Multidrug resistant

Bull Cancer, 1994 May, 81(5), 409 - 13
{Value of rhodamine 123 in the detection of minor amounts of multidrug resistant cells}; Maynadie M et al.; The neoplasias resistance to chemotherapy is mainly due to multidrug resistance phenomenon (MDR) mediated by an ATP-dependent efflux pump called P-glycoprotein . This function can be reversed by many multidrug reversing agents so numerous chemotherapy regimens have been initiated in malignancies . To make sure the success of these protocols it is necessary to detect as soon and as certain as possible the presence of resistant cells among malignant population . We have chosen to evaluate functional test using rhodamine 123 in this aim in view . We have made various mixtures of resistant ans sensitive cells of three cell lines . Rhodamine 123 allows to detect 1% of resistant cells among sensitive cells . Influence of dead cells on the interpretation is discussed.

Bull Cancer, 1994 May, 81(5), 400 - 8
{Structure and function of P-glycoprotein}; Robert J; P-glycoprotein is a membrane ATPase transporter responsible for multidrug resistance . Its primary structure is known from cDNA sequencing, but its tri-dimension structure remains hypothetical . Its physiological role in detoxification, as well as its involvement in anticancer drug transport, are no longer questioned, but its molecular mechanism of action remains unknown . It appears likely that it extrudes the drugs laterally, in the plane of the membrane, that several distinct drug or modulator binding sites exist, that its activity is regulated by phosphorylation . Purification and reconstitution of P-glycoprotein will probably allow to better understand its mechanism of action.

Bull Cancer, 1994 May, 81(5), 392 - 9
{The multidrug resistance phenotype: some morphological and biological characteristics except efflux pump}; Jardillier JC et al.; Recent data from the literature together with personal results strongly suggest that multidrug resistance phenotype is overwhelming the sole expression of P170 glycoprotein efflux pump . Morphological alterations have been put in evidence in MDR cells after transmission and scanning electron microscopy . They include presence of osmiophilic vesicles and modifications of nuclear and nucleolar chromatin . Biological characteristics include the hypersecretory pattern of lysosomal enzymes from MDR cells . Such a fact could be more or less related to the increased occurrence of mdr1 RNA in metastasis, especially in breast cancers, compared to primary tumors . If the P170-mediated efflux is one of the key mechanism of MDR, a decreased influx of anticancer drugs cannot be excluded . Liposomes, for instance made of cardiolipin, are thus able to increase the intracellular drug uptake of vinblastine without any action upon efflux mechanism.

Bull Cancer, 1994 May, 81(5), 386 - 91
{Pharmacological control of P-glycoprotein expression}; Muller C et al.; Calcium channel inhibitors, such as verapamil, have been identified as having the ability to modulate the multidrug-resistant (MDR) phenotype due to overexpression of P-glycoprotein (Pgp) . We have studied the effect of verapamil on Pgp expression levels in a cell line originating from acute myeloblastic leukemia and resistant to adriamycin, K562/ADR . In this line, the addition of 15 microM verapamil in the culture medium gives a 3-fold decrease of Pgp expression after 72 hours of treatment . Similar results have been obtained for two other MDR cell lines, which suggest that this phenomenon is not specific of a single model . The level of mdr1 mRNAs is decreased in the presence of verapamil (with a maximum effect obtained at the 24th hour), which suggests that the mechanism of action of verapamil is transcriptional and/or post-transcriptional . We have also studied the effect of verapamil on the level of expression of mdr1 mRNAs in non-drug selected cells such as the HEL line (human acute myeloblastic leukemia) and the parental K562 line, which present a very low level of expression of Pgp, detectable only by PCR . In these lines, verapamil treatment has no effect on the level of expression of mdr1 mRNAs . The effect of verapamil is therefore restricted to drug-selected lines presenting high levels of Pgp expression . The impact of the negative regulation of Pgp expression on the MDR phenotype has been studied in the K562/ADR line . When the cells are treated for 72 h by verapamil, there is a decrease of resistance and an increase of intracellular accumulation of anticancer agents such as daunorubicin or vinblastine . Negative regulation of Pgp expression appears therefore as a possible strategy for MDR phenotype reversal . The effect of verapamil, whose molecular mechanism of action is being studied, could constitute a basis for this strategy.

Bull Cancer, 1994 May, 81(5), 381 - 5
{Cellular resistance to DNA-topoisomerase II inhibitors}; Jacquemin-Sablon A et al.; Chinese hamster lung cells resistant to 9-OH-ellipticine (DC-3F/9-OH-E) present a complex phenotype . These cells, which are about 150-fold resistant to 9-OH-E, display a cross-resistance to other topo-II inhibitors, such as m-AMSA or VP-16, which stabilize the cleavable complex . In addition, these cells display also a cross-resistance to suramin, which is also a topo-II inhibitor, but does not stabilize the cleavable complex . Finally, DC-3F/9-OH-E present a multidrug-resistant phenotype (MDR) which confers a cross-resistance to natural products such as actinomycin D, taxol or vincristine, due to a decrease of cellular accumulation of these drugs . Analysis of expression of the genes encoding topo-II alpha and beta, and the evaluation of both enzyme forms by immunoblotting, revealed that DC-3F cells contained about 20-fold less of the beta form than of the alpha form . The alpha form was decreased by about 4-5-fold in DC-3F/9-OH-E, whereas the beta form became undetectable . Purification and characterization of topo-II activities in sensitive and resistant cells is presently in progress . Analysis of the expression of pgp1, 2, 3 genes, involved in the MDR phenotype in hamster, by Northern blotting or by immunoblotting, has shown that the MDR phenotype in DC-3F/9-OH-E cells is due to the overexpression of pgp1 gene . In these cells, pgp3 expression is positively regulated by myc oncogene expression . Overexpression of the myc gene is followed by an overexpression of the pgp3 gene and is associated to a reversal of the MDR phenotype.

Biochem Pharmacol, 1994 Apr 29, 47(9), 1601 - 6
Analysis of MRP mRNA in mitoxantrone-selected, multidrug-resistant human tumor cells; Futscher BW et al.; MRP, a gene recently isolated from a non-P-glycoprotein-mediated multidrug-resistant small cell lung cancer cell line, is a candidate multidrug-resistance gene . Mitoxantrone, an anthracenedione antitumor agent, frequently selects for non-P-glycoprotein-mediated multidrug resistance in in vitro models . To determine whether mitoxantrone-selected multidrug resistance was due to overexpression of MRP, we examined the expression of MRP in four mitoxantrone-selected, multidrug-resistant human tumor cell lines, using a reverse transcriptase/polymerase chain reaction assay . Results from these experiments suggest that overexpression of MRP does not appear to play a primary role in mitoxantrone-selected multidrug resistance in these cell lines, and that other novel drug-resistance mechanisms are likely.

J Biol Chem, 1994 Apr 29, 269(17), 12797 - 803
Solubilization and characterization of the overexpressed PDR5 multidrug resistance nucleotide triphosphatase of yeast; Decottignies A et al.; A 160-kDa plasma membrane protein of the yeast Saccharomyces cerevisiae was overexpressed by mutating the PDR1 or the PDR3 transcription factor gene . The protein is the membrane-bound ATP binding cassette transporter PDR5 (Balzi, E., Wang, M., Leterme, S., Van Dyck, L., and Goffeau, A . (1994) J . Biol . Chem . 269, 2206-2214) . PDR5 was solubilized with n-dodecyl-beta-D-malto-side and separated from the PMA1 plasma membrane H(+)-ATPase by glycerol gradient centrifugation . The PDR5 protein hydrolyzes nucleoside diphosphates and triphosphates . This activity is sensitive to low concentrations of vanadate, of oligomycin, and of a variety of hydrophobic compounds . Many of these properties liken PDR5 to the purified mammalian P-glycoprotein responsible for multidrug resistance.

N Engl J Med, 1994 Apr 28, 330(17), 1179 - 84
The effect of directly observed therapy on the rates of drug resistance and relapse in tuberculosis; Weis SE et al.; BACKGROUND . Tuberculosis has reemerged as an important public health problem, and the frequency of drug resistance is increasing . A major reason for the development of resistant infections and relapse is poor compliance with medical regimens . In Tarrant County, Texas, we initiated a program of universal directly observed treatment for tuberculosis . We report the effect of the program on the rates of primary and acquired drug resistance and relapse among patients with tuberculosis . METHODS . We collected information on all patients with positive cultures for Mycobacterium tuberculosis in Tarrant County from January 1, 1980, through December 31, 1992 . Through October 1986, patients received a traditional, unsupervised drug regimen . Beginning in November 1986, nearly all patients received therapy under direct observation by health care personnel . RESULTS . A total of 407 episodes in which patients received traditional treatment for tuberculosis (January 1980 through October 1986) were compared with 581 episodes in which therapy was directly observed (November 1986 through December 1992) . Despite higher rates of intravenous drug use and homelessness and an increasing rate of tuberculosis during this 13-year period, the frequency of primary drug resistance decreased from 13.0 percent to 6.7 percent (P < 0.001) after the institution of direct observation of therapy, and the frequency of acquired resistance declined from 14.0 percent to 2.1 percent (P < 0.001) . The relapse rate decreased from 20.9 percent to 5.5 percent (P < 0.001), and the number of relapses with multidrug-resistant organisms decreased from 25 to 5 (P < 0.001) . CONCLUSIONS . The administration of therapy for M . tuberculosis infection under direct observation leads to significant reductions in the frequency of primary drug resistance, acquired drug resistance, and relapse.

Proc Natl Acad Sci U S A, 1994 Apr 26, 91(9), 3497 - 504
Cell biological mechanisms of multidrug resistance in tumors; Simon SM et al.; Multidrug resistance (MDR) is a generic term for the variety of strategies tumor cells use to evade the cytotoxic effects of anticancer drugs . MDR is characterized by a decreased sensitivity of tumor cells not only to the drug employed for chemotherapy but also to a broad spectrum of drugs with neither obvious structural homology nor common targets . This pleiotropic resistance is one of the major obstacles to the successful treatment of tumors . MDR may result from structural or functional changes at the plasma membrane or within the cytoplasm, cellular compartments, or nucleus . Molecular mechanisms of MDR are discussed in terms of modifications in detoxification and DNA repair pathways, changes in cellular sites of drug sequestration, decreases in drug-target affinity, synthesis of specific drug inhibitors within cells, altered or inappropriate targeting of proteins, and accelerated removal or secretion of drugs.

Presse Med, 1994 Apr 23, 23(16), 731 - 3
{Association of tuberculosis and HIV infection}; Perronne C; Eight million people contract tuberculosis every year, 95% of them in developing countries, and one-third of the world's population is infected with Mycobacterium tuberculosis . Annually, tuberculosis causes three million deaths (in Africa 26% of the avoidable deaths) . The main cause of dissemination is the absence of early diagnosis and insufficient treatment . Today, 3% of the new cases of tuberculosis are related to infection with the human immunodeficiency virus (HIV), a proportion which is rising rapidly . HIV infection does not change the classic rules of treatment; rifampicin, isoniazid, ethambutol and pyrazinamide for 2 months followed by at least 4 more months with a two-drug regimen (rifampicin and isoniazid) . No-compliance is the major cause of recurrence, together with the risk of infection with another strain of M . tuberculosis . Certain authors suggest that in Africa, due to poor compliance and the lack of a sufficient provision of major antituberculous agents, treatment should be continued for life in HIV positive patients . Others propose chemotherapy for an HIV infected patients who are healthy carriers of M . tuberculosis . The risk of selecting mutant strains could be avoided by limiting prophylaxis to non-febrile patients . Nevertheless, the long-term effect of generalized chemoprophylaxis on the epidemiology of resistant strains is unknown . The only method of screening for healthy carriers is the tuberculin skin test but interpretation is complicated by prior BCG vaccination and now by HIV infection . There are two crucial steps required to control tuberculosis in this era of the tuberculosis-HIV partnership . First, patients should have easy and cost-free access to antituberculous drugs and second, compliance must be improved . Certain barriers have been lifted, including the requirement of patient identification to obtain free drugs . Hospital staffs must renew their efforts and attempt to follow-up their patients to assure compliance after discharge . All these measures will be difficult to implement but are the price we must pay to eradicate a new rise in the incidence of tuberculosis and the risk of multidrug-resistant strains . The only alternative may well be a return to pre-antibiotic days.

J Biol Chem, 1994 Apr 22, 269(16), 11751 - 9
Benzo{a}pyrene-resistant MCF-7 human breast cancer cells . A unique aryl hydrocarbon-nonresponsive clone; Moore M et al.; Wild-type MCF-7 human breast cancer cells were cultured for 3 months in 1 microM benzo{a}pyrene (BaP), and resistant clones were screened for inducibility of CYP1A1 gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) . One of the BaP-resistant (BaPR) clones exhibited unique genotypic expression which distinguished it from both wild-type and drug-resistant (AdrR) variant MCF-7 cells . Glutathione levels, glutathione S-transferase activities, estrogen receptor levels, estrogen responsiveness, and expression of the multidrug-resistant MDR1 and MRP mRNA levels were similar in the wild-type and BaPR cells, whereas these parameters were reported to be altered in AdrR cells . In contrast, TCDD induced CYP1A1 gene expression and inhibited selected estrogen-induced responses in wild-type but not BaPR MCF-7 cells . Treatment of wild-type and BaPR cells with {3H}TCDD resulted in formation of the radiolabeled aryl hydrocarbon (Ah) 6 S nuclear receptor complex in both cell lines . The loss of Ah responsiveness in the BaPR variant cells correlated with the failure of the nuclear or transformed cytosolic Ah receptor complex to bind genomic dioxin-responsive elements as determined in gel retardation assays.

J Biol Chem, 1994 Apr 22, 269(16), 12332 - 8
Human leukemia K562 cell mutant (K562/OA200) selected for resistance to okadaic acid (protein phosphatase inhibitor) lacks protein kinase C-epsilon, exhibits multidrug resistance phenotype, and expresses drug pump P-glycoprotein; Zheng B et al.; A human leukemia K562 cell mutant (K562/OA200) selected for resistance to okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A (PP1/PP2A), has been established . In wild type cells, the cytotoxicity of OA was associated with mitotic arrest and concentration- and time-dependent DNA fragmentation, a hallmark of apoptosis . The mutant was 100-fold more resistant to OA in terms of effects on these parameters . Although the synthesis of several proteins was altered, enzyme assay and immunoblot analysis indicated that the levels of PP1 and PP2A were unchanged in the mutant . Protein kinase C (PKC) assays and immunoblot analysis of calcium-dependent (cPKC) and calcium-independent (nPKC) isoforms revealed that nPKC-epsilon was strikingly absent in the mutant, which otherwise expressed in comparable amounts all other isotypes (cPKC-alpha, cPKC-beta, and nPKC-zeta) also present in the wild type . Northern blot analysis confirmed an absence of PKC-epsilon mRNA in the mutant cells . The OA200 cells were cross-resistant not only to another PP1/PP2A inhibitor, calyculin A, but also to structurally unrelated anticancer drugs (such as vinblastine and taxol) and furthermore, overexpressed the verapamil-sensitive drug pump P-glycoprotein at both the protein and mRNA levels . The mutant, however, was not cross-resistant to several PKC inhibitors tested including cardiotoxin, mastoparan, staurosporine, and an alkylphospholipid . Cardiotoxin, at a subtoxic concentration, enhanced by 6-fold vinblastine cytotoxicity in OA200 cells . These findings indicate that the multidrug resistance phenotype can be induced by cytotoxic agents other than conventional anticancer drugs, show that the development of multidrug resistance is not necessarily associated with increased cPKC activity, and identify certain PKC inhibitors that have potential as resistance modulators.

Science, 1994 Apr 15, 264(5157), 375 - 82
Inactivation of antibiotics and the dissemination of resistance genes; Davies J; The emergence of multidrug-resistant bacteria is a phenomenon of concern to the clinician and the pharmaceutical industry, as it is the major cause of failure in the treatment of infectious diseases . The most common mechanism of resistance in pathogenic bacteria to antibiotics of the aminoglycoside, beta-lactam (penicillins and cephalosporins), and chloramphenicol types involves the enzymic inactivation of the antibiotic by hydrolysis or by formation of inactive derivatives . Such resistance determinants most probably were acquired by pathogenic bacteria from a pool of resistance genes in other microbial genera, including antibiotic-producing organisms . The resistance gene sequences were subsequently integrated by site-specific recombination into several classes of naturally occurring gene expression cassettes (typically "integrons") and disseminated within the microbial population by a variety of gene transfer mechanisms . Although bacterial conjugation once was believed to be restricted in host range, it now appears that this mechanism of transfer permits genetic exchange between many different bacterial genera in nature.

Int J Cancer, 1994 Apr 15, 57(2), 229 - 39
Reversal of multidrug resistance by tyrosine-kinase inhibitors in a non-P-glycoprotein-mediated multidrug-resistant cell line; Takeda Y et al.; We have already established a human leukemia sub-line resistant to the growth-inhibitory effect of TPA (12-O-tetradecanoylphorbol 13-acetate) (K562/TPA) derived from K562 . K562/TPA was found to be a non-P-glycoprotein-mediated multidrug-resistant cell line, in which intracellular drug accumulation was not reduced . In K562/TPA, adriamycin (ADM) was distributed mainly in the cytoplasm and was scarcely observed in the nucleus . We determined the relative levels of multidrug-resistance-associated protein (MRP), which was recently identified as the novel transporter . The relative levels of MRP in K562/TPA were the same as in K562 . Although the catalytic activity of K562/TPA topoisomerase II was about half that of the parental cells, resistance to other drugs could not be explained by topoisomerase-II activity . To elucidate the mechanism of drug resistance in K562/TPA, we tried to find chemicals that would reverse the drug resistance . Tyrosine-kinase inhibitors enhanced the cytotoxicity of anti-neoplastic drugs against K562/TPA . Therefore we examined the modification of nuclear ADM accumulation in K562/TPA by one of these tyrosine-kinase inhibitors, genistein . Although the amount of ADM was decreased in the nuclei of K562/TPA cells, it was significantly increased after incubation in the presence of genistein . The formation of DNA single-strand breaks by ADM, etoposide, and ACNU was significantly lower in K562/TPA than in K562, but was significantly increased in the presence of genistein . These results suggest that genistein could overcome drug resistance by enhancing the accumulation of drug into the nuclear fraction of K562/TPA.

Proc Natl Acad Sci U S A, 1994 Apr 12, 91(8), 3176 - 80
Drug-selected coexpression of human glucocerebrosidase and P-glycoprotein using a bicistronic vector; Aran JM et al.; Bicistronic cassettes under control of a single promoter have recently been suggested as useful tools for coordinate expression of two different foreign proteins in mammalian cells . Using the long 5' untranslated region of encephalomyocarditis virus as translational enhancer of the second gene, a bicistronic unit composed of cDNA for human P-glycoprotein {the product of the multidrug resistance gene, MDR1 (also called PGY1)} as selectable marker and cDNA for human glucocerebrosidase (GC; EC 3.2.1.45) (a membrane-associated lysosomal hydrolase) was constructed . NIH 3T3 cells transfected with a Harvey murine sarcoma virus retroviral vector carrying this bicistronic cassette (pHaMCG) express active P-glycoprotein and GC and expression of both proteins augments coordinately with selection for increased colchicine resistance . Percoll gradient analysis of homogenates showed that GC was targeted to the lysosomal fraction . The ability to select for expression of GC with natural product drugs after introduction of the pHaMCG retroviral vector may be useful in gene therapy strategies for Gaucher disease.

Biochemistry, 1994 Apr 12, 33(14), 4163 - 74
Detection of oligomeric and monomeric forms of P-glycoprotein in multidrug resistant cells; Poruchynsky MS et al.; P-glycoprotein (P-gp) is thought to function as a drug efflux pump in multidrug resistant (MDR) cells . The functional form of P-gp in its native state is not known . Previous results from radiation target size analysis have suggested that P-gp occurs as dimers in MDR cell plasma membranes {Boscoboinik et al . (1990) Biochim . Biophys . Acta 1027, 225-228} . In this study, we used sucrose gradient velocity sedimentation to determine if P-gp oligomers could be retrieved from detergent extracts of hamster and human MDR cell lines . The proportion of P-gp recovered as higher order oligomers was dependent on the detergents used for solubilization of the cells . When a detergent such as CHAPS was used, 50% or more of the P-gp sedimented as higher order oligomers . In contrast, in the presence of SDS, only monomers were retrieved, but naturally occurring oligomers could be preserved if the cells were treated with a cross-linker prior to detergent solubilization . The oligomers and monomers were both able to bind the photoactive analog of ATP (8-azido{alpha-32P}ATP) or the drug {3H}azidopine in membrane preparations . P-gp is a phosphoprotein, and its phosphorylated state is thought to be important for function . When MDR cells were labeled with {32P}orthophosphate in vivo, we observed that the monomer and dimer were more highly phosphorylated than the larger oligomers, suggesting that these different forms of P-gp may be functionally distinct . The assembly of oligomers appears to occur in an early bisynthetic compartment, and asparagine-linked glycosylation is not required for their formation . Our findings indicate that oligomers of P-gp exist in MDR cells and raise the possibility that the dynamics of oligomer formation and dissociation may be important in the mechanism of action of P-gp.

J Biol Chem, 1994 Apr 8, 269(14), 10739 - 46
Reversible transcriptional activation of mdr1 by sodium butyrate treatment of human colon cancer cells; Morrow CS et al.; We investigated the mechanism of sodium butyrate (NaB)-mediated induction of mdr1 mRNA in parental (wild type) and multidrug-resistant (Ad1000) SW620 colon cancer cell lines . NaB treatment resulted in reversible, time-dependent increases in nuclear run-on transcription of endogenous mdr1 in these cell lines that paralleled the reversible increases of mdr1 mRNA in both timing and magnitude . In contrast, NaB treatment had no effect on mdr1 mRNA stability . Thus, the effects of NaB on mdr1 mRNA levels are fully attributable to altered mdr1 transcription . Furthermore, NaB induces the expression of transiently transfected chloramphenicol acetyltransferase reporter plasmids that are under the transcriptional control of the mdr1 promoter (mdrCAT vectors) . Transfections using mdrCAT vectors modified by deletion and site-directed mutagenesis of the mdr1 promoter indicate that NaB-mediated induction of these vectors is at least partially dependent upon sequences present in the basal mdr1 promoter between -89 and +11 relative to the start site of transcription . The Y-box motif located between -82 and -73 contributes to NaB inducibility of mdrCAT vector expression in Ad1000 SW620 cells.

Anticancer Drug Des, 1994 Apr, 9(2), 129 - 37
Synthesis and biological activity of 3'-deamino-3'-haloanthracyclines; Demetzos C et al.; 4'-O-Acetyl-3'-deamino-3'-chloro and 4'-O-acetyl-3'-deamino-3'-bromoepidaunorubicin analogs have been synthesized by condensation of daunomycinone with the corresponding hexopyranosyl chlorides . The glycosides obtained were less potent than adriamycin (ADR) in inhibiting the proliferation of tumor cells in vitro, but they have shown promising activity against a variety of multidrug resistant (MDR) cell lines.

Am J Trop Med Hyg, 1994 Apr, 50(4), 522 - 6
New, antimalarial, tricyclic 1,2,4-trioxanes: evaluations in mice and monkeys; Posner GH et al.; We have concluded initial preclinical studies with synthetic trioxanes numbered 3-9 and have compared them with artemisinin (numbered 1) using CD-1 mice infected with Plasmodium berghei . Based on their antimalarial effectiveness in mice, two of these synthetic trioxanes were selected for evaluation in Aotus monkeys infected with multidrug-resistant (MDR) P . falciparum . Trioxane numbered 8 (12 and 48 mg/kg), trioxane numbered 9 (12 and 48 mg/kg) and arteether (numbered 2, 48 mg/kg) were administered intramuscularly in three 12-hr doses to A . lemurinus lemurinus (Panamanian owl monkeys) infected with the Vietnam Smith/RE strain of P . falciparum and monitored for parasitemia . Trioxane numbered 8 at 12 mg/kg cleared parasitemia in two monkeys, but recrudescence occurred in one animal . Treatment of the recrudescent infection with 48 mg/kg was curative . Infections in two monkeys treated initially with 48 mg/kg were cured (six-month follow-up) . Trioxane numbered 9 produced a similar outcome: 12 mg/kg suppressed parasitemia in two monkeys but was not curative; however, 48 mg/kg cured infections in all four monkeys treated . These preliminary observations show synthetic trioxanes numbered 8 and 9 to be as effective as arteether (numbered 2) against MDR in P . falciparum in the Aotus monkey.

J Clin Oncol, 1994 Apr, 12(4), 835 - 42
Phase I trial of doxorubicin with cyclosporine as a modulator of multidrug resistance; Bartlett NL et al.; PURPOSE: To study the effects of cyclosporine (CsA), a modulator of multidrug resistance (MDR), on the pharmacokinetics and toxicities of doxorubicin . PATIENTS AND METHODS: Nineteen patients with incurable malignancies entered this phase I trial . Initially patients received doxorubicin alone (60 or 75 mg/m2) as a 48-hour continuous intravenous (i.v.) infusion . Patients whose tumors did not respond received CsA as a 2-hour loading dose of 6 mg/kg and a 48-hour continuous infusion of 18 mg/kg/d with doxorubicin . Target CsA levels were 3,000 to 4,800 ng/mL (2.5 to 4.0 mumol/L) . Doxorubicin doses were reduced to 40% of the prior dose without CsA, and then escalated until myelosuppression equivalent to that resulting from doxorubicin alone was observed . Doxorubicin pharmacokinetics were analyzed with and without CsA . RESULTS: Thirteen patients received both doxorubicin alone and the combination of doxorubicin and CsA . Mean CsA levels were more than 2,000 ng/mL for all cycles and more than 3,000 ng/mL for 68% of cycles . Dose escalation of doxorubicin with CsA was stopped at 60% of the doxorubicin alone dose, as four of five patients at this dose level had WBC nadirs equivalent to those seen with doxorubicin alone . Nonhematologic toxicities were mild . Reversible hyperbilirubinemia occurred in 68% of doxorubicin/CsA courses . The addition of CsA to doxorubicin increased grade 1 and 2 nausea (87% v 47%) and vomiting (50% v 10%) compared with doxorubicin alone . There was no significant nephrotoxicity . Paired pharmacokinetics were studied in 12 patients . The addition of CsA increased the dose-adjusted area under the curve (AUC) of doxorubicin by 55%, and of its metabolite doxorubicinol by 350% . CONCLUSION: CsA inhibits the clearance of both doxorubicin and doxorubicinol . Equivalent myelosuppression was observed when the dose of doxorubicin with CsA was 60% of the dose of doxorubicin without CsA . Understanding these pharmacokinetic interactions is essential for the design and interpretation of clinical trials of MDR modulation, and should be studied with more potent MDR modulators.

Blood, 1994 Apr 1, 83(7), 1731 - 7
Interleukin-4 induces programmed cell death (apoptosis) in cases of high-risk acute lymphoblastic leukemia; Manabe A et al.; We investigated the effects of interleukin-4 (IL-4) on the survival of leukemic and normal B-cell progenitors cultured on bone marrow stroma . IL-4 (at 100 U/mL) was cytotoxic in 16 of 21 cases of B-lineage acute lymphoblastic leukemia, causing reductions in CD19+ cell numbers that ranged from 50% to greater than 99% (median 83.5%) of those in parallel cultures not exposed to the cytokine . All nine cases with the t(9;22)(q34;q11) or the t(4;11)(q21;q23), chromosomal features that are often associated with multidrug resistance and a fatal outcome, were susceptible to IL-4 toxicity . IL-4 cytotoxicity resulted from induction of programmed cell death (apoptosis); there was no evidence of cell killing mediated by T, natural killer, or stromal cells . IL-4 cytotoxicity extended to a proportion of normal B-cell progenitors . After 7 days of culture with IL-4 at 100 U/mL, fewer CD19+, CD34+ normal lymphoblasts (the most immature subset) survived: in five experiments the mean (+/- SEM) reduction in cell recoveries caused by IL-4 was 60.0% +/- 6.0% . By contrast, reductions in recovery of more differentiated bone marrow B cells (CD19+, CD34-, surface Ig+) were low (6.6% +/- 2.2%; P < .001 by t-test) . Our findings indicate that IL-4 is cytotoxic for human B-cell precursors and support clinical testing of IL-4 in cases of high-risk lymphoblastic leukemia resistant to conventional therapy.

Cancer Res, 1994 Apr 1, 54(7), 1649 - 52
Expression complementary DNA library transfer establishes mrp as a multidrug resistance gene; Kruh GD et al.; The emergence of drug-resistant cancer cells is a major obstacle to cancer treatment . Resistant cells often display a multidrug-resistant phenotype that reduces the promise of combination chemotherapy, the classic approach to the prevention of drug resistance . mdr1, a member of the ABC cassette superfamily of transporters which encodes an energy-dependent drug efflux pump, is the only gene known to confer the multidrug-resistant phenotype . Other multidrug resistance mechanisms must exist, since cell lines which have this phenotype in the absence of mdr1 overexpression have been described . We report here the application of a novel approach involving expression complementary DNA library transfer to the identification of drug-resistant genes . Using this approach we establish that mrp, a member of the ABC cassette superfamily of transporters, is capable of conferring a multidrug-resistant phenotype . This approach should be useful in the identification of other novel resistance genes.

J Infect Dis, 1994 Apr, 169(4), 722 - 9
Combination therapy with zidovudine and didanosine selects for drug-resistant human immunodeficiency virus type 1 strains with unique patterns of pol gene mutations; Shafer RW et al.; Drug resistance conferred by specific human immunodeficiency virus type 1 (HIV-1) pol gene mutations has been associated with clinical progression in HIV-infected patients receiving anti-retroviral therapy . This study examined drug susceptibilities and pol mutations of HIV-1 strains from patients treated for 1 year with zidovudine, didanosine (ddI), or zidovudine and ddI . Ten (42%) of 24 patients receiving combination therapy versus 8/26 (31%) receiving only zidovudine had HIV-1 strains with phenotypic zidovudine resistance or a zidovudine resistance pol mutation at codon 215 (P = .6) . In contrast, a ddI resistance mutation at codon 74 was less common among patients receiving combination therapy (2/24) than among those receiving ddI only (17/26; P < .001) . Two patients receiving combination therapy developed resistance to zidovudine and ddI; they had HIV strains with amino acid mutations at codons 62, 75, 77, 116, and 151 . Combination therapy with zidovudine and ddI selects for zidovudine-resistant HIV-1 strains lacking a ddI resistance mutation and for multidrug-resistant strains containing novel pol mutations.

Ann Trop Med Parasitol, 1994 Apr, 88(2), 137 - 44
In vitro susceptibility of Cambodian isolates of Plasmodium falciparum to halofantrine, pyronaridine and artemisinin derivatives; Basco LK et al.; Multidrug-resistant Plasmodium falciparum is widespread in Cambodia . The in vitro susceptibilities of 14 Cambodian isolates to chloroquine, quinine, mefloquine, halofantrine, pyrimethamine, cycloguanil, pyronaridine, artemisinin, arteether, artemether and artelinate were studied using a semi-microtest on day 0 and after 15-30 days of culture . The culture-adapted isolates were all resistant to chloroquine, pyrimethamine and cycloguanil . The susceptibility to quinine was generally low . Three isolates were resistant to mefloquine . A comparison of susceptibility to cycloguanil, quinine, and mefloquine prior to and after culture adaptation showed a trend toward a higher resistance level in some isolates . Halofantrine, pyronaridine and artemisinin derivatives were highly active against the multidrug-resistant Cambodian isolates, with very similar 50% inhibitory concentrations (IC50) . These results confirm the presence of multidrug-resistant P . falciparum isolates in Cambodia and indicate that quinine- and mefloquine-resistant populations of the parasite may already exist in the field . The high in vitro activities of halofantrine, pyronaridine and artemisinin derivatives indicate their potential usefulness for the treatment of multidrug-resistant malaria.

Lymphokine Cytokine Res, 1994 Apr, 13(2), 125 - 31
Enhanced expression of HLA-A,B,C and inducibility of TAP-1, TAP-2, and HLA-A,B,C by interferon-gamma in a multidrug-resistant small cell lung cancer line; Fisk B et al.; Recent evidence suggests that deficient HLA Class I expression in SCLC lines may be due, in part, to down-regulation of TAP-1 and TAP-2 expression, and, thus, deficient antigen processing . Given the capability of the multidrug transporter mediating MDR, P-gp, to transport peptides, we hypothesized that P-gp may substitute for TAP-1/TAP-2 and enhance antigen processing in SCLC . To investigate this, we studied the H69 line (parent SCLC) and VPR-2 (MDR subline selected in etoposide, P-gp +) . HLA-A,B,C expression was significantly increased in VPR-2 cells relative to H69, and was much more inducible with IFN-gamma . TAP-1 and TAP-2 were expressed at low levels in both lines . Differential induction of TAP-1 expression with IFN-gamma exposure was observed, with a dramatic increase in VPR-2 cells, and no change in H69 . TAP-2 expression was enhanced in both lines with IFN-gamma, but to a greater degree in VPR-2 . VPR-2 cells were resistant to LAK killing relative to H69, and were minimally sensitized with IFN-gamma . In contrast, IFN-gamma enhanced susceptibility of H69 to LAK killing 3-fold . The direct correlation between enhancement of HLA-A,B,C expression by IFN-gamma and the differential inducibility of TAP-1 and TAP-2 expression in P-gp-SCLC lines is novel . Relative LAK sensitivity of H69 and its increase by IFN-gamma may have clinical implications.

Am J Infect Control, 1994 Apr, 22(2), 65 - 74
Evaluation of single-use masks and respirators for protection of health care workers against mycobacterial aerosols; Chen SK et al.; BACKGROUND: The recent increase in multidrug-resistant tuberculosis has spawned a major controversy concerning the degree of respiratory protection needed by health care workers, particularly during sputum-inducing procedures . The objective of this study was to measure the filtration efficiencies of a single-use submicron surgical mask, two disposable dust/mist respirators, a dust/mist/fume respirator, and a high-efficiency particulate air respirator against aerosolized mycobacteria . Facial fit was not addressed . METHODS: In a specially designed enclosed test apparatus, an aerosol was generated with a Collison nebulizer from a known concentration of Mycobacteria chelonae, used as a surrogate for Mycobacterium tuberculosis . Aerosol concentrations were measured with Anderson samplers upstream and downstream of the test masks and respirators, which were heat sealed to a metal plate . RESULTS: Mean efficiencies ranged from approximately 97% for the surgical mask and a dust/mist respirator to more than 99.99% for the high-efficiency particulate air respirator . Measurements of filter efficiency with an Aerodynamic Particle Sizer for the M . chelonae aerosol and independent challenge tests with latex spheres correlated closely with measurements of M . Chelonae collection efficiency determined with Andersen samplers . CONCLUSIONS: Analysis of variance and Tukey's method for multiple comparisons indicated that the dust/mist/fume respirator and the HEPA respirator collected M . chelonae with significantly greater efficiency than did either the surgical mask or the dust/mist respirator . Even the least efficient mask tested, however, had a filter efficiency of more than 97% against particles averaging less than 1 micron in aerodynamic diameter.

Br J Haematol, 1994 Apr, 86(4), 792 - 7
Gene amplification in non-Hodgkin's lymphoma; Ben-Yehuda D et al.; Among 426 consecutively ascertained and karyotypically abnormal non-Hodgkin's lymphoma (NHL) tumours, cytological evidence for gene amplification in the form of homogeneously staining regions (HSRs) was encountered in nine cases of large cell diffuse lymphoma (LC-DL) . The mean age of patients with HSRs was 62.9 years and four died within a year of diagnosis . To identify candidate gene(s) amplified in these tumours, we performed a Southern blot analysis of tumour DNA using probes for 23 known protooncogenes and the multidrug resistance gene, PGY1 . Besides a two-fold amplification of the BCL2 gene in two cases, no evidence for overt amplification of any of the genes assayed was found . To confirm DNA amplification in these specimens we performed the DNA in-gel renaturation assay . Evidence for presence of amplified DNA fragments was obtained in four of seven specimens . These results suggest amplification of a novel gene(s) . To our knowledge, this is the first formal study of gene amplification in a large consecutively ascertained series of fresh lymphoma biopsies.

Neuropathol Appl Neurobiol, 1994 Apr, 20(2), 118 - 21
Detection of multidrug resistance gene product (P-glycoprotein) expression in ependymomas; Geddes JF et al.; A neurosurgical series of 33 ependymal tumours was examined for expression of the membrane transport molecule P-glycoprotein, which is linked with the development of multidrug resistance in many human tumours . We employed the monoclonal antibodies JSB1 and C219, raised to two different epitopes of the P-glycoprotein molecule, and found P-glycoprotein expression both in normal ependyma and in 29 of the tumours . This is the first time that ependymal tumours have been demonstrated to express the protein, and we conclude that its expression may contribute to the reported failure of adjuvant chemotherapy to improve outcome in ependymomas.

Proc Natl Sci Counc Repub China B, 1994 Apr, 18(2), 64 - 70
The induction of multidrug resistance in human cervical carcinoma cell lines by estrogenic hormones; Biing JT et al.; The role of estrogenic hormones on the induction of drug resistance was studied in cervical cancer cell lines, SiHa and Caski . After the cells were inoculated with estradiol (E2) or diethylstilbestrol (DES) in various dosages, the cell survival rates with adriamycin treatment were examined by MTT (3-{4,5-dimethyl-thiazole-2-yl}-2,5-diphenyl-tetrazolium-bromide) test and the intracellular accumulation of adriamycin was evaluated by flow cytometry . In the same condition, the expression of multidrug resistance gene-1 (mdr-1 gene) was detected either by Northern blot hybridization for mdr-1 mRNA or by immunoblot for P-glycoprotein 170 . The data in this study indicated that estrogenic hormones had the capacity to induce drug resistance in cervical carcinoma cell lines . When cells were treated with estrogenic hormones and adriamycin simultaneously, the intracellular accumulation of adriamycin declined and corresponded with the drug resistance . Since the expression of the mdr-1 gene induced by E2 or DES results in drug resistance, it is suggested that the mdr-1 gene in SiHa cells may contain the estrogenic responsive element (ERE) in its regulatory region . However, the mechanism of drug resistance induced by estrogenic hormones in Caski cells is different from SiHa cells due to the absence of mdr-1 gene expression . Despite that, this experiment may provide a model to investigate the relationships between estrogenic hormones and drug resistance in other female genital cancers.

Anticancer Drugs, 1994 Apr, 5(2), 229 - 38
Extent and persistence of P-glycoprotein inhibition in multidrug-resistant P388 cells after exposure to resistance-modifying agents; Boesch D et al.; The low daunomycin (DAU) retention in P388 cells displaying P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) can be increased by the presence of various resistance-modifying agents (RMAs) . Taking the DAU retention restoration as an indicator of Pgp function inhibition and using a few RMAs, including SDZ PSC 833, SDZ 280-446, cyclosporin A (CsA) and verapamil, we compared different conditions of MDR cell exposure to the RMA . The 'co + post-RMA' treatments (RMA present during both DAU uptake and efflux phases) generally led to higher DAU retention levels than the 'co-RMA' treatments (RMA present during the DAU uptake phase only) . The magnitude and persistence of Pgp function inhibition induced by the RMA was further examined by only pulsing the cells with the RMA and growing them in RMA-free medium before the DAU retention assay ('pre-RMA' treatment) . While recovery of Pgp function was nearly complete within minutes after a pulse exposure to verapamil, this took increasing times with CsA, SDZ 280-446 and SDZ PSC 833, the latter RMA leaving traces of inhibition of Pgp function even 2 days after the pulse exposure of the MDR-P388 cells . The persistence of Pgp inhibition conferred by some RMAs being much longer than by others, this feature should be taken into account when designing chemotherapy protocols in the clinic.

Hematol Oncol Clin North Am, 1994 Apr, 8(2), 383 - 410
Multidrug resistance . Clinical opportunities in diagnosis and circumvention; Chan HS et al.; Increased P-glycoprotein expression has been shown to be the molecular cause of multidrug resistance in tumor cell lines . Sensitive immunohistochemical and molecular biologic techniques have been developed to detect P-glycoprotein/mdr1 mRNA expression in clinical samples of tumors . We have reviewed the tools now available for assessment of P-glycoprotein expression in the clinic, the current evidence for a relevant role of the protein in mediation of resistance to chemotherapy, and one strategy used to overcome therapeutic failures due to multidrug resistance . It is now recognized that low levels of increased P-glycoprotein/mdr1 mRNA can occur at diagnosis and during the course of treatment in some cases of acute myelogenous leukemia, non-Hodgkin's lymphoma, multiple myeloma, breast carcinoma, rhabdomyosarcoma and undifferentiated sarcoma of children, neuroblastoma, and retinoblastoma, and these relatively low levels of mdr1 overexpression appear to be associated with poor prognosis . In contrast, it has not been established whether a multidrug resistance mechanism is the rate-limiting factor in response to chemotherapy in carcinomas that arise from tissues normally expressing increased P-glycoprotein . Clinical trials have been initiated to determine whether pharmacologic chemosensitization improves the outcome of chemotherapy-treated malignancies . Preliminary results suggest that chemosensitizers can modulate the effects of increased P-glycoprotein in low-expressing tumors for which effective multiagent chemotherapy is available . Further research is needed for more potent chemosensitizers or combinations of agents that can be used more effectively . The successful circumvention of chemotherapy failure by chemosensitizers will ultimately establish the clinical relevance of the P-glycoprotein efflux mechanism.

Leuk Res, 1994 Apr, 18(4), 283 - 91
Transport and metabolism of polyamines in wild and multidrug resistant human leukemia (K 562) cells; Khan NA et al.; Multidrug resistance (MDR) can be defined as the resistance of cancer cells not just to chemotherapeutic agents to which they have been exposed but also to other apparently unrelated compounds . This MDR phenotype is commonly associated with the high expression of levels of 170 kDa P-glycoprotein, encoded by MDR genes . In the present study, the uptake kinetics of polyamines and their biosynthesis were studied in wild and multidrug resistant (MDR) K 562 cells in culture . The rate (Vmax) of polyamine uptake was significantly lower in MDR cells than that in wild type cells, whereas the Km for the uptake was not significantly different in these cells, suggesting that polyamine transporter is not modified in MDR cells, though their different physiological state influences the uptake process . In a 32 h chase, the transported radioactive polyamines were gradually interconverted . {14C}putrescine was converted into {14C}spermidine following between 15 min and 32 h of culture, and into {14C}-spermine after 16 h of culture, in both the cell types; however, the levels of interconverted radioactive polyamines were always lower in MDR cells as compared with wild type cells . Similarly, internalized {14C}spermidine was converted into {14C}spermine, but not into {14C}putrescine in both the cells types . {14C}spermidine is metabolized into {14C}spermine after 4 h of culture in wild type cells, whereas in MDR cells the interconversion of {14C}spermidine into {14C}spermine is seen only after 16 h of culture . Blocking of the transmembrane drug efflux pump, expressed in the MDR cells, by preincubation in the presence of verapamil, did not influence the uptake of either of the two polyamines (putrescine and spermidine) by MDR cells . On the contrary, this kind of preincubation of wild type cells in the presence of verapamil significantly increased the uptake of these two polyamines . The levels of intracellular polyamine contents in MDR cells were always lower than those in the parental cell line . These results demonstrate that MDR cells are defective in both the uptake of polyamines and their biosynthesis as compared with wild type cells.

Leuk Res, 1994 Apr, 18(4), 233 - 43
Clinical relevance of P-glycoprotein expression in haematological malignancies; Nooter K et al.; Although, generally speaking, haematological malignancies are chemotherapy-responsive tumours and high remission induction rates are obtained, disease-related death is the rule rather than the exception . The appearance of cell populations, resistant to multidrug-based chemotherapy, constitutes the major problem to achieve cures in these patients . Advances in cell biology have partly contributed to the elucidation of different multidrug resistance (MDR) mechanisms, which enable cells to survive the cytotoxic effects of multiple chemotherapeutic agents . Of these resistance mechanisms, the one that is referred to as classical MDR is the most extensively studied, both in the laboratory as well as in patients, and here we will focus on its clinical relevance in haematological malignancies . The classical MDR phenotype is caused by enhanced cellular drug efflux due to increased activity of a membrane-bound glycoprotein (P-glycoprotein) drug pump, that can pump out anthracyclines, anthracenediones, vinca alkaloids and epipodophyllotoxins, thereby actively lowering the intracellular drug concentrations to sublethal levels . As soon as molecular probes for the detection of MDR cells became available, clinical studies were initiated to answer three main questions . Do human tumor cells express P-glycoprotein? If so, is the expression indicative of a bad prognosis, c.q . resistant disease? And last but not least, can we interfere with the P-glycoprotein drug pump in the patient? Clinical data indicate that classical MDR may be involved in the development of drug resistance, especially in some haematological malignancies, such as acute myelocytic leukaemia (AML), non-Hodgkin's lymphomas (NHL), and multiple myelomas (MM) . In almost all types of haematological malignancies, either untreated or treated, elevated P-glycoprotein levels have been reported, ranging from low to high . However, the acquisition of clinical MDR associated with P-glycoprotein expression occurs only in those diseases (for example, AML and MM) that are heavily treated with MDR-related drugs, probably by selection of pre-existing P-glycoprotein-expressing malignant cells . Since P-glycoprotein is found to be expressed on the membrane of normal haemopoietic progenitor cells as well, it seems likely that P-glycoprotein-positive haematological tumours develop by malignant transformation of P-glycoprotein-expressing normal haemopoietic counterparts . Especially for AML, convincing data have been reported in the literature to show that P-glycoprotein expression at diagnosis is a bad prognostic factor that predicts refractoriness . Using in vitro model systems for classical MDR, a large number of agents have been identified that can circumvent P-glycoprotein-mediated drug resistance, the so-called resistance modifying agents (RMA).(ABSTRACT TRUNCATED AT 400 WORDS)

Eur J Biochem, 1994 Apr 1, 221(1), 363 - 73
The multidrug-resistance-reverser verapamil interferes with cellular P-glycoprotein-mediated pumping of daunorubicin as a non-competing substrate; Spoelstra EC et al.; We examined P-glycoprotein-mediated verapamil transport, using two drug-sensitive and multi-drug resistant cell-line couples, i.e . A2780, 2780AD and SW-1573, SW-1573/1R500 . The interaction of 3H-labeled verapamil with cells was measured using a flow-through system . The verapamil-containing medium was pumped over the cells and monitored on-line for radioactivity . In the P-glycoprotein-expressing cells, verapamil accumulation was increased by vinblastine and some known multidrug resistant (MDR) modifiers . Subsequent removal of these modifiers caused release of verapamil into the medium against a verapamil concentration gradient . In this manner, we obtained evidence that verapamil is actively transported by the MDR-related P-glycoprotein . Using the flow-through system, we also exposed the cells to flowing culture medium containing daunorubicin, and measured the inhibition of daunorubicin efflux by verapamil . We found that, although the active efflux of daunorubicin was maximally blocked by verapamil short-term, longer-term active efflux of daunorubicin resumed . At a daunorubicin concentration in the flowing medium of 5 microM, increasing the verapamil concentration resulted in the same short-term effects, but in a significantly longer period of a maximal inhibition of daunorubicin efflux from the cells . At a daunorubicin concentration of 20 microM, increasing the verapamil concentration affected neither the short-term nor the long-term effects . These and other observations are in agreement with a model in which daunorubicin and verapamil are non-competing substrates for P-glycoprotein . In conclusion, we obtained evidence that verapamil is actively transported by the MDR-related P-glycoprotein and that verapamil and daunorubicin are non-competing substrates for P-glycoprotein . Consequently, the effectiveness of verapamil as an MDR antagonist may be compromised because it is extruded by P-glycoprotein.

Biochem J, 1994 Apr 1, 299 ( Pt 1), 309 - 15
Phosphorylation by protein kinase C and cyclic AMP-dependent protein kinase of synthetic peptides derived from the linker region of human P-glycoprotein; Chambers TC et al.; Specific sites in the linker region of human P-glycoprotein phosphorylated by protein kinase C (PKC) were identified by means of a synthetic peptide substrate, PG-2, corresponding to residues 656-689 from this region of the molecule . As PG-2 has several sequences of the type recognized by the cyclic AMP-dependent protein kinase (PKA), PG-2 was also tested as a substrate for PKA . PG-2 was phosphorylated by purified PKC in a Ca2+/phospholipid-dependent manner, with a Km of 1.3 microM, and to a maximum stoichiometry of 2.9 +/- 0.1 mol of phosphate/mol of peptide . Sequence analysis of tryptic fragments of PG-2 phosphorylated by PKC identified Ser-661, Ser-667 and Ser-671 as the three sites of phosphorylation . PG-2 was also found to be phosphorylated by purified PKA in a cyclic AMP-dependent manner, with a Km of 21 microM, and to a maximum stoichiometry of 2.6 +/- 0.2 mol of phosphate/mol of peptide . Ser-667, Ser-671 and Ser-683 were phosphorylated by PKA . Truncated peptides of PG-2 were utilized to confirm that Ser-661 was PKC-specific and Ser-683 was PKA-specific . Further studies showed that PG-2 acted as a competitive substrate for the P-glycoprotein kinase present in membranes from multidrug-resistant human KB cells . The membrane kinase phosphorylated PG-2 mainly on Ser-661, Ser-667 and Ser-671 . These results show that human P-glycoprotein can be phosphorylated by at least two protein kinases, stimulated by different second-messenger systems, which exhibit both overlapping and unique specificities for phosphorylation of multiple sites in the linker region of the molecule.

Gan To Kagaku Ryoho, 1994 Apr, 21(5), 665 - 70
{Study for modifying activity of solvents on antitumor activity of paclitaxel}; Fujimoto S; Paclitaxel, a novel diterpenoid compound, has been used by dissolving in Cremophor EL (polyoxyethylene castor oil) due to its poor aqueous solubility . Cremophor EL was shown to reverse multidrug resistant phenotypes of various cell lines as well as to reverse cross-resistance to paclitaxel of a multidrug resistant cell line in vitro . Thus, a study was carried out to determine the modifying activity of Cremophor EL on the antitumor activity of paclitaxel against P388 leukemia, adriamycin-resistant subline (P388/ADM) and vincristine-resistant subline (P388/VCR) in vivo . Dimethyl sulfoxide (DMSO) was used as a counterpart solvent . The results showed that, although no significant antitumor activity was observed by paclitaxel in both solvents against P388/ADM, a significantly higher antitumor activity was induced by paclitaxel dissolved in Cremophor EL-based solvent compared with DMSO-based solvent against P388/VCR . However, more significant difference in the antitumor activity of paclitaxel against P388 parental line was observed between two solvents and both resistant sublines showed an obvious cross-resistance to paclitaxel . Therefore, it appeared that cross-resistance reversing activity of Cremophor EL is not so high as to be detectable at in vivo level.

Gan To Kagaku Ryoho, 1994 Apr, 21(5), 583 - 90
{Microtubules and antineoplastic drugs}; Arioka H et al.; Microtubules, which are composed of polymerized tubulin dimers, play an important role in various cell functions . For example, they maintain cell shape, form mitotic spindles in M phase of cell cycle, and carry an axonal transport in nerve cells . Microtubules have also been an important target of cancer chemotherapy . Vinca alkaloids depolymerize microtubules, the mechanisms of which action have extensively been investigated recently . Clinical trials of vinorelbine (navelbine), a new semisynthetic vinca alkaloid, are ongoing in Japan . One of advantages of the drug is reduced risk of neurotoxicity . Estramustine may act on microtubule-associated proteins (MAPs) as well as tubulin . It shows additive or synergistic cytotoxicity preclinically when used in combination with vinblastine . This combination was active against hormone-refractory prostate cancer . Another novel drug rhizoxin, which has a similar mechanism of action to that of vinca alkaloids, is also a promising cytotoxic agent and is examined clinically in Europe . Taxanes, which include paclitaxel (Taxol) and taxotere, are interesting drugs because they promote polymerization of tubulin and stabilize microtubules . They show promising antitumor activity against breast, ovarian and lung cancers . Phase I and II trials are ongoing in Japan . Paclitaxel may also potentiate cytotoxicity of radiation . There are several mechanisms of resistance to microtubule-acting drugs . One is multidrug resistance mediated by P-glycoprotein . Other mechanisms include mutation of tubulin.

J Clin Oncol, 1994 Apr, 12(4), 812 - 9
Clinical and pharmacologic study of multidrug resistance reversal with vinblastine and bepridil; Linn SC et al.; PURPOSE: To achieve an adequate plasma concentration of bepridil, a calcium channel blocker, which reverts multidrug resistance (MDR) in vitro, when administered in combination with vinblastine in patients with advanced colorectal cancer, a tumor characterized by high MDR1 gene expression . To study the pharmacokinetics of both drugs, tolerability and antitumor activity in relation to the MDR1 expression in tumor tissue . PATIENTS AND METHODS: Sixteen colorectal cancer patients entered the study . Bepridil was administered by central venous catheter as 5-mg/kg bolus over 30 minutes, followed by 12 mg/kg for 12 hours and 5 mg/kg for 24 hours . Vinblastine 5 mg/m2 was administered as an intravenous (i.v.) bolus 24.5 hours after the start of bepridil . MDR1/P-glycoprotein (Pgp) expression was assessed in 14 tumor samples by immunohistochemistry and RNase protection assay . RESULTS: The bepridil plasma level was greater than 2 mumol/L at the time of vinblastine administration in all patients investigated . At the dose used in the study, bepridil produced a QTc-prolongation more than 50 ms, which prevented further dose escalation . However, cardiac toxicity was asymptomatic in all treated patients, and other side effects were mild . MDR1/Pgp expression was positive in nine of 14 cases . Of fifteen patients assessable for response, one complete remission of 8 months' duration and 14 progressions were observed . The responding patient attained complete remission again when re-treated on progression with vinblastine alone . CONCLUSION: Bepridil plasma concentrations needed in vitro to modulate MDR could be achieved in this study with tolerable toxicity; however, despite most tumors being MDR1/Pgp-positive, no response was obtained that could be attributed to the drug combination . Mechanisms of drug resistance other than MDR are probably implicated in drug resistance of colorectal cancer.

Int J Cancer, 1994 Apr 1, 57(1), 104 - 10
The protein kinase C inhibitor CGP 41251, a staurosporine derivative with antitumor activity, reverses multidrug resistance; Utz I et al.; Multidrug resistance (MDR) is frequently associated with overexpression of a 170-kDa P-glycoprotein (Pgp) . Data suggest altered protein kinase C (PKC) activity in cells expressing the multidrug-resistant phenotype . The staurosporine derivative CGP 41251, an experimental anticancer drug, has been shown to exert selectivity for inhibition of protein kinase C activity and to exhibit antitumor activity in vitro and in vivo . Here we show that CGP 41251 is also able to reverse MDR . After treatment of the multidrug-resistant human lymphoblastoid cell line CCRF-VCR1000 with 500 nM Adriamycin, cell proliferation was reduced to 81% of untreated controls . A combination of 500 nM Adriamycin with a non-toxic concentration of 150 nM CGP 41251 (IC50 for inhibition of cell proliferation 420 nM CGP 41251) inhibits cell proliferation of CCRF-VCR1000 cells to 29% of untreated controls . In sensitive CCRF-CEM cells no enhancement of Adriamycin-induced cytotoxicity was observed upon addition of 150 nM CGP 41251 . Strong synergism of the inhibition of cell proliferation was also observed after concomitant treatment of KB-8511 cells with CGP 41251 and Vinblastine or Adriamycin . Drug-sensitive KB-31 cells could not be further sensitized to Adriamycin or Vinblastine with CGP 41251 doses above 100 nM . Pretreatment with 50-1000 nM CGP 41251 for 30 min led to a dose-dependent increase in the intracellular accumulation of rhodamine 123, a substrate of P-glycoprotein . Treatment of multidrug-resistant CCRF-VCR1000 cells with CGP 41251 for 10 min was sufficient to inhibit the efflux of rhodamine 123 . Preincubation with CGP 41251 for 12 or 24 hr did not alter multidrug resistance gene (mdrI)-mRNA levels . CGP 41251, a drug with antitumor efficacy in experimental systems, might offer an attractive combination partner for the treatment of tumors expressing the MDR phenotype.

Br J Cancer, 1994 Apr, 69(4), 680 - 6
Increased mdr1 gene transcript levels in high-grade carcinoma of the bladder determined by quantitative PCR-based assay; Clifford SC et al.; Overexpression of the multidrug resistance (mdr1) gene has been implicated in resistance to a number of the chemotherapeutic agents currently used in the treatment of bladder cancer (doxorubicin, vincristine and epirubicin) . We report the development and validation of a quantitative assay for the determination of mdr1 gene transcript levels based on reverse transcription and the polymerase chain reaction (PCR), sensitive to less than 2-fold variations in transcript levels . Using these techniques, mdr1 mRNA levels were investigated in 32 primary untreated transitional cell carcinomas of the bladder . mdr1 mRNA was detected in all samples, with levels varying between individual tumours over a 63-fold range . These variations were not associated with the proliferative status of the tumour . mdr1 mRNA levels were significantly higher in poorly differentiated high-grade (G3) tumours than in well- and moderately differentiated low-grade (G1 and G2) tumours (P = 0.0057) . The results suggest that this relationship may extend to mdr1 mRNA levels being an indicator of poor prognosis, as anticipated on the basis of the observed relationship to tumour stage and grade . No evidence was found to implicate mdr1 mRNA levels as a predictor of tumour recurrence or progression . Given that mdr1 mRNA levels are increased in a proportion of high-grade bladder tumours that are routinely subjected to chemotherapy, we discuss the possibility that mdr1 mRNA levels may be clinically significant as determinants of chemotherapeutic response and outcome in bladder cancer.

Pathol Biol (Paris), 1994 Apr, 42(4), 328 - 37
Cross resistance relevance of the chemical structure of different anthracyclines in multidrug resistant cells; Tapiero H et al.; Positively charged doxorubicin (DOX) and non-positively charged anthracyclines, aclarubicin (ACR) and morpholino-carminomycin (KRN 8602), have been investigated with respect to pharmacological parameters, cytotoxicity, DNA damage and repair in DOX-sensitive and -resistant murine and human cells . Friend leukemia cells (FLC) resistant to high concentrations of doxorubicin (DOX-RFLC3) or daunorubicin (DNR-RFLC3) (1771 and 1543 fold resistance respectively) express less than 10 fold resistance to aclarubicin (ACR) . In these cells, the intracellular accumulation of ACR is similar in sensitive and resistant cells . Resistance to ACR was not observed in either DOX-RFLC1 or DNR1 with a lower level of resistance (27 fold) . Increased expression of a 170,000-dalton surface antigen (gp-170) was found to be correlated with the level of resistance . However, when the selective agent in ACR, despite the low level of resistance (2.8 fold) both high expression of gp 170 and resistance to DOX (77 fold) or DNR (62 fold) are observed . It is assumed therefore that induction of multidrug resistance phenotype can be achieved by compounds which do not display cross resistance with DOX or DNR . Reduced levels or absence of cross-resistance can be related to the electrical charge of the compound . This assumption is supported by further studies on DOX-sensitive or -resistant human K562 cells exposed to another non-positively charged anthracycline, KRN 8602 . In the continuous presence of drug, K562/DOX were less resistant to KRN 8602 (2.9 fold) than to DOX (31 fold) . After short time exposure followed by growth in drug-free medium, absence of cross-resistance to KRN 8602 has been observed in K562/DOX . Furthermore, accumulation experiments showed that high intracellular drug concentrations were rapidly achieved (within 15 min) in both DOX-sensitive and -resistant cells . In cells exposed to DOX, DNA single-strand break (DNA-SSBs) frequencies were related to time and drug concentration while those produced by KRN 8602 or ACR were maximal after short time incubation . DNA-SSBs produced by these anthracyclines are not repaired when cells are incubated in drug free medium . In DOX resistant cells, DNA-SSBs produced by DOX were repaired whereas those produced by ACR or KRN 8602 were not . It is suggested, therefore, that absence of cross resistance to various anthracyclines is related to differences in the chemical electrical charge, which may influence drug accumulation and DNA repair in resistant cells.

Mol Pharmacol, 1994 Apr, 45(4), 773 - 6
Direct demonstration of high affinity interactions of immunosuppressant drugs with the drug binding site of the human P-glycoprotein; Rao US et al.; The interactions between the human P-glycoprotein (Pgp) and two different types of immunosuppressant drugs known to modulate multidrug resistance in tumor cells have been directly investigated using our newly developed drug-stimulated ATPase assay for Pgp function . The macrolides FK506 and FK520 stimulate the Pgp-ATPase activity with affinities in the 100 nM range, nearly 10 times higher than that of verapamil, a well known Pgp substrate . On the other hand, the cyclic peptides cyclosporin A and dihydrocyclosporin C do not stimulate the Pgp-ATPase activity at all . They do, however, act as potent competitive inhibitors of verapamil-stimulated Pgp-ATPase activity, with affinity constants in the 20-25 nM range . Thus, although these two classes of immunosuppressant drugs affect the Pgp in different ways, they both probably interact with high affinity at the transported drug binding site(s) of the Pgp, which would explain their ability to resensitize multidrug-resistant cells to the killing action of certain antitumor drugs . Possible implications of these findings for Pgp function, cancer chemotherapy, and immunosuppression are discussed.

Mol Cell Biol, 1994 Apr, 14(4), 2419 - 28
Expression of the plasmodial pfmdr1 gene in mammalian cells is associated with increased susceptibility to chloroquine; van Es HH et al.; Chloroquine (CQ)-resistant (CQR) Plasmodium falciparum malaria parasites show a strong decrease in CQ accumulation in comparison with chloroquine-sensitive parasites . Controversy exists over the role of the plasmodial pfmdr1 gene in the CQR phenotype . pfmdr1 is a member of the superfamily of ATP-binding cassette transporters . Other members of this family are the mammalian multidrug resistance genes and the CFTR gene . We have expressed the pfmdr1-encoded protein, Pgh1, in CHO cells and Xenopus oocytes . CHO cells expressing the Pgh1 protein demonstrated an increased, verapamil-insensitive susceptibility to CQ . Conversely, no increase in drug susceptibility to primaquine, quinine, adriamycin, or colchicine was observed in Pgh1-expressing cells . CQ uptake experiments revealed an increased, ATP-dependent accumulation of CQ in Pgh1-expressing cells over the level in nonexpressing control cells . The increased CQ accumulation in Pgh1-expressing cells coincided with an enhanced in vivo inhibition of lysosomal alpha-galactosidase by CQ . CHO cells expressing Pgh1 carrying two of the CQR-associated Pgh1 amino acid changes (S1034C and N1042D) did not display an increased CQ sensitivity . Immunofluorescence experiments revealed an intracellular localization of both mutant and wild-type forms of Pgh1 . We conclude from our results that wild-type Pgh1 protein can mediate an increased intracellular accumulation of CQ and that this function is impaired in CQR-associated mutant forms of the protein . We speculate that the Pgh1 protein plays an important role in CQ import in CQ-sensitive malaria parasites.

Biochem Biophys Res Commun, 1994 Mar 30, 199(3), 1428 - 35
Involvement of a DNA binding protein, MDR-NF1/YB-1, in human MDR1 gene expression by actinomycin D; Asakuno K et al.; The human multidrug resistance 1 (MDR1) gene is an SOS gene that responds to environmental stress including various anticancer agents . The chloramphenicol acetyltransferase (CAT) gene was linked to various lengths of MDR1 promoter, and these constructs were integrated into the genome of human cancer KB cells . Using these cell lines, we previously demonstrated that various environmental stimuli lead to an increased abundance of both CAT enzymatic activity and CAT mRNA in a sequence dependent manner . We examined the molecular mechanism of this stress response using actinomycin D, a potent RNA synthesis inhibitor . We found that CAT activity was significantly increased more than 10 fold by actinomycin D itself without comparable elevation of CAT mRNA . CAT induction was, however, lost in the presence of a deletion from position -136 to -76 . Gel mobility shift assays showed that the specific DNA binding activity of the transacting protein, MDR-NF1/YB-1, which binds to the inverted CCAAT box, was augmented in nuclear extracts from the cells treated with actinomycin D . We also found that actinomycin D increased the steady state levels of MDR-NF1/YB-1 mRNA, which encodes the inverted CCAAT box binding protein . These results indicate that MDR-NF1/YB-1 mediates the response of the MDR1 gene to environmental stress.

Biochem Biophys Res Commun, 1994 Mar 30, 199(3), 1181 - 7
Mycobacterium tuberculosis induces expression of P-glycoprotein in promonocytic U1 cells chronically infected with HIV type 1; Gollapudi S et al.; A productive infection with HIV-1 is associated with an increased expression of a 170 kd plasma membrane P-glycoprotein (P-gp), that functions as a metabolically active drug efflux pump, in human T and macrophage cell lines . In this investigation we show that phagocytosis of M . tuberculosis by U1 cells, that are chronically infected with HIV-1 but produce minimal or no virus, resulted in an expression of P-gp that was associated with increased production of HIV-1 p24 antigen . In addition, U1 cells that had phagocytosed M . tuberculosis accumulated significantly less intracellular isoniazid (INH) as compared to U1 cells . Furthermore, verapamil, that binds to P-gp, increased the intracellular accumulation of INH and the sensitivity of M . tuberculosis to INH . These data suggest induction of P-gp expression may be one of the host mechanisms for the development of multidrug resistant M . tuberculosis in HIV 1 infection.

J Biol Chem, 1994 Mar 25, 269(12), 8667 - 74
Pyridine nucleotide redox potential modulates cystic fibrosis transmembrane conductance regulator Cl- conductance; Stutts MJ et al.; Cl- conductance of the apical membrane of airway epithelial cells has properties of a passive diffusion mechanism but is decreased by inhibition of oxidative metabolism . Recent reports that cAMP-dependent Cl- conductance also requires ATP at the intracellular domains of the cystic fibrosis transmembrane conductance regulator (CFTR) suggests that ATP concentration could mediate metabolic regulation of Cl- conductance . However, metabolic inhibitors affect processes other than ATP free energy levels, including notably the metabolic pathways that set the redox potential of pyridine nucleotides within the cell . We have investigated the possibility that CFTR-mediated Cl- conductance is affected by the ratio of oxidized to reduced intracellular pyridine nucleotides . CFTR was expressed in airway and heterologous cells and studied under whole cell voltage clamp conditions, which permitted the intracellular NAD(P)+/NAD(P)H ratio to be varied independently of ATP concentration . In three cell types expressing CFTR, whole cell dialysis with reduced pyridine nucleotides inhibited activation of Cl- currents by forskolin and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), whereas dialysis with oxidized pyridines increased both basal and stimulated CFTR-mediated Cl- conductance . In cell-attached membrane patches, the open probability of 5-6-picosiemens Cl- channels that had been activated by forskolin and CPT-cAMP was further and reversibly increased by permeant oxidants . Neither swelling-induced whole cell K+ currents in CFTR-expressing cells nor swelling-induced whole cell Cl- currents in multidrug resistance protein-expressing cells were affected by NADPH . Pyridine nucleotide redox potential had little effect on phosphorylation of histone by protein kinase A . We conclude that CFTR Cl- conductance function can be modulated by pyridine nucleotide redox potential . This effect points to the existence of a mechanism or mechanisms by which cytosolic nucleotides other than ATP can affect plasma membrane Cl- conductance and may help explain how a passive ion conductance is linked to cellular energy metabolism.

N Z Med J, 1994 Mar 23, 107(974), 99 - 101
Drug resistant tuberculosis in Auckland 1988-92; Bradley A et al.; AIM . To report the prevalence of drug-resistant Mycobacterium tuberculosis in Auckland . METHOD . Review of all M tuberculosis culture positive cases from January 1988 to December 1992 . Ethnicity was recorded from the national Paxus medical information data base and drug sensitivity results from the Green Lane Hospital tuberculosis reference laboratory . The clinical details of all patients with drug resistant tuberculosis were extracted from hospital case records . RESULTS . Of the 417 patients with positive cultures, 43 (10%) had isolates resistant to one or more antituberculous drugs . Only 4 patients had multidrug-resistant organisms (defined as resistance to at least isoniazid and rifampicin) and there is no evidence that this is an increasing problem . Resistance rates were highest in those previously treated for M tuberculosis, and those born in, or likely to have acquired their organism from, Samoa, all other Pacific Islands, or South East Asia . CONCLUSIONS . Drug resistant M tuberculosis is a problem in Auckland but rates and patterns are little different from 1980-1982 . Multidrug-resistance is not yet a problem in Auckland, as it is in the United States of America . Ways of maintaining this situation are discussed.

FEBS Lett, 1994 Mar 21, 341(2-3), 295 - 8
Partial restoration of the actin cytoskeleton in transformed Syrian hamster fibroblasts selected for low levels of 'typical' multidrug resistance; Erokhina MV et al.; Two independent colchicine (CLC)-resistant sublines of Rous sarcoma virus-transformed Syrian hamster fibroblasts were isolated . Each subline represented variants with 11- and 12.4-fold resistance, respectively, their 23- and 23.7-fold resistant descendants, as well as variants cultured in CLC-free medium for 10 months without loss of resistance . All variants demonstrated 'typical' multidrug resistance . The parental cells contained actin in dispersed form, as determined by rhodamine-phalloidin staining . In contrast, already in 11- and 12.4-fold resistant sublines up to 30% of cells demonstrated restored stress fibers . Cultivation in CLC-free medium leads to the accumulation of cells with a partially restored actin cytoskeleton . Putative mechanisms of up-regulation of stress fiber assembly in cells with P-glycoprotein-mediated multidrug resistance are discussed.

J Biol Chem, 1994 Mar 18, 269(11), 7976 - 81
Alterations in Ca2+ transport ATPase and P-glycoprotein expression can mediate resistance to thapsigargin; Gutheil JC et al.; Resistance to the intracellular Ca2+ pump inhibitor thapsigargin (TG) is associated with overexpression of both Ca2+ transport ATPase and the multidrug resistance (mdr) transporter P-glycoprotein (pgp) . This is supported by increased resistance to TG following transfection of a functional pgp1 cDNA, and reversal of TG resistance with known inhibitors of pgp function . However, pgp is unlikely to represent the only mechanism of resistance to TG . Cell lines selected for high levels of resistance to TG (250-fold) show only a 3.7-fold increase in pgp expression and a 2-fold increase in cross-resistance to other drugs of the mdr class . Overexpression of endogenous Ca2+ transport ATPase may represent a second mechanism of resistance to TG . Increased Ca2+ ATPase expression (3-fold) is seen in cells made resistant to TG, and TG resistance increases with the transfection of a specific Ca2+ ATPase cDNA into DC-3F cells . If these transfectants are then made resistant to TG, both the endogenous Ca2+ ATPase and the exogenously transfected Ca2+ ATPase become overexpressed . These studies suggest that while TG may be a substrate for pgp, acquired resistance to TG can involve alterations in both pgp and Ca2+ ATPase expression . Additional, as yet unidentified, mechanisms of resistance may be involved in resistance to TG.

Cancer Res, 1994 Mar 15, 54(6), 1479 - 84
Antitumor activity of free and liposome-entrapped annamycin, a lipophilic anthracycline antibiotic with non-cross-resistance properties; Zou Y et al.; The lipophilic anthracycline antibiotic annamycin (Ann) was entrapped in liposomes of different size {median diameter: 1.64 microns, multilamellar liposomal Ann (L-Ann); 0.030 micron, small unilamellar Ann (S-Ann)} with > 90% entrapment efficiency and tested in vitro against four pairs of sensitive and multidrug-resistant (MDR) tumor cell lines and in vivo by the i.v . route in five tumor models: advanced s.c . B16 melanoma; s.c . M5076 reticulosarcoma; lung metastases of Lewis lung carcinoma; and s.c . KB and KB-V1 xenografts in nude mice . Predetermined optimal doses of the different formulations were used and the results were compared with doxorubicin (Dox) . In vitro, Ann, either in suspension in 10% dimethyl sulfoxide (F-Ann) (1 mg/ml) or entrapped in liposomes, was able to partially overcome resistance in all four pairs of sensitive and MDR KB, 8226, P388, and CEM cell lines (resistance indexes 63, 269, 333, and 356 for Dox versus 4, 5, 19, and 8.7 for L-Ann, respectively) . In vivo, both F-Ann and liposome-entrapped Ann were slightly more effective than Dox in inhibiting the growth of advanced s.c . B16 melanoma tumors . L-Ann was markedly more effective than Dox and moderately more effective than F-Ann in prolonging the life span of animals bearing s.c . M5076 and lung metastases of Lewis lung carcinoma tumors . All drugs were equally effective at optimal doses in delaying the growth of s.c . KB xenografts, whereas all Ann formulations were markedly more effective than Dox in delaying the growth of s.c . KB-V1 (MDR) xenografts . In all in vivo experiments, S-Ann was consistently more effective than L-Ann and L-Ann was more effective than F-Ann . These results indicate that (a) Ann is more effective than Dox by the i.v . route against several tumor models and that MDR tumors are partially not cross-resistant to Ann both in vitro and in vivo, (b) liposomes enhance the in vivo antitumor properties of Ann, and (c) small liposomes are more effective than large liposomes in enhancing Ann antitumor activity.

Blood, 1994 Mar 15, 83(6), 1619 - 25
Analysis of treatment failure in patients with minimally differentiated acute myeloid leukemia (AML-M0); Stasi R et al.; Reports of treatment of patients with minimally differentiated acute myeloid leukemia (AML-M0) are limited, heterogeneous, and controversial . We verified the prognosis of this subtype by analyzing the results of 189 consecutive patients with de novo AML . Fifteen cases fitting the criteria of AML-M0 were identified . No clinical features distinguished them from other patients with AML . The median age was 61 years (range 27 to 70), with a leukocyte count ranging from 0.6 to 185 x 10(9)/L . In all cases the leukemic cells expressed CD34 and reacted with at least one of the antibodies to early myeloid antigens, ie, CD13, CD33, or myeloperoxidase . Immunophenotypic analysis also showed positivity for CD7 in seven samples and the multidrug-resistance P-glycoprotein (P-170) in six . Cytogenetic analysis was abnormal in 12 of the 13 patients in whom an adequate number of mitoses could be evaluated . No single abnormality prevailed, the most common findings being trisomy 8 (three cases) and aberrations of chromosome 7 (two cases) . Antileukemic treatment differed according to age, but for remission induction, all patients received a combination of cytosine arabinoside and an anthracycline or mitoxantrone . The prognosis of patients with AML-M0 was remarkably poor as compared with the other French-American-British subtypes . Whereas the overall rate of complete remission (CR) was 58% with a median survival of 63 weeks, only 6 of the 15 patients with AML-M0 achieved a CR, and the median survival of this group was 16 weeks (range 3 to 39) . The major determinant of treatment failure was unresponsiveness to chemotherapy, as only one patient died of infection during the hypoplastic phase . The CR duration of responders was short, ranging from 3 to 22 weeks, and no second remissions were observed . We conclude that conventional combination chemotherapy yields disappointing results in AML-M0 . The reason for this may be the convergence of various unfavorable prognostic factors, such as (1) the high incidence of cytogenetic abnormalities; (2) the lack of differentiation features and the expression of immaturity markers such as CD34 and CD7; and (3) the frequent expression of P-170 . Nonconventional therapeutic approaches should be developed to alter the prognosis of this form of leukemia.

Biochem Biophys Res Commun, 1994 Mar 15, 199(2), 855 - 61
Increased P-type ATPase activity in Leishmania tropica resistant to methotrexate; Sanchez A et al.; In the present report, we show evidence that the membrane from the protozoan parasite Leishmania tropica (LRC-L39), in vitro resistant to 1 mM of methotrexate (MTX), has a significative increased ATPase activity with respect to wild-type line . This ATPase activity is vanadate sensitive, a characteristic of the P-type ATPases included in the ATP-binding casette (ABC) superfamily of transporters, such as P-glycoprotein involved in the multidrug resistance in mammalian cells . Also, this ATPase activity is not modified by MTX, ammonium molybdate and other detergents such as Triton X-100, Brij 58 and lysophosphatidylcholine . However, unlike the P-glycoprotein, we have observed that the ATPase activity is not stimulated by the drugs verapamil and puromycin . This significative ATPase activity could be related to the overexpressed putative P-glycoprotein, with unknown function in these MTX-resistant parasites.

Cancer Res, 1994 Mar 15, 54(6), 1536 - 41
MDR1 gene-specific monoclonal antibody C494 cross-reacts with pyruvate carboxylase; Rao VV et al.; Overexpression of P-glycoprotein, the plasma membrane protein product of the MDR1 gene, is a major determinant in the development of resistance to a large number of cancer chemotherapeutic agents . A battery of antibodies, including the MDR1 gene-specific monoclonal antibody (mAb) C494, is used to evaluate human tissues in clinical multidrug resistance surveillance and modulation trials . In rat liver fractions, we report that mAb C494 strongly cross-reacted with a nonmembranous M(r) approximately 130,000 protein, comigrating with core-glycosylated human MDR1 on 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis . By immunoblotting and microsequence analysis, this protein was identified as pyruvate carboxylase (PC), an abundant mitochondrial enzyme . A search of the National Center for Biotechnology Information data base, using the epitope-specific sequence of mAb C494, revealed that PC (mouse) contains four of the five most reactive amino acids (TLEG), located near the COOH-terminal end of PC at positions 1167-1170 . mAb C494 specifically reacted with PC purified from bovine liver; immunoreactivity was completely abolished by preincubating mAb C494 in the presence of excess synthetic C494 epitope-specific peptide . Furthermore, in cryosections of human skeletal muscle, a tissue known not to express P-glycoprotein, peptide-displaceable immunohistochemical staining with mAb C494 showed a distinct mitochondrial pattern specific to type 1 fibers . Variable immunostaining results were obtained with formaldehyde-fixed, paraffin-embedded muscle and isolated liver mitochondrial preparations . In summary, mAb C494 cross-reacted strongly with rat, bovine, and human PC . Caution is warranted in interpretation of immunoblots and immunohistochemical sections with this putative MDR1 gene-specific mAb.

J Biol Chem, 1994 Mar 11, 269(10), 7145 - 9
Relationships between rhodamine 123 transport, cell volume, and ion-channel function of P-glycoprotein; Altenberg GA et al.; The P-glycoprotein (Pgp), a plasma membrane protein overexpressed in multidrug-resistant tumor cells, is thought to be both an ATPase that actively exports cytotoxic drugs and a Cl- channel activated by cell swelling . The partial reversal of multidrug resistance by Cl- transport blockers suggests a possible role for Cl- in Pgp-mediated drug transport . We used multidrug-resistant Chinese hamster fibroblasts and human breast cancer cells expressing Pgp to study the roles of Cl- (and also Na+ and HCO3-/CO2) on Pgp-mediated efflux of the fluorescent dye rhodamine 123 (R123) . In Pgp-expressing Chinese hamster fibroblasts, exposed to isosmotic solutions, the unidirectional efflux of R123 was not measurably changed by a approximately 60-min removal of Cl- (or by exposure to Na(+)-free, or nominally HCO3-/CO2-free medium); short term (2-3 min) ion substitutions were also ineffective . In human breast cancer cells transfected with human mdr1 cDNA, hyposmotic solutions activated a Cl- current but had no effect on the Pgp-mediated unidirectional efflux of R123 . Additionally, in human breast cancer cells, the intracellular presence of R123 did not prevent activation of the Cl- current by hyposmotic solution . The lack of detectable effect of removal of Cl-, Na+, or HCO3- on Pgp-mediated R123 transport rules out direct coupling between substrate transport and transport of either of these ions by Pgp . The persistence of Pgp-mediated R123 efflux in osmotically swollen cells indicates that activation of the Pgp-associated Cl- current does not hinder the Pgp pump function . The lack of effect of R123 on swelling-activated Cl- current denotes that Pgp-mediated transport of organic substrates and Pgp-associated Cl- currents can occur at the same time in a single cell . These results underscore the dissociation between Pgp-mediated active drug transport and electrodiffusive Cl- transport.

JAMA, 1994 Mar 2, 271(9), 665 - 71
Nationwide survey of drug-resistant tuberculosis in the United States; Bloch AB et al.; OBJECTIVE--To determine antituberculosis drug resistance patterns, geographic distribution, demographic characteristics, and risk factors of reported tuberculosis (TB) patients in the United States . DESIGN--Survey of reported TB cases in the United States . For culture-positive cases reported to the Centers for Disease Control and Prevention, we asked health departments to provide drug susceptibility test results from initial Mycobacterium tuberculosis isolates . STUDY POPULATION--Culture-positive TB cases in the United States reported during the first quarter of 1991 . MAIN OUTCOME MEASURES--Individual TB case reports submitted to the Centers for Disease Control and Prevention and drug susceptibility test results . RESULTS--Resistance to one or more antituberculosis drugs was found in 14.2% of cases . Resistance to isoniazid and/or rifampin was found in 9.5% of cases whose isolates were tested against one or both drugs; such cases were found in 107 counties in 33 states . Resistance to both isoniazid and rifampin (multidrug-resistant {MDR} TB) was found in 3.5% of cases whose isolates were tested against both drugs; such cases were found in 35 counties in 13 states . New York City accounted for 61.4% of the nation's MDR TB cases . The 3-month population-based incidence rate of MDR TB in New York City was 52.4 times (95% confidence interval {CI}, 35.5 to 78.3) that of the rest of the nation (9.559 vs 0.182 cases per million population) . Compared with the rate in non-Hispanic whites in the rest of the nation (0.032 cases per million), the relative risk of MDR TB in New York City non-Hispanic whites was 39.0 (95% CI, 8.1 to 164.5), 299.3 (95% CI, 112.5 to 927.1) in Hispanics, 420.9 (95% CI, 121.0 to 1515.8) in Asian/Pacific Islanders, and 701.0 (95% CI, 296.4 to 2018.1) in non-Hispanic blacks . CONCLUSIONS--With nearly 10% of TB patients resistant to isoniazid and/or rifampin, greater use of four-drug regimens and directly observed therapy is indicated . Aggressive intervention to prevent the further spread of MDR TB is needed to find every TB patient and to provide optimal patient management to ensure completion of chemotherapy.

Trends Biochem Sci, 1994 Mar, 19(3), 119 - 23
Multidrug resistance pumps in bacteria: variations on a theme; Lewis K; Multidrug resistance pumps (MDRs) arise from three different gene families and are widespread in bacteria . For example, in Escherichia coli alone, there seem to be seven distinct MDRs . The most common belong to the major facilitator family of membrane translocases; this type of MDR is closely related to specific antibiotic extrusion pumps such as the tetracycline/H+ antiporter . This similarity in design, and the high incidence of apparently independent evolution of MDRs, suggests that the property of multidrug resistance might have resulted from a loss of specificity in a specific hydrophobic-drug efflux pump.

Trends Pharmacol Sci, 1994 Mar, 15(3), 83 - 9
Tamoxifen: new membrane-mediated mechanisms of action and therapeutic advances; Wiseman H; Tamoxifen protects membranes and lipoprotein particles against oxidative damage . This antioxidant action is likely to contribute to the observed cardioprotective action of tamoxifen and supports the use of this compound in treating and even preventing breast cancer . Membrane-mediated mechanisms of tamoxifen action, through a putative modulation of membrane fluidity, are likely to play an important role in its anticancer action and its ability to reverse multidrug resistance, and could also lead to clinical uses as an anti-Candida and anti-viral agent . In this review, Helen Wiseman discusses the interaction of tamoxifen with membranes and lipoprotein particles, and considers the possible clinical implications.

J Infect Dis, 1994 Mar, 169(3), 595 - 603
Mefloquine prophylaxis prevents malaria during pregnancy: a double-blind, placebo-controlled study; Nosten F et al.; A double-blind, placebo-controlled study of mefloquine antimalarial prophylaxis in pregnancy (> 20 weeks of gestation) was conducted in 339 Karen women living in an area of multidrug-resistant malaria transmission on the Thai-Burmese border . Mefloquine gave > or = 86% (95% confidence interval {CI}, 59%-94%) protection against Plasmodium falciparum and complete protection against Plasmodium vivax infections . Mefloquine prophylaxis was well tolerated; use of an initial loading dose (10 mg/kg) was associated with transient dizziness, but there were no other significant adverse effects on the mother, the pregnancy, or infant survival or development (followed for 2 years) . Falciparum malaria was associated with maternal anemia and a mean reduction in birth weight in gravidae I, II, and III of 225 g (95% CI, 26-423) . Maternal anemia at delivery (hematocrit < 30%) was associated with increased infant mortality: 26% versus 15% (relative risk, 1.9; 95% CI, 1.1-3.2) . Mefloquine is safe and effective for antimalarial prophylaxis in the second half of pregnancy.

Br J Cancer, 1994 Mar, 69(3), 609 - 12
Immunohistochemical detection of mutant p53 protein in epithelial ovarian cancer using polyclonal antibody CMI: correlation with histopathology and clinical features; Renninson J et al.; Approximately 30-50% of cases of ovarian adenocarcinoma harbour mutations in the p53 tumour-suppressor gene associated with elevated levels of the protein detected by immunohistochemical staining . To investigate any relation between the presence of mutant p53 and clinicopathological features of disease, we examined a series of 50 cases of epithelial ovarian adenocarcinoma for expression of p53 by immunohistological staining on fixed, paraffin-embedded tissue sections using the polyclonal antibody CM1, and by direct nucleotide sequencing of polymerase chain reaction-amplified DNA from selected cases . Of the 50 cases examined, 28 (56%) were p53 positive and there was no significant correlation between p53 status and differentiation stage, clinical (FIGO) stage, multidrug resistance (mdr-1 P-glycoprotein) expression or response to treatment . However, we observed a statistically significant difference between the high prevalence of p53-positive serous tumours (18 out of 23) and the lower prevalence of p53-positive cases in mucinous tumours (3 of 12) suggesting that factors related to disease aetiology, associated with these histological subtypes, may determine the prevalence of functional inactivation of the p53 tumour-suppressor gene in ovarian adenocarcinoma.

Cancer Res, 1994 Mar 1, 54(5), 1271 - 5
Reversal of drug sensitivity in multidrug-resistant tumor cells by an MDR1 (PGY1) ribozyme; Kobayashi H et al.; In order to reverse P-glycoprotein-mediated drug resistance in a specific manner, we designed two hammerhead ribozymes which can cleave the GUC sequence in codon 179 and 196 of MDR1 (PGY1) mRNA . The ribozymes were directly synthesized using a set of primers, one containing a bacteriophage T7 RNA polymerase promoter . A target MDR1 RNA was created by a reverse transcription polymerase chain reaction using a MOLT-3 human acute leukemia cell line resistant to trimetrexate (TMQ) (MOLT-3/TMQ800), which displayed MDR1 overexpression . In a cell-free system, both ribozymes cleaved a target piece of MDR1 RNA into 2 fragments at the specific sites at a physiological pH and temperature . The cleavage reaction was dependent on time, ribozyme:substrate ratio, and magnesium concentration . The 196 MDR1 ribozyme was more active than the 179 MDR1 ribozyme . The 196 MDR1 ribozyme was then cloned into a human expression vector, and MOLT-3/TMQ800 cells were transfected . The original MOLT-3/TMQ800 cells were nearly 700-fold resistant to vincristine, whereas the transfectant cells selected in G418 became only 20- to 30-fold resistant . The level of resistance and the amount of MDR1 RNA expressed appeared to correlate inversely with the amount of ribozyme expression . A disabled 196 MDR1 ribozyme was capable of neither specific cleavage in vitro nor decreasing MDR1 expression in transfectant cells . These results indicate that it was the ribozyme activity and not antisense activity which was responsible for decreased MDR1 RNA . This approach may be applicable to cancer patients as a specific means to reverse tumors with P-glycoprotein-mediated MDR phenotype back to a drug-sensitive one.

Blood, 1994 Mar 1, 83(5), 1337 - 47
Synergistic reversal of multidrug-resistance phenotype in acute myeloid leukemia cells by cyclosporin A and cremophor EL; Ross DD et al.; Cremophor (Crem) EL, the vehicle for intravenous delivery of cyclosporin A (CsA), has been reported to counteract multidrug resistance (MDR) in P-glycoprotein (Pgp)-over-expressing cell lines . Because of this, we sought to determine whether Crem functions independently as a modulator of MDR in blast cells obtained from acute myelogenous leukemia (AML) patients, and the nature of its interaction in combination with CsA in reversing an MDR phenotype . In the phenotypically classical MDR AML cell lines HL-60/Vinc (overexpresses Pgp) or HL-60/AR (does not overexpress Pgp), the dose causing half-maximum enhancement (D50) of daunorubicin (DNR, 1 micrograms/mL, 3 hours) accumulation was achieved by the combination of CsA and Crem (CsA/Crem) at 1.2 mumol/L CsA . In contrast, the D50 for Crem alone was approached at an amount that would be needed to suspend 6.2 mumol/L CsA for HL-60/Vinc, and 81 mumol/L CsA for HL-60/AR . The D50 concentrations for CsA alone (dissolved in ethanol, which does not alter DNR accumulation) were also higher than those for CsA/Crem, being 6.5 mumol/L for HL-60/Vinc, and 3.1 mumol/L for HL-60/AR . The maximum absolute level of enhancement of DNR accumulation (Emax) in each cell line was approximately equivalent for CsA/Crem or CsA alone, and was equal to the 3 hr intracellular DNR accumulation observed in parental, drug sensitive HL-60/W cells . For Crem alone, HL-60/AR and HL-60/Vinc cells showed markedly different responses: HL-60/Vinc cells attained intracellular DNR content comparable to HL-60/W, whereas HL-60/AR cells achieved only approximately 35% of this level . Multiple-drug effects were analyzed by calculation of the Combination Index (Chou and Talalay, Adv Enzyme Regul 22:27, 1984), which indicated that CsA and Crem are synergistic in causing enhancement of DNR accumulation in these MDR HL-60 cell lines . In blasts from AML patients, 5 mumol/L CsA/Crem or an equivalent amount of Crem alone each caused significant (P < .001) enhancement of DNR accumulation (60 AML-patient marrow samples) or DNR retention (51 AML-patient marrows) . Similarly, CsA/Crem or Crem alone caused significant (P < .01) enhancement of the cytotoxicity of DNR in 36 AML blast cell specimens . The degree of enhancement of accumulation/retention or cytotoxicity by CsA/Crem was approximately equivalent to that obtained with Crem alone . These studies indicate that Crem can reverse an MDR phenotype in patient AML blast cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Trop Med Parasitol, 1994 Mar, 45(1), 45 - 6
In vitro activity of the enantiomers of N-desbutyl derivative of halofantrine; Basco LK et al.; The in vitro activity of the enantiomers of N-desbutylhalofantrine, the major human metabolite of halofantrine, was compared using the semi-microtest against the multidrug-resistant Plasmodium falciparum FCM 29/Cameroon clone . The mean 50% inhibitory concentration (IC50) values (+/- standard deviation) of the enantiomers were equivalent (2.07 +/- 0.41 and 1.70 +/- 0.33 nmol/L) . The enantiomers of the metabolite of halofantrine, as well as those of the parent compound, have the same antimalarial activity . Since (+)-halofantrine and enantiomer-1 of the metabolite attain higher plasma concentrations in man, our study suggests that these enantiomers may be more active in vivo.

Br J Haematol, 1994 Mar, 86(3), 547 - 54
Characterization and modulation of drug transport kinetics in K562 c1.6 daunorubicin-resistant cell line; Jiang XR et al.; The effects of cyclosporin A (CSA) and cellular energy depletion on daunorubicin (DAU) transport kinetics were investigated in a human erythroid leukaemia cell line K562 c1.6 selected for resistance to daunorubicin . K562 c1.6/DAU resistant cells displayed high levels of P-glycoprotein and a high level of multidrug resistance against several antitumour drugs . The resistance factors of K562 c1.6/DAU cells to DAU, doxorubicin, vinblastine and etoposide were 106, 114, 85 and 13 respectively . A 1.6-fold decrease (P < 0.01, n = 8) in DAU accumulation and a 4-fold increase (P < 0.001, n = 8) in DAU efflux were shown in the resistant cells when compared to K562 c1.6 drug-sensitive parental cells . K562 c1.6/DAU cells were also shown to reach a DAU saturation level (SL) 8-fold faster (P < 0.001, n = 8) than the parental cells . Addition of CSA to the resistant cells led to a dose-dependent increase in cellular DAU retention, while no such effect was observed in the sensitive cells by the introduction of CSA . Resistance to the antitumour drugs could be reduced to various extents by CSA . The patterns of changes and modulations of DAU transport kinetics, as well as chemosensitivity in K562 c1.6/DAU cells were found to be similar to a vinblastine-resistant leukaemia cell line CEM/VLB100 . However, K562 c1.6/DAU cells were more resistant to DAU, doxorubicin and etoposide than the CEM/VLB100 cells . An increase in DAU accumulation, intracellular SL and the time to reach 90% saturation level (SL90), and a decrease in DAU efflux in the resistant but not the sensitive cells were found in response to ATP depletion by sodium azide . These effects could be completely reversed by addition of glucose . Our results suggest that the presence of an energy-dependent effluxing mechanism responsible for the decreased drug accumulation and enhanced drug efflux may make a major contribution to the mechanism of resistance in K562 c1.6/DAU resistant cells.

Anticancer Res, 1994 Mar-Apr, 14(2A), 433 - 5
Molecular interrelationships in multidrug resistance (review); Kellen JA; A brief review of the "state of the art" (1993) knowledge concerning the interrelationships of oncogenes and tumour suppressor gene p53 in multidrug resistance is presented . Gene products which induce malignant transformation and uncontrolled proliferation may play a role in activating gene(s) involved in multidrug resistance . In view of the fact that drug resistance remains the major reason for chemotherapy failure in cancer therapy, a better understanding of the genetic connections which influence the final outcome of cancer could help rationalizing treatment.

Anticancer Res, 1994 Mar-Apr, 14(2A), 397 - 403
Sensitivity of multidrug-resistant MCF-7 cells to a transferrin-doxorubicin conjugate; Lemieux P et al.; Two multidrug-resistant breast cancer cell lines (MCF-7/AdrVp and MCF-7/D.40) each expressing a different membrane protein, involved in the drug resistance, have been treated with a transferrin-doxorubicin conjugate . Conjugates have shown an increase of activity over free doxorubicin on these resistant cell lines . Growth inhibition of doxorubicin-resistant cells, as evaluated by the MTT-assay, was higher for conjugates than for free doxorubicin especially for a 4-day contact period . I D 50 were twice and 10-fold lower for the conjugate than for free doxorubicin on resistant cells . MCF-7/AdrVp seemed to be particularly affected by the conjugate even if its intracellular content of doxorubicin was similar . With the Trf-Dox conjugate, an inverted correlation does exist between the drug-DNA content and the cytotoxicity of the conjugate . Verapamil influenced the uptake of free doxorubicin but not the uptake of Trf-Dox conjugate, thus showing a different mechanism of entry.

Lung Cancer, 1994 Mar, 10 Suppl 1, S67 - 72
Fundamental bases of combined therapy in lung cancer: cell resistance to chemotherapy and radiotherapy; Duchesne GM; This overview briefly examines the mechanisms of drug resistance in lung cancer, including multidrug resistance and its atypical phenotypes, the role of cytoplasmic protectors such as glutathione, and resistance at the level of the DNA through topoisomerases, gene amplification or mutation, and DNA repair . Understanding of radioresistance is less advanced, but resistance may arise through limitation of the amount of DNA damage inflicted or by its subsequent modification by intracellular protectors or DNA repair . The mechanisms of radioresistance are generally distinct from those of chemoresistance providing a rationale for the use of combined modality therapy.

Haematologica, 1994 Mar-Apr, 79(2), 119 - 26
p170-dependent multidrug resistance . Restoring full sensitivity to idarubicin with verapamil and cyclosporin A derivatives; Michieli M et al.; BACKGROUND . Cell sensitivity to anthracyclines and other drugs depends on several factors, including overexpression of a 170Kd transmembrane glycoprotein (P170) that enhances drug efflux from the cells . Since the result of treatment is negatively related to the expression of P170 in leukemia, malignant lymphoma and other tumors, it is important to investigate drugs and methods that can modify multidrug resistance (MDR) . MATERIALS AND METHODS . Using an MTT-microcultured tetrazolium colorimetric method, we assayed sensitivity to daunorubicin (DNR) and to its 4-demethoxy derivative idarubicin (IDA) in two MDR cell lines (CEM VLB and LOVO DX) and in their respective non-MDR parental lines (CEM and LOVO 109), with and without three MDR modifiers, namely the D-isomer of verapamil (DVRP), cyclosporin A (CyA) and the new CyA derivative SDZ PSC 833 . RESULTS . We showed that down-modulation of resistance with MDR modifiers was greater for DNR than for IDA in MDR cells . However, we also demonstrated that restoration of full sensitivity could only be achieved for IDA, not for DNR . DVRP and CyA in combination were more effective than either compound alone and could abolish P170-related resistance to IDA at concentrations of 1-2 microM and 1.6 microM, respectively . SDZ PSC 833 alone was even more effective and set MDR to zero at a concentration ranging between 0.8 and 1.6 microM . CONCLUSIONS . These data suggest that combinations of IDA and MDR modifiers may improve the results of cancer and leukemia treatment and that they are worth investigating in vivo, with attention to possible effects on drug pharmacokinetics and on normal tissue damage.

Br J Haematol, 1994 Mar, 86(3), 540 - 6
Quantitation of multidrug resistant MDR1 transcript in acute myeloid leukaemia by non-isotopic quantitative cDNA-polymerase chain reaction; Lyttelton MP et al.; Drug resistance in acute myeloid leukaemia (AML) may be caused by overexpression of the P glycoprotein (PGP), an efflux pump encoded by the multidrug resistance mdr 1 gene . Previous studies have suggested that increased PGP expression in the leukaemic blasts is of prognostic significance, and that use of PGP antagonists may be beneficial in treatment . We describe preliminary results with a non-isotopic quantitative MDR 1 cDNA-PCR assay, using an artificial RNA construct sharing primer recognition sites with the target MDR 1 mRNA (MDR 1 nucleic acids 483-504 and 624-644) as an internal control . KB 3.1 parent and KB 8.5 MDR positive cell lines expressed 0.004 and 1.96 molecules MDR 1 mRNA/pg total RNA . Semiquantitative screening of 60 RNA samples from 53 AML cases detected MDR 1 transcript ranging from 0 to 1.81 molecules per pg RNA . The median value at presentation (33 patients) was 0.055 and was higher in 14 patients at relapse (0.13) and in seven patients with refractory disease (0.14) . Quantitation of MDR 1 transcript in serial samples in seven treated patients between presentation and relapse showed the decrease in three patients (0.18-0.02 x) to be as marked as the increase in three other patients (3-16 x) . The method described is well suited for the study of clinical samples because it is sensitive, specific, rapid and requires small amounts of clinical material.

Anticancer Res, 1994 Mar-Apr, 14(2A), 581 - 5
Inhibition of rhodamine 123 secretion by cyclosporin A as a model of P-glycoprotein mediated transport in liver; Stapf V et al.; The interaction between P-glycoprotein modulators and P-glycoprotein mediated transport was investigated using rhodamine 123 in the isolated perfused rat liver of a mutant (TR-) rat strain . TR- rats, deficient in the canalicular multispecific anion transport system, are unable to extrude organic anions (glucuronides) and therefore excrete solely unconjugated rhodamine 123 via P-glycoprotein . Cyclosporin A, a modulator of multidrug resistance in tumor cells, inhibited the biliary secretion of rhodamine 123 dose dependently in a non-competitive manner . Both cyclosporin A and rhodamine inhibited photoaffinity labeling of immunoprecipitated P-glycoprotein with azidopine, indicating binding to hepatic P-glycoprotein . Our results indicate that monitoring the biliary rhodamine 123 secretion in the isolated perfused liver of TR- rats offers a new system for testing modulators of P-glycoprotein like cyclosporin A.

Anticancer Res, 1994 Mar-Apr, 14(2A), 571 - 6
Multidrug resistance of murine leukemia cells characterization and correlation with cytochrome P-450 dependent activities, cytosolic calcium and cell cycle state; Pfeil D et al.; This paper reports studies on P388 leukemic cells sensitive and resistant to ADR and VCR . The P450 dependent enzyme system and the cytosolic calcium were estimated and are discussed in relation to MDR . It could be shown, in comparison to sensitive P388 cells, that the plasma membrane permeability for fluorescent dyes like rhodamine 123 and fura-2 and the biotransformation of xenobiotics are changed in resistant cells . Whereas the transport behaviour for the dyes is similarly induced in resistant cells independent of the drug which made the MDR, the P450 dependent enzyme activities are strongly increased in P388/ADR in comparison to the P388 and P388/VCR . The cell cycle analysis shows the same effect . Many more cells of P388/ADR are in the S-phase in comparison to P388 or P388/VCR . The LI is also increased in this direction . Therefore, it can be concluded that, depending on the kind of drug which made the MDR, different biochemical mechanisms are activated.

Yeast, 1994 Mar, 10(3), 377 - 83
Mapping and sequencing of two yeast genes belonging to the ATP-binding cassette superfamily; Dean M et al.; ATP-binding cassette (ABC) transporters share significant sequence identity within their ATP-binding domains . Degenerate oligonucleotides based on highly conserved portions of the ATP-binding domain genes were used to clone portions of two members of the ABC gene superfamily from Saccharomyces cerevisiae DNA . These genes were designated MDL1 and MDL2 (for multidrug resistance-like) . Each MDL gene is predicted to encode a single set of transmembrane domains and a single ATP-binding domain, thus the MDL gene products are 'half-molecule' ABC proteins . The two genes were mapped to precise regions on chromosomes XII and XVI and show a considerable similarity to the mammalian P-glycoprotein/multidrug resistance (MDR) and peptide transporter (TAP) genes . Preliminary analysis of null mutants constructed by gene replacement has indicated that the MDL genes are not essential for viability of yeast . The sequences have been deposited in the GenBank data library under Accession Numbers L16958 (Locus YSCBCSA) and L16959 (Locus YSCBCSB).

Cell Mol Biol (Noisy-le-grand), 1994 Mar, 40(2), 137 - 45
Actinomycin D causes multidrug resistance and differentiation in a human rhabdomyosarcoma cell line; Melguizo C et al.; The emergence of drug-resistant tumor cells remains a major problem in cancer chemotherapy . Resistance to multiple unrelated antineoplastic drugs may be related, in part, to expression of the P-glycoprotein . The cell line RD, derived from an embryonic rhabdomyosarcoma tumor, was used as an in vitro model to examine the development of drug resistance . A cell line resistant to actinomycin D (RD-DAC) was developed by growing RD in increasing concentrations of the drug . The ID50 (concentration of drug needed to induce a 50% reduction in cell growth) of the resultant line to actinomycin D was more than 15 times that of the parental line . The resistant line was cross-resistant to vincristine and doxorubicin . Resistance to actinomycin D resulted in increased P-glycoprotein expression, which was associated with a change in desmin and vimentin expression . These results suggest that exposure to chemotherapeutic drugs can induce not only classical multidrug resistance, but also a process of cellular differentiation in rhabdomyosarcoma cells.

Bull Math Biol, 1994 Mar, 56(2), 207 - 23
A mathematical model for the inhibition of the multidrug resistance-associated P-glycoprotein pump; Michelson S et al.; An extension of an earlier model of the p170 glycoprotein pump is presented . In an earlier work (Michelson and Slate, Bull . math . Biol . 54, 1023-1038, 1992), the pump was modeled using an energy-dependent model of facilitated diffusion . In this paper we add an inhibitor to the model . New equations are derived which represent either competitive or non-competitive inhibition in the pumping action of the glycoprotein . Numerical simulations were run which provide a response surface (initial loading concentration of inhibitor and its ability to compete with an ideal anti-cancer drug vs a summary measure of cytoplasmic exposure) for each scenario . The importance of the exposure profile, how it is related to ultimate tumor cell survival, and the binding requirements for developing multidrug resistance inhibitors are discussed.

Hokkaido Igaku Zasshi, 1994 Mar, 69(2), 354 - 71
{Features of filamentous cytoskeletons in acquired multidrug-resistance of HL-60 human leukemia cell line}; Kawamura K; Acquired resistance to chemotherapeutic drugs by tumor cells is an important obstacle to effective therapy of human malignancy . These resistance cell lines originated from human or rodent have been characterized by increased expression of MDR (Multidrug-resistance) gene and P-glycoprotein which plays as efflux pump of drugs from cells . These multidrug-resistance sublines also have been reported increased activities of protein kinases and glutathione S-transferase-pi . Although there have been extensive biophysical and biochemical characterization of the differences between parental lines and MDR tumor cell sublines, morphologic observations have been limited . In this study, filamentous cytoskeletons which involve many biological phenomena such as maintenance of cell morphology, mitosis, cellular movement, transport, and adhesion, were observed by confocal laser microscopy . To compare the expression of each cytoskeletons, fluorescent intensities of cells stained for each cytoskeletons were measured by confocal laser microscopic system . Utilizing this methodology, higher microtubular expression was observed in HL-60/ADR and K562/ADR than in their parental lines, but no significant differences of actin and vimentin were observed . Phosphorylation by protein kinases has been established as a key factor in the regulation of cytoskeletal function . But little is known about the role of protein phosphorylation in cytoskeletal function . Since increased activities of PKC and PTK were detected in HL-60/ADR, the effect of PKC inhibitor, staurosporine (STR), or PTK inhibitor, genistein (GNS), on cell growth was detected . STR and GNS reduced the resistance to Adriamycin in HL-60/ADR . Furthermore, STR and GNS disrupted the filamentous structure of microtubules in HL-60/ADR, and suppressed the expression of microtubules to 37%, and 49%, respectively . In contrast, PKC activator, phorbol ester (TPA), caused stronger microtubular assembling in HL-60/ADR, and increased the expression of microtubules to 134% . Resulting from this study, it is likely that acquired MDR of HL-60 and K562 was associated with increased expression of microtubules, and microtubular assembling or disassembling was considered to be regulated in part by PKC and PTK.

Leukemia, 1994 Mar, 8(3), 388 - 94
Prognostic value of cell marker analysis in de novo acute myeloid leukemia; Del Poeta G et al.; Clinical and cytologic characteristics were correlated to immunologic markers in 154 patients with newly diagnosed acute myeloid leukemia (AML) . The panel of monoclonal antibodies (MoAbs) was selected to identify differentiation-associated antigens of both the myeloid and the lymphoid lineages (CD13, CD33, CD14, CD15, CD7, CD34, CD10, HLA-DR, CD19, CD2, CD5, TdT) . The expression of multidrug resistance P-glycoprotein (P-170) was also evaluated in 117 patients . Differences in antigenic expression was observed among the various French-American-British (FAB) subgroups . HLA-DR was poorly expressed on the blasts of acute promyelocytic leukemia (M3), and was always found in FAB M5 . CD34 was detectable in all M0 cases and only in one M3 (p < 0.001) . Lymphoid-associated antigens were positive in 74 cases (48.1%) . In particular, CD7 was found in 49 patients (31.8%), and TdT in 30 (21.3%), 15 samples displaying coexpression of these two antigens . The incidence of CD7+ cases was particularly elevated in M0 and M5 AML (p = 0.005) . It significantly correlated with the expression of CD34, HLA-DR, P-170 (p < 0.001, p = 0.018 and p = 0.034 respectively), and with a leukocyte count > 50 x 10(9)/l (p = 0.038) . Sixty-nine (59%) samples demonstrated P-170 positivity . Again, this phenotype was particularly expressed in the poorly differentiated forms (M5, M0 and M1) and showed significant correlation with the immaturity markers CD34, CD7 and HLA-DR (p = 0.013, p = 0.022 and p = 0.001, respectively) . Expression of individual antigens correlated with prognosis . Refractoriness to first line therapy was associated with CD7 expression (p = 0.002) and P-170 (p = 0.001) . The CD7 marker was also significantly associated with a very low overall survival (p < 0.001) and continuous complete remission (p < 0.001) . CD14 expression also significantly predicted lower survival rates (p = 0.033) . The combination (CD7+ CD14+) identified a subset of patients with a particularly adverse outcome . The prognostic value of CD7 expression, alone or in combination with other markers, was confirmed in multivariate analysis.

J Nucl Med, 1994 Mar, 35(3), 510 - 5
Expression of recombinant human multidrug resistance P-glycoprotein in insect cells confers decreased accumulation of technetium-99m-sestamibi; Rao VV et al.; The multidrug-resistant P-glycoprotein is a M(r) 170,000 plasma membrane protein encoded by the mammalian multidrug resistance gene (MDR) which appears to function as an efflux transporter of a variety of potent chemotherapeutic agents . METHODS: To directly demonstrate that 99mTc-sestamibi is recognized by the human P-glycoprotein, we overexpressed recombinant human MDR1 P-glycoprotein in host Sf9 insect cells using a baculoviral vector and correlated expression of the gene product with 99mTc-sestamibi accumulation . RESULTS: In parental Sf9 cells and in wild-type baculoviral infected (control) cells, 99mTc-sestamibi accumulation asymptotically approached a plateau of 650 fmoles (mg protein)-1 (nMo)-1 and 337 fmoles (mg protein)-1 (nMo)-1, respectively . In MDR1 baculoviral infected cells, P-glycoprotein expression was maximal at 72 hr postinfection, while 99mTc-sestamibi accumulation was reduced to 12 fmole (mg protein)-1 (nMo)-1 . Verapamil (500 microM), the classical MDR modulator, produced an approximately 300% enhancement of 99mTc-sestamibi accumulation in Sf9 cells expressing MDR1 P-glycoprotein, but only a 50% enhancement in parental Sf9 cells, consistent with verapamil-induced inhibition of P-glycoprotein-mediated 99mTc-sestamibi efflux . CONCLUSIONS: These data demonstrate that the recombinant protein is transiently expressed in a functional state capable of drug transport in Sf9 cell membranes and that 99mTc-sestamibi is a transport substrate recognized by the human MDR1 P-glycoprotein . Technetium-99m-sestamibi may prove useful for functionally characterizing P-glycoprotein expression in human tumors in vivo.

Int J Cancer, 1994 Mar 1, 56(5), 749 - 54
Verapamil decreases P-glycoprotein expression in multidrug-resistant human leukemic cell lines; Muller C et al.; We studied the effect of verapamil on Pgp expression (Pgp) in MDR human leukemia cell lines, K562/ADR and CEM VLB100 . In the K562/ADR cell line, addition of verapamil to the culture medium (15 microM concentration) resulted in a 3-fold decrease in Pgp expression after 72 hr exposure . The effect of verapamil was reversible, and Pgp expression reached the level of untreated controls 24 hr after discontinuation of verapamil . Similar results were obtained with the human vinblastine-resistant cell line, CEM VLB100 . On the contrary, no effect on Pgp expression was observed when the cells were treated with nifedipine or diltiazem (2 other calcium-channel blockers), even at doses that inhibited cell proliferation . The level of Pgp mRNA in the presence of verapamil was measured by Northern blot and was also decreased 2-fold (with the maximum reached within 24 hr), suggesting a transcriptional or post-transcriptional mechanism for verapamil . We further established that the effect of verapamil on Pgp expression led to an increase in DNR and VLB accumulation and cytotoxicity . These results suggest that verapamil acts specifically on Pgp expression in these drug-selected leukemic cells . The identification of a potentially novel mechanism of action may provide new insights as to how chemosensitization may be more effectively applied in vivo.

Bull Cancer, 1994 Mar, 81(3), 203 - 11
{Quantitative cytological study of the activity of a new resistance modulator, S 9788, on human leukemic cells using multiparametric image analysis}; Oum'Hamed Z et al.; The triazinoaminopiperidine derivative S 9788 is a new multidrug resistance modulator . The modulating activity of S 9788, comparatively to those of verapamil and the combination of S 9788 and verapamil, was demonstrated on the human leukemic T cell line CCRF-CEM resistant (about 6000 fold) to vinblastine using Microculture Tetrazolium Assay . S 9788 at 5 microM, strongly potentialized the cytotoxic activity of vinblastine but the reversion of resistance remained partial . Verapamil and the combination S 9788-verapamil, tested at equimolar concentrations, were respectively 1000 and two times less active than S 9788 alone . The impact of S 9788, verapamil and their combination on the cytological modifications bound to vinblastine resistance of CEM cells was evaluated by multiparametric quantitative cytological analysis (21 nuclear parameters measured) using a SAMBA 2005 cell image processor . Treatments with the different modulators, in absence or in presence of vinblastine, had no significant effects on the morphology of sensitive CEM cells . On vinblastine resistant CEM cells, S 9788 and the combination S 9788-verapamil induced significant cytological modifications . These modifications were characterized by a partial reversion of some parameters (more specifically nuclear texture parameters) to values close to those observed in parental sensitive cells and permitted an automatic classification of these treated resistant cells in cells of "sensitive" type with a percentage superior to 50% . In conclusion, the reversion of resistance induced by S 9788 on CEM cells resistant to vinblastine does not fit only with a biological phenomenon like the efflux of cytotoxic agents but is associated with a set of cellular alterations involved in multidrug resistance.

Curr Biol, 1994 Mar 1, 4(3), 259 - 60
P-glycoprotein . To flip or not to flip?
Higgins CF.
The phenotype of mice homozygous for mutations of the mdr2 gene suggests that the mdr2 protein, which is closely related to the multidrug resistance P-glycoprotein, has a role in phospholipid transport.

Cancer Res, 1994 Mar 1, 54(5), 1355 - 9
Expression of multidrug resistance gene and localization of P-glycoprotein in human primary ovarian cancer; Arao S et al.; Resistance to chemotherapy is the major obstacle to controlling malignant tumors . To characterize multidrug resistance phenotype in human primary ovarian cancer without chemotherapy, expressions of the mdr1 gene in 52 cases of ovarian cancer (44 common epithelial, 5 nonepithelial, and 3 metastatic cancers) were analyzed by polymerase chain reaction of RNA after reverse transcription . Furthermore, localization of P-glycoprotein, which is encoded by the mdr1 gene, was studied immunohistochemically . Although overall expression of the mdr1 gene was relatively low, its expression level was the highest in well-differentiated cancer tissues . Serous and mucinous adenocarcinomas showed higher levels of expression compared with clear cell and endometrioid carcinomas . P-glycoprotein was positive on luminal surfaces of lining cells of ovarian cancer and on those of inclusion cysts from which epithelial ovarian cancer is considered to develop . Thus, some ovarian cancer cases before chemotherapy are intrinsically multidrug resistant, which can be determined by mdr1 gene expression, and this phenotype should be taken into account for effective chemotherapy of ovarian epithelial carcinomas.

J Biol Chem, 1994 Feb 25, 269(8), 6133 - 9
Diversity of multidrug resistance in mammalian cells; Devine SE et al.; Mammalian cells displaying the multidrug resistance (mdr) phenotype are refractory to the toxic effects of a group of unrelated natural product drugs, many of which are used for cancer chemotherapy . The pattern of cross-resistance can be extremely variable among independently selected cell lines, even though such cells are often exposed to only a single drug . The overexpression of P-glycoprotein (pgp), a 150-180-kDa drug efflux pump, has been shown to confer mdr to otherwise drug-sensitive cells; however, the variable nature of cross-resistance indicates that normal pgps alone are unlikely to account for all of the observed cross-resistance phenotypes . In this report, we examined possible factors contributing to cross-resistance diversity in mammalian cells . We show that drug-resistant Chinese hamster lung cells selected during relatively short periods of drug exposure in vitro (less than 6-8 weeks) routinely overexpressed endogenous pgps and predominantly showed a cross-resistance pattern that was similar to that conferred by the introduction and overexpression of the hamster wild-type pgp1 cDNA alone . Longer drug exposure periods at higher drug concentrations, however, led to the selection of cell lines with altered cross-resistance properties . Like the short term clones, these cell lines all overexpressed endogenous pgp . In one case, the altered phenotype was shown to be caused by the acquisition of point mutations within codons 338 and 339 of the pgp1 gene, leading to two adjacent amino acid substitutions within the encoded pgp . Although the basis for the remaining altered phenotypes remains unknown, these results indicate that additional genetic alterations beyond those responsible for the initial acquisition of mdr emerge in the face of increased selective pressure, thus further modifying or complementing the cross-resistance phenotype initially conferred by wild-type pgp.

Biochim Biophys Acta, 1994 Feb 23, 1190(1), 72 - 84
Interaction of multidrug-resistant Chinese hamster ovary cells with the peptide ionophore gramicidin D; Loe DW et al.; A major form of multidrug resistance results from the overexpression of P-glycoprotein, a 170 kDa membrane protein . Multidrug resistant (MDR) Chinese hamster ovary (CHO) cells and mdrl transfectants displayed cross-resistance to the channel-forming peptide ionophore gramicidin D, which was reversed by various chemosensitizers, thus directly implicating P-glycoprotein as the mediator of resistance . However, gramicidin D was not able to inhibit {3H}azidopine photolabelling of P-glycoprotein . MDR cells were not resistant to other pore-forming ionophores, but showed a modest level of cross-resistance to the mobile ionophore valinomycin . There was no difference in 125I-gramicidin D uptake by resistant and sensitive cells . Resistant cells showed lower 86Rb+ uptake, relative to the drug-sensitive parent . Addition of GmD increased both the rate and the level of 86Rb+ uptake in sensitive cells, but had no effect on MDR cells . MDR cells also showed much lower rates of gramicidin D-dependent 86Rb+ efflux than sensitive cells, and this was greatly increased by verapamil . These results suggest that P-glycoprotein interferes with the formation of ion-conducting gramicidin D channels . In contrast, valinomycin had the same effect on gramicidin D-dependent cation efflux in MDR and sensitive cells . Gramicidin D is thus unique among the ionophores is being a substrate for P-glycoprotein, which appears to greatly reduce the formation of active dimeric channels in the plasma membrane of MDR cells.

Proc Natl Acad Sci U S A, 1994 Feb 15, 91(4), 1275 - 9
Consequences of altered isoprenylation targets on a-factor export and bioactivity; Caldwell GA et al.; Cysteine-containing amino acid sequences (CAAX, CC, and CXC; C is cysteine, A is any aliphatic amino acid, and X is any amino acid) are targets for the attachment of C15 (farnesyl) and C20 (geranylgeranyl) isoprenoids to peptides and proteins by specific prenyltransferases . Although much work has centered on the enzymatic mechanisms of these enzymes, the biological consequences of the differential isoprenylation they catalyze remain to be elucidated . Farnesylation of the a-factor mating pheromone of Saccharomyces cerevisiae is a known prerequisite for its biological activity and its secretion through a pathway utilizing the yeast STE6 protein, a homolog of the mammalian multidrug resistance (MDR) P-glycoprotein . We generated specific mutations in the a-factor gene to encode isoprenylation targets for geranylgeranylation {Cys-Val-Ile-Leu (CVIL) and Ser-Val-Cys-Cys (SVCC)} in place of the natural farnesylation motif {Cys-Val-Ile-Ala (CVIA)} . The a-factors containing these modified prenylation sites were successfully exported by a STE6-dependent mechanism . Furthermore, these peptides, as well as synthetic geranylgeranyl a-factor, retained bioactivity . Chromatographic comparisons of synthetic and biosynthetic pheromones suggest that, in vivo, a peptide substrate containing the geranylgeranylation target CVIL can be both farnesylated and geranylgeranylated . These results clearly demonstrate that in vivo (i) different prenyltransferases may recognize the same substrate; (ii) both farnesylated and geranylgeranylated a-factor peptides are substrates for export via STE6, a MDR-like protein; and (iii) farnesylated and geranylgeranylated pheromones are both biologically active.

J Biol Chem, 1994 Feb 11, 269(6), 4180 - 6
Molecular cloning and expression of the Saccharomyces cerevisiae STS1 gene product . A yeast ABC transporter conferring mycotoxin resistance; Bissinger PH et al.; We have cloned a yeast gene that confers a multidrug resistance phenotype on Saccharomyces cerevisiae when present in multiple copies . The STS1 (for Sporidesmin Toxicity Suppressor) gene encodes a 1511-residue protein whose predicted structural organization is characterized by 12 alpha-helical membrane segments and two domains containing consensus sites for ATP binding, indicating that STS1 is a new yeast ATP-binding cassette (ABC) transporter . A chromosomal deletion of STS1 leads to viable delta sts1 cells of both mating types, suggesting that STS1 is not essential for cell growth . However, delta sts1 cells exhibit supersensitivity to sporidesmin and to other structurally unrelated drugs such as cycloheximide . Conversely, overexpression of STS1 leads to increased resistance to the same drugs . Although Northern analysis showed that STS1 mRNA is present in all yeast cell types, its drastically reduced level in alpha-factor-arrested cells indicates that expression of STS1 is regulated by mating pheromones . Subcellular fractionation and immunoblotting using monoclonal antibodies, which recognize a fully functional epitope-tagged Sts1 protein, showed that Sts1 is a 175-kDa membrane protein localized mainly to intracellular membranes.

J Biol Chem, 1994 Feb 4, 269(5), 3745 - 54
ATPase activity of purified and reconstituted P-glycoprotein from Chinese hamster ovary cells; Shapiro AB et al.; P-glycoprotein was purified from multidrug-resistant Chinese hamster ovary CHRB30 cells by a combination of anion exchange and immunoaffinity chromatography . The P-glycoprotein was about 90% pure and had a Vmax for ATP hydrolysis in detergent solution of 321 nmol/min/mg with a Km of 0.94 mM . The ATPase activity was inhibited by low concentrations of vanadate and N-ethylmaleimide, but unaffected by azide or ouabain . When the purified P-glycoprotein was reconstituted into phospholipid bilayer membranes, the ATPase activity became highly stimulated by several chemosensitizers and drugs involved with multidrug resistance . Verapamil, a potent chemosensitizer, increased the Vmax for ATP hydrolysis by 22-fold and the Km for ATP by 5.4-fold . This effect of verapamil on P-glycoprotein has not previously been observed . These results demonstrate that purified P-glycoprotein has an intrinsic ATPase activity with unique properties . This activity appears sufficient to account for the ATP-dependent reduction in intracellular drug accumulation of P-glycoprotein-expressing multidrug-resistant cells.

Am J Clin Oncol, 1994 Feb, 17(1), 10 - 3
A phase II trial of vinblastine plus dipyridamole in advanced renal cell carcinoma . A Hoosier Oncology Group Study; Murphy BR et al.; One potential explanation for why renal cell carcinoma is usually poorly responsive to chemotherapy is intrinsic multidrug resistance . Dipyridamole (DP) is one of several agents known to bind to P-glycoprotein and potentially reverse multidrug resistance in vitro, and dosages needed to obtain appropriate levels in vivo appear to be well tolerated . The current study was undertaken to evaluate the combination of vinblastine (VLB) and DP in patients with advanced renal cell carcinoma and no prior chemotherapy . From August 1989 through December 1989, 15 patients with inoperable recurrent or metastatic renal cell carcinoma were treated with VLB 0.2 mg/kg (i.v . slow push) and concurrently received DP75 mg p.o . q.i.d . starting 48 hours before and continuing 48 hours after VLB administration . Courses were repeated every 3 weeks for a maximum of 8 weeks, or until disease progression or undue toxicities ensued . The predominant toxicities seen were mild neurotoxicity and leukopenia . Only 1 patient had grade IV leukopenia, and no lethal toxicities occurred . No objective responses were seen; 2 patients had stable disease for 29 and 30+ months . The median survival was 9 months (range: 2.5-30+) . We conclude that at the dose and schedule used in this study, the combination of VLB and DP may be administered with acceptable toxicities, but is ineffective in the treatment of advanced renal cell carcinoma.

Cancer Res, 1994 Feb 1, 54(3), 756 - 62
Altered subcellular distribution of topoisomerase II alpha in a drug-resistant human small cell lung cancer cell line; Feldhoff PW et al.; A drug-resistant human small cell lung cancer cell line, H209/V6, selected in the presence of increasing concentrations of 9-(4,6-O-ethylidene-beta-D-glucopyranosyl)-4'-demethylepipodophylloto xin (VP-16) from parental H209 cells, is 22-, 9-, and 4-fold resistant to VP-16, 4'-(9-acridinyl-amino)methanesulfon-m-anisidide, and doxorubicin, respectively, but not cross-resistant to 1,4-dihydroxy-5,8-bis((2-{(2-hydroxyethyl)amino} ethyl}-amino)-9,10-anthracenedione . These cells do not overexpress P-glycoprotein or the multidrug resistance-associated protein . Immunoblotting demonstrates that H209 cells contain the M(r) 170,000 isoform of topoisomerase II (topo II), while H209/V6 cells have a M(r) 160,000 enzyme but none of the M(r) 170,000 isoform . The cell lines have equal amounts of topo II beta . The H209/V6 cells have a 5-fold decrease in total immunoreactive topo II alpha . The catalytic and VP-16-induced DNA cleavage activities of the topo II present in 0.35 M NaCl nuclear extracts are decreased 2- to 3-fold in the drug-resistant cell line . This decrease in enzymatic activity is not consistent with either the 22-fold VP-16 resistance of the H209/V6 cell line or the approximately 5-fold decrease in immunoreactive topo II alpha in the cells . The M(r) 160,000 isoform from the H209/V6 cell line and the M(r) 170,000 enzyme from the parental cell line were purified so that the enzymatic activity of the 2 isoforms could be evaluated . The catalytic activities of the purified isoforms were found to be very similar . The drug-induced DNA cleavage activity of the M(r) 160,000 enzyme was reduced compared to the M(r) 170,000 enzyme . However, as with the nuclear extracts, the differences in enzymatic activity of the purified enzymes are considerably less than the level of drug resistance . Investigations of the subcellular localization of topo II by immunocytochemical techniques and cytoplasm/nuclear fractionation studies demonstrated that the M(r) 160,000 topo II alpha-related enzyme is primarily localized in the cytoplasm, while the M(r) 170,000 topo II alpha enzyme and topo II beta are located in the nucleus . These data imply that the deleted sequence in the M(r) 160,000 enzyme is not necessary for catalytic activity but is required to facilitate nuclear localization.

Proc Natl Acad Sci U S A, 1994 Feb 1, 91(3), 1143 - 7
Selection for mefloquine resistance in Plasmodium falciparum is linked to amplification of the pfmdr1 gene and cross-resistance to halofantrine and quinine; Cowman AF et al.; Two chloroquine-resistant cloned isolates of Plasmodium falciparum were subjected to mefloquine selection to test if this resulted in alterations in chloroquine sensitivity and amplification of the pfmdr1 gene . The mefloquine-resistant lines derived by this selection were shown to have amplified and overexpressed the pfmdr1 gene and its protein product (Pgh1) . Macrorestriction maps of chromosome 5, where pfmdr1 is encoded, showed that this chromosome has increased in size in response to mefloquine selection, indicating the presence of a gene(s) in this area of the genome that confers a selective advantage in the presence of mefloquine . Concomitant with the increase in mefloquine resistance was a corresponding increase in the level of resistance to halofantrine and quinine, suggesting a true multidrug-resistance phenotype . The mefloquine-selected parasite lines also showed an inverse relationship between the level of chloroquine resistance and increased pfmdr1 gene copy number . These results have important implications for the derivation of amplified copies of the pfmdr1 gene in field isolates, as they suggest that quinine pressure may be involved.

Proc Natl Acad Sci U S A, 1994 Feb 1, 91(3), 1128 - 32
Intracellular pH and the control of multidrug resistance; Simon S et al.; Many anticancer drugs are classified as either weak bases or molecules whose binding to cellular structures is pH dependent . Accumulation of these drugs within tumor cells should be affected by transmembrane pH gradients . Indeed, development of multidrug resistance (MDR) in tumor cells has been correlated with an alkaline shift of cytosolic pH . To examine the role of pH in drug partitioning, the distribution of two drugs, doxorubicin and daunomycin, was monitored in fibroblasts and myeloma cells . In both cell types the drugs rapidly accumulated within the cells . The highest concentrations were measured in the most acidic compartments--e.g., lysosomes . Modifying the cellular pH in drug-sensitive cells to mimic reported shifts in MDR caused an immediate change in the cellular drug concentration . Drug accumulation was enhanced by acidic shifts and reversed by alkaline shifts . All of these effects were rapid and reversible . These results demonstrate that the alkaline shift observed in MDR is sufficient to prevent the accumulation of chemotherapeutic drugs independent of active drug efflux.

Br J Cancer, 1994 Feb, 69(2), 315 - 9
L1210 cells selected for resistance to methoxymorpholinyl doxorubicin appear specifically resistant to this class of morpholinyl derivatives; Geroni C et al.; We investigated the mechanism of resistance in murine L1210 leukaemia cells selected after treatment with FCE 23762 methoxymorpholinyl doxorubicin: (MMRDX), a methoxymorpholinyl derivative of doxorubicin active in vitro and in vivo on multidrug-resistant (mdr) cells, currently undergoing phase I clinical trials . The resistant subline obtained after repeated in vitro treatments, L1210/MMRDX, is resistant in vitro and in vivo to all tested methoxymorpholinyl derivatives and to cyanomorpholinyl doxorubicin, but shows resistance to morpholinyl derivatives only in vivo or following their activation with rat S9-liver fractions in vitro . L1210/MMRDX cells are sensitive to classic mdr- and altered topoisomerase (AT)-mdr-associated drugs . These cells do not appear to overexpress the mdr1 gene, nor do they exhibit impaired intracellular drug accumulation and efflux or altered levels of glutathione and glutathione S-transferase . The extent of DNA single-strand break formation and, after microsomal activation, of DNA interstrand cross-links after treatment with MMRDX was similar in the parent and the resistant subline . The mechanism of resistance in L1210/MMRDX cells remains to be identified but may prove a novel one, highly specific for this class of mdr-active anthracyclines.

J Biochem (Tokyo), 1994 Feb, 115(2), 213 - 8
ATP-dependent uptake of anti-neoplastic agents by acidic organelles; Moriyama Y et al.; Daunomycin, an anti-neoplastic agent, is known to be sequestered by acidic organelles in normal and multidrug-resistant cells {Willingham, M.C., Cornwell, M.M., Cardarelli, C.O., Gottesman, M.M., & Pastan, I . (1986) Cancer Res . 46, 5941-5946} . We studied the mechanism of accumulation of daunomycin into acidic organelles using chromaffin granule vesicles and proteoliposomes reconstituted with purified F-type H(+)-ATPase as model systems . Radiolabeled daunomycin was taken up by chromaffin vesicles upon addition of ATP . Its ATP-dependent uptake was stimulated about 1.4- to 1.8-fold by valinomycin plus K+, but was inhibited by ammonium chloride (10 mM) and nigericin plus K+ . Quinidine (5 microM), verapamil (5 microM), or vanadate (0.5 mM), inhibitors of P-glycoprotein, had no effect on its uptake . Daunomycin was also taken up by liposomes reconstituted with F-type H(+)-ATPase . Furthermore, doxorubicin and vinblastine were taken up by these vesicles, whereas colchicine and rhodamine 123 were not . The accumulations of daunomycin and doxorubicin in acidic organelles of cultured cells were decreased by inhibiting vacuolar ATPase by addition of bafilomycin A1 or concanamycin A, or by increasing the internal pH by addition of nigericin . Melittin and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide dissipated the delta pH and inhibited accumulation of daunomycin in the membrane vesicles and acidic organelles in cultured cells . These results indicate that the delta pH established by vacuolar-type ATPase drives the uptake of daunomycin, doxorubicin or vinblastine into acidic organelles, and that no specific transporters are involved in their uptakes.

Antimicrob Agents Chemother, 1994 Feb, 38(2), 170 - 3
In vivo activity of paromomycin against susceptible and multidrug-resistant Mycobacterium tuberculosis and M . avium complex strains; Kanyok TP et al.; Encouraged by in vitro results, we have assessed the in vivo activity of paromomycin (PRM) against Mycobacterium tuberculosis, multidrug-resistant (MDR) M . tuberculosis (resistant to isoniazid, rifampin, and streptomycin), and Mycobacterium avium complex in C57BL/6 mice and their beige counterparts . In all these experiments, PRM was effective in preventing mortality from a mycobacterial infection and was significantly more active than the drug-free control (P < 0.0005) in reducing the CFU relative to the mean log CFU in the lungs, livers, and spleens of infected animals . In the drug-susceptible M . tuberculosis experiment, PRM given at 100 and 200 mg/kg of body weight was significantly less active than isoniazid at 25 mg/kg (P < 0.0005) in reducing the mean log CFU in the lungs, livers, and spleens of infected mice . In the MDR M . tuberculosis experiment, PRM given at 200 mg/kg was effective, relative to the drug-free control, in reducing the mean log CFU of an isolate of M . tuberculosis resistant to isoniazid, rifampin, and streptomycin . In the M . avium complex experiment, PRM given at 200 mg/kg was as effective as amikacin at 50 mg/kg in reducing the mean log CFU in the lungs, livers, and spleens of infected mice . On the basis of our experiments, we believe that PRM has promising activity in vivo in the treatment of infections caused by M . tuberculosis, MDR M . tuberculosis, and M . avium complex.

Surg Oncol, 1994 Feb, 3(1), 17 - 25
Hepatic arterial infusion of verapamil and doxorubicin with complete hepatic venous isolation and extracorporeal chemofiltration: pharmacological evaluation of reduction in systemic drug exposure and assessment of hepatic toxicity; Fuhrman GM et al.; Tumour resistance to chemotherapeutic drugs through expression of the multidrug resistance phenotype is a major impediment in the treatment of hepatic malignancies . We performed hepatic arterial infusion of verapamil (at a dose known to block P-glycoprotein activity) and doxorubicin combined with complete hepatic venous isolation and extracorporeal chemofiltration in non-tumour-bearing pigs with normal livers to evaluate the pharmacology and toxicology of this drug combination . The complete hepatic venous isolation-chemofiltration system significantly reduced system exposure to both verapamil and doxorubicin (P < 0.01) . Hepatic arterial infusion of verapamil (2 mg/kg) alone did not result in hepatocellular toxicity . However, the combination of verapamil and doxorubicin (3 mg/kg or 5 mg/kg) produced significant elevations in liver enzymes (P < 0.01), and gross histological evidence of liver damage in 90% of the treated animals . The results of this study indicate that hepatic arterial infusion of verapamil and doxorubicin, in an attempt to improve treatment response in unresectable liver tumours expressing the multidrug resistance phenotype, may not be tolerated by patients with limited hepatic reserve.

Jpn J Cancer Res, 1994 Feb, 85(2), 135 - 8
Circumvention of atypical multidrug resistance with tumor necrosis factor; Cimoli G et al.; Some "multidrug-resistant" (MDR) cell lines are not associated with a defect in drug accumulation or with the overexpression of P-glycoprotein . These cell lines are defined as "atypical MDR" (at-MDR) and they often express altered or mutated topoisomerase II . We investigated the ability of tumor necrosis factor to reverse at-MDR (in the human ovarian cancer cell line A2780 DX3) on the basis of its efficacy in potentiating in vitro topoisomerase II-targeted drugs, and because there is convincing evidence that the synergy is due to an increased number of topoisomerase-associated strand-breaks as well as to an increased level of extractable topoisomerase.

Am J Trop Med Hyg, 1994 Feb, 50(2), 187 - 92
Efficacy and tolerance of extended-dose halofantrine for drug-resistant falciparum malaria in Thailand; Watt G et al.; New treatments for malaria are urgently needed in areas such as Thailand where highly drug-resistant strains of Plasmodium falciparum are prevalent . Mefloquine is rapidly losing efficacy and conventional doses of halofantrine are infective . We therefore used pharmacokinetic stimulation to design an extended-dose halofantrine regimen and tested it in 26 soldiers stationed along the Thai-Cambodian border . Halofantrine was given after meals as three doses of 500 mg each at 4-hr intervals on the first day, followed by 500 mg a day for six days (total dose 4.5 g) . Twenty-six soldiers treated with quinine-tetracycline for seven days (Q7T7) served as controls . There were no significant differences in efficacy between halofantrine and Q7T7 (P > 0.1) as assessed by cure rate (92% versus 85%), mean parasite clearance time (82 hr versus 81 hr), or mean fever clearance time (93 hr versus 99 hr) . Halofantrine was better tolerated than Q7T7 . The side effects score was lower (2 versus 11; P < 0.001), there were less days on which side effects occurred (2.0 days versus 5.5 days; P < 0.001), and fewer patients had adverse effects on every treatment day (4% versus 42%; P < 0.01) . High-dose halofantrine is as effective and better tolerated than quinine-tetracycline for multidrug-resistant falciparum malaria.

J Infect Dis, 1994 Feb, 169(2), 467 - 70
Clindamycin in combination with chloroquine or quinine is an effective therapy for uncomplicated Plasmodium falciparum malaria in children from Gabon; Kremsner PG et al.; Multidrug resistance of Plasmodium falciparum is becoming common in Africa . In a randomized trial, four short-term regimens were compared for treating uncomplicated P . falciparum malaria in children 4-15 years old in Gabon . One hundred thirty patients received chloroquine (25 mg/kg over 48 h; group C), chloroquine (as above) plus clindamycin (5 mg/kg every 12 h for 6 doses; group CCl), quinine (12 mg/kg every 12 h for 6 doses; group Q), or quinine (as above) plus clindamycin (as above; group QCl) . In group C, only 9% of patients were cured by day 28, 44% showed recrudescent malaria (RI), and 47% showed intermediate or high-grade resistance (RII/RIII) . In group CCl, 70% of patients were cured and 30% showed recrudescences . In group Q, 32% were cured and 68% showed recrudescences . In group QCl, 88% were cured and 12% showed recrudescences after day 14 . All treatment regimens were well tolerated . Thus, the combination of clindamycin with chloroquine or quinine enhances parasite clearance and improves response to therapy.

Cell Biol Int, 1994 Feb, 18(2), 79 - 84
Effect of pentoxifylline, a multidrug resistance reversal agent, on haemopoietic stem cell homing; Gude RP et al.; This paper investigates the mechanism of mouse haemopoietic stem cell homing into the cytoskeleton depolymerizing agent Pentoxifylline was used, and shown to inhibit stem cell homing . The inhibition was reversible after 6 hours . The results obtained suggest that the haemopoietic stem cell homing receptor is anchored to cytoskeletal support intracellularly.

Semin Diagn Pathol, 1994 Feb, 11(1), 39 - 46
The molecular biology of childhood rhabdomyosarcoma; Parham DM; Rhabdomyosarcomas comprise a biologically heterogeneous group of childhood malignancies that are histologically characterized by varying degrees of differentiation, ranging from uncommitted primitive mesenchymal cells to fetal myotubes . This differentiation pattern is reflected in the behavior of the tumor cells in vitro and can be manipulated by the addition of various biological and chemical agents to the intercellular milieu . Recent studies have documented some of the genetic and phenotypic events that occur under these conditions . In addition, the use of cell lines has facilitated the identification of characteristic karyotypic abnormalities and DNA transcriptional activators that are of potential diagnostic importance . Alterations of tumor-suppressor and multidrug-resistance genes have also been described, and they portend identification of susceptible families and potential therapeutic strategies to deal with this clinically aggressive family of childhood neoplasia . These developments herald an increasing utility of molecular biology in the diagnosis and treatment of pediatric sarcomas and necessitate pathologists' familiarity with this subject.

Gan To Kagaku Ryoho, 1994 Feb, 21(3), 395 - 402
{Enhancement of cellular accumulation of cyclosporine by anti-P-glycoprotein monoclonal antibody MRK-16, and their synergistic modulation of multidrug resistance}; Naito M et al.; Drug resistance is a major obstacle to successful cancer chemotherapy . P-glycoprotein, which transports various antitumor agents outside the resistant tumor cells, plays a key role in multidrug resistance . We found that MRK-16, a monoclonal antibody against P-glycoprotein, and cyclosporine, synergistically enhanced the antitumor effects of vincristine and adriamycin in multidrug-resistant K562/ADM cells . On the other hand, the combined use of MRK-16 with verapamil or FK-506 did not show such synergistic effects . Drug accumulation studies revealed that MRK-16 remarkably increased the accumulation of cyclosporine, but not verapamil, in K562/ADM cells . This increased accumulation of cyclosporine by MRK-16 in K562/ADM cells directly resulted in the enhanced accumulation of vincristine and adriamycin in the cells . The synergistic effect of MRK-16 and cyclosporine was further confirmed by isobologram analysis in three different highly multidrug-resistant tumor cells . Moreover, while MRK-16 alone did not enhance the sensitivity of the KB-8-5 cells moderately resistant to vincristine, it increased two-fold the reversing effect of cyclosporine at 1 microM, an achievable blood concentration . Since MRK-16 alone showed therapeutic effects against multidrug-resistant tumors, the combined use of MRK-16, cyclosporine and antitumor agents would provide therapeutic benefits for the treatment of resistant tumors.

Gan To Kagaku Ryoho, 1994 Feb, 21(3), 307 - 13
{Effect of a new anticancer drug, docetaxel (RP56976), on human leukemia cell lines}; Murata K et al.; The antitumor effect of a new derivative of taxol, docetaxel (RP56976), was examined in K562 human tumor system in vitro . K562 cells presenting a multidrug resistant phenotype showed cross resistance to docetaxel . However, the resistance levels of docetaxel in these cell lines were much lower than for adriamycin and vincristine . Flow cytometric analysis showed the accumulation of cells into G2/M phase after 18hrs . Wright-Giemsa staining showed a marked increase of metaphase population . Docetaxel was shown to promote the assembly of microtubule protein without GTP in vitro, but no inhibitory effect on DNA, RNA and protein synthesis . Moreover, topoisomerase activities were not affected by docetaxel . These results indicate that docetaxel acts as a strong mitotic inhibitor in cancer chemotherapy.

Leukemia, 1994 Feb, 8(2), 327 - 35
Mdr1 gene expression in childhood acute lymphoblastic leukemias and lymphomas: a critical evaluation by four techniques; Brophy NA et al.; Expression of the multidrug resistance gene mdr1 is reported to be an important determinant of responsiveness to therapy and survival in some cancers . Many different methods have been used to evaluate mdr1 expression in these studies . This paper compares four methods for determination of mdr1 expression . We studied the mdr1 gene expression in 36 freshly established cell lines from 28 children with acute lymphoblastic leukemia (16 T-ALL, six BCP-ALL, two B-ALL (L3), two biphenotypic leukemias, two Burkitt's lymphomas) . Leukemic specimens were obtained at the time of diagnosis in 16 cases, and after chemotherapy in 20 cases . In all the samples, mdr1 mRNA was measured by slot blotting and reverse transcriptase polymerase chain reaction (rt-PCR), and the presence of the mdr1 product, P-glycoprotein, was detected by immunohistochemistry with the MRK-16 monoclonal antibody . In situ mdr1 RNA hybridization was performed in 30 cases . Complete agreement was noted between all the techniques in 14 cases (39%) . Results differed on a single test result in another 39% of the cases . These 78% of cases were considered assessable, and the consensus result was presumed to be correct . By this consensus criterion, immunohistochemistry yields both false negative (11%), and false positive (11%) results . RNA slot blotting has a high (21%) false positive rate . In situ mRNA hybridization and rt-PCR have the highest concordance, 80% . The 28 patients from whom these cell lines were derived appear to represent a very poor prognosis group, since there are only two patients (with Burkitt's lymphoma) who are long-term survivors . Nonetheless, a complete clinical response to therapy was correlated with absence of mdr1 expression in assessable cases (p = 0.04) . These four methods of determining mdr1 expression often yield discordant results . Therefore, the use of at least two methods for evaluating mdr1 expression is advisable . Rt-PCR is recommended because of its relative simplicity and specificity . This should be supplemented by a technique (immunohistochemistry or flow cytometry) able to detect heterogeneity of P-glycoprotein expression among cells.

Leukemia, 1994 Feb, 8(2), 289 - 91
Antisense inhibition of myeloperoxidase increases the sensitivity of the HL-60 cell line to vincristine; Schlaifer D et al.; Myeloperoxidase (MPO), a heme-peroxidase found in the HL-60 myeloblastic cell line, is involved in vincristine (VCR) metabolism and the inactivation of this drug . We have examined whether decreased MPO activity correlated with increased sensitivity to VCR toxicity in myeloid leukemia cells . We have used MPO antisense RNA to reduce 60% of the MPO activity in the HL-60 cells . The MPO-deficient HL-60 cell line, C15, was significantly more sensitive to VCR than the parental MPO-positive cell line . Both cell lines were negative for P170-glycoprotein expression . Conversely, an MPO-positive C15 subclone was more resistant to VCR than the MPO-deficient C15 cell line . No significant differences in cytotoxic effects were observed between MPO-positive and MPO-deficient cells, following treatment with either daunorubicin or actinomycin D, two multidrug resistance-related drugs . These results strongly support an important role for MPO in VCR resistance in HL-60 cells . Antisense manipulation of the MPO content of myeloid cells could be of potential interest in leukemia treatment.

J Urol, 1994 Feb, 151(2), 485 - 91
Ketoconazole effectively reverses multidrug resistance in highly resistant KB cells; Siegsmund MJ et al.; The antifungal agent ketoconazole was found to overcome resistance to vinblastine and doxorubicin in multidrug resistant KB-V1 cells in vitro . These cells are several hundred-fold more resistant than the parental cell line KB-3-1 . Ketoconazole had little or no effect on the parental KB-3-1 cells . The concentrations used to overcome drug resistance in vitro have already been safely used in vivo for treatment of fungal infections and in the monotherapy of hormone independent prostate carcinomas to block adrenal androgen production . Because of a possible beneficial effect of a combination of ketoconazole and a chemotherapeutic drug in multidrug resistant cancers, we examined a panel of 11 prostate carcinoma tissues for the expression of the MDR1 gene by an RNA-PCR assay . MDR1 expression was detectable, albeit at low levels, in 8 of the 11 tumors, suggesting a possible role of this gene in the drug resistance of prostate carcinomas . Our data suggest that ketoconazole might be useful in overcoming multidrug resistance in concentrations that are achievable in humans.

Bull Cancer, 1994 Feb, 81(2), 93 - 103
{Pharmacological properties of S9788, a new modulator of multidrug resistance}; Anthracycline antibiotics in cancer therapy . Focus on drug resistance; University of Texas, M.D . Anderson Cancer Center, Houston30 years ago an anthracycline antibiotic was shown to have antineoplastic activity . This led to the development of well over 1000 analogues with a vast spectrum of biochemical characteristics . Many biological actions have been described . The original anthracyclines are active against many types of cancer and are an integral part of several curative combinations . They are ineffective against other tumours . Although some analogues show an altered spectrum of activity or an improved therapeutic index relative to the older agents, it is not clear that cardiotoxicity can be totally avoided with these agents . Primary and secondary resistance to anthracyclines remain major clinical problems . Pharmacokinetic studies have been of limited help in explaining this . Overexpression of a surface-membrane permeability glycoprotein (Pgp) was identified in ovarian cancer of patients who had clinical multidrug resistance in 1985 . This led the way for the discovery of a number of resistance mechanisms in vitro . Some of these have been found in more than 1 type of cell line, and more than 1 mechanism may exist in a single cell . Additional resistance proteins have been identified, qualitative and quantitative alterations of topoisomerase II have been described, and some mechanisms in other systems have not yet been identified . Some of these may prove to be important in clinical drug resistance . Drugs such as calcium antagonists and cyclosporin, studied initially for their ability to block the Pgp pump, appear to be heterogeneous in this capacity and may have additional sites of action . It will be critical for clinical studies to define the precise resistance mechanism(s) that must be reversed . To date this has been difficult, even in trials ostensibly dealing with the original Pgp . Liposomes can potentially alter toxicity and target drug delivery to specific sites . In addition, they may permit the use of lipophilic drugs that would otherwise be difficult to administer systemically . Resistant tumours may be sensitive to anthracyclines delivered by liposomes . To reduce cardiac toxicity, administering doxorubicin (adriamycin) by slow infusion through a central-venous line should be considered whenever feasible . Monitoring of cardiac ejection fraction and the use of endomyocardial biopsy will permit patients to be treated safely after they reach the dose threshold at which heart failure begins to be a potential risk . A number of structurally modified anthracyclines with the potential advantages of decreased cardiotoxicity and avoidance of multidrug resistance mechanisms are entering clinical trials . Meanwhile, the vast weight of clinical experience leaves doxorubicin as a well tolerated and effective choice for most potentially anthracycline-sensitive tumours.

Jpn J Cancer Res, 1994 Feb, 85(2), 194 - 203
Cyclosporin A enhances susceptibility of multi-drug resistant human cancer cells to anti-P-glycoprotein antibody-dependent cytotoxicity of monocytes, but not of lymphocytes; Yano S et al.; Cyclosporin A (CsA) was previously found to bind to P-glycoprotein expressed on multidrug-resistant (MDR) cancer cells . In the present study, the effect of CsA on anti-P-glycoprotein monoclonal antibody (mAb)-dependent cell-mediated cytotoxicity (ADCC) against human MDR cells was examined . The ADCC reaction was assessed by 4-h 51Cr-release assay . Highly purified lymphocytes (> 99%) and monocytes (> 99%) obtained from blood mononuclear cells (MNC) of healthy donors were used as effector cells . CsA decreased the cytotoxic activity of MNC against MDR cells, but enhanced their ADCC activity in the presence of anti-P-glycoprotein mAb MRK16 . Lymphocyte-mediated ADCC and natural killer activity against MDR cells were also suppressed by addition of CsA . CsA induced a significant dose-dependent increase in monocyte-mediated ADCC activity . Interestingly, pretreatment of MDR cancer cells, but not of monocytes, with CsA significantly enhanced ADCC activity mediated by monocytes, but not by lymphocytes . A CsA analog (PSC833) and FK-506, but not verapamil also increased the sensitivity of MDR cells to ADCC by monocytes . CsA did not affect the binding of monocytes to MDR cells in the presence of MRK16 mAb . These results indicate that CsA may directly enhance the susceptibility of MDR cancer cells to the monocyte-mediated ADCC reaction.

Am J Pathol, 1994 Feb, 144(2), 227 - 36
New immunohistochemical "sandwich" staining method for mdr1 P-glycoprotein detection with JSB-1 monoclonal antibody in formalin-fixed, paraffin-embedded human tissues; Toth K et al.; We have developed a new immunoperoxidase "sandwich" staining method for amplified detection of P-glycoprotein (Pgp) that is suitable for use on formalin-fixed, paraffin-embedded (conventional) tissue sections . This was accomplished by substantially changing the procedure described by Chan (1988) so as to increase specific staining intensity and to decrease nonspecific background staining . To determine the most appropriate primary antibody for the assay, we compared the immunoreactivity of JSB-1, C494, and C219 monoclonal antibodies recognizing internal epitopes of Pgp, and MRK16 and 4E3 monoclonal antibodies recognizing external epitopes of Pgp . Paraffin sections of Pgp-positive normal human tissues (adrenal, liver, kidney, and brain), of renal tumors, and of cell pellets of sensitive and multidrug resistant human tumor cell lines (MCF-7, KB) were used for comparisons . Immunostaining was excellent with JSB-1, moderate with C494, and very weak with C219 . MRK16 and 4E3 showed no reaction . Nonspecific background staining was reduced by 1) omitting immunoglobulin G from secondary antibodies; 2) decreasing the concentration of peroxidase-antiperoxidase complex; and 3) utilizing casein solution for blocking and washing . Pretreatment of sections before immunostaining was also simplified . Using JSB-1, the threshold for detection of elevated Pgp corresponded to less than two-fold relative resistance to doxorubicin . Applying this method, we found two of 26 non-small cell lung cancers were positive for Pgp, consistent with previous results of others using frozen sections . This new immunoperoxidase sandwich staining method using JSB-1 now allows reliable Pgp detection in sections of formalin-fixed, paraffin-embedded (archived) surgical specimens and small biopsy materials commonly used for diagnostic purposes.

Blood, 1994 Feb 1, 83(3), 724 - 36
Circulating monoclonal B cells expressing P glycoprotein may be a reservoir of multidrug-resistant disease in multiple myeloma; Pilarski LM et al.; Multiple myeloma is basically an incurable cancer . Most patients respond initially to chemotherapy with reduction in bone marrow (BM) plasma cells and monoclonal Ig levels, but the disease nearly always recurs and becomes refractory to therapy . The objective of this study was to characterize the expression of the multidrug transport pump, P-glycoprotein 170 (P-gp), in myeloma . The great majority of B cells from peripheral blood mononuclear cells (PBMCs) in myeloma express P-gp, detected by the monoclonal antibody MRK-16 . P-gp+ blood B cells exhibit extensive DNA hyperdiploidy, suggesting replicative abnormality characteristic of malignant growth . We speculate these represent a stem cell population in myeloma . The proportion of B cells expressing P-gp was comparable among untreated myeloma patients and those treated with chemotherapy, biologic response modifiers, or off treatment . Among BM cells, P-gp was absent or low in untreated myeloma patients but was expressed at high levels on BM cells from patients previously treated with chemotherapy . For untreated patients the majority of B/plasma cells expressing P-gp are located in PBMCs, not the BM cells . Flow cytometric analysis of rhodamine 123 dye efflux indicated a functional P-gp that was efficiently blocked by verapamil or cyclosporin A (CsA) . Both the CD11bhi CD19+ B cells and the T cells in myeloma PBMCs had active CsA-inhibited dye efflux, but monocytes lacked the ability to efflux dye . Nearly all CD38hi plasma cells from myeloma BM cells retained dye, indicating their lack of a functional transport pump . Thus, PBMC B cells may be the predominant set of drug-resistant tumor cells . Myeloma PBMC B cells were cultured with Adriamycin with or without CsA and drug toxicity evaluated by the induction of apoptosis, using flow cytometry to quantitate DNA disruption . No apoptosis was detectable at 0.01 microgram/mL adriamycin, the in vivo steady-state level, with or without CsA . With 0.1 microgram adriamycin, no apoptosis was detectable in the absence of CsA, but with CsA, 66% of B cells initiated DNA disruption, whereas most T cells were spared . This work suggests that currently used drug dosages are too low to effect P-gp+ B-cell death, even in the presence of CsA . We suggest that blood B cells comprise a highly drug-resistant subset of the myeloma B lineage that escapes conventional chemotherapy and may underlie the almost uniform fatal relapse in myeloma patients.

Cancer Lett, 1994 Jan 30, 76(2-3), 139 - 45
Effect of Calphostin C (PKC inhibitor) on daunorubicin resistance in P388/ADR and HL60/AR cells: reversal of drug resistance possibly via P-glycoprotein; Gupta S et al.; Calphostin C is a potent and specific inhibitor of protein kinase C (PKC) . In this investigation we examined the effect of Calphostin C (without prior exposure to light) on daunorubicin (DNR) accumulation and sensitivity to DNR in multidrug-resistant (MDR) murine leukemia P388/ADR and human myeloid leukemia HL60/AR cells . P388/ADR cells overexpress P-glycoprotein, whereas HL60/AR cells lack any expression of P-glycoprotein (both at mRNA and protein levels) . Calphostin C, in a concentration-dependent manner, increased the accumulation of DNR in P388/ADR cells and partially reversed (threefold) the DNR resistance in P388/ADR cells but had no effect on either of the parameters in HL60/AR cells . Calphostin C-induced increased accumulation of DNR in P388/ADR cells was due to increased uptake and decreased efflux of DNR . Furthermore, Calphostin C increased the uptake and decreased the efflux of rhodamine 123 (a substrate for P-gp) in P388/ADR cells but had no such effect in P388 cells . In addition, Calphostin C without exposure to light did not inhibit PKC activity in any of the cell lines studied . Taken together, these data suggest that Calphostin C may reverse drug resistance via P-glycoprotein independently of its effect on PKC activity . Therefore, any data regarding the effect of Calphostin C on the reversal of MDR should be interpreted in the light of these findings.

Biochem Biophys Res Commun, 1994 Jan 28, 198(2), 804 - 10
Identification of a P-glycoprotein-related protein (mini-P-glycoprotein) which is overexpressed in multidrug resistant cells; Kawai K et al.; Drug resistant variant cell lines were selected in vitro for resistance to adriamycin or vincristine, P388/ADR and P388/VCR-600, respectively, from P388 murine leukemia cells . These cells were demonstrated by immunoblot studies with P-glycoprotein specific monoclonal antibody (C219) to overexpress not only P-glycoprotein but also approximately 65 kDa protein, and the expression levels were found to correlate with the degree of resistance . Furthermore, in Northern blot analysis with an MDR1 cDNA as a probe, an overexpressed transcript with the length of about 2.4 kilobases which might encode this previously uncharacterized protein was also detected in the multidrug resistant cell line . Thus, it is suggested that this protein might be associated with multidrug resistant phenotype and be related to P-glycoprotein.

J Biol Chem, 1994 Jan 21, 269(3), 2206 - 14
PDR5, a novel yeast multidrug resistance conferring transporter controlled by the transcription regulator PDR1; Balzi E et al.; The complete sequence of the pleiotropic drug resistance gene PDR5 from Saccharomyces cerevisiae is reported and analyzed . PDR5 encodes a 160-kDa protein with a predicted duplicated six membrane-span domain and a repeated putative ATP-binding domain . PDR5 shares this structural feature with the mammalian multidrug resistance pumps as well as the functional capacity of conferring resistance to various inhibitors upon amplification (Leppert, G., McDevitt, R., Falco, S . C., Van Dyk, T . K., Ficke, M . B., and Golin, J . (1990) Genetics 125, 13-20) . The yeast PDR5 is thus a new member of the ABC (ATP-binding cassette) protein superfamily . Mutations in another yeast pleiotropic drug resistance gene, PDR1, encoding a putative transcription regulator (Balzi, E., Chen, W., Ulaszewski, S., Capieaux, E., and Goffeau, A . (1987) J . Biol . Chem . 262, 16871-16879), increase markedly the mRNA levels of the PDR5 and STE6 genes . The multidrug resistance mutations pdr1-3 and pdr1-6 also lead to considerable overexpression of the PDR5 plasma membrane protein.

Biochem Pharmacol, 1994 Jan 20, 47(2), 257 - 66
Identification of a multidrug resistance modulator with clinical potential by analysis of synergistic activity in vitro, toxicity in vivo and growth delay in a solid human tumour xenograft; Plumb JA et al.; Circumvention of multidrug resistance in vitro by resistance modulators is well documented but their clinical use may be limited by effects on normal tissues . We have compared four resistance modifiers, both in terms of modulation of doxorubicin sensitivity in vitro and toxicity in vivo, in order to determine whether it is possible to select agents with clinical potential . Verapamil, D-verapamil and quinidine are all maximally active in the multidrug resistant cell line at about 7 microM and are not cytotoxic at this concentration . The tiapamil analogue Ro11-2933 is a highly potent resistance modulator such that at only 2 microM sensitization is greater than is seen with the other modulators at 7 microM . Since the ID50 concentration for Ro11-2933 is 17.7 microM (5-12-fold less than the other modifiers) we have used isobologram analysis to demonstrate that the interaction with doxorubicin is supra-additive and cannot be explained by additive toxicity . This method of analysis also revealed that when resistance modulation is related to the cytotoxicity of the modulator itself, all four modulators show comparable activity . On the other hand, measurement of the acute toxicity in mice of the modulators did reveal differences . The LD10 for verapamil (51 mg/kg) was about one third of that for quinidine (185 mg/kg) and this is consistent with the known maximum tolerated plasma levels in patients . Furthermore, whilst epirubicin alone was unable to reduce the growth rate of a multidrug resistant human tumour xenograft, the addition of quinidine, but not verapamil, at the maximum tolerated dose did do so . D-Verapamil was only about half as toxic as racemic verapamil and this too is consistent with clinical observations . The LD10 for Ro11-2933 (152 mg/kg) was comparable with that for quinidine . In the human tumour xenograft model maximal growth inhibition was observed with the combination of epirubicin and Ro11-2933 (45 mg/kg) and this degree of growth inhibition was comparable to that obtained with epirubicin alone in the drug sensitive xerografts . Ro11-2933 had no measurable effects on the plasma or tumour pharmacokinetics of epirubicin . These results suggest that it is possible to predict the clinical potential of a resistance modulator . Furthermore, Ro11-2933 is a promising agent for use in the clinic since maximal resistance modulation in vivo is observed at about one third of the LD10 dose.

J Natl Cancer Inst, 1994 Jan 19, 86(2), 110 - 7
A 190-kilodalton protein overexpressed in non-P-glycoprotein-containing multidrug-resistant cells and its relationship to the MRP gene; Barrand MA et al.; BACKGROUND: A 190k (190-kilodalton) membrane protein has been identified in several multidrug-resistant (MDR) cell lines that show decreased drug accumulation without expression of P-glycoprotein . It is not clear whether this 190k protein is involved directly in drug efflux . Recently, a gene for a putative transporter protein, MRP (multidrug resistance-associated protein) has been sequenced and localized to chromosome 16 . The protein encoded by this gene contains a 7-amino-acid sequence present in the synthetic peptide used to generate the antiserum recognizing the 190k protein . PURPOSE: The study was undertaken to clarify the relationship of the 190k protein to MRP gene expression in non-P-glycoprotein-containing MDR cells of the large-cell and adenocarcinoma lung cancer lines, COR-L23 and MOR . METHODS: Expression of the 190k protein was determined by Western blot analysis and that of the MRP gene by polymerase chain reaction amplification of complementary DNA reverse transcribed from RNA . Abnormalities of chromosome 16 were investigated in chromosome spreads by fluorescence in situ hybridization . RESULTS: The amount of detectable 190k protein is closely associated with degree of drug resistance . Cell lines surviving in higher drug concentrations have greater amounts of protein, and revertant lines grown without drug for up to 28 weeks show reduced expression of the protein together with enhanced drug sensitivity . The 190k protein appears to be one of the major proteins differentially expressed in membranes of drug-resistant cells . The amount of MRP messenger RNA correlates closely with that of the 190k protein . The MDR cells contain amplified chromosome 16 material with many double minutes in the large-cell lung tumor lines and an enlarged chromosome 16 in the adenocarcinoma lines . CONCLUSION: The 190k protein detected immunologically is likely to be the protein, encoded by the MRP gene, which becomes overexpressed in these cells as a consequence of chromosomal amplification and fragmentation . IMPLICATION: Though associated with drug resistance, enhanced drug efflux, and decreased drug accumulation in cell lines, the role of this protein in clinical resistance has yet to be determined.

J Natl Cancer Inst, 1994 Jan 19, 86(2), 91 - 8
Surface-epitope masking: a strategy for the development of monoclonal antibodies specific for molecules expressed on the cell surface; Shen R et al.; BACKGROUND: Producing monoclonal antibodies against specific targets, including tumor-specific antigens, is a tedious and extremely inefficient process . PURPOSE: Our purpose was to determine whether DNA transfection combined with an immunologic masking tactic could be used to efficiently generate hybridomas that secrete monoclonal antibodies . The quest was for monoclonal antibodies that would react with molecules existing on the surface of genetically altered cells . METHODS: We developed a masking technique called surface-epitope masking (SEM) . The SEM procedure involves the selective blocking of surface antigens present in a genetically engineered cell (referred to as a "tester") with high-titer polyclonal antibodies that have been produced against the untransfected parental cell (referred to as a "driver") . Surface-epitope-masked tester cells were injected into BALB/c mice; immune spleen cells then taken from these mice were fused with myeloma cells . RESULTS: This process resulted in the efficient generation of hybridomas that secreted monoclonal antibodies that reacted with cell-surface antigens on transfected tester cells and with additional cell types that expressed the same surface molecules . In one case, CREF-Trans 6 cells were engineered to express a typical multidrug-resistant (MDR) phenotype . Using CREF-Trans 6:MDR cells as a tester cell line, we utilized the SEM procedure to produce monoclonal antibodies that displayed surface reactivity to both CREF-Trans 6:MDR cells and MDR human breast carcinoma (MCF7) cells . In a second case, human prostatic carcinoma CREF-Trans 6 cells, which were DNA transfected and derived from nude mouse tumors, were used as the tester cell line . The SEM procedure was again used to produce monoclonal antibodies . These antibodies were designed to and did react with: (a) tumor-associated antigens on the surface of the original LNCaP cell line used to obtain human prostatic carcinoma DNA, (b) primary and secondary nude mouse transfectants derived from tumors, and (c) two additional human prostatic carcinoma cell lines, DU-145 and PC-3 . CONCLUSIONS: The SEM approach was used for the efficient and selective development of monoclonal antibodies that react with cell-surface molecules with both known and unknown functions . IMPLICATIONS: The SEM procedure should be useful in producing monoclonal antibodies and identifying genes associated with important cellular processes, including immunologic recognition, tumorigenesis, metastasis, atypical multidrug resistance, and autoimmune diseases.

Cancer Res, 1994 Jan 15, 54(2), 357 - 61
Overexpression of multidrug resistance-associated protein (MRP) increases resistance to natural product drugs; Grant CE et al.; Amplification of the gene encoding multidrug resistance-associated protein (MRP) and overexpression of its cognate mRNA have been detected in multidrug-resistant cell lines derived from several different tumor types . To establish whether or not the increase in MRP is responsible for drug resistance in these cell lines, we have transfected HeLa cells with MRP expression vectors . The transfectants display an increase in resistance to doxorubicin that is proportional to the levels of a M(r) 190,000, integral membrane protein recognized by anti-MRP antibodies . The transfectants are also resistant to vincristine and VP-16 but not to cisplatin . The results demonstrate that MRP overexpression confers a multidrug resistance phenotype similar to that formerly associated exclusively with elevated levels of P-glycoprotein.

Cancer Lett, 1994 Jan 15, 76(1), 51 - 6
Increase of cell viability and P-glycoprotein expression in Chinese hamster ovary cells after 24 hours' exposure to vincristine; Vickers SE et al.; Cell viability and P-glycoprotein expression in Chinese hamster ovary cells have been measured following incubation in a range of vincristine concentrations for 24 h . An increase of 12% in cell viability was observed with increasing drug concentrations in a drug-sensitive cell line (E29) and a lesser increase of 5% in a multidrug-resistant cell line (VRA15) . The increased cell viability corresponded to a rise of P-glycoprotein level . Slot blots showed that there was an increase in P-glycoprotein mRNA with all drug concentrations tested in E29, although there was only partial correlation of mRNA levels with P-glycoprotein levels viability.

Eur J Biochem, 1994 Jan 15, 219(1-2), 521 - 8
Constitutive expression of functional P-glycoprotein in rat hepatoma cells; Fardel O et al.; P-glycoprotein is a plasma-membrane glycoprotein involved in multidrug resistance . P-glycoprotein overexpression has been demonstrated to occur in tumor cells after cytotoxic drug exposure, but also in some cancers including hepatocellular carcinomas before any chemotherapeutic treatment . In order to better analyze this constitutive type of tumoral drug resistance, we have investigated P-glycoprotein expression and function in rat liver tumors induced experimentally by administration of diethylnitrosamine and in two cell clones derived from one of these tumors designated as RHC1 and RHC2 . High levels of P-glycoprotein mRNAs were found in both liver tumor samples and the two hepatoma cell clones as assessed by Northern blotting; both RHC1 and RHC2 cells displayed altered liver functions commonly observed in rat hepatoma cells, particularly the decreased expression of albumin and overexpression of the fetal glutathione S-transferase 7-7 . The use of specific multidrug resistance (mdr) probes revealed a major induction of the mdr1 gene in liver tumor samples while RHC1 and RHC2 cells expressed both mdr1 and mdr3 genes without displaying a major alteration in the number of mdr gene copies as assessed by Southern blotting . High amounts of P-glycoprotein were also demonstrated in RHC1 and RHC2 cells by Western blotting . These cells were strongly resistant to doxorubicin and vinblastine, two anticancer drugs transported by P-glycoprotein . Doxorubicin intracellular retention was low in RHC1 and RHC2 cells, but was strongly enhanced in the presence of verapamil, a known modulator agent of P-glycoprotein; low retention appeared to occur via a drug efflux mechanism, indicating that P-glycoprotein was fully active . These results show that rat hepatoma cells can display elevated levels of functional P-glycoprotein without any prior cytotoxic drug selection and suggest that these cells represent a useful model for analyzing P-glycoprotein regulation in intrinsically clinical drug-resistant cancers.

Cancer, 1994 Jan 15, 73(2), 298 - 303
Immunohistochemical study of expression and cellular localization of the multidrug resistance gene product P-glycoprotein in primary liver carcinoma; Itsubo M et al.; BACKGROUND . Expression and cellular localization of the multidrug resistance gene product P-glycoprotein, which plays an important role in multidrug resistance to cancer chemotherapy, were immunohistochemically studied in paraffin sections from 55 patients with primary liver carcinoma . METHODS . Tumor samples from 43 patients with hepatocellular carcinoma (HCC) and from 12 with cholangiocellular carcinoma (CCC) were obtained at autopsy or at surgical resection . Immunohistochemical study was performed by the avidin-biotin-peroxidase technique using monoclonal antibody JSB-1 directed against P-glycoprotein . RESULTS . Anti-P-glycoprotein immunostaining was observed in 67.4% of cases with HCC and in 66.7% of cases with CCC . When P-glycoprotein was detected in HCC, it was localized on the contact surface among tumor cells regardless of histologic type and on the cellular surface facing the blood space in the trabecular type and on the luminal surface in the pseudoglandular type . In CCC, relatively weak immunoreactivity for P-glycoprotein was localized in the cytoplasm of tumor cell both in glandular and nonglandular types, and stronger immunoreactivity was sometimes seen on the luminal surface of glandular type . The incidence of expression of P-glycoprotein was not influenced by previous cancer chemotherapy both in HCC and CCC . In the trabecular type of HCC, however, all cases with P-glycoprotein expression on the cellular surface facing the blood space were from the patients treated with antitumor agents . CONCLUSIONS . Resistance to cancer chemotherapy in primary liver carcinoma results from P-glycoprotein protein expression.

Cancer Res, 1994 Jan 15, 54(2), 441 - 7
Effect of tamoxifen on the multidrug-resistant phenotype in human breast cancer cells: isobologram, drug accumulation, and M(r) 170,000 glycoprotein (gp170) binding studies; Leonessa F et al.; We have performed isobologram analyses of the ability of tamoxifen (TAM) to alter the response to Adriamycin (ADR) and vinblastine (VBL) in human breast cancer cells . MCF-7 cells express functional receptors for estrogen and progesterone but do not express detectable levels of M(r) 170,000 glycoprotein (gp170) . CL 10.3 and MCF-7ADR cells are MCF-7 variants which express gp170 . CL 10.3 but not MCF-7ADR cells express functional steroid hormone receptors . Tamoxifen (1-2.5 microM) interacts synergistically with ADR and VBL in CL 10.3 and MCF-7ADR cells . TAM increases the cytotoxicity of VBL and ADR and the intracellular levels of {3H}VBL by approximately 2-3-fold . TAM also prevents the binding of {3H}azidopine to gp170 . The ability of TAM to concurrently increase the cytotoxic effects of ADR and VBL, increase VBL accumulation, and inhibit the binding of azidopine to gp170 strongly implies that the synergistic effects of TAM are mediated through its effects on gp170 . TAM produces an antagonistic to additive interaction with ADR and VBL in MCF-7 cells, and at high concentrations (5 microM) the synergy apparent in CL 10.3 and MCF-7ADR cells is lost . While TAM clearly has a significant potential for use as a chemosensitizing agent, the design of clinical trials may require careful consideration.

J Mol Biol, 1994 Jan 14, 235(2), 554 - 64
Transmembrane aromatic amino acid distribution in P-glycoprotein . A functional role in broad substrate specificity; Pawagi AB et al.; Multidrug resistance (MDR) in cancer cells is associated with overexpression of P-glycoprotein (Pgp), a membrane protein which interacts with structurally diverse hydrophobic molecules of high membrane affinity . In an analysis of the molecular basis for this broad range of substrate specificity, we found that the transmembrane (TM) regions of Pgp are rich in highly conserved aromatic amino acid residues . Computer-generated three-dimensional model structures showed that a typical substrate, rhodamine 123, can intercalate between three to four phenylalanine side-chains in any of several Pgp TM helices with minimal protrusion of the drug into bulk lipid, and that five to six (of the 12 Pgp putative TM segments) helices can facilitate transport through creation of a sterically compatible pore . In contrast to the case for proteins involved in the transport of membrane-impermeable, relatively polar substrates, the "transport path" for Pgp substrates need not be polar, and may involve either an internal channel occupied largely by aromatic side-chains, or external gaps along TM helix-lipid interfaces . Weakly polar interactions between drug cationic sites and Pgp aromatic residues contribute additionally to overall protein/drug binding . The ability of Pgp to recognize and efflux structurally diverse molecules suggests that rather than a unique structure, the Pgp channel may maintain the intrinsic capacity to undergo wide-ranging drug-dependent dynamic reorganization.

J Biol Chem, 1994 Jan 14, 269(2), 1432 - 6
Volume-sensitive chloride currents in four epithelial cell lines are not directly correlated to the expression of the MDR-1 gene; Rasola A et al.; It has been shown recently that heterologous expression of human MDR-1 gene, which is responsible for multidrug resistance during cancer therapy, causes appearance of volume-sensitive Cl- currents, thus suggesting that the product of the MDR-1 gene (the P-glycoprotein) has a Cl- channel activity (Valverde, M . A., Diaz, M., Sepulveda, M . A., Gill, D . R., Hyde, S . C., and Higgins, C . F . (1992) Nature 355, 830-833) . In the present work, we have tested four epithelial cell lines both for the expression of MDR-1 gene and for the presence of volume-sensitive Cl- currents . LoVo/H and LoVo/Dx cells derive from a human colon adenocarcinoma, the latter cell line being resistant to high concentrations of the antitumoral drug doxorubicin . 9HTEo- cells were obtained by transformation of human tracheal epithelium . The 9HTEo-/Dx cell line was established from these cells by selection in doxorubicin . As expected, higher levels of P-glycoprotein expression were detected in LoVo/Dx and 9HTEo-/Dx by means of reverse transcriptase polymerase chain reaction technique, indirect immunofluorescence, and Western immunoblot assays . In contrast with these data, the size of swelling-induced Cl- current was the same in the sensitive cell line and in its drug-resistant counterpart . Actually, the Cl- conductance of 9HTEo- and 9HTEo-/Dx was 4-fold higher than that of either LoVo/H or LoVo/Dx cells . This indicates that the amplitude of this conductance is not directly related to the expression of the MDR-1 gene.

Biochim Biophys Acta, 1994 Jan 3, 1189(1), 1 - 6
Reversal agent inhibition of the multidrug resistance pump in human leukemic lymphoblasts; Wigler PW et al.; Multidrug resistant cancer cells of the MDR-1 phenotype utilize an ATP-dependent pump to excrete toxic drugs . Rhodamine 123 (R123) is a fluorescent substrate of the MDR pump . An assay for the ATP-dependent initial efflux of R123 from CEM/VLB100 human leukemic lymphoblasts has been developed . The MDR-1 cells were treated with a reversal agent and preloaded with 40.0 nM R123 in buffer at 30 degrees C that contained sodium azide and 2-deoxyglucose . The cells were rinsed with cold buffer and resuspended in L-glutamine/glucose solution at 23 degrees C . The cell suspension was passed through a filter and R123 in the filtrate was detected at 2-s intervals by fluorescence . Efflux of R123 was inhibited by the reversal agents amiodarone, cyclosporin A, Ro11-2933 (DMDP), quinidine, and the optical isomers of propranolol . The MDR pump is stereospecific for the (R)-diastereomer quinidine; however, the (S)-diastereomer quinine is a relatively weak inhibitor of the pump . Cyclosporin A was the most potent inhibitor tested against the efflux of R123 by the MDR pump.

Int J Cancer, 1994 Jan 2, 56(1), 113 - 8
Dipyridamole modulates multidrug resistance and intracellular as well as nuclear levels of doxorubicin in B16 melanoma cells; Damle BD et al.; Simultaneous occurrence of resistance to many chemotherapeutic agents, termed multidrug resistance (MDR), is a complex phenotype . MDR occurs due to several reasons, including over-expression of a 170-kDa membrane-bound protein, called P-glycoprotein (P-gp), which apparently participates in active drug efflux . Multidrug-resistant cells also frequently exhibit an altered pattern of intracellular drug distribution, resulting in a reduction in the nuclear level of drugs such as doxorubicin (DOX) . In this study, the effect of dipyridamole (DP) on drug resistance and on intracellular as well as nuclear levels of DOX in multidrug-resistant melanoma cells has been examined . For this purpose, drug-sensitive murine melanoma cells (B16V) and their multidrug-resistant variant cells, (B16VDXR; selected for resistance to DOX) which over-produce P-gp, were employed . B16VDXR cells were cross-resistant to several anti-cancer agents including etoposide (VP-16) and mitoxantrone (Mitox) . DP (10 microM) significantly potentiated the cytotoxicity of DOX, VP-16 and Mitox towards multidrug-resistant B16VDXR cells but not in parental drug-sensitive B16V cells . The presence of DP resulted in a 3.7-fold increase in the total cellular level and a 4.2-fold increase in the nuclear content of DOX in the resistant cells . Isobologram analysis indicates that DP at several pharmacologically relevant concentrations synergistically potentiates the activity of DOX in B16VDXR cells.

J Comput Biol, 1994 Winter, 1(4), 257 - 69
A quantitative comparison of DNA sequence assembly programs; Miller MJ et al.; We have compared 11 sequence assembly programs for the accuracy and reproducibility with which they assemble DNA fragments into a completed sequence . To test the assemblers under controlled conditions, the rat multidrug resistance (RATMDRM) gene sequence was randomly divided into overlapping 200- to 400-base fragments . Various degrees of error, in the form of miss-identified bases, missed bases, and duplicated bases, were randomly added to these fragments . The probability of an error, and the type of error, was modified using an error distribution template that was developed by comparing the original fragments used to sequence RATMDRM with the final, edited sequence stored in GenBank . From 0 to 15% error was then added to independent sets of fragments, and assemblage was attempted . The quality of the assemblages was evaluated by comparing the number of differences between the assembled sequence and the original sequence . Tests were also done to determine if the order in which fragments were added to a project affected the final sequence and if the quality of assemblage was sequence dependent . Similar results were also obtained using other, unrelated sequences . The programs could be roughly divided into three groups based on the accuracy and reproducibility of assembly . Three (GCG, FAB, and AutoAssembler) consistently produced consensus sequences of low error and high reproducibility . Intermediate results were obtained with five other programs (Sequencher, AssemblyLIGN, XBAP, SeqMan, and AutoAssembler in a mode that made use of an external special processor) . Less satisfactory results were obtained with the remaining three programs (GeneWorks, GENeration, and PC/Gene) . The ability of the programs to edit the assembled sequence was also compared . Five of the programs were able to display and edit automatic sequencer trace files . The Sequencher program had a particularly well-designed sequence editor that allowed rapid examination and correction of assembly errors.

Intervirology, 1994, 37(6), 307 - 14
Failure of antiretroviral therapy: role of viral and cellular factors; Cinatl J Jr et al.; Effective therapy of human immunodeficiency virus (HIV) infection is mainly based on inhibition of reverse transcriptase by nucleoside analogues such as zidovudine (azidothymidine; AZT), didanosine, and zalcitabine . A major problem associated with long-term AZT therapy is the waning efficacy ('clinical resistance') over time . Clinical isolates of HIV-1 with reduced susceptibility to AZT can be recovered from HIV-infected individuals under prolonged treatment . However, the clinical importance of AZT resistance is uncertain . Other factors such as increased virus burden, increased virulence, and AZT toxicity could contribute, singly or in combination, to the loss of therapeutic benefit . Recent observations based on experimental models and clinical trials suggest that cellular mechanisms ('cellular resistance') may account for clinical resistance to antiviral agents . In vitro experiments demonstrated that in analogy to antitumoral therapy, the acquisition of multidrug resistance, i.e., resistance of cells to multiple, structurally unrelated chemotherapeutic agents, may play a role in the failure of long-term antiretroviral therapy . The 'cellular resistance' may contribute directly to the failure of antiviral therapy by the generation of subtherapeutic levels of antiviral compounds and/or their active forms . Indirectly, such subtherapeutic concentrations of active substances which permit limited replication of virus may represent a selective pressure for emergence and development of a resistant virus population . Hence it is of great importance to investigate the role of cellular factors in 'clinical resistance' to AZT and other anti-HIV agents . More detailed knowledge of cellular interactions and antiviral agents could help to improve or develop new strategies for antiviral therapy regimens.

J Am Dent Assoc, 1994 Jan, 125(1), 42 - 9
MDR-TB . Another challenge from the microbial world; Shearer BG; Beginning in 1985, the long decline in TB cases was dramatically reversed; from 1985 through 1992 reported cases increased 20.1 percent nationally . Two characteristics of this resurgent epidemic are unique: its prevalence among immunocompromised HIV-infected people and the emergence of multidrug-resistant TB . Current epidemiological trends, demographics and treatment approaches are discussed, as well as the implications MDR-TB holds for dentistry.

Leukemia, 1994 Jan, 8(1), 160 - 4
Comparative effects of quinine and cinchonine in reversing multidrug resistance on human leukemic cell line K562/ADM; Genne P et al.; We have previously suggested that quinine and cinchonine could be good candidates for clinical circumvention of multidrug resistance (MDR) in hematological malignancies because of their tolerance and their retained efficacy in serum . In the present study, we have used the well-characterized multidrug resistant human leukemic cell line K562/ADM to compare the effect in vitro of quinine and cinchonine on doxorubicin, mitoxantrone, and vincristine uptake and cytotoxicity . In serum-free medium, quinine induced a dose-dependent increase of doxorubicin uptake reaching about 200% at 40 microM, while it had a slight and no effect on mitoxantrone and vincristine uptake respectively . In the same conditions, cinchonine induced a rapid and significant increase in the accumulation of the three drugs, reaching a plateau phase between 5 and 10 microM . Quinine and cinchonine induced both potentiation of doxorubicin, vincristine and mitoxantrone cytotoxicity in K562/ADM cells . However, quinine reached a plateau phase at 10 microM, while cinchonine had a maximal effect at 5 microM and was significantly more potent at low concentrations . When diluted in plasma, cinchonine was less bound to proteins than quinine . The free fraction of alkaloids was 37-55% for cinchonine and 20-30% for quinine . Cinchonine-induced enhancement of vincristine cellular accumulation was little modified by plasma proteins . When incubated in whole blood, the fraction of cinchonine trapped in red blood cells was rapidly and completely exchangeable with plasma . We conclude that cinchonine is a stronger inhibitor of MDR than quinine.

Cancer Chemother Pharmacol, 1994, 33(4), 317 - 24
Differential effectiveness of a range of novel drug-resistance modulators, relative to verapamil, in influencing vinblastine or teniposide cytotoxicity in human lymphoblastoid CCRF-CEM sublines expressing classic or atypical multidrug resistance; Hill BT et al.; A series of five potential modulators of resistance were tested for their relative ability, as compared with verapamil, to sensitize CEM lymphoblastoid leukemia drug-resistant tumor sublines expressing either the classic or the atypical multidrug-resistance (MDR) phenotype to vinblastine or teniposide . Maximal non-cytotoxic concentrations of each modulator were tested and sensitization induces (SIs) were derived by comparing the drug concentration required to inhibit growth by 50% in their presence or absence . Like verapamil (10 microM) itself, three of the other modulators tested, namely, S9788 (4 microM), flunarizine (20 microM) and quinidine (30 microM), resulted in 2- to 3-fold sensitization of vinblastine against the parental CEM cells, and comparable effects were noted in the CEM/VM-1 cells, which were not cross-resistant to vinblastine . In contrast, cyclosporin A (0.5 microM) and B859-35 (2 microM) did not enhance vinblastine growth inhibition in these lines . However, the greatest sensitization with all the modulators was noted in the classic MDR VBL1000 cells, with SIs ranging from 40- to 350-fold, except for cyclosporin A, which proved ineffective at the concentration tested (SI, 2.6) . The greatest extent of differential sensitization of these VBL1000 tumor cells occurred with quinidine or B859-35, which proved significantly more effective than verapamil alone . Combinations of modulators resulted in additive effects, with B859-35 plus cyclosporin A proving superior to B859-35 plus verapamil . In contrast, none of these compounds proved effective as a sensitizer to teniposide . The growth-inhibitory effects of this drug were not modified significantly in either the 92-fold teniposide-resistant VM-1 cells or in the parental cells . Addition of verapamil itself also failed to modulate teniposide growth inhibition in the VBL1000 cells, which express significant cross-resistance to this drug (36-fold) . However, SI values of 3- to 5-fold were obtained using quinidine or B859-35 . These results serve (a) to emphasise the need to monitor the effects of modulators not only on drug-resistant cells but also on their drug-sensitive counterparts so as to ensure differential sensitization such that normal sensitive tissues are not likely to be adversely influenced and (b) to highlight the observation that the extent of modulation differs depending not only on the antitumor drug used but also on the mechanism of drug resistance expressed . This in vitro model system appears to provide a useful screening system for resistance modulators and certainly could be used in attempts to identify alternative agents that may influence teniposide sensitivity in these drug-resistant sublines.

Cancer Chemother Pharmacol, 1994, 33(4), 265 - 72
Evaluation of a novel bis-naphthalimide anticancer agent, DMP 840, against human xenografts derived from adult, juvenile, and pediatric cancers; Houghton PJ et al.; The new bis-naphthalimide antitumor agent (R,R)2,2'-{1,2-ethanediylbis{imino(1-methyl-2.1-ethanediyl)}-bis(5 -nitro 1H-benz{de}-isoquinoline-1,3-2H) dione} dimethanesulfonate (DMP 840) was evaluated against parental and multidrug-resistant human KB cell lines in vitro and against these lines growing as xenografts in immune-deprived mice . In vitro, KB8-5 cells were 50-fold resistant to vincristine but only 16-fold resistant to DMP 840 as measured by clonogenic survival . For in vivo evaluation, DMP 840 was given by i.v . injection daily for 9 days or for 5 days/week for 2 consecutive weeks {(dx5)2} . In contrast to the cross-resistance of KB cell lines in vitro, both KB3-1 and KB8-5 tumors were highly and equally sensitive to DMP 840; only KB3-1 xenografts demonstrated sensitivity to vincristine, which was consistent with the in vitro results . DMP 840 was also evaluated against a panel of human tumors comprising colon adenocarcinoma and rhabdomyosarcoma xenografts . Against eight lines of colon adenocarcinoma, DMP 840 caused a high frequency of partial and complete regressions in two lines and significant inhibition of growth in two lines . DMP 840 caused complete regressions in five of six lines of advanced rhabdomyosarcomas, demonstrating a broad range of effective dose levels . The pattern of activity against this tumor panel was similar but not identical to that of two inhibitors of topoisomerase I . There was no cross-resistance to DMP 840 in xenografts selected for resistance to vincristine or in a rhabdomyosarcoma selected for resistance to the topoisomerase I inhibitor topotecan . In contrast, a colon tumor selected for topotecan resistance was completely resistant to DMP 840 . Slight cross-resistance to DMP 840 was demonstrated in a rhabdomyosarcoma xenograft that was selected for primary resistance to melphalan and was cross-resistant to topoisomerase I inhibitors . The pattern of activity and cross-resistance in these tumors was compared with that shown by two agents that inhibit topoisomerase I: topotecan and CPT-11.

Chest, 1994 Jan, 105(1), 45 - 8
The third epidemic--multidrug-resistant tuberculosis; Neville K et al.; We recently observed a striking increase in multidrug-resistant tuberculosis (MDR-TB) among patients admitted to the Chest Service at Bellevue Hospital Center in New York . We reviewed the laboratory susceptibility test results of 4,681 tuberculosis (TB) cases over the past 20 years, Combined resistance to isoniazid and rifampin increased from 2.5 percent in 1971 to 16 percent in 1991 with higher rates noted for individual drugs . We reviewed the medical records of 100 patients with drug-resistant TB, finding that these individuals were predominantly less than 40 years of age, minority, male, jobless, undomiciled, with a high percentage of drug abuse and human immunodeficiency virus infection . We conclude that the epidemics of AIDS and TB are complicated by a third epidemic of MDR-TB . This third epidemic requires urgent attention to achieve more rapid diagnosis, to develop new therapeutic regimens, and to address the social and hospital environment ot care for these individuals.

Cancer Res, 1994 Jan 1, 54(1), 75 - 84
Inhibition of microtubules and cell cycle arrest by a new 1-deaza-7,8-dihydropteridine antitumor drug, CI 980, and by its chiral isomer, NSC 613863; de Ines C et al.; CI 980 (NSC 613862; {S-(-)}) and NSC 613863 {R-(+)} are the two chiral isomers of ethyl 5-amino 1,2-dihydro-2-methyl-3-phenylpyrido{3,4-b}pyrazin-7-ylcar bamate (NSC 370147), which is a mitotic inhibitor with in vivo and in vitro activity against murine multidrug-resistant sublines . We have characterized the inhibition of in vitro microtubule assembly by the S (CI 980) and R (NSC 613863) enantiomers, their actions on the cytoplasmic microtubule network of epithelial-like PtK2 cells, and on the cell cycle of different human and murine leukemias and PtK2 cells . Assembly of purified tubulin, or tubulin plus microtubule-associated proteins, into microtubules was substoichiometrically inhibited by both compounds, which also induced a slow depolymerization of preassembled microtubules . Half inhibitory concentrations were 0.4-0.7 microM and 1.6-2.1 microM for the S and R isomers, respectively . Excess of both drugs induced polymerization of liganded tubulin into abnormal polymers similar to colchicine . The cytoplasmic microtubules of PtK2 cells were disrupted by both compounds in a concentration- and time-dependent manner, which was observed by indirect immunofluorescence microscopy and quantified by an enzyme-linked immunoassay of cytoskeletal tubulin . Half inhibitory concentrations were 6 nM S isomer, 100 nM R isomer, and 1 microM colchicine . Twenty nM S isomer or 500-700 nM R isomer gave nearly maximal effect . At these concentrations, half maximal microtubule depolymerization took place after 2 h of treatment . After drug removal, slow microtubule assembly and nearly complete reorganization of the cytoplasmic microtubules of PtK2 cells were observed (24 h) . One nM S enantiomer or 25 nM R enantiomer induced mitotic arrest in 8 h in U937, HL60, and EL4 leukemias . PtK2 cells also stopped in mitosis after a 24-h incubation with 50 nM R isomer or 5 nM S isomer . The inhibition of cell division was irreversible in the leukemic cells, while PtK2 cells partially resumed growth . Although the interactions of CI 980 with microtubules in vitro are not very different from other drugs, it is a most potent cellular microtubule and mitotic inhibitor.

Ann Intern Med, 1994 Jan 1, 120(1), 71 - 9
Tuberculosis and the health care worker: a historical perspective; Sepkowitz KA; Many hospital outbreaks of tuberculosis have occurred in recent years in the United States, resulting in tuberculosis infection and disease among health care workers and patients . Several hospital workers have died of nosocomially acquired multidrug-resistant tuberculosis . Assuring the safety of the health care worker with respect to tuberculosis has become an urgent priority . A review of the medical literature of the past 100 years reveals that our current view of tuberculosis care as an occupational hazard emerged only in the 1950s, after a fierce and extensive debate . Many authorities had felt that care of the tuberculous patient conferred a health advantage to the care provider . This paper reviews this debate and considers steps taken decades ago, before our current environmental interventions were available to ensure the safety of the health care worker.

J Cancer Res Clin Oncol, 1994, 120(8), 471 - 8
Influence of cytokines on mdr1 expression in human colon carcinoma cell lines: increased cytotoxicity of MDR relevant drugs; Walther W et al.; We investigated the effects of tumor necrosis factor (TNF) alpha, interferon (IFN) gamma and interleukin-2 (IL-2) on the mdr1 gene expression in four human colon carcinoma cell lines (Lo Vo, HT 115, SW 480, and LS 174T) at different times (8, 24, 48, and 72 h) . We found no significant changes in mdr1 expression after 8 h and 24 h of cytokine treatment in all four lines . After 48 h and 72 h, however, a marked reduction of mdr1 expression in Lo Vo, HT 115, and SW 480 cells and an unaffected expression in LS 174T cells was observed . We examined whether the cytokine-mediated reduction of mdr1 expression correlates to the multidrug resistance (MDR) phenotype . In those cell lines showing a decreased mdr1 expression after a long-term cytokine pretreatment we found a dramatic enhancement of cytotoxicity of the MDR relevant drugs vincristine and doxorubicin, whereas LS 174T cells remained resistant . By contrast, the simultaneous application of cytokines and cytostatics caused no additive or synergistic effects . We conclude that in certain colon carcinoma cell lines a decreased mdr1 expression caused by prolonged cytokine pretreatment correlates with an enhanced cytotoxicity of drugs susceptible to MDR as an MDR-overcoming effect.

Cancer Chemother Pharmacol, 1994, 34(2), 96 - 102
Reversal of the human and murine multidrug-resistance phenotype with megestrol acetate; Wang L et al.; MA is an orally active PG derivative with an excellent safety profile that is used primarily for the treatment of carcinomas of the breast and endometrium . We investigated the potential application of MA as an MDR-reversal agent using cell culture and human tumor xenograft models . The reversing activity of MA in vitro was compared with that of PG and VER in two human MDR cell lines, the colon carcinoma HCT-116/VM46 and the breast carcinoma MCF-7/ADR, and in a murine cell line, J774.2 . At concentrations as low as 3 microM, MA was capable of partially restoring sensitivity to Act D in the HCT-116/VM46 cells and sensitivity to DOX in the MCF-7/ADR cells . Although less effective than VER, MA was about 2.5 times more potent than PG in reversing MDR at equimolar concentrations . Increased accumulation of DOX in drug-resistant cells that were treated simultaneously with MA was observed by flow cytometry . In vivo, using established human colon and breast carcinoma xenografts implanted s.c . in athymic mice, the combined therapy with MA and DOX resulted in enhanced antitumor activity relative to that of DOX alone in the MDR sublines . These results suggest that MA may be a promising clinical MDR-reversing agent.

Cancer Chemother Pharmacol, 1994, 34(2), 109 - 18
Cellular pharmacology of the partially non-cross-resistant anthracycline annamycin entrapped in liposomes in KB and KB-V1 cells; Perez-Soler R et al.; The in vitro cytotoxicity, cellular pharmacology, and DNA lesions induced by the lipophilic anthracycline annamycin (Ann) were studied in KB and KB-V1 (multidrug-resistant) cells . Ann was tested in suspension in saline and 10% dimethylsulfoxide (DMSO: final concentration, 0.05%-0.5%) or entrapped in multilamellar liposomes (median size, 1.57 microns) . Doxorubicin (Dox) was about twice as cytotoxic as Ann or liposome-entrapped Ann (L-Ann) against KB cells . Both Ann and L-Ann displayed a partial lack of cross-resistance with Dox (resistance indices: > 60 for Dox, 4.7 for Ann, 4.0 for L-Ann) . Accumulation of Ann in KB and KB-V1 cells was consistently about 2-3 and 10-20 times higher, respectively, than that of Dox . Cellular retention of Ann in KB and KB-V1 cells was about 2 and 30 times higher, respectively, than that of Dox as a result of the different efflux patterns of the two drugs: Dox was not effluxed from KB cells but was significantly effluxed from KB-V1 cells (66% at 1 h, whereas Ann efflux was similar in both cell lines (about 50% at 1 h) . Dox retention in KB-V1 cells was increased by a factor of 2 in the presence of verapamil or cyclosporine A, but Ann retention was not . In addition, accumulation of Dox in KB-V1 cells was enhanced by the metabolic inhibitor deoxyglucose/azide and the membrane carboxylic ionophore monensin, whereas accumulation of Ann was not affected by either agent . All these findings indicate significant differences in the cellular transmembrane transport systems between Dox and Ann and suggest that Ann efflux is not mediated by P-glycoprotein . Liposome entrapment reduced by a factor of 1.3-2.0 the cellular accumulation of Ann without affecting its cytotoxicity . As compared with Dox, both Ann and L-Ann induced 3 times more DNA double- and single-strand breaks in KB cells . In KB-V1 cells, Dox did not induce DNA damage, whereas the extent of DNA breaks induced by both Ann and L-Ann was similar to that induced by Dox in KB cells . Our results indicate (1) that the lack of cross-resistance between Ann and Dox is associated with a markedly enhanced accumulation and retention of Ann in KB-V1 cells and (2) that the type of liposomes used does not significantly affect the cellular effects of Ann.

J Cancer Res Clin Oncol, 1994, 120(7), 422 - 6
Modulation of doxorubicin-toxicity by tamoxifen in multidrug-resistant tumor cells in vitro and in vivo; Pommerenke E et al.; Modulation of the resistance of tumors offers new strategies to improve the therapeutical treatment of cancer . In this report, the anti-oestrogen tamoxifen was investigated in multidrug-resistant tumor cells in vitro and in vivo . The doxorubicin-resistance of L 1210/DOX-tumor cells, which express the multidrug-resistance phenotype, could be completely circumvented by addition of 1 microgram/ml tamoxifen . In contrast, no increased effect could be observed in the parental L 1210 tumor cells or in cytosine arabinoside-resistant L 1210 cells not expressing the multidrug-resistance phenotype . Thus, the enhancing effect of tamoxifen was restricted only to the multidrug-resistant L 1210/DOX tumor cells . Similar to the in vitro experiments, a significant reduction in the growth in solid tumors of mice by the combined treatment of doxorubicin and tamoxifen was again observed only in the multidrug-resistant L 1210/DOX tumors.

Eur J Cancer, 1994, 30A(1), 89 - 93
Enhanced cytotoxicity of doxorubicin encapsulated in polyisohexylcyanoacrylate nanospheres against multidrug-resistant tumour cells in culture; Bennis S et al.; We have studied the cytotoxicity and accumulation of doxorubicin encapsulated in polyisohexylcyanoacrylate nanospheres in a model of doxorubicin-resistant rat glioblastoma variants differing by their degree of resistance to this drug . We observed that the particulate form of doxorubicin was always more cytotoxic than free doxorubicin, whereas coadministration of drug-unloaded nanospheres with free doxorubicin did not modify significantly doxorubicin cytotoxicity . In C6 0.001 cells, which were 6-fold resistant and present a pure multidrug-resistant phenotype, the reversal of doxorubicin resistance was complete . In C6 0.1 cells, which were 60-fold resistant, as with C6 1V cells (selected with vincristine), the reversal of doxorubicin resistance was almost complete, with a residual resistance factor of 2-3 . In C6 0.5 cells, which were 600-fold resistant to doxorubicin, the reversal of resistance was only partial and, in all cases, not above the expected participation of P-glycoprotein to the phenotype of resistance . Intracellular drug accumulation after 2-h exposure to 17.2 mumol/l doxorubicin was systematically reduced by a factor of 2-3 when doxorubicin was incubated under the form of nanospheres; doxorubicin accumulation after a 2-h exposure to IC50 was also highly reduced in all cell lines for doxorubicin-loaded nanospheres . This work shows that association of doxorubicin with nanoparticles could provide a useful tool for circumventing multidrug resistance, probably by a bypass of P-glycoprotein rather than by an inhibition of this pump.

Antimicrob Agents Chemother, 1994 Jan, 38(1), 96 - 103
In vitro antiplasmodial, antiamoebic, and cytotoxic activities of a series of bisbenzylisoquinoline alkaloids; Marshall SJ et al.; Twenty-four bisbenzylisoquinoline alkaloids were screened for antiplasmoidal, antiamoebic, and cytotoxic activities by use of in vitro microtests . Eight of the alkaloids had antiplasmodial activity, with a 50% inhibitory concentration (IC50) of less than 1 microM against a multidrug-resistant strain of Plasmodium falciparum (chloroquine had an IC50 of 0.2 microM) . The three alkaloids most active against Entamoeba histolytica, aromoline, isotrilobine, and insularine, had IC50s of 5 to 11.1 microM (metronidazole had an IC50 of 1.87 microM) . None of the 24 bisbenzylisoquinoline alkaloids exhibited significant cytotoxicity against the KB cell line, the most toxic being berbamine, with an IC50 of 17.8 microM (the IC50 of podophyllotoxin was 0.008 microM) . Bisbenzylisoquinoline alkaloids merit further investigation as potential novel antimalarial agents.

Cancer Chemother Pharmacol, 1994, 33(6), 504 - 8
Uptake of doxorubicin from loaded nanoparticles in multidrug-resistant leukemic murine cells; Colin de Verdiere A et al.; Previous studies have clearly demonstrated that polyisobutylcyanoacrylate (PIBCA) doxorubicin-loaded nanoparticles (NS-Dox PIBCA) can overcome the resistance of P388/ADR cells . In the present paper, we found that overcoming multidrug resistance with the aid of doxorubicin (Dox) loaded onto these nanoparticles was associated with an increased intracellular drug content . Indeed, after a 6-h incubation period, the amount of cell-associated drug was 5 times higher when the cells were incubated with NS-Dox PIBCA as compared with free Dox . Further experiments, such as uptake studies in the presence of cytochalasin B or efflux studies, indicated a possible mechanism of nanoparticle/cell interaction . These results suggested that nanoparticles did not enter the cells by an endocytic process, in contrast to a previous hypothesis.

Anal Biochem, 1994 Jan, 216(1), 97 - 109
Determination of multidrug resistance levels in cultured mammalian cells using ornithine decarboxylase activity; Assaraf YG et al.; We describe an ornithine decarboxylase (ODC) activity-based assay for the quantitation of multidrug resistance (MDR) and its reversal by MDR modulators in cultured mammalian cells . ODC catalyzes the first and rate-limiting step in polyamine biosynthesis . The activity of this enzyme rises rapidly after growth initiation, such as after addition of serum-containing medium to quiescent mammalian cells . This increase in enzyme activity is prevented when growth is arrested, such as after treatment with cytotoxic drugs . In this assay cultures of drug-sensitive animal and human carcinoma cells as well as their MDR sublines were exposed to various concentrations of different cytotoxic agents for 6-48 h . A dose-dependent decrease in ODC activity was obtained with a variety of chemotherapeutic agents including anthracyclines, vinca alkaloids, epipodophyllotoxins, actinomycin D, antifolates, and cisplatinum . Anticancer drug resistance levels were calculated as the 50% inhibitory concentration of ODC activity obtained with drug-resistant cells divided by that obtained with sensitive cells . These cytotoxicity determinations correlated favorably with those obtained by the well-established colony formation assay . The ODC assay also proved useful in the assessment of MDR reversal with modulators of the MDR phenotype . Therefore, these studies show that the ODC assay could be useful for the reliable determination of drug resistance levels in cultured mammalian cells and for the assessment of drug resistance reversal by various modulators of the MDR phenotype.

Bull World Health Organ, 1994, 72(1), 73 - 81
Falciparum malaria in eastern Thailand: a randomized trial of the efficacy of a single dose of mefloquine; Fontanet AL et al.; Reported are the results of a randomized trial of a single dose of mefloquine (15 mg/kg or 25 mg/kg body weight) for the treatment of uncomplicated multidrug-resistant falciparum malaria . Of the 110 adult patients enrolled in the study 57 were randomly assigned to the 15 mg/kg group and 53 to the 25 mg/kg group . The baseline characteristics of the patients did not differ significantly in the two groups, except that those in the 15 mg/kg group had lower haemoglobin levels . Adverse effects following treatment were commoner in the 25 mg/kg group, but not significantly so . Seven patients (6%) did not complete the 42-day follow-up . The parasitological failure rates in the 15 and 25 mg/kg groups were, respectively, 50% (28/56) and 43% (25/53) on day 28, and 62% (33/53) and 56% (28/50) on day 42 . Treatment failures were not correlated with the serum mefloquine concentrations on day 2, and 13 out of 19 patients with serum mefloquine concentrations > 2000 micrograms/l on day 2 showed an R response during the follow-up . The mean ratio between the concentrations of the (SR)-(-) and (RS)-(+) enantiomers of mefloquine on day 2 was 3.37, indicating that there are differences in their pharmacokinetics . Re-treatment of patients who showed an R response with seven days of quinine (30 mg.kg-1.day-1)+tetracycline (25 mg.kg-1.day-1) was successful in 93% of the casesPIP: During November-December, 1991, clinicians administered either 15 mg/kg mefloquine or 25 mg/kg mefloquine to 57 and 53 patients, respectively, to treat falciparum malaria . The patients were at the referral hospital in Site 8, a Khmer refugee camp on the border between Thailand and Cambodia . The 2 groups responded essentially the same, except the 15 mg/kg group had a lower mean hemoglobin level than the 25 mg/kg group (9.4 g/dl vs . 10 g/dl; p = .03) . Parasitological failure rates on day 28 and 42 were not significantly different between the 2 groups . On day 28, they were 50% for the 15 mg/kg group and 43.4% for the 25 mg/kg group . They were especially high at day 42 (62% and 56%, respectively) . Patients in the 25 mg/kg group were more likely to experience adverse effects of mefloquine (13% vs . 7% for vomiting, 26% vs . 19% for diarrhea, and dizziness 74% vs . 70%), but the differences were not significant . The mean serum mefloquine level was basically the same for both groups (2165 mcg/l in 15 mg/kg group and 2284 mcg/l in the 25 mg/kg group) . 13 of 19 patients tested had serum mefloquine concentrations greater than 2000 mcg/1 on day 2, yet they experienced parasitological failure . This concentration traditionally indicated successful treatment . People who vomited within an hour of receiving mefloquine had much lower serum mefloquine levels than those who did not vomit (1289 mcg/l vs . 2300 mcg/l; p = .03) . Patients who experienced dizziness on day 2 had much higher serum mefloquine levels than those who were not dizzy (2394 mcg/l vs . 1371 mcg/l; p = .006) . On day 2, the mean ratio between the levels of the (SR)-(-) and (RS)-(+) enantiomers of mefloquine stood at 3.37, suggesting that differences exist in their pharmacokinetics . The 7-day retreatment regimen with quinine and tetracycline was successful in 93% of the 50 patients who experienced parasitological failure .

Med Pediatr Oncol, 1994, 22(5), 299 - 308
Clinical relevance of in vitro drug resistance testing in childhood acute lymphoblastic leukemia: the state of the art; Pieters R et al.; Nowadays about two-thirds of children with acute lymphoblastic leukemia (ALL) can be cured with chemotherapy, but one-third die from the disease . The clinical response of leukemic cells to chemotherapy is roughly due to two factors: the effective drug levels reaching the cells and the resistance of these cells to the drugs . The clinical value of cellular drug resistance in children with ALL is not known . We developed an in vitro assay to study drug resistance in these children . In this article, the main results obtained with this MTT assay on samples from 137 children with ALL are summarized: (1) patients whose cells are resistant to several drugs at initial diagnosis have a poor prognosis; (2) relapsed leukemias show a considerable drug resistance which might partly explain the poor prognosis . Relapsed cases differ in their type and degree of resistance; (3) the poor outcome of high risk groups as defined by age and immunophenotype can partly be explained by specific patterns of drug resistance; (4) P-glycoprotein-mediated multidrug resistance is not an important cause of resistance in childhood ALL; and (5) no relation exists between the activities of the purine enzymes HGPRT, 5'NT, ADA, and PNP and drug resistance in childhood ALL . The conclusion is that in vitro drug resistance data have clinical relevance and can be used to develop more effective and less toxic treatment strategies in childhood ALL.

World J Urol, 1994, 12(2), 104 - 11
From laboratory expertise to clinical practice: multidrug-resistance-based gene therapy becomes available for urologists; Mickisch GH et al.; Many human tumors such as bladder carcinoma that are initially responsive to chemotherapy eventually fail to respond to treatment . For most drugs, dose escalation that may be required for a cure cannot be achieved because sensitive tissues such as bone marrow limit cytotoxic therapy . Approaches to prevent or circumvent myelosuppression are therefore a high priority of research on dose intensification protocols . One such strategy is to protect bone marrow cells by virtue of expression of the multidrug-resistance (MDR1) gene encoding for P-glycoprotein . In our first set of experiments, we transplanted bone marrow cells derived from transgenic mice that constitutively express MDR1 to lethally irradiated recipients (n = 36) . From 6 weeks to 10 months after the transplant, all animals contained MDR1 DNA in spleen and bone marrow specimens as indicated by Southern-blot analysis and expressed MDR1 RNA in bone marrow samples as detected by slot-blot analysis . In addition, these animals were resistant to the myelosuppressive effect of doxorubicin, daunomycin, taxol, vinblastine, vincristine, etoposide, and actinomycin D, whereas control animals that were reconstituted with normal bone marrow reacted with a significant decrease in their white blood counts . In a second set of experiments, we retrovirally transfected a construct consisting of a murine long-terminal repeat (LTR) promoter and the human MDR1 gene into CD34-positive bone marrow stem cells from rhesus monkeys using the same technique as in the ongoing clinical ADA gene-therapy protocol . Upon transplantation, high-level and long-lasting expression of the human MDR1 gene was observed in recipient monkeys.(ABSTRACT TRUNCATED AT 250 WORDS)

Leuk Lymphoma, 1994, 13 Suppl 1, 27 - 30
Expression of resistance genes in acute leukemia; Zhou DC et al.; To evaluate the frequency and the prognostic value of different mechanisms of drug resistance in acute leukemias, we investigated the expression of mdr1 by immunocytochemistry, mRNA slot blot or RT-PCR in 182 cases of adult acute myeloid and 37 cases of adult lymphoblastic leukemia . Before treatment, 39% of de novo AML, 38% of secondary AML, and 7% of de novo ALL exhibited a high level of mdr1 mRNA . After chemotherapy, the frequency of mdr1 gene expression in ALL raised dramatically to 60% (P < 0.005), while no significant change was found for AML cases . In 91 patients treated with MDR-related drugs, mdr1 gene expression was related to the failure of chemotherapy (P < 0.0001) . The overexpression of multidrug resistance-associated protein (mrp) and anionic glutathione S-transferase (GST pi) was also investigated in 38 and 61 AML patients respectively . An overexpression of mrp gene was noted in 39% of the cases . For GST pi gene, the frequency of overexpression was 28% . A positive and significative correlation was found among mdr1, mrp and GST pi genes expression.

Cancer Chemother Pharmacol, 1994, 34(5), 423 - 30
Reversal of multidrug resistance by bis(phenylalkyl)amines and structurally related compounds; Ramu A et al.; We have previously reported that multidrug (MDR)-reversal activity can be exerted by compounds in which two ring structures of certain types are connected by one alkyl bridge to a secondary or tertiary amine group . In the present investigation we studied the MDR-reversal activity of compounds in which the two ring structures were connected by separate alkyl bridges to the amine group . The structure-activity relationship of these compounds verified previous findings on the structural features that support MDR-reversal activity as well as the features that reduce such activity . In addition, the present study reveals additional chemical groups and ring structures that support MDR-reversal activity as well as those that reduce it.

Bull N Y Acad Med, 1994 Summer, 71(1), 18 - 36
Human immunodeficiency virus infection and tuberculosis: an analysis and a course of action; Bryt AB et al.; Tuberculosis, once on the steady decline in the western world, has resurfaced with renewed vigor in the wake of the human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) epidemic . People with HIV infection are both more likely to contract primary tuberculosis and at greater risk for reactivation of latent tuberculosis . Tuberculous disease may present with atypical signs and symptoms in HIV-infected hosts because of alterations in the immune system . Superimposed on the virulent interaction of HIV and tuberculosis is the emerging problem of multidrug resistant strains that often resist currently available therapies . HIV-positive health professionals working in high-risk environments pose a special problem, while populations unable to comply with currently available pharmacological therapies pose another . We have many tools available to combat the resurgence of tuberculosis, but new methods of diagnosis and new approaches to treatment are sorely needed.

Ann Hematol, 1994, 69 Suppl 1, S1 - 6
Role of protein kinases in antitumor drug resistance; Grunicke H et al.; The activity of several proteins involved in the development of antitumor drug resistance is regulated by protein phosphorylation . These proteins include the mdr-1-encoded P-glycoprotein (Pgp) and topoisomerase II (topo II) . The corresponding evidence is reviewed and attempts to modulate multidrug resistance (MDR) by protein kinase C inhibitors are described . The expression of several proteins which are essential in drug resistance is regulated at the transcriptional level, involving protein phosphorylation by members of the protein kinase C (PKC) family, casein kinase II (CKII), and others . These proteins include mdr-1-encoded P-glycoprotein, metallothionein, glutathione S-transferase (GST), dTMP synthase, and the proteins Fos and Jun . The corresponding genes are under positive regulation of ras, which in turn requires the activation of a protein kinase cascade for its function . Protein kinases are therefore potentially useful targets in reducing the expression of proteins involved in the development of multifactorial drug resistance caused by the expression of transforming ras-genes . Attempts to inhibit the ras-induced fos expression by an inhibitor of protein kinase C (ilmofosine) are described . Protein kinase inhibitors are also able to synergistically enhance the cytotoxicity of cis-platinum, which is discussed as resulting from a reduction of PKC-dependent fos expression.

Annu Rev Public Health, 1994, 15, 303 - 23
The re-emergence of tuberculosis; Porter JD et al.; PIP: Tuberculosis (TB) is reemerging as a major public health problem worldwide . The emergence of multidrug resistance and the interaction between TB and HIV infection are responsible for this surge . Immunologists, epidemiologists, economists, clinicians, policymakers, and heads of TB control programs attended a 1993 public health conference called Tuberculosis--Back to the Future, at the London School of Hygiene and Tropical Medicine . They agreed that urgent research is needed on diagnostics, new drugs, vaccines, and control strategies to address the reemergence of TB . They also called for a much higher investment in existing cost effective methods for TB control . Research scientists and staff of national TB control programs must work closely together to assure success of any strategy . Before the HIV epidemic, the risk of TB infection in developing countries fell 1-5% per year . The HIV epidemic has reversed this trend . It also fell in developed countries . In the US, the risk began to increase in 1986 in New York City due to increased immigration from TB endemic countries, interaction between TB and HIV, and decay in the health infrastructure supporting TB control programs . Treatment failures or relapses due to disorganized treatment programs account for most multidrug resistance in the US as well as in other countries . Research areas of diagnostics improvement include clinical evaluation, microscopy, culture, and radiographic examination . Drug research areas are in new drugs to improve patient compliance; investigation of the mechanism of virulence of Mycobacterium tuberculosis to understand mycobacterial persistence, thus helping in the development of new drugs; and operations research of rational use of drugs by clinicians . Vaccine research is perhaps the most important research, especially since the current vaccine is not always effective . TB control program and operational issues include case finding and treatment, chemoprophylaxis, treatment regimens, operational research, supervision, and drug supply .

Yao Xue Xue Bao, 1994, 29(1), 20 - 3
{The correlation between multidrug resistance and glutathione (GSH) in EAC, EAC/ADR and EAC/ADR(R) cell lines}; Wang QD et al.; Determination of GSH was performed by a modified fluorometric method . The experiments showed that the GSH content in resistant cell line (EAC/ADR) was 4 fold higher than that in sensitive cell line (EAC), being 1.25 +/- 0.35 microgram/10(6) cells in EAC/ADR and 0.31 +/- 0.12 microgram/10(6) cells in EAC . When the drug treatment was discontinued for 36 weeks, the sensitivity was partially recovered and the GSH level in EAC/ADR(R) cell parallelly decreased . In addition, vincristine, mitomycin C, actinomycin D and VP-16 showed no effect on the GSH content in the EAC/ADR cell.

Lijec Vjesn, 1994 Jan-Feb, 116(1-2), 41 - 5
{Multiple drug resistance}; Svoboda-Beusan I; The multidrug-resistance (MDR) phenotype expressed in mammalian cell lines is complex . A cell selected with a single agent can acquire cross-resistance to a remarkably wide range of compounds which have no structural or functional similarities . The basis for cross-resistance seems to be a decreased net cellular accumulation of the drugs involved, and has been attributed to alterations in plasma membrane . An over-expressed plasma membrane P glycoprotein of relative molecular mass of 170 kD (Pgp) is consistently found in different MDR human and animal cell lines, and in transplantable tumors . Consequently, it has been postulated that Pgp directly or indirectly mediates MDR . This paper reviews the current knowledge on etiology, pathogenesis, diagnosis and therapy of MDR.

Cancer Chemother Pharmacol, 1994, 34(3), 242 - 8
Reduced levels of topoisomerase II alpha and II beta in a multidrug-resistant lung-cancer cell line; Evans CD et al.; We have previously shown that the doxorubicin-selected multidrug-resistant small-cell lung-cancer cell line H69AR is resistant to VP-16-induced single-strand DNA breaks as compared with its parental H69 cell line . Levels of immunoreactive topoisomerase II alpha are also reduced in H69AR cells . In the present study, we found that cleaved complex formation in the presence of VP-16 was decreased in H69AR cells as compared with H69 cells . In addition, the resistant cells contained lower levels of both topoisomerase II alpha and topoisomerase II beta protein and mRNA . However, these changes were not accompanied by a decrease in the P4-unknotting (strand-passing) activity of 0.67 M NaCl nuclear extracts of H69AR cells, nor was there any difference in VP-16 inhibition of unknotting activity in the H69 and H69AR nuclear extracts . These data suggest that reduced levels of topoisomerase II alpha and II beta may contribute to the resistance of H69AR cells to VP-16 and other drugs that target these isoenzymes.

Eur J Cancer, 1994, 30A(9), 1360 - 9
A comparison of rhodamine 123 accumulation and efflux in cells with P-glycoprotein-mediated and MRP-associated multidrug resistance phenotypes; Twentyman PR et al.; Rhodamine 123 (Rh123) is a fluorescent dye which locates in the mitochondria of cells . It is a substrate for P-glycoprotein (Pgp) and can, therefore, be used as a molecular probe in studies of the multidrug resistance (MDR) phenotype . However, not all MDR cells overexpress Pgp . In some, the MDR phenotype is associated with expression of an alternative transporter molecule, the multidrug resistance-associated protein (MRP) . We have studied the accumulation and efflux of Rh123 in MDR cells having both Pgp-mediated and MRP-associated phenotypes . In the mouse tumour parental cell line, EMT6/P, Rh123 accumulates rapidly to reach plateau levels by 90 min . Confocal microscopy confirms a localisation to the mitochondria . In the MDR subline, EMT6/AR1.0, which overexpresses Pgp and which is 10-fold resistant to Rh123 cytotoxicity, accumulation is dramatically reduced . Efflux of Rh123 from both resistant and parental lines is rapid but can be inhibited by reduced temperature or by the presence of cyclosporin A (5 micrograms/ml) . Efflux from the parental line is probably due to the presence of very low, but detectable, levels of Pgp but the existence of other mechanisms cannot be ruled out . In contrast, the human lung cancer parental cell line COR-L23/P, and its MRP-associated (but Pgp-negative) MDR subline, COR-L23/R (which is 23-fold resistant to Rh123 cytotoxicity), accumulate Rh123 at similar rates for the first 30 min . The curves then diverge so that, at 180 min, the resistant cells contain only 70% of the Rh123 of parental cells . Confocal microscopy demonstrates a similar distribution of fluorescence in resistant and parental cells . Essentially no efflux of Rh123 occurs from parental cells, whereas 70% of the content is lost from resistant cells over a period of 150 min . Such efflux may again be inhibited by reduced temperature but cyclosporin A (5 micrograms/ml) has little effect . These observations should be borne in mind when interpreting Rh123 efflux data in terms of MDR mechanisms.

Oncol Res, 1994, 6(3), 119 - 27
Analysis of MRP gene expression and function in HL60 cells isolated for resistance to adriamycin; Krishnamachary N et al.; In an effort to define clearly the basis of non-P-glycoprotein multidrug resistance in HL60/ADR cells, we have analyzed expression of MRP mRNA levels and the MRP-encoded protein in resistant cells and also in resistant cells that have undergone a reversion to drug sensitivity . The results demonstrate that an MRP cDNA containing 5'-end coding sequences reacts with a 6-kb RNA, which is overexpressed in the resistant isolate . As resistant cells revert to drug sensitivity there is essentially a complete loss of the 6-kb RNA . Southern blot analysis indicates that the MRP gene is amplified compared to the copy number found in sensitive cells . Revertant cells no longer contain amplified MRP sequences . Western blot analysis has been conducted using an antibody prepared against the carboxyl terminus (15 amino acids) of the deduced sequence of the MRP-encoded protein . The antibody is reactive with a 190-kDa protein (P190) and with two closely migrating proteins of 65 and 70 kDa (P70), which are overexpressed in plasma membranes and endoplasmic reticulum of resistant cells . Both proteins are greatly reduced in revertant cells . Growth of cells in the presence of tunicamycin demonstrates that both P190 and P70 are glycosylated, with the deglycosylated forms migrating in polyacrylamide gels as proteins of 165 kDa and 45 kDa, respectively . Additional antisera have also been prepared against sequence domains contained in the C-terminal region of P190 . These antisera are reactive with both P190 and P70 . Antisera directed against sequences of the amino terminal region of P190 do not react with P70.(ABSTRACT TRUNCATED AT 250 WORDS)

Acta Oncol, 1994, 33(7), 787 - 91
Reversal of multidrug resistance by new dihydropyridines with low calcium antagonist activity; Capolongo L et al.; The clinical use of Ca++ antagonist agents as modulators of multidrug resistance is limited by their strong vasodilator activity . This study reports data obtained by testing a series of new 1,4 dihydropyridine derivatives (DHPs) for their in vitro resistance modulating activity and their Ca++ antagonist effect . All the tested DHPs are active to increase doxorubicin activity with dose modifying factor values ranging between 2 and 47 on P388/DX cells and 12 and 36 on LoVo/DX cells . Their resistance modulating action is exerted through an increase of DX intracellular level . The Ca++ antagonist activity of DHPs, evaluated as capacity to inhibit the KCl-induced contractions in isolated Guinea pig ileum strips, is not related to their resistance modulating activity . This finding makes it possible to select, for further in vivo evaluations, compounds IX, X and XI, which have strong ability to overcome multidrug resistance and low Ca++ antagonist effect.

Acta Oncol, 1994, 33(7), 773 - 7
Expression of the multidrug-resistance gene product in untreated human breast cancer and its relationship to prognostic markers; De La Torre M et al.; The immunohistochemical expression of the 170-kDa permeability glycoprotein (P-gp) was investigated in 41 primary untreated breast carcinomas, using the monoclonal antibodies C219 and MRK16 . DNA ploidy by flow cytometry and estrogen (ER) and progesterone receptor (PgR) contents were also determined . P-gp expression, as revealed by C219 or MRK16, was observed in 6 (14%) of the investigated cancers . P-gp expression had a tendency to occur in non-diploid, high-grade tumors as well as in patients with lymph node negative disease . However, except for lymph node status, these associations were not statistically significant . No positive statistical relationships were observed between other prognostic parameters (age, tumor size, and receptor status) and P-gp expression . Considering the great heterogeneity observed in previous studies and the low expression of P-gp observed hereby, the utility of P-gp immunostaining as a guide for therapy planning in patients with breast cancer remains uncertain.

Acta Clin Belg, 1994, 49(5), 214 - 9
{Resistance to chemotherapy: current progress}; Andre M et al.; Chemotherapy has become a very important part of the therapy for several cancers . Complete remission, and sometime cure, can be achieved with this treatment modality . However, when relapse occur, a second remission is seldom obtained . This failure is believed to be linked, at least in part, to the development of drug resistant tumor cells . Our understanding of the mechanisms underlying this resistance, especially regarding the multidrug resistance (MDR) phenomenon, has improved over the recent years . New strategies are being developed to circumvent drug resistance and come now to clinical trials.

Cancer Chemother Pharmacol, 1994, 35(2), 169 - 73
Preclinical pharmacology of 1069C85, a novel tubulin binder; Raynaud FI et al.; The compound 1069C85, methyl N-{6-(3,4,5-trimethoxybenzyloxy)imidazo(1,2b)-pyridazin-2-yl } carbamate, is a novel synthetic tubulin binder currently undergoing phase I clinical trial . It was developed with a view to overcoming multidrug resistance and is given orally . Cytotoxicity studies in vitro against human ovarian carcinoma cell lines showed a lack of cross-resistance with cisplatin and no cross-resistance in two doxorubicin-resistant cell lines that exhibit high levels of resistance to both paclitaxel and vinblastine . Pharmacokinetic studies in BALB/c mice showed the oral bioavailability to be 20%, with 35% of the drug being excreted unchanged in the faeces over the first 24 h . Maximal plasma concentrations (Cmax) were achieved within 2 h of oral administration as compared with 7.5 min following i.v . injection . At a dose of 20 mg/kg, the tumour drug concentration exceeded the plasma Cmax by a factor of 1.5 and was within the in vitro cytotoxic concentration range . The drug showed a linear relationship between the dose and the area under the plasma concentration versus time curve (AUC) for doses of up to 20 mg/kg, above which no further increase in AUC was observed, possibly due to saturable absorption . 1069C85 is highly protein-bound (85%-99%) and appears to be subject to metabolism . The demonstration of cytotoxic activity against multidrug-resistant human tumour cell lines and the detection of potentially cytotoxic levels in an experimental tumour following oral administration support the choice of 1069C85 for clinical development.

Cancer Chemother Pharmacol, 1994, 35(1), 84 - 8
A new procedure for the preparation of liposomal doxorubicin: biological activity in multidrug-resistant tumor cells; Thierry AR et al.; We describe a new procedure for the preparation of liposomal doxorubicin . Doxorubicin can be efficiently complexed to preformed or lyophilized cardiolipin-containing liposomes . Complex formation was performed by vigorous vortexing . As much as 96.8% of the initial drug quantity may be bound to those liposomes under optimal incubation conditions (4 h at 37 degrees C) . The binding study showed the presence of two levels of specific binding (dissociation constants, 28 +/- 8 microM and 1.0 +/- 0.3 mM) . The drug is firmly integrated in the liposome-membrane lipid bilayer rather than binding at the surface . Cytotoxicity studies using tumor cells revealed efficient drug delivery using liposome-complexed doxorubicin . This new liposomal doxorubicin preparation reverses multidrug resistance in MCF-7/ADR and CH LZ cells at levels equivalent to that obtained with a previously described liposome-encapsulated doxorubicin preparation, showing that the drug is integrated as well in the liposome carrier and is transported as well into cells . Increased concentration of liposomes at the subcytotoxic level in liposome-complexed doxorubicin enhances drug cytotoxicity in multidrug-resistant CH LZ cells as compared with liposome-encapsulated drug . This new preparation for liposomal doxorubicin may be carried out immediately prior to clinical administration, offering advantages in terms of cost and stability.

Cancer Chemother Pharmacol, 1994, 35(1), 53 - 8
Modulation of anthracycline accumulation and metabolism in rat hepatocytes in culture by three revertants of multidrug resistance; Le Bot MA et al.; The aim of this study was to compare the action of three multidrug resistance (MDR) modulators, cyclosporine A, S 9788, and verapamil, on the efflux of two anthracyclines, doxorubicin and daunorubicin, and of daunorubicinol, the C-13 alcohol metabolite of daunorubicin . Rat-hepatocyte primary cultures have been used as a model of P-glycoprotein (Pgp) expression . This model allows the study of MDR at different levels of Pgp expression, which increases in parallel with the time in culture; furthermore, the hepatocytes are capable of metabolizing drugs, which enables the determination of the role of Pgp on metabolite efflux . All modulators tested were incubated for 6 h at concentrations of 1, 5, and 15 microM with doxorubicin (0.5 microM) and at 1 and 15 microM with daunorubicin (0.5 microM) on hepatocytes grown for 4 and 48 h in culture . Daunorubicinol (0.5 microM) was tested with modulators at 48 h of culture . In fresh hepatocytes, the three MDR modulators did not induce an increase in the intracellular retention of anthracycline as compared with controls (no MDR modulator) . At 48 h of culture, the three test drugs increased doxorubicin intracellular accumulation . In contrast, daunorubicin retention was not modified, but that of its metabolites was increased . Within the concentration range tested, cyclosporine was the most potent modulator without dose-dependent activity . The activity rank order was cyclosporine > S 9788 > verapamil . Cyclosporine and S 9788 were as active in coincubation as in preincubation with anthracyclines . Verapamil had no action when incubated before the addition of anthracyclines . Cyclosporine and S 9788 had an effect on the intracellular retention of daunorubicinol used alone whereas verapamil did not . The action of cyclosporine and S 9788 on the retention of daunorubicinol proves that at least a part of the efflux of C-13 alcohol metabolites of anthracyclines is mediated by Pgp . This study shows that S 9788, cyclosporine, and verapamil are MDR modulators in hepatocytes with high-level Pgp expression . This study also demonstrates that hepatocytes are a potent tool for the study of the action of new MDR modulators on cytostatic drugs as well as on their metabolites.

Acta Neurochir Suppl (Wien), 1994, 60, 257 - 60
P-glycoprotein expression in brain capillary endothelial cells after focal ischemia in rat; Samoto K et al.; We investigated the time kinetics of P-glycoprotein (P-gp), a membrane bound drug efflux pump for many anti-cancer drugs in multidrug resistant cells, using a rat ischemic brain model . Frozen sections of the brain were studied immunohistochemically with anti-Factor VIII antibody for endothelial cells, with anti-glial fibrillary acidic protein (GFAP) antibody for reactive astrocytes, and with MC6-4 monoclonal antibody for P-gp . A putative blood-brain barrier (BBB) marker, gamma-glutamyl transpeptidase (gamma-GTP), and the progression of the brain edema were also studied . P-gp positive endothelial cells disappeared in the ischemic lesion by post-ischemic Day 3 . Factor VIII-positive regenerating capillaries were first observed on Day 3 without P-gp expression when the brain edema reached a maximum . P-gp positive endothelial cells began to reappear on Day 5, and were detected in all endothelial cells by Day 8 . The time kinetics of P-gp expression in the endothelial cells showed a similar pattern as that of gamma-GTP, and its induction is associated with GFAP-positive reactive astrocytes . These results suggest that P-gp might play an important role in maintaining the BBB function in conjunction with glial cells.

Yao Xue Xue Bao, 1994, 29(4), 246 - 51
{Vincristine-resistant human KB cell line and mechanism of multidrug resistance}; Zhang XH et al.; A multidrug-resistant (mdr) clone of human cancer KB cells was isolated by stepwise selection on exposure to increasing doses of vincristine . The final clone, KBv200, obtained after ethylmethane sulfonate (EMS) mutagenesis showed 175-fold higher resistance to vincristine than did KB cells . The cells were also cross-resistant to taxol, colchicine and adriamycin . Cellular accumulation of vincristine in KBv200 was decreased to less than one-fifth of that in KB . To determine the presence of mdr 1 mRNA in KBv200 and KB, total cellular RNAs from each cell line were analyzed by means of slot blot hybridization . The result showed that the mdr 1 gene had been highly expressed in KBv200 . In addition, verapamil, a calcium channel blockers, was shown to increase VCR accumulation in KBv200 and reverse the vincristine resistance . All these results demonstrate that the mechanism of KBv200 cell resistance to multiple drugs resulted from increased expression of mdr 1 gene and brought about over production of P-glycoprotein and increased the efflux of drugs.

Invest New Drugs, 1994, 12(1), 1 - 13
Multidrug resistance in cancer chemotherapy; Patel NH et al.; Resistance to chemotherapy is the single most important reason for treatment failure in cancer patients . Over the past 15 years, we have gained significant insight into one of the mechanisms responsible for this process: multidrug resistance (MDR) . Far from being a phenomenon limited to the laboratory, multidrug resistance has been identified in a wide variety of newly diagnosed and recurrent human tumors . A number of compounds can block p-glycoprotein and overcome MDR in vitro and in vivo . Current strategies to block MDR are discussed in this review . Future research in this area will focus on the identification of more selective and potent MDR reversing agents and the development of entirely new approaches to overcoming multidrug resistance such as monoclonal antibodies, immunotoxins, and gene therapy.

Virchows Arch, 1994, 425(2), 133 - 8
Ultrastructural localization of P-glycoprotein on capillary endothelial cells in human gliomas; Tanaka Y et al.; The P-glycoprotein (P-Gp) encoded by the human multidrug-resistance gene MDR1 has been suggested to play certain roles in the blood-brain barrier (BBB) . However, the detailed mechanism of the activity of P-Gp in multidrug-resistance (MDR) remains unclear in human glioma . We examined the localization of P-Gp in human glioma by immunohistochemical (IHC) and immunoelectron microscopic (IEM) methods with anti P-Gp monoclonal antibodies (C219, MRK16) . We also examined MDR1 expression in primary glioma and xenografts by reverse transcription-polymerase chain reaction (RT-PCR) with human MDR1-specific primers . The IHC study showed no P-Gp expression on tumour cells but it was present on capillary endothelial cells and IEM analysis showed definitive localization on their luminal surface . MDR1 gene expression was detected in eight primary glioma and three normal brain specimens by RT-PCR, but not in glioma xenografts . The lack of MDR1 expression in these cells appears to be a consequence of the replacement of the original human stroma, including blood vessels, by murine stroma in glioma xenografts . The unique distribution of P-Gp on the capillary blood vessels was confirmed in human glioma by the results of immunohistochemical and molecular biological studies.

Oncol Res, 1994, 6(2), 71 - 7
Transactivation of the human multidrug resistance (MDR1) gene promoter by p53 mutants; Nguyen KT et al.; Multidrug resistance in human cancer is associated with overexpression of the MDR1 gene, which encodes a plasma membrane energy-dependent efflux pump termed P-glycoprotein (or the multidrug transporter), which confers cross-resistance to multiple hydrophobic natural product cytotoxic drugs . We have previously shown in cotransfection experiments that activity of the human MDR1 gene promoter is modulated by Ras and p53, suggesting that expression of the MDR1 gene may be associated with the activation of oncogenes and/or functional loss of tumor suppressor genes during oncogenesis . To further characterize the effects of p53 on the MDR1 promoter, we have shown in the current study that the region of the promoter that is required for transactivation by p53 mutants overlaps with the region that is essential for basal promoter activity . In addition, we also have shown that several different p53 mutants transactivate the MDR1 promoter in several different cell types, including embryo fibroblasts derived from the p53-deficient (p53-l-) mice generated by gene targeting.

Oncol Res, 1994, 6(2), 59 - 70
Functional role of phosphorylation of the multidrug transporter (P-glycoprotein) by protein kinase C in multidrug-resistant MCF-7 cells; Aftab DT et al.; Multidrug resistance (MDR) is the phenomenon in which cells become resistant to several classes of structurally and functionally diverse drugs after exposure to a single cytotoxic agent . One form of MDR is associated with the overexpression of a large plasma membrane phosphoglycoprotein, P-glycoprotein, which acts as an energy-requiring drug transport pump . Protein kinase C may participate in MDR through posttranslational modification of P-glycoprotein . The purpose of this study is to critically evaluate P-glycoprotein as a substrate for protein kinase C and to determine whether phosphorylation leads to changes in drug transport . Protein kinase C from rat brain phosphorylated immunoprecipitated P-glycoprotein in a manner dependent on the activation of the exogenous kinase . Phorbol 12-myristate 13-acetate (PMA) increased the phosphorylation of P-glycoprotein 6-fold and selectively decreased the accumulation of vinblastine in resistant MCF-7/AdrR cells . PMA selectively decreased the cellular association of vinblastine with MDR cells after brief periods of incubation, but only after critical concentrations of drug were achieved . The actions of PMA did not require new synthesis of P-glycoprotein . PMA had similar effects in MCF-7/BC-19, a cell line transfected with a cDNA for P-glycoprotein . Staurosporine inhibited the effects of PMA on the phosphorylation of P-glycoprotein and on the accumulation of vinblastine . These data demonstrated that immunoprecipitated P-glycoprotein can be a substrate for protein kinase C, and that phosphorylation of the transporter is associated with significant changes in drug transport.

Oncol Res, 1994, 6(2), 49 - 57
Dipyridamole reverses the resistance to topoisomerase II inhibitors but not to antimicrotubule agents in multidrug-resistant melanoma cells; Damle B et al.; The influence of dipyridamole (DP) on the cytotoxicity and cellular disposition of several DNA topoisomerase II (topo II) inhibitors and antimitotic agents in multidrug-resistant B16VDXR cells was examined . B16VDXR cells, derived from parental B16V cells by step-wise treatment with doxorubicin (DOX), overexpress a 170 kDa P-glycoprotein (P-gp) . Additionally, the resistance to DOX in B16VDXR cells is associated with decreased frequency of DNA strand breaks compared to that in the drug-sensitive B16V cells . DP (10 microM) significantly (P < 0.01) potentiated the cytotoxicity of DOX (6.4-fold), mitoxantrone (2.3-fold), and etoposide (14-fold) in the drug-resistant B16VDXR cells . This was accompanied by a 3.7-fold and 4.2-fold increase in the total intracellular and nuclear levels of DOX, respectively . Surprisingly, no significant change in the intracellular and nuclear levels or the efflux of etoposide was observed in B16VDXR cells . Combination index (CI) analysis, however, indicated that DP interacted synergistically with DOX as well as etoposide . Further, it was intriguing to observe that DP (10 microM) failed to modulate the resistance to vincristine, vinblastine, and taxol . This was despite a significant increase in the accumulation of vinblastine (3.3-fold) and taxol (3.9-fold) in B16VDXR cells in the presence of DP (10 microM) . The observed pattern of chemosensitization suggests that in addition to interaction with P-gp, the multidrug-resistance modulating activity of DP may involve P-gp independent mechanism(s) . The possibilities include that (i) DP interacts with topo II or (ii) DP promotes the formation and/or obstructs the repair of DNA strand breaks caused by topo II inhibitors.

Eur J Cancer, 1994, 30A(7), 1002 - 7
Expression of P-glycoprotein and in vitro or in vivo resistance to doxorubicin and cisplatin in breast and ovarian cancers; Veneroni S et al.; The expression of P-glycoprotein (P-gp) was studied by immunocytohistochemistry, using the C219 monoclonal antibody, in 39 locally advanced breast cancers and 20 ovarian cancers from previously untreated patients . P-gp was expressed in 46 and 35% of breast and ovarian tumours, respectively . A significant association was observed in both tumour types between P-gp expression and in vitro resistance to doxorubicin . We also observed a higher clinical response rate to doxorubicin +/- vincristine in patients with breast cancers not expressing P-gp . Conversely, no correlation was found between P-gp expression and in vitro resistance to cisplatin or in vivo response to cisplatin +/- cyclophosphamide treatment in ovarian cancers . Our results support the relevance of P-gp expression as a specific indicator of resistance to certain drugs, such as doxorubicin and vincristine, involved in the phenomenon of multidrug resistance in breast and ovarian cancer cells.

Acta Oncol, 1994, 33(6), 631 - 7
In vivo reversal of multidrug resistance by two new dihydropyridine derivatives, S16317 and S16324; Kraus-Berthier L et al.; Two new dihydropyridine derivatives with low calcium channel affinity, S16317 and S16324, were found to fully overcome multidrug resistance in vitro . These two compounds increased doxorubicin cytotoxicity on the human COLO 320DM cell line and completely reversed the vincristine resistance of murine P388/VCR cells . In vivo, S16324 administered p.o . (200 mg/kg on days 1 to 4) or i.p . (50 mg/kg on days 1, 5, 9) in combination with vincristine (i.p.) restored the antitumor activity of vincristine in P388/VCR-bearing mice . S16317 showed a reversing activity when administered p.o., i.v . (days 1 to 4) or i.p . (days 1, 5, 9) at the same dose (25 mg/kg), suggesting a remarkable bioavailability . Moreover, these two compounds potentiated the antitumor activity of vincristine in the sensitive P388 leukemia, increasing the number of long-term survivors . These results suggest that combination chemotherapy using S16317 or S16324 would be effective not only in circumventing multidrug resistance but also in preventing the emergency of a population of resistant tumor cells in sensitive tumors.

Acta Clin Belg, 1994, 49(3-4), 138 - 47
Is BCG vaccination against tuberculosis still indicated?
Vanderschueren S, Peetermans W, Bobbaers H.
More than 70 years after the introduction of BCG vaccination into clinical practice many questions remain to be answered . Major trials produced conflicting results regarding the degree of protection of this vaccine against tuberculosis . Several factors which may contribute to the inconsistent results of these trials, are discussed . In developing countries continuation of routine BCG vaccination of infants is highly recommended considering the ease of administration, low cost, wide availability and safety on the one hand and the protection provided particularly against haematogenous spread of tuberculosis on the other hand . In developed countries the vaccine is reserved for high risk groups since the prevalence of tuberculosis in the general population declined dramatically during the past decades . However, as the incidence of tuberculosis in the Western world no longer decreases steeply and indeed increases again in some countries, and because (para-) medical personnel risks to be among the prime victims of this re-emergence of tuberculosis, rigorously sustained preventive measures to protect this professional category deserve renewed interest . Among those, BCG vaccination can be considered, especially in case of a high prevalence of multidrug-resistant tuberculosis, but also for the Belgian situation with a high degree of non-immune health care workers . Therefore, we believe that BCG vaccination still has a future.

J Cancer Res Clin Oncol, 1994, 120(10), 599 - 604
Novobiocin modulates colchicine sensitivity in parental and multidrug-resistant B16 melanoma cells; Nordenberg J et al.; The effect of the antibiotic agent novobiocin on the sensitivity of melanoma cells to colchicine and vinblastine was examined in drug-sensitive and drug-resistant B16 melanoma cells . A cell line COL/R was selected for colchicine resistance . The COL/R cell line (resistant to 80 ng/ml colchicine) was found to possess the multidrug-resistant (MDR) phenotype . The cells were shown to be cross-resistant to vinblastine and Adriamycin and to overexpress P glycoprotein . P glycoprotein activity was assessed by using the rhodamine 123 accumulation test . Rhodamine accumulation was markedly decreased in COL/R cells as compared to the parental B16 cells . Verapamil reversed drug resistance and increased rhodamine accumulation in COL/R cells . Novobiocin in combination with colchicine or vinblastine synergistically inhibited the proliferation of parental B16 cells . In COL/R cells, novobiocin markedly decreased colchicine resistance and increased rhodamine accumulation . These data show that novobiocin increases the sensitivity of both parental and MDR melanoma cells to microtubule-disrupting cytotoxic drugs.

Drugs Exp Clin Res, 1994, 20(1), 13 - 8
Reversal effects of several Ca(2+)-entry blockers, neuroleptics and local anaesthetics on P-glycoprotein-mediated vincristine resistance of L1210/VCR mouse leukaemic cell line; Barancik M et al.; The ability of several Ca(2+)-entry blockers, neuroleptics and local anaesthetics to depress the P-glycoprotein-mediated resistance to vincristine was studied in vitro using the L1210/VCR cell line . This cell line was obtained by long-term adaptation of the L1210 mouse leukaemic cell line on vincristine and showed an overexpression of P-glycoprotein and accompanying multidrug resistance (MDR) which was defined as a cell resistance to several cytostatics such as vincristine, vinblastine and actinomycin D . Efficiency of the drugs applied to reverse this resistance was as follows: for Ca(2+)-entry blockers: verapamil (VER) > or = galopamil (GAL) > flunarizine (FLU) >> diltiazem (DIL) > nimodipine (NIM) > or = nifedipine (NIM); for neuroleptics: trifluoperazine (TFP) > chlorpromazine (CHP) > thioridazine (TRD) > perphenazine (PER); for local anaesthetics: carbanilate-Ca7 > cinchocaine (CIN) >> carbanilate-Ca3 > articaine (ART) > carbanilate CAO > lidocaine (LID) . Quaternary cabanilate derivatives (Ca7Q and Ca3Q) with permanent positive charge were found to be unable to reverse the vincristine resistance of L1210/VCR cells . No reasonable correlation between the ability of calcium-entry blockers (DIL, VER, GAL, NIF, NIM and FLU) to reduce the viability of L1210/VCR cells growing in the medium supplemented with vincristine and their reported affinity to the L-type of calcium channel was observed . On the other hand, significant positive correlations were observed between both the inhibitory action of local anaesthetics on propagation of action potential in rat sciatic nerve and the ability of drugs to interact with calmodulin and the ability of the respective drug to reverse the resistance of L1210 cells to vincristine.

Cancer Chemother Pharmacol, 1994, 34(6), 459 - 64
Quercetin potentiates the effect of adriamycin in a multidrug-resistant MCF-7 human breast-cancer cell line: P-glycoprotein as a possible target; Scambia G et al.; This study demonstrates that the flavonoid quercetin (Q), a plant-derived compound with low toxicity in vivo, greatly potentiates the growth-inhibitory activity of Adriamycin (ADR) on MCF-7 ADR-resistant human breast cancer cells . The effect of Q was dose-dependent at concentrations ranging between 1 and 10 microM . Since ADR resistance in these cells is associated with the expression of high levels of P-glycoprotein (Pgp), we evaluated the effect of Q and related flavonoids of Pgp activity in cytofluorographic efflux experiments with the fluorescent dye rhodamine 123 (Rh 123) . Our results indicate that Q and 3-OMe Q (3',4',7-trimethoxyquercetin) but not the 3-rhamnosylglucoside of Q (rutin) inhibit the Pgp pump-efflux activity in a dose-related manner . Moreover, 10 microM Q reduces the expression of the immunoreactive Pgp in MCF-7 ADR-resistant cells as evaluated by cytofluorimetric assay . In conclusion, these findings provide a further biological basis for the potential therapeutic application of Q as an anti-cancer drug either alone or in combination with ADR in multidrug-resistant breast tumor cells.

Oncol Res, 1994, 6(1), 33 - 7
Comparison of the effects of genistein and amsacrine on leukemia cell proliferation; Finlay GJ et al.; Genistein is an inhibitor of the enzymes protein tyrosine kinase and topoisomerase-II . It induces G2-phase arrest in human Jurkat and murine P388 leukemia cells at concentrations at which it is also cytotoxic . The effects of genistein have been investigated on Jurkat and P388 leukemia sublines that manifest multidrug resistance . Cells that possess altered topoisomerase-II activity ("atypical" multidrug resistance) are resistant to both the G2 phase-arresting and cytotoxic effects of genistein . The ability of genistein to impede progression through the cell cycle and kill cells is similar to that of amsacrine, a classical topoisomerase-II poison . This result identifies topoisomerase-II rather than tyrosine kinase activity as the target of genistein-mediated cytotoxicity and G2-phase arrest.

Eur J Cancer, 1994, 30A(5), 687 - 90
Paclitaxel-induced cytotoxicity--the effects of cremophor EL (castor oil) on two human breast cancer cell lines with acquired multidrug resistant phenotype and induced expression of the permeability glycoprotein; Fjallskog ML et al.; Paclitaxel (Taxol) is a new cytotoxic agent with considerable activity in phase II studies on metastatic breast cancer . Paclitaxel for clinical use is dissolved in the solvents cremophor EL and ethanol . In this study, we added paclitaxel, formulated either in cremophor EL and ethanol or only in ethanol, in increasing concentrations to two parental human breast cancer cell lines (ZR 75-1 and HS 578T) and their corresponding sublines with acquired doxorubicin resistance and P-glycoprotein expression . Paclitaxel dissolved either in ethanol or ethanol plus cremophor EL, resulted in steep and almost identical dose-response curves for the parental lines ZR 75-1 and HS 578T, respectively, independent of the solvent used . When paclitaxel was formulated only in ethanol the effects on the corresponding doxorubicin-resistant sublines were significantly reduced compared with paclitaxel dissolved in ethanol plus cremophor EL . These effects by cremophor EL may partly explain some of the antitumoral effects observed by paclitaxel in anthracycline failing patients.

J Cancer Res Clin Oncol, 1994, 120(9), 533 - 8
The chemosensitizer cyclosporin A enhances the toxic side-effects of doxorubicin in the rat; Van de Vrie W et al.; The feasibility of using chemosensitizers in the circumvention of P-glycoprotein-mediated multidrug resistance has been shown in many studies . We recently reported on the chemosensitizing effect of cyclosporin A (CsA) on doxorubicin in a rat solid tumour model . Using the same experimental design we investigated the side-effects of the combination treatment . During the 35-day experiment doxorubicin treatment caused dose-dependent weight loss, which was enhanced by combination treatment with CsA . The main doxorubicin-related side-effects were myelosuppression (transient leucopenia and thrombopenia) and nephrotoxicity . Damage to the kidney was severe, leading to a nephrotic syndrome and resulting in ascites, pleural effusion, hypercholesterolaemia and hypertriglyceridaemia . These toxicities were enhanced by the addition of the chemosensitizer CsA . Mild doxorubicin-related cardiomyopathy and minimal hepatotoxicity were seen on histological examination . There were no signs of enhanced toxicity of the combination treatment in tissues with known high expression levels of P-glycoprotein, like the liver, adrenal gland and large intestine . CsA had a low toxicity profile, as it only caused a transient rise in bilirubin . In conclusion, the chemosensitizer CsA enhanced the side-effects of the anticancer drug doxorubicin without altering the toxicity pattern . There was no evidence of a therapeutic gain by adding CsA to doxorubicin, compared to single-agent treatment with doxorubicin in 25%-33% higher doses, because of the enhanced toxicity of the combination treatment.

Cancer Chemother Pharmacol, 1994, 34(4), 307 - 16
Chemomodulation of drugs involved in multidrug resistance in chronic lymphatic leukemia of the B-cell type; Reichle A et al.; Reduced drug accumulation may be one reason for intrinsic drug resistance in chronic lymphatic leukemia of the B-cell type (B-CLL) . Immunophenotyped leukemic human B-cells from 38 cases of B-CLL were characterized for P-glycoprotein (PGP) content . In all, 30 cases of B-CLL were additionally analyzed for further parameters: accumulation of daunorubicin (DNR, n = 20) and rhodamine 123 (Rh123, n = 30) in both the presence and the absence of verapamil (VRP) . Also, 16 cases of B-CLL were analyzed for vincristine (VCR) accumulation with or without VRP . Concerning the relative expression of PGP, these 16 cases of B-CLL were representative for the whole group of 30 cases . Spontaneous accumulation of Rh123 and VCR varied over a wide range: accumulation of Rh123, by a factor of 11.8; accumulation of VCR, by a factor of 9.7; and accumulation of DNR, by a factor of 3.6 . VRP modulated the accumulation of RH123 in 16/30 cases (53%), that of VCR in 69% of cases, and that of DNR in 11% of cases . The maximal VRP-mediated increases in accumulation amounted to factors of 1.3 for DNR, 2.3 for Rh123, and 7.8 for VCR . Spontaneous drug accumulation did not correlate with the extent of chemomodulation . The amount of PGP in B-CLL cells (all cases studied were PGP-positive) did not correlate with drug accumulation or with the extent of VRP-mediated chemomodulation . Thus, high expression of PGP is only partially responsible for defective drug accumulation in B-CLL . Only the degree of chemomodulation by VRP is predictive for the extent of the PGP-related functional drug accumulation defect . Thus, in B-CLL, PGP-independent drug accumulation defects seem to be as important as those mediated by PGP . The extent of this drug accumulation defect varies for the different drugs in the following order VCR > Rh123 > DNR . The relevance of PGP-mediated and -independent drug accumulation defects in vivo may depend to a large extent on the drug being used and on the individual cell type . Both types of defect in drug accumulation are of high importance when regimens include VCR a drug commonly used in second-line chemotherapy of B-CLL . Both defects in drug accumulation may be responsible for intrinsic VCR resistance in B-CLL.

Wien Klin Wochenschr, 1994, 106(8), 242 - 6
{Functional detection of P-glycoprotein expressing cells with flow cytometry}; Ludescher C et al.; Classical multidrug resistance (MDR) is associated with the overexpression of a membrane glycoprotein termed P-glycoprotein (P-gp) that transports a variety of apparently unrelated anti-cancer drugs out of cells . Based on the fluorescent properties of the dye rhodamine 123 (Rh 123) we aimed to develop a functional flow cytometric assay for the detection of MDR-expressing cells . Using drug sensitive cell lines (KB-3-1) and MDR mutants (KB-8-5, KB-C1) experimental conditions were established enabling demonstration of significant differences in Rh 123 accumulation/efflux . Using two-colour flow cytometry we subsequently analysed 42 consecutive patients suffering from B-cell chronic lymphocytic leukemia (B-CLL) . 34/42 (81%) cases showed a marked Rh 123 efflux which was completely abolished in the presence of the MDR inhibitor verapamil . The percentage of Rh 123 effluxing cells ranged from 10 to 70% with a median of 32% . In 26 cases extracted RNA was analysed by quantitative polymerase chain reaction to evaluate the expression of MDR 1 mRNA . In 25 of 26 (96%) cases MDR 1 mRNA was detectable . The levels of MDR 1 mRNA expression correlated well with the results of the Rh 123 efflux assay (r = 0.72; p < 0.0001) . In addition, 37 cases of acute myeloid leukemia were analysed and demonstrated Rh 123 efflux in 18/37 (49%) cases . In this entity the percentage of Rh 123 effluxing cells ranged from 12 to 80% (median 45%) . Additionally, we evaluated the Rh 123 efflux/retention in peripheral blood cells of healthy volunteers.(ABSTRACT TRUNCATED AT 250 WORDS)

Cancer Chemother Pharmacol, 1994, 34(3), 183 - 90
Characterization of an etoposide-resistant human ovarian cancer cell line; Kubota N et al.; Etoposide (VP-16) is one of the most important anticancer agents available and is used in many chemotherapeutic regimens . To characterize resistance to this drug, we established a VP-16-resistant human ovarian cancer cell line, SKOV3/VP, by continuous stepwise exposure of SKOV3 cells to VP-16 . The degree of resistance to VP-16 of SKOV3/VP was about 25 times that of the parent cell line (SKOV3), and SKOV3/VP showed cross-resistance to teniposide, adriamycin, CPT-11, and vincristine . The accumulation of {3H}-VP-16 observed in SKOV3/VP cells was about half that seen in SKOV3 cells, and the accumulation of Adriamycin by this resistant cell line was also lower than that of its parent . Overexpression of neither the multidrug resistance gene mdr-1, the multidrug-resistance-associated protein (mrp) gene, nor P-glycoprotein was detected using reverse transcriptase-polymerase chain reaction analysis and flow cytometry with MRK-16, a monoclonal antibody against P-glycoprotein . The topoisomerase II activity of nuclear extracts from SKOV3/VP cells was lower than that from the parental cells, as was the amount of DNA topoisomerase II, demonstrated by immunoblotting . These results suggest that the mechanism responsible for the multidrug resistance of this cell line may be attributable to changes on its DNA topoisomerase II and to its reduced accumulation of the drugs as compared with the parental line SKOV3.

Cancer Chemother Pharmacol, 1994, 34(2), 125 - 32
Inhibition of P-glycoprotein-mediated vinblastine transport across HCT-8 intestinal carcinoma monolayers by verapamil, cyclosporine A and SDZ PSC 833 in dependence on extracellular pH; Zacherl J et al.; The ability of the multidrug resistance modifiers R- and R,S-verapamil (VPL), cyclosporine A (CsA) and its non-immunosuppressive derivative SDZ PSC 833 (PSC 833) to inhibit P-glycoprotein (P-gp)-mediated transepithelial flux of tritiated vinblastine was investigated using tight and highly resistant (R > 1,400 omega cm2) monolayer cultures of intestinal adenocarcinoma-derived HCT-8 cells grown on permeable tissue-culture inserts . Apical addition of these chemosensitizers inhibited drug flux (137 pmol h-1 cm-2; range, 133-142 pmol h-1 cm-2) in the basal to apical secretory direction at clinically relevant concentrations, with PSC 833 showing the highest activity, exhibiting inhibition at concentrations as low as 10 ng/ml (9 nM) . Acidification of the modulator-containing apical compartment to an extracellular pH (pHo) of 6.8 had no influence on MDR reversal by CsA at 1 microgram/ml (0.9 microM; flux inhibition, 52%) or by PSC 833 at 100 ng/ml (0.09 microM; flux inhibition, 60%), in contrast to R,S- and R-VPL, which showed decreased inhibition and caused less accumulation of vinblastine in HCT-8 cells under this condition (flux inhibition of 35% and 23%, respectively, at pHo 6.8 vs 50% and 43%, respectively, at pHo 7.5) . P-gp-mediated rhodamine 123 efflux from dye-loaded single-cell suspensions of HCT-8 cells as measured by flow cytometry was not impeded at pHo 6.8 in comparison with pHo 7.5 in standard medium, but at low pHo the inhibitory activity of R-VPL (29% vs 60% rhodamine 123 efflux inhibition) was diminished significantly, again without a reduction in the effect of PSC 833 (rhodamine 123 flux inhibition, 75%) . In conclusion, drug extrusion across polarised monolayers, which offer a relevant model for normal epithelia and tumour border areas, is inhibited by the apical presence of R,S- and R-VPL, CsA and PSC 833 at similar concentrations described for single-cell suspensions, resulting in increased (2.2- to 3.7-fold) intracellular drug accumulation . Functional apical P-gp expression, the absence of paracellular leakage and modulator-sensitive rhodamine 123 efflux in single HCT-8 cells indicate a P-gp-mediated transcellular efflux in HCT-8 monolayers . In addition to its high MDR-reversing capacity, the inhibitory activity of PSC 833 is not affected by acidic extracellular conditions, which reduce the VPL-induced drug retention significantly . As far as MDR contributes to the overall cellular drug resistance of solid tumours with hypoxic and acidic microenvironments, PSC 833 holds the greatest promise for clinical reversal of unresponsiveness to the respective group of chemotherapeutics.

Acta Clin Belg, 1994, 49(1), 5 - 11
Restriction fragment length polymorphism analysis of drug resistant Mycobacterium tuberculosis strains isolated in Belgium; Rigouts L et al.; The recently identified insertion element IS6110 is present in most strains of the Mycobacterium tuberculosis complex . Restriction fragment length polymorphism (RFLP) based on IS6110 generates strain specific fingerprints and allows typing of M . tuberculosis strains . We present here the results of a RFLP study on 12 multi drug and 10 single drug resistant, recent isolates of M . tuberculosis received from different clinical microbiology laboratories in Belgian hospitals . All isolates originating from different patients yielded distinct RFLP patterns possessing 2 to 13 copies of IS6110 . There was no correlation between the number or location of IS6110 copies and the drug resistance patterns . These results illustrate the existence of a broad polymorphism among Belgian isolates . Though this preliminary study did not reveal an outbreak or a micro-epidemic, we consider that the establishment of a DNA fingerprint bank in Belgium will be extremely helpful for tracing recent sources of infection, for the control of a possible spread of multidrug resistant organisms, and for the surveillance of tuberculosis in general.

Cancer Chemother Pharmacol, 1994, 34(1), 81 - 5
Dominant expression of multidrug resistance in intraspecific murine lymphoma hybrid cells; Palyi I et al.; Cultured P388/VCR mouse lymphoma cells resistant to vincristine (VCR) and to 5-bromodeoxyuridine (BUdR) and deficient in thymidine kinase (TK-) were fused with P388/DAG cells resistant to 1.2:5,6-dianhydrogalactitol (DAG), an anticancer alkylating agent, and to 6-thioguanine (6-TG) and deficient in hypoxanthine phosphoribosyl-transferase (HPRT-) . The hybrid cells expressed multidrug resistance (MDR), i.e., resistance to VCR and cross-resistance to Adriamycin (ADM) and actinomycin D (Act . D), in a dominant manner . The presence of glycoprotein p170, the MDR gene product, was detected in the hybrid cells . Resistance to DAG was also expressed dominantly, whereas cross-resistance to dibromodulcitol (DBD), a chemically related anticancer drug, was slight.

Cancer Chemother Pharmacol, 1994, 33(6), 465 - 71
Functional studies with a full-length P-glycoprotein cDNA encoded by the hamster pgp1 gene; Devine SE et al.; Hamster cells grown in culture may, like human and mouse cells, develop multidrug resistance (MDR) when exposed to certain cytotoxic chemotherapeutic agents . Several phenotypic features that are characteristic of MDR have been described; these include (1) resistance to many structurally and functionally unrelated drugs that have different cellular targets and modes of action; (2) reversal of MDR by certain agents, including verapamil and cyclosporin A; and (3) reduced intracellular drug accumulation relative to that of drug-sensitive cells . In this report we show that the introduction and overexpression of the hamster pgp1 cDNA confers to otherwise drug-sensitive cells an MDR phenotype with these features . Moreover, pgp1 transfectants showed varying degrees of resistance to anthracycline analogues, indicating that structural analogues of commonly used anticancer agents are capable of circumventing drug resistance conferred by pgp.

Cancer Invest, 1994, 12(2), 138 - 44
Two multidrug-resistant Friend leukemic cell lines selected with different drugs exhibit overproduction of different P-glycoproteins; Savaraj N et al.; Two Friend leukemic multidrug-resistance (MDR) cell lines were established by exposure to stepwise increased concentrations of rhodamine-123 (RHO) (cell line RR-30) or Adriamycin (ADR) (cell line ARN-15) . RR-30 displays preferential resistance to RHO, whereas ARN-15 is more resistant to ADR . The levels of resistance to other MDR drugs and reversibility by verapamil between these two MDR cell lines were somewhat different . Southern blot, RNase protection, and Western blot analysis using gene-specific probes demonstrated that RR-30 and ARN-15 cells preferentially amplified the mdr1 and mdr3 genes, respectively, leading to overexpression of the corresponding P-glycoproteins (p-gp) . Our results suggest that members of the mdr gene family can be amplified independently by using different selecting agents, which could be responsible for the differences in the sensitivities to these selecting agents as well as to these MDR drugs.

J Cancer Res Clin Oncol, 1994, 120(5), 263 - 71
Secondary combined resistance to the multidrug-resistance-reversing activity of cyclosporin A in the cell line F4-6RADR-CsA; Dietel M et al.; Multidrug-resistant tumor cells can be resensitized by combined application of the selecting cytostatic drug and a chemosensitizer, such as cyclosporin A (CsA) or a calcium channel blocker . Since clinical trials on the circumvention of multidrug resistance (MDR) with chemosensitizers report disparate results, we investigated whether tumor cells of the MDR phenotype can develop additional resistance to the cytostatic chemosensitizer combination . Thus, the Adriamycin(ADR)-selected, P-glycoprotein-positive MDR Friend leukemia cell line F4-6RADR was exposed to stepwise increased concentrations of CsA at a constant level of 0.05 microgram/ml ADR . The initial CsA concentration (plus 0.05 microgram/ml ADR) to inhibit cell growth of F4-6RADR cells by 50% (IC50) was 0.04 microgram/ml . By continuous incubation for more than 6 months, the IC50 for CsA (at constant ADR) was elevated to 3.6 micrograms/ml (90-fold), thus generating the variant F4-6RADR-CsA . The F4-6RADR-CsA cells were cross-resistant for cyclosporin H (CsH), a non-immunosuppressive derivative of CsA . As shown by immunocytochemistry as well as by the polymerase chain reaction and by Western blotting including densitometry, P-glycoprotein was preserved in the F4-6RADR-CsA variant and was expressed at a 4-fold higher level than in F4-6RADR cells . Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis could detect no new proteins in F4-6RADR-CsA as compared to F4-6RADR . Interestingly, resistance of F4-6RADR-CsA cells remained reversible for the calcium antagonists verapamil and dihydropyridine B859-35 (dexniguldipine-HCl), indicating that CsA and these compounds interfere with the P glycoprotein function by different pharmacodynamic mechanisms . Transport studies with {14C}ADR, performed in the presence and absence of chemosensitizers, confirmed the good correlation of P-glycoprotein function with the pattern of resistance found in proliferation assays . Cellular accumulation of {3H}cyclosporin was reduced to 71% of that of the F4-6 controls in F4-6RADR-CsA cells, but remained at the level of controls in F4-6RADR cells . Results indicate that increased amounts of the P-glycoprotein--besides other, perhaps more important mechanisms that are as yet unknown--partially mediate CsA resistance in F4-6RADR-CsA cells . We have designated this new form of resistance "secondary combined resistance" (SCR) . The results suggest that at least some clinical cases of insensitivity to chemosensitizers or of relapse after reversing therapy could be explained by SCR, and that resensitizing treatment of tumor patients should be based on the consideration of several chemosensitizers of different pharmacodynamics.

J Neurobiol, 1994 Jan, 25(1), 23 - 34
A putative nicotine pump at the metabolic blood-brain barrier of the tobacco hornworm; Murray CL et al.; In mammals, P-glycoprotein immunostaining at the blood-brain barrier has implicated the multidrug pump in the restricted movement of many cytotoxic agents into the central nervous system (CNS) . Since many insects require a sophisticated blood-brain barrier system to protect their CNS from plant-derived neurotoxins, we have investigated the possibility that a P-glycoprotein homolog constitutes a component of the insect blood-brain barrier . We have used the nicotine-resistant tobacco hornworm (Manduca sexta) to address this issue . Manduca has been previously shown, in physiological studies, to have an alkaloid (nicotine/morphine/atropine) pump at its excretory malpighian tubules . We show (1) that the tubules are P-glycoprotein immunopositive, (2) that Manduca has a metabolic blood-brain barrier for nicotine, (3) that the barrier co-localizes with P-glycoprotein immunostaining, and (4) that detoxifying enzymes as well as the nicotine pump are likely to account for the metabolic blood-brain barrier to nicotine . These findings may provide insights on two major fronts, the troublesome problem of multi-insecticide resistance, a phenomenon that parallels multidrug resistance in tumor cells, and the problem of tolerance to addictive neuroactive drugs like nicotine or morphine.

Med Pediatr Oncol, 1994, 22(4), 244 - 9
Cytosine arabinoside and mitoxantrone treatment of relapsed or refractory childhood leukemia: initial response and relationship to multidrug resistance gene 1; Wells RJ et al.; The objective of this study was to determine the response rate and toxicity of high-dose cytosine arabinoside (AC) and mitoxantrone (M) in relapsed or refractory childhood acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) and to correlate response with the expression of the multidrug resistance gene 1 (mdr1) . Twenty-nine patients were treated with AC 1.0 g/m2 infused over 2 h every 12 h for eight doses (days 1-4) and M 12 mg/m2 infused over 1 h (days 3-6) . Mdr1 expression was determined by a polymerase chain reaction (pcr) assay . Ten of 15 patients (67%) with AML obtained a complete remission (CR) of 3 to 30+ months duration . Eight of 14 (57%) ALL patients obtained a CR of 1 to 23+ months duration . The major toxicities were hematopoietic and infectious . Seventy-nine per cent of patients developed a documented infection during induction . Mdr1 did not correlate with a lower induction rate . This AC/M regimen is active in childhood AML and ALL.

Jpn J Cancer Res, 1994 Jan, 85(1), 13 - 6
5'-Deoxy-5-fluorouridine increases daunorubicin uptake in multidrug-resistant cells and its activity is related with P-gp 170 expression; van der Heyden S et al.; Most anticancer agents fail to induce clear responses in the treatment of colorectal cancer . This can be explained by involvement of overexpression of the membrane glycoprotein, P-gp 170, which is associated with multidrug resistance (MDR), and/or with involvement of ras . Fluoropyrimidines are amongst the few options in the chemotherapeutic treatment of colorectal cancers . 5'-Deoxy-5-fluorouridine (dFUrd) needs intracellular activation via 5-fluorouracil into 5-fluoro-2'-deoxyuridine-5'-monophosphate and 5-fluorouridine-5'-triphosphate . In the present study, the cytotoxic activity of dFUrd in vitro and dFUrd combined with daunorubicin (DNR) was assessed with the (3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium) bromide assay in cells with increased P-gp 170 expression versus controls . Surprisingly, dFUrd was most active in cells with high P-gp 170 expression, a finding which can not be explained by intracellular metabolic activity only . The results also show that dFUrd improves the DNR uptake in MDR-positive cells, and this is related with increased cytotoxicity of the anthracycline.

Oncology, 1994 Jan-Feb, 51(1), 2 - 8
Characterization of vincristine-resistant HOB1 lymphoma cell line showing the classical MDR phenotype and altered expression of membrane glycoproteins; Lee WP et al.; A multidrug-resistant (MDR) cell line isolated from HOB1 lymphoma cells was characterized . The MDR phenotype in this cell line was typified by resistance to vincristine with varying degrees of cross-resistance to Adriamycin, colcemid and actinomycin D . Decreased intracellular {3H}vincristine with concurrent increase in the expression of a 170-kDa membrane glycoprotein (P-glycoprotein) suggested a plausible underlying mechanism for the development of resistance . Amplification of the mdr1 gene as well as a homogeneous staining region on the long arm of the 7th chromosome was observed . Moreover, metabolic studies with {14C}glucosamine or {14C}mannose indicated differential expressions of membrane glycoproteins between the drug-sensitive parental and drug-resistant descendant cells . It is concluded that the development of drug resistance in HOB1 lymphoma cells was strongly correlated with the overexpression of P-glycoprotein.

Cancer Res, 1994 Jan 1, 54(1), 152 - 8
Multidrug resistance-associated protein gene overexpression and reduced drug sensitivity of topoisomerase II in a human breast carcinoma MCF7 cell line selected for etoposide resistance; Schneider E et al.; A human breast cancer cell line (MCF7/WT) was selected for resistance to etoposide (VP-16) by stepwise exposure to 2-fold increasing concentrations of this agent . The resulting cell line (MCF7/VP) was 28-, 21-, and 9-fold resistant to VP-16, VM-26, and doxorubicin, respectively . MCF7/VP cells also exhibited low-level cross-resistance to 4'-(9-acridinylamino)-methanesulfon-m-anisidide, mitoxantrone, and vincristine and no cross-resistance to genistein and camptothecin . Furthermore, these cells were collaterally sensitive to the alkylating agents melphalan and chlorambucil . DNA topoisomerase II levels were similar in both wild-type MCF7/WT and drug-resistant MCF7/VP cells . In contrast, topoisomerase II from MCF7/VP cells appeared to be 7-fold less sensitive to drug-induced cleavable complex formation in whole cells and 3-fold less sensitive in nuclear extracts than topoisomerase II from MCF7/WT cells . Although this suggested that the resistant cells may contain a qualitatively altered topoisomerase II, no mutations were detected in either the ATP-binding nor the putative breakage/resealing regions of either DNA topoisomerase II alpha or II beta . In addition, the steady-state intracellular VP-16 concentration was reduced by 2-fold in the resistant cells, in the absence of detectable mdr1/P-gp expression and without any change in drug efflux . In contrast, expression of the gene encoding the MRP was increased at least 10-fold in resistant MCF7/VP cells as compared to sensitive MCF7/WT cells . These results suggest that resistance to epipodophyllotoxins in MCF7/VP cells is multifactorial, involving a reduction in intracellular drug concentration, possibly as a consequence of MRP overexpression, and an altered DNA topoisomerase II drug sensitivity.

Tsitologiia, 1994, 36(7), 687 - 95
{The isolation and genetic characteristics of the new cell line of mouse myeloma spEBR-5 selected for ethidium bromide resistance and characterized by multiple drug resistance}; Berezkina EV et al.; Stable mutant cells spEBR-5, resistant to ethidium bromide in concentration of 5 micrograms/ml, have been isolated by multistep selection in mouse myeloma cells sp2/0-Ag14 . The spEBR-5 cells, selected with ethidium bromide, appeared to be cross resistant to unrelated drugs in connection with mdr/lb gene amplification and overexpression . Five specific chromosomal markers were detected in the karyotype of spEBR-5 cells as a result of chromosome structural rearrangement . No cytological manifestation of gene amplification such as HCR of chromosomes or DMs was found . A diffuse location of amplified sequences in chromosome(s) is suggested . The new mutant cell lines spEBR-5 can be used as a model for investigation of multidrug resistance mechanisms.

Invest New Drugs, 1994, 12(3), 185 - 95
Reversal of multidrug resistance in murine fibrosarcoma cells by thioxanthene flupentixol; Fan D et al.; The purpose of this study was to identify calcium channel and calmodulin antagonists effective in increasing the cytotoxic effects of several chemotherapeutic drugs against UV-2237 murine fibrosarcoma MDR cells . Among 8 compounds tested at nontoxic concentrations, flupentixol, a piperazine-substituted thioxanthene, was the most potent in enhancing the cytotoxicity of anticancer drugs commonly associated with the multidrug resistant (MDR) phenotype, such as Adriamycin, actinomycin D, vinblastine, and vincristine, but not 5-fluorouracil, a drug usually unaffected by MDR . The chemosensitizing effects of flupentixol were produced by increasing intracellular drug accumulation via a mechanism unrelated to the binding of the plasma membrane P-glycoprotein.

Invest New Drugs, 1994, 12(3), 169 - 82
Differential cytotoxic effects of docetaxel in a range of mammalian tumor cell lines and certain drug resistant sublines in vitro; Hill BT et al.; The in vitro cytotoxic effects of docetaxel (Taxotere; RP56976, NSC688503) proved both time and concentration dependent . Amongst thirteen human cell lines from various tumor types, exposure to increasing concentrations of docetaxel over 24 hrs resulted in a plateau-shaped dose response curve, suggesting that increased cell kill becomes more dependent on increased exposure duration than on concentration . IC50 concentrations (reducing survival by 50%) ranged from 0.13-3.3 ng/ml, with three neuroblastoma lines proving most sensitive and three breast and two colon carcinoma lines showing least sensitivity . There was significant cross-resistance to docetaxel in the classic multidrug resistant (MDR) Chinese hamster ovarian (CHO) CHRC5 line and the human lymphoblastoid CCRF-CEMVLB1000 line, as well as in two vincristine (VCR)-selected MDR MCF-7 sublines . All four of these MDR sublines overexpress P-glycoprotein (Pgp), as did a 6-fold docetaxel-selected resistant CHO subline . As an apparent corollary, in two human teratoma lines selected for etoposide resistance and showing some cross-resistance to VCR and in two CHO sublines expressing low levels of VCR resistance, yet all proving Pgp positive, no docetaxel cross-resistance was identified . Verapamil modulated docetaxel resistance only in sublines expressing resistance to the drug and overexpressing Pgp . Four other human tumor sublines selected for resistance to 5-fluorouracil, cisplatin or teniposide, showed a lack of cross-resistance to docetaxel . Furthermore, cross-resistance to docetaxel was not apparant in four epipodophyllotoxin-selected resistant sublines with alterations in topoisomerase II, indicating its effectiveness against tumor cells expressing the topoisomerase II-related MDR phenotype . Our observation that docetaxel cross-resistance was not automatically expressed by classic MDR tumour cells appears of interest and of potential clinical relevance.

Breast Cancer Res Treat, 1994, 31(2-3), 263 - 71
Transcriptional regulation of multidrug resistance in breast cancer; Glazer RI et al.; The development of cross-resistance to many natural product anticancer drugs, termed multidrug resistance (MDR), is one of the major reasons why cancer chemotherapy ultimately fails . This type of MDR is often associated with over-expression of the MDR1 gene product, P-glycoprotein (Pgp), a multifunctional drug transporter . The expression of MDR in breast tumors is related to their origination from a tissue that constitutively expresses Pgp as well as to the development of resistance during successive courses of chemotherapy . Therefore, understanding the mechanisms that regulate the transcriptional activation of MDR1 may afford a means of reducing or eliminating MDR . We have found that MDR1 expression can be modulated by type I cAMP-dependent protein kinase (PKA), opening up the possibility of modulating MDR by selectively down-regulating the activity of PKA-dependent transcription factors which upregulate MDR1 expression . High levels of type I PKA occurs in primary breast carcinomas and patients exhibiting this phenotype show decreased survival . The selective type I cAMP-dependent protein kinase (PKA) inhibitors, 8-Cl-cAMP and Rp8-Cl-cAMP{S} may be particularly useful for downregulating PKA-dependent MDR-associated transcription factors, and we have found these compounds to downregulate transient expression of a reporter gene under the control of several MDR1 promoter elements . Thus, investigations of this nature should not only lead to a greater understanding of the mechanisms governing the expression of MDR, but also provide a focus for pharmacologic intervention by a new class of inhibitors.

Adv Clin Chem, 1994, 31, 1 - 61
Multidrug resistance in the laboratory and clinic; Bellamy WT et al.; Multidrug resistance represents a major obstacle in the successful therapy of neoplastic diseases . Studies have demonstrated that this form of drug resistance occurs in cultured tumor cell lines as well as in human cancers . P-glycoprotein appears to play an important role in such cells by acting as an energy-dependent efflux pump to remove various natural-product drugs from the cell before they have a chance to exert their cytotoxic effects . Using the tools of molecular biology, studies are beginning to reveal the true incidence of multidrug resistance, as mediated by the MDR1 gene, in the clinical setting . It has been demonstrated, at least in the laboratory, that resistance mediated by P-glycoprotein may be modulated by a wide variety of compounds, including verapamil and cyclosporine A . These are compounds which, by themselves, generally have little or no effect on the tumor cells, but when used in conjunction with antineoplastic agents act to decrease, and in some instances eliminate, drug resistance . The mechanism(s) by which these agents act to reverse resistance is not fully understood . Clinical trials to modulate P-glycoprotein activity are now under way to determine whether such strategies will be feasible . The detection of the P-glycoprotein in patient samples is very important in the design of these studies, as it appears that drug-resistant cells lacking P-glycoprotein will be unaffected by agents such as verapamil . Clinical studies are needed in which patients are stratified into chemotherapy protocols based on levels of MDR1 mRNA or P-glycoprotein expression in the primary tumors . Several research areas have been identified that are important to the transfer of the discovery of the MDR1 gene and its protein product from the research laboratory to the clinical environment . There is an immediate need for comprehensive information on the prevalence and levels of expression of the human MDR genes and their protein products in human organs and tissues . Data are needed on P-glycoprotein levels in specific subpopulations (e.g., according to age, sex, race, and diet), and the study of the heterogeneity and variability of expression of P-glycoprotein in normal human tissues should be given high priority . Since early studies have indicated some successes in identifying patients with classic multidrug resistance who might be responsive to chemosensitization, it can be anticipated that clinical research will accelerate in this area . The next wave of clinical studies will provide clinical investigators with opportunities to develop and evaluate P-glycoprotein tests and correlate test results with clinical outcomes.

Invest New Drugs, 1994, 12(2), 93 - 7
Growth-inhibitory properties of novel anthracyclines in human leukemic cell lines expressing either Pgp-MDR or at-MDR; Mariani M et al.; The objective of the experiments reported in this paper was the identification of promising anthracycline analogs on the basis of lack of cross-resistance against tumor cells presenting either P-glycoprotein multidrug resistance (Pgp-MDR) or the altered topoisomerase multidrug resistant (at-MDR) phenotype . Differently modified anthracycline analogs known to be active against MDR cells were assayed in vitro against CEM human leukemic cells, and the sublines CEM/VLB100 and CEM/VM-1 exhibiting respectively the Pgp-MDR and the at-MDR phenotype . Two classes of molecules, in which the -NH2 group in C-3' position is substituted with a morpholino, methoxymorpholino (morpholinyl-anthracycline), or an alkylating moiety, present equivalent efficacy in the drug-sensitive and the two drug-resistant sublines . These results indicate that such molecules may exert their cytotoxic effect through a mode of action different from that of "classical" anthracyclines and is not mediated through topoisomerase II inhibition . Both molecules represent novel concepts in the field of new anthracyclines derivatives.

Br J Neurosurg, 1994, 8(5), 585 - 91
Multidrug resistance gene (MDR 1) expression in neuro-axial tumours of children and young adults; Billson AL et al.; Drug resistance in many cancers outside the CNS has been associated with over-expression of the multidrug resistance gene (MDR1), which codes for the transmembrane efflux pump P-glycoprotein (Pgp) . To determine whether tumours of the neuroaxis over-express MDR1 and to identify the site of Pgp expression we examined 50 tumour specimens from 46 children and young adults using immunocytochemistry . Pgp was not expressed by any neoplastic cells, but was detected in the endothelium of tumour blood vessels in 35 of the 50 samples (70%) . 11/35 (31%) were Pgp positive in the majority of vessels, 11/35 (31%) in a proportion, but < 50% of vessels, and 13/35 (37%) in one or two vessels . Pgp was also detected in surrounding normal brain capillaries . MDR1 may play a role in the chemoresistance of neuro-axial tumours either by its expression in the normal blood-brain barrier or by forming a blood-tumour barrier . The proportion of vessels expressing Pgp may determine the degree of resistance.

Neoplasma, 1994, 41(5), 297 - 303
Effect of pentoxifylline on P-glycoprotein mediated vincristine resistance of L1210 mouse leukemic cell line; Breier A et al.; Effect of pentoxifylline (PTX) on vincristine (VCR) resistance of multidrug resistant L1210/VCR mouse leukemic cell line was studied . Reversal effect of PTX (in concentration 50-150 mg/l) on vincristine resistance, i.e . potentiation of vincristine cytotoxicity on L1210/VCR cells by PTX was found . PTX alone in the above concentration did not exert any significant cytotoxic effect on sensitive or resistant cell lines in the absence of vincristine . Resistance of L1210/VCR cell line was found previously to be accompanied with overexpression of drug transporting P-glycoprotein . Indeed, lower level of 3H-vincristine accumulation by resistant L1210/VCR cell line in comparison with sensitive L1210 cell line was observed . Accumulation of 3H-vincristine by L1210/VCR cell line was significantly increased in the presence of PTX . PTX in the same condition did not exert any considerable effect on accumulation of 3H-vincristine by nonresistant L1210 cells . Observable morphological damage was observed in L1210/VCR cells cultivated in medium containing vincristine (0.2 mg/l) and pentoxifylline (100 mg/l) in comparison with the non-damaged cells in the presence of vincristine or pentoxifylline alone . The results obtained indicate that pentoxifylline may be considered as a reversal agent in multidrug resistance.

Eur J Cancer, 1994, 30A(11), 1705 - 9
Expression of the multidrug resistance-associated protein (MRP) gene in human lung tumours and normal tissue as determined by in situ hybridisation; Thomas GA et al.; In a number of cell lines with a multidrug resistant phenotype, there is no overexpression of the putative efflux pump, P-glycoprotein . Some such lines do, however, overexpress the MRP gene which encodes a protein bearing considerable amino acid homology to P-glycoprotein . We have used in situ hybridisation to study expression of the MRP gene in human cell lines, lung tumours (representing all the major histologies) and normal lung tissue . Considerable heterogeneity of expression was seen in parental cell line COR-L23/P whereas relatively uniform high-level expression was seen in the resistant line COR-L23/R . Normal bronchial epithelium was strongly positive, but the major epithelial component of all eight lung tumours studied showed only a negative to weak signal . However, the leading edge of the tumours consistently produced a more intense signal similar to that in normal epithelium . Areas of lymphocytic infiltrate were more strongly positive than the tumour epithelium . These results suggest that expression of the MRP gene may be a significant factor determining response of lung tumours to chemotherapy, but that considerable caution is needed in the interpretation of expression studies carried out on homogenised tissue biopsies.

Eur J Cancer, 1994, 30A(10), 1541 - 5
Point mutations in the mdr1 promoter of human osteosarcomas are associated with in vitro responsiveness to multidrug resistance relevant drugs; Stein U et al.; Among human sarcomas, osteosarcomas usually display high intrinsic mdr1 expression while malignant fibrous histiocytomas (MFH) do not . A comparative polymerase chain reaction (PCR)-based sequence analysis of the mdr1 promoter revealed point mutations in seven out of nine osteosarcomas at nucleotides +103 (2 cases T-->C) and +137 (5 cases G-->T) . No changes were seen in eight MFHs . When COS cells transfected with CAT constructs containing the T-->C chloramphenicol acetyltransferase mutant mdr1 promoters were treated with vincristine or doxorubicin, expression of the CAT gene was enhanced to a higher extent than with constructs containing wild-type or G-->T-mutant mdr1 promoters . We suggest that there is a correlation between the type of mdr1 promoter mutation and responsiveness to MDR relevant drugs.

Wien Klin Wochenschr, 1994, 106(22), 704 - 8
{Possibilities and limits of cytostatic chemotherapy in pancreatic carcinoma}; Scheithauer W; The investigation of chemotherapy for pancreatic cancer has been hampered by the fact that most patients present with severe general illness and thus tolerate this form of treatment poorly . Furthermore, because of the relative inaccessibility of the pancreas it has been difficult to monitor objective response rates . In patients with disseminated disease only few drugs have shown antitumour activity and the response rates generally do not exceed 20% . Several combination regimens have been tested . Of those assessed in randomized trials, the median survival has ranged from 2 to 6.5 months, which is not significantly better than with supportive therapy alone . Endocrine treatment measures and certain experimental treatment approaches such as reversal of multidrug resistance, photodynamic therapy and radioimmunotherapy may represent promising fields for future research . Encouraging preliminary results, however, warrant confirmation in controlled clinical trials . In patients with unresectable though localized pancreatic tumours the use of conventional external-beam radiation therapy plus 5-fluorouracil can significantly increase survival . Combined radiochemotherapy also seems to play an important role in the postoperative adjuvant treatment of potentially curative tumours, increasing the long-term results of surgical management.

World J Urol, 1994, 12(4), 214 - 23
Chemoresistance of renal cell carcinoma: 1986-1994; Mickisch GH; Multidrug resistance (MDR) in a variety of human tumors such as renal cell carcinoma (RCC) is thought to be caused by expression of the mdr1 gene and may be reversed by applying chemosensitizers such as Dexverapamil that inhibit the mdr1 gene product P-glycoprotein . On the basis of our preclinical analysis, we initiated a clinical (GCP) study with vinblastine (VBL), the most effective--if at all--chemotherapeutic agent; dexverapamil; and dexamethasone in patients with RCC . All patients had histologically proven RCC that was metastatic and progressive at study entry . The statistical design featured a preliminary study of two cycles of VBL alone followed by tumor evaluation . If no response was documented, with all patients thus serving as their own control, dexverapamil and dexamethasone were added for a minimum of three cycles of combination therapy . Having obtained institutional permission by the ethical review committee (MEC 124, 106-1993/12), we enrolled 24 patients on this protocol starting on May 3, 1993 . In the preliminary study, 1 complete response (CR) was achieved with VBL alone, and myelotoxicity led to an adequate dose reduction from 2 mg/m2 VBL per day given as a 5-day continuous infusion (days 1-5) in 6/10 yet evaluable patients to 1.4 mg/m2 per day . In 8/11 yet evaluable patients, dexverapamil doses reached > or = 3000 mg/day by 7-day oral uptake (days 0-6, supported by 20 mg dexamethasone given twice daily), which is significantly higher than those previously reported . The combination of VBL given at 1.4 mg/m2 per day plus, dexverapamil given at 3000 mg per day was felt to be safe and well tolerated . Nine patients were yet evaluable for response . One partial response and three minor responses were noted in this heavily pretreated study population . It appears that this innovative approach may have some activity in RCC and may eventually lead to a rational treatment modality . Careful evaluation in ongoing studies is warranted.

Breast Cancer Res Treat, 1994, 32(1), 57 - 65
Flow cytometry: potential utility in monitoring drug effects in breast cancer; Koester SK et al.; Flow cytometric analysis of DNA ploidy and S-phase fraction are well recognized prognostic indicators in breast cancer . The present paper deals with the widening of the applications of flow cytometry to monitoring the effectiveness of antiestrogen therapy, detecting clonal selection and emergence of drug resistance, and monitoring chemosensitizing properties of drugs . Antiestrogen activity can be studied by DNA flow cytometry to address clinical research problems such as patient-specific pharmacokinetics, dosing compliance, and acquired antiestrogen resistance . Patient plasma specimens containing various concentrations of triphenylethylenes can be monitored for drug-induced effects using cell cycle measurements and correlated to in vivo drug levels . DNA flow cytometry has also been instrumental in the study of the effects of prolonged low-dose (0.5 microM for > 100 days) tamoxifen treatment on human estrogen receptor negative MDA-MB-231 cells, where it was shown that tamoxifen may significantly alter cell cycle kinetics and tumorigenicity of these cells, selecting a new, more aggressive, and rapidly growing clone . Lastly, it has been shown that the chemosensitizing properties of another triphenylethylene antiestrogen, toremifene, on estrogen receptor negative, multidrug resistant MDA-MB-231-A1 human breast cancer cells can be studied using flow cytometric analysis . Toremifene (and its metabolites N-desmethyltoremifene and toremifene IV) are able to "resensitize" MDA-MB-231-A1 cells to vinblastine and doxorubicin, as reflected in a marked shift of cells to G2/M phase of the cell cycle . Flow cytometry is a widely available technique that might be applied clinically to monitor, at the cellular level, drug effects on tumors, including the modulators of drug resistance.

Cancer Detect Prev, 1994, 18(5), 407 - 13
Multidrug resistance gene and P-glycoprotein expression in anaplastic carcinoma of the thyroid; Yamashita T et al.; The resistance of malignant tumors to chemotherapy with anticancer drugs has been considered to be due partly to overexpression of the multidrug resistance gene (mdr1) and its gene product, P-glycoprotein (P-GP), which acts as a drug efflux pump for several chemotherapeutic agents . In order to elucidate the mechanism of anticancer drug resistance in anaplastic thyroid carcinoma with very poor prognosis, we examined the expression of mdr1 mRNA and P-GP, and analyzed their relationships to chemotherapy response . Twenty surgical samples from 16 patients with anaplastic thyroid carcinoma were used . The mdr1 mRNA expression was examined by reverse transcription and polymerase chain reaction, and P-GP expression was evaluated by an immunohistochemical method using JSB-1 monoclonal antibody . Of the 20 clinical samples, expression of mdr1 mRNA and P-GP was observed in three and four samples, respectively . Three of the patients from whom the samples were obtained had been given anticancer drugs before biopsy . Of 12 patients who received chemotherapy for clinically evaluable diseases, 2 responded well, but 10 showed no response . All except one patient died of cancer progression . There was no relationship between the response to chemotherapy and the expression of mdr1 and P-GP . The expression of mdr1 mRNA and/or P-GP was observed in 5 of 16 patients with anaplastic thyroid carcinoma . However, the appearance of anticancer drug resistance in anaplastic thyroid carcinoma may not be explained solely by the expression of mdr1 and P-GP.

Cancer Biother, 1994 Summer, 9(2), 143 - 51
In vitro effects of pentoxifylline and doxorubicin on cell survival and DNA damage in sensitive and MDR-P388 leukemia cells; Viladkar A et al.; The utility of chemosensitizers to improve efficacy of chemotherapy is now gaining importance . This report investigated whether an active hemorheological agent, pentoxifylline (PTX), can circumvent drug resistance in parental (P388/S) and multidrug resistant (P388/DOX) P388 leukemia cells . For detection of doxorubicin (DOX) resistance and reversal of this resistance by PTX, the incorporation of nucleic acid precursor was measured after addition of DOX and PTX, respectively . The effect of PTX on the induction of DNA strand breaks by DOX was also examined . Increased fragmentation of DNA was illustrated in P388/DOX leukemia cells exposed to the combination of DOX and PTX . The most prominent feature of the multidrug-resistant cell is the reduced accumulation of the drug intracellularly . P388/DOX cells showed less accumulation of DOX in the cell as compared to that of the parental cell line . Further studies demonstrated that PTX significantly enhanced the intracellular accumulation of DOX in both the cell lines . These studies warrant the use of PTX as an adjuvant in cancer chemotherapy.

J Neurooncol, 1994, 20(2), 165 - 76
Drug resistance in brain tumors; Feun LG et al.; Resistance to chemotherapy in brain tumors is complex and may involve multiple mechanisms . For commonly used drugs, such as nitrosoureas and platinum compounds, major mechanisms may involve increaded DNA repair or removal of the drug-DNA adducts . For water soluble nitrosoureas and also for platinum compounds, other mechanisms, such as alteration in drug transport, may be important . Another major mechanism may involve glutathione and glutathione-S-transferase pathways . For vinca alkaloids and epipodophyllotoxins p-glycoprotein mediated MDR appears to be the major feature in drug resistance . In addition, alteration of tubulin and topoisomerase II have been described in resistance to vinca alkaloids and epipodophyllotoxins respectively . Recently, increased multidrug resistance associated protein gene expression has been found in glioma cells and brain tumor samples; its clinical significance requires further investigation.

Genet Anal Tech Appl, 1994, 11(3), 69 - 76
Use of chromosome microdissection, the polymerase chain reaction, and dot blot hybridization to analyze double minute chromosomes; Sognier MA et al.; The potential usefulness of chromosome microdissection, the polymerase chain reaction (PCR), and dot blot hybridization as a quick screening method for determining the genetic composition of double minute chromosomes (DMs) was evaluated . DMs or abnormally banding regions (ABRs) were microdissected from multidrug-resistant hamster cell lines and amplified with PCR using primers specific for the hamster multidrug-resistance (MDR) gene, pgp 1 . The microdissected-PCR-amplified products were shown to (a) hybridize to a 32P-labeled pCHP1 probe for the hamster MDR gene by using dot blot or Southern blot analysis and also (b) hybridize back to the chromosome region from which they were originally dissected by using fluorescent in situ hybridization . Microdissected/PCR-amplified DMs were also shown to hybridize to ABRs . When microdissected DMs and ABRs were amplified using hamster specific Alu primers, the resulting material was shown to hybridize with probes for hamster MDR and Alu . These results suggest that the DMs contained in these MDR hamster cell lines contain Alu-like sequences and the chromosome microdissection-PCR-hybridization approach might be used as a quick screening method for identifying genes amplified in DMs and ABRs in cell lines and human tumor samples.

J Neurooncol, 1994, 22(3), 239 - 43
Multidrug resistance in human cancer; Lehnert M; Resistance to cytotoxic chemotherapy continues to be a major obstacle to more effective treatment of human cancers . A particular problem in clinical cancer chemotherapy is the phenomenon of simultaneous resistance of cancers to a variety of unrelated cytotoxic agents . Such resistance to multiple drugs is observed much more often than resistance to individual compounds . A similar experimental phenomenon has been termed multidrug resistance or MDR . Much has been learned in recent years about molecular mechanisms which can lead to MDR in cancer cells and a number of studies has been performed to evaluate the clinical relevance of such mechanisms . In particular, P-glycoprotein-associated MDR (MDR1) has received a lot of attention . This review will discuss (i) some principal aspects of drug resistance in cancer with particular emphasis on MDR1; (ii) available data on drug resistance mechanisms in brain tumors; and (iii) our current knowledge on the putative role of P-glycoprotein in the blood-brain barrier.

Jpn J Physiol, 1994, 44 Suppl 2, S9 - 15
Regulation of volume activated chloride channels by protein kinase C-mediated phosphorylation of P-glycoprotein; Hardy SP et al.; The multidrug resistance P-glycoprotein (Pgp) transports hydrophobic drugs out of cells and has been recently associated with volume-activated chloride channels . Activation of these channels by hypotonic swelling was seen to be prevented by protein kinase C (PKC) in cells expressing high levels of Pgp by transfection . HeLa cells possess equivalent chloride currents yet they are not regulated by PKC . HeLa cells do not express Pgp as assessed by Western blotting . Following transfection of HeLa cells with cDNA encoding for Pgp, PKC-dependent suppression of volume activated chloride currents was observed . PKC regulation in transiently transfected HeLa cells was abolished by alanine replacement of the serine/threonine residues in the consensus phosphorylation sites of the linker region of Pgp . Replacement of these residues with glutamate, to mimic the effect of phosphorylation, mimicked the effects of PKC on channel activation . These results indicate that overexpression of Pgp confers PKC-regulation of endogenous volume-activated chloride channels . More generally they favour a model in which Pgp acts as a regulator of volume-activated chloride channels.

Zhen Ci Yan Jiu, 1994, 19(2), 69 - 71
{Effects of moxibustion on the function of MDR gene product, P-glycoprotein (P-170)}; Zhang T et al.; The experiments were performed on BABL/c mice with S-180R adriamycin resistant tumor cells . This animal model was used to analyze the drug accumulation in the S-180R cells by flow cytometer . The drug accumulation presents the pump activity of multidrug resistance (MDR) gene product P-glycoprotein (P-170) in the cell membrane, A weak inhibition was found when moxibustion at Guan-Yan point alone . And a very significant inhibition was observed in the presence of low-dosage of verapamil, but not at high dose . This study may develop a new way to research the mechanism of acupuncture and moxibustion at molecular level, and may be useful to overcome the anticancer drug resistance.

Fundam Clin Pharmacol, 1994, 8(6), 491 - 502
Clinical application of mefloquine pharmacokinetics in the treatment of P falciparum malaria; Karbwang J et al.; Malaria remains a major public health problem in large areas of the world . One of the major factors responsible for the resurgence is the emergence of Plasmodium falciparum, resistant to available antimalarials . An antimalarial, mefloquine, has been considered since its introduction as a promising alternative antimalarial drug to overcome the situation of widespread multidrug resistant P falciparum . Pharmacokinetic studies of mefloquine have been investigated in several groups of subjects either as mefloquine alone or as combined regimens . The oral absorption of mefloquine is relatively rapid, reaching peak concentrations within 24 hours . Metabolism takes place in the liver, with carboxymefloquine as a major metabolite . Mefloquine has a large apparent volume of distribution of 200 L and is highly bound (98%) to plasma proteins . The elimination is slow; the terminal half-life is 13 10 to 14 days in Thai patients with falciparum malaria . Vomiting within 1 hour of drug administration has an influence on blood concentrations of mefloquine and this may result in treatment failure . The whole blood concentrations of mefloquine on the first two days of treatment are important determinants of parasitological response . There appear to be no pharmacokinetic interactions between mefloquine and the other two components of Fansimef in patients with uncomplicated falciparum malaria . The advantage of this combination over mefloquine alone in multidrug resistant P falciparum is still debatable . However, recent data seem to support the higher efficacy of Fansimef over mefloquine alone . Concurrent administration of antibiotics, ie ampicillin and tetracycline with mefloquine results in a significant increase in maximum concentration, reduction of the apparent volume of distribution and shortening of the terminal elimination half-life of mefloquine . An antiemetic drug metoclopramide accelerates the absorption of mefloquine and increases the maximum concentration . In contrast, mefloquine concentrations are decreased in the presence of an antimalarial, artesunate . Primaquine has no effect on the pharmacokinetics of mefloquine when given concurrently.

Yao Xue Xue Bao, 1994, 29(12), 891 - 8
{Apoptosis resistance and its reversal in harringtonine resistant cell line}; Fang M et al.; Harringtonine (HT), a domestic antitumor drug extracted from Cephalotaxus hainanensis Li showed high chemotherapeutic efficacy on human acute granulocytic leukemia and acute myelocytic leukemia in clinics . Apoptosis of HL-60 cells can be induced by HT effectively; but for cells resistant to harringtonine, apoptosis can not be induced, even if the drug (HT) concentration is over 100 times of IC50 value . Although apoptosis occurred when its multidrugs resistance had been reversed by verapamil, compared with sensitive HL-60 cells, the time at which apoptosis happened delayed and the drug dosage increased . All these suggest that apoptotic resistance might be one of the marks of drug resistance in tumor cells, and apoptosis related factors could play a role in the formation of multidrug resistance.

Oncol Res, 1994, 6(9), 429 - 38
Changes in protein kinase C subspecies protein expression and activity in a series of multidrug-resistant human KB carcinoma cell lines; Drew L et al.; We have investigated the relationship between protein kinase C (PKC), levels of resistance and drug used for selection in a series of human KB carcinoma cell lines by comparing protein kinase C activity and PKC alpha, beta I, beta II, gamma, delta, epsilon, and zeta subspecies protein expression . PKC alpha protein expression was increased by 600% and 375% in KB-A1 and KB-C1 lines respectively over the parent KB-3-1 line; only KB-A1 cells showed increased PKC delta expression . Expression of other PKC subspecies was equal to that of KB-3-1 cells . There was considerable variation between the different KB cell lines in total cytosolic PKC activity, the KB-A1 and KB-C1 lines showing 400% and 350% increases respectively, KB-V1 and KB-8-5-11 about 180%, and KB-8-5 no increase relative to the parent KB-3-1 line . For calcium-independent PKC activity, the KB-C1 and KB-A1 lines only were increased over the KB-3-1 line . Immunoprecipitation with antisera to PKC subspecies confirmed that the increase in KB-A1 cytosolic total PKC activity was due largely to PKC alpha and partially to PKC delta . Membrane-associated PKC activity was increased by 500% and 350% in KB-A1 and KB-C1 lines respectively, by 250% and 270% in KB-V1 and KB-8-5-11, and not increased in KB-8-5 cells relative to the KB-3-1 cells . For KB-C1, KB-8-5-11, and KB-8-5 lines, which show decreasing resistance to colchicine, our results suggest a correlation between PKC and multidrug resistance in cells selected for resistance to this drug . There is no correlation between PKC and multidrug resistance for cells selected in different drugs . Our study therefore suggests that specific PKC subspecies are associated with the MDR phenotype of some KB cell lines, but that the extent of PKC involvement depends on the type of drug used for selection and its concentration.

Eur J Cancer, 1994, 30A(8), 1117 - 23
Influence of dexniguldipine-HC1 on rhodamine-123 accumulation in a multidrug-resistant leukaemia cell line: comparison with other chemosensitisers; Boer R et al.; In the clinical therapy of cancer, resistance to many cytostatic drugs is a major cause of treatment failure . Among other mechanisms, the expression and pumping activity of P-glycoprotein (PGP) in the membrane of resistant cancer cells is responsible for the reduced uptake of cytostatics . The blockade or inhibition of PGP activity by chemosensitisers seems to be a tenable way to restore sensitivity to antineoplastic drugs and therapeutic efficacy . In the present work the influence of the new chemosensitiser dexniguldipine on rhodamine-123 accumulation in multidrug-resistant leukaemia cells was investigated . Dexniguldipine increases cellular rhodamine-123 accumulation dose-dependently.pEC50 values (-log concentration of drug showing a half maximal effect) in accumulation studies are dependent on pH of the test system and are in the range of 6.5 (pH 7.2) to 7.2 (pH 8.0) for dexniguldipine . In comparison with other chemosensitisers such as SDZ PSC 833, cyclosporin A, verapamil, dipyridamole, quinidine and amiodarone, dexniguldipine is the most potent drug in this test system . In addition to equilibrium measurements of rhodamine-123 accumulation, efflux of rhodamine-123 was analysed in the absence and presence of chemosensitisers . A clear dose-dependency was seen and, moreover, a dramatic decrease in efflux rates was achieved in the presence of chemosensitisers . The described system can be used to investigate PGP-mediated drug transport on a pharmacological and biochemical basis.

J Cancer Res Clin Oncol, 1994, 120(9), 539 - 44
Competitive nested polymerase chain reaction for quantification of human MDR1 gene expression; Grunebach F et al.; Tumor cell resistance to cytotoxic drugs is considered one of the major obstacles to successful chemotherapy . Multidrug resistance (MDR) describes the simultaneous expression of cellular resistance to a wide range of structurally and functionally unrelated drugs . The development of the multidrug resistance phenotype is accompanied by multiple morphological and biochemical changes: (a) increased glutathione levels in the cytoplasm, (b) modified levels of enzymes in the nucleus, particularly topoisomerase II, (c) increased DNA repair capacity and (d) overexpression of the (human) MDR1 gene encoding a transmembrane efflux pump (P-glycoprotein, gp-170), which leads to decreased intracellular accumulation and therefore to resistance to a variety of cytotoxic drugs . In this report we describe a competitive polymerase chain reaction (PCR) assay for the absolute quantification of MDR1 mRNA . This assay uses a transcript generated in vitro as an internal standard which is later coamplified together with the MDR1 cDNA . Both cDNAs exhibit the same MDR1 primer sites but differ in the length of the amplicon . For a second round of amplification we applied nested MDR1 primers and were successful in improving the sensitivity of this competitive PCR system . This test for characterizing the MDR1 expression offers high sensitivity and specificity and is therefore of great clinical relevance . It should be useful in improving monitoring and design of chemotherapy.

Cancer Chemother Pharmacol, 1994, 34(1), 44 - 50
V79 Chinese hamster lung cells resistant to the bis-alkylator bizelesin are multidrug-resistant; Butryn RK et al.; Bizelesin (U-77779) is a highly potent bis-alkylating antitumor agent that is effective against several tumor systems in vitro and in vivo . V79 cells that were 125- to 250-fold resistant to bizelesin developed after constant exposure to gradually increasing concentrations of the drug . Resistant cells exhibited a multidrug-resistant phenotype and genotype as indicated by cross-resistant to several structurally and functionally unrelated drugs, e.g., colchicine, actinomycin D, and Adriamycin, and overexpression of mdr mRNA . Very low levels of cross-resistance to the alkylating agents cisplatin and melphalan were seen . Multidrug-resistant mouse leukemia (P388/Adriamycin-resistant) and human (KB/vinblastine-resistant) cells were also resistant to bizelesin . Bizelesin resistance was unstable and decreased when cells were grown in the absence of the drug . Resistant and sensitive cell lines had similar levels of glutathione, and bizelesin cytotoxicity for resistant cells was not markedly affected by treatment with buthionine sulfoximine . Cross-resistance between bizelesin and several of its analogs is reported.

Anticancer Res, 1994 Jan-Feb, 14(1A), 13 - 9
Intracellular expression of P-glycoprotein in a human colon tumor cell line; Duensing TD et al.; A number of human tumor cell lines originating in tissues which normally express high levels of P-glycoprotein were examined for the expression of mdr-1 RNA and P-glycoprotein and their sensitivity to doxorubicin and vincristine . There was a wide variation in expression levels and in drug sensitivity among the cell lines . In the human colon tumor cell line, LS 174T, high levels of P-glycoprotein and mdr-1 RNA were observed, but this line was very sensitive to doxorubicin and vincristine . Immunoprecipitation of P-glycoprotein from preparations of membrane and cytoplasmic proteins and immunofluorescence studies using anti-P-glycoprotein antibodies revealed that P-glycoprotein in these cells is not associated with the plasma membrane, but is, instead, found intracellularly . These results emphasize the need for functional analysis of P-glycoprotein in cells that express this mediator of multidrug resistance.

Cytometry, 1994 Jan 1, 15(1), 64 - 72
Improved flow-cytometric detection of low P-glycoprotein expression in leukaemic blasts by histogram subtraction analysis; Muller MR et al.; Expression of the drug efflux pump P-glycoprotein (PGP) was determined by flow cytometry in human lung cancer cell lines and in leukaemic blasts derived from 60 patients with acute myeloid leukaemia (AML) . Cells from the PGP-negative parent cell line H69/P and the multidrug resistant (MDR)-variant H69/LX4 could be clearly distinguished by immunostaining with the anti-PGP monoclonal antibody MRK16 . In leukaemic blasts, the differences in fluorescence intensities between samples incubated with the idiotypic nonspecific (control sample) and specific antibody (test sample) were small, resulting in nondisjunct distributions . Only in a few leukaemia specimens were PGP-expressing cells detectable by simple subtraction of histograms using a threshold . Therefore, an improved histogram subtraction analysis, based on curve fitting and a statistical test, was applied to distinguish antigen-positive from antigen-negative cells . Moreover, a multiparametric staining procedure employing propidium iodide (PI) and Hoechst 33342 was used to reduce staining artefacts . By this approach, leukaemic cells with low expression of PGP were detected in 39 out of 60 cases . Subpopulations with strong PGP expression, resulting in disjunct fluorescence distributions, were not observed . Only in 5 out of 60 specimens were PGP expressing cells detected by a conventional subtraction of histograms using a threshold . Comparison of data obtained with or without the multiparametric gating procedure indicated that the increase in sensitivity was mainly due to the application of the data analysis . However, exclusion of cell debris using PI and Hoechst staining properties reduced the deviation of data from mean values . No relation between PGP expression and cell cycle position was observed in either cell lines or in leukaemic blasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Cell Biol, 1994 Jan, 14(1), 277 - 86
Functional expression of P-glycoprotein in Saccharomyces cerevisiae confers cellular resistance to the immunosuppressive and antifungal agent FK520; Raymond M et al.; We have recently reported that expression in yeast cells of P-glycoprotein (P-gp) encoded by the mouse multidrug resistance mdr3 gene (Mdr3) can complement a null ste6 mutation (M . Raymond, P . Gros, M . Whiteway, and D . Y . Thomas, Science 256:232-234, 1992) . Here we show that Mdr3 behaves as a fully functional drug transporter in this heterologous expression system . Photolabelling experiments indicate that Mdr3 synthesized in yeast cells binds the drug analog {125I}iodoaryl azidoprazosin, this binding being competed for by vinblastine and tetraphenylphosphonium bromide, two known multidrug resistance drugs . Spheroplasts expressing wild-type Mdr3 (Ser-939) exhibit an ATP-dependent and verapamil-sensitive decreased accumulation of {3H}vinblastine as compared with spheroplasts expressing a mutant form of Mdr3 with impaired transport activity (Phe-939) . Expression of Mdr3 in yeast cells can confer resistance to growth inhibition by the antifungal and immunosuppressive agent FK520, suggesting that this compound is a substrate for P-gp in yeast cells . Replacement of Ser-939 in Mdr3 by a series of amino acid substitutions is shown to modulate both the level of cellular resistance to FK520 and the mating efficiency of yeast mdr3 transformants . The effects of these mutations on the function of Mdr3 in yeast cells are similar to those observed in mammalian cells with respect to drug resistance and transport, indicating that transport of a-factor and FK520 in yeast cells is mechanistically similar to drug transport in mammalian cells . The ability of P-gp to confer cellular resistance to FK520 in yeast cells establishes a dominant phenotype that can be assayed for the positive selection of intragenic revertants of P-gp inactive mutants, an important tool for the structure-function analysis of mammalian P-gp in yeast cells.

Gene, 1993 Dec 22, 136(1-2), 231 - 6
Cloning and organization of the abc and mdl genes of Escherichia coli: relationship to eukaryotic multidrug resistance; Allikmets R et al.; Using degenerate oligodeoxyribonucleotides from conserved regions of the gene family encoding ATP-binding domain of the active transporter, two new Escherichia coli genes were identified . The first of the genes, named mdl (multidrug resistance-like), is located at min 10.2 of the E . coli chromosome and encodes two ATP-binding motifs and two hydrophobic (transmembrane) domains . The ATP-binding domains of mdl show 35-38% amino acid (aa) identity with members of the eukaryotic P-glycoprotein/multidrug resistance family . To date, 25 members of the ATP-transporter/permease gene family have been characterized in E . coli . Comparison of the ATP-binding domains from this family indicates that mdl is part of a distinct subfamily of sequences that includes hlyB, msbA, and cvaB . Gene-disruption studies revealed that mdl is not essential for cell growth . The second open reading frame, named abc (ATP-binding cassette), is located at min 4.9 of the chromosome, encodes a single ATP-binding domain, and is most homologous to ftsE, a cell division control gene of E . coli . The abc gene product also shows aa sequence homology to several E . coli permeases.

Eur J Biochem, 1993 Dec 15, 218(3), 871 - 82
A plasma membrane 'vacuum cleaner' for daunorubicin in non-P-glycoprotein multidrug-resistant SW-1573 human non-small cell lung carcinoma cells . A study using fluorescence resonance energy transfer; Mulder HS et al.; A multidrug resistant (MDR) human non-small cell lung carcinoma cell line, SW-1573/2R120 (2R120), not containing the drug-efflux pump P-glycoprotein (PgP), has been studied for the transport of daunorubicin (DN) across the cellular plasma membrane . Earlier, reduced initial DN-uptake rates and lower cellular DN steady-state concentrations were found for this cell line, when it was compared to the SW-1573 wild-type cell line . This finding was an indication for the presence of another cellular drug-efflux pump . However, we found similar DN-efflux rates in drug-free medium for the two cell lines, while for Pgp-containing MDR SW-1573/2R160 (2R160) cells the efflux rate was increased compared to wild-type cells . In order to elucidate differences in DN transport across the cellular plasma membrane, the association of DN with plasma membranes of intact cells was investigated, using fluorescence-resonance-energy transfer . For this purpose, the plasma-membrane probe 1-(4-trimethyl-ammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was chosen since, because of the overlap between the emission spectrum of TMA-DPH and the excitation spectrum of DN, transfer of energy can be achieved from TMA-DPH to DN . Cells were loaded with TMA-DPH and, after addition of 10 microM DN, the TMA-DPH fluorescence was quenched . Rapid initial quenching proved to be similar in the MDR 2R160 (Pgp-containing) cells and in the SW-1573 wild-type cells (21 +/- 1% and 20 +/- 2%, respectively), but was less in the MDR 2R120 cells not containing Pgp (14 +/- 1%) . This finding correlated with a lowered amount of DN dissolved in the plasma membrane of 2R120 cells . We interpret these data to be the result of a 'vacuum-cleaner' pumping system other than Pgp which removes DN from a plasma membrane compartment and equilibrates relatively slowly with the interior of the cell.

Cancer Res, 1993 Dec 15, 53(24), 5946 - 53
Teniposide-resistant CEM cells, which express mutant DNA topoisomerase II alpha, when treated with non-complex-stabilizing inhibitors of the enzyme, display no cross-resistance and reveal aberrant functions of the mutant enzyme; Chen M et al.; We have examined the effects of a group of DNA topoisomerase II (topo II) inhibitors, merbarone, aclarubicin, SN22995, RP60475F, and fostriecin, in CCRF-CEM cells and two sublines, CEM/VM-1 and CEM/VM-1-5, that were selected for increasing resistance to teniposide (VM-26) . The teniposide-resistant sublines have been termed "at-MDR" for altered topo II-associated multidrug resistance . These topo II inhibitors differ from the "classic" inhibitors such as teniposide in that they do not stabilize DNA-topo II complexes . In this study, we found that our at-MDR cell lines express little or no cross-resistance to these "non-classic" topo II inhibitors . Merbarone and SN22995 inhibited VM-26-mediated DNA-topo II complexes in CEM cells only when they were added before VM-26 . Since they did not deplete topo II protein, it suggested that these drugs may inhibit topo II activity before the enzyme binds to DNA, thereby preventing stabilization of VM-26-mediated topo II-DNA complexes . Continuous exposure of CEM cells to merbarone, SN22995, or VM-26 caused G2 arrest, as determined by flow cytometry . Likewise, at-MDR cells continuously treated with VM-26 also arrested in G2 . By contrast, treatment of at-MDR cells with either merbarone or SN22995 produced a qualitatively different pattern; the at-MDR cells first accumulated in G2 but then escaped the G2 block and proceeded into mitosis with elongated and intertwined chromosomes but failed to divide . Their DNA was re-replicated, however, and the cells eventually accumulated at the 8N DNA stage . Given that both wild-type and mutant topo II alpha alleles are expressed in the at-MDR cells (B . Y . Bugg, M . K . Danks, W . T . Beck, and D . P . Suttle . Expression of a mutant DNA topoisomerase II in CCRF-CEM human leukemic cells selected for resistance to teniposide . Proc . Natl . Acad . Sci . USA, 88: 7654-7658, 1991), we hypothesize that drugs such as merbarone may inhibit the activity of wild-type topo II alpha, allowing the aberrant activity of the mutant enzyme to be revealed during chromosome condensation and sister chromatid segregation.

Cancer, 1993 Dec 15, 72(12), 3553 - 63
Clinical trials of agents that reverse multidrug resistance . A literature review; Raderer M et al.; BACKGROUND . The discovery of the P-170 glycoprotein as a mediator of multidrug resistance (MDR) represents one of the most important research accomplishments in antineoplastic pharmacology during the last decade . Demonstration of P-170 in epithelial tissues, untreated and chemotherapeutically pretreated human malignancies, and identification of various agents capable of reversing resistance in vitro generated enthusiasm for clinical studies throughout the world . The authors provide an overview of the current status of clinical investigations of MDR1 reversing agents in hematologic and solid malignancies . METHODS . The authors performed an extensive literature search and selected more than 70 articles concerning the potential clinical relevance of P-glycoprotein/MDR1 modulating agents . Information abstracted included type of reverting agent and chemotherapeutic regimen, number of patients, tumor type, histologic proof of P-glycoprotein expression, and objective response rates . RESULTS . Proof of the involvement of MDR1 in clinical drug resistance has been slow to accumulate, primarily because of difficulties in adapting assays of MDR1 expression and in planning appropriate trials . Pilot studies have shown that verapamil, cyclosporine, and other chemosensitizers may reverse resistance in a subset of patients, but significant (cardiovascular) side effects are common . For leukemias, lymphomas, and multiple myeloma, response rates of 60-80% may be achieved with the potential for cure, whereas in solid tumors, only a few patients appear to benefit . CONCLUSIONS . Because of predominantly negative results and unanswered fundamental questions regarding the biology of P-glycoprotein, additional clinical trials with less toxic modulators or their combination are appropriate to delineate optimal strategies for MDR1 reversal and to define the spectrum of responsive tumors . Additional attention also must be given to the coexistence of other resistance mechanisms that may offer separate opportunities for modulation.

Cancer Res, 1993 Dec 15, 53(24), 5994 - 6000
Effects of nonionic detergents on P-glycoprotein drug binding and reversal of multidrug resistance; Zordan-Nudo T et al.; Multidrug-resistant cells are thought to maintain low intracellular cytotoxic drug concentration though the active efflux of drugs across the cell membrane . It is presently believed that P-glycoprotein mediates this energy-dependent drug efflux by interacting directly with various lipophilic compounds . In this report, we have used {3H}azidopine in a photoaffinity labeling assay to study the effect of detergents and denaturing agents on P-glycoprotein drug binding in intact cells . Nonionic detergents such as Triton X-100 or Nonidet P-40 at very low concentrations were found to completely abolish azidopine photolabeling to P-glycoprotein and are able to reverse the multidrug resistance phenotype . In contrast, high concentrations of the denaturing agent urea or the zwitterionic detergent 1-{(3-cholamidopropyl)dimethylamino}-1-propanesulfonate did not inhibit azidopine photolabeling to P-glycoprotein . A comparison between verapamil and Triton X-100 revealed that the latter was more effective in inhibiting azidopine photolabeling to P-glycoprotein while verapamil was more effective in potentiating {3H}vinblastine accumulation in drug-resistant cells . Drug transport studies showed that {3H}Triton X-100 accumulated in both drug-sensitive and -resistant cells, and its accumulation was not modulated by excess vinblastine, verapamil, or colchicine . Taken together, these findings suggest that low concentrations of Triton X-100 reverse the multidrug resistance phenotype by inhibiting P-glycoprotein drug binding . In addition, it is also suggested that the site(s) of P-glycoprotein drug binding is localized to sequences found within the lipid bilayer.

Cancer Res, 1993 Dec 15, 53(24), 5977 - 81
Flavonol-stimulated efflux of 7,12-dimethylbenz(a)anthracene in multidrug-resistant breast cancer cells; Phang JM et al.; We used a series of P-glycoprotein (P-gp) expressing multidrug-resistant (MDR) cells, developed from human breast cancer MCF-7 cells by exposure to Adriamycin, to investigate the effects of flavonoids on P-gp-mediated efflux mechanisms for chemical carcinogens . We previously showed that MDR cells derived from exposure to Adriamycin are cross-resistant to a chemical carcinogen, benzo(a)pyrene, due to its cellular efflux by the P-gp-mediated putative drug efflux pump . Our current studies extended this observation to another polycyclic aromatic hydrocarbon, 7,12-dimethylbenz(a)anthracene, known to induce mammary tumors in animals . In our attempt to find naturally occurring dietary compounds which may stimulate the P-gp-mediated efflux of carcinogens, we found that certain flavonols, kaempferol, quercetin, and galangin, are potent stimulators of the P-gp-mediated efflux of 7,12-dimethylbenz(a)-anthracene . The increased efflux decreased the cellular burden of 7,12-dimethylbenz(a)anthracene . Since these flavonol compounds are widely distributed in fruits and vegetables, their stimulatory effect on P-gp may be a mechanism relevant to carcinogenesis and the observed lowered cancer risk in humans with higher dietary intake of fruits and vegetables.

J Natl Cancer Inst, 1993 Dec 15, 85(24), 2023 - 9
Response of small-cell lung cancer xenografts to chemotherapy: multidrug resistance and direct clinical correlates; Poupon MF et al.; BACKGROUND: Patients with small-cell lung carcinomas (SCLCs) initially respond to combination chemotherapy . Only a few benefit in terms of long-term survival because most relapse . Such outcome may be attributable to development of multidrug resistance . PURPOSE: The response of SCLC to chemotherapy was examined in terms of (a) patient survival, (b) drug sensitivity of tumors in patients and of tumor xenografts in nude mice, and (c) expression of multidrug resistance gene MDR1 and GST-pi gene . METHODS: Tumor samples obtained from seven untreated patients and from one patient both before and after chemotherapy were transplanted into nude mice . The patients were treated with a combination of cyclophosphamide (C'), cisplatin (C), doxorubicin (A), and etoposide (V) (C'CAV) or C'AV and radiotherapy . Drug sensitivity of SCLCs was tested in nude mice that had received tumor xenografts from these seven patients . The expression of MDR1 and GST-pi genes was assessed in the mRNA extracted from xenografts by Northern blot analysis . P-glycoprotein was quantified by enzyme immunoassay . RESULTS: The patients' responses to C'CAV closely correlated with those of the corresponding xenografts . The tumors of the two patients who showed long-term survival after C'CAV completely regressed when they were transplanted into nude mice and subsequently treated with C'CAV . Despite initial complete response, the remaining five patients died during year 1 . A high percentage of mice receiving the tumor grafts from these five patients showed only partial tumor regression after C'CAV treatment . The MDR1 transcript was detected in all five of these xenografts . Four of five xenografts were from untreated patients, and the fifth was from a treated patient . MDR1 mRNA expression was absent in the tumor of this fifth patient before chemotherapy, but both the mice receiving the corresponding xenograft and the patient showed expression of MDR1 after C'CAV treatment . MDR1 mRNA expression was absent in the tumor xenografts obtained from two patients with long-term survival . Expression of P-glycoprotein correlated with MDR1 mRNA expression . All xenografts except one expressed the GST-pi gene . CONCLUSIONS: The absence of MDR1 gene expression during chemotherapy for SCLC indicates a favorable prognosis, gene expression is often coincident with ineffective chemotherapy, and tumor xenografts can be appropriately used to predict response to chemotherapy . IMPLICATIONS: Failure of chemotherapy to control SCLC seems to be related to an acquired multidrug resistance involving the MDR1-mediated mechanism . Therapeutic benefit could therefore be expected from chemotherapy combined with inhibitors of MDR1.

Biochim Biophys Acta, 1993 Dec 12, 1153(2), 225 - 36
The role of drug-lipid interactions in the biological activity of modulators of multi-drug resistance; Wadkins RM et al.; Of the compounds that have now been shown to circumvent acquired cellular multidrug resistance, little or no structure-activity relationship has been found, although their proposed mechanism of action is through modulation of function of p-glycoprotein . While it has been suggested that this inhibition is a direct binding to p-glycoprotein, we show here that such a model seriously neglects the effects many of these compounds have on lipid physical properties . We have characterized the interactions between 16 structurally diverse pharmacological agents (nine of which are known to reverse multidrug resistance) and a variety of lipids . Potent modulators inhibit the membrane binding of rhodamine 6G, and we have observed a correlation of the measured Ki values with the effectiveness of the compounds in situ . We have determined the effects of the compounds on detergent micellization, and have shown substantial changes on the critical micelle concentration of detergents in the presence of modulators . Finally, we have examined the changes in model membrane 'viscosity' induced by the compounds . These results indicate that both direct p-glycoprotein and indirect lipid interactions of modulators should be considered in the mechanism by which these compounds reverse multidrug resistance.

JEMS, 1994 Jan, 19(1), 70 - 3
TB: return of an old scourge; Gaston B; In recent years, several hundred health care workers nationwide have contracted tuberculosis after being infected on the job; at least 16 of those workers have developed multidrug-resistant strains of the disease, and at least five of them have died . TB is alive and well--and EMS providers need to know what it is and how to protect themselves from it.

Biochem Pharmacol, 1993 Dec 3, 46(11), 1999 - 2005
Biochemical characterization of a mitomycin C resistant colon cancer cell line variant; Perry RR et al.; Resistance may limit the clinical usefulness of a variety of chemotherapeutic drugs including mitomycin C (MMC) . The MMC-sensitive HT-29 colon cancer cell line and its MMC-resistant subline, HT-29R13, were studied in vitro under aerobic conditions to help characterize the mechanisms associated with MMC resistance . HT-29R13 cells exhibit approximately 2-fold resistance to MMC compared with HT-29 cells and lack the typical multidrug-resistance pattern; resistance is stable in the absence of drug exposure . Levels of glutathione (GSH) and total glutathione-S-transferase (GST) activity were not different between the two cell lines; however, levels of GSH reductase and GSH peroxidase were increased significantly in HT-29R13 . Although total GST activity was unchanged, GST-pi and GST-alpha isoenzyme expression as measured using western blot were increased significantly in HT-29R13 compared with HT-29 . DT-diaphorase levels and topoisomerase II activity were decreased significantly in HT-29R13 . Both cell lines had equal P-glycoprotein expression . Multiple drug resistance mechanisms are present in HT-29R13 including decreased drug activation (decreased DT-diaphorase), increased drug detoxification (increased GST-pi and GST-alpha, GSH reductase, GSH peroxidase), and decreased accessibility of DNA targets (decreased topoisomerase II) . Further work will be necessary to determine the degree to which each of these mechanisms contribute to MMC resistance in this model.

Hinyokika Kiyo, 1993 Dec, 39(12), 1227 - 32
{Circumvention of the multidrug-resistance in renal cancer by bisbenzylisoquinoline}; Kakehi Y et al.; A bisbenzylisoquinoline alkaloid, cepharanthine, significantly enhanced vinblastine, adriamycin and etoposide sensitivities in P-glycoprotein positive renal cancer cells . However, it did not show any enhancing effect on cisplatin sensitivity . Four patients with metastatic renal cell carcinomas were treated with intraarterial chemotherapy using vinblastine and/or adriamycin in combination with cepharanthine for their metastatic lesions (3 bone and 1 contralateral kidney metastases) . A partial response was observed in 1 patient with femoral bone metastasis and a minor response in 1 patient with lumbar bone metastasis, although they were also treated with interferons . No adverse effects associated with cepharanthine were seen except in one patient complaining of redness and burning sense of the skin probably due to its vaso-dilatation effect.

Mol Cell Biol, 1993 Dec, 13(12), 7380 - 92
Activation of the mouse mdr3 gene by insertion of retroviruses in multidrug-resistant P388 tumor cells; Lepage P et al.; In multidrug-resistant (MDR) derivatives of the mouse lymphoid tumor P388, the emergence of MDR is associated with overexpression and transcriptional activation of the mdr3 gene, either in the absence of (P388/VCR-10) or concomitant with (P388/ADM-2) gene amplification . In both instances, Northern (RNA) blotting analyses have suggested the presence of altered mdr3 transcripts in these cells, possibly originating from novel transcription initiation sites . The mechanisms underlying mdr3 overexpression in these cells have been investigated . In P388/VCR-10 cells, Southern blotting analyses together with genomic DNA cloning and nucleotide sequencing have demonstrated the presence of an intact mouse mammary tumor virus (MMTV) within the boundaries of intron 1 of mdr3 . cDNA cloning and nucleotide sequencing indicated that this integration event results in the synthesis and overexpression of a hybrid MMTV-mdr3 mRNA which initiates within the U3 region of the 5' long terminal repeat (LTR) of the provirus . Consequently, this mRNA lacks the normal exon 1 of mdr3 but contains (i) MMTV LTR-derived sequences at its 5' end, (ii) a novel mdr3 exon, mapping within the boundaries of intron 1 downstream of the MMTV integration site and generated by alternative splicing, and (iii) an otherwise intact 3' portion of mdr3 starting at exon 2 . A similar type of analysis of P388/ADM-2 cells revealed that mdr3 overexpression in these cells is associated with the integration of an intracisternal A particle (IAP) within an L1Md repetitive element, immediately upstream of mdr3 . The IAP insertion results in the overexpression of hybrid IAP-mdr3 mRNA transcripts that initiate within the 3' LTR of the IAP and which contain IAP LTR-derived sequences at the 5' end spliced 14 nucleotides upstream of the normal exon 1 of mdr3 . Taken together, these results indicate that independent retroviral insertions were the initial mutagenic event responsible for mdr3 overexpression and survival during drug selection of these cell lines . Amplification of the rearranged and activated mdr3 gene copy occurred during further selection for high-level drug resistance in P388/ADM-2 cells.

J Clin Oncol, 1993 Dec, 11(12), 2417 - 26
Phase I/pharmacokinetic study of high-dose progesterone and doxorubicin; Christen RD et al.; PURPOSE: We developed a new formulation of progesterone that permits administration of up to 10 g of progesterone as a continuous intravenous infusion over 24 hours and conducted a phase I clinical trial to determine whether progesterone could modulate the in vivo cytotoxicity of the P-glycoprotein substrate doxorubicin . PATIENTS AND METHODS: Thirty-four patients with advanced malignancies were treated with increasing doses of progesterone and a fixed dose of 60 mg/m2 of doxorubicin given as an intravenous bolus 2 hours after starting a 24-hour intravenous infusion of progesterone . RESULTS: Progesterone enhanced doxorubicin-induced myelotoxicity in a dose-dependent fashion without altering the pharmacokinetics of doxorubicin . The steady-state plasma concentration of progesterone at a dose level of 4 g was 4.1 +/- 0.9 mumol/L, which was higher than the minimal concentration required to reverse multidrug resistance (MDR) in vitro . CONCLUSION: Progesterone enhanced the hematologic toxicity of doxorubicin without altering its pharmacokinetics, suggesting that progesterone could modulate P-glycoprotein at the level of pluripotent hematopoietic stem cells . Adequate tissue concentrations of progesterone could be achieved in vivo to modulate doxorubicin toxicity in the bone marrow and thus potentially in tumor tissue as well . Selectivity may potentially be gained by using hematopoietic growth factors to offset the enhanced hematologic toxicity of doxorubicin while leaving the enhancement of toxicity to tumor cells unchanged.

Cancer, 1993 Dec 1, 72(11 Suppl), 3484 - 8
Reversal of multidrug resistance to cancer chemotherapy; Leyland-Jones B et al.; In the past few years, the role of membrane-bound transport genes in human disease has been increasingly recognized and understood . Of these genes, the p170 membrane glycoprotein may function as an outward transport pump for many cancer chemotherapeutic drugs associated with the multidrug resistance phenotype . This article reviews the different classes of the current major modulators of multidrug resistance.

Blood, 1993 Dec 1, 82(11), 3452 - 9
p53 gene mutation in B-cell chronic lymphocytic leukemia is associated with drug resistance and is independent of MDR1/MDR3 gene expression; el Rouby S et al.; We studied 53 patients with B-cell chronic lymphocytic leukemia (B-CLL) and found mutations of the p53 gene in 15% . Patients with p53 gene mutations were found to have an aggressive form of B-CLL disease characterized by advanced Rai stage, rapid lymphocyte doubling time (LDT), and resistance to chemotherapy . While 27 of 29 treated patients (93%) without p53 mutations achieved a partial remission, only one of seven treated patients (14%) with p53 mutations achieved a partial remission (P = .00009) . Adjusting for prognostic factors (age, sex, race, and Rai stage), patients with p53 gene mutations had a 13-fold greater risk of death than patients without p53 mutations (P = .013) . In addition to examining the clinical relevance of p53 gene mutations in B-CLL, we investigated the possible role of p53 gene regulation in the expression of the multidrug resistance genes MDR1 and MDR3 . We quantitated MDR1 and MDR3 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) . Expression of both the MDR1 and MDR3 genes was independent of p53 gene mutation or prior drug treatment, and did not predict for clinical response . Our findings indicate that p53 gene mutations in B-CLL are associated with a poor clinical outcome and may be a prognostic indicator for drug resistance.

Br J Haematol, 1993 Dec, 85(4), 826 - 8
Retinoic acid-induced apoptosis and regression of a refractory Epstein-Barr virus-containing T cell lymphoma expressing multidrug-resistance phenotypes; Su IJ et al.; The virus-associated T cell leukaemias/lymphomas are characterized by a poor prognosis primarily because of the rapid emergence of drug resistance which may lead to failure of subsequent chemotherapy . We report here a case of Epstein-Barr virus-associated T cell lymphoma which relapsed soon after chemotherapy and radiotherapy . The neoplastic cells of the relapsed tumour expressed high levels of multi-drug resistance gene (mdr1)-related P-glycoprotein and glutathione-S-transferase-pi, both of which were absent in the pre-chemotherapy tumour tissues . Empirical treatment with oral 13-cis-retinoic acid (RA) was then given with subsequent complete disappearance of the tumour . The therapeutic effect of RA appears to act through an apoptotic process . In accordance with our previous report of a successful salvage of a refractory Ki-1 large cell lymphoma . RA appears to be a potentially useful drug for some specific type T-cell lymphomas.

Shi Yan Sheng Wu Xue Bao, 1993 Dec, 26(4), 429 - 39
{Expression of human multidrug resistance gene (mdr1) cDNA in murine ES cells and in chimeric mice}; Tsung HC et al.; Human multidrug resistance gene (mdr1) was introduced into mouse embryonic stem cells (ES-5 line) by calcium phosphate mediated transfection, and transfected ES-5 cells were then selected by stepwise increase in colchicine concentration (30, 50, 100, and 200 ng/ml respectively) . Finally, we obtained 4 clones that could be stably grown in culture medium with colchicine at 200 ng/ml and designated as ES-mdr1 clones A, B, C, and D . Southern blot analysis of DNA from ES-mdr1 A and D cells digested by Hind III and hybridized with mdr1 cDNA 5 A probe was shown in Fig . 3 . Characteristic 4.8 and 2.4 kb fragments of mdr1 gene were found as expected and their amplification under increased concentration of colchicine in culture medium was also evident from the figure . Slot blot and Northern analysis of total RNA and poly A+ RNA extracted from ES-mdr1 cells were shown in Fig . 4 and 5, demonstrating that ES-mdr1 cells could express mdr1 mRNA . Indirect immunofluorescence analysis with antibodies against p170 glycoprotein indicated that p170 protein translated from mdr1 mRNA was present at the surface of ES-mdr1 cells (Plate I, Fig . 2) . The biological characteristics of ES-mdr1 cells cultured in medium containing 200 ng/ml colchicine were investigated . The cells maintained their undifferentiated morphology and grew in nests (Plate I, Fig . 1), like the parental ES-5 cells . When ES-mdr1 cells were cultured in suspension in vitro, these cells were still capable of producing simple and cystic embryoid bodies . ES-mdr1 cells injected subcutaneously into 129 mice formed tumor-like outgrowths giving a great variety of cell types (Plate I, Fig . 4) . These results indicated that the integration and expression of human mdr1 gene and selection against colchicine did not affect the pluripotency of ES-mdr1 cells both in vitro and in vivo . However, ES-mdr1 cells, unlike their parental ES-5 cells, could no longer be induced to differentiate by either RA or HMBA (Plate I, Figs . 3a, 3b), indicating that the human mdr1 gene transfected ES cells had changed their competence of inducible response to differentiation in vitro . The details and possible significance of such change require further studies . From the above preliminary data, we are of the opinion that ES-mdr1 cells may serve as a model to study the mode of action of p170 glycoprotein at cellular level and to screen possible means to counteract the action of mdr1 gene.(ABSTRACT TRUNCATED AT 400 WORDS)

Anticancer Drugs, 1993 Dec, 4(6), 641 - 50
Effect of various chemosensitizers on chemoresistance to adriamycin in MIP-101 cell line, a colon carcinoma cell line: analysis of glutathione and related enzymes; Mestdagh N et al.; We evaluated the multidrug resistance modulating effect of verapamil, buthionine sulfoximine, trifluoperazine and tamoxifen in a human colorectal cell line resistant de novo to adriamycin . We studied the effect of these chemosensitizers on glutathione content, glutathione reductase, transferases, peroxidases, gamma-glutamyl transpeptidase and glucose-6-phosphate dehydrogenase activities . The ratio of the activities between resistant and sensitive cells treated by these compounds as compared with the ratio of the untreated cells decreases, thus contributing to the reversal of chemoresistance . This study implies a role of glutathione and related enzymes in chemoresistance to adriamycin, although this is certainly not the sole mechanism.

FASEB J, 1993 Dec, 7(15), 1499 - 506
P-glycoprotein regulates chemosensitivity in early developmental stages of the mouse; Elbling L et al.; The multidrug resistance (MDR) P-glycoprotein (P-gp) is an active transporter associated with chemoresistance of tumor cells . A fundamental aspect not yet entirely clarified is the physiological role of MDR-P-gp in normal mammalian tissues . In this paper we report that multidrug (chemo)resistance is already present in mouse oocytes and early cleavage embryos . Expression of MDR-specific P-gp is detectable by antibody (C219) staining from the primary oocyte onward to the eight-cell embryo . MDR-mRNA is demonstrated in mature oocytes using an Mdr1-specific cDNA probe . Functional activity of P-gp is shown by the efficacy of MDR reversers (verapamil or quinidine) in enhancement of: 1) drug accumulation (daunomycin) in all stages investigated, 2) drug cytotoxicity (daunomycin or mitomycin c-induced developmental impairment) in two-cell embryos cultured for 24 h, and 3) drug cytokinesis-blocking activity (cytochalasin D; our recent findings demonstrate cytochalasins to be substrates for P-gp and to indicate the presence of MDR by their microfilament-disrupting action on cycling cells) in four- and eight-cell embryos cultured for 24 h . Furthermore, functional involvement of P-gp in vivo is demonstrated . Concurrent administration of verapamil increases doxorubicin-induced developmental impairment in the zygote stage during the first cleavage cycle in pregnant females . Results provide evidence that MDR-P-gp has an efficient protective function in early reproduction.

Leuk Res, 1993 Dec, 17(12), 1021 - 9
Staining with Hoechst 33342 and rhodamine 123: an attempt to detect multidrug resistant phenotype cells in leukemia; Lahmy S et al.; Development of resistance is the major cause of failure in chemotherapeutic treatments . We have previously shown that the level of labeling with Hoechst 33342 and rhodamine 123 in established cell lines was decreased in cells with 'classic' MDR phenotype . This functional test was carried out using fluorescence image cytometry on living cells . We applied this protocol to patients with chronic lymphocytic leukemia . Although a large variability of the labeling is observed in cells from healthy donors, this approach seems to be useful for early detection of P-gp-dependent resistance in leukemia cells and for identification of new reversing agents on patient lymphocytes.

Trends Biotechnol, 1993 Dec, 11(12), 511 - 6
Multidrug resistance during cancer chemotherapy--biotechnological solutions to a clinical problem; Pearson CK et al.; Tumour cells can be resistant to a variety of chemotherapeutic drugs of different structure (multidrug resistance) by expressing a transmembrane pump (P-glycoprotein) on their cell surface . This situation can lead to a failure of cancer chemotherapy as the P-glycoprotein acts by actively pumping the drugs out of cells, thus lowering the intracellular concentration of the drug and, hence, its cytotoxic effectiveness . This review summarizes present and proposed approaches to preventing or circumventing the action of this drug-transporting protein.

Tokai J Exp Clin Med, 1993 Dec, 18(3-6), 99 - 106
In vivo acquired drug resistance and multidrug resistance gene (MDR1) expression in the KB carcinoma cell line xenotransplanted in nude mice; Abe Y et al.; We studied the correlation between in vivo responsiveness of KB xenografts to anticancer drugs and the expression level of the human multidrug resistance gene (MDR1) encoding P-Glycoprotein (P-Gp) . We established KB xenografts (xeKB3-1 and xeKB8-5) by inoculating these in vitro lines into nude mice . The responsiveness was evaluated by an in vivo chemosensitivity assay (T/C; sensitive, < 50%) . Xenograft xeKB3-1 was sensitive to vincristine (VCR) (T/C, 48%), and xeKB8-5 was resistant to VCR (T/C, 72%) . We selected a VCR-resistant variant (xeKB3-1-R, T/C, 76%) by treating xeKB3-1 with VCR (1.2 mg/kg, x3) in vivo . The MDR1 expression was evaluated by a semi-quantitative assay using reverse transcription-polymerase chain reaction . A MDR1 expression pattern in xeKB3-1 and xeKB8-5 in vivo was the same as that to KB3-1 and KB8-5 in vitro . The xenograft xeKB3-1-R expressed definitive but significantly lower levels of MDR1 than xeKB8-5 . These results suggest that acquired drug resistance is related to minimally enhanced expression of the P-Gp protein/MDR1 gene in KB xenografts in vivo.

Am J Physiol, 1993 Dec, 265(6 Pt 1), C1711 - 5
Induction of multidrug resistance downregulates the expression of CFTR in colon epithelial cells; Breuer W et al.; The epithelial cell line HT-29, which constitutively expresses the cystic fibrosis transmembrane conductance regulator (CFTR), was induced to become drug resistant by cultivation in the presence of colchicine . The gradual acquisition of drug resistance was associated with a corresponding increase in the expression of the multidrug resistance P-glycoprotein (P-gp) and a marked (> 80%) decrease in the constitutive levels of CFTR protein, as determined by immunoblotting . The reduction in CFTR content occurred at the onset of acquisition of drug resistance when P-gp expression was still relatively low . Reversal of drug resistance by removal of colchicine from the culture medium led to a 70% decrease in P-gp levels and a concomitant 40% increase in CFTR . The levels of other membrane proteins such as Na(+)-K(+)-ATPase and alkaline phosphatase remained relatively constant (< 26% variation) . We propose that a selective downregulation of CFTR is elicited by acquisition of the multidrug resistance (MDR) phenotype and that induction of P-gp expression leads to a reversible repression of CFTR biosynthesis . These findings provide an experimental foundation for the complementary patterns of expression of the CFTR and MDR1 genes observed in vivo.

Leuk Res, 1993 Dec, 17(12), 1031 - 5
Detection of P glycoprotein activity on normal and leukemic CD34+ cells; Drenou B et al.; Rhodamine 123 is transported by the transmembrane efflux pump P glycoprotein (Pgp) . We used this fluorescent dye to study multidrug resistance (MDR) activity in normal and leukemic CD34+ cells . These immature cells had a high degree of MDR activity . Among leukemic cells, CD34+ leukemias had significantly higher MDR activity as compared to CD34- leukemias . Heterogeneous results in cell subpopulations, however, indicate that prognosis should be interpreted in the light of MDR analysis.

Biochim Biophys Acta, 1993 Nov 28, 1158(3), 201 - 8
Progesterone and its metabolites: the potent inhibitors of the transporting activity of P-glycoprotein in the adrenal gland; Ichikawa-Haraguchi M et al.; P-glycoprotein (P-gp) is a transmembrane glycoprotein responsible for the multidrug resistant (MDR) phenotype in various cancer cells . It has been shown that P-gp transports various kinds of anti-cancer agents as well as hydrophobic chemicals . Although P-gp is also expressed in normal human tissues, such as liver, kidney, and adrenal gland, its function and transporting substrates in these tissues are still unknown . In previous work, we demonstrated that some compounds in human plasma modulate the transporting activity of P-gp . We also found that P-gp is expressed at a high level in the bovine adrenal gland and that this tissue contains large amount of compounds which inhibit the transporting activity of P-gp . We purified such compounds from the adrenal gland by monitoring the ability to enhance the accumulation of {3H}vincristine in MDR cells . Two major compounds were purified and identified as progesterone and pregnenolone by nuclear magnetic resonance (NMR) analysis . Progesterone was the most potent and abundant compound that inhibited the transporting activity of P-gp among the compounds extracted from bovine adrenal gland with methanol . We also found that six authentic progesterone metabolites in the 5 beta-metabolic pathway but none in the 5 alpha-metabolic pathway were able to enhance the accumulation of {3H}vincristine in MDR cells and to inhibit {3H}azidopine photolabeling of P-gp in the adrenal gland . These results indicate that some progesterone metabolites can interact with P-gp and that stereoisomerism around carbon 5 of the progesterone metabolites is important for them to be recognized by P-gp.

Biochem Pharmacol, 1993 Nov 17, 46(10), 1841 - 8
Crosstalk between epidermal growth factor receptor and P-glycoprotein in actinomycin D-resistant Chinese hamster lung cells; Meyers MB et al.; Multidrug-resistant cells can manifest an increase in epidermal growth factor (EGF) receptor number along with increased P-glycoprotein (Pgp) synthesis . An interrelationship of the two membrane proteins in actinomycin D-resistant Chinese hamster lung cells (DC-3F/AD X) in terms of the effect of EGF on Pgp phosphorylation was investigated . EGF was not a mitogen for the resistant cells, nor was it mitogenic for DC-3F, the parental drug-sensitive line . Brief treatment of DC-3F/AD X cells with EGF resulted in a 30-50% decrease in the level of Pgp phosphorylation, and treatment of the cells with okadaic acid, a specific inhibitor of protein phosphatases-1 and -2A (PP1 and 2A), increased Pgp phosphorylation . Okadaic acid also increased phosphorylation of Pgp in plasma membranes isolated from DC-3F/AD X cells by 30-40% . Protein phosphatase activity in extracts of cells grown in EGF-containing medium was greater by 30% than that of cells grown in standard medium, and okadaic acid inhibited the increases . The results suggested that EGF activated PP1 and PP2A in DC-3F/AD X cells and that Pgp was a substrate for the phosphatases . The properties of Pgp may be modulated by the signalling system transduced by ligand-activated EGF receptor.

Cancer Res, 1993 Nov 15, 53(22), 5487 - 93
Reversal of etoposide resistance in non-P-glycoprotein expressing multidrug resistant tumor cell lines by novobiocin; Rappa G et al.; Previous reports from this laboratory have demonstrated that novobiocin produces supraadditive cytotoxicity and increases the formation of drug-stabilized topoisomerase II-DNA covalent complexes in WEHI-3B myelomonocytic leukemia and A549 lung carcinoma cells when combined with etoposide (VP-16) . Inhibition of the efflux of VP-16 by novobiocin is responsible for the increase in VP-16 accumulation, which in turn leads to increased formation of VP-16-stabilized topoisomerase II-DNA covalent complexes and increased cytotoxicity . We now report that novobiocin synergistically enhanced the sensitivity of the multidrug resistant variants, WEHI-3B/NOVO and A549(VP)28, to VP-16, causing almost complete reversal of the resistance to the epipodophyllotoxin . These two tumor cell variants are resistant to several topoisomerase II-targeted drugs, particularly VP-16, but not to Vinca alkaloids; this finding corresponds to the fact that they do not overexpress the P-glycoprotein . The effects of novobiocin in these resistant sublines are mediated through the intracellular accumulation of VP-16, resulting in an increase in the formation of lethal VP-16-induced topoisomerase II-DNA covalent complexes . In the P-glycoprotein expressing multidrug resistant HCT116(VM)34 colon carcinoma and L1210/VMDRC0.06 leukemia cell lines, the latter being transfected with the human mdr-1 gene, novobiocin did not potentiate the cytotoxic activity of VP-16 nor increase the intracellular accumulation of VP-16 and the formation of covalent complexes, whereas their normal counterparts were sensitive to the potentiating activity of novobiocin when used in combination with VP-16 . These results indicate that the action of novobiocin on the intracellular transport of VP-16 is not directed at the level of the P-glycoprotein, but that the action of novobiocin is antagonized by the presence of the P-glycoprotein . Since novobiocin is a clinically available antibiotic, has numerous structural analogues available for comparative studies, and has a relatively low toxicity profile, this drug, as well as structurally related agents, would appear to have significant clinical potential in combination with an epipodophyllotoxin for the treatment of non-P-glycoprotein expressing multidrug resistant tumors.

Cancer Res, 1993 Nov 15, 53(22), 5475 - 82
Specific targeting and killing activities of anti-P-glycoprotein monoclonal antibody MRK16 directed against intrinsically multidrug-resistant human colorectal carcinoma cell lines in the nude mouse model; Iwahashi T et al.; Anti-P-glycoprotein (P-gp) monoclonal antibody, MRK16, and its F(ab')2 fragment were evaluated for its therapeutic efficacy to P-gp-mediated multidrug resistant human colorectal carcinoma cell lines in a nude mouse model . In a blood clearance experiment, 125I-labeled MRK16 had a half-life (16 h) 7 times longer than its F(ab')2 fragment (half-life of 1.8 h) in circulation in nude mice, and approximately 16 and 5% of MRK16 were retained on days 10 and 20 after injection, respectively . In biodistribution experiments using nude mice bearing HCT-15, an intrinsically resistant cell line, 125I-labeled MRK16 accumulated at the tumor site significantly higher than its F(ab')2 fragment as revealed by the percentage of injected dose/g of tissue values (7.4 versus 0.6%) on day 3 after injection . In contrast, the tissue to blood ratio at the tumor site of the MRK16 was significantly lower than that of its F(ab')2 fragment (1.2 versus 10.5) . Specific targeting of the MRK16 F(ab')2 fragment to the P-gp-positive tumor (HCT-15) but not to the P-gp-negative tumor (COLO 205) was observed in the nude mice bearing both tumors . In the therapeutic efficacy tests, when administered i.v . 3 times on days 1, 4, and 7 after tumor s.c . inoculation, MRK16 alone showed the significant inhibition of tumor growth of P-gp-positive cell lines, HCT-15, DLD-1, SW480, and SW1417 in contrast to cases of P-gp-negative cell lines, COLO 205 and KM20L2 . This inhibitory effect of MRK16 was enhanced in combination with Adriamycin, which alone hardly inhibited the tumor growth . However, MRK16 F(ab')2 fragment alone, even at 1 mg/mouse, had little inhibitory effect on the growth of HCT-15 in the same treatment schedule . When administered at early palpable stage, the degree of HCT-15 tumor growth suppression depended on the number of MRK16 injections . At more progressed stages, treatment with MRK16 alone showed little antitumor activity but when combined with Adriamycin resulted in significant suppression of tumor growth . The present results suggest that MRK16 may be useful for in vivo immunoscintigraphy and immunotherapy of multidrug-resistant colorectal carcinoma.

Cancer Res, 1993 Nov 15, 53(22), 5382 - 5
17 beta-estradiol glucuronide: an inducer of cholestasis and a physiological substrate for the multidrug resistance transporter; Gosland M et al.; The multidrug resistance (MDR) gene family has been shown to be highly expressed in several normal tissues including the canalicular membrane of the hepatocyte . We report that a cholestatic estrogen metabolite, 17 beta-estradiol glucuronide (E217G), is a substrate for the MDR transporter, P-glycoprotein . In cytotoxicity studies, the MDR sarcoma cell line Dx5 was 4.7-fold resistant to E217G, and the K562/R7 leukemia MDR cell line was 5.0-fold resistant to E217G relative to their parental cell lines . There was also a 2- to 3-fold accumulation defect of {3H}E217G in the MDR cells relative to their parental cell lines . E217G (100 microM) modulated resistance ot doxorubicin, taxol, vinblastine, and etoposide in the Dx5 cells, completely reversing the 30- to 60-fold resistance observed with these agents . E217G had no effect on the toxicity of these compounds in the parental cell line (MES-SA) . In contrast, MDR cells were not resistant to the noncholestatic estrogen metabolite, estriol 3-glucuronide, and this metabolite did not modulate resistance to MDR substrates . ATP-dependent transport of {3H}E217G in rat canalicular membranes was inhibited by several MDR substrates including vinblastine, etoposide, verapamil, cyclosporine, and PSC-833.

Biochem Biophys Res Commun, 1993 Nov 15, 196(3), 1034 - 41
Absence of cooperativity for MgATP and verapamil effects on the ATPase activity of P-glycoprotein containing membrane vesicles; Garrigos M et al.; Purified membrane vesicles were prepared from Chinese Hamster lung fibroblasts expressing high amounts of P-glycoprotein (P-gp), which is responsible for the multidrug resistance . P-gp ATPase activity, characterized in the presence or absence of verapamil, had a Michaelian behavior for its MgATP dependence . Thus only one MgATP molecule should be sufficient for the catalytic cycle . With increasing verapamil concentrations, a bell-shape curve was observed for ATPase activity, with half-activation and -inhibition concentrations of 1.2 microM and 490 microM, respectively . No cooperativity for verapamil was detected . These results strongly suggest that P-gp functions as an active transporter, with a coupling stoichiometry of one MgATP molecule hydrolysed for one verapamil molecule transported.

J Biol Chem, 1993 Nov 15, 268(32), 24197 - 202
Functional reconstitution of drug transport and ATPase activity in proteoliposomes containing partially purified P-glycoprotein; Sharom FJ et al.; P-glycoprotein, the multidrug transporter, is proposed to act as an ATP-driven drug efflux pump . We previously reported the partial purification of P-glycoprotein from multidrug-resistant cells (Doige, C . A., Yu, X., and Sharom, F . J . (1992) Biochim . Biophys . Acta 1109, 149-160) . We now report the reconstitution of this preparation into phospholipid bilayers using rapid detergent removal by gel filtration chromatography . The resulting proteoliposomes displayed ATP-dependent {3H}colchicine uptake over a time period of 0-4 min . No drug uptake was observed for liposomes of lipid alone, or liposomes reconstituted with a similar extract from drug-sensitive cells . Drug uptake was osmotically sensitive, and abolished by detergent permeabilization, indicating that it represented true transport into the vesicle lumen . Steady-state levels of drug uptake increased with drug concentration, approaching saturation at approximately 150 microM colchicine, with half-maximal accumulation at 50 microM . Drug was accumulated actively against a 5.6-fold concentration gradient . Multidrug resistance spectrum drugs and chemosensitizers inhibited colchicine uptake by P-glycoprotein proteoliposomes, whereas cytosine arabinoside and methotrexate had no effect . Reconstituted liposomes showed high levels of ATPase activity, which was stimulated over 2-fold by verapamil and trifluoperazine . These results suggest that P-glycoprotein functions as an active drug transporter with constitutive ATPase activity.

Blood, 1993 Nov 15, 82(10), 3157 - 62
Predominance of functional multidrug resistance (MDR-1) phenotype in CD34+ acute myeloid leukemia cells; te Boekhorst PA et al.; The expression of the MDR-1-encoded P-170 glycoprotein (P-170) associated with clinical multidrug resistance (MDR) was investigated in 52 consecutive patients with untreated acute myeloid leukemia (AML) . P-170 expression was analyzed in correlation with CD34 expression and clinical response . Thirty of 52 patients expressed P-170 (58%) . Eight of 30 P-170+ as compared with 16 of 22 P-170- patients achieved a complete remission (CR) (27% v 73%, P = .003) . In 21 of 30 P-170+ patients, expression of the CD34 antigen was observed in greater than 10% of the blast cells, as compared with 14 of 22 P-170- patients (70% v 64%, P > .05) . The CR rate of CD34+ and CD34- patients was 31% and 76%, respectively (P = .006) . In AMLs that simultaneously expressed both P-170 and CD34, the CR rate was worse as compared with those negative for P-170 and CD34 (5% v 63%, P = .004) . In 12 patients (8 P-170+, 4 P-170-) CD34 and P-170 expression were further characterized by double fluorescence studies . It was shown that P-170+ cells were largely, but not exclusively, restricted to the CD34+ cell population . For the 8 P-170+ AML samples, the median ratio of P-170+/P-170- in CD34+ cells was 4.845 (range, 0.60 to 25.00) as compared with 0.135 (range, 0.02 to 0.67) in CD34- cells . In these 12 AML samples, the presence of functional resistance as defined by reduced daunorubicin accumulation was evaluated in CD34+ and CD34- AML cells . In 8 of 8 P-170+ patients, intracellular daunorubicin accumulation in CD34+ AML blast cells was lower than in CD34- cells, and it increased after cyclosporin addition . No difference of intracellular daunorubicin accumulation was observed between CD34+ and CD34- AML cells of 4 P-170- patients . These data indicate that P-170 expression in AML with a heterogeneous CD34+ phenotype seems predominantly present in CD34+ AML blast cells.

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