) genes did not relate to MMC resistance . Gene expression analysis for NQO1 (DTD gene) and GSTpi predicted which enzyme activity predominated in a cell line, except K562 and K562/DOX . For tumors with DTD activity only, MMC given by itself was most active . In cell lines in which DTD action was predominant, tumor selectivity was achieved by enhancing DTD-mediated activation with m-iodobenzylguanidine and hyperglycemia, which reduced the intra-tumoral pH . KW2149, a novel MMC analogue activated by glutathione, was most active against tumors in which GSTpi predominated . These various enzyme-specific effects could be observed even in cell lines derived from tumors with multidrug resistance . Such MMC treatment based on cell enzymology may enhance significantly MMC efficacy, helping to overcome multidrug resistance.

Am J Physiol, 1997 Aug, 273(2 Pt 1), C687 - 702
Experimentally induced changes in the endocytic traffic of P-glycoprotein alter drug resistance of cancer cells; Kim H et al.; The MDR-1 gene product, plasma membrane glycoprotein or P-glycoprotein (PGP), has been shown to confer drug resistance to cancer cells by acting as an energy-dependent drug-efflux pump . We have examined the endocytic traffic of PGP in human multidrug-resistant cells and tested whether the traffic and the steady-state intracellular localization of PGP can be experimentally modulated . Here we show that 1) under steady state approximately 70% of cellular PGP is on the surface whereas approximately 30% is intracellular, 2) surface PGP undergoes constitutive endocytosis and recycling, 3) endocytosis of PGP involves clathrin and adaptin complex 2-dependent mechanism, and 4) PGP cycles through a Rab5-responsive endosomal compartment . Biochemical (such as antibody crosslinking of PGP or treatment of cells with chloroquine) and molecular (such as overexpression of Rab5) treatments were used to modulate the endocytic/ recycling traffic of PGP . Such treatments resulted in the redistribution of PGP from the cell surface to intracellular compartments . Cells with such "mislocalized" PGP showed a decrease in multidrug resistance, suggesting that clinically relevant strategies can be attempted by modulating PGP's temporal and spatial distribution within cancer cells.

Infect Control Hosp Epidemiol, 1997 Aug, 18(8), 542 - 7
The cost of selected tuberculosis control measures at hospitals with a history of Mycobacterium tuberculosis outbreaks; Kellerman S et al.; OBJECTIVE: To determine the cost of nonrespirator-related tuberculosis (TB) control measures at several hospitals, following publication of the Centers for Disease Control and Prevention (CDC)'s revised TB infection control guidelines . DESIGN: Infection control (IC) and TB coordinators obtained cost information on tuberculin skin-test (TST) programs, addition of IC and employee health service (EHS) personnel, and the retrofit or new construction of environmental controls . SETTING: Four hospitals with, and one community hospital without, prior nosocomial multidrug-resistant TB transmission . RESULTS: During the study period, the TST program costs remained constant at four of five hospitals and increased at one hospital (median 1994 TST program cost: $5,568; range, $2,393-$44,902) . Additional IC or EHS personnel were hired at four of five hospitals (median cost increase, $125,500; range, $63,000-$228,000) . The median cost of new construction or new equipment purchases (ie, sputum induction booths, ultraviolet lights, or portable high-efficiency particulate air filters) at study hospitals was $163,000 (range, $45,000-$524,000) and $70,000 (range, $31,000-$93,000), respectively . CONCLUSIONS: Costs associated with implementing control measures similar to those recommended in the CDC TB IC guidelines varied widely by hospital . Engineering controls involved the largest capital outlay, but increases in personnel were the largest continuing cost . These costs represent improvements made to upgrade selected aspects of hospital TB control programs, not the cost of an optimal TB control program.

Semin Oncol, 1997 Aug, 24(4 Suppl 10), S10 - 18-S10-21
Docetaxel (Taxotere) for the treatment of anthracycline-resistant breast cancer; Ravdin PM; Until the introduction of the taxoids, docetaxel (Taxotere; Rhone-Poulenc Rorer, Antony, France) and paclitaxel (Taxol; Bristol-Myers Squibb Oncology, Princeton, NJ), in the 1990s, anthracyclines were widely recognized as the best single agents for the treatment of breast cancer . However, even when anthracyclines are used in combination regimens with response rates of over 50%, including complete responses in 17% of patients, few women (3%) with metastatic disease remain disease free at 5 years after treatment . The low level of sustained responses is largely due to the phenomenon of drug resistance . Anthracycline resistance often involves multidrug resistance efflux mechanisms, but also can involve factors affecting topoisomerase II and apoptosis . When combining other cytotoxic agents with anthracyclines, it is of value to use non-cross-resistant drugs so that the induction of anthracycline-resistance mechanisms does not also affect the efficacy of other agents in the combination therapy . Clinical studies have shown that docetaxel, which is highly active against metastatic breast cancer as a single agent, has a high level of non-cross-resistance with anthracyclines . The overall response rate to docetaxel monotherapy in patients with anthracycline-resistant or refractory metastatic disease has been shown to be 41% . The response rate to first-line docetaxel monotherapy for metastatic breast cancer has been shown to be 61%, suggesting that two thirds of the activity of docetaxel is retained in anthracycline-resistant disease . Treatment with a simultaneous combination of docetaxel and doxorubicin has been found to be very active, with a response rate of 89%, and trials to exploit the lack of cross-resistance between these agents, in sequential regimens and adjuvant therapies, are under way.

Semin Oncol, 1997 Aug, 24(4 Suppl 10), S10 - 11-S10-17
Anthracycline resistance: the problem and its current definition; Gianni L; Anthracyclines were introduced for the treatment of breast cancer in the 1970s and were considered the most active single agents until the recent introduction of the taxoids . Although incorporation of anthracyclines into combination regimens has been shown to improve clinical outcomes, the duration of response and survival in women with metastatic disease is still modest, and 50% of women treated with adjuvant chemotherapy eventually relapse . Intrinsic and acquired drug resistance, leading to untreatable disease, are fundamental reasons for clinical failure in breast cancer, but the clinical relevance of the various known mechanisms of drug resistance is not clear . P-glycoprotein (Pgp)-mediated multidrug resistance, the most studied form of anthracycline resistance, can be inhibited by a variety of chemicals . While in vitro studies have demonstrated the efficacy of some Pgp inhibitors, and led to the development of more clinically acceptable agents, clinical studies have not shown a consistent advantage in using Pgp inhibitors . Since Pgp is a physiologic efflux mechanism, consideration also should be given to the possible consequences of its inhibition . Studies with Pgp knock-out transgenic mice suggest that Pgp is not essential for life, but that Pgp inhibition may make some tissues, such as the brain, more vulnerable to cytotoxic drugs . Correlating overexpression of the MDR1 gene in human tumor samples with treatment failure has not proved straightforward, and further studies are needed to clarify the contribution of multidrug resistance mechanisms to clinical anthracycline resistance . Mechanisms other than drug efflux pumps, which may contribute to anthracycline resistance, include changes in topoisomerase II, the major cellular target of anthracyclines . There remains a gulf between the laboratory definitions of drug resistance, which can be elucidated in great detail, and the clinical definition, which is based on the time to treatment failure . New drugs still need to be assessed empirically in the clinic, and these results may then be correlated with laboratory findings . We cannot yet reliably predict clinical efficacy, cross-resistance, or the mechanisms responsible for treatment failure from laboratory studies.

Histochem Cell Biol, 1997 Aug, 108(2), 179 - 82
The multidrug-resistance P-glycoprotein (Pgp, MDR1) is an early marker of blood-brain barrier development in the microvessels of the developing human brain; Schumacher U et al.; The multidrug-resistance P-glycoprotein (Pgp) was initially identified as an energy-dependent proton pump, which transports a variety of non-related compounds out of chemotherapy-resistant cancer cells . Molecular biological investigations using knockout mice for the mouse homologue of the human Pgp showed that these mice partially lack a functioning blood-brain barrier, indicating that Pgp has an important role in the blood-brain barrier as its normal function . The presence of Pgp expression in formalin-fixed and wax-processed tissue sections can be assessed using the monoclonal antibody, JSB-1 . Since no data on the developmental expression of Pgp are available, we stained a developmental series of human brain sections with JSB-1 . Our results indicate that Pgp expression in endothelia of brain microvessels occurs regularly in embryos of about 30-mm crown-rump length (CRL) . Strong reactivity is seen in blood vessels of fetuses from 123-mm CRL . There is also reactivity in pial blood vessels but not in choroid plexus blood vessels known to be without a blood-brain barrier . Pgp expression is therefore an early marker of the blood-brain barrier in the developing human brain.

Mol Pharmacol, 1997 Aug, 52(2), 323 - 34
Quantitative structure-activity relationship of multidrug resistance reversal agents; Klopman G et al.; Multidrug resistance (MDR) is one of the major obstacles to long term successful cancer chemotherapy . The use of MDR reversal (MDRR) agents is a promising approach to overcome the undesired MDR phenotype . To design more effective MDRR agents that are urgently needed for clinical use, a data set of 609 diverse compounds tested for MDRR activity against P388/ADR-resistant cell lines was submitted to the MULTICASE computer program for structure-activity analysis . Some substructural features related to MDRR activity were identified . For example, the CH2-CH2-N-CH2-CH2 group was found in most of the active compounds, and the activity was further enhanced by the presence of (di)methoxylphenyl groups, whereas the presence of a stable quaternary ammonium salt, a carboxylic, a phenol, or an aniline group was found to be detrimental to activity . Possible explanations for these observations are proposed . Some physicochemical properties, e.g., the partition coefficient (log P) and the graph index (which in some sense measures the "complexity" of a molecule) were also found to be relevant to activity . Their role in MDRR was also rationalized . Based on our quantitative structure-activity relationship study of MDRR agents, some compounds with desired substructural features and activity were identified from the MACCS-II and National Cancer Institute DIS databases and tested experimentally . Our study may also help the rational design of anti-cancer drugs . Based on this study and on observations by other researchers, we postulate that P-glycoprotein-mediated resistance to paclitaxel could probably be eliminated by proper substitution of its benzamido and phenyl groups . Several novel compounds with the paclitaxel skeleton are proposed, which may lead to a new generation of paclitaxel anti-cancer drugs with less MDR potential.

Genome Res, 1997 Aug, 7(8), 802 - 19
Multiplex sequencing of 1.5 Mb of the Mycobacterium leprae genome; Smith DR et al.; The nucleotide sequence of 1.5 Mb of genomic DNA from Mycobacterium leprae was determined using computer-assisted multiplex sequencing technology . This brings the 2.8-Mb M . leprae genome sequence to approximately 66% completion . The sequences, derived from 43 recombinant cosmids, contain 1046 putative protein-coding genes, 44 repetitive regions, 3 tRNAs, and 15 tRNAs . The gene density of one per 1.4 kb is slightly lower than that of Mycoplasma (1.2 kb) . Of the protein coding genes, 44% have significant matches to genes with well-defined functions . Comparison of 1157 M . leprae and 1564 Mycobacterium tuberculosis proteins shows a complex mosaic of homologous genomic blocks with up to 22 adjacent proteins in conserved map order . Matches to known enzymatic, antigenic, membrane, cell wall, cell division, multidrug resistance, and virulence proteins suggest therapeutic and vaccine targets . Unusual features of the M . leprae genome include large polyketide synthase (pks) operons, inteins, and highly fragmented pseudogenes.

Drugs Aging, 1997 Aug, 11(2), 152 - 64
Treatment of multiple myeloma in elderly patients . New developments; Ossenkoppele GJ; The median of survival among patients with multiple myeloma (MM) is about 30 months from the onset of treatment . Tumour burden and a range of other parameters, such as C-reactive protein levels, the plasma cell labelling index and beta2-microglobulin levels, can be used to assign patients to favourable and unfavourable prognostic groups . Conventional chemotherapy consists of melphalan and prednisone, and is as effective as moderately intensive cytotoxic drug regimens . Although second-line chemotherapy is initially effective, all patients eventually die . Maintenance therapy will interferon-alpha prolongs the plateau phase of the disease, but its effects on overall survival are minimal . One of the promising developments in the treatment of MM has been the introduction of high dosage chemotherapy, which can now be safely administered when stem cells are used for haematological recovery . Autologous bone marrow transplantation has been shown to produce a significant improvement in survival compared with conventional therapy . Several studies are under way that are examining the effects of multiple courses of high dosage chemotherapy together with peripheral stem cell support . Purging of autologous stem cell harvests will be performed in the near future to minimise contamination with myeloma cells . It is now feasible to use high dosage chemotherapy, with the support of granulocyte colony-stimulating factor-stimulated whole blood, in selected elderly patients . Besides the promising development of intensive therapy, a number of other treatment strategies have emerged, including treatment with monoclonal antibodies against interleukin-6 and multidrug resistance-modulating agents . Better supportive care can be provided for some patients by using epoetin (recombinant human erythropoietin), and the sequelae of lytic bone lesions can be ameliorated through the use of bisphosphonatesPublication Types:

	Review
	Review, Tutorial

What is Food Microbiology?, What Is Staphylococcus Aureus?, What Is Cell Biology?, What Is Genetics?, What Is Biofilter?, n, Microbe, n, Microorganism, e, Microbiology, r, Bacteria, c, Bacteriology, e, Escherichia coli, o, Pseudomonas aeruginosa, n, Bacteriological, e, Antibiotics, r, Pseudomonas aeruginosa, o, Prokaryotes, c, Prokaryotes, i, S. cerevisiae, n, Lactobacillus, s, Bacteria, o, Cryptococci, n, Antibiotics, o, Streptococcal, o, Phage, i, Escherichia coli, s, Multidrug resistant, r, S. cerevisiae, i, Microorganisms, r, Vibriosis, c, Streptomycin, i, Bacillus subtilis

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005