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 migh