Multidrug resistant

Antisense Nucleic Acid Drug Dev, 2000 Dec, 10(6), 443 - 52
Targeting the human mdr1 gene by 125I-labeled triplex-forming oligonucleotides; Sedelnikova OA et al.; Antigene radiotherapy is our approach to targeting specific sites in the genome by combining the highly localized DNA damage produced by the decay of Auger electron emitters, such as 125I, with the sequence-specific action of triplex-forming oligonucleotides (TFO) . As a model, we used the multidrug resistance gene (mdr1) overexpressed and amplified nearly 100 times in the human KB-V1 carcinoma cell line . Phosphodiester pyrrazolopyrimidine dG (PPG)-modified TFO complementary to the polypurine-polypyrimidine region of the mdr1 gene were synthesized and labeled with 125I-dCTP at the C5 position of two cytosines by the primer extension method . 125I-TFO were delivered into KB-V1 cells with several delivery systems . DNA from the 125I-TFO-treated cells was recovered and analyzed for sequence-specific cleavage in the mdr1 target by Southern hybridization . Experiments with plasmid DNA containing the mdr1 polypurine-polypyrimidine region and with purified genomic DNA confirmed the ability of the designed 125I-TFO to bind to and introduce double-strand breaks into the target sequence . We showed that 125I-TFO in nanomolar concentrations can recognize and cleave a target sequence in the mdr1 gene in situ, that is, within isolated nuclei and intact digitonin-permeabilized cells . Our results demonstrate the ability of 125I-TFO to target specific sequences in their natural environment, that is, within the eukaryotic nucleus . The nearly 100-fold amplification of the mdr1 gene in KB-V1 cells affords a very useful cell culture model for evaluation of methods to produce sequence-specific DNA double-strand breaks for gene-specific radiotherapy.

Trans R Soc Trop Med Hyg, 2000 Nov-Dec, 94(6), 689 - 93
Randomized comparison of mefloquine-artesunate versus quinine in the treatment of multidrug-resistant falciparum malaria in pregnancy; McGready R et al.; Since no effective malaria prevention measures have been identified for pregnant women living on the western border of Thailand, prompt diagnosis and efficient treatment are paramount, although drug resistance in Plasmodium falciparum has narrowed the treatment options . An open randomized comparison of supervised quinine (10 mg salt/kg every 8 h) for 7 days (Q7) versus mefloquine 25 mg base/kg (total dose) plus artesunate 4 mg/kg per day for 3 days (MAS3) was conducted in 1995-97 in 108 Karen women with acute uncomplicated falciparum malaria in the second or third trimesters of pregnancy . The MAS3 regimen was more effective than the Q7 regimen: day 63 cure rates were 98.2% (95% CI 94.7-100) (n = 65) for MAS3 and 67.0% (95% CI 43x3-90x8) (n = 41) for Q7, P = 0x001 . The MAS3 regimen was also associated with less gametocyte carriage; the average person-gametocyte-weeks for MAS3 was 2.3 (95% CI 0-11) and for Q7 was 46x9 (95% CI 26-78) per 1000 person-weeks, respectively (P < 0.001) . MAS3 was significantly better tolerated . These evident advantages must be balanced against a possible increased risk of stillbirth with the use of mefloquine in pregnancy . Further randomized studies assessing the safety and efficacy of other artemisinin-containing combination regimens in pregnancy are needed urgently.

Trans R Soc Trop Med Hyg, 2000 Nov-Dec, 94(6), 603 - 7
Tuberculosis treatment failure and drug resistance--same strain or reinfection?
Sonnenberg P, Murray J, Shearer S, Glynn JR, Kambashi B, Godfrey-Faussett P.
Tuberculosis patients may have Mycobacterium tuberculosis in their sputum at the end of treatment, and may show new drug resistance, due to either inadequate treatment of the original episode or reinfection with a new strain during therapy . In a cohort study of mineworkers with tuberculosis in South Africa, 57 of 438 patients had positive sputum cultures 6 months after recruitment in 1995 . Of the 31 patients who initially had fully sensitive strains, 3 developed multidrug resistance (MDR) and 3 single-drug resistance (SDR) . Of the 6 who started with SDR, 3 became MDR . HIV infection was not associated with drug resistance at enrollment or 6 months later . We compared pairs of DNA fingerprints from isolates of M . tuberculosis at recruitment and 6 months later in the 48 patients for whom we had both available . In 45, the pairs were identical . In 1 patient, although both isolates were fully sensitive, the later fingerprint had 1 less band (transposition) . In 2 pairs, the fingerprint patterns were completely different: one seemed to be the result of laboratory error and the other was a true reinfection with an MDR strain . Despite a high risk of infection, with a moderate proportion of background drug-resistant strains (11% SDR, 6% MDR), reinfection is not a common cause of treatment failure or drug resistance at 6 months.

J Nucl Med, 2001 Jan, 42(1), 17 - 20
Paclitaxel-Based chemotherapy for non-small cell lung cancer: predicting the response with 99mTc-tetrofosmin chest imaging; Kao CH et al.; The purpose of this study was to retrospectively predict the chemotherapeutic response to paclitaxel for non-small cell lung cancer (NSCLC) using 99mTc-tetrofosmin (TF) uptake and to detect the expression of 170-kDa multidrug resistance-mediated P-glycoprotein (MDR-Pgp) . METHODS: Before chemotherapy with paclitaxel, 20 patients with stage IIIb or IV NSCLC were enrolled in this study to undergo early and delayed 99mTc-TF chest imaging for calculating tumor-to-normal lung ratios (T/NL) and retention indices (RI) for assessment of the MDR-Pgp in NSCLC . RESULTS: The early and delayed mean T/NLs were 1.59 +/- 0.25 and 1.50 +/- 0.25, respectively, for 10 patients with a good response and 1.09 +/- 0.09 and 1.03 +/- 0.05, respectively, for 10 patients with a poor response . The differences were shown to be significant (P < 0.001) by independent Student t tests . However, no significant differences (P = 0.801) between good-response patients (-5.70% +/- 3.67%) and poor-response patients (-5.23% +/- 4.51%) were found in RI . In addition, other prognostic factors (age, sex, tumor size, stage, and cell type) were not significantly different between good-response patients and poor-response patients . CONCLUSION: 99mTc-TF chest images are potential tools for understanding MDR-Pgp expression in NSCLC and for predicting the chemotherapeutic response to paclitaxel.

Arch Immunol Ther Exp (Warsz), 2000, 48(6), 547 - 51
Bacteriophage therapy of bacterial infections: an update of our institute's experience; Weber-Dabrowska B et al.; 1307 patients with suppurative bacterial infections caused by multidrug-resistant bacteria of different species were treated with specific bacteriophages (BP) . BP therapy was highly effective; full recovery was noted in 1123 cases (85.9%) . In 134 cases (10.9%) transient improvement was observed and only in 50 cases (3.8%) was BP treatment found to be ineffective . The results confirm the high effectiveness of BP therapy in combating bacterial infections which do not respond to treatment with the available antibiotics.

Lancet, 2001 Jan 6, 357(9249), 42 - 3
Multidrug-resistance protein 1 in focal cortical dysplasia; Sisodiya SM et al.; Drug resistance in epilepsy is poorly understood . We used routine immunohistochemistry to assess overexpression of a multidrug-resistance protein in dysplastic neurons, glia, and around vessels in surgically resected epileptogenic human brain tissue . We showed non-tumoral overexpression of this multidrug-resistance protein, which might contribute to drug resistance in epilepsy caused by focal cortical dysplasia.

Int J Hematol, 2000 Dec, 72(4), 418 - 24
Multidrug resistance of acute leukemia and a strategy to overcome it; Motoji T et al.; A representative cause of multidrug resistance (MDR) is expression of the MDR gene (mdr1) and its product P-glycoprotein (P-gp) . The function of P-gp is thought to be the extrusion of anticancer drugs from the cell against a concentration gradient . In acute myelogenous leukemia (AML), P-gp expression in leukemic blast cells at initial presentation has been reported to be 20% to 40% . The remission rate of acute leukemia patients is significantly lower in P-gp+ patients than in P-gp- patients . A significantly shorter survival and relapse-free survival in P-gp+ AML patients compared with P-gp- patients has been reported . Intracellular daunorubicin/Rhodamine123 content in P-gp+ leukemic blast cells is significantly lower than in P-gp- leukemic blast cells . By using a leukemic blast colony assay, lowered sensitivity to the anticancer drug was revealed not only in leukemic blast cells but also in leukemic progenitors . One method to overcome MDR with a possibility of clinical application is to use drugs that interfere with the function of P-gp . The addition of MS-209 in vitro as an MDR-reversing agent significantly enhanced the intracellular daunorubicin/Rhodamine 123 accumulation and the retention of P-gp+ leukemic blast cells to a level similar to that of P-gp- blast cells . Recent clinical trials using MDR-reversing agents have demonstrated some encouraging results in P-gp+ patients but not in P-gp- patients.

Genes Immun, 2000 Aug, 1(6), 371 - 9
P-glycoprotein (encoded by multidrug resistance genes) is not required for interleukin-2 secretion in mice and humans; Gollapudi S et al.; P-glycoprotein (encoded by multidrug resistance genes), a member of the ATP-binding cassette transporter protein superfamily, has been shown to play a role in the secretion of cytokines . This conclusion was based upon the inhibition of cytokine secretion by anti-P-gp monoclonal antibodies . In this study, we show that anti-CD3-stimulated lymphocytes from wild-type, mdr1a knock out and mdr1ab double knock out mice produce similar amounts of IL-2, IFN-gamma, IL-4, and IL-10 . In addition, Jurkat T cells that lack P-gp and MDR1-transfected Jurkat T cells (JurkatP-gp) as well as purified human peripheral blood CD4+ P-gp+ and CD4+ P-gp- and CD8+ P-gp+ and CD8+ P-gp- T cell subsets produced comparable amounts of IL-2 . These data show that P-gp is not required for secretion of IL-2, IFN-gamma, IL-4, and IL-10 secretion in mice and IL-2 secretion in humans.

Cancer Res, 2001 Jan 1, 61(1), 392 - 9
D-24851, a novel synthetic microtubule inhibitor, exerts curative antitumoral activity in vivo, shows efficacy toward multidrug-resistant tumor cells, and lacks neurotoxicity; Bacher G et al.; N-(pyridin-4-yl)-{1-(4-chlorbenzyl)-indol-3-yl}-glyoxyl-amid (D-24851) is a novel synthetic compound that was identified in a cell-based screening assay to discover cytotoxic drugs . D-24851 destabilizes microtubules and blocks cell cycle transition specifically at G2-M phase . The binding site of D-24851 does not overlap with the tubulin binding sites of known microtubule-destabilizing agents like vincristine or colchicine . In vitro, D-24851 has potent cytotoxic activity toward a panel of established human tumor cell lines including SKOV3 ovarian cancer, U87 glioblastoma, and ASPC-1 pancreatic cancer cells . In vivo, oral D-24851 treatment induced complete tumor regressions (cures) in rats bearing Yoshida AH13 sarcomas . Of importance is that the administration of curative doses of D-24851 to the animals revealed no systemic toxicity in terms of body weight loss and neurotoxicity in contrast to the administration of paclitaxel or vincristine . Interestingly, multidrug-resistant cell lines generated by vincristine-driven selection or transfection with the Mr 170,000 P-glycoprotein encoding cDNA were rendered resistant toward paclitaxel, vincristine, or doxorubicin but not towards D-24851 when compared with the parental cells . Because of its synthetic nature, its oral applicability, its potent in vitro and in vivo antitumoral activity, its efficacy against multidrug-resistant tumors, and the lack of neurotoxicity, D-24851 may have significant potential for the treatment of various malignancies.

Cancer Res, 2001 Jan 1, 61(1), 172 - 7
Simultaneous treatment with 1-beta-D-arabinofuranosylcytosine and daunorubicin induces cross-resistance to both drugs due to a combination-specific mechanism in HL60 cells; Takemura H et al.; We have established a human myelogenous leukemia cell line (HL60/AD) that is 10-fold cross-resistant to both 1-beta-D-arabinofuranosylcytosine (ara-C) and daunorubicin; the cell line was isolated from HL60 by simultaneous treatment with these two agents at low drug concentrations attainable in clinical trials . HL60/AD was found to have multiple resistance mechanisms . With regard to ara-C, HL60/AD cells showed decreased deoxycytidine kinase activity but did not show elevation of cytidine deaminase activity or a decrease in ara-C influx . With regard to daunorubicin, a decrease in topoisomerase II activity was found . A decrease in intracellular accumulation of daunorubicin was also found . P-glycoprotein was not detected, but the multidrug resistance-associated protein was expressed . Furthermore, an increase of total cellular glutathione (GSH) content was found . Interestingly, the resistance of HL60/AD cells not only to daunorubicin but also to ara-C was markedly reversed by treatment with L-buthionine-(S,R)-sulfoximine (BSO), a potent inhibitor of GSH synthesis . After exposure of HL60/AD to ara-C, mitochondrial membrane potential and reactive oxygen intermediates showed no significant change, but a considerable loss of mitochondrial membrane potential and an increase in reactive oxygen intermediate generation were caused by pre-incubation with BSO . Neither elevation of GSH nor reversal of resistance by BSO was found in ara-C-resistant HL60 cells that were selected only with ara-C . These findings suggest that in addition to the summation of the mechanisms of resistance to each agent reported previously, an increased level of GSH plays an important role in the cross-resistance induced in HL60/AD cells by simultaneous exposure to both drugs.

Ann N Y Acad Sci, 2000, 922, 188 - 94
Transport of topoisomerase I inhibitors by the breast cancer resistance protein . Potential clinical implications; Schellens JH et al.; The multidrug resistance protein BCRP (breast cancer resistance protein) is a member of the ATP-binding cassette family of drug transporters . Overexpression of BCRP caused by exposure of cells to mitoxantrone (MX) or doxorubicin/verapamil resulted in a resistance pattern that is different from what is generally seen in the case of P-glycoprotein and MRP1 overexpression . Recently, the BCRP gene has been described in ovarian, breast, colon, and gastric cancer and fibrosarcoma cell lines . Our human tumor cells T8 and MX3, derived from the ovarian cancer cell line IGROV1 by stepwise increased exposure to topotecan and MX, are resistant to topotecan, CPT11, SN38, and 9-aminocamptothecin as well as MX . Increased energy-dependent efflux of affected drugs was noted . BCRP is a very efficient transporter of topotecan . Our recent studies, using the monoclonal antibody (mAb) BXP34, revealed that BCRP is located in the plasma membrane of the T8 and MX3 cell lines . Preliminary results of staining of human tumor cells showed low or absent levels of BCRP in a panel of solid tumors and acute myeloid leukemia cells.

Cas Lek Cesk, 2000 Oct 11, 139(20), 620 - 2
{Resistance to cytostatics in hematologic neoplasm cells . Part III}; Kodydkova K et al.; Resistance to cytotoxic drugs is a serious drawback in the treatment of patient with tumours, both of the haemopoietic and non-haemopoietic origin . The cytotoxic effect of drugs on the malignant cells manifests as the process of apoptosis . In the resistant malignant cells, apoptosis becomes prevented by several mechanisms . The multidrug resistance (MDR) is one of the principal mechanisms when the cancer cells develop resistance to multiple chemically and functionally unrelated cytotoxic compounds . The decrease of the cytotoxic drug concentration at the molecular target site may come from activation of some efflux membrane systems participating in the transport of cytotoxic drugs out of the cell (e.g . Pgp, MDR, and LRP) . Another mechanism of resistance is the increased enzymatic detoxification of the drug by glutathion-S-transferase system . Changes in the molecular target of the cytotoxic drug such as topoisomerase molecule can be also responsible for the resistance . At least two additional mechanisms of resistance of tumour cells were identified . Resistance can come from either deregulation of proapoptotic mechanisms in tumour cells or by increased activity of reparation processes which control the damaged molecule of DNA . Several methods that detect the cause of resistance in distinct cell populations have been developed . The great effort is now focused on both the detection of mechanisms of the resistance and on the clinical procedures of overcoming the resistance to cytotoxic drugs.

Cas Lek Cesk, 2000 Sep 27, 139(19), 591 - 5
{Resistance of hematologic neoplasm cells to cytostatics . Part II}; Kodydkova K et al.; Resistance to cytotoxic drugs is a serious drawback in the treatment of patients with tumours, both of the haemopoietic and non-haemopoietic origin . The cytotoxic effect of drugs on the malignant cells manifests as the process of apoptosis . In the resistant malignant cells, apoptosis becomes prevented by several mechanisms . The multidrug resistance (MDR) is one of the principal mechanisms when the cancer cells develop resistance to multiple chemically and functionally unrelated cytotoxic compounds . The decrease of the cytotoxic drug concentration at the molecular target site may come from activation of some efflux membrane systems participating in the transport of cytotoxic drugs out of the cell (e.g . Pgp, MDR, and LRP) . Another mechanism of resistance is the increased enzymatic detoxification of the drug by glutathion-s-transferase system . Changes in the molecular target of the cytotoxic drug such as topoisomerase molecule can be also responsible for the resistance . At least two additional mechanisms of resistance of tumour cells were identified . Resistance can come from either deregulation of proapoptotic mechanisms in tumour cells or by increased activity of reparation processes which control the damaged molecule of DNA . Several methods that detect the cause of resistance in distinct cell populations have been developed . The great effort is now focused on both the detection of mechanisms of the resistance and on the clinical procedures of overcoming the resistance to cytotoxic drugs.

J Int Med Res, 2000 Nov-Dec, 28(6), 300 - 6
Drug-resistant tuberculosis at the University Hospital in Gaziantep, South-eastern Turkey; Balci I et al.; We aimed to determine the present status of drug resistance of Mycobacterium tuberculosis at the Gaziantep University Hospital in south-east Turkey . Data for 1995 to 1999 were retrospectively evaluated with respect to smear-positive cases, first positive culture for Mycobacterium tuberculosis for each patient and drug-susceptibility tests for the major antituberculous drugs . Cultures were done using the Bactec 460 TB method . A total of 106 (40.2%) strains were resistant to at least one drug . Single drug resistance was observed in 47 strains (17.8%) and resistance to two or three drugs was found in 28 and 29 strains (10.6 and 11.0%), respectively . Two strains (0.8%) were resistant to all four drugs . While multidrug resistance was observed in 52 (19.7%) strains, resistance to isoniazid + rifampin was observed in 20 (7.6%) strains . This retrospective study showed that combined drug resistance of M . tuberculosis is highly prevalent in southeastern Turkey . Possible reasons for the failure of current control policies were considered.

Curr Genet, 2000 Dec, 38(5), 248 - 55
Isolation and molecular characterization of the carboxy-terminal pdr3 mutants in Saccharomyces cerevisiae; Simonics T et al.; Multidrug resistance in Saccharomyces cerevisiae mainly results from the overexpression of genes coding for the membrane efflux pumps, the major facilitators and the ABC binding cassette transporters, under the control of key transcription regulators encoded by the PDR1 and PDR3 genes . Pdr3p transcriptional activator contains a weak activation domain near the N-terminal zinc finger, a central regulatory domain, and a strong activation domain near the carboxyl terminus . Here we report the results of the mutational analysis of the C-terminal region of Pdr3p . After in vitro mutagenesis of the PDR3 gene six single amino acid substitutions were identified and resulted in resistance to cycloheximide, sulfomethuron methyl, 4-nitroquinoline oxide, fluconazole, mucidin, chloramphenicol and oligomycin . All the C-terminal pdr3 mutant alleles also conferred multidrug resistance in the presence of the wild-type PDR3 gene . The pdr3 mutations resulted in overexpression of both the PDR3 and PDR5 genes as revealed by transactivation experiments involving the PDR3-lacZ and PDR5-lacZ fusion genes and Western blot analyses using antibodies against Pdr5p . Most of the C-terminal pdr3 mutations were found in two sequence stretches exhibiting a high degree of amino acid identity with Pdr1p indicating that they might play a significant role in protein-protein interactions during the initiation of transcription of genes involved in multidrug resistance.

Rev Physiol Biochem Pharmacol, 2001, 142, 1 - 96
Modulation of protein kinase C in antitumor treatment; Hofmann J; PKC isoenzymes were found to be involved in proliferation, antitumor drug resistance and apoptosis . Therefore, it has been tried to exploit PKC as a target for antitumor treatment . PKC alpha activity was found to be elevated, for example, in breast cancers and malignant gliomas, whereas it seems to be underexpressed in many colon cancers . So it can be expected that inhibition of PKC activity will not show similar antitumor activity in all tumors . In some tumors it seems to be essential to inhibit PKC to reduce growth . However, for inhibition of tumor proliferation it may be an advantage to induce apoptosis . In this case an activation of PKC delta should be achieved . The situation is complicated by the facts that bryostatin leads to the activation of PKC and later to a downmodulation and that the PKC inhibitors available to date are not specific for one PKC isoenzyme . For these reasons, PKC modulation led to many contradicting results . Despite these problems, PKC modulators such as miltefosine, bryostatin, safingol, CGP41251 and UCN-01 are used in the clinic or are in clinical evaluation . The question is whether PKC is the major or the only target of these compounds, because they also interfere with other targets . PKC may also be involved in apoptosis . Oncogenes and growth factors can induce cell proliferation and cell survival, however, they can also induce apoptosis, depending on the cell type or conditions in which the cells or grown . PKC participates in these signalling pathways and cross-talks . Induction of apoptosis is also dependent on many additional factors, such as p53, bcl-2, mdm2, etc . Therefore, there are also many contradicting results on PKC modulation of apoptosis . Similar controversial data have been reported about MDR1-mediated multidrug resistance . At present it seems that PKC inhibition alone without direct interaction with PGP will not lead to successful reversal of PGP-mediated drug efflux . One possibility to improve chemotherapy would be to combine established antitumor drugs with modulators of PKC . However, here also very contrasting results were obtained . Many indicate that inhibition, others, that activation of PKC enhances the antiproliferative activity of anticancer drugs . The problem is that the exact functions of the different PKC isoenzymes are not clear at present . So further investigations into the role of PKC isoenzymes in the complex and interacting signalling pathways are essential . It is a major challenge in the future to reveal whether modulation of PKC can be used for the improvement of cancer therapy.

Eur J Nucl Med, 2000 Dec, 27(12), 1845 - 63
Use of myocardial imaging agents for tumour diagnosis--a success story?
Schomacker K, Schicha H.
The search for new radiopharmaceuticals for tumour diagnosis usually proceeds on the basis of rational concepts drawing on the latest advances in molecular biology . Using this approach, radioactive peptide hormones, antibodies and oligonucleotides have been developed that are used increasingly in nuclear medicine for diagnostic and therapeutic purposes . This article, however, focusses on a group of radiopharmaceuticals whose use in tumour diagnosis was not the outcome of a methodical development programme but rather the result of a chance discovery . These radiopharmaceuticals, thallium-201 and technetium-99m labelled 2-methoxyisobutylisonitrile (MIBI), tetrofosmin and furifosmin, were first developed through extensive research efforts for cardiac imaging, but during their worldwide application for myocardial scintigraphy they were accidentally found to accumulate in tumours . Intensive studies were then begun on cell cultures in an attempt to discover the cause of their uptake into tumours . The aim was to compare the effectiveness of the radiopharmaceuticals for tumour diagnosis in a range of indications and to investigate the various mechanisms by which they are taken up into tumours . While the more favourable radiophysical properties of 99mTc-MIBI render it superior to 201Tl for many diagnostic purposes, neither 99mTc-tetrofosmin nor 99mTc-furifosmin has yet proved suitable for clinical routine examinations, although the former has found limited application . In the case of 99mTc complexes, the breakthrough came with the experimental finding that these substances are substrates of P-glycoprotein, a product of the human multidrug resistance gene (MDR1) . The concentration of 99mTc complexes in tumour cells is a function of a passive, membrane potential-dependent influx into and a P-glycoprotein-controlled efflux out of the tumour cell . Preliminary studies suggest that in vivo detection of MDR may even be possible . There is also evidence that the P-glycoprotein-mediated transport system can be blocked competitively . However, it will be some time before a system can be developed for detection of MDR on a routine basis.

Eur J Nucl Med, 2000 Dec, 27(12), 1786 - 92
Comparison of the accumulation and efflux kinetics of technetium-99m sestamibi and technetium-99m tetrofosmin in an MRP-expressing tumour cell line; Utsunomiya K et al.; The potential clinical use of technetium-99m labeled sestamibi (Tc-MIBI) and tetrofosmin (Tc-Tfos) to image tumours is currently being evaluated . In this study . the accumulation and efflux of Tc-MIBI and Tc-Tfos in the nasopharyngeal carcinoma cell line CNE-1 were examined in the presence or absence of various inhibitors of P-glycoprotein (PGP) and/or multidrug resistance associated protein (MRP) activity {GG918, PSC833, verapamil (Vrp), cyclosporin A (CsA) and buthionine sulfoximine (BSO)} . Reverse-transcriptase polymerase chain reaction analysis and immunodetection of the CNE-1 cells detected expression of MRP, MRPI and MRP2 but not PGP . Tc-MIBI and Tc-Tfos accumulation was increased (P < 0.0001) and efflux decreased (P < 0.05) in the presence of BSO, CsA, Vrp and PSC833 but not GG918, which is a specific inhibitor of PGP . The absolute accumulation of Tc-MIBI was approximately twofold higher than that seen with Tc-Tfos, whereas the addition of inhibitors caused a much greater suppression of Tc-Tfos transport (>2 times greater than for Tc-MIBI) . However, no qualitative differences in inhibitors were seen between Tc-MIBI and Tc-Tfos . These results suggest that both Tc-MIBI and Tc-Tfos are substrates for the MRP transporter and that PSC833, Vrp, CsA and BSO but not GG918 can inhibit MRP activity . These results indicate that Tc-MIBI and Tc-Tfos may be suitable imaging agents for detecting MRP-mediated drug resistance in human cancers.

Zhonghua Xue Ye Xue Za Zhi, 1998 Sep, 19(9), 470 - 2
{Study on the relationship between DNA topoisomerase II activity and multidrug resistance in leukemic cells}; Zheng W et al.; OBJECTIVE: To explore the relationship between DNA topoisomerase II (Topo II) activity and multidrug resistance in acute leukemia(AL) . METHODS: Topo II activities were measured in peripheral blood or bone marrow leukemia cells from 17 pretreated patients and 20 after combination chemotherapy by fluorometric assay . In the same time, multidrug resistant p-glycoprotein expression was detected by immunohistochemistry using JSB-1 monoclonal antibody and mdr-1 gene expression was assayed by RT-PCR in 18 post-chemotherapy patients . RESULTS: Topo II activities were significantly higher in pretreated AL patients (0.6415 +/- 0.0561) than in healthy blood donors (0.2304 +/- 0.0591)(P < 0.005) and significantly lower in post-chemotherapy patients (0.3563 +/- 0.1011) than in pretreated ones (P < 0.005) . Among 15 poorly responsed patients, 12 were p170 positive and mdr-1 overexpression, 3 case were p170 negative . Topo II activity was also decreased in these poor responsers . CONCLUSION: Decreased Topo II activity may be another factor in multidrug resistance.

Leukemia, 2000 Dec, 14(12), 2085 - 94
Phase I study of cinchonine, a multidrug resistance reversing agent, combined with the CHVP regimen in relapsed and refractory lymphoproliferative syndromes; Solary E et al.; Overexpression of P-glycoprotein (P-gp) in cancer cells reduces intracellular accumulation of various anticancer drugs including anthracyclines and vinca alkaloids . This multidrug resistance (MDR) phenotype can be reversed in vitro by a number of non-cytotoxic drugs . We have identified the quinine's isomer cinchonine as a potent MDR reversing agent, both in vitro and in animal models . Here, we report an open phase I dose escalation trial in patients with refractory or relapsed malignant lymphoid diseases . Cinchonine dihydrochloride was administered by continuous i.v . infusion for 48 h and escalated over five dose levels ranging from 15 to 35 mg/kg/d . Cinchonine infusion started 24 h before i.v . doxorubicin (25 mg/m2), vinblastine (6 mg/m2), cyclophosphamide (600 mg/m2) and methylprednisolone (1 mg/kg/d) (CHVP regimen) and lasted for 24 h after chemotherapy infusion . Thirty-four patients received 87 cycles of CHVP/cinchonine . The MTD of cinchonine administered by continuous i.v . infusion was 30 mg/kg/d . Prolonged cardiac repolarization was the main dose-limiting toxicity . No ventricular arrhythmia including 'torsade de pointes' was observed . An MDR reversing activity was identified in the serum from every patient and correlated with cinchonine serum level . When infused at 30 mg/kg/d, cinchonine demonstrated a limited influence on doxorubicin pharmacokinetic . We conclude that i.v . infusion of cinchonine might be started 12 h before MDR-related chemotherapy infusion and requires continuous cardiac monitoring but no reduction of cytotoxic drug doses.

J Neurochem, 2001 Jan, 76(2), 627 - 36
The multidrug resistance protein MRP1 mediates the release of glutathione disulfide from rat astrocytes during oxidative stress; Hirrlinger J et al.; The release of glutathione disulfide has been considered an important process for the maintenance of a reduced thiol redox potential in cells during oxidative stress . In cultured rat astrocytes, permanent hydrogen peroxide-induced oxidative stress caused a rapid increase in intracellular glutathione disulfide, which was followed by the appearance of glutathione disulfide in the medium . Under these conditions, the viability of the cells was not compromised . In the presence of cyclosporin A and the quinoline-derivative MK571, inhibitors of multidrug resistance proteins (MRP1 and MRP2), glutathione disulfide accumulated in cells and the release of glutathione disulfide from astrocytes during H2O2 stress was potently inhibited, suggesting a contribution of MRP1 or MRP2 in the release of glutathione disulfide from astrocytes . Using RT-PCR we amplified a cDNA from astroglial RNA with a high degree of homology to MRP1 from humans and mouse . In contrast, no fragment was amplified by using primers specific for rat MRP2 . In addition, the presence of MRP1 protein in astrocytes was demonstrated by its immunolocalization in cells expressing the astroglial marker protein glial fibrillary acidic protein . Our data identify rat astrocytes as a MRP1-expressin, brain cell type and demonstrate that this transporter participates in the release of glutathione disulfide from astrocytes during oxidative stress.

J Pharmacol Exp Ther, 2001 Mar, 296(3), 723 - 35
In vitro substrate identification studies for p-glycoprotein-mediated transport: species difference and predictability of in vivo results; Yamazaki M et al.; Two different cellular assay models were assessed as in vitro systems for P-glycoprotein (P-gp) substrate identification: cellular accumulation studies with KB-V1, a human MDR1 P-gp-overexpressing multidrug-resistant human epidermoid carcinoma cell line; and transcellular transport studies with L-MDR1 (or L-mdr1a), a human MDR1 (or mouse mdr1a)-transfected porcine renal epithelial cell line . The in vitro-in vivo correlation for P-gp-mediated transport activity was also examined by comparing in vitro data obtained from L-mdr1a cell studies and in vivo data from mdr1a (-/-)/(+/+) CF-1 mice studies for several compounds . The results are summarized as follows: 1) two in vitro assay systems routinely identified the substrate for human MDR1 P-gp-mediated transport with similar quantitative results; 2) in vitro studies with L-MDR1 and L-mdr1a cells demonstrated that the P-gp substrate susceptibility is different between human and mouse for certain compounds (species difference); and 3) in vivo brain concentration ratios of mdr1a (-/-) to (+/+) CF-1 mice, either at a certain time point or up to 60 min, correlated well with the in vitro transcellular transport ratios from L-mdr1a cells (r(2) = 0.968 and 0.926, respectively) . This indicates that, at least in mice, the in vitro data are valid predictors of the in vivo contribution of P-gp: the contribution of P-gp to the distribution of the compound to the brain up to 60 min post i.v . administration . These results provide a rationale for predicting in vivo relevance of P-gp in human from in vitro data using human P-gp-expressing cells.

J Neurochem, 2001 Feb, 76(4), 966 - 74
A bioreversible prodrug approach designed to shift mechanism of brain uptake for amino-acid-containing anticancer agents; Killian DM et al.; By derivatization at the N-terminus of amino acid-based anticancer agents (e.g . melphalan and acivicin) to form a drug delivery system (TDDS), we demonstrate a change in the mechanism of brain uptake from the large neutral amino acid transporter (LAT) pathway to passive . An in situ rat brain perfusion technique was used to determine the brain capillary permeability-surface area (PA) product for {(14)C}L-Leu as control (5.18 +/- 0.32 x 10(-2) mL/s/g), which was inhibited competitively (to 7-18% of control) by an excess concentration of the amino-acid-containing anticancer agents, acivicin and melphalan . However, TDDS did not compete for LAT-mediated brain uptake of the radiotracer {(14)C}L-Leu . Brain uptake of TDDS was determined after in situ brain perfusion followed by RP-HPLC along with LC-MS/MS detection of the analytes in brain samples . The PA product for CH(3)-TDDS containing melphalan (5.09 +/- 2.0 x 10(-2) mL/s/g) shows that these agents rapidly cross the blood-brain barrier . Furthermore, competition studies of CH(3)-TDDS with {(3)H}verapamil suggest that the TDDS interacts significantly with the multidrug resistant efflux system (P-glycoprotein) at the blood-brain barrier . Therefore, TDDS were shown to lack LAT-mediated brain uptake . The drug delivery systems, however, showed uptake predominantly via the passive route along with recognition by the multidrug resistant efflux protein at the cerebrovasculature.

Clin Infect Dis, 2001 Feb 15, 32(4), 643 - 6 Epub 2001 Feb 09.
Novel treatment of meningitis caused by multidrug-resistant Mycobacterium tuberculosis with intrathecal levofloxacin and amikacin: case report; Berning SE et al.; We report the case of a 25-year-old HIV-negative man with disseminated multidrug-resistant tuberculosis (MDRTB), who-on a retreatment regimen-experienced total resolution of TB miliary disease, but progressive TB meningitis . Therefore, intrathecal treatment with amikacin and levofloxacin was instituted, with successful clinical and microbiological results.

Am J Hum Genet, 2001 Mar, 68(3), 642 - 52 Epub 2001 Feb 09.
Compound heterozygosity for a recurrent 16.5-kb Alu-mediated deletion mutation and single-base-pair substitutions in the ABCC6 gene results in pseudoxanthoma elasticum; Ringpfeil F et al.; Pseudoxanthoma elasticum (PXE) is a systemic heritable disorder affecting the elastic structures in the skin, eyes, and cardiovascular system, with considerable morbidity and mortality . Recently, mutations in the ABCC6 gene (also referred to as "MRP6" or "eMOAT") encoding multidrug-resistance protein 6 (MRP6), a putative transmembrane ABC transporter protein of unknown function, have been disclosed . Most of the genetic lesions delineated thus far consist of single-base-pair substitutions resulting in nonsense, missense, or splice-site mutations . In this study, we examined four multiplex families with PXE inherited in an autosomal recessive pattern . In each family, the proband was a compound heterozygote for a single-base-pair-substitution mutation and a novel, approximately 16.5-kb deletion mutation spanning the site of the single-base-pair substitution in trans . The deletion mutation was shown to extend from intron 22 to intron 29, resulting in out-of-frame deletion of 1,213 nucleotides from the corresponding mRNA and causing elimination of 505 amino acids from the MRP6 polypeptide . The deletion breakpoints were precisely the same in all four families, which were of different ethnic backgrounds, and haplotype analysis by 13 microsatellite markers suggested that the deletion had occurred independently . Deletion breakpoints within introns 22 and 29 were embedded within AluSx repeat sequences, specifically in a 16-bp segment of DNA, suggesting Alu-mediated homologous recombination as a mechanism.

Biochem Biophys Res Commun, 2001 Feb 16, 281(1), 109 - 14
Down-regulation of catalase gene expression in the doxorubicin-resistant AML subline AML-2/DX100; Kim HS et al.; A major obstacle to successful cancer chemotherapy is the development of multidrug resistance (MDR) . The previous study revealed that a doxorubicin-resistant AML subline (AML-2/DX100) overexpressed an MDR-associated protein (MRP) but not P-glycoprotein . The AML-2/DX100 also showed various levels of resistance to daunorubicin and vincristine but was paradoxically sensitive to hydrogen peroxide (5-fold), t-butyl hydroperoxide (3-fold), and paraquat (2-fold) when compared to the drug-sensitive parental AML-2 cells (AML-2/WT) . We compared the activities of antioxidant enzymes to detoxify reactive oxygen species (ROS), including superoxide dismutases, glutathione S-transferase, catalase, glutathione reductase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase in both AML-2/WT and AML-2/DX100 . Interestingly, of these antioxidant enzymes, catalase activity of AML-2/DX100 decreased significantly to about one-third that of AML-2/WT (P < 0.000005) . The decreased activity of catalase was due to reduced expression of the catalase gene; confirmed by Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses . The decreased activity of catalase was maintained even in the absence of doxorubicin for 3 months as well as by the treatment of probenecid, an MRP inhibitor . In addition, there was no difference in catalase activity between HL-60 and another MRP-overexpressing subline HL-60/Adr . Taken together, the paradoxical increase in the sensitivity of an MRP-overexpressing AML-2/DX100 in response to peroxides and paraquat is due to the down-regulation of catalase gene expression, which totally independent of overexpression of MRP . It is therefore possible that decreased catalase activity could be exploited as an Achilles' heel in resistant cells such as this.

Curr Infect Dis Rep, 2001 Feb, 3(1), 68 - 76
Recent Advances in the Prophylaxis and Treatment of Malaria; Labbe AC et al.; Increases in international travel and escalating drug resistance are putting put a growing number of travelers at risk of contracting malaria . Resistance to chloroquine and proguanil and real and perceived intolerance to standard agents, such as mefloquine, has highlighted the need for new antimalarials to prevent and treat malaria . Promising new agents to prevent malaria include the combination of atovaquone and proguanil, primaquine, and a related 8-aminoquinoline, tafenoquine . These agents are active against the liver stage of the malaria parasite, and therefore can be discontinued shortly after the traveler leaves the malaria-endemic area; this offers a clear advantage, in terms of adherence to a treatment regimen . For treatment of multidrug-resistant Plasmodium falciparum malaria, the combination of artemisinin derivatives plus mefloquine, or atovaquone plus proguanil, are the most active drug regimens.

JAMA, 2001 Feb 14, 285(6), 777 - 84
Virologic and regimen termination surrogate end points in AIDS clinical trials; Gilbert PB et al.; Suppression of plasma human immunodeficiency virus (HIV) RNA levels has been widely accepted as an appropriate surrogate end point for HIV disease progression, and it is currently used as the primary end point to determine efficacy in many antiretroviral trials . However, this end point does not always measure other important effects of treatment, such as inducement of multidrug resistance, which depletes future therapy options, and toxic effects . An alternative that directly factors in these treatment costs is a composite regimen termination end point, defined as a protocol-determined change in regimen due to either virologic failure or treatment-related toxic effects . Pros and cons for using purely virologic vs various composite primary end points are discussed . Conclusions include (1) a trial's clinical objective guides the choice of primary end point, (2) a purely virologic end point is often preferable, (3) it may be important to analyze both end point types in interpreting study results, and (4) long-term clinical outcome studies are needed for identifying the most predictive surrogate end points.

J Acquir Immune Defic Syndr, 2001 Jan 1, 26(1), 36 - 43
Patterns of resistance mutations to antiretroviral drugs in extensively treated HIV-1-infected patients with failure of highly active antiretroviral therapy; Rousseau MN et al.; Resistance-mutation patterns in the reverse transcriptase (RT) and protease genes of HIV-1 were analyzed in 22 patients who had been extensively pretreated and who failed to respond to highly active antiretroviral therapy (HAART) . The number of mutations ranged from 8 to 19 (median, 13): 4 to 12 (median, 6) mutations in the RT gene, and 4 to 8 (median, 7) mutations in the protease gene . In the RT gene, the most frequent resistance mutations were found at codons 215 (100%), 41 (95%), 67 (91%), and 210 (77%) . Multidrug-resistant mutation patterns including Q151M and insertion mutations at codon 69, which confer cross-resistance to the different nucleoside analogue RT inhibitors were detected in 1 and 3 patients, respectively; 1 patient with insertion mutation displayed a NGQGV {corrected} sequence at codons 67 to 70 . In the protease gene, the most frequent mutations were found at codons 63 (95%), 10 (86%), 90(86%), 71(77%), 46 (50%), 36 (45%), and 84 (45%) . Genotypic resistance to zidovudine, saquinavir, and indinavir was found in 100% of the patients . All patients showed also resistance or possible resistance to stavudine, abacavir, ritonavir, and nelfinavir . Mutations conferring genotypic resistance to nonnucleoside analogue RT inhibitors (NNRTIs) were found in 12 (80%) of the NNRTI-experienced patients and 1 of 7 NNRTI-naive patients . Our results indicate that failure of HAART in the patients extensively pretreated results from the multiplicity of RT and protease mutations that confer genotypic resistance to almost all available antiretroviral drugs . In these patients, genotypic resistance tests confirm the lack of alternative salvage therapy strategies based on the currently available antiretroviral drugs.

Chemotherapy, 2001 Mar-Apr, 47(2), 136 - 42
Inhibition of telomerase activity as a measure of tumor cell killing by cisplatin in squamous cell carcinoma cell line; Mese H et al.; BACKGROUND: Telomerase is a ribonucleoprotein enzyme which is involved in the maintenance of chromosome ends . Telomerase activity is frequently associated with malignant phenotypes, and it can be considered a ubiquitous tumor marker . In this study we describe an approach for developing in vitro chemosensitivity assays based on the assessment of telomerase activity in squamous cell carcinoma to treatment with anticancer drugs . METHODS: We used A431/CDDP1 and A431/CDDP2, two previously established cisplatin (CDDP)-resistant A431 sublines . These cell lines were not multidrug resistant but specifically resistant to the CDDP drug family . The telomeric repeat amplification protocol (TRAP assay) was used to measure telomerase activity in CDDP-resistant cell lines and to examine the effect of CDDP and other anticancer drugs on enzyme levels . We next analyzed the relations between telomerase activity and cytotoxic effects of CDDP in human squamous cell carcinoma (HSC2, HSC3, HSC4 and KB cell lines) . RESULTS: The telomerase activity of A431/CDDP1 and A431/CDDP2 was significantly higher than that of the parent A431 cell (A431/P) treated with CDDP and carboplatin at IC(50) . On the other hand, telomerase activity in these cell lines was not influenced by treatment with 5-fluorouracil, bleomycin, Adriamycin or taxol . The regression line derived from the quantitative analysis of the telomeric ladder versus IC(50) of CDDP concentrations was analyzed . Telomerase activity was found to be positively correlated with the IC(50) values for CDDP . CONCLUSIONS: The results of the present studies on in vitro squamous cell carcinoma cell lines indicate that telomerase activity inhibition can be used as a marker of tumor cell killing by CDDP . Therefore, the present investigation provides a rational basis for an in vitro chemosensitivity assay based on telomerase activity evaluation .

Pathol Oncol Res, 1997, 3(2), 147 - 158
Molecular Events as Targets of Anticancer Drug Therapy; Aszalos A et al.; The aim of this review is to introduce some molecular targets for cancer chemotherapy, with comments on their mode of action, preclinical and clinical results . The representatives of the following groups are covered: phosphorylation inhibitors, protein kinase modulators, receptor antagonists, immunomodulators, differentiating agents, multidrug resistance modulation, telomerase inhibitors, and bioreductive agents.

Pathol Oncol Res, 1995, 1(1), 64 - 70
Modulation of Multidrug Resistance in Cancer by Immunosuppresive Agents . Preclinical Studies; Aszalos A; This is a brief summary of the status of known immunosuppressive drugs describing their potential and mode of action to reverse the function of the MDR1 gene product, the P glycoprotein . Different aspects of these immunosuppressors have been reviewed in the recent literature . This summary will focus only on those studies which relate to the effect of these drugs on the P-glycoprotein . In addition, studies which may explain the mode of action, but do not deal directly with P-glycoprotein, are also summarized.

Curr Med Chem, 2001 Jan, 8(1), 51 - 64
Analysis of drug transport kinetics in multidrug-resistant cells: implications for drug action; Garnier-Suillerot A et al.; Multidrug resistance (MDR) in model systems is known to be conferred by two different integral proteins--the 170-kDa P-glycoprotein (P-gp) and the 190-kDa multidrug resistance-associated protein (MRP1)--that pump drugs out of MDR cells . The intracellular level of a drug, which influences the drug's cytotoxic effect, is a function of the amount of drug transported inside the cell (influx) and the amount of drug expelled from the cell (efflux) . One possible pharmacological approach to overcoming drug resistance is the use of specific inhibitors that enhance the cytotoxicity of known antineoplastic agents . Many compounds have been proven to be very efficient in inhibiting P-gp activity, but only some of them can inhibit MRP1 . However, the clinical results obtained so far by this approach have been rather disappointing . The other likely approach is based on the design and synthesis of new non-cross-resistant drugs whose physicochemical properties favor the uptake of such drug by resistant cells . Our recent studies have shown that whereas the P-gp- and MRP1-mediated efflux of different anthracycline-based drugs may not differ considerably, their kinetics of uptake do . Thus, the high uptake of drug by cells may lead to concentrations at the cellular target site high enough to achieve the needed cytotoxicity against MDR cells . Therefore, increased drug lipophilicity might be one factor in improving drug cytotoxicity in MDR cells . In vitro studies have shown that idarubicin, an analogue of daunorub cin, is more effective than daunorubicin and doxorubicin against MDR tumor cell lines and that this increased effectiveness is related in part to the increased lipophilicity of idarubicin . Other studies have also confirmed the strong impact of lipophilicity on the uptake and retention of anthracyclines in MDR cells.

Curr Med Chem, 2001 Jan, 8(1), 39 - 50
Reversal of multidrug resistance by the P-glycoprotein modulator, LY335979, from the bench to the clinic; Dantzig AH et al.; Multidrug resistance may be conferred by P-glycoprotein (Pgp, ABCB1) or the multidrug resistance associated protein (MRP) . These membrane proteins are members of the ATP binding cassette transporter superfamily and are responsible for the removal from the cell of several anticancer agents including doxorubicin . Modulators can inhibit these transporters . LY335979 is among the most potent modulators of Pgp with a Ki of 59 nM . LY335979 is selective for Pgp, and does not modulate MRP-mediated resistance by MRP1 (ABCC1) and MRP2 (ABCC2) . LY335979 significantly enhanced the survival of mice implanted with Pgp-expressing murine leukemia (P388/ADR) when administered in combination with either daunorubicin, doxorubicin or etoposide . Coadministration of LY335979 with paclitaxel compared to paclitaxel alone significantly reduced the tumor mass of the Pgp-expressing UCLA-P3.003VLB lung carcinoma in a xenograph model and delayed the development of tumors in mice implanted with the parental drug-sensitive UCLA-P3 tumor . LY335979 was without significant effect on the pharmacokinetics of these anticancer agents . This may be due impart to its poor inhibition of four major cytochrome P450 isozymes important in metabolizing doxorubicin and other oncolytics . The selectivity and potency of this modulator allows the clinical evaluation of the role of Pgp in multidrug resistance . LY335979 is currently in clinical trials.

Int J Oncol, 2001 Feb, 18(2), 375 - 81
Wild-type p53 marginally induces endogenous MDR-1 mRNA without causing a measurable drug resistance in human cancer cells; Nicoletti MI et al.; The notion that wt p53 downregulates MDR-1 links p53 mutations to multidrug resistant phenotype . Alternatively, it has been envisioned that wt p53 protects cells against DNA damaging drugs by inducing MDR-1 . Opposing conclusions on the relationship between MDR-1 and p53 have been predominantly based on the effects of p53 on MDR-1 promoter-constructs . We found that introduction of wt p53 slightly induced MDR-1 mRNA in three cell lines having endogenous mt p53 . Wt p53-mediated induction of endogenous MDR-1 may represent a rudiment of cellular protection against toxic compounds earlier in evolution . Marked induction of p21WAF1/CIP1 (p21) mRNA was observed in all cell lines; and lower levels of wt p53 were required to induce p21 than MDR-1 . Pgp was undetectable and wt p53 did not increase resistance to an MDR-1 substrate, suggesting the changes in MDR-1 mRNA may be functionally insignificant . Unlike endogenous MDR-1, the expression of an MDR-1 promoter (-434/+147 fragment) - luciferase construct was unchanged or even inhibited by wt p53 that may be secondary to wt p53-mediated cytotoxicity . Thus, partial promoter constructs may not accurately represent endogenous MDR-1.

Int J Oncol, 2001 Feb, 18(2), 323 - 9
DNA (cytosine) methyltransferase overexpression is associated with acquired drug resistance of murine neuroblastoma cells; Wang C et al.; We have previously reported that C-1300 murine neuroblastoma (rMNB) cells made resistant to the nucleoside analogue, (Z)-5'-fluoro-4', 5'-didehydro-5'deoxyadenosine (MDL), an irreversible inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase have an increased expression of the S-adenosylmethionine (AdoMet) synthetase gene . Results of the immunoblot analysis of DNA (cytosine) methyltransferase with anti-human DNA (cytosine) methyltransferase specific polyclonal antibody demonstrated a significant increase ( approximately 2-fold, p<0.01) in expression of DNA (cytosine) methyltransferase protein in rMNB/MDL cells compared to wild-type C1300 MNB (wMNB) cells . To rule out the possibility that multidrug resistance (MDR) genes are involved in development of acquired drug resistance in murine neuroblastoma (rMNB/MDL) cells made resistant to MDL, the expression of Mdr1a, Mdr1b, Mdr2 (multidrug resistance/P-glycoprotein), and Mrp-1 (multidrug resistance associated protein) was examined in rMNB-MDL cells . The analysis of Mdr and Mrp-1 expression was performed by RT-PCR using PCR specific primers to respective genes . No significant difference was observed in the expression of MDR1a, Mdr1b and Mrp-1 genes between wMNB and rMNB-MDL cells, however, a slight decrease was noticed in Mdr1 expression in some samples . Expression of the Mdr2 (human MDR3) gene, which is not associated with the acquired drug resistance phenotype, was significantly decreased in rMNB-MDL cells . These findings were also confirmed by the immunoblot analyses using specific monoclonal antibodies to Mdr1/3 proteins . Expression of N-Myc gene--a prognostic factor in neuroblastoma tumors was also not altered in rMNB-MDL cells . Results of the present study suggest that acquired drug resistance in rMNB-MDL cells to MDL is associated to the overexpression of DNA (cytosine) methyltransferase, and could be due to genetic or epigenetic changes in particular to DNA hypermethylation in response to an increased AdoMet synthetase gene expression.

Am J Infect Control, 2001 Feb, 29(1), 41 - 7
Drug-resistant tuberculosis in Taipei, 1996-1999; Wang PD et al.; OBJECTIVES: To determine the trends, patterns, and risk factors associated with drug-resistant tuberculosis, we conducted a hospital-based retrospective study in Taipei . METHODS: Clinical and bacteriologic data were routinely collected from 453 patients with a diagnosis of tuberculosis who were treated at Taipei Municipal Chronic Disease Hospital from January 1996 through December 1999 for whom drug-susceptibility testing was done . RESULTS: Resistance to at least one drug was identified in 154 (34%) out of the 453 patients, and 34 (7.5%) patients were resistant to at least isoniazid and rifampin . Among the 199 patients with recurrent tuberculosis, 98 (49.2%) had isolates that showed resistance to at least one drug . Among the 254 new patients, 56 (22.0%) had isolates that were drug resistant . For all 453 patients, resistance to rifampin was most common (17.4%), followed by resistance to isoniazid (13.9%), streptomycin (13.7%), ethambutol (8.2%), and kanamycin (3.5%) . A history of previous tuberculosis therapy (odds ratio = 9.4; 95% CI, 2.9-28) and being born outside of Taiwan (odds ratio 3.3; 95% CI, 1.1-34) were significant risk factors for multidrug resistance . CONCLUSIONS: Our data suggest that the Taipei tuberculosis control program should be rapidly strengthened by expanded use of directly observed therapy and more careful bacteriologic and clinical follow-up, particularly in cases of recurrence and in persons born outside of Taiwan in tuberculosis endemic areas . Our results also indicate that the regular measuring of rates of drug resistance and the monitoring and guiding of tuberculosis treatment programs could increase the therapeutic response rate and prevent the appearance of newly acquired resistance in patients with tuberculosis . In addition, with high rifampin resistance (17.4%), the regulated market for rifampin is essential in Taiwan.

J Cell Sci, 2001 Feb, 114(Pt 4), 677 - 84
Protective role of Bcl2 in metabolic oxidative stress-induced cell death; Lee YJ et al.; Previous studies have shown that overexpression of Bcl2 protects cells from glucose deprivation-induced cell death in multidrug-resistant human breast carcinoma, MCF-7/ADR cells . In this study, we further investigated the protective role of Bcl2 in glucose deprivation-induced cytotoxicity . Although Bcl2 did not prevent a 3.2-fold increase in the level of hydroperoxide during glucose deprivation, it led to a compartmentalization of hydroperoxide molecules in the mitochondria . It also inhibited glucose deprivation-induced cytochrome c release from the mitochondria . It is possible that overexpression of Bcl2 prevents glucose deprivation-induced ceramide generation, probably by preventing the leakage of hydroperoxide from the mitochondria . We also observed that glucose deprivation induced a sixfold increase in oxidized glutathione content, as well as in thiol precursor content . Overexpression of Bcl2 suppressed an increase in oxidized glutathione content and thiol precursor content . Our results indicate that Bcl2 protects cells from metabolic oxidative stress-induced damage by inhibiting the leakage of hydroperoxide from the mitochondria and subsequently preventing ceramide generation . Preventing ceramide generation inhibits the signal transduction pathway and results in the suppression of cytochrome c release from the mitochondria.

Clin Infect Dis, 2001 Feb 1, 32(3), 373 - 80 Epub 2001 Jan 26.
Clinical outcomes of Estonian patients with primary multidrug-resistant versus drug-susceptible tuberculosis; Lockman S et al.; Little is known about the clinical outcomes of patients with primary multidrug-resistant (MDR) tuberculosis . Clinical outcomes among 46 patients in Estonia with primary MDR tuberculosis and 46 patients with pansusceptible tuberculosis were compared . Patients with MDR tuberculosis were more likely than those with pansensitive tuberculosis to have treatment failure (odds ratio, 8.9; 95% confidence interval {CI}, 3.0-26.3) after adjusting for medical problems and weeks of effective treatment, often with second-line drugs . Ten patients (22%) with MDR tuberculosis and 2 (4%) with susceptible tuberculosis died of tuberculosis (P=.03) . MDR tuberculosis (hazard ratio {HR}, 7.8; 95% CI, 1.6-37.4), number of medical problems (HR, 2.5; 95% CI, 1.5-4.4), and male sex (HR, 5.8; 95% CI, 1.1-29.6) were associated with death due to tuberculosis in multivariable analysis . Human immunodeficiency virus test results were negative for all 55 patients tested . These findings underscore the urgent need for increased attention to prevention and treatment of MDR tuberculosis globally.

Mol Cell Biol Res Commun, 2000 Aug, 4(2), 90 - 7
Endotoxin downregulates hepatic expression of P-glycoprotein and MRP2 in 2-acetylaminofluorene-treated rats; Tang W et al.; In liver, the ATP-dependent transporters P-glycoprotein (PGP) and multidrug resistance protein-2 (MRP2) are involved in the secretion of numerous drugs and toxins in bile . Although constitutive levels of PGP and MRP-2 are decreased in rat liver after exposure to endotoxin, it is possible that induced forms of these transporters may be alternately affected . In vitro, the hepatocarcinogen, 2-acetylaminofluorene (AAF) induces expression of PGP and MRP2 . Thus, we examined the influence of endotoxin on the expression of PGP and MRP2 in AAF-treated rats . Expression of PGP and MRP2 was analyzed on Westerns and by RT-PCR in livers obtained from endotoxin and control groups . In vivo, AAF treatment significantly induced PGP/mdr1 expression and imposed a significant reduction in the expression of spgp . MRP2 protein and mRNA levels were not altered by AAF administration . Endotoxin administration to both AAF-treated and non-AAF-treated rats elicited significant reductions in the protein and mRNA expression of MRP2 and PGP (P < 0.05) . Our data indicate that endotoxin suppresses the overexpression of PGP and constitutive expression of MRP2 in AAF-treated rats . Furthermore, in vivo administration of AAF, which maximally induces PGP does not induce MRP2 .

J Hosp Infect, 2001 Feb, 47(2), 91 - 7
Investigation and control of a large outbreak of multi-drug resistant tuberculosis at a central Lisbon hospital; Hannan MM et al.; An increase in the number of new cases of tuberculosis (TB) combined with poor clinical outcome was identified among HIV-infected injecting drug users attending a large HIV unit in central Lisbon . A retrospective epidemiological and laboratory study was conducted to review all newly diagnosed cases of TB from 1995 to 1996 in the HIV unit . Results showed that from 1995 to 1996, 63% (109/173) of the Mycobacterium tuberculosis isolates from HIV-infected patients were resistant to one or more anti-tuberculosis drugs; 89% (95) of these were multidrug-resistant, i.e., resistant to at least isoniazid and rifampicin . Eighty percent of the multidrug-resistant strains (MDR) available for restriction fragment length polymorphism (RFLP) DNA fingerprinting clustered into one of two large clusters . Epidemiological data support the conclusion that the transmission of MDR-TB occurred among HIV-infected injecting drug users exposed to infectious TB cases on open wards in the HIV unit . Improved infection control measures on the HIV unit and the use of empirical therapy with six drugs once patients were suspected to have TB, reduced the incidence of MDR-TB from 42% of TB cases in 1996 to 11% in 1999 .

J Med Chem, 2001 Jan 18, 44(2), 180 - 5
Cytotoxic responses to aromatic ring and configurational variations in alpha-conidendrin, podophyllotoxin, and sikkimotoxin derivatives; Dantzig A et al.; Derivatives of alpha-conidendrin, podophyllotoxin, and sikkimotoxin were prepared to evaluate the cytotoxic contributions of C-4 configuration and pendant and fused arene substitutions . Dimethyl-alpha-conidendryl alcohol (5), 9-deoxypodophyllol (6), and 9-deoxysikkimol (17) were dehydrated to their respective oxolane derivatives 4, 3, and 9 . Diols 5 and 6 were converted via oxabicyclo{3.2.1}octanols 10 and 14 to target oxolanes 8 and 7 where C-4 had been inverted relative to that in 3 and 4 . Cytotoxicities of the five oxolanes were determined in two drug-sensitive human leukemia and two multidrug-resistant cell lines expressing P-glycoprotein or multidrug-resistance associated protein (MRP) . Changing the pendant arene configuration or replacing a m-methoxy by hydrogen resulted in a 100-fold cytotoxicity loss . Replacing a methylenedioxy group in the fused arene by two methoxy substituents reduced cytotoxicity by 10-fold . Drug-resistant cell lines were equally resistant to compounds 3, 4, 8, and 9 indicating that these four compounds do not serve as substrates of the transport proteins P-glycoprotein and MRP.

Cytometry, 2001 Mar 1, 43(3), 189 - 94
The drug resistance proteins, multidrug resistance-associated protein and P-glycoprotein, do not confer resistance to Fas-induced cell death; Cullen K et al.; BACKGROUND: Multidrug resistance (MDR) is mediated by the drug resistance proteins, the multidrug resistance-associated protein (MRP) and P-glycoprotein, both of which confer resistance by the active efflux of chemotherapeutic drugs from the cell . Reduced Fas (CD95/APO-1) expression and resistance to Fas-mediated apoptosis have also been correlated with P-glycoprotein-mediated MDR . METHODS: We investigated cell surface Fas expression (using anti-Fas monoclonal antibody DX2.1) in a series of MRP-expressing drug-resistant leukemia sublines, and P-glycoprotein-expressing leukemia sublines, and their susceptibility to apoptosis induced by anti-Fas treatment (CH-11 monoclonal antibody) . Caspase-3 activation was detected by Western blot and apoptosis was determined by flow cytometry with 7-aminoactinomycin D (7-AAD) staining of cells . RESULTS: Fas expression was not reduced in either the MRP- or P-glycoprotein-expressing drug-resistant cell lines, although expression was reduced by 15% in one low-level drug-resistant subline . Expression of MRP or P-glycoprotein did not confer resistance to caspase-3 activation or to anti-Fas-induced cell death . CONCLUSIONS: MDR mediated by the drug transport proteins MRP and P-glycoprotein does not correlate with resistance to Fas-mediated cell death or resistance to caspase-3 activation .

Cytometry, 2001 Mar 1, 43(3), 170 - 4
Paclitaxel sensitization of multidrug-resistant cells to chemotherapy is independent of the cell cycle; Locke V et al.; BACKGROUND: Recent studies have shown that paclitaxel (Taxol) is an active chemotherapeutic in the treatment of small cell lung cancer . Paclitaxel binds to tubulin and prevents depolymerization . This causes cells to arrest in the G(2)/M phase of the cell cycle, resulting in sensitization of cells to drug or radiation treatment . METHODS: A drug-resistant H69 small cell lung cancer subline was established . Cytotoxicity of cisplatin and chlorambucil was determined using the MTT cell viability assay and distribution of DNA in the cell cycle . DNA distribution was analyzed by flow cytometry after treatment with paclitaxel or the other tubulin-binding drugs, vinblastine and navelbine . RESULTS: The H69-EPR drug-resistant subline was resistant to epirubicin (sixfold) and was cross-resistant to cisplatin (7.5-fold) and chlorambucil (7.5-fold) . Pretreatment with paclitaxel or vinblastine, but not navelbine, sensitized the subline to cisplatin and chlorambucil (P < 0.05), with no effect on parental H69 cells . Sensitization was dose dependent and occurred at doses below those that caused a G(2)/M block in the cell cycle . CONCLUSION: Sensitization of drug-resistant cells by paclitaxel was not associated with its ability to cause a G(2)/M block in the cell cycle . Sensitization by paclitaxel and vinblastine, but not navelbine, which preferentially targets mitotic tubulin, suggests that sensitization may involve changes in the tubulin-dependent intracellular transport processes rather than changes in mitotic tubulin and the G(2)/M block .

Br J Haematol, 2001 Feb, 112(2), 308 - 14
Calcein assay for multidrug resistance reliably predicts therapy response and survival rate in acute myeloid leukaemia; Karaszi E et al.; In this study, we evaluated the suitability of the calcein assay as a routine clinical laboratory method for the identification of multidrug-resistant phenotype in acute leukaemia . This study presents the results of the calcein tests obtained in two large haematological centres in Hungary . Assays were performed with blast cells of 93 de novo acute leukaemia patients, including 65 patients with acute myeloid leukaemia (AML) . Results were expressed as multidrug resistance activity factor (MAF) values . AML patients were divided into responders and non-responders and MAF values were calculated for each group . In both centres, responder patients displayed significantly lower MAF values than non-responders (P = 0.0045 and P = 0.0454) . Cut-off values were established between the MAFR + SEM and MAFNR - SEM values . On the basis of these cut-off levels, multidrug resistance (MDR) negativity showed a 72% predictive value for the response to chemotherapy, whereas MDR positivity was found to have an average predictive value of 69% for therapy failure . MDR activity was a prognostic factor for survival rate and the test was suitable for detecting patients at relapse . The calcein assay can be used as a quantitative, standardized, inexpensive screening test in a routine clinical laboratory setting . The assay detects both P-glycoprotein and multidrug resistance-associated protein activities, and identifies AML patients with unfavourable therapy responses.

BJU Int, 2001 Feb, 87(3), 245 - 50
Cellular resistance to mitomycin C is associated with overexpression of MDR-1 in a urothelial cancer cell line (MGH-U1); Hayes MC et al.; OBJECTIVE: To compare multidrug resistance (MDR)-1 and MDR-3 gene expression in a new urothelial cancer cell line (MGHU-1, with resistance to mitomycin C) against controls and the established (epirubicin-resistant) MDR clone, and to correlate MDR with cytotoxicity data . MATERIALS AND METHODS: Resistance to mitomycin C was induced by the long-term exposure of wild-type MGHU-1 cells to increasing concentrations (20-400 nmol/L) of mitomycin C . The cytotoxicity of mitomycin C or epirubicin was then compared in MGHU-1, MGHU-MMC (mitomycin C-resistant) and MGHU-1R (established MDR) cells, using the tetrazolium biomass assay . The expression of MDR-1 and -3 was investigated by the reverse transcriptase-polymerase chain reaction, using cDNA-specific primers after titration, and compared with DNA and negative controls . RESULTS: MDR-1 and -3 were significantly and equally overexpressed in MGHU-1R, and associated with a dramatic increase in the 50% inhibitory drug concentration (P < 0.001) for mitomycin C and epirubicin against controls . In MGHU-MMC, the overexpression of MDR-1 was three times greater than that of MDR-3 . The cytotoxicity profile for both agents was very similar to that of MGHU-1R . Trace amounts of MDR-1, but not MDR-3, were identified in the MGHU-1 wild-type . CONCLUSIONS: Urothelial cancer cell resistance to mitomycin C is associated with cross-resistance to epirubicin and overexpression of MDR-1, suggesting that mitomycin C falls within the MDR category . Clinical application of this methodology may allow patients to be identified who are unlikely to benefit from intravesical chemotherapy.

Int J Biochem Cell Biol, 2001 Jan, 33(1), 11 - 7
Proteins expressed in osteosarcoma and serum levels as prognostic factors; Trieb K et al.; Osteosarcoma is the most frequent highly malignant bone-tumor with a peak manifestation during the second and third decade of life . Although survival rate increased up to 60-70% within the last 20 years, the problem of non-response to chemotherapy remains . Initial tumor size and response to neoadjuvant chemotherapy are the most accepted prognostic factors used for postoperative stratification of chemotherapy . The identification of patients with a bad response to therapy at the time of diagnosis would facilitate already a preoperative stratification of chemotherapy or a more aggressive regime to increase survival . This review reflects on recently described molecular markers but not on clinical parameters in human osteosarcoma with respect to their prognostic potential . This includes p53, the p-glycoprotein, the multidrug resistance gene, the humen epidermal growth factor receptor and metallothionein expression . Heat shock proteins have recently become important in osteosarcoma because of their prognostic value and their role in drug resistance . A short overview of serological markers is also given . Further results on drug resistance and survival may be provided by ongoing studies, which investigate the role of proteins of the apoptotic and antiapoptotic families in human osteosarcoma.

Eur J Cancer, 2001 Jan, 37(1), 122 - 30
Apoptotic effects of thiazolobenzimidazole derivatives on sensitive and multidrug resistant leukaemic cells; Grimaudo S et al.; We investigated the cytotoxic activity of eight thiazolobenzimidazole derivatives on sensitive HL60 and multidrug-resistant (MDR) (HL60R) leukaemia cell lines . The antitumour effects of these compounds were compared with those of RS-TBZ, a thiazolobenzimidazole derivative, previously described in our reports, that was able to induce apoptosis more markedly in MDR cells than in the parental sensitive cell lines . Only two compounds in this study proved to have interesting effects: (a) the S-enantiomer of TBZ, that was able to induce apoptosis in MDR cells in a slightly more selective manner than TBZ (racemic form); and (b) TBZ-4-OCH3 (TBZ-4-OCH3), that showed cytotoxic and apoptotic effects on sensitive and resistant leukaemia cells greater than TBZ, without cytotoxic effects on normal haemopoietic progenitor cells . Moreover, we observed that TBZ-4-OCH3 was also active in cells expressing Bcr-Abl, an oncogene that confers resistance to apoptosis induced by several stimuli, including cytotoxic agents . The inhibition of caspase-9 and caspase-3 by specific polypeptide inhibitors decreased the apoptotic effects of TBZ-4-OCH3 in HL60 cells indicating that apoptosis induced by this compound was, at least partly, caspase-mediated . On the contrary, the blocking of FL-associated cell surface antigen (Fas) using a specific Fas-blocking monoclonal antibody did not affect the level of apoptosis induced by TBZ-4-OCH3 suggesting that the Fas pathway was not involved . In addition, the caspase 8 inhibitor was unable to inhibit the apoptotic activity of TBZ-4-OCH3 . The very low toxicity shown by TBZ-4-OCH3 in normal haemopoietic progenitor cells and its high activity in sensitive and MDR neoplastic cells suggest a possible clinical use for this new compound.

Hepatol Res, 2001 Feb, 19(2), 103 - 107
Characterization of ATP-dependent monoglucuronosyl bilirubin transport across rat canalicular membrane vesicles; Kamisako T et al.; Bilirubin conjugates are secreted from hepatocytes into bile . Monoglucuronosyl bilirubin is an endogenous substrate for the multidrug resistance protein 2, which is located in rat hepatocyte canalicular membrane . We have characterized this ATP-dependent transport using rat canalicular membrane vesicles . Monoglucuronosyl bilirubin, 3H-labeled in the glucuronosyl moiety, was synthesized enzymatically using recombinant UDP-glycosyltransferase 1A1, and was stabilized with ascorbate . The rate for ATP-dependent transport of monoglucuronosyl bilirubin (at 0.5 microM) was 7.3+/-1.1 pmol mg protein(-1) min(-1) and the K(m) value was 1.3+/-0.4 microM . This is the first time to demonstrate this kinetic constant of monoglucuronosyl bilirubin for the rat hepatocyte canalicular membrane . The K(m) value is similar to one for recombinant rat multidrug resistance protein 2.

Toxicology, 2001 Jan 2, 156(2-3), 109 - 17
Unaltered expression of multidrug resistance transporters in polycyclic aromatic hydrocarbon-resistant rat liver cells; Payen L et al.; Rat liver epithelial cells resistant to the chemical carcinogen 3MC, termed F258/3MC cells and generated by long-term exposure of parental F258 cells to the PAH, were characterized, especially with respect to expression of multidrug resistance transporters such as P-glycoprotein, MRP1 and MRP2 . F258/3MC cells were found to be cross-resistant to other PAHs such as BP and dimethylbenz(a)anthracene but remained sensitive to known substrates of multidrug resistance efflux pumps such as doxorubicin and vincristine . They did not display either decreased cellular PAH accumulation or increased PAH efflux . In addition, P-glycoprotein and MRP2 mRNA levels were not, or only barely detected, in F258/3MC cells and in their parental counterparts whereas these PAH-resistant and sensitive cells showed closed levels of MRP1 mRNAs and activity . Moreover, P-gp- and MRP1-overexpressing cells were shown to display similar accumulation and efflux of BP than those found in P-gp- and MRP1-negative control cells . These data therefore suggest that multidrug resistance transporters do not contribute to PAH resistance in PAH-selected liver cells.

Toxicology, 2001 Jan 2, 156(2-3), 81 - 91
Sequencing and tissue distribution of the canine MRP2 gene compared with MRP1 and MDR1; Conrad S et al.; The effects of xenobiotic drugs and toxic compounds depend largely on their kinetic properties, which can be influenced by transmembrane drug transporters like MDR1/P-glycoprotein and the drug-conjugate transporters multidrug resistance protein (MRP) 1 and 2 . As the dog is a preferential species used in the pharmacological and toxicological evaluation of new drugs, we sequenced the canine MRP2 cDNA and investigated its expression in various canine tissues compared with the related transporters MRP1 and P-glycoprotein . The tissue distribution pattern of these ABC-transporters differs partially from the distribution described in humans . So we found relatively high renal and low hepatic canine MRP2 expression levels, relatively high hepatic canine MRP1 expression levels, and quite high levels of MRP1 and P-glycoprotein in the dog brain . The knowledge of the tissue distribution pattern of these transporters will aid to interpret pharmacokinetic and toxicokinetic data gained from dog studies and to extrapolate them to humans.

J Pharmacol Exp Ther, 2001 Feb, 296(2), 584 - 91
Kinetic profiling of P-glycoprotein-mediated drug efflux in rat and human intestinal epithelia; Stephens RH et al.; Intestinal drug efflux mediated by P-glycoprotein and other ABC transporters is widely accepted as a reason for low or variable oral absorption . However, little is known about species and regional differences in P-glycoprotein so the functional and predictive relevance of observations made in cell models such as Caco-2 is uncertain . The aim of this study was to define the kinetics of drug efflux in rat and human intestinal tissues in vitro using the "reference" substrates digoxin and vinblastine . The expression and functional role of other ABC transporters in the transport of these compounds was also investigated . Saturable, verapamil-sensitive efflux of digoxin was observed in all intestinal regions . Apparent affinity of the efflux process varied within a relatively narrow range (50-92 microM), increasing in rat from small to large intestine . In contrast, maximal transporter activity varied over a 4- to 5-fold range with ileum > jejunum > colon . Similar regional differences in efflux were also observed with vinblastine . Maximal efflux levels were similar in Caco-2 and ileum for both substrates, suggesting that Caco-2 may quantitatively predict small intestinal drug efflux . Digoxin efflux kinetics was virtually identical in rat and human colon . Inhibitor studies showed that digoxin and vinblastine efflux in intestinal tissues was mediated by P-glycoprotein, although a minor component could be attributed to multidrug resistance-related protein (MRP)-like transporters in Caco-2 . This study has analyzed the differential functional expression of drug efflux along the gastrointestinal tract . Such data will be critical in developing predictive models of P-glycoprotein-mediated efflux using information gathered from in vitro systems.

J Immunol, 2001 Feb 15, 166(4), 2451 - 9
Specific MDR1 P-glycoprotein blockade inhibits human alloimmune T cell activation in vitro; Frank MH et al.; MDR1 P-glycoprotein (P-gp), the multidrug resistance-associated transmembrane transporter, is physiologically expressed by human peripheral immune cells, but its role in cell-mediated immunity remains poorly understood . Here, we demonstrate a novel role for P-gp in alloantigen-dependent human T cell activation . The pharmacologic P-gp inhibitor tamoxifen (1-10 microM) and the MDR1 P-gp-specific mAb Hyb-241 (1-20 microg/ml), which detected surface P-gp on 21% of human CD3(+) T cells and 84% of CD14(+) APCs in our studies, inhibited alloantigen-dependent, but not mitogen-dependent, T cell proliferation in a dose-dependent manner from 40-90% (p < 0.01) . The specific inhibitory effect on alloimmune T cell activation was associated with >85% inhibition (p < 0.01) of IL-2, IFN-gamma, and TNF-alpha production in 48-h MLR coculture supernatants . Addition of recombinant human IL-2 (0.1-10 ng/ml) restored proliferation in tamoxifen-treated cocultures . Pretreatment of purified CD4(+) T cells with Hyb-241 mAb before coculture resulted in inhibition of CD4(+) T cellular IFN-gamma secretion . Also, blockade of P-gp on allogeneic APCs inhibited IL-12 secretion . Taken together these results demonstrate that P-gp is functional on both CD4(+) T cells and CD14(+) APCs, and that P-gp blockade may attenuate both IFN-gamma and IL-12 through a positive feedback loop . Our results define a novel role for P-gp in alloimmunity and thus raise the intriguing possibility that P-gp may represent a novel therapeutic target in allograft rejection.

Br J Pharmacol, 2001 Feb, 132(3), 778 - 84
The sulphonylurea glibenclamide inhibits multidrug resistance protein (MRP1) activity in human lung cancer cells; Payen L et al.; 1 . Glibenclamide, a sulphonylurea widely used for the treatment of non-insulin-dependent diabetes mellitus, has been shown to inhibit the activities of various ATP-binding cassette (ABC) transporters . In the present study, its effects towards multidrug resistance protein 1 (MRP1), an ABC efflux pump conferring multidrug resistance and handling organic anions, were investigated . 2 . Intracellular accumulation of calcein, an anionic dye substrate for MRP1, was strongly increased by glibenclamide in a dose-dependent manner in MRP1-overexpressing lung tumour GLC4/Sb30 cells through inhibition of MRP1-related calcein efflux . By contrast, glibenclamide did not alter calcein levels in parental control GLC4 cells . Another sulphonylurea, tolbutamide, was however without effect on calcein accumulation in both GLC4/Sb30 and GLC4 cells . 3 . Glibenclamide used at 12.5 microM was, moreover, found to strongly enhance the sensitivity of GLC4/Sb30 cells towards vincristine, an anticancer drug handled by MRP1 . 4 . Efflux of carboxy-2',7'-dichlorofluorescein, an anionic dye handled by the ABC transporter MRP2 sharing numerous substrates with MRP1 and expressed at high levels in liver, was also strongly inhibited by glibenclamide in isolated rat hepatocytes . 5 . In summary, glibenclamide reversed MRP1-mediated drug resistance likely through inhibiting MRP1 activity and blocked organic anion efflux from MRP2-expressing hepatocytes . Such effects associated with the known inhibitory properties of glibenclamide towards various others ABC proteins suggest that this sulphonylurea is a general inhibitor of ABC transporters.

Br J Pharmacol, 2001 Feb, 132(3), 722 - 8
Ivermectin excretion by isolated functionally intact brain endothelial capillaries; Nobmann S et al.; 1 . Functionally intact brain endothelial capillaries were isolated from porcine brain . p-Glycoprotein was localized at the lumenal membrane of intact capillaries by immunohistochemistry using a murine monoclonal antibody and a secondary FITC fluorescent labelled anti-mouse IgG . Western blot staining of p-glycoprotein in isolated endothelial cells confirmed the immunohistochemistry . 2 . Excretion of the fluorescent labelled anthelmintic drug Ivermectin (BODIPY-Ivermectin) was studied in the isolated brain endothelial capillaries . Drug accumulation in the capillary lumen was visualized by fluorescence confocal laser scanning microscopy and was measured by image analysis . Secretion of BODIPY-Ivermectin into the capillary lumen exhibited characteristics of specific and energy-dependent transport . Steady state lumenal fluorescence intensity averaged 1.6 times cellular fluorescence and was reduced 3--4 times below cellular levels when metabolism was inhibited by NaCN . 3 . BODIPY-Ivermectin secretion was inhibited in a concentration-dependent manner by unlabeled Ivermectin . In addition, lumenal but not cellular fluorescence intensity was significantly decreased when capillaries were incubated with PSC-833, Cyclosporin A or Verapamil, all inhibitors of p-glycoprotein . Conversely, unlabelled Ivermectin reduced the p-glycoprotein (Pgp)-mediated secretion of a fluorescent derivative of Verapamil, (BODIPY-Verapamil) . 4 . BODIPY-Ivermectin secretion was not affected in the presence of Leucotriene C(4) (LTC(4)), a potent inhibitor of multidrug resistance related protein (mrp)-mediated transport processes . In addition, excretion of Fluorescein-Methotrexate, an mrp-substrate, was not inhibited by Ivermectin . 5 . Uptake experiments with isolated porcine brain capillary cells showing increased cellular uptake of BODIPY-Ivermectin in the presence of unlabelled drug or PSC-833 supported the findings of a Pgp interaction in intact capillaries . 6 . The data are consistent with BODIPY-Ivermectin and Ivermectin being transported across the lumenal membrane of brain capillaries . For the first time Pgp-interaction of Ivermectin at the blood brain barrier is demonstrated on a cellular level in an intact vascular tissue.

Antimicrob Agents Chemother, 2001 Feb, 45(2), 439 - 46
High-affinity binding of silybin derivatives to the nucleotide-binding domain of a Leishmania tropica P-glycoprotein-like transporter and chemosensitization of a multidrug-resistant parasite to daunomycin; Perez-Victoria JM et al.; In order to overcome the multidrug resistance mediated by P-glycoprotein-like transporters in Leishmania spp., we have studied the effects produced by derivatives of the flavanolignan silybin and related compounds lacking the monolignol unit on (i) the affinity of binding to a recombinant C-terminal nucleotide-binding domain of the L . tropica P-glycoprotein-like transporter and (ii) the sensitization to daunomycin on promastigote forms of a multidrug-resistant L . tropica line overexpressing the transporter . Oxidation of the flavanonol silybin to the corresponding flavonol dehydrosilybin, the presence of the monolignol unit, and the addition of a hydrophobic substituent such as dimethylallyl, especially at position 8 of ring A, considerably increased the binding affinity . The in vitro binding affinity of these compounds for the recombinant cytosolic domain correlated with their modulation of drug resistance phenotype . In particular, 8-(3,3-dimethylallyl)-dehydrosilybin effectively sensitized multidrug-resistant Leishmania spp . to daunomycin . The cytosolic domains are therefore attractive targets for the rational design of inhibitors against P-glycoprotein-like transporters.

J Clin Microbiol, 2001 Feb, 39(2), 816 - 9
Fatal pulmonary infection due to multidrug-resistant Mycobacterium abscessus in a patient with cystic fibrosis; Sanguinetti M et al.; We report a case of fatal pulmonary infection caused by Mycobacterium abscessus in a young patient with cystic fibrosis, who underwent bipulmonary transplantation after a 1-year history of severe lung disease . Fifteen days after surgery he developed septic fever with progressive deterioration in lung function . M . abscessus, initially isolated from a pleural fluid specimen, was then recovered from repeated blood samples, suggesting a disseminated nature of the mycobacterial disease . Drug susceptibility testing assay, performed on two sequential isolates of the microorganism, showed a pattern of multidrug resistance . Despite aggressive therapy with several antimycobacterial drugs, including clarithromycin, the infection persisted, and the patient died.

J Clin Microbiol, 2001 Feb, 39(2), 636 - 41
Analysis for a limited number of gene codons can predict drug resistance of Mycobacterium tuberculosis in a high-incidence community; Van Rie A et al.; Correct and rapid diagnosis is essential in the management of multidrug-resistant tuberculosis (MDR-TB) . In this population-based study of 61 patients with drug-resistant tuberculosis, we evaluated the frequency of mutations and compared the performance of genotypic (mutation analysis by dot blot hybridization) and phenotypic (indirect proportion method) drug resistance tests . Three selected codons (rpoB531, rpoB526, and katG315) allowed identification of 90% of MDR-TB cases . Ninety percent of rifampin, streptomycin, and ethambutol resistance and 75% of isoniazid resistance were detected by screening for six codons: rpoB531, rpoB526, rrs-513, rpsL43, embB306, and katG315 . The performance (reproducibility, sensitivity, and specificity) of the genotypic method was superior to that of the routine phenotypic method, with the exception of sensitivity for isoniazid resistance . A commercialized molecular genetic test for a limited number of target loci might be a good alternative for a drug resistance screening test in the context of an MDR "DOTS-plus" strategy.

J Clin Microbiol, 2001 Feb, 39(2), 581 - 5
Molecular epidemiology of penicillin-susceptible, multidrug-resistant serotype 6B pneumococci isolated from children in Greece; Syrogiannopoulos GA et al.; Since January 1996, and over a 3-year time span, a significant spread of serotype 6B multidrug-resistant (MDR) pneumococci, susceptible to penicillin and resistant to erythromycin, clindamycin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole, was noted in young carriers living in central and southern Greece . Using restriction fragment end labeling and penicillin binding protein (PBP) genotyping, we studied 41 serotype 6B penicillin-susceptible MDR pneumococci isolated during two independent studies in Greece . Forty (98%) of these 41 isolates were strongly related, representing a single lineage (genetic relatedness, > or = 91%) . The Greek isolates were closely related (genetic relatedness, approximately 91%) to the penicillin-resistant MDR clone of serotype 6B that spread from Spain to Iceland in the late 1980s . Moreover, the Greek group of isolates was genetically distinct (genetic relatedness, < or = 83%) from other penicillin-susceptible or -resistant serotype 6B strains from various parts of the world . All serotype 6B penicillin-susceptible MDR isolates displayed a penicillin-susceptible PBP 1A-2B-2X genotype . Our findings suggest that the penicillin-susceptible MDR 6B clone that was found in Greece between the years 1996 and 1999 represents the ancestor of the pandemic penicillin-resistant MDR clone 6B.

J Bacteriol, 2001 Feb, 183(4), 1455 - 8
Overexpression of the response regulator evgA of the two-component signal transduction system modulates multidrug resistance conferred by multidrug resistance transporters; Nishino K et al.; Overexpression of evgA, a response regulator of a two-component system, increased multidrug efflux in Escherichia coli . Since overexpression of the emrKY operon, which is controlled by evgAS, could account only for deoxycholate resistance, the evgAS locus apparently controls expression of at least one other multidrug efflux operon.

Chest, 2001 Jan, 119(1), 176 - 80
Mycobacterium tuberculosis disease in Somali immigrants in Minnesota; Kempainen R et al.; STUDY OBJECTIVE: To characterize pulmonary and extrapulmonary Mycobacterium tuberculosis cases in the Somali community in Minnesota . DESIGN: Retrospective chart review of active tuberculosis cases in Somalis reported to the Minnesota Department of Health between January 1993 and June 1998 . PATIENTS: Ethnic Somalis in the state of Minnesota with M tuberculosis diagnosed by positive culture or radiographic findings consistent with tuberculosis and clinical improvement when treated with antituberculous drugs . RESULTS: Eighty-two Somali patients were diagnosed with tuberculosis during the study period . Extrapulmonary disease (typically lymphadenopathy) was present in 46% (n = 38) . The 1997 incidence of tuberculosis in Minnesota's Somali population was estimated at 170 cases per 100,000 population compared with a national incidence of 20.5 per 100,000 among African Americans and 2.5 per 100,000 among whites . Ninety percent of Somali patients were < 40 years of age; 63% were diagnosed within 1 year of immigration, and > 90% had positive results with the purified protein derivative skin test . M tuberculosis was confirmed in 24 of 25 isolates from extrapulmonary cases . Multidrug resistance was present in 3.4%, and only two patients had AIDS . CONCLUSIONS: Somalis have a high incidence of active disease, with frequent extrapulmonary involvement in the absence of AIDS, clinical presentation shortly after immigration, and infrequent infection with resistant organisms . Health-care providers should maintain an increased awareness for tuberculosis when evaluating Somali immigrants.

Blood, 2001 Feb 1, 97(3), 759 - 66
Retrovirus insertion and transcriptional activation of the multidrug-resistance gene in leukemias treated by a chemotherapeutic agent in vivo; Nagayama J et al.; To understand the molecular basis for multidrug-resistant (MDR) cancer cells in vivo, this study analyzed molecular changes of the mdr1a gene region in leukemia cells in mice during continuous treatment with vincristine . An inverse insertion of murine leukemia retrovirus (MuLV) into the 5'-flanking region of the mdr1a gene was found . This insertion was concomitantly accompanied by up-regulation of the mdr1a gene and the loss of chemosensitivity . Deletion of long-terminal repeat (LTR) sequences dramatically decreased the mdr1a promoter-driven reporter activity . The MuLV LTR insertion appears to exert its enhancer activity on mdr1a transcription during the appearance of MDR leukemia cells . Two mechanisms were postulated to explain the mdr1a gene activation by retrovirus insertion during in vivo chemotreatment: de novo insertion of MuLV induced by vincristine treatment and selection of a small fraction of pre-existing cells carrying MuLV insertion during vincristine treatment . No rearranged sequence was detected by polymerase chain reaction in parental cells . This result argued for the first mechanism . The randomly altered distribution of MuLV during repetitive chemotreatment might also be consistent with this hypothesis . On the other hand, the retrovirus insertion was detected at the same site of the mdr1a promoter region in 2 independent experiments, which suggests the second mechanism . It should be noted that in vivo chemotreatment using vincristine could generate the mdr1a-overexpressing cells through retrovirus insertion and the enhancer effect of the LTR.

J Clin Oncol, 2001 Feb 1, 19(3), 832 - 42
Phase I study of infusional paclitaxel in combination with the P-glycoprotein antagonist PSC 833; Chico I et al.; PURPOSE: PSC 833 (valspodar) is a second-generation P-glycoprotein (Pgp) antagonist developed to reverse multidrug resistance . We conducted a phase I study of a 7-day oral administration of PSC 833 in combination with paclitaxel, administered as a 96-hour continuous infusion . PATIENTS AND METHODS: Fifty patients with advanced cancer were enrolled onto the trial . PSC 833 was administered orally for 7 days, beginning 72 hours before the start of the paclitaxel infusion . Paclitaxel dose reductions were planned because of the pharmacokinetic interactions known to occur with PSC 833 . RESULTS: In combination with PSC 833, maximum-tolerated doses were defined as paclitaxel 13.1 mg/m(2)/d continuous intravenous infusion (CIVI) for 4 days without filgrastim, and paclitaxel 17.5 mg/m(2)/d CIVI for 4 days with filgrastim support . Dose-limiting toxicity for the combination was neutropenia . Statistical analysis of cohorts revealed similar mean steady-state concentrations (C(pss)) and areas under the concentration-versus-time curve (AUCs) when patients received paclitaxel doses of 13.1 or 17.5 mg/m(2)/d for 4 days with PSC 833, as when they received a paclitaxel dose of 35 mg/m(2)/d for 4 days without PSC 833 . However, the effect of PSC 833 on paclitaxel pharmacokinetics varied greatly among individual patients, although a surrogate assay using CD56+ cells suggested inhibition of Pgp was complete or nearly complete at low concentrations of PSC 833 . Responses occurred in three of four patients with non-small-cell lung cancer, and clinical benefit occurred in five of 10 patients with ovarian carcinoma . CONCLUSION: PSC 833 in combination with paclitaxel can be administered safely to patients provided the paclitaxel dose is reduced to compensate for the pharmacokinetic interaction . Surrogate studies with CD56+ cells indicate that the maximum-tolerated dose for PSC 833 gives serum levels much higher than those required to block Pgp . The variability in paclitaxel pharmacokinetics, despite complete inhibition of Pgp in the surrogate assay, suggests that other mechanisms, most likely related to P450, contribute to the pharmacokinetic interaction . Future development of combinations such as this should include strategies to predict pharmacokinetics of the chemotherapeutic agent . This in turn will facilitate dosing to achieve comparable CPss and AUCs.

Br J Cancer, 2000 Apr, 82(7), 1327 - 31
Osteoblastic differentiation and P-glycoprotein multidrug resistance in a murine osteosarcoma model; Takeshita H et al.; A recent study of multidrug resistance (MDR) 1 gene transfected osteosarcoma cells found a cause-effect relationship between increased expression of P-glycoprotein (P-gp) and a low aggressive phenotype . However, several experimental and clinical studies have observed contradictory findings in that P-gp expression has been associated with tumour progression . In the present study, we characterized P-gp-positive and P-gp-negative single-cell clones of a murine osteosarcoma, to further investigate the relationship between P-gp expression and changes in cell phenotype . Although these clones were all selected by doxorubicin (DOX) exposure, they were heterogeneous with respect to MDR1 gene expression . The P-gp-positive clones revealed MDR phenotype, whereas the P-gp-negative clones showed no resistance to drugs . Morphological and functional analysis showed that both the P-gp-positive and P-gp-negative clones were more differentiated than the parent cells in terms of enhanced activity of cellular alkaline phosphatase, an increase in well-organized actin stress fibres and enhanced osteogenic activity . Moreover, these subclones all displayed a decrease in malignant potential such as oncogenic activity, tumour growth rate and metastatic ability, regardless of their P-gp status . These results indicate that the observed osteoblastic differentiation and less aggressive phenotype in DOX-selected osteosarcoma cells may not only be explained by the direct effect of P-gp, and accordingly, consideration of the effect of DOX, as well as P-gp, appears to be important.

Cancer Chemother Pharmacol, 2000, 45(4), 305 - 11
Effect of high-dose cyclosporine on etoposide pharmacodynamics in a trial to reverse P-glycoprotein (MDR1 gene) mediated drug resistance; Lum BL et al.; PURPOSE: The consequences of using cyclosporine (CsA) therapy to modulate P-glycoprotein-mediated multidrug resistance include increased myelosuppression, hyperbilirubinemia, and altered disposition of the cytotoxin . The purpose of this study was to analyze further the relationship between the degree of leukopenia, and etoposide pharmacokinetic factors . METHODS: Each patient initially received intravenously-administered etoposide alone (150-200 mg/m2/d x 3) . Later it was given in combination with CsA administered at escalating loading doses (range 2-7 mg/kg) as a 2 hour intravenous (IV) infusion followed by a 3 day continuous infusion, at doses ranging from 5 to 21 mg/ kg/day . Serial plasma etoposide concentration-time samples were assayed by high-performance liquid chromatography (HPLC) . The area under the curve (AUC) of unbound etoposide was calculated from the total plasma etoposide AUC using a previous published equation {22} where % unbound etoposide = (1.4 x total bilirubin) - (6.8 x serum albumin) + 34.4 . The percent decrease in white blood cell (WBC) count and the total or unbound etoposide AUC relationship was fitted to a sigmoid Emax model adapted for paired observations, where: % Decrease in WBC count =E(max) x PDRV(H+Z x delta)/(PDRV50 + Z x beta) + PDRVH + Z x delta In this equation, Z was the variable describing the two treatment groups (0 = no CsA and 1 = CsA) . The fitted parameters were PDRV50, the pharmacodynamic response variable (PDRV) producing 50% of the maximal response; parameter beta, which describes the effect of the treatment group on the PDRV50; parameter H (Hill constant), which defines the slope of the response curve and parameter delta, which describes the effect of the treatment group on parameter H . RESULTS: CsA at a median concentration of 1,938 microg/ml resulted in a median increase in the total plasma etoposide AUC by 103% and the calculated unbound plasma etoposide AUC by 104% . This paralleled a 12% greater median percent decrease in WBC count during etoposide + CsA treatment (72% vs . 84%, P = 0.03) . The percent decrease in WBC count and total or unbound etoposide AUC relationship was fitted to the sigmoid Emax model . The model using the unbound etoposide AUC described the data adequately (r = 0.790) and was precise, with a mean absolute error of 6.4% (95% confidence interval: -4.9, 7.8) . The fitted parameter-estimates suggested that at equivalent unbound etoposide AUC values above 10 microg x h/ml, the sigmoid Emax model predicted a 5% greater WBC count suppression when CsA was added to the treatment regimen . CONCLUSION: These findings suggest that a small degree of the enhanced myelosuppression observed with CsA combined with etoposide might be attributable to inhibition of P-glycoprotein in bone marrow precursor cells . However, the majority of the effect observed appears to be due to pharmacokinetic interactions, which result in increases in unbound etoposide.

Biochem Biophys Res Commun, 2000 Apr 13, 270(2), 608 - 15
Functional comparison between YCF1 and MRP1 expressed in Sf21 insect cells; Ren XQ et al.; YCF1 is a yeast vacuole membrane transporter involved in resistance to Cd(2+) and to exogenous glutathione S-conjugate precursors . MRP1 contributes to multidrug resistance (MDR) in tumor cells . MRP1 and YCF1 have extensive amino acid sequence homology (63% amino acid similarity) . We expressed MRP1 or YCF1 in insect cell membranes and compared their functions to know more about their structure-function relationships . YCF1 and MRP1 with His epitopes were expressed in Sf21 insect cells; both of them in the plasma membrane . The ATP-dependent transport of {(3)H}LTC(4) in Sf/YCF1-His vesicles was osmotically sensitive and showed saturable kinetics with an apparent K(m) of 758 nM for LTC(4) and 94 microM for ATP which were similar to those in yeast cells . The K(m) of YCF1 for LTC(4) (758 nM) was sevenfold higher than that of MRP1 (108 nM) . MK-571 and ONO-1078, reversing agents for MRP1-mediated MDR, considerably inhibited the transport of LTC(4) by both YCF1 and MRP1 . However, PAK-104P, a pyridine analog that reverses MDR associated with P-gp and MRP1, inhibited the transporting activity of MRP1 stronger than that of YCF1 . KE1, another MDR reversing agent, moderately inhibited the transport of LTC(4) by MRP1 but not that of YCF1 . In conclusion, we successfully expressed yeast YCF1 in Sf21 insect cells and found that the localization of the protein was different from that in yeast . The function of YCF1 in Sf21 insect cells was similar but not identical to that of MRP1 .

Biochem Biophys Res Commun, 2000 Apr 13, 270(2), 415 - 20
Resistance to apoptosis is correlated with the reduced caspase-3 activation and enhanced expression of antiapoptotic proteins in human cervical multidrug-resistant cells; Ding Z et al.; Recent studies have indicated that induction of apoptosis is the primary cytotoxic mechanism of most cancer chemotherapeutic agents, and abnormalities in the control of apoptosis can affect the sensitivity of malignant cells to multiple drugs . Here, we treated cells with cisplatin and other apoptotic stimuli and found that multidrug-resistant (MDR) endocervical HEN-16-2/CDDP cells, compared with drug-sensitive parental cells, were significantly more resistant to apoptosis and exhibited decreased proteolytic activation of caspase-3 . The latter was further demonstrated by decreased cleavage of its substrate poly(ADP-ribose) polymerase (PARP) . Further, Western blot analysis showed that MDR HEN-16-2/CDDP cells had significantly higher levels of the apoptosis-inhibiting proteins BAG-1 p50 and p33 isoforms and Bcl-X(L) . This study provided the first evidence that overexpression of antiapoptotic BAG-1 p50 and p33 and Bcl-X(L) may cause resistance to apoptosis through reduction of caspase-3 activity in human cervical cells having an MDR phenotype .

J Biol Chem, 2001 Apr 13, 276(15), 11653 - 61 Epub 2001 Jan 11.
Characterization of the catalytic cycle of ATP hydrolysis by human P-glycoprotein . The two ATP hydrolysis events in a single catalytic cycle are kinetically similar but affect different functional outcomes; Sauna ZE et al.; P-glycoprotein (Pgp) is a plasma membrane protein whose overexpression confers multidrug resistance to tumor cells by extruding amphipathic natural product cytotoxic drugs using the energy of ATP . An elucidation of the catalytic cycle of Pgp would help design rational strategies to combat multidrug resistance and to further our understanding of the mechanism of ATP-binding cassette transporters . We have recently reported (Sauna, Z . E., and Ambudkar, S . V . (2000) Proc . Natl . Acad . Sci . U . S . A . 97, 2515-2520) that there are two independent ATP hydrolysis events in a single catalytic cycle of Pgp . In this study we exploit the vanadate (Vi)-induced transition state conformation of Pgp (Pgp.ADP.Vi) to address the question of what are the effects of ATP hydrolysis on the nucleotide-binding site . We find that at the end of the first hydrolysis event there is a drastic decrease in the affinity of nucleotide for Pgp coincident with decreased substrate binding . Release of occluded dinucleotide is adequate for the next hydrolysis event to occur but is not sufficient for the recovery of substrate binding . Whereas the two hydrolysis events have different functional outcomes vis a vis the substrate, they show comparable t(12) for both incorporation and release of nucleotide, and the affinities for {alpha-(32)P}8-azido-ATP during Vi-induced trapping are identical . In addition, the incorporation of {alpha-(32)P}8-azido-ADP in two ATP sites during both hydrolysis events is also similar . These data demonstrate that during individual hydrolysis events, the ATP sites are recruited in a random manner, and only one site is utilized at any given time because of the conformational change in the catalytic site that drastically reduces the affinity of the second ATP site for nucleotide binding . In aggregate, these findings provide an explanation for the alternate catalysis of ATP hydrolysis and offer a mechanistic framework to elucidate events at both the substrate- and nucleotide-binding sites in the catalytic cycle of Pgp.

J Biol Chem, 2001 Mar 23, 276(12), 8812 - 9 Epub 2000 Dec 29.
Cross-talk between transcriptional regulators of multidrug resistance in Saccharomyces cerevisiae; Zhang X et al.; Multiple or pleiotropic drug resistance often arises in the yeast Saccharomyces cerevisiae due to genetic alterations of the functional state of the Cys(6)-Zn(II)(2) transcription factors Pdr1p and Pdr3p . Single amino acid substitutions give rise to hyperactive forms of these regulatory proteins, which in turn cause overproduction of downstream target genes that directly mediate multidrug resistance . Previous work has identified a novel Cys(6)-Zn(II)(2) transcription factor designated Yrr1p as mutant forms of this protein confer high level resistance to the cell cycle inhibitor reveromycin A and DNA damaging agent 4-nitroquinoline-N-oxide . In the present study, we demonstrate that Yrr1p also mediates oligomycin resistance through activation of the ATP-binding cassette transporter-encoding gene YOR1 . Additionally, insertion of triplicated copies of the hemagglutinin epitope in the C-terminal region of Yrr1p causes the protein to behave as a hyperactive regulator of transcription . We have found that YRR1 expression is both controlled in a Pdr1p/Pdr3p-dependent manner and autoregulated . Chromatin immunoprecipitation experiments also show that Yrr1p associates with target promoters in vivo . Together these data argue that the signal generated by activation of Pdr1p and/or Pdr3p can be amplified through the action of these transcriptional regulatory proteins on downstream target genes, like YRR1, that also encode transcription factors.

Clin Cancer Res, 2000 Dec, 6(12), 4932 - 8
Response and determinants of sensitivity to paclitaxel in human non-small cell lung cancer tumors heterotransplanted in nude mice; Perez-Soler R et al.; The lack of tumor models that can reliably predict for response to anticancer agents remains a major deficiency in the field of experimental cancer therapy . Although heterotransplants of certain human solid tumors can be successfully grown in nude mice, they have never been appropriately explored for prediction of in vivo chemosensitivity to anticancer agents . We determined the tumor response rate and studied the influence of several biological and molecular tumor parameters on the in vivo sensitivity to paclitaxel in a series of heterotransplanted human non-small cell lung cancer (NSCLC) tumors . One hundred consecutive resected NSCLC tumors were heterotransplanted s.c . in nude mice . The in vivo sensitivity to i.v . paclitaxel (60 mg/kg every 3 weeks) was studied in 34 successfully grown heterotransplants . Treatment started when the tumors reached a size of 5 mm in diameter, and strict standard clinical criteria (>50% shrinkage in tumor weight or cross-sectional surface) were used to define tumor response . Baseline multidrug resistance protein (MRP), Her-2/neu, and epidermal growth factor receptor (EGFR) expression, and pre- and posttherapy bax and bcl-2 expression were determined by Western blot analysis . p53 status was determined by sequencing . The overall take rate was 46% (95% confidence interval, 36-56%) and was significantly higher (P < 0.05) for squamous carcinoma tumors (75%) than for adenocarcinoma tumors (30%) and bronchoalveolar tumors (23%) . The heterotransplants were morphologically very similar to the original tumors . The response rate to paclitaxel was 21% (95% confidence interval, 9-38%) . Baseline tumor parameters associated with response were no Her-2/neu expression (none of the responding tumors expressed Her-2/neu versus 48% of the nonresponding tumors, P = 0.05) and baseline bcl-2 expression (all responding tumors expressed bcl-2 versus only 43% of the nonresponding tumors, P = 0.02) . There was a trend toward a higher response rate in bax-positive tumors, and MRP- and EGFR-negative tumors, but it was not statistically significant . The response was independent of baseline p53 status and baseline mitotic index . Responding tumors had a higher bax/bcl-2 ratio 24 h after therapy, but the difference was only marginally significant (2.8 for responding tumors versus 1.1 for nonresponding tumors, P = 0.07) . The extent of mitotic arrest at 24 h after therapy was not associated with response . Human NSCLC heterotransplants are morphologically identical to the original tumors and have a response rate to paclitaxel that is equivalent to that reported in Phase II studies in patients with advanced NSCLC treated with single-agent paclitaxel . NSCLC heterotransplants deserve to be explored to evaluate new agents for lung cancer and to predict clinical response on an individual basis in selected groups of patients.

Clin Cancer Res, 2000 Dec, 6(12), 4618 - 27
MDR1 gene overexpression and altered degree of methylation at the promoter region in bladder cancer during chemotherapeutic treatment; Tada Y et al.; Overexpression of the multidrug resistance 1 (MDR1) gene is closely associated with the clinical outcome of hematopoietic malignancies, but the alteration of its expression during chemotherapeutic treatment and the precise mechanism underlying MDR1 gene overexpression in solid tumors remains unclear . We determined the expression and degree of methylation at the promoter of the MDR1 gene in bladder cancer . The mRNA levels of the MDR1 gene were found to be markedly enhanced, 3.5- to 5.7-fold higher in bladder cancers after chemotherapeutic treatment than those in untreated primary tumors . The MDR1 gene was overexpressed in recurrent tumors in 89% of patients who showed rerecurrence, whereas overexpression was observed in 25% of the patients without re-recurrence . A statistically significant inverse correlation existed between MDR1 expression and the methylation of 5'CpG sites at the promoter in patients with bladder cancer after chemotherapeutic treatment, with the degree of methylation at several CpG sites, rather than other specific sites, involved in this regulation . Consistent with the increase in MDR1 expression, the frequency of patients with a hypermethylated promoter decreased to 50 and 17% after intravesical and systemic chemotherapy, respectively . Thus, overexpression of the MDR1 gene might be a prognostic marker for intravesical recurrence, whereas methylation of the promoter region negatively regulates MDR1 expression and the appearance of multidrug resistance mediated by P-glycoprotein in bladder cancers.

AIDS, 2000 Dec 22, 14(18), 2857 - 67
Virological and immunological effects of treatment interruptions in HIV-1 infected patients with treatment failure; Miller V et al.; OBJECTIVE: To analyse the immunological and virological effects of treatment interruptions in HIV-1-infected patients with treatment failure and multidrug-resistant virus . METHODS: Drug susceptibility was assessed using Antivirogram and genotypic analysis was based on population and clonal sequencing for 48 patients who had interrupted treatment (> or = 2 months) . RESULTS: Treatment interruption resulted in viral load increases (mean 0.7 log 10 copies/ ml; P = 0.0001) and CD4 cell count decreases (mean 89 x 10(6) cells/l; P = 0.0001) . A complete shift to wild-type virus at the phenotypic, genotypic and clonal level was observed in 28/45 patients . These patients differed from those that did not show a shift to wild type in baseline CD4 cell counts (192 versus 59 x 10(6) cells/l; P= 0.007) and in the relationship between baseline viral load and CD4 cell count (no correlation versus a significant negative correlation; P= 0.008) . Response to re-initiation of treatment fell with increasing viral load {relative hazard (RH) 0.33; P= 0.001} and with increasing total number of drugs with reduced susceptibility (RH 0.51; P = 0.0003); it improved with the number of new drugs received (RH 2.12; P = 0.0002) and a shift to wild type (RH 5.22, P = 0.006) . CONCLUSIONS: Changes in surrogate markers suggest that treatment provided benefit in spite of virological failure and resistant virus . Although patients with a shift to wildtype virus responded better in the short term to treatment re-initiation, the long-term effects are not known and the risk of immune deterioration needs to be carefully considered.

Schweiz Med Wochenschr, 2000 Dec 9, 130(49), 1909 - 13
Drug-resistant tuberculosis: resistance mechanisms and rapid susceptibility testing; Pfyffer GE; Globally, the emergence of multidrug-resistant strains of Mycobacterium tuberculosis is an increasing problem which adversely affects patient care and public health . In contrast to other bacteria, resistance of M . tuberculosis is exclusively associated with chromosomal mutations . Recently developed molecular biological techniques have significantly helped in understanding the basis of drug action and resistance mechanisms in this organism . The information gained at the molecular level will help to develop efficient future diagnostic strategies and create novel drugs, both of which will ultimately have a direct impact on treatment programmes.

Int J Cancer, 2001 Jan 1, 91(1), 126 - 31
Reversal of LRP-associated drug resistance in colon carcinoma SW-620 cells; Kitazono M et al.; Resistance to multiple drugs is mediated by lung resistance-related protein (LRP) as well as P-glycoprotein (P-gp) and multidrug resistance protein (MRP) . The levels of expression of LRP mRNA and LRP in a human colon carcinoma cell line, SW-620, were increased by the differentiation-inducing agent, sodium butyrate (NaB) . Treatment of SW-620 cells with NaB for 2 weeks conferred resistance to adriamycin (ADM) and VP-16 . The resistance was almost completely reversed by PAK-104P, a pyridine analog, but not by cepharanthine . ADM accumulated mainly in the nuclei of SW-620 cells not treated with NaB and in the cytoplasm of SW-620 cells treated with NaB . When the NaB-treated SW-620 cells were incubated with ADM in the presence of PAK-104P, the accumulation of ADM in nuclei was substantially increased . Isolated nuclei from untreated cells accumulated more ADM than nuclei from NaB-treated cells . Efflux of ADM from the nuclei isolated from NaB-treated cells was enhanced . PAK-104P and an antibody against LRP increased the accumulation of ADM in the isolated nuclei from NaB-treated cells, and inhibited the enhanced efflux of ADM from the nuclei . These findings suggest that at least in part, PAK-104P reverses LRP-mediated drug resistance by inhibiting the efflux of ADM from nuclei . PAK-104P may be useful for reversing MDR in tumors that overexpress LRP.

Curr Opin Oncol, 2001 Jan, 13(1), 21 - 6
Drug resistance mechanisms in acute leukemia; Chauncey TR; Markers of anticancer drug resistance are predictive of treatment response and outcome in patients with acute myeloid leukemia . Immunologic detection of the drug efflux pumps, P-glycoprotein (Pgp) and multidrug resistance-associated protein 1 (MRP1), correlate with functional assays of drug resistance . These accumulation defects also appear operable in acute lymphoblastic leukemia . Many of the efflux pumps identified share significant structural homology with the large superfamily of ATP-binding cassette transporters . Other markers such as lung-resistance protein, bcl-2, and breast cancer-resistance protein, have been described in acute myeloid leukemia patients although their pathophysiology and clinical relevance are less clear and the methodology for their quantification are not well standardized . Preclinical studies have shown that small molecules capable of reversing efflux can restore drug sensitivity in resistant tumor models . Although initial clinical studies were limited by both potency and specificity of the reverser, later studies with more effective reversers have in many instances been limited by pharmacokinetic interactions exacerbating the clinical toxicities of chemotherapy . Although one large randomized study has demonstrated a proven survival advantage without increased toxicity using cyclosporine, the inconsistent results with other modulators raise doubt as to the utility and overall strategy of using drug efflux blockers in patients with established Pgp overexpression . Many of these patients have additional resistance mechanisms, and achieving meaningful clinical responses will likely require more complex clinical strategies . Preventing or delaying development of drug resistance in chemosensitive patients represents another therapeutic strategy to be tested.

Trends Cell Biol, 2001 Jan, 11(1), 10 - 12
Peptide-based targeting of fluorophores to peroxisomes in living cells; Pap EH et al.; Peptides carrying different fluorophores can be designed to incorporate spontaneously into living cells when added to the medium . By incorporating the peroxisome-targeting sequence PTS1, the peptide is recognized by the protein-import machinery of peroxisomes and, as a result, can accumulate in these organelles . Depending on the cell type, an inhibitor of the multidrug-resistance protein might be required to ensure strong accumulation . In this update, we discuss the potential of these peptide-linked fluorophores in solving issues related to organelle function and dynamics.

Pharm Res, 2000 Oct, 17(10), 1168 - 74
Mechanisms of transport and structure-permeability relationship of sulfasalazine and its analogs in Caco-2 cell monolayers; Liang E et al.; PURPOSE: To investigate the mechanisms involved in transport of sulfasalazine in Caco-2 cells . METHODS: Permeability coefficients of sulfasalazine and its analogs across Caco-2 cell monolayers were measured as a function of direction of transport, energy and concentration dependence, and in the presence of inhibitors of various cellular efflux pumps and transporters . RESULTS: Permeability coefficients of sulfasalazine across Caco-2 cell monolayers were approximately 342-, 261-, and 176-fold higher from basolateral to apical direction (BL-->AP) than from apical to basolateral direction (AP-->BL) at 100, 200, and 500 microM, respectively . Carrier permeability coefficient, non-saturable membrane permeability coefficient, and Michaelis constant were estimated to be 1.4x10(-5) cm/s, 1.9x10(-8) cm/s, and 369 microM, respectively . The efflux of sulfasalazine was completely blocked at 4 degrees C and in the presence of an uncoupler of oxidative phosphorylation . Using cellular efflux inhibitors, the permeability of sulfasalazine was shown to depend on multidrug resistance-associated protein and anion sensitive transport mechanisms . Structure-permeability studies showed that the affinity of sulfasalazine for the cellular efflux pumps and transporters in Caco-2 cells depended strongly on the carboxylic acid functional group . CONCLUSIONS: The permeability of sulfasalazine across Caco-2 cell monolayer is very low due to its strong interaction with multiple cellular efflux pumps and transporters . This may partially explain its low absorption in vivo.

Biol Pharm Bull, 2000 Dec, 23(12), 1528 - 31
Inhibitory effect of human immunodeficiency virus protease inhibitors on multidrug resistance transporter P-glycoproteins; Shiraki N et al.; The objective of this study was to determine whether human immunodeficiency virus (HIV) protease inhibitors (saquinavir, ritonavir and nelfinavir) interact with other HIV protease inhibitors and/or HIV reverse transcriptase inhibitors (zidovudine, didanosine, lamivudine, zalcitabine and sanilvudine) . We measured transport of nelfinavir, an HIV protease inhibitor which is known as a substrate for the multidrug resistance transporter P-glycoprotein (P-gp), in an epithelial monolayer model and Ki for P-gp of some drugs by a calcein flux assay . Transport in a basal to apical direction was 2-fold greater than apical to basal flux for nelfinavir, Ki for P-gp of a potent P-gp inhibitor cyclosporin A was 1.09 microM and those of ritonavir and nelfinavir were 111 microM and 28.6 microM, whereas all HIV reverse transcriptase inhibitors gave high K1 values . These data show that nelfinavir, which is a substrate for P-gp, inhibits a P-gp function as a drug efflux pump and that HIV reverse transcriptase inhibitors do not inhibit P-gp.

Int J Tuberc Lung Dis, 2000 Dec, 4(12 Suppl 2), S171 - 5
Access to newer laboratory procedures: a call for action; Hale YM et al.; Healthy People 2010, an initiative from the federal government, calls for action from tuberculosis controllers and tuberculosis laboratories in the fight to eliminate tuberculosis . Many patients, such as immunocompromised patients and those infected with multidrug-resistant tuberculosis strains, pose a challenge for care and diagnosis . Fortunately, many changes have occurred in the last decade to facilitate more rapid and accurate testing to assist with the care of these patients . California, Florida, New York and Texas have almost 50% of the tuberculosis cases in the United States, and their public health laboratories utilize different approaches to meet the same goal of rapid and accurate testing of specimens . With the targets of Healthy People 2010 (e.g., to reduce the average time for a laboratory to confirm and report tuberculosis cases to 2 days for 75% of cases) already looming on the horizon, innovative methods for achieving these goals should be evaluated . Using these public health laboratories as models, rapid, gold-standard testing methods should be provided to all patients in the United States . Soon it will be the year 2010..., are you ready to swiftly move forward?

J Exp Clin Cancer Res, 2000 Sep, 19(3), 335 - 48
Evolutionary malignant resistance of cells to damaging factors as common biological defence mechanism in neoplastic development . Review of conception; Monceviciute-Eringiene E; Cells have some inborn resistance to harmful factors, which could be called physiological or natural resistance . The mechanisms of multixenobiotic resistance (MXR) and multidrug resistance (MDR) have common features in the formation of acquired resistance in microorganisms, carcinogenesis, tumour metastases and chemotherapy or irradiation . ATP-dependent membrane P-glycoprotein, as an MDR efflux pump, glutathione S-transferases and other products of evolutionary resistance-related genes arised for exportation and detoxification of cytotoxic xenobiotics and drugs are transmitted from bacteria to man . On the one hand, this evolutionary MXR as a common biological defence mechanism is a "driving" power to conserve homeostasis of cells, tissues and organs . On the other hand, mutation, selection and simplification of properties are the causes of functional and morphological changes in tumour cells which regress to a more primitive mode of existence (atavism) for adaptation to survival . In the present work are presented data on the forms of E . coli resistant to antibiotics and of sarcoma 45 resistant to alkylic preparations . They may be helpful in revealing the causes of resistance and acquired accelerated growth of cells . The development of tumours as fibromas 14-15 years following injection of a vital dye trypan blue into human skin supports our conception that neoplastic growth is a particular case of the evolutionary resistance of cells adapted to the damaging factors . So, tumour cells adopting the enhancement mechanisms of general biological persistent resistance, i . e . undergoing repeated cycles of malignancy enhancement, adapt themselves to survive under the changed unfavourable conditions.

Int J Tuberc Lung Dis, 2000 Dec, 4(12), 1149 - 55
Primary drug-resistant tuberculosis in children; Schaaf HS et al.; SETTING: The Western Cape Province of South Africa, an area with a high tuberculosis (TB) incidence where initial isoniazid (INH) resistance and multidrug resistance (MDR) among adults was 3.9% and 1.1%, respectively, during 1992-1993 . OBJECTIVE: To determine the drug resistance incidence among children as compared to adults, to compare the clinical features of drug-resistant and drug-susceptible TB, and the degree of INH resistance in isoniazid-resistant isolates . METHODS: All Mycobacterium tuberculosis cultures obtained from children (0-13 years) at a regional hospital were prospectively collected from August 1994 to April 1998 and susceptibility testing done on each child's specimens . Degree of INH resistance was determined in available resistant isolates . The children's clinical records were reviewed . RESULTS: Susceptibility results were available in 306/338 children with cultures of M . tuberculosis; 21 isolates (6.9%) were INH-resistant, and seven were MDR . Taking into account study limitations, the incidence of INH resistance was 5.6% and MDR 1% in children aged <5 years . Clinical features were similar in children with drug-susceptible and drug-resistant TB . CONCLUSION: The incidence of drug resistance in childhood tuberculosis in Western Cape is low, and probably reflects the level of primary drug resistance amongst organisms currently circulating in the community.

Int J Tuberc Lung Dis, 2000 Dec, 4(12), 1104 - 10
Pulmonary tuberculosis in prisons of the ex-USSR state Georgia: results of a nation-wide prevalence survey among sentenced inmates; Aerts A et al.; SETTING: The penitentiary system of the ex-USSR state of Georgia . OBJECTIVES: To measure the prevalence of active pulmonary tuberculosis and drug-resistant disease among prisoners . To identify factors associated with active tuberculosis and multidrug resistance (MDR) . DESIGN: A comprehensive multiphasic screening survey for tuberculosis . RESULTS: The prevalence of smear- or culture-positive tuberculosis was 5995 per 100,000 prisoners (n = 448 cases among 7473 inmates) . Of all the strains, 215 (77.9%) were resistant to at least one drug and 37 (13.0%) were MDR . Independent risk markers associated smear- or culture-positive tuberculosis with included prison stay of 2 years or more, body mass index <20 kg/m2, accommodation in a large size prison, previous anti-tuberculosis treatment, cough of 2 weeks or more and loss of appetite . Risk markers associated with MDR were a prison stay of less than 2 years and being over 25 years of age . CONCLUSIONS: In Georgia, the excess risk for tuberculosis among prisoners is unprecedented, suggesting that prisons represent a significant reservoir of tuberculosis . Only a comprehensive strategy for tuberculosis control in prisons, including prison reform, control of anti-tuberculosis drugs, and prompt and efficient diagnosis and treatment of patients can have an impact on the tuberculosis burden in the prison system.

J Drug Target, 2000, 8(4), 247 - 56
The effect of fatty acids and analogues upon intracellular levels of doxorubicin in cells displaying P-glycoprotein mediated multidrug resistance; Abulrob AN et al.; Multidrug resistance mediated by overexpression of P-glycoprotein (P-gp) is a major obstacle in the chemotherapeutic management of cancer . The objectives of the current work were to examine if fatty acids affect the intracellular transport and dynamics of doxorubicin in drug-resistant cancer cell lines, and to assess if such effects were mediated through modulation of P-gp efflux pump activity . Among the range of fatty acids tested in this study, eicosapentaenoic acid diester (EPADI) increased doxorubicin accumulation {A} to 137% and retention {R} to 212% in doxorubicin-resistant MCF-7/ADR breast carcinoma cells, and {A} to 147% and {R} to 163% in vinblastine-resistant KBVI nasopharyngeal carcinoma cells . Consistent with EPADI-induced increases in intracellular doxorubicin concentrations, EPADI (10 microg/ml) sensitized MCF-7/ADR cells to the cytotoxic effects of doxorubicin (1 microg/ml) as assessed by MTT assay (viability < 50% of control), while EPADI itself displayed no cytotoxicity . The combination of EPADI (10 microg/ml) with verapamil (1 microM) resulted in a considerable increase in the {A} and {R} of the model P-gp substrate rhodamine-123 within drug-resistant cells compared to when either agent were used alone . KBV1 cells treated with combination of EPADI (10 microg/ml) and verapamil (1 microM) achieved 160% and 1120% greater {A} and {R} of rhodamine-123, respectively, compared to untreated cells . The P-gp modulatory effects of EPADI either alone, or as part of a combination with more potent inhibitors, should be further investigated.

J Nat Prod, 2000 Dec, 63(12), 1638 - 40
In vitro antiplasmodial, antiamoebic, and cytotoxic activities of some monomeric isoquinoline alkaloids; Wright CW et al.; Twenty-one alkaloids have been assessed for activities against Plasmodium falciparum (multidrug- resistant strain K1) in vitro; 18 of these are reported for the first time . Two protoberberine alkaloids, dehydrodiscretine and berberine, were found to have antiplasmodial IC(50) values less than 1 M, while seven alkaloids-allocrytopine, columbamine, dehydroocoteine, jatrorrhizine, norcorydine, thalifendine, and ushinsunine-had values between 1 and 10 M . These results are discussed in the context of structure-activity relationships . Compounds were also assessed for antiamoebic and cytotoxic activities, but none was significantly active except for berberine, which was moderately cytotoxic.

Bioorg Med Chem Lett, 2001 Jan 8, 11(1), 75 - 7
B-ring substituted 5,7-dihydroxyflavonols with high-affinity binding to P-glycoprotein responsible for cell multidrug resistance; Boumendjel A et al.; Starting from the interaction of galangin (3,5,7-trihydroxyflavone) with a cytosolic nucleotide-binding domain of P-glycoprotein, a series of flavonol derivatives was synthesized and tested for their binding affinity towards the same target . The 5,7-dihydroxy-4'-iodoflavonol and 5,7-dihydroxy-4'-n-octylflavonol derivatives displayed much higher binding affinities, with respective increases of 6- and 93-fold as compared to galangin.

Cancer Treat Rev, 2000 Dec, 26(6), 449 - 62
The blood-brain barrier and oncology: new insights into function and modulation; Bart J et al.; The efficacy of chemotherapy for malignant primary or metastatic brain tumours is still poor . This is at least partly due to the presence of the blood-brain barrier (BBB) . The functionality of the BBB can be explained by physicochemical features and efflux pump mechanisms . An overview of the literature is presented with emphasis on oncology.The BBB consists of capillary endothelial cells that lack fenestrations and are connected together with continuous tight junctions, with a high electrical resistance . Permeability of tight junctions can be increased in vitro by contraction of the cytoskeleton, caused by bradykinin agonists . Different efflux pumps are present in the BBB . Examples are P-glycoprotein (P-gp), organic anion transporters, (OAT) and multidrug-resistance-associated proteins (MRP)(1 and 3) . These pumps act as a multi-specific efflux pump for various chemotherapeutic drugs . Experiments have shown that P-gp can be inhibited by different non-chemotherapeutic substrates such as cyclosporin A . The functionality in vivo of P-gp can be measured with positron emission tomography and {(11)C}-verapamil or with single photon emission computer tomography and(99m)Tc-sestamibi . MRP(1)and MRP(3)act as organic anion transporters that in vitro act as efflux pumps for substances that are conjugated or co-transported with glutathione and glucuronide, respectively . Methotrexate has been recently demonstrated to be transported by MRP(1)and MRP(3) . Results of studies which demonstrate the clinical relevance and applicability of BBB modulators are eagerly awaited .

Biochem Pharmacol, 2001 Jan 1, 61(1), 61 - 6
Characterization of multidrug transporters in a normal renal tubular cell line resistant to doxorubicin . Multidrug transporters in the LLC-PK(1) cell line and its resistant counterpart; Decorti G et al.; LLC-PK(1) is a proximal tubular cell line derived from normal pig kidney which has a structure and function similar to those of renal proximal tubular cells and which expresses baseline levels of P-glycoprotein . We isolated by drug selection a doxorubicin-resistant cell line (LLC-PK(1)/ADR) that exhibited a multidrug-resistant phenotype; this cell line was characterized by reduced intracellular drug concentrations, an increased drug extrusion, and increased expression of a 170-kDa P-glycoprotein detected by Western blot analysis with monoclonal antibody C219 . In addition, an increased expression of MDR1 mRNA was seen by reverse transcriptase-polymerase chain reaction . These results suggest that it is possible to induce the overexpression of P-glycoprotein by chronic treatment with doxorubicin in a normal cell line that physiologically expresses low levels of this protein . This multi-resistant cell line could provide an interesting model for studying the role of P-glycoprotein and the consequence of its induction in a normal tissue.

Biochem Pharmacol, 2001 Jan 1, 61(1), 39 - 47
Reduced cellular transport and activation of fluoropyrimidine nucleosides and resistance in human lymphocytic cell lines selected for arabinosylcytosine resistance; Agarwal RP et al.; Arabinofuranosylcytosine (araC) resistant H9-araC0.05 and H9-araC0.5 sublines were obtained following in vitro exposure of H9 cells to 0 . 05 and 0.5 microM araC, respectively . These cell lines were 83.3- and 266.7-fold, 21- and 80-fold, and 2.4- and 4.0-fold more resistant to 5-fluorouridine (FUR), 5-fluoro-2'-deoxyuridine (FdUR), and 5-fluorouracil (FU), respectively . Compared with H9 cells, the cellular accumulation of FUR was 2.2 and 0.2%, FdUR 15.6 and 0.9%, and FU 56.9 and 66.5% in H9-araC0.05 and H9-araC0.5 cells, respectively . An araC resistant HL60 cell line (promyelocytic cell line) was 5.0- and 1.7-fold resistant to FUR and FdUR, respectively, but displayed no resistance to FU . The lower FUR and FdUR nucleotide levels in the resistant cells were a result of reduced cellular transport and uridine kinase (UR kinase) and thymidine kinase (TK) activities . Compared with the parental cell line, the p-nitrobenzyl thioinosine (an inhibitor of nucleoside transport) binding sites also were lower in the araC resistant cells . There was no difference in the expression of multidrug-resistant protein and thymidylate synthase mRNA in the parental and the resistant cell lines . Data presented here suggest that araC exposure of H9 cells, in addition to araC resistance, induced/selected cells that were resistant to FUR and FdUR . These cells had altered cellular drug transport and lower TK and UR kinase activities . Further studies to understand molecular mechanisms of this phenomenon are warranted.

Leuk Res, 2001 Jan, 25(1), 85 - 93
Modulator activity of PSC 833 and cyclosporin-A in vincristine and doxorubicin-selected multidrug resistant murine leukemic cells; Lopes EC et al.; Multidrug resistance (MDR) lines from a murine T-cell leukemia were selected in increasing vincristine (VCR) or doxorubicin (DOX) concentrations . Daunorubicin (DNR) efflux was evidenced after 25 additional passages with constant 160 ng ml(-1) of either VCR or DOX, an effect that was inhibited by verapamil, cyclosporin-A (CsA) and PSC 833 . The expression of Pgp was not evidenced in the resistant cell lines using anti-human Pgp antibodies . Cell proliferation assay showed that cell lines resistant to VCR (LBR-V160) or DOX (LBR-D160) required higher doses of either drug to produce GI50 compared with control cell line obtained after culture in the absence of VCR or DOX . When resistant cell lines were maintained during 60 days in the absence of either VCR or DOX, MDR phenotype reversal was obtained in LBR-D160 while LBR-V160 remained resistant to the drug, as shown by cell proliferation assays and by drug efflux pump functionality . When VCR or DOX were used together with either CsA or PSC 833, the latter was more effective to produce reversal of resistance than the former, whereas CsA presented greater cytotoxic effect than PSC 833 for sensitive and resistant cells . Cross-resistance was found between VCR, DOX and other antineoplasic agents on murine leukemic cell line . VCR was more effective to induce MDR since the resistant cell lines were more stable to the MDR phenotype.

Leuk Res, 2001 Jan, 25(1), 69 - 75
Drug resistance does not correlate with resistance to Fas-mediated apoptosis; Cullen KV et al.; Recent reports have correlated multidrug resistance (MDR) and P-glycoprotein expression with decreased Fas expression and resistance to Fas-mediated apoptosis . We report the MRP-overexpressing MDR subline CEM/E1000 has the same Fas expression (MFI 74.3 +/- 0.7) as the parental CCRF-CEM T-cell leukaemia cells (MFI 70.0 +/- 3.6; P>0.05), and that the level of apoptosis induced by anti-Fas antibody or drug was similar in both cell lines . Further the P-glycoprotein-expressing CEM/VLB(100) subline of the CCRF-CEM cells showed increased Fas expression (MFI 114.8 +/- 3.6; P<0.001) and no resistance to Fas-mediated apoptosis . This questions the hypothesis that selection of drug resistance results in resistance to Fas-mediated apoptosis, with important implications for the rational use of immunotherapy in the treatment of drug resistant cancer.

Leuk Res, 2001 Jan, 25(1), 23 - 32
Blasts from elderly acute myeloid leukemia patients are characterized by low levels of culture- and drug-induced apoptosis; Garrido SM et al.; Increasing evidence suggests that the biology of acute myeloid leukemia (AML) may differ between older and younger patients, with a higher incidence of antecedent myelodysplasia, unfavorable cytogenetic abnormalities, and multidrug resistance seen in the elderly . Abrogation of apoptosis in response to cytotoxic medications is associated with drug resistance in AML, as is expression of bcl-2, an important anti-apoptotic protein . We hypothesized that blasts from elderly (> or = 55 years) and young adult AML patients might have different levels of apoptotic and cell cycle responses to chemotherapeutic agents, as well as different levels of proliferation and of bcl-2 protein expression . Therefore, we cultured bone marrow leukemia samples from previously untreated elderly (n=33) and young (n=21) AML patients for 48 h and then measured apoptosis, bcl-2 protein levels, cell cycle distributions, and expression of a proliferation marker, proliferating cellular nuclear antigen (PCNA) in multi-parametric flow cytometry assays . In some experiments, leukemia samples were exposed to cytarabine (Ara-C) or daunomycin (DNR) for the last 16-18 h of the culture period . In comparison to samples from young patients, cultured samples from elderly AML patients had a higher fraction of viable cells, as measured by Trypan blue exclusion, higher PCNA expression, and significantly less culture-induced and drug-induced apoptosis . The mean apoptosis after culture was 13% for elderly AML samples, versus 20% for young AML samples (P=0.009) . Similarly, the mean apoptosis after Ara-C was lower in elderly than in young AML samples, 13 versus 28% (P=0.001), as was the mean apoptosis after DNR, 15 versus 26% (P=0.012) . Diminished apoptotic responses in elderly AML cells were not consistently associated with high bcl-2 levels at thaw or bcl-2 levels increased by culture . These data suggest that new therapies should be developed to overcome abrogated apoptosis, particularly in elderly AML patients.

Curr Biol, 2000 Dec 14-28, 10(24), 1587 - 90
Endocytosis and the development of cell polarity in yeast require a dynamic F-actin cytoskeleton; Ayscough KR; Studies using drugs that cause the disassembly of filamentous actin (F-actin) have demonstrated the importance of an intact actin cytoskeleton for polarised secretion by yeast cells {1,2} . To address the level of dynamic turnover needed for such processes, however, drugs or mutants that confer stabilising properties on F-actin are needed . Jasplakinolide is the only readily available drug that stabilises F-actin structures both in vivo and in vitro {3-6} . Yeast strains have been generated in which two of the ABC multidrug resistance transporter genes have been deleted, rendering normally jasplakinolide-resistant yeast cells sensitive to its effects . Treatment of these cells with jasplakinolide caused rapid and dramatic effects on the actin cytoskeleton, resulting in the accumulation of single large actin structures in cells . These structures, however, still contained components that are normally associated with cortical actin patches . A dynamic actin cytoskeleton was found to be critical for the generation of cell polarity and endocytosis.

J Exp Biol, 2001 Jan, 204(Pt 2), 217 - 27
Enhanced xenobiotic transporter expression in normal teleost hepatocytes: response to environmental and chemotherapeutic toxins; Albertus JA et al.; Many aquatic organisms are resistant to environmental pollutants, probably because their inherent multi-drug-resistant protein extrusion pump (pgp) can be co-opted to handle man-made pollutants . This mechanism of multixenobiotic resistance is similar to the mechanism of multidrug resistance exhibited in chemotherapy-resistant human tumor cells . In the present study, a variety of techniques were used to characterize this toxin defense system in killifish (Fundulus heteroclitus) hepatocytes . The cellular localization and activity of the putative drug efflux system were evaluated . In addition, in vitro and in vivo studies were used to examine the range of expression of this putative drug transporter in the presence of environmental and chemotherapeutic toxins . The broad range of pgp expression generally observed in transformed mammalian cells was found in normal cells of our teleost model . Our findings suggest that the expression of the pgp gene in the killifish could be an excellent indicator of toxin levels or stressors in the environment.

Mol Microbiol, 2001 Jan, 39(2), 304 - 12
Pse1/Kap121-dependent nuclear localization of the major yeast multidrug resistance (MDR) transcription factor Pdr1; Delahodde A et al.; Pdr1 and Pdr3 are two very similar transcription factors that mainly control membrane biogenesis by adjusting the production of different membrane proteins, such as different ABC or major facilitator superfamily (MFS) transporters . We observed that the pse1-1 mutation in the importin/beta-karyopherin Pse1/Kap121 specifically induced the cytoplasmic localization of Pdr1, but not that of Pdr3 . Interactions between Pse1 and Pdr1 could be observed in vivo, and a short peptide of 44 amino acids from Pdr1 was shown to contain the information necessary and sufficient for Pse1-dependent nuclear import . This Pdr1-NLS sequence, absent in Pdr3, although rich in serine and tyrosine, is different from the Pse1-dependent nuclear localization signal (NLS) of Pho4 . Furthermore, we showed that Pse1/Kap121 is likely to be the sole import receptor for the regulator Pdr1 . Together, these new observations underscore the diversity of cellular processes that address to the nucleus two very similar transcription factors involved in the control of the same phenotype, thus securing their function in the cell.

Microsc Res Tech, 2001 Jan 1, 52(1), 83 - 8
Brain drug delivery, drug metabolism, and multidrug resistance at the choroid plexus; Ghersi-Egea JF et al.; The choroid plexuses (CPs) have the capability to modulate drug delivery to the cerebrospinal fluid (CSF) and to participate in the overall cerebral biodisposition of drugs . The specific morphological properties of the choroidal epithelium and the existence of a CSF pathway for drug distribution to different targets in the central nervous system suggest that the CP-CSF route is more significant than previously thought for brain drug delivery . In contrast to its role in CSF penetration of drugs, CP is also involved in brain protection in that it has the capacity to clear the CSF from numerous potentially harmful CSF-borne exogenous and endogenous organic compounds into the blood . Furthermore, CP harbors a large panel of drug-metabolizing enzymes as well as transport proteins of the multidrug resistance phenotype, which modulate the cerebral bioavailability of drugs and toxins . The use of an in vitro model of the choroidal epithelium suitable for drug transport studies has allowed the demonstration of the choroidal epithelium acting as an effective metabolic blood-CSF barrier toward some xenobiotics, and that a vectorial, blood-facing efflux of conjugated metabolites occurs at the choroidal epithelium . This efflux involves a specific transporter with characteristics similar to those of the multidrug resistance associated protein (MRP) family members . Indeed, at least one member, MRP1, is largely expressed at the CP epithelium, and localizes at the basolateral membrane . These metabolic and transport features of the choroidal epithelium point out the CP as a major detoxification site within the brain .

Microsc Res Tech, 2001 Jan 1, 52(1), 60 - 4
Organic anion transport across the choroid plexus; Gao B et al.; Several organic anion transport systems have recently been identified and localized at the apical and basolateral plasma membrane domains of choroid plexus epithelial cells . These organic anion transporters include (1) indirectly coupled Na(+)/dicarboxylate cotransport and dicarboxylate/organic anion exchange, which is represented on the molecular level by a member of the "kidney"-type organic anion transporter (OAT) family at the apical plasma membrane domain; (2) the organic anion transporting polypeptide 1 (Oatp1) and Oatp2, which both mediate typical "liver"-like organic anion transport activities at the apical and basolateral plasma membrane domains, respectively; and (3) the multidrug resistance protein Mrp1/MRP1 at the basolateral plasma membrane domain, and the P-glycoprotein Mdr1/MDR1 at an apical and subapical membrane vesicle compartment . This cellular transport polarity can account, at least in part, for the previously suggested physiologic transport properties of the choroid plexus epithelium and provides a framework for the identification and localization of additional organic anion transporters involved in the absorption and/or excretion of drugs and drug metabolites at the choroid plexus .

Genes Chromosomes Cancer, 2001 Feb, 30(2), 136 - 42
Identification of chromosomal loci associated with non-P-glycoprotein-mediated multidrug resistance to topoisomerase II inhibitor in lung adenocarcinoma cell line by comparative genomic hybridization; Struski S et al.; In order to identify genomic changes associated with an etoposide resistance acquisition, we used comparative genomic hybridization (CGH) to compare a human lung adenocarcinoma cell line, A549 wild type, and three sublines, A549-VP1-3, exposed to increasing concentrations of the topoisomerase II inhibitor, VP16 . R-banding karyotype, fluorescence in situ hybridization (FISH), and Southern blot for the MLL gene were also performed . The CGH analysis showed that the A549-VP3 cell line shared chemoresistance-specific abnormalities (amplification of 11q23-qter, loss of chromosome 17, and deletions of 2p14-pter and 2q23-q24) . FISH analysis confirmed the loss of one chromosome 17 in the three resistant sublines and revealed an increased fragmentation of chromosome 2 in more than two segments, depending on the etoposide concentration . FISH with an MLL gene probe showed additional signals of MLL (from three in the A549-WT to seven in the A549-VP3 cell line) translocated onto several other chromosomes . Southern blot indicated an amplification of the MLL gene, dependent on the etoposide concentration, without gene rearrangement . The CGH results are suggestive of loci that could be associated with the acquisition of an etoposide-chemoresistant phenotype . Deletion of the 2p region has already been reported, without any candidate gene being identified . The role of MLL in leukemogenesis has previously been demonstrated, but its role in the development of other tumors or its significance in the chemoresistance process remains to be elucidated .

J Infect Dis, 2001 Feb 1, 183(3), 461 - 8 Epub 2000 Dec 27.
Epidemiologic differences between United States- and foreign-born tuberculosis patients in Houston, Texas; El Sahly HM et al.; The proportion of foreign-born tuberculosis patients in the United States is increasing . To analyze the epidemiology of tuberculosis in foreign-born people, culture-positive patients with tuberculosis in Houston, Texas, were interviewed from October 1995 through September 1998, and their isolates were molecularly characterized . Of the 1131 patients included in the study, 795 (70.3%) were US born and 336 (29.7%) were foreign born . The decrease in tuberculosis case rate among US-born people was 3.5 times that of foreign-born people . Significantly more US-born than foreign-born patients belonged to strain clusters (71.3% vs . 29.5%; P<.001) . Risk factors associated with strain clustering were as follows: black ethnicity, low income, and homelessness in US-born patients and homelessness in foreign-born patients . Isolates from foreign-born patients were more likely to be resistant to >/=1 drug (15.4% vs . 8.4%; P=.001) and to be multidrug resistant (2.4% vs . 0.7%; P=.027) than isolates from US-born patients . These observations warrant increased emphasis on this distinct subpopulation of tuberculosis patients.

J Antibiot (Tokyo), 2000 Oct, 53(10), 1201 - 6
Reversal of multidrug resistance by 7-O-benzoylpyripyropene A in multidrug-resistant tumor cells; Rho MC et al.; 7-O-Benzoylpyripyropene A (7-O-BzP), a semi-synthetic analog of pyripyropene, was investigated for its reversing effect on multidrug-resistant (MDR) tumor cells . 7-O-BzP (6.25 microg/ml) completely reversed resistance against vincristine and adriamycin in vincristine-resistant KB cells (VJ-300) and adriamycin-resistant P388 cells (P388/ADR), respectively . 7-O-BzP alone had no effect on the growth of drug sensitive and drug-resistant cells . 7-O-BzP (6.25 microg/ml) significantly enhanced accumulation of {3H}vincristine in VJ-300 cells and completely inhibited the binding of {3H}azidopine to the P-glycoprotein in VJ-300 cells and P388/ADR cells . The result suggests that 7-O-BzP effectively reverses P-glycoprotein-related MDR by interacting directly with P-glycoprotein in drug resistant VJ-300 and P388/ADR cells.

Trans R Soc Trop Med Hyg, 2000 Sep-Oct, 94(5), 545 - 8
Artemether-lumefantrine for the treatment of multidrug-resistant falciparum malaria; van Vugt M et al.; The efficacy and safety of the 6-dose regimen of artemether-lumefantrine were assessed in an open randomized trial in children and adults presenting with acute, uncomplicated Plasmodium falciparum malaria in Thailand between November 1997 and March 1998 . 200 patients were enrolled in 2 centres: 150 received artemether-lumefantrine (i.e., a median total dose of 9.6 mg/kg {interquartile range 8.7-10.7} and 57.9 mg/kg of lumefantrine {52.4-64.0}) and 50 the standard combination of artesunate (12 mg/kg over 3 d) and mefloquine (25 mg/kg) . All patients had rapid initial clinical and parasitological responses . The 28 d cure rates were high: 97.7% (95% confidence interval {95% CI} 93.5-99.5%) for artemether-lumefantrine and 100% (95% CI 92.5-100%) for artesunate-mefloquine . The 6-dose regimen of artemether-lumefantrine was better tolerated than, and as effective as, artesunate-mefloquine, the current standard treatment in this area of multidrug-resistant P . falciparum malaria.

Trans R Soc Trop Med Hyg, 2000 Sep-Oct, 94(5), 537 - 44
Plasmodium falciparum antimalarial drug susceptibility on the north-western border of Thailand during five years of extensive use of artesunate-mefloquine; Brockman A et al.; Following a marked decline in the efficacy in vivo of mefloquine between 1990 and 1994, a combination of artesunate (4 mg/kg/d for 3 d) and mefloquine (25 mg/kg) has been used as first line treatment of uncomplicated falciparum malaria in camps for displaced persons located along the north-western border of Thailand . Antimalarial drug susceptibility of fresh isolates of Plasmodium falciparum from this population was evaluated using a radioisotope microdilution assay between 1995 and 1999 . In total, 268 isolates were collected, of which 189 were from primary infections and 79 from recrudescent infections . The geometric mean 50% inhibitory concentration (IC50) values from primary infections were: dihydroartemisinin 1.2 ng/mL, artesunate 1.6 ng/mL, artemether 4.8 ng/mL, atovaquone 0.4 ng/mL, lumefantrine 32 ng/mL, chloroquine 149 ng/mL, quinine 354 ng/mL, mefloquine 27 ng/mL and halofantrine 4.1 ng/mL . A significant positive correlation was found between the susceptibility in vitro to artesunate and quinine (r = 0.43, P < 0.001), mefloquine (r = 0.46, P < 0.001), and halofantrine (r = 0.51, P < 0.001) . These levels of resistance in vitro are among the highest reported and confirm continuing high level multidrug resistance in this area . Despite intensive use of the combination between 1995 and 1999 there has been a significant improvement in mefloquine sensitivity (P < 0.001) and artesunate sensitivity (P < 0.001) . This supports observations in vivo that the combination of artesunate and mefloquine has reversed the previous decline in mefloquine sensitivity.

J Biol Chem, 2001 Mar 23, 276(12), 8657 - 64 Epub 2000 Dec 19.
Correlation between steady-state ATP hydrolysis and vanadate-induced ADP trapping in Human P-glycoprotein . Evidence for ADP release as the rate-limiting step in the catalytic cycle and its modulation by substrates; Kerr KM et al.; P-glycoprotein (Pgp) is a transmembrane protein conferring multidrug resistance to cells by extruding a variety of amphipathic cytotoxic agents using energy from ATP hydrolysis . The objective of this study was to understand how substrates affect the catalytic cycle of ATP hydrolysis by Pgp . The ATPase activity of purified and reconstituted recombinant human Pgp was measured using a continuous cycling assay . Pgp hydrolyzes ATP in the absence of drug at a basal rate of 0.5 micromol x min x mg(-1) with a K(m) for ATP of 0.33 mm . This basal rate can be either increased or decreased depending on the Pgp substrate used, without an effect on the K(m) for ATP or 8-azidoATP and K(i) for ADP, suggesting that substrates do not affect nucleotide binding to Pgp . Although inhibitors of Pgp activity, cyclosporin A, its analog PSC833, and rapamycin decrease the rate of ATP hydrolysis with respect to the basal rate, they do not completely inhibit the activity . Therefore, these drugs can be classified as substrates . Vanadate (Vi)-induced trapping of {alpha-(32)P}8-azidoADP was used to probe the effect of substrates on the transition state of the ATP hydrolysis reaction . The K(m) for {alpha-(32)P}8-azidoATP (20 microm) is decreased in the presence of Vi; however, it is not changed by drugs such as verapamil or cyclosporin A . Strikingly, the extent of Vi-induced {alpha-(32)P}8-azidoADP trapping correlates directly with the fold stimulation of ATPase activity at steady state . Furthermore, P(i) exhibits very low affinity for Pgp (K(i) approximately 30 mm for Vi-induced 8-azidoADP trapping) . In aggregate, these data demonstrate that the release of Vi trapped {alpha-(32)P}8-azidoADP from Pgp is the rate-limiting step in the steady-state reaction . We suggest that substrates modulate the rate of ATPase activity of Pgp by controlling the rate of dissociation of ADP following ATP hydrolysis and that ADP release is the rate-limiting step in the normal catalytic cycle of Pgp.

Anticancer Res, 2000 Sep- Oct, 20(5B), 3533 - 8
Modulation of P-glycoprotein-mediated doxorubicin resistance in canine cell lines; Page RL et al.; OBJECTIVE: To characterize the chemosensitivity of wild type and multidrug resistant canine cell lines and determine the relative potency of the P-glycoprotein (Pgp) modulators verapamil, tamoxifen and a cyclosporin-A analog (PSC833) . METHODS: The dose required to reduce cell proliferation to 50% of control (ED50) for doxorubicin (DOX) and cisplatin was determined for canine cell lines 4TG11-50c, OS2.4wt, OS2.4DX and the human cell line MCF7/DX . The effect of Pgp chemomodulators on cytotoxicity was quantified by determining the dose modifying factor {DMF = (ED50 of Dox alone)/(ED50 of Dox + Modulator)} . Relative potency of modulators was defined as DMFMOD1/DMFMOD2 . Pgp function was assessed by DiOC2 dye retention and by accumulation of DOX after chemomodulator addition . RESULTS: All cell lines were equally cisplatin sensitive but varied in doxorubicin resistance . PSC833 was 12X, 5X and 2X more potent than tamoxifen in 4TG11-50c, OS2.4WT and OS2.4DX, respectively . Dye retention was a better indicator of chemomodulator-enhanced cytotoxicity than was DOX accumulation . CONCLUSIONS: Pgp inhibition is cell line, modulator and concentration dependent but the cytotoxic potency of a modulator may be predicted by the extent of dye retention in canine drug resistant cell lines.

Anticancer Res, 2000 Sep- Oct, 20(5B), 3403 - 10
Cytofluorimetric analysis of a renal tubular cell line and its resistant counterpart; Rosati A et al.; P-glycoprotein (P-gp) and multidrug resistance related protein (MRP) overexpression is often responsible of the development of multidrug resistance in cancer therapy . These proteins are also expressed in normal tissues, where their physiological role is related to the extrusion of endogenous toxins or to secretory function in liver and kidney . The LLC-PK1 cell line is derived from normal pig proximal renal tubule and physiologically expresses low levels of P-gp and MRP . A resistant cell line (LLC-PK1/ADR) has been established in our laboratory by chronic exposure to increasing doses of doxorubicin . Cytofluorimetric analysis of P-gp and MRP expression performed by C219 and MRPm6 immunofluorescence detection showed that these cells overexpress P-gp but not MRP . The uptake of doxorubicin and rhodamine 123 has been quantified in LLC-PK1 and LLC-PK1/ADR cells and compared with data obtained using other tumor cell lines commonly used as reference for studying P-gp or MRP overexpression . P388 sensitive cells and its resistant counterpart P388/ADR cells, which overexpress P-gp and PANC-1 cells, which express high levels of MRP were used . A lower fluorescence intensity was evident with both doxorubicin and rhodamine 123 in LLC-PK1/ADR as well as in P388/ADR cells, that overexpresses P-gp, in comparison with the parental lines . The uptake was increased by a pretreatment with verapamil . Verapamil was completely ineffective on PANC-1 cells, confirming a selective effect of this inhibitor on P-gp . Propidium iodide staining, performed after doxorubicin treatment, confirmed a higher cytotoxicity of the antineoplastic drug in the LLC-PK1 cells compared with the resistant counterpart.

Lancet, 2000 Dec 2, 356(9245), 1888 - 94
Atovaquone-proguanil versus chloroquine-proguanil for malaria prophylaxis in non-immune travellers: a randomised, double-blind study . Malarone International Study Team; Hogh B et al.; BACKGROUND: Chloroquine plus proguanil is widely used for malaria chemoprophylaxis despite low effectiveness in areas where multidrug-resistant malaria occurs . Studies have shown that atovaquone and proguanil hydrochloride is safe and effective for prevention of falciparum malaria in lifelong residents of malaria-endemic countries, but little is known about non-immune travellers . METHODS: In a double-blind equivalence trial, 1083 participants travelling to a malaria-endemic area were randomly assigned to two treatment groups: atovaquone-proguanil plus placebos for chloroquine and proguanil, or chloroquine, proguanil, and placebo for atovaquone-proguanil . Follow-up was by telephone 7 and 60 days after travel and at a clinic at 28 days . Serum samples were tested for antibodies to a malaria circumsporozoite protein . Blood and serum samples of participants with a potential malaria diagnosis were tested in a reference laboratory . FINDINGS: 7 days after travel, at least one adverse event was reported by 311 (61%) of 511 participants who received atovaquone-proguanil and 329 (64%) of 511 who received chloroquine-proguanil . People receiving atovaquone-proguanil had a lower frequency of treatment-related gastrointestinal adverse events (59 {12%} vs 100 {20%}, p=0.001), and of treatment-related adverse events of moderate or severe intensity (37 {7%} vs 56 {11%}, p=0.05) . There were fewer treatment-related adverse events that caused prophylaxis to be discontinued in the atovaquone-proguanil group than in the chloroquine-proguanil group (one {0.2%} vs ten {2%}, p=0.015) . INTERPRETATION: Overall the two preparations were similarly tolerated . However, significantly fewer adverse gastrointestinal events were observed in the atovaquone-proguanil group in than in the chloroquine-proguanil group.

Cancer Detect Prev, 2000, 24(5), 485 - 95
Synergistic potentiating effect of D(+)-mannose, orotic, and hippuric acid sodium salt on selective toxicity of a mixture of 13 substances of the circulatory system in culture for various tumor cell lines; Kulcsar G; Despite global immune system abnormalities in the autoimmune deficiency syndrome, the incidence of only a few tumor types increases, and the degree of immunosuppression does not seem to be critical in the development of these tumors, indicating that the immune system does not prevent tumor development . Consequently, because tumors do not develop in most individuals, other defense systems may exist . We demonstrated previously that 13 substances in the circulatory system acting synergistically induced apoptosis in vitro and in vivo in different tumor cell lines, but not in normal cells and animals . We investigated another 17 compounds and five ions in the circulatory system to determine their participation in the defense provided by the 13 substances . Three of the 17 substances but no ions had a potentiating effect on the mixture of substances used previously . The new 16-component mixture suppressed in vitro growth of six human and murine tumor cell lines, including multidrug-resistant tumor cells, without cytotoxic effects in two normal cell lines . The selectivity also was demonstrated by investigating the mixture's effect over time on tumor and normal cell growth.

Drugs, 2000 Nov, 60(5), 985 - 95
Drug treatment of tropical parasitic infections: recent achievements and developments; Stephenson I et al.; Drug development offers potential solutions to a number of tropical health diseases, although the expense of pharmaceutical research and lack of return on investment has limited the production of new agents . The greatest successes have been through the development of single dose therapy and mass treatment control programmes for a number of diseases . We review some of the current treatment regimens for malaria, intestinal helminth infection, onchocerciasis, filariasis and schistosomiasis, and their use in clinical practice . Geographical spread and emergence of drug resistant parasites have hindered the control of malaria, the most important global parasitic infection . Artemisinin compounds have proved effective antimalarial agents producing rapid reduction of parasite load and can be used in combination treatment regimens to combat multidrug resistance . Intestinal helminth infections are widespread, giving rise to nutritional deficiencies and impaired childhood cognitive development . Pregnant women in developing countries are at increased risk of morbidity . Treatment with a single dose benzimidazole such as albendazole or mebendazole has beneficial effects on morbidity and rates of transmission . Diethylcarbamazine has been used in the treatment of onchocerciasis and human filariasis . A complicated escalating dose regimen over several weeks is associated with systemic and allergic reactions and may require corticosteroid cover . Simplified regimens for mass population treatment with ivermectin have proved useful and been used in combination with single dose albendazole and diethylcarbamazine . The African Programme for Onchocerciasis Control in West and Central Africa has been one of the most successful mass control programmes virtually eliminating new infections by a combination of chemotherapy, education and vector control . Schistosomiasis is of increasing importance as a result of the creation of new snail habitats by agricultural and economic development . Praziquantel has become the most widely available and effective chemotherapy for schistosomiasis . There have been a number of reports of persistent schistosome egg shedding after treatment posing concerns about the emergence of drug resistance . Eflornithine has been successfully used in patients with human trypanosomiasis failing melarsoprol therapy however expense and availability have limited its potential . Mass control treatment programmes have targeted schoolchildren, adolescents and pregnant women . The integration of schistosomiasis, onchocerciasis, filariasis and helminth control programmes has been considered as a cost-effective method of delivering treatment . It is likely that future control will be based on this optimisation and integration of existing regimens, rather than the development of new agents.

Bioorg Med Chem Lett, 2000 Dec 4, 10(23), 2669 - 73
Programmed cell death (PCD) associated with the stilbene motif of arotinoids: discovery of novel apoptosis inducer agents possessing activity on multidrug resistant tumor cells; Simoni D et al.; Considering that the stereochemistry of the C9-C10 alkenyl portion of natural 9-cis-RA, as the one of the olefinic moiety of the previously described isoxazole retinoid 4, seems of particular importance for their apoptotic activity, we prepared a novel class of TTNPB analogues bearing both the cis or trans configuration of the alkenyl portion . The compounds were evaluated in vitro for their cytotoxic and apoptotic activities . We discovered that the cis-TTNPB 9c possesses apoptotic activity comparable with that of the retinoid 4 . Moreover, the amino arotinoid 16c showed potent apoptotic activity in HL60 promyelocytic leukemia cells . Interestingly, 16c proved to be a particularly potent apoptosis-inducing agent active in multidrug resistant (MDR) cell lines . Therefore, to the best of our knowledge, 16c may represent the first known aminoarotinoid endowed with potent apoptotic activity in MDR cells . Taken together, these results seem to point out that the cis-stilbene motif of arotinoids may be at least an important feature in conferring cytotoxic and apoptotic activity to this class of compounds.

Bioorg Med Chem Lett, 2000 Dec 4, 10(23), 2629 - 32
New multidrug resistance modulators from Atractylodis Lanceae Rhizoma; Murakami N et al.; Three new oligoacylated sucroses designated atractysucroses-I, -II, and -III were obtained from Atractylodis Lanceae Rhizoma as multidrug resistance (MDR) modulating substances against human carcinoma cell lines and their chemical structures were characterized as a mixture of acyl groups . Atractysucrose-I modulates MDR in KB-C2 cells as strongly as verapamil . This is the first example of an MDR-modulator classified as a carbohydrate.

Bioorg Med Chem Lett, 2000 Dec 4, 10(23), 2603 - 5
2,4,5-Trisubstituted imidazoles: novel nontoxic modulators of P-glycoprotein mediated multidrug resistance . Part 2; Zhang C et al.; Solution-phase combinatorial chemistry was applied to the optimization and development of clinical candidate OC144-093 (22), a novel and nontoxic modulator of P-glycoprotein mediated multidrug resistance.

Bioorg Med Chem Lett, 2000 Dec 4, 10(23), 2599 - 601
2,4,5-Trisubstituted imidazoles: novel nontoxic modulators of P-glycoprotein mediated multidrug resistance . Part 1; Sarshar S et al.; N-4,5-Di-(4-dialkylamino)phenyl imidazoles (A) are potent modulators of P-glycoprotein mediated multidrug resistance . This manuscript describes the discovery and lead optimization of this novel class of inhibitors.

J Biol Chem, 2001 Mar 23, 276(12), 8648 - 56 Epub 2000 Dec 13.
Enhanced multispecificity of arabidopsis vacuolar multidrug resistance-associated protein-type ATP-binding cassette transporter, AtMRP2; Liu G et al.; Recent investigations have established that Arabidopsis thaliana contains a family of genes encoding ATP-binding cassette transporters belonging to the multidrug resistance-associated protein (MRP) family . So named because of the phenotypes conferred by their animal prototypes, many MRPs are MgATP-energized pumps active in the transport of glutathione (GS) conjugates and other bulky amphipathic anions across membranes . Here we show that Arabidopsis MRP2 (AtMRP2) localizes to the vacuolar membrane fraction from seedlings and is not only competent in the transport of GS conjugates but also glucuronate conjugates after heterologous expression in yeast . Based on the stimulatory action of the model GS conjugate 2,4-dinitrophenyl-GS (DNP-GS) on uptake of the model glucuronide 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) and vice versa, double-label experiments demonstrating that the two substrates are subject to simultaneous transport by AtMRP2 and preloading experiments suggesting that the effects seen result from cis, not trans, interactions, it is inferred that some GS conjugates and some glucuronides reciprocally activate each other's transport via distinct but coupled binding sites . The results of parallel experiments on AtMRP1 and representative yeast and mammalian MRPs indicate that these properties are specific to AtMRP2 . The effects exerted by DNP-GS on AtMRP2 are not, however, common to all GS conjugates and not simulated by oxidized glutathione or reduced glutathione . Decyl-GS, metolachlor-GS, and oxidized glutathione, although competitive with DNP-GS, do not promote E(2)17betaG uptake by AtMRP2 . Reduced glutathione, although subject to transport by AtMRP2 and able to markedly promote E(2)17betaG uptake, neither competes with DNP-GS for uptake nor is subject to E(2)17betaG-promoted uptake . A multisite model comprising three or four semi-autonomous transport pathways plus distinct but tightly coupled binding sites is invoked for AtMRP2.

Hepatology, 2001 Jan, 33(1), 140 - 7
Differential regulation of hepatic bile salt and organic anion transporters in pregnant and postpartum rats and the role of prolactin; Cao J et al.; We characterized expression and activity of the bile salt transporters Na(+)/taurocholate (TC) cotransporting polypeptide (Ntcp), and bile salt export pump (Bsep), and the expression of organic anion transporting polypeptides 1 and 2 (Oatp1 and 2) and multidrug resistance associated protein-2 (Mrp2) in pregnancy and throughout lactation in rats . The V(max) for Na(+)/TC cotransport in basolateral liver plasma membrane was increased 1.7-fold in 2 days postpartum relative to control and pregnant rats . This correlated well with an increase in Ntcp messenger RNA (mRNA) and a 2-fold increase in Ntcp protein . Ntcp mRNA remained significantly elevated until 14 days postpartum but had begun to decline by 21 days postpartum . The maximal secretory rate (nmol/min/g liver) for TC in the single pass isolated perfused liver was also increased by 10%, 31%, and 24% at 2, 14, and 21 days postpartum and correlated with increased expression of Ntcp and Bsep mRNA and protein . Infusion of ovine prolactin (oPRL) to ovariectomized rats increased expression of both Ntcp and Bsep mRNA and protein . These data indicate a coordinate increased expression of bile salt transporters postpartum and by PRL . Mrp2 mRNA was stable in pregnancy and postpartum, whereas Mrp2 protein expression decreased significantly in pregnancy, but returned to control levels postpartum . Organic anion transporting polypeptide 2 (Oatp2) mRNA was decreased in pregnancy and increased postpartum, but changes in Oatp2 protein were not significant . Oatp1 mRNA and protein were unchanged in pregnancy and postpartum.

Mol Ther, 2000 Dec, 2(6), 609 - 18
MDR1 gene expression in NOD/SCID repopulating cells after retroviral gene transfer under clinically relevant conditions; Schilz AJ et al.; We have adapted a recently published protocol for retroviral gene transfer into hematopoietic cells {A . J . Schilz et al . (1998) Blood 92: 3163-3171} with respect to clinical requirements such as large-volume vector stock generation, adequate cell source, high cell numbers, and serum-free conditions . We present data on transduction efficacy and expression of the multidrug resistance 1 (MDR1) gene in human CD34(+) cells from mobilized peripheral blood (PB) mediated by a gibbon ape leukemia virus (GALV)-pseudotyped retroviral vector . Using a 1-day cytokine-mediated prestimulation, consisting of human interleukin (IL)-3, IL-6, stem cell factor (SCF), Flt-3 ligand (FL), and thrombopoietin (TPO), followed by a 3-day transduction procedure, we were able to detect up to 51% CD34(+) cells expressing MDR1 . Xenotransplantation of transduced cells into NOD/LtSz-scid/scid (NOD/SCID) mice resulted in a mean engraftment level of 23% (0.1 to 87%) . As shown by quantitative PCR analysis, a mean of 12.7% (range 0.3 to 55%) of the engrafted human cells in the bone marrow of chimeric mice contained the MDR1 cDNA . Furthermore, enhanced expression of MDR1 above control levels was detected in up to 15% of the engrafted human cell population . Our data suggest that NOD/SCID repopulating cells derived from mobilized PB can be transduced efficiently with existing retroviral vector systems under clinically applicable conditions.

Jpn J Cancer Res, 2000 Dec, 91(12), 1326 - 32
Cytotoxicity of cis-{((1R,2R)-1,2-cyclohexanediamine-N,N')bis(myristato)}-platinum (II) suspended in Lipiodol in a newly established cisplatin-resistant rat hepatoma cell line; Kishimoto S et al.; The cytotoxic activity of cis-{((1R,2R)-1,2-cyclohexanediamine-N, N')bis(myristato)}platinum (II) (SM-11355) was evaluated in a cisplatin (CDDP)-resistant tumor cell line, and compared with that of CDDP . H4-II-E / CDDP with acquired resistance to CDDP was established by continuous exposure of a rat hepatic tumor line, H4-II-E, to increasing concentrations of CDDP over 12 months . Compared with the parental cell line, this cell line exhibited an 8.8-fold increase in resistance to CDDP and was not cross-resistant to 1,2-diaminocyclohexane platinum (II) dichloride (DPC) . There were no differences in sensitivity to six non-platinum antitumor drugs between H4-II-E and H4-II-E / CDDP, which suggests that H4-II-E / CDDP is not multidrug-resistant . Intracellular platinum accumulation and the formation of a platinum-DNA adduct following CDDP exposure were significantly reduced in H4-II-E / CDDP compared to the parental cell line . The acquired CDDP resistance in H4-II-E / CDDP appeared to be predominantly due to reduced CDDP uptake . H4-II-E / CDDP was also resistant to CDDP suspended in Lipiodol (CDDP / Lipiodol), but was not cross-resistant to SM-11355 suspended in Lipiodol (SM-11355 / Lipiodol) . Also, there were no differences in intracellular platinum accumulation or the formation of platinum-DNA adducts after SM-11355 / Lipiodol exposure between H4-II-E and H4-II-E / CDDP . These results suggest that acquired CDDP resistance in H4-II-E / CDDP does not influence the cytotoxic activity of SM-11355 / Lipiodol.

Br J Haematol, 2000 Nov, 111(2), 565 - 9
Contrasting in vitro effects for the combination of fludarabine, cytosine arabinoside (Ara-C) and granulocyte colony-stimulating factor (FLAG) compared with daunorubicin and Ara-C in P-glycoprotein-positive and P-glycoprotein-negative acute myeloblastic leukaemia; Higashi Y et al.; It has been suggested that the FLAG remission induction regimen comprising fludarabine (F-ara), cytosine arabinoside (Ara-C) and granulocyte colony-stimulating factor (G-CSF) may be capable of overcoming P-glycoprotein (P-gp)-related multidrug resistance (MDR) in patients with acute myeloblastic leukaemia (AML) . We have investigated the in vitro response of P-gp-positive and -negative AML clones to FLAG and compared this with their response to treatment with Ara-C and daunorubicin (DNR) . Twenty-four cryopreserved samples from patients with AML were studied using a flow cytometric technique for the enumeration of viable (7-amino actinomycin D negative) cells . Samples consisted of 12 P-gp-positive and 12 P-gp-negative cases, as measured by the MRK16 antibody . The results were analysed by calculating the comparative drug resistance (CDR), i.e . the percentage cell death caused by Ara-C + DNR subtracted from the percentage cell death, caused by FLAG after 48 h incubation in suspension culture . P-gp-positive clones were shown to have a significantly higher CDR than P-gp-negative clones (P = 0 . 001) . Furthermore, a significant positive correlation (r2 = 0.40, P < 0.01) was found between P-gp protein expression and CDR . However, P-gp function, measured using cyclosporin modulation of rhodamine 123 (R123) uptake, was not associated with the CDR, demonstrating that there are other properties of P-gp, besides its role in drug efflux, that modulate the responsiveness of AML blasts to chemotherapy . These results are consistent with a potential benefit for FLAG in P-gp-positive AML, but not P-gp-negative AML, compared with standard anthracycline and Ara-C therapy.

Eur J Pharm Sci, 2000 Nov, 12(1), 69 - 77
P-Glycoprotein in cell cultures: a combined approach to study expression, localisation, and functionality in the confocal microscope; Hammerle SP et al.; Madin Darby canine kidney (MDCK) cells transfected with the multidrug resistance mdr1 gene, MDR1-MDCK (Pastan et al., 1988, Proc . Natl . Acad . Sci . USA 85 4486-4470), were used in a combined approach to study expression, localisation and functionality of the P-glycoprotein (P-gp) membrane transporter in the same cell culture preparations . Cells were characterised with regard to their growth curve, transepithelial electrical resistance (TEER), and cytoarchitecture . Efflux of the P-gp substrate rhodamine123 (rho123) was monitored with confocal laser scanning microscopy (CLSM) . The transfected cells grew in multilayers . After reaching confluence they exhibited a complete tight junction (TJ) network . P-gp was strongly expressed at the uppermost apical surface of the multilayer already after 4 days in culture . The lower cell layers were not clearly polarised . P-gp-mediated transport could be followed by efflux of the fluorescent rho123 from the cells into the apical extracellular space . Verapamil, a P-gp inhibitor, significantly decreased efflux . For MDCK parent cells the rho123 assay was negative up to about day 20, and only at later times (day 25) low P-gp activity was detected . These results clearly show that despite the fact that the transfected cells form irregular layers, they provide a good model for screening of P-gp substrates and inhibitors.

Proc Natl Acad Sci U S A, 2000 Dec 19, 97(26), 14295 - 300
Explaining the high mutation rates of cancer cells to drug and multidrug resistance by chromosome reassortments that are catalyzed by aneuploidy; Duesberg P et al.; The mutation rates of cancer cells to drug and multidrug resistance are paradoxically high, i.e., 10(-3) to 10(-6), compared with those altering phenotypes of recessive genes in normal diploid cells of about 10(-12) . Here the hypothesis was investigated that these mutations are due to chromosome reassortments that are catalyzed by aneuploidy . Aneuploidy, an abnormal number of chromosomes, is the most common genetic abnormality of cancer cells and is known to change phenotypes (e.g., Down's syndrome) . Moreover, we have shown recently that aneuploidy autocatalyzes reassortments of up to 2% per chromosome per mitosis because it unbalances spindle proteins, even centrosome numbers, via gene dosage . The hypothesis predicts that a selected phenotype is associated with multiple unselected ones, because chromosome reassortments unbalance simultaneously thousands of regulatory and structural genes . It also predicts variants of a selected phenotype based on variant reassortments . To test our hypothesis we have investigated in parallel the mutation rates of highly aneuploid and of normal diploid Chinese hamster cells to resistance against puromycin, cytosine arabinoside, colcemid, and methotrexate . The mutation rates of aneuploid cells ranged from 10(-4) to 10(-6), but no drug-resistant mutants were obtained from diploid cells in our conditions . Further selection increased drug resistance at similar mutation rates . Mutants selected from cloned cells for resistance against one drug displayed different unselected phenotypes, e.g., polygonal or fusiform cellular morphology, flat or three-dimensional colonies, and resistances against other unrelated drugs . Thus our hypothesis offers a unifying explanation for the high mutation rates of aneuploid cancer cells and for the association of selected with unselected phenotypes, e.g., multidrug resistance . It also predicts drug-specific chromosome combinations that could become a basis for selecting alternative chemotherapy against drug-resistant cancer.

Antimicrob Agents Chemother, 2001 Jan, 45(1), 73 - 8
Role of P glycoprotein in the course and treatment of Encephalitozoon microsporidiosis; Leitch GJ et al.; Encephalitozoon microsporidia are obligate intracellular protozoan parasites that proliferate and differentiate within a parasitophorous vacuole inside host cells that are usually epithelial in nature . Isolates of the three species of the Encephalitozoon microsporidia, E . cuniculi, E . hellem, and E . intestinalis, were obtained from AIDS patients and cultured in green monkey (E6) kidney cells . Anti-P-glycoprotein (anti-Pgp) and anti-multidrug resistance-associated protein (anti-MRP) monoclonal antibodies were used to probe for multidrug resistance (MDR) pump epitopes and verapamil- or cyclosporin A- and probenecid-modulated intracellular calcein fluorescence were used to assess the expression of Pgp and MRP respectively in uninfected and infected cells . Pgp, but not MRP, was detected immunocytochemically and by verapamil- and cyclosporin A-potentiated intracellular fluorescence in both host cells and parasite developing stages . When an in vitro infection assay was employed, verapamil and cyclosporin A acted as chemosensitizing agents for the antiparasitic drug albendazole . These observations suggest that inhibiting host cell and perhaps parasite MDR pumps may increase the efficacy of antiparasitic agents in these and other microsporidia species.

Biochem Biophys Res Commun, 2000 Dec 20, 279(2), 369 - 77
Multidrug resistance P-glycoprotein is not involved in cholesterol esterification; Issandou M et al.; The aim of the present paper is to reinvestigate the role of multidrug resistance P-glycoprotein MDR1 and MDR-associated protein (MRP1) in cholesterol esterification using well-characterized inhibitors . Using specific substrate efflux assay, we show that GF120918 (0.2 microM) and probenecid (5 mM) were specific inhibitors of MDR1 and MRP1, respectively . In HepG2 cells, neither of them affect the esterification of cholesterol derived from the uptake of cholesterol-rich lipoprotein, while both verapamil (100 microM) and progesterone (100 microM) were able to inhibit cholesterol esterification . Similar results were obtained with verapamil, progesterone, and GF120918 in the MDR1-overexpressing cells MCF7/ADR . The capacity of progesterone to reduce cholesterol esterification is not correlated with its ability to inhibit MDR1 but is rather due to direct inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) . We conclude that the esterification of cholesterol is not correlated with MDR1 or MRP1 activity, thus excluding their role in the intracellular transport of endocytosis-derived cholesterol .

Epidemiol Infect, 2000 Oct, 125(2), 463 - 4
Tuberculosis drug resistance in England and Wales . How much is 'home-grown'?
Hayward AC, Herbert J, Watson JM.
The fact that a substantial proportion of tuberculosis drug resistance (especially multidrug resistance) is due to transmission of resistant strains or treatment failure in the UK underlines the need to strengthen control in this country . Early identification and effective treatment of cases (including measures to support adherence and appropriate use of initial treatment regimes involving at least four drugs in those at risk of isoniazid resistance) would help to control the problem {1}.

J Bacteriol, 2001 Jan, 183(1), 235 - 49
Characterization of In53, a class 1 plasmid- and composite transposon-located integron of Escherichia coli which carries an unusual array of gene cassettes; Naas T et al.; Further characterization of the genetic environment of the gene encoding the Escherichia coli extended-spectrum beta-lactamase, bla(VEB-1), revealed the presence of a plasmid-located class 1 integron, In53, which carried eight functional resistance gene cassettes in addition to bla(VEB-1) . While the aadB and the arr-2 gene cassettes were identical to those previously described, the remaining cassettes were novel: (i) a novel nonenzymatic chloramphenicol resistance gene of the cmlA family, (ii) a qac allele encoding a member of the small multidrug resistance family of proteins, (iii) a cassette, aacA1b/orfG, which encodes a novel 6'-N-acetyltransferase, and (iv) a fused gene cassette, oxa10/aadA1, which is made of two cassettes previously described as single cassettes . In addition, oxa10 and aadA1 genes were expressed from their own promoter sequence present upstream of the oxa10 cassette . arr-2 coded for a protein that shared 54% amino acid identity with the rifampin ADP-ribosylating transferase encoded by the arr-1 gene from Mycobacterium smegmatis DSM43756 . While in M . smegmatis, the main inactivated compound was 23-ribosyl-rifampin, the inactivated antibiotic recovered from E . coli culture was 23-O-ADP-ribosyl-rifampin . The integrase gene of In53 was interrupted by an IS26 insertion sequence, which was also present in the 3' conserved segment . Thus, In53 is a truncated integron located on a composite transposon, named Tn2000, bounded by two IS26 elements in opposite orientations . Target site duplication at both ends of the transposon indicated that the integron likely was inserted into the plasmid through a transpositional process . This is the first description of an integron located on a composite transposon.

Cell, 2000 Nov 22, 103(5), 757 - 68
The leukotriene C(4) transporter MRP1 regulates CCL19 (MIP-3beta, ELC)-dependent mobilization of dendritic cells to lymph nodes; Robbiani DF et al.; Adaptive immune responses begin after antigen-bearing dendritic cells (DCs) traffic from peripheral tissues to lymph nodes . Here, we show that DC migration from skin to lymph nodes utilizes the leukotriene C(4) (LTC(4)) transporter multidrug resistance-associated protein 1 (MRP1) . DC mobilization from the epidermis and trafficking into lymphatic vessels was greatly reduced in MRP1(-/-) mice, but migration was restored by exogenous cysteinyl leukotrienes LTC(4) or LTD(4) . In vitro, these cysteinyl leukotrienes promoted optimal chemotaxis to the chemokine CCL19, but not to other related chemokines . Antagonism of CCL19 in vivo prevented DC migration out of the epidermis . Thus, MRP-1 regulates DC migration to lymph nodes, apparently by transporting LTC(4), which in turn promotes chemotaxis to CCL19 and mobilization of DCs from the epidermis.

J Biol Chem, 2001 Mar 16, 276(11), 7952 - 6 Epub 2000 Dec 13.
Role of multidrug resistance protein 1 (MRP1) and glutathione S-transferase A1-1 in alkylating agent resistance . Kinetics of glutathione conjugate formation and efflux govern differential cellular sensitivity to chlorambucil versus melphalan toxicity; Paumi CM et al.; We investigated the role of phase II (conjugation) and phase III (efflux) detoxification of the anticancer drugs melphalan (MLP) and chlorambucil (CHB) . Although both drugs are substrates of Alpha-class glutathione S-transferases (GST) and the monoglutathionyl conjugates formed in these enzymatic reactions are transported by MRP1, we found that GSTA1-1 and MRP1 acted in synergy to confer resistance to CHB but not to MLP (Morrow, C . S., Smitherman, P . K., Diah, S . K., Schneider, E., and Townsend, A . J . (1998) J . Biol . Chem . 273, 20114-20120) . To explain this selectivity of MRP1/GST-mediated resistance, we report results of side-by-side experiments comparing the kinetics of MLP- versus CHB-glutathione conjugate: formation, product inhibition of GSTA1-1 catalysis, and transport by MRP1 . The monoglutathionyl conjugate of CHB, CHB-SG, is a very strong competitive inhibitor of GSTA1-1 (K(i) 0.14 microM) that is >30-fold more potent than that of the corresponding conjugate of MLP, MLP-SG (K(i) 4.7 microM) . The efficiency of GSTA1-1-mediated monoglutathionyl conjugate formation is more than 4-fold higher for CHB than MLP . Lastly, both CHB-SG and MLP-SG are efficiently transported by MRP1 with similar V(max) although the K(m) for CHB-SG (0.37 microm) is significantly lower than for MLP-SG (1.1 microM) . These results indicate that MRP1 is required for GSTA1-1-mediated resistance to CHB in order to relieve potent product inhibition of the enzyme by intracellular CHB-SG formed . The kinetic properties of MRP1 are well suited to eliminate CHB-SG at pharmacologically relevant concentrations . For MLP detoxification, where product inhibition of GSTA1-1 is less important, GSTA1-1 does not confer resistance because of the relatively poorer catalytic efficiency of MLP-SG formation . Similar analyses can be useful for predicting the pharmacological and toxicological consequences of MRP and GST expression on cellular sensitivity to various other electrophilic xenobiotics.

Haematologica, 2000 Dec, 85(12), 1261 - 7
The relevance of multidrug resistance-associated P-glycoprotein expression in the treatment response of B-cell chronic lymphocytic leukemia; Svoboda-Beusan I et al.; BACKGROUND AND OBJECTIVES: The aim of this study was to determine whether expression of P-glycoprotein (Pgp) is an intrinsic feature of B-lymphocytes in B-cell chronic lymphocytic leukemia (B-CLL) and how it correlates with hematologic indices and tumor load in the disease . Furthermore, the change of Pgp expression under cytotoxic treatment and its correlation to treatment outcome were studied . DESIGN AND METHODS: In 42 B-CLL patients, of whom 13 were sequentially monitored, expression of extracellular (MRK-16) and intracellular (C-219) Pgp epitopes on peripheral blood lymphocytes was determined by flow cytometry analysis and quantified by ratio of the mean fluorescence (RMF) in flow cytometry analysis . RESULTS: Median RMF values in B-CLL patients were higher than in age-matched controls . Pgp expression did not correlate with any of the hematologic features or clinical stage of the disease . Patients who received some type of cytoreductive treatment prior to the study had lower Pgp values for both measured epitopes (median RMF for C-219 and MRK-16 of 1.99 and 2.03 in comparison to those of non-treated patients: 3.11 and 2.88, respectively) . In 13 patients monitored during treatment the decrease in RMF was noted after treatment with chlorambucil, with RMF values for both Pgp epitopes decreasing in responders . This was in contrast to unchanged or even increased RMF values in those patients who did not respond to therapy . INTERPRETATION AND CONCLUSIONS: Our study confirms the importance of quantitative evaluation of Pgp expression by flow cytometry . At the clinical level, cross-sectional, single test evaluation of Pgp is of limited value whereas sequential follow-up values correlate with treatment response.

Clin Infect Dis, 2001 Jan, 32(1), 159 - 61 Epub 2000 Dec 11.
High rate of tuberculosis reinfection during a nosocomial outbreak of multidrug-resistant tuberculosis caused by Mycobacterium bovis strain B; Rivero A et al.; We present a study of a nosocomial outbreak of multidrug-resistant tuberculosis caused by Mycobacterium bovis in 31 patients, 30 of whom were infected with human immunodeficiency virus; all 31 died of progressive tuberculosis . All M . bovis strains had identical spoligotyping patterns and showed resistance to 12 antituberculosis drugs . Reinfection was suggested in 11 cases and confirmed in 4 by molecular typing methods . The causative strain was named "B strain."

Biochem Biophys Res Commun, 2000 Dec 9, 279(1), 124 - 30
Carrier-mediated uptake of rhodamine 123: implications on its use for MDR research; Cho CW et al.; We have examined the effects of verapamil and PSC 833 on cellular uptake and release of rhodamine 123 (R123) in two human cancer cell lines . Both verapamil and PSC 833 were able to increase R123 accumulation in the multidrug resistant (MDR) MV522/Q6 and KB-8-5 lines in the release study . However, the effects of these drugs on R123 accumulation during accumulation study were quite different . Incubation with PSC 833 increased R123 accumulation in both MDR lines . By contrast, incubation with verapamil only increased R123 accumulation in the KB-8-5 line . The failure of verapamil to increase R123 accumulation in the MV522/Q6 cells can be attributed to the presence of a carrier system in the parent MV522 cells that recognizes both R123 and verapamil, but not PSC 833, as substrates . These results imply that performing R123 accumulation study without first ascertaining possible role of a carrier system for cellular uptake of R123 and putative P-gp modulators might inadvertently lead one to draw improper conclusions on P-gp activity .

Cas Lek Cesk, 2000 Sep 13, 139(18), 553 - 6
{Resistance of cells in hematopoietic malignancies to cytostatic agents . Part 1}; Kodydkova K et al.; Resistance to cytotoxic drugs is a serious drawback in the treatment of patients with tumours, both of the haemopoietic and non-haemopoietic origin . The cytotoxic effect of drugs on the malignant cells manifests as the process of apoptosis . In the resistant malignant cells, apoptosis becomes prevented by several mechanisms . The multidrug resistance (MDR) is one of the principal mechanisms when the cancer cells develop resistance to multiple chemically and functionally unrelated cytotoxic compounds . The decrease of the cytotoxic drug concentration at the molecular target site may come from activation of some efflux membrane systems participating in the transport of cytotoxic drugs out of the cell (e.g . Pgp, MDR, and LRP) . Another mechanism of resistance is the increased enzymatic detoxification of the drug by glutathion-S-transferase system . Changes in the molecular target of the cytotoxic drug such as topoisomerase molecule can be also responsible for the resistance . At least two additional mechanisms of resistance of tumour cells were identified . Resistance can come from either deregulation of proapoptic mechanisms in tumour cells or by increased activity of reparation processes which control the damaged molecule of DNA . Several methods that detect the cause of resistance in distinct cell populations have been developed . The great effort is now focused on both the detection of mechanisms of the resistance and on the clinical procedures of overcoming the resistance to cytotoxic drugs.

Biochem Pharmacol, 2000 Dec 15, 60(12), 1967 - 75
Characterization and inhibition by a wide range of xenobiotics of organic anion excretion by primary human hepatocytes; Payen L et al.; Organic anion secretion by human hepatocytes was characterized using primary liver parenchymal cell cultures and the anionic fluorescent dye carboxy-2',7'-dichlorofluorescein (CF) . Probenecid, a well-known common blocker of the membrane transport process for anions, was shown to increase CF accumulation in primary human hepatocytes by inhibiting cellular CF efflux in a dose-dependent manner, thereby establishing the presence of an efflux system for organic anions in cultured hepatocytes . Outwardly directed transport of CF from hepatocytes was found to be temperature-dependent; it was not altered by changes in the ionic composition of the incubation medium used in efflux experiments . In addition to probenecid, various structurally and functionally unrelated xenobiotics such as glibenclamide, rifampicin, vinblastine, MK-571, indomethacin, and cyclosporin A were shown to inhibit secretion of CF by primary human hepatocytes, thus suggesting that organic anion excretion by human liver may be impaired by various drugs . Northern blot and Western blot analyses of the expression of multidrug resistance proteins (MRP), such as MRP1 and MRP2, which are known to mediate cellular outwardly directed transport of organic anions indicated that MRP2 was present at substantial levels in cultured human hepatocytes as well as in their in vivo counterparts, whereas MRP1 expression was only barely detectable . These results therefore suggest that MRP2, unlike MRP1, may contribute to the organic anion efflux system displayed by primary human hepatocytes and inhibited by a wide range of xenobiotics.

Biochem Pharmacol, 2000 Dec 15, 60(12), 1915 - 23
Lack of glutathione conjugation to adriamycin in human breast cancer MCF-7/DOX cells . Inhibition of glutathione S-transferase p1-1 by glutathione conjugates from anthracyclines; Gaudiano G et al.; One of the proposed mechanisms for multidrug resistance relies on the ability of resistant tumor cells to efficiently promote glutathione S-transferase (GST)-catalyzed GSH conjugation of the antitumor drug . This type of conjugation, observed in several families of drugs, has never been documented satisfactorily for anthracyclines . Adriamycin-resistant human breast cancer MCF-7/DOX cells, presenting a comparable GSH concentration, but a 14-fold increase of the GST P1-1 activity relative to the sensitive MCF-7 cells, have been treated with adriamycin in the presence of verapamil, an inhibitor of the 170 P-glycoprotein (P-gp) drug transport protein, and scrutinized for any production of GSH-adriamycin conjugates . HPLC analysis of cell content and culture broths have shown unequivocally that no GSH conjugates are present either inside the cell or in the culture broth . The only anthracycline present inside the cells after 24 hr of incubation was > 98% pure adriamycin . Confocal laser scanning microscopic observation showed that in MCF-7/DOX cells adriamycin was localized mostly in the Golgi apparatus rather than in the nucleus, the preferred site of accumulation for sensitive MCF-7 cells . These findings rule out GSH conjugation or any other significant biochemical transformation as the basis for resistance to adriamycin and as a ground for the anomalous localization of the drug in the cell . Adriamycin, daunomycin, and menogaril did not undergo meaningful conjugation to GSH in the presence of GST P1-1 at pH 7.2 . Indeed, their synthetic C(7)-aglycon-GSH conjugates exerted a strong inhibitory effect on GST P1-1, with K(i) at 25 degrees in the 1-2 microM range, scarcely dependent on their stereochemistry at C(7).

Mol Carcinog, 2000 Nov, 29(3), 170 - 8
Role of multidrug-resistance protein 2 in glutathione S-transferase P1-1-mediated resistance to 4-nitroquinoline 1-oxide toxicities in HepG2 cells; Morrow CS et al.; Previous studies in our laboratory have shown that the phase III efflux transporter multidrug-resistance protein (MRP)1 can act synergistically with the phase II conjugating glutathione S-transferases (GST) to confer resistance to the toxicities of some electrophilic drugs and carcinogens . To determine whether the distinct efflux transporter MRP2 could also potentiate GST-mediated protection from electrophilic toxins, we examined the effect of regulatable GSTP1-1 expression in MRP2-rich HepG2 cells on 4-nitroquinoline 1-oxide (4NQO)-induced cytotoxicity and genotoxicity (nucleic-acid adduct formation) . Expression of GSTP1-1 was associated with a fourfold to tenfold protection from 4NQO-induced cytotoxicity . Inhibition of MRP2-mediated efflux activity by sulfinpyrazone or cyclosporin A completely reversed GSTP1-1-associated resistance-a result indicating that GSTP1-1-mediated cytoprotection is absolutely dependent on MRP2 efflux activity . Moreover, MRP2 efflux activity also augmented GSTP1-1-mediated protection from 4NQO-induced nucleic-acid adduct formation . We conclude that MRP2-mediated efflux of the glutathione conjugate of 4NQO and/or another toxic derivative of 4NQO is required to support GSTP1-1-associated protection from 4NQO toxicities in HepG2 cells.

Med Pediatr Oncol, 2000 Dec, 35(6), 619 - 22
Multidrug resistance-associated protein 1 (MRP1) expression in neuroblastoma cell lines and primary tumors; Goto H et al.; BACKGROUND AND PROCEDURE: MRP1 expression by neuroblastomas was evaluated by Northern blot analysis in 21 cell lines and 90 primary untreated tumors . Cytotoxicity assay in cell lines was performed for five anticancer drugs used in treating neuroblastoma . RESULTS: MRP1 expression did not correlate with drug resistance or with MYCN RNA expression in cell lines . MRP1 expression was higher in drug-sensitive cell lines established after chemotherapy relative to cell lines at diagnosis, but highly drug-resistant cell lines showed low MRP1 expression . Positive expression of MRP1 RNA in primary tumors was associated with a poorer survival relative to MRP1-negative tumors . However, MRP1 expression levels did not correlate with age, stage, MYCN amplification, or MYCN expression, and higher MRP1 expression was not associated with a worse outcome . CONCLUSIONS: In neuroblastoma, positive MRP1 RNA expression at diagnosis has prognostic significance, but high drug resistance is conferred by mechanisms other than MRP1 .

Med Pediatr Oncol, 2000 Dec, 35(6), 585 - 9
Expression of N-myc and MRP genes and their relationship to N-myc gene dosage and tumor formation in a murine neuroblastoma model; Norris MD et al.; BACKGROUND: Although the association between N-myc gene amplification and poor clinical outcome in neuroblastoma is well established, the mechanism by which amplification influences prognosis is not well defined . PROCEDURE: We used a human N-myc transgenic mouse model to investigate the role of N-myc in neuroblastoma, including its relationship to the multidrug-resistance-associated protein (MRP) gene . We developed a rapid real-time PCR method to distinguish homozygous and hemizygous N-myc mice that is comparable to Southern analysis . RESULTS: A highly significant correlation (P < 0.0001) between N-myc and MRP expression was demonstrated in murine tumors . Amplification of the transgene was observed in the majority of tumors, highlighting the clinical relevance of this model . However, no correlation between N-myc expression and transgene dosage or tumor latency was observed . CONCLUSIONS: The data suggest that increased N-myc dosage contributes to increased tumor incidence and decreased latency by mechanisms independent of N-myc expression .

Med Pediatr Oncol, 2000 Dec, 35(6), 563 - 8
p53 mutations and loss of p53 function confer multidrug resistance in neuroblastoma; Keshelava N et al.; BACKGROUND: Neuroblastomas often acquire sustained drug resistance during therapy . Sensitivities to carboplatin, etoposide, or melphalan were determined for 18 neuroblastoma cell lines; eight were sensitive and ten were resistant . As p53 mutations are rare in neuroblastomas studied at diagnosis, we determined if acquired p53 mutations and loss of function conferred multidrug resistance . RESULTS: Loss of p53 function (p53-LOF), defined as a failure to induce p21 and/or MDM2 in response to melphalan, was seen in 1/8 drug-sensitive and 6/10 drug-resistant cell lines . In four cell lines p53-LOF was associated with mutations in the DNA binding region of p53, while three cell lines with LOF and four cell lines with functional p53 had no evidence of p53 muta-tions . Nonfunctional and mutated p53 was detected in one resistant cell line, while a sensitive cell line derived from the same patient prior to treatment had functional and wild type (wt) p53 . We transfected HPV 16 E6 (which mediates degradation of p53, causing LOF) into two drug-sensitive neuroblastoma cell lines with functional p53 . LC(90) values of HPV 16 E6 transfected cell lines were 3-7-fold (melphalan), 8-109-fold (carboplatin), and 2-158-fold (etoposide) greater than that of LXSN-transfected controls . CONCLUSIONS: These data suggest that some neuroblastomas acquire p53 mutations during therapy, which is associated with a loss of p53 function, and can confer high-level multidrug resistance .

J Natl Cancer Inst, 2000 Dec 6, 92(23), 1934 - 40
Analysis of the MRP4 drug resistance profile in transfected NIH3T3 cells; Lee K et al.; BACKGROUND: Multidrug resistance-associated protein (MRP) 1 and canalicular multispecific organic anion transporter (cMOAT or MRP2) are adenosine triphosphate-binding cassette transporters that confer resistance to anticancer agents . In addition to these two transporters, there are at least four other human MRP subfamily members (MRP3 through MRP6) . We and others reported previously that MRP3 is capable of conferring resistance to certain anticancer agents . In this study, we investigated whether MRP4 (MOAT-B), whose transcript accumulates to the highest levels in prostate tissue, has the capacity to confer drug resistance . METHODS: MRP4-transfected NIH3T3 cells were generated, and their drug sensitivity was analyzed . The subcellular localization of MRP4 was assessed by immunohistochemical analysis in transfected cells and in prostate tissue . Statistical tests were two-sided . RESULTS: MRP4 was detected as a 170-kd protein that was localized in the plasma membrane and cytoplasm of transfected cells . The MRP4 transfectants displayed 5.5-fold increased resistance to methotrexate in short-term drug-exposure assays (P=.022) and exhibited decreased cellular accumulation of this agent at 4 hours (P=.006) and 24 hours (P<.001) . In continuous-exposure assays, however, the MRP4 transfectants did not display increased resistance for either methotrexate or natural product cytotoxic agents (anthracyclines, etoposide, vinca alkaloids, and paclitaxel {Taxol}) . However, the transfectants did show increased resistance (2.3-fold) for the anti-acquired immunodeficiency syndrome nucleoside analogue 9-(2-phosphonylmethoxyethyl)adenine (PMEA) (P=.022) in continuous-exposure assays . Consistent with MRP4's plasma membrane localization in transfected cells, analysis of prostate tissue showed that MRP4 protein was localized primarily in the basolateral plasma membranes of tubuloacinar cells . CONCLUSIONS: These results indicate that MRP4 confers resistance to short-term methotrexate and continuous PMEA treatment . Given its structure, drug resistance profile and subcellular localization, MRP4 probably functions as an amphipathic anion efflux pump whose substrate range includes glutamate and phosphate conjugates.

J Infect Dis, 2001 Jan 1, 183(1), 23 - 35 Epub 2000 Dec 08.
Early human immunodeficiency virus (HIV) infection in the HIV Network for Prevention Trials Vaccine Preparedness Cohort: risk behaviors, symptoms, and early plasma and genital tract virus load; Celum CL et al.; Risk behaviors, symptoms, and virologic characteristics were studied among 103 human immunodeficiency virus (HIV) seroconverters in vaccine preparedness cohorts during 1995-1998 . Overall, 83% of subjects were men who had sex with men; most reported multiple risk episodes and symptoms (84%, > or =1 symptom) during seroconversion . Acute HIV was diagnosed in only 8 of 50 who sought medical care . Median initial pretreatment plasma virus load was 25,800 copies/mL (range, undetectable-262,000 copies/mL) a mean of 4 months after seroconversion, and 9.7% had nucleoside-associated mutations; none had multidrug resistance . Semen virus load was more variable, 1.3 log(10) lower and modestly correlated (r=.28; 95% confidence interval, 0.16-0.42) with plasma among untreated men . When the plasma RNA level was <5000 copies/mL, 32% of untreated men, 13% on nucleoside regimens, and 7% on protease inhibitor-containing regimens had detectable seminal RNA . Acute HIV was seldom diagnosed, representing missed opportunities for early treatment and prevention . Most subjects had several relatively stable virus loads before initiation of antiretrovirals, indicating feasibility of assessing HIV vaccines on virus set point in efficacy trials.

Clin Cancer Res, 2000 Nov, 6(11), 4201 - 4
Phase I trial of paclitaxel plus megestrol acetate in patients with paclitaxel-refractory ovarian cancer; Markman M et al.; Increased expression of P-glycoprotein has been proposed as one important mechanism for inherent or acquired drug resistance of malignant disease to cytotoxic chemotherapy . In experimental systems, hormonal agents, including megestrol acetate (MA), have been shown to be capable of reversing P-glycoprotein-mediated multidrug resistance to natural products, including paclitaxel . Because paclitaxel is one of the most active cytotoxic agents in ovarian cancer (OC), we sought to determine whether retreating patients with well-defined paclitaxel-resistant OC with a combination of paclitaxel and MA would result in clinically relevant reversal of that resistant state . In this Phase I trial, 44 patients with OC or primary peritoneal carcinoma received paclitaxel (135-175 mg/m2 over 3 h) plus an oral loading dose (800-9600 mg over 24 h) and subsequent maintenance dose (800-3200 mg/day x 3 days) of micronized MA . Both the loading dose and maintenance therapy were delivered in four equal daily doses . Therapy was repeated every 21 days, assuming recovery from the toxicity of the prior course . There were no intrapatient dose escalations . The major toxicity of the regimen was peripheral neuropathy (32% of patients; 11% grade 2-3), although four individuals developed major venous blood clots and one suffered a stroke . Four patients exhibited biological evidence of antineoplastic effects, although only one patient experienced improvement in clinically relevant symptoms . Although pharmacokinetic studies were not performed as a component of this study, prior evaluation of MA pharmacokinetics and in vitro data examining the concentrations of the agent required to reverse P-glycoprotein-mediated paclitaxel resistance suggest that the majority of our patient population achieved levels of MA theoretically capable of producing this desired effect . We conclude that the level of activity and toxicity pattern observed in this trial, associated with the combination of paclitaxel and MA, does not provide strong support for further exploration of the regimen as a treatment strategy to overcome paclitaxel resistance in OC.

Clin Cancer Res, 2000 Nov, 6(11), 4186 - 91
Phase I trial of XR9576 in healthy volunteers demonstrates modulation of P-glycoprotein in CD56+ lymphocytes after oral and intravenous administration; Stewart A et al.; XR9576 is a novel inhibitor of P-glycoprotein (P-gp) that has been shown to reverse P-gp-dependent multidrug-resistance in tumor cell lines and tumor-bearing animals . Here we report the first i.v . and p.o . administration to healthy volunteers of XR9576 in dose-escalating studies with the aim of investigating its effects on safety, its pharmacokinetics, and a surrogate marker of efficacy . XR9576 was administered as a single dose-upward titration of 0.1, 0.2, 0.5, 1.0, and 2 mg/kg XR9576 i.v . or 50, 100, 200, 500, and 750 mg/volunteer p.o . The surrogate marker for in vivo efficacy examined the accumulation of the P-gp substrate Rhodamine-123 (Rh-123) in P-gp-expressing CD56+ lymphocytes by flow cytometry . Addition of Rh-123 to blood samples from subjects given XR9576 or a placebo demonstrated drug-dependent modulation of P-gp activity . Even at the lowest doses, significant effects were observed on Rh-123 accumulation in CD56+ cells . Maximal effects were seen during the i.v . infusion or 4-6 h after oral administration . As the dose was increased, a concomitant rise in the level and duration of P-gp blockade was observed . A dose of 2.0 mg/kg i.v . and > or = 200 mg/volunteer p.o . gave approximately 100% inhibition of P-gp for in excess of 24 h . All doses of XR9576 were well tolerated . Inhibition increased with XR9576 plasma concentration, and maximal activity was achieved at 150-200 ng/ml XR9576 . In conclusion, XR9576 has demonstrated sustained inhibition of P-gp after i.v . and oral administration and, supported by the elimination half-life of about 24 h, XR9576 is being taken into Phase II as a once-daily agent.

Gynecol Oncol, 2000 Dec, 79(3), 430 - 7
Tumor suppressor gene, cell surface adhesion molecule, and multidrug resistance in Müllerian serous carcinomas: clinical divergence without immunophenotypic differences; Soslow RA et al.; OBJECTIVES: We hypothesize that differences in the expression of selected tumor suppressor genes, cell surface adhesion molecules, and multidrug resistance glycoproteins could account for some of the reported differences between uterine serous carcinoma (USC) and extrauterine serous carcinomas (ESC), including ovarian and primary peritoneal carcinoma (OSC and PSC, respectively) . METHODS: We studied the expression of the following antigens in 20 USCs, 20 OSCs, and 10 PSCs: p53 and mdm-2 (tumor suppressor genes), CD44 and CD44v6 (cell surface adhesion molecules), and the p-glycoprotein (a multidrug resistance protein recognized by two antibodies, C494 and JSB1) . We further studied chemotherapeutic drug resistance by examining reports prepared using the Oncotech Extreme Drug Resistance Assay from 24 of the 50 study patients . Clinical data were obtained from medical record review . RESULTS: USC, OSC, and PSC patients were similar with respect to mean age at diagnosis, mean gravidity, mean parity, personal history of breast cancer, percentage treated with chemotherapy, and survival at 3 and 5 years postdiagnosis . Significant clinical differences included a high prevalence of nulliparity in OSC (P = 0.05), a low prevalence of Caucasian race in USC (P = 0.008), a paucity of stage I patients in OSC and PSC (P = 0.03), a high prevalence of familial breast cancer in OSC (P = 0.06), and superior 2-year survival in OSC (P = 0.02) . Seventy-five percent of USCs, 52% of OSCs, and 60% of PSCs expressed p53 . Five percent of USCs, 19% of OSCs, and 0% of PSCs expressed mdm-2 . Forty percent of USCs, 33% of OSCs, and 10% of PSCs expressed CD44 . None of the USCs, OSCs, or PSCs expressed CD44v6 . Sixty-one percent of USCs and OSCs and 82% of PSCs expressed C494 while 17% of USCs, 19% of OSCs, and 20% of PSCs expressed JSB1 . None of these apparent differences was statistically significant . USC, OSC, and PSCs patients did not demonstrate significant differences with respect to extreme drug resistance . However, the following trends were noted (P = 0.06): more prevalent low drug resistance for cyclophosphamide in OSC compared with USC and more prevalent extreme drug resistance for etoposide in OSC compared with USC . CONCLUSIONS: Therefore, despite significant clincial differences, the USCs and ESCs in our series do not differ significantly with respect to the expression of the tumor suppressor genes, cell surface adhesion molecules, and drug resistance proteins studied . It is premature, however, to recommend that USCs and ESCs should be treated identically .

J Biol Chem, 2001 Mar 2, 276(9), 6404 - 11 Epub 2000 Dec 01.
Glutathione stimulates sulfated estrogen transport by multidrug resistance protein 1; Qian YM et al.; Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette (ABC) transporter that transports a range of hydrophobic xenobiotics, as well as relatively hydrophilic organic anion conjugates . The protein is present at high levels in testicular Leydig and Sertoli cells . Studies with knockout mice suggest that MRP1 may protect germ cells from exposure to some cytotoxic xenobiotics, but potential endobiotic substrates in this organ have not been identified . Previously, we have shown certain D-ring, but not A-ring, estrogen glucuronides can act as competitive inhibitors of MRP1 mediated transport, suggesting that they are potential substrates for the protein . In the case of 17 beta-estradiol-17 beta-d-glucuronide, this has been confirmed by direct transport studies . The Leydig cell is the major site of estrogen conjugation in the testis . However, the principal products of conjugation are A-ring estrogen sulfates, which are then effluxed from the cell by an unknown transporter . To determine whether MRP1/mrp1 could fulfill this function, we used membrane vesicles from MRP1-transfected HeLa cells to assess this possibility . We found that estradiol and estrone 3-sulfate alone were poor competitors of MRP1-mediated transport of the cysteinyl leukotriene, leukotriene C(4) . However, in the presence of reduced glutathione (GSH), their inhibitory potency was markedly increased . Direct transport studies using {(3)H}estrone 3-sulfate confirmed that the conjugated estrogen could be efficiently transported (K(m) = 0.73 microm, V(max) = 440 pmol mg(-)1 protein min(-)1), but only in the presence of either GSH or the nonreducing alkyl derivative, S-methyl GSH . In contrast to previous studies using vincristine as a substrate, we detected no reciprocal increase in MRP1-mediated GSH transport . These results provide the first example of GSH-stimulated, MRP1-mediated transport of a potential endogenous substrate and expand the range of MRP1 substrates whose transport is stimulated by GSH to include certain hydrophilic conjugated endobiotics, in addition to previously identified hydrophobic xenobiotics.

J Clin Microbiol, 2000 Dec, 38(12), 4599 - 603
Evaluation of Etest for susceptibility testing of multidrug-resistant isolates of Mycobacterium tuberculosis; Hazbon MH et al.; To prescribe effective treatment schemes for patients with tuberculosis, more-efficient susceptibility testing techniques for Mycobacterium tuberculosis are needed, especially in regions with multidrug resistance . Etest (AB BIODISK, Solna, Sweden) is a simple technique that provides quantitative drug susceptibility results for M . tuberculosis in 5 to 10 days from a culture grown at low cost . The performance of Etest was compared to that of the reference proportion method, using 95 M . tuberculosis clinical isolates of which 42.1% (40 of 95) were resistant to at least one antibiotic by the reference method . Overall agreement between Etest and the reference method was 98.9% (94 of 95) for detection of multidrug resistance; for resistance to individual drugs, agreement was 97.9% (93 of 95) for rifampin, 96.0% (92 of 95) for ethambutol, 94.7% (90 of 95) for isoniazid, and 85.3% (81 of 95) for streptomycin . This study supports the utility of Etest for timely detection of drug resistance in M . tuberculosis and for use in tuberculosis control programs.

Clin Chem Lab Med, 2000 Sep, 38(9), 893 - 7
The human multidrug resistance-associated protein (MRP) gene family: from biological function to drug molecular design; Ishikawa T et al.; The ATP-binding cassette transmembrane proteins play an important role in transport of drugs as well as of biologically active endogenous substances . The human multidrug resistance-associated protein (MRP) subfamily consists of at least six members, exhibiting a wide spectrum of biological functions . MRP1 operates as an ATP-dependent primary active transporter for substrates conjugated with glucuronide, sulfate or glutathione . Leukotriene C4 is an important endogenous substrate for MRP1 . Glutathione serves as a cofactor in MRP1-mediated drug transport as well . Genes encoding both MRP1 and the catalytic subunit of gamma-glutamylcysteine synthetase (gamma-GCS) are coordinately regulated in cultured cancer cell lines as well as colorectal cancer tissues from colon cancer patients . The induction of MRP1 and gamma-GCS expression by oxidative stress varies among different cell lines, and p53 mutations are associated with elevated levels of induction . To modulate the transport function of MRP1, we have synthesized novel glutathione derivatives as photoreactive biochemical probes targeting the transporter protein . GIF-0019 restored the cellular sensitivity of MRP1-overexpressing drug-resistant cancer cells to anticancer prostaglandins in vitro, which was characterized by enhanced mRNA levels of the cyclin-dependent kinase inhibitor p21, suppressed c-myc expression and G1 arrest.

Clin Chem Lab Med, 2000 Sep, 38(9), 877 - 81
P-glycoprotein and bioavailability-implication of polymorphism; Liu Y et al.; P-Glycoprotein (P-gp) may have a significant impact on systemic and tissue/cellular bioavailability of drugs because it functions as an "anti-absorption" mechanism that effluxes drug molecules out of the lipid bilayer and cytoplasm . The ability to reduce bioavailability at the tissue/cellular level was first discovered during the investigation of the causes of multidrug resistance (MDR) in cancer chemotherapy . Initially, it was thought that MDR is only caused by P-gp . Recently, many other transporters such as multidrug resistance-related protein (MRP) have also been identified . The ability of P-gp to impact systemic drug bioavailability was only recently recognized . Dr . Alfred Schinkel's group was first to show a significant improvement in the systemic bioavailability of several drugs in the MDR1 knockout mice . The same group also discovered that the blood-brain barrier (BBB) has a very high expression level of P-gp, and that this protein is necessary to restrict the entrance of various drug molecules into the central nervous system (CNS) . Polymorphism in the normal human cells has not been reported, but it has been discovered in human cancer cells . Functional implication of P-gp polymorphism in changing the tissue bioavailability has been studied in rodents . These studies strongly support the role of P-gp in restricting tissue bioavailability of anticancer drugs . These studies also support the effectiveness of P-gp in limiting CNS toxicity of the cytotoxic drugs . The functional implication of P-gp on systemic bioavailability is much less well defined in humans, although it appears to be quite obvious in MDR1 knockout mice . Pharmacokinetic models clearly suggest that a change in the absorption rate will have a significant impact on systemic blood level of a drug . However, whether functionally significant polymorphisms of P-gp exist in humans has not been determined . If they do exist, they will surely impact on both systemic bioavailability and drug interaction potentials of many drugs . In the drug development process, several models may be used to select a lead compound that may or may not interact with P-gp, depending on whether the interaction is desirable . Several inhibitors of P-gp are currently on the clinical trial stage . Natural inhibitors of P-gp have also been discovered . There is no doubt that these new developments will have significant impact on the bioavailability of a variety of anticancer and CNS drugs in the next decades.

Oncology, 2000 Nov, 59(4), 329 - 35
Cellular efflux pump and interaction between cisplatin and paclitaxel in ovarian cancer cells; Kamazawa S et al.; OBJECTIVE: The aim of this study was to evaluate the combination effect of paclitaxel (PTX) and cisplatin (CDDP) and to determine the mechanisms of interaction between these agents . METHODS AND RESULTS: We used human ovarian adenocarcinoma cell lines, namely a parent cell line (KF), a CDDP-resistant cell line (KFr) and a PTX-resistant cell line (KFTx).The combination effect of PTX and CDDP was synergistic on KF and KFTx and additive on KFr . The incidence of anaphase or telophase, evaluated by immunofluorescence microscopy, decreased with PTX and significantly decreased with PTX and CDDP in KF and KFTx . The concentration of PTX, which was measured by high-performance liquid chromatography, was higher in KF and KFTx cells treated with a combination of PTX and CDDP than those treated with PTX alone . Multidrug resistance gene mRNA appeared in KFTx and its expression decreased after exposure to PTX and CDDP . After exposure to CDDP, the expression of multidrug resistance-associated protein (MRP) and the concentration of glutathione increased in KF, but not in KFr or KFTx . MRP expression slightly increased in KF and KFTx after exposure to PTX . In contrast, its expression decreased in KFr . CONCLUSION: The present study suggests that CDDP enhances PTX accumulation and that the interaction of these agents is synergistic in CDDP-sensitive cells .

FEMS Microbiol Lett, 2000 Dec 1, 193(1), 19 - 23
Contribution of the multidrug efflux pump LfrA to innate mycobacterial drug resistance; Sander P et al.; Multidrug resistance (MDR) in bacteria has been associated with efflux pumps that export structurally unrelated compounds and decrease cytoplasmic drug accumulation . To investigate MDR in mycobacteria, we studied the Mycobacterium smegmatis mutant mc(2)11, which is resistant to doxorubicin, tetracycline, rhodamine, ethidium bromide and the hydrophilic fluoroquinolones . A genomic library constructed from this mutant was used to select clones conferring resistance to doxorubicin . Surprisingly, the clone selected encodes the efflux pump LfrA, which has been reported to confer resistance to hydrophilic fluoroquinolones, ethidium bromide, rhodamine, and acriflavine . To define the contribution of LfrA to the innate mycobacterial drug resistance and to the MDR phenotype in mc(2)11, the lfrA gene was disrupted in both the mc(2)11 mutant and the mc(2)155 wild-type parent . LfrA disruption of the wild-type strain decreased resistance to ethidium bromide and acriflavine, and increased accumulation of ethidium bromide . However, disruption of lfrA gene results only in a 2-fold decrease in minimal inhibitory concentrations (MICs) for ciprofloxacin, doxorubicin, rhodamine, and accumulation of {(14)C}ciprofloxacin was unchanged . LfrA disruption of the MDR strain mc(2)11 produced a similar phenotype . Thus, LfrA contributes significantly to the intrinsic MICs of M . smegmatis for ethidium bromide and acriflavine, but not for ciprofloxacin, doxorubicin or rhodamine.

Med Clin (Barc), 2000 Oct 7, 115(11), 401 - 4
{Genotypic resistance to antiretroviral drugs in patients with therapeutic failure to highly active antiretroviral therapy}; Gutierrez F et al.; BACKGROUND: To assess genotypic resistance mutations in patients with virological failure with highly active antiretroviral therapy (HAART) METHODS: Genotyping of reverse transcriptase (RT) and protease (PRO) HIV-1 genes were carried out in 33 adherent patients failing on HAART . RESULTS: Resistance mutations were found in 32 of the 33; 27 of them (81.8%) being primary mutations: 26 (78.8%) in the RT gene and 60 (60.6%) in the PRO gene . Overall, 66.6% had genotypic resistance to two drugs and 60.6% showed resistance to drugs belonging to the two main classes of antiretroviral drugs . At the time of treatment failure, 72.7% had on their therapeutic regimen one antiretroviral drug to which they had resistance mutations, 48.5% had genotypic resistance to two drugs of the therapeutic regimen and 21.2% to three drugs . CONCLUSIONS: Most adherent patients failing on HAART carry drug resistant genotypes . These patients may constitute a reservoir of multidrug resistant HIV that may limit treatment options in the future.

J Biol Chem, 2001 Feb 16, 276(7), 4819 - 27 Epub 2000 Nov 28.
Aromatic hydrocarbon receptor (AhR).AhR nuclear translocator- and p53-mediated induction of the murine multidrug resistance mdr1 gene by 3-methylcholanthrene and benzo(a)pyrene in hepatoma cells; Mathieu MC et al.; The mouse multidrug resistance gene family consists of three genes (mdr1, mdr2, and mdr3) encoding P-glycoprotein . We show that the expression of mdr1 is increased at the transcriptional level upon treatment of the hepatoma cell line Hepa-1c1c7 with the polycyclic aromatic hydrocarbon 3-methylcholanthrene (3-MC) . This increase is not observed in the aromatic hydrocarbon receptor (AhR)-defective TAOc1BP(r)c1 and the AhR nuclear translocator (Arnt)-defective BP(r)c1 variants, demonstrating that the induction of mdr1 by 3-MC requires AhR.Arnt . We show that the mdr1 promoter (-1165 to +84) is able to activate the expression of a reporter gene in response to 3-MC in Hepa-1c1c7 but not in BP(r)c1 cells . Deletion analysis indicated that the region from -245 to -141 contains cis-acting sequences mediating the induction, including a potential p53 binding sequence . 3-MC treatment of the cells increased the levels of p53 and induced p53 binding to the mdr1 promoter in an AhR.Arnt-dependent manner . Mutations in the p53 binding site abrogated induction of mdr1 by 3-MC, indicating that p53 binding to the mdr1 promoter is essential for the induction . Benzo(a)pyrene, a polycyclic aromatic hydrocarbon and AhR ligand, which, like 3-MC, is oxidized by metabolizing enzymes regulated by AhR.Arnt, also activated p53 and induced mdr1 transcription . 2,3,7,8-Tetrachlorodibenzo-p-dioxin, an AhR ligand resistant to metabolic breakdown, had no effect . These results indicate that the transcriptional induction of mdr1 by 3-MC and benzo(a)pyrene is directly mediated by p53 but that the metabolic activation of these compounds into reactive species is necessary to trigger p53 activation . The ability of the anticancer drug and potent genotoxic agent daunorubicin to induce mdr1 independently of AhR.Arnt further supports the proposition that mdr1 is transcriptionally up-regulated by p53 in response to DNA damage.

Curr Infect Dis Rep, 2000 Jun, 2(3), 215 - 223
Nosocomial or Healthcare Facility-Related Pneumonia in Adults; Balaguera HU et al.; Nosocomial or hospital-acquired pneumonia (HAP) is a dynamic disease with multiple etiologic agents and a changing natural history . The emergence and spread of multidrug-resistant bacterial pathogens is a current concern . Because of the parallels between HAP and pneumonia occurring in patients in subacute or chronic care facilities, we suggest the use of a more inclusive term for these patients: healthcare facility-related pneumonia . This article focuses on current controversies in the pathogenesis, diagnosis, management, and prevention of bacterial HAP in adults . We endorse early, appropriate antibiotic therapy based on disease severity and the use of strategies to prevent infection, improve patient outcome, and reduce hospital costs.

Curr Infect Dis Rep . 2000 Feb;2(1):78.
Probiotics in the Treatment of Diarrheal Diseases; Reid G; Infections of the gastrointestinal tract are a major health problem worldwide, and many result in death, especially in the Third World . The approach to treatment and prevention of these infections has revolved around antibiotic administration for many years, but the emergence of multidrug-resistant pathogens is forcing people to look at alternatives to antibiotics . For more than 100 years, probiotic remedies (viable organisms that benefit the host by improving the microbial balance) have been used to fight infection . Now, substantial scientific and clinical evidence indicates that certain well-selected probiotic organisms can reduce the risk and duration of diarrhea.

Curr Infect Dis Rep, 1999 Aug, 1(3), 289 - 297
Antiretroviral Drug Resistance in HIV-1; Hanna GJ et al.; Progress in understanding antiretroviral resistance has evolved rapidly in recent years . Specific resistance mutations have been associated with virologic failure of different nucleoside reverse transcriptase inhibitors (NRTIs) . These mutations vary in the extent of cross resistance they confer to other drugs in the same class . In addition, two novel mutational patterns conferring resistance to multiple NRTIs have been recognized . Considerable class-specific cross resistance also exists among viruses with reduced susceptibility to nonnucleoside reverse transcriptase inhibitors (NNRTIs) . Among protease inhibitors, low level resistance that arises early during virologic failure may be drug specific in some situations, but high level resistance seen later during suboptimal therapy is likely to confer cross resistance to the entire class . Prevalence of drug resistance in infected patients appears to be considerable, and transmission of multidrug-resistant virus has been documented . Current methods of testing for resistance are promising, but they have significant limitations and require further clinical validation . The best approach to prevent resistance is to start treatment early during infection with a regimen that engenders good compliance and is potent enough to decrease viral load to below detection limits of the most sensitive assay available . Once resistance arises, salvage regimens in general have compromised efficacy and should be planned with attention to the patient's prior drug treatment history and the viruses' suspected or demonstrated resistance patterns.

Mol Pharmacol, 2000 Dec, 58(6), 1563 - 9
Reversal of P-glycoprotein and multidrug-resistance protein-mediated drug resistance in KB cells by 5-O-benzoylated taxinine K; Okumura H et al.; A newly synthesized taxoid originally from the Japanese yew Taxus cuspidata, 5-O-benzoylated taxinine K (BTK) was examined for its ability to reverse P-glycoprotein (P-gp) and multidrug resistance protein (MRP)-mediated multidrug resistance . BTK reversed the resistance to paclitaxel, doxorubicin (ADM), and vincristine (VCR) of KB-8-5 and KB-C2 cells that overexpress P-gp by directly interacting with P-gp . BTK also moderately reversed the resistance to ADM of KB/MRP cells that overexpress MRP . However, BTK neither inhibited the transporting activity of MRP nor reduced intracellular glutathione levels in KB/MRP cells . BTK shifted the distribution of ADM in KB/MRP cells from punctate cytoplasmic compartments to the nucleoplasm and cytoplasm by inhibiting acidification of cytoplasmic organelles . These two functions of BTK make it able to reverse both P-gp- and MRP-mediated MDR . BTK in combination with ADM should be useful for treating patients with tumors that overexpress both P-gp and MRP.

Mol Pharmacol, 2000 Dec, 58(6), 1357 - 67
Xenobiotic transport across isolated brain microvessels studied by confocal microscopy; Miller DS et al.; To identify specific transporters that drive xenobiotics from central nervous system to blood, the accumulation of fluorescent drugs was studied in isolated capillaries from rat and pig brain using confocal microscopy and quantitative image analysis . Luminal accumulation of daunomycin and of fluorescent derivatives of cyclosporine A (CSA) and ivermectin was concentrative, specific, and energy-dependent (inhibition by NaCN) . Transport was reduced by PSC 833, ivermectin, verapamil, CSA, and vanadate, but not by leukotriene C(4) (LTC(4)), indicating the involvement of P-glycoprotein . Luminal accumulation of the fluorescent organic anions sulforhodamine 101 and fluorescein methotrexate was also concentrative, specific, and energy-dependent . LTC(4), chlorodinitrobenzene, and vanadate reduced transport of these compounds, but PSC 833 and verapamil did not, indicating the involvement of a multidrug resistance-associated protein (Mrp) . Immunostaining localized P-glycoprotein and Mrp2 to the luminal surface of the capillary endothelium and quantitative polymerase chain reaction showed Mrp1 and Mrp2 expression . Finally, the HIV protease inhibitors saquinavir and ritonavir were potent inhibitors of transport mediated by both P-glycoprotein and Mrp . These results validate a new method for studying drug transport in isolated brain capillaries and implicate both P-glycoprotein and one or more members of the Mrp family in drug transport from central nervous system to blood.

Hepatology, 2000 Dec, 32(6), 1317 - 28
Impaired protein maturation of the conjugate export pump multidrug resistance protein 2 as a consequence of a deletion mutation in Dubin-Johnson syndrome; Keitel V et al.; The Dubin-Johnson syndrome is an inherited disorder characterized by conjugated hyperbilirubinemia . The deficient hepatobiliary transport of anionic conjugates is caused by the absence of a functional multidrug-resistance protein 2 (MRP2, symbol ABCC2) from the apical (canalicular) membrane of hepatocytes . Mechanisms underlying this deficiency may include rapid degradation of mutated MRP2 messenger RNA (mRNA) or impaired MRP2 protein maturation and trafficking . We investigated the consequences of the mutation MRP2Delta(R,M), which leads to the loss of 2 amino acids from the second ATP-binding domain of MRP2 . The MRP2Delta(R,M) mutation is associated with the absence of the MRP2 glycoprotein from the apical membrane of hepatocytes . Transfection of mutated MRP2 complementary DNA (cDNA) led to an MRP2Delta(R,M) protein that was only core glycosylated, sensitive to endoglycosidase H digestion, and located in the endoplasmic reticulum (ER) of transfected HEK293 and HepG2 cells . This indicated that deletion of Arg1392 and Met1393 leads to impaired maturation and trafficking of the protein from the ER to the Golgi complex . Inhibition of proteasome function resulted in a paranuclear accumulation of the MRP2Delta(R,M) protein, suggesting that proteasomes are involved in the degradation of the mutant protein . This is the first mutation in Dubin-Johnson syndrome shown to cause deficient MRP2 maturation and impaired sorting of this glycoprotein to the apical membrane.

J Pharm Pharmacol, 2000 Oct, 52(10), 1171 - 8
Thermal dependence of multidrug-resistant-modulator efficiency: a study in anionic liposomes; Castaing M et al.; This study was designed to test the hypothesis that there exists a correlation between the ability of lipophilic drugs to mediate the reversal of multidrug-resistance (MDR) by interacting with the membrane phospholipids and the metabolic level in tissues . The permeation properties of five MDR-modulators were studied by quantifying their ability to induce the leakage of Sulphan blue through unilamellar liposomes, over the temperature range 27-42 degrees C . The dye leakage induced by a non-ionic detergent (Triton X-100), two calcium blockers (diltiazem and verapamil) and two antiparasitic agents (thioacridine derivative and mepacrine) was temperature-dependent . The permeation process was a co-operative one (1.1 < Hill coefficient < 7.5) and the permeation doses inducing 50% dye leakage (PD50) were 1.5 - 14.9 mM . The permeation ability of the MDR-modulators (log(1/PD50)) decreased significantly as the net electric charge (z) increased . The passive dye leakage (deltaG < 0) was found to be an endothermic process (deltaH > 0), favoured by an increase in the membrane disorder (deltaS > 0) . The apparent enthalpy factor (deltaH50) associated with 50% dye leakage increased with the net electric charge of the compound, and this energetically non-favoured event was entirely offset by the concomitant increase in the entropy factor (deltaS50) . The apparent permeation enthalpy (deltaH50) and entropy (deltaS50) showed the lowest values for Triton X-100 (deltaH50 = 7.1 +/- 0.53 kJ mol(-1), deltaS50 = 76.9 +/- 1.86 Jmol(-1) K(-1)), and the highest values for mepacrine (deltaH50 = 79.5 +/- 3.80 kJmol(-1), deltaS50 = 306.7 +/- 5-97 J mol(-1) K(-1)) . When the temperature was increased from 27 to 42 degrees C, the apparent Gibbs free energy (deltaG50) of the dye leakage induced by Triton X-100 decreased by less than 10% of the initial value, and that induced by mepacrine decreased by nearly 40% . The results provide evidence that in tissues with high metabolic levels and therefore high temperatures, MDR-reversal is likely to be enhanced via favourable drug-membrane interactions controlled by the electric charge of the modulators.

Arch Pharm (Weinheim), 2000 Oct, 333(10), 329 - 36
Synthesis of a series of 3-cyanopropionamides and 4-imino-gamma-butyrolactams and evaluation of their function as modulators of multidrug resistance; Ros F et al.; The synthesis of the 3-cyanopropionamides 3a and 3b, of the 2,2-dimethyl-3-cyanopropionamides 4a-4c and of the 4-imino-gamma-butyrolactams 5a and 5b (cyclic functional isomers of 3-cyanopropionamides) is described . The amides 3a and 3b were obtained by aminolysis of the corresponding acid chlorides, which are accessible via hydrolysis of the ethyl esters to the acids . This methodology was not used for the synthesis of the amides 4a-4c owing to steric hindrance to hydrolysis in the corresponding ethyl esters . These nonreactive esters, accessible by alkylation of 1-cyano carbanions with ethyl bromodimethylacetate, could be directly converted into the amides 4a-4c by aminolysis with the lithium amide of 3,4-dimethoxy-N-methylphenethylamine . Instead of open-chain amides, the lactams 5a and 5b are obtained when the lithium amide of 3,4-dimethoxyphenethylamine (i.e., of a primary rather than secondary amine) is used for the aminolysis . The synthesized compounds were tested for their ability to decrease the resistance to vincristine in a multidrug-resistant subline of murine leukemic lymphoblasts that are 300-fold resistant to the antiproliferative drug . The amides 4a and 4c, and lactam 5a, all of which have a highly branched carbon backbone, were active . Lactam 5a reduced the vincristine resistance by 90% at a 2-microM concentration.

Med Hypotheses, 2000 Dec, 55(6), 470 - 3
Tissue factor expression and multidrug resistance in cancer: two aspects of a common cellular response to a hostile milieu; Lwaleed BA et al.; Tissue factor (TF) is the main physiological initiator of blood coagulation and its activation or de-encryption within plasma membranes is important for trapping, extravasation and angiogenesis in the development and spread of solid malignancies . Multidrug resistance is also an adaptive response in malignant (and normal) cells . It is often mediated by the over-expression of the P-glycoprotein (P-gP) efflux pump.Both TF and P-gP tend to be expressed together, perhaps as part of a coherent 'crisis management' response of cells to environmental change or challenge . An associated feature in such a response appears to be the reversal of normal phospholipid charge asymmetry in the plasma membrane bilayer.Responses to environmental stimuli affect function in normal and malignant tissue . Uniting the study of TF expression or de-encryption and MDR-1 phenotype would be biologically enlightening and might ultimately influence clinical cancer management and the control of thrombotic problems associated with treatment .

Breast Cancer, 1997 Dec 25, 4(4), 259 - 263
Immunohistochemical Detection of P-glycoprotein in Breast Cancer and Its Significance as a Prognostic Factor; Tsukamoto F et al.; Overexpression of P-glycoprotein (Pgp) in tumors is one of the major mechanisms which mediates the multidrug resistance (MDR) phenotype . To evaluate the prognostic significance of Pgp in breast cancer, Pgp expression was examined in paraffin-embedded tissue sections of 94 breast cancer specimens by immunohistochemistry . Tissue specimens were obtained by mastectomy without preoperative chemotherapy . UIC2 monoclonal antibody which recognizes an extracellular epitope of human Pgp was employed . Of the 94 breast cancer specimens, 35(37.2%)were positive for Pgp expression . Pgp expression had no correlation with menopausal or hormone receptor status, axillary Iymph node involvement or tumor size . However, a significant correlation was observed between Pgp expression and disease relapse (p=0.0322) . Pgp-positive patients showed a significantly shorter disease-free survival period than Pgp-negative patients by the Kaplan-Meier method (p=0.0433) . These results suggest that immunohistochemical detection of Pgp in breast cancer tissue may have prognostic value after radical operation.

Breast Cancer, 1997 Dec 25, 4(4), 210 - 212
Multidrug Resistance and Prodrug Activation for Cancer Gene Therapy of Breast Cancer; Fujii T et al.; Current approaches to the treatment of cancer (radiation and chemotherapy) are limited by the toxicity to normal tissues and the sensitivity of tumor cells to these treatments . The use of gene delivery vector systems for the introduction of genetic elements into normal and neoplastic tissues which code for proteins that protect the normal chemotherapy-sensitive tissues and sensitize the tumor cells to radiation and chemotherapy, may contribute to an overall improvement in the outcome of cancer treatment programs . This chapter will review the clinical trials either completed or about to be initiated which are designed to accomplish these goals.

Blood, 2000 Dec 1, 96(12), 3948 - 52
Level of minimal residual disease after consolidation therapy predicts outcome in acute myeloid leukemia; Venditti A et al.; We used flow cytometry to quantify minimal residual disease (MRD) in 56 patients with acute myeloid leukemia (AML) expressing a leukemia-associated phenotype . Thirty-four patients aged 18 to 60 years were entered into the AML-10 protocol (induction, consolidation, and autologous stem-cell transplantation {ASCT}), whereas 22 patients older than 60 years received the AML-13 protocol (induction, consolidation, and consolidation II) . After induction, the level of MRD that was best associated with treatment outcome was 4.5 x 10(-4) residual leukemic cells . However, the outcome in patients with at least 4.5 x 10(-4) cells (n = 26) was not significantly different from that in patients with fewer leukemic cells (n = 30); there were 15 (58%) relapses in the first group and 12 (40%) relapses in the second . After consolidation, the most predictive MRD cutoff value was 3.5 x 10(-4) cells: 22 patients had an MRD level of 3.5 x 10(-4) cells or higher and 17 (77%) of these patients had relapse, compared with 5 of 29 patients (17%) with lower MRD levels (P <.001) . An MRD level of 3.5 x 10(-4) cells or higher after consolidation was significantly correlated with poor or intermediate-risk cytogenetic findings, a multidrug resistance 1 (MDR1) phenotype, short duration of overall survival, and short duration of relapse-free survival (P =.014,.031,.00022, and.00014, respectively) . In multivariate analysis, this MRD status was significantly associated with a high frequency of relapse (P <.001) and a short duration of overall (P =.025) and relapse-free survival (P =.007) . ASCT did not alter the prognostic effect of high MRD levels after consolidation: the relapse rate after transplantation was 70% . Thus, we found that an MRD level of 3.5 x 10(-4) cells or higher at the end of consolidation strongly predicts relapse and is significantly associated with an MDR1 phenotype and intermediate or unfavorable cytogenetic findings . (Blood . 2000;96:3948-3952)

Can J Public Health, 2000 Sep-Oct, 91(5), 366 - 70
Drug resistance study of Mycobacterium tuberculosis in Canada, February 1, 1993 to January 31, 1994; Farzad E et al.; OBJECTIVE: To estimate the prevalence of resistance of Mycobacterium tuberculosis to first-line antituberculosis drugs in Canada . METHODS: M . tuberculosis isolates from one third of all culture-positive tuberculosis (TB) cases diagnosed between February 1, 1993 to January 31, 1994 in Canada were collected prospectively . Proportion of drug-resistant isolates and the factors related to drug resistance were measured . RESULTS: Of 458 study cases, 40 (8.7%) had resistance to at least one first-line antituberculosis drug, of which 5.9% had mono-resistance, 0.7% had multidrug-resistance(MDR-TB)--i.e., resistance to at least isoniazid and rifampin--and 2.2% had other patterns . The overall prevalence of resistance among the foreign-born cases was 10.6% with the highest level among those who resided in Canada for less than four years (15.5%) . CONCLUSIONS: Canada has a relatively low prevalence of antituberculosis drug resistance and a very low prevalence of MDR-TB . Some new immigrants to Canada may be at higher risk for drug resistance and their initial treatment needs to be tailored accordingly.

J Med Chem, 2000 Nov 16, 43(23), 4534 - 41
Classification of multidrug-resistance reversal agents using structure-based descriptors and linear discriminant analysis; Bakken GA et al.; Linear discriminant analysis is used to generate models to classify multidrug-resistance reversal agents based on activity . Models are generated and evaluated using multidrug-resistance reversal activity values for 609 compounds measured using adriamycin-resistant P388 murine leukemia cells . Structure-based descriptors numerically encode molecular features which are used in model formation . Two types of models are generated: one type to classify compounds as inactive, moderately active, and active (three-class problem) and one type to classify compounds as inactive or active without considering the moderately active class (two-class problem) . Two activity distributions are considered, where the separation between inactive and active compounds is different . When the separation between inactive and active classes is small, a model based on nine topological descriptors is developed that produces a classification rate of 83.1% correct for an external prediction set . Larger separation between active and inactive classes raises the prediction set classification rate to 92.0% correct using a model with six topological descriptors . Models are further validated through Monte Carlo experiments in which models are generated after class labels have been scrambled . The classification rates achieved demonstrate that the models developed could serve as a screening mechanism to identify potentially useful MDRR agents from large libraries of compounds.

Leuk Res, 2000 Nov, 24(11), 917 - 25
Potentiation of chemosensitivity in multidrug-resistant human leukemia CEM cells by inhibition of DNA-dependent protein kinase using wortmannin; Kim SH et al.; DNA-dependent protein kinase (DNA-PK) is activated by DNA strand breaks and participates in DNA repair . Its regulatory subunit, Ku autoantigen, binds to DNA and recruits the catalytic subunit (DNA-PKcs) . We show here a new role of DNA-PK in the development of multidrug resistance (MDR) . The Ku-DNA binding activity, the levels of Ku70/Ku80 and DNA-PKcs in MDR variants, CEM/VLB(10-2), CEM/VLB(55-8) and CEM/VLB100 were higher than those in their parental drug-sensitive CEM cells in a drug resistance-dependent fashion . Also, CEM/VLB100 cells showed about 3-fold increase of DNA-PK enzyme activity as compared with CEM cells . Similar results were observed in another MDR cell line, FM3A/M mouse mammary carcinoma cells . Moreover, we observed that CEM/VLB100 cells were about 11-fold sensitive to wortmannin, which inhibits DNA-PK, compared with the CEM cells, and sensitized the MDR cells when combined with either bleomycin or vincristine, but have a little effect on CEM cells . Wortmannin was shown to inhibit DNA-PK and Ku-DNA binding activity in CEM/VLB100 cells dose dependently but had a little or no effect on their parental cells . Our results suggested that enhanced expression of DNA-PK participates in the development of MDR, and the use of DNA-PK inhibitors such as wortmannin is likely to improve the effectiveness of anticancer drugs and thus could partially overcome drug resistance in MDR cells, through its ability to inhibit Ku/DNA-PK activity.

Curr Opin Oncol, 2000 Nov, 12(6), 550 - 6
Lung resistance-related protein/major vault protein and vaults in multidrug-resistant cancer; Scheffer GL et al.; Tumor cells that are insensitive to anticancer drugs frequently have a multidrug-resistant (MDR) phenotype . Proteins that can be involved in this phenomenon are transport-associated proteins such as P-glycoprotein, multidrug-resistance protein 1, breast cancer resistance protein, and lung resistance-related protein (LRP) . LRP was identified as the major vault protein (MVP), the main component of multimeric vault particles . With the recent identification of the two minor vault proteins as telomerase-associated protein (TEP1) and vault-poly (ADP-ribose) polymerase (VPARP), and with high-resolution three-dimensional imaging, the composition of vaults is almost unraveled . Although the first direct evidence for a causal relationship between LRP/MVP expression and drug resistance has been obtained, many functional aspects of vaults in normal physiology and in MDR still need to be clarified . The current clinical data on LRP/MVP detection indicate that LRP/MVP expression can be of high clinical value to predict the response to chemotherapy of several tumor types.

Trends Biochem Sci, 2000 Nov, 25(11), 530 - 4
Multidrug resistance: a role for cholesterol efflux pathways?
Liscovitch M, Lavie Y.
Multidrug resistance (MDR) severely impairs the efficacy of cancer chemotherapy . Several protein transporters that mediate drug export have been identified, but additional adaptations appear to be necessary for full-fledged drug resistance . The cell surface density of caveolae and the expression of the caveolar coat protein caveolin are dramatically increased in MDR cancer cells . Acquisition of MDR might thus be accompanied by upregulation of caveolin-dependent cholesterol efflux pathways, raising the possibility that these same pathways are utilized for delivering drugs from intracellular compartments to the plasma membrane, where drugs can be extruded from the cells by drug efflux ATPases . The upregulation of caveolin mandates a phenotypic change of MDR cells in terms of their cholesterol homeostasis and is accompanied by loss of important features of the transformed phenotype of MDR cancer cells.

Antimicrob Agents Chemother, 2000 Dec, 44(12), 3414 - 24
Mefloquine pharmacokinetic-pharmacodynamic models: implications for dosing and resistance; Simpson JA et al.; Antimalarial resistance develops and spreads when spontaneously occurring mutant malaria parasites are selected by concentrations of antimalarial drug which are sufficient to eradicate the more sensitive parasites but not those with the resistance mutation(s) . Mefloquine, a slowly eliminated quinoline-methanol compound, is the most widely used drug for the treatment of multidrug-resistant falciparum malaria . It has been used at doses ranging between 15 and 25 mg of base/kg of body weight . Resistance to mefloquine has developed rapidly on the borders of Thailand, where the drug has been deployed since 1984 . Mathematical modeling with population pharmacokinetic and in vivo and in vitro pharmacodynamic data from this region confirms that, early in the evolution of resistance, conventional assessments of the therapeutic response </=28 days after treatment underestimate considerably the level of resistance . Longer follow-up is required . The model indicates that initial deployment of a lower (15-mg/kg) dose of mefloquine provides a greater opportunity for the selection of resistant mutants and would be expected to lead more rapidly to resistance than de novo use of the higher (25-mg/kg) dose.

Antimicrob Agents Chemother, 2000 Dec, 44(12), 3285 - 7
Early bactericidal activity of paromomycin (aminosidine) in patients with smear-positive pulmonary tuberculosis; Donald PR et al.; The early bactericidal activity of the aminoglycoside paromomycin (aminosidine) in doses of 7.5 and 15 mg/kg of body weight was measured in 22 patients with previously untreated smear-positive pulmonary tuberculosis . The fall in log(10) CFU per milliliter of sputum per day during the first 2 days of treatment for 7 patients receiving a paromomycin dosage of 7.5 mg/kg/day was 0.066, with a standard deviation (SD) of 0.216 and confidence limits from -0.134 to 0.266, and that for 15 patients receiving 15 mg/kg/day was 0.0924, with an SD of 0.140 and confidence limits from 0.015 to 0.170 . The difference between the mean and zero was not significant for the 7 . 5-mg/kg dose group but was significant for the 15-mg/kg dose group (t = 2.55, P = 0.023) . Since paromomycin has no cross-resistance with streptomycin and has no greater toxicity than other aminoglycosides, these results suggest that it has the potential to substitute for streptomycin in antituberculosis regimens and may be a particularly valuable addition to the drug armamentarium for the management of multidrug-resistant tuberculosis.

Mol Ther, 2000 Nov, 2(5), 435 - 45
Context dependence of different modules for posttranscriptional enhancement of gene expression from retroviral vectors; Schambach A et al.; We present a systematic comparison of three modules that enhance expression from retroviral gene transfer vectors at a posttranscriptional level: (i) splice signals (SS) that create an intron in the 5' untranslated region; (ii) constitutive RNA transport elements (CTE), originally discovered in D-type retroviruses; and (iii) the posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE) . Here we show that enhancement of expression depends not only on the specific element, but also on the gene of interest, implying context-dependent activity of the RNA elements . Interestingly, different results were obtained for genes that normally require or do not require such control elements . Expression of the HIV-1 gag-protease gene, which normally depends on the viral export factor Rev, was strongly enhanced by an oligomeric CTE, while WPRE had only a marginal effect . On the other hand, both CTE and WPRE compensated for the lack of an intron in the expression of human beta-globin . In this case, the strongest stimulation of RNA production was observed when functional SS were combined with the WPRE . Both CTE and, in particular, WPRE also enhanced expression of cDNAs that do not normally require any such element (green fluorescent protein, human multidrug resistance-1) . In this study, functional SS and WPRE acted in an additive manner, resulting in a 10-fold higher level of expression . Our results indicate that the described modules act on different levels of RNA processing, transport, and translation and that the correct choice of a posttranscriptional enhancer configuration depends on the type of cDNA to be expressed.

J Cell Sci, 2000 Dec, 113 Pt 24, 4451 - 61
Characterization of the amino-terminal regions in the human multidrug resistance protein (MRP1); Bakos E et al.; The human multidrug resistance protein (MRP1) contributes to drug resistance in cancer cells . In addition to an MDR1-like core, MRP1 contains an N-terminal membrane-bound (TMD(0)) region and a cytoplasmic linker (L(0)), both characteristic of several members of the MRP family . In order to study the role of the TMD(0) and L(0) regions, we constructed various truncated and mutated MRP1, and chimeric MRP1-MDR1 molecules, which were expressed in insect (Sf9) and polarized mammalian (MDCKII) cells . The function of the various proteins was examined in isolated membrane vesicles by measuring the transport of leukotriene C(4) and other glutathione conjugates, and by vanadate-dependent nucleotide occlusion . Cellular localization, and glutathione-conjugate and drug transport, were also studied in MDCKII cells . We found that chimeric proteins consisting of N-terminal fragments of MRP1 fused to the N terminus of MDR1 preserved the transport, nucleotide occlusion and apical membrane routing of wild-type MDR1 . As shown before, MRP1 without TMD(0)L(0) (Delta MRP1), was non-functional and localized intracellularly, so we investigated the coexpression of Delta MRP1 with the isolated L(0) region . Coexpression yielded a functional MRP1 molecule in Sf9 cells and routing to the lateral membrane in MDCKII cells . Interestingly, the L(0) peptide was found to be associated with membranes in Sf9 cells and could only be solubilized by urea or detergent . A 10-amino-acid deletion in a predicted amphipathic region of L(0) abolished its attachment to the membrane and eliminated MRP1 transport function, but did not affect membrane routing . Taken together, these experiments suggest that the L(0) region forms a distinct domain within MRP1, which interacts with hydrophobic membrane regions and with the core region of MRP1.

Gene, 2000 Oct 31, 257(2), 243 - 9
Repression of chick multidrug resistance-associated protein 1 (chMRP1) gene expression by estrogen; Hagen SG et al.; Although a number of genes have been identified whose transcriptional activities are stimulated by estrogen, relatively few have been discovered that are repressed . In an effort to determine whether estrogen can directly repress gene expression, attempts were made to identify genes that are direct targets of the estrogen receptor and whose activities are repressed by it . Because the development and differentiation of the chick oviduct are exquisitely dependent upon estrogen, this seemed an appropriate model system for testing this hypothesis . RNA was isolated from estrogen-treated and estrogen-withdrawn chick oviducts and was subjected to differential display analysis . Surprisingly, one of the products repressed by estrogen encoded the chick homolog of the multidrug resistance-associated protein 1 (MRP1) gene . Further cloning resulted in a chick MRP1 (chMRP1) cDNA clone that is 72% identical with human MRP1 . Translation of the chMRP1 sequence indicates a 77% amino acid identity with both the human and mouse MRP1 proteins . Treatment of estrogen-withdrawn chicks with 17beta-estradiol decreased chMRP1 mRNA levels to 50% within 30 min and to 70% by 1h, which is comparable to the level observed with chronic repression by estrogen . ChMRP1 mRNA is present in many other tissues, including the heart, lung, brain, kidney, skeletal muscle, and intestine, but is undetectable in the liver . This study indicates that in estrogen-responsive tissues such as chick oviduct, the regulation of chMRP1 gene expression is controlled by estrogen.

Curr Gastroenterol Rep, 2000 Dec, 2(6), 440 - 5
Clinical pharmacology of inflammatory bowel disease therapies; Sandborn WJ et al.; Knowledge about the clinical pharmacology of medical therapy of inflammatory bowel disease has incrementally advanced . Small studies with mesalamine have suggested that intestinal mucosal concentrations of mesalamine may predict clinical response to mesalamine therapy . Increased expression of glucocorticoid receptor beta and increased expression of the multidrug resistance drug pump P-glycoprotein 170 have been proposed as markers of drug resistance to glucocorticoids . A baseline determination of thiopurine methyltransferase phenotype or genotype may predict early leukopenia in patients treated with azathioprine or 6- mercaptopurine . Serial measurement of erythrocyte 6-thioguanine nucleotides may be useful in tailoring the dose of these medications . A loading dose of intravenous azathioprine does not accelerate the time to response in patients with steroid-treated Crohn's disease; however, standard azathioprine may work more quickly than previously reported . Methotrexate, 15 to 25 mg/wk, is effective for the treatment of Crohn's disease (active or in remission), and there is no significant difference in the erythrocyte concentrations of methotrexate polyglutamate in patients with inflammatory bowel disease receiving 15 mg, compared with 25 mg, subcutaneously on a weekly basis.

Int J Oncol, 2000 Dec, 17(6), 1077 - 86
Doxorubicin-resistant variants of human prostate cancer cell lines DU 145, PC-3, PPC-1, and TSU-PR1: characterization of biochemical determinants of antineoplastic drug sensitivity; David-Beabes GL et al.; Intrinsic and acquired antineoplastic drug resistance remain a major problem for advanced prostate cancer treatment . In order to characterize mechanisms of anti-neoplastic drug resistance in human prostate cancer cell lines, resistant sublines of four of the commonly studied prostate cancer cell lines (DU 145, PC-3, PPC-1, and TSU-PR1) were selected following exposure to increasing concentrations of doxorubicin (from 10-1000 nM) . Sensitivity patterns of the parent and doxorubicin-resistant sublines to various anti-neoplastic drugs, including adriamycin, amsacrine, etoposide, camptothecin, vinblastine, vincristine, fluorodeoxyuridine, and melphalan, were determined using a sulforhodamine B growth inhibition assay . The expression of three well-described antineoplastic drug resistance proteins, P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and lung resistance protein (LRP), was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR) assays specific for each of the mRNA species, and using immunocytochemical staining procedures specific for each of the polypeptides . All four of the doxorubicin-selected prostate cancer cell lines exhibited a multidrug resistance phenotype; administration of verapamil restored doxorubicin sensitivity for each of the drug resistant sublines . Although significant MDR1 expression was not detected in any of the parent cell lines before drug exposure by RT-PCR analysis or by immunocytochemistry, both MDR1 mRNA and P-gp protein were expressed by the TSU-PR1 Adr 1000 subline . In contrast, MRP mRNA and protein were present in each of the prostate cancer cell lines before doxorubicin-selection, and an increase in MRP expression appeared to accompany the acquisition of drug resistance in DU 145, PC-3, and PPC-1 doxorubicin-resistant sublines . LRP was variably expressed by each of the parent and resistant cell lines . These data suggest that drug resistance in human prostate cancer may be multifactorial, with MRP and LRP frequently expressed in prostate cancer cells before antineoplastic drug treatment and P-gp expression occasionally acquired after drug exposure.

Biochem Pharmacol, 2000 Dec 1, 60(11), 1611 - 9
Increased mRNA levels of xeroderma pigmentosum complementation group B (XPB) and Cockayne's syndrome complementation group B (CSB) without increased mRNA levels of multidrug-resistance gene (MDR1) or metallothionein-II (MT-II) in platinum-resistant human ovarian cancer tissues; Dabholkar M et al.; Tumor tissue specimens from human ovarian cancer patients were assessed for relative mRNA abundance levels of several genes thought to be involved in the development of in vitro drug resistance in this disease . Higher mRNA levels of Xeroderma pigmentosum group B (XPB), which links DNA repair with DNA transcription, and of Cockayne's syndrome group B (CSB), which is essential for gene-specific repair, were observed in tumor tissues that were clinically resistant to platinum-based chemotherapy, as compared with tissues from patients responding to therapy . In a cohort of 27 patients, mRNA levels of XPB averaged 5-fold higher in platinum-resistant tumors (P = 0.001); and for CSB, mRNA levels averaged 6-fold higher but with greater variability (P = 0.033) . Concurrently, these platinum-resistant tumor tissues did not exhibit significantly higher mRNA levels for the MDR1 (multidrug-resistance) gene (P = 0.134) or of the metallothionein-II (MT-II) gene (P = 0.598) . Since these platinum-resistant tumors also show higher mRNA levels of ERCC1 and XPA, platinum resistance appears to be associated with concurrent up-regulation of four genes (XPA, ERCC1, XPB, and CSB) . These four genes participate in DNA damage excision activity, gene-specific repair, and linkage of DNA repair with DNA transcription . These data suggest that concurrent up-regulation of genes involved in nucleotide excision repair may be important in clinical resistance to platinum-based chemotherapy in this disease.

Emerg Infect Dis, 2000 Nov-Dec, 6(6), 566 - 8
Recent trends in tuberculosis, Japan; Mori T; Despite a decline after World War II, the rate of tuberculosis in Japan remains high . Infection is heavily concentrated in the > or =60-year age group, and 82% of patients are > or =40 years of age . The success rate for treatment of smear-positive patients is 78% . Multidrug-resistant strains of Mycobacterium tuberculosis are rare.

Semin Liver Dis, 2000, 20(3), 273 - 92
Hepatic transport of bile salts; Kullak-Ublick GA et al.; The vectorial secretion of bile salts from blood into bile is a major driving force for bile formation . The basolateral hepatocyte membrane extracts bile salts from sinusoidal blood via Na(+)-dependent and Na(+)-independent membrane transporters . Na(+)-dependent uptake of bile salts is mediated by the Na(+)-taurocholate co-transporting polypeptide, a 51-kDa protein that is exclusively expressed in hepatocytes . Na(+)-independent uptake of bile salts is mediated by the organic anion transporting polypeptides, a superfamily of multispecific bile salt and amphipathic substrate transporters . Within the hepatocyte, bile salts are bound to cytosolic proteins and traverse the cell mainly by diffusion . Transport across the canalicular membrane is the rate-limiting step in overall hepatocellular bile salt excretion and is mediated by the bile salt export pump (BSEP), a homologue of the P-glycoproteins or multidrug resistance gene products . BSEP is a vulnerable target for inhibition by estrogen metabolites, drugs such as cyclosporine A, and abnormal bile salt metabolites, all of which can cause retention of bile salts and consequently intrahepatic cholestasis . Canalicular efflux of divalent sulfated or glucuronidated bile salts is mediated by the multidrug resistance protein 2 (MRP2), which is strongly decreased in cholestasis . Decreased MRP2 expression leads to compensatory increases in the basolateral expression of MRP1 and MRP3, which mediate the sinusoidal efflux of divalent bile salt conjugates and other organic anions . Thus, the hepatocyte can regulate expression levels of individual bile salt transporters during cholestasis to evade hepatotoxic injury.

Semin Liver Dis, 2000, 20(3), 265 - 72
Hepatic secretion of conjugated drugs and endogenous substances; Keppler D et al.; Conjugate export pumps of the multidrug resistance protein (MRP) family mediate the ATP-dependent secretion of anionic conjugates across the canalicular and the basolateral hepatocyte membrane into bile and sinusoidal blood, respectively . Xenobiotic and endogenous lipophilic substances may be conjugated with glutathione, glucuronate, sulfate, or other negatively charged groups and thus become substrates for export pumps of the MRP family . The apical isoform, MRP2 (gene symbol ABCC2), has been localized to the apical membrane of several polarized epithelia and particularly to the canalicular membrane of hepatocytes . Absence of functionally active MRP2 glycoprotein from this membrane domain prevents the secretion of many anionic conjugates into bile . Prototypic endogenous substrates of high affinity for recombinant human MRP2 include bisglucuronosyl bilirubin, monoglucuronosyl bilirubin, and the glutathione S-conjugate leukotriene C4 . Several mutations in the human MRP2 gene have been identified that lead to the absence of MRP2 from the canalicular membrane and to the conjugated hyperbilirubinemia of Dubin-Johnson syndrome . MRP2-mediated conjugate export represents a decisive final step in the detoxification of drugs, toxins, and endogenous substances . The basolateral isoform, MRP3 (gene symbol ABCC3), is upregulated in MRP2 deficiency and in extrahepatic cholestasis . MRP3 mediates the ATP-dependent transport of anionic conjugates, particularly of glucuronides and sulfoconjugates, across the basolateral hepatocyte membrane into sinusoidal blood . The inverse regulation of MRP3 and MRP2 expression under many conditions is consistent with their distinct localization and with a compensatory role of MRP3 in the hepatic secretion of anionic conjugates during impaired transport into bile.

Semin Liver Dis, 2000, 20(3), 245 - 50
Foreword: from classic bile physiology to cloned transporters; Jansen PL; Uptake of drugs and metabolites from the circulation into the liver is facilitated by transporter proteins in the basolateral membrane of the hepatocyte . Among these proteins are the sodium taurocholate cotransporting protein, various multispecific transporters for organic anions and cations, transporters for glucose, amino acids, and prostaglandins . The canalicular membrane contains a number of ATP-dependent transporters belonging to the families of P-glycoproteins and multidrug resistance-associated proteins . Transport across the canalicular membrane represents the rate-determining step in the secretion of compounds from blood to bile . Mutations of genes encoding these canalicular transporters are associated with liver diseases such as progressive familial intrahepatic cholestasis and Dubin-Johnson syndrome . Wilson's disease appears to be due to a defect of a copper-transporting P-type ATPase . Also, bile ductuli contribute to bile formation . Mutations in the CFTR gene, encoding a chloride channel in bile duct epithelial cells, leads to the hepatic component of cystic fibrosis.

Mol Carcinog, 2000 Oct, 29(2), 103 - 11
Elevated expression of hepatic proliferative markers during early hepatocarcinogenesis in hepatitis-B virus transgenic mice lacking mdr1a-encoded P-glycoprotein; Bao JJ et al.; Recent studies have shown that expression levels of the multidrug resistance gene MDR1, which encodes the drug transporter P-glycoprotein, correlate with prognostic outcomes of certain tumor types . These findings suggest that expression of MDR1 may affect tumor behaviors . To address this issue further, we investigated the expression of mdr1a, a human MDR1 homolog, on the development of hepatocellular carcinoma in a transgenic mouse model carrying the liver-targeted expression of human hepatitis-B virus (HBV) surface antigen . The pathogenetic program was compared in HBV mice carrying either mdr1a(+/+) or mdr1a(-/-) . We found that the expressions of proliferative activity markers, Ki67 nuclear antigen, and proliferating cell nuclear antigen were elevated in mdr1a(-/-) mice younger than 10 wk in comparison with those in the same age group of wild-type animals . Replication in the hepatic population as determined by bromodeoxyuridine incorporation tended to support observation that mdr1a(-/-) mice exhibited elevated labeling indices in this age group . Moreover, histologic staining and flow-cytometric analysis showed that the mdr1a(-/-) animals exhibited a higher cell population with polyploidy than did the mdr1a(+/+) counterparts of the same age . However, no significant differences in the expression of the liver-injury markers serum alanine transaminase and aspartate transaminase were observed . Although our results showed that absence of mdr1a expression is correlated with modest enhanced proliferative characteristics in the livers at stage before the development of hepatocellular carcinoma, the overall life spans between these two strains of mice were not significantly different . The implication of these findings to the role of P-glycoprotein in tumor development and cancer chemotherapy is discussed.

J Photochem Photobiol B, 2000 Jun, 56(1), 68 - 77
Dipyridamole and its derivatives modify the kinetics of the electron transport in reaction centers from Rhodobacter sphaeroides; Knox PP et al.; A well known vasodilator dipyridamole (DIP), 2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido{5,4-d}pyrim idine, and its derivatives have recently been shown as potential co-activators (modulators) in the phenomenon of multidrug resistance (MDR) in cancer therapy . They inhibit the specific function of a transmembrane P-glycoprotein responsible for the ex-flux of anti-cancer drugs from tumor cells . To clarify molecular mechanisms of the anti-MDR activity of DIP and its two derivatives, RA25 and RA47, we have studied their effects on electron transport in reaction centers (RC) from purple photosynthetic bacteria Rb . sphaeroides, using RC as a model system . Increasing concentrations of DIP and RA47 progressively accelerate the back electron transfer from the primary quinone acceptor QA to the bacteriochlorophyll dimer Bchl2 (Bchl2+ -QA- recombination) . In the absence of o-phenantroline, when both quinone acceptors QA and QB are involved in the electron transport, RA47 is more effective than DIP . DIP stabilizes the electron on the secondary quinone acceptor QB, the effect manifested as the retardation of Bchl2+ -QB- recombination . Effects of RA25 are negligible in all cases . The drugs are proposed to change the electron transport affecting the RC structural dynamics and the stabilization of the electron on quinone acceptors through modification of H-bonds in the system.

Physiol Res, 2000, 49(4), 447 - 53
Glutathione S-transferase does not play a role in multidrug resistance of L1210/VCR cell line; Bohacova V et al.; Multidrug resistance of cancer cells is often accompanied by the (over)expression of integral plasma membrane P-glycoprotein, an ATP-dependent transport pump for diverse unrelated compounds . The glutathione detoxification system represents another mechanism that may be involved in multidrug resistance . In the multidrug-resistant L1210/VCR cell line obtained by long-term adaptation of parental L1210 cells to vincristine, an increased expression of P-glycoprotein has previously been established . In this paper, we investigated if the glutathione detoxification system is also involved in the multidrug resistance of these cells . L1210/VCR cells with resistance induced by adaptation to vincristine were also found to be cross-resistant to vinblastine, actinomycin D, mitomycin C, doxorubicin and cyclophosphamide . The resistance of the above cells to vincristine and doxorubicin was accompanied by a depression of drug accumulation (which has not yet been established for other drug) . L1210/VCR cells are able to survive better than sensitive cells under conditions when glutathione was depleted by L-buthionine sulfoximine . Nevertheless, L-buthionine sulfoximine did not influence the resistance of L1210/VCR cells to vincristine . Moreover, the presence of sublethal concentrations of cytostatics neither changed the IC50 value of resistant cells to L-buthionine sulfoximine nor the cytoplasmic activity of glutathione S-transferase, the crucial enzyme of glutathione detoxification system . All the above findings indicate that the glutathione detoxification system is not involved in the mechanisms that ensure the multidrug resistance phenotype of L1210/VCR cells.

Int J Cancer, 2000 Dec 1, 88(5), 810 - 9
Mechanism of action of aragusterol a (YTA0040), a potent anti-tumor marine steroid targeting the G(1) phase of the cell cycle; Fukuoka K et al.; Aragusterol A (YTA0040), isolated from the Okinawan marine sponge of the genus Xestospongia, is a potent anti-tumor marine steroid that possesses a unique structural component . This compound showed broad-spectrum anti-proliferative activity against a panel of 14 human cancer cell lines (IC(50) = 0.01-1.6 microM) . P-glycoprotein-mediated, multidrug-resistant cells showed cross-resistance to YTA0040 cells, whereas cisplatin-resistant non-small-cell lung-cancer (NSCLC) sublines showed a collateral sensitivity to YTA0040 . In transplantable murine tumor models, YTA0040 displayed a broad spectrum and high degree of anti-tumor activity when administered i.p . or p.o . (life span T/C = 135-234%) . In P388 murine leukemia cells, YTA0040 caused dose- and time-dependent suppression of nucleic acid and protein synthesis, with protein synthesis being more potently and rapidly inhibited than nucleic acid synthesis . Flow-cytometric analysis revealed that YTA0040 blocked the entry of human NSCLC-derived A549 cells into S phase, leading to arrest in the G(1) phase of the cell cycle . Western blot analysis demonstrated that YTA0040 caused a dose-dependent decrease in the levels of expression of hyperphosphorylated pRb and cyclin A in A549 cells . The level of p53 protein expression was decreased by YTA0040 treatment . A higher concentration of YTA0040 down-regulated the levels of expression of CDK2, CDK4, cyclin D1 and cyclin E . These findings indicated that YTA0040 arrested human NSCLC cells in late G(1) phase of the cell cycle through inhibition of pRb phosphorylation . Inhibition of pRb phosphorylation by YTA0040 resulted from down-regulation of levels of expression of the CDKs and cyclins involved in the G(1)/S transition and not from induction of p53 and/or the CDK inhibitor p21 .

Biochem Biophys Res Commun, 2000 Nov 11, 278(1), 125 - 33
Cloning and initial analysis of the human multidrug resistance-related MVP/LRP gene promoter; Lange C et al.; The lung resistance-related protein (LRP) was identified as the human major vault protein (MVP), and is overexpressed in various multidrug-resistant cancer cell lines and clinical samples . We characterized DNA sequences upstream to the transcription initiation site of the MVP gene in the human non-small cell lung cancer cell line SW-1573 . A 1.9-kb and a shortened 0.7-kb fragment of the 5'-upstream genomic region show strong promoter activity in chloramphenicol acetyltransferase (CAT) reporter assays . The promoter is TATA-less and contains an inverted CCAAT-box and a Sp1 site located near to a p53 binding motif . An alternative 3'-splice site of intron 1 results in a splicing variant within the 5'-untranslated region of MVP mRNA .

World J Surg, 2000 Oct, 24(10), 1183 - 6
Molecular analysis of mechanisms regulating drug sensitivity and the development of new chemotherapy strategies for genitourinary carcinomas; Naito S et al.; The emergence of drug-resistant tumors during treatment remains one of the major obstacles in cancer chemotherapy . Overexpression of P-glycoprotein encoded by the multidrug resistance 1 (MDR1) gene or multidrug resistance-associated protein (MRP) (or both) and decreased expression of DNA topoisomerase II are responsible for expression of the multidrug resistance (MDR) phenotype . The expression of P-glycoprotein is also often observed in untreated cancers showing spontaneous MDR, such as renal cell carcinoma . Regarding cisplatin resistance, decreased cisplatin accumulation, an increase in cisplatin detoxification by glutathione-related enzymes or metallothionein (or both), and increased repair of DNA damage are all considered to play an important role . The combination of reversal agents targeting such drug resistance markers may be a way to improve the outcome of chemotherapy . Regarding the presently available reversal agents, however, clinically relevant chemosensitizing doses cannot be given to humans without inducing significant toxicity . The development of new agents that reverse drug resistance without causing significant toxicity and their clinical application based on the mechanisms regulating drug sensitivity may therefore be a potentially effective new treatment strategy for genitourinary carcinomas.

Lancet, 2000 Jul 22, 356(9226), 297 - 302
Effects of artesunate-mefloquine combination on incidence of Plasmodium falciparum malaria and mefloquine resistance in western Thailand: a prospective study; Nosten F et al.; BACKGROUND: Worsening drug resistance in Plasmodium falciparum malaria is a major threat to health in tropical countries . We did a prospective study of malaria incidence and treatment in an area of highly multidrug-resistant P . falciparum malaria . METHODS: We assessed incidence of P . falciparum malaria and the in-vivo responses to mefloquine treatment over 13 years in two large camps for displaced Karen people on the northwest border of Thailand . During this time, the standard mefloquine dose was first increased, and then combined artesunate and mefloquine was introduced as first-line treatment for uncomplicated P . falciparum malaria . FINDINGS: Early detection and treatment controlled P . falciparum malaria initially while mefloquine was effective (cure rate with mefloquine {15 mg/kg} and sulphadoxine-pyrimethamine in 1985, 98% {95% CI 97-100}), but as mefloquine resistance developed, the cure rate fell (71% {67-77} in 1990) . A similar pattern was seen for high-dose (25 mg/kg) mefloquine monotherapy from 1990-94 . Since the general deployment of the artesunate-mefloquine combination in 1994, the cure rate increased again to almost 100% from 1998 onwards, and there has been a sustained decline in the incidence of P . falciparum malaria in the study area . In-vitro susceptibility of P . falciparum to mefloquine has improved significantly (p=0.003) . INTERPRETATION: In this area of low malaria transmission, early diagnosis and treatment with combined artesunate and mefloquine has reduced the incidence of P . falciparum malaria and halted the progression of mefloquine resistance . We recommend that antimalarial drugs should be combined with artemisinin or a derivative to protect them against resistance.

J Cell Sci, 2000 Dec, 113 Pt 23, 4241 - 51
Reversible induction of rat hepatoma cell polarity with bile acids; Ng KH et al.; A dynamic model for inducing and isolating polarized cell colonies from differentiated rat hepatoma was established with chenodeoxycholic acid (CDCA) . Cells were treated with 75 microM CDCA in a 1% solvent mix (DMSO/ethanol: 0.5%/0.5%) for 11 days and positive Fao-BA1 and C2rev7-BA1 clones were isolated, respectively, from Fao and C2rev7 . Cell polarization in these two clones was demonstrated by (i) the detection of (gamma)-glutamyl transpeptidase activity (gamma)-GT) and the presence of specific proteins, namely aminopeptidase N (APN), bile acid export pump (Bsep), multidrug resistance-associated protein 2 (Mrp2) at the canalicular pole, (ii) the expression of tight junction (ZO-1) and basolateral (1-18) marker proteins, (iii) the presence of regular microvilli in the cavities sealed by tight junctions, and (iv) functional bile canaliculi-like structures with the capacity to metabolise and secrete carboxyfluorescein diacetate dye . The polarized phenotype was maintained for more than 200 cell generations in the presence of CDCA and could be modulated by cell density or omitting the inducing agent . Hence this cellular model is well suited for studies on hepatic differentiation, polarization and bile salt trafficking with therapeutic implications.

J Infect Dis, 2000 Dec, 182(6), 1788 - 90 Epub 2000 Oct 26.
Mutations at amino acid position 315 of the katG gene are associated with high-level resistance to isoniazid, other drug resistance, and successful transmission of Mycobacterium tuberculosis in the Netherlands; van Soolingen D et al.; The prevalence of mutations at amino acid (aa) position 315 in the katG gene of isoniazid (INH)-resistant Mycobacterium tuberculosis isolates in The Netherlands and the mutation's association with the level of INH resistance, multidrug resistance, and transmission were determined . Of 4288 M . tuberculosis isolates with available laboratory results, 295 (7%) exhibited INH resistance . Of 148 aa 315 mutants, 89% had MICs of 5-10 microg/mL, whereas 75% of the other 130 INH-resistant strains had MICs of 0.5-1 microg/mL . Of the aa 315 mutants, 33% exhibited monodrug resistance, compared with 69% of other INH-resistant strains (P<.0001) . Multidrug resistance was found among 14% of the aa 315 mutants and 7% of the other INH-resistant strains (P>.05) . The probability of being in an IS6110 DNA restriction fragment length polymorphism cluster was similar for aa 315 mutants and INH-susceptible strains, but the probability was reduced in other INH-resistant strains . Thus, aa 315 mutants lead to secondary cases of tuberculosis as often as INH-susceptible strains do.

J Infect Dis, 2000 Dec, 182(6), 1765 - 8 Epub 2000 Oct 17.
A deletion mutation in region V of the cytomegalovirus DNA polymerase sequence confers multidrug resistance; Chou S et al.; A patient with AIDS and cytomegalovirus (CMV) retinitis received ganciclovir and foscarnet for 20 and 5 months, respectively, with evidence of periodic disease progression . After this therapy, a CMV isolate from the patient was resistant to ganciclovir, foscarnet, and cidofovir . Sequence analysis showed a known ganciclovir resistance mutation in the viral UL97 phosphotransferase (L595F) and a new mutation in conserved region V of the DNA polymerase gene (pol) sequence (codons 981-982 deleted) . The pol mutation was transferred to a laboratory CMV strain (Towne) by homologous recombination and selection with either ganciclovir or foscarnet . Recombinant viruses containing this deletion showed a 6-8-fold increased ganciclovir resistance and a 3-5-fold increased resistance to both foscarnet and cidofovir, compared with the wild-type CMV . A single mutation in region V of CMV pol can, therefore, confer multiple drug resistance in a clinical isolate.

Leukemia, 2000 Nov, 14(11), 1915 - 20
Decitabine (5-Aza-2'-deoxycytidine) decreased DNA methylation and expression of MDR-1 gene in K562/ADM cells; Ando T et al.; Multidrug resistance (MDR) is a major problem in patients with hematological malignancies . Although drug-resistance is known to be induced by the expression of P-glycoprotein (P-gp) encoded by the MDR-1 gene, little is known about the mechanisms regulating this gene . Herein, we studied the DNA methylation patterns at the enhancer and repressor binding sites of the MDR-1 gene using the human erythroleukemia cell line K562 and its multidrug resistant derivative K562/ADM (adriamycin) . Direct DNA sequence analysis demonstrated methylation to be present at the repressor site (minus 110 GC-box) of the MDR-1 gene in K562/ADM cells, but not in parental K562 cells . Methylation-specific PCR (MSP) analysis yielded similar results . Treatment of K562/ADM cells with 5-Aza-2'-deoxycytidine (decitabine; DAC), an inhibitor of DNA methyltransferase, caused demethylation of the repressor binding site of MDR-1 gene, as assessed by MSP, and also decreased P-gp expression, as assessed by flow cytometric and Northern blot analysis . Although it is generally accepted that DAC upregulates gene expression by demethylating the activator binding sites, our present results suggest that DAC induces down-regulation of P-gp expression as a result of demethylation at the repressor binding site in K562/ADM cells . In this regard, methylation-dependent regulation of the MDR-1 gene in K562/ADM cells is unique.

Anticancer Res, 2000 Sep-Oct, 20(5A), 3221 - 5
Characterization of chemoresistance mechanisms in a series of cisplatin-resistant transitional carcinoma cell lines; Hour TC et al.; We explored the mechanisms of cisplatin resistance in a series of bladder transitional carcinoma cells that are either sensitive or progressively resistant to cisplatin . Resistant lines were raised by chronic exposure of the parental cells to progressively increased concentrations of cisplatin . The cisplatin IC50s of the sensitive and the three resistant cells were 4.3, 25.0, 40.4, and 52.2 microM, respectively . The expressions of glutathione S-transferase pi (GST-pi) and multidrug resistance-associated protein (MRP) were enhanced in a dose-response manner as cells acquired progressive cisplatin resistance . Expression of mdr-1 transcript was detected in the three resistant lines but not in the sensitive line . Glutathione contents were increased in resistant cells, yet the trend of increase did not reach statistical significance (p = 0.061) . In conclusion, transitional carcinoma cells may gain cisplatin resistance through multiple pathways including up-regulation of GST-pi, MRP and possibly mdr-1 . Glutathione contents may play a less significant role in cisplatin chemoresistance.

Anticancer Res, 2000 Sep-Oct, 20(5A), 2891 - 7
The human multidrug resistance gene (MDR-1): immunocytochemical detection of its expression in oral SCC; Lo Muzio L et al.; A large number of oral cancer patients show poor or partial response to chemotherapy and the mechanisms are poorly understood . At present, an MDR-1 product, the P-170 glycoprotein, is the best known of the P-170 family and is involved in resistance to natural product-based chemotherapeutics, including taxanes, anthracyclines, vinca alkaloids, podophyllotoxins and camptothecins . Although several reports suggest that P-170 is clinically relevant in haematological malignancies, its role in solid tumours is not well understood . Its overexpression has been found to be correlated with the poor outcome observed in patients treated with chemotherapy and presenting drug resistance . The aim of this study was to detect the protein expression patterns of MDR-1 product by immunohistochemistry in formalin-fixed-paraffin-embedded tissues . For these reasons, 30 oral SCC and 6 healthy oral mucosa specimens were tested with anti-P-170 antibodies using standard streptavidin-biotin-peroxide technique . Immunohistochemistry demonstrated that 4 cases (66.6%) of normal oral mucosa and 24 cases (80%) of oral SCC showed positivity . Four cases (13.4%) showed strong positivity in tumour areas and complete negativity in normal epithelial cells adjacent to the tumour . No staining was observed in stromal structures, with the exception of the lymphocytic compartment that showed a strong staining as reported in literature for CD56+ and CD8+ cells . Four G1 tumours (33%) and 2 G3 tumour (33%) showed strong positivity in areas with a higher degree of differentiation . P-170 positivity in normal epithelial cells of smoker patients, in differentiated area of neoplasia and negativity or zonal positivity in undifferentiated area of tumour suggested that activation of the MDR-1 gene or selection of intrinsically multidrug resistance neoplastic cells may occur at early stages of tumorigenesis of oral cancers, before the real evidence of cellular transformation . Thus the contact with possible chemical carcinogens, such as those of tobacco smoke, may induce activation of MDR-1 gene . This study was conducted only on untreated carcinomas so for this reason it cannot indicate the real incidence of acquired multidrug resistance . The data of MDR-1 product expression by immunohistochemistry in oral SCC might suggest that an overexpression of this protein could constitute a hallmark of potential more aggressive phenotype for this type of neoplasia and a rapid method for pre-screening tumours for a constitutive multidrug resistance in order to orientate the cancer treatment.

Biochem Biophys Res Commun, 2000 Nov 2, 277(3), 757 - 63
Enhanced drug resistance in cells coexpressing ErbB2 with EGF receptor or ErbB3; Chen X et al.; Overexpression of ErbB2 has been found in approximately 25-30% of human breast cancers and has been shown to render the cancer cells more resistant to chemotherapy . However, it is not clear whether ErbB2 overexpression renders the cells more resistant to specific anti-cancer drugs or renders the cells more resistant to a broad range of anti-cancer drugs . It is not clear how the function of ErbB2 in drug resistance is related to expression and activation of the other ErbB receptors . In this communication, we showed that several breast cancer cell lines including BT20, BT474, MCF-7, MDA-MB-453, and SKBR-3 cells had a similar pattern of resistance to a broad range of anti-cancer drugs including 5-Fluorouracil, Cytoxan, Doxorubincin, Taxol, and Vinorelbin, suggesting a mechanism of multidrug resistance . High expression of P-glycoprotein and the ErbB receptors contribute to drug resistance of these breast cancer cells; however, overexpression of ErbB2 alone is not a major factor in determining drug resistance . To further determine the role of the ErbB receptors in drug resistance, we selected various NIH 3T3 cell lines that specifically expressed EGF receptor (EGFR), ErbB2, ErbB3, EGFR/ErbB2, EGFR/ErbB3, or ErbB2/ErbB3 . A cytotoxicity assay showed that expression of ErbB2 alone did not significantly enhance drug resistance, whereas coexpression of either EGFR or ErbB3 with ErbB2 significantly enhanced drug resistance . Moreover, ErbB2 was highly phosphorylated in NIH 3T3 cells that coexpress ErbB2 with either EGFR or ErbB3, but not in NIH 3T3 cells that express ErbB2 alone . Together, our results suggest that coexpression of EGFR or ErbB3 with ErbB2 induces high phosphorylation of ErbB2 and renders the cells more resistant to various anti-cancer drugs .

J Clin Microbiol, 2000 Nov, 38(11), 3919 - 25
Genetic diversity of protease and reverse transcriptase sequences in non-subtype-B human immunodeficiency virus type 1 strains: evidence of many minor drug resistance mutations in treatment-naive patients; Vergne L et al.; Most human immunodeficiency virus (HIV) drug susceptibility studies have involved subtype B strains . Little information on the impact of viral diversity on natural susceptibility to antiretroviral drugs has been reported . However, the prevalence of non-subtype-B (non-B) HIV type 1 (HIV-1) strains continues to increase in industrialized countries, and antiretroviral treatments have recently become available in certain developing countries where non-B subtypes predominate . We sequenced the protease and reverse transcriptase (RT) genes of 142 HIV-1 isolates from antiretroviral-naive patients: 4 belonged to group O and 138 belonged to group M (9 subtype A, 13 subtype B, 2 subtype C, 5 subtype D, 2 subtype F1, 9 subtype F2, 4 subtype G, 5 subtype J, 2 subtype K, 3 subtype CRF01-AE, 67 subtype CRF02-AG, and 17 unclassified isolates) . No major mutations associated with resistance to nucleoside reverse transcriptase inhibitors (NRTIs) or protease inhibitors were detected . Major mutations linked to resistance to non-NRTI agents were detected in all group O isolates (A98G and Y181C) and in one subtype J virus (V108I) . In contrast, many accessory mutations were found, especially in the protease gene . Only 5.6% of the 142 strains, all belonging to subtype B or D, had no mutations in the protease gene . Sixty percent had one mutation, 22.5% had two mutations, 9.8% had three mutations, and 2.1% (all group O strains) had four mutations . In order of decreasing frequency, the following mutations were identified in the protease gene: M36I (86.6%), L10I/V (26%), L63P (12.6%), K20M/R (11.2%), V77I (5.6%), A71V (2.8%), L33F (0.7%), and M46I (0.7%) . R211K, an accessory mutation associated with NRTI resistance, was also observed in 43.6% of the samples . Phenotypic and clinical studies are now required to determine whether multidrug-resistant viruses emerge more rapidly during antiretroviral therapy when minor resistance-conferring mutations are present before treatment initiation.

EMBO J, 2000 Nov 1, 19(21), 5752 - 61
Role of a dipeptide insertion between codons 69 and 70 of HIV-1 reverse transcriptase in the mechanism of AZT resistance; Mas A et al.; The 3'-azido-3'-deoxythymidine (AZT)-resistant pheno type of a heavily mutated human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) carrying a dipeptide (Ser-Ser) insertion between codons 69 and 70 as well as other mutations related to resistance to RT inhibitors has been studied . Recombinant virus carrying this variant RT (termed SS RT) showed reduced susceptibility to all nucleoside RT inhibitors in clinical use, particularly to AZT . In the presence of ATP, recombinant SS RT had an increased ability to remove the 3'-terminal nucleotide from AZT- terminated primers and extend the unblocked primer, compared with wild-type HIV-1 RT (BH10 isolate) . Insertion of two serines in the sequence context of BH10 RT did not affect the ATP-dependent phosphorolytic activity of the enzyme, and had no influence in resistance to RT inhibitors . However, SS RT mutants lacking the dipeptide insertion or bearing a four-serine insertion showed reduced ATP-dependent phosphorolytic activity that correlated with increased AZT sensitivity, as determined using a recombinant virus assay . Therefore, the insertion appears to be critical to enhance AZT resistance in the sequence context of multidrug-resistant HIV-1 RT.

Cancer Res, 2000 Oct 15, 60(20), 5761 - 6
Extensive contribution of the multidrug transporters P-glycoprotein and Mrp1 to basal drug resistance; Allen JD et al.; Despite accumulating evidence that multidrug resistance transporter proteins play a part in drug resistance in some clinical cancers, it remains unclear whether the relatively low levels of multidrug resistance transporter expression found in most untreated tumors could substantially affect their basal sensitivity to antineoplastic drugs . To shed light on this problem, the drug sensitivities of wild-type mouse cell lines were compared with those of lines in which the Mdr1a and Mdr1b genes encoding P-glycoprotein (P-gp) were inactivated and lines in which the Mrp1 gene was inactivated in addition to Mdr1a and Mdr1b . These models permit a clean dissection of the contribution of each transporter to drug resistance at expression levels similar to those in normal tissues and avoid complications that might arise from previous exposure of cell lines to drug selection . For substrate drugs, we found that these contributions can indeed be very substantial . Lines lacking functional P-gp were, on average, markedly more sensitive to paclitaxel (16-fold), anthracyclines (4-fold) and Vinca alkaloids (3-fold) . Lines lacking both P-gp and Mrp1 were (compared with wild-type lines) hypersensitive to an even broader array of drugs, including epipodophyllotoxins (4-7-fold), anthracyclines (6-7-fold), camptothecins (3-fold), arsenite (4-fold) and Vinca alkaloids, especially vincristine (28-fold) . Thus, even very low levels of P-gp and Mrp1 expression that may be difficult to detect in tumors could significantly affect their innate sensitivity to a wide range of clinically important substrate drugs . An implication is that the use of resistance reversal agents to sensitize drug-naive tumors may be appropriate in more cases than is presently appreciated.

Life Sci, 2000 Sep 15, 67(17), 2117 - 24
Inhibition of P-glycoprotein expression and reversal of drug resistance of human hepatoma HepG2 cells by multidrug resistance gene (mdr1) antisense RNA; Chan JY et al.; The development of multiple drug resistance in tumor cells is a significant problem in cancer therapy . In human, one of the reasons causing the resistance is due to the overexpression of the mdr1 gene product, P-glycoprotein . In our study, we had developed multiple drug resistant HepG2 cell line (HepG2/DR) . To reverse the resistance, HepG2-DR cells were treated with antisense RNA against mdr1 gene . Total RNA and protein were extracted from the transfected cells . Northern analysis showed that mRNA level of mdr1 was decreased whereas a reduction in P-glycoprotein was detected by Western blot . By using flow cytometry, the ability of intracellular doxorubicin retention increased and drug efflux decreased in the treated cells . The result also showed that the cellular sensitivity to doxorubicin, vincristine and methotrexate measured in IC50 increased 83.3% 84.6% and 50% respectively . All these findings suggested that the expression of p-glycoprotein was successfully inhibited by antisense RNA and the drug resistance was reduced.

J Biol Chem, 2001 Jan 12, 276(2), 1138 - 45
Multiple Yap1p-binding sites mediate induction of the yeast major facilitator FLR1 gene in response to drugs, oxidants, and alkylating agents; Nguyen DT et al.; The bZip transcription factor Yap1p plays an important role in oxidative stress response and multidrug resistance in Saccharomyces cerevisiae . We have previously demonstrated that the FLR1 gene, encoding a multidrug transporter of the major facilitator superfamily, is a transcriptional target of Yap1p . The FLR1 promoter contains three potential Yap1p response elements (YREs) at positions -148 (YRE1), -167 (YRE2), and -364 (YRE3) . To address the function of these YREs, the three sites have been individually mutated and tested in transactivation assays . Our results show that (i) each of the three YREs is functional and important for the optimal transactivation of FLR1 by Yap1p and that (ii) the three YREs are not functionally equivalent, mutation of YRE3 being the most deleterious, followed by YRE2 and YRE1 . Simultaneous mutation of the three YREs abolished transactivation of the promoter by Yap1p, demonstrating that the three sites are essential for the regulation of FLR1 by Yap1p . Gel retardation assays confirmed that Yap1p differentially binds to the three YREs (YRE3 > YRE2 > YRE1) . We show that the transcription of FLR1 is induced upon cell treatment with the oxidizing agents diamide, diethylmaleate, hydrogen peroxide, and tert-butyl hydroperoxide, the antimitotic drug benomyl, and the alkylating agent methylmethane sulfonate and that this induction is mediated by Yap1p through the three YREs . Finally, we show that FLR1 overexpression confers resistance to diamide, diethylmaleate, and menadione but hypersensitivity to H(2)O(2), demonstrating that the Flr1p transporter participates in Yap1p-mediated oxidative stress response in S . cerevisiae.

Int J Tuberc Lung Dis, 2000 Oct, 4(10), 947 - 55
Surveillance of drug-resistant tuberculosis and molecular evaluation of transmission of resistant strains in refugee and non-refugee populations in North-Eastern Kenya; Githui WA et al.; SETTING: Three refugee camp complex clinics and an adjacent non-refugee treatment centre in North-Eastern Kenya . OBJECTIVES: To use conventional and molecular epidemiology tools to determine: 1) the prevalence of drug resistance in newly diagnosed patients with smear-positive pulmonary tuberculosis in refugee and non-refugee populations; 2) risk factors for resistance in the two populations; and 3) whether IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping showed similarities in DNA fingerprinting patterns of drug-resistant isolates that could infer transmission within and between the two populations . RESULTS: Of 241 isolates from the camps, 44 (18.3%) were resistant to one or more drugs, seven of which (2.9%) were multidrug-resistant TB (MDR-TB) . Of 88 isolates from the non-refugees, five (5.7%) were resistant to one or more drugs without MDR-TB . Drug resistance was higher in the camps than in the non-refugee population (OR = 3.7; 95%CI 1.42-9.68; P < 0.007) . Resistance was significantly higher in one camp compared with the other two, despite a comparable ethnic distribution . Unusually, females were more associated with drug resistance than their male counterparts in both populations (OR = 2.3; 95%CI 1.2-4.8; P = 0.008) . There was evidence of transmission of streptomycin-resistant strains in the refugee population . DNA fingerprints of resistant strains from the non-refugee population were unique and different from those in the refugee camps . CONCLUSION: The observed high levels of drug resistance and MDR-TB, combined with evidence of transmission of strains resistant to streptomycin in the refugee population, suggest a need for strengthened TB control programmes in settings with a high risk of developing drug-resistant strains.

Int J Tuberc Lung Dis, 2000 Oct, 4(10), 931 - 9
Causes and costs of hospitalization of tuberculosis patients in the United States; Taylor Z et al.; OBJECTIVE: To examine the costs, lengths of stay and patient characteristics associated with tuberculosis (TB) hospitalizations . METHODS: A prospective cohort study of 1493 TB patients followed from diagnosis to completion of therapy at 10 public health programs and area hospitals in the US . The main outcome measures were the following: 1) occurrence, 2) cost, and 3) length of stay of TB-related hospitalizations . RESULTS: There were 821 TB-related hospitalizations among the study participants; 678 (83%) were initial hospitalizations and 143 (17%) were hospitalizations during the treatment of TB . Patients infected with human immunodeficiency virus (HIV) (OR 1.8, 95% CI 1.2-2.6), and homeless patients (OR, 1.7 95% CI 1.1-2.8) were at increased risk of being hospitalized at diagnosis . Homeless patients (RR 2.5, 95%CI 1.5-4.3), patients who used alcohol excessively (RR 1.9, 95% CI 1.2-3.0), and patients with multidrug-resistant TB (RR 5.7, 95% CI 2.7-11.8) were at increased risk of hospitalization during treatment . The median length of stay varied from 9 to 17 days, and median costs per hospitalization varied from $6441 to $12968 among the sites . CONCLUSION: Important social factors, HIV infection, and local hospitalization practice patterns contribute significantly to the high cost of TB-related hospitalizations . Efforts to address these specific factors are needed to reduce the cost of preventable hospitalizations.

Int J Tuberc Lung Dis, 2000 Oct, 4(10), 904 - 10
The impact of directly-observed treatment on the epidemiology of tuberculosis in Beijing; Zhang LX et al.; SETTING: Fully supervised chemotherapy, or directly observed treatment (DOT), for newly detected smear-positive cases in Beijing, has been successfully implemented for two decades . OBJECTIVE: To evaluate the progress made in tuberculosis control, and in particular to evaluate the impact of DOT on tuberculosis epidemiology in Beijing . DESIGN: Epidemiological parameters on tuberculosis, consisting of mortality, prevalence, notification rate, tuberculous meningitis in children and initial drug resistance rate, were collected and analysed . Their trends were evaluated and compared with DOT implemented for new smear-positive cases in Beijing from 1978 to 1996 . RESULTS: The coverage of DOT for new smear-positive pulmonary tuberculosis cases has increased from 10% in 1978 to more than 90% since 1990 . Since DOT was introduced in 1978, mortality from tuberculosis has declined by an average of more than 7% per year . The reduction rate of 17.2%, and the rates of chronic cases and tuberculous meningitis in children decreased dramatically . The rate of newly registered smear-positive cases decreased from 18.9/100000 in 1986 to 7.3/100000 in 1996, giving an average annual reduction rate of 9.1 during this period . Initial resistance to isoniazid and streptomycin decreased from respectively 13.9% and 12.3% in 1978-1979 to 4.2% and 5.8% by 1996 . The level of multidrug resistance was low and stable, at 0.8% in 1996 . CONCLUSION: The experience of the Beijing tuberculosis control programme convincingly demonstrates that it is possible to improve the epidemiological situation rapidly in a low-income country, at very low cost and in a manner that is self-sufficient and sustainable.

Gene, 2000 Oct 17, 257(1), 67 - 75
Rab6c, a new member of the rab gene family, is involved in drug resistance in MCF7/AdrR cells; Shan J et al.; A new Rab6 homolog cDNA, Rab6c, was discovered by a hypermethylated DNA fragment probe that was isolated from a human multidrug resistant (MDR) breast cancer cell line, MCF7/AdrR, by the methylation sensitive-representational difference analysis (MS-RDA) technique . Rab6c was found to be under-expressed in MCF7/AdrR and MES-SA/Dx5 (a human MDR uterine sarcoma cell line) compared with their non-MDR parental cell lines . MCF7/AdrR cells expressing the exogenous Rab6c exhibited less resistance to several anti-cancer drugs, such as doxorubicin (DOX), taxol, vinblastine, and vincristine, than the control cells containing the empty vector . Flow cytometry experiments confirmed that the transfectants' diminished resistance to DOX was caused by increased drug accumulation induced by the exogenous Rab6c . These results indicate that Rab6c is involved in drug resistance in MCF7/AdrR cells.

Cancer Chemother Pharmacol, 2000, 46(4), 329 - 37
Multiple factors other than p53 influence colon cancer sensitivity to paclitaxel; Sharma N et al.; PURPOSE: To determine factors which influence the sensitivity of human colorectal carcinoma cell lines to paclitaxel . METHODS: The paclitaxel sensitivity of ten human colorectal carcinoma cell lines, and a panel of RKO colon carcinoma cell lines, isogenic except for p53 status, were studied . The inhibitory concentrations causing a 50% decrease in growth (IC50) were assayed after 3, 24, and 96 h after paclitaxel exposure . The doubling time (DT) and cell cycle parameters of cells were also measured . The expression of the multidrug resistance glycoprotein-1 (MDR-1), bcl-2 and bax was quantitatively assessed by immunoblotting . RESULTS: Mean IC50 values at 24 and 96 h drug exposure were about 1.5 logs lower than the IC50 values at 3 h, regardless of the p53 status . No difference was found between the IC50 values of wild-type and mutant p53 cells, or among the RKO panel of cells . Correlation analysis showed that: (1) resistance was associated with longer DTs, but this was generally abated by a 96-h exposure; (2) with a 3-h exposure, the combination of MDR, bcl-2 and bax parameters with DT (DT + MDR + bcl-2 bax) best correlated with IC50 values (r = 0.77); (3) with a 96-h exposure, in spite of the generally decreased IC50 values, a combination of MDR-1, bcl-2 and bax parameters (MDR + bcl-2-bax) best correlated with the IC50 values (r = 0.71) . CONCLUSIONS: These results suggest that the exposure duration, DT, and expression of MDR-1, bcl-2 and bax each contribute to paclitaxel sensitivity of human colorectal carcinoma cells . In assessing paclitaxel drug resistance, multiple factors should always be considered . There may be a therapeutic window for taxanes in colon cancer by optimizing pharmacokinetics and modulating MDR-1 and bcl-2 resistance factors.

Cancer Chemother Pharmacol, 2000, 46(4), 305 - 12
In vitro evaluation of newly developed chalcone analogues in human cancer cells; De Vincenzo R et al.; PURPOSE: Among flavonoids, chalcones have been identified as interesting compounds having chemopreventive and antitumor properties . We studied a panel of newly developed chalcone analogues (S1-S10) using MDA-MB 231 and MCF-7 ADRr breast cancer cells and the T-leukemic Jurkat cell line . Quercetin was used as the reference compound . METHODS: Antiproliferative activity was evaluated by cell counts performed after 72 h of exposure to the drugs . DNA analysis and redox activity were evaluated using flow cytometry . Apoptosis was assessed by morphological analysis, using YOYO-1 as DNA dye; p-glycoprotein function was ascertained by quantitating the efflux of rhodamine 123 . RESULTS: All cells were sensitive to chalcone analogues yielding IC50 in micromolar concentrations with the following order regardless of the multidrug resistance (MDR) status: S1 > S2 > quercetin . S1 and S2, the most active compounds, were selected to evaluate their effect on the cell cycle, apoptosis, redox activity, and modulation of the p-glycoprotein function . No significant perturbation in cell cycle was seen with concentration up to 1 microM after 24 h . After 72 h a slight increase in G2/M block and DNA fragmentation occurred at 10 microM . Morphological analysis of apoptosis showed that chalcone analogues induced apoptosis to a higher extent than quercetin . Redox analysis demonstrated that all substances were able to increase intracellular thiol levels, which returned to baseline value after 24 h for all drugs except quercetin . Production of reactive oxygen species was essentially unaffected by all compounds . Finally, in MDR-positive MCF-7 ADRr cells chalcone analogues were unable to modulate p-glycoprotein function while quercetin was able to . CONCLUSIONS: Newly developed S1 and S2 chalcones have a different but higher antitumor activity than quercetin and could be considered as potential new anticancer drugs.

Medicina (B Aires), 2000, 60(3), 357 - 60
{Coxitis due to multidrug resistant Mycobacterium tuberculosis in a HIV negative patient}; Palmero DJ et al.; A case of an HIV negative female patient with coxofemoral arthritis of tuberculous etiology, multidrug-resistant strain, and connective tissue disease associated to glucocorticoid therapy is reported . The patient was treated with cycloserine, ethambutol, p-aminosalicylic acid and ofloxacin, with improvement of the joint lesions . Previous publications on this subject are reviewed.

Jpn J Cancer Res, 2000 Oct, 91(10), 1001 - 6
The specific expression of three novel splice variant forms of human metalloprotease-like disintegrin-like cysteine-rich protein 2 gene inBrain tissues and gliomas; Harada T et al.; We have previously identified 67 exons on a yeast artificial chromosome contig spanning 1.5 Mb around the multidrug resistance 1 gene region of human chromosome 7q21.1 . In this study, we identified three novel cytoplasmic variants (MDC2-gamma, MDC2-delta, and MDC2-epsilon) of the human metalloprotease-like disintegrin-like cysteine-rich protein 2 (MDC2) among these exons by screening a human brain cDNA library and also by using a reverse transcription polymerase chain reaction . Genomic sequence analysis strongly supported the idea that the variations in the cytoplasmic domain were generated by alternative splicing . The expression of MDC2 variant forms in human brain tissue and gliomas was examined by reverse transcription polymerase chain reaction and RNase protection assay . MDC2-epsilon was expressed only in the cortical and hippocampal regions in human brain, but not in gliomas . In contrast, MDC2-gamma was a major form expressed in human gliomas . Specific expression of these cytoplasmic variants of MDC2 in human brain and its malignancies is discussed.

Thorax, 2000 Nov, 55(11), 962 - 3
Resource implications of patients with multidrug resistant tuberculosis; White VL et al.; BACKGROUND: Multidrug resistant tuberculosis (MDR TB) requires a complex drug regimen and lengthy multidisciplinary care . The financial cost of successful management of each case is potentially large . METHODS: The costs of managing nine HIV negative patients with pulmonary MDR TB were compared with 18 age group and ethnicity matched controls with fully sensitive disease . Calculations included: cost of outpatient visits and inpatient stays including negative pressure isolation; costs of drug provision and toxicity monitoring; costs of additional procedures and multidisciplinary referrals . RESULTS: The mean cost of managing a case of pulmonary MDR TB was in excess of 60,000 pounds sterling and for sensitive disease it was 6040 pounds sterling . CONCLUSIONS: Clinicians and healthcare commissioning authorities may both be underestimating the costs of managing MDR TB, and accordingly the consequences for units dealing with such cases may be serious . Funding of care for MDR TB in the UK requires strategic decisions at regional or governmental level.

J Biol Chem, 2001 Jan 12, 276(2), 1479 - 85
A transcriptional regulator of a pristinamycin resistance gene in Streptomyces coelicolor; Folcher M et al.; Pip is a pristinamycin-induced transcriptional regulator protein detected in many Streptomyces species by its ability to specifically bind sequence motifs within the promoter of a Streptomyces pristinaespiralis multidrug resistance gene (ptr) . To investigate the possible role of Pip in regulating multidrug resistance, it was purified from a genetically characterized species, Streptomyces coelicolor, utilizing an affinity matrix of the ptr promoter conjugated to magnetic beads . Reverse genetics identified the corresponding locus and confirmed that it encoded Pip, a protein belonging to the TetR family of procaryotic transcriptional repressors . Pip binding motifs were located upstream of the adjacent gene pep, encoding a major facilitator antiporter homologous to ptr . In vivo analysis of antibiotic susceptibility profiles demonstrated that pep conferred elevated levels of resistance only to pristinamycin I (PI), a streptogramin B antibiotic having clinical importance . Purified recombinant Pip was a dimer (in the presence or absence of PI) and displayed a high affinity for its palindromic binding motifs within the ptr promoter and the upstream region of pep . The Pip/ptr promoter complex was dissociated by PI but not by any of the other nonstreptogramin antibiotics that were described previously as transcriptional inducers . These procaryotic regulatory elements served as the basis for the development of systems allowing repression or induction of cloned genes in mammalian and plant cells in response to streptogramin antibiotics (including pristinamycin, virginiamycin, and Synercid(R)).

Blood, 2000 Nov 1, 96(9), 3286 - 9
Drug-resistant human cytomegalovirus infection in children after allogeneic stem cell transplantation may have different clinical outcomes; Eckle T et al.; Three seropositive pediatric recipients of allogeneic stem cell transplantation out of a group of 42 patients receiving T-cell-depleted, unrelated transplants and 37 patients receiving T-cell-depleted, haploidentical transplants were monitored longitudinally for human cytomegalovirus (HCMV) infection and the emergence of antiviral drug resistance . Early in the posttransplant course, all 3 patients developed HCMV mutations conferring drug resistance to ganciclovir . One child additionally developed multidrug resistance to foscarnet and cidofovir, with mutations in the viral phosphotransferase gene (UL97) and the DNA-polymerase gene (UL54) being found . These data show that resistant HCMV infection does not necessarily correlate with a severe clinical outcome . The early detection of genotypic resistance up to 129 days before the emergence of phenotypic resistance and the dissociation of resistance patterns among different body sites emphasize the importance of genotypic analyses of different DNA specimens for an efficient antiviral therapy . T-cell-depleted children having transplantation might be at an increased risk for the development of drug resistance.

Semin Oncol, 2000 Oct, 27(5), 540 - 59
New approaches to acute lymphoblastic leukemia in adults: where do we go?
Hoelzer D, Gokbuget N.
The optimization of conventional treatment approaches, such as chemotherapy, stem cell transplantation (SCT), and supportive care, and the exploration of new approaches will hopefully further improve the outcome of adults with acute lymphoblastic leukemia (ALL) . Subgroup-adjusted treatment has already greatly improved treatment outcomes in T- and mature B-cell ALL . These approaches should be further refined, for example, in T-ALL with cyclophosphamide and cytarabine, in pro-B ALL with high-dose cytarabine (HdAC), in B-precursor ALL with high-dose methotrexate (HdM) and 6-mercaptopurine (6-MP), and in mature B-ALL with HdM and HdAC . The indications for SCT will be extended to include elderly patients undergoing allogeneic mini-transplants, and tumor eradication will be improved by better conditioning regimens such as radioimmunoconjugates and methods to induce the graft-versus-leukemia (GvL) effect, such as donor leukocyte infusions (DLI) or allogeneic mini-transplants applied after autologous transplants . Molecular therapeutic approaches, for example, those directed against the fusion protein BCR-ABL with ABL-tyrosine kinase inhibitor, are on the way to creating a new avenue for the treatment of ALL . In the future, drug resistance should be exploited as a pretherapeutic test for treatment strategies, but whether multidrug resistance modulation with available drugs will be used in ALL remains open . Evaluation of the pharmacokinetics of cytostatic drugs and the pharmacogenomics of cytostatic agents in adult ALL may contribute to the development of individualized treatment strategies with higher efficacy and lower toxicity . Minimal residual disease (MRD) evaluation is attractive in adult ALL, because it can be determined in a very high percentage of patients . It has been shown to be predictive for relapse and might be of benefit for redefinition of complete remission (CR), for determination of the efficacy of single treatment elements, and for treatment tailoring during the course of disease . New treatment approaches include also several forms of immunotherapy for B- as well as T-lineage ALL; after the demonstration that such approaches are also effective in ALL, their optimal place in the treatment strategy for adult ALL can be determined.

Morphologie, 2000 Jun, 84(265), 11 - 5
Nucleus labeling or membrane labeling for studying the proliferation of drug treated cells?
Boutonnat J, Barbier M, Ronot X, Seigneurin D.
Proliferation and multidrug resistance status are key predictors of therapeutic outcome in acute myeloid leukemia (AML) . This study compared cell cycle analysis (nuclear labeling) with cell division analysis (membrane labeling, PKH67) for studying the proliferation of cells cultured with daunorubicin (DNR) and/or Cytarabine (Ara-C), drugs commonly used in AML treatment . PKHs are a family of lipophilic, fluorescent membrane intercalating dyes . When labeled cells divide, the resulting daughter cells receive half the label, reducing fluorescence intensity to one-half that of the parent cells . DNR has the disadvantage of overlapping the spectrum of propidium iodide (PI), which is the most commonly used marker of membrane integrity . In this study, necrosis was evaluated using TOTO-3, a marker of nucleic acids emitting fluorescence above 645 nm and which incorporates cells with disrupted membranes . Comparison of cell cycle analysis with cell division for studying proliferation revealed that PKH67 was a marker of choice for analyzing the mitotic process in cells cultured with drugs.

Pharm, Sci . Technol . Today . 2000 Oct 1, 3(10), 366 - 368
Monitor: PROFILE; Zhang Y et al.; P-glycoprotein and multidrug resistance-associated protein transporters in the blood-brain barrier: another important brick in the wall

J Pharmacol Exp Ther, 2000 Nov, 295(2), 512 - 8
Altered hepatobiliary disposition of acetaminophen glucuronide in isolated perfused livers from multidrug resistance-associated protein 2-deficient TR(-) rats; Xiong H et al.; Previous studies have demonstrated that phenobarbital treatment impairs the biliary excretion of acetaminophen glucuronide (AG), although the transport system(s) responsible for AG excretion into bile has not been identified . Initial studies in rat canalicular liver plasma membrane vesicles indicated that AG uptake was stimulated modestly by ATP, but not by membrane potential, HCO(3)(-), or pH gradients . To examine the role of the ATP-dependent canalicular transporter multidrug resistance-associated protein 2 (Mrp2)/canalicular multispecific organic anion transporter (cMOAT) in the biliary excretion of AG, the hepatobiliary disposition of acetaminophen, AG, and acetaminophen sulfate (AS) was examined in isolated perfused livers from control and TR(-) (Mrp2-deficient) Wistar rats . Mean bile flow in TR(-) livers was approximately 0.3 microl/min/g of liver ( approximately 4-fold lower than control) . AG biliary excretion was decreased (>300-fold) to negligible levels in TR(-) rat livers, indicating that AG is an Mrp2 substrate . Similarly, AS biliary excretion in TR(-) livers was decreased ( approximately 5-fold); however, concentrations were still measurable, suggesting that multiple mechanisms, including Mrp2-mediated active transport, may be involved in AS biliary excretion . AG and AS perfusate concentrations were significantly higher in livers from TR(-) compared with control rats . Pharmacokinetic modeling of the data revealed that the rate constant for basolateral egress of AG increased significantly from 0.028 to 0.206 min(-1), consistent with up-regulation of a basolateral organic anion transporter in Mrp2-deficient rat livers . In conclusion, these data indicate that AG biliary excretion is mediated by Mrp2, and clearly demonstrate that substrate disposition may be influenced by alterations in complementary transport systems in transport-deficient animals.

Graefes Arch Clin Exp Ophthalmol, 2000 Sep, 238(9), 727 - 32
Multidrug resistance-associated proteins in glaucoma surgery; Esser J et al.; BACKGROUND: Multidrug resistance (MDR) describes the phenomenon of cross-resistance between different cytostatic agents which are structurally and functionally dissimilar . Two recently discovered proteins, lung resistance protein (LRP) and the multidrug resistance-related protein (MRP) have been implicated in the development of MDR . Since resistance to chemotherapeutic agents is a common problem in filtration surgery, especially in cases of complicated glaucoma, we decided to investigate the presence of MRP and LRP in surgically removed Tenon specimens from glaucoma patients . METHODS: The presence of MRP and LRP in surgically removed Tenon tissue (n=15) was analyzed by immunohistochemistry . The expression by cultured Tenon fibroblasts was assessed by reverse-transcriptase polymerase chain reaction (RT-PCR) and fluorocytometry . RESULTS: LRP expression was detected in 8 of 10 Tenon specimens . Positive staining for MRP was obtained in 5 of 10 specimens . Negative controls with non-immune mouse IgG did not display any specific staining . RT-PCR and fluorocytometry revealed constitutive expression of MRP and LRP, at the RNA and protein level respectively, that was unaltered by pretreatment of the cells with mitomycin C or 5-fluorouracil . CONCLUSION: Our results demonstrate, that besides P-glycoprotein, other components of the MDR-system are present in conjunctival fibroblasts . Future developments in the use of chemotherapeutic agents in association with of filtration surgery need to take account of the presence of these counteracting mechanisms.

Biochemistry, 2000 Oct 24, 39(42), 13026 - 33
Multidrug resistance protein MRP1 reconstituted into lipid vesicles: secondary structure and nucleotide-induced tertiary structure changes; Manciu L et al.; Multidrug resistance protein MRP1 is an ATP-dependent drug efflux pump that confers resistance in human cancer cells to various chemotherapeutic drugs . We have reconstituted purified MRP1 in lipid vesicles . The reconstituted protein conserves ATPase and drug transport activity . Structural analysis of MRP1 was investigated by infrared spectroscopy for the first time . This technique offers a unique opportunity to determine structural parameters characterizing a membrane protein in its lipid environment . Addition of different ligands (MgATP, MgATPgammaS, MgADP and P(i), and MgADP) did not significantly affect the MRP1 secondary structure, which is made of 46% alpha-helix, 26% beta-sheet, 12% beta-turns, and 17% random coil . Binding of MgATP increased the protein accessibility to the solvent, suggesting a modification in the tertiary organization of the protein . Hydrolysis of MgATP to MgADP and P(i) did not significantly change the global accessibility of the protein . Release of P(i), after hydrolysis, caused a decrease in the accessibility of MRP1 to the water phase which brings the protein back to its initial conformation . All together, the data demonstrate that MRP1 adopts different structures during its catalytic cycle.

Cancer Biother Radiopharm, 2000 Aug, 15(4), 339 - 46
In vitro comparison of sestamibi, tetrofosmin, and furifosmin as agents for functional imaging of multidrug resistance in tumors; Muzzammil T et al.; Sestamibi, tetrofosmin, and furifosmin are 99mTc-labeled myocardial perfusion imaging agents which have been shown to be substrates for P-glycoprotein (Pgp), the multidrug-resistance transporter which is overexpressed in some tumors . The three tracers were directly compared in vitro in the human breast cancer cell line MCF7-WT and two multidrug-resistant variants, MCF7-BC19 (MDR1 gene transfected) and MCF7-AdrR (doxorubicin selected) . Tracer accumulation over the course of 60 minutes was determined . Dose-response curves were generated for two modulators of Pgp function, GG918 and PSC833 . The general shape of accumulation curves for the three tracers in MCF7-WT cells was similar, with accumulation levels being sestamibi > tetrofosmin > furifosmin . Accumulation of sestamibi and furifosmin in MCF7-BC19 cells was reduced to 10% and 21% of MCF7-WT levels, respectively, but this accumulation deficit could be completely reversed by addition of 0.1 microM GG918 or 2 microM PSC833 . Accumulation of sestamibi and tetrofosmin in MCF7-AdrR cells was 1.6% and 12% of MCF7-WT levels, respectively, and could only be enhanced to 30% and 45% of MCF7-WT levels by addition of GG918 or PSC833 . In contrast, furifosmin showed similar levels of accumulation in MCF7-WT and MCF7-BC19 cells, slightly lower levels in MCF7-AdrR cells, and no consistent response to Pgp modulators . These results support the continued investigation of sestamibi and tetrofosmin as agents for functional imaging of multidrug resistance in human cancer.

Cancer Biother Radiopharm, 2000 Aug, 15(4), 327 - 37
Scintigraphic detection of multidrug resistance in cancer; Del Vecchio S et al.; One of the most extensively studied mechanisms of drug resistance involves the drug efflux pump P-glycoprotein (Pgp) . The availability of radiolabeled substrates of Pgp such as 99mTc-MIBI and analogous 99mTc-labeled agents allows the clinical assessment of Pgp function in cancer patients . The consistency of the results from different institutions and trials strongly support the clinical application of this imaging technique for individual tailoring of chemotherapeutic regimens and for designing clinical trials with Pgp modulators.

Gastroenterology, 2000 Oct, 119(4), 1113 - 22
Molecular identification and functional characterization of Mdr1a in rat cholangiocytes; Gigliozzi A et al.; BACKGROUND & AIMS: The multidrug resistance P-glycoprotein 170 gene products (mdr1a and 1b) are glycosylated plasma membrane proteins that function as adenosine triphosphate-dependent transmembrane export pumps for lipophilic xenobiotics of widely different structure . We assessed whether these P-glycoproteins are functionally expressed in cholangiocytes . METHODS: A reverse-transcription polymerase chain reaction was performed on RNA from a normal rat cholangiocyte cell line using mdr1-specific primers . Northern and Western blot analyses were performed on cholangiocytes immunoisolated from 2-week bile duct-ligated rats and cholangiocytes and isolated cholangiocyte membrane subfractions, respectively . Functional assays were performed in isolated bile duct units from bile duct-ligated rats and incubated with rhodamine 123, a P-glycoprotein substrate, with or without the P-glycoprotein inhibitors verapamil or GF120918 . RESULTS: A 400-base pair fragment with 99% homology to the cytosolic domain of rat intestinal mdr1a (5' 1953-2350 3') was identified that hybridized to a 5.2-kilobase RNA transcript in a normal rat cholangiocyte cell line, isolated rat cholangiocytes, and ileum . Western analysis localized mdr1 to the apical membrane of cholangiocytes . Confocal microscopy showed active secretion of rhodamine 123 into the lumen of isolated bile duct units that was abolished by vanadate and P-glycoprotein competitive antagonists, verapamil and GF120918, in a dose-dependent manner . CONCLUSIONS: These findings provide the first molecular and functional evidence for the expression of mdr1a on the luminal membrane of cholangiocytes, where it may have a protective role.

Pancreas, 2000 Oct, 21(3), 240 - 7
Expression of the multidrug-resistance 1 (MDR1) gene and prognosis in human pancreatic cancer; Lu Z et al.; Multidrug-resistance 1 (MDR1) encodes a 170 kDa transmembrane glycoprotein (P-glycoprotein), which acts as a drug-efflux pump . In the present study, we analyzed the expression of MDR1/P-glycoprotein in human pancreatic cancer and correlated the results with clinical parameters . Pancreatic cancer tissue samples were obtained from 67 patients (30 female, 37 male) who underwent surgery . Normal pancreatic tissues obtained from 15 previously healthy organ donors (4 female, 11 male) served as controls . MDR1 mRNA levels were analyzed by Northern blotting, and the exact site of MDR1 mRNA expression was determined by in situ hybridization and immunohistochemistry . Northern blot analysis indicated that in comparison with the normal pancreas, MDR1 mRNA levels were only increased 1.4-fold (p = 0.03) in the pancreatic cancer samples . However, there was a 2.9-fold (p < 0.01) increase in MDR1 mRNA levels when only the samples that exhibited increased expression (38%) were analyzed . In situ hybridization and immunohistochemical analysis showed that MDR1 was highly expressed in the cancer cells of these samples . Statistical analysis revealed that patients with high MDR1/P-glycoprotein expression had a shorter postoperative survival time compared with patients with weak to moderate expression of MDR1 . On the basis of in situ hybridization, survival in the intense group was 11.6 (n = 12) versus 14.2 months (n = 42) in the mild to moderate group . On the basis of immunohistochemistry, survival in the intense group was 7.5 months (n = 10) versus 14.1 months (n = 40) in the mild to moderate group . Surprisingly, survival of patients with high expression of MDR1/P-glycoprotein was not significantly different from that of patients without detectable MDR1/P-glycoprotein expression . These findings suggest that both strong expression of MDR1/P-glycoprotein and lack of expression seem to influence tumor growth via known and yet unknown mechanisms.

Cancer J, 2000 Jul-Aug, 6(4), 256 - 65
Phase I and pharmacokinetic study of novobiocin in combination with VP-16 in patients with refractory malignancies; Murren JR et al.; PURPOSE: The coumarin antibiotic novobiocin potentiates the activity of etoposide (VP-16) in vitro by increasing intracellular accumulation of VP-16 . The drug efflux pump inhibited by novobiocin appears to be distinct from both of the major proteins associated with the multidrug resistance phenotype in human cancers, the 170-kDa P-glycoprotein and the 190-kDa multidrug resistance protein . In a recent study, we found that novobiocin augmented VP-16 accumulation ex vivo in 16 of 24 fresh tumor samples at concentrations that could be achieved in vivo . Therefore, we conducted a clinical trial to determine the maximum tolerated dose and the pharmacokinetics of novobiocin when given in combination with VP-16 . PATIENTS AND METHODS: Patients with refractory cancer were treated with VP-16 on days 1, 3, and 5 . Antiemetics, consisting of ondansetron and dexamethasone, were given 60 minutes before the VP-16 was administered . Novobiocin was given orally 30 minutes before the VP-16, and the dose was escalated in successive groups of patients according to a standard dose escalation design . Treatment cycles were repeated every 4 weeks . Plasma concentrations of novobiocin were determined during the first treatment cycle by high-performance liquid chromatography . RESULTS: Thirty-three patients were treated for a total of 69 cycles . Eleven patients were treated with a starting dose of VP-16 of 120 mg/m2, and three of these patients experienced neutropenic fever . The dose of VP-16 was reduced to 100 mg/m2, and an additional 22 patients were enrolled . The dose of novobiocin ranged from 3 to 9 g . At a novobiocin dose of at least 5.5 g, plasma concentrations of at least 150 microM were sustained for 24 hours . Dose-limiting toxicities consisted of neutropenic fever and reversible hyperbilirubinemia . Nausea, which was a limiting toxicity in other trials of novobiocin, was well controlled with the use of serotonergic antiemetics . Diarrhea was common but mild in most patients . DISCUSSION: In previously treated patients, the recommended dose of novobiocin in this schedule is 7 g/m2/day . Novobiocin does not appear to augment the toxicity of VP-16 to the bone marrow or the gastrointestinal mucosa . Plasma concentrations of novobiocin equivalent to the levels required to modulate VP-16 in vitro are readily achievable for total but not unbound free drug.

Anticancer Drugs, 2000 Aug, 11(7), 583 - 90
A bioassay for the activity of PSC 833 in human serum for modulation of P-glycoprotein-mediated multidrug resistance; Uchiyama-Kokubu N et al.; We established a rapid and sensitive ex vivo bioassay to detect the multidrug resistance (MDR)-inhibitory activity of SDZ PSC 833 ({3'-keto-Bmt1}-{Val2}-cyclosporin (PSC 833)) in two RPMI 8226 human myeloma sublines (parent 8226 and doxorubicin-resistant subline Dox6) in 75% human serum . In vitro sensitivity of the tumor to doxorubicin was determined by 3-h drug exposure growth inhibition assay (MTT assay) . PSC 833 in serum restored the IC50 of doxorubicin in the P-glycoprotein (P-gp)-positive resistant subline to the same level as in the sensitive cells at 1 microg/ml, which has been shown to be an achievable concentration in clinical trials . In addition, the cytotoxic effect of doxorubicin was enhanced by PSC 833 in the sera of the patient in whom the blood level was 705.7 ng/ml . However, 10 microg/ml PSC 833 in serum does not cause a complete recovery in the IC90 of doxorubicin in the resistant sublines . This MDR-inhibitory activity was supported by the finding that PSC 833 in serum does not increase accumulation of rhodamine 123 in doxorubicin-resistant cells in an in vitro functional assay . The present study provides evidence that PSC 833 in human serum is effective to modulate P-gp-mediated MDR but insufficient for the reversal of MDR from the clinicopharmacological point of view.

Acta Clin Belg, 2000 Jul-Aug, 55(4), 215 - 21
Repetitive analyses of P-glycoprotein in chronic myeloid leukaemia; De Moerloose B et al.; P-glycoprotein, a pump located in the plasma cell membrane, extrudes several clinically important drugs from the cell, and hence causes multidrug resistance . Reversing clinical drug resistance is possible by using agents that inhibit the activity of P-glycoprotein . We describe the results of sequential flow cytometric determinations of P-glycoprotein expression and activity in two patients suffering from acute lymphoblastic transformation of chronic myeloid leukaemia . Neither P-glycoprotein expression, nor its activity could be detected in the initial sample of the first patient . In the second patient, no P-glycoprotein expression was found at diagnosis . However, after chemotherapy containing P-glycoprotein substrates, a significant expression was found in both patients and the functional flow cytometric test was positive . In order to achieve an accurate selection of patients that might benefit from the clinical use of P-gp inhibitors, repeated analyses are indicated in each patient suffering from acute leukaemia, during the course of the illness.

Cancer Invest, 2000, 18(7), 614 - 25
Multidrug resistance phenotype and paclitaxel (Taxol) sensitivity in human renal carcinoma cell lines of different histologic types; Reinecke P et al.; We compared the effects of paclitaxel (Taxol) in human renal cell carcinoma (RCC) of different histologic types . The growth inhibitory effects of paclitaxel on 34 human RCC cell lines of strictly defined different histologic types were determined by 3-{4,5-dimethylthiazolyl}-2,5-diphenyltetrazoliumbromide (MTT) assays . Paclitaxel-induced morphologic alterations were visualized by light and immunofluorescence and by transmission electron microscopy . The expression and function of P-glycoprotein and multidrug resistance-associated protein (MRP) were defined by reverse transcriptase polymerase chain reaction and fluorescence-activated cell sorting (FACS) analysis, respectively . Modulation of P-glycoprotein function was performed by verapamil or Cremophor EL . A significant (p < 0.05) dose-dependent paclitaxel-induced growth inhibition could be demonstrated in all cell lines, with the effects of paclitaxel dissolved in Cremophor EL/ethanol (= Taxol) exceeding the effects of paclitaxel dissolved in dimethyl sulfoxide . The extent of response markedly varied between the different cell lines, although chromophilic RCCs exhibited a more pronounced response to Taxol (IC50: 0.03-0.38 microM) than clear cell RCCs (IC50: 0.01-36.69 microM) . Exposure to paclitaxel/Taxol induced an increase of microtubule bundles in the clear cell and the chromophobe RCCs but not in the chromophilic RCCs . The expression of the MRP was low in RCC cell lines and was not found to be related to paclitaxel/Taxol sensitivity . In contrast, the expression level of P-glycoprotein was much more pronounced and showed a positive correlation (p < 0.05) with the response to paclitaxel . Reversal of P-glycoprotein function by verapamil or Cremophor EL enhanced the growth inhibitory effects of paclitaxel and further supported the role of P-glycoprotein for paclitaxel sensitivity of human RCCs . Paclitaxel/Taxol effectively inhibits proliferation of human RCCs in vitro, irrespective of their histologic types . Moreover, expression and function of P-glycoprotein markedly contribute to paclitaxel responsiveness, although other as yet undefined drug resistance mechanisms are effective in human RCCs as well.

Cancer Invest, 2000, 18(7), 609 - 13
P-glycoprotein expression and multidrug resistance in cutaneous T-cell lymphoma; Jillella AP et al.; Advanced-stage cutaneous T-cell lymphoma (CTCL) is generally resistant to standard chemotherapy . Because P-glycoprotein (P-gp) has been detected in other types of resistant solid tumors, leukemias, and lymphomas, we analyzed P-gp expression in CTCL . Twenty-seven patients with CTCL and circulating Sezary cells in the peripheral blood as observed on a peripheral smear treated at the Yale Photopheresis Center between 1987 and 1993 were identified . Twenty-five of these patients had skin biopsies evaluated for expression of P-gp using JSB-1 (Accurate Chemical), MRK-16 (gift of T . Tsuruo), and UIC-2 (gift of E . Metchner) . P-gp expression was considered present if immunoreactivity was noted with two of the three antibodies . Eighteen of 25 patients (72%) evaluated exhibited expression . The patients were treated with various combinations of drugs consisting of topical and systemic steroids electron beam therapy, psoralens in combination with UV light A (PUVA), systemic chemotherapy, and photopheresis before testing the tissue for P-gp expression . Treatment with systemic chemotherapy in P-gp-positive patients produced responses in 3 and no responses in 11 patients (4 were lost to follow-up) . Seven patients did not express P-gp: One patient responded to treatment, five did not respond, and one patient was lost to follow-up . These results demonstrate that P-gp is frequently expressed in CTCL . P-gp expression in our study was not a useful predictor of drug resistance.

Rev Panam Salud Publica, 2000 Sep, 8(3), 151 - 5
Patients with tuberculosis in Bolivia: why do they die?
Olle-Goig JE.
The objective of this research was to analyze why patients with tuberculosis (TB) die and to evaluate whether there are factors contributing to their fatal outcome that could be corrected . A cross-sectional observational study was conducted of the patients with active TB or its sequelae admitted to the TB ward of the main public hospital in the city of Santa Cruz, Bolivia, over a 29-month period, from October 1993 through February 1996 . The available records of the patients who died during hospitalization were reviewed . Out of 597 patients, 94 of them (15.7%) died . We examined the records of 90 of these 94 patients . Their mean age was 35.1 years (standard deviation, 16.7 years), and 45 of the patients (50.0%) were male . On admission 42 of the 90 patients (46.7%) had never been treated for TB or had received anti-TB treatment for less than one month, 23 (25.6%) had returned after having abandoned their TB treatment, 8 (8.9%) had had an erroneous diagnosis, 6 (6.7%) had tuberculosis sequelae, 6 (6.7%) were undergoing tuberculosis treatment, and 5 (5.6%) were known to have multidrug-resistant TB . Of the 90 patients, 83 (92.2%) had pulmonary tuberculosis (median lobes affected, 4), 6 (6.7%) had pleural tuberculosis, and 12 (13.3%) had extrapulmonary tuberculosis (some patients had more than one form of TB) . Patients died a median of 5.5 days after entering the TB ward . The causes of death were: hemoptysis, 6 patients (6.7%); other tuberculosis-related causes, 65 patients (72.2%); drug reactions, 6 patients (6.7%); nontuberculosis causes, 6 patients (6.7%); and undetermined causes, 7 patients (7.8%) . Factors possibly contributing to death were late diagnosis (38.9%), errors in follow-up (14.4%), and errors in treatment (24.4%) . In conclusion, most patients with active or inactive TB admitted to our ward died as a consequence of tuberculosis . There were several factors possibly contributing to their fatal outcome that could be corrected.

J Natl Cancer Inst, 2000 Oct 18, 92(20), 1651 - 6
Role of breast cancer resistance protein in the bioavailability and fetal penetration of topotecan; Jonker JW et al.; BACKGROUND AND METHODS: Breast cancer resistance protein (BCRP/MXR/ABCP) is a multidrug-resistance protein that is a member of the adenosine triphosphate-binding cassette family of drug transporters . BCRP can render tumor cells resistant to the anticancer drugs topotecan, mitoxantrone, doxorubicin, and daunorubicin . To investigate the physiologic role of BCRP, we used polarized mammalian cell lines to determine the direction of BCRP drug transport . We also used the BCRP inhibitor GF120918 to assess the role of BCRP in protecting mice against xenobiotic drugs . Bcrp1, the murine homologue of BCRP, was expressed in the polarized mammalian cell lines LLC-PK1 and MDCK-II, and the direction of Bcrp1-mediated transport of topotecan and mitoxantrone was determined . To avoid the confounding drug transport provided by P-glycoprotein (P-gp), the roles of Bcrp1 in the bioavailability of topotecan and the effect of GF120918 were studied in both wild-type and P-gp-deficient mice and their fetuses . RESULTS: Bcrp1 mediated apically directed transport of drugs in polarized cell lines . When both topotecan and GF120918 were administered orally, the bioavailability (i.e., the extent to which a drug becomes available to a target tissue after administration) of topotecan in plasma was dramatically increased in P-gp-deficient mice (greater than sixfold) and wild-type mice (greater than ninefold), compared with the control (i.e., vehicle-treated) mice . Furthermore, treatment with GF120918 decreased plasma clearance and hepatobiliary excretion of topotecan and increased (re-)uptake by the small intestine . In pregnant GF120918-treated, P-gp-deficient mice, relative fetal penetration of topotecan was twofold higher than that in pregnant vehicle-treated mice, suggesting a function for BCRP in the maternal-fetal barrier of the placenta . CONCLUSIONS: Bcrp1 mediates apically directed drug transport, appears to reduce drug bioavailability, and protects fetuses against drugs . We propose that strategic application of BCRP inhibitors may thus lead to more effective oral chemotherapy with topotecan or other BCRP substrate drugs.

Antimicrob Agents Chemother, 2000 Nov, 44(11), 3210 - 2
Resistance to methotrexate due to AcrAB-dependent export from Escherichia coli; Kopytek SJ et al.; Many laboratory strains of Escherichia coli are resistant to methotrexate (MTX), a folate analogue that binds dihydrofolate reductase (DHFR) . Mutations that inactivate either tolC or acrA confer MTX sensitivity . Further, overexpression of a fusion protein with DHFR activity reverses this sensitivity by titrating out intracellular MTX . These results suggest that MTX accumulates in cells where mutations in acrA or tolC have inactivated the TolC-dependent AcrAB multidrug resistance efflux pump.

Antimicrob Agents Chemother, 2000 Nov, 44(11), 3079 - 86
Cloning and characterization of SmeDEF, a novel multidrug efflux pump from Stenotrophomonas maltophilia; Alonso A et al.; Stenotrophomonas maltophilia is a nosocomial bacterial pathogen intrinsically resistant to several antibiotics . The mechanisms involved in this intrinsic multiresistance phenotype are poorly understood . A library of chromosomal DNA from a spontaneous multidrug-resistant S . maltophilia D457R mutant (A . Alonso and J . L . Martinez, Antimicrob . Agents Chemother . 41:1140-1142, 1997) was screened for complementation of erythromycin susceptibility on an antibiotic-hypersusceptible Escherichia coli DeltaacrAB strain . Cloning and further analysis revealed that a 6-kbp region constituting a transcriptional unit was capable of complementing the antibiotic-susceptible phenotype of an E . coli DeltaacrAB strain . We identified three open reading frames, smeD, smeE and smeF, which code for members of the membrane fusion protein, resistance nodulation division, and outer membrane factor families, respectively . Drug susceptibility assays indicated that the SmeDEF system cloned in E . coli mediates resistance to a wide range of antibiotics . Ethidium bromide and norfloxacin accumulation experiments in the presence and in the absence of carbonyl cyanide m-chlorophenylhydrazone showed that this system constitutes a drug efflux pump dependent on the membrane proton motive force . The presence of high levels of smeDEF mRNA in the multiresistant D457R mutant was consistent with the high levels of SmeF (formerly Omp54) observed in the same strain . In contrast, transcription levels of smeDEF in the D457 strain were tiny, which correlates with the low levels of SmeF observed for this strain . Also, for both the D457 and D457R strains, we observed growth phase-dependent regulation in which the highest level of transcription corresponded to early exponential phase, with transcription decreasing throughout the growth curve to undetectable levels at 24 h.

Crit Rev Oncol Hematol, 2000 Nov-Dec, 36(2-3), 193 - 207
Multicellular resistance: a paradigm for clinical resistance?
Desoize B, Jardillier J.
Research on resistance to cancer treatment was mainly focused for 20 years on multidrug resistance (MDR) . No useful method of reversing MDR, suitable for clinical use, has yet emerged from this large quantity of work . The reason could be an inadequate evaluation of the target . When grown in spheroids, cancer cells exhibit a phenomenon known as 'multicellular resistance' (MCR) . Tumours in patients seem to present the same characteristics . The mechanisms underlying MCR can be classified into two forms: contact resistance and resistance inherent in the spheroid structure . Mechanisms of MCR include: inhibition of apoptosis, high proportion of quiescent cells, modulation of protein expression (including topoisomerases and repair enzymes), potential permeability problems, presence of a hypoxic and necrotic centre and other possible mechanisms that remain to be discovered . A new therapeutic class of drugs is required to overcome MCR . Compounds, which are able to disrupt communication and binding between tumour cells and their microenvironment, seem to be able to circumvent MCR . Interesting results are obtained in vitro and in vivo in mice with specific antibodies or peptides recognised by cell binding proteins . Interestingly, these compounds also appear to be able to inhibit metastasis . Hyaluronidase has already been used with anticancer drugs in patients and was shown to increase drug potency . The explanation given is that it improves drug penetration into spheroids . We now hypothesise that hyaluronidase, in fact, decreases MCR and thus could be the first member of a new therapeutic class.

Eur J Pharm Sci, 2000 Oct, 11(4), 265 - 83
Multidrug resistance (MDR) in cancer . Mechanisms, reversal using modulators of MDR and the role of MDR modulators in influencing the pharmacokinetics of anticancer drugs; Krishna R et al.; In recent years, there has been an increased understanding of P-glycoprotein (P-GP)-mediated pharmacokinetic interactions . In addition, its role in modifying the bioavailability of orally administered drugs via induction or inhibition has been also been demonstrated in various studies . This overview presents a background on some of the commonly documented mechanisms of multidrug resistance (MDR), reversal using modulators of MDR, followed by a discussion on the functional aspects of P-GP in the context of the pharmacokinetic interactions when multiple agents are coadministered . While adverse pharmacokinetic interactions have been documented with first and second generation MDR modulators, certain newer agents of the third generation class of compounds have been less susceptible in eliciting pharmacokinetic interactions . Although the review focuses on P-GP and the pharmacology of MDR reversal using MDR modulators, relevance of these drug transport proteins in the context of pharmacokinetic implications (drug absorption, distribution, clearance, and interactions) will also be discussed.

Methods Find Exp Clin Pharmacol, 2000 Jun, 22(5), 281 - 4
Enhancement of doxorubicin activity in multidrug-resistant cells by mefloquine; Fujita R et al.; We studied the effect of the antimalarial drug mefloquine on the resistance of K562 cells to doxorubicin . Mefloquine synergistically potentiated the cytotoxicity of doxorubicin for doxorubicin-resistant K562 cells (K562/DXR) at a concentration of 0.5-3 microM, but had hardly any synergistic effect in the parental cell line (K562) at the same concentration . Mefloquine was more potent than verapamil, a known modulator of multidrug-resistance . Since doxorubicin resistance in these cells is associated with the expression of high levels of P-glycoprotein, we evaluated the effect of mefloquine and of P-glycoprotein activity in cytofluorographic efflux experiments with the fluorescent dye rhodamine 123 . Our results indicate that mefloquine inhibits the P-glycoprotein pump-efflux activity in a dose-related manner . Moreover, mefloquine reduces the expression of the immunoreactive P-glycoprotein in K562/DXR cells as evaluated by cytofluorimetric assay . Taken together, the results indicate that mefloquine reverses the multidrug-resistance phenotype through direct interaction with P-glycoprotein.

Ann Med, 2000 Sep, 32(6), 408 - 16
Electrophysiologically guided ablation of the pulmonary veins for the curative treatment of atrial fibrillation; Shah DC et al.; Catheter ablation of triggers that induce paroxysms of atrial fibrillation (AF) is an emerging curative therapy for this most common of supraventricular arrhythmias . In a series of 225 consecutive patients with multidrug resistant AF, 96% of triggering foci originated from one or several pulmonary veins (PV) independent of ambient ectopy or structural heart disease . This article describes an ablation procedure that is guided by activation mapping tailored to each individual PV, including criteria to define an arrhythmogenic PV, the use of provocative manoeuvres, the role of circumferential mapping catheters to provide information on the extent, distribution and activation of PV muscle as well as the monitoring of distal PV potentials (PVP) during ablation . Radiofrequency ablation to eliminate distal PVPs is performed by targeting the proximal PVP during sinus rhythm (right PV) or left atrial pacing (left PV) . This end-point predicts a successful outcome more often than acute ectopy suppression . Complete elimination of AF is presently achieved in 70% of the patients, resulting in the elimination of antiarrhythmic treatment and suspension of anticoagulant treatment . It is anticipated that continued technological development will improve and facilitate this technique of curative treatment of AF.

Biochem Biophys Res Commun, 2000 Oct 5, 276(3), 909 - 16
Cholesterol interaction with the daunorubicin binding site of P-glycoprotein; Wang E et al.; The inherent complexities of cholesterol disposition and metabolism preclude a single transmembrane active transport avenue for this steroid-precursor, cell-membrane constituent . Yet the ABC (ATP binding cassette) transporters are inextricably linked to elements of cholesterol disposition . Recent observations have suggested that, under certain settings, the ABC transporter P-glycoprotein (P-gp) performs a direct role in cholesterol disposition . The gene product of MDR1 (multidrug resistance transporter), P-glycoprotein also confers protection against xenobiotics . Using a whole cell assay in which the retention of a marker substrate is evaluated and quantified, we studied the ability of cholesterol to inhibit directly the function of this transporter . In a NIH-G185 cell line presenting an overexpressed amount of the human transporter P-gp, cholesterol caused dramatic inhibition of daunorubicin transport with an IC(50) of about 8 microM yet had no effect on the parent cell line nor rhodamine 123 transport . Additionally, using the ATP-hydrolysis assay, we showed that cholesterol increases P-gp-mediated ATP hydrolysis by approximately 1.6-fold with a K(s) of 5 microM . Suggesting that cholesterol directly interacts with the substrate binding site of P-gp, these results are consistent with cholesterol being transported by MDR1 P-gp .

J Cell Physiol, 2000 Nov, 185(2), 293 - 301
Induction of MRP1 and gamma-glutamylcysteine synthetase gene expression by interleukin 1beta is mediated by nitric oxide-related signalings in human colorectal cancer cells; Ikegami Y et al.; Treatment of human colorectal cancer cells HT29 with interleukin 1beta (IL-1beta) induces expression of the multidrug resistance protein (MRP1) gene encoding the ATP-dependent glutathione S-conjugate export (GS-X) pump and the gamma-glutamylcysteine synthetase (gamma-GCSh) gene encoding heavy (catalytic) subunit of gamma-glutamylcysteine synthetase, the rate-limiting enzyme for the biosynthesis of glutathione (GSH) . The induction can be suppressed by N(G)-methyl-L-arginine, a specific inhibitor of nitric oxide synthase (NOS) . These results suggest that IL-1beta-mediated MRP1 and gamma-GCSh induction involve nitric oxide (NO) -related signaling . Further supports to the involvement of NO in the induction of MRP1 and gamma-GCSh expression are made by the following observations . (i) Expression of MRP1 and gamma-GCSh genes were induced by treating the cells with NO donors, i.e., S-nitro-N-acetyl-D,L-penicillamide (SNAP) and S-nitroso-L-glutathione, in a concentration-dependent manner . (ii) Ectopic expression of inducible NOS (iNOS) activity by transfecting expressible recombinant iNOS cDNA encoding functional iNOS but not the nonfunctional version resulted in elevated expression of MRP1 and gamma-GCSh . We also demonstrated that HT-29 cells treated with either 1L-1beta or SNAP induced ceramide production, and addition of C2 or C6 ceramides into cultured HT-29 cells resulted in induction of gamma-GCSh but not MRP1 expression . Collectively, our results demonstrate that induction of MRP1 and gamma-GCSh by IL-1beta is regulated, at least in part, by an NO-related signaling, and induction of gamma-GCSh is by NO-related ceramide signaling .

J Gastroenterol, 2000, 35(9), 659 - 64
Recent advances in bilirubin metabolism research: the molecular mechanism of hepatocyte bilirubin transport and its clinical relevance; Kamisako T et al.; Bilirubin is taken up from blood into hepatocytes by sinosuidal membrane transporters and then excreted into bile through the bile canalicular membrane mainly as bilirubin glucuronides . (1) Mechanism of bilirubin uptake into hepatocytes: many organic anions are incorporated into hepatocytes by organic anion transporting polypeptides (rat, oatp1, oatp2, oatp3; human, OATP), liver-specific transporter (rlst/HLST), and/or by organic anion transporters (OAT2, OAT3) . Oatp1 and HLST transport bilirubin monoglucuronide . However, a transporter of unconjugated bilirubin in the sinusoidal membrane has not as yet been identified . Unconjugated bilirubin may also go across the hepatocyte sinusoidal membrane by a diffusion process . (2) Intrahepatic transport and conjugation of bilirubin: ligandin carries bilirubin to the endoplasmic reticulum (ER) of hepatocytes . In the ER, bilirubin is conjugated by bilirubin uridine diphosphate (UDP)-glycosyltransferase (bilirubin UGT; UGT1A1) to form mono- and diglucuronides of bilirubin . (3) Transport mechanism of bilirubin glucuronides across the hepatocyte canalicular membrane: at the canalicular membrane, bilirubin glucuronides are excreted into bile by multidrug resistance-associated protein 2 (MRP2), a member of the ATP-binding cassette transporter family . (4) Regurgitation of bilirubin glucuronides into blood: MRP3, which is located in the lateral membrane, transports bilirubin glucuronides into blood under conditions of impaired biliary bilirubin excretion.

Biochem Pharmacol, 2000 Nov 15, 60(10), 1485 - 9
Multidrug resistance protein functionality: no effect of intracellular or extracellular pH changes; Marbeuf-Gueye C et al.; A major problem in the treatment of cancer is cellular resistance to cytotoxic drugs . In tumor cells in vitro, the development of multidrug resistance is usually accompanied by increased expression of drug transporters, either P-glycoprotein (P-gp) or multidrug resistance-associated protein (MRP(1)) . Both proteins belong to the superfamily of ATP-binding cassette (ABC) transporter proteins and mediate the transport of a broad range of drugs . Altenberg et al . (Proc Natl Acad Sci USA90: 9735-9738, 1993) have shown that changes in intra- or extracellular pH do not mediate P-gp-dependent multidrug resistance . Therefore, we similarly studied whether changes in intra- or extracellular pH could mediate MRP(1)-dependent multidrug resistance . In particular, we measured the MRP(1)-mediated efflux of hydroxyrubicin from GLC4/ADR cells . Since hydroxyrubicin is a fully neutral anthracycline derivative that has no deprotonable function at pH lower than 10 and so cannot accumulate in non-nuclear compartments under the influence of pH or transmembrane gradients, we hypothesized that any modifications of its kinetics of efflux as a function of pH can be assigned to a modification of the transporter efficiency . However, as our data show, modifications of extra- and/or intracellular pH yielded no modification of the MRP(1)-mediated efflux of hydroxyrubicin.

J Biol Chem, 2001 Jan 5, 276(1), 413 - 20
2-acetylaminofluorene up-regulates rat mdr1b expression through generating reactive oxygen species that activate NF-kappa B pathway; Deng L et al.; Overexpression of multidrug resistance genes and their encoded P-glycoproteins is a major mechanism for the development of multidrug resistance in cancer cells . The hepatocarcinogen 2-acetylaminofluorene (2-AAF) efficiently activates rat mdr1b expression . However, the underlying mechanisms are largely unknown . In this study, we demonstrated that a NF-kappa B site on the mdr1b promoter was required for this induction . Overexpression of antisense p65 and I kappa B alpha partially abolished the induction . We then delineated the pathway through which 2-AAF activates NF-kappa B . 2-AAF treatment led to the increase of intracellular reactive oxygen species (ROS) which causes activation of IKK kinases, degradation of I kappa B beta (but not I kappa B alpha), and increase in NF-kappa B DNA binding activity . Consistent with the idea that ROS may participate in mdr1b regulation, antioxidant N-acetylcysteine inhibited the induction of mdr1b by 2-AAF . Overproduction of a physiological antioxidant glutathione (GSH) blocked the activation of IKK kinase complex and NF-kappa B DNA binding . Based on these results, we conclude that 2-AAF up-regulates mdr1b through the generation of ROS, activation of IKK kinase, degradation of I kappa B beta, and subsequent activation of NF-kappa B . This is the first report that reveals the specific cis-elements and signaling pathway responsible for the induction of mdr1b by the chemical carcinogen 2-AAF.

J UOEH, 2000 Sep 1, 22(3), 269 - 82
{Molecular mechanisms of multidrug resistance in Mycobacterium tuberculosis}; Taniguchi H; Control of tuberculosis caused by multidrug-resistant (MDR) Mycobacterium (M.) tuberculosis has become one of the major problems throughout the world . Understanding of the molecular mechanisms of resistance may help in the development of novel methods for the rapid and precise detection of drug-resistant M . tuberculosis . Eight agents have been recommended to treat tuberculosis . Isoniazid (INH), rifampicin (RFP), pyrazinamide (PZA), streptomycin(SM), and ethambutol (EB) are used as the first line agents, and the others are the second line agents . MDR M . tuberculosis strains are resistant both to INH and RFP which have the most effective bactericidal activity to M . tuberculosis . Nearly 95% of RFP resistant strains possess a mutation on the rpoB gene encoding a DNA-dependent RNA polymerase . INH particularly shows an inhibition of the cell wall synthesis of M . tuberculosis and approximately 90% of INH resistant strains have a mutation on the inhA, katG, and ahpG gene encoding enzymes related to a mycolic acid synthesis of cell wall . PZA resistant strains have a mutation on the pncA gene encoding a pyrazinamidase which degradates pyrazinamide to a bactericidal substance, pyrazinoic acid . SM resistant strains have a mutation on the rrs and rpsL gene encoding a 16S rRNA and a S12 ribosomal subunit protein, respectively . EB resistant strains have a mutation on the embB gene encoding a arabinosyl transferase which catalyzes cell wall synthesis . Resistant mechanisms of second-line agents have also been identified . Recently, rapid detection methods for RFP and INH resistant mutations have been developed on the basis of these studies.

Biochim Biophys Acta, 2000 Sep 29, 1468(1-2), 73 - 86
Effect of ethylene oxide and propylene oxide block copolymers on the permeability of bilayer lipid membranes to small solutes including doxorubicin; Erukova VY et al.; The effects of ethylene oxide and propylene oxide block copolymers (pluronics) on the permeability of several weak acids and bases through bilayer lipid membranes have been studied by the methods of monitoring (1) pH shifts near planar bilayers, (2) doxorubicin fluorescence quenching inside liposomes, and (3) current transients in the presence of hydrophobic anions . It has been shown that pluronics facilitate the permeation of comparatively large molecules (such as 2-n-undecylmalonic acid and doxorubicin) across lipid bilayers, while the permeation of small solutes (such as ammonium and acetic acid) remains unaffected . Pluronics also accelerate the translocation of large hydrophobic anions (tetraphenylborate) . The effect of pluronics correlates with the content of propylene oxide units: it is enhanced when the portion of polypropylene oxide block in the copolymer is increased . The action of the pluronic on lipid membrane permeability differs from the effect of the conventional detergent Triton X-100, which does not affect doxorubicin transport if added at concentrations similar to those used for pluronics . It has been proposed that pluronics accelerate the processes of solute diffusion within lipid bilayers (in a structure-dependent manner) rather than influencing the rate of solute adsorption/desorption on the membrane surface . We suppose that the effect of pluronics on doxorubicin permeation across lipid bilayers along with the known effect on the multidrug resistance protein determines its influence on the therapeutic activity of anthracycline drugs.

Cancer Res, 2000 Sep 15, 60(18), 5269 - 77
Specific detection of multidrug resistance proteins MRP1, MRP2, MRP3, MRP5, and MDR3 P-glycoprotein with a panel of monoclonal antibodies; Scheffer GL et al.; Tumor cells may display a multidrug resistance phenotype by overexpression of ATP binding cassette transporter genes such as multidrug resistance (MDR) 1 P-glycoprotein (P-gp) or the multidrug resistance protein 1 (MRP1) . MDR3 P-gp is a close homologue of MDR1 P-gp, but its role in MDR is probably minor and remains to be established . The MRP1 protein belongs to a family of at least six members . Three of these, i.e., MRP1, MRP2, and MRP3, can transport MDR drugs and could be involved in MDR . The substrate specificity of the other family members remains to be defined . Specific monoclonal antibodies are required for wide-scale studies on the putative contribution of these closely related transporter proteins to MDR . In this report, we describe the extensive characterization of a panel of monoclonal antibodies (Mabs) detecting several MDR-related transporter proteins in both human and animal tissues . The panel consists of P3II-1 and P3II-26 for MDR3 P-gp; MRPr1, MRPm6, MRPm5, and MIB6 for MRP1; M2I-4, M2II-12, M2III-5 and M2III-6 for MRP2; M3II-9 and M3II-21 for MRP3; and M5I-1 and M5II-54 for MRP5 . All Mabs in the panel appeared to be fully specific for their cognate antigens, both in Western blots and cytospin preparations, as revealed by lack of cross-reactivity with any of the other family members . Indeed, all Mabs were very effective in detecting their respective antigens in cytospins of transfected cell lines, whereas in flow cytometric and immunohistochemical analyses, distinct differences in reactivity and suitability were noted . These Mabs should become valuable tools in studying the physiological functions of these transporter proteins, in screening procedures for the absence of these proteins in hereditary metabolic (liver) diseases, and in studying the possible contributions of these molecules to MDR in cancer patients.

Pneumologia, 2000 Apr-Jun, 49(2), 129 - 36
{Therapeutic pneumothorax--an effective adjuvant method in treating multidrug-resistant tuberculosis}; Strambu I; Treatment of multidrug-resistant TB is difficult, even when all alternative drugs are at hand, due to important side effects and high cost, buying seldom efficient . Therapeutic pneumothorax (TP) proved its efficacy in pre-antibiotic era, so one can assume that it can be applied with the same success in multidrug resistant TB . Three MDR TB cases are presented (resistance to 2, 3 and 4 drugs), with unilateral lesions, in which TP was applied as a help for the drug treatment which was guided by the antibiogram . Favorable results were obtained in all 3 cases, with constant bacteriological negativity . In one of the cases (with 4 drugs resistance), treatment was completed after 3 months by a lobectomy; they were all considered healed after 18 months . In conclusion, in some cases of MDR TB, the TP (if there are no pleural adherences) may lead to good results . This presentation aims to remind the pulmonologists the favourable effects of TP in selected cases.

J Indian Med Assoc, 2000 Mar, 98(3), 123 - 5
Tuberculosis control in India--past, present and future; Prabhakar R; Tuberculosis (TB) continues to be a major health problem in India . There has been no perceptible change in the epidemiology of TB since the National Sample Survey 1956-57 . Detection of cases has been low over the years since the inception of National TB control programme in 1962 due to passive case finding and high drop out rates among sputum positive patients . Shortening the course of chemotherapy with regimens containing bactericidal and sterilising drugs helped in improving the treatment adherence of patients and cutting down the chain of transmission substantially . Further it is advisable to implement properly the directly observed short course treatment (DOTS) as per WHO guidelines to prevent the development of multidrug resistant TB (MDRTB) . Proper management of RNTCP and prevention of MDRTB are all the more important in areas where there is high prevalence of HIV/AIDS co-infected with TB . New vaccine development is also a priority area for research . There is an urgent need for health systems research built into the ongoing programme with proper managerial inputs.

J Indian Med Assoc, 2000 Mar, 98(3), 100 - 2
Tuberculosis: the global epidemic; Davies PD; The modern era of tuberculosis(TB) began in the mid 1980s . In 1993, WHO took the unique step of declaring TB to be a world emergency . Despite this intervention it is estimated that deaths from TB will increase from 3 million a year currently to 5 million by the year 2050 . There are 4 principal reasons: World population's increase, co-infection with HIV/AIDS, poverty and programme decline . Other causes contributing to the global epidemic are multidrug resistant TB, immigration, and indifference . The practical solution must concentrate on the completed correct treatment of the disease particularly in those who are sputum smear positive . For this reason WHO is vigorously promoting the Directly Observed Therapy Short course (DOTS) campaign . Doctors treating TB should ideally be part of the public health system . They should have access to first class bacteriological services, good quality of drugs and should make sure that the patient receives the drugs under supervision . Though the reasons for increasing TB are multifactorial it is within the capability of the world to re-exert control providing that the political will is present.

J Clin Microbiol, 2000 Oct, 38(10), 3686 - 8
Comparison of phenotypic and genotypic methods for pyrazinamide susceptibility testing with Mycobacterium tuberculosis; Davies AP et al.; Mycobacterium tuberculosis converts pyrazinamide to its active form by using the enzyme pyrazinamidase . This enzyme is coded for on the pncA gene, and mutations in the pncA gene result in absence of active enzyme, conferring resistance to the drug pyrazinamide . We investigated 27 strains of Mycobacterium tuberculosis suspected of being multidrug resistant . Each isolate was tested for susceptibility to pyrazinamide by the BACTEC 460TB method, and 19 were pyrazinamide resistant . The presence of active pyrazinamidase enzyme was sought by using the Wayne assay, which was positive in all of the sensitive isolates and four of the resistant isolates . The pncA gene was amplified by PCR, and mutations were sought by single-strand conformation polymorphism (SSCP) analysis . We identified four isolates which were phenotypically resistant to pyrazinamide, but which had active pyrazinamide enzyme on the Wayne assay and normal pncA gene SSCP . MICs measured by BACTEC 460TB and susceptibility testing at a lower pH of 5.5 confirmed genuine resistance . The pncA gene was sequenced in these four isolates and found not to have any mutations . This implies that an alternative mechanism of resistance exists in these strains . We conclude that genotypic assessment of pyrazinamide resistance is unreliable, because it depends on the identification of a single resistance mechanism . Phenotypic methods such as the BACTEC 460TB technique remain the best methods for pyrazinamide susceptibility testing.

Phytochemistry, 2000 Aug, 54(8), 839 - 45
Neutral taxoids from Taxus cuspidata as modulators of multidrug-resistant tumor cells; Kosugi K et al.; Two taxoids, taxinine NN-7 (1) and 3,11-cyclotaxinine NN-2 (2), were isolated from the neutral fraction of the EtOAc extract of a mixture of needles and young stems of Taxus cuspidata . The structures were determined by spectroscopic analysis . Both compounds showed some activity as modulators of multidrug-resistant tumor cells.

J Biol Chem, 2000 Dec 15, 275(50), 39272 - 8
Identification of residues within the drug-binding domain of the human multidrug resistance P-glycoprotein by cysteine-scanning mutagenesis and reaction with dibromobimane; Loo TW et al.; P-glycoprotein (P-gp) can transport a wide variety of cytotoxic compounds that have diverse structures . Therefore, the drug-binding domain of the human multidrug resistance P-gp likely consists of residues from multiple transmembrane (TM) segments . In this study, we completed cysteine-scanning mutagenesis of all the predicted TM segments of P-gp (TMs 1-5 and 7-10) and tested for inhibition by a thiol-reactive substrate (dibromobimane) to identify residues within the drug-binding domain . The activities of 189 mutants were analyzed . Verapamil-stimulated ATPase activities of seven mutants (Y118C and V125C (TM2), S222C (TM4), I306C (TM5), S766C (TM9), and I868C and G872C (TM10)) were inhibited by more than 50% by dibromobimane . The activities of mutants S222C (TM4), I306C (TM5), I868C (TM10), and G872C (TM10), but not that of mutants Y118C (TM2), V125C (TM2), and S776C (TM9), were protected from inhibition by dibromobimane by pretreatment with verapamil, vinblastine, or colchicine . These results and those from previous studies (Loo, T . W . and Clarke, D . M . (1997) J . Biol . Chem . 272, 31945-31948; Loo, T . W . and Clarke, D . M . (1999) J . Biol . Chem . 274, 35388-35392) indicate that the drug-binding domain of P-gp consists of residues in TMs 4, 5, 6, 10, 11, and 12.

Heredity, 2000 Aug, 85 ( Pt 2), 184 - 90
Genetics of alpha-amanitin resistance in a natural population of Drosophila melanogaster; Begun DJ et al.; The genetic basis of variation in resistance to natural toxins is of interest for both ecological and evolutionary genetics . The wide variety of larval resources used by Drosophila, both within and between species, makes flies an excellent system for studying causes and consequences of selection resulting from exposure to natural toxins associated with different resources . In this study we carry out a genetic analysis of alpha-amanitin resistance in a population sample of Drosophila melanogaster . Data from mapping crosses of chromosome III support a role for a naturally occurring polymorphism in a multidrug resistance gene (Mdr65A) in alpha-amanitin resistance . However, there are no amino acid differences between resistant and sensitive chromosomes at Mdr65A . Therefore, if Mdr65A mutants contribute to the difference between alpha-amanitin-resistant and alpha-amanitin-sensitive third chromosome lines, the underlying cause is a gene regulatory mutation.

Stem Cells, 1998, 16 Suppl 1, 247 - 50
Results of retroviral and adenoviral approaches to cancer gene therapy; Yin LH et al.; Genetic modification for cancer treatment has involved the introduction of chemotherapy protection and sensitization genes into normal and tumor cells, respectively, for the purpose of improving the outcome of conventional approaches to the treatment of solid tumor neoplasms . This paper will review the use of multidrug resistance-1 retroviral vectors and cytosine deaminase adenoviral prodrug activation vectors for this purpose.

Biochemistry, 2000 Oct 3, 39(39), 11921 - 7
Investigation of the role of glutamine-471 and glutamine-1114 in the two catalytic sites of P-glycoprotein; Urbatsch IL et al.; P-glycoprotein, also known as multidrug resistance protein, pumps drugs out of cells using ATP hydrolysis as the energy source . Glutamine-471 and the corresponding glutamine-1114 in the two catalytic sites of P-glycoprotein are conserved in ABC transporters . X-ray structures show that they lie close to the bound nucleotide . Proposed functional roles are (1) activation of the attacking water for ATP hydrolysis, (2) coordination of the essential Mg(2+) cofactor in Mg nucleotide, and (3) signal communication between catalytic site reaction chemistry and drug-binding sites . We made mutations Q471A, Q471E, Q1114A, and Q1114E in mouse MDR3 P-glycoprotein . Pure mutant and wild-type proteins were prepared and subjected to enzymatic and biochemical characterization . We conclude from the results that the primary role of this glutamine residue is in interdomain signal communication . Coordination of the Mg(2+) cofactor is not a critical functional role, neither is activation of the attacking water molecule, although an auxiliary role in positioning the water cannot be ruled out . We found that equivalent mutations (Ala or Glu) in either of the two P-glycoprotein catalytic sites produced the same effects, implying functional symmetry of the two sites.

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