Chest, 2000 Jan, 117(1), 110 - 6 Delays in tuberculosis isolation and suspicion among persons hospitalized with HIV-related pneumonia; Bennett CL et al.; BACKGROUND: Despite awareness of HIV-related tuberculosis (TB), nosocomial outbreaks of multidrug-resistant TB among HIV-infected individuals occur . OBJECTIVE: To investigate delays in TB isolation and suspicion among HIV-infected inpatients discharged with TB or Pneumocystis carinii pneumonia (PCP), common HIV-related pneumonias . DESIGN: Cohort study during 1995 to 1997 . SETTING: For PCP, 1,227 persons who received care at 44 New York City, Chicago, and Los Angeles hospitals . For TB, 89 patients who received care at five Chicago hospitals . MEASUREMENTS: Two-day rates of TB isolation/suspicion . RESULTS: For HIV-related PCP, Los Angeles hospitals had the lowest 2-day rates of isolation/suspicion of TB (24.3%/26.6% vs 65.5%/66.4% for New York City and 62.8%/58.3% for Chicago, respectively; p < 0.001 for overall comparison by chi(2) test for each outcome measure) . Within cities, hospital isolation/suspicion rates varied from < 35 to > 70% (p < 0.001 for interhospital comparisons in each city) . The Chicago hospital with a nosocomial outbreak of multidrug-resistant TB from 1994 to 1995 isolated 60% of HIV-infected individuals who were discharged with a diagnosis of HIV-related TB and 52% discharged with HIV-related PCP, rates that were among the lowest of all Chicago hospitals in both data sets . CONCLUSION: Low 2-day rates of TB isolation/suspicion among HIV-related PCP patients were frequent . One Chicago hospital with low 2-day rates of TB isolation/suspicion among persons with HIV-related PCP also had low 2-day rates of isolation/suspicion among confirmed TB patients . That hospital experienced a nosocomial multidrug-resistant TB outbreak . Educational efforts on the benefits of early TB suspicion/isolation among HIV-infected pneumonia patients are needed. Planta Med, 1999 Dec, 65(8), 690 - 4 Antiplasmodial activity of extracts and alkaloids of three Alstonia species from Thailand; Keawpradub N et al.; Methanol extracts prepared from various parts of Alstonia scholaris, A . macrophylla and A . glaucescens, collected from Thailand, have been assessed for antiplasmodial activity against multidrug-resistant K1 strain of Plasmodium falciparum cultured in human erythrocytes . Pronounced antiplasmodial activity was exhibited by methanol extract of the root bark of A . macrophylla with an IC50 value of 5.7 micrograms/ml . Thirteen indole alkaloids were isolated from the active extract . These alkaloids and a semisynthetic bisindole O-acetylmacralstonine were subsequently tested against the K1 strain of P . falciparum . Pronounced antiplasmodial activity was observed mainly among the bisindole alkaloids, particularly villalstonine and macrocarpamine with IC50 values of 0.27 and 0.36 microM, respectively . The potent alkaloids were further tested against T9-96, the chloroquine-sensitive strain of P . falciparum . It has been found that the active alkaloids, in contrast to chloroquine, have significantly higher affinity to the K1 strain than to the T9-96 strain. J UOEH, 1999 Dec 1, 21(4), 265 - 76 Studies on the multidrug resistance gene expression in the livers of rats associated with different susceptibility of hepatocarcinogenesis; Gotoh S et al.; Inbred carcinogen-resistant DRH rat strain developed from the closed colony Donryu rats on the basis of selective markers such as poor induction of gamma-glutamyltranspeptidase and marked reduced incidences of liver tumors during 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) administration, which took more than 10 years . Previously, we observed that the Donryu rat liver was quite sensitive to both DNA-damaging and cytotoxic effects of 3'-Me-DAB, while the DRH rat liver showed tolerance to 3'-Me-DAB under the same conditions . In the present study, we examined mRNA levels related to the cytotoxic drug resistance mechanism in the livers of DRH and Donryu rats using RT-PCR . Contrary to our expectation, we observed rather similar levels of mRNAs between the two rat strains under the following conditions: i) mdr1 mRNA induction after 3'-Me-DAB administration . ii) MLP-2 mRNA reduction by 3'-Me-DAB administration, iii) MLP-2 mRNA induction after cholestasis and iv) constitutive levels of cMOAT gene expression . On the other hand, the levels of p53 mRNA and p53 protein in the Donryu rat liver were higher than those in DRH rat liver during 3'-Me-DAB administration, suggesting that the former were more sensitive to 3'-Me-DAB than DRH rat under these conditions . In conclusion, we failed to demonstrate the difference in the cytotoxic drug resistance mechanism between DRH and Donryu rats at least under the conditions examined in this study. Anticancer Drugs, 1999 Nov, 10(10), 921 - 8 In vitro cellular accumulation and cytotoxicity of liposomal and conventional formulations of daunorubicin and doxorubicin in resistant K562 cells; Wang Y et al.; Previous investigations have indicated the possibility to circumvent multidrug resistance (MDR) by incorporation of an anthracycline into liposomes . We examined the in vitro cytotoxicity and cellular drug accumulation of the anthracyclines daunorubicin and doxorubicin compared with the commercially available liposomal formulations DaunoXome and Caelyx in human myelogenous leukemia K562 cells . The drug-sensitive parental K562/K line was compared with the P-glykoprotein (P-gp)-expressing cell lines K562/Dnr and K562/Vcr . Two cell lines with reduced levels of topoisomerase II (K562/Nov and K562/Ida) were also included . The cytotoxicity was determined by fluorometric microculture cytotoxicity assay and the cellular drug levels were determined by high performance liquid chromatograghy . There was a strong inverse correlation between P-gp levels and cellular drug accumulation (rho = -0.83, p = 0.04) and cytotoxicity (rho = -0.95, p = 0.01) of daunorubicin . Also the cytotoxicity of DaunoXome and doxorubicin was related to P-gp levels (rho = -0.96, p = 0.01 and rho = -0.90, p = 0.07, respectively) . Caelyx did not show any cytotoxic effect due to impaired cellular uptake of the pegylated liposome . Regardless of the P-gp levels of the treated cells, DaunoXome showed the same cytotoxic effect despite lower intracellular accumulation (range 22-47%), compared with conventional daunorubicin. Anticancer Res, 1999 Sep-Oct, 19(5B), 4311 - 4 Intracellular accumulation and retention of calcein in Ehrlich ascites tumor cells and their adriamycin-resistant strain; Asaumi J et al.; In this study, we observed the intracellular accumulation and retention of calcein in Ehrlich ascites tumor cells (wild type EAT cells) and their adriamycin (ADR)-resistant strain to further study the mechanisms of ADR accumulation . In the wild type EAT cells, intracellular accumulation of calcein increased rapidly, but it was not incorporated into the cells in the ADR-resistant strain . This suggests that the efflux system or the inhibition system of influx, which does not exist in the wild type EAT cells, involves in the ADR-resistant strain . Calcein incorporated into the cells was effluxed from the cells in both strains at almost the same rate . This suggests that the both strains may use a similar efflux system against calcein . P-gp extrudes calcein-AM directly from the cell membrane, but not calcein . Calcein is effluxed by multidrug resistant protein (MRP) or multi-specific organic anion transporter (MOAT) . Therefore, the mechanism, which extrudes calcein of both strains, may be the other efflux system included MRP or MOAT apart from P-gp . In the same medium condition as treating in the calcein efflux experiments, about 20% amount of ADR incorporated into the cells was effluxed in the ADR-resistant cells, but not in the wild type cells . Since the efflux rates in the ADR differed from those in calcein in the same medium condition, the mechanism of the ADR efflux might differ from that of calcein efflux in the ADR-resistant strain. Anticancer Res, 1999 Sep-Oct, 19(5B), 4085 - 90 Psammaplin A, a natural phenolic compound, has inhibitory effect on human topoisomerase II and is cytotoxic to cancer cells; Kim D et al.; We evaluated the topoisomerase II (Topo II) inhibitory activity of psammaplin A (PSA), a naturally occurring biphenolic compound, and its cytotoxicity to some human cancer cells including P-glycoprotein (Pgp)-overexpressing multidrug resistant (MDR) cell line . PSA completely inhibited the DNA relaxation activity of Topo II at 75.0 microM . It also completely inhibited the DNA decatenation activity of Topo II at 75.0 microM, and showed about 50% inhibitory activity at 18.8 microM . In the cytotoxicity assay, the effective concentrations that cause 50% inhibition of cell growth (EC50) were 0.48, 0.39, 1.83 and 3.76 microM to A549, SK-OV-3, HCT15 and HCT15/CL02 (MDR cell line established from HCT15 cells) cancer cells, respectively . In the presence of 8.0 microM of verapamil (VER), a well-known MDR modulator, the EC50 of PSA to HCT15/CL02 cells was reduced about 2.1 fold . Meanwhile, the EC50s of standard Topo II inhibitory drugs such as doxorubicin, etoposide and mitoxantrone to HCT15/CL02 cells were reduced about 8.5, 9.3 and 8.1 fold in the presence of 8.0 microM VER, respectively . From the results, we conclude that PSA has Topo II inhibitory activity, and its cytotoxicity to cancer cells is not so strongly affected by Pgp-associated MDR phenotype in comparison with some Topo II inhibitory anticancer drugs used in the clinic. Int J Cancer, 2000 Jan 15, 85(2), 267 - 74 Redox regulation of P-glycoprotein-mediated multidrug resistance in multicellular prostate tumor spheroids; Wartenberg M et al.; Multicellular prostate tumor spheroids develop intrinsic P-glycoprotein (Pgp)-mediated multidrug resistance with the appearance of quiescent cell areas . We have investigated the effect of intracellular reactive oxygen species (ROS) on Pgp expression in large, quiescent and drug-resistant multicellular spheroids (diameter 250 +/- 50microm) . Using the ROS-sensitive fluorescence dye 2;7;-dichlorodihydrofluorescein diacetate (H(2)DCFDA), we demonstrated that these tumor spheroids are characterized by reduced intracellular ROS compared with drug-sensitive small spheroids (diameter 60 +/- 20microm) consisting predominantly of proliferating cells . The prooxidants hydrogen peroxide, menadione and glyceraldehyde raised ROS in large tumor spheroids and significantly down-regulated Pgp within 24 hr . Comparable effects were achieved with the known Pgp-reversing agents sodium orthovanadate, quinidine and cyclosporin A but not with verapamil . Consequently, the retention and toxicity of the anthracycline doxorubicin was increased in tumor spheroids treated with prooxidants . Co-administration of prooxidants and the free radical scavenger ebselen did not alter Pgp levels, indicating that down-regulation of Pgp is mediated via ROS . Down-regulation of Pgp by H(2)O(2) was abolished when either forskolin, 8-Br-cAMP or IBMX, which raise intracellular cAMP levels, was co-administered, indicating that Pgp expression is regulated by protein kinase A (PKA) . Furthermore, Pgp was down-regulated by the PKA inhibitors Rp-cAMPs and H89 . Since prooxidants stimulated the growth of multicellular spheroids and down-regulated the cyclin-dependent kinase inhibitor p27(kip1), we conclude that ROS-mediated Pgp down-regulation may be paralleled by recruitment of drug-resistant quiescent cells in the depth of the tumor tissue for cell-cycle activity . Anal Biochem, 2000 Jan 15, 277(2), 247 - 53 Yeast two-hybrid assay for examining human immunodeficiency virus protease heterodimer formation with dominant-negative inhibitors and multidrug-resistant variants; Todd S et al.; The yeast two-hybrid assay was used to study the dimerization of engineered and naturally occurring variants of human immunodeficiency virus (HIV) protease (PR) monomers . Defective monomers that were previously shown to exhibit a dominant-negative (D-N) effect in cultured mammalian cells were tested for their ability to interact in the two-hybrid assay . Similarly, monomers with dimer-interface substitutions and monomers harboring in vivo selected mutations that confer multidrug resistance (mdr) in an AIDS patient were tested for interaction in yeast . Dimer formation between wt monomers with catalytic aspartates was not detected in yeast, whereas the dimerization of PR monomers harboring the acid active site substitution D25N was readily demonstrated . The use of inactive monomers harboring the D25N substitution as a genetic background for studying additional HIV PR mutations allowed for the probing of interactions between monomers with mdr-associated mutations with those based on the HIV-1 HXB2R sequence . The HTLVIII/HIV-1 HXB2R clone has been the basis for a large number of HIV-related plasmids, primers, antibodies, and other specific reagents throughout the HIV research community . The results of our assay suggest that HXB2R-based D-N PR inhibitors associate with variant monomers based on the recently obtained nucleotide sequence from an AIDS patient with a multidrug-resistant virus . These results further encourage the use of D-N PR inhibitors as antiviral agents which may complement existing small-molecule combination therapies . J Inorg Biochem, 1999 Oct, 77(1-2), 89 - 93 The role of DNA mismatch repair in cisplatin mutagenicity; Lin X et al.; Cisplatin (DDP) is used with varying success for the treatment of a wide spectrum of human cancers . The most abundant lesions produced in DNA are intrastrand crosslinks, which are believed to account for not only the cytotoxic action but also the mutagenicity of the drug . The molecular basis for the mutagenicity of DDP adducts is believed to be related to bypass replication across the adducts by DNA polymerase . This results in misincorporation of non-complimentary bases by polymerase beta which, if left unpaired, will generate point or frameshift mutations . An important replication-associated correction function is provided by the post-replicative DNA mismatch repair (MMR) system . Loss of MMR activity is well documented to result in increased mutation rates and instability of genomic DNA . Inactivation of the MMR system also augments the intrinsic mutagenicity of DDP and enhances the risk of developing cells resistant to other drugs commonly used in combination with DDP . A future challenge will be to assess the clinical significance of the presence of MMR-deficient cells in tumors, and investigate new approaches to circumvent such multidrug resistance. Biochemistry, 2000 Jan 11, 39(1), 194 - 204 Identification and characterization of the MDR1 promoter-enhancing factor 1 (MEF1) in the multidrug resistant HL60/VCR human acute myeloid leukemia cell line; Ogretmen B et al.; In this report, the molecular mechanisms involved in the overexpression of MDR1 mRNA in the multidrug resistant variant of the HL60 human acute myeloid leukemia cell line, HL60/VCR, were investigated . RT-PCR and nuclear run-on assays revealed that the expression of MDR1 mRNA is regulated by increased transcriptional initiation in HL60/VCR cells . Transient transfections with a 241 bp MDR1 promoter (spanning the -198 to +43 region) DNA fragment/pGL3-basic plasmid construct resulted in about 6-fold increased luciferase activity in HL60/VCR but not in HL60 cells . Moreover, ds CAAT-oligomer from the MDR1 promoter cloned upstream of the SV-40 promoter in the pGL3-promoter plasmid caused about a 7-fold increase in luciferase activity compared with plasmid constructs containing CAAT-deleted, GC-box, and nonspecific oligomers in HL60/VCR transfectants . These results were confirmed by transfecting HL60/VCR cells with the pGL3-basic plasmid containing a 237 bp mutated MDR1 proximal promoter lacking the CAAT sequence in which no change in luciferase activity was observed . However, a 5-6-fold increase in luciferase activity was measured in these cells when transfected with the wt MDR1 promoter DNA/pGL3-basic plasmid constructs . These results show that the CAAT-region is involved in upregulating the MDR1 promoter in HL60/VCR cells . A nuclear factor binding to the CAAT-region of the MDR1 promoter specifically was detected in electrophoretic mobility shift assays (EMSAs) in HL60/VCR but not in HL60 extracts . Two MDR1 promoter-associated polypeptides with molecular masses of about 130 and 162 kDa were identified in HL60/VCR cells by electroelution, specific DNA-affinity chromatography, and silver staining . Interestingly, cross-linking and Southwestern analysis indicate that only the 130 kDa protein, which we refer to as MDR1-promoter enhancing factor 1 (MEF1), has a strong DNA-binding ability, interacting with the 5'-GTCAATCC-3' element of the MDR1 promoter, as determined by DNase I protection assay . These data reveal that MEF1 upregulates the MDR1 promoter activity. Nutr Cancer, 1999, 35(1), 80 - 6 Chemopreventive effects of tea extracts and various components on human pancreatic and prostate tumor cells in vitro; Lyn-Cook BD et al.; Pancreatic and prostate cancers pose serious problems to human health . To determine the potential for chemopreventive intervention against pancreatic and prostate cancers, black and green tea extracts and components of these extracts were examined in vitro for their effect on tumor cell growth . Components included a mixture of polyphenols from green tea (GTP), mixtures of polyphenols (BTP) and of theaflavins (MF) from black tea, and the purified components epicatechin-3-gallate (ECG) and epigallocatechin-3-gallate (EGCG) . Two human cell lines, pancreatic adenocarcinoma (HPAC) and prostate tumor (LNCaP), were exposed to these agents for 24 hours . Results showed inhibition (approx 90%) of cell growth in pancreatic tumor cells by black and green tea extracts (0.02%) . GTP (10 micrograms/ml) and MF (100 micrograms/ml) significantly inhibited growth (approx 90%); ECG and EGCG inhibited growth as well (approx 95%) . Black and green tea extracts, GTP, and EGCG decreased the expression of the K-ras gene, as determined by reverse transcription-polymerase chain reaction . Green and black tea extracts decreased the multidrug-resistant gene (mdr-1), although GTP and EGCG increased expression . Similar data were obtained in the prostate cell line LNCaP . All agents significantly inhibited growth . These agents increased expression of the mdr-1 gene . This study suggests that components from black and green tea extracts can modulate the expression of genes known to play a role in the carcinogenesis process and, therefore, may be potential agents for chemoprevention against pancreatic cancer. Infection, 1999 Nov-Dec, 27(6), 331 - 4 Negative predictors of survival in HIV-infected patients with culture-confirmed pulmonary tuberculosis; Palmieri F et al.; We performed a retrospective study based on chart review of 118 HIV-infected patients with culture-confirmed pulmonary TB, in which M . tuberculosis isolates were tested for drug susceptibility . Patients were enrolled in the period January 1987 to December 1996 and followed until September 1997 . The median survival for the entire cohort was 15.2 months with a 1-year survival rate of 57% . Prior AIDS-defining illness, low CD4 count (< 200/mm3), not having received antituberculous therapy with at least two drugs to which M . tuberculosis was susceptible in vitro, starting within four weeks of diagnosis, treatment duration of less than three weeks and multidrug resistant tuberculosis were each independently associated with decreased survival in multivariate analysis. Clin Neurol Neurosurg, 1999 Dec, 101(4), 238 - 44 The effects of anticancer drugs in combination with nimodipine and verapamil on cultured cells of glioblastoma multiforme; Durmaz R et al.; The presence of the cellular multidrug resistance (MDR1) gene and its product, P-glycoprotein (Pgp), is thought to be a mechanism for the failure of chemotherapy in cancer patients . Calcium channel blockers have been shown to sensitise cancer cells to anticancer drugs by reversing Pgp expression in cell lines . The interactions between anticancer drugs such as carmustine (BCNU), vincristine (VCR) and procarbazine (PCB) and calcium channel blockers such as nimodipine and verapamil on cultured cells of glioblastoma from eight patients were therefore tested . Pgp expression was examined immunohistochemically using C219 monoclonal antibody in cytospin preparation . The cytotoxicity of the drugs was screened using microculture tetrazolium assay . The cells from five patients showed positive immunoreaction for Pgp . Nimodipine showed growth-inhibitory activity against glioblastoma cells at a rate of 16.55-26.88% (P < 0.05), but a similar effect was not observed with verapamil . While antiproliferative effects of BCNU were around 20.91-45.09% (P < 0.05) on the cells from seven patients, VCR was the most effective agent in inhibition of cell growth at a rate of 26.43-48.47% (P < 0.05) . The response of the cells from five patients to PCB was from 11.98 to 16.32% (P < 0.05) . When used together, nimodipine further enriched cytotoxicity of the anticancer drugs up to 11.14-40.85% (P < 0.05) without relation to Pgp expression . In conclusion, the enhancement of cytotoxicity of anticancer drugs by nimodipine suggests that there might be a synergy between anticancer drugs and nimodipine in the inhibition of glioma cell growth. Semin Hematol, 1999 Oct, 36(4 Suppl 8), 16 - 25 New developments in the treatment of acute myeloid leukemia: focus on topotecan; Kantarjian H; Although the prognosis for adults with acute myelogenous leukemia (AML) has improved over the past 10 years, overall results remain modest . Current research areas in the treatment of AML include dose-intensive therapy and stem-cell transplantation (SCT), immunotherapy, modulation of leukemia resistance (eg, multidrug resistance {MDR} Inhibitors), differentiation therapy (eg, retinolds), exploitation of different disease pathophysiology (eg, angiogenesis inhibitors, apoptosis-inducing agents), targeted therapy (eg, monoclonal antibodies, gene therapy), and the development of additional active chemotherapeutic agents with different mechanisms of action . Topotecan, a topoisomerase-I Inhibitor, may potentially enhance the activity of standard induction chemotherapy with cytosine arabinoside (cytarabine) and topoisomerase-II inhibitors . Topotecan is being investigated as salvage and front-line therapy for AML in combination with etoposide, cytarabine, or cyclophosphamide . The role that topotecan will eventually play In the treatment of AML is not yet clear, but encouraging results from triple combination induction therapy In patients with unfavorable prognoses warrant further investigation. Biomaterials, 2000 Jan, 21(1), 1 - 7 Reversion of multidrug resistance by co-encapsulation of doxorubicin and cyclosporin A in polyalkylcyanoacrylate nanoparticles; Soma CE et al.; Individual and combined polyalkylcyanoacrylate nanoparticle formulation of cyclosporin A and doxorubicin were prepared and evaluated in an attempt to show improved growth inhibition efficacy in a resistant cell culture line . The drug loaded nanoparticles were prepared using the well established emulsion polymerization process without using any modification for the hydrophilic doxorubicin drug whereas the incorporation of cyclosporin A needed to wait a moment after the polymerization reaction started . This was necessary to avoid cyclosporin A precipitation and polymer aggregation . Cyclosporin A release from the nanoparticles was rapid probably because the drug was adsorbed onto the nanoparticles surface rather than embedded into the polymeric core . Doxorubicin displayed also a burst effect but with a slower second phase probably related with the nanoparticles bioerosion rate owing to its entrapment in the polymeric network . Finally, it was shown in resistant cell culture experiments that the association of both cyclosporin A and doxorubicin within a single nanoparticle formulation elicited the most effective growth rate inhibition as compared to other combinations of both drugs while using a lower amount of polymer compared to separated nanoparticle formulations . This result was probably due to the synergistic effect achieved by combining the chemo-sensitizing compound cyclosporin A, with an effective cytotoxic drug like doxorubicin. Pediatr Int, 1999 Dec, 41(6), 641 - 7 Expression of multidrug resistance-related genes does not contribute to risk factors in newly diagnosed childhood acute lymphoblastic leukemia; Kakihara T et al.; BACKGROUND: The present study was designed to evaluate the association of multidrug resistance gene (MDR1), multidrug resistance-associated protein (MRP) gene and lung resistance protein (LRP) gene expression (mRNA levels) with risk factors such as phenotype, age and leukocyte count in newly diagnosed childhood acute lymphoblastic leukemia, because biological mechanisms contributing to risk factors were not known . METHODS: Expression of the MDR1, MRP and LRP genes in leukemic cells from 40 pediatric patients was quantitated using the reverse transcriptase polymerase chain reaction method . The associations of expression of these genes with age and leukocyte count, which are known as risk factors for acute lymphoblastic leukemia (ALL), were analyzed . Moreover, the associations with clinically classified risk-related groups (high-risk ALL and low-risk ALL) were also investigated . RESULTS: No positive correlations between expression of these genes and age or leukocyte count were found . The expression of these genes was not different among clinically classified risk-related groups . CONCLUSIONS: Expression (mRNA levels) of the MDR1, MRP and LRP genes did not contribute to risk factors in newly diagnosed childhood acute lymphoblastic leukemia. Cancer, 2000 Jan 1, 88(1), 79 - 87 Paclitaxel and tamoxifen: An active regimen for patients with metastatic melanoma; Nathan FE et al.; BACKGROUND: In early trials of paclitaxel administered as a 24-hour infusion, an overall response rate of 16% was reported for patients with metastatic melanoma . Paclitaxel is a natural product-based agent and is thus subject to the problem of multidrug resistance (MDR) . Tamoxifen is an agent that can abrogate MDR and potentially enhance the effect of paclitaxel . A Phase II trial of the combination was undertaken with previously treated patients . METHODS: Patients with metastatic cutaneous or mucosal melanoma who were previously treated with the Dartmouth chemotherapy regimen (dacarbazine, carmustine, cisplatin, and tamoxifen) were evaluated . Paclitaxel was administered at a dose of 225 mg/m(2) intravenously over 3 hours every 3 weeks . All patients also took tamoxifen 40 mg orally daily . Treatment continued until disease progression . RESULTS: Twenty-one patients completed at least two cycles of paclitaxel and were evaluable for response . Five responses were observed, 1 complete response, and 4 partial responses, for an overall response rate of 24% . The combination was well tolerated . The most common nonhematologic side effects were myalgia and paresthesia . Hematologic toxicity was mild . No patients developed neutropenic fever . CONCLUSIONS: This is the first report of a Phase II trial evaluating paclitaxel as a 3-hour infusion in melanoma patients . The 3-hour infusion is well tolerated and results in little myelosuppression and minimal neurotoxicity . The contribution of tamoxifen is difficult to evaluate because plasma levels were not measured . It is possible that a higher response rate might be observed with larger doses of tamoxifen . Further investigation of paclitaxel in the treatment of patients with metastatic melanoma is warranted . Mol Pharmacol, 2000 Jan, 57(1), 188 - 97 Altered expression of hepatic cytochromes P-450 in mice deficient in one or more mdr1 genes; Schuetz EG et al.; We hypothesized that the drug efflux protein P-glycoprotein (Pgp), the product of the multidrug resistance gene MDR1, might influence hepatic expression of CYP3A or other cytochromes P-450 (P-450s) because Pgp can transport endogenous regulators of these cytochromes . We began with variants of a CF-1 mouse strain containing a defective mdr1a gene that is inherited in a Mendelian fashion . The amount of CYP3A protein in liver was inversely related to the gene dose of the normal mdr1a allele in these mice . mdr1a knockout mice of either mixed (FVB x 129/Ola) or pure FVB genetic background and housed in Amsterdam display an increased expression of CYP2B and CYP3A proteins . However, because mdr1a ablation causes a compensatory increase in hepatic mdr1b (which can efflux intracellular glucocorticoids), we reasoned that mdr1b might mask the overall effect of mdr1a absence on P-450 gene expression . Targeted inactivation of the mdr1b gene increased P-450 expression, but the effect was modest compared with mdr1a ablation . Mice nullizygous for both mdr1a and mdr1b-type Pgps and kept in Amsterdam had dramatically increased levels of CYP3A protein as well as other P-450s examined and of the electron donor P-450 reductase . Consistent with the protein results, CYP3A catalytic activity measured as midazolam 1'- and 4-hydroxylation in liver microsomes from these knockout mice revealed a rank order of activities with mdr1a/1b > mdr1a > mdr1b > (+/+) mice . In contrast to results in mice housed in Amsterdam, in the genetically identical mdr1a or mdr1a/1b (-/-) male mice housed in the United States, hepatic P-450 expression was unaffected by mdr1 genotype or actually showed a slight decrease in mdr1a (-/-) mice . These results provide a revealing picture of mdr1-type Pgp as an upstream regulator of hepatic P-450 expression, and demonstrate that these pharmacologically relevant phenotypes in knockout mice depend not only on the genetic make-up of the mice but also on the environment. Mol Pharmacol, 2000 Jan, 57(1), 59 - 67 Endothelin B receptor-mediated regulation of ATP-driven drug secretion in renal proximal tubule; Masereeuw R et al.; In the kidney, endothelins (ETs) are important regulators of blood flow, glomerular hemodynamics, and sodium and water homeostasis . They have been implicated in the pathophysiology of acute ischemic renal failure, nephrotoxicity by cyclosporine, cisplatin and radiocontrast agents, and vascular rejection of kidney transplants . Here, we used intact killifish renal proximal tubules, fluorescent substrates for Mrp2 (fluorescein-methotrexate, FL-MTX) and P-glycoprotein (a fluorescent CSA derivative, NBD-CSA), and confocal microscopy to reveal a new role for renal ET: regulation of ATP-driven drug transport in proximal tubule . Subnanomolar to nanomolar concentrations of ET-1 rapidly reduced the cell-to-tubular lumen transport of both fluorescent compounds . These effects were prevented by an ET(B) receptor antagonist but not by an ET(A) receptor antagonist . Immunostaining with an antibody to mammalian ET(B) receptors showed specific localization to the basolateral membrane of the fish tubular epithelial cells . ET-1 effects on transport were blocked by protein kinase C-selective inhibitors, implicating protein kinase C in ET-1 signaling . Finally, the nephrotoxic radiocontrast agent iohexol reduced cell-to-lumen FL-MTX and NBD-CSA transport, and these effects were abolished by an ET(B) receptor antagonist . These are the first results linking ET to the control of xenobiotic transport and the first demonstrating control of renal multidrug resistance-associated protein 2 and P-glycoprotein by a hormone. Bioorg Med Chem Lett, 1999 Dec 20, 9(24), 3411 - 6 N'-{2-(2-thiophene)ethyl}-N'-{2-(5-bromopyridyl)} thiourea as a potent inhibitor of NNI-resistant and multidrug-resistant human immunodeficiency virus-1; Uckun FM et al.; The thiophene-ethyl thiourea (TET) compound N'-{2-(2-thiophene)ethyl}-N'-{2-(5-bromopyridyl)}-thiourea (compound HI-443) was five times more potent than trovirdine, 1250 times more potent than nevirapine, 100 times more potent than delavirdine, 75 times more potent than MKC-442, and 50 times more potent than AZT against the multidrug resistant HIV-1 strain RT-MDR with a V106A mutation . HI-443 was almost as potent against the NNI-resistant HIV-1 strain A17 with a Y181C mutation as it was against HTLV(IIIB) . The activity of HI-443 against A17 was ten times more potent than that of trovirdine, 2083 times more potent than that of nevirapine, and 1042 times more potent than that of delavirdine . HI-443 inhibited the replication of the NNI-resistant HIV-1 strain A17 variant with Y181C plus K103N mutations in RT with an IC50 value of 3.3 microM, whereas the IC50 values of trovirdine, nevirapine, and delavirdine were all >100 microM . These findings establish the novel thiophene containing thiourea compound HI-443 as a novel NNI with potent antiviral activity against NNI-sensitive, NNI-resistant and multidrug-resistant strains of HIV-1. Bioorg Med Chem Lett, 1999 Dec 20, 9(24), 3403 - 6 Highly increased cellular accumulation of vincristine, a useful hydrophobic antitumor-drug, in multidrug-resistant solid cancer cells induced by a simply reduced taxinine; Sako M et al.; Regio- and/or chemo-selective reductions of taxinine (1a), a taxane diterpenoid readily obtainable from the needles of a Japanese yew (Taxus cuspidata), at the 5-O-cinnamoyl and 4-exo-methylene moieties have been accomplished by the catalytic hydrogenation over Pd/C or Rh/C to obtain 5-O-phenylpropionylated taxinine A (1b), 5-O-cyclohexylpropionylated taxinine A (1c), and 5-O-phenylpropionylated 4,20-dihydrotaxinine A (2a) in almost quantitative yields, respectively . Among them, taxoid 1b was found to be highly effective in increasing the cellular accumulation of vincristine in the multidrug-resistant human ovarian cancer cells compared with the cases of verapamil and the previously reported taxoids. Neurosci Res, 1999 Nov, 35(2), 155 - 64 Induction of blood-brain barrier properties in immortalized bovine brain endothelial cells by astrocytic factors; Sobue K et al.; The blood-brain barrier (B-BB) protects the free passage of substances into the brain and maintains the homeostasis of the central nervous system . It is commonly accepted that astrocytes surrounding brain endothelial cells influence the B-BB formation and the exhibition of B-BB function of capillaries . To begin the in vitro study on the B-BB, it is essential to obtain a homogenous and sufficient supply of brain endothelial cells as well as astrocytes . We thus immortalized the bovine brain endothelial cell (BBEC) by transfection of the SV40 large T antigen and obtained a single clone, t-BBEC-117, which retained the brain endothelial cell phenotype . Astrocyte in co-culture was found to tighten the intercellular contacts of the immortal cells resulting in a reduced L-glucose permeability, and its conditioned medium (CM) augmented a B-BB phenotype, alkaline phosphatase (ALP) activity . Among known astrocytic factors, only fibroblast growth factor-basic (bFGF) could mimic the actions of astrocytes as measured by L-glucose permeability and ALP activity . Moreover, anti-bFGF antibody canceled 90% of ALP activation by astrocyte CM . Basic FGF, however, failed to induce other B-BB phenotypes such as the expressions of multidrug resistance (mdr) and glucose transporter (GLUT-1) genes . These data suggest that bFGF is one of the most plausible astrocytic factors to induce the B-BB properties of immortal brain endothelial cells together with some unknown factors in the astrocyte CM. Eur J Pharm Biopharm, 2000 Jan, 49(1), 11 - 5 HPMA copolymer-anticancer drug-OV-TL16 antibody conjugates . 3 . The effect of free and polymer-bound adriamycin on the expression of some genes in the OVCAR-3 human ovarian carcinoma cell line; Kunath K et al.; The effect of an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-Adriamycin-OV-TLl6 antibody conjugate {P(GFLG)-ADR-Ab} on OVCAR-3 human ovarian carcinoma cells was studied . A nontargeted HPMA copolymer-ADR conjugate (P(GFLG)-ADR) and free ADR were the controls . The IC(50) doses were 0.65, 3.0, and 65 microM for free ADR, targeted P(GFLG)-ADR-Ab conjugate, and nontargeted P(GFLG)-ADR conjugate, respectively . These differences reflect the different mechanisms of cell entry of the compounds evaluated . Free ADR and HPMA copolymer-ADR conjugates had different impacts on the expression of MDR1, MRP, c-fos, c-jun, and bcl-2 genes which encode the P-glycoprotein (MDR1) and the multidrug resistance-associated protein (MRP) efflux pumps, and play an important role in cell death signaling pathways (c-fos, c-jun, and bcl-2) . Whereas high doses of free ADR induced MDR1 gene expression, HPMA copolymer-bound ADR appeared to be without effect . On the contrary, expression of the MRP gene was not influenced by free ADR, whereas HPMA copolymer-ADR conjugates seemed to suppress the gene expression in a concentration-dependent manner . There were differences in the expression of c-fos, c-jun, and bcl-2 genes after the incubation of OVCAR-3 cells with free and HPMA copolymer-bound ADR indicating differences in activation of cell death signaling pathways. In Vitro Cell Dev Biol Anim, 1999 Nov-Dec, 35(10), 580 - 92 Evaluation of an in vitro coculture model for the blood-brain barrier: comparison of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources; Scism JL et al.; Cocultures of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources, American Type Culture Collection and European Collection of Animal Cell Cultures, were evaluated as an in vitro model for the blood-brain barrier . Monolayers of endothelial cells grown in the presence or absence of glial cells were examined for transendothelial electrical resistance, sucrose permeability, morphology, multidrug resistance-associated protein expression, and P-glycoprotein expression and function . Coculture of glial cells with endothelial cells increased electrical resistance and decreased sucrose permeability across European endothelial cell monolayers, but had no effect on American endothelial cells . Coculture of European glial cells with endothelial cells caused cell flattening and decreased cell stacking with both European and American endothelial cells . No P-glycoprotein or multidrug resistance-associated protein was immunodetected in endothelial cells grown in glial cell-conditioned medium . Functional P-glycoprotein was demonstrated in American endothelial cells selected in vinblastine-containing medium over eight passages, but these cells did not form a tight endothelium . In conclusion, while European glial cells confer blood-brain barrier-like morphology and barrier integrity to European endothelial cells in coculture, the European endothelial-glial cell coculture model does not express P-glycoprotein, normally found at the blood-brain barrier . Further, the response of endothelial cells to glial factors was dependent on cell source, implying heterogeneity among cell populations . On the basis of these observations, the umbilical vein endothelial cell-glial cell coculture model does not appear to be a viable model for predicting blood-brain barrier penetration of drug molecules. Cell Prolif, 1999 Aug, 32(4), 203 - 13 Optimized fluorescent probe combinations for evaluation of proliferation and necrosis in anthracycline-treated leukaemic cell lines; Boutonnat J et al.; Proliferation and multidrug resistance status are key predictors of therapeutic outcome in acute myeloid leukaemia (AML) . Anthracyclines such as daunorubicin (DNR) are typically used to treat AML and can induce drug resistance . The goal of the studies described here was to select a combination of fluorescent probes that could be used in combination with flow cytometry to monitor cell proliferation vs . cell death/necrosis as a function of anthracycline uptake . Propidium iodide (PI), the most commonly used marker of membrane integrity, cannot be used to evaluate necrosis in DNR-containing cells because of spectral overlap . A membrane integrity probe compatible with the use of a dye dilution method using PKH67 to study cell proliferation was also selected . The results show that DAPI and Cascade Blue (CB), like PI, were able to detect necrotic cells when no DNR was present, although CB gave less resolution between viable and necrotic cells than PI or DAPI . In the presence of DNR, DAPI cannot be used owing to the fluorescence quenching by DNR . However, it was found that a combination of DNR, CB, and PKH67 allows simultaneous identification of chemoresistant cells, based on reduced DNR accumulation, necrotic cells based on CB incorporation, and proliferating cells based on partitioning of PKH67 fluorescence between daughter cells . It was also found that unless a marker of necrosis is used in combination with the dye dilution assay, a moderate decrease of fluorescence as a result of necrosis may be incorrectly interpreted as proliferation. Bioorg Med Chem Lett, 1999 Dec 6, 9(23), 3381 - 6 Reversal of resistance in multidrug resistance protein (MRP1)-overexpressing cells by LY329146; Norman BH et al.; The benzothiophene LY329146 reverses the drug resistance phenotype in multidrug resistance protein (MRP1)-overexpressing cells when dosed in combination with MRP1-associated oncolytics doxorubicin and vincristine . Additionally, LY329146 inhibited MRP1-mediated uptake of the MRP1 substrate LTC4 into membrane vesicles prepared from MRP1-overexpressing cells. Rev Mal Respir, 1999 Nov, 16(5), 842 - 5 {Breast abscess and pregnancy toxemia revealing multidrug resistant tuberculosis}; Gach O et al.; We report here the case of a 33-year-old woman admitted in hospital for eclampsia . An infectious course led to the diagnosis of tuberculosis breast abscess with laryngitis and tuberculous bilateral excavated bronchopneumonia . The isolated strain demonstrated resistance to the principals antituberculous agents . However outcome was favorable after cesarean and treatment adapted to sensitivity studies. Anal Cell Pathol, 1999, 18(4), 175 - 81 Textural analysis of nuclear mitotic apparatus antigen (NuMA) spatial distribution in interphase nuclei from human drug-resistant CEM lymphoblasts; Rafki-Beljebbar N et al.; In tumour cell lines, the resistance of cancer cells to a variety of structurally unrelated chemotherapeutic drugs is termed multidrug-resistance or MDR . We reported previously {6} that MDR leukemic cells displayed nuclear texture changes, as assessed by image cytometry . The nature of these changes remained uncertain but they could be associated with alterations of the nuclear matrix which could serve an important role in DNA organization and chromatin structure . Therefore, we have compared the textural features observed in G0/G1 nuclei from human leukemic CEM cells and their MDR variant CEM-VLB, after staining of either DNA by Feulgen method or nuclear matrix by immunodetection of NuMA antigen on DNase treated samples . Chromatin or NuMA distributions within the nucleus were evaluated by image cytometry . Changes in textural parameters indicate that modifications of NuMA distribution observed in MDR cells are parallel to those observed at the whole chromatin level (i.e., a more decondensed and coarse texture with increase of Energy and Long-run sections and decrease of Contrast and Short-run sections) . Moreover, Optical Densities measurements indicate that MDR cells seem to contain less NuMA, a datum confirmed by immunoblotting of nuclear proteins . In conclusion, chromatin changes observed by image cytometry in drug-resistant human leukemic CEM cells appear associated with modifications of the nuclear matrix structure. Biochem Pharmacol, 2000 Feb 1, 59(3), 293 - 300 Development of daunorubicin resistance in tumour cells by induction of carbonyl reduction; Ax W et al.; A resistant descendant of the human stomach carcinoma cell line EPG85-257 was selected in the presence of increasing concentrations of daunorubicin (DRC) . To avoid the expression and activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP), cells were cultured in the presence of verapamil . The resulting cells were used to evaluate an induced carbonyl reduction as a new determinant in DRC resistance . The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide) toxicity assay was performed to estimate sensitivity to DRC in both cell lines . IC50 values of DRC increased almost 8-fold in the resistant descendants compared to the parental cell line . P-gp transcripts were detectable in both cell lines at only very low levels, and no significant alterations between sensitive and resistant cells were observed . MRP mRNA expression was markedly higher compared to P-gp mRNA, but, as was the case with P-gp, MRP mRNA levels in sensitive and resistant cells showed no alteration . This was probably due to the effect of the presence of verapamil during cell selection . Another known drug resistance factor, the lung resistance-related protein (LRP), was not at all detectable . Interestingly, resistant cells possessed 6-fold higher levels of DRC carbonyl-reducing activity, leading to the less toxic 13-hydroxy metabolite daunorubicinol (DRCOL) . The 6-fold higher DRCOL formation roughly parallels the 8-fold increase in DRC IC50 values during cell selection, and therefore may account for DRC resistance in these cells . The determination of specific carbonyl reducing enzymes, known to be involved in DRC detoxification, revealed that mRNA expression of carbonyl reductase (EC 1.1.1.184), aldose reductase (EC 1.1.1.21), and dihydrodiol dehydrogenase 2 (EC 1.3.1.20) increased in the resistant descendant . In contrast, the phase II-conjugating enzyme activities of glutathione S-transferases were significantly lower in resistant than in sensitive cells, whereas those of glucuronosyl transferase were not detectable in either cell line . Apparently, conjugating enzymes are not involved in DRC resistance in human stomach carcinoma cells . These studies indicate that DRC resistance in human stomach carcinoma cells may appear as a result of an induction of metabolic DRC inactivation via carbonyl reduction to the less active 13-hydroxy metabolite DRCOL. J Infect Dis, 2000 Jan, 181(1), 390 - 4 Granulocyte-macrophage colony-stimulating factor augments phagocytosis of Mycobacterium avium complex by human immunodeficiency virus type 1-infected monocytes/macrophages in vitro and in vivo; Kedzierska K et al.; The role of human immunodeficiency virus type 1 (HIV-1) infection on the ability of human monocytes/macrophages to phagocytose Mycobacterium avium complex (MAC) in vivo and in vitro and the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on this function were investigated . By use of a flow cytometric assay to quantify phagocytosis, HIV-1 infection was found to impair the ability of monocyte-derived macrophages to phagocytose MAC in vitro, whereas GM-CSF significantly improved this defect . Phagocytosis was not altered by exposure to a mutant form of GM-CSF (E21R) binding only to the alpha chain of the GM-CSF receptor, suggesting that signaling by GM-CSF that leads to augmentation of phagocytosis is via the beta chain of the receptor . In a patient with AIDS and disseminated multidrug-resistant MAC infection, GM-CSF treatment improved phagocytosis of MAC by peripheral blood monocytes and reduced bacteremia . These results imply that GM-CSF therapy may be useful in restoring antimycobacterial function by human monocytes/macrophages. Antimicrob Agents Chemother, 2000 Jan, 44(1), 103 - 10 Genotypic analysis of Mycobacterium tuberculosis in two distinct populations using molecular beacons: implications for rapid susceptibility testing; Piatek AS et al.; Past genotypic studies of Mycobacterium tuberculosis may have incorrectly estimated the importance of specific drug resistance mutations due to a number of sampling biases including an overrepresentation of multidrug-resistant (MDR) isolates . An accurate assessment of resistance mutations is crucial for understanding basic resistance mechanisms and designing genotypic drug resistance assays . We developed a rapid closed-tube PCR assay using fluorogenic reporter molecules called molecular beacons to detect reportedly common M . tuberculosis mutations associated with resistance to isoniazid and rifampin . The assay was used in a comparative genotypic investigation of two different study populations to determine whether these known mutations account for most cases of clinical drug resistance . We analyzed samples from a reference laboratory in Madrid, Spain, which receives an overrepresentation of MDR isolates similar to prior studies and from a community medical center in New York where almost all of the resistant isolates and an equal number of susceptible controls were available . The ability of the molecular beacon assay to predict resistance to isoniazid and rifampin was also assessed . The overall sensitivity and specificity of the assay for isoniazid resistance were 85 and 100%, respectively, and those for rifampin resistance were 98 and 100%, respectively . Rifampin resistance mutations were detected equally well in isolates from both study populations; however, isoniazid resistance mutations were detected in 94% of the isolates from Madrid but in only 76% of the isolates from New York (P = 0.02) . In New York, isoniazid resistance mutations were significantly more common in the MDR isolates (94%) than in single-drug-resistant isolates (44%; P < 0.001) . No association between previously described mutations in the kasA gene and isoniazid resistance was found . The first mutations that cause isoniazid resistance may often occur in sequences that have not been commonly associated with isoniazid resistance, possibly in other as yet uncharacterized genes . The molecular beacon assay was simple, rapid, and highly sensitive for the detection of rifampin-resistant M . tuberculosis isolates and for the detection of isoniazid resistance in MDR isolates. Tumour Biol, 2000 Jan-Feb, 21(1), 54 - 62 Vanadate is toxic to adherent- growing multidrug-resistant cells; Capella LS et al.; The development of multidrug resistance (MDR) is the primary cause of failure of cancer chemotherapy and circumventing this problem is a major challenge in oncology . Vanadate is known to inhibit the ATPase activity of the P-glycoprotein and multidrug-resistant associated protein . In the present study we show that adherent MDR cells are more sensitive to vanadate than adherent non-MDR ones, but the same is not true for suspension-growing cells . Vanadate induced stress fiber in the non-MDR adherent MDCK cell line, but destroyed the actin fibers of MDCK/60 and MA104 cells, two adherent MDR cell lines, suggesting that the sensitivity of these cells to vanadate is related to their actin cytoskeleton . The results suggest that vanadate may be used as an adjuvant in the chemotherapy of solid tumors, not only as an ATPase inhibitor but also because of its effect in the MDR cell cytoskeleton . Int J Cancer, 1999 Dec 10, 83(6), 790 - 7 p21Waf1/Cip1 acts in synergy with bcl-2 to confer multidrug resistance in a camptothecin-selected human lung-cancer cell line; Zhang Y et al.; The realization that chemotherapeutic agents induce apoptosis raises the concern that tumors resistant to chemotherapy are unable to initiate the apoptotic program . In the present study, we examined the apoptosis-resistance mechanism of a multidrug-resistant cell line, A549/CPT, which was established from the human lung-cancer cell line A549 by in vitro selection with gradually increased camptothecin (CPT) concentrations . We found that A549/CPT cells were resistant to anti-cancer drug-induced apoptosis in which caspase-3-like protease activity was attenuated remarkably, compared with parental A549 cells . We observed 2 mechanisms associated with apoptosis resistance in A549/CPT cells: over-expression of anti-apoptotic bcl-2 and elevated expression of p21Waf1/Cip1 . Transfection of either bcl-2 or p21Waf1/Cip1 cDNA into parental A549 cells resulted in resistance to apoptosis . Furthermore, the co-treatment of p21Waf1/Cip1 and bcl-2 anti-sense oligodeoxy-nucleotides restored drug susceptibility in A549/CPT cells more effectively than either one of them alone . These results indicate that co-induction of bcl-2 and p21Waf1/Cip1 in A549/CPT cells may be involved in acquired drug resistance by inhibiting caspase-mediated apoptosis . Agents aimed at preventing both bcl-2 and p21Waf1/Cip1 expression may increase the efficiency of chemotherapy. Br J Haematol, 1999 Dec, 107(4), 861 - 9 Pgp-positive leukaemic cells have increased mtDNA but no increased rate of proliferation; Jia L et al.; Cells of solid tumours tend to rely on glycolysis for energy . On the other hand, increased glycolysis in solid tumour cells expressing the multidrug resistance protein MDR-1 has been associated with increased malignancy in tumours . We have previously shown that cells of the MDR-1-positive CEM/VLB100 leukaemic cell line have increased mitochondrial electron transport chain (mtETC) activity compared with parental CEM cells . In the present study we used infrared (IR) spectroscopy to demonstrate that the mitochondrial DNA (mtDNA) content in the CEM/VLB100 cell line was significantly increased compared to that in the parental CEM cells . The increase in mtDNA was not accompanied by an increase in mitochondrial protein as both lipid and protein levels were decreased in CEM/VLB100 mitochondria . The ATP content was similar in these two cell lines . However, the ATP-dependent membrane efflux pump function in CEM/VLB100 cells was significantly reduced when mitochondrial ATP synthesis was inhibited by oligomycin, a specific inhibitor of mitochondrial F0F1-ATPase . Proliferation of CEM/VLB100 cells was significantly decreased compared to parental CEM cells, and was independent of p53 expression . Thus, we conclude that: (1) IR spectroscopy is a potential powerful technique for detecting mtDNA, protein and lipid contents simultaneously; (2) leukaemic cells mainly rely on mtDNA for energy; (3) increased expression of an ATP-dependent membrane efflux pump such as Pgp may up-regulate ATP generation and mtDNA content . These metabolic perturbations may exist merely to serve the efflux pump and do not result in an increase in leukaemic cell proliferation . In addition, the associated reduction in mitochondrial lipid and protein may contribute to sensitize the cells to cytochrome c release. Cancer Res, 1999 Dec 1, 59(23), 5964 - 7 Expression of multidrug resistance protein-3 (multispecific organic anion transporter-D) in human embryonic kidney 293 cells confers resistance to anticancer agents; Zeng H et al.; Multidrug resistance-associated protein (MRP)1 and canalicular multispecific organic anion transporter (cMOAT)/MRP2 are ATP-binding cassette (ABC) transporters that confer resistance to natural product cytotoxic drugs . We recently described the complete coding sequences of four human MRP/cMOAT subfamily members and found that, among these proteins, MRP3/MOAT-D is most closely related to MRP1 (58% identity; M . G . Belinsky and G . D . Kruh, Br . J . Cancer, 80: 1342-1349, 1999) . In the present study, we sought to determine whether MRP3 is capable of conferring resistance to cytotoxic drugs . To address this question, human embryonic kidney 293 cells were transfected with an MRP3 expression vector, and the drug resistance phenotype of the transfected cells was analyzed . The MRP3-transfected cells displayed approximately 4-fold resistance to etoposide and approximately 2-fold resistance to vincristine, compared with control transfected cells . In addition, approximately 1.7-fold resistance was observed for the antimetabolite methotrexate . Increased resistance was not observed for several other natural product agents, including anthracyclines and Taxol . The MRP-transfected cells exhibited reduced accumulation of radiolabeled etoposide, consistent with the operation of a plasma membrane efflux pump . These results indicate that MRP3 confers resistance to some anticancer agents but that its resistance pattern is distinct from the resistance patterns of other ABC transporters involved in resistance to natural product chemotherapeutic agents. Cancer Res, 1999 Dec 1, 59(23), 5938 - 46 Camptothecin resistance: role of the ATP-binding cassette (ABC), mitoxantrone-resistance half-transporter (MXR), and potential for glucuronidation in MXR-expressing cells; Brangi M et al.; The mitoxantrone resistance (MXR) gene encodes a recently characterized ATP-binding cassette half-transporter that confers multidrug resistance . We studied resistance to the camptothecins in two sublines expressing high levels of MXR: S1-M1-80 cells derived from parental S1 colon cancer cells and MCF-7 AdVp3,000 isolated from parental MCF-7 breast cancer cells . Both cell lines were 400- to 1,000-fold more resistant to topotecan, 9-amino-20(S)-camptothecin, and the active metabolite of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38), than their parental cell lines . The cell lines demonstrated much less resistance to camptothecin and to several camptothecin analogues . Reduced accumulation and energy-dependent efflux of topotecan was demonstrated by confocal microscopy . A significant reduction in cleavable complexes in the resistant cells could be observed after SN-38 treatment but not after camptothecin treatment . In addition to topotecan and SN-38, MXR-overexpressing cells are highly resistant to mitoxantrone and epirubicin . Because these compounds are susceptible to glucuronidation, we examined UDP-glucurono-syltransferase (UGT) activity in parental and resistant cells by TLC . Glucuronides were found at equal levels in both parental and resistant colon cancer cell lines for epirubicin and to a lesser extent for SN-38 and mitoxantrone . Low levels of glucuronidation could also be detected in the resistant breast cancer cells . These results were confirmed by analysis of the UGT1A family mRNAs . We thus conclude that colon and breast cancer cells have a capacity for glucuronidation that could contribute to intrinsic drug resistance in colon cancer cells and may be acquired in breast cancer cells . The lack of selection for higher levels of UGT capacity in the colon cells suggests that high levels of expression of MXR alone are sufficient to confer resistance to the camptothecins. Med Clin (Barc), 1999 Nov 6, 113(15), 572 - 4 {Resistance of Mycobacterium tuberculosis in Ferrol, Spain . Associated factors}; Garcia Rodriguez JF et al.; OBJECTIVE: To know the frequency of resistances of Mycobacterium tuberculosis and the associated factors . PATIENTS AND METHODS: Prospective study of the sensitivity of Mycobacterium tuberculosis by means of the method of the proportions of Canetti in the Hospital Arquitecto Marcide-Profesor Novoa Santos (Ferrol, Spain) among 1991 and 1998 . A descriptive and multiple regression analyses were performed . RESULTS: Were studied 355 strains . Primary resistance: Isoniazid 1.1%, Streptomycin 1.1% . Secondary resistance: Isoniazid 11.6%, in the 5.2% existed multidrug-resistance . The risk factors for drug-resistant tuberculosis were previous treatment (odds ratio {OR} = 10.9; 95% CI, 2.9-39.4) and age higher than 40 years (OR = 3.9; 95% CI, 1.1-14.5) . CONCLUSIONS: A low frequency of resistance was observed . The factors associated with the resistances were previous treatment and age. J Pharmacol Exp Ther, 2000 Jan, 292(1), 265 - 70 Function and expression of multidrug resistance-associated protein family in human colon adenocarcinoma cells (Caco-2); Hirohashi T et al.; Several organic anions are actively extruded from intestinal epithelial cells into the lumen and vascular sides . To examine the role of the multidrug resistance-associated protein (MRP) family in the intestinal efflux of organic anions, the function and expression of these proteins were investigated with Caco-2, a human adenocarcinoma cell line that retains many of the characteristics of normal enterocytes . {(3)H}2,4-Dinitrophenyl-S-glutathione (DNP-SG) and {(3)H}17beta-estradiol 17-beta-D-glucuronide (E(2)17betaG), typical substrates for MRP1 and cMOAT (canalicular multispecific organic anion transporter)/MRP2, were taken up into brush-border membrane vesicles (BBMVs) from Caco-2 in an ATP-dependent manner, with K(m) values of 16.9 +/- 7.2 and 9.4 +/- 1.2 microM, respectively . The uptake of {(3)H}DNP-SG into BBMVs was osmotically sensitive and stimulated to some extent by other nucleotide triphosphates (GTP, CTP, and UTP) but not by ADP or AMP . An ATPase inhibitor, vanadate, inhibited the ATP-dependent uptake of {(3)H}DNP-SG to some extent . Reverse-transcriptase polymerase chain reaction resulted in the amplification of MRP1, MRP3, and MRP5 . Northern blot analysis indicated extensive expression of cMOAT/MRP2 and MRP3 and only minimal expression of MRP1 and MRP5 . Although cMOAT/MRP2 was continuously expressed throughout the culture period, MRP3 was not expressed immediately after the confluent state was reached . Collectively, the presence of ATP-dependent transport systems for DNP-SG and E(2)17betaG was demonstrated in Caco-2 cells . Because cMOAT/MRP2 and MRP3 may be expressed on brush-border and basolateral membranes in epithelial cells, respectively, the transport activity associated with BBMVs may result from the function of cMOAT/MRP2. Br J Cancer, 1999 Dec, 81(8), 1304 - 10 Reversal of MDR1-associated resistance to topotecan by PAK-200S, a new dihydropyridine analogue, in human cancer cell lines; Vanhoefer U et al.; Recent data suggest that expression of the membrane P170-glycoprotein (P-gp) may confer resistance to the topoisomerase-I-interactive agent topotecan . The present study describes the cellular effects of a new dihydropyridine analogue, PAK-200S, on P-gp-mediated resistance to topotecan in human breast and ovarian tumour cells . PAK-200S at a non-cytotoxic concentration of 2.0 microM completely reversed resistance to topotecan in P-gp-expressing MCF-7/adr (breast) and A2780/Dx5 (ovarian) tumour cells, respectively, with no effects on parental cells . Cellular pharmacokinetic studies by reversed-phase high-performance liquid chromatography analysis showed significantly lower cellular drug concentrations of the pharmacologically active closed-ring lactone of topotecan in multidrug-resistant cells than in parental cells . PAK-200S was effective in restoring the cellular lactone concentrations of topotecan in resistant MCF-7/adr cells to levels comparable to those obtained in parental cells . Furthermore, exposure of MCF-7/adr cells to topotecan in the presence of PAK-200S significantly increased the induction of protein-linked DNA breaks . PAK-200S did not alter nuclear topoisomerase I-mediated ex vivo pBR322 DNA plasmid unwinding activity and topoisomerase-I protein expression . These results suggest that reversal of P-gp-mediated resistance to topotecan by PAK-200S was related to the restoration of cellular drug concentrations of the active lactone form of topotecan rather than a direct effect on topoisomerase-I function. Semin Oncol, 1999 Oct, 26(5 Suppl 17), 24 - 7 Approaches to the treatment of patients with hormone-sensitive prostate cancer; DiPaola RS; Androgen ablation therapy provides effective palliation for patients with advanced cancer of the prostate for only a short duration because the tumor eventually develops resistance . Among the many potential molecular mechanisms involved in the development of tumor resistance to both androgen ablation therapy and chemotherapy, mutations in the p53 tumor suppressor gene, overexpression of the antiapoptotic protein bcl-2, and overexpression of the multidrug resistance protein probably play a role . Because hormone-resistant tumors demonstrate greater expression of bcl-2 and because transfection of the bcl-2 gene into hormone-sensitive tumor cells confers resistance to both hormone therapy and chemotherapy, efforts to abrogate bcl-2 in prostate tumors represent one approach to improve clinical results . Of several agents recently shown to reduce prostate-specific antigen levels in phase II studies, 13-cis-retinoic acid and interferon-alpha can reduce the expression of bcl-2 and overcome bcl-2-mediated resistance to paclitaxel in resistant cell lines . For these reasons, our current studies test the hypothesis that reducing the expression of bcl-2 with 13-cis-retinoic acid and interferon-alpha in combination with taxanes will improve clinical results . Additionally, other studies test the hypothesis that treatment early, before the development of resistance mechanisms, in hormone-sensitive disease will improve results . Studies with docetaxel (Taxotere; Rhone-Poulenc Rorer, Collegeville, PA) and with estramustine combination therapy are also ongoing. Antimicrob Agents Chemother, 2000 Jan, 44(1), 134 - 8 Chemical specificity of the PDR5 multidrug resistance gene product of Saccharomyces cerevisiae based on studies with tri-n-alkyltin chlorides; Golin J et al.; To understand the chemical basis of action for the PDR5-encoded multidrug resistance transporter of Saccharomyces cerevisiae, we compared the relative hypersensitivities of the wild-type (RW2802) and null mutant strains toward a series of tri-n-alkyltin compounds . These compounds differ from each other in a systematic fashion-either by hydrocarbon chain length or by anion composition . Using zone-of-inhibition and fixed-concentration assays, we found that the ethyl, propyl, and butyl compounds are strong PDR5 substrates, whereas the methyl and pentyl compounds are weak . We conclude that hydrophobicity and anion makeup are relatively unimportant factors in determining whether a tri-n-alkyltin compound is a good PDR5 substrate but that the dissociation of the compound and the molecular size are significant. Leukemia, 1999 Dec, 13(12), 2023 - 30 Relationship between the intracellular daunorubicin concentration, expression of major vault protein/lung resistance protein and resistance to anthracyclines in childhood acute lymphoblastic leukemia; Den Boer ML et al.; In vitro resistance to anthracyclines is related to a poor prognosis in childhood acute lymphoblastic leukemia (ALL), but the underlying mechanisms are poorly understood . Using flow cytometry, we studied the contribution of daunorubicin (DNR) accumulation and retention, cell size, expression of the major vault protein/lung resistance protein (LRP), P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) to the cytotoxicity of DNR (by MTT assay) in childhood ALL . The accumulated and retained DNR content was not related to the degree of DNR resistance, nor did the content differ between 53 initial and 20 relapse ALL samples (P >0 . 05), although the latter were median two-fold more resistant to DNR (P = 0.004) . Leukemic cell volume correlated with resistance to the anthracyclines DNR (Rs 0.32, P = 0.012) and idarubicin (Rs 0.46, P = 0.011) but not to other classes of drugs such as prednisolone, vincristine, L-asparaginase and etoposide . Relapsed patients had 1 . 5-fold larger cells than patients at initial diagnosis of ALL (P = 0 . 001) . After cell volume correction, the intracellular DNR concentration was lower in relapsed compared with initial ALL cells (eg 60 min accumulation, P = 0.003) . Moreover, the intracellular DNR concentration inversely correlated with DNR resistance, both in the accumulation (Rs -0.44, P < 0.001) and retention (Rs -0.33, P = 0 . 016) test condition . The accumulated DNR concentration inversely correlated with expression of LRP (Rs -0.36, P = 0.012) but not with P-gp and MRP . Expression of LRP, but not of P-gp and MRP, significantly correlated with DNR resistance in childhood ALL (Rs 0 . 33, P = 0.03) . In conclusion, the intracellular DNR concentration and the expression level of LRP may contribute to DNR resistance in childhood ALL . The strength of the correlations also indicates that resistance to anthracyclines can not be explained by one single mechanism. Leukemia, 1999 Dec, 13(12), 1943 - 53 Clinical significance of CD95, Bcl-2 and Bax expression and CD95 function in adult de novo acute myeloid leukemia in context of P-glycoprotein function, maturation stage, and cytogenetics; Wuchter C et al.; Resistance to chemotherapy-induced apoptosis and a multidrug-resistance (MDR) phenotype, mainly mediated by P-glycoprotein (P-gp), contribute to chemotherapy failure in hematologic malignancies . To study apoptosis-regulating factors in acute myeloid leukemia (AML), we investigated cell samples of adults with de novo AML by flow cytometry for constitutive expression levels of the apoptosis-related molecules CD95 (n = 135), Bcl-2 (n = 131), and Bax (n = 66), as well as spontaneous apoptosis in vitro (n = 104) and susceptibility to anti-CD95-induced apoptosis (CD95 sensitivity) (n = 93) . We correlated these findings with P-gp function as detected by the rhodamine123-efflux test (n = 121), immunophenotype, FAB morphology, cytogenetics, and clinical data of the examined patients . Immature FAB M0/1 AML cells expressed significantly more Bcl-2 (P < 0.0002) and less CD95 (P < 0.0003) compared with AML cells of the more mature FAB M2-5 subtypes . No maturation-dependent difference in Bax expression was observed . FAB M2-5 AML cells were more susceptible to anti-CD95-induced apoptosis (P < 0.008) and showed a lower P-gp function (P < 0.002) than FAB M0/1 AML cells . Leukemic cells of AML patients who achieved a complete remission (CR) after induction chemotherapy expressed less Bcl-2 than non-responder (NR) (69 CR, 23 NR; P = 0.05) . CR was associated with a higher extent of spontaneous apoptosis in vitro (58 CR, 17 NR; P=0.05) and a tendency towards a higher CD95 expression (73 CR, 23 NR; P = 0.08) compared to NR . CR also correlated with a low P-gp function (70 CR, 21 NR; P = 0.008) and a tendency towards CD34 negativity (73 CR, 23 NR; P = 0.08) . No correlation between Bax expression and response to induction chemotherapy (49 CR, 12 NR) was observed . In stepwise logistic regression analyses, P-gp function and the extent of spontaneous apoptosis in vitro as well as CD95 sensitivity but not Bcl-2, CD95, Bax, and CD34 expression levels emerged as significant markers for response to induction chemotherapy . We conclude that the constitutive expression of CD95 and Bcl-2, as well as CD95 sensitivity and P-gp function but not constitutive Bax expression depend on the maturation stage of leukemic cells in adult de novo AML . P-gp function, the extent of spontaneous apoptosis in vitro and CD95 sensitivity are more predictive for response to induction chemotherapy in adult de novoAML than the constitutive expression levels of the apoptosis-related molecules CD95, Bcl-2 and Bax. Biochem Biophys Res Commun, 1999 Dec 20, 266(2), 492 - 6 Inhibition of P-glycoprotein activity and chemosensitization of multidrug-resistant ovarian carcinoma 2780AD cells by hexanoylglucosylceramide; Veldman RJ et al.; In the present study we show that neutral hexanoyl-(glyco)sphingolipids inhibit P-glycoprotein (Pgp) activity in human ovarian 2780AD cells . By contrast, hexanoylceramide and the gangliosides GM(3) and GM(2) had no effect on Pgp activity, whereas sphingosine had a stimulating effect . In the case of hexanoylglucosylceramide, inhibition of Pgp activity by was reflected by a regained doxorubicin sensitivity of cells, which were grown in medium supplemented with the lipid . Our results lead to the conclusion that a direct transmodulation of Pgp activity by glycolipids occurs, depending on lipid headgroup structure, which can result in reduced resistance to the chemotherapeutic agent doxorubicin . Parasitol Res, 1999 Dec, 85(12), 1007 - 11 Effects of the multidrug-resistance-reversing agents verapamil and CL 347,099 on the efficacy of ivermectin or moxidectin against unselected and drug-selected strains of Haemonchus contortus in jirds (Meriones unguiculatus); Molento MB et al.; The development of anthelmintic resistance is making parasite control in small ruminants problematic . Following the discovery that the drug transporter P-glycoprotein may be involved in macrocyclic lactone resistance in Haemonchus contortus, we determined the effect of two multidrug-resistance modulators, verapamil and CL347,099, on the efficacy of ivermectin and moxidectin against unselected and drug-selected strains of H . contortus . CL347,099 is an analog of verapamil that has multidrug-resistance properties but weaker calcium-channel-blocking activity than the parent drug . The combinations of verapamil with either ivermectin or moxidectin significantly reduced worm counts of the selected strains as compared with the untreated controls, whereas ivermectin or moxidectin alone did not significantly reduce worm counts as compared with the untreated controls . The CL347,099 plus moxidectin combination was significantly more efficacious than moxidectin alone against the ivermectin-selected strain . The drug-combination regimes were without adverse effect on the jirds . However, higher levels of verapamil (> or =40 mg/kg) produced some toxicity. Parasitology, 1999 Nov, 119 ( Pt 5), 435 - 40 Inoculum effect leads to overestimation of in vitro resistance for artemisinin derivatives and standard antimalarials: a Gambian field study; Duraisingh MT et al.; Artemisinin (QHS) and its derivatives are new antimalarials which are effective against Plasmodium falciparum parasites resistant to chloroquine (CQ) . As these drugs are introduced it is imperative that resistance is monitored . In this paper we demonstrate that the inoculum size used in in vitro testing influences the measured in vitro susceptibility to QHS and its derivative dihydroartemisinin (DHA) and to mefloquine (MEF) and CQ over the range of parasitaemias routinely used in testing with the WHO in vitro microtest . An increase in parasitaemia and/or haematocrit was accompanied by a decrease in the measured sensitivity of 2 laboratory lines . In the context of a field study testing in vitro susceptibility of parasite isolates from patients with uncomplicated malaria in Fajara, The Gambia we demonstrate that failure to control for inoculum size significantly overestimates the level of resistance to QHS and DHA as well as MEF, halofantrine (HAL) and quinine (QUIN) . When controlling for the inoculum effect, cross-resistance was observed between QHS, MEF and HAL suggesting the presence of a multidrug resistance-like mechanism . These studies underline the importance of inoculum size in in vitro susceptibility testing. Life Sci, 1999, 65(22), 2343 - 9 Nuclear transport as an ultimate step of multidrug resistance; Bobichon H et al.; Adriamycin (ADM) incorporation into nuclei of whole multidrug resistant (MDR) CEM cells is lower than into sensitive ones (S), that is mostly thought to be the consequence of a decrease of drug related to the activity of the multidrug resistance plasma membrane protein P 170 . Isolated nuclei of the lymphoblastic tumor cell line CEM, which structures were controlled by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal microscopy, where incubated with 10(-6) mole/l of ADM . Incorporation into DNA was quantified by spectrofluorimetry . It was lower and slower into MDR nuclei than into S ones . Different modulators of active transport influence drug transfer into S nuclei and had no effect in MDR nuclei . The nuclear transfer into S nuclei appeared divided into two components: one was decreased by WGA, increased by cytosolic factors and an other part was purely passive in an identical intensity to MDR nuclei . Resistance of MDR nuclei seemed indebt to a defect, in these cells, of factors that mediate and/or activate nuclear transport of drug. Pharm Res, 1999 Oct, 16(10), 1550 - 6 Inhibition of P-glycoprotein by D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS); Dintaman JM et al.; PURPOSE: To investigate whether d-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) functions as an inhibitor of P-glycoprotein (P-gp), the multidrug resistance transporter . METHODS: Two assays were used to measure the function of TPGS on P-gp function . First, we examined the ability of TPGS to modulate the cytotoxicity of established, cytotoxic, P-glycoprotein substrates . Parental NIH 3T3 cells and NIH 3T3 cells transfected with the human MDR1 cDNA (G185) were exposed to doxorubicin, paclitaxel, colchicine, vinblastine and 5-fluorouracil (5FU) in the presence or absence of TPGS . Cytotoxicity was assessed with the MTT assay . Second, polarized transport of the P-gp substrates rhodamine 123 (R123), paclitaxel and vinblastine was measured using the human intestinal HCT-8 and Caco-2 cell lines grown in Transwell dishes . Drug flux was measured by liquid scintillation counting or fluorescence spectroscopy of the media . RESULTS: G185 cells were 27-135 fold more resistant to the cytotoxic drugs doxorubicin, vinblastine, colchicine and paclitaxel than the parental NIH 3T3 cells . In contrast 5FU, which is not a P-gp substrate, is equally cytotoxic to parental and G185 cells . Co-administration of TPGS enhanced the cytotoxicity of doxorubicin, vinblastine, paclitaxel, and colchicine in the G185 cells to levels comparable to the parental cells . TPGS did not increase the cytotoxicity of 5FU in the G185 cells . Using a polarized epithelial cell transport assay, TPGS blocked P-gp mediated transport of R123 and paclitaxel in a dose responsive manner . CONCLUSIONS: These data demonstrate that TPGS acts as a reversal agent for P-glycoprotein mediated multidrug resistance and inhibits P-gp mediated drug transport . These results suggest that enhanced oral bioavailability of drugs co-administered with TPGS may, in part, be due to inhibition of P-glycoprotein in the intestine. Drugs, 1999, 58 Suppl 2, 52 - 4 Quinolones in the treatment of typhoid fever; Akalin HE; Typhoid fever is a severe systemic disease . Treatment with appropriate antibiotics is essential for enteric fever . Development and rapid dissemination of resistance to chloramphenicol, ampicillin, and cotrimoxazole have complicated the treatment of enteric fever . Therapeutic options for the treatment of multidrug-resistant strains are limited to third generation cephalosporins or fluoroquinolone antibiotics . Recent clinical experiences have shown that quinolones are the drugs of choice for treatment of enteric fever . Studies have shown that shorter courses may be sufficient to cure uncomplicated typhoid fever. Drugs, 1999, 58 Suppl 2, 19 - 22 Activity of quinolones against mycobacteria; Jacobs MR; The fluoroquinolones have been shown to be active in vitro against many mycobacterial species, including most strains of Mycobacterium tuberculosis complex and M . fortuitum, and some strains of M . kansasii, M . avium-intracellulare (MAI) complex and M . leprae . Ciprofloxacin, ofloxacin and sparfloxacin are the best studied of these agents to date, and are among the most active of this group against M . tuberculosis and other mycobacteria . Treatment of patients with multidrug-resistant pulmonary tuberculosis using ofloxacin has resulted in the selection of quinolone-resistant mutants in a few patients . Many strains of MAI, however, are resistant to fluoroquinolones, and structure-activity relationships and DNA gyrase studies have been undertaken to identify the moieties associated with activity and the lack thereof . The genetic and molecular basis of quinolone resistance in mycobacteria has revealed both the recent progress made in these areas and the limitations of the quinolones against this genus . Considerable progress will need to be made in resolving these issues in order for the quinolones to become clinically useful antimycobacterial agents. J Antimicrob Chemother, 1999 Nov, 44(5), 647 - 52 Comparative antimycobacterial activities of ofloxacin, ciprofloxacin and grepafloxacin; Vacher S et al.; Infections caused by non-tuberculous mycobacteria and multidrug-resistant Mycobacterium tuberculosis are difficult to treat . New compounds potentially active against these bacteria are therefore constantly being sought . Among them is grepafloxacin, a new C5 fluoroquinolone . A panel of 130 isolates of mycobacteria including 33 M . tuberculosis isolates and 97 isolates of different species of atypical mycobacteria were analysed for susceptibility to grepafloxacin, ofloxacin and ciprofloxacin . The MICs of these fluoroquinolones were determined using the agar-dilution method . Different mycobacterial species showed different degrees of susceptibility to grepafloxacin, ofloxacin and ciprofloxacin but little difference was observed between the MICs of the three antibiotics against strains of the same mycobacterial species . In addition, to evaluate the intracellular activity of these drugs, six strains of mycobacteria were studied using a human-macrophage infection model . Preliminary results of macrophage experiments showed that grepafloxacin was more active than ofloxacin and ciprofloxacin, particularly against Mycobacterium kansasii and, to a lesser degree, against Mycobacterium avium complex and Mycobacterium marinum . However, the three fluoroquinolones had comparable activities against M . tuberculosis. Parasitol Res, 1999 Nov, 85(11), 920 - 2 Enhanced absorption of pour-on ivermectin formulation in rats by co-administration of the multidrug-resistant-reversing agent verapamil; Alvinerie M et al.; The effect of verapamil, a multidrug-resistance (Mdr)-reversing agent on the absorption of a pour-on formulation of ivermectin was evaluated in rats . Absorption of ivermectin was effectively enhanced (40%) by the presence of verapamil, suggesting that absorption of ivermectin involves Mdr-P-glycoprotein and that verapamil should act as a competitive inhibitor for the transport and extrusion of ivermectin by P-glycoprotein . This hypothesis is consistent with other studies describing verapamil as a blocking agent of P-glycoprotein involved in the efflux of ivermectin in a resistant strain of Haemonchus contortus. Clin Cancer Res, 1999 Nov, 5(11), 3445 - 53 Discovery of differentially expressed genes associated with paclitaxel resistance using cDNA array technology: analysis of interleukin (IL) 6, IL-8, and monocyte chemotactic protein 1 in the paclitaxel-resistant phenotype; Duan Z et al.; In an attempt to define the molecular changes associated with the paclitaxel-resistant phenotype in human cancer, a paclitaxel-resistant ovarian cancer cell line, SKOV-3TR, was established through stepwise selection in increasing paclitaxel concentrations . SKOV-3TR was cross- resistant to doxorubicin and vincristine and overexpressed multidrug resistance gene 1 but not multidrug resistance associated protein . SKOV-3TR and the paclitaxel-sensitive SKOV-3 parent line were characterized using human cDNA array technology that examined expression of a wide variety of genes involved in cell growth, signal transduction, cell death, and immune function . cDNA probes from reverse transcribed mRNAs of both paclitaxel-resistant and parent cells were compared to identify genes differentially expressed in the paclitaxel-resistant cells . Of 588 different human cDNA transcripts compared, 6 genes were found to be markedly decreased, and 12 genes increased in the resistant subline . Northern analysis and/or reverse transcription-PCR confirmed that 12 of these 18 genes were over- or underexpressed in SKOV-3TR . In addition, at least eight of the genes were found differentially expressed in several other paclitaxel- and/or doxorubicin-resistant cell lines, both those with increased multidrug resistance expression and those without . Included in the set of overexpressed genes were the cytokines/chemokines interleukin 6, interleukin 8, and monocyte chemotactic protein 1 . ELISA assays confirm that mRNA overexpression of these cytokine/chemokines was associated with the increased secretion of these molecules in the tissue culture supernatant . Evaluation of supernatants from an expanded collection of paclitaxel- and Adriamycin-resistant cell lines demonstrated that all of the resistant lines had significant overexpression of at least one cytokine/chemokine as compared with their drug-sensitive parent line . The overexpression of these cytokines seemed to be stable and associated with a drug-resistant phenotype with only a modest induction of cytokine expression in the parent line with short-term paclitaxel exposure . These findings suggest that the development of paclitaxel resistance is accompanied by multiple changes in gene expression including stable alterations in selective chemokine and cytokine expression . The role these associated genetic changes have in the drug-resistant phenotype is discussed. Folia Microbiol (Praha), 1999, 44(2), 171 - 6 Use of mutated PDR3 gene as a dominant selectable marker in transformation of prototrophic yeast strains; Lackova D et al.; For successful transformation of prototrophic industrial yeast strains dominant selectable markers are necessary . In the present study we show the applicability of a selection system based on the phenotype of multidrug resistance . The mutant pdr3-9 allele on centromeric or episomal vector, encoding a more efficient transcriptional activator with Y276H amino acid substitution, was used as a dominant selectable marker for selection of transformants . The pdr3-9 allele conferred resistance of transformed cells to cycloheximide, chloramphenicol, mucidin and oligomycin both in the absence and in the presence of a chromosomal copy of the PDR3 gene . Both multicopy YEp352/pdr3-9 and centromeric pFL38/pdr3-9 vectors bearing the mutant pdr3-9 allele have proved to be a valuable tool for a direct selection of transformants of industrial strains of Saccharomyces cerevisiae. Am J Trop Med Hyg, 1999 Nov, 61(5), 807 - 13 Molecular epidemiology of malaria in Yaounde, Cameroon V . analysis of the omega repetitive region of the plasmodium falciparum CG2 gene and chloroquine resistance; Basco LK et al.; A novel Plasmodium falciparum gene, denoted cg2 gene, has been recently discovered, and a distinct genotype, characterized by 12 point mutations and 3 size polymorphisms, has been shown to be associated with chloroquine resistance in laboratory-adapted parasite strains . One of the polymorphic regions, denoted the omega region, consists of 16 tandem repeat units in chloroquine-resistant strains, while the chloroquine-sensitive strains have either < or = 15 or > or = 17 repeat units . In this study, the in vivo and in vitro responses were compared with the number of repeat units in the omega region of the cg2 gene for 75 Cameroonian isolates determined either by DNA sequencing or agarose gel electrophoresis . The 16-repeat units that characterize the resistant strains were found in 10 chloroquine-sensitive isolates (50% inhibitory concentration {IC50} < 100 nM) and 30 chloroquine-resistant isolates (IC50 > or = 100 nM) . Thirty-five isolates (28 chloroquine-sensitive isolates and 7 chloroquine-resistant isolates) displayed < or = 15 or > or = 17 repeat units . Of the 18 patients responding with treatment failure, 15 were infected with parasites carrying 16 repeat units . Twenty-eight patients (11 with isolates carrying 16 repeat units and 17 with isolates carrying < or = 15 or > or = 17 repeat units) showed an adequate clinical response . The sensitivity, specificity, and predictive value were 81% (83%), 74% (61%), and 75% (58%), respectively compared with in vitro (or in vivo) responses . Neither the level of IC50 nor the key P . falciparum multidrug resistance gene 1 (pfmdr 1) allele at position 86 was associated with the number of omega repeat units . Although in vitro and in vivo resistance to chloroquine was statistically associated with the presence of 16 repeat units in the omega region (P < 0.05), the number of omega repeat units did not adequately discriminate patients infected with chloroquine-resistant parasites from those infected with chloroquine-sensitive parasites . Further studies on the cg2 gene are needed to determine whether cg2 gene is a reliable genetic marker for chloroquine resistance. Am J Trop Med Hyg, 1999 Nov, 61(5), 780 - 3 Analysis of mefloquine resistance and amplification of pfmdr1 in multidrug-resistant Plasmodium falciparum isolates from Thailand; Chaiyaroj SC et al.; Resistance to quinoline-containing compound has been associated with the Plasmodium falciparum multidrug resistance 1 (pfmdr1) gene . We analyzed wild P . falciparum isolates with high levels of chloroquine and mefloquine resistance for their macrorestriction maps of chromosome 5 and sequence of pfmdr1 . Two types of chromosome 5 amplification were found . Eleven of 62 resistant isolates displayed Bgl 1 fragments larger than 100 kb . Twenty-nine isolates possessed multiple copies of the fragments . We failed to detect any amplification of this region on chromosome 5 in 22 mefloquine-resistant isolates, suggesting that other mechanisms can mediate the mefloquine-resistant phenotype . There was no direct association between pfmdr1 mutations and chloroquine sensitivity . Resistant lines could have Asn-86 and Tyr-184 or Phe-184, the predicted sequence of those chloroquine-sensitive isolates . No mutation at Asn-1042 and Asp-1246 was detected among these chloroquine-resistant isolates . Therefore, a few base substitutions in the pfmdr1 gene may not be sufficient to account for all chloroquine-resistant phenotypes. Int J Cancer, 2000 Jan 1, 85(1), 131 - 41 Increased intracellular drug accumulation and complete chemosensitization achieved in multidrug-resistant solid tumors by co-administering valspodar (PSC 833) with sterically stabilized liposomal doxorubicin; Krishna R et al.; We have previously demonstrated that liposome encapsulation of doxorubicin (DOX) can alleviate adverse interactions with non-encapsulated DOX and the cyclosporine multidrug-resistant (MDR) modulator Valspodar . We have now investigated the behavior of different liposomal DOX formulations in MDA435LCC6/MDR-1 human breast cancer solid tumor xenograft models to identify liposome characteristics associated with enhanced therapeutic activity and the mechanism whereby increased chemosensitization is achieved . Toxicity studies incorporating conventional phosphatidylcholine (PC)/cholesterol (chol) and sterically stabilized (polyethylene glycol 2000 {PEG}-containing) formulations of DOX indicated that whereas PC/Chol DOX was approximately 3-fold more toxic in the presence of Valspodar, PEG containing distearoylglycerophosphocholine (DSPC)/Chol DOX was minimally affected . In mice bearing MDR tumors, co-administration of Valspodar and egg phosphocholine (EPC)/Chol DOX resulted in modest MDR modulation and efficacy, whereas the sterically stabilized formulation induced reductions in tumor growth equivalent to that achieved for drug-sensitive tumors treated with non-encapsulated DOX . Pharmacokinetic studies revealed a 2.5-fold increase in plasma DOX area under the curve (AUC) upon co-administration of Valspodar with EPC/Chol DOX whereas no such alterations were observed with the sterically stabilized liposomes . Compared to non-encapsulated DOX combined with Valspodar, improvements in efficacy and toxicity correlated with the extent to which liposomal DOX formulations were able to circumvent pharmacokinetic interactions . Confocal microscopy demonstrated that Valspodar increased cell-associated DOX which correlated with the level of anti-tumor efficacy . J Biol Chem, 1999 Dec 10, 274(50), 35388 - 92 Identification of residues in the drug-binding domain of human P-glycoprotein . Analysis of transmembrane segment 11 by cysteine-scanning mutagenesis and inhibition by dibromobimane; Loo TW et al.; The drug-binding domain of the human multidrug resistance P-glycoprotein (P-gp) probably consists of residues from multiple transmembrane (TM) segments . In this study, we tested whether the amino acids in TM11 participate in binding drug substrates . Each residue in TM11 was initially altered by site-directed mutagenesis and assayed for drug-stimulated ATPase activity in the presence of verapamil, vinblastine, or colchicine . Mutants G939V, F942A, T945A, Q946A, A947L, Y953A, A954L, and G955V had altered drug-stimulated ATPase activities . Direct evidence for binding of drug substrate was then determined by cysteine-scanning mutagenesis of the residues in TM11 and inhibition of drug-stimulated ATPase activity by dibromobimane, a thiol-reactive substrate . Dibromobimane inhibited the drug-stimulated ATPase activities of two mutants, F942C and T945C, by more than 75% . These results suggest that residues Phe(942) and Thr(945) in TM11, together with residues previously identified in TM6 (Leu(339) and Ala(342)) and TM12 (Leu(975), Val(982), and Ala(985)) (Loo, T . W., and Clarke, D . M . (1997) J . Biol . Chem . 272, 31945-31948) form part of the drug-binding domain of P-gp. Exp Cell Res, 1999 Dec 15, 253(2), 396 - 402 Role of P-glycoprotein in human natural killer-like cell line-mediated cytotoxicity; Takahashi M et al.; Natural killer (NK) cells express the highest amount of P-glycoprotein (Pgp), a product of the multidrug resistance (MDR) 1 gene, among lymphoid cells, and our previous studies demonstrated that Pgp is required for NK cell-mediated cytotoxicity . In this study we examined the role of Pgp in NK cell-mediated cytotoxicity using a human NK-like cell line, i.e., YTN cells and two MDR reversing agents, nicardipine and its structural analog, AHC-93 . These two agents inhibited the Pgp function (rhodamine-123 excretion) as well as cell-mediated cytotoxicity, confirming that Pgp is critical for NK cell-mediated cytotoxicity . As revealed by video-rate ultraviolet laser-scanning confocal microscopy, AHC-93 did not inhibit the increase in the intracellular calcium concentration upon binding to target cells, whereas nicardipine did, as reported previously . These two reagents relocated acridine orange dye from lysosomes to the cytoplasm at concentrations similar to those required for the inhibition of cell-mediated cytotoxicity . These results suggest that Pgp is directly or indirectly involved in pH regulation in lysosomes, but not in calcium homeostasis . Hum Gene Ther, 1999 Nov 20, 10(17), 2811 - 21 Selection of keratinocytes transduced with the multidrug resistance gene in an in vitro skin model presents a strategy for enhancing gene expression in vivo; Pfutzner W et al.; In gene therapy studies, achieving prolonged, high-level gene expression in a significant percentage of cells has been difficult . One solution to enhance expression would be to select for cells expressing both the desired gene and a linked selectable marker gene in a bicistronic vector . As a potential target tissue, the skin is easily accessible for safe topical application of a selecting agent that could lead to significant gene expression in a high percentage of keratinocytes . To test the feasibility of such an approach, a skin raft culture model was developed . Human keratinocytes were transduced with the multidrug resistance (MDR) gene, which confers resistance to a variety of cytostatic and antimitotic compounds, such as colchicine . While growing on acellular dermis, transduced keratinocytes were treated with various doses of colchicine (10-50 ng/ml) . Colchicine treatment increased the percentage of keratinocytes expressing MDR to almost 100% in raft cultures, Significantly, keratinocytes in colchicine-treated, MDR-transduced raft cultures were able to proliferate normally and form a stratified, differentiated epidermis . This model suggests that topical selection for MDR-expressing keratinocytes in vivo should be feasible without hampering the biologic integrity of skin . Thus, topical selection leading to enhanced expression of a desired gene, linked to a resistance gene, holds future promise for skin gene therapy. Br J Haematol, 1999 Dec, 107(3), 600 - 4 Molecular heterogeneity of the NUP98/HOXA9 fusion transcript in myelodysplastic syndromes associated with t(7;11)(p15;p15); Hatano Y et al.; The reciprocal translocation t(7;11)(p15;p15) has been reported as occurring mainly in acute myelogenous leukaemia (AML) and the acute phase of chronic myelogenous leukaemia (CML) . This translocation in AML involves both the nucleoporin gene NUP98 on 11p15 and the homeobox gene HOXA9 on 7p15 . The invariant chimaeric NUP98/HOXA9 transcripts are a result of the fact that each breakpoint of the NUP98 and the corresponding breakpoint of the HOXA9 gene cluster occur within the same intron . Only one patient with myelodysplastic syndromes (MDS) carrying this chromosome aberration has been reported, but this study did not involve molecular analysis . We describe two patients with MDS associated with t(7;11): patient 1 was a Japanese man diagnosed with chronic myelomonocytic leukaemia; patient 2 was a Japanese woman with refractory anaemia with excess of blasts in transformation . Within a year both patients developed AML and showed multidrug resistance to chemotherapy . Southern blot analysis showed rearrangements of the NUP98 gene of the two patients and the HOXA9 gene of patient 2 . Patient 1 had two types of the novel NUP98/HOXA9 fusion transcripts . Each of them lacked the common 141 bp NUP98 exon which was contained in the NUP98/HOXA9 fusion transcripts detected in patient 2 and the reported AML cases . These data indicated that t(7;11) could determine the development of various myeloid leukaemias and that the resultant chimaeric transcripts are heterogenous. Antimicrob Agents Chemother, 1999 Dec, 43(12), 2943 - 9 The pfmdr1 gene is associated with a multidrug-resistant phenotype in Plasmodium falciparum from the western border of Thailand; Price RN et al.; On the western border of Thailand, Plasmodium falciparum has become resistant to almost all antimalarial agents . The molecular basis of resistance in these parasite populations has not been well characterized . This study assessed genetic polymorphisms in the pfmdr1 gene in 54 parasites collected from the western border of Thailand to determine the relationship of pfmdr1 copy number and codon mutations with parasite sensitivities to mefloquine, chloroquine, halofantrine, quinine, and artesunate assessed in vitro . A point mutation at codon 86 (resulting in a change of Asn to Tyr) was associated with a significantly lower 50% inhibitory concentration (IC(50)) of mefloquine (median, 9 ng/ml versus 52.4 ng/ml; P = 0.003) . Overall 35% of the isolates (19 of 54) had an increase in pfmdr1 copy number, and all 19 carried the wild-type allele at codon 86 . Increased pfmdr1 copy number was associated with higher IC(50)s of mefloquine (P = 0.04) and artesunate (P = 0.005), independent of polymorphism at codon 86 . The relationship between pfmdr1 and resistance to structurally distinct antimalarial agents confirms the presence of a true multidrug-resistant phenotype. Cancer Res, 1999 Nov 15, 59(22), 5751 - 7 CHS 828, a novel pyridyl cyanoguanidine with potent antitumor activity in vitro and in vivo; Hjarnaa PJ et al.; A new class of recently discovered antineoplastic agents, the pyridyl cyanoguanidines, exert a potent antitumor activity in rodents after oral administration . Optimization in vitro and in vivo has resulted in the selection of the lead candidate CHS 828 (N-(6-chlorophenoxyhexyl)-N'cyano-N"-4-pyridylguanidine) . CHS 828 was found to exert potent cytotoxic effects in human breast and lung cancer cell lines, with lesser effects on normal fibroblasts and endothelial cells . In a study using a panel of cell lines with different resistance patterns, the effects of CHS 828 showed a low correlation with the activity patterns of known anticancer agents, and no sensitivity to known mechanisms of multidrug resistance was observed . In nude mice bearing human tumor xenografts, CHS 828, at doses from 20 to 50 mg/kg/day p.o., inhibited the growth of MCF-7 breast cancer tumors and caused regression of NYH small cell lung cancer tumors . Oral administration of CHS 828 once weekly improved efficacy without increasing toxicity . CHS 828 was found to compare favorably with established chemotherapeutic agents such as cyclophosphamide, etoposide, methotrexate, and paclitaxel . In mice with NYH tumors, long-term survival (>6 months) was observed after treatment with CHS 828 was stopped . In conclusion, CHS 828 is an effective new antitumor agent, with a potentially new mechanism of action . CHS 828 is presently being tested in Phase I clinical trials in collaboration with the European Organization for Research and Treatment of Cancer. Ann Oncol, 1999, 10 Suppl 5, S83 - 6 Multidrug resistance in non-small-cell lung cancer; Scagliotti GV et al.; Resistance to cytotoxic drugs is an important cause of treatment failure . The causes are complex and may be determined by a combination of the tumour characteristics, such as the proportion of resting cells, adequacy of blood supply, and specific cellular mechanisms, as in the multidrug resistance phenotype . In lung cancer four types of multidrug resistance have been defined on the basis of the cellular drug targets involved, i.e., classical multidrug resistance (MDR), non-P-glycoprotein MDR (also called MRP), atypical MDR (mediated through altered expression of topoisomerases II) and lung resistance-related protein . In lung cancer the role of the different forms of multidrug resistance is complex and only partially understood. Biochim Biophys Acta, 1999 Dec 6, 1461(2), 377 - 94 Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance; Konig J et al.; The membrane proteins mediating the ATP-dependent transport of lipophilic substances conjugated to glutathione, glucuronate, or sulfate have been identified as members of the multidrug resistance protein (MRP) family . Several isoforms of these conjugate export pumps with different kinetic properties and domain-specific localization in polarized human cells have been cloned and characterized . Orthologs of the human MRP isoforms have been detected in many different organisms . Studies in mutant rats lacking the apical isoform MRP2 (symbol ABCC2) indicate that anionic conjugates of endogenous and exogenous substances cannot exit from cells at a sufficient rate unless an export pump of the MRP family is present in the plasma membrane . Several mutations in the human MRP2 gene have been identified which lead to the absence of the MRP2 protein from the hepatocyte canalicular membrane and to the conjugated hyperbilirubinemia of Dubin-Johnson syndrome . Overexpression of recombinant MRP2 confers resistance to multiple chemotherapeutic agents . Because of its function in the terminal excretion of cytotoxic and carcinogenic substances, MRP2 as well as other members of the MRP family, play an important role in detoxification and chemoprevention. Biochim Biophys Acta, 1999 Dec 6, 1461(2), 359 - 76 Structural, mechanistic and clinical aspects of MRP1; Hipfner DR et al.; The cDNA encoding ATP-binding cassette (ABC) multidrug resistance protein MRP1 was originally cloned from a drug-selected lung cancer cell line resistant to multiple natural product chemotherapeutic agents . MRP1 is the founder of a branch of the ABC superfamily whose members (from species as diverse as plants and yeast to mammals) share several distinguishing structural features that may contribute to functional and mechanistic similarities among this subgroup of transport proteins . In addition to its role in resistance to natural product drugs, MRP1 (and related proteins) functions as a primary active transporter of structurally diverse organic anions, many of which are formed by the biotransformation of various endo- and xenobiotics by Phase II conjugating enzymes, such as the glutathione S-transferases . MRP1 is involved in a number of glutathione-related cellular processes . Glutathione also appears to play a key role in MRP1-mediated drug resistance . This article reviews the discovery of MRP1 and its relationships with other ABC superfamily members, and summarizes current knowledge of the structure, transport functions and relevance of this protein to in vitro and clinical multidrug resistance. Biochim Biophys Acta, 1999 Dec 6, 1461(2), 347 - 57 The multidrug resistance protein family; Borst P et al.; The human multidrug resistance protein (MRP) family contains at least six members: MRP1, the godfather of the family and well known as the multidrug resistance protein, and five homologs, called MRP2-6 . In this review, we summarize what is known about the protein structure, the expression in tissues, the routing in cells, the physiological functions, the substrate specificity, and the role in multidrug resistance of the individual members of the MRP family. Biochim Biophys Acta, 1999 Dec 6, 1461(2), 315 - 25 Determining the structure and mechanism of the human multidrug resistance P-glycoprotein using cysteine-scanning mutagenesis and thiol-modification techniques; Loo TW et al.; The multidrug resistance P-glycoprotein is an ATP-dependent drug pump that extrudes a broad range of hydrophobic compounds out of cells . Its physiological role is likely to protect us from exogenous and endogenous toxins . The protein is important because it contributes to the phenomenon of multidrug resistance during AIDS and cancer chemotherapy . We have used cysteine-scanning mutagenesis and thiol-modification techniques to map the topology of the protein, show that both nucleotide-binding domains are essential for activity, examine packing of the transmembrane segments, map the drug-binding site, and show that there is cross-talk between the ATP-binding sites and the transmembrane segments. Biochim Biophys Acta, 1999 Dec 6, 1461(2), 305 - 13 Comparative aspects of the function and mechanism of SUR1 and MDR1 proteins; Ueda K et al.; ATP-binding cassette (ABC) superfamily proteins have divergent functions and can be classified as transporters, channels, and receptors, although their predicted secondary structures are very much alike . Prominent members include the sulfonylurea receptor (SUR1) and the multidrug transporter (MDR1) . SUR1 is a subunit of the pancreatic beta-cell K(ATP) channel and plays a key role in the regulation of glucose-induced insulin secretion . SUR1 binds ATP at NBF1, and ADP at NBF2 and the two NBFs work cooperatively . The pore-forming subunit of the pancreatic beta-cell K(ATP) channel, Kir6.2, is a member of the inwardly rectifying K(+) channel family, and also binds ATP . In this article, we present a model in which the activity of the K(ATP) channel is determined by the balance of the action of ADP, which activates the channel through SUR1, and the action of ATP, which stabilizes the long closed state by binding to Kir6.2 . The concentration of ATP could also affect the channel activity through binding to NBF1 of SUR1 . MDR1, on the other hand, is an ATP-dependent efflux pump which extrudes cytotoxic drugs from cells before they can reach their intracellular targets, and in this way confers multidrug resistance to cancer cells . Both NBFs of MDR1 can hydrolyze nucleotides, and their ATPase activity is necessary for drug transport . The interaction of SUR1 with nucleotides is quite different from that of MDR1 . Variations in the interactions with nucleotides of ABC proteins may account for the differences in their functions. Biochem Biophys Res Commun, 1999 Dec 9, 266(1), 167 - 73 Treatment of multidrug resistant (MDR1) murine leukemia with P-glycoprotein substrates accelerates the course of the disease; Yang JM et al.; The prognosis of patients with tumors expressing P-glycoprotein (P-gp), the MDR1 gene product, is generally poor . It is assumed that this is due to decreased tumor responsiveness that results from decreased drug accumulation . We observed that treatment of animals bearing MDR1-transfected leukemic cells with P-gp substrates (i.e., drugs that are transported by P-gp) significantly worsened host survival compared to treatment with vehicle or non-P-gp substrates . This effect was seen with cancer chemotherapeutic agents (paclitaxel and vincristine) and with the MDR modulator, trans-flupenthixol . To determine the mechanism(s) underlying this observation, we studied alterations in pharmacokinetics, pharmacodynamics, and metastasis . We found that the drug-induced acceleration of disease was associated with increased metastases . P-gp(+) cells treated with P-gp substrates demonstrated several pro-metastatic features, including membrane ruffling and invasion through a hepatocyte monolayer . These results suggest that the treatment of MDR tumors with P-gp substrates may produce changes in malignant behavior that could adversely affect therapeutic outcomes . Pediatr Neurol, 1999 Oct, 21(4), 731 - 4 Tuberous sclerosis associated with MDR1 gene expression and drug-resistant epilepsy; Lazarowski A et al.; Intractable seizures are the most common manifestation in severe cases of tuberous sclerosis . Multidrug resistance type 1 (MDR1) gene expression is directly linked to the resistance of tumor cells to chemotherapy as the major cause of treatment failure, but it has not been reported in tuberous sclerosis cells nor has the relationship between the MDR1 gene and antiepileptic drugs been described . A 4-month-old female is described with poorly controlled seizures secondary to tuberous sclerosis . The patient was treated with antiepileptic drugs, including phenytoin, phenobarbital, and lorazepam, without improvement of symptoms . Phenytoin blood levels were invariably subtherapeutic and ranged from 0.45 to 3.55 microg/mL, despite several consecutive intravenous loading doses . Surgical treatment with total resection of the brain lesions was performed as a last resort . Immunohistochemical analysis of the resected tissues revealed high levels of P-glycoprotein 170 expression, the product of the MDR1 gene . Both MDR1 gene expression and persistently low phenytoin levels likely share a common pathway liable to induce drug-resistant epilepsy. Gastroenterology, 1999 Dec, 117(6), 1370 - 9 Hepatocanalicular bile salt export pump deficiency in patients with progressive familial intrahepatic cholestasis; Jansen PL et al.; BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis (PFIC), an inherited liver disease of childhood, is characterized by cholestasis and either normal or increased serum gamma-glutamyltransferase activity . Patients with normal gamma-glutamyltransferase activity have mutations of the FIC1 locus on chromosome 18q21 or mutations of the BSEP gene on chromosome 2q24 . Also, patients with bile acid synthesis defects have low gamma-glutamyltransferase activity . We investigated expression of the bile salt export pump (BSEP) in liver samples from patients with a PFIC phenotype and correlated this with BSEP gene mutations . METHODS: BSEP and multidrug resistance protein 2 (MRP2) expressions were studied by immunohistochemistry in liver specimens of 28 patients and BSEP gene mutation analysis in 19 patients . Bile salt kinetics were studied in 1 patient . RESULTS: Sixteen of 28 liver samples showed no canalicular BSEP staining . Staining for MRP2 showed a normal canalicular pattern in all but 1 of these samples . Ten of 19 patients showed BSEP gene mutations; BSEP protein expression was lacking in all 10 patients . No mutations were found in 9 of 19 patients, and in all except 1, BSEP protein expression was normal . Bile salt concentration in bile of BSEP-negative/MRP2-positive PFIC patients was 0.2 +/- 0.2 mmol/L (n = 9; <1% of normal) and in BSEP-positive PFIC patients 18.1 +/- 9.9 mmol/L (n = 3; 40% of normal) . The kinetic study confirmed the dramatic decrease of bile salt secretion in BSEP-negative patients . CONCLUSIONS: The findings show a close correlation between BSEP gene mutations and canalicular BSEP expression . Biliary secretion of bile salts is greatly reduced in BSEP-negative patients. J Nat Prod, 1999 Nov, 62(11), 1545 - 50 Cytotoxic sesquiterpenoids from Ratibida columnifera; Cui B et al.; Bioassay-directed fractionation of the flowers and leaves of Ratibida columnifera using a hormone-dependent human prostate (LNCaP) cancer cell line led to the isolation of 10 cytotoxic substances, composed of five novel xanthanolide derivatives (2-4, 7, and 8), a novel nerolidol derivative (9), and three known sesquiterpene lactones, 9alpha-hydroxy-seco-ratiferolide-5alpha-O-angelate+ ++ (1), 9alpha-hydroxy-seco-ratiferolide-5alpha-O-(2-methylbut yrate) (5), 9-oxo-seco-ratiferolide-5alpha-O-(2-methylbutyrate) (6), as well as a known flavonoid, hispidulin (10) . On the basis of its cytotoxicity profile, compound 5 was selected for further biological evaluation, and was found to induce G1 arrest and slow S traverse time in parental wild type p53 A2780S cells, but only G2/M arrest in p53 mutant A2780R cells, with strong apoptosis shown for both cell lines . The activity of 5 was not mediated by the multidrug resistance (MDR) pump, and it was not active against several anticancer molecular targets (i.e., tubulin polymerization/depolymerization, topoisomerases, and DNA intercalation) . While these results indicate that compound 5 acts as a cytotoxic agent via a novel mechanism, this substance was inactive in in vivo evaluations using the murine lu