Clin Cancer Res, 1999 Mar, 5(3), 673 - 80 Expression of multidrug resistance protein-related genes in lung cancer: correlation with drug response; Young LC et al.; Recently, cDNAs have been identified that encode four human proteins (MRP2-5) with structural similarity to the multidrug resistance protein (MRP) . Preliminary studies have shown that levels of mRNAs encoding MRP2, MRP3, and MRP5, are increased in some drug-selected cell lines, but the correlation of MRP2-5 mRNA levels with drug resistance has not been examined . Using a collection of small cell lung cancer (SCLC) and non-SCLC patient samples and unselected cell lines established from patients at various stages of treatment, we examined the expression of MRP2, MRP3, MRP4, and MRP5, as well as MDR1 and MRP, by PCR . The levels of individual mRNAs were correlated with the sensitivity of these cell lines to doxorubicin (DOX), vincristine, VP-16, and cis-diamminedichloroplatinum(II), as determined by a modified MTT assay . Using both SCLC and non-SCLC cell lines, we confirmed the previously observed correlation of MRP mRNA levels with resistance to DOX (B . G . Campling et al., Clin . Cancer Res., 3:115-122, 1997) and found a strong correlation of MRP3 mRNA levels with resistance of the cell lines to DOX . In addition, the mRNA levels of both MRP and MRP3 correlated with resistance of the cell lines to vincristine, VP-16, and cis-diamminedichloroplatinum(II) . These findings are consistent with the suggestion that MRP3, like MRP, may contribute to the drug resistance phenotype of lung cancer cells. Cancer Chemother Pharmacol, 1999, 43(5), 371 - 8 The bisindolylmaleimide protein kinase C inhibitor, Ro 32-2241, reverses multidrug resistance in KB tumour cells; Merritt JE et al.; Ro 32-2241 is a bisindolylmaleimide that selectively inhibits protein kinase C (PKC) as compared with other protein kinases . Experiments were carried out to examine its potential as a multidrug resistance-reversing agent . Ro 32-2241 inhibited efflux, and increased accumulation, of {3H}-daunomycin in multidrug-resistant (MDR) KB-8-5 and KB-8-5-11 cells and had no effect on drug-sensitive KB-3-1 cells . Ro 32-2241 completely reversed the doxorubicin resistance of KB-8-5 and KB-8-5-11 cells, showing no effect on the sensitivity of drug-sensitive KB-3-1 cells . The potency of Ro 32-2241 was comparable with that of cyclosporin A and better than that of verapamil, known modulators of multidrug resistance . Ro 32-2241 also completely reversed the taxol resistance of KB-8-5 cells and partially reversed the resistance of KB-8-5-11 cells . Vinblastine resistance was also partially reversed . Mechanistic experiments were carried out to determine whether Ro 32-2241 interacted with P-glycoprotein (Pgp) directly . Increased efflux of {14C}-Ro 32-2241 was seen with the more resistant KB-8-5-11 cells (although the percentage effluxed was very low as compared with {3H}-daunomycin), suggesting that Ro 32-2241 can act as a substrate for Pgp . Direct interaction of Ro 32-2241 with Pgp was confirmed by demonstration that it inhibited binding of {3H}-azidopine to Pgp in KB-8-5-11 membranes . In conclusion, Ro 32-2241, acting directly on Pgp (rather than, or in addition to, an effect on PKC), is effective in reducing or reversing resistance to doxorubicin, taxol and vinblastine in human tumour cells with a clinically relevant degree of MDR . However, results of in vivo experiments conducted to investigate the effects of Ro 32-2241 on resistance to doxorubicin suggest that it may not be possible to achieve sufficiently high levels of Ro 32-2241 in vivo to modulate MDR. Br J Cancer, 1999 Mar, 79(7-8), 1090 - 7 Dual mechanism of daunorubicin-induced cell death in both sensitive and MDR-resistant HL-60 cells; Come MG et al.; Exposure of some acute myeloid leukaemia (AML) cells to daunorubicin leads to rapid cell death, whereas other AML cells show natural drug resistance . This has been attributed to expression of functional P-glycoprotein resulting in reduced drug accumulation . However, it has also been proposed that P-glycoprotein-expressing multidrug-resistant (MDR) cells are inherently defective for apoptosis . To distinguish between these different possibilities, we have compared the cell death process in a human AML cell line (HL-60) with a MDR subline (HL-60/Vinc) at doses that yield either similar intracellular daunorubicin concentrations or comparable cytotoxicity . Adjustment of the dose to obtain the same intracellular drug accumulation in the two cell lines did not result in equal cytotoxicity, suggesting the presence of additional resistance mechanisms in the P-glycoprotein-expressing HL-60/Vinc cells . However, at equitoxic doses, similar cell death pathways were observed . In HL-60 cells, daunorubicin induced rapid apoptosis at 0.5-1 microM and delayed mitotic cell death at 0.1 microM . These concentrations are within the clinical dose range . Similarly, HL-60/Vinc cells underwent apoptosis at 50-100 microM daunorubicin and mitotic cell death at 10 microM . These results show, for the first time, that anthracyclines can induce cell death by a dual mechanism in both sensitive and MDR cells . Our results also show that not only the cytotoxicity, but also the kinetics and mechanism of cell death, are dose dependent . Interestingly, regrowth was observed only in association with delayed cell death and the formation of enlarged, often polyploid, cells with micronucleation, suggesting that morphological criteria may be useful to evaluate treatment efficacy in patients with myeloid leukaemias. Br J Cancer, 1999 Mar, 79(7-8), 1053 - 60 Selective inhibition of MDR1 P-glycoprotein-mediated transport by the acridone carboxamide derivative GG918; Wallstab A et al.; The acridone carboxamide derivative GG918 (N- inverted question mark4-{2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl}-pheny l inverted question mark-9,10dihydro-5-methoxy-9-oxo-4-acridine carboxamide) is a potent inhibitor of MDR1 P-glycoprotein-mediated multidrug resistance . Direct measurements of ATP-dependent MDR1 P-glycoprotein-mediated transport in plasma membrane vesicles from human and rat hepatocyte canalicular membranes indicated 50% inhibition at GG918 concentrations between 8 nM and 80 nM using N-pentyl-{3H}quinidinium, {'4C}doxorubicin and {3H}daunorubicin as substrates . The inhibition constant K for GG918 was 35 nM in rat hepatocyte canalicular membrane vesicles with {3H}daunorubicin as the substrate . Photoaffinity labelling of canalicular and recombinant rat Mdr1b P-glycoprotein by {3H}azidopine was suppressed by 10 muM and 40 muM GG918 . The high selectivity of GG918-induced inhibition was demonstrated in canalicular membrane vesicles and by analysis of the hepatobiliary elimination in rats using {3H}daunorubicin, {3H}taurocholate and {3H}cysteinyl leukotrienes as substrates for three distinct ATP-dependent export pumps . Almost complete inhibition of {3H}daunorubicin transport was observed at GG918 concentrations that did not affect the other hepatocyte canalicular export pumps . The high potency and selectivity of GG918 for the inhibition of human MDR1 and rat Mdr1b P-glycoprotein may serve to interfere with this type of multidrug resistance and provides a tool for studies on the function of these ATP-dependent transport proteins. Bioorg Med Chem Lett, 1999 Feb 22, 9(4), 595 - 600 Reversal of P-glycoprotein mediated multidrug resistance by novel anthranilamide derivatives; Roe M et al.; We have synthesised and evaluated a series of anthranilamide based modulators of P-glycoprotein . These studies have identified XR9576(2), a potent inhibitor of P-glycoprotein in vitro and in vivo . The general synthesis and the SAR of these compounds are described. Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3900 - 5 Choroid plexus epithelial expression of MDR1 P glycoprotein and multidrug resistance-associated protein contribute to the blood-cerebrospinal-fluid drug-permeability barrier; Rao VV et al.; The blood-brain barrier and a blood-cerebrospinal-fluid (CSF) barrier function together to isolate the brain from circulating drugs, toxins, and xenobiotics . The blood-CSF drug-permeability barrier is localized to the epithelium of the choroid plexus (CP) . However, the molecular mechanisms regulating drug permeability across the CP epithelium are defined poorly . Herein, we describe a drug-permeability barrier in human and rodent CP mediated by epithelial-specific expression of the MDR1 (multidrug resistance) P glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP) . Noninvasive single-photon-emission computed tomography with 99mTc-sestamibi, a membrane-permeant radiopharmaceutical whose transport is mediated by both Pgp and MRP, shows a large blood-to-CSF concentration gradient across intact CP epithelium in humans in vivo . In rats, pharmacokinetic analysis with 99mTc-sestamibi determined the concentration gradient to be greater than 100-fold . In membrane fractions of isolated native CP from rat, mouse, and human, the 170-kDa Pgp and 190-kDa MRP are identified readily . Furthermore, the murine proteins are absent in CP isolated from their respective mdr1a/1b(-/-) and mrp(-/-) gene knockout littermates . As determined by immunohistochemical and drug-transport analysis of native CP and polarized epithelial cell cultures derived from neonatal rat CP, Pgp localizes subapically, conferring an apical-to-basal transepithelial permeation barrier to radiolabeled drugs . Conversely, MRP localizes basolaterally, conferring an opposing basal-to-apical drug-permeation barrier . Together, these transporters may coordinate secretion and reabsorption of natural product substrates and therapeutic drugs, including chemotherapeutic agents, antipsychotics, and HIV protease inhibitors, into and out of the central nervous system. Nucl Med Biol, 1999 Jan, 26(1), 35 - 41 Measurement of P-glycoprotein expression in human neuroblastoma xenografts using in vitro quantitative autoradiography; Fonti R et al.; P-glycoprotein (P-gp) has a role in multidrug resistance (MDR) encountered in human cancers . In this study, we used the colchicine-resistant cell line BE(2)-C/CHCb(0.2), a strain of neuroblastoma cell line BE(2)-C, as a model to measure variations of P-gp expression in cells grown in vitro and in vivo . Cells were cultured in the medium supplemented with colchicine . At the beginning of the study the drug was withdrawn and, after 22 days, added back to the culture medium . Cells were harvested at various time points and xenografted in nude mice . P-gp content in cells was measured by self-competitive binding assay and in tumors, by quantitative autoradiography (QAR) . Both assays were carried out using 125I-labeled monoclonal antibody MRK16, reactive with P-gp . Concentration of P-gp in cells varied from a maximum of 1,361 pmol/g in the presence of colchicine to a minimum of 374 pmol/g in the absence of colchicine in the culture medium . P-gp concentration in the tumors ranged from 929 to 188 pmol/g, which correlated with P-gp content in the cells at the time of their injection in the mice . QAR is an accurate and reliable method to quantify P-gp expression in tumors . Changes in colchicine concentration in the ambient medium of BE(2)-C/CHCb(0.2) cells growing in vitro resulted in a change in phenotype of P-gp expression, which was stable under conditions of in vivo growth over approximately 9 cell divisions in nude mice xenografts . Therefore, P-gp content in xenografts depends only on the level of resistance of the cells at the time of their injection in the mice. Hepatology, 1999 Apr, 29(4), 1156 - 63 Characterization of the human multidrug resistance protein isoform MRP3 localized to the basolateral hepatocyte membrane; Konig J et al.; Several members of the multidrug resistance protein (MRP) family are expressed in the liver . Adenosine triphosphate (ATP)-dependent transport of glutathione and glucuronoside conjugates across the hepatocyte canalicular membrane is mediated by the apical MRP isoform, MRP2 (APMRP), also known as canalicular multispecific organic anion transporter (cMOAT) . We have cloned an additional MRP isoform, MRP3, from human liver and localized it to the basolateral membrane domain of hepatocytes . Basolateral MRP (BLMRP) is composed of 1,527 amino acids and encoded by 4,581 base pairs of complementary DNA . Northern blotting of various human tissues indicated an expression of MRP3 in the liver, colon, pancreas, and, at a lower level, in the kidney . The amino acid identity of MRP3 with MRP1 and MRP2 is 58% and 48%, respectively . These three isoforms, encoded by genes on different chromosomes, have a similar predicted topology of transmembrane segments and ATP-binding domains . Antibodies raised against two peptide sequences of MRP3 that are not shared by other MRP family members detected recombinant MRP3 expressed in polarized MDCK cells . Both antibodies served to localize MRP3 to the basolateral membrane of hepatocytes . Double-label immunofluorescence microscopy confirmed that MRP3 was not detectable in the canalicular membrane domain . A particularly strong expression of the MRP3 protein was observed in the basolateral hepatocyte membrane of two patients with Dubin-Johnson syndrome who are deficient in MRP2 . These results indicate that the basolateral MRP isoform, MRP3, may be upregulated when the canalicular secretion of anionic conjugates by MRP2 is impaired. Int J Tuberc Lung Dis, 1999 Mar, 3(3), 214 - 8 Phase II trial of recombinant interferon-alpha2b in patients with advanced intractable multidrug-resistant pulmonary tuberculosis: long-term follow-up; Palmero D et al.; SETTING: Multidrug-resistant tuberculosis patients without human immunodeficiency virus (HIV) infection, with Mycobacterium tuberculosis resistant to almost all of the available drugs . OBJECTIVE: Limited phase II trial with recombinant interferon-alpha2b in five chronic multidrug-resistant tuberculosis patients . METHODS: Three million units of r-IFN-alpha2b were administered subcutaneously every week for 12 weeks . Before and after treatment, and during a 30-month follow-up period, the patients underwent clinical and radiological examination, together with bacteriological, immunological and routine laboratory testing . RESULTS: Two of the five patients became long-term sputum smear and culture negative after r-IFN-alpha2b therapy; one of the patients showed clinical improvement and negative smear after therapy, but remained culture positive . The other two patients showed no response . CONCLUSION: The results of this trial suggest that r-IFN-alpha2b should be evaluated further in multidrug-resistant tuberculosis in prospective controlled trials. Int J Tuberc Lung Dis, 1999 Mar, 3(3), 207 - 13 Outbreak of multiple drug-resistant tuberculosis in Lisbon: detection by restriction fragment length polymorphism analysis; Portugal I et al.; SETTING: Multidrug-resistant tuberculosis (MDR-TB) mainly among human immunodeficiency virus (HIV) seropositive patients in Lisbon hospitals in 1996-1997 . OBJECTIVE: Detection of transmission of MDR-TB strains and epidemic outbreaks in several hospital units in the city of Lisbon, including a prison hospital . DESIGN: Use of restriction fragment length polymorphism (RFLP) to fingerprint isolates of Mycobacterium tuberculosis resistant to isoniazid, rifampicin, and one other drug . RESULTS: A total of 43 MDR-TB strains were typed . Sixty-seven per cent of the patients were HIV positive, 12% were HIV negative, and the remainder had unknown HIV status . About 88% of the isolates were grouped in three genetically similar clusters, suggesting possible recent transmission . A predominant cluster (cluster A), corresponding to 72% of the cases, was found, 45% of which came from the prison hospital . Strains from this cluster were resistant to isoniazid, rifampicin, streptomycin, and sometimes ethambutol . A retrospective epidemiological investigation was conducted with respect to all patients in cluster A, and epidemiological links were established between them . CONCLUSION: Our results suggest recent transmission of MDR-TB, mainly in HIV-positive patients, in Lisbon hospitals . Moreover, the predominant MDR-TB clustered strains were not confined to HIV-infected individuals, as they were also isolated in some immunocompetent patients. Int J Tuberc Lung Dis, 1999 Jan, 3(1), 4 - 11 Low levels of drug resistance amidst rapidly increasing tuberculosis and human immunodeficiency virus co-epidemics in Botswana; Kenyon TA et al.; SETTING: Botswana, southern Africa, where the tuberculosis (TB) case rate increased by 120% from 1989 to 1996 in spite of a decade of implementation of the directly observed therapy, short-course (DOTS) strategy . OBJECTIVE: To determine prevalence of and risk factors for drug-resistant tuberculosis in an epidemic setting . DESIGN: Systematic national random survey of newly diagnosed pulmonary TB and all patients with TB requiring retreatment during 1995-1996 . Interviews were conducted, human immunodeficiency virus (HIV) testing was offered, and drug susceptibility testing was performed for isoniazid, rifampicin, streptomycin and ethambutol . RESULTS: Resistance to at least one drug was identified in 16 (3.7%) new cases and 18 (14.9%) retreatment cases . One (0.2%) new and seven (5.8%) retreatment cases had resistance to at least both isoniazid and rifampicin (multidrug-resistant TB) . Retreatment cases with multidrug-resistant TB were significantly more likely to have worked in the mines in South Africa than were cases with fully susceptible isolates (6/7 {85.7%} versus 32/ 103 {31.1%}, odds ratio 13.3, 95% confidence interval 1.5-311.0, P = 0.007) . Of 240 patients tested for HIV, 117 (48.8%) were positive; prevalence was similar among new and retreatment cases, and was not a risk factor for drug resistance in either group . CONCLUSION: During the HIV and TB co-epidemics in sub-Saharan Africa, DOTS may help to control drug-resistant TB . However, the TB case rate can be expected to continue to climb in spite of the implementation of the DOTS strategy. Eur J Biochem, 1999 Feb, 259(3), 841 - 50 Stimulation of P-glycoprotein-mediated drug transport by prazosin and progesterone . Evidence for a third drug-binding site; Shapiro AB et al.; P-glycoprotein is a plasma membrane protein of mammalian cells that confers multidrug resistance by acting as a broad-specificity, ATP-dependent efflux transporter of diverse lipophilic neutral or cationic compounds . Previously, we identified two positively cooperative drug-binding sites of P-glycoprotein involved in transport {Shapiro, A . B . & Ling, V . (1997) Eur . J . Biochem . 250, 130-137} . The H site is selective for Hoechst 33342 and colchicine . The R site is selective for rhodamine 123 and anthracyclines . Substrate binding to one site stimulates transport by the other . In this paper, we show that prazosin and progesterone stimulate the transport of both Hoechst 33342 and rhodamine 123 . Rhodamine 123 and prazosin (or progesterone) in combination stimulate Hoechst 33342 transport in an additive manner . In contrast, Hoechst 33342 and either prazosin or progesterone interfere with each other, so that the stimulatory effect of the combination on rhodamine 123 transport is less than that of each individually . Non-P-glycoprotein-specific effects of prazosin on membrane fluidity and permeability were excluded . These results indicate the existence of a third drug-binding site on P-glycoprotein with a positive allosteric effect on drug transport by the H and R sites . This allosteric site appears to be one of the sites of photoaffinity labeling of P-glycoprotein by {125I}iodoarylazidoprazosin {Safa, A . R., Agresti, M., Bryk, D . & Tamai, I . (1994) Biochemistry 33, 256-265} and is likely not to be capable of drug transport. Lab Invest, 1999 Mar, 79(3), 271 - 80 MDR1 gene expression in primary and advanced breast cancer; Yang X et al.; P-glycoprotein (Pgp)-associated multidrug resistance (MDR) is related to intrinsic and acquired cross resistance to anthracyclines, vinca alkaloids, and other antineoplastic antibiotics . Expression of MDR1 is widely considered to play an important role in conferring resistance to adjuvant chemotherapy in women with breast tumor cells in women with disseminated disease, although data supporting this view is, at best, conflicting . The expression of MDR1 gene and its gene product, P-glycoprotein, was investigated in primary and advanced breast cancers (both previously untreated and previously treated on specific treatment protocols) to assess the role of P-glycoprotein in determining responsiveness to adjuvant chemotherapy . Expression was assessed by immunohistochemistry, reverse transcription-PCR (RT-PCR), Northern Blot and Western Blot . MDR1 mRNA was detected in 40% of the breast cancers tested by RT-PCR with 40 cycles of PCR amplification . When reducing the PCR amplification cycles to 28, the MDR1 gene expression signal disappeared from breast cancers of the highest expressers; however, known MDR1 positive control normal tissues, such as adrenal, kidney, and liver continued to show an expression product . Western and Northern blots failed to demonstrate the MDR1 gene product, P-glycoprotein, in these breast cancers . In contrast, physiologic levels of P-glycoprotein was clearly detected in normal adrenal, kidney, and liver by these techniques . Immunohistochemistry confirmed that breast carcinoma cells lacked P-glycoprotein expression; however, interstitial mononuclear cells, morphologically consistent with lymphocytes or macrophages did show immunostaining in some of these breast tumors . MDR1 gene expression identified by RT-PCR was not correlated either with response to paclitaxel therapy (29 patients able to be evaluated, p = 0.34, Fisher Exact Test) or overall survival (32 breast cancer patients with clinical follow-up information, p = 0.336, log rank) . In conclusion, P-glycoprotein was not expressed in breast carcinoma cells at significant levels, although it was expressed in stomal lymphocytes or macrophages . These results suggest that P-glycoprotein does not play a significant role in multidrug resistance of breast cancer. Int J Tuberc Lung Dis, 1999 Feb, 3(2), 143 - 8 Drug susceptibility of Mycobacterium tuberculosis in a rural area of Bangladesh and its relevance to the national treatment regimens; Van Deun A et al.; SETTING: Greater Mymensingh District, a rural area of Bangladesh, at the start of the National Tuberculosis Programme (NTP) . OBJECTIVES: To determine the prevalence of initial and acquired drug resistance of Mycobacterium tuberculosis, and to assess the appropriateness of the NTP's standard regimens . DESIGN: Sampling of pre-treatment sputum from all newly registered smear-positive cases in five centres covering the area . Culture and susceptibility testing in a supra-national reference laboratory . RESULTS: Initial resistance to isoniazid (H) was 5.4%, and to rifampicin (R) 0.5% . Acquired H and R resistance were 25.9% and 7.4%, respectively . Multidrug resistance (MDR) was observed in one new case only and in 5.6% of previously treated patients . Changing the present NTP indication for retreatment regimen to one month of previous H intake would increase coverage of H-resistant cases from 52% to 89%, adding 6% to drug costs . CONCLUSION: The prevalence of drug resistance is surprisingly low in Bangladesh, but could rise with improving economic conditions . The NTP regimens for smear-positive cases are appropriate, all the more so since the human immunodeficiency virus is virtually absent . Indications for the retreatment regimen should be extended to include all patients treated for at least one month with any drug . The NTP regimen for smear-negative cases runs the risk of leading to MDR under present field conditions. Bioorg Med Chem Lett, 1999 Feb 8, 9(3), 389 - 94 Modulation of multidrug resistance in tumor cells by taxinine derivatives; Hosoyama H et al.; Among a series of taxinine (1) and its designed derivatives (2-33), two taxoids (29 and 33) increased cellular accumulation of vincristine in multidrug-resistant tumor cells more potently than verapamil, while the activities of eight taxoids (11, 14-16, 22, and 30-32) were comparable with that of verapamil . These results reveal that some taxinine derivatives are good modifiers of multidrug resistance in tumor cells. Cytometry, 1999 Feb 15, 38(1), 24 - 9 Correlation of rhodamine 123 efflux by neoplastic plasma cells with clinical and biological characteristics of multiple myeloma; Perez-Simon JA et al.; Although a variable proportion of multiple myeloma patients can achieve response with conventional chemotherapy, residual tumor cells, which are refractory, finally reemerge leading to disease progression . The expression of the multidrug resistance protein (MDR1) has been one of the most extensively explored mechanisms of drug resistance and has been related to a poor response to chemotherapy in several human tumors . Nevertheless, a careful analysis of the literature on MDR1 expression in multiple myeloma (MM) shows the existence of disturbing discrepancies as regards both the incidence of MDR1 over-expression and its clinical value . A prerequisite for the assessment of MDR1 in tumor cells should be the identification of the neoplastic cells present in the sample . This is particularly important in MM, where the percentage of tumor cells in bone marrow (BM) is relatively low . In the present study we have analyzed the functional expression of MDR1 in BM plasma cells (PC), from a group of 40 untreated MM patients . For that purpose, the rhodamine 123 efflux assay was used in combination with specific staining for plasma cells (CD38 strong+) . The mean fluorescence channel (MFC) of rhodamine 123 in myelomatous PC from MM patients was 311 and 110 after incubating cells with this fluorochrome for 15 and 60 min, respectively . The median percentage of rhodamine 123 elimination by BM PC was of 61% (range: 0.29 to 88%) . Upon analyzing the relationship between the ability of myelomatous PC to eliminate rhodamine 123 and other clinical and biological disease characteristics we found that, within the group of patients displaying high MDR1 expression (>61% rhodamine efflux), there was a higher incidence of cases with bone disease (P = 0.014) and advanced clinical stages (P = 0.031), greater calcium (P = 0.007) and creatinine serum levels (P = 0.061), and lower levels of albumin in serum (P = 0.015) . All these parameters are usually associated with a poor prognosis . When we analyzed the possible relationship between the ability of BM PC to eliminate rhodamine 123 and the presence of numerical chromosome abnormalities we observed that a low MDR1 expression was related to a higher incidence of trisomies of chromosomes 6 and 17, although these differences did not reach statistical significance (P = 0.06) . In spite of these associations, from the prognostic point of view, MDR1 expression did not correlate with other relevant prognostic factors, response to treatment (P = 0.38) or overall survival (P = 0.12). Eur Urol, 1999 Apr, 35(4), 327 - 35 Estramustine reversal of resistance to intravesical epirubicin chemotherapy; Jennings AM et al.; OBJECTIVE: Failure of epirubicin treatment of superficial bladder cancer implies multidrug resistance (MDR) which is common . MDR is characterised by decreased cellular levels of drug . TCC cell lines sensitive to epirubicin and resistant to both epirubicin and mitomycin C were used to investigate augmented therapy by adding the MDR reversing agent estramustine to an in vitro model . METHODS: Cells were cultured as monolayers . Fluorescence analysis was performed by flow cytometry and confocal microscopy . Cells were exposed to epirubicin 20 microg/ml for 2 h and increasing amounts of estramustine . Cytotoxicity was determined under similar exposure conditions and MTT culture (dye reduction by live cells) allowed viable biomass to be read optically . RESULTS: Resistant cells accumulated eight times less epirubicin than sensitive cells . Confocal microscopy confirmed this for nuclear uptake . Accumulation in resistant cells can be increased to near-sensitive cell levels using 40 microg/ml estramustine . Image analysis of confocal fluorescence showed a shift from cytoplasm to nucleus . This correlated with increased cytotoxicity . CONCLUSION: Estramustine plus epirubicin chemotherapy can overcome MDR and may achieve more successful tumour killing in vivo . It may also prevent emergence of resistance . Primary TCC culture examination permits detection of sensitive and resistant cells and may predict outcome allowing a more logical treatment selection. Nucl Med Commun, 1999 Feb, 20(2), 115 - 22 99Tcm-sestamibi imaging of inhibition of the multidrug resistance transporter in a mouse xenograft model of human breast cancer; Muzzammil T et al.; Sestamibi is known to be a substrate for P-glycoprotein, the membrane transporter which confers multidrug resistance by pumping certain chemotherapeutic agents out of tumour cells . In this study, the utility of sestamibi imaging for detecting inhibition of P-glycoprotein (Pgp) function by two potent, second-generation chemosensitizers, PSC833 and GG918, was assessed in a mouse xenograft model of multidrug-resistant human breast cancer, MCF7-AdrR . Preliminary in vitro studies confirmed that MCF7-AdrR cells accumulate only low levels of sestamibi and that both sensitizers inhibit transporter function to a similar extent, resulting in 20-fold higher accumulation of sestamibi . MCF7-AdrR cells were grown as a xenograft in SCID mice and sestamibi kinetics in the tumour were analysed by dynamic imaging (30 frames at a rate of one frame per minute) . Administration of either chemosensitizer produced a dose-dependent slowing of the efflux rate of sestamibi compared to untreated tumours . The same effect was evident in two additional parameters: percent activity remaining at 30 min determined by imaging and sestamibi levels measured in excised tissues . These results show that sestamibi imaging can detect inhibition of Pgp function and suggest that this approach could be used clinically to document effective delivery of chemosensitizers. Arch Biochem Biophys, 1999 Apr 1, 364(1), 99 - 106 Functional characterization of the carnitine transporter defective in primary carnitine deficiency; Scaglia F et al.; Primary carnitine deficiency is an autosomal recessive disorder caused by defective carnitine transport which impairs fatty acid oxidation and manifests as nonketotic hypoglycemia or skeletal or heart myopathy . Here we report the functional characterization of this transporter in human fibroblasts . Carnitine enters normal cells by saturable and unsaturable routes, the latter corresponding to Na+-independent uptake . Saturable carnitine transport was absent in cells from patients with primary carnitine deficiency . In control cells, saturable carnitine transport was energized by the electrochemical gradient of Na+ . Carnitine uptake was not inhibited by amino acid substrates of transport systems A, ASC, and X-AG, but was inhibited competitively (in potency order) by butyrobetaine > carnitine > palmitoylcarnitine = acetylcarnitine > betaine . Carnitine uptake was also noncompetitively inhibited by verapamil and quinidine, inhibitors of the multidrug resistance family of membrane transporters, suggesting that the carnitine transporter may share a functional motif with this class of transporters . A high-affinity carnitine transporter was present in kidney 293 cells, but not in HepG2 liver cells, whose carnitine transporter had a Km in the millimolar range . These result indicate the presence of multiple types of carnitine transporters in human cells . Pflugers Arch, 1999 Apr, 437(5), 652 - 60 P-glycoprotein inhibition by glibenclamide and related compounds; Golstein PE et al.; Glibenclamide is well known to interact with the sulphonylurea receptor (SUR) and has been shown more recently to inhibit the cystic fibrosis transmembrane conductance regulator protein (CFTR), both proteins that are members of the ABC {adenosine 5'-triphosphate (ATP)-binding cassette} transporters . The effect of glibenclamide and two synthetic sulphonylcyanoguanidine derivatives (dubbed BM-208 and BM-223) was examined on P-glycoprotein, the major ABC transporter responsible for multidrug resistance (MDR) in cancer cells . To this end, we employed different cell lines that do or do not express P-glycoprotein, as confirmed by Western blotting: first, a tumour cell line (VBL600) selected from a human T-cell line (CEM) derived from an acute leukaemia; second, an epithelial cell line derived from a rat colonic adenocarcinoma (CC531(mdr+)) and finally, a non tumour epithelial cell line derived from the proximal tubule of the opossum kidney (OK) . Glibenclamide and the two related derivatives inhibited P-glycoprotein because firstly, they acutely increased {3H}colchicine accumulation in P-glycoprotein-expressing cell lines only; secondly BM-223 reversed the MDR phenomenon, quite similarly to verapamil, by enhancing the cytotoxicity of colchicine, taxol and vinblastine and thirdly, BM-208 and BM-223 blocked the photoaffinity-labelling of P-glycoprotein by {3H}azidopine . Furthermore, glibenclamide is itself a substrate for P-glycoprotein, since the cellular accumulation of {3H}glibenclamide was low and substantially increased by addition of P-glycoprotein substrates (e . g., vinblastine and cyclosporine) only in the P-glycoprotein-expressing cell lines . We conclude that glibenclamide and two sulphonylcyanoguanidine derivatives inhibit P-glycoprotein and that sulphonylurea drugs would appear to be general inhibitors of ABC transporters, suggesting an interaction with some conserved motif. Mol Cell Biol, 1999 Apr, 19(4), 2998 - 3009 Mutational disruption of plasma membrane trafficking of Saccharomyces cerevisiae Yor1p, a homologue of mammalian multidrug resistance protein; Katzmann DJ et al.; The ATP binding cassette (ABC) transporter protein Yor1p was identified on the basis of its ability to elevate oligomycin resistance when it was overproduced from a high-copy-number plasmid . Analysis of the predicted amino acid sequence of Yor1p indicated that this protein was a new member of a subfamily of ABC transporter proteins defined by the multidrug resistance protein (MRP) . In this work, Yor1p is demonstrated to localize to the Saccharomyces cerevisiae plasma membrane by both indirect immunofluorescence and biochemical fractionation studies . Several mutations were generated in the amino-terminal nucleotide binding domain (NBD1) of Yor1p to test if the high degree of sequence conservation in this region of the protein was important for function . Deletion of a phenylalanine residue at Yor1p position 670 led to a mutant protein that appeared to be retained in the endoplasmic reticulum (ER) and that was unstable . As shown by others, deletion of the analogous residue from a second mammalian MRP family member, the cystic fibrosis transmembrane conductance regulator (CFTR), also led to retention of this normally plasma membrane-localized protein in the ER . Changes in the spacing between or the sequences flanking functional motifs of Yor1p NBD1 led to defective trafficking or decreased activity of the mutant proteins . Analyses of the degradation of wild-type and DeltaF670 Yor1p indicated that the half-life of DeltaF670 Yor1p was dramatically shortened . While the vacuole was the primary site for turnover of wild-type Yor1p, degradation of DeltaF670 Yor1p was found to be more complex with both proteasomal and vacuolar contributions. Jpn J Cancer Res, 1998 Dec, 89(12), 1276 - 83 Expression of MRP and cMOAT in childhood neuroblastomas and malignant liver tumors and its relevance to clinical behavior; Matsunaga T et al.; Advanced neuroblastoma and malignant liver tumor are representative childhood cancers for which combined chemotherapy including cisplatin and doxorubicin is routinely performed . The prognosis of patients with tumors which develop multiple drug resistance (MDR) is unfavorable . To elucidate the role of multidrug resistance-associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT) in the clinical behavior of the tumors, we examined 42 neuroblastomas and 10 malignant liver tumors for the expressions of MRP and cMOAT by quantitative RNA-polymerase chain reaction (PCR) . The amplification and expression of N-myc oncogene in the neuroblastomas were also investigated . We found a close association between MRP and N-myc expression in each neuroblastoma sample but no significant relationship between MRP expression and the patients' outcome . The forced expression of N-myc failed to enhance the expression of MRP in N-myc transfected neuroblastoma cell lines . cMOAT was rarely expressed in the neuroblastomas, but was frequently expressed in the malignant liver tumors . The expression of MRP and cMOAT in the childhood liver tumors was more common and higher, especially in advanced cases with a poor outcome, than that observed in normal liver or in 9 hepatocellular carcinomas from adult patients . The enhanced expression of these genes might be characteristic of childhood malignant liver tumors and related to their clinical chemoresistance. Transpl Int, 1999, 12(1), 10 - 7 P-glycoprotein expression is not a useful predictor of acute or chronic kidney graft rejection; Melk A et al.; Because of the role of P-glycoprotein (P-gp) in multidrug resistance (MDR), it has been suggested that P-gp might play a role in acute and chronic rejection after organ transplantation . The purpose of the present work was to investigate a possible relationship between graft outcome and P-gp expression on peripheral mononuclear cells of renal transplant recipients . We determined P-gp expression in 27 patients with long-term, stable graft function (ST) and in 15 patients with chronic deterioration of graft function (CR) . In addition, 40 patients were studied prior to, and at intervals after, transplantation with 21 healthy individuals serving as controls . P-gp values were highest in healthy controls and in ST patients . We found no correlation between P-gp values and acute rejection . CR patients tended to have lower levels of P-gp expression . Our results contradict the opinion that an overexpression of P-gp induces acute or chronic rejection by inhibiting the efficacy of immunosuppressive treatment. Eur J Nucl Med, 1999 Mar, 26(3), 283 - 93 Visualization of multidrug resistance in vivo; Hendrikse NH et al.; Various mechanisms are involved in multidrug resistance (MDR) for chemotherapeutic drugs, such as the drug efflux pumps, P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP) . In this review the mechanisms involved in MDR are described and results are reviewed with particular attention to the in vivo imaging of Pgp and MRP . Various detection assays provide information about the presence of drug efflux pumps at the mRNA and protein levels . However, these methods do not yield information about the dynamic function of Pgp and MRP in vivo . For the study of Pgp- and MRP-mediated transport, single-photon emission tomography (SPET) and positron emission tomography (PET) are available . Technetium-99m sestamibi is a substrate for Pgp and MRP, and has been used in clinical studies for tumour imaging, and to visualize blockade of Pgp-mediated transport after modulation of the Pgp pump . Other 99mTc radiopharmaceuticals, such as 99mTc-tetrofosmin and several 99Tc-Q complexes, are also substrates for Pgp, but to date only results from in vitro and animal studies are available for these compounds . Several agents, including {11C}colchicine, {11C}verapamil and {11C}daunorubicin, have been evaluated for the quantification of Pgp-mediated transport with PET in vivo . The results suggest that radiolabelled colchicine, verapamil and daunorubicin are feasible substrates with which to image Pgp function in tumours . Uptake of {11C}colchicine and {11C}verapamil is relatively high in the chest area, reducing the value of both tracers for monitoring Pgp-mediated drug transport in tumours located in this region . In addition, it has to be borne in mind that only comparison of Pgp-mediated transport of radioalabelled substrates in the absence and in the presence of Pgp blockade gives quantitative information on Pgp-mediated pharmacokinetics . Leukotrienes are specific substrates for MRP . Therefore, N-{11C}acetyl-leukotriene E4 provides an opportunity to study MRP function non-invasively . Results obtained in MRP2 mutated GY/TR rats have demonstrated visualization of MRP-mediated transport . This tracer permits the study of MRP transport function abnormalities in vivo, e.g . in Dubin-Johnson patients, who are MRP2 gene deficient . Results obtained show the feasibility of using SPET and PET to study the functionality of MDR transporters in vivo. Int J Cancer, 1999 Mar 31, 81(1), 81 - 9 Characterization of intracellular pH gradients in human multidrug-resistant tumor cells by means of scanning microspectrofluorometry and dual-emission-ratio probes; Belhoussine R et al.; Multidrug-resistant cells are believed to contain a plasma-membrane-efflux pump which is hypothesized to expel anticancer drugs from the cytosol to the cell exterior . Many of these drugs are classified as weak bases whose binding to intracellular targets is pH-dependent . Slight alterations in intracellular pH gradients have been shown to affect accumulation, endocytosis and secretion of drugs . In this study, we developed a new method based on confocal spectral imaging analysis to determine intracellular pH gradients in sensitive and MDR tumor cells . Fluorescein isothiocyanate (FITC) and tetramethylrhodamine conjugated to dextran (FRD) and SNAFL-calcein-AM were used to determine pH in acidic compartments . Carboxy-SNARF1-AM was used to examine cytosolic pH . We observed that sensitive (HL60, K562, CEM and MCF7) cells exhibit lower acidity of the subcellular organelles than that corresponding to drug-resistant derivatives . Moreover, results obtained with carboxy-SNARF1-AM show that resistant cells display a more alkaline cytosolic pH . This results in a considerably larger pH gradient between the vesicular compartments and the cytosol of resistant cells than of sensitive cells . The lower pH gradient observed in sensitive cells may be related to a disruption in the organization of the trans-Golgi network (TGN) . In drug-resistant cells, the organization of TGN appears compact . In addition, confocal microscopic analysis of cells labelled with FRD and SNAFL-calcein showed that sensitive cells contain a lower number of acidified vesicles . This suggest a diminished capacity of these cells to remove protonated drugs from the cytoplasm to secretory compartments followed by their secretion through the activity of the secretory and recycling pathways. J Chemother, 1999 Feb, 11(1), 61 - 8 Cytotoxicity and adjuvant activity of cationic photosensitizers in a multidrug resistant cell line; Wainwright M et al.; The toxicities and phototoxicities of methylene blue (MB), toluidine blue O (TBO) and Victoria blue BO (VBBO) in a murine mammary tumour cell line (EMT6-S) and a multidrug resistant (MDR) sub-line (EMT6-R) were measured and their efficacy against the resistant sub-line was compared to that of doxorubicin and cis-platinum . The MDR cell line was considerably more susceptible to VBBO than to the conventional agent doxorubicin . VBBO was also phototoxic whereas illumination did not alter the activity of doxorubicin or of cisplatin . Both TBO and MB showed limited light activation (2-fold) in both the sensitive and resistant cell lines . Pre-treatment with VBBO prior to exposure to doxorubicin caused a two-fold increase in doxorubicin toxicity in both cell lines . MB and TBO, however, increased doxorubicin toxicity in EMT6-R cells x2 and x3 respectively, but had less effect on the sensitive cell line (increase x1.4 and x2 respectively) . Thus MB and TBO may act on the MDR cell line via a different mechanism to that of VBBO. Cancer Lett, 1999 Jan 8, 135(1), 113 - 9 Enhancement of glucuronosyl etoposide transport by glutathione in multidrug resistance-associated protein-overexpressing cells; Sakamoto H et al.; Multidrug resistance-associated protein (MRP) has been shown to transport glutathione (GSH) S-conjugates such as leukotriene C4 (LTC4) and S-(2,4-dinitrophenyl)-glutathione (DNP-SG) . On the other hand, it has while it has been reported that MRP-overexpressing cells exhibit decreased sensitivity to drugs which do not form GSH S-conjugates . In this study, we found that GSH affects the transport of glucuronosyl etoposide as a major metabolite of etoposide in MRP-overexpressing KB/VP-4 cells . The relative resistance level of KB/VP-4 cells to etoposide was 70-fold that of wild-type KB cells . Membrane vesicles prepared from KB/VP-4 cells exhibited markedly enhanced ATP-dependent transport of glucuronosyl etoposide as well as LTC4 . Transport of glucuronosyl etoposide was augmented in the presence of GSH . Treatment of KB/VP-4 cells with buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, resulted in about 75% depletion of cellular GSH levels, a four-fold increase of the sensitivity to etoposide and depression of glucuronosyl etoposide efflux . These results suggest that GSH plays a role in the enhancement of MRP-mediated glucuronosyl etoposide transport. Cancer Lett, 1999 Jan 8, 135(1), 97 - 105 The study of innate drug resistance of human hepatocellular carcinoma Bel7402 cell line; Huang M et al.; The innate drug resistance of human hepatocellular carcinoma (HCC) Bel7402 cell line was studied in vitro . MTT assay showed that Bel7402 cells were innately resistant to doxorubicin (Dox), and even more resistant to vincristine (VCR) . This resistance could be effectively reversed by verapamil (Ver), one of the classical multidrug resistance (MDR) modulating agents . However, the differences in 5-fluorouracil (5-FU) toxicity between these two cell lines is much less and the resistance of Bel7402 cells could only be slightly reversed by Ver, which may be experimental noise . Immunocytochemical staining using anti-p-glycoprotein monoclonal antibody JSB-1 indicated that the expression of the P-glycoprotein (P-gp) in the innate Bel7402 cells was elevated compared with the sensitive KB cells . The accumulation of Dox in innate resistant Bel7402 cells was 50.7% lower than that in sensitive KB cells by using spectrofluometric analyses, and the accumulation of Dox increased 1.6 fold in Bel7402 cells in the presence of Ver . The susceptibility of Dox-induced apoptosis was also increased in the presence of Ver by using flow cytometric assay and DNA fragmentation quantitative assay as well as by Hoechst 33258 staining . It appears that the innate Bel7402 cells might be useful in screening new antitumour drugs or new chemosensitisers which could overcome the innate or acquired resistant mechanism, and the toxicity and reversal effects with 5-FU are different from those known to be P-gp substrates such as VCR, Dox, and taxol. Biochem Pharmacol, 1998 May 1, 55(9), 1385 - 90 Requirement of expression of P-glycoprotein on human natural killer leukemia cells for cell-mediated cytotoxicity; Yamashiro T et al.; The requirement of P-glycoprotein, a product of the multidrug resistance (MDR)1 gene, for natural killer (NK) cell-mediated cytotoxicity was examined by using a human NK-like cell line, YTN, which is cytotoxic toward JY cells . YTN cells express P-glycoprotein, a judged by flow cytometry and polymerase chain reaction of reverse-transcribed mRNA . YTN cell-mediated cytotoxicity was inhibited by MDR-reversing reagents as well as the F(ab')2 fragment of a monoclonal antibody against P-glycoprotein . Furthermore, antisense oligonucleotides for MDR1 mRNA inhibited expression of P-glycoprotein as well as YTN cell-mediated cytotoxicity . Thus, this study provides firm evidence that P-glycoprotein plays an essential role in cell-mediated cytotoxicity. J Biol Chem, 1999 Mar 19, 274(12), 8269 - 81 Retinal stimulates ATP hydrolysis by purified and reconstituted ABCR, the photoreceptor-specific ATP-binding cassette transporter responsible for Stargardt disease; Sun H et al.; Many substrates for P-glycoprotein, an ABC transporter that mediates multidrug resistance in mammalian cells, have been shown to stimulate its ATPase activity in vitro . In the present study, we used this property as a criterion to search for natural and artificial substrates and/or allosteric regulators of ABCR, the rod photoreceptor-specific ABC transporter responsible for Stargardt disease, an early onset macular degeneration . ABCR was immunoaffinity purified to apparent homogeneity from bovine rod outer segments and reconstituted into liposomes . All-trans-retinal, a candidate ligand, stimulates the ATPase activity of ABCR 3-4-fold, with a half-maximal effect at 10-15 microM . 11-cis- and 13-cis-retinal show similar activity . All-trans-retinal stimulates the ATPase activity of ABCR with Michaelis-Menten behavior indicative of simple noncooperative binding that is associated with a rate-limiting enzyme-substrate intermediate in the pathway of ATP hydrolysis . Among 37 structurally diverse non-retinoid compounds, including nine previously characterized substrates or sensitizers of P-glycoprotein, only four show significant ATPase stimulation when tested at 20 microM . The dose-response curves of these four compounds are indicative of multiple binding sites and/or modes of interaction with ABCR . Two of these compounds, amiodarone and digitonin, can act synergistically with all-trans-retinal, implying that they interact with a site or sites on ABCR different from the one with which all-trans-retinal interacts . Unlike retinal, amiodarone appears to interact with both free and ATP-bound ABCR . Together with clinical observations on Stargardt disease and the localization of ABCR to rod outer segment disc membranes, these data suggest that retinoids, and most likely retinal, are the natural substrates for transport by ABCR in rod outer segments . These observations have significant implications for understanding the visual cycle and the pathogenesis of Stargardt disease and for the identification of compounds that could modify the natural history of Stargardt disease or other retinopathies associated with impaired ABCR function. Life Sci, 1999, 64(9), 763 - 74 Evidence for a multidrug resistance-associated protein 1 (MRP1)-related transport system in cultured rat liver biliary epithelial cells; Courtois A et al.; Cellular accumulation and efflux of the anionic fluorescent dye carboxy-2',7'-dichlorofluorescein (CF) were studied in rat liver SDVI cells thought to derive from primitive bile ductules, in order to characterize carrier-related membrane transport of organic anions in epithelial cells . Probenecid, a common blocker of anion transport, was found to strongly enhance CF levels in SDVI cells in a dose-dependent manner through inhibition of dye efflux . Such an outwardly-directed transport was demonstrated to be temperature-dependent and down-regulated by various metabolic inhibitors, therefore outlining its requirement for energy; it was shown to be Na+- and membrane potential-independent and inhibited by anionic drugs such as indomethacin, indoprofen and rifamycin B . These functional features are closed to those described for multidrug resistance-associated protein 1 (MRP1) that was furthermore demonstrated, in contrast to P-glycoprotein, to be expressed in SDVI cells and to lower CF accumulation in MRP1-overexpressing drug-resistant tumor cells . These data therefore suggest that active membrane transport of organic anions such as CF occurs in epithelial cells like cultured liver biliary SDVI cells through a MRP1-related efflux system. J Clin Microbiol, 1999 Apr, 37(4), 1197 - 9 rpoB mutations in multidrug-resistant strains of Mycobacterium tuberculosis isolated in Italy; Pozzi G et al.; Mutations of rpoB associated with rifampin resistance were studied in 37 multidrug-resistant (MDR) clinical strains of Mycobacterium tuberculosis isolated in Italy . At least one mutated codon was found in each MDR strain . It was always a single-base substitution leading to an amino acid change . Nine different rpoB alleles, three of which had not been reported before, were found . The relative frequencies of specific mutations in this sample were different from those previously reported from different geographical areas, since 22 strains (59.5%) carried the mutated codon TTG in position 531 (Ser-->Leu) and 11 (29.7%) had GAC in position 526 (His-->Asp). Semin Oncol, 1999 Feb, 26(1), 6 - 20 Dose-intensive therapy for adult acute lymphoblastic leukemia; Finiewicz KJ et al.; Major challenges remain to be overcome to increase the cure rate for acute lymphoblastic leukemia (ALL), especially for middle-aged and older adults . Despite high rates of complete remission (CR), many patients relapse after chemotherapy alone . Dose-intensive therapy and stem-cell transplantation (SCT) have been able to rescue some of these patients . However, many patients presently are being cured using intensive consolidation chemotherapy during first remission (CRI) and at a lower cost and toxicity than with SCT . The use of SCT in CRI should be governed by an assessment of known risk factors . Among younger adults in the prime transplant age group (< 50 years), there is no advantage to allogeneic (allo)-SCT across the board, but it is recommended for those with Philadelphia chromosome-positive (Ph+) ALL or pro-B ALL with t(4;11) and possibly for those with B-lineage ALL and initial WBC counts > 100,000/microL . There is as yet no evidence that allo-SCT can improve the already high cure rate achieved with chemotherapy alone in favorable subsets such as T-cell ALL . There appears to be no advantage to autologous (auto)-SCT over chemotherapy for consolidation of either high-risk or standard-risk patients in CRI . The argument that early use of auto-SCT shortens the duration of treatment and thereby improves the quality of life is not persuasive, as there is little morbidity from maintenance chemotherapy . Patients who receive a modern, intensive multiagent chemotherapy program for CRI but later relapse are unlikely to be cured with additional chemotherapy alone . High-grade multidrug resistance develops rapidly . These patients should undergo allo-SCT if possible . Unfortunately, allo-SCT is available to only a minority of such patients because of the lack of a donor or insurance coverage, or the presence of comorbid conditions or older age . The use of alternative donors (either matched, unrelated donors or partially human leukocyte antigen {HLA} matched family members) is appropriate in this circumstance . Auto-SCT with or without previously used purging methods is ineffective for patients with advanced ALL. Zhonghua Jie He He Hu Xi Za Zhi, 1997 Feb, 20(1), 30 - 2 {Relationship of mdr1 gene expression and multidrug resistance of primary lung cancer}; Bai L et al.; OBJECTIVE: To evaluate the expression of mdr1 gene in various type of primary lung cancer . METHOD: A serious of 48 resected primary lung cancer tissues from 5 post-chemotherapy and 43 untreated patients and 33 adjacent normal lung tissues were analyzed for mdr1 gene by RT-PCR method . RESULTS: Fifteen of 48 tumor tissues were found to have overexpression of mdr1 gene, comprising 6 of 12 adenocarcinomas, 7 of 22 squamous cell carcinomas, 1 of 2 sarcocarcinomas, 1 of 12 small cell carcinomas . Of 5 post-chemotherapy specimens, only one case showed positive . In addition, 3 of 33 adjacent normal tissues also showed mdr1-positive . Overexpression of mdr1 gene did not be associated with tumor size, metastasis and stage of lung cancer . The relapse rate with mdr1-positive cases was higher (80%) than that with mdr1-negative (58%) by one year follow-up of these patients . CONCLUSION: Overexpression of mdr1 gene can be detected in untreated primary lung cancer, specially in non-small cell lung cancer and may be regarded as one poorly prognostic marker. Exp Parasitol, 1999 Mar, 91(3), 250 - 7 Haemonchus contortus: sequence heterogeneity of internucleotide binding domains from P-glycoproteins; Sangster NC et al.; P-Glycoproteins are transmembrane proteins associated with acquired multidrug resistance in mammalian cells and some protozoan parasites by a process of active drug export . P-glycoproteins contain two nucleotide binding domains which couple ATP to the drug transport process . The region between the nucleotide binding domains of P-glycoproteins, termed the internucleotide binding domain (IBD), was PCR-amplified from adult and larval cDNA libraries using degenerate primers . The 11 clones isolated by this method fall into several distinct groups, with one group of alleles displaying between 82 and 99% identity at the nucleotide level . This sets a baseline for sequence variation of transcribed alleles from a parasitic nematode . Northern blotting showed that P-glycoprotein genes are transcribed in a developmentally regulated fashion in Haemonchus contortus . Southern blots of H . contortus drug-resistant isolates with an IBD probe revealed a pattern consistent with the involvement of P-glycoprotein in resistance to avermectin/milbemycin anthelmintics . Am J Trop Med Hyg, 1999 Feb, 60(2), 238 - 43 A randomized, double-blind, comparative trial of a new oral combination of artemether and benflumetol (CGP 56697) with mefloquine in the treatment of acute Plasmodium falciparum malaria in Thailand; Looareesuwan S et al.; CGP 56697, a new oral fixed combination of artemether and benflumetol, was tested in a double-blinded, randomized trial in 252 adult patients treated either with CGP 56697 (4 x 4 tablets each containing 20 mg of artemether and 120 mg of benflumetol, given at 0, 8, 24, and 48 hr), or with mefloquine (three tablets of 250 mg at initial diagnosis, followed by two tablets of 250 mg at 8 hr) . Baseline data of the two groups were comparable . The 28-day cure rate with CGP 56697 was lower than with mefloquine (69.3% versus 82.4%; P = 0.002) . However, CGP 56697 was more effective than mefloquine in parasite clearance time (43 hr versus 66 hr; P < 0.001) fever clearance time (32 hr versus 54 hr; P < 0.005), and gametocyte clearance time (152 hr versus 331 hr; P < 0.001) . This study revealed that CGP 56697 is effective against multidrug-resistant Plasmodium falciparum malaria in Thailand, but higher doses will probably be needed to improve the cure rate. Cancer Chemother Pharmacol, 1999, 43(4), 302 - 8 Mechanism of cytotoxicity of N-{2-(dimethylamino)ethyl} acridine-4-carboxamide and of its 7-chloro derivative: the roles of topoisomerases I and II; Bridewell DJ et al.; DACA {N-{2-(dimethylamino)ethyl}acridine-4-carboxamide}, an acridine derivative that is highly active against solid tumours in mice, is currently in clinical trial . The ability of DACA to overcome "atypical" (topoisomerase II-mediated) multidrug resistance has been hypothesised to stem from its dual topoisomerase I/II specificity . We investigated the topoisomerase specificity of DACA and its 7-chloro derivative (C1-DACA) using camptothecin and amsacrine as control compounds . In cell-free assays employing supercoiled plasmid DNA, C1-DACA at 5 microM induced topoisomerase I-mediated DNA breakage, indicating cleavable complex formation (poisoning), and at 10 microM it inhibited relaxation of DNA, consistent with suppression (self-inhibition) of poisoning . In this assay, DACA provided no evidence of poisoning of this enzyme but inhibited its function at concentrations above 10 microM . In DNA cleavage assays utilising purified topoisomerase II, DACA induced breakage of supercoiled plasmid DNA at 5 microM whereas C1-DACA showed very weak poisoning at 1 microM and inhibition at 5 microM . Under conditions required for the assay of DNA relaxation, C1-DACA, but not DACA, inhibited topoisomerase II action at 5 microM . The actions of DACA and C1-DACA could also be distinguished by their ability to form DNA-protein cross-links in H460 human lung carcinoma cells as measured by precipitation of DNA-protein complexes with sodium dodecyl sulfate and potassium chloride . Both drugs stimulated the formation of complexes at low concentrations but inhibited formation at high concentrations . In survival assays with H460 cells, both drugs demonstrated biphasic responses with self-inhibition of cytotoxicity at intermediate drug concentrations . It was concluded that although both drugs have dual topoisomerase I/II specificity, DACA preferentially poisons topoisomerase II and C1-DACA preferentially poisons topoisomerase I . In addition, drug-induced inhibition of topoisomerase action at higher drug concentrations may mask poisoning in the cell-free assays as well as masking cytotoxicity in cultured cells . A model in which drug binding occludes topoisomerase-binding sites on the DNA can explain this self-inhibition of cytotoxic action. J Clin Oncol, 1999 Mar, 17(3), 1061 - 70 Mechanisms of action of and resistance to antitubulin agents: microtubule dynamics, drug transport, and cell death; Dumontet C et al.; PURPOSE: To analyze the available data concerning mechanisms of action of and mechanisms of resistance to the antitubulin agents, vinca alkaloids and taxanes, and more recently described compounds . DESIGN: We conducted a review of the literature on classic and recent antitubulin agents, focusing particularly on the relationships between antitubulin agents and their intracellular target, the soluble tubulin/microtubule complex . RESULTS AND CONCLUSION: Although it is widely accepted that antitubulin agents block cell division by inhibition of the mitotic spindle, the mechanism of action of antitubulin agents on microtubules remains to be determined . The classic approach is that vinca alkaloids depolymerize microtubules, thereby increasing the soluble tubulin pool, whereas taxanes stabilize microtubules and increase the microtubular mass . More recent data suggest that both classes of agents have a similar mechanism of action, involving the inhibition of microtubule dynamics . These data suggest that vinca alkaloids and taxanes may act synergistically as antitumor agents and may be administered as combination chemotherapy in the clinic . However, enhanced myeloid and neurologic toxicity, as well as a strong dependence on the sequence of administration, presently exclude these combinations outside the context of clinical trials . Although the multidrug resistance phenotype mediated by Pgp appears to be an important mechanism of resistance to these agents, alterations of microtubule structure resulting in altered microtubule dynamics and/or altered binding of antitubulin agents may constitute a significant mechanism of drug resistance. Cancer Res, 1999 Mar 1, 59(5), 1036 - 40 A novel taxane with improved tolerability and therapeutic activity in a panel of human tumor xenografts; Polizzi D et al.; Clinically available taxanes represent one of the most promising class of antitumor agents, despite several problems with their solubility and toxicity . In an attempt to improve the pharmacological profile of taxanes, a new series of analogues was synthesized from 14beta-hydroxy-10-deacetylbaccatin III and tested in a panel of human tumor cell lines . On the basis of the pattern of cytotoxicity and lack of cross-resistance in tumor cell lines expressing the typical multidrug-resistant phenotype, a compound (IDN5109) was selected for preclinical development . A comparative efficacy study of IDN5109 and paclitaxel was performed using a large panel of human tumor xenografts, characterized by intrinsic (seven tumors) or acquired (four tumors) resistance to cisplatin or doxorubicin, including four ovarian, one breast, one cervical, three lung, one colon, and one prostatic carcinoma . Drugs were delivered i.v . according to the same schedule (four times every 4th day) . IDN5109 achieved a very high level of activity (percentage tumor weight inhibition >70%; log10 cell kill >1) in all but one of the tested tumors . Compared to paclitaxel, IDN5109 exhibited a significantly superior activity in six tumors (including the four tumors that were resistant to paclitaxel) and a comparable activity against the other five paclitaxel-responsive tumors . Additional advantages of IDN5109 over paclitaxel were also suggested by its toxicity profile . IDN5109 was not only less toxic (maximal tolerated doses were 90 and 54 mg/kg for IDN5109 and paclitaxel, respectively), but it also appeared to be endowed with a reduced neurotoxic potential and an improved profile of tolerability compared to the parent drug . Furthermore, the best antitumor efficacy was often already reached with doses lower than the maximal tolerated dose, suggesting an improved therapeutic index for the new drug . In conclusion, the results support the preclinical interest of IDN5109 in terms of the toxicity profile and of the efficacy with particular reference to the ability to overcome multiple mechanisms of drug resistance. Cancer Res, 1999 Mar 1, 59(5), 1021 - 8 Multiple mechanisms confer drug resistance to mitoxantrone in the human 8226 myeloma cell line; Hazlehurst LA et al.; Selection for in vitro drug resistance can result in a complex phenotype with more than one mechanism of resistance emerging concurrently or sequentially . We examined emerging mechanisms of drug resistance during selection with mitoxantrone in the human myeloma cell line 8226 . A novel transport mechanism appeared early in the selection process that was associated with a 10-fold resistance to mitoxantrone in the 8226/MR4 cell line . The reduction in intracellular drug concentration was ATP-dependent and ouabain-insensitive . The 8226/MR4 cell line was 34-fold cross-resistant to the fluorescent aza-anthrapyrazole BBR 3390 . The resistance to BBR 3390 coincided with a 50% reduction in intracellular drug concentration . Confocal microscopy using BBR 3390 revealed a 64% decrease in the nuclear:cytoplasmic ratio in the drug-resistant cell line . The reduction in intracellular drug concentration of both mitoxantrone and BBR 3390 was reversed by a novel chemosensitizing agent, fumitremorgin C . In contrast, fumitremorgin C had no effect on resistance to mitoxantrone or BBR 3390 in the P-glycoprotein-positive 8226/DOX6 cell line . Increasing the degree of resistance to mitoxantrone in the 8226 cell line from 10 to 37 times (8226/MR20) did not further reduce the intracellular drug concentration . However, the 8226/MR20 cell line exhibited 88 and 70% reductions in topoisomerase II beta and alpha expression, respectively, compared with the parental drug sensitive cell line . This decrease in topoisomerase expression and activity was not observed in the low-level drug-resistant, 8226/MR4 cell line . These data demonstrate that low-level mitoxantrone resistance is due to the presence of a novel, energy-dependent drug efflux pump similar to P-glycoprotein and multidrug resistance-associated protein . Reversal of resistance by blocking drug efflux with fumitremorgin C should allow for functional analysis of this novel transporter in cancer cell lines or clinical tumor samples . Increased resistance to mitoxantrone may result from reduced intracellular drug accumulation, altered nuclear/cytoplasmic drug distribution, and alterations in topoisomerase II activity. J Natl Cancer Inst, 1999 Mar 3, 91(5), 429 - 33 Atypical multidrug resistance: breast cancer resistance protein messenger RNA expression in mitoxantrone-selected cell lines; Ross DD et al.; BACKGROUND: Human cancer cell lines grown in the presence of the cytotoxic agent mitoxantrone frequently develop resistance associated with a reduction in intracellular drug accumulation without increased expression of the known drug resistance transporters P-glycoprotein and multidrug resistance protein (also known as multidrug resistance-associated protein) . Breast cancer resistance protein (BCRP) is a recently described adenosine triphosphate-binding cassette transporter associated with resistance to mitoxantrone and anthracyclines . This study was undertaken to test the prevalence of BCRP overexpression in cell lines selected for growth in the presence of mitoxantrone . METHODS: Total cellular RNA or poly A+ RNA and genomic DNA were isolated from parental and drug-selected cell lines . Expression of BCRP messenger RNA (mRNA) and amplification of the BCRP gene were analyzed by northern and Southern blot hybridization, respectively . RESULTS: A variety of drug-resistant human cancer cell lines derived by selection with mitoxantrone markedly overexpressed BCRP mRNA; these cell lines included sublines of human breast carcinoma (MCF-7), colon carcinoma (S1 and HT29), gastric carcinoma (EPG85-257), fibrosarcoma (EPF86-079), and myeloma (8226) origins . Analysis of genomic DNA from BCRP-overexpressing MCF-7/MX cells demonstrated that the BCRP gene was also amplified in these cells . CONCLUSIONS: Overexpression of BCRP mRNA is frequently observed in multidrug-resistant cell lines selected with mitoxantrone, suggesting that BCRP is likely to be a major cellular defense mechanism elicited in response to exposure to this drug . It is likely that BCRP is the putative "mitoxantrone transporter" hypothesized to be present in these cell lines. Br J Cancer, 1999 Feb, 79(5-6), 831 - 7 Induction of MDR1 gene expression by anthracycline analogues in a human drug resistant leukaemia cell line; Hu XF et al.; The effects of 4-demethoxydaunorubicin (idarubicin, IDA) and MX2, a new morpholino-anthracycline, on up-regulation of the MDR1 gene in the low-level multidrug resistant (MDR) cell line CEM/A7R were compared at similar concentrations (IC10, IC50 and IC90) over a short time exposure (4 and 24 h) . The chemosensitivity of each drug was determined by a 3-day cell growth inhibition assay . Compared with epirubicin (EPI), IDA and MX2 were 17- and eightfold more effective in the CEM/A7R line respectively . No cross-resistance to 5-FU was seen in the CEM/A7R line . Verapamil (5 microM) and PSC 833 (1 microM), which dramatically reversed resistance to EPI in the CEM/A7R line, had no sensitizing effect on the resistance of this line to MX2, but slightly decreased resistance to IDA . The sensitivity to 5-FU was unchanged by these modulators . The induction of MDR1 mRNA expression by IDA, MX2 and 5-FU was analysed by Northern blotting and semiquantitatively assessed by scanning Northern blots on a phosphorimager . The relative level of MDR1 expression was expressed as a ratio of MDR1 mRNA to the internal RNA control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) . IDA, MX2 and 5-FU differentially up-regulated MDR1 mRNA in the CEM/A7R line in a dose-dependent manner . Both IDA and MX2 induced MDR1 expression within 4 h . 5-FU up-regulated MDR1 expression only when drug exposure was prolonged to 24 h . Based on MRK 16 binding, flow cytometric analysis of P-glycoprotein (Pgp) expression paralleled the increase in MDR1 mRNA levels . For the three anthracyclines, the increase in MDR1 expression was stable in cells grown in the absence of drug for more than 3 weeks after drug treatment . The induction of MDR1 expression by 5-FU was transient, associated with a rapid decrease in the increased Pgp levels which returned to baseline 72 h after the removal of 5-FU . This study demonstrates that MDR1 expression can be induced by analogues of anthracyclines not pumped by Pgp, and that this induction appears to be stable despite a 3-week drug-free period. Br J Cancer, 1999 Feb, 79(5-6), 807 - 13 GR-891: a novel 5-fluorouracil acyclonucleoside prodrug for differentiation therapy in rhabdomyosarcoma cells; Marchal JA et al.; Differentiation therapy provides an alternative treatment of cancer that overcomes the undesirable effects of classical chemotherapy, i.e . cytotoxicity and resistance to drugs . This new approach to cancer therapy focuses on the development of specific agents designed to selectively engage the process of terminal differentiation, leading to the elimination of tumorigenic cells and recovery of normal cell homeostasis . A series of new anti-cancer pyrimidine acyclonucleoside-like compounds were designed and synthesized by structural modifications of 5-fluorouracil, a drug which causes considerable cell toxicity and morbidity, and we evaluated their applicability for differentiation therapy in human rhabdomyosarcoma cells . We tested the pyrimidine derivative GR-891, (RS)-1-{{3-(2-hydroxyethoxy)-1-isopropoxy}propyl}-5-fluorouracil, an active drug which shows low toxicity in vivo and releases acrolein which is an aldehyde with anti-tumour activity . Both GR-891 and 5-fluorouracil caused time- and dose-dependent growth inhibition in vitro; however, GR-891 showed no cytotoxicity at low doses (22.5 micromol l(-1) and 45 micromol l(-1)) and induced terminal myogenic differentiation in RD cells (a rhabdomyosarcoma cell line) treated for 6 days . Changes in morphological features and in protein organization indicated re-entry in the pathway of muscular maturation . Moreover, GR-891 increased adhesion capability mediated by the expression of fibronectin, and did not induce overexpression of P-glycoprotein, the mdr1 gene product, implicated in multidrug resistance . New acyclonucleoside-like compounds such as GR-891 have important potential advantages over 5-fluorouracil because of their lower toxicity and their ability to induce myogenic differentiation in rhabdomyosarcoma cells . Our results suggest that this drug may be useful for differentiation therapy in this type of tumour. Blood, 1999 Mar 15, 93(6), 1831 - 7 Cytokine-based tumor cell vaccine is equally effective against parental and isogenic multidrug-resistant myeloma cells: the role of cytotoxic T lymphocytes; Shtil AA et al.; Tumor cells that survive initial courses of chemotherapy may do so by acquiring a multidrug-resistant phenotype . This particular mechanism of drug resistance may also confer resistance to physiological effectors of apoptosis that could potentially reduce the efficacy of immune therapies that use these pathways of cell death . We have previously demonstrated high efficacy for a cytokine-based tumor cell vaccine in a murine MPC11 myeloma model . In the present study, the effects of this vaccination were compared in MPC11 cells and their isogenic sublines selected for mdr1/P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) . Immunization with MPC11 cells expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-12 (IL-12) led to long-lasting protection of mice against subcutaneous (sc) challenge with both parental cells or their MDR variants . Similarly, immunization with GM-CSF/IL-12-transfected MDR sublines caused rejection of transplantation of both parental cells and the MDR sublines . Whereas MPC11 cells and their MDR variants were resistant to APO-1/CD95/Fas ligand, the immunization generated potent granzyme B/perforin-secreting cytotoxic T lymphocytes (CTLs) that were similarly effective against both parental and isogenic MDR cells . We conclude that MDR mediated by mdr1/Pgp did not interfere with lysis by pore-forming CTLs . Immunotherapy based on pore-forming CTLs may be an attractive approach to the treatment of drug-resistant myeloma. Curr Opin Microbiol, 1998 Oct, 1(5), 535 - 46 New antivirals - mechanism of action and resistance development; Balzarini J et al.; In recent years, several novel treatment modalities emerged for a number of virus infections, including lamivudine for hepatitis B virus, abacavir, adefovir dipivoxyl and apropovir disprometil for human immunodeficiency virus, cidofovir for cytomegalovirus, and famciclovir (the oral prodrug of penciclovir) and cidofovir for other herpesviruses (i.e . herpes simplex virus and varicella-zoster virus) . For all drugs, resistance eventually develops upon prolonged administration to the infected individuals, albeit at a varying extent . In addition, new mutations related to multidrug resistance have recently been identified. Kekkaku, 1999 Jan, 74(1), 71 - 5 {Clinical evaluation of new quinolones as antituberculosis drugs}; Kawahara S et al.; Compared with the recent rapid advances in the diagnosis of tuberculosis, advances in the treatment of tuberculosis have been quite slow . For example, as long as six months are required for initial treatment, even with addition of pyrazinamide in the first two months to isoniazid, rifampicin, and streptomycin or ethambutol . Moreover, it is not always easy to treat patients who cannot receive standard agents including isoniazid and rifampicin due to adverse effects of these drugs or drug resistance . For these reasons, the development of new agents with potent antituberculosis activities and fewer adverse effects is urgently desired . However, at present, few new antituberculosis agents are being developed, and new quinolones are considered the most promising new antituberculosis agents due to their lack of cross-resistance to previously existing antituberculosis agents, their excellent in vitro and in vivo antituberculous activities, and good pharmacokinetics . We therefore reviewed experimental studies and clinical reports useful for evaluation of potential clinical use of new quinolones as antituberculosis drugs . Our conclusions are summarized below: 1) Of nine new quinolones on the market, ofloxacin, ciprofloxacin, aparfloxacin and levofloxacin have excellent in vitro and in vivo antituberculous activities without cross-resistance to previously existing antituberculosis agents . 2) Ofloxacin appears to be useful clinically for intractable multidrug-resistant tuberculosis . The incidence and severity of adverse effects of ofloxacin were very low on longterm administration . 3) Ofloxacin resistance emerged from two to four months after initiation of administration of ofloxacin, and ofloxacin exhibited cross-resistance to other new quinolones . 4) The usefulness of new quinolones for initial treatment of tuberculosis is unclear . 5) New quinolones should be used to treat patients with drug-resistant tuberculosis and those patients for whom adverse effects have limited the use of standard agents . However, monotherapy with new quinolones is not recommended, due to the significant risk of emergence of quinolone resistance . 6) Not only ofloxacin, ciprofloxacin, sparfloxacin and levofloxacin, which are on the market, but also gatifloxacin, CS-940, Du-6859a and other newly developed new quinolones are candidates for new antituberculosis agents . However, careful study not only of antituberculous activities and pharmacokinetic but also drug interactions and chronic cumulative toxicities due to long-term administration are needed prior to clinical application of these drugs. Kekkaku, 1999 Jan, 74(1), 47 - 52 {View of development of fluoroquinolones}; Namba K; The global resurgence of tuberculosis and the emergence of multidrug-resistant tuberculosis have emphasized an urgent need for new, effective antimycobacterial drugs . A new antimycobacterial drugs should have a different mechanism of action to the standard drugs and should not show cross-resistance with them . Favorable pharmacokinetic properties, low incidence of side effects and low cost are the characteristics that would make antimycobacterial drugs suitable for extensive use . The fluoroquinolones would appear to fulfill most of the criteria for an ideal class of antimycobacterial drugs . The fluoroquinolones have been proposed for the treatment of Mycobacterium tuberculosis (M . tuberculosis) infections, with the results of in vitro, animal model and clinical studies suggesting that ofloxacin, ciprofloxacin, sparfloxacin and levofloxacin are the most promising of these drugs . However, these fluoroquinolones should not be used as first-line drugs, but rather, they should be reserved for treatment of tuberculosis that is resistant to rifampicin and isoniazid . Recently, a new investigational fluoroquinolone derivative, AM-1155, DU-6859a and CS-940, have excellent in vitro activity against M . tuberculosis, further studies are required to assess its clinical activity . We discussed the future of view of development of fluoroquinolones for mycobacterial diseases on the basis of structure-activity and structure-side-effect relationship studies . The comparative analysis enabled us to elucidate the structural requirements for the antimycobacterial activity and side-effects of fluoroquinolones . In addition, we summarized the newer methods for high-throughput screening of compounds against M . tuberculosis and discussed the problem of development of fluoroquinolones for mycobacterial diseases. Curr Pharm Des, 1999 Mar, 5(3), 217 - 27 Ring-B modified anthracyclines; Suarato A et al.; One of the most investigated classes of antitumor drugs is represented by anthracyclines . Over thirty years since the original discovery of daunorubicin and doxorubicin thousands of anthracycline analogues have been synthesized and tested to identify compounds superior to the parent drugs in terms of increased therapeutic effectiveness, reduced toxicity or both . Previous structure-activity studies had shown that minor modifications of the anthracycline structure can result not only in active agents, but, more importantly, analogues with reduced cardiotoxicity and activity on multi drug resistance . The fact that 4-demethoxydaunorubicin showed higher potency than daunorubicin and a reduced cardiotoxicity, prompted us to explore novel analogues with altered substitution patterns on the anthraquinone system, particularly ring-B . In this review we will describe total synthesis and antitumor activity of three classes of derivatives: whereby one hydroxyl group in ring-B was either removed or replaced with nitro or amino groups . While these modifications yielded anthracyclines with a promising pharmacological activity, they did not modify activity on multidrug resistant (MDR) tumors . On the other hand, introduction of morpholino group in the sugar part of these new molecules, dramatically increased activity on MDR tumors . We conclude that activity on MDR tumors is not bound to modifications in the aglycone moiety of anthracyclines. J Biol Chem, 1999 Mar 12, 274(11), 6979 - 91 Multidrug resistance (MDR1) P-glycoprotein enhances esterification of plasma membrane cholesterol; Luker GD et al.; Class I P-glycoproteins (Pgp) confer multidrug resistance in tumors, but the physiologic function of Pgp in normal tissues remains uncertain . In cells derived from tissues that normally express Pgp, recent data suggest a possible role for Pgp in cholesterol trafficking from the plasma membrane to the endoplasmic reticulum . We investigated the esterification of plasma membrane cholesterol under basal conditions and in response to sphingomyelinase treatment in transfected and drug-selected cell lines expressing differing amounts of functional class I Pgp . Compared with parental NIH 3T3 fibroblasts, cells transfected with human multidrug resistance (MDR1) Pgp esterified more cholesterol both without and with sphingomyelinase . Esterification also was greater in drug-selected Dox 6 myeloma cells than parental 8226 cells, which express low and non-immunodetectable amounts of Pgp, respectively . However, no differences in total plasma membrane cholesterol were detected . Transfection of fibroblasts with the multidrug resistance-associated protein (MRP) did not alter esterification, showing that cholesterol trafficking was not generally affected by ATP-binding cassette transporters . Steroidal (progesterone, dehydroepiandrosterone) and non-steroidal antagonists (verapamil, PSC 833, LY335979, and GF120918) were evaluated for effects on both cholesterol trafficking and the net content of 99mTc-Sestamibi, a reporter of drug transport activity mediated by Pgp . In Pgp-expressing cells treated with nonselective and selective inhibitors, both the kinetics and efficacy of inhibition of cholesterol esterification differed from the antagonism of drug transport mediated by Pgp . Thus, although the data show that greater expression of class I Pgp within a given cell type is associated with enhanced esterification of plasma membrane cholesterol in support of a physiologic function for Pgp in facilitating cholesterol trafficking, the molecular mechanism is dissociated from the conventional drug transport activity of Pgp. J Lipid Res, 1999 Mar, 40(3), 439 - 46 Characterization of a leukotriene C4 export mechanism in human platelets: possible involvement of multidrug resistance-associated protein 1; Sjolinder M et al.; Platelets express leukotriene (LT) C4 synthase and can thus participate in the formation of bioactive LTC4 . To further elucidate the relevance of this capability, we have now determined the capacity of human platelets to export LTC4 . Endogenously formed LTC4 was efficiently released from human platelets after incubation with LTA4 at 37 degrees C, whereas only 15% of produced LTC4 was exported when the cells were incubated at 0 degrees C . The activation energy of the process was calculated to 49.9 +/- 7.7 kJ/mol, indicating carrier-mediated LTC4 export . This was also supported by the finding that the transport was saturable, reaching a maximal export rate of 470 +/- 147 pmol LTC4/min x 10(9) platelets . Furthermore, markedly suppressed LTC4 transport was induced by a combination of the metabolic inhibitors antimycin A and 2-deoxyglucose, suggesting energy-dependent export.The presence in platelets of multidrug resistance-associated protein 1 (MRP1), a protein described to be an energy-dependent LTC4 transporter in various cell types, was demonstrated at the mRNA and protein level . Additional support for a role of MRP1 in platelet LTC4 export was obtained by the findings that the process was inhibited by probenecid and the 5-lipoxygenase-activating protein (FLAP) inhibitor, MK-886 . The present findings further support the physiological relevance of platelet LTC4 production. Am J Hum Genet, 1999 Mar, 64(3), 739 - 46 Genomic structure of the canalicular multispecific organic anion-transporter gene (MRP2/cMOAT) and mutations in the ATP-binding-cassette region in Dubin-Johnson syndrome; Toh S et al.; Dubin-Johnson syndrome (DJS) is an autosomal recessive disease characterized by conjugated hyperbilirubinemia . Previous studies of the defects in the human canalicular multispecific organic anion transporter gene (MRP2/cMOAT) in patients with DJS have suggested that the gene defects are responsible for DJS . In this study, we determined the exon/intron structure of the human MRP2/cMOAT gene and further characterized mutations in patients with DJS . The human MRP2/cMOAT gene contains 32 exons, and it has a structure that is highly conserved with that of another ATP-binding-cassette gene, that for a multidrug resistance-associated protein . We then identified three mutations, including two novel ones . All mutations identified to date are in the cytoplasmic domain, which includes the two ATP-binding cassettes and the linker region, or adjacent putative transmembrane domain . Our results confirm that MRP2/cMOAT is the gene responsible for DJS . The finding that mutations are concentrated in the first ATP-binding-cassette domain strongly suggests that a disruption of this region is a critical route to loss of function. Biosci Biotechnol Biochem, 1999 Jan, 63(1), 162 - 7 Functional analysis of the promoter of the yeast SNQ2 gene encoding a multidrug resistance transporter that confers the resistance to 4-nitroquinoline N-oxide; Cui Z et al.; The yeast gene SNQ2, which encodes a multidrug resistance ABC superfamily protein, is required for resistance to the mutagen 4-nitroquinoline N-oxide (4-NQO) . The expression of the SNQ2 gene is under the control of a regulatory network that involves the transcription factor Yrr1p, as well as Pdr1p/Pdr3p (Cui et al., Mol . Microbiol., 29, 1307-1315 (1998)) . By 5'-deletion analysis of the promoter by using SNQ2-lacZ fusion constructs, four regions: -745 to -639 (region I), -639 to -578 (region II), -548 to -533 (region III) and -533 to -485 (region IV) were found to be important for SNQ2 expression . Genetic analysis suggested that the site in region IV was responsible for the Yrr1p-mediated SNQ2 expression . A consensus motif known for the binding of Pdr1p/Pdr3p (PDRE) was not found in region IV. Mol Pharmacol, 1999 Mar, 55(3), 535 - 40 Suppression of replication of multidrug-resistant HIV type 1 variants by combinations of thymidylate synthase inhibitors with zidovudine or stavudine; Gao WY et al.; The replication of recombinant multidrug-resistant HIV-1 clones modeled on clinically derived resistant HIV-1 strains from patients receiving long-term combination therapy with zidovudine (AZT) plus 2',3'-dideoxycytidine was found to regain sensitivity to AZT and stavudine (D4T) as a consequence of a pharmacologically induced decrease in de novo dTMP synthesis . The host-cell system used was phytohemagglutinin-stimulated peripheral blood mononuclear cells; dTMP and dTTP depletion were induced by single exposures to a low level of the thymidylate synthase inhibitor 5-fluorouracil (5-FU) or its deoxynucleoside, 2'-deoxy-5-fluorouridine . The host-cell response to the latter was biphasic: a very rapid decrease in the rate of de novo dTMP formation and, consequently, in intracellular dTTP pools, followed by slower recovery in both indices over 3 to 24 h . With the additional presence of AZT or D4T, however, replication of the multidrug-resistant HIV-1 strains remained inhibited, indicating dependence of HIV DNA chain termination by AZT-5'-monophosphate or 2',3'-didehydro-2', 3'-dideoxythymidine-5'-monophosphate in these resistant strains on simultaneous inhibition of host-cell de novo synthesis of thymidine nucleotides . No effect on viability of control (uninfected) phytohemagglutinin-stimulated/peripheral blood mononuclear cells was noted on 6-day exposures to 5-FU or 2'-deoxy-5-fluorouridine alone or in combination with AZT or D4T, even at drug levels severalfold higher than those used in the viral inhibition studies . These studies may provide useful information for the potential clinical use of AZT/5-FU or D4T/5-FU combinations for the prevention or reversal of multidrug resistance associated with long-term dideoxynucleoside combination therapy. Hepatology, 1999 Mar, 29(3), 814 - 21 Changes in the localization of the rat canalicular conjugate export pump Mrp2 in phalloidin-induced cholestasis; Rost D et al.; Administration of phalloidin, one of the toxic peptides of the mushroom Amanita phalloides, leads to rapid and sustained cholestasis in rats . Although attributed to the interaction of phalloidin with microfilaments, the events leading to cholestasis are incompletely understood . The adenosine triphosphate (ATP)-dependent, apical conjugate export pump, termed multidrug resistance protein 2 (Mrp2) or canalicular multispecific organic anion transporter, is the major driving force for bile salt-independent bile flow . We investigated the role of Mrp2 in phalloidin-induced cholestasis . Bile flow decreased to 53% and 31% of control at 15 and 30 minutes after phalloidin (0.5 mg/kg), respectively . Mrp2-mediated {3H}leukotriene excretion into bile during the initial 45 minutes was reduced to 44% of control when {3H}LTC4 was injected 15 minutes after phalloidin treatment . Mrp2 was progressively lost from the hepatocyte canalicular membrane and detected predominantly on intracellular membrane structures together with other canalicular proteins including P-glycoproteins, ecto-ATPase, and dipeptidyl-peptidase IV . By contrast, structures involved in intercellular adhesion (zonula occludens, zonula adhaerens, and desmosomes) as well as intermediate filaments of the cytokeratin type appeared largely unaffected within 30 minutes after phalloidin . In line with the immunofluorescence analysis, immunoblots indicated a loss of Mrp2 and P-glycoproteins from the canalicular membrane and a 3- and 4.6-fold increase of these transport proteins in the microsomal fraction, respectively . Our results indicate that phalloidin induces marked alterations of the hepatocyte canalicular architecture and a loss of Mrp2 together with other proteins from the canalicular membrane . The resulting cholestasis can therefore be explained in part by the loss of export pumps, including Mrp2, from the canalicular membrane. Br J Haematol, 1999 Feb, 104(2), 328 - 35 P-glycoprotein, lung resistance-related protein and multidrug resistance associated protein in de novo acute non-lymphocytic leukaemias: biological and clinical implications; Michieli M et al.; P-glycoprotein (PGP), lung resistance-related protein (LRP) and multidrug resistance associated protein (MRP) expression and the blast cells' intracellular daunorubicin accumulation (IDA) were evaluated in 96 previously untreated cases of de novo acute non-lymphocytic leukaemia (ANLL) . 47/96 patients (49%) were classified as PGP+ 44/ 96 (46%) as LRP+, and 8/96 (8%) as MRP+ . The more frequent MDR clusters were PGP-/LRP-/MRP- (32/96 cases, 33%) and the PGP+/LRP+/MRP- (27/96 cases, 28%) followed by PGP+/LRP-/MRP- (15/96 cases, 16%) and PGP-/LRP+/MRP- (14/96 cases, 14%) . A favourable karyotype was observed more frequently in PGP- and LRP-cases . A highly significant correlation was found between either PGP or LRP overexpression and leukaemic blast cell IDA . All the patients received standard induction and consolidation treatments containing MDR-related (idarubicin, mitoxantrone, etoposide) and other (arabinosyl cytosine) drugs . Multivariate analysis showed that PGP overexpression was significantly associated with a poor response to treatment, both in terms of primary resistance or shorter survival . Other independent prognostic factors were age and cytogenetics . LRP overexpression did not reach statistical significance, although for LRP+ cases the trend was unfavourable . Due to small numbers, no conclusion could be made regarding MRP overexpression, but 5/8 cases showed unfavourable karyotypic abnormalities, 8/8 had a defective IDA and 6/8 failed to achieve remission . This study showed that both PGP and LRP overexpression are common features in de novo ANLL at onset whereas MRP overexpression is more rare . It suggested that overexpression of one of the MDR related proteins was associated with a defective IDA, and confirmed that, in addition to age and cytogenetics, PGP retains an independent prognostic value . It also suggested that LRP did not affect clinical outcome when patients were treated with idarubicin or mitoxantrone and arabinosyl cytosine. Br J Haematol, 1999 Feb, 104(2), 321 - 7 Different expression of glutathione S-transferase alpha, mu and pi in childhood acute lymphoblastic and myeloid leukaemia; Den Boer ML et al.; Expression of three major classes of glutathione S-transferases (GSTs), i.e . alpha, mu and pi class, P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) were studied in childhood acute lymphoblastic leukaemia (ALL), acute myeloid leukaemia (AML) and normal peripheral blood lymphocytes by flow cytometry . In vitro cytotoxicity of 4-hydroxy-ifosfamide (IFOS), daunorubicin (DNR) and prednisolone (PRED) was assessed by the MTT assay . Expression of alpha, mu and pi class GST did not significantly differ between leukaemic cells from 100 initial and 14 unrelated relapse ALL patients (GSTalpha P=026; GSTmu P=O009; GSTpi P=0.13) . The expression of GSTalpha (1.4-fold, P=0.0004), GSTpi (13-fold, P = 0001) and to a lesser extent also GSTmu (1.1-fold, P=0.03) was higher in ALL compared with normal peripheral blood lymphocytes . Expression of GSTmu and GST7pi was significantly higher in 18 AML compared with 100 ALL patients at initial diagnosis (respectively 1.3-fold, P=0.0005 and 2-fold, P<0.0001) . In contrast, GSTalpha was median 2-fold lower expressed in the AML samples (P< 0.0001) . Expression levels of alpha, mu and pi class GSTs were not related to the degree of resistance to IFOS, DNR and PRED nor to immunophenotype, white blood cell count or age at presentation of childhood ALL . One exception was a remarkably low expression of GSTalpha in IFOS-sensitive samples compared with a heterogenous expression in IFOS-resistant samples (P= 0.02) . Expression of GSTpi, but not of GSTalpha or GSTmu, weakly correlated with the expression of MRP (Rs 0.36, P = 0.002, n = 74) but not with P-gp . However, a high expression of both GSTpi and MRP was not associated with in vitro resistance to IFOS, DNR or PRED . The present data suggest that expression of GSTs is not linked to the degree of resistance to IFOS, DNR and PRED or clinical risk factors in childhood ALL . Whether the high expression of GSTmu and GSTpi in AML cells contributes to the relative resistance to IFOS, DNR and PRED compared with ALL samples (P < or = 0.0001) warrants further study. Br J Haematol, 1999 Feb, 104(2), 307 - 12 Use of standardized flow cytometric determinants of multidrug resistance to analyse response to remission induction chemotherapy in patients with acute myeloblastic leukaemia; Pallis M et al.; We have used a combination of flow cytometric assays to define multidrug resistance (MDR) positive and negative blasts in cryopreserved samples from 47 MRC trial patients with acute myeloblastic leukaemia (AML) . Our primary test is a standardized assay for daunorubicin accumulation . Confirmatory assays for MDR comprised the cyclosporin modulation assay for rhodamine-123 uptake as a measure of functional P-glycoprotein and the measurement of lung resistance protein and multidrug resistance associated protein (with LRP-56 and MRPr1 respectively) . 57% of samples had both low accumulation and at least one positive confirmatory test . 32% were MDR negative in all four assays . 15% of patients had primary chemo-resistant disease . Resistant disease rates were 22% for confirmed MDR-positive patients and 0% for confirmed MDR-negative patients (P=0.07) . Complete remission was achieved in 74% of patients, with rates of 63% in confirmed MDR-positive patients and 93% in confirmed MDR-negative patients (P=0.06) . The use of a standardized method for daunorubicin uptake, combined with the use of confirmatory tests, should reduce the uncertainty that is currently characteristic of MDR evaluation in leukaemia . In comparison with daunorubicin uptake, p-gp expression, measured using MRK-16 antibody, was more closely associated with remission rates (P =0.01) . This suggests an additional role for p-glycoprotein in mediating drug resistance beyond that of a drug efflux pump. Biochem Biophys Res Commun, 1999 Feb 24, 255(3), 618 - 24 Direct interaction between a quinoline derivative, MS-209, and multidrug resistance protein (MRP) in human gastric cancer cells; Nakamura T et al.; MS-209 is a novel quinoline derivative reversing P-glycoprotein-mediated multidrug resistance (MDR) . We investigated the interaction between MS-209 and multidrug resistance protein (MRP) in MRP-overexpressing human gastric cancer cells . We measured {3H}leukotriene C4 uptake into the membrane vesicles of the cells and intracellular calcein and {3H}vincristine accumulation with or without MS-209 . In multi-drug-resistant MKN45R0.8 cells selected by doxorubicin, MS-209 dose dependently reduced MRP-mediated {3H}leukotriene C4 uptake and increased calcein accumulation . In both resistant and unselected cell lines expressing the MRP gene, MS-209 increased {3H}vincristine accumulation in proportion with the level of MRP mRNA expression and enhanced the cytotoxicity of etoposide, doxorubicin, and vincristine . The reversal effects correlated with the level of MRP mRNA expression in these cells . Our results indicate that MS-209 effectively reverses intrinsic and acquired MRP-mediated MDR of gastric cancer cells by interacting directly with MRP . Biochem Biophys Res Commun, 1999 Feb 16, 255(2), 289 - 94 Structural and functional analysis of the LaMDR1 multidrug resistance gene in Leishmania amazonensis; Katakura K et al.; We determined primary sequences of the LaMDR1 gene in Leishmania amazonensis, a protozoan parasite that causes cutaneous leishmaniasis . The longest open reading frame encodes 1341 amino acids for a protein consisting of two similar halves, each containing six putative transmembrane domains and one ATP-binding domain . The protein has no potential N-glycosylation sites at the extracellular region . The LaMDR1 protein was 91 and 78% identical to the closely related ldmdr1 in L . donovani and lemdr1 in L . enriettii, respectively, revealing less conservation in the C-terminal than in the N-terminal transmembrane domains . Transfection of LaMDR1 conferred a multidrug resistance phenotype to wild-type promastigotes, which exhibited a significant level of resistance to vinblastine, doxorubicin, and actinomycin D, but not to puromycin and colchicine . This drug specificity of LaMDR1 was overlapping with but distinct from that of ldmdr1, suggesting functional diversity of MDR1 proteins among different Leishmania species . Int J Cancer, 1999 Mar 1, 80(5), 773 - 80 Monocyte chemoattractant protein-1 gene modification of multidrug-resistant human lung cancer enhances antimetastatic effect of therapy with anti-P-glycoprotein antibody in SCID mice; Nokihara H et al.; Distant metastases and multidrug resistance are critical problems in the therapy of human small cell lung cancer (SCLC) . In this study, we investigated whether transduction of the monocyte chemoattractant protein-1 (MCP-1) gene into multidrug-resistant (MDR) human lung cancer cells affected the formation of metastases or their inhibition by the anti-P-glycoprotein (P-gp) monoclonal antibody (MAb) MRK16 . MDR human SCLC (H69/VP) cells were transduced with the human MCP-1 gene inserted into the expression vector BCMGSNeo . MCP-1 gene transduction had no effect on drug sensitivity, the expression of surface antigens or the in vitro proliferation of H69/VP cells . Using the metastatic model of NK cell-depleted SCID mice, H69/VP cells transduced with the MCP-1 gene were inoculated intravenously (i.v.) and formed metastatic colonies in the liver, kidneys and lymph nodes, similar to those formed by parent or mock-transduced cells . However, systemic treatment of the mice with MRK16 reduced the metastases of H69/VP cells in the liver, kidneys and lymph nodes, and was significantly more effective in inhibiting the metastases of MCP-1 producing H69/VP than those of mock-transduced cells . MCP-1 gene transduction significantly prolonged the survival of tumor-bearing mice treated with MRK16 . Our findings suggest that local production of MCP-1 in the tumor site increases the anti-P-gp antibody-dependent cell-mediated cytotoxicity, and the MCP-1 gene-induced modification of MDR human SCLC cells thereby enhances the antimetastatic effect of therapy with anti-P-gp antibody . Thus, the accumulation of effector cells in the tumor site is a very important factor in the therapy using the anti-P-gp antibody. J Cell Physiol, 1999 Feb, 178(2), 247 - 57 Lysosomotropic agents increase vinblastine efflux from mouse MDR proximal kidney cells exhibiting vectorial drug transport; Lacave R et al.; Vinblastine (VBL) transport and efflux were studied in mouse proximal tubule PKSV-PR cells and in their multidrug-resistant derivatives PKSV-PRcol50 cells . The PKSV-PRcol50 cells produced more mdr1b transcripts and had higher resistance to various drugs . PKSV-PRcol50 cells had a predominantly basal-to-apical flux of {3H}VBL, 2.7 times larger than that in PKSV-PR cells . This flux was partially inhibited by verapamil (VRP) (10 microM) and cyclosporin A (CsA) (200 nM) . {3H}VBL efflux was also greater in PKSV-PRcol50 than in PKSV-PR cells . Treatment with NH4Cl (30 mM), a lysosomotropic weak base, and concanamycin A (CCM A) (20 nM), an inhibitor of the vacuolar H+/ATPase, further increased {3H}VBL efflux from PKSV-PRcol50 cells . The cytoplasmic pH (pHcyt) of these drug-resistant cells transiently increased in the presence of NH4Cl deltapHcyt: +0.4) . CCM A caused a moderate, delayed increase in pHcyt (deltapHcyt: +0.1) and made the acidic intralysosomal compartment more alkaline (deltapHlys: +1.3) . VRP and CsA prevented the NH4Cl- and CCM A-induced {3H}VBL efflux from PKSV-PRcol50 cells . However, VRP (10 microM) did not significantly affect pHcyt of PKSV-PRcol50 cells, the NH4Cl-and CCM A-induced pHcyt responses, and the effect of CCMA on pHlys . Thus, lysosomotropic agents may affect the kinetics of {3H}VBL efflux . Our results also suggest that the inhibitory action of VRP on VBL efflux was not directly mediated by a pH-dependent process in these drug-resistant renal proximal tubule cells. Biochem Pharmacol, 1999 Mar 15, 57(6), 589 - 95 Chemotherapy by slowing glucosphingolipid synthesis; Radin NS; The hypothesis offered here is that many different illnesses could be treated by slowing the synthesis of glucosphingolipids (GSLs) with a suitable inhibitor . In people with inadequate hydrolases for the GSLs (e.g . Gaucher's disease), the lipids accumulate to a pathological degree . It should be possible to eliminate the accumulation by slowing the synthesis of the GSLs to match the ability of the patient to degrade them . In people with cancer, the tumors secrete excessive amounts of GSLs, which block the ability of the immune system to attack the tumor cells . By blocking the synthesis of tumor GSLs, it should be possible to enable the patient to generate antibodies and activated T cells that can destroy the tumor . Tumors exhibiting multidrug resistance may do so by synthesizing GSLs even faster than usual . It should be possible to restore the sensitivity of the tumor to anti-cancer drugs by inhibiting their synthesis of GSLs . Metastasis of tumors also appears to require the formation of GSLs, so an inhibitor should help block tumor dissemination . Diabetics tend to have high levels of blood glucose, which acts to stimulate kidney growth via more rapid synthesis of GSLs . This pathological growth can be blocked by inhibiting the formation of kidney GSLs . Viruses, bacteria, and bacterial toxins have been found to bind to specific GSLs of human and animal cells . Presumably, this binding leads to the damaging process of infection . It should be possible to treat such infections by depleting the host's body of its GSLs. J Natl Cancer Inst, 1999 Feb 3, 91(3), 236 - 44 The ras oncogene-mediated sensitization of human cells to topoisomerase II inhibitor-induced apoptosis; Koo HM et al.; BACKGROUND: Among the inhibitors of the enzyme topoisomerase II (an important target for chemotherapeutic drugs) tested in the National Cancer Institute's In Vitro Antineoplastic Drug Screen, NSC 284682 (3'-hydroxydaunorubicin) and NSC 659687 {9-hydroxy-5,6-dimethyl-1-(N-{2(dimethylamino)ethyl}carbamoyl)-6H-pyrido -(4,3-b)carbazole} were the only compounds that were more cytotoxic to tumor cells harboring an activated ras oncogene than to tumor cells bearing wild-type ras alleles . Expression of the multidrug resistance proteins P-glycoprotein and MRP (multidrug resistance-associated protein) facilitates tumor cell resistance to topoisomerase II inhibitors . We investigated whether tumor cells with activated ras oncogenes showed enhanced sensitivity to other topoisomerase II inhibitors in the absence of the multidrug-resistant phenotype . METHODS: We studied 20 topoisomerase II inhibitors and individual cell lines with or without activated ras oncogenes and with varying degrees of multidrug resistance . RESULTS: In the absence of multidrug resistance, human tumor cell lines with activated ras oncogenes were uniformly more sensitive to most topoisomerase II inhibitors than were cell lines containing wild-type ras alleles . The compounds NSC 284682 and NSC 659687 were especially effective irrespective of the multidrug resistant phenotype . The ras oncogene-mediated sensitization to topoisomerase II inhibitors was far more prominent with the non-DNA-intercalating epipodophyllotoxins than with the DNA-intercalating inhibitors . This difference in sensitization appears to be related to a difference in apoptotic sensitivity, since the level of DNA damage generated by etoposide (an epipodophyllotoxin derivative) in immortalized human kidney epithelial cells expressing an activated ras oncogene was similar to that in the parental cells, but apoptosis was enhanced only in the former cells . CONCLUSIONS: Activated ras oncogenes appear to enhance the sensitivity of human tumor cells to topoisomerase II inhibitors by potentiating an apoptotic response . Epipodophyllotoxin-derived topoisomerase II inhibitors should be more effective than the DNA-intercalating inhibitors against tumor cells with activated ras oncogenes. Biochim Biophys Acta, 1999 Feb 24, 1453(2), 304 - 10 Expression of atrC - encoding a novel member of the ATP binding cassette transporter family in Aspergillus nidulans - is sensitive to cycloheximide; Angermayr K et al.; A new member of the ABC superfamily of transmembrane proteins in Aspergillus nidulans has been cloned and characterized . The topology of conserved motifs subgroups AtrC in the P-glycoprotein cluster of ABC permeases, the members of this subfamily, are known to participate in multidrug resistance (MDR) in diverse organisms . Alignment results display significant amino acid similarity to AfuMDR1 and AflMDR1 from Aspergillus fumigatus and flavus, respectively . Northern analysis reveals that atrC mRNA levels are 10-fold increased in response to cycloheximide . Evidence for the existence of eight additional hitherto unpublished ABC transporter proteins in A . nidulans is provided. Mol Carcinog, 1999 Jan, 24(1), 25 - 8 Regulation of metastasis-related gene expression by p53: a potential clinical implication; Sun Y et al.; Tumor metastasis is the main cause of mortality and treatment failure in cancer patients . It is a complex biological process regulated by alternations in expression of many genes . The p53 tumor suppressor gene has been shown to regulate expression of some metastasis-related genes . p53 transcriptionally activates expression of the genes encoding epidermal growth factor receptor, matrix metalloproteinase (MMP)-2, cathepsin D, and thrombospondin-1 but represses expression of the genes encoding basic fibroblast growth factor and multidrug resistance-1 . Decreased expression of E-cadherin is associated with p53 alternations . Because these p53-regulatory genes either promote or inhibit tumor metastasis, the net effect of p53 expression on tumor metastasis depends upon the pattern of expression of these genes in a particular tumor . Because radiotherapy has been shown to increase tumor metastasis in both animal and human studies and because p53 is activated by radiation or DNA-damaging reagents, here we propose the working hypothesis that p53 may promote tumor metastasis upon induction by local radiotherapy or chemotherapy in some tumor types . For patients whose tumors contain wild-type p53, MMP inhibitors might be given with or before radiotherapy or chemotherapy to prevent an increase in tumor metastasis . Special caution should be taken with patients with cancers such as nasopharyngeal carcinoma in which p53 mutation is infrequent and radiotherapy is the main choice of treatment . To test our hypothesis, three studies are proposed and could serve as an initial step in understanding the complex biological process following radiation-induced p53 activation and its roles in regulation of tumor metastasis. Cancer Res, 1999 Feb 15, 59(4), 880 - 5 SDZ PSC 833, the cyclosporine A analogue and multidrug resistance modulator, activates ceramide synthesis and increases vinblastine sensitivity in drug-sensitive and drug-resistant cancer cells; Cabot MC et al.; Resistance to chemotherapy is the major cause of cancer treatment failure . Insight into the mechanism of action of agents that modulate multidrug resistance (MDR) is instrumental for the design of more effective treatment modalities . Here we show, using KB-V-1 MDR human epidermoid carcinoma cells and {3H}palmitic acid as metabolic tracer, that the MDR modulator SDZ PSC 833 (PSC 833) activates ceramide synthesis . In a short time course experiment, ceramide was generated as early as 15 min (40% increase) after the addition of PSC 833 (5.0 microM), and by 3 h, {3H}ceramide was >3-fold that of control cells . A 24-h dose-response experiment showed that at 1.0 and 10 microM PSC 833, ceramide levels were 2.5- and 13.6-fold higher, respectively, than in untreated cells . Concomitant with the increase in cellular ceramide was a progressive decrease in cell survival, suggesting that ceramide elicited a cytotoxic response . Analysis of DNA in cells treated with PSC 833 showed oligonucleosomal DNA fragmentation, characteristic of apoptosis . The inclusion of fumonisin B1, a ceramide synthase inhibitor, blocked PSC 833-induced ceramide generation . Assessment of ceramide mass by TLC lipid charring confirmed that PSC 833 markedly enhanced ceramide synthesis, not only in KB-V-1 cells but also in wild-type KB-3-1 cells . The capacity of PSC 833 to reverse drug resistance was demonstrated with vinblas