Multidrug resistant

Int J Tuberc Lung Dis, 1998 Jun, 2(6), 490 - 8
Drug-resistant tuberculosis in the Dominican Republic: results of a nationwide survey; Espinal MA et al.; SETTING: The Dominican Republic . OBJECTIVE: To assess the extent of drug-resistant tuberculosis (TB) following the guidelines of the World Health Organization (WHO)/International Union Against Tuberculosis and Lung Disease (IUATLD) new global surveillance project on drug resistance in TB . METHODS: Using a multi-step proportional weighted approach, a sample of 688 sequential cases of smear positive pulmonary TB diagnosed between April 1994 and April 1995 was studied in six of the country's eight health regions . Pre-treatment sputum samples were cultured on Loewenstein-Jensen medium and drug susceptibility tests were performed using the economic variant of the proportion method . RESULTS: Of 420 cases with drug susceptibility results, resistance to one or more drugs was observed in 43.8%; resistance was found in 52.1% of 117 TB cases with a history of previous antituberculosis treatment and in 40.6% of 303 new TB cases . In five of the six health regions surveyed, > or = 41% of strains were resistant to one or more drugs . Multidrug resistance (MDR) to isoniazid and rifampicin with or without resistance to other drugs was found in 43 (10.2%) of 420 cases, including 6.6% of new TB cases . In five of the six health regions > or = 8% of strains were classified as MDR . Independent predictors of MDR-TB included being in the age group 25 to 44 years (odds ratio {OR} = 4.2, 95% confidence interval {Cl} 1.5, 11.6; P = 0.005), being aged 45 years and over (OR = 4.5, 95% CI 1.4, 14.4; P = 0.009), and having a prior history of TB (OR = 3.7, 95% CI 1.9, 7.4; P = 0.0001) . CONCLUSION: The proportion of Mycobacterium tuberculosis strains resistant to one or more anti-TB drugs in the Dominican Republic is among the highest observed world-wide . The severity of the problem urgently requires the full implementation of TB control strategies endorsed by the WHO and the IUATID, which include political commitment to a National TB Program, case detection utilizing sputum-smear microscopy, directly observed treatment, regular drug supply, and standardised recording and reporting systems . Also, the sale of TB drugs in the private market should be controlled.

Int J Tuberc Lung Dis, 1998 Jun, 2(6), 484 - 9
Multidrug-resistant tuberculosis in the Florence province from 1992 to 1995; Nutini S et al.; SETTING: Epidemiological data on the frequency of drug-resistant tuberculosis is not available in Italy . OBJECTIVES: Evaluation of the rate of multidrug-resistant tuberculosis in the Province of Florence, Italy . DESIGN: Retrospective analysis of all sensitivity tests performed with the Bactec method on initial mycobacterial isolates, from 1 January 1992 to 31 December 1995, in the Province of Florence . RESULTS: The following rates of resistance were found in the 433 samples tested: isoniazid + rifampicin 2.5%, at least one drug 13.8%, isoniazid 10.6%, rifampicin 3.6%, streptomycin 3.6%, pyrazinamide 1.7% and ethambutol 0.6% . Resistance was higher in foreign-born individuals from high prevalence countries than in the Italian-born population, whereas resistance to streptomycin was more frequent in the latter . The yearly rates of resistance showed no significant variation in the period examined . Clinical data were available in 231 patients: the rate of resistance to at least one drug and to isoniazid + rifampicin were 10.8% and 0%, respectively, in never treated patients, and 28.5% and 7.1%, respectively, in previously treated patients . CONCLUSION: These data show higher multidrug resistance rates than those found in other European countries such as England and Wales, France and Switzerland . This result suggests the need to establish official guidelines for the correct treatment of tuberculosis in Italy, in order to prevent the onset of drug resistance, and to establish a national surveillance system for mycobacterial resistance.

Clin Cancer Res, 1998 Jun, 4(6), 1563 - 6
Potential interactions between antitubulin agents and temperature: implications for modulation of multidrug resistance; Dumontet C et al.; We analyzed the effect of high temperature (a 1-h incubation at 43 degrees C) on the accumulation and cytotoxicity of vinblastine and docetaxel in two model cell lines, K562 and MESSA, and their multidrug resistance (MDR) counterparts, K562/R7 and MESSA/Dx5 . High temperature increased the amount of intracellular vinblastine and docetaxel significantly in MESSA cell and, to a much lesser extent, in K562 cells . MDR-positive cells retained a profound drug accumulation defect at 43 degrees C . Hyperthermia enhanced the cytotoxic effect of vinblastine (but not docetaxel) in both K562 and MESSA cells, but not in the MDR-positive variants . PSC833, a potent modulator of P-glycoprotein, induced high levels of drug accumulation in the two MDR-positive cell lines at both 37 degrees C and at 43 degrees C . PSC833 also significantly reduced the resistance levels of the two MDR-positive lines at both 37 degrees C and at 43 degrees C . The effect of hyperthermia on drug accumulation thus seems to depend on the cell line, whereas the effect on cytotoxicity depends on the type of compound . The MDR phenotype remains a therapeutic obstacle at 43 degrees C but is accessible to modulation.

Clin Cancer Res, 1998 Jun, 4(6), 1393 - 403
The expression of drug resistance gene products during the progression of human prostate cancer; Sullivan GF et al.; Prostate cancer progresses from a localized disease to a widely disseminated malignancy . Each step along this progression pathway involves multiple genetic alterations that impart a survival advantage to the tumor cell over its normal counterparts and may confer resistance to therapy . Because metastatic prostate cancer is one of the most therapy-resistant human neoplasms, we studied the expression of certain molecular determinants of drug resistance in the context of tumor progression . Paraffin-embedded formalin-fixed resected prostates were chosen based on Gleason grade and surgical stage . Immunohistochemistry was used to detect the expression of multidrug resistance protein (MRP), topoisomerase II alpha, p53, glutathione S-transferase pi, Bcl-2, and P-glycoprotein in these specimens . We found that all of the proteins were expressed in resected prostate except for P-glycoprotein . The expression of MRP, topoisomerase II alpha, p53, and Bcl-2 increased with the Gleason grade . In addition, the expression of MRP, topoisomerase II alpha, and p53 increased with the surgical stage . In contrast, the glutathione S-transferase pi and Bcl-2 expression decreased with the increasing surgical stage . Stage was the strongest indicator of protein expression . These results suggest that drug resistance gene products are expressed in prostate cancer at the time of surgical resection . Thus, although the emergence of the "pan-resistance" phenotype in prostate cancer may partly be a function of the selection pressure exerted by therapeutic interventions, certain determinants of chemoresistance may be caused by genetic changes accompanying tumorigenesis.

Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 7024 - 9
The drug efflux protein, P-glycoprotein, additionally protects drug-resistant tumor cells from multiple forms of caspase-dependent apoptosis; Smyth MJ et al.; Multidrug resistance mediated by the drug efflux protein, P-glycoprotein (P-gp), is one mechanism that tumor cells use to escape death induced by chemotherapeutic agents . However, the mechanism by which P-gp confers resistance to a large variety of structurally diverse molecules has remained elusive . In this study, classical multidrug resistant human CEM and K562 tumor cell lines expressing high levels of P-gp were less sensitive to multiple forms of caspase-dependent cell death, including that mediated by cytotoxic drugs and ligation of Fas . The DNA fragmentation and membrane damage inflicted by these stimuli were defined as caspase dependent by various soluble peptide fluoromethylketone caspase inhibitors . Inhibition of P-gp function by the anti-P-gp mAb MRK-16 or verapamil could reverse resistance to these forms of cell death . Inhibition of P-gp function also enhanced drug or Fas-mediated activation of caspase-3 in drug-resistant CEM cells . By contrast, caspase-independent cell death events in the same cells, including those mediated by pore-forming proteins or intact NK cells, were not affected by P-gp expression . These observations suggest that, in addition to effluxing drugs, P-gp may play a specific role in regulating some caspase-dependent apoptotic pathways.

J Pharmacol Exp Ther, 1998 Jun, 285(3), 1260 - 5
Characterization of efflux transport of organic anions in a mouse brain capillary endothelial cell line; Kusuhara H et al.; Cumulative evidence suggests that several organic anions are actively effluxed from the brain to the blood across the blood-brain barrier (BBB) . We examined the possibility of the presence of primary active transporters for organic anions (multidrug resistance associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT)) on the BBB by measuring the ATP-dependent uptake of 2,4-dinitrophenyl-S-glutathione (DNP-SG) and leukotriene C4 (LTC4) into membrane vesicles prepared from a cell line derived from mouse brain capillary endothelial cells (MBEC4) . The ATP-dependent uptake of DNP-SG into the membrane vesicles was osmotically sensitive and was also supported by GTP, but not by AMP or ADP . An ATPase inhibitor, vanadate, blocked the ATP-dependent uptake of DNP-SG . The ATP-dependent uptake process was saturable, with Km values of 0.56 and 0.22 microM, and Vmax values of 5.5 and 27.5 pmol/min/mg protein for DNP-SG and LTC4, respectively . Northern and Western blot analyses showed the expression of murine MRP but not cMOAT in MBEC4 cells . Western blot analysis of the rat cerebral endothelial cells indicated the expression of protein(s) that is detectable with MRPr1, an antibody against MRP . These results, together with previous findings that both DNP-SG and LTC4 are good ligands for MRP, suggest that MRP is responsible for the unidirectional, energy-dependent efflux of organic anions from the brain into the circulating blood across the BBB.

Mol Pharmacol, 1998 Jun, 53(6), 1068 - 75
Hepatic expression of multidrug resistance-associated protein-like proteins maintained in eisai hyperbilirubinemic rats; Hirohashi T et al.; The biliary excretion of several organic anions is mediated by the canalicular multispecific organic anion transporter (cMOAT), which is hereditarily defective in mutant rats such as Eisai hyperbilirubinemic rats (EHBR) . In addition, using a kinetic study with isolated canalicular membrane vesicles, we recently suggested the presence of ATP-dependent organic anion transporter(s) other than cMOAT in EHBR {Pharm Res (NY) 12:1746-1755 (1995); J Pharmacol Exp Ther 282:866-872 (1997)} . The aim of this study is to provide a molecular basis for the presence of multiplicity in the biliary excretion of organic anions in rats . Based on the homology with human multidrug resistance-associated protein (hMRP), two cDNA fragments encoding the carboxyl-terminal ATP-binding cassette region were amplified by reverse transcription-polymerase chain reaction from EHBR liver . These fragments exhibited approximately 70% amino acid identity with hMRP and rat cMOAT;, therefore, they were designated MRP-like proteins (MLP-1 and MLP-2) . The cloned full length cDNA of MLP-1 and -2 from the Sprague-Dawley (SD) rat liver and colon cDNA library was composed of 1502 and 1523 amino acids, respectively, had the characteristics of ATP-binding cassette transporters, and exhibited homology with hMRP and rat cMOAT . Northern blot analysis indicated that MLP-1 is expressed predominantly in the liver in both SD rats and EHBR, whereas hepatic expression of MLP-2 was observed only in EHBR . In addition, MLP-2 was markedly induced by ligation of the bile duct in SD rat liver . In both SD rats and EHBR, MLP-2 was expressed predominantly in the duodenum, jejunum, and colon . These findings suggest that MLP-1 and MLP-2 might be novel members of the MRP family responsible for the excretion of organic anions from these epithelial cells, and that MLP-2 is an inducible one.

Mol Pharmacol, 1998 Jun, 53(6), 1062 - 7
Adenosine triphosphate-dependent transport of anionic conjugates by the rabbit multidrug resistance-associated protein Mrp2 expressed in insect cells; van Aubel RA et al.; The multidrug resistance-associated protein Mrp2 is expressed in liver, kidney, and small intestine and mediates ATP-dependent transport of conjugated organic anions across the apical membrane of epithelial cells . We recently cloned a rabbit cDNA encoding a protein that on basis of highest amino acid homology and tissue distribution was considered to be the rabbit homolog of rat Mrp2 . To investigate whether rabbit Mrp2 mediates ATP-dependent transport similar to rat Mrp2, we expressed rabbit Mrp2 in Spodoptera frugiperda (Sf9) cells using recombinant baculovirus . Mrp2 was expressed as an underglycosylated protein in Sf9 cells and to a higher level compared with rabbit liver and renal proximal tubules . Both 17beta-estradiol-17-beta-D-glucuronide ({3H}E217betaG, 50 nM) and {3H}leukotriene C4 (3 nM) were taken up by Sf9-Mrp2 membrane vesicles in an ATP-dependent fashion . Uptake of {3H}E217betaG was dependent on the osmolarity of the medium and saturable for ATP (Km = 623 microM) . Leukotriene C4, MK571, phenolphthalein glucuronide, and fluorescein-methotrexate were good inhibitors of {3H}E217betaG transport . The inhibitory potency of cyclosporin A and methotrexate was moderate, whereas fluorescein, alpha-naphthyl-beta-D-glucuronide, and p-nitrophenyl-beta-D-glucuronide did not inhibit transport . In conclusion, we show direct ATP-dependent transport by recombinant rabbit Mrp2 and provide new data on Mrp2 inhibitor specificity.

J Cell Sci, 1998 Apr, 111 ( Pt 8), 1137 - 45
Cyclic AMP stimulates sorting of the canalicular organic anion transporter (Mrp2/cMoat) to the apical domain in hepatocyte couplets; Roelofsen H et al.; The canalicular membrane of rat hepatocytes contains an ATP-dependent multispecific organic anion transporter, also named multidrug resistance protein 2, that is responsible for the biliary secretion of several amphiphilic organic anions . This transport function is markedly diminished in mutant rats that lack the transport protein . To assess the role of vesicle traffic in the regulation of canalicular organic anion transport, we have examined the redistribution of the transporter to the canalicular membrane and the effect of cAMP on this process in isolated hepatocyte couplets, which retain secretory polarity . The partial disruption of cell-cell contact, due to the isolation procedure, leaves the couplet with both remnant apical membranes, as a source of apical proteins, and an intact apical domain and lumen, to which these proteins are targeted . The changes in distribution of the transporter were correlated to the apical excretion of a fluorescent substrate, glutathione-methylfluorescein . The data obtained in this study show that the transport protein, endocytosed from apical membrane remnants, first is redistributed along the basolateral plasma membrane . Then it is transcytosed to the remaining apical pole in a microtubule-dependent fashion, followed by the fusion of transporter-containing vesicles with the apical membrane . The cAMP analog dibutyrylcAMP stimulates all three steps, resulting in increased apically located transport protein, glutathione-methylfluorescein transport activity and apical membrane circumference . These findings indicate that the organic anion transport capacity of the apical membrane in hepatocyte couplets is regulated by cAMP-stimulated sorting of the multidrug resistance protein 2 to the apical membrane . The relevance of this phenomenon for the intact liver is discussed.

Anticancer Drugs, 1998 Mar, 9(3), 263 - 71
Antitumor activity of KW-2170, a novel pyrazoloacridone derivative; Ashizawa T et al.; 5-(3-Aminopropyl)amino-7,10-dihydroxy-2-(2-hydroxethyl)-aminoethyl -6H-pyrazolo{4,5,1-de}acridin-6-one dihydroxy-chloride (KW-2170), a novel derivative of pyrazoloacridone, was selected and evaluated for its antitumor activity and toxicity in mice . KW-2170 exhibited antitumor activity superior to adriamycin (ADM) against Sarcoma 180, breast carcinoma MM102 and fibrosarcoma Meth A inoculated s.c . in mice . Its therapeutic index (LD10/ED50) was higher than that of ADM on two murine carcinoma models, MM102 and Meth A . KW-2170 showed significant antitumor activity against 17 human tumor xenografts of a total of 24 tumors tested and the total tumor response rate by treatment with KW-2170 was significantly higher than that by ADM (70.8 versus 58.3%) . In particular, human lung carcinoma was highly sensitive to KW-2170, and a marked tumor regression was observed on Lu-65 and Lu-99 human lung carcinoma xenograft models . Ovary and pancreas carcinomas were also sensitive to the drug . Additionally, its therapeutic index was also high on these human carcinoma models in comparison with that of ADM . The best antitumor efficacy of KW-2170 was observed by a weekly treatment schedule followed by a single treatment schedule and a successive administration schedule also tended to be toxic to the hosts . KW-2170 exhibited very low cross-resistance against four lines of multidrug resistant tumors expressing high levels of P-glycoprotein, and the drug showed significant antitumor activity against ADM-resistant human ovary carcinoma A2780/ADM and against nasopharynx carcinoma KB-A1 xenografts which were not sensitive to ADM . These results indicate that KW-2170 has a very potent antitumor activity and is feasible as a new antitumor drug against ADM-refractory solid tumors in clinics.

Anticancer Drugs, 1998 Mar, 9(3), 255 - 61
The bisbenzylisoquinoline alkaloids, tetrandine and fangchinoline, enhance the cytotoxicity of multidrug resistance-related drugs via modulation of P-glycoprotein; Choi SU et al.; The occurrence of resistance to chemotherapeutic drugs is a major problem for successful cancer treatment and reducing drug accumulation by P-glycoprotein (P-gp) is one of the major mechanisms of multidrug resistance (MDR) . The present study was performed to evaluate the MDR-reversal abilities of two bisbenzylisoquinoline alkaloids, tetrandine (TET) and fangchinoline (FAN), compared with verapamil (VER), a well-known P-gp modulator . TET (3.0 microM), FAN (3.0 microM) and VER (10.0 microM) reduced the paclitaxel (TAX) concentration required to achieve 50% inhibition of cell growth (EC50) to HCT15 (P-gp-positive) cells about 3100-, 1900- and 410-fold, and these compounds also reduced the EC50 value of actinomycin D (AMD) about 36.0-, 45.9- and 18.2-fold in the cells, respectively . Meanwhile, TET, FAN and VER had no effect on the cytotoxicity of the drugs to SK-OV-3 (P-gp-negative) cells . On the other hand, TET (3.0 microM), FAN (3.0 microM) and VER (10.0 microM) similarly enhanced the accumulation rates of rhodamine 123, a well known P-gp substrate, in HCT15 cells (200-250%) . After efflux for 2 h with fresh medium, TET and FAN also enhanced the residual rate of rhodamine 123 about 5.0- and 2.6-fold in comparison with control, respectively . TET, FAN and VER could not affect the accumulation and residual rate of rhodamine 123 in SK-OV-3 cells . From the result, we conclude that TET and FAN enhanced the cytotoxicity of MDR-related drugs via modulation of P-gp.

Semin Oncol, 1998 Apr, 25(2 Suppl 6), 58 - 61
Megestrol acetate as a biomodulator; Chang AY; Megestrol acetate is a synthetic analog of progesterone . In general, megestrol acetate exerts its progesterone-like hormonal effect by binding to the progesterone receptor . It has been recognized that megestrol acetate can increase weight and improve some aspects of quality of life in cancer patients and in patients with the acquired immunodeficiency syndrome . This effect occurs in patients with or without concurrent chemotherapy or radiotherapy, through an as yet unknown mechanism . Recently, megestrol acetate has been shown to reverse, at least partially, multidrug resistance to doxorubicin and/or vincristine in cancer cell lines . This potentially beneficial effect has not yet been studied in clinical trials . This biological activity is thought to be mediated through the unique binding of megestrol acetate to p-glycoprotein . At least in vitro megestrol acetate can also enhance the cytotoxic effect of these two chemotherapeutic agents in some MDR-nonexpressing cell lines . These findings suggest that megestrol acetate deserves further preclinical and clinical studies to evaluate its potential role of enhancing cytotoxicity of chemotherapy and improving the quality of life of cancer patients.

Antimicrob Agents Chemother, 1998 Jun, 42(6), 1524 - 6
Contribution of beta-lactamases to beta-lactam susceptibilities of susceptible and multidrug-resistant Mycobacterium tuberculosis clinical isolates; Segura C et al.; The beta-lactamases in 154 clinical Mycobacterium tuberculosis strains were studied . Susceptibilities to beta-lactam antibiotics, their combination with clavulanate (2:1), and two fluoroquinolones were determined in 24 M . tuberculosis strains susceptible to antimycobacterial drugs and in nine multiresistant strains . All 154 M . tuberculosis isolates showed a single chromosomal beta-lactamase pattern (pI 4.9 and 5.1) . M . tuberculosis beta-lactamase hydrolyzes cefotaxime with a maximum rate of 22.5 +/- 2.19 IU/liter (strain 1382) . Neither amoxicillin, carbenicillin, cefotaxime, ceftriaxone, nor aztreonam was active alone . Except for aztreonam, beta-lactam combinations with clavulanate produced better antimycobacterial activity.

Antimicrob Agents Chemother, 1998 Jun, 42(6), 1375 - 81
Recombinant expression and characterization of the major beta-lactamase of Mycobacterium tuberculosis; Voladri RK et al.; New antibiotic regimens are needed for the treatment of multidrug-resistant tuberculosis . Mycobacterium tuberculosis has a thick peptidoglycan layer, and the penicillin-binding proteins involved in its biosynthesis are inhibited by clinically relevant concentrations of beta-lactam antibiotics . beta-Lactamase production appears to be the major mechanism by which M . tuberculosis expresses beta-lactam resistance . beta-Lactamases from the broth supernatant of 3- to 4-week-old cultures of M . tuberculosis H37Ra were partially purified by sequential gel filtration chromatography and chromatofocusing . Three peaks of beta-lactamase activity with pI values of 5.1, 4.9, and 4.5, respectively, and which accounted for 10, 78, and 12% of the total postchromatofocusing beta-lactamase activity, respectively, were identified . The beta-lactamases with pI values of 5.1 and 4.9 were kinetically indistinguishable and exhibited predominant penicillinase activity . In contrast, the beta-lactamase with a pI value of 4.5 showed relatively greater cephalosporinase activity . An open reading frame in cosmid Y49 of the DNA library of M . tuberculosis H37Rv with homology to known class A beta-lactamases was amplified from chromosomal DNA of M . tuberculosis H37Ra by PCR and was overexpressed in Escherichia coli . The recombinant enzyme was kinetically similar to the pI 5.1 and 4.9 enzymes purified directly from M . tuberculosis . It exhibited predominant penicillinase activity and was especially active against azlocillin . It was inhibited by clavulanic acid and m-aminophenylboronic acid but not by EDTA . We conclude that the major beta-lactamase of M . tuberculosis is a class A beta-lactamase with predominant penicillinase activity . A second, minor beta-lactamase with relatively greater cephalosporinase activity is also present.

Eur J Cancer, 1998 Jan, 34(1), 168 - 74
In vitro modulation of doxorubicin and docetaxel antitumoral activity by methyl-beta-cyclodextrin; Grosse PY et al.; Methyl-beta-cyclodextrin (MEBCD) was investigated for its effect on the antitumoral activity of various antineoplastic agents (doxorubicin (DOX), docetaxel (DXL), 5-fluorouracil (5-FU) and cisplatin (CDDP)) in three different human parental sensitive cancer cell lines (K562 S, MCF7 S and A2780 S) and their multidrug resistant variant sublines (K562 R, MCF7 R and A2780 R) . At non-cytotoxic concentrations, MEBCD was able to increase significantly DOX and DXL cytotoxic activity in all the cell lines tested . The sensitisation ratios (IC50 drug control/IC50 drug-MEBCD treated) ranged from 3l1 to 14.3 . Moreover, intracellular DOX accumulation, determined by high-performance liquid chromatography, was also increased when cells were treated with MEBCD combined with DOX (approximately 2-3 fold) . The effects of MEBCD in resistant sublines were greater than in their parental sensitive cell lines . Other experiments demonstrated that the action of the MEBCD was independent of DOX . These data provided a basis for the potential therapeutic application of MEBCD in cancer therapy.

Eur J Cancer, 1998 Jan, 34(1), 81 - 6
Expression of the multidrug resistance protein (MRP) in squamous cell carcinoma of the oesophagus and response to pre-operative chemotherapy; Nooter K et al.; One of the major problems in the treatment of squamous cell carcinoma of the oesophagus (ESCC) is the unresponsiveness to cytotoxic drugs . So far, the mechanisms underlying the intrinsic drug resistance of ESCC remain unclear . The aim of this study was to determine the role of the newly recognised drug resistance protein, the multidrug resistance protein (MRP), in ESCC drug resistance . Tumour biopsies from ESCCs were analysed by RNase protection assay (RPA) as well as by immunohistochemistry (IHC) for the presence of MRP mRNA or protein, respectively . The ESCC samples were obtained from patients participating in a prospective randomised clinical phase III trial, evaluating pre-operative chemotherapy (cisplatin and etoposide) followed by surgery versus surgery alone in patients with operable ESCC . For most patients, tumour biopsies taken at diagnosis by endoscopy as well as surgically resected primary tumours were available . Of 58 ESCC patients enrolled, 28 received chemotherapy before surgical resection of their tumours, and 30 were treated with surgery alone . 12 patients (3 complete and 9 partial responses; 43%) showed a major response after chemotherapy, 10 patients (36%) had stable disease (SD), and 6 (21%) progressive disease (PD) . On 14 surgically resected, untreated, primary ESCCs, the IHC scores correlated with the MRP mRNA levels, quantitated by RPA (multiple testing, P < 0.01) . MRP expression was detected by IHC in the vast majority (52/58; 90%) of the diagnostic biopsies . MRP expression did not differ significantly between CR + PR, and patients with SD or PD . In addition, multivariate analysis by logistic regression did not show any effect of tumour cell differentiation or UICC tumour stage on the outcome of pre-operative chemotherapy in relation to MRP expression . However, a difference became apparent (Sign-test, P < 0.05) for higher MRP expression in tumours from patients with PR or SD, when comparing MRP levels in paired tumour samples before and after chemotherapy, suggesting that chemotherapy selected for drug-resistant cell clones.

J Exp Med, 1998 May 18, 187(10), 1583 - 98
Defective acidification in human breast tumor cells and implications for chemotherapy; Altan N et al.; Multidrug resistance (MDR) is a significant problem in the treatment of cancer . Chemotherapeutic drugs distribute through the cyto- and nucleoplasm of drug-sensitive cells but are excluded from the nucleus in drug-resistant cells, concentrating in cytoplasmic organelles . Weak base chemotherapeutic drugs (e.g., anthracyclines and vinca alkaloids) should concentrate in acidic organelles . This report presents a quantification of the pH for identified compartments of the MCF-7 human breast tumor cell line and demonstrates that (a) the chemotherapeutic Adriamycin concentrates in acidified organelles of drug-resistant but not drug-sensitive cells; (b) the lysosomes and recycling endosomes are not acidified in drug-sensitive cells; (c) the cytosol of drug-sensitive cells is 0.4 pH units more acidic than the cytosol of resistant cells; and (d) disrupting the acidification of the organelles of resistant cells with monensin, bafilomycin A1, or concanamycin A is sufficient to change the Adriamycin distribution to that found in drug-sensitive cells, rendering the cell vulnerable once again to chemotherapy . These results suggest that acidification of organelles is causally related to drug resistance and is consistent with the hypothesis that sequestration of drugs in acidic organelles and subsequent extrusion from the cell through the secretory pathways contribute to chemotherapeutic resistance.

Biochemistry, 1998 May 5, 37(18), 6503 - 12
Proximity of the nucleotide binding domains of the P-glycoprotein multidrug transporter to the membrane surface: a resonance energy transfer study; Liu R et al.; Very little structural information is available for P-glycoprotein (Pgp), which has been implicated in the multidrug resistance of human tumors because of its ability to act as an ATP-driven efflux pump for hydrophobic compounds . Highly purified Pgp has been labeled on two cysteine residues with the fluorescence probe NBD-Cl (7-chloro-4-nitro-2,1,3-benzoxadiazole) . We show that NBD labels the same cysteine residues as MIANS {2-(4-maleimidoanilino)naphthalene-6-sulfonic acid}; they are located within the Walker A motif of the nucleotide binding domain, close to the site where ATP binds . NBD- and MIANS-labeled Pgps were reconstituted by detergent dilution into phospholipid vesicles containing increasing mole fractions of rhodamine- or NBD-labeled phosphatidylethanolamine (PE), respectively . The fluorescence of the NBD-Pgp and MIANS-Pgp donors was quenched in a concentration-dependent manner by the rhodamine-PE and NBD-PE acceptors . Using two different methods to analyze Forster resonance energy transfer, the distance of the Pgp-bound probes from the lipid-water interfacial region of the bilayer was estimated to be 31-35 A . This distance is compatible with the low-resolution structure of Pgp determined by electron microscopy, and indicates that the nucleotide binding domains lie close to the membrane surface . The experimental data fitted very well to theoretical quench curves for a single protein-bound fluor, suggesting that the two nucleotide binding domains are located equidistant from the bilayer . Following the addition of ATP to MIANS-Pgp, the NBD-PE quench curve no longer conformed to the models . These results imply that Pgp interacts differently with PE when it is in the ATP-bound form.

Rapid Commun Mass Spectrom, 1998, 12(10), 620 - 4
Cellular uptake profile of paclitaxel using liquid chromatography tandem mass spectrometry; Kerns EH et al.; A new method for studying cellular uptake has been developed . This method is based on selected reaction monitoring liquid chromatography tandem mass spectrometry analysis of preparations from cell culture . The limit of detection for paclitaxel was approximately 0.1 microM intracellular concentration . This method has been utilized to study the uptake of paclitaxel and an analog (BMS-190616) in normal and multidrug resistant (MDR) cell lines . Paclitaxel and the analog, that had been noted to overcome MDR in animal models, were incubated with normal cells (HCT116) and MDR cells (HCT116(VM)46) at therapeutic concentrations . Intracellular drug concentrations were assayed at intervals from 0 to 1.0 h . Results show that paclitaxel accumulates to a level 12 times greater and BMS-190616 to a level 5 times greater in the normal cells as compared to MDR cells suggesting that paclitaxel is more sensitive to MDR than the analog . Furthermore, the steady state level of BMS-190616 was 4 fold greater than paclitaxel in the MDR cell line suggesting that at least part of this compound's increased therapeutic effect can be attributed to processes of uptake and efflux at the cellular level . These data show that the method is rapid, sensitive and presents a unique advantage over traditional radioisotopic methods in that it can readily be employed on a range of analogs without any additional synthetic effort.

Emerg Infect Dis, 1998 Apr-Jun, 4(2), 195 - 209
Multidrug-resistant Mycobacterium tuberculosis: molecular perspectives; Rattan A et al.; Multidrug-resistant strains of Mycobacterium tuberculosis seriously threaten tuberculosis (TB) control and prevention efforts . Molecular studies of the mechanism of action of antitubercular drugs have elucidated the genetic basis of drug resistance in M . tuberculosis . Drug resistance in M . tuberculosis is attributed primarily to the accumulation of mutations in the drug target genes; these mutations lead either to an altered target (e.g., RNA polymerase and catalase-peroxidase in rifampicin and isoniazid resistance, respectively) or to a change in titration of the drug (e.g., InhA in isoniazid resistance) . Development of specific mechanism-based inhibitors and techniques to rapidly detect multidrug resistance will require further studies addressing the drug and drug-target interaction.

J Cell Biochem, 1998 Jun 15, 69(4), 463 - 9
Upregulation of P-glycoprotein in rat hepatoma rho(o) cells: implications for drug-DNA interactions; Pillay V et al.; Rat hepatoma cells lacking mitochondrial DNA (rho(o) cells) were used as a model system to examine the possible roles of mitochondrial DNA as a target for the DNA-acting anticancer drug Adriamycin (doxorubicin) . The rho(o) cells were 45-fold less sensitive to Adriamycin than the parental rho+ cells containing mitochondrial DNA . Other non-DNA-acting drugs also exhibited similar behaviour, and this was shown to be due to a multidrug resistance (MDR) phenotype in the rho(o) cells . This was indicated by confocal microscopy where rho+ cells exhibited thirteenfold higher cellular levels of Adriamycin than rho(o) cells . Upregulation (tenfold) of P-glycoprotein in rho(o) cells was also confirmed by Northern dot blot analysis . Since the MDR phenotype is present in rho(o) cells and upregulation of P-glycoprotein is maintained in these cells, rho(o) cells are not a good model system for drug-DNA studies (where the drug is susceptible to extrusion by P-glycoprotein), and any such results obtained with this system must be treated with considerable caution.

Nucl Med Biol, 1998 Apr, 25(3), 225 - 32
A novel areneisonitrile Tc complex inhibits the transport activity of MDR P-glycoprotein; Rao VV et al.; P-glycoprotein (Pgp), the product of the multidrug resistance (MDR1) gene, has been an important cancer target for development of MDR modulators that act to inhibit Pgp efflux transport activity . From a series of novel substituted areneisonitrile analogues of Tc-sestamibi, a known Pgp transport substrate, emerged the hexakis(3,4,5-trimethoxyphenylisonitrile)Tc(I) complex (Tc-TMPI) as a potential modulator of Pgp . Tracer 99mTc-TMPI showed net cellular accumulation in inverse proportion to expression of Pgp and enhancement upon addition of classic MDR modulators . At pharmacological concentrations, the carrier-added 94Tc-TMPI complex showed potent inhibition of Pgp-mediated 99mTc-sestamibi transport (EC50, 1.1 +/- 0.2 microM) and displacement of a Pgp-specific photolabel in a concentration-dependent manner . We conclude that 99Tc-TMPI directly inhibited Pgp transport activity and serves as a convenient template for development of nonradioactive Re(I) analogues as novel MDR modulators.

Leuk Res, 1998 Mar, 22(3), 249 - 56
Multidrug-resistant acute leukemia cells are responsive to prolonged exposure of daunorubicin: implications for liposome-encapsulated daunorubicin; Verdonck LF et al.; We examined the cytotoxic effects of free daunorubicin (DNR) and liposome-encapsulated DNR on multidrug-resistant (MDR1) leukemia cells of patients with acute leukemias who had failed primary induction treatment that included DNR . This was analyzed ex-vivo with DNR concentrations and exposure times that normally can be achieved in-vivo for both drugs with induction treatment . The leukemic blasts of patients both with drug-resistant AML and drug-resistant ALL were, ex-vivo, very sensitive to DNR concentrations and exposure times that can be achieved in-vivo by liposome-encapsulated DNR . However, under identical conditions, free DNR and liposome-encapsulated DNR had a similar cytotoxic profile, arguing against a unique mechanism of cytotoxicity by the liposomal constructure . These data suggest that liposome-encapsulated DNR may be preferable to free DNR for the treatment of acute leukemias.

Cancer Lett, 1998 May 15, 127(1-2), 107 - 12
Alpha-tocopherol antagonizes the multidrug-resistance-reversal activity of cyclosporin A, verapamil, GF120918, clofazimine and B669; Van Rensburg CE et al.; The effects of the membrane-stabilizing agent, alpha-tocopherol (25 microg/ml), on the chemosensitizing interactions of cyclosporin A (5 microg/ml), verapamil (2 microg/ml), clofazimine (1 microg/ml), B669 (0.5 microg/ml) and GF120918 (0.015 microg/ml) with a P-glycoprotein-expressing human lung cancer cell line (H69/LX4) have been investigated in vitro . In an assay of cell proliferation, all the chemosensitizing agents restored the sensitivity of H69/LX4 cells to doxorubicin and vinblastine . The inclusion of alpha-tocopherol (25 microg/ml) antagonized the multidrug-resistance (MDR)-modifying activity of all five chemosensitizing agents, effectively preventing restoration of sensitivity to both doxorubicin and vinblastine in H69/LX4 cells.

Pharm Res, 1998 May, 15(5), 706 - 11
Decreased expression and activity of P-glycoprotein in rat liver during acute inflammation; Piquette-Miller M et al.; PURPOSE: Drug disposition is often altered in inflammatory disease . Although the influence of inflammation on hepatic drug metabolism and protein binding has been well studied, its impact on drug transport has largely been overlooked . The multidrug resistance (MDR) gene product, P-glycoprotein (P-gp) is involved in the active secretion of a large variety of drugs . Our goal was to ascertain the influence of acute inflammation (AI) on the expression and functional activity of P-gp . METHODS: AI was induced in rats through turpentine or lipopolysaccharide (LPS) administration . Expression of P-gp in liver was detected at the level of protein on Western blots using the monoclonal antibody C-219 and at the level of mRNA using an RNase protection assay . P-gp mediated transport activity was assessed by measuring the verapamil-inhibitable efflux of rhodamine 123 (R123) in freshly isolated hepatocytes . RESULTS: Turpentine-induced AI significantly decreased the hepatic protein expression of P-gp isoforms by 50-70% and caused a significant 45-65% reduction in the P-gp mediated efflux of R123 . Diminished mRNA levels of all three MDR isoforms were seen . LPS-induced AI similarly resulted in significantly reduced levels and activity of P-gp in liver . Although differences in the constitutive levels of P-gp were seen between male and female rats, the influence of AI on P-gp expression and activity was not gender specific . CONCLUSIONS: Experimentally-induced inflammation decreases the in vivo expression and activity of P-gp in liver . This is the first evidence that expression of P-gp is modulated in response to experimentally-induced inflammation.

Cancer Chemother Pharmacol, 1998, 42(1), 9 - 16
Reversal of multidrug resistance by a liposome-MDR1 ribozyme complex; Masuda Y et al.; PURPOSE: Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy . We examined whether cationic liposome-mediated transfer of a ribozyme could reverse MDR . METHODS: A ribozyme which cleaved codon 196 of MDR1 mRNA was constructed from synthetic oligonucleotides . The MDR1 ribozyme was mixed with N-(1-(2,3-dileoyloxy)propyl)-N,N,N-trimethylammonium methyl sulfate (DOTAP) to form a liposomal complex . The complex was used to treat two P-glycoprotein-producing MDR cell lines: MCF-7/R human breast cancer cells resistant to doxorubicin and MOLT-3/TMQ800 human ALL cells resistant to trimetrexate (TMQ) . In order to investigate the differential sensitivity of these two cell lines to the liposome-ribozyme complex, cellular pharmacological studies including phase-contrast and confocal microscopic studies were performed . RESULTS: Treatment with the liposome-ribozyme complex resulted in reversal of vincristine (VCR) resistance in MCF-7/R cells, but not in MOLT-3/TMQ800 cells . In MCF-7/R cells the treatment resulted in decreases in MDR1 mRNA expression and P-glycoprotein production, whereas no changes in these parameters were seen in MOLT-3/TMQ800 cells . Phase-contrast microscopy revealed that in MCF-7/R cells treatment with DOTAP led to the formation of cytoplasmic vacuoles, and treatment with latex beads resulted in the development of a shiny material in the cytoplasm . In contrast, in MOLT-3/TMQ800 cells hardly any morphological changes occurred . Confocal microscopic imaging showed cytoplasmic fluorescence in MCF-7/R cells after treatment with DOTAP/FITC-dextran or FITC-conjugated latex beads . In MOLT-3/TMQ800 cells no fluorescence was detected . Treatment with cytochalasin B abolished fluorescence in MCF-7/R cells after treatment with DOTAP/FITC-dextran or FITC-conjugated latex beads . These studies show that MCF-7/R cells have high endocytotic activity whereas MOLT-3/TMQ800 cells have little activity . CONCLUSIONS: Endocytotic activity was correlated with the success of cationic liposome-mediated transfer of MDR1 ribozyme . Determination of endocytotic activity of target tumor cells may be predictive of efficacy of liposome-mediated gene transfer.

Planta Med, 1998 May, 64(4), 335 - 8
Mode of action of torilin in multidrug-resistant cancer cell lines; Kim SE et al.; The mechanism of action of multidrug-resistance reversing activity of torilin was studied . In vitro experiments for the accumulation and efflux of vinblastine clearly indicated that MDR reversing effects of torilin would directly be associated with the increase of the intracellular accumulation of anticancer drugs by blocking the drug efflux . Furthermore, torilin increased the membrane ATPase activity from KB-V1 cells, suggesting that torilin might function by inhibiting drug transport mediated by P-glycoprotein.

Planta Med, 1998 May, 64(4), 332 - 4
Torilin, a sesquiterpene from Torilis japonica, reverses multidrug-resistance in cancer cells; Kim SE et al.; A sesquiterpene compound reversing multidrug-resistance in cancer cells was isolated from the fruits of Torilis japonica and the structure was identified as torilin . Torilin potentiated the cytotoxicities of adriamycin, vinblastine, taxol and colchicine against multidrug-resistant KB-V1 and MCF7/ADR cells.

Cell Physiol Biochem, 1998, 8(3), 138 - 50
pH regulation in sensitive and multidrug resistant Ehrlich ascites tumor cells; Litman T et al.; Maintenance and regulation of intracellular pH (pHi) was studied in wild-type Ehrlich ascites tumor cells (EHR2) and five progressively daunorubicin-resistant, P-glycoprotein (P-gp)-expressing strains, the maximally resistant of which is EHR2/1.3 . Steady-state pHi was similar in cells expressing different amounts of P-gp, in the absence and presence of glucose . In EHR2/1.3, glucose-induced acidification was reduced, and proton efflux was increased, compared to the wild-type EHR2, differences which were not caused by increased activity of a Na+/H+ exchanger in the resistant cells . Comparing all six cell lines, no evidence was found for a correlation between the amount of P-gp in the membrane and pHi regulation, which was also unaffected by P-gp modulators . However, a correlation was seen between relative resistance/daunorubicin accumulation and acid extrusion rate, which is likely to be due to aspects of development of drug resistance other than P-gp.

Anticancer Res, 1998 Mar-Apr, 18(2A), 1159 - 66
Cytoplasmic localization of anthracycline antitumor drugs conjugated with reduced glutathione: a possible correlation with multidrug resistance mechanisms; Serafino A et al.; The accumulation of adriamycin (ADR), daunomycin (DAUNO) and their glutathione (GSH)-conjugates, recently obtained by anaerobic reaction of the parent anthracyclines with reduced GSH, was examined in drug-sensitive and multidrug resistant (MDR) cell lines using confocal laser scanning microscopy . In all drug-sensitive lines used (TVM-A12 and TVM-A197 human melanoma cells, K562 lymphoblastoid cells and MCF-7 human breast cancer cells) ADR and DAUNO were mostly located in the nuclei, while their GSH-conjugates were found only in the cytoplasm, predominantly in the Golgi region . On the contrary, in MDR MCF-7/Dox cells, both conjugated and non conjugated anthracyclines gave fluorescence only in the cytoplasm, mostly in the Golgi region, the intensity of the fluorescence being stronger in cells pretreated with verapamil . Viability assay showed that the GSH-conjugate are significantly less cytotoxic than the parent anthracyclines in sensitive cells and showed the same scarce cytotoxicity in MDR MCF-7/Dox cells . These results demonstrate that conjugation of anthracycline antitumor drugs with GSH prevents their access to the nucleus and decreases their cytotoxicity . Furthermore, the observations on MCF-7/Dox suggest that GSH-conjugation of anthracycline might occur in resistant cells and can be in part responsible for the MDR in breast cancer cells.

Anticancer Res, 1998 Mar-Apr, 18(2A), 1091 - 7
Multiwavelength videomicrofluorometric study of some human leukemic lymphoblasts: effect of adriamycin on some biological parameter; Rocchi E et al.; The development of multidrug resistance (MDR) in heterogeneous cell sensitive and resistant populations to a variety of clinically important cytotoxic drugs poses a major obstacle to cancer chemotherapy . The MDR phenotype is characterized by a decrease the intracellular drug accumulation and by an overexpression of the MDR1 gene which encodes the membrane protein, P-glycoprotein (Pgp) . To evaluate the MDR phenotype, rationale investigations of the cytotoxic processes and effect,s of Adriamycin (ADR) were done to obtain information on individual cells . Such information could be obtained through a multiparametric approach involving multiwavelength microfluorometry and numerical image analysis on single living cells . To achieve this, cells should be simultaneously stained with Hoechst 33342 (nuclear staining), Rhodamine 123 (mitochondria staining) and Nile Red (cell contour delineation) . Changes in the biological parameters accessible from R123, Ho33342 and C-SNARF-1/AM (probe used for the pHi measurements) labelling were found more informative than changes in morphological parameters for the discrimination of sensitive and resistant cells . Furthermore, this approach allows the discrimination between two resistant cell lines expressing different mechanisms of resistance.

Anticancer Res, 1998 Mar-Apr, 18(2A), 1005 - 10
Chromosome mediated gene transfer of drug resistance to mitoxantrone; Hazlehurst LA et al.; The anthracenedione, mitoxantrone, frequently selects for a unique drug resistance phenotype that is not mediated by either MDR 1, MRP, or altered DNA topoisomerase II . In this study, we demonstrate that mitoxantrone resistance is likely to be multifactorial with at least one resistance mechanism being the result of a dominant genetic event . This finding was demonstrated by conducting chromosome transfer experiments from human breast cancer cell lines that were either sensitive (MCF7/S) or resistant to mitoxantrone (MCF7/Mitox) . Chromosomes transferred from MCF7/Mitox cells into CHO-K1 cells resulted in the isolation of multiple clones resistant to mitoxantrone . In contrast, chromosomes transferred from the drug sensitive MCF7/S, parent cell line did not confer drug resistance in the rodent CHO-K1 recipient cell line . Both Alu-PCR analysis and Southern blot analysis demonstrated human DNA in the CHO-K1 cells receiving chromosomes from the MCF7/Mitox cells . Unlike the MCF7/Mitox cell line, the drug resistant, CHO-K1 chromosome transferrant clones did not have a decrease in total drug accumulation . We conclude that chromosome transfer from the MCF7/Mitox cell line into CHO-K1 cells, confers a non-transport mediated mechanism of drug resistance that is a dominant genetic event . These studies provide evidence of the genetic multifactorial nature of multidrug resistance in cells selected with mitoxantrone in-vitro.

Anticancer Res, 1998 Mar-Apr, 18(2A), 739 - 42
Avoidance of doxorubicin resistance in osteosarcoma cells using a new quinoline derivative, MS-209; Takeshita H et al.; P-glycoprotein (Pgp), a membrane drug efflux pump, is thought to be responsible for the observed drug resistance in osteosarcoma . We have recently developed Pgp-positive, multidrug resistant (MDR) murine osteosarcoma cell lines, which may be suitable models for the study of drug resistance in osteosarcoma . In this study, we investigated the effect of a newly synthesized quinoline compound, MS-209, on the reversal of doxorubicin (DOX) resistance in these cell lines . Three different types of resistance modifying agents (RMAs) as well as MS-209 were studied . These included the calcium channel blocker verapamil, and the immunosuppressive agents cyclosporin A and FK506 . The reversal effects of the RMAs on DOX resistance were assessed by the MTT assay . In the absence of RMAs, the MDR osteosarcoma cells were 20-fold more resistant to DOX than the parental cells . When MS-209 was added at a final concentration of 0.1 to 3 microM to the MDR cells, 3-to 74-fold sensitization was observed . A complete reversal (37-fold sensitization) of the resistance was obtained at 1 microM MS-209 . This concentration of MS-209 was 3-, 8- and 28-fold more effective than the same concentration of FK506, verapamil and cyclosporin A, respectively . These results indicate that MS-209 may be a more effective RMA, and that DOX resistance in osteosarcoma cells could be reversed by comparatively low doses of MS-209.

Anticancer Res, 1998 Mar-Apr, 18(2A), 701 - 5
Expression analysis of protein kinase C isozymes and multidrug resistance associated genes in ovarian cancer cells; Beck JF et al.; We recently demonstrated a correlation between the expression levels of the PKC eta isozyme and the MDR1 or MRP genes in blasts from AML patients, and in primary breast cancers . In order to extend these findings we analysed ovarian cancer cells from 14 ascites aspirates from 8 patients using a cDNA-PCR approach . 5 patients were examined in follow up studies . 4 out of these 5 patients received continuous chemotherapy . The relative increases in MDR1, MRP, LRP or PKC eta mRNA expression levels were monitored . In one of these patients combined significant increase in MDR1, MRP, LRP and PKC was seen . One follow up sample was obtained after chemotherapy was discontinued . In this case significant relative decreases of MDR1, LRP and PKC eta mRNA expression levels were found . Furthermore, a significant positive correlation was determined for the relative mRNA expression levels of MRP and PKC eta . These results point to a multifactorial emergence of MDR in this type of tumor with a possible involvement of the PKC eta isozyme.

Ann Dermatol Venereol, 1996, 123(9), 574 - 6
{Aggressive cutaneous T-cell lymphoma associated with the presence of Epstein-Barr virus . 2 cases}; Mouly F et al.; INTRODUCTION: The factors of prognosis of the cutaneous T-cell lymphomas are less well known as those of the B-cell lymphomas and the role of the Epstein-Barr virus (EBV) is not yet definitively evaluated . CASE REPORTS: Two male patients aged 62 and 82 years had a mycosis fungoides with a lethal outcome . The first patient had mutilating facial tumors; the RNA m of EBV and the genome of EBV were demonstrated in the diseased skin . The second patient had an erythrodermic course with enlarged peripheral lymph nodes and circulating Sezary's cells; the genome of EBV was demonstrated by PCR in the diseased skin . DISCUSSION: The role of the EBV has already been demonstrated in peripheral aggressive T-cell lymphomas . In the mycosis fungoides, the EBV is associated with the lesions in 0 to 32 p . cent according to the published series . EBV associated T-cell lymphomas have a poor survival rate and the EBV infection may be associated with the expression of the multidrug resistant gene-1 (MDR-1) and the risk of a terminal hemophagocytosis . In our both patients the presence of the EBV in the lymphocytes of the skin lesions is also an argument in favour of the pathogenic role of the virus.

Gene Ther, 1998 Mar, 5(3), 403 - 8
Feasibility of multidrug resistance (MDR-1) gene transfer in patients undergoing high-dose therapy and peripheral blood stem cell transplantation for lymphoma; Devereux S et al.; We have performed a pilot study of MDR-1 gene transfer in patients receiving CD34-selected peripheral blood stem cell (PBSC) transplant for lymphoma . To ensure minimum engraftment thresholds and facilitate CD34 purification, mobilisation of > 2 x 10(6) CD34 cells/kg was a condition for recruitment . Of 11 patients counselled for study entry, only five achieved this target in a single apheresis . In three consenting patients, purified CD34 cells were exposed to A12M1 MDR-1 retroviral supernatant for 6 h, cryopreserved then thawed and readministered following ablative chemotherapy . No delay in engraftment was observed, although one patient received additional back-up cells . Gene transfer was demonstrated by polymerase chain reaction (PCR) for vector-derived MDR-1 cDNA sequence in all cases . Analysis of peripheral blood and bone marrow cells after transplant has, however, shown no evidence of in vivo gene transfer with a follow-up of 12, 15 and 18 months . The effect of MDR-1 substrate drugs has not yet been tested as all patients remain in clinical and radiological remission of their lymphoma . These results confirm the difficulty of achieving in vivo gene transfer in human haemopoietic cells and indicate major logistical constraints in PBSC mobilisation in patients with relapsed and resistant disease in whom initial studies are appropriate.

Ann Trop Med Parasitol, 1998 Jan, 92(1), 31 - 6
Plasma concentrations of artemether and its major plasma metabolite, dihydroartemisinin, following a 5-day regimen of oral artemether, in patients with uncomplicated falciparum malaria; Karbwang J et al.; Plasma concentrations of artemether and its active plasma metabolite (dihydroartemisinin) were measured in 49 male, Thai patients with acute, uncomplicated, multidrug-resistant, Plasmodium falciparum malaria, following their treatment with oral artemether (300 mg on the first day, then 100 mg daily for another 4 days) . Four patients recrudesced (on days 19-22) . After the first dose, artemether became undetectable in < or = 18 h and this drug was also undetectable in samples collected immediately before each dose . Although dihydroartemisinin followed similar trends, three patients had detectable plasma concentrations of this metabolite 24 h after the first dose (i.e . immediately before the second dose) . Median (range) values for plasma concentrations of dihydroartemisinin 6 h {354 (150-751) v . 196 (178-220) ng/ml} and 12 h {158 (25-420) v . 54 (25-115) ng/ml} after the initial dose, estimated antimalarial activities (calculated as dihydroartemisinin equivalents) 6 h {331 (78.2-644.1) v . 23 (183.3-270) nmol/litre} and 12 h {98.3 (10-192.2) v . 56.7 (9.8-59.4) nmol/litre} after the initial dose, and the corresponding 'areas under the curves' (AUC) {3684 (1562-8216) v . 834 (1401-2030) ng.h/ml} were all significantly higher in the patients with sensitive responses than in those who recrudesced.

N Engl J Med, 1998 Jun 4, 338(23), 1641 - 9
Global surveillance for antituberculosis-drug resistance, 1994-1997 . World Health Organization-International Union against Tuberculosis and Lung Disease Working Group on Anti-Tuberculosis Drug Resistance Surveillance; Pablos-Mendez A et al.; BACKGROUND: Drug-resistant tuberculosis threatens efforts to control the disease . This report describes the prevalence of resistance to four first-line drugs in 35 countries participating in the World Health Organization-International Union against Tuberculosis and Lung Disease Global Project on Anti-Tuberculosis Drug Resistance Surveillance between 1994 and 1997 . METHODS: The data are from cross-sectional surveys and surveillance reports . Participating countries followed guidelines to ensure the use of representative samples, accurate histories of treatment, standardized laboratory methods, and common definitions . A network of reference laboratories provided quality assurance . The median number of patients studied in each country or region was 555 (range, 59 to 14,344) . RESULTS: Among patients with no prior treatment, a median of 9.9 percent of Mycobacterium tuberculosis strains were resistant to at least one drug (range, 2 to 41 percent); resistance to isoniazid (7.3 percent) or streptomycin (6.5 percent) was more common than resistance to rifampin (1.8 percent) or ethambutol (1.0 percent) . The prevalence of primary multidrug resistance was 1.4 percent (range, 0 to 14.4 percent) . Among patients with histories of treatment for one month or more {corrected}, the prevalence of resistance to any of the four drugs was 36.0 percent (range, 5.3 to 100 percent), and the prevalence of multidrug resistance was 13 percent (range, 0 to 54 percent) . The overall prevalences were 12.6 percent for resistance to any of the four drugs {corrected} (range, 2.3 to 42.4 percent) and 2.2 percent for multidrug resistance (range, 0 to 22.1 percent) . Particularly high prevalences of multidrug resistance were found in the former Soviet Union, Asia, the Dominican Republic, and Argentina . CONCLUSIONS: Resistance to antituberculosis drugs was found in all 35 countries and regions surveyed, suggesting that it is a global problem.

BMJ, 1998 May 9, 316(7142), 1423 - 5
Drug resistant tuberculosis in prisons in Azerbaijan: case study; Coninx R et al.; OBJECTIVES: To document the existence of drug resistance in a tuberculosis treatment programme that adheres strictly to the DOTS principles (directly observed treatment, short course) and to determine the extent of drug resistance in a prison setting in one of the republics of the former Soviet Union . DESIGN: Case study . SETTING: Central Penitentiary Hospital in Baku, the referral centre for tuberculosis patients from all prisons in Azerbaijan . SUBJECTS: Prisoners with tuberculosis: 28 selected patients not responding clinically or bacteriologically to the standard treatment (group 1) and 38 consecutive patients at admission to the programme (group 2) . MAIN OUTCOME MEASURES: Drug resistance of Mycobacterium tuberculosis strains grown from sputum . RESULTS: All the non-responding patients (group 1) had strains resistant to at least one drug . 25 (89%) of the non-responding patients and nine (24%) of the consecutive patients had M tuberculosis strains resistant to both rifampicin and isoniazid . A further 17 patients in group 2 had strains resistant to one or more first line drugs . CONCLUSIONS: Drug resistant M tuberculosis strains are common in prisons in Azerbaijan . Tuberculosis problems tend to be worse in prisons, but prisoners and former prisoners may have an important role in the transmission of tuberculosis, particularly of drug resistant forms, in the community . National programmes to control tuberculosis will have to take into account and address the problems in prisons to ensure their successPIP: Tuberculosis is a significant health problem in Azerbaijan . In prisons, this problem is compounded by overcrowding, poor general health, a high representation of risk groups, late case finding, and incomplete treatments . The present study investigated the extent of drug resistance at the Central Penitentiary Hospital in Baku--the country's only treatment center for prisoners with tuberculosis . This International Committee of the Red Cross program, established in 1995, uses the directly observed treatment, short course (DOTS) strategy . Sputum samples were collected from two groups of prisoners: 1) 28 patients who failed to respond, clinically or bacteriologically, after a minimum of 8 weeks to the treatment regimen recommended by the World Health Organization and 2) 38 patients consecutively enrolled over a 4-week period from whom sputum was taken before the start of treatment . Mycobacterium tuberculosis was isolated from all 66 sputum specimens . In the first group, 25 strains (98%) were multidrug resistant (to rifampicin and isoniazid) . Such resistance occurred in all new cases and 14 (82%) of the 17 failure or relapse cases . In the second group, 9 strains (24%) were multidrug resistant and only 12 (32%) were fully susceptible . This resistance was found in 3 strains (15%) among the 20 new cases and in 6 strains (33%) among the 18 cases of treatment failure or relapse . These findings suggest that prisoners may have an important future role in the transmission of tuberculosis, especially multidrug resistant forms, in the former Soviet Union .

Int J Oncol, 1998 May, 12(5), 1143 - 9
Multidrug resistance modulation in rhabdomyosarcoma and neuroblastoma cell lines; Cowie FJ et al.; Four rhabdomyosarcoma and three neuroblastoma cell lines were characterised for the presence of P-glycoprotein and MDR-1 expression using immunohistochemistry, northern analysis, RT-PCR and in situ mRNA hybridisation . None of the rhabdomyosarcoma lines were unequivocally positive in contrast to all three neuroblastoma lines . Chemosensitivity to cytotoxic agents was determined using the MTT assay and chemosensitisation by cyclosporin and verapamil was evaluated . In a single rhabdomyosarcoma line (HX 170) there was sensitisation to etoposide using verapamil but not to other drugs or using cyclosporin A . In contrast, in all three neuroblastoma lines both cyclosporin and verapamil sensitised to vincristine and doxorubicin . No evidence of sensitisation to etoposide was apparent . The sensitisation was most marked for vincristine, using either modulator and therefore the influence of modulator scheduling was evaluated with this drug in the neuroblastoma line SK N BE . Prolonged pre-exposure to modulator did not appear necessary and maximum sensitisation was apparent where either cyclosporin or verapamil was added 1-3 h prior to and post vincristine . Continuity of exposure was important and even a break of 30 min appeared to reduce sensitisation . These data confirm the potential for chemosensitisation in MDR-1 positive neuroblastoma cell lines and provide some basis for rational schedule design in clinical practice . Because of the probability that vincristine resistance is predominantly related to MDR-1 and less multifactorial than for other drugs such as doxorubicin or etoposide, this agent should be considered for inclusion in any clinical evaluation of MDR reversal strategies.

Int J Oncol, 1998 May, 12(5), 1137 - 42
P-glycoprotein-mediated multidrug resistance is modulated by pretreatment with chemosensitizers in HCT-8 carcinoma cells in vitro; Haberl I et al.; The effects of pretreatment with the multidrug resistance (MDR) modulators verapamil (VPM), tamoxifen (TMX), cyclosporin A (CsA), and SDZ PSC833 (PSC) on drug sensitivity of the P-glycoprotein (Pgp) expressing human ileocecal carcinoma cell line HCT-8 is described . Following pretreatment of 2, 16 and 48 h with the individual modulators, rhodamine 123 efflux (RHO), transepithelial vinblastine transport (VIN) across treated HCT-8 monolayers, and chemosensitivity to doxorubicin (DOX) were determined and compared to Pgp protein expression and phosphorylation . After 2 h, VPM, TMX, CsA and PSC inhibited RHO efflux and VIN transport and increased the chemosensitivity of HCT-8 to DOX significantly . Prolonged exposure failed to further increase inhibition of Pgp-mediated transport, but in contrast maximized phosphorylation of Pgp (16 h) and Pgp protein expression (48 h), respectively.

Leuk Lymphoma, 1998 Feb, 28(5-6), 469 - 82
Prognostic value of P-glycoprotein and leukocyte differentiation antigens in chronic myeloid leukemia; Stavrovskaya A et al.; P-glycoprotein (Pgp) mediated multidrug resistance is often the cause of therapy failure in some tumors . Pgp expression was shown to have prognostic value in several hematological malignancies, especially in acute myeloblastic leukemia (AML) and acute lymphoblastic leukemia (ALL) . In chronic myeloid leukemia (CML) Pgp is expressed by peripheral blood (PB) cells more often in the terminal disease stages (20-50% of patients have Pgp+ phenotype) . Sequential studies show that Pgp+ cells often disappear from the PB during the course of therapy . Nevertheless Pgp expression has some prognostic value in blast crisis (BC) predicting shorter BC, while CD13 has the same predictive value in BC . 10% of patients formed a distinct group with large numbers of Pgp+CD34+ blasts in the PB and also had shorter BC . Cases with inactive Pgp were found in chronic and accelerated phases of CML but not in BC.

Int J Tuberc Lung Dis, 1998 May, 2(5), 390 - 6
Use of DNA fingerprinting for primary surveillance of nosocomial tuberculosis in a large urban hospital: detection of outbreaks in homeless people and migrant workers; Lemaitre N et al.; SETTING: A large urban teaching hospital in the southeast of Paris . OBJECTIVE: Primary surveillance of nosocomial transmission of tuberculosis (TB) by systematic restriction fragment length polymorphism analysis (RFLP) of isolates (n = 161) recovered from smear-positive pulmonary TB patients identified from 1 March 1993 to 28 February 1994, and from all TB patients (with any form of tuberculous infection) identified from 1 March 1994 to 30 April 1995 . RESULTS: Systematic RFLP analysis revealed 12 clusters of patients (n = 40) infected by strains of Mycobacterium tuberculosis showing matching RFLP patterns . None of the isolates were multidrug-resistant . Compared with non-clustered patients, clustered patients were more likely to be homeless (55% vs 19%, P < or = 0.001), or Africans living in hostels for migrant workers (20% vs 6%, P = 0.01), and had fewer previous admissions to hospital (12% vs 28%, P = 0.05) . Further epidemiological investigations showed that the clustered TB cases actually resulted not from nosocomial transmission, but from transmission in the community, very likely in homeless shelters and hostels for migrant workers . CONCLUSION: No nosocomial transmission of TB was identified among the patients included during the study period . Systematic RFLP analysis using hospital-based sampling can detect the spread of TB in specific environments in the community where transmission is occurring.

Oncol Res, 1998, 10(1), 29 - 41
Characterization of 2-chloro-N10-substituted phenoxazines for reversing multidrug resistance in cancer cells; Thimmaiah KN et al.; Twenty-one 2-chloro-N10-substituted phenoxazines have been synthesized and characterized as potential modulators of multidrug resistance (MDR) . Many of the compounds, at a concentration of 100 microM, enhanced accumulation of vinblastine (VLB) in drug-resistant KB8-5 cells to a greater extent than the same concentration of verapamil (VRP) . However, the effects on VLB accumulation were specific, because these derivatives had little activity in the parental drug-sensitive line KB3-1 . The compounds slowed the efflux of VLB from KB8-5 cells, suggesting that the chlorophenoxazines, like VRP, can inhibit P-glycoprotein (P-gp)-mediated efflux of VLB from this cell line . Two of the chlorophenoxazine derivatives, and also VRP, were able to stimulate the vanadate-sensitive ATPase activity attributable to P-gp in membranes isolated from MDR1 baculovirus-infected Sf9 cells . This result suggests that these modulators exert their effect by directly interacting with P-gp . Apart from the parent unsubstituted molecule, 2-chlorophenoxazine, there was a good correlation between log10P and the ability of the compounds to enhance VLB accumulation in KB8-5 . This suggests that lipophilicity of a modulator is important, but is not the sole determinant of potency . Within this series of compounds, the optimal structural features for MDR modulation include a hydrophobic phenoxazine ring with a -Cl atom in the C-2 position and a tertiary amine group four carbons from the tricyclic ring . Many of the agents at the IC10 concentration completely reversed the 37-fold VLB resistance in KB8-5 cells . The most active agents in KB8-5 were able to partially reverse VLB resistance in an MDR colon carcinoma cell line GC3/c1 and completely reversed the 86-fold VLB resistance in the MDR1-overexpressing breast carcinoma cell line BC19/3 . These same agents could only partially sensitize BC19/3 cells to taxol and doxorubicin, suggesting that the chlorophenoxazine derivatives show some specificity for modulating VLB resistance.

Orv Hetil, 1998 May 10, 139(19), 1139 - 45
{Tuberculosis at the threshold of the 21st century}; Hutas I; The author summarizes the most important results of the present history of tuberculosis obtained both in Hungary and abroad . He deals with the state of the art of the tuberculosis epidemics all over the world and in Hungary, presenting also the potential genetic risks . He also discusses the risk groups with special regard to the extremely large regional differences, searching for the underlying causes, as well as to that of the relationships between the epidemic and the social factors . He reviews the up-to-date methods of the diagnostics of Mycobacterium tuberculosis . He evaluates the significance of mass screening in the early diagnosis of tuberculosis and lung cancer in Hungary . He gives an overview of the current strategy of treatment, of the principals of directly observed therapy short course (DOTS) and the international results obtained . In the Hungarian surveillance programme, these principles are adapted under domestic conditions . Based on the data from Hungary and abroad, he presents the risk of development of multidrug-resistant strains and the mechanism of their development, presuming that genetic methods will also play a role in the future management of tuberculosis.

Nucleic Acids Res, 1998 Jun 15, 26(12), 3001 - 5
Binding of the modified daunorubicin WP401 adjacent to a T-G base pair induces the reverse Watson-Crick conformation: crystal structures of the WP401-TGGCCG and WP401-CGG{br5C}CG complexes; Dutta R et al.; 2'-Bromo-4'-epi-daunorubicin (alpha-manno configuration, denoted WP401) is a new anthracycline drug that exhibits promising activity toward multidrug-resistant cancer cells . We carried out X-ray diffraction analyses of the complexes formed in the presence of formaldehyde between WP401 and two DNA hexamers, TGGCCG and CGG{br5C}CG . The two complexes crystallized in different crystal lattices with respective crystal data of space group P4322, a = b = 37.20 A, c = 70.53 A and space group P43212, a = b = 37.23 A, c = 61 . 96 A . These new crystal forms are different from the P41212 form of other daunorubicin/doxorubicin complexes studied previously . The refined crystal structures at approximately 2.0 A resolution revealed that the entire 2:1 drug-DNA complex is in the asymmetrical unit . Two WP401 drug molecules bind to the duplex, with the aglycones intercalated between the CpG or TpG steps and their modified daunosamines in the minor groove . As observed earlier, in the presence of formaldehyde, WP401 more readily forms a covalent adduct with (C/T)GG*:CCG than with (C/T)GC:G*CG (G* is the crosslink site), the opposite of what is seen for daunorubicin and doxorubicin . Surprisingly, the two T-G mismatched base pairs in the WP401-TGGCCG complex adopt the reverse Watson-Crick conformation, instead of the wobble conformation . The unusual T-G reverse Watson-Crick conformation may be required in order to maintain favorable stacking interactions between the base pair and the aglycone of WP401 . Our results show that chemical modifications like bromo or iodo substitution on anthracycline drugs have significant effects on their DNA binding properties.

Int J Cancer, 1998 May 29, 76(5), 729 - 37
Drug binding to P-glycoprotein is inhibited in normal tissues following SDZ-PSC 833 treatment; Jette L et al.; Rats were treated with daily injections of SDZ-PSC 833 (PSC) to study the interaction of this potent modulator of multidrug resistance (MDR) with P-glycoprotein (P-gp) expressed in normal tissues . After 2 days of treatment, the level of P-gp expression, detected by Western blot analysis, was not modified in renal brush border membranes (BBMs) and brain capillaries . However, the amount of P-gp detected with the photoaffinity probe {125I}-arylazidoprazosin (IAAP) was decreased in both tissues, suggesting that the drug binding properties of P-gp were altered by PSC treatment . This effect was further characterized by treating rats with PSC for 10 days . Following these treatments, the amount of immunodetected P-gp was increased in renal BBMs and brain capillaries . However, no increase in P-gp expression was observed in photolabeling experiments, suggesting that induced P-gp was not functional . In vitro experiments performed with renal BBMs showed that the inhibition of P-gp photolabeling by cyclosporin A (CsA), verapamil and vinblastine could be reversed by performing washing steps to remove these drugs before incubating the samples with IAAP . However, the inhibition mediated by PSC was less reversible since photolabeling of P-gp remained inhibited following the washing steps . Pre-incubation of intact CHRC5 cells with PSC, CsA and verapamil also inhibited P-gp photolabeling and increased rhodamine 123 accumulation . For PSC pre-treated samples, these effects were not completely reversed following washing, but were abolished for CsA and Ver pre-treated samples . Our results suggest that PSC could block P-gp function by a different mechanism from that of CsA and verapamil, involving modification of the drug binding sites.

Int J Cancer, 1998 May 29, 76(5), 702 - 8
Induction of broad drug resistance in small cell lung cancer cells and its reversal by paclitaxel; Su GM et al.; The H82 "variant" and the H69 "classic" small cell lung cancer (SCLC) cell lines were treated with low levels of epirubicin (69 and 14 nM) which caused little cell death but produced the H82/E8 and H69/E8 extended-multidrug resistant sublines . Both were resistant to drugs associated with multidrug resistance (MDR), and to chlorambucil (9.5- and 5.6-fold, respectively) and cisplatin (2.3- and 8.5-fold, respectively) . There was increased expression of the multidrug resistance-associated protein (MRP1) in the H82/E8 subline while P-glycoprotein expression was not detected in any cells or sublines . Treatment of the H82 cells for 1 hr with 69 nM epirubicin increased MRP1-mRNA expression within 4 hr and this was associated with an increase in the resistance to epirubicin, chlorambucil, cisplatin and paclitaxel . Further, a 1 hr treatment with non-cytotoxic doses of chlorambucil (2.5 microM), cisplatin (1.3 microM) or paclitaxel (13 nM), drugs not normally associated with MRP1-mediated MDR, also increased MRP1-mRNA expression in the H82 cells with paclitaxel causing the highest increase (4.5-fold) . For chlorambucil treatment, this increased MRPI-mRNA expression was accompanied by increased drug resistance while paclitaxel treatment had no effect on drug resistance in the H82 cells . For the drug resistant H82/E8 subline, these drug treatments had no effect on the MRP1-mRNA expression and little effect on increasing the subline drug resistance . However, pretreatment with paclitaxel sensitised the H82/E8 subline to chlorambucil and cisplatin returning the subline to the sensitivity of the H82 cell line . We conclude that treatment with low levels of MDR and non-MDR drugs can induce extended-multidrug resistance in SCLC cells, a process that probably involves the co-ordinate upregulation of MRP1 and other resistance mechanisms . The results also suggest paclitaxel may have a role as a response modifier in the treatment of refractory SCLC.

Free Radic Biol Med, 1998 Apr, 24(6), 913 - 23
Antiproliferative effect of the piperidine nitroxide TEMPOL on neoplastic and nonneoplastic mammalian cell lines; Gariboldi MB et al.; The stable nitroxide 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) is widely used as a probe in biophysical studies and as an antioxidant in several experimental models . The potential cytotoxic effects of TEMPOL were tested on a panel of human and rodent cell lines, and the nitroxide proved to be significantly more effective in inhibiting the growth of neoplastic than nonneoplastic cell lines after a 96-h exposure . More detailed studies on MCF-7/WT cells indicate that at least 24 h are necessary for TEMPOL to induce irreversible cell damage, which seems to be related to the reactivity of the nitroxyl group . This observation, together with the antagonistic effect of N-acetylcysteine, suggests an involvement of free radical-mediated processes . Cell cycle studies indicate a biphasic effect of TEMPOL, with a short-term accumulation of the cells in the G1 phase and a later increase in G2/M phase; the pattern of DNA fragmentation observed in TEMPOL-treated cells points to an apoptotic mode of cell death . In conclusion, our data suggest that, while the possible cytotoxic effects of TEMPOL should not be overlooked when using this compound as a biophysical probe or antioxidant, these same properties could be exploited as a novel approach to cancer chemotherapy, especially in tumor cells exhibiting unfavorable characteristics, such as a multidrug-resistant phenotype or loss of hormone receptors.

Clin Cancer Res, 1998 May, 4(5), 1305 - 14
Bryostatin 1 down-regulates mdr1 and potentiates vincristine cytotoxicity in diffuse large cell lymphoma xenografts; Al-Katib AM et al.; The down-regulation of multidrug resistance (mdr1) gene expression as detected by competitive reverse transcription-PCR and the antitumor activity of bryostatin 1 (Bryo1) are investigated in a newly established cell line from a patient with relapsed diffuse large cell lymphoma (DLCL) . The cell line (WSU-DLCL2) grows in liquid culture and forms s.c . tumors in mice with severe combined immune deficiency . WSU-DLCL2 is a mature B-cell line (IgG lambda) that is negative for EBV nuclear antigen, expresses the multidrug resistance phenotype, and has t(14;18)(q32;q21) plus other chromosomal aberrations . Exposure of the WSU-DLCL2 cells to Bryo1 in culture reversed the multidrug resistance phenotype within 24 h . A functional assay revealed a 4-fold increase in {3H}vincristine accumulation in Bryo1-treated cells compared with control . Vincristine (VCR), doxorubicin, Bryo1, and 1-beta-D-arabinofuranosylcytosine showed no clinically significant activity when given alone to WSU-DLCL2-bearing severe combined immune deficiency mice . However, when given 24 h before each cytotoxic agent, Bryo1 substantially increased the antitumor activity of VCR but not 1-beta-D-arabinofuranosylcytosine . There was a statistically significant (P < 0.001) decrease in the expression of P-glycoprotein in WSU-DLCL2 tumors taken from Bryo1-treated animals compared with untreated controls . In vivo, a competitive reverse transcription-PCR assay revealed decreased mdr1 RNA expression 24 h after Bryo1 treatment . These findings taken together indicate that Bryo1-induced down-regulation of mdr1 might be one mechanism by which Bryo1 potentiates VCR activity . The sequential use of both agents resulted in clinically significant antitumor activity in this lymphoma model.

Receptors Channels, 1997, 5(3-4), 175 - 83
Drug accumulation in the presence of the multidrug resistance pump: dissociation between verapamil accumulation and the action of P-glycoprotein; Ayesh S et al.; We studied the interaction between the multidrug transporter, P-glycoprotein, and two compounds that interact with it: vinblastine, a classical substrate of the pump, and verapamil, a classical reverser . Steady-state levels of accumulation of these two drugs were determined in a multidrug resistant P388 leukemia cell line, P388/ADR . The time course of accumulation of these drugs, and the effect of energy starvation and the presence of chloroquine on the level of their steady-state accumulation were quite disparate . Vinblastine inhibited the accumulation of verapamil whereas it enhanced the accumulation of daunomycin, another classic substrate of P-glycoprotein . Verapamil did not compete with the intracellular binding sites of vinblastine . In all these aspects, vinblastine behaved as a typical substrate of P-glycoprotein but verapamil did not . Our data suggest that verapamil is a reverser of P-glycoprotein but that its intracellular accumulation is not affected by this membrane-bound transporter.

Neoplasma, 1997, 44(6), 366 - 9
Human multidrug-resistant (MRP,p190) myeloid leukemia HL-60/ADR cells in vitro: resistance to the mevalonate pathway inhibitor lovastatin; Hunakova L et al.; Mevalonate pathway inhibitor lovastatin inhibited proliferation of human multidrug-resistant promyelocytic leukemia HL-60/ADR cells in vitro, with MRP-gene coded p190 mediated drug resistance, to a markedly lesser extent than that of the parental drug sensitive HL-60 cells and also that of the other human multidrug resistant (MDR-1, P-glycoprotein) myeloid leukemia cell line HL-60/VCR . The sensitivity of the examined human leukemia cell lines to the cytostatic activity of lovastatin correlated approximately with the potential of lovastatin to induce the characteristic cell cycle alteration (i.e . the accumulation of lovastatin-treated cells in the G0/G1 phase of the cell cycle) . The P-glycoprotein positive HL-60/VCR cells and the parental drug sensitive HL-60 cells were more sensitive to this cell cycle alteration than the HL-60/ADR multidrug resistant leukemia cells with MRP drug resistance . Lovastatin (72 hours, 20 micromol) induced apoptosis and cell necrosis in HL-60 cells, apoptosis but not cell necrosis in HL-60/VCR cells and neither apoptosis nor necrosis in HL-60/ADR cells.

Blood, 1998 Jun 1, 91(11), 4106 - 17
Rhodamine-123 staining in hematopoietic stem cells of young mice indicates mitochondrial activation rather than dye efflux; Kim M et al.; Low-intensity fluorescence of rhodamine-123 (Rh-123) discriminates a quiescent hematopoietic stem cell (HSC) population in mouse bone marrow, which provides stable, long-term hematopoiesis after transplantation . Rh-123 labels mitochondria with increasing intensity proportional to cellular activation, however the intensity of staining also correlates with the multidrug resistance (MDR) phenotype, as Rh-123 is a substrate for P-glycoprotein (P-gp) . To address the mechanisms of long-term repopulating HSC discrimination by Rh-123, mouse bone marrow stem and progenitor cells were isolated based on surface antigen expression and subsequently separated into subsets using various fluorescent probes sensitive to mitochondrial characteristics and/or MDR function . We determined the cell cycle status of the separated populations and tested for HSC function using transplantation assays . Based on blocking studies using MDR modulators, we observed little efflux of Rh-123 from HSC obtained from young (3- to 4-week-old) mice, but significant efflux from HSC derived from older animals . A fluorescent MDR substrate (Bodipy-verapamil, BodVer) and Rh-123 both segregated quiescent cells into a dim-staining population, however Rh-123-based separations resulted in better enrichment of HSC function . Similar experiments using two other fluorescent probes with specificity for either mitochondrial mass or membrane potential indicated that mitochondrial activation is more important than either mitochondrial mass or MDR function in defining HSC in young mice . This conclusion was supported by morphologic studies of cell subsets separated by Rh-123 staining.

Eur J Nucl Med, 1998 Apr, 25(4), 401 - 9
Application of SPET using technetium-99m sestamibi in brain tumours and comparison with expression of the MDR-1 gene: is it possible to predict the response to chemotherapy in patients with gliomas by means of 99mTc-sestamibi SPET?
Yokogami K, Kawano H, Moriyama T, Uehara H, Sameshima T, Oku T, Goya T, Wakisaka S, Nagamachi S, Jinnouchi S, Tamura S.
Technetium-99m sestamibi (MIBI) is thought to be passively taken up by metabolically active tumour cells and effluxed from them by P-glycoprotein (Pgp) . This 170-kDa membrane-bound protein, encoded by the MDR-1 gene, acts as an energy-dependent efflux pump for several antineoplastic agents, resulting in multidrug resistance . For this reason, it is of interest whether the tumour's response to chemotherapy can be predicted by MIBI single-photon emission tomography (SPET) . In this study, MIBI SPET was compared with thallium-201 (Tl) SPET using magnetic resonance imaging as a guide in 16 patients with untreated brain tumours {ten glioblastomas (GBs), two anaplastic astrocytomas (AAs), two low-grade gliomas (LGASs) and two metastatic brain tumours) and in four patients who had received treatment for with brain tumours (two GBs, two AAs) . In addition, we investigated the expression of the MDR-1 gene and its product Pgp in the same patients, and compared the results with MIBI SPET findings . MIBI, as well as Tl, was highly accumulated and retained in the enhanced region of malignant gliomas . In addition, MIBI SPET yielded sharp and well-contrasted images, and the margin of the tumour was more clearly defined than with Tl SPET due to a good signal-to-noise ratio . Follow-up MIBI SPET in patients who had received therapy showed marked uptake in a patient with malignant transformation, who deteriorated clinically . Patients with no uptake on MIBI SPET showed no sign of recurrence . Semiquantitative analysis of untreated patients showed a relationship between the early uptake index (UI, ratio of average count/pixel in the lesion to that in the contralateral area on early images) and the degree of malignancy (early UI = 1.08+/-0.06 in LGASs, 4.10+/-0.84 in AAs, 5.71+/-3.47 in GBs, and 7.52+/-1.52 in metastatic brain tumours) . The retention index (RI, ratio of delayed to early UI) of MIBI was significantly lower than that of Tl in metastatic brain tumours (P<0.05), but not in malignant gliomas . Histological and biological investigation of gliomas showed that the MDR-1 gene and its product Pgp were expressed only in normal endothelial cells and not in tumour cells or proliferating endothelial cells; Pgp tended to decrease as the degree of malignancy rose . Hence, the presence of Pgp and the grade of malignancy were inversely related in gliomas . By contrast, immunohistochemical study showed strong accumulation of Pgp in metastatic brain tumour cells . These histopathological findings and MIBI SPET findings are compatible with experimental data; MIBI was washed out by Pgp . The main cause of chemoresistance is probably not an increasing drug efflux by Pgp in gliomas . Thus, MIBI SPET is useful for detecting the active lesions, but may not be useful for predicting the response to chemotherapy in gliomas.

J Biol Chem, 1998 Apr 24, 273(17), 10733 - 40
Multidrug resistance protein . Identification of regions required for active transport of leukotriene C4; Gao M et al.; Multidrug resistance protein (MRP) is a broad specificity, primary active transporter of organic anion conjugates that confers a multidrug resistance phenotype when transfected into drug-sensitive cells . The protein was the first example of a subgroup of the ATP-binding cassette superfamily whose members have three membrane-spanning domains (MSDs) and two nucleotide binding domains . The role(s) of the third MSD of MRP and its related transporters is not known . To begin to address this question, we examined the ability of various MRP fragments, expressed individually and in combination, to transport the MRP substrate, leukotriene C4 (LTC4) . We found that elimination of the entire NH2-terminal MSD or just the first putative transmembrane helix, or substitution of the MSD with the comparable region of the functionally and structurally related transporter, the canalicular multispecific organic anion transporter (cMOAT/MRP2), had little effect on protein accumulation in the membrane . However, all three modifications decreased LTC4 transport activity by at least 90% . Transport activity could be reconstituted by co-expression of the NH2-terminal MSD with a fragment corresponding to the remainder of the MRP molecule, but this required both the region encoding the transmembrane helices of the NH2-terminal MSD and the cytoplasmic region linking it to the next MSD . In contrast, a major part of the cytoplasmic region linking the NH2-proximal nucleotide binding domain of the protein to the COOH-proximal MSD was not required for active transport of LTC4.

Ann Oncol, 1998 Jan, 9(1), 85 - 93
No evidence of significant activity of the multidrug resistance gene product in primary human breast cancer; Hegewisch-Becker S et al.; BACKGROUND: The discovery of the multidrug resistance (MDR1) gene product P-glycoprotein (P-gp) has been widely seen as an important milestone in our understanding of the mechanisms underlying the clinical phenomenon of the emergence of resistant cells . MDR1 expression has been shown for numerous solid tumors and for virtually all hematologic malignancies . Nevertheless, results regarding MDR1/P-gp expression in human breast cancer have been controversial and the results of clinical trials on modulation of P-gp activity have not been encouraging . PATIENTS AND METHODS: MDR1/P-gp expression and the function of the P-gp pump were investigated in 61 tumor samples from patients with primary breast cancers by multiparameter analysis using MDR1-RT-PCR, immunohistochemistry with two MAbs (UIC2 and MRK16) and the rhodamine 123 (Rh123) efflux assay . The cellular composition of the tumor cell suspension was analyzed by using specific MAbs against the P-gp expressing lymphocyte subsets CD4, CD8 and CD56, as well as against the HER-2/neu gene product, which was used to identify breast carcinoma cells . RESULTS: UIC2 and MRK16 revealed a staining positivity in 72% and 75% of samples, respectively . A positive MDR1-RT-PCR signal was detected in 62% of the samples . Nevertheless, no correlation between immunohistochemistry and RT-PCR could be established . Furthermore, there was no correlation between HER-2/neu expression and MDR1-RT-PCR or P-gp immunohistochemical assays . A contamination by CD8+ and CD4+ lymphocytes was established in 100% and 84% of tumor cell suspensions, respectively . As assessed by the Rh123 efflux assay CD8+ and the CD4+ lymphocytes exhibited marked P-glycoprotein activity, whereas such activity was not detectable in a single instance for the breast carcinoma cells . In MDR1-RT-PCR positive samples, contamination by CD8 lymphocytes averaged 4.3%, while the contamination of CDS cells in the MDR1 mRNA-negative samples was only 2.4% (P = 0.007) . This signal vanished after elimination of the lymphocyte subpopulations by T-cell rosetting . CONCLUSIONS: In primary breast cancer detection of MDR1 gene expression by means of RT-PCR or immunohistochemical assays is not indicative for the MDR phenotype, since there is no evidence of significant activity of the P-gp pump.

Am J Respir Crit Care Med, 1998 May, 157(5 Pt 1), 1609 - 15
Preliminary results of collapse therapy with plombage for pulmonary disease caused by multidrug-resistant mycobacteria; Jouveshomme S et al.; Seven patients underwent collapse therapy with polystyrene sphere plombage for pulmonary disease caused by multidrug-resistant mycobacteria . Four patients were infected with multidrug-resistant strains of Mycobacterium tuberculosis, two with Mycobacterium xenopi, one with Mycobacterium avium . All patients were heavily pretreated before surgery, had extensive, bilateral cavitary disease and were considered unsuitable for resection because of extensive disease or functional respiratory impairment . Six patients had active disease at time of surgery . Collapse therapy with insertion of six to 18 spheres resulted in long-standing bacteriological conversion in six patients . Collapse therapy was unilateral in six and bilateral in one . No immediate postoperative complication or death was observed . Hospital stay was short (mean 12 d) . Collapse therapy is a conservative alternative therapy in patients with pulmonary disease caused by multidrug-resistant mycobacteria at high risk of treatment failure considered unsuitable for pulmonary resection.

Genes Dev, 1998 May 15, 12(10), 1453 - 63
Regulation of the fission yeast transcription factor Pap1 by oxidative stress: requirement for the nuclear export factor Crm1 (Exportin) and the stress-activated MAP kinase Sty1/Spc1; Toone WM et al.; The fission yeast Sty1 stress-activated MAP kinase is crucial for the cellular response to a variety of stress conditions . Accordingly, sty1- cells are defective in their response to nutrient limitation, lose viability in stationary phase, and are hypersensitive to osmotic stress, oxidative stress, and UV treatment . Some of these phenotypes are caused by Sty1-dependent regulation of the Atf1 transcription factor, which controls both meiosis-specific and osmotic stress-responsive genes . However, in this report we demonstrate that the cellular response to oxidative stress and to treatment with a variety of cytotoxic agents is the result of Sty1 regulation of the Pap1 transcription factor, a bZip protein with structural and DNA binding similarities to the mammalian c-Jun protein . We show that both Sty1 and Pap1 are required for the expression of a number of genes involved in the oxidative stress response and for the expression of two genes, hba2+/bfr1+ and pmd1+, which encode energy-dependent transport proteins involved in multidrug resistance . Furthermore, we demonstrate that Pap1 is regulated by stress-dependent changes in subcellular localization . On imposition of oxidative stress, the Pap1 protein relocalizes from the cytoplasm to the nucleus in a process that is dependent on the Sty1 kinase . This relocalization is the result of regulated protein export, rather than import, and involves the Crm1 (exportin) nuclear export factor and the dcd1+/pim1+ gene that encodes an Ran nucleotide exchange factor.

J Pharm Pharmacol, 1998 Mar, 50(3), 343 - 9
Effect of the number of samples on Bayesian and non-linear least-squares individualization: a study of cyclosporin treatment of haematological patients with multidrug resistance; Wu G et al.; We have studied whether the prediction of drug concentrations improves as the number of samples used for individualization is increased, and whether the Bayesian method of individualization is superior to the non-linear least-squares method . Data were obtained from ten adult haematological patients with multidrug resistance who were treated with cyclosporin . The predictions of blood-cyclosporin concentrations were made using the Abbott PKS program . The number of samples used for individualization was increased from 1 to 30 for the Bayesian method and from 4 to 30 for the non-linear least-squares method . Linear regression, percentage prediction error, and absolute and relative predictive performance were used to evaluate the predictions . The results show that the Bayesian method affords greater precision than the non-linear least-squares method, but that the non-linear least-squares method is more accurate and results in less bias . Whereas for linear regression predictions improve as the number of samples is increased, other evaluations show improvement in the range from 5 to 11 samples; linear regression, percentage prediction errors and prediction bias support the opinion that the Bayesian method progressively becomes the non-linear least-squares method as the number of samples used for individualization is increased, but the accuracy and precision of prediction do not support this opinion . The study supports the statement that Bayes' law requires parameters from an infinite population, otherwise the advantage of the Bayesian method might be marginal.

Am J Trop Med Hyg, 1998 May, 58(5), 630 - 7
Characterization of Plasmodium falciparum isolated from the Amazon region of Brazil: evidence for quinine resistance; Zalis MG et al.; The prevalence and severity of drug-resistant malaria is emerging rapidly in the Amazon basin of Brazil . In support of clinical trials using the new antimalarial drug combination of atovaquone and proguanil, we performed in vitro drug sensitivities, molecular characterization of parasite populations using the circumsporozoite protein, merozoite surface antigen-1 (MSA-1), and MSA-2 markers, and an analysis of the Plasmodium falciparum multidrug resistance (pfmdr1) gene sequence and copy number in 26 isolates of P . falciparum obtained in a gold-mining endemic area in Peixoto de Azevedo, Mato Grosso State . All 26 isolates were found to be resistant to chloroquine (50% inhibitory concentration {IC50} = 100-620 nM) and sensitive to mefloquine (IC50 < 23 nM) and halofantrine (IC50 < 6 nM) . The isolates also show reduced susceptibility to quinine (IC50 = 48-280 nM) . Sequence analysis of the pfmdr1 gene revealed Asn, Phe, Cys, Asp, and Tyr in positions 86, 184, 1034, 1042, and 1246, respectively . These point mutations were similar to that previously described in other Brazilian isolates . Southern blot analysis revealed no amplification of the pfmdr1 gene . These results suggest that three different mechanisms for drug resistance exist for chloroquine, mefloquine, and quinine.

J Med Invest, 1998 Feb, 44(3-4), 185 - 91
The role of cyclosporin A on antibody-dependent monocyte-mediated cytotoxicity against human multidrug-resistant cancer cells; Yano S et al.; A P-glycoprotein (P-gp) inhibitor, cyclosporin A (CsA) was found to enhance the susceptibility of multidrug resistant (MDR) cancer cells to anti-P-gp antibody-dependent cellular cytolysis (ADCC) by monocytes, but the exact mechanism is unknown . In this study, we examined whether CsA enhanced the susceptibility of MDR cells through its inhibitory effect of P-gp function by using anti-ganglioside GM2 (GM2) monoclonal antibody (Ab), KM966, instead of anti-P-gp Ab, MRK16 . Monocyte-ADCC induced by both KM966 and MRK16 against P-gp positive human MDR ovarian cancer cells was significantly augmented by addition of CsA . KM966, but not MRK16, induced monocyte-ADCC against P-gp negative human ovarian cancer cells and CsA enhanced this ADCC activity, indicating that suppressive effect of P-gp function by CsA was not essential to the enhancement of ADCC . Moreover, pretreatment of tumor cells with CsA augmented their susceptibility to monocyte-ADCC irrespective of P-gp expression . Interestingly, KM966 or MRK16 induced monocyte-ADCC against various human lung cancer cells expressing either GM2 or P-gp, but CsA did not affect these ADCC . These findings suggest that CsA may enhance the susceptibility to the monocyte-ADCC of ovarian cancer cells, but not of lung cancer cells, irrespective of its suppressive effect of P-gp function.

Exp Cell Res, 1998 May 1, 240(2), 312 - 20
An active efflux system for heavy metals in cisplatin-resistant human KB carcinoma cells; Chen ZS et al.; The mechanism for cisplatin resistance in cisplatin-resistant KCP-4 cells was studied . Although multidrug resistance-associated protein (MRP) was not detected in KCP-4 cells, the cells were more resistant to heavy metals than multidrug-resistant C-A120 cells that overexpressed MRP . KCP-4 cells expressed metallothionein, but it was scarcely involved in cisplatin resistance in these cells . KCP-4 cells did not express canalicular multispecific organic anion transporter (cMOAT) . The glutathione (GSH) level was 4.7-fold higher in KCP-4 cells than in KB-3-1 cells . When the GSH level in KCP-4 cells was decreased by treating the cells with buthionine sulfoximine and nitrofurantoin, the accumulation of and sensitivity to cisplatin in the cells were increased . C-A120 cells were only 3.0-fold more resistant to cisplatin than KB-3-1 cells and this resistance was not affected by the increased glutathione level . The accumulation of platinum in C-A120 and KCP-4 cells was 68.5 and 20.4% of that in KB-3-1 cells, respectively, while the intracellular levels of antimony potassium tartrate in C-A120 and KCP-4 cells were 13.2 and 9.9% of that in KB-3-1 cells, respectively . The ATP-dependent efflux of antimony was enhanced in both C-A120 and KCP-4 cells . These results, taken together, suggest an efflux pump for heavy metals different from MRP and cMOAT is involved in cisplatin resistance in KCP-4 cells.

Zhonghua Jie He He Hu Xi Za Zhi, 1996 Dec, 19(6), 338 - 41
{Application of PCR-SSCP technique in detection of rpoB gene mutation in rifampin-resistant Mycobacterium tuberculosis}; He X et al.; OBJECTIVE: To evaluate rpoB gene mutation in rifampin-resistant Mycobacterium tuberculosis and its relationship with rifampin resistance . METHODS: Forty clinical isolates of Mycobacterium tuberculosis were analyzed by PCR-SSCP technique, with H37Rv reference strains as control group . RESULTS: The sensitivity of amplication products of 411bp and 258bp were found to be 5 pg/microliters, 500 organisms per milliliter and 1 pg/microliter, 500 organisms per milliliter respectively . rpoB gene belongs to genus specificity . Characteristics of SSCP graph of 258bp fragment: Ten sensitive strains were the same as H37 Rv . Thirty strains of rifampin-resistant or multidrug resistance, including rifampin, were different from H37Rv except for three strains . Positive rate was 90%, while specificity 100% . CONCLUSIONS: Results showed that PCR-SSCP technique could detect rPOB gene mutation, which might associate with rifampin resistance and be helpful to rapid detection and research of rifampin-resistant Mycobacterium tuberculosis.

Blood, 1998 May 1, 91(9), 3163 - 71
Inhibition of P-glycoprotein and recovery of drug sensitivity of human acute leukemic blast cells by multidrug resistance gene (mdr1) antisense oligonucleotides; Motomura S et al.; To overcome the problem of multidrug resistance, we investigated the effectiveness of phosphrothioate antisense oligonucleotides (MDR1-AS) in suppressing multidrug resistance gene (mdr1) expression in drug-resistant acute myelogenous leukemia (AML) blast cells and the K562 adriamycin-resistant cell line K562/ADM . The percentage of cells with the mdr1 gene product P-glycoprotein (P-gp) was decreased from 100% to 26% by 20 micromol/L MDR1-AS in the K562/ADM cells, and from 48.1% to 10.2% by 2.5 micromol/L MDR1-AS in the AML blast cells . Western blot analysis also showed a decrease in the amount of P-gp in the MDR1-AS-treated K562/ADM cells . This effect was specific to MDR1-AS, and not observed with sense or random control oligonucleotides . The expression of mdr1 mRNA in K562/ADM and AML blast cells treated with MDR1-AS was decreased compared with the random control . Intracellular rhodamine retention and {3H}daunorubicin also increased after antisense treatment . Chemosensitivity to daunorubicin increased in MDR1-AS-treated blast cells up to 5.9-fold in the K562/ADM cells and 3.0- to 6.4-fold in the AML blast cells . The expression of mdr1 mRNA derived from colony cells decreased in the MDR1-AS-treated groups . No inhibitory effect of the oligonucleotides on normal bone marrow progenitors was observed . These findings suggest that MDR1-AS is useful to overcome multidrug resistance in the treatment of leukemia.

Biochim Biophys Acta, 1998 May 8, 1380(3), 329 - 35
Cytotoxicity and DNA binding characteristics of dextran-conjugated doxorubicins; Yang M et al.; The antitumor antibiotic doxorubicin was conjugated with polymeric dextrans of various molecular weights and the cytotoxicity of the conjugates against human carcinoma KB-3-1 cells and its multidrug-resistant subclone KB-V-1 cells was measured by tetrazolium salt MTT assay . The conjugates were much less toxic to the KB-3-1 cells than the free doxorubicin but exhibited similar toxicity to the KB-V-1 cells . The conjugate-DNA interactions were monitored in real-time using an optical biosensor based on evanescent wave detection to obtain the association (ka) and dissociation (kd) rate constants as well as the equilibrium binding constants (KA) of the bindings . Both ka and kd values for the conjugates are more than three magnitudes smaller than those for free doxorubicin, while the KA values of the conjugate-DNA complexes are only about 10 times smaller than that of the free doxorubicin-DNA complex . The results indicate that the cytotoxicity and the DNA-binding kinetics of doxorubicin may be modified with dextran conjugation . The KA values obtained from the biosensor measurements were in close agreement with those determined in solution by fluorescent titration method, verifying the utility of the label-free biosensing measurements as an efficient method for studying ligand-DNA interactions .

Rinsho Byori, 1998 Apr, 46(4), 380 - 90
{Quantitative analysis of multidrug resistance phenotype in hematological malignancies}; Kobayashi H et al.; Cells with multidrug resistance (MDR) phenotype express P-glycoprotein (P-gp) on the cell membrane, which functions as a drug-efflux pump . To quantify the expression of the gene encoding P-gp (multidrug resistance 1; MDR1) and assess P-gp function, we developed competitive reverse transcription-polymerase chain reaction (RT-PCR) assay using heterologous competitor RNA and flow cytometric analysis using rhodamine 123 (Rh123; an artificial substrate for P-gp), respectively . First, we adjusted the assays by analyzing leukemia sublines showing various levels of MDR1 expression . The MDR1 expression in leukemia sublines quantified by competitive RT-PCR assay showed linearity over a wide range, and the results were parallel with those of MDR1 expression measured by Northern blot analysis, the P-gp antigen expression measured by flow cytometric analysis using MRK16, P-gp function analysis by Rh123 dye-efflux assay, and MDR phenotype . Then, we applied these assays to leukemic cells from patients with hematological malignancies . All 69 samples from 64 patients were successfully assayed, and the range of MDR1 expression was from 1.6 to 100 amol/microgram RNA . Since subpopulations of normal lymphocytes show a low degree of P-gp function, strict gating of leukemia cells was mandatory for dye-efflux assay of clinical samples . MDR1 expression in normal lymphocytes was below 8 amol/microgram RNA . By comparison to MDR1 expression quantified by competitive RT-PCR assay with P-gp function assessed by Rh123 dye-efflux assay in gated leukemic cells, more than 8 amol/microgram RNA was regarded as positive MDR1 expression in the leukemic cells themselves . These data suggest that these assays are suitable for evaluating P-gp expression and function in clinical samples when the proper cut-off value is used and may provide insights into the contribution of P-gp to drug resistance in hematological malignancies.

Chin Med J (Engl), 1997 Mar, 110(3), 167 - 72
Establishment of doxorubicin-resistant human bladder cancer cell line (BUI-87/ADMR) and its mechanism of multidrug resistance; Guo H et al.; OBJECTIVE: To establish a doxorubicin-resistant human bladder cancer cell line, BIU-87/ADMR, and to study its biological characteristics and mechanism of drug resistance . METHODS: A human bladder cancer cell line resistant to doxorubicin, BIU-87/ADMR, has been established in vitro by exposing BIU-87 parent cells to progressively increasing concentrations of the drug over a period of 8 months . The cell line has been characterized in terms of growth kinetics, morphology, cross-resistance to other anticancerous agents, pharmacokinetics of daunorubicin and expression of P-glycoprotein (P-gp) which is closely related to the MDR phenotype . RESULTS: The BIU-87/ADMR cell line was 6.3 times more resistant to doxorubicin than the parent BIU-87 . It exhibited cross-resistance to doxorubicin derivatives (epirubicin, daunorubicin), vincristine and etoposide, but not to cisplatin and mitomycin C . Compared to the parent cells, the resistant cells have a slower growth rate and lower confluent density . Unlike the parent BIU-87, about 75% of the BIU-87/ADMR cells showed a positive reaction with monoclonal antibody against P-gp, JSB-1 . Intracellular drug accumulation studies with fluorescence spectrometry indicated that the resistance exhibited by the BIU-87/ ADMR line was mainly caused by an increased active efflux . CONCLUSIONS: The results suggest that MDR is an important phenomenon in bladder cancer and that more than one pathway of MDR may be present in human bladder cancer cell lines . BIU-87/ADMR may be a useful model for the development of new chemotherapeutic strategies in overcoming drug-resistance in the treatment of bladder cancer.

Haematologica, 1998 Apr, 83(4), 290 - 7
P-glycoprotein (PGP), and not lung resistance-related protein (LRP), is a negative prognostic factor in secondary leukemias; Damiani D et al.; BACKGROUND AND OBJECTIVE: In cell lines, there is an ongoing debate about the role of the lung resistance-related protein (LRP) whereas the role played by P-glycoprotein (PGP) in determining a multidrug resistance is well known . The aim of this study was to evaluate the frequency and the role of a PGP and an LRP overexpression in affecting the intracellular daunorubicin accumulation (IDA) and in predicting the therapy outcome on a subset of overt secondary acute non lymphocytic leukemias (ANLL) . An adjunctive point was to evaluate the efficacy of the reversal agent SDZ PSC 833 (PSC) in counteracting impaired IDA . DESIGN AND METHODS: By flow cytometry, PGP and LRP expression and the IDA were evaluated on 54 overt secondary ANLL PGP and LRP overexpressions were respectively defined by an MRK-16 mean fluorescence index (MFI) > or = 6 (PGP+) and by an LRP-56 MFI > or = 5 i.e . by MRK-16 and LRP-56 MFIs higher than the one observed in normal leukocytes . The blasts' IDA was studied after a two-hour incubation in 1000 ng/mL daunorubicin in the presence or in the absence of the MDR reversal agent SDZ PSC 833 (PSC) 1.6 mumol . RESULTS: A PGP overexpression was detected in 40/54 (74%) cases while an LRP overexpression was observed on 33/54 (61%) cases . No differences were found in terms of PGP and LRP expressions between ANLL developing after chemo/radiotherapy (therapy-related ANLL) or evolving from a myelodysplastic syndrome (MDS-related ANLL) . Compared to the PGP-, the PGP+ cases showed a significantly lower mean IDA (DNR NMFI 196 +/- 46 vs . 267 +/- 53, p < 0.001) . The co-incubation of DNR with the PSC significantly increased only the mean IDA of the PGP+ cases, that grew from a DNR NMFI of 196 +/- 46 to a DNR NMFI of 284 +/- 67 (p < 0.0001) . With respect to normal leukocytes, even the PGP- cases had an impaired IDA suggesting that other mechanisms, including an LRP overexpression, could affect the IDA . A strongly negative correlation was observed between PGP overexpression and therapy outcome, in fact, 8/10 (80%) PGP- but only 2/27 (7%) PGP+ patients obtained complete remission (p = 0.0002) . Moreover, 7/33 (21%) cases showing an impaired IDA (NMFI < 280) but 4/4 (100%) with NMFI > 280 had complete remission (p = 0.006) . No correlation was found between therapy response and LRP or CD34 expression . INTERPRETATION AND CONCLUSIONS: This data suggests that an important role in determining therapy outcome is played by PGP in secondary leukemias . Even if the LRP is frequently overexpressed in secondary leukemias and is likely to contribute to the reduction of the intracellular drug accumulation, the role played by LRP in determining the therapy-outcome has still to be cleared.

Zhonghua Nei Ke Za Zhi, 1996 Sep, 35(9), 591 - 4
{Detection of multidrug resistance in patients with leukemia by using flow cytometry and RNA in situ hybridization}; Li S et al.; Multidrug resistance (MDR) in leukemia and the reversal of MDR by cyclosporin A (CsA) in vitro have been studied through intracellular accumulation of daunorubicin (DNR) in leukemic myeloblasts . The study was carried out with real-time flow cytometry and relative expression levels of MDR, which is estimated by RNA in situ hybridization in 26 patients suffering from leukemia . The change of intracellular DNR accumulation in vitro after adding CsA was also analyzed . The results showed that intracellular DNR accumulation in newly diagnosed and treated patients (MDR1 negative) with remission increased significantly than that in refractory and relapsing patients (P < 0.01) . Good reversal effect was obtained in refractory patients and MDR1 positive relapsing patients by adding CsA (P < 0.01) . It is suggested that MDR detection by the two above-mentioned methods could help to project the chemotherapy schedule clinically, CsA had obvious reversal effect on the drug-resistant leukemic cells in vitro.

J Antibiot (Tokyo), 1998 Mar, 51(3), 353 - 8
Aureobasidins as new inhibitors of P-glycoprotein in multidrug resistant tumor cells; Kurome T et al.; Cyclic depsipeptide antibiotic aureobasidin A (AbA) and its analogs were tested for the inhibitory activity of P-glycoprotein in multidrug resistant cancer cells as well as for the antifungal activity . Some analogs with lower antifungal activity than AbA showed higher inhibition of P-glycoproteins indicating difference of the structure-activity relationships between the two activities . Among AbA analogs tested, {D-beta-hydroxy-methylvalyl9}-AbA newly prepared by chemical synthesis, which had much lower antifungal activity than AbA, showed 10-fold higher inhibitory activity of P-glycoprotein than AbA.

Biochem Biophys Res Commun, 1998 Apr 28, 245(3), 918 - 22
Resistance to apoptosis induced by topoisomerase I inhibitors in multidrug-resistant HL60 leukemic cells; Palissot V et al.; The induction of apoptosis by topoisomerase I inhibitors, camptothecin and SN38, was evaluated in drug-sensitive HL60 and multidrug-resistant (MDR) HL60-Vinc leukemic cells . MDR cells displayed a partial resistance to these apoptotic stimuli and this phenomenon was not modulated by verapamil . Basal free calcium concentrations were similar in both cell sublines and were not modified during treatment . Cytoplasmic pH was more acidic in sensitive cells than in MDR cells . Moreover, a significant acidification was obtained during the early stage of apoptosis in sensitive HL60 cells only . Basal Bcl-2 protein expression was found to be greater in MDR than in sensitive cells and was not modulated by apoptosis inducers . This increase of Bcl-2 in MDR cells could be due to the selection process as vincristine enhances Bcl-2 phosphorylation and expression in HL60 sensitive cells . MDR HL60-Vincristine cells therefore display a resistance to apoptosis induced by non-MDR drugs, possibly by Bcl-2 overexpression and inability of these drugs to mediate intracellular pH changes in these drug-resistant cells.

Pathol Res Pract, 1998, 194(3), 149 - 55
Multidrug resistance in glioblastoma . Chemosensitivity testing and immunohistochemical demonstration of P-glycoprotein; Leweke F et al.; Chemosensitivity of previously untreated glioblastomas to mitoxantrone, methotrexate, ACNU and BCNU was tested on cultured tissue . Sixteen of 62 tumors were partially chemosensitive in vitro . The monoclonal antibody C 219 was used to demonstrate the presence of p-glycoprotein in the 16 sensitive and five highly resistant glioblastomas . All 21 tumors identically expressed p-glycoprotein . These results show that untreated glioblastomas primarily express p-glycoprotein even if they are at least partially chemosensitive in vitro . Therefore, immunohistochemical demonstration of p-glycoprotein with the monoclonal antibody C 219 can not provide reliable information on short term resistance of the individual tumors to antineoplastic drugs . P-glycoprotein expression could, however, help to explain the disappointing overall long-term efficacy of chemotherapy by showing the existence of cell populations with early drug resistance in these tumors.

J Clin Oncol, 1998 May, 16(5), 1677 - 83
Tumor clearance of technetium 99m-sestamibi as a predictor of response to neoadjuvant chemotherapy for locally advanced breast cancer; Ciarmiello A et al.; PURPOSE: Since we have previously shown that the efflux rate of technetium 99m (99mTc) sestamibi, a transport substrate of P-glycoprotein (Pgp), is directly correlated with Pgp levels in untreated breast carcinoma, we tested whether tumor clearance of 9mTc-sestamibi may be predictive of therapeutic response to neoadjuvant chemotherapy in patients with locally advanced breast cancer . PATIENTS AND METHODS: Thirty-nine patients with stage III disease, median tumor diameter 5.8 cm (range, 3 to 10) were enrolled onto this prospective clinical trial and underwent 99mTc-sestamibi scan before neoadjuvant chemotherapy . Patients were injected intravenously (i.v.) with 740 MBq of 99mTc-sestamibi; a 15-minute dynamic study was performed, and static planar images were obtained at 0.5, 1, 2, and 4 hours . The time to half clearance of 99mTc-sestamibi was calculated in each patient from decay corrected time-activity curves using a monoexponential fitting . Patients were treated with epirubicin 150 mg/m2 i.v . every 2 weeks for three courses and then underwent surgery within 3 weeks from the completion of chemotherapy . Residual tumor was assessed by pathologic examination of mastectomy specimens . RESULTS: Seventeen of 39 patients showed a rapid tumor clearance of 9mTc-sestamibi (time to half clearance {t1/2} < or = 204 minutes) and 15 of these 17 (88%) showed a highly cellular macroscopic residual tumor at histology that indicated lack of tumor response to neoadjuvant chemotherapy . In contrast, only eight of 22 (36%) with prolonged retention of 99mTc-sestamibi (t1/2 > 204 minutes) showed residual macroscopic tumor at histology (Fisher's exact test, P < .01) . CONCLUSION: A rapid tumor clearance of 99mTc-sestamibi may predict lack of tumor response to neoadjuvant chemotherapy with drugs affected by the multidrug-resistant phenotype in patients with locally advanced breast carcinoma.

Toxicology, 1998 Feb 20, 126(1), 23 - 31
Decreased hepatobiliary secretion of inorganic mercury, its deposition and toxicity in the Eisai hyperbilirubinemic rat with no hepatic canalicular organic anion transporter; Sugawara N et al.; Eisai hyperbilirubinemic (EHB) rats, a new mutant strain inbred from Sprague-Dawley (SD) rats, show no inherent expression of the canalicular multidrug resistance protein (cMrp) and lack canalicular multispecific organic anion transporter (cMOAT) activity . A sample of 203Hg (40 microCi with 40 microg Hg/kg) was injected intravenously (i.v.) into four male SD and EHB rats . Biliary excretion of reduced-glutathione (GSH) was 426 and 2 microg/bile for 15 min in the SD and EHB rats, respectively . Biliary excretion of 203Hg for 45 min in EHB rats significantly decreased to 1/4 of that of the SD rats . However, there was no difference in the hepatic uptake of 203Hg between the two strains . Other rats were injected subcutaneously (s.c.) with HgCl2 solution (at 0.2 and 1.6 mg/kg) containing 203Hg . Some 4 days after the injection of 0.2 mg/kg, about 3 and 13% of the total dose was found in the liver in SD and EHB rats, respectively . The hepatic supernatant Hg was recovered mainly in the void volume of a Sephadex column . Some 2 days after the injection of 1.6 mg/kg, these values were 3 and 23% in SD and EHB rats, respectively . The increased retention stimulated hepatic metallothionein (MT) induction and increased the proportion of Hg in the MT region on the Sephadex column . On the other hand, biliary excretion of 203Hg for 15 min in EHB rats was about 1/6-1/4 of that in SD rats . With the injection of 1.6 mg/kg, hepatic and renal functions worsened in EHB rats . In particular, severe necrosis was found in the renal tubules . Our results suggest that biliary secretion of inorganic Hg may be partly regulated by the ATP-dependent transport system, the glutathione S-conjugate export pump (GS-X pump) composed of Mrp and MOAT . Significantly decreased excretion stimulates hepatic retention of inorganic Hg . However, the hepatic lesions are less predictive . The MT induction may reduce the toxicity of metal to the liver cells.

Haematologica, 1998 Jan, 83(1), 27 - 33
Short course infusional idarubicin plus intermittent cytarabine and etoposide for refractory hematologic malignancies: clinical and preliminary pharmacological results; Bassan R et al.; BACKGROUND AND OBJECTIVE: Idarubicin (IDA) is relatively immune to the multidrug resistance P-gp mechanism that is frequently expressed in recurrent and refractory hematologic malignancies . Owing to rapid metabolism in vivo, a continuous infusion (CI) of IDA might prolong exposure time to the parent drug rather than its more P-gp susceptible alcohol metabolite . For this reason we developed a brief retreatment schedule incorporating CI IDA in order to obtain clinical as well as preliminary pharmacological data in patients with refractory leukemias and lymphomas . DESIGN AND METHODS: Eligible patients had either advanced-stage acute myeloid or lymphoid leukemias (AML, ALL) or high-grade non-Hodgkin's lymphomas (NHL) which failed curative-intent frontline or salvage regimens in use at our institution during the study period (July-October 1992) . CI IDA 5 mg/m2/d was employed together with intermittent (every 8 hours) intermediate-dose cytarabine (500 mg/m2) and etoposide (200 mg/m2); all drugs were given for 2-4 days . A preliminary pharmacokinetic evaluation of CI IDA was carried out in three patients, including a comparison with bolus delivery in one . The in vitro effects of CI-type vs bolus-type IDA delivery in terms of intracellular IDA accumulation and related pro-apoptotic activity were assessed in P-gp- and P-gp+ human leukemic CEM cells by means of cytofluorimetry (IDA fluorescence intensity = FI, annexin V expression), with and without the addition of P-gp inhibitor cyclosporin A (CsA) . RESULTS: Complete (2) or partial (4) responses were achieved in a total of 12 patients (17% and 33%, respectively), despite prior treatments with anthracyclines (100% of cases) and cytarabine-etoposide (33% of cases) . Hematological toxicity caused the duration of treatment to be reduced from 4 days to 2 days after the first 4 patients . The procedural death rate was 42% (5/12), which was probably related in part to the sum of adverse prognostic characteristics: median patient age 55 years, two-thirds of cases having previously failed second/third-line regimens . The pharmacokinetic study showed an increased plasma AUC value with CI IDA in one patient (2.9-fold increase vs bolus delivery) due to the prolonged presence of low IDA plasma levels (10-20 ng/mL vs 50 ng/mL), as seen in two other cases as well . On the other hand, the in vitro study did not prove to be in favor of CI IDA because the FI threshold (> 1500 units) associated with increased apoptosis of P-gp+ cells (> 10%) was achieved only with bolus-type IDA exposure (50 ng/mL for 30') plus CsA . INTERPRETATION AND CONCLUSIONS: This short regimen demonstrated activity against end-stage leukemias and lymphomas and might prove to be more effective and less toxic in younger patients and in those with less advanced disease . In view of the results from plasma pharmacokinetics and in vitro intracellular IDA accumulation and apoptosis assays in lymphoblastic CEM cells, CI IDA 5 mg/m2/day may not represent a better therapeutic option than a rapid bolus injection, particularly in P-gp+ neoplasms . If obtaining an adequate intracellular drug concentration is the primary treatment goal, a higher CI IDA dosage, the addition of a P-gp down-regulator such as CsA and others, and in vivo study focusing on tumor samples from patients could all be helpful.

Haematologica, 1998 Jan, 83(1), 13 - 20
Intracellular expression of P-170 glycoprotein in peripheral blood mononuclear cell subsets from healthy donors and HIV-infected patients; Malorni W et al.; BACKGROUND AND OBJECTIVE: P-glycoprotein (P-gp) is a transmembrane efflux pump that actively extrude a variety of unrelated drugs from cancer cells, leading to the so-called multidrug resistance (MDR) phenomenon . However, P-gp has also been found in normal bone marrow and peripheral blood mononuclear cells (PBMC) . Recently, the presence of P-glycoprotein in PBMC from human immunodeficiency virus (HIV)-infected patients has also been investigated and a phenotype-associated P-gp expression has been detected . DESIGN AND METHODS: A total of thirty-eight HIV-1 positive patients with a mean age of 34 years (range, 24-41 years) were studied after an informed consent . Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on a Ficoll/Hypaque and P-glycoprotein expression was investigated on lymphocyte population by single and double-color immunofluorescence techniques . We investigated: i) both surface and intracellular expression of the P-gp molecule in different PBMC subsets, ii) P-gp expression modifications occurring during HIV infection, and iii) the effect of HIV-gp120 on the expression of P-gp by T lymphocyte subsets from healthy donors . RESULTS: Our experimental findings indicate that: a) P-gp glycoprotein can be detected on an intracellular level in different PBMC subpopulations (mainly CD8+ T lymphocytes, CD16+ NK cells and CD14+ monocytes); b) this intracellular expression is decreased in specific PBMC subsets (i.e . T-CD8+ and NK-CD16+) from HIV-infected patients and c) a rearrangement was obtained when CD4+ and CD8+ lymphocytes from healthy donors were exposed in vitro to the HIV-binding glycoprotein gp120 . INTERPRETATION AND CONCLUSIONS: Our results indicate that P-gp glycoprotein can also be expressed intracellularly and can be rearranged in PBMC subsets from HIV-infected patients.

Biochemistry, 1998 Apr 7, 37(14), 5010 - 9
Human P-glycoprotein exhibits reduced affinity for substrates during a catalytic transition state; Ramachandra M et al.; Human P-glycoprotein (Pgp), a plasma membrane protein that confers multidrug resistance, functions as an ATP-dependent drug efflux pump . Pgp contains two ATP binding/utilization sites and exhibits ATPase activity that is stimulated in the presence of substrates and modulating agents . The mechanism of coupling of ATP hydrolysis to drug transport is not known . To understand the role of ATP hydrolysis in drug binding, it is necessary to develop methods for purifying and reconstituting Pgp that retains properties including stimulation of ATPase activity by known substrates to an extent similar to that in the native membrane . In this study, (His)6-tagged Pgp was expressed in Trichoplusia ni (High Five) cells using the recombinant baculovirus system and purified by metal affinity chromatography . Upon reconstitution into phospholipid vesicles, purified Pgp exhibited specific binding to analogues of substrates and ATP in affinity labeling experiments and displayed a high level of drug-stimulated ATPase activity (specific activity ranging from 4.5 to 6.5 micromol min-1 mg-1) . The ATPase activity was inhibited by ADP in a competitive manner, and by vanadate and N-ethylmaleimide at low concentrations . Vanadate which is known to inhibit ATPase activity by trapping MgADP at the catalytic site inhibited photoaffinity labeling of Pgp with substrate analogues, {125I}iodoarylazidoprazosin and {3H}azidopine, only under ATP hydrolysis conditions . Because vanadate-trapped Pgp is known to resemble the ADP and phosphate-bound catalytic transition state, our findings indicate that ATP hydrolysis results in a conformation with reduced affinity for substrates . A catalytic transition conformation with reduced affinity would essentially result in substrate dissociation and supports a model for drug transport in which an ATP hydrolysis-induced conformational change leads to drug release toward the extracellular medium.

J Biol Chem, 1998 Apr 10, 273(15), 8971 - 4
Vaults are up-regulated in multidrug-resistant cancer cell lines; Kickhoefer VA et al.; Vaults are 13-MDa ribonucleoprotein particles composed largely of a 104-kDa protein, termed major vault protein or MVP, and a small vault RNA, vRNA . While MVP levels have been found to increase up to 15-fold in non-P-glycoprotein multidrug-resistant cell lines, the levels of vault particles have not been investigated . As both the function of vault particles and the mechanism of drug resistance in non-P-glycoprotein cells are unknown, we decided to determine whether vault synthesis was coupled to MDR . By cloning the human gene for vRNA and careful quantitation of the MVP and vRNA levels in MDR cells, we find that vRNA is in considerable excess to MVP . Sedimentation measurements of vault particles in multidrug resistance cells have indeed revealed up to a 15-fold increase in vault synthesis, coupled with a comparable shift of associated vRNA, demonstrating that vault formation is limited by expression of MVP or the minor vault proteins . The observation that vault synthesis is linked directly to multidrug resistance supports a direct role for vaults in drug resistance.

Biochem Biophys Res Commun, 1998 Apr 7, 245(1), 85 - 9
Induction of multidrug resistance gene expression in rat liver cells in response to acute treatment by the DNA-damaging agent methyl methanesulfonate; Fardel O et al.; Expression of multidrug resistance (mdr) genes encoding the P-glycoprotein (P-gp) drug efflux pump was analysed in cultured rat liver epithelial cells acutely treated by the DNA-damaging agent methyl methanesulfonate (MMS) . Exposure to this alkylating agent used at 30 microg/ml for 12 or 24 h was shown to enhance mdr mRNA levels in rat liver cells without alteration of cell viability . Induction of mdr transcripts occurred through increased expression of the mdr1b gene as indicated by reverse transcriptase-polymerase chain reaction analysis using rat mdr gene-specific primers and was not associated with up-regulation of cytochrome P-450 1A1, thereby suggesting that this detoxifying enzyme and P-gp were not coordinately regulated by MMS . In addition, the DNA-damaging agent was found to enhance in a dose-dependent manner cellular efflux of the P-gp substrate rhodamine 123, which was inhibited by the P-gp inhibitor verapamil, thus providing evidence that exposure to MMS led to increased P-gp-related drug transport in rat liver cells . The up-regulation of functional P-gp expression occurring in MMS-treated liver cells may be interpreted as a part of the cellular response to DNA damage .

Drug Metab Dispos, 1998 Apr, 26(4), 360 - 6
Overlapping substrate specificities of cytochrome P450 3A and P-glycoprotein for a novel cysteine protease inhibitor; Zhang Y et al.; K02 (morpholine-urea-Phe-Hphe-vinylsulfone), a newly developed peptidomimetic, acts as a potent cysteine protease inhibitor, especially of cathepsins B and L (which are associated with cancer progression) and cruzain (a cysteine protease of Trypanosoma cruzi, which is responsible for Chagas' disease) . Here we investigated features of the disposition of K02 using in vitro systems, characterizing the interaction of the drug with human cytochrome P450 (CYP) 3A and P-glycoprotein (P-gp), a mediator of multidrug resistance (MDR) to cancer chemotherapy and a countertransporter in the intestine that limits oral drug bioavailability . P-gp functions as an ATP-dependent drug efflux pump to reduce intracellular cytotoxic concentrations . An HPLC assay was developed to analyze K02 and its metabolites formed in human liver microsomes . Three major primary metabolites were determined by LC/MS/MS to be hydroxylated products of the parent compound . A rabbit anti-CYP3A polyclonal antibody (200 microl antibody/mg microsomal protein) produced 75-94% inhibition of the formation of these three hydroxylated metabolites . Ketoconazole (5 microM), a selective CYP3A inhibitor, produced up to 75% inhibition, whereas other CYP-specific inhibitors, i.e . quinidine (CYP2D6), 7,8-benzoflavone (CYP1A2), and sulfaphenazole (CYP2C9), showed no significant effects . An identical metabolite formation profile for K02 was observed with cDNA-expressed human CYP3A4 (Gentest) . These data demonstrate that K02 is a substrate for CYP3A . Formation of 1'-hydroxymidazolam, the primary human midazolam metabolite, was markedly inhibited by K02 via competitive processes, which suggests the potential for drug-drug interactions of K02 with other CYP3A substrates . K02 significantly inhibited the photoaffinity labeling of P-gp with azidopine and LU-49888, a photoaffinity analogue of verapamil . Transport studies with {14C}K02, using MDR1-transfected Madin-Darby canine kidney cell monolayers in the Transwell system, demonstrated that the basolateral-to-apical flux of K02 across MDR1-transfected Madin-Darby canine kidney cells was markedly greater than the apical-to-basolateral flux (ratio of 63 with 10 microM {14C}K02) . This suggests that K02 is also a P-gp substrate . These studies are important for formulating strategies to increase the absorption and/or decrease the elimination of K02 and to optimize its delivery to malignant cells and parasite-infected host cells.

Drug Metab Dispos, 1998 Apr, 26(4), 313 - 7
Metabolism of beta-arteether to dihydroqinghaosu by human liver microsomes and recombinant cytochrome P450; Grace JM et al.; beta-Arteether (AE) is an endoperoxide sesquiterpene lactone derivative currently being developed for the treatment of severe, complicated malaria caused by multidrug-resistant Plasmodium falciparum . Studies were undertaken to determine which form(s) of human cytochrome P-450 catalyze the conversion of beta-arteether to its deethylated metabolite, dihydroqinghaosu (DQHS), itself a potent antimalarial compound . In human liver microsomes, AE was metabolized to DQHS with a Km of 53.7 +/- 29.5 microM and a Vmax of 1.64 +/- 1 . 78 nmol DQHS/min/mg protein . AE biotransformation to DQHS was inhibited by ketoconazole and troleandomycin . Ketoconazole was a competitive inhibitor, with an apparent Ki of 0.33 +/- 0.11 microM . Because AE is being developed for patients who fail primary treatment, it is possible that AE may be involved in life-threatening drug-drug interactions, such as the associated cardiotoxicity of mefloquine and quinidine . Coincubation of AE with other antimalarials showed mefloquine and quinidine to be competitive inhibitors with a mean Ki of 41 and 111 microM, respectively . Metabolism of AE using human recombinant P450s provided evidence that cytochrome P450s 2B6, 3A4, and 3A5 were the primary isozymes responsible for its deethylation . CYP3A4 metabolized AE to dihydroqinghaosu at a rate approximately 10 times that of CYP2B6 and approximately 4.5-fold greater than that of CYP3A5 . These results demonstrate that CYP3A4 is the primary isozyme involved in the metabolism of AE to its active metabolite, DQHS, with secondary contributions by CYP2B6 and -3A5.

Biochem J, 1998 Apr 1, 331 ( Pt 1), 99 - 103
ATP-dependent transport of unconjugated bilirubin by rat liver canalicular plasma membrane vesicles; Pascolo L et al.; The transport of highly purified 3H-labelled unconjugated bilirubin (UCB) was investigated in rat liver plasma membrane vesicles enriched in the canalicular domain and found to be stimulated (more than 5-fold) by the addition of ATP . Other nucleotides, such as AMP, ADP, GTP and a non-hydrolysable ATP analogue (adenosine 5'-{alpha, beta-methylene} triphosphate), did not stimulate {3H}UCB transport, indicating that ATP hydrolysis was necessary for the stimulatory effect . {3H}UCB uptake occurred into an osmotically sensitive space . At an unbound bilirubin concentration ({Bf}) below saturation of the aqueous phase (no more than 70 nM UCB), the ATP-dependent transport followed saturation kinetics with respect to {Bf}, with a Km of 26+/-8 nM and a Vmax of 117+/-11 pmol per 15 s per mg of protein . Unlabelled UCB inhibited the uptake of {3H}UCB, indicating that UCB was the transported species . Inhibitors of ATPase activity such as vanadate or diethyl pyrocarbonate decreased the ATP effect (59+/-11% and 100% respectively) . Daunomycin, a known substrate for multidrug resistance protein-1, and taurocholate did not inhibit the ATP-dependent {3H}UCB transport, suggesting that neither mdr-1 nor the canalicular bile acid transporter is involved in the canalicular transport of UCB . {3H}UCB uptake (both with and without ATP) in canalicular vesicles obtained from TR- rats was comparable to that in vesicles obtained from Wistar rats, indicating that the canalicular multispecific organic anion transporter, cMOAT, does not account for UCB transport . These results indicate that UCB is transported across the canalicular membrane of the liver cell by an ATP-dependent mechanism involving an as yet unidentified transporter.

J Pharmacol Exp Ther, 1998 May, 285(2), 438 - 43
Effect of the Mdr1a P-glycoprotein gene disruption on the tissue distribution of SDZ PSC 833, a multidrug resistance-reversing agent, in mice; Desrayaud S et al.; The involvement of mdr1a P-glycoprotein (P-gP) on the tissue distribution of the multidrug resistance-reversing agent SDZ PSC 833 was assessed by use of mdr1a (-/-) mice . The mdr1a (-/-) and wild-type mdr1a (+/+) mice received a 4-h constantrate i.v . infusion (2 micrograms/min) of {14C}SDZ PSC 833 . Mice were sacrificed at 0, 0.5, 1, 2 and 4 h during infusion and at 0.5, 1, 3, 8 and 24 h after stopping the infusion . Blood and tissues were analyzed on total (14C) and parental SDZ PSC 833 concentrations . Mdr1a (-/-) mice exhibited increased SDZ PSC 833 accumulation in cerebrum, cerebellum and somewhat in testes and small intestine compared with the wild-type mice . The difference between mdr1a (-/-) and (+/+) brain (cerebrum and cerebellum) penetration depended on SDZ PSC 833 blood concentrations, because this cyclosporin analog apparently governs its own brain penetration by inhibiting the P-glycoprotein pump in mdr1a (+/+) mice . Thus the mdr1a (-/-)/(+/+) ratio of brain concentrations tended to decrease and increase at high and low blood concentrations, respectively . These findings clearly demonstrate the interaction of SDZ PSC 833 with the P-glycoprotein present at the blood-brain barrier . The SDZ PSC 833 distribution in other mdr1a P-glycoprotein-expressed tissues, as well as its metabolism and elimination, was not affected by the mdr1a gene disruption . This suggests that factors other than mdr1a P-gP are involved in the disposition of this multidrug resistance-reversing agent.

Med Clin (Barc), 1998 Jan 24, 110(2), 51 - 5
{Incidence and current clinical spectrum of tuberculosis in a metropolitan area in the south of Spain}; Garcia Ordonez MA et al.; BACKGROUND: To study the incidence and clinical spectrum of tuberculosis in the metropolitan area of Malaga (Spain) . METHODS: Prospective study which includes all patients who had a diagnosis of tuberculosis within the referral area of "Carlos Haya" Malaga Regional Hospital from March 1, 1993 to February 28, 1994 . RESULTS: During the study period, there were 138 cases of tuberculosis, with an incidence of 43.7 cases/10(5) inhabitants . Ninety one cases (66%) were male, and the mean age (SD) was 33.2 (18.3), with 88% being less than 55 years old . Thirty six patients (26.1%) were HIV-infected . Extrapulmonary tuberculosis made up 27.5% of the cases, and was more frequent in HIV-infected patients (p < 0.01; odds ratio: 2.9; 95% CI: 1.2-7.1) . The mean (SD) time to diagnosis was 54.3 (76) days . The diagnosis was microbiologically confirmed in 106 cases (76.8%), histologically in 14 cases (10.1%), and the remaining 18 cases (13.1%) were clinically diagnosed . The global rate of resistance was 10.8% . The rate of primary resistance was 4.6%, and the rate of multidrug-resistant tuberculosis was 3.1% . Eighty nine patients (77.4%) were cured, six patients (5.2%) stopped the treatment, 3 (2.6%) had relapses and 1 (0.9%) was considered a therapeutic failure; 16.7% of patients were lost for follow-up . Sixteen patients died and in nine of them (6.5%) the death was attributed to tuberculosis . CONCLUSIONS: The incidence of tuberculosis in Malaga urban area is high . It mainly affects young males of unfavored socio-economic classes . HIV-infected patients account for a high percentage of the cases . The high number of productive cases and the long time to diagnosis evidence the shortcomings of our sanitary system . These facts, together with the high rate of non-compliance, of treatment may explain the seriousness of the current situation in our country.

Clin Cancer Res, 1998 Feb, 4(2), 389 - 98
Levels of multidrug resistance (MDR1) P-glycoprotein expression by human breast cancer correlate with in vitro resistance to taxol and doxorubicin; Mechetner E et al.; To determine whether multidrug resistance (MDR1) P-glycoprotein (Pgp) expression correlated with clinical MDR1-related drug resistance, we established a protocol for quantitative measurement of Pgp expression and in vitro drug resistance in doxorubicin resistant MCF7 breast cancer cell lines and 359 freshly resected specimens of breast carcinoma . Pgp expression was detected with 4E3, UIC2, and JSB-1 monoclonal antibodies using flow cytometry and immunohistochemistry (IHC) . Pgp function was determined using PSC833 in a drug resistance-reversal assay and with a three-dimensional agarose-based extreme drug resistance assay . MCF7 calibrator cell lines expressed Pgp, which was functional and in proportion to the degree of drug resistance . Flow cytometry, UIC2 shift assays, IHC scores, and determination of absorbance products by image analysis were all highly correlated (r > 0.9) . Overall Pgp expression increased from 11% in untreated patients to 30% in patients who had previously received chemotherapy . Compared with Pgp-negative tumors, a significant increase in doxorubicin and Taxol resistance was seen for breast cancers that expressed Pgp, regardless of prior treatment . A strong correlation between the degree of Pgp expression and in vitro resistance to Taxol and doxorubicin (but not to 5-fluorouracil) was found when either IHC scores or image analysis-based methods were used to quantify Pgp expression (n = 185, P < 0.0001) . The degree of Pgp expression strongly correlated with the degree of drug resistance in the clinical specimens studied . These data suggest that (a) Pgp contributes to clinical MDR1-related drug resistance, and (b) both intrinsic and acquired expression of Pgp in breast cancer may contribute in part to therapeutic failure and relapse.

J Clin Microbiol, 1998 May, 36(5), 1422 - 4
Recurrent catheter-related infection caused by a single clone of Mycobacterium chelonae with two colonial morphotypes; Hsueh PR et al.; We describe herein a recurrent catheter-related (Port-A-Cath; Smiths Industries Medical Systems {SIMS} Deltec, Inc., St . Paul, Minn.) infection caused by multidrug-resistant Mycobacterium chelonae with two colonial morphotypes in a 53-year-old woman with gastric adenocarcinoma . Four isolates recovered from this patient within a 3-month period were found to belong to a single clone on the basis of the isolates' identical antibiotypes as determined by the E test and their identical random amplified polymorphic DNA patterns.

J Clin Microbiol, 1998 May, 36(5), 1214 - 9
Use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide for rapid detection of rifampin-resistant Mycobacterium tuberculosis; Mshana RN et al.; We describe a test which uses the ability of viable cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to detect resistance to a bactericidal drug, rifampin, in in vitro-cultured Mycobacterium tuberculosis . The assay shows a linear relationship between the number of viable bacteria and the ability to reduce MTT . Dead mycobacteria were unable to reduce MTT . Rifampin-sensitive M . bovis (BCG) and M . tuberculosis exposed to rifampin showed a rifampin concentration-dependent inhibition of the ability to reduce MTT, while the resistant strains were unaffected . The inhibition of MTT reduction after treatment with rifampin paralleled the reduction in the number of CFU . By using mixing experiments in which the population percentages of rifampin-sensitive and -resistant strains were varied, the assay could detect the presence of rifampin resistance in the mixture when at least 1% of the bacterial population was composed of drug-resistant strains . The assay is cheap, can be visually read, and requires less than 3 days to obtain susceptibility results . The total time required to obtain results, from the time sputum is received in the laboratory, is, in most cases, less than 4 to 5 weeks, which is the time required for primary culture of the bacteria . The MTT assay could, in combination with a test to detect resistance to isoniazid, be a cheap and rapid screening method for multidrug-resistant M . tuberculosis that is affordable even by low-income countries where tuberculosis is a major public health problem.

J Histochem Cytochem, 1998 Apr, 46(4), 513 - 7
Different pattern of MRP localization in ciliated and basal cells from human bronchial epithelium; Brechot JM et al.; The MRP (multidrug resistance-associated protein) transmembrane transporter, which actively transports a wide variety of lipophilic substrates out of cancer cells, has been suggested to play a major role in cell detoxification via efflux of glutathione conjugates . Because bronchial epithelial cells are constantly exposed to environmental pollutants, MRP might be a particularly important defense mechanism against xenobiotics . This study was therefore designed to investigate MRP localization by immunohistochemistry in bronchial epithelial cells collected by scraping from surgical specimens . In parallel, MRP mRNA was detected by reverse transcriptase chain reaction (rt-PCR) in bronchial cell lysates . However, the pattern of protein expression differed markedly according to cell type . In ciliated epithelial cells, immunostaining was restricted to the basolateral surface, without any labeling at the apical surface, which is at variance with the localization of CFTR and MDR1 proteins, other members of the same family of transporters . In basal cells, MRP was present over the entire circumference of the plasma membrane . Basal cells were identified by their morphology and specifically after incubation with an anticytokeratin 17 monoclonal antibody . In conclusion, the different patterns of localization suggest specific roles for MRP in basal and ciliated cells.

Biochem Biophys Res Commun, 1998 Apr 17, 245(2), 325 - 31
Sequence analysis and functional characterization of the 5'-flanking region of the rat multidrug resistance protein 2 (mrp2) gene; Kauffmann HM et al.; Gene expression of the canalicular conjugate transporter mrp2 is inducible by treatment with the DNA-damaging agents 2-acetylaminofluorene (50 and 100 microM), and cisplatin (20 microM) in primary rat hepatocytes as well as in the rat hepatoma cell line H4IIE . Furthermore, phenobarbital (1 and 2 mM) induces mrp2 gene expression, probably explaining the increase in bile-salt-independent bile flow caused by phenobarbital, while the cholestatic drug ethinyl estradiol (10(-6) M) leads to an increase in mrp2 mRNA but decreases Mrp2 protein level probably via a posttranscriptional mechanism . The 5'-flanking region of the rat mrp2 gene was sequenced and cloned into a luciferase reporter vector . Transient transfection assays with reporter vectors containing unidirectionally deleted 5'-flanking regions using H4IIE cells indicate that two different sequences of 17 and 37 bases comprising a Y-Box and a GC-Box are required for mrp2 gene basal expression . Sequences mediating 2-AAF induction are located within a region 250 bases upstream of the translation start site while the inducing effect of phenobarbital seems to be mediated by another domain located further upstream.

Genomics, 1998 Apr 1, 49(1), 14 - 22
Localization of 67 exons on a YAC contig spanning 1.5 Mb around the multidrug resistance gene region of human chromosome 7q21.1; Torigoe K et al.; A contig of 21 nonchimeric yeast artificial chromosomes (YACs) was previously assembled across 1.5 Mb of the multidrug resistance (MDR) gene (PGY1 and PGY3) region of human chromosome 7q21.1 . This region of the human genome has now been subjected to exon amplification to detect the presence of additional genes . Exon trapping was performed directly on the YACs . Sixty-seven gene fragments were isolated and characterized by sequence analysis and comparison with public databases . The localization of these exons in the 1.5-Mb region was determined by hybridization to YAC clones, and they were localized in 11 subregions of YAC contigs . The exon collection includes 21 exons that were identical to known cDNA sequences of PGY1, PGY3, sorcin (SRI), the cDNA similar to the delta subunit of the human amiloride-sensitive Na- channel (SCNED), and 4 cDNAs with unknown function; 43 exons that showed homology/similarity to known cDNA sequences of mouse DMP1, rat COT, mouse and human NADHD, human MDC, 3 cDNAs encoding possible membrane proteins, and 21 other cDNAs; and 3 exons that shared no homology/similarity with any sequence in public databases . The nucleotide sequences of all the PGY1 and PGY3 exons were identical to the corresponding cDNA sequences previously determined, and these exons were localized to the expected positions on the appropriate YAC clones . No other member of the MDR gene family thus appeared to be present in the 1.5-Mb region . The integrated physical and exon maps should prove valuable for both fine mapping and determination of a complete gene map of this segment of the genome.

J Immunol, 1998 Feb 1, 160(3), 1122 - 31
Antagonism of immunostimulatory CpG-oligodeoxynucleotides by quinacrine, chloroquine, and structurally related compounds; Macfarlane DE et al.; Phosphorothioate oligodeoxynucleotides containing CpG (CpG-ODN) activate immune responses . We report that quinacrine, chloroquine, and structurally related compounds completely inhibit the antiapoptotic effect of CpG-ODN on WEHI 231 murine B lymphoma cells and inhibit CpG-ODN-induced secretion of IL-6 by WEHI 231 . They also inhibit IL-6 synthesis and thymidine uptake by human unfractionated PBMC induced by CpG-ODN . The compounds did not inhibit LPS-induced responses . Half-maximal inhibition required 10 nM quinacrine or 100 nM chloroquine . Inhibition was noncompetitive with respect to CpG-ODN . Quinine, quinidine, and primaquine were much less powerful . Quinacrine was effective even when added after the CpG-ODN . Near-toxic concentrations of ammonia plus bafilomycin A1 (used to inhibit vesicular acidification) did not reduce the efficacy of the quinacrine, but the effects of both quinacrine and chloroquine were enhanced by inhibition of the multidrug resistance efflux pump by verapamil . Agents that bind to DNA, including propidium iodide, Hoechst dye 33258, and coralyne chloride did not inhibit CpG-ODN effect, nor did 4-bromophenacyl bromide, an inhibitor of phospholipase A2 . Examination of the structure-activity relationship of seventy 4-aminoquinoline and 9-aminoacridine analogues reveals that increased activity was conferred by bulky hydrophobic substituents on positions 2 and 6 of the quinoline nucleus . No correlation was found between published antimalarial activity and ability to block CpG-ODN-induced effects . These results are discussed in the light of the ability of quinacrine and chloroquine to induce remission of rheumatoid arthritis and lupus erythematosus.

Eur J Pharmacol, 1998 Feb 19, 343(2-3), 313 - 21
The ability of verapamil to restore intracellular accumulation of anthracyclines in multidrug resistant cells depends on the kinetics of their uptake; Mankhetkorn S et al.; The basic distinguishing feature of all cells expressing functional P-glycoprotein-multidrug resistance is a decrease of steady state drug levels as compared to those in drug-sensitive controls . A variety of small molecules, such as verapamil and cyclosporin A, bind to P-glycoprotein and inhibit its ability to pump out antitumor drugs . The kinetics of P-glycoprotein-mediated efflux of various anthracycline derivatives was measured in multidrug-resistant (MDR) K562 cells in the presence of verapamil . Used for the purpose were daunorubicin, idarubicin and 8-S-fluoro-idarubicin which have the same pKa of deprotonation equal to 8.4, but different lipophilicity, 4'-epi-2'-bromo-daunorubicin which has a lipophilicity which is comparable to that of daunorubicin but a pKa equal to 6.3, pirarubicin with pKa equal to 7.7 and lipophilicity different from that of these derivatives were used . Our data show (1) that verapamil is unable to completely block the P-glycoprotein-mediated efflux of anthracyclines and that 10% of its functionality remains even with high verapamil concentrations, (2) that the ability of verapamil to restore intracellular accumulation of anthracyclines in MDR cells depends on the kinetics of their uptake . With fast kinetics uptake, as is the case for idarubicin, 8-S-fluoro-idarubicin, 4'-epi-2'-bromo-daunorubicin and pirarubicin (which have either a low pKa and/or high lipophilicity), verapamil can restore in multidrug resistant cells an intracellular drug level which is comparable to that observed in sensitive cells . This is not possible when the kinetics of uptake is low as is the case for daunorubicin . Cyclosporin A is a more potent modulator and is able to fully restore daunorubicin accumulation in multidrug resistant cells.

Cancer Lett, 1997 Nov 11, 119(2), 177 - 84
Wild-type p53 gene increases MDR1 gene expression but decreases drug resistance in an MDR cell line KBV200; Li ZH et al.; Inactivation of p53 gene and overexpression of MDR1 gene are both associated with drug resistance . Previous studies have suggested that p53 gene can modulate the expression activity of MDR1 gene promoter in a promoter-CAT system . In the present study, wild-type p53 gene cDNA was introduced into a multidrug-resistant cell line, KBv200, in which endogenous p53 gene is aberrant . In wt-p53 transfected cells, the expression of MDRI gene was significantly increased, accumulation of adriamycin (ADM) was decreased, and the sensitivity to vincristine (VCR), ADM and 5-fluorouracil (5-FU) was increased compared with the parent KBv200 cells . After treatment with ADM and VCR, the p53-transfectants were more susceptible to apoptosis . The results suggest that the increase in drug sensitivity of the cells may be, at least in part, due to p53-dependent apoptosis induced by anticancer agents.

Appl Radiat Isot, 1998 Jul, 49(7), 811 - 3
Enzymatic synthesis of {4-methoxy-11C}daunorubicin for functional imaging of P-glycoprotein with PET; Eriks-Fluks E et al.; One of the mechanisms for multidrug resistance (MDR) of tumors is an overexpression of the P-glycoprotein (P-gp) . The cytostatic agent daunorubicin was labeled with carbon-11 to probe P-gp with PET . An enzymatic route for the conversion of carminomycin to {4-methoxy-11C}daunorubicin ({4-methoxy-11C}DNR) was investigated, since attempts failed to prepare daunorubicin chemically using {11C}methyl iodide . In the enzymatic synthesis methylation was accomplished by S-adenosyl-L-{methyl-11C}methionine ({11C}SAM), which was synthesized from L-{methyl-11C}methionine . This methylation is catalyzed by carminomycin-4-O-methyltransferase (CMT) . The overall radiochemical yield of {4-methoxy-11C}DNR is 1% (EOB), with a total synthesis time of 75 min . In conclusion, {4-methoxy-11C}DNR can be successfully prepared from carminomycin and {11C}SAM using enzymes.

Pathol Biol (Paris), 1997 Oct, 45(8), 637 - 42
{Multi-drug resistance and myelodysplastic syndromes: a possible role for remission inducing agents?}; Wattel E et al.; Expression of P-glycoprotein (PGP), the product of the multidrug resistance gene (mdr1), is common in myelodysplastic syndromes (MDSs) and explains in part the low rate of complete remissions (CRs) obtained after aggressive chemotherapy . Reversion of the mdr phenotype to restore chemosensitivity has been the focus of many studies over the last ten years . Two phase III studies evaluated quinine for obtaining reversion of mdr gene expression in MDSs treated by aggressive chemotherapy . Results suggested better response rates and longer survival times in quinine-treated MDR-positive patients . However, the toxicity of quinine warrants further work aimed at developing other mdr phenotype reversion-inducing agents . Some such agents have proved superior over quinine in in vitro studies . Reversion of other mechanisms underlying chemoresistance in MDSs is a promising avenue of research.

Exp Nephrol, 1998 Mar-Apr, 6(2), 89 - 97
P glycoprotein: a new mechanism to control drug-induced nephrotoxicity; del Moral RG et al.; The role of P glycoprotein (P-gp) in kidney is now being explored, and under physiological conditions, this protein is thought to be an excretory pump of cationic xenobiotics and metabolites . Functionally, two different types of P-gp have been described, but only the class I has been related to drug transport, and its overexpression confers the multidrug resistance phenotype in tumoral cells . It has been proposed that P-gp is involved in the energy-dependent transport of substrates through the cell membrane (toxic metabolites, toxins, nutrients, ions, peptides, etc.)--like a 'hydrophobic molecule vacuum cleaner' . Several physiological functions have been attributed to P-gp: defense against xenobiotic aggression and transmembrane transport of prenylcysteine methyl esters, removing these cytotoxic metabolites from cells . A variety of substrates ranging from chemotherapeutics to steroid hormones, antibiotics, and calcium channel blockers can be transported by P-gp, suggesting the possible involvement of this protein in other unknown functions . Results from our group and others have suggested that overexpression of P-gp in renal tubular and mesangial cells prevents pharmacological nephrotoxicity by cyclosporin A (CsA) . On the other hand CsA, a substrate of the pump, could act as a blocker in tubular cells by competitive inhibition . One relevant aspect in kidney is the possible relationship between P-gp and protein kinase C . Several reports suggest that protein kinase C may play a role in inducing the P-gp overexpression in cells under xenobiotic pressure, through activation of the ras oncoprotein family . This could be mediated directly by angiotensin II as a ras activator . This way, the detoxicant function of P-gp against products of the ras catabolism could mediate their accumulation when the 'vacuum cleaner' function is blocked by CsA or tacrolimus, contributing to the initial development of fibroblastic activation that leads to interstitial fibrosis associated with nephrotoxicity by these immunosuppressor drugs . In conclusion, P-gp expression could be an important component of a complex detoxifying system in kidney against xenobiotics or regulating the traffic of endogenous metabolites responsible for the susceptibility of subjects to the development of nephrotoxicity against different drugs.

Br J Cancer, 1998 Apr, 77(7), 1155 - 63
Phase II trial of dexverapamil and epirubicin in patients with non-responsive metastatic breast cancer; Lehnert M et al.; Agents capable of reversing P-glycoprotein-associated multidrug resistance have usually failed to enhance chemotherapy activity in patients with solid tumours . Based on its toxicity profile and experimental potency, dexverapamil, the R-enantiomer of verapamil, is considered to be promising for clinical use as a chemosensitizer . The purpose of this early phase II trial was to evaluate the effects of dexverapamil on epirubicin toxicity, activity and pharmacokinetics in patients with metastatic breast cancer . A two-stage design was applied . Patients first received epirubicin alone at 120 mg m(-2) i.v . over 15 min, repeated every 21 days . Patients with refractory disease continued to receive epirubicin at the same dose and schedule but supplemented with oral dexverapamil 300 mg every 6 h x 13 doses . The Gehan design was applied to the dexverapamil/epirubicin cohort of patients . Thirty-nine patients were entered on study, 25 proceeded to receive epirubicin plus dexverapamil . Dexverapamil did not increase epirubicin toxicity . The dose intensity of epirubicin was similar when used alone or with dexverapamil . In nine intrapatient comparisons, the area under the plasma concentration-time curve (AUC) of epirubicin was significantly reduced by dexverapamil (mean 2968 vs 1901 microg ml{-1} h{-1}, P= 0.02) . The mean trough plasma levels of dexverapamil and its major metabolite nor-dexverapamil were 1.2 and 1.5 microM respectively . The addition of dexverapamil to epirubicin induced partial responses in 4 of 23 patients evaluable for tumour response (17%, CI 5-39%, s.e.P 0.079) . The remissions lasted 3, 8, 11 and 11+ months . These data suggest that the concept of enhancing chemotherapy activity by adding chemosensitizers may function not only in haematological malignancies but also in selected solid tumours . An increase in the AUC and toxicity of cytotoxic agents does not seem to be a prerequisite for chemosensitizers to enhance anti-tumour activity.

Br J Cancer, 1998 Apr, 77(7), 1089 - 96
Expression of gamma-glutamylcysteine synthetase (gamma-GCS) and multidrug resistance-associated protein (MRP), but not human canalicular multispecific organic anion transporter (cMOAT), genes correlates with exposure of human lung cancers to platinum drugs; Oguri T et al.; We examined the steady-state levels of mRNA for gamma-glutamylcysteine synthetase (gamma-GCS), multidrug resistance-associated protein (MRP) and human canalicular multispecific organic anion transporter (cMOAT) in human lung cancer specimens to elucidate their roles in relation to platinum drug resistance in vivo . Seventy-six autopsy samples (38 primary tumours and their corresponding normal lung tissues) obtained from 38 patients were analysed using the quantitative reverse transcription polymerase chain reaction (RT-PCR) method . Both subunits (heavy and light subunits) of gamma-GCS expression levels of normal lung and tumour tissues exposed to platinum drugs during life were significantly higher than those of non-exposed tissues, whereas only the MRP expression levels of tumours were elevated in association with ante-mortem platinum drug exposure . The gamma-GCS and MRP expression levels correlated significantly . The cMOAT expression levels did not correlate with ante-mortem platinum drug exposure . Next, we monitored gamma-GCS heavy subunit expression levels in peripheral mononuclear cells of eight previously untreated lung cancer patients after platinum drug administration, which revealed that these drugs induced gamma-GCS expression in vivo . These results suggest that gamma-GCS expression is induced by platinum drugs in vivo and/or the physiological stress response to xenobiotics.

Anticancer Res, 1998 Jan-Feb, 18(1A), 379 - 84
Tumor cell membrane as a potential target for methyl-beta-cyclodextrin; 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 DOX intracellular accumulation and efflux, in comparison to verapamil in a sensitive parental and multidrug-resistant human cancer cell line (HL-60 S and HL-60 R) . Moreover, cell membrane and nuclear modifications induced by MEBCD were investigated . At concentration of 10 mumol for 10(6) cells, MEBCD combined with doxorubicin (DOX), was able to significantly enhance the intracellular concentration of DOX in HL-60 S and HL-60 R cell lines during the period of exposure . In the resistant subline, MEBCD activity was higher than that of verapamil . Moreover, treatment of cells with MEBCD resulted in a modification in cell membrane integrity and cell morphology, but had no own activity in the distribution of the cells within cell cycle.

Anticancer Res, 1998 Jan-Feb, 18(1A), 129 - 37
A new calyculin derivative from the sponge Theonella swinhoei is a novel and potent inhibitor of tumor cell proliferation; Steube KG et al.; A novel calyculin derivative was isolated from the marine sponge Theonella swinhoei . Using human and animal tumor cell lines and freshly explanted peripheral blood cells, we investigated several biological effects of this natural product (i.e . cell growth, cytotoxicity, induction of differentiation and apoptosis) . The new calyculin exhibited a dose-dependent cytotoxicity against various cell lines from different species and tissues . The ID50 values ranged between 20 and 90 ng/ml . Viability of a multidrug resistant HELA subclone was not affected . Apoptosis of the Hodgkin's lymphoma cell line HDLM-2 induced by antiserum was not prevented by the substance . A reduced drug sensitivity of the monocytic cell line MONOMAC-6 could be observed after induction of differentiation of these cells by phorbol ester and lipopolysaccharide . Even so, non-dividing peripheral blood cells were also resistant to the action of the calyculin derivative, suggesting that the cytotoxin may act preferentially on proliferating cells rather than on quiescent cells . Our data introduce a new calyculin as a marine natural product with interesting features stimulating further studies as a chemotherapeutic or investigational drug.

Mol Biochem Parasitol, 1998 Mar 15, 91(2), 327 - 35
Ivermectin resistance in nematodes may be caused by alteration of P-glycoprotein homolog; Xu M et al.; Resistance to ivermectin and related drugs is an increasing problem for parasite control . The mechanism of ivermectin resistance in nematode parasites is currently unknown . Some P-glycoproteins and multidrug resistance proteins have been found to act as membrane transporters which pump drugs from the cell . A disruption of the mdrla gene, which encodes a P-glycoprotein in mice, results in hypersensitivity to ivermectin . Genes encoding members of the P-glycoprotein family are known to exist in nematodes but the involvement of P-glycoprotein in nematode ivermectin-resistance has not been described . Our data suggest that a P-glycoprotein may play a role in ivermectin resistance in the sheep nematode parasite Haemonchus contortus . A full length P-glycoprotein cDNA from H . contortus has been cloned and sequenced . Analysis of the sequence showed 61-65% homology to other P-glycoprotein/multidrug resistant protein sequences, such as mice, human and Caenorhabditis elegans . Expression of P-glycoprotein mRNA was higher in ivermectin-selected than unselected strains of H . contortus . An alteration in the restriction pattern was also found for the genomic locus of P-glycoprotein derived from ivermectin-selected strains of H . contortus compared with unselected strains . P-glycoprotein gene structure and/or its transcription are altered in ivermectin-selected H . contortus . The multidrug resistance reversing agent, verapamil, increased the efficacy of ivermectin and moxidectin against a moxidectin-selected strain of this nematode in jirds (Meriones unguiculatus) . These data indicate that a P-glycoprotein may be involved in resistance to ivermectin and other macrocyclic lactones in H . contortus.

Pol J Pharmacol, 1997 Nov-Dec, 49(6), 387 - 94
Participation of the multispecific organic anion transporter in hepatobiliary excretion of glutathione S-conjugates, drugs and other xenobiotics; Makowski P et al.; Polarized liver cells, hepatocytes, are involved in carbohydrate, protein and fat metabolism, breakdown of hemoglobin and production of bile . They are also involved in overall detoxification processes in an organism associated with the transport of bile salts, cholesterol, phospholipids, endo- and xenobiotics, end-products of cellular metabolism and ions through the canalicular region of the hepatocyte plasma membrane . Uptake of the above-mentioned compounds into hepatocytes through the basolateral region of plasma membrane is followed by their chemical modification by enzymes of detoxification phase I (e.g . cytochromes P-450) and phase II (e.g . glutathione S-transferases) . Canalicular transport participates in phase III of detoxification, and the molecular machinery involved in this process is localized in the canalicular region of the plasma membrane . Canalicular transport includes the following transport systems: a specific canalicular transporter for bile salts, a multidrug resistance 2 P-glycoprotein (MDR2) participating in the transport of lipids, a multidrug resistance 3 P-glycoprotein (MDR3) responsible for the transport of organic cations and the multispecific organic anion transporter (cMOAT) involved in the transport of non-bile acid organic anions . The cMOAT transport system is discussed in this detailed review.

Somat Cell Mol Genet, 1997 Jul, 23(4), 259 - 74
Maintenance of hypomethylation status and preferential expression of exogenous human MDR1/PGY1 gene in mouse L cells by YAC mediated transfer; Kusaba H et al.; Selection of cells for resistance to vincristine or doxorubicin often induces overexpression of the multidrug resistance (MDR) genes, which encode the cell surface P-glycoproteins, as a result of gene amplification, transcriptional activation, or mRNA stabilization . The LMD1 and LMD4 cell lines were established after the transfer into mouse L cells of two independent yeast artificial chromosome clones containing 300 and 850 kb, respectively, of the human MDR locus . The human MDR1/PGY1 gene, but not the endogenous mouse mdr1a and mdr1b genes, was overexpressed as a result of gene amplification and transcriptional activation in various sublines of LMD1 and LMD4 cells selected for resistance to vincristine . Then we asked why human MDR1/PGY1 gene, but not mouse relevant gene, was expressed . Determination of the methylation status of cytosine residues at Msp I/Hap II cleavage sites (CCGG) in the promoter regions of human MDR1/PGY1 and mouse mdr1a revealed hypomethylation and hypermethylation of the human and mouse genes, respectively in LMD1, LMD4, and their vincristine-resistant derivatives . Various vincristine-resistant sublines were also established after exposure of LMD1 cells for 48 h to 5-aza-2'-deoxycytidine, an inhibitor of DNA methyltransferase . These sublines exhibited overexpression of mouse mdr1a and mdr1b, but not of human MDR1/PGY1, as well as hypomethylation of the mouse mdr1a promoter region . Thus, the selective expression of human or mouse MDR genes in this cell system appears to be related to the methylation status of the respective gene promoter regions.

Biochim Biophys Acta, 1998 Mar 6, 1370(1), 31 - 40
Comparison of the interaction of doxorubicin, daunorubicin, idarubicin and idarubicinol with large unilamellar vesicles . Circular dichroism study; Gallois L et al.; Doxorubicin, daunorubicin and other anthracycline antibiotics constitute one of the most important groups of drugs used today in cancer chemotherapy . The details of the drug interactions with membranes are of particular importance in the understanding of their kinetics of passive diffusion through the membrane which is itself basic in the context of multidrug resistance (MDR) of cancer cells . Anthracyclines are amphiphilic molecules possessing dihydroxyanthraquinone ring system which is neutral under the physiological conditions . Their lipophilicity depends on the substituents . The amino sugar moiety bears the positive electrostatic charge localised at the protonated amino nitrogen . The four anthracyclines used in this study doxorubicin, daunorubicin, idarubicin and idarubicinol (an idarubicin metabolite readily formed inside the cells) have the same amino sugar moiety, daunosamine, with pKa of 8.4 . Thus, all drugs studied will exhibit very similar electrostatic interactions with membranes, while the major differences in overall drug-membrane behaviour will result from their hydrophobic features . Circular dichroism (CD) spectroscopy was used to understand more precisely the conformational aspects of the drug-membrane systems . Large unilamellar vesicles (LUV) consisting of phosphatidylcholine, phosphatidic acid (PA) and cholesterol, were used . The anthracycline-LUV interactions depend on the molar ratio of phospholipids per drug . At low molar ratios drug:PA, these interactions depend also on the anthracycline lipophilicity . Thus, both doxorubicin and daunorubicin bind to membranes as monomers and their CD signal in the visible is positive . However, doxorubicin with its very low lipophilicity binds to the LUV through electrostatic interactions, with the dihydroxyanthraquinone moiety being in the aqueous phase, while daunorubicin, which is more lipophilic is unable to bind only through electrostatic interactions and actually the hydrophobic interactions are the only detected . The highly hydrophobic idarubicin, forms within the bilayer a rather complex entity involving 2-3 molecules of idarubicin associated in the right-handed conformation, one cholesterol molecule and also molecule(s) of phosphatidic acid, as this special oligomeric species is not detected in the absence of negatively-charged phospholipids . Idarubicinol differs from idarubicin with CH(13)-OH instead of C(13)=O and its interactions with LUV are distinctly different . Its CD signal in the visible becomes negative and no self associations of the molecule within the bilayer could be detected . The variation of the sign of the Cotton effect (positive to negative) may derive from the changes in the C(6a)-C(7)-O(7)-C(1') dihedral angle . It is noteworthy that C(13)-OH group, which strongly favours formation of the dimeric species in aqueous solutions when compared to idarubicin prevent association inside the LUV bilayer . At high ratios of phospholipids per drug all of them are embedded within the bilayer as monomer .

Cancer Lett, 1998 Apr 10, 126(1), 89 - 95
Expression of multidrug resistance-associated protein (MRP) in head and neck squamous cell carcinoma; Tsuzuki H et al.; We investigated the expression of multidrug resistance-associated protein (MRP) in 115 cases of head and neck squamous cell carcinoma (H&NSCC) by immunohistochemistry and examined the relationship between MRP expression and clinical factors . Thirty-four (30%) of 115 cases of H&NSCC had expression of MRP . The clinical stage was inversely associated with the expression of MRP (P = 0.0090), but not with age, sex, tumor size, metastasis, recurrence, death from disease or overall survival rate for 5 years . In vitro chemosensitivity to five chemotherapeutic agents (cis-diamminedichloroplatinum, 5-fluorouracil, peplomycin, mitomycin C and Adriamycin) was tested by ATP assay and no correlation between the sensitivity of tumor cells to the cytotoxicity of any drug and MRP expression was found . These results suggest that the resistance to anticancer drugs is not dependent only on the expression of MRP in H&NSCC.

Cancer Lett, 1998 Apr 10, 126(1), 75 - 81
Actin organization associated with the expression of multidrug resistant phenotype in osteosarcoma cells and the effect of actin depolymerization on drug resistance; Takeshita H et al.; We have previously reported that P-glycoprotein (Pgp)-overexpressing multidrug resistant (MDR) osteosarcoma cells were functionally more differentiated than their parent cells . The present study showed that in the parent cells, the actin filaments were sparsely distributed or were diffusely spread throughout the cytoplasm, whereas the MDR osteosarcoma cells exhibited a remarkable increase in well-organized actin stress fibers . Furthermore, dihydrocytochalasin B, a specific inhibitor of actin polymerization, dramatically disrupted this network of stress fibers, increased the intracellular accumulation of doxorubicin (DOX) and modified the resistance against DOX . These results indicate that the organization of actin filaments associated with cellular differentiation may be involved in the expression of Pgp function in the MDR osteosarcoma cells.

Southeast Asian J Trop Med Public Health, 1997 Sep, 28(3), 465 - 71
A comparative study of artesunate and artemether in combination with mefloquine on multidrug resistant falciparum malaria in eastern Thailand; Thimasarn K et al.; Plasmodium falciparum in Thailand is highly resistant to chloroquine, sulfadoxine-pyrimethamine and there is increasing resistance to quinine and mefloquine . The use of qinghaosu derivatives alone or in combination with mefloquine has been shown successfully effective against multidrug resistant P . falciparum in many clinical trials . However their applications with ambulatory treatment should be assessed . 394 uncomplicated falciparum malaria cases studied at Trat and Chanthaburi malaria clinics, eastern Thailand, were allocated at random to receive either one of the seven following regimens: A) artesunate 600 mg over 2 days and mefloquine 1,250 mg in divided doses . B) artemether 640 mg over 2 days and mefloquine 1,250 mg in divided doses . C) artesunate alone 700 mg over 5 days period . D) artemether alone 800 mg over 5 days period . E) quinine plus tetracycline for 7 days . F) mefloquine 1,250 mg in divided doses and G) artesunate 600 mg over 2 days period and mefloquine 750 mg . The follow-up was on Days 1, 2, 7, 14, 21 and 28 . Patients tolerated all regimens very well and there was no serious side effects . The adverse effects did not differ among the seven regimens . The cure rates were 98.7, 97.1, 97.9, 96.7, 92.3, 100 and 95.2%, respectively . There was no significant difference of cure rates among various regimens . A total of 16 P . vivax and 1 P . malariae reinfections were reported among the study groups during the second half of the follow-up period, 14 of which were from the groups administered short action drugs (artesunate, artemether or quinine) . The results suggested that either artesunate 600 mg or artemether 640 mg in combination with mefloquine 1,250 mg over a period of two days should be considered as alternative regimens for treating uncomplicated multi-drug resistant falciparum malaria.

Lipids, 1998 Mar, 33(3), 295 - 301
Cytotoxicity of tocopherols and their quinones in drug-sensitive and multidrug-resistant leukemia cells; Cornwell DG et al.; Cytotoxicities of tocopherols (alpha-T, gamma-T, delta-t), their para (alpha-TQ, gamma-TQ, delta-TQ)- and ortho (Tocored)-quinone oxidation products, the synthetic quinone analog of gamma-TQ containing a methyl group substituted for the phytyl side-chain (TMCQ) and the synthetic quinone analog of Tocored containing a methyl group substituted for the phytyl side-chain (PR) were measured in acute lymphoblastic leukemia cell lines that are drug-sensitive (CEM) and multidrug-resistant (CEM/VLB100) . Among tocopherols, only delta-T exhibited cytotoxicity . Among para quinones, alpha-TQ showed no cytotoxicity, while gamma-TQ and delta-TQ were highly cytotoxic in both CEM and CEM/VLB100 cell lines (LD50 < 10 muM) . delta-TQ and gamma-TQ were more cytotoxic than the widely studied chemotherapeutic agent doxorubicin, which also showed selective cytotoxicity to CEM cells . The orthoquinone Tocored was less cytotoxic than doxorubicin in drug-sensitive cells but more cytotoxic than doxorubicin in multidrug-resistant cells . Cytotoxicity was not a function of the phytyl side-chain since both TMCQ and PR were cytotoxic in leukemia cells . Cytotoxic para and ortho quinones were electrophiles that formed adducts with nucleophilic thiol groups in glutathione and 2-mercaptoethanol . Cytotoxicity was enhanced when the glutathione pool was depleted by preincubation with buthionine-{S,R}-sulfoximine, but cytotoxicity was diminished by the addition of N-acetylcysteine to cultures . alpha-T also diminished the cytotoxicity of para- and orthoquinones . Buthionine-{S,R}-sulfoximine did not block the inhibitory effect of either N-acetylcysteine or alpha-T, showing that these agents did not act solely by maintaining the glutathione pool as an essential antioxidant system . In conclusion, tocopherylquinones represent a new class of alkylating electrophilic quinones that function as highly cytotoxic agents and escape multidrug resistance in acute lymphoblastic leukemia cell lines.

Antimicrob Agents Chemother, 1998 Apr, 42(4), 843 - 8
Cyclosporin analogs inhibit in vitro growth of Cryptosporidium parvum; Perkins ME et al.; Cyclosporine and nonimmunosuppressive cyclosporin (CS) analogs were demonstrated to be potent inhibitors of the growth of the intracellular parasite Cryptosporidium parvum in short-term (48-h) in vitro cultures . Fifty-percent inhibitory concentrations (IC50s) were 0.4 microM for SDZ 033-243, 1.0 microM for SDZ PSC-833, and 1.5 microM for cyclosporine . Two other analogs were less effective than cyclosporine: the IC50 of SDZ 205-549 was 5 microM, and that of SDZ 209-313 was 7 microM . These were much lower than the IC50 of 85 microM of paromomycin, a standard positive control for in vitro drug assays for this parasite . In addition, intracellular growth of excysted sporozoites that had been incubated for 1 h in cyclosporine was significantly reduced, suggesting that the drug can inhibit sporozoite invasion . The cellular activities of the CS analogs used have been characterized for mammalian cells and protozoa . The two analogs that were most active in inhibiting C . parvum, SDZ PSC-833 and SDZ 033-243, bind weakly to cyclophilin, a peptidyl proline isomerase which is the primary target of cyclosporine and CS analogs . However, they are potent modifiers of the activity of the P glycoproteins/ multidrug resistance (MDR) transporters, members of the ATP-binding cassette (ABC) superfamily . Hence, both cyclophilin and some ABC transporters may be targets for this class of drugs, although drugs that preferentially interact with the latter are more potent . Cyclosporine (0.5 microM) had no significant chemosensitizing activity . That is, it did not significantly increase sensitivity to paromomycin, suggesting that an ABC transporter is not critical in the efflux of this drug . Cyclosporine at concentrations up to 50 microM was not toxic to host Caco-2 cells in the CellTiter 96 assay . The results of this study complement those of studies of the inhibitory effect of cyclosporine and CS analogs on other apicomplexan parasites, Plasmodium falciparum, Plasmodium vivax, and Toxoplasma gondii.

Int J Tuberc Lung Dis, 1998 Apr, 2(4), 312 - 6
The bacteriology of pulmonary tuberculosis in a population with high human immunodeficiency virus seroprevalence; Karstaedt AS et al.; SETTING: A public sector urban university hospital in Soweto, South Africa . OBJECTIVE: To describe the utility of sputum smear microscopy and the prevalence of Mycobacterium tuberculosis resistance to antituberculosis drugs according to human immunodeficiency virus (HIV) serostatus in adults . DESIGN: A retrospective descriptive study of consecutive cases using a record review . RESULTS: We studied 412 adults with culture-proven pulmonary tuberculosis, of whom 185 (44.9%) were HIV-seropositive and had a significantly lower sputum smear positivity than HIV seronegatives (68% versus 79%, P < 0.05) . Smear positivity was significantly higher in HIV-infected patients with CD4 counts < or = 50/mm3 compared to those with CD4 counts of 201-300/mm3 (P < 0.05) . In patients with and those without a history of previous treatment for tuberculosis, resistance to one or more antituberculosis drugs was found in 32.2% and 13.6% of cases, respectively, while resistance to both isoniazid and rifampicin (multidrug-resistant tuberculosis {MDR}) was found in 15.3% and 4.5% of patients, respectively . There was no significant difference in resistance between HIV-positive and seronegative patients . CONCLUSION: A strong tuberculosis control programme and good surveillance will be required to prevent the further spread of MDR tuberculosis . Surveys such as these are useful for monitoring control programmes.

Int J Tuberc Lung Dis, 1998 Apr, 2(4), 265 - 71
Fluoroquinolones: a new treatment for tuberculosis?
Gillespie SH, Kennedy N.
The fluoroquinolones have secured an important place in the management of bacterial infection, they are well absorbed orally, are found in respiratory secretions in higher concentrations than in serum and are concentrated inside macrophages . The agents are well tolerated and have an excellent safety record in long-term therapy . No new antituberculosis agents have been developed since the introduction of rifampicin into clinical use, so fluoroquinolones have been investigated for potential efficacy in tuberculosis . In vitro studies have shown that they are active against Mycobacterium tuberculosis at achievable concentrations . Treatment studies in mice have demonstrated efficacy . Few clinical studies have been performed in humans, but ciprofloxacin has demonstrated significant early bactericidal activity . Regimens including a fluoroquinolone have been comparable to other standard regimens, although the outcome in human immunodeficiency virus (HIV) seropositive patients was significantly poorer . There is still insufficient clinical data to use fluoroquinolones in first-line treatment of tuberculosis, but they may find a role in the management of multidrug-resistant infections or in patients with adverse reactions to other agents.

Annu Rev Physiol, 1998, 60, 243 - 66
Organic cation transporters in intestine, kidney, liver, and brain; Koepsell H; This review focuses on sodium-independent transport systems for organic cations in small intestine, liver, kidney, and brain . The roles of P-glycoproteins (MDR) and anion transporters (OATP) in organic cation transport are reported, and two members of the new transporter family OCT are described . The OCT transporters belong to a superfamily that includes multidrug-resistance proteins, facilitative diffusion systems, and proton antiporters . They mediate electrogenic transport of small organic cations with different molecular structures, independently of sodium and proton gradients . The current knowledge of the distribution and functional properties of cloned cation transport systems and of cation transport measured in intact plasma membranes is used to postulate identical or homologous transporters in intestine, liver, kidney, and brain.

Leukemia, 1998 Apr, 12(4), 627 - 32
Use of the comet test in the evaluation of multidrug resistance of human cell lines; Mattii L et al.; The comet test is a reported method for measuring DNA damage in individual mammalian cells . In the present report, the ability of this test to detect multidrug resistance (MDR) was evaluated . For this purpose, two human leukemia, well-characterized parental cell lines, HL60 and CEM, and their derived multidrug-resistant cells, HL60/DNR and CEM/VBL, were cultured with or without different anti-cancer agents . To evaluate the comet test, two DNA-damaging agents were used: daunorubicin (DNR), which is involved in MDR, and ambamustine (AMBA), which is independent from MDR . Moreover, in order to evaluate the specificity of the comet test, the activity of vinblastine (VBL), an MDR-related, DNA-independent anti-cancer drug, was also tested . Finally, the specificity of the comet test in detecting MDR was confirmed by culturing parental or resistant cells with DNR with or without the revertant agent verapamil (VER) . Results confirm that the comet test is able to predict cellular chemoresistance when DNA damaging agents are tested . Finally, experiments on the role of the comet test in evaluating certain aspects of DNA repair are discussed.

Ned Tijdschr Geneeskd, 1998 Jan 24, 142(4), 180 - 4
{Characterization of 100 patients with tuberculosis treated in the Academic Medical Center in Amsterdam}; Juffermans N et al.; OBJECTIVE: To inventory both clinical and demographical data of patients with tuberculosis in the AMC, as well as diagnostic procedures, response to therapy and rate of resistance . DESIGN: Retrospective, descriptive . SETTING: The Academic Medical Center . Amsterdam, the Netherlands . METHOD: The medical records of all patients with a bacteriologically confirmed infection with Mycobacterium tuberculosis complex between January 1993 and December 1995 were studied . RESULTS: 70 out of 100 patients with tuberculosis were not born in the Netherlands . Out of 50 patients tested 18 were HIV positive . The most common abnormality seen on X-rays of non-HIV positive patients were caverns . The X-ray of HIV positive patients showed no abnormalities in 39%; there was no correlation with CD4 cell count . In 74% of the patients with pulmonary tuberculosis the diagnosis was made by culture of the sputum . Treatment consisted of INH, rifampicin, pyrazinamide and ethambutol . Twelve patients were infected with resistant strains, of which two were multidrug resistant . Four patients died of tuberculosis . CONCLUSION: Tuberculosis was seen mostly among immigrants . Only half the patients with tuberculosis were tested for presence of HIV antibodies . Culture and staining of sputum played a key role in the diagnosis of tuberculosis . Multiresistant tuberculosis was present in 2% of the patients . Death due to tuberculosis in this population was 4%.

Biochem Mol Biol Int, 1998 Mar, 44(3), 443 - 52
Differential expression of multidrug resistance (mdr) and canalicular multispecific organic anion transporter (cMOAT) genes following extrahepatic biliary obstruction in rats; Kagawa T et al.; The hepatic canalicular membrane has transporters that play an important role as efflux pumps in the excretion of endogenous bile constituents or xenobiotics into bile canaliculi . To elucidate functional significance of canalicular transporters in the mechanism of cholestasis, mRNA expression levels of multidrug resistance (mdr) 1b, mdr2 and canalicular multispecific organic anion transporter (cMOAT) genes were analyzed by Southern blotting of reverse-transcribed PCR products of liver mRNA obtained from cholestatic rats that had been subjected to bile duct ligation . Both mdr1b and mdr2 mRNA expression increased after ligation . Immunohistochemical study revealed that the products of both mdr1b and mdr2 were present on the canaliculi, and that their levels increased after ligation . In contrast, cMOAT mRNA expression was not increased, but rather attenuated by ligation . The expression of canalicular transporters was differentially regulated in extrahepatic biliary obstruction, indicating the different roles are played by mdr and cMOAT in the pathogenesis of cholestasis.

Cancer Chemother Pharmacol, 1998, 41(6), 517 - 21
Effect of the multidrug resistance modulator valspodar on serum cortisol levels in rabbits; Cufer T et al.; PURPOSE: To contribute to a better understanding of the physiological role of P-glycoprotein (P-gp) in the adrenal gland, we initiated our studies in rabbits . The aim of our study was to explore the effect of the selective multidrug resistance (MDR) modulator PSC 833 (valspodar) on serum cortisol in rabbits . METHODS: Baseline and corticotropin-stimulated serum cortisol levels were measured before and after valspodar treatment in adult male rabbits . Seven rabbits were treated with 50 mg/kg per dose and seven, with 75 mg/kg per dose of valspodar subcutaneously . Serum cortisol levels were determined by radioimmunoassay adjusted for expected values . RESULTS: Serum cortisol levels (baseline as well as corticotropin-stimulated) increased after both valspodar treatment regimens . The increase was dose-dependent and was higher for the baseline than for the corticotropin-stimulated values . Serum valspodar levels exceeding 1000 ng/ml were achieved in all except one animal in each group . We hypothesize that the increased serum cortisol levels were due to increased adrenocorticotropic hormone (ACTH) secretion after valspodar treatment, but, unfortunately, we could not measure ACTH properly in rabbits by means of the commercially available kits . CONCLUSIONS: Our study indicates that P-gp is not involved in steroid hormone secretion in the adrenal gland . This is evident from observations that serum cortisol levels were found to have increased rather than decreased in rabbits treated with a P-gp blocker and that the treated animals appeared healthy and normal . Since P-gp was found to play an important role in protection against xenobiotics in some other organs, further studies to explore the protective role of P-gp in the adrenal gland are warranted.

Hum Gene Ther, 1998 Mar 20, 9(5), 649 - 57
Efficient transduction of human hematopoietic cells with the human multidrug resistance gene 1 via SV40 pseudovirions; Rund D et al.; Transduction of MDR1 may be of use in chemoprotection of normal bone marrow (BM) cells during treatment of malignancies, or as a selectable marker for the transfer of other genes into the BM, a critical target for the cure of many diseases . To that aim, the human multidrug resistance gene MDR1 was cloned into an SV40 pseudoviral vector containing the SV40 origin of replication (ori) and encapsidation signal (ses), and the plasmid was encapsidated in COS cells as SV40/MDR1 pseudovirions . Expression of the human MDR1 gene was demonstrated in murine MEL cells infected with SV40/MDR1 pseudovirions, using a monoclonal antibody (MPK16) specific for the human 170-kD P-glycoprotein . Functional P-glycoprotein was demonstrated by resistance to colchicine in NIH-3T3 cells infected with SV40/MDR1 pseudovirions . Activity of P-glycoprotein was assayed by rhodamine-123 dye exclusion and fluorescence-activated cell sorter analysis (FACS) in various cell types including hematopoietic cells . Highly efficient gene transfer and expression was demonstrated in all murine and human cell types tested, including primary human BM cells . Using multiplicities of infection (moi) of 1-2, over 95% of cells were found to become MDR1+ . The percent of MDR1+ cells was proportional to the moi . We conclude that the SV40 pseudoviral vector is efficient for gene transmission into human hematopoietic cells.

Kidney Int Suppl, 1998 Apr, 65, S11 - 7
P-glycoprotein functions and substrates: possible roles of MDR1 gene in the kidney; Ernest S et al.; There is a renewed attention on the multidrug resistance genes and their products, P-glycoproteins, since recent molecular and functional studies revealed unexpected functions in normal tissues . There are two types of human P-glycoprotein: Type I, encoded by the MDR1 gene, present in excretory organs and in non-polarized cells; and Type II, encoded by MDR2, present in the canalicular membrane of hepatocytes . MDR1 Pgp transports xenobiotics, peptides, steroids, and phospholipids, and is also a regulator of swelling-activated chloride channels . MDR2 Pgp is exclusively a phosphatidylcholine translocase . In the kidney, the MDR1 gene and protein are expressed in mesangial, proximal tubule, thick loop of Henle, and collecting duct cells . In mesangial and proximal tubule cells Pgp transports xenobiotics . Concomitant exposure of kidney cells to two Pgp substrates results in increased cell toxicity . Extracts from supernatants of mesangial cell cultures inhibit Pgp-mediated transport, suggesting that a mesangial-cell metabolite could be a substrate of Pgp . Active vitamin D3 and platelet activating factor inhibit Pgp transport and are possible endogenous substrates in proximal tubule and mesangial cells, respectively . Pgp could be also a regulator of swelling-activated chloride channels present in the kidney.

J Clin Invest, 1998 Apr 1, 101(7), 1310 - 9
Drug export activity of the human canalicular multispecific organic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA; Evers R et al.; The canalicular (apical) membrane of the hepatocyte contains an ATP-dependent transport system for organic anions, known as the multispecific organic anion transporter (cMOAT) . The deduced amino acid sequence of cMOAT is 49% identical to that of the human multidrug resistance- associated protein (MRP) MRP1, and cMOAT and MRP1 are members of the same sub-family of adenine nucleotide binding cassette transporters . In contrast to MRP1, cMOAT was predominantly found intracellularly in nonpolarized cells, suggesting that cMOAT requires a polarized cell for plasma membrane routing . Therefore, we expressed cMOAT cDNA in polarized kidney epithelial MDCK cell lines . When these cells are grown in a monolayer, cMOAT localizes to the apical plasma membrane . We demonstrate that cMOAT causes transport of the organic anions S-(2,4-dinitrophenyl)-glutathione, the glutathione conjugate of ethacrynic acid, and S-(PGA1)-glutathione, a substrate not shown to be transported by organic anion transporters previously . Transport is inhibited only inefficiently by compounds known to block MRP1 . We also show that cMOAT causes transport of the anticancer drug vinblastine to the apical side of a cell monolayer . We conclude that cMOAT is a 5'-adenosine triphosphate binding cassette transporter that potentially might be involved in drug resistance in mammalian cells.

J Biol Chem, 1998 Mar 27, 273(13), 7285 - 92
Transcriptional analysis of the EhPgp5 promoter of Entamoeba histolytica multidrug-resistant mutant; Perez DG et al.; We report here the cloning and transcriptional characterization of the EhPgp5 multidrug resistance gene promoter isolated from the drug-resistant clone C2 of Entamoeba histolytica . The EhPgp5 promoter has the TATA-like motif at -31 base pairs; transcription initiates three nucleotides upstream from the ATG in trophozoites grown in 225 microM emetine (clone C2(225)), whereas in those grown without the drug (clone C2) a product with no open reading frame was detected . The promoter was active in transfected clone C2 trophozoites, its activity increased when trophozoites were cultured in 40 microM emetine, while it was turned off in the drug-sensitive clone A . The first -235 base pair kept full promoter activity, suggesting that it has important drug responsive elements . Gel shift assays detected the complex Ib in clone C2, which was augmented in clone C2(225) . Competition experiments suggested that complex Ib may be constituted by HOX and AP-1 like factors in clone C2, whereas in clone C2(225), complex Ib was only competed by the HOX sequence . Complexes Ie, detected in clones A and C2 but not in C2(225), and Ia, present in all clones, were competed by the TATA box oligonucleotide . Our results suggest that proteins forming complexes Ib and Ie may be participating in the regulation of the EhPgp5 gene expression.

J Biol Chem, 1998 Mar 27, 273(13), 7277 - 84
Transcriptional analysis of the EhPgp1 promoter of Entamoeba histolytica multidrug-resistant mutant; Gomez C et al.; We present here the cloning and characterization of the EhPgp1 multidrug resistance gene promoter isolated from the Entamoeba histolytica drug-resistant mutant clone C2 . The EhPgp1 promoter lacks the typical TATA box and the transcriptional initiation sequences described for other E . histolytica promoters . The major transcription initiation site of the EhPgp1 gene was located at the ATG start codon . The EhPgp1 core promoter located within the first 244 base pairs showed a higher chloramphenicol acetyltransferase expression in the transfected trophozoites of clone C2 than in those of the sensitive clone A . Gel shift assays revealed three specific DNA-protein complexes (Ia, IIa, and IIIc) using nuclear extracts from clone C2, whereas three main complexes (If, IIf, and IIg) were limited to clone A . Competition assays suggested the presence of C/EBP-like and OCT-like proteins in complexes Ia and IIa, respectively, probably involved in the expression of the EhPgp1 gene, whereas complex IIIc was competed by GATA-1, C/EBP, OCT, and HOX oligonucleotides . Thus, differential DNA-protein complexes may be formed by transcriptional factors involved in the regulation of the EhPgp1 gene expression.

Int J Oncol, 1998 Apr, 12(4), 871 - 82
Glutathione-related mechanisms in cellular resistance to anticancer drugs; Zhang K et al.; Glutathione conjugation and transport of glutathione conjugates of anticancer drugs out of cells have been shown to work as a system in the detoxification of many anticancer drugs . The major components of this system include glutathione (GSH), GSH-related enzymes and glutathione conjugate export pump (GS-X pump) . GSH can combine with anticancer drugs to form less toxic and more water soluble GSH conjugates, the conjugation reaction is catalysed by glutathione S-transferases (GSTs) . The GSH conjugates of anticancer drugs can be exported from cells by GS-X pump or multidrug resistance-associated protein (MRP) . GSH, glutathione-related enzymes and GS-X pump or MRP have been found to be increased or overexpressed in many drug resistant cells . Increased detoxification of anticancer drugs by this system may confer drug resistance . Inhibition of this detoxification system is a strategy for modulation of drug resistance.

J Pharm Sci, 1998 Apr, 87(4), 403 - 10
Human intestinal permeability; Lennernas H; This review focuses on permeability measurements in humans, briefly discussing different perfusion techniques, the relevance of human Peff values, and various aspects of in vivo transport mechanisms . In addition, human Peff values are compared with corresponding data from three preclinical transport models . The regional human jejunal perfusion technique has been validated in several important ways . One of the most important findings is that there is a good correlation between the measured human effective permeability values and the extent of absorption of drugs in humans determined by pharmacokinetic studies . Estimations of the absorption half-lives from the measured Peff agree very well with the time to maximal amount of the dose absorbed achieved after an oral dose in humans . We have also shown that it is possible to determine the Peff for carrier-mediated transported compounds and to classify them according to the proposed biopharmaceutical classification system (BCS) . Furthermore, human in vivo permeabilities can be predicted using preclinical permeability models, such as in situ perfusion of rat jejunum, the Caco-2 model, and excised intestinal segments in the Ussing chamber . The permeability of passively transported compounds can be predicted with a particularly high degree of accuracy . However, special care must be taken for drugs with a carrier-mediated transport mechanism, and a scaling factor has to be used . Finally, the data obtained in vivo in humans emphasize the need for more clinical studies investigating the effect of physiological in vivo factors and molecular mechanisms influencing the transport of drugs across the intestinal and as well as other membrane barriers . It will also be important to study the effect of antitransport mechanisms (multidrug resistance, MDR), such as efflux by P-glycoprotein(s) and gut wall metabolism, for example CYP 3A4, on bioavailability.

Invest New Drugs, 1997, 15(4), 311 - 8
Potent induction of human colon cancer cell uptake of chemotherapeutic drugs by N-myristoylated protein kinase C-alpha (PKC-alpha) pseudosubstrate peptides through a P-glycoprotein-independent mechanism; Bergman PJ et al.; Phorbol ester protein kinase C (PKC) activators and PKC isozyme over-expression have been shown to significantly reduce intracellular accumulation of chemotherapeutic drugs, in association with the induction of multidrug resistance (MDR) in drug-sensitive cancer cells and enhancement of drug resistance in MDR cancer cells . These observations constitute solid evidence that PKC plays a significant role in the MDR phenotype of cancer cells . PKC-catalyzed phosphorylation of the drug-efflux pump P-glycoprotein was recently ruled out as a contributing factor in MDR . At present, the sole drug transport-related event that has been identified as a component of the role of PKC in MDR is PKC-induced expression of the P-glycoprotein-encoding gene mdr1 . The objective of this study was to test the hypothesis that PKC can modulate the uptake of chemotherapeutic drugs in cancer cells independently of P-glycoprotein . We analyzed the effects of selective PKC activators/inhibitors on the uptake of radiolabelled cytotoxic drugs by cultured human colon cancer cells that lacked P-glycoprotein activity and did not express the drug efflux pump at the level of message (mdr1) or protein . We found that the selective PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) significantly reduced uptake of {14C} Adriamycin and {3H} vincristine in human colon cancer cells devoid of P-glycoprotein activity, and that PKC-inhibitory N-myristoylated PKC-alpha pseudosubstrate synthetic peptides potently and selectively induced uptake of the cytotoxic drugs in the phorbol ester-treated and non-treated colon cancer cells . TPA treatment of the cells did not induce expression of either P-glycoprotein or its message mdr1 . In contrast with {14C}Adriamycin and {3H} vincristine uptake, {3H} 5-fluorouracil uptake by the cells was unaffected by TPA and reduced by the PKC-inhibitory peptides . These results indicate that PKC activation can significantly reduce the uptake of multiple cytotoxic drugs by cancer cells independently of P-glycoprotein, and that N-myristoylated PKC-alpha pseudosubstrate peptides potently and selectively induce uptake of multiple cytotoxic drugs in cultured human colon cancer cells by a novel mechanism that does not involve P-glycoprotein and may involve PKC isozyme inhibition . Thus, N-myristoylated PKC-alpha pseudosubstrate peptides may offer a basis for the development of agents that reverse intrinsic drug resistance in human colon cancer.

Blood, 1998 Apr 1, 91(7), 2467 - 74
Enhanced MDR1 gene expression in human T-cell leukemia virus-I-infected patients offers new prospects for therapy; Lau A et al.; Overexpression of P-glycoprotein (P-gp), the protein product of the multidrug resistance gene (MDR1), confers a drug resistant phenotype on cells . This phenotype is reminiscent of human T-cell leukemia virus (HTLV)-transformed leukemic cells, for which no consistently effective chemotherapeutic regime has been found . The presence of an active multiple drug resistance (MDR) phenotype in freshly isolated peripheral blood mononuclear cells (PBMC) from HTLV-I-infected subjects was investigated . Significant P-gp-mediated efflux activity and enhanced MDR1 mRNA expression was observed in nine of 10 HTLV-infected subjects . The development of MDR phenotypes was found to be independent of disease type or status with significant MDR activities being observed in adult T-cell leukemia (ATL), HTLV-associated myelopathy (HAM)/tropical spastic paraparesis (TSP), and asymptomatic HTLV-infected individuals . P-gp-mediated drug efflux was also found to be restricted to CD3+ T-cell populations . Furthermore, we show the novel finding that the MDR1 gene promoter is transcriptionally activated by the HTLV-I tax protein, suggesting a molecular basis for the development of drug resistance in HTLV-I infections . These observations open up the possibility of new chemotherapeutic approaches to HTLV-associated diseases through the use of P-gp inhibitors.

Int J Oncol, 1998 Mar, 12(3), 711 - 5
Role of the vacuolar H+-ATPase in daunorubicin distribution in etoposide-resistant MCF7 cells overexpressing the multidrug-resistance associated protein; Benderra Z et al.; Some multidrug-resistant cell lines efflux anticancer drugs but do not overexpress the well-known P-glycoprotein pump or Pgp . A 190 kDa or multidrug-resistant associated protein (MRP) has been identified and described as an MDR mediator . Many studies on cells overexpressing MRP and Pgp, show a concentration of the drug inside cytoplasmic vesicles followed by an exocytotic process . We studied daunorubicin (DNR) subcellular distribution in the presence of an H+-ATPase pump inhibitor 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD) and verapamil (VPL) in two human breast adenocarcinoma MCF7 etoposide-resistant and adriamycin-resistant cell lines, overexpressing respectively MRP (MCF7/VP) and Pgp (MCF7/ADR) . Nucleo-cytoplasmic distribution of daunorubicin was carried out using scanning confocal microspectrofluorometry . This technique allows the determination of nuclear accumulation of anthracyclines . Our results show that NBD was able to increase the nuclear accumulation of DNR in MCF7/VP but not in MCF7/ADR cells . Similarly, NBD could reverse DNR resistance in MCF7/VP cells but had no effect on DNR cytotoxicity in MCF7/ADR cells . VPL caused a significant increase in nuclear accumulation of DNR in MCF7/VP and MCF7/ADR cells . Incubation of MCF7/VP and MCF7/ADR cells with VPL, increased the sensitivity of these cells . These data demonstrate clearly that even if vesicular sequestration can happen in cells overexpressing MRP and Pgp proteins, only the MRP protein is able to extrude the drug through intracellular vesicles and efflux . In cells overexpressing Pgp, drug efflux probably takes place directly at the membrane level.

FEBS Lett, 1998 Mar 20, 425(1), 40 - 4
On the target of a novel class of antibiotics, oxazolidinones, active against multidrug-resistant Gram-positive bacteria; Burghardt H et al.; Oxazolidinones are a promising new class of synthetic antibiotics active against multidrug-resistant Gram-positive bacteria . To elucidate their mode of action, the effect of DuP 721 on individual steps of protein translation was studied . The drug does not interfere with translation initiation at the stage of mRNA binding or formation of 30S pre-initiation complexes . However, it inhibits the puromycin-mediated release of {35S}formyl-methionine from 70S initiation complexes in a dose-dependent manner . Inhibition involves binding of the oxazolidinone to the large ribosomal subunit and is twice as high with 50S subunits from Gram-positive as with those from Gram-negative bacteria.

Biochim Biophys Acta, 1998 Mar 5, 1401(3), 265 - 76
Novel cellular determinants for reversal of multidrug resistance in cells expressing P170-glycoprotein; Yin MB et al.; The newly synthesized calcium channel blocker, Ro44-5912, significantly potentiates doxorubicin (Dox)-induced cytotoxicity at non-cytotoxic concentrations in Dox-resistant human ovarian cell line, A2780/DX5, overexpressing P170-glycoprotein (Pgp) . Induction of DNA single- and double-strand breaks (ssbs and dsbs) was measured using alkaline elution and constant-field gel electrophoresis (CFGE) assays . The results indicate that potentiation of the cytotoxicity of Dox by Ro44-5912 was accompanied by significant increases in both, Dox-induced DNA ssbs and dsbs in the resistant cells . Pulsed-field gel electrophoresis (PFGE) analysis showed that Dox induced DNA fragments in the 50-800 kilobase (kb) and 0.8-5.7 megabase (Mb) ranges . The majority of the newly synthesized DNA fragments were in the 50-800 kb range . Ro44-5912 treatment resulted in significant potentiation of DNA fragmentation in the 50-800 kb range with a minor increase in 0.8-5.7 Mb DNA fragments, suggesting that the modulator functions by potentiating nascent DNA fragmentation in the resistant cells . Exposure to Dox with Ro44-5912 was associated with a prolonged blockage of cells in the S-phase . In contrast, exposure to Dox alone resulted in temporary blockage of cells in G2/M phase (approximately 24 h) followed by restoration of cell proliferation and normal DNA histograms at 48 h after 2 h drug exposure . Incorporation of BrdUrd by flow cytometric analysis was inhibited by Dox in the presence of Ro44-5912, showing that there is a block of DNA replication . An increased damage in newly synthesized DNA could concur with a blocked DNA replication . Moreover, slowing progression through the S-phase in cells exposed to Dox in combination with Ro44-5912 is accompanied by increased sensitivity of Dox poisons, indicating a correlation of specific S-phase perturbation with the reversal of Dox resistance by Ro44-5912 in cells expressing Pgp . The results suggest that drug-induced augmentation of nascent DNA fragmentation and specific cell-cycle perturbation are potentially important molecular determinants for reversal of multidrug resistance in addition to restoration of intracellular drug retention.

Schweiz Med Wochenschr, 1998 Feb 14, 128(7), 264 - 7
{Pulmonary tuberculosis with resistance to 4 antitubercular drugs}; Reichlin S et al.; A 32-year-old immigrant from Pakistan was admitted to our hospital with cavernous pulmonary tuberculosis . He gave a history of several 1 to 2-months courses of antimycobacterial treatment administered earlier in Pakistan . We initiated combined therapy including isoniazid, rifampin, pyrazinamide, and ethambutol . Subsequently, results of susceptibility testing from M . tuberculosis-complex strains isolated before the onset of treatment documented the presence of resistance against both isoniazid and rifampin which may have been primary or acquired drug resistances . During the course of treatment, two additional resistances to pyrazinamide and ethambutol developed which were probably due to the initial therapy with only two active antimycobacterial agents . The emergence of multidrug-resistant strains of M . tuberculosis complex is a world-wide problem . Our case indicates that multiresistance must be considered in every patient presenting with tuberculosis . If there is a strong suspicion of multidrug-resistant tuberculosis, initial treatment with a combination of 5-6 antimycobacterial agents seems advisable until the results of susceptibility testing become available.

Int J Urol, 1998 Jan, 5(1), 1 - 15
Circumvention of multidrug resistance in genitourinary tumors; van Brussel JP et al.; Chemotherapy is the principal strategy to systemically challenge metastasized cancers of genitourinary origin . Unfortunately, the efficacy of chemotherapy is often hampered by multidrug resistance, the resistance to a variety of structurally and functionally distinct cytotoxic agents . Multidrug resistance can be either intrinsic or acquired, and can be caused by several mechanisms . The so-called classical multidrug resistance, mediated by the MDR1 gene product P-glycoprotein, has been held mainly responsible for inferring the multidrug resistance phenotype on urologic malignancies . However, several other multidrug resistance pathways have been identified . Multidrug resistance can be caused by the membrane-bound multidrug-resistance-associated protein, the detoxifying glutathione metabolism, the antiapoptotic protein BCL2, and changes in levels or activity of the topoisomerase enzymes . Strategies to overcome multidrug resistance of genitourinary tumors have arisen from the better understanding of the biologic and molecular mechanisms of multidrug resistance, and have been studied in experimental and clinical settings . However, attempts to modulate multidrug resistance in clinical renal cell, bladder, prostate, and testicular cancer have not been very rewarding so far, despite the optimism that had arisen from experimental data . Nevertheless, application of novel therapies to reverse multidrug resistance and to increase efficacy of chemotherapy for urologic cancers should be further pursued, within the setting of controlled clinical trials, to improve on current strategies.

J Nurs Educ, 1998 Mar, 37(3), 101 - 8
Actions taken by nursing education programs in the United States to prevent tuberculosis transmission in nursing students; Moore PV; Measures to prevent tuberculosis include education and skin testing of at-risk groups, including health care workers . This study focused on policies and practices related to tuberculosis in nursing education programs, especially skin testing and instruction . Data were collected from a stratified random sample of nursing administrators in associate and baccalaureate degree programs in the United States using an instrument adapted from a medical school study . Several factors may have contributed to fewer skin test conversions in nursing programs than in medical schools . Although most nursing education programs considered skin testing a priority, there were inconsistencies related to skin testing type and process when compared with recent Centers for Disease Control and Prevention guidelines . Major content gaps related to multidrug-resistant tuberculosis and the differences between pulmonary and extrapulmonary symptomatology were found.

J Biol Chem, 1998 Mar 13, 273(11), 5997 - 6000
Direct involvement of the Y-box binding protein YB-1 in genotoxic stress-induced activation of the human multidrug resistance 1 gene; Ohga T et al.; The human multidrug resistance 1 (MDR1) gene encoding P-glycoprotein is often overexpressed in various human tumors after chemotherapy . During treatment with various chemotherapeutic agents, the MDR1 gene is activated at the transcriptional level and/or amplified, resulting in overexpression . Our previous studies demonstrated that an inverted CCAAT box (Y-box) might be a critical cis-regulatory element regulating UV or drug-induced MDR1 gene expression . We have now established various cell lines from human head and neck cancer KB cells which were stably transfected with the chloramphenicol acetyltransferase (CAT) reporter gene driven by various MDR1 promoter deletion constructs . Transient transfection of antisense YB-1 expression constructs resulted in a decrease of both YB-1 protein levels and DNA binding activity to the inverted CCAAT box, as determined by Western blot and gel mobility shift assays . The limited expression and binding activity due to expression of antisense YB-1 constructs were also observed when cells were treated with UV . CAT activity of constructs containing the Y-box was enhanced after treatment with UV irradiation as well as genotoxic agents such as cisplatin and etoposide . Moreover, this activation was reduced by 50-80% by transfection of antisense YB-1 expression constructs . In contrast, transfection of antisense YB-1 expression constructs had no effect on CAT activity driven by MDR1 promoter constructs not containing the Y-box . These data indicate that YB-1 is directly involved in MDR1 gene activation in response to genotoxic stress.

Blood, 1998 Mar 15, 91(6), 2092 - 8
Relationship between major vault protein/lung resistance protein, multidrug resistance-associated protein, P-glycoprotein expression, and drug resistance in childhood leukemia; den Boer ML et al.; Cellular drug resistance is related to a poor prognosis in childhood leukemia, but little is known about the underlying mechanisms . We studied the expression of P-glycoprotein (P-gp), multidrug resistance (MDR)-associated protein (MRP), and major vault protein/lung resistance protein (LRP) in 141 children with acute lymphoblastic leukemia (ALL) and 27 with acute myeloid leukemia (AML) by flow cytometry . The expression was compared between different types of leukemia and was studied in relation with clinical risk indicators and in vitro cytotoxicity of the MDR-related drugs daunorubicin (DNR), vincristine (VCR), and etoposide (VP16) and the non-MDR-related drugs prednisolone (PRD) and L-asparaginase (ASP) . In ALL, P-gp, MRP, and LRP expression did not differ between 112 initial and 29 unrelated relapse samples nor between paired initial and relapse samples from 9 patients . In multiple relapse samples, LRP expression was 1.6-fold higher compared with both initial (P = .026) and first relapse samples (P = .050), which was not observed for P-gp and MRP . LRP expression was weakly but significantly related to in vitro resistance to DNR (Spearman's rank correlation coefficient 0.25, P = .016) but not to VCR, VP16, PRD, and ASP . No significant correlations were found between P-gp or MRP expression and in vitro drug resistance . Samples with a marked expression of two or three resistance proteins did not show increased resistance to the tested drugs compared with the remaining samples . The expression of P-gp, MRP, and LRP was not higher in initial ALL patients with prognostically unfavorable immunophenotype, white blood cell count, or age . The expression of P-gp and MRP in 20 initial AML samples did not differ or was even lower compared with 112 initial ALL samples . However, LRP expression was twofold higher in the AML samples (P < .001), which are more resistant to a variety of drugs compared with ALL samples . In conclusion, P-gp and MRP are unlikely to be involved in drug resistance in childhood leukemia . LRP might contribute to drug resistance but only in specific subsets of children with leukemia.

Int J Cancer, 1998 Mar 30, 76(1), 55 - 62
Transport of glutathione conjugates into secretory vesicles is mediated by the multidrug-resistance protein 1; Van Luyn MJ et al.; Intracellular glutathione-conjugate transport was evaluated in the human small cell lung carcinoma cell line GLC4 with low multidrug resistance protein (MRP1) expression and its 300x doxorubicin-resistant, MRP1-over-expressing, GLC4-Adr subline . Transport of non-toxic concentrations of monochlorobimane and 5-chloro-methyl fluorescein diacetate was evaluated using fluorescence microscopy . After exposure to these compounds, fluorescence was observed especially in intracellular vesicles in GLC4-Adr . Immunotransmission electron microscopy showed that MRP1 was present in the vesicle membranes and plasma membrane, while inside the vesicles the glutathione conjugate of 1-chloro-2,4-dinitrobenzene could be detected . Experiments with brefeldin A, which induces arrest in vesicle release from the Golgi complex, indicated that these vesicles may originate from the trans-Golgi network . In GLC4-Adr cells, doxorubicin also was transported in vesicles, with an arrest in vesicle release from the Golgi complex . Our results indicate that MRP1 functions as a glutathione-conjugate transporter not only at the plasma membrane but also in intracellular secretory vesicles.

Crit Rev Clin Lab Sci, 1998 Feb, 35(1), 1 - 57
In vivo model systems in P-glycoprotein-mediated multidrug resistance; van de Vrie W et al.; In this article we review the in vivo model systems that have been developed for studying P-glycoprotein-mediated multidrug resistance (MDR) in the preclinical setting . Rodents have two mdr genes, both of which confer the MDR phenotype: mdr 1a and mdr 1b . At gene level they show strong homology to the human MDR1 gene and the tissue distribution of their gene product is very similar to P-glycoprotein expression in humans . In vivo studies have shown the physiological roles of P-glycoprotein, including protection of the organism from damage by xenobiotics . Tumors with intrinsic P-glycoprotein expression, induced MDR or transfected with an mdr gene, can be used as syngeneic or xenogenic tumor models . Ascites, leukemia, and solid MDR tumor models have been developed . Molecular engineering has resulted in transgenic mice that express the human MDR1 gene in their bone marrow and in knockout mice missing a murine mdr gene . The data on pharmacokinetics, efficacy, and toxicity of chemosensitizers of P-glycoprotein in vivo are described . Results from studies using monoclonal antibodies directed against P-glycoprotein and other miscellaneous approaches for modulation of MDR are mentioned . The importance of in vivo studies prior to clinical trials is being stressed and potential pitfalls due to differences between species are discussed.

Virchows Arch, 1998 Mar, 432(3), 279 - 87
Generation of a monoclonal antibody to P-glycoprotein peptides using tuberculin-PPD as a carrier; Bashir I et al.; A novel immunization protocol together with stringent selection criteria have been employed to generate a new murine monoclonal antibody ("D8", isotype IgG1, kappa) which specifically recognizes the human p170 drug resistance glycoprotein . This antibody is directed towards a defined peptide sequence located in the -COOH terminal region of the first external loop of the molecule . It is reactive with its epitope within the intact native glycoprotein in formalin-fixed and conventionally processed histological tissues, in flow-cytometric preparations and by Western blotting . The antibody precipitates its target peptide sequence from solution, and thus may be a useful reagent with which to establish an ELISA, RIMA or other similar assay . The peptide epitope recognized by this monoclonal antibody is restricted to the human MDR1 gene product and is not contained within the rodent homologue of the P-170 molecule . Immunohistochemistry has consistently failed to detect this epitope in rodent tissues, thus confirming that it does not exhibit the cross-reactivity of other currently available anti-P-glycoprotein monoclonal antibodies . The experience of this study emphasizes the value of the tuberculin-PPD (purified protein derivative) immunization protocol as a powerful strategy when generating monoclonal antibodies to small synthetic peptides . The resulting monoclonal antibody (D8) will be an invaluable reagent with which to analyse P-170 glycoprotein expression when assessing the role of multidrug resistance in human cancers.

J Antibiot (Tokyo), 1998 Jan, 51(1), 68 - 72
Enhancement of drug accumulation by andrastin A produced by Penicillium sp . FO-3929 in vincristine-resistant KB cells; Rho MC et al.; In the course of our screening for compounds that reverse multidrug resistance, we found that the cytotoxicity of vincristine was enhanced 1.5-20-fold depending on the concentration of andrastin A in vincristine-resistant KB cells (VJ-300) . Andrastin A alone had no effect on the growth of drug sensitive KB cells and VJ-300 cells . On the other hand, andrastin A (25 and 50 micrograms/ml) significantly enhanced accumulation of {3H}vincristine in VJ-300 cells . Andrastin A (50 micrograms/ml) completely inhibited the binding of {3H}azidopine to the P-glycoprotein in VJ-300 cells . The result suggests that andrastin A directly interacts with P-glycoprotein and inhibits the efflux of antitumor agents in drug resistant cells.

Biochim Biophys Acta, 1998 Feb 2, 1369(1), 85 - 93
Effects of cardiovascular drugs on ATPase activity of P-glycoprotein in plasma membranes and in purified reconstituted form; Rebbeor JF et al.; Drug interactions with P-glycoprotein (Pgp) were quantitatively assessed using ATPase assay . Two experimental systems were used, (i) plasma membranes isolated from a multidrug-resistant cell line, which contained 30% Pgp as fraction of total membrane protein, and (ii) purified reconstituted Pgp . The cardioactive drugs verapamil, quinidine, diltiazem, nifedipine, and a series of digitalis analogs, interacted directly with Pgp as shown on ATPase in both systems . Apparent affinities of drug binding were calculated . Direct competition was shown between digitoxin and verapamil . Drug-drug interaction in vivo at the level of Pgp is expected from the results . This approach seems well-suited for empirical determination of drug interactions with Pgp, and prediction of drug-drug interactions.

Biochim Biophys Acta, 1998 Feb 2, 1369(1), 7 - 13
Cloning of AtMRP1, an Arabidopsis thaliana cDNA encoding a homologue of the mammalian multidrug resistance-associated protein; Marin E et al.; A cDNA encoding a putative ATP-binding cassette (ABC) transporter from Arabidopsis was cloned and sequenced based on an EST clone homologous to ABC sequences in other species . The cDNA is 5.5 kb long and contains an ORF encoding a 1623 amino acids protein . This sequence is the first MRP-like protein found in plants.

J Med Chem, 1998 Mar 12, 41(6), 981 - 7
Design, synthesis, and evaluation of the multidrug resistance-reversing activity of D-glucose mimetics of hapalosin; Dinh TQ et al.; When five substituents of hapalosin were placed on D-glucose, molecular modeling revealed that the substituents on mimetics 2 and 3 occupy similar spatial positions as the corresponding substituents on hapalosin . Mimetic 3 and all the glucopyranoside intermediates generated in its synthesis were assessed for their ability to reverse multidrug resistance (MDR) mediated by P-glycoprotein (P-gp) or the multidrug resistance-associated protein (MRP) . None of the sugar compounds were as effective as hapalosin in inhibiting P-gp in cytotoxicity and drug accumulation assays using MCF-7/ADR cells . By contrast, four D-glucose compounds exhibited similar efficacy as hapalosin in antagonizing MRP in cytotoxicity assays with HL-60/ADR cells.

Oncol Rep, 1998 Jan-Feb, 5(1), 249 - 55
Effects of tubulin-inhibiting agents in human lung and breast cancer cell lines with different multidrug resistance phenotypes; van Ark-Otte J et al.; Drug sensitivity was studied for the tubulin inhibitors taxol, taxotere, rhizoxin and for doxorubucin and cisplatin, in human lung and breast cancer cell lines, including drug-selected cell lines, overexpressing the membrane transporter P-glycoprotein (Pgp) or the multidrug resistance protein (MRP) . All tubulin-inhibiting agents were more potent than doxorubicin and cisplatin in all cell lines . In the drug resistance-selected cell lines (doxorubicin or mitoxantrone resistant) there was cross-resistance between the tubulin inhibitors and the selecting agent; however, MRP overexpressing cells were relatively less resistant to taxanes than the Pgp overexpressing cells . Polymerization of microtubules after exposure to taxol was observed in drug sensitive cell lines, but not in resistant cell lines, even at high taxol concentrations and after long exposure times . In the Pgp overexpressing cell lines, steady accumulation of 14C-taxol was defective and could be reverted by verapamil . MRP overexpressing cells did not have a significant accumulation defect of taxol, compared to the parental cell lines, and verapamil did not have any effect . These data confirm that the Pgp overexpression is an important mechanism of resistance to taxanes and rhizoxin in human lung and breast tumor cells . However, the presence of mechanisms other than transport defects may play an important role in non-Pgp expressing cells, and these may include an altered function of tubulins.

Mol Biol Cell, 1998 Feb, 9(2), 523 - 43
Genetic separation of FK506 susceptibility and drug transport in the yeast Pdr5 ATP-binding cassette multidrug resistance transporter; Egner R et al.; Overexpression of the yeast Pdr5 ATP-binding cassette transporter leads to pleiotropic drug resistance to a variety of structurally unrelated cytotoxic compounds . To identify Pdr5 residues involved in substrate recognition and/or drug transport, we used a combination of random in vitro mutagenesis and phenotypic screening to isolate novel mutant Pdr5 transporters with altered substrate specificity . A plasmid library containing randomly mutagenized PDR5 genes was transformed into appropriate drug-sensitive yeast cells followed by phenotypic selection of Pdr5 mutants . Selected mutant Pdr5 transporters were analyzed with respect to their expression levels, subcellular localization, drug resistance profiles to cycloheximide, rhodamines, antifungal azoles, steroids, and sensitivity to the inhibitor FK506 . DNA sequencing of six PDR5 mutant genes identified amino acids important for substrate recognition, drug transport, and specific inhibition of the Pdr5 transporter . Mutations were found in each nucleotide-binding domain, the transmembrane domain 10, and, most surprisingly, even in predicted extracellular hydrophilic loops . At least some point mutations identified appear to influence folding of Pdr5, suggesting that the folded structure is a major substrate specificity determinant . Surprisingly, a S1360F exchange in transmembrane domain 10 not only caused limited substrate specificity, but also abolished Pdr5 susceptibility to inhibition by the immunosuppressant FK506 . This is the first report of a mutation in a yeast ATP-binding cassette transporter that allows for the functional separation of substrate transport and inhibitor susceptibility.

J Cell Physiol, 1998 May, 175(2), 141 - 8
Insulin-like growth factor-I promotes multidrug resistance in MCLM colon cancer cells; Guo YS et al.; Insulin-like growth factor-I (IGF-I) is known as a potent mitogen for a variety of cell types, including colon cancer cell lines . The objective of this study was to determine the effect of IGF-I on cell death induced by cytotoxic agents actinomycin D (Act-D), lovastatin (LOV), and doxorubicin (DOX) in the MCLM mouse colon cancer cell line, and the mechanisms involved . Subconfluent monolayer MCLM cells were treated with IGF-I (25 ng/ml) for 12 h in serum-free media . Various concentrations of cytotoxic agents then were added to the cells that were incubated continually at 37 degrees C for 24 h . Cell survival was determined with the MTT (3-{4-5-dimenthylthiazol-2-yl}-2,5-diphenyltetrazolium bromide) assay, which assesses mitochondrial function in living cells . The mRNA expression for multidrug resistance gene-1 (mdr-1), c-H-ras, and manganese superoxide dismutase (MnSOD) in cells treated with IGF-I was examined by Northern blot or RNase protection assays . The levels of p-glycoprotein, a drug efflux pump encoded by the mdr-1 gene, were assessed by Western immunoblotting . Results demonstrated that 1) IGF-I significantly inhibited the cell death and apoptosis of MCLM cells treated with Act-D, LOV, or DOX; 2) IGF-I increased mRNA expression for mdr-1, c-H-ras, and MnSOD; 3) the p-glycoproteins in cells treated with IGF-I or stably transfected with c-H-ras were elevated when compared with control . These results suggest that IGF-I protects MCLM cells against death induced by cytotoxic agents; this acquired drug resistance may be mediated by multiple mechanisms, including promoting expression of mdr-1, c-H-ras, and MnSOD; whereas, the p-glycoprotein level stimulated by IGF-I may result partly from the increase of c-H-ras in the cells.

Clin Infect Dis, 1998 Mar, 26(3), 659 - 63
Perspectives on switching oral acyclovir from prescription to over-the-counter status: report of a consensus panel; Sande MA et al.; The proposed switching of oral acyclovir from prescription to over-the-counter (OTC) status for the 5-day episodic treatment of genital herpes was considered by a consensus panel . It was concluded that self-diagnosis/misdiagnosis, misuse, and adverse drug effects were potential problems with the OTC use of acyclovir . While acyclovir reduces asymptomatic shedding of herpes simplex virus type 2, the reduction in transmission of virus potentially resulting from increased acyclovir use was felt to be of unknown extent but likely to be of benefit overall . The availability of acyclovir would likely be improved . There were differences in opinion as to whether widespread availability of acyclovir (prescription or OTC) may speed the development of viral resistance . However, all panel members felt that granting OTC status may set an undesirable precedent for the switch from prescription to OTC use of other systemically administered antiinfective agents . The effect of this precedent, in terms of accelerating development of multidrug-resistant bacteria, was a major concern of all panel members . The consensus was that the switch of acyclovir to OTC status could not be supported.

J Pharm Sci, 1998 Mar, 87(3), 300 - 5
Functional expression of P-glycoprotein in the hepatic canalicular membrane of developing rats; Kamath AV et al.; P-glycoprotein (P-gp), the multidrug resistance gene product, is expressed in a normal liver exclusively on the canalicular membrane of the hepatocyte . The objective of this study was to examine the effect of age on the P-gp transport system using canalicular membrane (cLPM) vesicles isolated from the liver of developing (22 days old) and adult rats . No differences in protein yield, intravesicular volumes, and enrichments of cLPM enzymes or enzymes representing contamination of subcellular organelles were found for vesicles isolated from both groups, demonstrating the isolation of similar cLPM vesicle preparations . The transport of daunomycin (DNM), a P-gp substrate, was used to study age-related functional differences in P-gp . DNM uptake in the presence of ATP was greater than uptake in the absence of ATP in both young and adult cLPM vesicles, showing that P-gp is functional in both groups . In young and adult groups only ATP was a potent stimulator of transport when compared with ATP degradation products and a nonhydrolyzable ATP analogue . Although ATP-dependent uptake tended to be greater in the adult compared to the young, there was no statistically significant difference in DNM kinetics (Vmax, km, gamma) between groups . Canalicular membrane from the young rats showed decreased fluidity, as assessed by the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene; however there was no significant difference between groups . Examination of P-gp expression using the monoclonal antibody C219 revealed similar levels of expression in the young as in the adult . Our results suggest that P-gp in the bile canaliculus of developing rats is functional with similar levels of function and expression as observed in the adult.

Eur J Biochem, 1998 Feb 15, 252(1), 140 - 6
Comparison of the kinetics of active efflux of 99mTc-MIBI in cells with P-glycoprotein-mediated and multidrug-resistance protein-associated multidrug-resistance phenotypes; Vergote J et al.; The overexpression of two membrane glycoproteins, P-glycoprotein and multidrug-resistance protein (MRP1) is a major cause of resistance to chemotherapeutic agents in the treatment of human cancers . Both proteins confer a similar multidrug-resistant (MDR) phenotype . 99mTc-MIBI, a myocardial imaging agent, which is also useful for the detection of a variety of tumours, has been shown to be a substrate for P-glycoprotein and MRP1 . It thus may provide additional information about the P-glycoprotein and MRP1 status of tumour cells . In order to obtain information on the substrate specificity of these proteins, we have studied the transport kinetics of Tc-MIBI in two cell lines, K562/ADR and GLC4/ADR, which overexpress P-glycoprotein and MRP1, respectively . The mean active efflux coefficient ka, which is proportional to the ratio of maximal efflux rate VM to the apparent Michaelis-Menten constant Km, used to characterise the efficiency of the active efflux, was very similar being 1.9 +/- 0.6 x 10(-11) s(-1) x cells x ml and 1.3 +/- 0.5 x 10(-11) s(-1) x cells x ml for drug-resistant K562 and GLC4, respectively . These values are 50-100-times lower than for daunorubicin and other anthracycline derivatives, strongly suggesting that the efficiency of both transporters to pump Tc-MIBI is by far less than that to efflux anthracyclines . Our data show that (a) P-glycoprotein and MRP transporter efficiencies to wash out Tc-MIBI are similar, in spite of a different suspected mechanism of its transport and (b) that both transporters are less efficient to pump Tc-MIBI than to pump anthracyclines (the ka parameter is about 100-times lower for TC-MIBI than for anthracycline).

Micron, 1997 Oct, 28(5), 389 - 95
Intracellular mapping of 4'-deoxy-4'-iododoxorubicin in sensitive and multidrug resistant cells by electron spectroscopic imaging; Diociaiuti M et al.; Electron spectroscopic imaging (ESI) was employed to study, with high spatial resolution, the intracellular distribution of the halogenated derivative of doxorubicin 4'-deoxy-4'-iododoxorubicin (IDX) in sensitive and multidrug resistant human breast carcinoma cells . Both ESI and electron energy loss spectroscopy (EELS) observations confirmed results obtained with flow cytometry (FC), laser scanning confocal microscopy (LSCM) and secondary ion mass spectrometry (SIMS) . Moreover, ESI allowed us to obtain a more detailed intracellular localization of IDX . Our results confirm that nuclear DNA represents the main intracellular target for IDX and that the Golgi apparatus is involved in the intracellular transport of the drug.

Palliat Med, 1997 Nov, 11(6), 469 - 74
Tuberculosis in the hospice--a cause for concern?
McKeogh MM.
Open pulmonary tuberculosis has been increasingly seen in HIV-infected patients in this hospice . Multidrug-resistant tuberculosis is a new and serious threat and two cases have occurred in our hospice in the past two years . This infection poses a health risk to staff, patients, relatives and volunteers . Palliative care teams in the hospice and community must have an index of suspicion for this infection, take active measures to ensure patient compliance with tuberculosis treatment and be prepared to implement infection control guidelines when needed.

Proteins, 1998 Feb 15, 30(3), 275 - 86
A model for the nucleotide-binding domains of ABC transporters based on the large domain of aspartate aminotransferase; Hoedemaeker FJ et al.; ABC transporters are a large superfamily of integral membrane proteins involved inATP-dependent transport across biological membranes . Members of this superfamily play roles in a number of phenomena of biomedical interest, including cystic fibrosis (CFTR) and multidrug resistance (P-glycoprotein, MRP) . Most ABC transporters are predicted to consist of four domains, two membrane-spanning domains and two cytoplasmic domains . The latter contain conserved nucleotide-binding motifs . Attempts to determine the structure of ABC transporters and of their separate domains are in progress but have not yet been successful . To aid structure determination and possibly learn more about the domain boundaries, we set out to model nucleotide-binding domains (NBDs) of ABC transporters based on a known structure . Previous attempts to predict the 3D structure of NBDs were based solely on sequence similarity with known nucleotide-binding folds . We have analyzed the sequences of a number of nucleotide-binding domains with the algorithm THREADER, developed by D.T . Jones, and a possible fold was found in the structure of aspartate aminotransferase . We present a model for the N-terminal NBD of CFTR, based on the large domain of the A chain of aspartate aminotransferase . The model is refined using multiple sequence alignment, secondary structure prediction, and 3D-1D profiles . Our model seems to be in good agreement with known properties of nucleotide-binding domains and has some appealing characteristics compared with the previous models.

Eur J Cancer, 1997 Oct, 33(12), 2031 - 6
The prognostic value of MDR1 gene expression in primary untreated neuroblastoma; Haber M et al.; The contribution of MDR1 gene expression to the biology of childhood neuroblastoma is unclear . To clarify the role of MDR1 in this malignancy, we examined the relationship between MDR1 expression and patient outcome in subsets of 60 primary untreated neuroblastomas for which MYCN gene copy number and expression of the multidrug resistance-associated-protein (MRP) gene had been previously characterised . In contrast to MRP gene expression, MDR1 expression was lower in tumours with MYCN gene amplification compared with those without amplification . Strong correlations between MDR1 and MRP gene expression, and between MDR1 and MYCN gene expression, were observed in tumours lacking MYCN gene amplification (P < 0.0005) . In these single-copy tumours, very high MDR1 gene expression was significantly associated with poor outcome (P < 0.05) . Very high MDR1 expression was also strongly predictive of poor outcome in older children (P < 0.0001), but not in infants . These findings suggest a clinical role for the MDR1 gene in specific subgroups of primary neuroblastoma.

Eur J Cancer, 1997 Oct, 33(12), 1911 - 6
Evidence that the MYCN oncogene regulates MRP gene expression in neuroblastoma; Norris MD et al.; We have recently shown that expression of the multidrug resistance-associated protein (MRP) gene is a powerful prognostic indicator in childhood neuroblastoma and have suggested that the MYCN oncogene may regulate MRP gene expression . To address this hypothesis, we have examined the relationship between MYCN and MRP gene expression in neuroblastoma tumours and cell lines . MYCN and MRP gene expression were highly correlated in 60 primary untreated tumours both with (P = 0.01) and without MYCN gene amplification (P < 0.0001) . Like MRP, high MYCN gene expression was significantly associated with reduced survival, both in the overall study population and in older children without MYCN gene amplification (relative hazards = 13.33 and 19.61, respectively) . Inhibition of MYCN, through the introduction of MYCN antisense RNA constructs into human neuroblastoma cells in vitro, resulted in decreased MRP gene expression, determined both by RNA-PCR and Western analysis . The data are consistent with MYCN influencing neuroblastoma outcome by regulating MRP gene expression.

Biochemistry, 1998 Feb 24, 37(8), 2305 - 13
Epitope insertion favors a six transmembrane domain model for the carboxy-terminal portion of the multidrug resistance-associated protein; Kast C et al.; The overexpression of the multidrug resistance protein, MRP, in mammalian cells is associated with pleiotropic resistance to cytotoxic drugs . MRP is an integral membrane protein which belongs to the family of ATP-binding cassette transporters . Secondary structure predictions combined with biochemical analyses suggest that MRP encodes 11 transmembrane (TM) domains in the amino-terminal half of the protein and four or six transmembrane domains in the carboxy-terminal half of the protein . To gain insight into the membrane topology of the carboxy-terminal half of MRP, small, antigenic hemagglutinin (HA) epitopes (YPYDVPDYAS) were inserted within six predicted hydrophilic subfragments of this region (938, 1001, 1084, 1175, 1222, 1295) . These epitope-tagged MRP variants were expressed in HeLa cells to evaluate their ability to confer resistance to the drug etoposide (VP-16) . Insertion of the HA epitopes at positions 938, 1001, and 1222 resulted in functional proteins, while epitope insertion at positions 1084, 1175, and 1295 abrogated MRP function . The intracellular versus extracellular location of the HA epitopes present in biologically active MRP variants was then established in intact and permeabilized cells by immunofluorescence using an anti-HA antibody . Epitopes inserted at positions 1001 and 1222 were located on the extracellular side of the plasma membrane, while the epitope inserted at position 938 was located intracellularly . These results are consistent with a six TM rather than a four TM domain model for the membrane portion of the carboxy-terminal half of MRP.

Biochemistry, 1998 Feb 24, 37(8), 2243 - 50
Kinetic analysis of calcein and calcein-acetoxymethylester efflux mediated by the multidrug resistance protein and P-glycoprotein; Essodaigui M et al.; Multidrug resistance protein (MRP) and P-glycoprotein (Pgp) are both members of the superfamily of ATP binding cassette plasma membrane drug transport proteins, which may be partly responsible for multidrug resistance of tumor cells . Although MRP has been identified as an organic anion transporter and Pgp as a transporter of certain positively charged compounds, there is considerable overlap in resistance spectrum, suggesting that both proteins transport important anticancer agents such as doxorubicin, etoposide, and vincristine . To obtain more insight in the handling of drugs by both proteins, we performed a detailed kinetic analysis of the efflux of calcein-acetoxymethyl ester (CAL-AM), a common neutral substrate for both proteins and compared it with the kinetics of efflux of calcein (CAL) which is only effluxed by MRP . CAL, the hydrolysis product of the nonfluorescent CAL-AM, is negatively charged and highly fluorescent . For this purpose Pgp+ K562/ADR and MRP+ GLC4/ADR tumor cells were incubated with CAL-AM in ATP-rich or ATP-depleted buffer, and the calcein formation was followed in time by fluorescence development . The intracellular CAL could be distinguished from effluxed (extracellular) CAL by addition to the medium of Co2+, which completely quenched the extracellular CAL fluorescence . The results showed that the Vmax for efflux of CAL-AM and CAL by MRP were very similar (1.0-1.2 x 10(5) molecules/cell/s) but that the Km for CAL-AM was much lower (0.05 microM) than for CAL (268 microM) . The latter therefore is much less efficiently transported by MRP than CAL-AM . The Km for CAL-AM transport by Pgp (0.12 microM) was similar to that for MRP . Compared to previously published data for anthracyclines, the kinetic data for MRP-mediated CAL-AM pumping are most similar to those for the neutral hydroxydaunorubicin . These data give a quantitative account of transport properties of MRP for two related but differently charged compounds.

J Biol Chem, 1998 Feb 27, 273(9), 5294 - 9
Glucose deprivation-induced cytotoxicity and alterations in mitogen-activated protein kinase activation are mediated by oxidative stress in multidrug-resistant human breast carcinoma cells; Lee YJ et al.; We previously observed that glucose deprivation induces cell death in multidrug-resistant human breast carcinoma cells (MCF-7/ADR) . As a follow up we wished to test the hypothesis that metabolic oxidative stress was the causative process or at least the link between causative processes behind the cytotoxicity . In the studies described here, we demonstrate that mitogen-activated protein kinase (MAPK) was activated within 3 min of being in glucose-free medium and remained activated for 3 h . Glucose deprivation for 2-4 h also caused oxidative stress as evidenced by a 3-fold greater steady state concentration of oxidized glutathione and a 3-fold increase in pro-oxidant production . Glucose and glutamate treatment rapidly suppressed MAPK activation and rescued cells from cytotoxicity . Glutamate and the peroxide scavenger, pyruvate, rescued the cells from cell killing as well as suppressed pro-oxidant production . In addition the thiol antioxidant, N-acetyl-L-cysteine, rescued cells from glucose deprivation-induced cytotoxicity and suppressed MAPK activation . These results suggest that glucose deprivation-induced cytotoxicity and alterations in MAPK signal transduction are mediated by oxidative stress in MCF-7/ADR . These results also support the speculation that a common mechanism of glucose deprivation-induced cytotoxicity in mammalian cells may involve metabolic oxidative stress.

Cytometry, 1998 Mar 1, 31(3), 187 - 98
A novel bioassay for P-glycoprotein functionality using cytochalasin D; Elbling L et al.; The functional contribution of both P-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP) to multidrug resistance (MDR) in tumor cells is commonly determined by drug cytotoxicity and/or accumulation/efflux tests . We report on a bioassay developed for the specific detection of functional P-gp levels and the efficacy of related chemosensitizers (CD-P-gp-assay) . The assay is based on the flow cytometric measurement of changes in the > or = G2M cell cycle compartment which are due to the induction of polykaryons after exposure of proliferating cells to three defined cytochalasin D (CD) concentrations with and without verapamil . As demonstrated in 13 well-characterized MDR cell models (20 resistant sublines), there is a significant correlation between cytokinesis-blocking CD doses, as well as responsiveness to chemosensitizers and MDR1 gene expression (mRNA and P-gp) allowing discrimination between different levels of P-gp-MDR . CD-P-gp-assay specificity was assessed by testing 23 compounds: 19 known as potent inhibitors of P-gp-MDR, some of them, though to a lesser extent, also of MRP-MDR; 1 inhibiting MRP-but not P-gp-MDR; 3 inactive in both types of MDR . A modulation of CD activity was confined exclusively to both P-gp-expressing cell lines and P-gp chemosensitizers . CD cytoskeletal activity measured by FACS is a specific and sensitive tool with which to detect functional P-gp and related chemosensitizers.

Cancer Res, 1998 Mar 15, 58(6), 1111 - 5
Eleutherobin, a novel cytotoxic agent that induces tubulin polymerization, is similar to paclitaxel (Taxol); Long BH et al.; Eleutherobin is a novel natural product isolated from a marine soft coral that is extremely potent for inducing tubulin polymerization in vitro and is cytotoxic for cancer cells with an IC50 similar to that of paclitaxel . This compound is cross-resistant along with other multidrug-resistant agents against P-glycoprotein-expressing cells and is cross-resistant with paclitaxel against a cell line that has altered tubulin . In mechanistic studies, eleutherobin shares with paclitaxel the ability to induce tubulin polymerization in vitro and is most likely cytotoxic by virtue of this mechanism . Human colon carcinoma cells exposed to eleutherobin contain multiple micronuclei and microtubule bundles, and they arrest in mitosis, depending on concentration, cell line, and length of exposure . These morphological abnormalities appearing in cultured cells are indistinguishable from those induced by paclitaxel . Electron microscopy reveals that eleutherobin induces homogeneous populations of long, rigid microtubules similar to those formed by paclitaxel . Thus, eleutherobin is a new chemotype with a mechanism of action similar to that of paclitaxel and, as such, has promising potential as a new anticancer agent.

Biochem Pharmacol, 1998 Mar 1, 55(5), 605 - 15
Frequent coexpression of MRP/GS-X pump and gamma-glutamylcysteine synthetase mRNA in drug-resistant cells, untreated tumor cells, and normal mouse tissues; Kuo MT et al.; Expression of the multidrug-resistance protein gene MRP, which confers non-P-glycoprotein-mediated multidrug resistance, has been found in many drug-resistant variants and tumor samples . Recent studies have demonstrated that MRP functions as an ATP-dependent transporter functionally related to the previously described glutathione-conjugate (GS-X) pump . We have shown recently that the MRP and gamma-glutamylcysteine synthetase (gamma-GCS) heavy subunit mRNA levels are coordinately overexpressed in cisplatin (CP)-resistant human leukemia cells (Ishikawa et al., J Biol Chem 271: 14981-14988, 1996) and frequently co-elevated in human colorectal tumors (Kuo et al., Cancer Res 56: 3642-3644, 1996) . In the present study, we showed the coexpression patterns of thirteen additional human drug-resistant cell lines representing different tumor cell origins selected with different agents, except for one doxorubicin-selected line which demonstrated minor elevation in MRP mRNA with no detectable increase in gamma-GCS mRNA, suggesting that the increase of MRP mRNA preceded the increase in gamma-GCS mRNA . Furthermore, in seventeen randomly selected untreated tumor cell lines, the overall correlation coefficient between MRP and gamma-GCS mRNA levels was 0.861 . In normal mice, the correlation coefficient of mrp and gamma-gcs mRNA was 0.662 in fourteen tissues (kidney and liver were not included) analyzed . Kidney and liver expressed low levels of mrp relative to gamma-gcs; however, these two tissues expressed high levels of a functionally related mrp homologue, mrp2 (cMoat or cMrp), which may have compensated for the underexpressed mrp in maintaining the total GS-X pump activities . Altogether, these results demonstrated the frequent coexpression of these two genes in various cell settings.

Biochem Biophys Res Commun, 1998 Mar 17, 244(2), 481 - 8
Bromocriptine modulates P-glycoprotein function; Orlowski S et al.; The multidrug resistance (MDR)-associated P-glycoprotein (P-gp) is a membrane transporter which carries, at the expense of MgATP hydrolysis, many amphiphilic molecules, such as the MDR-related cytotoxic drugs vincristine and vinblastine, and the MDR-reversing agents verapamil and progesterone . We have tested the effects on P-gp function of bromocriptine (BCT), an ergot alkaloid known as a D2 dopaminergic receptor agonist . BCT (at 4 microM) partially reverses the P-gp-mediated vincristine resistance of the Chinese hamster lung fibroblasts DC-3F/ADX, a MDR cell line . P-gp containing membrane vesicles prepared from the DC-3F/ADX cells exhibit, in the absence of any added drug, a basal MgATPase activity due to P-gp . BCT inhibits this basal ATPase activity, with a half-inhibiting concentration of 0.30 +/- 0.15 microM . BCT also inhibits the verapamil-induced P-gp ATPase stimulation competitively (Ki approximately 0.2 microM), and the progesterone-induced P-gp ATPase stimulation non-competitively (Ki approximately 0.07-0.10 microM) . BCT also non-competitively inhibits the vinblastine-dependent P-gp ATPase activity within the same concentration range . Hydroxylated metabolites of BCT have different effects on P-gp ATPase, only the monohydroxylated being able to modulate both the basal and the drug-stimulated ATPase activities . In conclusion, these effects of BCT on P-gp function can be linked to a specific interaction with P-gp, probably involving inhibition of P-gp-mediated drug transport.

Biochem Pharmacol, 1998 Feb 15, 55(4), 523 - 31
Inhibition of the membrane translocation and activation of protein kinase C, and potentiation of doxorubicin-induced apoptosis of hepatocellular carcinoma cells by tamoxifen; Cheng AL et al.; Hepatocellular carcinoma (HCC) is characterized by high drug resistance to currently available chemotherapeutic agents . In a prospective clinical study, we have demonstrated that high-dose tamoxifen significantly enhanced the therapeutic efficacy of doxorubicin in patients with far-advanced HCC . In a search for a possible mechanism, we found that tamoxifen at a clinically achievable concentration (2.5 microM) significantly enhanced doxorubicin-induced cytotoxicity and apoptosis of Hep-3B cells, a multidrug resistance (MDR)-1 expressing HCC cell line . This synergistic cytotoxic effect of tamoxifen, at this concentration, however, was not mediated by MDR inhibition . Instead, as evidenced by both western blot and immunofluorescence studies, tamoxifen inhibited the cytoplasmic-membrane translocation of protein kinase C (PKC)-alpha . 12-O-Tetradecanoylphorbol-13-acetate (TPA) restored the membrane translocation of PKC-alpha and abrogated the synergistic cytotoxicity of tamoxifen . We also showed that tamoxifen, at this concentration, did not directly affect the enzyme activity of PKC . Further, membrane translocation of other membrane-bound proteins, such as Ras protein, was similarly inhibited by tamoxifen, but could not be restored by the addition of TPA . Together, these data suggested that tamoxifen may act on the cytoplasmic membrane, and thereby inhibit PKC-alpha translocation to the membrane where it is activated . We hypothesize that high-dose tamoxifen may be an effective modulator of doxorubicin in the treatment of HCC, and suggest that biochemical modulation of PKC as a measure to improve systemic chemotherapy for HCC deserves further investigation.

Br J Cancer, 1998 Mar, 77(5), 694 - 702
Comparative evaluation by semiquantitative reverse transcriptase polymerase chain reaction of MDR1, MRP and GSTp gene expression in breast carcinomas; Lacave R et al.; Identification and quantitative evaluation of drug resistance markers are essential to assess the impact of multidrug resistance (MDR) in clinical oncology . The MDR1 gene confers pleiotropic drug resistance in tumour cells, but other molecular mechanisms are also involved in drug resistance . In particular, the clinical pattern of expression of the other MDR-related genes is unclear and their interrelationships are still unknown . Here, we report standardization of the procedures used to determine a reliable method of semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) using a standard series of drug-sensitive and increasingly resistant cell lines to evaluate the expression of three MDR-related genes, i.e . MDR1 (multidrug resistance gene 1), MRP (multidrug resistance related protein) and GSTp (glutathione-S-transferase p), reported to be endogenous standard genes for normalization of mRNAs . A total of 74 breast cancer surgical biopsies, obtained before any treatment, were evaluated by this method . When compared with classical clinical and laboratory findings, GSTp mRNA level was higher in diploid tumours . However, the main finding of our study suggests a clear relationship between two of these MDR-related gene expressions, namely GSTp and MRP . This finding provides new insight into human breast tumours, which may possibly be linked to the glutathione conjugate carrier function of MRP . Well defined semiquantitative RT-PCR procedures can therefore constitute a powerful tool to investigate MDR phenotype at mRNA levels of different related genes in small and precious tumour biopsy specimens.

Monaldi Arch Chest Dis, 1997 Oct, 52(5), 450 - 4
Antituberculosis drug-resistance surveillance as a tool for tuberculosis control programmes: a retrospective study; La Raja M et al.; The aim of this study was to verify the suitability of antituberculosis (antiTB) drug-resistance surveillance as a tool for tuberculosis (TB) control programmes at local level . A retrospective study reviewing laboratory records and medical records of TB patients referred to Udine Hospital between 1981 and 1995 was analysed . The initial susceptibility pattern for each Mycobacterium tuberculosis isolate was recorded . It was found that between 1981 and 1995, 899 M . tuberculosis strains underwent susceptibility testing for four first-line drugs . Over a period of 15 yrs the annual number of M . tuberculosis strains initially decreased and then stabilized . Overall, 15.3% of the 899 strains showed initial resistance to at least one first-line drug, and 2.8% to two or more first-line drugs . Streptomycin-resistant strains were the most commonly observed (10.8%), with resistance to isoniazid, rifampicin and ethambutal shown to be 6.4, 1.0 and 0.4%, respectively . Multidrug resistant (MDR)-TB was observed in only five cases . An additional four cases eventually developed secondary MDR-TB during the follow-up . The proportion of resistant strains did not vary significantly over time . Recurrent TB disease was significantly associated with resistant strains (odds ratio = 3.59, p < 0.01) . Only one patient had a documented human immunodeficiency virus (HIV)-positive serology . All six patients who developed MDR-TB during or after treatment, were suffering either from chronic alcoholism or from a psychotic disorder . In the study it was shown that recurrent tuberculosis cases, tuberculosis patients with behavioural problems (i.e . alcoholism, psychiatric disorder) and patients presenting with primary resistant Mycobacterium tuberculosis strains are at risk of multidrug-resistant tuberculosis and may thus benefit from the directly observed treatment approach, which has been proposed as a mainstay in tuberculosis control programme strategy.

Anticancer Drugs, 1998 Feb, 9(2), 157 - 65
Reversal of multidrug resistance by novel verapamil analogs in cancer cells; Choi SU et al.; The present study was performed to evaluate the ability of KR-30032 and KR-30035 to overcome multidrug resistance (MDR) by measuring the cytotoxicity and the accumulation rate of rhodamine . Additionally, the adverse cardiac toxicity of KR-30032 and KR-30035 was evaluated by measuring the changes of tension in isolated rat aorta and left ventricular pressure (LVP) in guinea pig heart . KR-30035 potentiated the paclitaxel-induced cytotoxicity to HCT15 {P-glycoprotein (P-gp)-expressed cells} to over 15-fold greater than that of verapamil and KR-30032 was equipotent with verapamil (EC50: 0.07, 5.0 and 3.3 nM at 1.0 microg/ml) . KR-30032 and KR-30035 were without effect on cytotoxicity to SK-OV-3 cells (P-gp-non-expressing cells), as well as to tamoxifen-induced cytotoxicity in the above cell types . Maximal rhodamine accumulation rates with KR-30032, KR-30035 and verapamil were 290, 291 and 271% in HCT15 cells; and 451, 970 and 440% in HCT15/CL02 cells, respectively . KR-30032 and KR-30035 were 20- to 25-fold less potent than verapamil in relaxing aorta (EC50: 8.13, 6.40 and 0.32 microM, respectively) and were 12- to 35-fold less potent than verapamil in decreasing LVP in isolated hearts (EC50: 41.8, 14.1 and 1.2 microM, respectively) . The results of this study suggest that KR-30032 and KR-30035 are active modulators of MDR with potentially minimal cardiovascular toxicity.

Anticancer Drugs, 1998 Feb, 9(2), 135 - 40
A phase I/II trial of paclitaxel for non-Hodgkin's lymphoma followed by paclitaxel plus quinine in drug-resistant disease; Miller TP et al.; Patients with non-Hodgkin's lymphoma (NHL) recurrent after chemotherapy exhibit clinical characteristics compatible with the phenomenon of multidrug resistance (MDR) and frequently have detectable levels of P-glycoprotein (P-gp) . Paclitaxel has been used in recurrent NHL with limited success . To test whether clinical resistance to paclitaxel can be reversed, we treated patients having paclitaxel-resistant NHL with paclitaxel plus quinine and measured the effects of quinine on paclitaxel pharmacokinetics . Eligible patients had recurrent and measurable NHL . Patients initially received paclitaxel, 120 mg/m2 (dose determined by a phase I trial of paclitaxel plus quinine), as a 20-24 h infusion every 3 weeks until there was evidence of clinical resistance . Patients then received paclitaxel at the same dose rate plus oral quinine at a fixed dose rate of 400 mg three times each day . Paclitaxel pharmacokinetics were studied in each patient using paired samples from plasma obtained at the end of the 24 h paclitaxel infusion as an estimate of the steady-state drug level . Of 14 patients treated with paclitaxel alone, one patient obtained a partial response (7%) . At the time of disease progression, one patient (same patient) obtained a partial response with paclitaxel plus quinine (7%) . Steady-state paclitaxel levels were obtained in 12 patients . In 11 of 12 patients the steady-state paclitaxel level was substantially lower with the addition of quinine . The average ratio of end of infusion plasma levels (paclitaxel alone/paclitaxel plus quinine) was 0.6 (range 0.31-0.97) indicating a 40% decrease in paclitaxel levels with the addition of quinine (p=0.001) . We conclude that paclitaxel given by this dose and schedule has modest activity in recurrent NHL . The addition of quinine to paclitaxel also has limited activity, but the combination did reverse paclitaxel resistance in one patient, adding support to the hypothesis that clinical drug resistance can be overcome with chemosensitizers in individual patients . Pharmacokinetic studies indicate that the reversal of drug resistance in this study cannot be attributed to changes in clearance of paclitaxel (which appears to increase with quinine), but more likely to the sensitization of lymphoma cells.

Br J Haematol, 1998 Mar, 100(3), 509 - 15
Multidrug resistance analysis in lymphoproliferative disease of large granular lymphocytes; Lamy T et al.; Multi-drug resistance (MDR) phenotype contributes to the ineffectiveness of chemotherapy . P-glycoprotein (PgP) and lung resistance protein (LRP) are proteins implicated in chemoresistance . We analysed the expression of PgP and LRP respectively in 17 and 15 cases of lymphoproliferative disease of granular lymphocytes (LDGL) including 10 cases of clonal large granular lymphocytic (LGL) leukaemia, six cases of oligoclonal (n = 5) and polyclonal (n = 1) CD3+ lymphoproliferation and one case of CD3- NK lymphocytosis . Functional PgP activity, as determined by Rh123 dye efflux assay, was found in all the patients . The mean percentage of effluxing cells was 47 +/- 22%, compared to 35 +/- 8% on normal lymphocytes (P<0.04) . The efflux was blocked in the presence of verapamil, a PgP revertant agent . A high proportion of CD57+ cells (66 +/- 10%) from these patients expelled Rh123 . Functional PgP activity was associated with expression of MDR1 mRNA . By using immunocytochemistry, LRP expression was detected in 11/15 patients (73%) . 7/10 LGL leukaemia patients presented a LRP+/Efflux+ phenotype and 5/7 had LRP+/Efflux+/MDR1 mRNA+ phenotype . These findings suggest that the PgP+/LRP+ phenotype is frequently observed in LDGL . Its clinical relevance in aggressive cases remains to be determined.

Hum Gene Ther, 1998 Feb 10, 9(3), 287 - 93
Co-expression of human adenosine deaminase and multidrug resistance using a bicistronic retroviral vector; Zhou Y et al.; Current gene therapy protocols designed to treat adenosine deaminase (ADA) deficiency and other metabolic disorders suffer from low-efficiency delivery to target cells and a lack of long-term stability in expression of the therapeutic proteins . These problems may be resolved by use of an in vivo dominant selection . The multidrug transporter (MDR1) has been suggested as a useful selective marker for gene therapy . In this work, we co-expressed ADA and MDR1 cDNA in a retroviral vector using an internal ribosome entry site (IRES) from encephalomyocarditis virus . This system produced a bicistronic mRNA containing both ADA and MDR1, which enables co-expression of ADA and MDR1, and also allows the two proteins to be translated separately . After in vitro selection using a cytotoxic MDR1 substrate, vincristine, we demonstrated that functional ADA was co-expressed with MDR1 in proportion to the expression level of MDR1, whereas MDR1 expression was proportional to the stringency of the vincristine selection . Because the efficiency of IRES-dependent translation was much lower than that of cap-dependent translation in this system, we observed lower expression of the genes positioned after the IRES . This asymmetric expression caused a lower viral titer when MDR1 was placed downstream from the IRES, but it also provided a way of modulating the relative expression of ADA and MDR1 . The retroviral system described in this work may serve as a useful tool to evaluate the strategies involving in vivo dominant selection for gene therapy of ADA-deficient patients.

Am J Trop Med Hyg, 1998 Feb, 58(2), 195 - 203
Transmission intensity and Plasmodium falciparum diversity on the northwestern border of Thailand; Paul RE et al.; Genetic analysis of the number of Plasmodium falciparum genotypes per infected person in regions of holoendemic and hyperendemic malaria suggest that in areas of lower transmission intensity, significantly fewer parasite genotypes per infected person should be found . A predominance of single clone infections in the human population could generate the controversial clonal population structure proposed for P . falciparum by Tibayrenc and others . Characterization of P . falciparum from individuals on the Thai-Burmese border, an area of hypoendemic transmission, revealed a higher number of genotypes per infected person than that predicted . Possible reasons for this observation are discussed, with particular attention paid to human migration and multidrug resistance.

Clin Infect Dis, 1998 Feb, 26(2), 303 - 7
A continuing outbreak of multidrug-resistant tuberculosis, with transmission in a hospital nursery; Nivin B et al.; We investigated an increase in cases of multidrug-resistant tuberculosis (MDRTB) at a large urban facility where a prior nosocomial outbreak of MDRTB had occurred . Nosocomial transmission appeared to account for this outbreak as well, including a cluster of cases in a newborn nursery . Seven of 24 patients (29%) described in this investigation may have been exposed in the hospital nursery during an approximately 2-week period . We believe this to be the first documented outbreak of MDRTB in a hospital nursery . The transmission in the nursery demonstrates that the possibility of exposure to unrecognized active tuberculosis in nursery and hospital personnel is always present . Infection and active disease in the infants developed after a relatively short period of exposure . These findings underscore the need for adherence to published infection control guidelines in health care settings.

Cancer Res, 1998 Mar 1, 58(5), 947 - 55
Role of glutathione S-transferases in the resistance of human colon cancer cell lines to doxorubicin; Beaumont PO et al.; The anthracycline doxorubicin has little activity against colorectal cancers . It is hypothesized that this is attributable to a multifactorial resistance mechanism in which the glutathione S-transferases (GST) may play a role . We studied the relationship between GST expression and doxorubicin resistance in four human colon adenocarcinoma cell lines (HT-29, LoVo, SW620, and Caco-2), with the goal of modulating GST activity to overcome resistance . Caco-2 cells were the most resistant to doxorubicin, showing an IC50 value approximately 80- to 90-fold higher than HT-29 or LoVo and 600-fold higher than SW620 . Total GST catalytic activity was significantly higher in Caco-2 cells compared with the other lines . All four cell lines expressed GST-pi at the catalytic activity, protein, and mRNA levels; however, no significant differences were observed among the cell lines . GST-mu expression was not detectable at the protein and mRNA levels, and the four cell lines displayed very low catalytic activity toward a GST-mu-selective substrate . Caco-2 cells showed a unique, highly expressed GST-alpha-immunoreactive band that was not detected in the other lines; however, the glutathione peroxidase activity of Caco-2 cells was the lowest among the four cell lines . Neither ethacrynic acid nor glutathione analogues that function as GST class-selective inhibitors were able to potentiate the cytotoxic effects of doxorubicin in these colon cancer cell lines, as demonstrated in both microplate colorimetric and clonogenic assays . The multidrug resistance-associated protein and P-glycoprotein were either not detectable or expressed at such low levels that they are not likely to contribute to the differences in doxorubicin sensitivity observed among these cell lines.

Pharmacol Ther, 1998 Jan, 77(1), 1 - 28
A comparison of the phenomenology and genetics of multidrug resistance in cancer cells and quinoline resistance in Plasmodium falciparum; Bray PG et al.; Plasmodium falciparum is the causative agent of the most deadly form of human malaria . Chemotherapy traditionally has been the main line of defense against this parasite, and chloroquine, the drug of choice, has been one of the most successful drugs ever developed . Unfortunately, the evolution and spread of resistance to chloroquine and other quinoline-containing drugs means that these compounds are now virtually useless in many endemic areas . Future prospects for the use of quinoline compounds improved considerably when it was demonstrated that chloroquine resistance could be circumvented in vitro by a number of structurally and functionally unrelated compounds such as verapamil and desipramine . The phenomenon of resistance reversal by compounds such as verapamil is also a key feature of drug resistance in mammalian cells, and this has raised the possibility that the underlying mechanisms of drug resistance of the two cell types could be similar . This hypothesis has prompted a large number of studies into the genetics and biochemistry of resistance to quinoline-containing drugs in P . falciparum . Both the genetic and the biochemical studies have raised issues of controversy and stimulated much debate . These issues are discussed in this review, in the context of a comparison with the genetics and biochemistry of multidrug resistance in mammalian cells.

Blood, 1998 Mar 1, 91(5), 1749 - 56
Genetic polymorphism in MDR-1: a tool for examining allelic expression in normal cells, unselected and drug-selected cell lines, and human tumors; Mickley LA et al.; By using RNase protection analysis, residues 2677 and 2995 of MDR-1 were identified as sites of genetic polymorphism . Through use of oligonucleotide hybridization, the genomic content and expression of individual MDR-1 alleles were examined in normal tissues, unselected and drug selected cell lines, and malignant lymphomas . In normal tissues, unselected cell lines, and untreated malignant lymphoma samples, expression of MDR-1 from both alleles was similar . In contrast, in drug selected cell lines, and in relapsed malignant lymphoma samples, expression of one allele was found in a large percentage of samples . To understand how expression of one allele occurs, two multidrug resistant sublines were isolated by exposing a Burkitt lymphoma cell line to increasing concentrations of vincristine . The resistant sublines expressed only one allele and had a hybrid MDR-1 gene composed of non-MDR-1 sequences proximal to MDR-1 . Previous studies showing hybrid MDR-1 genes after rearrangements provided a potential explanation for activation and expression of one MDR-1 allele . We conclude that oligonucleotide hybridization can be used as a sensitive tool to examine relative allelic expression of MDR-1, and can identify abnormal expression from a single allele . Acquired drug resistance in vitro and in patients is often associated with expression of a single MDR-1 allele, and this can be a marker of a hybrid MDR-1 gene.

Blood, 1998 Mar 1, 91(5), 1508 - 13
Expression of the lung resistance protein predicts poor outcome in de novo acute myeloid leukemia; Filipits M et al.; The 110-kD lung resistance protein (LRP) is overexpressed in P-glycoprotein-negative multidrug-resistant cell lines and most likely involved in the multidrug resistance (MDR) of these cell lines . To determine the clinical significance of LRP, we have studied LRP expression of leukemic blasts and its association with clinical outcome in patients with de novo acute myeloid leukemia (AML) . LRP expression of leukemic blasts obtained from peripheral blood or bone marrow of previously untreated patients (n = 86) was determined by immunocytochemistry by means of monoclonal antibody LRP-56 . LRP expression at diagnosis was detected in 31 (36%) patients . LRP expression was independent of age and sex of the patients, French-American-British subtype, cytogenetic abnormalities, and lactate dehydrogenase levels, but correlated with white blood cell count (P = .01) . Eighty-two patients received standard induction chemotherapy that included cytarabine and MDR drugs (daunorubicin in most patients, additional etoposide in the majority of patients) . The complete remission rate of induction chemotherapy was 72% (95% confidence interval {CI} = 61% to 82%) for the total study population . The complete remission rate was 81% (95% CI = 67% to 91%) for patients without LRP expression but only 55% (95% CI = 36% to 74%) for patients with LRP expression (P = .01) . Overall survival and disease-free survival were estimated according to Kaplan-Meier in 82 and 59 patients, respectively . Overall survival was significantly longer in patients without LRP expression than in patients with LRP expression . At a median follow-up of 16 months, median overall survival was 17 months (95% CI = 12 to 38 months) for LRP-negative patients but only 8 months (95% CI = 4 to 12 months) for -positive patients (P = .006) . Disease-free survival was 9 months (95% CI = 7 to 11 months) for LRP-negative patients and 6 months (95% CI = 5 to 8 months) for -positive patients (P = .078) . Outcome was best in patients lacking both LRP and P-glycoprotein expression . In conclusion, LRP predicts for poor outcome and thus the LRP gene appears to be another clinically relevant drug resistance gene in AML.

J Clin Invest, 1998 Feb 1, 101(3), 703 - 10
A multidrug-resistance protein (MRP)-like transmembrane pump is highly expressed by resting murine T helper (Th) 2, but not Th1 cells, and is induced to equal expression levels in Th1 and Th2 cells after antigenic stimulation in vivo; Lohoff M et al.; A transmembrane pump for organic anions was identified in resting murine T helper (Th) 2, but not Th1 lymphocyte cell clones, as revealed by extrusion of a fluorescent dye . Dye extrusion inhibition studies suggested that the pump may be the multidrug-resistance protein (MRP) . The different expression of the pump in resting Th1 and Th2 cell clones correlated with their respective levels of MRP mRNA . The pump was inducible in Th1 cells by antigenic stimulation in vitro leading to equal expression in activated Th1 and Th2 cell clones . This suggested that dye extrusion might allow the detection of Th2 (resting or activated) or of activated Th1 cells ex vivo based on a functional parameter . To test this, mice were infected with Leishmania major parasites to activate L . major-specific T cells of either Th1 (C57BL/6 mice) or Th2 (BALB/c mice) phenotype: 2-3% of CD4+ lymph node T cells of both strains of mice extruded the dye, defining a cell subset that did not coincide with subsets defined by other activation markers . Fluorescence-activated cell-sorting revealed that the lymphokine response (Th1 or Th2, respectively) to L . major antigens was restricted to this dye-extruding subset.

Hum Mol Genet, 1998 Feb, 7(2), 203 - 7
Mutations in the canilicular multispecific organic anion transporter (cMOAT) gene, a novel ABC transporter, in patients with hyperbilirubinemia II/Dubin-Johnson syndrome; Wada M et al.; Members of the ATP-binding cassette (ABC) transporter superfamily are mutated to cause diseases that include cystic fibrosis, hyperinsulinemia, adrenoleukodystrophy, Stargardt disease and multidrug resistance . We recently isolated a novel human member of ABC transporter superfamily as the candidate transporter for the glucuronide and glutathione-conjugated antitumor agents, and found it highly homologous to the rat cmoat gene . consistent with recent findings of defects in the homologous cmoat gene in two rat models of hyperbilirubinemia (TR- and Eisai), we report two deletions and a missense mutation in the active transport family signature region in the gene in patients with hyperbilirubinemia II/Dubin-Johnson syndrome (DJS; MIM 237500), respectively . These results strongly implicate the cMOAT gene as responsible for the defects in DJS patients.

Int J Cancer, 1998 Mar 16, 75(6), 885 - 93
Detection of P-glycoprotein in the Golgi apparatus of drug-untreated human melanoma cells; Molinari A et al.; The intracellular location of the MDR1 gene product, known as P-glycoprotein (P-gp), has been detected by flow cytometry in 3 stabilized human melanoma cell lines which had never undergone cytotoxic drug treatment and did not express P-gp on the plasma membrane . In addition, MDR1 mRNA expression was revealed by RT-PCR in the same cell lines . Immunofluorescence microscopy, performed by using the same 2 monoclonal antibodies (MM4.17 and MRK-16) as employed in the flow-cytometric analysis, revealed the presence of P-gp intracytoplasmically, in a well-defined perinuclear region . Double immunofluorescence labelling and immunoelectron microscopy strongly suggested the location of the transporter molecule in the Golgi apparatus . The same observations have been obtained on a primary culture from a metastasis of human melanoma . Analysis of the expression of another membrane transport protein, the multidrug-resistance-related protein (MRP1), showed that it was present in the cytoplasm of all the melanoma cell lines examined . MRP1 also showed Golgi-like localization . The study by laser scanning confocal microscopy on the intracellular localization of the anti-tumoral agent doxorubicin (DOX) during the drug-uptake and -efflux phases, indicated the Golgi apparatus as a preferential accumulation site for the anthracyclinic antibiotic . P-gp function modulators (verapamil and cyclosporin A) were able to modify DOX intracytoplasmic distribution and to increase drug intracellular concentration and cytotoxic effect in melanoma cells . On the contrary, MRP1 modulators (probenecid and genistein) did not significantly influence either DOX efflux and distribution or the sensitivity of melanoma cells to the cytotoxic drug.

Int J Cancer, 1998 Mar 16, 75(6), 855 - 63
Development of an intrinsic P-glycoprotein-mediated doxorubicin resistance in quiescent cell layers of large, multicellular prostate tumor spheroids; Wartenberg M et al.; Growing multicellular prostate tumor spheroids develop quiescent cell subpopulations in central regions with features of intrinsic multicell-mediated drug resistance . Doxorubicin (dox) uptake was significantly reduced in large spheroids (diameter 400+/-70 microm), which consist predominantly of quiescent cells, as compared to small spheroids (diameter 100+/-50 microm), which consist entirely of proliferating cells . After removal of dox from the incubation medium, dox fluorescence declined more efficiently in large spheroids, which led to a decreased dox toxicity as revealed by colony-forming assays . Verapamil significantly increased dox retention in large spheroids and, consequently, augmented dox toxicity . At a depth 80 microm from the spheroid periphery, a significantly decreased dox fluorescence was observed in the deep, quiescent cell layers of large spheroids . The P-glycoprotein-mediated multidrug resistance (MDR)-reversing agents verapamil, cyclosporin A, quinidine, sodium orthovanadate and tamoxifen significantly increased dox fluorescence at this depth, whereas genistein, indomethacin, probenecid and brefeldin A, which reverse multidrug-resistance-associated protein (MRP) function, exerted no effect . Anti-P-glycoprotein immunohistochemistry of multicellular tumor spheroids revealed an increase of P-glycoprotein expression in large speroids as compared to small spheroids, which was most prominent in the Ki-67-negative, quiescent cell layers 60 to 100 microm distant from the periphery of the spheroid, indicating that the MDR phenotype is related to cell quiescence . This was corroborated by whole-cell patch-clamp experiments, where the C219 antibody, which is directed against the ATP-binding site of P-glycoprotein, significantly inhibited P-glycoprotein-associated, volume-activated chloride currents in quiescent, but not proliferating cells from multicellular tumor spheroids.

J Clin Pharmacol, 1997 Nov, 37(11), 1009 - 14
Optimizing the absorption of valspodar, a P-glycoprotein modulator, Part II: Quantifying its pharmacokinetic variability and refining the bioavailability estimate; Kovarik JM et al.; A randomized, sequential, bioreplication study was performed with 24 healthy volunteers to assess the pharmacokinetic variability of oral and intravenous administrations of valspodar, a multidrug resistance modulator for use as a chemotherapy adjunct . Subjects received 200 mg as a 2-hour intravenous infusion and 400 mg orally as microemulsion soft gelatin capsules each given on two separate occasions . Following replicate intravenous administrations, reproducibility in peak concentration and area-under-the-curve was demonstrated by interoccasion equivalence testing . Low to moderate inter- and intra-subject pharmacokinetic variability were observed with coefficients of variation ranged from 9% to 19% . Absolute bioavailability from the microemulsion formulation was 60% . Replicate oral administrations demonstrated uniform and repeatable absorption with interoccasion equivalence in peak concentration, time to reach the peak concentration, and area-under-the-curve . Coefficients of inter- and intra-subject variation for these parameters ranged from 10% to 20% indicating low to moderate pharmacokinetic variability . When variabilities were compared between the two routes of administration, the absence of differences indicated that the rate and extent of drug absorption from the microemulsion formulation was as uniform and repeatable as by intravenous infusion.

J Clin Pharmacol, 1997 Nov, 37(11), 1001 - 8
Optimizing the absorption of valspodar, a P-glycoprotein modulator, Part I: Selecting an oral formulation and exploring its clinical pharmacokinetics/dynamics; Mueller EA et al.; Valspodar is a cyclosporine D analog used as a chemotherapy adjunct for modifying multidrug resistance . Two studies were sequentially performed to select an optimal oral formulation and to characterize selected aspects of its clinical pharmacokinetics/dynamics . An initial four-way crossover study with 20 volunteers compared the pharmacokinetics of single fasting administrations of 200 mg by intravenous infusion and 600 mg orally as a conventional oral solution, a microemulsion oral solution, and a microemulsion soft gelatin capsule . The two microemulsion dosage forms demonstrated significantly faster and less variable rates of absorption compared with the conventional oral solution . The microemulsion dosage forms were bioequivalent with absolute bioavailability nearly double that of the conventional oral solution . Based on these results, the microemulsion capsule was further investigated in a four-way randomized crossover study with 24 volunteers who received single fasting administrations of 200, 400, and 600 mg and a 400-mg administration after a fat-rich meal . Dose proportionality in area under the curve (AUC) was demonstrated over this dose range . Administration after a fat-rich meal caused a slight time lag until absorption began, a delay in time to reach the peak concentration, and a moderate increase of 24% in AUC . Serial determinations of total bilirubin were explored as a potential pharmacodynamic marker for P-glycoprotein inhibition . A similar magnitude of reversible hyperbilirubinemia was seen at all dose levels suggesting that P-glycoprotein inhibition in the biliary canaliculi was maximal even at the lowest dose tested . The microemulsion formulation (oral solution or soft gelatin capsule) represents an improved and less variable oral delivery form providing dose-proportional drug exposure over a clinically relevant dose range.

Tsitologiia, 1997, 39(11), 1038 - 45
{Reorganization of elements of the cytoskeletal and vacuolar systems in tumor cells in the early stage of developing multiple drug resistance}; Erokhina M et al.; The multidrug resistance (MDR) is one of the main reasons for chemotherapeutic failures in cancer patients . The overexpression of MDR gene product (Pgp) leads to the appearance of resistant tumor cells . We investigated alterations of cell structures in cells with a low level MDR and Pgp expression . The Syrian hamster embryo fibroblasts transformed by the Raus sarcoma virus were used in our experiments . Resistant sublines were isolated by propagation of the cells in colchicine supplemented medium . We have found that the first steps of MDR are accompanied by significant alterations of cytoskeleton elements and vacuolar system, e.g . changes in actin stress-fibres organization, microtubule distribution, the Golgi complex structure, vesicular transport . We suppose that the above cell structural alterations are not accidental . Perhaps, they are also essential for evolution of multidrug resistant mechanisms.

J Exp Clin Cancer Res, 1997 Dec, 16(4), 419 - 24
Clinical approach to circumvention of multidrug resistance in refractory leukemic patients: association of cyclosporin A with etoposide; Maia RC et al.; Alternative therapy for refractory leukemic patients is being increasingly adopted . Circumvention of multidrug resistance represents a strategy that has been taken into account when conventional chemotherapy failed . In this work a group of 15 refractory, heavily pretreated, patients was enrolled in a circumvention protocol including etoposide (ETO) and cyclosporin A (CSA) . All patients received etoposide prior to this schedule . Toxicity to circumvention protocol was acceptable and only one serious side-effect was observed . Two hematological clinical responses were seen, both of which were positive to P-glycoprotein immunostaining and exhibited in vitro modulation by CSA in cultures using the thymidine incorporation assay . Three out of four patients negative for P-glycoprotein achieved a minor response . Three out of six clinical failures were also negative for Pgp immunostaining one of which exhibited sinergistic effect between ETO and CSA . Our study suggests that hematological response to ETO and CSA association can be obtained in intensely pretreated leukemic patients . Several factors may affect the response such as clinical status before this therapy . Additionally, it also suggests that not all CSA effects on the combination ETO-CSA can be attributed to Pgp modulation.

Biochem Biophys Res Commun, 1998 Feb 24, 243(3), 816 - 20
Expression of multidrug resistance-associated protein (MRP) in brain microvessel endothelial cells; Huai-Yun H et al.; Multidrug resistance-associated protein (MRP) is a recently identified drug efflux transport system that actively transports organic acids and selected glucuronide or glutathione conjugates out of the cell . The current study presents, for the first time, both functional and biochemical data demonstrating the presence of MRP in the brain microvessel endothelial cells that form the blood-brain barrier (BBB) . Using known MRP inhibitors, such as indomethacin and probenecid, fluorescein accumulation in primary cultured bovine brain microvessel endothelial cell (BBMEC) monolayers was significantly enhanced compared to control . The specificity of the MRP inhibitors on cellular fluorescein accumulation was confirmed using both MRP positive (Panc-1) and MRP negative (KBv) cell lines . Furthermore, western blot analysis using a specific antibody for MRP (MRPm6) and RT-PCR studies using a complementary sequence probe for human MRP demonstrate the expression of MRP in BBMEC . Previous studies have demonstrated the significance of the P-glycoprotein drug efflux transporter in the BBB . Given its function as a drug efflux transport system, it is anticipated that MRP in the BBB will also have an important role in limiting the exposure of the brain to many endogenous and exogenous compounds, including both toxic and therapeutic agents.

Chest, 1998 Feb, 113(2), 379 - 86
Time to detection of Mycobacterium tuberculosis in sputum culture correlates with outcome in patients receiving treatment for pulmonary tuberculosis; Epstein MD et al.; STUDY OBJECTIVE: The purpose of this study was to determine whether the time to detection (TTD) of Mycobacterium tuberculosis in sputum culture correlates with the response to antituberculous treatment in patients with pulmonary tuberculosis . STUDY DESIGN: Twenty-six consecutive patients were studied who had active pulmonary tuberculosis and sufficient sputum cultures and clinical follow-up to allow adequate assessment . RESULTS: Following initiation of antituberculous therapy, 13 patients (group 1, responders) had a complete response to treatment, and the TTD of M tuberculosis using the mycobacterial growth indicator tube increased steadily . The remaining 13 patients (group 2, nonresponders) had persistent evidence of active disease and demonstrated little or no increase in the TTD with treatment unless an additional therapeutic intervention was implemented (surgery, improved compliance with medications, or a change in medications) . The presence of HIV infection, intravenous drug use, multidrug resistance, treatment with second-line therapy, extensive radiographic involvement, and cavitary disease were associated with a delayed increase in the TTD . CONCLUSIONS: The TTD was superior to clinical, radiographic, or conventional bacteriologic evaluation in determining treatment outcome . The TTD closely correlates with the overall response to treatment for pulmonary tuberculosis and may represent a useful adjunct to predict outcome in these patients.

Leuk Lymphoma, 1997 Dec, 28(1-2), 23 - 31
The role of MDR-1 in refractory lymphoma; Sandor V et al.; Although lymphoma is one of the few solid tumours for which chemotherapy can be curative, the treatment of refractory lymphoma remains a major clinical problem . P-glycoprotein (Pgp), the drug efflux pump encoded by the MDR-1 gene is associated with multidrug resistance in several laboratory models of drug resistance, and a number of investigators have attempted to establish a role for Pgp in refractory lymphoma . Despite a considerable variability in the results of these studies investigating Pgp expression in lymphoma, the preponderance of the data suggests that Pgp may at least in part account for drug resistance in this disease . Several clinical trials using Pgp modulating compounds have attempted to reverse the drug resistant phenotype of refractory lymphoma . These studies, although difficult to interpret because of the effect of Pgp modulators on chemotherapeutic drug pharmacokinetics, also suggest a role for Pgp in mediating drug resistance in a subset of patients with refractory lymphoma . Studies with newer Pgp modulating agents with phase III designs will be needed before Pgp modulation can be considered for incorporation into routine oncologic practice.

Rev Mal Respir, 1997 Dec, 14 Suppl 5, S88 - 104
{Antitubercular chemotherapy}; Jouveshomme S et al.; Treatment of tuberculosis has three major goals: healing the patient, preventing selection of resistant strains and control transmission of tuberculosis . A 6 month regimen consisting of isoniazid, rifampin with addition of pyrazinamide for 2 months is the preferred treatment for pulmonary and extra-pulmonary tuberculosis . If resistance to isoniazid is suspected, ethambutol should be added until drug susceptibility studies become available . This treatment is effective in both HIV infected and uniinfected persons . Treatment failure is mostly related to lack of patient adherence to the drug regimen and to multidrug-resistant tuberculosis . The treatment of multidrug-resistant tuberculosis requires second line drugs which are less effective and poorly tolerated . Prevention of resistant tuberculosis needs adequate treatment of each case of tuberculosis and improving of the patient compliance.

Ann Oncol, 1997 Dec, 8(12), 1221 - 8
Comparative antitumor efficacy of docetaxel and paclitaxel in nude mice bearing human tumor xenografts that overexpress the multidrug resistance protein (MRP)
Vanhoefer U, Cao S, Harstrick A, Seeber S, Rustum YM.
BACKGROUND: Multidrug resistance has been associated with expression of the multidrug resistance protein (MRP) . Recently, MRP-expression has been detected in human tumor samples of patients with breast cancer and non-small-cell lung cancer . Since taxoids are the most active drugs in the treatment of both tumor entities, the antitumor efficacies of paclitaxel and docetaxel were compared in nude mice bearing human tumor xenografts that express MRP . MATERIALS AND METHODS: Athymic nude mice (nu/nu) bearing tumor xenografts of parental human sarcoma HT1080 or MRP-expressing HT1080/DR4 cells (as confirmed by Northern blot analysis) were treated with the maximum tolerated doses (MTD) of doxorubicin ({Dx} 10 mg/kg i.v . push), paclitaxel ({PC} 50 mg/kg three-hour i.v . infusion), or docetaxel ({DC} 40 mg/kg three-hour i.v . infusion) . In vitro, the activity of doxorubicin, paclitaxel and docetaxel was evaluated by the sulphorhodamine B (SRB) assay using the pyridine analogue PAK-104P (5 microM), a potent inhibitor of MRP-function . RESULTS: At their MTDs both taxoids showed significant activity against MRP-negative HT1080 xenografts with response rates of 80% (40% CR) for PC and 100% (60% CR) for DC . In contrast, DC was significantly more active than PC in nude mice bearing doxorubicin resistant MRP-expressing HT1080/DR4 tumor xenografts (overall response rates: 100% (60% CR) for DC; 10% (0% CR) for PC; 0% for Dx) . Since treatment of mice with the MTD of PC or DC yielded similar overall toxicity (maximum weight loss for HT1080: PC 8.6 +/- 2.2%; DC 7.5 +/- 2.2% and for HT1080/DR4: PC 11.6 +/- 3.0%; DC 7.6 +/- 1.8%, respectively), these results demonstrate the increase in the therapeutic index for docetaxel against MRP-expressing tumors . In vitro, HT1080/DR4 cells were 270-fold, 6.4-fold and 2.8-fold more resistant than parental cells to doxorubicin, PC and DC, respectively . Pyridine analogue PAK-104P completely restored drug sensitivity to PC and DC, while no effect of PAK-104P on parental HT1080 cells was observed . CONCLUSIONS: Both taxoids, when given at their MTDs, showed significant efficacy against parental HT1080 tumor xenografts . However, docetaxel at its MTD was significantly more active against MRP-expressing tumor xenografts than paclitaxel . Furthermore, in vitro resistance of HT1080/DR4 cells was higher for PC (6.4-fold) than for DC (2.8-fold) . Since PAK-104P completely restored sensitivity to both taxoids, the observed resistance appears to be related to MRP . These data suggest, that docetaxel is not as readily transported by MRP as paclitaxel leading to an increased therapeutic ratio in MRP-expressing tumors in vivo . Therefore, docetaxel may have therapeutic advantages in the clinical treatment of MRP-expressing tumors.

Int J Cancer, 1998 Mar 2, 75(5), 757 - 61
Co-ordinated over-expression of the MRP and gamma-glutamylcysteine synthetase genes, but not MDR1, correlates with doxorubicin resistance in human malignant mesothelioma cell lines; Ogretmen B et al.; While human malignant mesothelioma is extremely resistant to chemotherapy, its intrinsic resistance mechanisms remain largely unknown . In this study, we used normal human mesothelial cells and 5 human mesothelioma cell lines not previously exposed to chemotherapeutic agents to demonstrate that the mRNA for the multidrug resistance-associated protein (MRP) and gamma-glutamylcysteine synthetase (gamma-GCSh) heavy subunit genes, but not the P-glycoprotein (MDR1) gene, are co-ordinately over-expressed in mesothelioma cell lines . Expression of MRP as detected with an anti-MRP antibody correlated with decreased doxorubicin accumulation and resistance of mesothelioma cells to this drug . Our results strongly suggest roles for MRP and gamma-GCSh in chemoresistance in mesotheliomas.

Oncol Res, 1997, 9(9), 485 - 94
Cellular determinants of resistance to indolocarbazole analogue 6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13(beta-D-glucopyranosyl)- 5H-indolo{2,3-alpha}pyrrolo{3,4-c}carbazole-5,7(6H)-dione (NB-506), a novel potent topoisomerase I inhibitor, in multidrug-resistant human tumor cells; Vanhoefer U et al.; Membrane protein-associated alterations in cellular drug accumulation have been recently implicated in resistance to topoisomerase I (TOP-I)-interactive drugs . The present study investigated the cellular determinants of resistance to the indolocarbazole compound NB-506 {6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13(beta-D-glucopyranosyl)- 5H-indolo{2,3-alpha}pyrrolo{3,4-c}carbazole-5,7(6H)-dione}, a structurally novel TOP-I-interactive drug, in parental and multidrug-resistant tumor cells expressing either the P-170 glycoprotein (Pgp170) or multidrug resistance protein (MRP) . MRP-expressing 250-fold doxorubicin-resistant human fibrosarcoma HT1080/DR4 tumor cells were drug sensitive to NB-506 and camptothecin (CPT) (resistance factor: 0.7 and 0.8, respectively) with no alterations of TOP-I parameters including DNA relaxation, expression of TOP-I protein and mRNA . In contrast, doxorubicin-resistant human ovarian A2780/Dx5 tumor cells {pgp170 phenotype} were 6.2-fold resistant to NB-506, whereas resistance to CPT was 2.6-fold . HPLC analysis of cellular NB-506 accumulation showed no significant differences between A2780 and A2780/Dx5 cells (peak intracellular concentrations after 120-min exposure to 10 microM NB-506: 400+/-85.0 and 352+/-95.1 nmol NB-506/mg protein, respectively) . However, resistant A2780/Dx5 cells expressed a lower amount of TOP-I mRNA and 29% protein levels of TOP-I compared to parental A2780 cells, resulting in decreased TOP-I catalytic activity (3.17+/-0.02 vs . 1.16+/-0.15 rel.U/microg nuclear protein) and reduced induction of NB-506-mediated cleavable complex formation in A2780/Dx5 cells . Furthermore, the lower induction of NB-506-induced protein-linked DNA breaks (PLDB) in A2780/Dx5 cells correlated with significantly decreased DNA 12.2-440 kb size fragmentation in these cells . The present study demonstrates that expression of MRP and Pgp170 does not confer resistance to NB-506 . Resistance to indolocarbazole substance NB-506 in A2780/Dx5 cells was only related to downregulation of TOP-I associated with lower induction of cleavable complex formation and DNA fragmentation . The data reported herein may indicate that the new indolocarbazole compound NB-506 has potent antitumor efficacy in membrane-associated multidrug resistance.

Oncol Res, 1997, 9(9), 477 - 84
Inhibitory effect of alkylating modulators on the function of P-glycoprotein; Yang JM et al.; Modulators of P-glycoprotein (P-gp) are often themselves transported out of cells, thereby limiting their effectiveness . It may be possible to develop more effective modulators of multidrug resistance by designing drugs that irreversibly block the function of P-gp . Therefore, we studied the effect of the mustard derivatives of fluphenazine (FPN) and trans-flupenthixol (FPT) on P-gp function . Both fluphenazine-mustard (FPN-M) and trans-flupenthixol-mustard (FPT-M) possessed alkylating activity, as assayed using 4-(p-nitrobenzyl) pyridine . Multidrug-resistant MCF-7/AdrR cells were incubated with FPN or FPN-M, or FPT or FPT-M for 1 h, washed for varying number of times in phosphate-buffered saline (PBS), then resuspended in medium containing {3H}vinblastine (VBL), and assayed for steady-state accumulation of the drug . Washing had far less of an effect on the ability of FPN-M and FPT-M to increase VBL accumulation compared to their parent compounds . After eight washes in excess PBS, the cells initially exposed to FPN or FPT accumulated only 30% and 50% of the initially accumulated drug, whereas the FPN-M- or FPT-M-treated cells accumulated approximately 75% and 90% of the control, respectively . FPN-M and FPT-M also increased the uptake and decreased the efflux of VBL from MDR cells despite repeated washing . We also examined the effects of these modulators on sensitivity of MDR cells to cytotoxic agents . FPN-M and FPT-M sensitized MCF-7/AdrR cells to VBL and doxorubicin to a greater extent than their parent compounds . These studies point out the potential of "irreversible" P-gp modulators to produce prolonged chemosensitization.

Ann Acad Med Singapore, 1997 Sep, 26(5), 659 - 63
Management of multiple drug-resistant malaria in Viet Nam; Hien TT et al.; Malaria is still the most common infectious cause of mortality and morbidity in Viet Nam as it is in many developing countries in the tropics . The presence of resistance to available antimalarials and compliance in the target population are factors that influence the choice of drugs and regimens . In order to develop an ideal treatment for malaria, we conducted several clinical trials in patients with the disease in different settings . The results of these trials suggest that a combination of single dose artemisinin (or its derivatives) and mefloquine is the most effective, safe and practical treatment for acute non-complicated malaria due to multidrug-resistant Plasmodium falciparum . Concerning severe and complicated malaria, parenteral or rectal multi-doses of artemisinin or analogues are recommended due to their rapid parasite clearance time and other possible anti-cytoadherence effects . With its rapid parasite clearance, very early treatment of uncomplicated cases with artemisinin (and derivatives), especially at a health post level may help to prevent the development of complications, consequently reducing the number of severe cases and the malaria mortality rate.

Ann Acad Med Singapore, 1997 Sep, 26(5), 549 - 56
Drug-resistant tuberculosis in Singapore, 1995 to 1996; Boudville IC et al.; Singapore's tuberculosis incidence of 49 to 57 per 100,000 population for 1987 to 1996 presents a picture that is intermediate between developed and developing countries . The proportion of drug-resistant isolates has remained stable at 4.3% to 5.5% from 1992 to 1996 despite rising HIV rates . From 1995 to 1996, of the 199 consecutive drug-resistant isolates at the Central Tuberculosis Laboratory, 66% were mono-resistant, 22% dual-resistant and 12% resistant to more than two drugs . Isoniazid resistance was most prevalent, being found in 72% of isolates, followed by streptomycin resistance in 45% . Resistance to isoniazid and streptomycin (21%) was more common than to isoniazid and rifampicin, i.e . multidrug resistance (MDR) (14%) . The small numbers indicated by the low overall prevalence of resistance and the predominance of single-drug resistance support the current initial choice of the standard short course with its three-drug initial phase . Of the 170 cases with matching National Tuberculosis Registry data, 72% of drug-resistant cases represented initial and 28% acquired resistance; testifying to the effectiveness of present day treatment regimens in suppressing resistance when compliance is assured . Case-control analysis using 244 drug-sensitive controls randomly selected from notifications in 1995 to 1996 showed an odds ratio for drug-resistance between subjects with a previous history and no previous history of tuberculosis of 2.47 (95% CI 1.40 to 4.37; P = 0.0007) . With each increment in the number of episodes of tuberculosis experienced, there was a trend of resistance to progressively more drugs (P = 0.000004) . This association remained even when a logistic regression model including all predictor variables was fitted . No associations were found with age, history of contact with tuberculosis, cavitary disease and, most notably, with human immunodeficiency virus infection . This study reaffirms that a history of previous tuberculosis should increase clinicians' index of suspicion for drug resistance, the urgency with which culture and sensitivity results are sought and the vigour with which patients are followed-up and compliance monitored.

Anticancer Res, 1997 Nov-Dec, 17(6D), 4647 - 51
Glutathione system, topoisomerase II level and multidrug resistance phenotype in acute myelogenous leukemia before treatment and at relapse; Massaad-Massade L et al.; In order to better understand acquired resistance to antitumor agents in acute myelogenous leukemia (AML), we investigated various drug resistance mechanisms; namely, topoisomerase II (topo II), glutathione system and P-glycoprotein (P-gp) . Blast cells of 31 patients with AML, 21 before treatment (BT) and 10 at relapse (AR) were studied . Topo II was evaluated by Western blot analysis . Glutathione-S-transferase activity (GST) and glutathione content (GSH) were investigated by spectrophotometric assays . GST isoenzymes (-alpha, -mu and -pi) were tested by Western blot and by immunocytochemical staining . P-gp was evaluated by an immunocytochemical method using MRK 16 antibody . Our results showed that GST, GSH and GST-pi were similar in patients BT and AR GST-mu was detected in 13/21 AML BT and in 5/10 AML AR . GST-alpha expression was higher (p < 0.05) in AML AR (60 +/- 105 AU/mg) compared to AML BT (10 +/- 10 AU/mg) . A relationship was found between GST-pi quantitation evaluated by Western blot and immunocytochemical staining, whereas no correlation was observed for the other isoenzymes . Topo II was detected in only 4 AML BT and 3 AML AR . Eleven out of 21 AML BT and 3/10 AML AR expressed P-gp with immunohistochemical study . These results indicate that only the "glutathione system", especially the GST-alpha could be involved in drug resistance in AML.

Anticancer Res, 1997 Nov-Dec, 17(6D), 4595 - 8
Correlation between multidrug resistance and the degree of differentiation of non-small-cell bronchopulmonary carcinoma (NSCLC) in vitro and in vivo; Bosch S et al.; The correlation between the degree of expression of the multidrug resistance-1 (MDR-1) gene and the process of differentiation into non-small-cell, bronchopulmonary carcinoma was studied in vitro and in vivo . For this purpose, a technique for the quantitative analysis of MDR-1 gene expression was developed by competitive reverse-transcriptase polymerase chain reaction . The study of 9 epidermoid carcinomas with various degrees of differentiation did not enable us to establish a correlation in vivo in the patient . However, an in vitro study performed on a non-small-cell lung carcinoma cell line and two of its clones showed that MDR-1 gene expression increased with the degree of differentiation, which was confirmed in vivo when this line was xenografted into nude mice.

Anticancer Res, 1997 Nov-Dec, 17(6D), 4577 - 82
Novel multidrug-resistance modulators, KR-30026 and KR-30031, in cancer cells; Choi SU et al.; The present study was performed to evaluate the ability of KR-30026 and KR-30031 to overcome multidrug resistance (MDR) by measuring the cytotoxicity of paclitaxel and the rate of rhodamine accumulation, which were then compared with verapamil . KR-30026 potentiated the paclitaxel-induced cytotoxicity of HCT15 to over 60 fold greater than that of verapamil, and KR-30031 was equipotent with verapamil (EC50: 0.00066, 0.04 and 0.05 nM at 4.0 micrograms/ml, respectively) . KR-30026 and KR-30031 were without effect on paclitaxel-induced cytotoxicity to SK-OV-3 cells, as well as on tamoxifen-induced cytotoxicity to HCT15, HCT15/CL02 and SK-OV-3 cells . Maximal rhodamine accumulation by KR-30026, KR-30031 and verapamil were similar in HCT15 cells, while KR-30026 was more potent than verapamil in HCT15/CL02 cells (721 and 440%, respectively) . To evaluate the cardiac toxicity of KR-30026 and KR-30031, the changes of tension in isolated rat aorta and left ventricular pressure (LVP) in guinea pig heart were determined; KR-30026 and KR-30031 were 15-40 and 25-70 fold less potent than verapamil, respectively . These results suggest that KR-30026 and KR-30031 are active modulators of MDR with potentially minimal cardiovascular toxicity.

Anticancer Res, 1997 Nov-Dec, 17(6D), 4531 - 4
Multiple drug-resistant C6 glioma cells cross-resistant to irradiation; Denecke J et al.; Although many advances in antineoplastic therapy have taken place, a clinical breakthrough in the therapy of malignant gliomas is still required . One of the reasons for this is the poor response to cytotoxic drugs and irradiation . We established a subline of the rat glioma cell line C6, named C6,5 x 10(-7) Dox, by exposure to increasing doses of doxorubicin for 5 months . C6,5 x 10(-7) Dox cells expressed high levels of P-glycoprotein (Pgp), known to function as an energy-dependent efflux pump for lipophilic drugs causing the multidrug resistance phenotype . Pgp, which normally has a molecular weight of 170 to 180 kd, appears in C6,5 x 10(-7) Dox cells as two bands with a molecular weight of 140 and 120 kd in western blots . In addition to the typical cross-resistance to doxorubicin, daunorubicin, vincristine and etoposide, we observed a significant resistance of the C6,5 x 10(-7) Dox cell line to irradiation, which cannot be explained by Pgp-expression.

Anticancer Res, 1997 Nov-Dec, 17(6D), 4359 - 70
Human melanoma cell lines selected in vitro displaying various levels of drug resistance against cisplatin, fotemustine, vindesine or etoposide: modulation of proto-oncogene expression; Kern MA et al.; Melanoma cells often display a multidrug-resistant phenotype, but the mechanisms involved are largely unknown . In order to establish a reproducable model system for studying the exact mechanisms conferring chemoresistance, we selected drug-resistant sublines in vitro derived from one parental human melanoma (MeWo) cell line . Four commonly used chemotherapeutic drugs (vindesine, etoposide, fotemustine, cisplatin) with different modes of action were choosen and stable sublines exhibiting four different levels of resistance against each drug were selected by continuous exposure over two years . Analysis of the drug-resistant sublines regarding their pharmacological characteristics and cross-resistance pattern revealed an up to 26-fold increased relative resistance against the alkylating agent fotemustine (MeWoFOTE) and an up to 35.7-fold increased relative resistance against topoisomerase-II-inhibiting etoposide (MeWoETO) . Cisplatin selection (MeWoCIS) resulted in a 6-fold higher resistance compared to parental MeWo cells, whereas vindesine exposure (MeWoVIND) increased relative resistance up to 10.2-fold . Sublines selected separately for resistance to the DNA-damaging agents fotemustine, cisplatin and etoposide demonstrated strong cross-resistance . In comparison to the parental cell line drug-resistant sublines showed altered expression patterns of proto-oncogenes . Levels of p53 mRNA decreased with increasing resistance to vindesine, etoposide and fotemustine . Expression of bcl-2 family members (bax, bcl-x) was modulated by fotemustine, etoposide and cisplatin . In addition the expression of members of the fos (c-fos) and jun (c-jun, jun-D) gene family encoding transcription factors of the AP-1 complex was altered in all drug-resistant sublines . The pattern of expression varied with the inducing stimulus and this was paralleled by changes in the transactivation potential of AP-1 . Our results reinforce the central role of AP-l in drug resistance probably through its participation in a programmed cellular stress response.

Parasitol Res, 1998, 84(2), 106 - 11
Identification of three ABC transporter genes in Trypanosoma brucei spp; Maser P et al.; ABC transporters are key players in the multidrug resistance of cancer cells and yeast, and they appear to be involved in the drug resistance of various pathogenic protozoa . No member of this ubiquitous protein family has yet been described in Trypanosoma brucei spp., the causative agents of African sleeping sickness and animal trypanosomiases . However, different cases of artificially induced drug resistance were shown to be linked to a reduction in net drug uptake . We used polymerase chain reaction with degenerate oligonucleotide primers corresponding to particularly conserved regions within the ATP-binding cassette to probe the genome of T . brucei spp . for the presence of ABC transporter genes . Three different sequence segments encoding ATP-binding cassettes were identified, which, upon Southern blotting, appeared to belong to distinct genes designated Tbabc1, Tbabc2, and Tbabc3 . They appear to be single-copy genes in both drug-susceptible and drug-resistant stocks of T . brucei spp., expressed in bloodstream forms as well as in the procyclic life stage . Whereas Tbabc3 shows moderate homology to various known ABC transporters, Tbabc1 and Tbabc2 are highly homologous to P-glycoprotein A of Leishmania tarentolae and to the multidrug resistance protein 1 of L . donovani, respectively.

Eur J Biochem, 1998 Jan 15, 251(1-2), 155 - 63
Multidrug-resistance drug-binding peptides generated by using a phage display library; Popkov M et al.; A phage display library of random decapeptides was used to generate peptide ligands that can bind multidrug-resistance (MDR) drugs mimicking, in this respect, the drug-binding activity of P-glycoprotein . Seven peptide sequences were identified that specifically bound doxorubicin . Five of these sequences expressed the core consensus motif WXXW . The displacement assay showed that the phages expressing these peptides bound MDR type drugs (vinblastine, doxorubicin, verapamil, and genistein) with the same selectivity as P-glycoprotein and did not interact with non-MDR type drugs, such as arabinosylcytosine (Ara-C) and melphalan . One of the selected peptides that showed a highest capacity for the binding (VCDWWGWGIC) was synthesized and displayed competition with the phage for doxorubicin binding . The structure modeling suggested that all the selected sequences contained a hydrophobic envelope in which MDR drugs could be docked with substantial energy minimization . Western blot analysis showed that monospecific antibody obtained against the phage expressing VCDWWGWGIC peptide could specifically recognize P-glycoprotein in the membrane fraction of MDR phenotype MCF-7ADR cells . The MDR drug-binding sequences generated during this work could provide an important tool for design and screening of new chemotherapeutic agents.

Br J Clin Pharmacol, 1998 Feb, 45(2), 173 - 5
Role of lipoproteins in the plasma binding of SDZ PSC 833, a novel multidrug resistance-reversing cyclosporin; Simon N et al.; AIMS: The plasma binding of the cyclosporin D analogue SDZ PSC 833 was investigated in vitro . METHODS: The plasma total binding constant (corresponding to the bound-to-free concentration or binding ratio) was determined at 37 degrees C by the erythrocyte partitioning technique on plasma samples from three healthy volunteers and three cancer patients . Lipoproteins were also removed from plasma samples from three healthy volunteers by a standard ultracentrifugal technique . RESULTS: SDZ PSC 833 plasma binding was 97.8 +/- 1.1% and 97.3 +/- 0.2% in samples from three healthy volunteers and three cancer patients respectively . More than 95% of blood SDZ PSC 833 was distributed in plasma . When the original plasma samples of three individuals were delipidated, SDZ PSC 833 binding was strongly decreased (58% bound to plasma proteins) and when lipoproteins were resuspended in the delipidated plasma samples to produce varying lipoprotein plasma concentrations, the binding increased continuously with the fraction of added lipoproteins . When lipoproteins were resuspended to restore the original lipoprotein plasma content, the % plasma-bound SDZ PSC 833 increased to 98.2%, close to the value observed with the original plasma (98.7%) . CONCLUSIONS: These results clearly indicate that SDZ PSC 833 plasma binding is mainly determined by lipoproteins and that in blood, most of SDZ PSC 833 is distributed in plasma.

Anticancer Drugs, 1998 Jan, 9(1), 58 - 66
Cytotoxicity and intracellular biotransformation of N-benzyladriamycin-14-valerate (AD 198) are modulated by changes in 14-O-acyl chain length; Lothstein L et al.; N-benzyladriamycin-14-valerate (AD 198) is pharmacologically superior to Adriamycin (ADR) based upon comparable cytotoxicity, decreased cardiotoxicity and the ability of AD 198 to circumvent multidrug resistance conferred by either P-glycoprotein overexpression or reduced topoisomerase II activity . AD 198, however, suffers from systemic lability of the 14-O-valerate moiety to enzymatic and non-enzymatic cleavage to yield N-benzyladriamycin (AD 288), which is more similar to ADR in activity . The purpose of this study was to determine whether stability of the ester linkage could be achieved while preserving the favorable characteristics of AD 198 by using a series of N-benzylated ADR congeners containing 14-O-acyl substitutions of incrementally shorter carbon chain lengths . Results from this study indicate that the linear five-carbon valerate substitution is the minimum length necessary to circumvent P-glycoprotein and prevent inhibition of topoisomerase II activity . In addition, although AD 198 is not a pro-drug of AD 288, intracellular 14-O-acyl cleavage appears to contribute to the cytotoxicity of AD 198.

Tsitologiia, 1997, 39(8), 747 - 54
{The genome structure and phenotypic characteristics of murine hybridoma 1F7 cells selected in the presence of adriamycin and ethidium bromide}; Meliksemian MB et al.; A study was made of geno- and phenotypic changes, associated with of multidrug resistance development in murine 1F7 hybridoma cells, selected for adriamycin and ethidium bromide resistance (1F7-EBR and 1F7-ADR) . In both cell lines overexpression of mdr1 gene was observed, while amplification of mdr1 gene was detected only in 1F7-ADR cells . Karyotypic analysis revealed in resistant cells the presence of a specific marker M45 absent from parental cells, thus suggesting its selective importance . The M45 length instability, as well as the presence of a distal typical homogeneously stained region in it provide an evidence for a link between M45 and mdr1 gene amplification . The frequency of double minute chromosomes in parental lines was the same as in multidrug resistant lines . In 1F7-ADR cells, in contrast with 1F7-EBR cells, the enhancement of immunoglobulin production and the increase in immunoglobulin gamma 2b heavy chain gene expression were observed, which correlated with a decline in DNA-topoisomerase II activity.

Acta Oncol, 1997, 36(7), 735 - 40
Does the multidrug-resistance modulator cyclosporin A increase the cardiotoxicity of high-dose anthracycline chemotherapy?
Eising EG, Gries P, Eggert J, Scheulen ME.
Cyclosporin A has heterogeneous effects on anthracycline-related cardiotoxicity and can prevent multidrug-resistance (MDR) . The aim of this study was to explore whether the coadministration of cyclosporin A is accompanied by an increase in cardiotoxicity . Forty-three patients (27 male, 16 female, age: 18-67 yrs {mean: 47.5 yrs, SD: 11.6 yrs}) received 177 radionuclide ventriculography examinations (RNV 177 at rest, 133 at stress) before and during chemotherapy with either doxorubicin (n = 23) or epirubicin (n = 20) . RNV studies were applied up to 11 times in the follow-up of the patients . A maximum of 10 courses of chemotherapy was performed . In the doxorubicin group only, the age of the patients and the cumulative dose of the chemotherapeutic agent had a significant negative impact on left ventricular ejection fractions, whereas cyclosporin A had a significant positive influence (multiple analysis of regression, p < 0.05) . Cyclosporin A did not cause any significant increase in cardiotoxicity in our patients.

J Neurochem, 1998 Mar, 70(3), 1151 - 9
Multidrug resistance-related transport proteins in isolated human brain microvessels and in cells cultured from these isolates; Seetharaman S et al.; The multidrug transporter, P-glycoprotein (Pgp), at the blood-brain barrier is thought to be important for limiting access of toxic agents to the brain, but controversy surrounds its cellular location, whether on endothelium or on adjacent astrocyte foot processes . In the present study, the distribution of protein and mRNA for Pgp and for another transporter, multidrug resistance-associated protein (MRP), is compared with that for the endothelial marker, platelet-endothelial cell adhesion molecule-1 (PECAM-1) and for the astrocyte-derived glial fibrillary acidic protein (GFAP) in microvessels isolated from human brain and in cells grown from these microvessels . Activities of the multidrug transporters are assessed in the cultured cells from the effects of transport inhibitors on intracellular {3H}vincristine accumulation . The isolated microvessels show strong immunocytochemical staining for Pgp and PECAM-1 and little or no staining for GFAP and MRP, and they contain mRNAs detectable by RT-PCR encoding only Pgp and PECAM-1, but not GFAP or MRP . Thus, Pgp may well be synthesised and expressed on cells within the microvessels rather than on adherent astrocyte foot processes . In cells grown from the microvessels, although PECAM-1 remains, Pgp expression decreases and MRP appears . Evidence suggests these multidrug transporters are functionally active in the cultured cells.

Pneumonol Alergol Pol, 1997, 65(3-4), 249 - 53
{Surgical treatment of patients with multi-resistant pulmonary tuberculosis--case reports}; Michalowska-Mitczuk D et al.; Two patients with multidrug-resistant pulmonary tuberculosis were surgically treated after 3 and 7 years of unsuccessful chemotherapy . There was pneumonectomy in one case and lobectomy with segmentectomy in the second . Pneumonectomy was complicated by bronchopleural fistula . Both patients become sputum culture negative after surgical treatment but first patient died 5 months after surgery because of acute hepatitis.

Br J Urol, 1998 Feb, 81(2), 234 - 40
Characterization and modulation of transitional cell carcinoma cell lines with acquired multidrug resistance; Yu DS et al.; OBJECTIVES: To characterize in vitro drug-induced multidrug resistance (MDR) in transitional cell carcinoma (TCC) cell lines, and to elucidate the possible mechanisms of acquired MDR and their modulation . MATERIALS AND METHODS: Two drug-resistant cell lines, TCC8702/A1000 (adriamycin 1000 ng/mL) and TCC8803/A200 (adriamycin 200 ng/mL), were established after long-term adriamycin treatment for at least 16 months . Their biological characteristics, including growth morphology, doubling time and cell cycle, were analysed . The drug-resistance pattern to various anticancer drugs was measured using a microplate cytotoxicity assay . The modulation of drug sensitivity by calcium-channel blockers and protein kinase C inhibitor was assessed among the different cancer cell lines . RESULTS: Both MDR sublines had lower growth rates, lower saturation densities and higher nuclear/cytoplasmic ratios than the parent cell lines . DNA staining and cell cycle analysis revealed that both TCC8702/A1000 and TCC8803/A200 cells had a decreased S-phase fraction and the TCC8803/A200 cells a changed stem line; both sublines showed increased expression of membranous glycoprotein gp-170 . The cytoplasmic content of glutathione and glucose-6-phosphate dehydrogenase were not related to the MDR development in the sublines . The drug-resistance index of TCC8702/A1000 to adriamycin was 121-fold higher than the native cell line and TCC8803/A200 was 189-fold higher . TCC8803/A200 also had a broader MDR to cisplatin, vinblastine and vincristine . Calcium-channel blockers (verapamil, quinidine) and protein kinase C inhibitors (tamoxifen) inhibited gp-170 activity and slowed the drug-efflux pump, with the acquired-MDR cells subsequently accumulating anticancer drugs . A calcium antagonist-based combination of modulators all presented synergistic cytotoxic enhancement of the anticancer drugs . Parent TCC cell lines had a poorer response to modulator treatment than their MDR sublines . CONCLUSION: Different MDR mechanisms and subsequent modulator responses exist between native and acquired drug resistance in TCC cells . Acquired MDR seems strongly related to increased gp-170 expression and responds well to calcium antagonists . This phenomenon may be applicable in clinical conditions.

Int J Tuberc Lung Dis, 1997 Dec, 1(6), 528 - 35
Tuberculosis among foreign-born persons in New York City, 1992-1994: implications for tuberculosis control; Tornieporth NG et al.; OBJECTIVE: To study the pattern of transmission of tuberculosis (TB) among foreign-born persons living in New York City . DESIGN: A retrospective multicenter study comparing 158 foreign-born patients to 231 US-born patients diagnosed with TB between 1992 and 1994 . The patients were stratified according to their Mycobacterium tuberculosis isolate DNA fingerprint patterns . RESULTS: Nineteen (16%) of 122 isolates from foreign-born TB patients and 75 (42%) of 180 isolates from US-born TB patients had DNA fingerprint patterns (cluster patterns) indicative of recent exogenous transmission (P < 0.001) . All cluster pattern strains from foreign-born cases were identical to those found among US-born patients . The likelihood of infection with a cluster pattern strain among foreign-born persons increased with duration of residence in the US, and was significantly associated with being homeless (P < 0.05), or having multidrug-resistant TB (P = 0.00072) . CONCLUSION: Although most (84%) cases of TB among foreign-born persons in New York City appear to result from reactivation of infections they acquired abroad, the ones who acquire new infections become infected with strains that are already circulating among the US-born TB patients in New York City, and they have risk factors similar to those faced by US-born tuberculosis patients.

Yeast, 1998 Jan 15, 14(1), 49 - 65
The Sge1 protein of Saccharomyces cerevisiae is a membrane-associated multidrug transporter; Ehrenhofer-Murray AE et al.; In this study, we report the further characterization of the Saccharomyces cerevisiae crystal violet-resistance protein Sge1 . Sge1 is a highly hydrophobic 59 kDa protein with 14 predicted membrane-spanning domains . It shares homologies with several drug-resistance proteins and sugar transporters of the major facilitator superfamily . Here, we have demonstrated that Sge1 is not only a crystal violet-resistance protein, but that it also confers resistance to ethidium bromide and methylmethane sulfonate . Disruption of SGE1 leads to increased sensitivity towards all three compounds, thus designating Sge1 as a multiple drug-resistance protein . Subcellular fractionation as well as immunolocalization on whole yeast cells demonstrated that Sge1 was tightly associated with the yeast plasma membrane . Furthermore, Sge1 was highly enriched in preparations of yeast plasma membranes . In analogy to other multidrug-resistance proteins, we suggest that Sge1 functions as a drug export permease.

Br J Cancer, 1998 Feb, 77(4), 556 - 61
Role of glutathione, glutathione S-transferases and multidrug resistance-related proteins in cisplatin sensitivity of head and neck cancer cell lines; Welters MJ et al.; Resistance to chemotherapy is a major problem in the treatment of patients with head and neck squamous cell carcinoma (HNSCC) . Important factors involved are drug detoxification by glutathione (GSH) and reduced drug accumulation due to active transport out of the cell by so-called 'multidrug resistance-related proteins' . We have studied a panel of eight HNSCC cell lines showing differences in sensitivity to the anti-cancer drug cisplatin . Our previous studies indicated that the IC50 values were inversely correlated with the intracellular accumulation of platinum (Pt) . In the present study, cellular GSH levels were found not to be related to the IC50 values . The expression levels of the enzymes glutathione S-transferase (GST) alpha, mu, and pi, the multidrug resistance-related proteins P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and the lung resistance protein (LRP) were determined semiquantitatively by means of immunocytochemistry . The levels of the GSTs, P-gp and LRP were not found to be correlated with the IC50 values of the HNSCC cell lines . Surprisingly, however, an inverse correlation was found between MRP levels and IC50 values . The MRP expression levels were in agreement with the results of the MRP functional assay, based on the transport of calcein across the cell membrane as performed for two of the cell lines . Further studies should prove whether other pump mechanisms or DNA repair are involved in the cisplatin accumulation and the subsequent HNSCC cell growth inhibition.

Cytometry, 1998 Feb 1, 31(2), 137 - 45
Doxorubicin distribution in multicellular prostate cancer spheroids evaluated by confocal laser scanning microscopy and the "optical probe technique"; Wartenberg M et al.; Multicell-mediated drug resistance is a major impediment for the effectiveness of chemotherapeutic approaches and has been shown to be a feature of many solid tumors . We used confocal laser scanning microscopy to evaluate the depth distribution of the fluorescent cytostatic drug doxorubicin (Dox) in two size classes of multicellular cancer spheroids (MCS) (psi150+/-50 microm and 350+/-50 microm) . MCS (psi150+/-50 microm) solely consist of proliferating cells, whereas in MCS (psi350+/-50 microm) peripheral proliferating cell layers are followed in the depth of the tissue by drug resistant quiescent cell areas . A technique was developed which allows noninvasively to trace fluorescence distributions down to a depth of approximately 180 microm in living MCS . This was achieved by confocal radial recordings of the mean Dox fluorescence in 600 microm2 regions of interest (ROI), equidistantly spaced (10 microm) from the center of MCS towards their periphery . The resulting fluorescence intensity profiles were subsequently corrected for absorbtion and light scattering in the depth of the tissue by a convenient algorithm . A 10 min incubation of MCS (psi150+/-50 microm) with Dox (10 microM) led to a peripheral accumulation, after 2 h Dox was homogeneously distributed within the whole MCS . In contrast, after Dox treatment of MCS (psi350+/-50 microm) for 2 h, the drug was accumulated within the peripheral proliferating cell rim of 78+/-8 microm, whereas deeper, quiescent cell layers remained unstained . When MCS were incubated with verapamil, cyclosporin A, orthovanadate, and quinidine, which are known to reverse P-glycoprotein (Pgp)-mediated multidrug resistance (MDR), Dox accumulated also in deeper cell layers . Genistein and indometacin which reverse multidrug resistance mediated by the multidrug resistance-associated protein (MRP) were without effects . The optical probe technique proved to be well suited to study MDR in a living three dimensional tissue context.

J Exp Biol, 1998 Jan, 201 ( Pt 1), 21 - 31
Alterations of ionic membrane permeabilities in multidrug-resistant neuroblastoma x glioma hybrid cells; Gerard V et al.; A population of NG108-15 neuroblastoma cells resistant to doxorubicin (NG/DOXR) was established . The cells exhibited a multidrug resistance phenotype with cross-resistance to vinblastin and colchicine, overexpression of a 170 kDa membrane protein identified as P-glycoprotein and reversal of resistance by verapamil and quinine . Compared with NG108-15 cells, NG/DOXR cells showed an increase in Na+ current density and a decrease in cyclic-AMP-activated Cl- current density with no change in K+- and volume-sensitive Cl- current densities . As previously observed in NG108-15 cells, the vacuolar-type H+-ATPase inhibitors bafilomycin A1 and nitrate induced membrane depolarizations in NG/DOXR cells . The resting potentials of sensitive and resistant cells were not significantly different, but the depolarizations evoked by these agents were significantly larger in NG/DOXR than in NG108-15 cells . The resting membrane potential of NG/DOXR cells, but not that of NG108-15 cells, was depolarized by verapamil, and this effect was abolished by bafilomycin . The volume-sensitive Cl- currents of drug-sensitive and drug-resistant cells were inhibited by a decrease in intracellular pH from 7.3 to 6.8 . Whereas bafilomycin prevents activation of Cl- currents in both drug-sensitive and drug-resistant cells, verapamil inhibited the Cl- current only in NG/DOXR cells . The results are discussed in terms of the roles of cytoplasmic pH and membrane potential in multidrug resistance.

Cancer, 1998 Feb 15, 82(4), 661 - 6
The expression of multidrug resistance protein in human gastrointestinal tract carcinomas; Takebayashi Y et al.; BACKGROUND: Multidrug resistance protein (MRP) is a membrane phosphoglycoprotein with an Mr of 190,000 that is involved in the non-P-glycoprotein mediated multidrug resistance of human tumor cells . The aim of this study was to determine the clinicopathologic relevance of MRP expression in human gastrointestinal tract carcinomas . METHODS: The authors prepared a rabbit antiserum against MRP that does not cross-react with P-glycoprotein and retrospectively examined the expression of MRP in 86 squamous cell carcinomas of the esophagus, 103 adenocarcinomas of the stomach, and 139 colorectal adenocarcinomas by immunohistochemistry . None of the patients in this study had received prior chemotherapy . RESULTS: The proportion of MRP positive samples in the squamous cell carcinomas of the esophagus (62.8%, 54 of 86) was significantly higher than that in the adenocarcinomas of the stomach (34.1%, 35 of 103) and the colorectal adenocarcinomas (40.3%, 56 of 139) (P <0.01) . The proportion of MRP positivity in the well-differentiated carcinomas was significantly higher than that in moderately or poorly differentiated carcinomas . MRP expression was independent of gender, lymph node metastasis, and tumor progression . CONCLUSIONS: These data indicate that the expression of MRP is correlated with the differentiation of carcinoma cells in the gastrointestinal tract and may be involved in the intrinsic drug resistance of well-differentiated squamous cell carcinoma of the esophagus.

Int J Urol, 1997 Nov, 4(6), 583 - 90
Multifactorial involvement of multidrug resistance-associated {correction of resistance} protein, DNA topoisomerase II and glutathione/glutathione-S-transferase in nonP-glycoprotein-mediated multidrug resistance in human bladder cancer cells; Kim WJ et al.; BACKGROUND: Multiple mechanisms are important in multidrug resistance in urothelial cancers . We investigated the acquisition of a multidrug resistance phenotype in human bladder cancer cells exposed to doxorubicin . METHODS: Human bladder cancer cell line 5637 and 2 doxorubicin drug-resistant sublines (5637/DR5.5 and 5637/DR50) were used . Measurements were made of the steady state mRNA levels of the multidrug resistance gene (mdr1), multidrug resistance-associated protein (MRP), glutathione-S-transferase-pi and DNA topoisomerase II (topo II) genes, P-glycoprotein (PgP) and MRP expression, glutathione (GSH) and GSH enzyme activity, and topo II catalytic activity . The pharmacokinetics were compared between the parent and the drug-resistant sublines . RESULTS: 5637/DR5.5 and 5637/DR50 cells were 7.6- and 16.2-fold more resistant to doxorubicin and 16.7- and 48.3-fold more resistant to etoposide, respectively, compared with 5637 cells . A dose escalation of doxorubicin increased the MRP expression, GSH levels and glutathione-S-transferase (GST) activity, although no PgP expression was observed in any cell line . Resistance was brought about by decreased drug accumulation through drug efflux, although intracellular daunorubicin concentrations were similar between DR5.5 and DR50 cells . Topo II catalytic activity was undetectable in DR50 cells, but maintained in both the parent and DR5.5 cells . CONCLUSION: Reduced drug accumulation in doxorubicin-resistant cells was mediated by MRP instead of PgP indicating that MRP-mediated drug efflux functions in a limited manner for drug resistance . An increase in drug efflux via MRP, reduced topo II activity, and increased GSH levels/GSH-related enzyme activities may play major roles in nonPgP-mediated multidrug resistance in urothelial cancers treated with anthracyclines.

Int J Clin Pharmacol Ther, 1998 Jan, 36(1), 58 - 60
Expression of the novel mitoxantrone resistance associated gene MXR7 in colorectal malignancies; Lage H et al.; It was shown previously that a glypican encoding gene (MXR7/GPC3/OCl-5) was associated with mitoxantrone resistance in vitro . This study describes and investigation of an association between multidrug resistance and MXR7 in surgical cryo-specimens of 51 gastrointestinal tumors . The mRNA expression levels differ widely according to tumor species . In primary colorectal cancers, the level of MXR7 mRNA expression correlated loosely with the mRNA expression level of the multidrug-resistance-associated protein (MRP) . In colorectal metastases an elevated MXR7 mRNkA expression level was observed when compared to primary colorectal carcinomas.

Int J Clin Pharmacol Ther, 1998 Jan, 36(1), 9 - 13
Pharmacological insights from P-glycoprotein knockout mice; Schinkel AH; The mdr1-type P-glycoproteins can confer multidrug resistance to tumor cells by actively pumping a wide variety of drugs from the cell . To counteract this drug resistance, P-glycocoprotein-blocking agents are currently administered to patients during chemotherapy . However, this may also affect the normal physiological function(s) of the mdr1-type P-glycocoproteins . In order to establish these functions, we have generated mice with a genetic deficiency in both of their mdr1-type P-glycocoprotein genes . Our results indicate the mdr1-type P-glycocoproteins are not essential for basic physiological functions . However, mice without mdr1-type P-glycocoproteins display drastic alterations in the pharmacological handling of drugs, demonstrating an important role for mdr1-type P-glycocoprotein in the blood-brain barrier, where it prevents the accumulation of many drugs in the brain . Moreover, we found that intestinal P-glycocoprotein has a prominent role in the extrusion of several drugs from the blood into the intestinal lumen, and in preventing drugs in the intestinal lumen from (re-)entering the bloodstream . The latter property can have important implications for the oral bioavailability of many drugs . Our results indicate that effective P-glycocoprotein-blocking agents should be used with caution, given the potentially extensive pharmacokinetic effects of treatment with these compounds.

Neurochem Res, 1998 Feb, 23(2), 203 - 9
Evaluation of the role of P-glycoprotein in ivermectin uptake by primary cultures of bovine brain microvessel endothelial cells; Rose JM et al.; The P-glycoprotein efflux system located on the apical membrane of brain capillary endothelial cells functions as part of the blood-brain barrier . In this study, primary cultures of bovine brain microvessel endothelial cells (BMECs) were investigated for the presence of a P-glycoprotein system and its contribution in regulating ivermectin distribution across the blood-brain barrier . Results of rhodamine 123 uptake studies with cyclosporin A and verapamil as substrates indicated that a functional efflux system was present on BMECs . Immunoblot analysis with the C219 monoclonal antibody to the product of the multidrug resistant member 1(MDR1) gene also confirmed the expression of MDR1 in the BMECs . Unbound ivermectin was shown to significantly increase the uptake of rhodamine 123 in BMECs, however, the drug only modestly enhanced the transcellular passage of rhodamine . The results of these studies affirmed that unbound ivermectin is an inhibitor of the MDR1 efflux system in BMECs.

Biol Reprod, 1998 Feb, 58(2), 330 - 7
Multidrug resistance gene expression correlates with progesterone production in dehydroepiandrosterone-induced polycystic and equine chorionic gonadotropin-stimulated ovaries of prepubertal rats; Lee GY et al.; Polycystic ovaries (PCO) can be induced in prepubertal rats by daily injection of dehydroepiandrosterone (DHEA) . There are high levels of progesterone, androgens, and estrogens in the cystic fluid of DHEA-treated rat ovaries . The purpose of this study was to investigate whether high levels of steroids in the PCO correlate with the expression of multidrug resistance gene product P-glycoprotein (Pgp) . Using C219, a monoclonal antibody that recognizes the 170-kDa ATP-dependent transmembrane pump, we localized Pgp on the plasma membrane of granulosa cells in cystic follicles but not of oocytes or thecal/interstitial cells . In normal prepubertal rats, Pgp was localized in progesterone-producing granulosa cells of the preovulatory follicles and in cells of the corpora lutea after eCG/hCG stimulation, but not in growing follicles, oocytes, or thecal/interstitial cells . Northern analysis of these tissues indicated strong expression of Pgp mRNA in the preovulatory follicles, cystic follicles, and corpora lutea . From these findings it seems that progesterone produced by the granulosa cells may act in an autocrine manner to induce the expression of Pgp . It may be possible that progesterone interacts with the Pgp of these granulosa cells to modulate steroid efflux.

Anticancer Drug Des, 1998 Jan, 13(1), 19 - 33
Antiproliferative activity of colchicine analogues on MDR-positive and MDR-negative human cancer cell lines; De Vincenzo R et al.; In this study the in vitro antitumor activity of a series of 20 colchicine analogues was tested and compared with colchicine and thiocolchicine on three different human cancer cell lines, two of which express the multidrug-resistance (MDR) phenotype . At concentrations from 1 nM to 100 microM, all compounds tested inhibited cancer cell proliferation . The IC50 values indicate that the three fluorinated analogues were the most active compounds, with a similar decreasing order of potency (IDN 5005 > IDN 5079 > IDN 5080) on the two MDR-expressing cell lines, whereas thiocolchicine was the most effective compound on the MDR-negative MDA-MB 231 cells . A strong correlation (r = 0.94; P = 0.004) was found between IC50 values obtained using the two MDR-positive cell lines . Conversely, IC50 values obtained in MDA-MB 231 cells did not show a significant correlation with MDR-positive cell lines, thereby suggesting some difference in the antiproliferative mechanism(s) of colchicine analogues . Cell cycle analysis of the most active analogues in breast cancer cells showed a relationship between cell cycle blocking activity and growth inhibition . The most active agents on the MDR-positive MCF7 ADRr cell line, after 24 h of culture, in terms of cell cycle blocking activity were the three fluorinated analogues . Interestingly, after 72 h, when the cell cycle block subsided, a consistent amount of DNA fragmentation was evident . The extent of cell cycle block, measured as the G2/G1 ratio, was significantly correlated with the apoptosis rate expressed as a percentage of DNA fragmentation on both cell lines, thereby suggesting that a large number of blocked cells underwent the apoptotic pathway.

Carcinogenesis, 1998 Jan, 19(1), 109 - 15
Multidrug resistance protein and glutathione S-transferase P1-1 act in synergy to confer protection from 4-nitroquinoline 1-oxide toxicity; Morrow CS et al.; Model cell lines developed from MCF7 breast carcinoma cells were used to examine the roles of glutathione S-transferase P1-1 (GSTP1-1) and multidrug resistance protein (MRP) in the protection of cells from 4-nitroquinoline 1-oxide (4NQO) toxicities . Increased expression of GSTP1-1 alone in MCF7 cells results in limited protection from the formation of 4NQO-derived covalent adducts of nucleic acids but affords no protection from 4NQO-mediated cytotoxicity . Increased expression of MRP alone conferred modest protection while co-expression of GSTP1-1 with MRP produced high-level protection from both 4NQO-derived adduct formation and 4NQO cytotoxicity . This synergistic resistance to 4NQO toxicities (both nucleic acid adduct formation and cytotoxicity) is associated with a GSTP1-1-dependent increase in 4NQO-glutathione (QO-SG) conjugate formation and a MRP-dependent increase in QO-SG efflux . These data indicate that MRP is an important export transporter for the glutathione conjugate of the carcinogen, 4NQO . Moreover, this MRP-dependent efflux activity is necessary to achieve the full protection from 4NQO toxicity-protection that is potentiated by GSTP1-1-mediated QO-SG formation.

Br J Cancer, 1998, 77(3), 412 - 6
Use of the comet assay for assessment of drug resistance and its modulation in vivo; Huang P et al.; Drug resistance is generally considered to be a major impediment to successful cancer chemotherapy, yet it is generally not possible to predict the degree or timing of the emergence of tumour resistance in most chemotherapy protocols . Recent developments with the single-cell gel electrophoresis or 'comet' assay for DNA damage at the single-cell level suggest that this technique might provide a method for identifying and potentially monitoring tumour cell responsiveness to many anti-cancer agents in situ . In principle, this assay could be applied to any accessible tumour being treated with chemotherapeutic agents that cause overt DNA damage . We have investigated that supposition using several rodent and human tumour cell lines exhibiting a spectrum of resistance to the DNA strand-breaking drug, etoposide . By assessing cells grown as monolayers, spheroids and xenografted tumours in immunodeficient mice, we found that the comet assay can provide not only an index of sensitivity to etoposide, but, additionally, can demonstrate the efficacy (or lack thereof) of multidrug resistance (MDR) reversing agents for cells in vitro, and tumours in vivo.

Br J Cancer, 1998, 77(3), 359 - 65
CPT-11 sensitivity in relation to the expression of P170-glycoprotein and multidrug resistance-associated protein; Jansen WJ et al.; The relevance of P170-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) for the sensitivity to CPT-11 was investigated in human malignant cell lines as well as in human tumour xenografts . In vitro, the P-gp-positive sublines BRO/mdr1.1 (transfected with MDR1) and 2780AD were slightly cross-resistant against carboxylesterase-activated CPT-11 . Cross-resistance against SN-38 was present in 2780AD cells, but not in BRO/mdr1.1 cells . The P-gp modulators BIBW22BS, verapamil and dexniguldipine partly reversed the resistance against CPT-11 in the P-gp-positive sublines . BIBW22BS was the most effective modulator in the reversal of the resistance against carboxylesterase-activated CPT-11 as well as against SN-38 in the 2780AD subline . In contrast to doxorubicin and vincristine, the BRO/mdr1.1 xenografts were at least as sensitive to CPT-11 as the BRO xenografts . The 2780AD xenografts were slightly less sensitive than the parent tumours, but there was no difference in topoisomerase I DNA unwinding activity . Therefore, the high retention of the multidrug-resistant phenotype of 2780AD cells in vivo may be the cause of the low cross-resistance against CPT-11 . The MRP-positive subline GLC4/ADR was cross-resistant against carboxylesterase-activated CPT-11 and SN-38 . GLC4/ADR cells, however, demonstrated a twofold lower topoisomerase I activity than GLC4 cells . Cross-resistance against the camptothecin derivatives was not apparent in the MRP-transfected subline of SW1573/S1 . In conclusion, P-gp-positive cells show a low cross-resistance against CPT-11/SN38, which is only apparent with high P-gp expression in vivo . MRP does not seem to play a role in the sensitivity to CPT-11.

Br J Cancer, 1998, 77(3), 353 - 8
99mTc-sestamibi is a substrate for P-glycoprotein and the multidrug resistance-associated protein; Hendrikse NH et al.; 99mTc-sestamibi (99mTc-MIBI) is a substrate for the P-glycoprotein (P-gp) pump but it is not known whether it is a substrate for the multidrug resistance-associated protein (MRP) pump . Therefore, 99mTc-MIBI was evaluated in the GLC4 cell line and its doxorubicin-resistant MRP-, but not P-gp-, overexpressing GLC4/ADR sublines as well as in the S1 cell line and its MRP-transfected subline S1-MRP . 99mTc-MIBI concentration decreased in the GLC4/ADR sublines with increasing MRP overexpression and was lower in S1-MRP than in S1 . 99mTc-MIBI plus vincristine increased 99mTc-MIBI concentration in GLC4 lines compared with 99mTc-MIBI alone . 99mTc-MIBI efflux raised with increasing MRP expression in the GLC4 lines . Glutathione depletion elevated 99mTc-MIBI concentration in GLC4/ADR150x . Cross resistance for 99Tc-MIBI, used to test cytotoxicity of the Tc compound, was observed in GLC4/ADR150x vs GLC4 . 99Tc-MIBI induced a synergistic effect on vincristine cytotoxicity in GLC4/ADR150x . These results show that 99mTc-MIBI is involved in MRP-mediated efflux . The fact that 99mTc-MIBI efflux is influenced by MDR1 and MRP expression must be taken into account when this gamma-rays-emitting complex is tested for tumour efflux measurements.

Proc Natl Acad Sci U S A, 1998 Jan 6, 95(1), 282 - 7
Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis; de Vree JM et al.; Class III multidrug resistance (MDR) P-glycoproteins (P-gp), mdr2 in mice and MDR3 in man, mediate the translocation of phosphatidylcholine across the canalicular membrane of the hepatocyte . Mice with a disrupted mdr2 gene completely lack biliary phospholipid excretion and develop progressive liver disease, characterized histologically by portal inflammation, proliferation of the bile duct epithelium, and fibrosis . This disease phenotype is very similar to a subtype of progressive familial intrahepatic cholestasis, hallmarked by a high serum gamma-glutamyltransferase (gamma-GT) activity . We report immunohistochemistry for MDR3 P-gp, reverse transcription-coupled PCR sequence analysis, and genomic DNA analysis of MDR3 from two progressive familial intrahepatic cholestasis patients with high serum gamma-GT . Canalicular staining for MDR3 P-gp was negative in liver tissue of both patients . Reverse transcription-coupled PCR sequencing of the first patient's sequence demonstrated a homozygous 7-bp deletion, starting at codon 132, which results in a frameshift and introduces a stop codon 29 codons downstream . The second patient is homozygous for a nonsense mutation in codon 957 (C --> T) that introduces a stop codon (TGA) . Our results demonstrate that mutations in the human MDR3 gene lead to progressive familial intrahepatic cholestasis with high serum gamma-GT . The histopathological picture in these patients is very similar to that in the corresponding mdr2(-/-) mouse, in which mdr2 P-gp deficiency induces complete absence of phospholipid in bile.

Proc Natl Acad Sci U S A, 1998 Jan 6, 95(1), 162 - 6
Hexamethylene bisacetamide induces programmed cell death (apoptosis) and down-regulates BCL-2 expression in human myeloma cells; Siegel DS et al.; Multiple myeloma (MM) is a B cell malignancy characterized by the expansion of monoclonal Ig-secreting plasma cells with low proliferative activity . It is postulated that inhibition of physiologic cell death is an underlying factor in the pathophysiology of MM . The development of chemoresistance is a common feature in patients with MM . In the present studies, hexamethylene bisacetamide (HMBA), a hybrid polar compound that is a potent inducer of terminal differentiation of various transformed cells, is shown to inhibit the growth of several human myeloma cell lines (ARP-1, U266, and RPMI 8226), including doxorubicin-resistant RPMI 8226 variants that overexpress the multidrug-resistance gene, MDR-1, and its product, p-glycoprotein . In addition to growth arrest and suppression of clonogenicity, HMBA induces apoptosis both in freshly isolated human myeloma cells and in cell lines, as determined by morphologic alterations, cell cycle distribution and endonucleosomal DNA fragmentation . Further, HMBA decreases BCL-2 protein expression in myeloma cells within 12-48 hr . Overexpression of BCL-2 protein in ARP-1 cells confers resistance to HMBA-induced apoptosis . Taken together, these data suggest that HMBA is a potent inducer of apoptosis in human myeloma cells, which may act through suppressing the anti-apoptotic function of the bcl-2 gene . HMBA, and related hybrid polar compounds, may prove useful in the management of this presently incurable disease.

Am J Pathol, 1998 Feb, 152(2), 373 - 8
Immunohistochemical detection of the human major vault protein LRP with two monoclonal antibodies in formalin-fixed, paraffin-embedded tissues; Schroeijers AB et al.; Multidrug resistant cancer cells frequently overexpress the 110-kd lung resistance-related protein (LRP) as detected with the monoclonal antibody (MAb) LRP-56 . Recently, we identified LRP as the major vault protein (MVP), which is the major constituent of vaults, multisubunit cellular organelles . Clinically, LRP/MVP expression in cancer at time of diagnosis provided a strong and independent prognostic factor for response to chemotherapy and outcome in different tumor types, notably acute myeloid leukemia and ovarian cancer . To facilitate additional immunohistopathological studies, we have optimized LRP/MVP detection in paraffin-embedded tissues using two monoclonal antibodies, LRP-56 and LMR-5 . Blocking experiments showed that LRP-56 and LMR-5 MAbs detect different epitopes of LRP/MVP . Immunohistochemical studies with both MAbs in a panel of human multidrug resistant tumor cell lines, normal tissues, and colorectal tumors showed that LRP/MVP expression can be reliably detected after formalin-fixation and paraffm-embedding using overnight incubation at 4 degrees C with the primary MAbs at 5- to 10-fold higher concentrations (ie, 1 to 10 microg/ml) as currently used with frozen sections . Both streptavidin-biotin complex and alkaline phosphatase-anti-alkaline phosphatase techniques could be successfully used for signal-amplification . Staining quality did not benefit from antigen-retrieval pretreatments . The optimized staining methodology facilitates studies in archival material on the putative role of LRP/MVP in clinical drug resistance.

Curr Opin Oncol, 1998 Jan, 10(1), 31 - 5
Resistance to chemotherapy in acute leukemia; Lowenberg B et al.; The successful treatment of acute myeloid leukemia (AML) is hampered by the development of chemotherapy resistant disease . One molecular mechanism of pleiotropic resistance, typical multidrug resistance (MDR), has been recognized as an independent adverse prognostic factor in AML . Therefore, uniform laboratory assays are needed to evaluate the effect of MDR expression on the clinical outcome of strategies to overcome resistance . One approach has been to administer high-dose therapy with stem cell support . In elderly patients, less toxic treatment is required, while the results of induction treatment in this age group need further improvement . The use of hematopoietic growth factors may reduce treatment-associated morbidity and mortality, but does not consistently enhance cell death . MDR reversal with cyclosporin or PSC833 has shown promising results in Phase I/II trials.

Kekkaku, 1997 Dec, 72(12), 659 - 72
{Rapid diagnosis of tuberculosis}; Abe C; Two systems, the newly developed Mycobacteria Growth Indicator Tube (MGIT) and biphasic Septi-Chek AFB based on liquid media, proved to be significantly better than the egg-based solid media for the isolation of mycobacteria from clinical specimens . The difference in the rates of isolation of bacteria between the two groups of media was more remarkable with smear-negative specimens . The isolation of the Mycobacterium tuberculosis complex by MGIT occurred 8 days previous to the isolation by the conventional Ogawa method . The mean time for detecting M . tuberculosis complex by Septi-Chek AFB was similar to those of the Ogawa method . A greater difference in isolation time was observed for mycobacteria other than M . tuberculosis (MOTT) isolates . These results indicate that the MGIT and Septi-Chek AFB systems based on liquid media are efficient for the recovery of mycobacteria . PCR and other nucleic acid amplification methods are widely used for the detection of M . tuberculosis in clinical specimens . Although the sensitivities of the Gen-Probe Amplified Mycobacteria Direct Test (MTD) and Amplicor Mycobacteria for the detection of the M . tuberculosis complex appear to be similar to the sensitivity of the culture method using the Septi-Chek AFB, the two methods should be quite useful for rapid detection of M . tuberculosis infections . On the other hand, two cooperative blind studies conducted between 6 to 9 laboratories to estimate the reliability and reproducibility of these two commercially available kits revealed the necessity of good laboratory practice and development of reference reagents to monitor the performance of the whole assay, including pretreatment of clinical specimens . Considerable progress has been made in recent years toward understanding the molecular basis of the resistance to antituberculosis drugs, isoniazid (katG, inhA, ahpC), rifampin (rpoB), pyrazinamide (pncA), streptomycin (rpsL, rrs), ethambutol (embB), and fluoroquinolones (gyrA) . Most cases of resistance are related usually to simple nucleotide substitutions rather than to acquisition of new genetic elements . Multidrug-resistant isolates of M . tuberculosis arise as a consequence of sequential accumulation of mutations conferring resistance to single therapeutic agents . The basis of resistance is not able to be explained yet in a substantial percentage of strains (> 90%) for other antituberculosis drugs than rifampin . Further studies are required to fully understand the molecular mechanisms of resistance.

Semin Cancer Biol, 1997 Jun, 8(3), 171 - 82
Search for specific inhibitors of multidrug resistance in cancer; Sarkadi B et al.; This paper deals with the theoretical background, methods and clinical significance of the search for specific inhibitors of multidrug resistance (MDR) in cancer . After discussing the basic features of the drug pump membrane proteins (MDR1 and MRP) responsible for this phenomenon, the possible mechanism of action of MDR inhibitors is reviewed . Modulators of drug pump expression may include inhibitors of the transcription and translation, as well as the processing and post-translational modifications of MDR1 and MRP . The function of the drug pumps can be effectively inhibited by substrate analogs, inhibitors of ATP-binding and utilization, specific monoclonal antibodies, and by various agents with still non-specified mechanisms . Basic methods are presented for the in vitro screening of MDR inhibitors in whole cells and in pump protein preparations, as well as for the in vivo assessment of the efficiency and safety of such inhibitors . The review also discusses some current problems and perspectives of MDR modulation in clinical tumor therapy.

Biochem Pharmacol, 1997 Nov 15, 54(10), 1151 - 8
Flip-flop of doxorubicin across erythrocyte and lipid membranes; Regev R et al.; Doxorubicin, an anticancer drug, is extruded from multidrug resistant (MDR) cells and from the brain by P-glycoprotein located in the plasma membrane and the blood-brain barrier, respectively . MDR-type drugs are hydrophobic and, as such, enter cells by diffusion through the membrane without the requirement for a specific transporter . The apparent contradiction between the presumably free influx of MDR-type drugs into MDR cells and the efficient removal of the drugs by P-glycoprotein, an enzyme with a limited ATPase activity, prompted us to examine the mechanism of passive transport within the membrane . The kinetics of doxorubicin transport demonstrated the presence of two similar sized drug pools located in the two leaflets of the membrane . The transbilayer movement of doxorubicin occurred by a flip-flop mechanism of the drug between the two membrane leaflets . At 37 degrees, the flip-flop exhibited a half-life of 0.7 min, in both erythrocyte membranes and cholesterol-containing lipid membranes . The flip-flop was inhibited by cholesterol and accelerated by high temperatures and the fluidizer benzyl alcohol . The rate of doxorubicin flux across membranes is determined by both the massive binding to the membranes and the slow flip-flop across the membrane . The long residence-time of the drug in the inner leaflet of the plasma membrane allows P-glycoprotein a better opportunity to remove it from the cell.

Trans R Soc Trop Med Hyg, 1997 Sep-Oct, 91(5), 590 - 1
Apoptosis related to chloroquine sensitivity of the human malaria parasite Plasmodium falciparum; Picot S et al.; As chemoresistance of Plasmodium falciparum to chloroquine has arisen, new ways of combating the infection are needed . Similarities exist between the multidrug resistance of mammalian cells and chloroquine resistance of P . falciparum, based on the occurrence of internucleosomal deoxyribonucleic acid (DNA) breakdown and the ability of some anticancer drugs and chloroquine to induce apoptosis . Using chloroquine, oligonucleosomal DNA fragmentation was observed with a sensitive strain of P . falciparum, but not with a resistant one . This suggests that apoptosis may be involved in the action of chloroquine on the parasite.

J Antimicrob Chemother, 1997 Dec, 40(6), 885 - 8
In-vitro activity of fluorinated quinolones and macrolides against drug-resistant Mycobacterium tuberculosis; Hoffner SE et al.; The increasing incidence of drug-resistant Mycobacterium tuberculosis necessitates therapeutic alternatives . The in-vitro activities of seven fluoroquinolone and macrolide compounds were tested against 23 clinical isolates of M . tuberculosis, including 17 multidrug-resistant strains . Sparfloxacin was the most active fluoroquinolone, with MICs of < or = 1 mg/L for all tested strains, followed by levofloxacin and ciprofloxacin . Trovafloxacin had no inhibitory activity at the concentrations tested . The MICs of the macrolides were generally higher, clarithromycin being the most active with MICs of < or = 8 mg/L for eight of the 23 strains.

J Nat Prod, 1998 Jan, 61(1), 112 - 5
Two pimarane diterpenoids from Ephemerantha lonchophylla and their evaluation as modulators of the multidrug resistance phenotype; Na GX et al.; Two new pimarane diterpenoids, lonchophylloids A (1) and B (2), were isolated from the stems of Ephemerantha lonchophylla . The structures of 1 and 2 were established predominantly through the application of extensive 1H-and 13C-NMR, 1D- and 2D-homonuclear and heteronuclear correlation NMR experiments, and X-ray diffraction methods . Consistent with structure--activity predictions, both compounds were capable of sensitizing cells that expressed the multidrug resistance phenotype to the toxicity of the anticancer drug doxorubicin.

J Nat Prod, 1998 Jan, 61(1), 108 - 11
A new sesquiterpene ester from Celastrus orbiculatus reversing multidrug resistance in cancer cells; Kim SE et al.; In a search for revertants of multidrug-resistance in cancer cells, a novel (1) and two known (2,3) sesquiterpene esters were isolated from the root of Celastrus orbiculatus . The structure of 1 was elucidated as 1 beta,2 beta-diacetoxy-6 alpha,9 alpha-bis(benzoyloxy)dihydro-beta-agarofuran . Compounds 1-3 partially or completely reversed resistance to adriamycin, vinblastine, and paclitaxel of multidrug-resistant KB-V1 and MCF7/ADR cells.

Br J Cancer, 1998, 77(2), 201 - 9
Multidrug resistance protein-mediated transport of chlorambucil and melphalan conjugated to glutathione; Barnouin K et al.; The human multidrug resistance protein (MRP1) confers resistance of cells to a number of different cytostatic drugs and functions as an export pump for glutathione S-conjugates, glucuronides and other amphiphilic anions . The present study details for the first time MRP1-mediated ATP-dependent transport of various glutathione S-conjugates of the bifunctional alkylating agents chlorambucil and melphalan . In membrane vesicles prepared from cells expressing recombinant MRP1, the conjugates were transported at rates in the following order: monoglutathionyl chlorambucil > bisglutathionyl chlorambucil > monohydroxy monoglutathionyl chlorambucil and monoglutathionyl melphalan > monohydroxy monoglutathionyl melphalan . In addition, we show that membranes from chlorambucil-resistant GST-alpha-overexpressing CHO cells as well as from their parental cells express the hamster homologue of MRP1 . With both CHO cell membrane preparations, we observed ATP-dependent transport of monoglutathionyl chlorambucil and of leukotriene C4, a glutathione S-conjugate and high-affinity substrate of MRP1 . The transport rates measured in the resistant cells were only two- to three-fold higher than those measured in the control cells . These results together with cytotoxicity assays comparing MRP1-overexpressing cell pairs with the CHO cell pair indicate that, although MRP1-mediated transport is active, it may not be the rate-limiting step in chlorambucil resistance in these cell lines.

Int J Oncol, 1998 Feb, 12(2), 383 - 6
Regulation of the MDR1 promoter by cyclic AMP-dependent protein kinase and transcription factor Sp1; Rohlff C et al.; The expression of multidrug-resistance (MDR) in breast carcinoma cell line MCF-7/ADR50 is primarily dependent on the transcriptional activation of the MDR1 gene . We now report that MDR in this cell line is partially reversed by the type I cAMP-dependent protein kinase (PKA) inhibitor, 8-Cl-cAMP . MDR1 promoter activity was also regulated through a PKA-dependent pathway and was inhibited by 8-Cl-cAMP, and stimulated by the enantiomeric agonist, SpcAMP{S} . MDR1 promoter activity through an Sp1 response element was stimulated by exogenous Sp1, a factor that we have shown to be activated by PKA . These results indicate that MDR1 promoter activity is linked to the cAMP/PKA signaling pathway, and that PKA antagonists may be useful for reversing the multidrug-resistant phenotype.

Int J Oncol, 1998 Feb, 12(2), 315 - 9
Differential expression and activity of P-glycoprotein and multidrug resistance-associated protein in CD34-positive KG1a leukemic cells; Fardel O et al.; CD34+ acute myeloid leukemias generally respond poorly to chemotherapy when compared to CD34- myeloid leukemias . In order to contribute to the analysis of the mechanisms involved in this drug resistance, expression and activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP), two drug efflux pumps conferring multidrug resistance, have been investigated in the CD34+ KG1a leukemic myeloid cell line and in two CD34- K562 and HL60 leukemic myeloid cell lines . Reverse transcription-polymerase chain reaction and dye efflux assays revealed that KG1a cells express P-gp but not MRP whereas neither P-gp nor MRP were detected in K562 and HL60 cells . In addition, KG1a cells were demonstrated to display resistance to anticancer drug substrates for P-gp such as vincristine and daunorubicin and to poorly accumulate vincristine . These results indicated that P-gp, in contrast to MRP, is expressed and functional in the drug-resistant CD34+ KG1a cell line, that may constitute a useful cellular model to analyze the constitutive chemoresistance of CD34+ acute myeloid leukemias.

Int J Oncol, 1998 Feb, 12(2), 287 - 91
Multidrug resistance mediated by overexpression of P-glycoprotein in human osteosarcoma in vivo; Suto R et al.; We examined the expression levels of P-glycoprotein (P-Gp)/the human multidrug resistance gene (MDR1) and in vivo chemosensitivity in the 7 osteosarcoma xenografts . Three of seven (43%) osteosarcoma xenografts expressed MDR1 by reverse transcriptase-polymerase chain reaction (RT-PCR) . The OSS-516R xenograft selected with vincristine (VCR) from the MDR1-negative xenograft OSS-516, which was sensitive to VCR and doxorubicin (DOX), acquired cross-resistance to DOX . In the OSS-516R, RT-PCR assay showed definite MDR1 expression and immunohistochemical analysis demonstrated P-Gp-positive tumor cells . These results suggest that P-Gp/MDR1 overexpression is related to multidrug resistance in human osteosarcoma in vivo.

Br J Cancer, 1998, 77(1), 87 - 91
Multiple gene expression analysis reveals distinct differences between G2 and G3 stage breast cancers, and correlations of PKC eta with MDR1, MRP and LRP gene expression; Beck J et al.; A possible link between protein kinase C (PKC) and P-glycoprotein (P-gp)-mediated-multidrug resistance (MDR) was assumed from studies on MDR cell lines selected in vitro . The functional relevance of PKC for the MDR phenotype remains unclear, and the involvement of a particular PKC isozyme in clinically occurring drug resistance is not known . Recently, we have demonstrated significant correlations between the expression levels of the PKC eta isozyme and the MDR1 or MRP (multidrug resistance-associated protein) genes in blasts from patients with acute myelogenous leukaemia (AML) and in ascites cell aspirates from ovarian cancer patients . To extend these findings to further types of human tumours we analysed specimens from 64 patients with primary breast cancer for their individual expression levels of several MDR-associated genes (MDR1, MRP, LRP (lung cancer resistance-related protein), topoisomerase (Topo) II alpha/IIbeta, cyclin A and the PKC isozyme genes (alpha, beta1, beta2, eta, theta, and mu) by a cDNA-PCR approach . We found significantly enhanced mean values for MRP, LRP and PKC eta gene expression, but significantly decreased Topo II alpha and cyclin A gene expression levels in G2 tumours compared with G3 . Remarkably, significant positive correlations between the MDR1, MRP or LRP gene expression levels and PKC eta were determined: MDR1/PKC eta (rs = +0.6451, P < 0.0001) n = 62; MRP/PKC eta (rs = +0.5454, P < 0.0001) n = 63; LRP/PKC eta (rs = +0.5436, P < 0.0001) n = 62; MRP/LRP (rs = +0.7703, P < 0.0001) and n = 62, MDR1/MRP (rs = +0.5042, P < 0.0001) n = 62 . Our findings point to the occurrence of a multifactorial MDR in the clinics and to PKC eta as a possible key regulatory factor for up-regulation of a series of MDR-associated genes in different types of tumours.

Am J Physiol, 1998 Jan, 274(1 Pt 1), C182 - 91
MDR1 in taste buds of rat vallate papilla: functional, immunohistochemical, and biochemical evidence; Jakob I et al.; Multidrug resistance P-glycoprotein (MDR1) is a membrane protein of 150-170 kDa that catalyzes the ATP-driven efflux of hydrophobic xenobiotics, including fluorescent dyes, from cells . Expressed in many epithelial tissues and in the endothelia of the blood-brain barrier, the MDR1 protein provides major routes of detoxification . We found that taste cells of the rat vallate papilla (VP; posterior tongue) had only a slow increase in fluorescence due to uptake of the hydrophobic dye calcein acetoxymethyl ester . However, the development of fluorescence was accelerated two- to threefold by substrates and/or inhibitors of MDR1, such as verapamil, tamoxifen, and cyclosporin A, and by addition of the transport-blocking antibody to MDR1, UIC2 . Western blots of vallate tissue rich in taste buds with the MDR1-specific monoclonal antibodies C219 and C494 revealed an immunoreactive protein at approximately 170 kDa . In contrast, the lingual epithelium surrounding the VP showed a much weaker band with these antibodies . Furthermore, using the antibodies C494 and UIC2 with tissue sections, MDR1-like immunoreactivity was found in taste cells . These results show that MDR1 is present and functional in vallate taste cells of the rat . MDR1-related transport may achieve active elimination of xenobiotics from the sensory cells and thereby protect the peripheral taste organs from potentially harmful molecules contained in an animal's food.

Int J Tuberc Lung Dis, 1997 Aug, 1(4), 319 - 25
Risk factors and outcome of human immunodeficiency virus-infected patients with sporadic multidrug-resistant tuberculosis in New York City; Mannheimer SB et al.; SETTING: An 880 bed university teaching hospital in New York City . OBJECTIVE: To assess risk factors and outcome for sporadic cases of multidrug-resistant tuberculosis (MDR-TB) in persons with human immunodeficiency virus (HIV) infection . DESIGN: In a retrospective cohort analysis, 13 HIV-positive patients with MDR-TB (cases) diagnosed between January 1991 and December 1993 were compared to 31 HIV-infected patients with susceptible or single drug-resistant tuberculosis (controls) diagnosed during the same time period to assess for differences in risk factors and outcome . RESULTS: Risk factors for MDR-TB included homosexual contact as a risk for HIV transmission, prior antiretroviral therapy and Pneumocystis carinii prophylaxis . Fatality rates were 62% for MDR-TB patients and 26% for controls (P < 0.04) . The median survival time was 5.8 months for cases and 9.8 months for controls . Risk factors associated with death included multidrug-resistance and CD4-lymphocyte counts below 200 . CONCLUSION: Sporadic MDR-TB infection in HIV-infected patients is associated with increased morbidity and mortality compared to infection with susceptible or single-drug-resistant TB . The median survival for HIV-infected patients with MDR-TB in this study is, however, two to three times longer than previously reported in MDR-TB outbreaks.

Int J Tuberc Lung Dis, 1997 Aug, 1(4), 314 - 8
Drug resistance among Mycobacterium tuberculosis isolates in Lebanon; Hamze MM et al.; OBJECTIVE: To determine the prevalence and patterns of drug resistance among Mycobacterium tuberculosis isolates recovered from tuberculosis (TB) cases in Lebanon . DESIGN: A total of 96 isolates were collected from the same number of TB cases between October 1994 and December 1995 . These isolates were obtained from cases with newly diagnosed (81.3%) and previously treated (18.7%) cases of TB, and tested against isoniazid (INH), rifampicin (RIF), streptomycin (STM) and ethambutol (ETH), using the BACTEC-TB susceptibility procedure and system . RESULTS: The male to female ratio was 2:1 and the mean ages of males and females were almost similar, 34.1 and 32.7 years, respectively . Resistance to one or more drugs was found in 25 of 96 (26%) isolates . The overall percentages of single drug resistance against INH, RIF, STM and ETH were 23.9%, 12.5%, 7.3% and 3.1%, respectively . These percentages were higher, for all drugs, in isolates recovered from previously treated compared to new cases of TB: INH (50% vs 17.9%), RIF (33.3% vs 7.7%), STM (22.2% vs 3.8%) and ETH (11.1% vs 1.3%) . Of the 25 resistant isolates, 11 were resistant to one drug only (10 to INH and 1 to STM), 10 were resistant to two drugs (7 to INH and RIF, 2 to INH and STM, 1 to STM and RIF), 2 were resistant to three drugs (1 to INH, RIF and ETH, 1 to INH, RIF and STM) and 2 were resistant to the four tested drugs . CONCLUSION: These data show that M . tuberculosis isolates in Lebanon have high rates of single and multidrug resistance, and speaks for the need to establish surveillance and monitoring programs in this country as part of the global effort to control TB.

Int J Tuberc Lung Dis, 1997 Aug, 1(4), 299 - 301
Multidrug-resistant tuberculosis in Denmark 1993-1995; Viskum K et al.; SETTING: All bacteriologically confirmed new cases of tuberculosis and treatment relapses in Denmark are examined for drug resistance . In the years 1993-1995, nine cases of multidrug-resistant tuberculosis (MDR-TB), all acquired outside Denmark, were identified among 1354 cases of tuberculosis . OBJECTIVE: To examine incidence, treatment and prognosis for patients with tuberculosis due to MDR Mycobacterium tuberculosis . DESIGN: Retrospective evaluation of routine data . RESULTS AND CONCLUSION: Multidrug resistance was present in less than one present of patients with tuberculosis . One patient died from tuberculosis without revision of treatment, and eight patients responded favourably to a regimen of pyrazinamide, streptomycin or amikacin, ofloxacin and cycloserine . In two patients, this regimen was supplemented with para-aminosalicylic acid and thiacetazone respectively . All patients needed prolonged hospitalization and had observed treatment . It is possible to cure such patients, but it is a lengthy and expensive process . It is expected that similar cases will be imported into the country and that they will occur within Denmark in the future.

Int J Tuberc Lung Dis, 1997 Jun, 1(3), 231 - 8
Quality assurance programme for drug susceptibility testing of Mycobacterium tuberculosis in the WHO/IUATLD Supranational Laboratory Network: first round of proficiency testing; Laszlo A et al.; SETTING: Quality assurance of the WHO/IUATLD global tuberculosis drug resistance surveillance programme . OBJECTIVE: To perform a proficiency test of drug susceptibility procedures within the WHO/IUATLD network of supranational reference laboratories (SRL) . DESIGN: Identical culture panels consisting of 20 clinical isolates of Mycobacterium tuberculosis containing both drug susceptible and drug resistant cultures were tested by the 16 laboratories of the network for resistance to streptomycin, isoniazid, rifampicin and ethambutol . The drug susceptibility testing procedures included the proportion, absolute concentration and resistance ratio methods as well as their variants, including the radiometric BACTEC 460 method . RESULTS: The first round of proficiency testing has shown that the specificity of drug susceptibility testing within the SRL network was significantly higher than its sensitivity . The testing of isoniazid and rifampicin shows a high degree of agreement between the labs, but discordant results can be obtained with streptomycin and ethambutol . CONCLUSION: Drug susceptibility procedures for the testing of isoniazid and rifampicin, the two anti tuberculosis drugs which define multidrug-resistant tuberculosis, are highly reliable within the SRL network . Procedures for drug susceptibility testing of streptomycin and ethambutol are still in need of standardization.

Int J Tuberc Lung Dis, 1997 Jun, 1(3), 220 - 4
A retrospective study of human immunodeficiency virus infection and drug-resistant tuberculosis in Durban, South Africa; Anastasis D et al.; SETTING: King George V hospital, a specialist referral hospital for tuberculosis (TB) patients in Durban, South Africa . OBJECTIVE: To investigate the relationship between drug-resistant TB and human immunodeficiency virus (HIV) infection . DESIGN: Retrospective descriptive study of 295 patient records, for the period January 1991 to April 1994, which were reviewed to collect data on HIV status, drug susceptibility and outcome as well as age, race, gender and previous TB treatment . RESULTS: Overall, 42 patients (14.2%) were HIV-seropositive while the rate of multidrug-resistant TB (MDR-TB) was 10.2% . Of those previously treated, 6.1% were HIV-seropositive while of those with no known history of previous TB, 5.4% were HIV-seropositive . A history of previous antituberculosis treatment was the strongest predictor for the presence of organisms resistant (Odds Ratio = 3.1; P = 0.0016) to one or more of the antituberculosis drugs . The prevalence of HIV infection was 13.1% in patients with drug-resistant TB and 14.9% in patients with drug-sensitive TB . CONCLUSION: The HIV epidemic has not exacerbated the occurrence of drug-resistant TB . History of previous TB treatment, and not HIV infection, was the principal factor associated with TB which is resistant to at least one primary anti-TB drug . However, as the HIV epidemic progresses in a milieu of high TB prevalence, the link with drug-resistant TB warrants constant monitoring and investigation.

Arch Pharm (Weinheim), 1997 Nov, 330(11), 343 - 7
Studies on propafenone-type modulators of multidrug-resistance IV1): synthesis and pharmacological activity of 5-hydroxy and 5-benzyloxy derivatives; Chiba P et al.; A series of 5-hydroxy and 5-benzyloxy analogs of the antiarrhythmic and multidrug resistance (MDR) modulating drug propafenone was synthesized and the MDR-modulating activity of the compounds was evaluated using a daunomycin efflux assay system . The key step of the synthesis is the selective reduction of the double bond in 1 without cleavage of the benzyl group thus leading to the phenol 3 . Alkylation with epichlorohydrine followed by nucleophilic epoxide ring opening gave the benzylated target compounds 5a-d . Subsequent cleavage of the benzyl group gave the 5-hydroxy analogs 6a-d . Structure activity relationship studies showed, that the 5-hydroxy derivates 6a-d fit the log P/log potency correlation line previously established for a series of propafenone analogs . In contrast, all four 5-benzyloxy analogs 5a-d showed almost identical EC50 values, independent of their log P value.

Biochem Biophys Res Commun, 1997 Dec 8, 241(1), 104 - 11
The quinoline-based drug, N-{4-{1-hydroxy-2-(dibutylamino)ethyl} quinolin-8-yl}-4-azidosalicylamide, photoaffinity labels the multidrug resistance protein (MRP) at a biologically relevant site; Vezmar M et al.; MRP is a member of the ABC trafficking proteins thought to mediate the transport of glutathione S-conjugates and amphiphilic natural products . However, unlike P-glycoprotein, the biochemical mechanism by which MRP mediates the resistance to cytotoxic drugs is not clear . In this report, we describe the interactions of a quinoline-based drug, N- inverted question mark4-{1-hydroxy-2-(dibutylamino)ethyl} quinolin-8-yl inverted question mark-4-azidosalicylamide (IAAQ), with MRP . Our results demonstrate the ability of IAAQ to photoaffinity label a 190 kDa protein in resistant Small Cell Lung Cancer cells (H69/AR) but not in the parental H69 cells . The photoaffinity labeling of the 190 kDa protein with IAAQ was both saturable and specific . The identity of the 190 kDa protein, as MRP, was confirmed by immunoprecipitation with the monoclonal antibody, QCRL-1 . Furthermore, a molar excess of LTC4, MK 571 or vinblastine inhibited the photoaffinity labeling of MRP with IAAQ in intact cells and plasma membranes . Cell growth and drug transport studies showed H69/AR cells to be less sensitive to and to accumulate less IAAQ than the parental H69 cells . In addition, MK 571 and doxorubicin increased the sensitivity to and the accumulation of IAAQ in H69/AR cells . Together, the results of this study show for the first time the direct binding of unaltered cytotoxic drug to MRP . Moreover, given the structural similarities between IAAQ and MK 571, we suggest that MK 571 modulates MRP-mediated resistance by direct binding to MRP .

J Clin Pathol, 1997 Jun, 50(6), 465 - 71
A new mdr-1 encoded P-170 specific monoclonal antibody: (6/1C) on paraffin wax embedded tissue without pretreatment of sections; Moran E et al.; AIMS: The generation and characterisation of a monoclonal antibody that specifically recognises the mdr-1 encoded protein, P-glycoprotein (P-170), on routinely processed formalin fixed, paraffin wax embedded tissue sections . METHODS: The monoclonal antibody, designated 6/1C, was produced following a combination of in vivo and in vitro immunisation regimens in Balb/c mice with a synthetic 12 amino acid peptide that corresponds to amino acids 21-32 (believed to be intracellularly located) of P-170 and has insignificant homology with the mdr-3 encoded P-170 . Antibody 6/1C was characterised by western blotting and immunocytochemistry on cytospins of paired multidrug resistant or sensitive cell lines, including mdr-1 and mdr-3 transfected cells, and by immunohistochemistry on normal and malignant formalin fixed paraffin wax embedded tissue sections . RESULTS: Antibody 6/1C showed a single band at 170 kDa on western blots of multidrug resistant cell lysates and mdr-1 transfected cell lysates that was absent on similar preparations of drug sensitive cells and mdr-3 transfected cells . Immunocytochemical studies on cytospins of multidrug resistant cells and mdr-1 transfected cells revealed strong inner plasma membrane/cytoplasmic staining . Staining was negligible on drug sensitive cells and cells transfected with the mdr-3 gene . Immunohistochemical studies on formalin fixed, paraffin wax embedded normal adult kidney, liver, and breast tissue and a range of fetal tissues exhibited staining patterns of a variety of secretory surfaces consistent with documented mdr-1 specific staining . Specific staining of malignant cells in similarly treated sections of breast tumours was seen also with antibody 6/1C . Staining on paraffin wax embedded tissue with this antibody did not require any pretreatment of tissue sections . CONCLUSIONS: This new monoclonal antibody, chosen for its specificity with the mdr-1 encoded P-170 and its reactivity on routinely fixed paraffin wax embedded tissue samples without pretreatment, appears to be useful for the investigation of P-170 in archival material . It is especially useful for retrospective studies on pretreatment and post-treatment tissue sections, and could help establish when and how rapidly mdr-1 associated drug resistance develops during chemotherapeutic regimens . Immunohistochemical assessment of P-170 expression in many cancers has potential for diagnostic purposes and may influence the choice of chemotherapeutic drugs used in the treatment of refractory tumours.

Hum Gene Ther, 1998 Jan 1, 9(1), 33 - 42
Dominant selection of hematopoietic progenitor cells with retroviral MDR1 co-expression vectors; Hildinger M et al.; When transferring the human multidrug resistance 1 (MDR1) cDNA, FMEV retroviral vectors mediate high-dose multidrug resistance and, thus, background-free selection in primary human hematopoietic progenitor cells . Here, we analyzed strategies for co-expression of a second gene from an FMEV:MDR1 vector . When linking the cDNAs with the internal ribosomal entry site (IRES) of poliovirus or retroviral splice signals, almost all multidrug-resistant hematopoietic colonies simultaneously coexpressed the 3' positioned second gene, neomycin-phosphotransferase (neoR) . The IRES strategy allowed functional co-transfer of a 4.2-kb lacZ-neoR fusion gene, resulting in a total proviral genome size of 11 kb, corresponding to the packaging limit of retroviral vectors . Preselection based on multidrug resistance elevated the expression of the second gene in IRES constructs, but not in splice vectors . Moreover, three intriguing observations were made . First, up to 30% of cells preselected for functional transfer of the 3' positioned cDNA (neoR) showed infunctional MDR1; this occurred irrespective of the linking principle and was associated with instability of the MDR1 transcription unit . Second, the levels of multidrug resistance achieved with the co-expression vectors were moderately lower (15-30% reduced) than those mediated by the monocistronic counterpart . Third, transduction with FMEV:MDR1 co-expression vectors still resulted in high-dose cancer drug resistance and background-free selection of hematopoietic progenitor cells (including primary human CD34+ colony-forming units) . Thus, for the first time, we describe MDR1 co-expression vectors that maintain their desired function in early and primary human hematopoietic cells . However, careful interpretation of the data reveals that further vector improvements are required to obtain clinically useful MDR1 co-expression vectors.

Int J Cancer, 1998 Jan 30, 75(3), 379 - 83
Therapeutic differentiation in a human rhabdomyosarcoma cell line selected for resistance to actinomycin D; Prados J et al.; Classical cytotoxic treatment of rhabdomyosarcoma (RMS) is accompanied often by significant morbidity and poor response . The use of cytotoxic agents may induce a multidrug resistance phenotype, which plays an important role in the sensitivity of tumoral cells to drugs . Using actinomycin D, a drug of choice in the treatment of RMS, we induced resistance in the TE.32.7 human RMS cell line . The TE.32.7-DAC-resistant cell line exhibited cross-resistance to vincristine and doxorubicin and showed mdr1/P-glycoprotein over-expression, suggesting that this mechanism was involved in the reduction in intracellular drug concentration and may be responsible for the failure of treatment of RMS with classical cycles of cytotoxics . Furthermore, this resistant cell line showed increased expression of the muscle differentiation markers desmin and alpha-actinin and ultrastructural changes which clearly indicated myogenic differentiation . Our findings suggest that, although this tumor is probably arrested along the normal myogenic pathway to maturation, induction of cell differentiation with anti-neoplastic drugs may be an alternative therapeutic approach . However, the failure of TE.32.7-DAC cells to completely re-enter the program of myogenic differentiation supports the hypothesis that multidrug resistance is a major obstacle in differentiation therapy for RMS.

Biochemistry, 1998 Jan 20, 37(3), 831 - 6
P-glycoprotein shows strong catalytic cooperativity between the two nucleotide sites; Senior AE et al.; P-Glycoprotein (Pgp) (also known as multidrug-resistance protein) contains two nucleotide binding sites, both of which are catalytic ATPase sites . The covalent reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) reacts in catalytic sites, and full inactivation of ATPase activity occurs at a reaction stoichiometry of 1 mol of NBD-Cl/mol of Pgp . We show that, at reaction stoichiometry of < or = 1 mol/mol, both nucleotide sites become labeled in relatively nonselective fashion . There is therefore strong interaction between the two nucleotide sites because (a) reaction of one site with NBD-Cl severely impedes reaction of reagent with the other site, and (b) reaction of one site inhibits steady-state ATPase, i.e . both sites are inhibited . Vanadate-trapping experiments revealed that when one nucleotide site was reacted with NBD-Cl, not even a single ATPase turnover event could occur in the other, intact, nucleotide site . The data demonstrate therefore that catalytic cooperativity between the two nucleotide sites in Pgp is extremely strong and mandatory for catalysis.

Eur J Biochem, 1997 Nov 15, 250(1), 115 - 21
P-glycoprotein-mediated Hoechst 33342 transport out of the lipid bilayer; Shapiro AB et al.; High-level expression of P-glycoprotein, a 170-kDa mammalian plasma membrane ATPase, is the cause of an important and widespread form of cancer multidrug resistance . P-glycoprotein reduces cellular accumulation of an enormous variety of lipophilic compounds . The basis for this broad substrate specificity is not well understood . We explored this issue by measuring the kinetics of transport of the lipophilic P-glycoprotein substrate Hoechst 33342 by P-glycoprotein-rich plasma membrane vesicles from CH(R)B30 cells . Hoechst 33342 is fluorescent when bound to the membrane, but not when in the aqueous medium, allowing movement of the dye out of the membrane to be quantitated by fluorescence intensity . The initial specific rate of transport was directly proportional to the amount of Hoechst 33342 in the lipid phase and inversely proportional to the concentration in the aqueous phase . This demonstrates that P-glycoprotein removes Hoechst 33342 from the lipid membrane, where it concentrates due to its hydrophobicity . Because the membrane concentration of hydrophobic P-glycoprotein substrates is high, it may be that P-glycoprotein need not recognize them with high affinity . Transport of hydrophobic substrates out of the lipid bilayer instead of the cytoplasm thus helps to explain the broad substrate specificity of P-glycoprotein.

Biochem J, 1997 Dec 15, 328 ( Pt 3), 897 - 904
Conformational changes of P-glycoprotein by nucleotide binding; Wang G et al.; P-glycoprotein (Pgp) is a membrane protein that transports chemotherapeutic drugs, causing multidrug resistance in human cancer cells . Pgp is a member of the ATP-binding cassette superfamily and functions as a transport ATPase . It has been suggested that the conformation of Pgp changes in the catalytic cycle . In this study, we tested this hypothesis by using limited proteolysis as a tool to detect different conformational states trapped by binding of nucleotide ligands and inhibitors . Pgp has high basal ATPase activity; that is, ATP hydrolysis by Pgp is not rigidly associated with drug transport . This activity provides a convenient method for studying the conformational change of Pgp induced by nucleotide ligands, in the absence of drug substrates which may generate complications due to their own binding . Inside-out membrane vesicles containing human Pgp were isolated from multidrug-resistant SKOV/VLB cells and treated with trypsin in the absence or presence of MgATP, Mg-adenosine 5'-{beta,gamma-imido}triphosphate (Mg-p{NH}ppA) and MgADP . Changes in the proteolysis profile of Pgp owing to binding of nucleotides were used to indicate the conformational changes in Pgp . We found that generation of tryptic fragments, including the loop linking transmembrane (TM) regions TM8 and TM9 of Pgp, were stimulated by the binding of Mg-p{NH}ppA, MgATP and MgADP, indicating that the Pgp conformation was changed by the binding of these nucleotides . The effects of nucleotides on Pgp conformation are directly associated with the binding and/or hydrolysis of these ligands . Four conformational states of Pgp were stabilized under different conditions with various ligands and inhibitors . We propose that cycling through these four states couples the Pgp-mediated MgATP hydrolysis to drug transport.

Hum Gene Ther, 1997 Nov 1, 8(16), 1901 - 10
Fibronectin fragment-facilitated retroviral transfer of the glutathione-S-transferase pi gene into CD34+ cells to protect them against alkylating agents; Kuga T et al.; To protect bone marrow cells from the toxicity of chemotherapy, a multidrug resistant gene or a dihydrofolate reductase gene has been introduced into stem cells . These genes, however, are not capable of conferring refractoriness to alkylating agents (AA), which are some of the most commonly used agents in chemotherapy regimens . In the present study, an attempt was made to endow human stem cell (CD34+ cells) with resistance to cyclophosphamide, a well-known AA, and adriamycin (ADM) by transducing the glutathione-S-transferase pi (GST-pi) gene whose product is thought to detoxify AA by conjugating them with glutathione and to remove a toxic peroxide formed by ADM . The gene transduction was carried out retrovirally with a virus titer of 1 x 10(5) FFU/ml, employing a recombinant fibronectin fragment; transduction efficiency was extremely low without the fragment . Incubation with interleukin-6 and stem cell factor enhanced the expression of fibronectin ligands VLA4 and VLA5 on CD34+ cells . This enhanced expression of VLA4 and VLA5 was considered to facilitate a close contact of the CD34+ cell to the retroviral vector via fibronectin fragments and the subsequent transduction process . The GST-pi gene-transduced CD34+ cells formed almost 3- and 2.5-fold more CFU-GM than neo gene-transduced CD34+ cells in the presence of 2.5 microg/ml of 4-hydroperoxycyclophosphamide (4-HC), an active form of cyclophosphamide, and 30 ng/ml ADM, respectively . The transfectants formed an appreciable number of colonies, even at higher concentrations of these drugs (5.0 microg/ml of 4-HC, 50 ng/ml of ADM) whereas neo gene-transduced or nontransduced CD34+ cells formed no colonies at all, indicating the possibility of selecting out the transfectants by exposing them to these anticancer drugs . Thus, we were able to demonstrate that transduction of the GST-pi gene confers resistance to cyclophosphamide as well as to ADM, and therefore this approach can be applied clinically for high-dose chemotherapy.

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