Multidrug resistant

Lancet, 1987 Dec 5, 2(8571), 1304 - 5
Reduced in-vitro susceptibility to mefloquine in West African isolates of Plasmodium falciparum; Oduola AM et al.; West African isolates of Plasmodium falciparum were more susceptible to chloroquine but less susceptible to mefloquine than were Southeast Asian isolates . The West African isolates were more sensitive to halofantrine than to mefloquine . Since neither mefloquine nor halofantrine has been used in West Africa, the findings suggest that P falciparum may be inherently resistant to mefloquine and that mefloquine should be introduced cautiously to West Africa . Moreover, halofantrine may be of greater value than mefloquine for controlling multidrug-resistant falciparum malaria in West Africa.

J Clin Oncol, 1987 Dec, 5(12), 1922 - 7
Intrinsic drug resistance in human kidney cancer is associated with expression of a human multidrug-resistance gene; Fojo AT et al.; The cloning of the cDNA for the mdr1 gene, whose expression is associated with the development of multidrug-resistance in cultured cells, has made it possible to explore the mechanism of multidrug resistance in human tumors . We have found that normal human kidney, six of eight adenocarcinomas of the kidney, and four cell lines derived from kidney adenocarcinomas express high levels of mdr1 mRNA . Two criteria suggest that primary multidrug resistance in human adenocarcinomas of the kidney results, at least in part, from expression of the mdr1 gene: (1) mdr1 mRNA levels are elevated in four unselected kidney adenocarcinoma cell lines that show a multidrug-resistant phenotype; and (2) multidrug resistance in these kidney cancer cell lines is reversed by verapamil and quinidine, agents known to reverse mdr1-associated drug resistance in cell lines selected for multidrug resistance in vitro . These results suggest that appropriate pharmacological intervention to reverse multidrug resistance might make adenocarcinomas of the kidney more sensitive to chemotherapy with agents such as Adriamycin (Adria Laboratories, Columbus, OH) and the vinca alkaloids.

Mol Cell Biol, 1987 Dec, 7(12), 4549 - 52
Decreased expression of the amplified mdr1 gene in revertants of multidrug-resistant human myelogenous leukemia K562 occurs without loss of amplified DNA; Sugimoto Y et al.; Amplification and increased expression of the mdr1 gene associated with multidrug resistance in human tumors were found in multidrug-resistant sublines of human myelogenous leukemia K562 selected with vincristine (K562/VCR) or adriamycin (K562/ADM) . In two revertant cell lines of K562/ADM, amplification of the mdr1 gene was maintained at the same level as in K562/ADM, but expression of the 4.5-kilobase mdr1 mRNA was greatly decreased, indicating that amplified genes may be inactivated at the level of transcription without a corresponding loss of amplified DNA.

Jpn J Cancer Res, 1987 Dec, 78(12), 1415 - 9
Detection of multidrug resistance markers, P-glycoprotein and mdr1 mRNA, in human leukemia cells; Tsuruo T et al.; We have examined the expression of P-glycoprotein in clinical leukemic cell samples by using a monoclonal antibody (MRK16) against P-glycoprotein . We found that leukemia cells isolated from 3 out of 6 patients with blast crisis of chronic myelogenous leukemia were reactive to MRK16 . These 3 cell lines expressed high levels of mdr1 mRNA, which codes for P-glycoprotein . The present result indicates that the clinically refractory state of the tumor may be predicted in part by determining P-glycoprotein expression using the monoclonal antibody against P-glycoprotein, and the mdr1 probe.

J Histochem Cytochem, 1987 Dec, 35(12), 1451 - 6
Immunocytochemical localization of P170 at the plasma membrane of multidrug-resistant human cells; Willingham MC et al.; P170 (P-glycoprotein) is a membrane protein found in high levels in multidrug-resistant cultured cell lines . We have localized this protein using monoclonal antibody MRK16 by immunofluorescence and electron microscopy in the multidrug-resistant human carcinoma cell line KB-C4 . The P170 determinant recognized by antibody MRK16 was found on drug-resistant KB-C4 cells, but not on parental drug-sensitive KB-3-1 cells . The determinant was present on the external surface of the plasma membrane and on the luminal side of Golgi stack membranes . P170 was excluded from coated pits at the plasma membrane and absent from endocytic vesicles and lysosomes . This determinant was detected only in small amounts in the endoplasmic reticulum . The high protein concentration of P170 in the plasma membrane is consistent with a role of this protein as a drug efflux pump at the cell surface.

Cancer Res, 1987 Nov 15, 47(22), 5982 - 8
Genetics of multidrug resistance: relationship of a cloned gene to the complete multidrug resistant phenotype; Croop JM et al.; Resistance to multiple chemotherapeutic agents remains the major cause of failure in cancer chemotherapy . Multidrug resistant cell lines developed in vitro have provided a useful model for analyzing this phenomenon . We describe a complementary DNA, lambda DR11, which is present in normal cells and overexpressed in multidrug resistant cell lines . We have placed this complementary DNA in an expression vector which uses the beta-actin promoter to drive transcription and introduced this vector via transfection into drug sensitive cells . Cells expressing increased levels of lambda DR11 are resistant to the same broad spectrum of chemotherapeutic agents which characterize the multidrug resistant phenotype . The expression of this complementary DNA in transfected clones is dependent upon the number of copies of lambda DR11 integrated in the genome as well as the amount of selective pressure placed on the clone during selection of the clone . Furthermore, the number of copies of lambda DR11 in the genome and the expression of lambda DR11 can be modulated by releasing an individual clone from selective pressure or by increasing the selective pressure on the clone . The endogenous sequences encoding the multidrug resistance gene are not amplified in transfected drug resistant clones . Finally, the drug resistant phenotype is reversed in the transfected clones by verapamil just as drug resistance is reversed in multidrug resistant cell lines.

Int J Cancer, 1987 Nov 15, 40(5), 635 - 42
Characterization of actinomycin-D-resistant CHO cell lines exhibiting a multidrug-resistance phenotype and amplified DNA sequences; Diddens H et al.; Actinomycin D (DACT)-resistant sublines of the Chinese hamster ovary cell line CHO-K1 were selected in vitro . Sublines were derived which expressed 5.2-fold (CHO 15DACT) and 35.8-fold (CHO 100DACT) resistance to DACT . The CHO 100DACT subline displayed marked cross-resistance to bleomycin, adriamycin, daunomycin, vinblastine, vincristine, VP 16 and VM 26 . No cross-resistance was found to cisplatin or methotrexate . The resistant cells exhibited enhanced (collateral) sensitivity to prednisolone . Combination of prednisolone with vincristine resulted in a pronounced synergistic effect on sensitive cells, whereas in resistant cells the combined effect of both drugs was merely additive . Resistant cells, viably stained with the DNA-specific dye Hoechst 33342, exhibited decreased fluorescence intensities compared to parental cells . In contrast to sensitive cells the resistant sublines did not accumulate the mitochondria-specific dye rhodamine 123 . Co-incubation with verapamil, however, effectively enhanced accumulation of the dye . The potential diagnostic value of these fluorescent compounds as marker dyes for the multidrug-resistance phenotype is discussed . Non-toxic doses of verapamil almost completely reversed the resistance to various drugs in CHO 100DACT cells . Specific DNA sequences were amplified in resistant cells, and the increase in resistance was paralleled by a concomitant increase in the copy number of these sequences, suggesting that the corresponding gene may be functionally linked to the multidrug-resistance phenotype.

Biochemistry, 1987 Nov 3, 26(22), 6900 - 4
Phosphorylation of the multidrug resistance associated glycoprotein; Mellado W et al.; Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistance phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons) . Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied . The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein . Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl2 was enhanced a minimum of 2-fold by 10 microM cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by {gamma-32P}ATP alone, suggesting that autophosphorylation was not involved . These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A . The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance.

Cancer Treat Rep, 1987 Nov, 71(11), 1093 - 4
Facilitation of emergence of multidrug-resistant state by alteration of tumor environment: implications from competitive ecology models; Michelson S; The presence of multidrug-resistant (MDR) cells in a solid tumor constitutes a major problem in cancer therapy . Current thinking suggests that the resistant phenotype arises de novo during the tumor's evolution via somatic mutation mechanisms . The proportion of MDR cells, once established, may be enriched during therapy as a consequence of differential cell kill . Michelson et al have developed mathematical models of these phenomena to gain an insight into the dynamics of clonal subpopulation emergence in general and MDR emergence in particular, and I now show that one unexpected consequence of therapy may be the facilitation of MDR emergence due to damage inflicted on the host . The therapeutic damage to the host is modeled as a decreased ability to carry a specific tumor burden.

Mol Biochem Parasitol, 1987 Nov, 26(1-2), 183 - 91
Flow cytofluorimetric analysis of drug accumulation by multidrug-resistant Trypanosoma brucei brucei and T . b . rhodesiense; Frommel TO et al.; Dual laser flow cytofluorimetry has been used to compare accumulation of compounds representing three major classes of trypanocidal drugs by drug sensitive and drug resistant clones of Trypanosoma brucei brucei and T . b . rhodesiense . Clones selected for resistance to melarsoprol were shown to be cross-resistant in vivo to two diamidines, pentamidine and Berenil, but not to suramin . At 35 degrees C, bloodstream forms of these multidrug-resistant clones accumulated lower intracellular concentrations of the diamidines 4',6-diamidino-2-phenyl-indole (DAPI) and Hoechst 33342, the phenanthridine ethidium bromide, and the acridine acriflavine than drug sensitive parasites . Accumulation of all four drugs was saturable . Drug concentrations giving half-maximal rates of accumulation were increased in the resistant clones relative to the sensitive parent clones . The rate of DAPI accumulation by both resistant and sensitive parasites was strongly temperature dependent and increased sharply above 27 degrees C . Two distinct populations were resolved in mixtures of sensitive and resistant clones exposed to DAPI . Resistant and sensitive cells accumulated identical intracellular concentrations of DAPI following brief treatment with the detergent Triton X-100 . The results suggest that alterations in the surface membrane of multidrug-resistant trypanosomes reduce accumulation of several drugs relative to drug sensitive parasites.

Mol Cell Biol, 1987 Nov, 7(11), 4075 - 81
Simultaneous expression of two P-glycoprotein genes in drug-sensitive Chinese hamster ovary cells; Endicott JA et al.; Overexpression of P-glycoprotein is characteristic of multidrug-resistant cells . We analyzed four P-glycoprotein transcripts that are simultaneously expressed in a drug-sensitive Chinese hamster ovary cell line . We concluded that these transcripts are encoded by two distinct members of a P-glycoprotein multigene family, each of which has two alternative polyadenylation sites . A comparison of the two hamster sequences with the single reported human and mouse P-glycoprotein cDNA sequences demonstrates that P-glycoprotein is a highly conserved protein, that the hamster multigene family is undergoing concerted evolution, and that differences between gene family members are maintained across species . These conserved differences suggest that there may be functional differences between P-glycoprotein molecules.

EMBO J, 1987 Nov, 6(11), 3325 - 31
The human mdr3 gene encodes a novel P-glycoprotein homologue and gives rise to alternatively spliced mRNAs in liver; Van der Bliek AM et al.; We have found cDNAs corresponding to a novel human P-glycoprotein gene in liver cDNA banks . The sequence of the 3' part of this cDNA reveals a domainal organization of the derived protein similar to that of the known P-glycoproteins and an 80% amino acid homology with the product of the human mdr1 gene (Chen et al., 1986) . The new gene lies within 500 kb from mdr1 as determined by pulsed field gradient gel electrophoresis and is designated mdr3, as it appears to correspond to the third of the three P-glycoprotein genes mapped in the hamster multidrug resistance domain . mdr3 yields a transcript of 4100 nucleotides, 400 nucleotides less than the mdr1 transcript; the difference is accounted for by the shorter 3'-untranslated region of the mdr3 mRNA . Our cDNAs provide evidence for alternative splicing of mdr3 pre-mRNAs . One alternative is an insert of seven amino acids between the two major blocks of the nucleotide binding site and another is a deletion of 43 or 47 amino acids covering the putative transmembrane segment 5a . We speculate that these alternatives superimposed on differential expression of P-glycoprotein homologues could provide an explanation for the large variation in cross-resistance patterns observed in cell lines selected for multidrug resistance with different cytostatic drugs.

Proc Natl Acad Sci U S A, 1987 Nov, 84(21), 7701 - 5
Carcinogen-induced mdr overexpression is associated with xenobiotic resistance in rat preneoplastic liver nodules and hepatocellular carcinomas; Fairchild CR et al.; We have previously reported the isolation of a human breast cancer cell line resistant to doxorubicin (adriamycin; AdrR MCF-7 cells) that has also developed the phenotype of multidrug resistance (MDR) . MDR in this cell line is associated with increased expression of mdr (P glycoprotein) gene sequences . The development of MDR in AdrR MCF-7 cells is also associated with changes in the expression of several phase I and phase II drug-detoxifying enzymes . These changes are remarkably similar to those associated with development of xenobiotic resistance in rat hyperplastic liver nodules, a well-studied model system of chemical carcinogenesis . Using an mdr-encoded cDNA sequence isolated from AdrR MCF-7 cells, we have examined the expression of mdr sequences in rat livers under a variety of experimental conditions . The expression of mdr increased 3-fold in regenerating liver . It was also elevated (3- to 12-fold) in several different samples of rat hyperplastic nodules and in four of five hepatomas that developed in this system . This suggests that overexpression of mdr, a gene previously associated with resistance to antineoplastic agents, may also be involved in the development of resistance to xenobiotics in rat hyperplastic nodules . In addition, although the acute administration of 2-acetylaminofluorene induced an 8-fold increase in hepatic mdr-encoded RNA, performance of a partial hepatectomy either before or after administration of 2-acetylaminofluorene resulted in a greater than 80-fold increase in mdr gene expression over that in normal untreated livers . This represents an important in vivo model system in which to study the acute regulation of this drug resistance gene.

Somat Cell Mol Genet, 1987 Nov, 13(6), 609 - 19
Cloning and characterization of DNA sequences amplified in multidrug-resistant Djungarian hamster and mouse cells; Gudkov AV et al.; Five recombinant phages containing different parts of genomic regions amplified in multidrug-resistant (MDR) cells have been isolated from genomic libraries of colchicine- and actinomycin D-resistant Djungarian hamster cells . Fragments of these clones together with a part of Chinese hamster mdr gene (plasmid pDR4.7 a gift of Dr . I . Roninson) were used as hybridization probes to study composition and variations of amplified DNA in a large number of MDR cell lines . Two of the six probes used (pC52 and pDR4.7) showed DNA amplification in a large number of MDR cell lines tested (commonly amplified clones) regardless of their origin (Djungarian hamster or mouse), type of selective agent used (colchicine, actinomycin D, or anthracyclines), and mode of selection (in vitro or in vivo) . These clones hybridized with two different RNA transcripts (pDR4.7, 5kb; pC52, 3.5 kb) that were overproduced in MDR cells . Degrees of amplification of both commonly amplified sequences correlated with levels of resistance in all but one of the cell lines . Other cloned sequences (sporadically amplified clones) were amplified to different extents (but never greater than the commonly amplified sequences) in some of the Djungarian hamster MDR cell lines . Such differential amplification is not the result of heterogeneity of cell population since 20 cell clones tested showed identical ratios of amplification of different amplified sequences . Sporadically amplified sequences usually coamplified with the commonly amplified ones at first steps of selection, but then they would cease to amplify and, at the later stages of selection, they could even be completely deamplified . It seems that disappearance of unnecessary parts of amplicons is a regular process accompanying stepwise gene amplification.

Cancer, 1987 Oct 15, 60(8 Suppl), 2075 - 80
Multidrug resistance in ovarian cancer; Fojo A et al.; The development of acquired resistance has limited the effectiveness of chemotherapy in the treatment of ovarian cancer . Experimental model systems were developed to study the mechanisms associated with primary resistance to chemotherapeutic agents and broad cross-resistance (multidrug resistance) which is characteristic of human ovarian cancer . Doxorubicin-resistant cell lines developed in vitro by exposure of a sensitive cell line to increasing concentrations of doxorubicin develop resistance on the basis of a decrease in drug accumulation and have increased expression of the mdr-1 gene . This gene encodes for a membrane glycoprotein and leads to a decreased drug accumulation in drug resistant cell lines . Cell lines established from patients refractory to doxorubicin-containing combinations, however, do not demonstrate a decrease in drug accumulation . Studies are in progress on the measurement of mdr-1 levels in tumors of patients undergoing treatment to determine whether agents, such as verapamil may be useful in the treatment of drug resistant gynecologic cancers . Human ovarian cancer cell lines from drug resistant patients also has been demonstrated to increase levels of glutathione . Lowering of glutathione levels with buthionine sulfoximine (BSO), which irreversibly inhibits the enzyme gamma-glutamyl cysteine synthetase, leads to a marked potentiation of the cytotoxicity of melphalan both in vitro and in vivo in a nude mouse model of human ovarian cancer . Based on those studies, BSO is undergoing toxicologic evaluation before initiation of clinical trials in drug resistant patients . Our studies demonstrate that drug resistance in human ovarian cancer is likely due to interaction of multiple factors . However, biochemical intervention in some of the key steps leading to drug resistance has been demonstrated experimentally feasible and indicates that pharmacologic reversal of drug resistance is a clinical possibility.

Cancer Res, 1987 Oct 15, 47(20), 5455 - 60
Pharmacological, molecular, and cytogenetic analysis of "atypical" multidrug-resistant human leukemic cells; Beck WT et al.; We previously described the cross-resistance patterns and cellular pharmacology of a human leukemic cell line, CEM/VM-1, selected for resistance to the epipodophyllotoxin teniposide (M . K . Danks et al., Cancer Res., 47: 1297-1301, 1987) . Compared to CEM/VLB100, which is a well characterized "classic" multidrug-resistant (MDR) cell line, the CEM/VM-1 cells display "atypical" multidrug resistance (at-MDR) in that they are cross-resistant to a wide variety of natural product antitumor drugs, except the Vinca alkaloids, and they are not impaired in their ability to accumulate radiolabeled epipodophyllotoxin . We have extended our characterization of this at-MDR cell line in the present study . In comparison to CEM/VLB100 cells, we found that CEM/VM-1 cells are not cross-resistant to either actinomycin D or colchicine . Verapamil and chloroquine, which enhance the cytotoxicity of vinblastine in CEM/VLB100 cells, had little or no ability to do so in the CEM/VM-1 cells . Membrane vesicles of the two resistant sublines were examined for overexpression of the MDR-associated plasma membrane protein (P-glycoprotein, Mr 170,000 protein, or 180,000 glycoprotein) by photoaffinity labeling with the vinblastine analogue N-(p-azido{3-125I}salicyl)-N'-beta-aminoethylvindesine . We were unable to visualize the MDR-associated protein in the CEM/VM-1 membranes with this photoaffinity probe under conditions in which the P-glycoprotein was readily seen in the membranes of CEM/VLB100 cells . Furthermore, no hybridization of the pMDR1 complementary DNA was seen in slot-blot analyses of the RNA from at-MDR cells, indicating that the mdr gene coding for P-glycoprotein is not overexpressed as is the case in the classic MDR cells . However, cytogenetic analysis indicated that the CEM/VM-1 cells contained an abnormally banded region on chromosome 13q, suggesting that a gene other than mdr may be amplified in these cells . Thus, despite the two cell lines having approximately equal degrees of resistance to epipodophyllotoxins, our data indicate that the mechanism(s) responsible for at-MDR is different from that for classic, P-glycoprotein-associated MDR.

J Biol Chem, 1987 Oct 5, 262(28), 13685 - 9
Biosynthesis of heterogeneous forms of multidrug resistance-associated glycoproteins; Greenberger LM et al.; Multidrug-resistant J774.2 mouse macrophage-like cells, selected for resistance to colchicine, vinblastine, or taxol, overexpress antigenically related glycoproteins with distinct electrophoretic mobilities . These plasma membrane glycoproteins are likely to play a pivotal role in the expression of the multidrug resistance phenotype . To determine how these multidrug resistance-associated glycoproteins differ, the biosynthesis and N-linked carbohydrate composition of these proteins were examined and compared . Vinblastineor colchicine-selected cells made a 125-kDa precursor that was rapidly processed (t1/2 approximately equal to 20 min) to mature forms of 135 and 140 kDa, respectively . Heterogeneity between the 135- and 140-kDa forms of the molecule can be attributed to N-linked carbohydrate . In contrast, taxol-selected cells made two precursors, 125 and 120 kDa, which appeared within 5 and 15 min after the onset of pulse labeling, respectively . They were processed to mature forms of 140 and 130 kDa . Since a single deglycosylated precursor or mature form was not observed after enzymatic removal of N-linked oligosaccharides, other differences, besides N-linked glycosylation, which occur in early processing compartments, are likely to account for the two multidrug resistance-associated glycoproteins in taxol-selected cells . These results demonstrate that a family of multidrug resistance-associated glycoproteins can be differentially expressed.

Genetika, 1987 Oct, 23(10), 1797 - 806
{Gene amplification in murine leukemia cells with multiple drug resistance acquired in vivo}; Demidova NS et al.; The P388rm and P388rx cell lines resistant to antracycline antibiotics were obtained as a result of chemotherapy of mice bearing P388 leukemia, by means of increasing dosages of rubomycin and ruboxyl, respectively . These cell lines possessed cross-resistance to vinblastine, vincristine, colchicine, actinomycin D and some other drugs . Multidrug resistance (MDR) of P388rm and P388rx is due to decreased uptake of different cytotoxic compounds by the cells . Development of resistance to rubomycin and ruboxyl was accompanied by the appearance of additional chromosomal structures--long homogeneously staining regions (HSRs), double minute chromosomes and others usually containing amplified DNA sequences . Southern blot-hybridization with cloned DNA fragments amplified in Djungarian and Chinese hamster cell lines having MDR has revealed in P388rm and P388rx cells approximately 50-fold amplification of mdr and pC52 genes . Thus, in mouse leukemia cells which acquired MDR in vivo, as a result of chemotherapy, amplification is observed of the same genes that undergo amplification during selection of cell cultures for MDR in vitro.

Br J Cancer, 1987 Oct, 56(4), 407 - 11
Occurrence of a multidrug-resistant phenotype in human lung xenografts; Mattern J et al.; The intrinsic sensitivity of a panel of 8 human epidermoid lung cancer xenografts to vincristine and actinomycin D has been examined and the cross-resistance patterns of the most vincristine-resistant and vincristine-sensitive tumour line were tested to a variety of other drugs, including radiation . The results demonstrate that xenograft lines derived from human lung tumours not previously treated with chemotherapy exhibit a similar general pattern of cross-resistance to the drugs vincristine, actinomycin D and adriamycin as is observed in human cell lines and in animal models selected for resistance to these drugs . It is also shown that intrinsic resistance to vincristine can be partially overcome by verapamil . This may indicate a potential role of this substance in circumventing clinically observed drug resistance.

J Natl Cancer Inst, 1987 Oct, 79(4), 831 - 6
Flow cytometric screening for selective toxicity to multidrug-resistant cells; Frankfurt OS; Mixed cultures originally containing doxorubicin {(DOX) NCS-123127}-sensitive leukemia P388 (P388/S) cells and DOX-resistant leukemia P388 (P388/R) cells were treated with drugs for 1 hour or with radiation, grown until the cell numbers had increased 250 times (8 cell doublings), and incubated with 2 micrograms daunorubicin (NCS-82151)/ml for 1 hour . The fluorescence of intracellular anthracycline measured on a flow cytometer was used as a marker to distinguish P388/S and P388/R cells . The proportions of low-fluorescent P388/R cells and high-fluorescent P388/S cells were determined from fluorescence histograms . Selective toxicity for multidrug-resistant P388/R cells was indicated by the decreased proportion of these cells in the mixed cultures . In untreated cultures grown from suspensions containing equal proportions of P388/R and P388/S cells, the mean proportion of P388/R cells after 4 days' growth was 34.4% . DOX eliminated P388/S cells from mixed cultures . Nitrogen mustard {(HN2) NCS-762} and x-rays were selectively toxic to P388/R cells . The selectivity of HN2 and x-rays was observed in a narrow dose range by the absence of selective inhibition at low doses, the decreased proportion of P388/R cells at moderate doses, and the killing of all cells in mixed cultures at high doses . Combination treatment of mixed cultures with DOX plus x-rays, or HN2 plus x-rays, produced complete inhibition of growth . Mixed cultures recovered after treatment with DOX plus HN2 contained only P388/R cells . Results obtained by colony-formation assay showed the same patterns of relative sensitivity for P388/R and P388/S cells as the data obtained by flow cytometry (FCM) selectivity assay . FCM analysis of mixed cultures detected selective toxicity for P388/R cells after treatments, characterized by approximately 1 log higher cell killing of P388/R cells than of P388/S cells . It was concluded that FCM analysis of mixed cultures provided a sensitive and reliable assay for fast screening of drugs for selective toxicity to multidrug-resistant cells.

J Cell Physiol, 1987 Oct, 133(1), 46 - 54
Isolation of a clone of F9 teratocarcinoma cells "naturally" resistant to G418; Edwards SA et al.; Resistance to the neomycin analogue G418 forms the basis of a dominant marker selection system for mammalian (and other) cells transfected with the bacterial neo gene . This system has been particularly effective because of the low incidence of spontaneous conversion to G418 resistance in mammalian cells; no case of resistance to the drug in the absence of the bacterial genes has yet been reported to our knowledge . During the course of transfection experiments, we recently isolated a clone of F9 teratocarcinoma cells which is drug resistant yet has no detectable integrated plasmid sequences, neo RNA transcripts, or aminoglycoside phosphotransferase activity . The G418-resistant clone (F9nr7) did not display enhanced resistance to other cytotoxic drugs tested: colchicine, actinomycin D, cycloheximide, and hygromycin B . Therefore, nr7 cells differ from multidrug-resistant phenotypes previously described . However, this clone is inhibited, relative to control cells, in its response to the differentiation-inducing drugs retinoic acid and dibutyryl cAMP, which suggests that some aspects of general drug metabolism may be altered in these cells.

Cancer Res, 1987 Oct 1, 47(19), 5141 - 8
Isolation of amplified and overexpressed DNA sequences from adriamycin-resistant human breast cancer cells; Fairchild CR et al.; An MCF-7 human breast cancer cell line was selected which was 200-fold more resistant to Adriamycin than the wild type cell line . This Adriamycin-resistant (AdrR) cell line exhibited a multidrug-resistant phenotype and was cross-resistant to a wide range of antineoplastic agents including Vinca alkaloids, anthracyclines, and epipodophyllotoxins . Cytogenetic analysis of the AdrR cell line showed the presence of homogeneously staining regions on several chromosomes which were not present in the parental cell line . Using the technique of in-gel renaturation, DNA sequences which were amplified 50- to 100-fold in the AdrR cell line and which covered a total of over 140 kilobases were isolated . In addition, AdrR cells were found to contain amplified and overexpressed sequences which were homologous to hamster P-glycoprotein gene sequences . A hamster cDNA P-glycoprotein gene probe was used to screen a lambda gt10 cDNA library made from human AdrR cell line mRNA and human cDNA sequences homologous to the P-glycoprotein gene were isolated . Hybridization studies with the cloned human cDNA (pADR1) showed that the AdrR MCF-7 cell line contained a 60-fold amplification of this DNA sequence and that polyadenylated mRNA from the AdrR cell line contained a 4.8-kilobase transcript which was overexpressed 45-fold . There was a direct correlation between DNA and RNA copy number of this sequence and level of resistance among several MCF-7 Adriamycin-resistant cell lines . In situ hybridization studies demonstrated that the human P-glycoprotein gene sequence was found on chromosome 7q21.1 in normal human lymphocytes and that amplified DNA sequences isolated from the AdrR MCF-7 cells by the in-gel hybridization technique were linked to the human P-glycoprotein sequences in the homogeneously staining regions in the AdrR cells.

J Med Chem, 1987 Sep, 30(9), 1576 - 81
Potential antitumor agents . 52 . Carbamate analogues of amsacrine with in vivo activity against multidrug-resistant P388 leukemia; Rewcastle GW et al.; Study of a series of aniline-substituted 9-anilinoacridines related to the antileukemic drug amsacrine showed that a 1'-carbamate group provided increased activity against the multidrug-resistant P388/ADR leukemia subline in vivo . Since activity against such resistant tumors is of great clinical significance, a series of acridine-substituted carbamate derivatives were evaluated against both wild-type and ADR/resistant P388 leukemia and the Lewis lung solid tumor in vivo . Structure-activity relationships for all three tumor lines were similar, with 3-halo-5-methyl and 3-halo-5-methoxy compounds proving the most active . This substitution pattern also provided the highest DNA binding . Such compounds (particularly the 3-chloro-5-methyl and 3-chloro-5-methoxy) have in vivo activity against wild-type P388 and Lewis lung comparable to that of the best amsacrine analogues previously developed (greater than 50% cures), as well as P388/ADR activity . This work essentially completes the development of the amsacrine series of antitumor agents.

Gan To Kagaku Ryoho, 1987 Sep, 14(9), 2617 - 25
{A molecular basis for multidrug-resistance and reversal of resistance with human malignant cells}; Akiyama S et al.; Development of cellular resistance to multiple types of anticancer agents has been recognized as one of the major obstacles for the effective cancer chemotherapy . Increased expression of mdr 1 mRNA seems to be a common mechanism for multidrug resistance (MDR) in human malignant cells . The product of the mdr 1 gene is P-glycoprotein . The predicted membrane orientation of the protein and homology with bacterial active transport proteins, and capability of the protein to bind hydrophobic anticancer agents are consistent with the function of P-glycoprotein as an energy-dependent efflux pump responsible for MDR phenotype . Most of the hydrophobic agents which overcome MDR are cationic and amphipathic . These agents interact with certain polar lipids and inhibit also the binding of hydrophobic anticancer agents with P-glycoprotein . They might directly bind to the binding site of anticancer agents on P-glycoprotein and competitively inhibit the binding of anticancer agents . Alternatively, they might bind to polar lipids of membrane vesicles and indirectly inhibit the binding ability of the protein to anticancer agents by perturbing the membrane function.

J Clin Oncol, 1987 Sep, 5(9), 1452 - 60
P-glycoprotein in human sarcoma: evidence for multidrug resistance; Gerlach JH et al.; Overexpression of an immunologically conserved, cell-surface glycoprotein (P-glycoprotein) is consistently associated with multidrug resistance in cell lines in vitro . A preliminary survey of specimens from 12 solid tumor types in our laboratories indicates significant overexpression of P-glycoprotein in some sarcomas . When tested by immunoblotting with monoclonal antibodies directed against P-glycoprotein; tumors from six of 25 sarcoma patients displayed elevated levels of P-glycoprotein . Three of the sarcoma patients exhibiting P-glycoprotein had not previously been exposed to chemotherapy, implying that overexpression of this marker and possible concomitant multidrug resistance may not depend only on selection during prior drug treatments . The P-glycoprotein overexpression in the sarcoma specimens is evidence for the presence of multidrug resistant cells in these tumors; thus, our data suggest that this mode of resistance may have clinical significance in sarcoma patients.

Mol Gen Mikrobiol Virusol, 1987 Aug, (8), 3 - 9
{Multiple drug resistance of tumor cells: manifestations, genetic basis, clinical aspects}; Gudkov AV; Data are reviewed concerning the results of study of multidrug-resistant (MDR) tumor cells . MDR often develops in the course of chemotherapy or in vitro selection of tumor cells by vincristine, adriamycin, actinomycin D, colchicine, etc . MDR cells are resistant to all these drugs though their targets and mechanisms of toxic action are quite different . Resistance is due to the decreased accumulation by MDR cells of these compounds . The genetic basis for MDR is amplification of a large genomic region that contains a number of genes coding for products and functions that are under extensive study . Specific karyotype and amplified DNA alterations occur during the development of MDR imitating the processes of appearance and variability of multigene families . The obtained data demonstrate the ways of overcoming of tumor multidrug resistance in clinic.

Biochem Pharmacol, 1987 Jul 15, 36(14), 2373 - 80
Sorcin (V19), a soluble acidic calcium-binding protein overproduced in multidrug-resistant cells . Identification of the protein by anti-sorcin antibody; Meyers MB et al.; Sorcin (soluble resistance-related calcium-binding protein), an acidic (pI = 5.7) protein (Mr approximately 20 kDa) previously designated V19, was originally identified in cells selected for high levels of resistance to vincristine . Two-dimensional gel electrophoresis and/or Western blot techniques now show sorcin to be overproduced in cells selected for resistance to actinomycin D (QUA/ADj), colchicine (CHRC5), and adriamycin (BE(2)-C/ADR) . Not all cell lines selected for resistance to these drugs overproduced sorcin; e.g . cells of an independently selected actinomycin D-resistant subline of QUA, QUA/ADsx, did not contain increased amounts of sorcin . Sorcin was purified by preparative gel electrophoresis from QUA/ADj cells and used to generate specific antiserum in chickens . By Western blot analyses the antiserum was shown to recognize sorcin in QUA/ADj and in vincristine-resistant mouse and Chinese hamster lung, colchicine-resistant Chinese hamster ovary, and adriamycin-resistant human neuroblastoma lines . Low level expression of the protein was detectable in control, drug-sensitive cells . Direct binding assays with 45Ca2+ showed that sorcin was a calcium-binding protein . QUA/ADj cells contained increased numbers of double minute chromosomes (DMs), cytogenetic indicators of gene amplification . As found for two other multidrug-resistant sublines, sorcin overproduction in QUA/ADj cells may be the result of amplification of the sorcin-encoding gene . The overproduction of this protein in multidrug-resistant cells of various species implies that sorcin plays a role in expression of the resistant phenotype.

Cancer Res, 1987 Jul 15, 47(14), 3736 - 41
Enhancement of voltage-gated Na+ channel current associated with multidrug resistance in human leukemia cells; Yamashita N et al.; Membrane currents were examined in a drug-sensitive human leukemia cell line (K562) and its multidrug-resistant cell line (K562/ADM) under the whole-cell variation of the patch electrode voltage clamp technique . Most K562 cells showed only the outward current, which was completely suppressed by internal Cs+ ions and 20 mM ethyleneglycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid . In contrast to K562 cells, most K562/ADM cells showed tetrodotoxin-sensitive, voltage-gated Na+ channel current in addition to the outward current . Na+ current was observed in four of 29 K562 cells examined, while it was observed in 29 of 33 K562/ADM cells . Two revertant cell lines (R1-3, R1-5) did not show Na+ current . It was concluded that the amplitude of voltage-gated Na+ current increases in association with multidrug resistance in human leukemia cells.

Biochem Pharmacol, 1987 Jul 1, 36(13), 2115 - 23
Action of calcium antagonists on multidrug resistant cells . Specific cytotoxicity independent of increased cancer drug accumulation; Cano-Gauci DF et al.; Previous studies have shown that calcium channel blockers can overcome, at least partially, multidrug resistance (MDR) . This study was undertaken to attempt to determine the mechanisms whereby these agents bring about this effect . Their influence on the uptake and retention of several cancer drugs and on the toxic actions of these compounds was assessed employing MDR cell lines from several species . The wild-type drug sensitive parent cells proved to be more susceptible than the multidrug resistant variants to the effects of calcium channel blockers on cancer drug accumulation . This was shown for verapamil, nifedipine and the calmodulin inhibitor trifluoperazine acting on human, mouse and Chinese hamster ovary (CHO) cell lines . The enhancement of drug accumulation by calcium antagonists was similar to that caused by non-ionic detergents . Furthermore, verapamil was unable to alter 45Ca2+ accumulation in sensitive or resistant cells, suggesting that these agents act in a calcium-independent manner . Verapamil accumulation in multidrug resistant cells was reduced compared to sensitive cells . In spite of this reduced accumulation, however, verapamil alone was much more toxic to multidrug resistant cells than to the sensitive cells . This suggests that calcium channel blockers are specifically toxic to MDR cells by virtue of an interaction with the MDR cell surface distinct from that involved in promoting cellular accumulation.

Arzneimittelforschung, 1987 Jul, 37(7), 862 - 7
Detection of murine S180 cells expressing a multidrug resistance phenotype using different in vitro test systems and a monoclonal antibody; Volm M et al.; Doxorubicin (adriamycin) preconditioned S180 cells were more resistant to doxorubicin . The resistance was detected by three different methods (short-term test, colony assay, tissue culture assay) . The doxorubicin-resistant S180 cells express the pleitrop drug resistance phenotype . There exists a multidrug resistance to doxorubicin, dactinomycin (actinomycin D), vincristine and colchicine . In addition, collateral sensitivity was found to fluorouracil (5-fluorouracil) and methotrexate . This multidrug phenotype is in accordance with the pleiotropic phenotype of colchicine-resistant CHO cells . Resistant S180 cells express a glycoprotein of Mr 170 kd determined by indirect immunofluorescence using a monoclonal antibody (Mab 265/F4) to the P-glycoprotein of colchicine-resistant CHO-cells . The P-glycoprotein could be an important prognostic factors for tumors with multidrug resistance.

Anticancer Res, 1987 Jul-Aug, 7(4B), 729 - 32
Antiblastic treatment does not affect N-myc gene amplification in neuroblastoma; Tonini GP et al.; Gene amplification has been found in the genome of cells growing in vivo and/or in vitro . In cell lines with acquired multidrug resistance gene amplification has been frequently detected . Moreover, extra-copies of cellular oncogenes have been located in tumor cells in vivo; particularly N-myc gene amplification was discovered in advanced stage of neuroblastoma (NB) . Neuroblastoma, a tumor of neural origin, has a high incidence in children . N-myc amplification has been demonstrated in untreated patient and a positive significant correlation with the progression of the disease has been established . In this paper we report on four NB patients treated with a polychemotherapeutic protocol and showing N-myc amplification . One patient examined before and after treatment displayed a slight change in N-myc gene copy numbers . It was shown that N-myc gene amplification is not affected by drug activities and that minimal residual of cells bearing N-myc amplification may remain in the tumor mass . N-myc amplification can also cause advantageous cell growth in the presence of drugs . The implications in the pharmacologic management of NB patient showing N-myc gene amplification is discussed.

FASEB J, 1987 Jul, 1(1), 51 - 4
ATP-binding properties of P glycoprotein from multidrug-resistant KB cells; Cornwell MM et al.; The photoaffinity reagent 8-azido-alpha-{32P}ATP was used to label a protein of 170 kDa in membrane vesicle preparations from a highly multidrug-resistant cell line, KB-V1, but not from the drug-sensitive parental cell line KB-3-1 . The 170-kDa labeled protein was immunoprecipitated with a monoclonal antibody (MRK-16) to P glycoprotein . Both ATP and GTP inhibited labeling by 8-azido-alpha-{32P}ATP . Labeling of P170 was not inhibited by 5 mM ADP, 5 mM ribose-5-phosphate, or 100 microM vinblastine . These data directly demonstrate that P glycoprotein has a nucleotide-binding site that could supply energy for drug transport.

Cancer Res, 1987 Jun 15, 47(12), 3186 - 9
Preclinical evaluation of illudins as anticancer agents; Kelner MJ et al.; Illudins are low molecular weight natural products which were previously evaluated as anticancer drugs using rodent tumor models . In the present studies, we used in vitro cultures of human cancer cells to reevaluate their potential as anticancer agents . Using continuous exposure, Illudins S and M were cytotoxic to human leukemia cells at concentrations of 6-100 nM, but dihydroilludin M was 3 orders of magnitude less toxic, thus identifying a ketone site as a structural feature critical for cytotoxicity . Cytokinetic studies showed that illudin S caused a complete block at the G1-S phase interface of the cell cycle . Kinetics of inhibition of radiolabeled thymidine, uridine, and leucine incorporation suggested a primary effect on DNA synthesis . In colony and liquid culture assays, cell killing was time dependent but near maximal with a 2-h exposure . Myeloid and T-lymphocyte leukemia cells were most sensitive (50% inhibitory concentration, 6-11 nM), but B-cell leukemia/lymphoma, melanoma, and ovarian carcinoma cells were at least 10 times more resistant . Bone marrow granulocyte/macrophage progenitors showed intermediate sensitivity . Illudin S was equally effective against CEM T-lymphocyte leukemia cells expressing the multidrug resistance phenotype associated with Mr 180,000 glycoprotein and the parental cell line . CEM cells resistant to doxorubicin, epipodophyllotoxins, and 1-beta-D-arabinofuranosylcytosine showed only a 2-fold increased resistance to illudin S . Illudins are novel and potent cytotoxins which may be preferentially active against human myeloid and T-cell leukemias, including cells resistant to more conventional chemotherapeutic agents . The present studies illustrate the breadth of information which can be obtained on a new agent using present in vitro screening procedures and human cells.

J Biol Chem, 1987 Jun 5, 262(16), 7884 - 8
Identification of the multidrug resistance-related membrane glycoprotein as an acceptor for calcium channel blockers; Safa AR et al.; A radioactive photoactive dihydropyridine calcium channel blocker, {3H}azidopine, was used to photoaffinity label plasma membranes of multidrug-resistant Chinese hamster lung cells selected for resistance to vincristine (DC-3F/VCRd-5L) or actinomycin D (DC-3F/ADX) . Sodium dodecyl sulfate-polyacrylamide gel electrophoretic fluorograms revealed the presence of an intensely radiolabeled 150-180-kDa doublet in the membranes from drug-resistant but not from the drug-sensitive parental (DC-3F) cells . A similar radiolabeled doublet was barely detected in a drug-sensitive partial revertant (DC-3F/ADX-U) cell line . The 150-180-kDa doublet exhibited a specific half-maximal saturable photolabeling at 1.07 X 10(-7) M {3H}azidopine . The dihydropyridine binding specificity was established by competitive blocking of specific photolabeling with nonradioactive azidopine as well as with nonphotoactive calcium channel blockers nimodipine, nitrendipine, and nifedipine . In addition, {3H}azidopine photolabeling was blocked by verapamil and diltiazem but was stimulated by excess prenylamine and bepridil suggesting a cross-specificity for up to four different classes of calcium channel blockers . The 150-180-kDa calcium channel blocker acceptor co-electrophoresed exactly with the 150-180-kDa surface membrane glycoprotein (gp150-180 or P-glycoprotein) Vinca alkaloid acceptor from multidrug-resistant cells and was immunoprecipitated by polyclonal antibody recognizing gp150-180 . {3H}Azidopine photolabeling of the 150-180-kDa component in the presence of excess vinblastine was reduced over 90%, confirming the identity or close relationship of the calcium channel blocker acceptor and the gp150-180 Vinca alkaloid acceptor . The {3H}azidopine photolabeling of gp150-180 also was reduced by excess actinomycin D, adriamycin, or colchicine, demonstrating a broad gp150-180 drug recognition capacity . The ability of gp150-180 to recognize multiple natural product cytotoxic drugs as well as calcium channel blockers suggests a direct function for gp150-180 in the multidrug resistance phenomenon and a role in the circumvention of that resistance by calcium channel blockers.

Cancer Res, 1987 Jun 1, 47(11), 2875 - 8
Chromosomal localization of three genes coamplified in the multidrug-resistant CHRC5 Chinese hamster ovary cell line; Jongsma AP et al.; At least five gene classes are amplified in the multidrug-resistant CHO cell line CHRC5 . Protein products have been identified for two classes; class 2 codes for the large membrane P-glycoprotein, whereas class 4 encodes the small cytoplasmic calcium-binding protein sorcin (V19) . By DNA analysis we have shown previously that these five genes are linked in two groups: class 1 + 2 + 3; and class 4 + 5 . By use of in situ hybridization with complementary DNAs derived from the resistant cell line we demonstrate here that genes from both linkage groups are amplified and situated together in each of two different chromosomal regions of the resistant Chinese hamster cell line . The positions of the amplicons correspond to cytogenetically identified homogeneously staining regions in an altered 7q+ chromosome and in a rearranged Z-7 {t(3;4)} chromosome . The native genes were mapped both in the CHRC5 line and in a normal diploid Chinese hamster cell strain, CHNF 86 . We confirm the position of the class 2 gene on 1q26 and we show that class 4 and 5 genes are located in the same region of 1q . We conclude that the gene classes 2, 4, and 5 are closely juxtaposed in the normal Chinese hamster genome and comprise one amplicon in resistant cells . Our results are compatible with the hypothesis that multidrug resistance is due to overexpression of P-glycoprotein genes and that the other genes amplified in the CHRC5 line are coamplified because they happen to lie close to the P-glycoprotein genes.

Cancer Res, 1987 Jun 1, 47(11), 2860 - 5
Phosphorylation of the Mr 170,000 to 180,000 glycoprotein specific to multidrug-resistant tumor cells: effects of verapamil, trifluoperazine, and phorbol esters; Hamada H et al.; An overexpression of plasma membrane glycoprotein with a relative molecular mass (Mr) of 170,000-180,000 is consistently found in different multidrug-resistant human and animal cell lines, although the functional role of the protein in multidrug resistance is not fully understood . It has been reported previously that the Mr 170,000-180,000 glycoprotein is involved, directly or indirectly, in the drug transport mechanism and the proliferation of multidrug-resistant tumor cells . In an attempt to clarify further the function of the Mr 170,000-180,000 glycoprotein, we have studied the phosphorylation state of the protein in intact K562/ADM cells and found that: the protein is phosphorylated in the basal state; verapamil and trifluoperazine, which inhibit the active drug efflux and restore drug sensitivity in resistant cells, caused an increase in the phosphorylation of the Mr 170,000-180,000 glycoprotein; 4 beta-phorbol 12 beta-myristate 13 alpha-acetate and 1-oleoyl 2-acetylglycerol enhanced phosphorylation of the protein; the protein was phosphorylated at serine residues; tryptic phosphopeptide mapping of the Mr 170,000-180,000 glycoprotein showed that 4 beta-phorbol 12 beta-myristate 13 alpha-acetate treatment induced an increase in phosphorylation at different sites of the protein from those induced by verapamil or trifluoperazine treatment, suggesting that the protein is phosphorylated by an array of complex regulation mechanisms . Phosphorylation of the Mr 170,000-180,000 glycoprotein might play a role in the regulation of processes affecting cellular function in multidrug resistance.

Proc Natl Acad Sci U S A, 1987 Jun, 84(12), 4288 - 92
A monoclonal antibody-Pseudomonas toxin conjugate that specifically kills multidrug-resistant cells; FitzGerald DJ et al.; One form of multidrug resistance is due to the expression of a 170-kDa energy-dependent drug efflux pump called P-glycoprotein in the plasma membranes of human cancer cells . We have prepared conjugates of Pseudomonas toxin with the anti-P-glycoprotein monoclonal antibody MRK-16 . These anti-P-glycoprotein-toxin conjugates specifically kill multidrug-resistant human KB cells . Similar conjugates could be useful in cancer therapy to reduce or eliminate multidrug-resistant tumor populations in tumors intrinsically resistant to chemotherapy or in populations that become resistant during combination chemotherapy.

Cancer Res, 1987 Jun 1, 47(11), 2961 - 6
Isolation of human KB cell lines resistant to epidermal growth factor-Pseudomonas exotoxin conjugates; Lyall RM et al.; Mutants of the human KB carcinoma cell line resistant to a cytotoxic conjugate of epidermal growth factor (EGF) and Pseudomonas exotoxin (PE) were selected . EGF-PE and the drug verapamil, which enhanced EGF-PE cytotoxicity, were used in the selection process . These mutants also showed some cross-resistance to PE . All of the EGF-PE resistant variants displayed lower levels of 125I-EGF binding, 20-50% of parental KB levels, without altered affinity for EGF and grew at a slower rate than the parental cell line KB-3-1 . These results indicate that EGF-PE resistant KB cells have a complex phenotype which includes a reduction in the number of EGF receptors and reduced sensitivity to unconjugated PE . Resistance to toxin-conjugates, although pleiotropic, is specific and does not lead to resistance to multiple other anticancer drugs, nor are independently selected multidrug resistant KB lines resistant to PE . These results argue that protocols for cancer treatment could effectively use specifically designed cytotoxic toxin conjugates as an adjunct to conventional chemotherapy.

Mol Gen Mikrobiol Virusol, 1987 Jun, (6), 33 - 8
{Pattern of evolution of amplicons during the development of multiple drug resistance in Djungarian hamster cells}; Gudkov AV et al.; Six cloned DNA fragments representing different portions of the genomic region amplified in multidrug resistant Djungarian hamster cells were used to study amplicon variations in a large number of the resistant cell lines . Expressed correlation exists between the degree of 3 cloned sequences amplification and the level of multidrug resistance . Three other cloned regions amplify coordinately with the latter ones at the initial steps of selection . Later their amplification halts and they mao even eliminate from amplicons of highly resistant cells . The rates and order of elimination of these sequences vary among different independently derived series of multidrug resistant cell lines.

Science, 1987 May 29, 236(4805), 1120 - 2
Expression of the multidrug-resistant gene in hepatocarcinogenesis and regenerating rat liver; Thorgeirsson SS et al.; Preneoplastic and neoplastic liver nodules and hepatocytes isolated from regenerating rat liver have been shown to be resistant to a broad range of carcinogenic agents . This phenomenon was studied by measuring the expression of the multidrug-resistant (mdr) gene in normal liver cells and in preneoplastic and neoplastic nodules and regenerating liver . Levels of messenger RNA for the mdr gene, which encodes P-glycoprotein, were elevated in both preneoplastic and neoplastic lesions . Expression of the mdr gene also reached high levels in regenerating rat liver 24 to 72 hours after partial hepatectomy . These results show that the expression of the mdr gene can be regulated in liver and is likely to be responsible for part of the multidrug-resistance phenotype of carcinogen-initiated hepatocytes and regenerating liver cells.

Cancer Res, 1987 May 15, 47(10), 2620 - 5
DNA-mediated transfer and cloning of a human multidrug-resistant gene of adriamycin-resistant myelogenous leukemia K562; Sugimoto Y et al.; We have successfully transferred and cloned a fragment of a human multidrug-resistant gene by using DNA-mediated gene transfer . Macromolecular DNA of human multidrug-resistant K562 cells was transfected to drug-sensitive mouse Ltk- cells to obtain a drug-resistant transfectant with a human resistant gene . Both primary and secondary transfectants showed similar patterns of cross-resistance to Adriamycin and vincristine . The mechanism of drug resistance of the transfectants was attributed to decreased retention of the drug . Three secondary transfectants obtained independently contained common Alu-containing EcoRI fragments 15, 6.5, 3.7, 2.6, and 1.9 kilobases long . The 2.6-kilobase EcoRI fragment was cloned from a lambda phage genomic library made from DNA of a secondary transfectant . The 2.6-kilobase fragment was detected in the primary and secondary transfectants but not in the parental Ltk-, Adriamycin-resistant Ltk-, and Adriamycin-resistant P388 cells . This sequence was found to be amplified in several multidrug-resistant cell lines such as Adriamycin-resistant ovarian carcinoma A2780 and colchicine-resistant KB carcinoma cells . The 2.6-kilobase fragment hybridized with a 4.5-kilobase mRNA which is overexpressed in the Adriamycin-resistant K562 cells and the Adriamycin-resistant A2780 cells but not detected in the parental K562 cells . The gene transferred and cloned in this study seems to be related to the P-glycoprotein gene as judged from the size of mRNA and its overexpression in some of the multidrug-resistant cell lines where P-glycoprotein was found to be highly expressed.

Cancer Res, 1987 May 15, 47(10), 2594 - 8
Multidrug resistance in a human small cell lung cancer cell line selected in adriamycin; Mirski SE et al.; A multidrug resistant variant (H69AR) of the human small cell lung cancer cell line NCI-H69 was obtained by culturing these cells in gradually increasing doses of Adriamycin up to 0.8 microM after a total of 14 months . H69AR expresses the multidrug resistant phenotype because it is cross-resistant to anthracycline analogues including daunomycin, epirubicin, menogaril, and mitoxantrone as well as to acivicin, etoposide, gramicidin D, colchicine, and the Vinca alkaloids, vincristine and vinblastine . H69AR is also similar to other multidrug resistant cell lines in that it displays little or no cross-resistance to bleomycin, 5-fluorouracil, and carboplatin . It has a slight collateral sensitivity to 1-dehydrotestosterone and lidocaine . H69AR has increased cell-cell adhesiveness compared to H69, but a similar growth rate in vitro and tumorigenicity in nude mice . When cultured in the absence of Adriamycin, there is a 40% decrease in resistance by 35 days of culture, compared to cells in continuous culture in drug, but no further decrease in resistance up to 181 days . Monoclonal antibodies to P-glycoprotein have no detectable reactivity with H69AR cells as determined by enzyme-linked immunosorbent assay and immunoblotting techniques . Thus, unlike most multidrug resistant cell lines, H69AR does not appear to express enhanced levels of P-glycoprotein . H69AR will provide a useful model for the study of multidrug resistance in human small cell lung cancer.

Cancer Res, 1987 May 1, 47(9), 2413 - 6
Effect of bisbenzylisoquinoline (biscoclaurine) alkaloids on multidrug resistance in KB human cancer cells; Shiraishi N et al.; Cepharanthine, a bisbenzylisoquinoline (biscoclaurine) alkaloid, completely overcomes resistance of a multidrug-resistant subline, ChR-24, derived from human KB carcinoma cells, to vincristine, actinomycin D, and daunomycin, and partially overcomes resistance to Adriamycin . Another biscoclaurine alkaloid, berbamine, partially overcomes resistance to these anticancer agents . Accumulation of {3H}daunomycin in ChR-24 cells is about 10% of that in both the parental KB and revertant cell line (Rev-2) which is derived from ChR-24 . Cepharanthine prominently increases the accumulation of daunomycin in resistant ChR-24 cells, but not in parental KB and Rev-2 cells . Enhanced efflux of daunomycin from the resistant cells is completely inhibited by cepharanthine . Cellular uptake of {3H}daunomycin is not significantly affected in the resistant cells by cepharanthine . Accumulation of {3H}cepharanthine is observed at similar levels in both KB and ChR-24 . Phosphatidylserine specifically inhibited the accumulation of {3H}cepharanthine in KB and ChR-24 cells when tested by adding various phospholipids such as phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin to culture medium . The enhanced accumulation of {3H}daunomycin in cepharanthine-treated ChR-24 cells is inhibited in the presence of 20 micrograms/ml phosphatidylserine . Cepharanthine may overcome multidrug resistance by binding to phosphatidylserine in the plasma membrane and perturbing membrane function.

Proc Natl Acad Sci U S A, 1987 May, 84(9), 3004 - 8
Expression of a full-length cDNA for the human "MDR1" gene confers resistance to colchicine, doxorubicin, and vinblastine; Ueda K et al.; Intrinsic and acquired multidrug resistance (MDR) is an important problem in cancer therapy . MDR in human KB carcinoma cells selected for resistance to colchicine, vinblastine, or doxorubicin (former generic name adriamycin) is associated with overexpression of the "MDR1" gene, which encodes P-glycoprotein . We previously have isolated an overlapping set of cDNA clones for the human MDR1 gene from multidrug-resistant KB cells . Here we report the construction of a full-length cDNA for the human MDR1 gene and show that this reconstructed cDNA, when inserted into a retroviral expression vector containing the long terminal repeats of Moloney leukemia virus or Harvey sarcoma virus, functions in mouse NIH 3T3 and human KB cells to confer the complete multidrug-resistance phenotype . These results suggest that the human MDR1 gene may be used as a positive selectable marker to introduce genes into human cells and to transform human cells to multidrug resistance without introducing nonhuman antigens.

Cancer Res, 1987 Apr 15, 47(8), 2103 - 6
P-glycoprotein expression in human breast cancer cells; Fuqua SA et al.; Multidrug resistance in Chinese hamster ovary cells is associated with the Mr 170,000 surface glycoprotein . Using our monoclonal antibody to this protein, we have isolated a complementary DNA clone from an expression vector library . This complementary DNA recognizes a 4.5-kilobase mRNA in drug-resistant but not-sensitive Chinese hamster ovary cells; it also recognizes a 5.0-kilobase mRNA in our Adriamycin-resistant subline of the MDA-231 human breast cancer cell line which is not expressed in the drug-sensitive parent line . Southern blot analysis shows that the P-glycoprotein sequences are greatly amplified in resistant Chinese hamster ovary cells but not in the resistant human breast cancer cells, indicating that amplification and expression of the Mr 170,000 P-glycoprotein gene are not necessarily coordinate events . Amplification of this gene may not be required for multidrug resistance in human cells.

Mol Gen Mikrobiol Virusol, 1987 Apr, (4), 14 - 9
{Cloning and characteristics of DNA sequences amplified in Djungarian hamster cells with multidrug resistance}; Chernova OB et al.; A number of DNA clones containing the amplified DNA sequences were isolated from the genomic library of multidrug-resistant (MDR) Djungarian hamster cells using the DNAC0t 10-250 hybridization probe . Five independent nonoverlapping clones were obtained that covered more than 100 kb of the amplified genomic region . These clones were used as hybridization probes in blot-hybridization with DNA from 7 independently derived MDR Djungarian hamster cell lines selected for the resistance to colchicine or actinomycin D . Some clones contained the DNA sequences amplified in all of the cell lines tested while the others contained the cell line specific amplified sequences . Hybridization in situ was used to localize the amplified DNA in metaphase chromosomes of a MDR cell line that contained about 140 copies of these sequences . The approximate size of an amplicon calculated on the basis of the obtained data is about 1-2 X 10(3) kb.

Cancer Res, 1987 Apr 1, 47(7), 1897 - 904
Development and characterization of a human myelogenous leukemia cell line resistant to 4'-(9-acridinylamino)-3-methanesulfon-m-anisidide; Beran M et al.; The human myelogenous leukemia cell line HL-60 was made resistant to amsacrine (m-AMSA) by repeated exposure in vitro to increasingly large doses of the drug . Resistance to m-AMSA developed in a triphasic process and was accompanied by a slightly slower growth rate and cloning efficiency and a more differentiated morphological phenotype . Extensive chromosomal rearrangement also took place . Among other chromosomal aberrations, one of the No . 6 homologues showed an added segment on the long arm in the form of an homogeneously staining region . One of the homologues of chromosome 14 in every cell showed a deletion of the distal end of the long arm that was replaced by an unidentified homogeneously staining segment . Membrane-associated 170 kd glycoprotein was not overexpressed in the resistant cells, which together with an absence of cross-resistance to Vinca alkaloids and anthracyclines points toward a mechanism of resistance different from multidrug resistance . The ability of resistant cells to respond to differentiation-inducing agents was not significantly changed as compared with that of the parental line . Growth of resistant cells in the absence of m-AMSA for over 200 population doublings within a period of more than 1.5 years did not result in reversion of the resistance, suggesting a stable genomic change . Resistance was not due to a decrease in the bioavailability of the drug . Uptake of {14C}m-AMSA by either whole cells or isolated nuclei of resistant cells exceeded that of the parental cell line, and outward transport of the drug was not more active; thus there were higher levels of intracellularly bound drug . The cell line represents an excellent model for studies of the mechanisms of resistance to m-AMSA and its modulation in human myelogenous leukemia.

Jpn J Cancer Res, 1987 Apr, 78(4), 397 - 404
Kinetic analysis of active efflux of vincristine from multidrug-resistant P388 leukemia cells; Inaba M et al.; Kinetic analysis of vincristine transport in parental and multidrug-resistant P388 leukemia cells was attempted by indirect assessment of its efflux . Practically, the initial velocity and steady-state level of vincristine uptake by ATP-depleted cells, and its steady-state level in untreated cells, were measured . As a result, a saturable process of not only influx but also efflux of vincristine was observed for the first time with both cell lines, suggesting the existence of a carrier-mediated system for influx and efflux . With increasing extracellular drug concentrations, the contribution of the mediated transport to the total flux was decreased and that of the unsaturable process, that is, simple diffusion, was increased . It should be particularly noted that the Km and Vmax values of efflux in the resistant cells were significantly less and greater, respectively, than those of the sensitive cells, providing a biochemical basis for enhanced efflux as a mechanism of multidrug-resistance . No significant difference in kinetic parameters of vincristine influx and intracellular binding contributing to resistance was found between the two cell lines.

Gan To Kagaku Ryoho, 1987 Mar, 14(3 Pt 2), 807 - 14
{Studies on the mechanism of multidrug resistance}; Inaba M; We have introduced two different approaches which we have introduced recently in an attempt to understand the mechanism of multidrug resistance, which was shown to be successfully reversed using non-antitumor analogs of anthracycline and vinca alkaloid . This approach was adopted on the basis of our hypothesis that these substances can inhibit the accelerated drug efflux of the resistant cells . On the other hand, we found that such efflux consists of a saturable as well as an unsaturable process using our newly-designed kinetic approach . In addition, greater Vmax and lower Km were found in the saturable process of efflux in resistant cells as compared with those in sensitive cells . In conclusion, these results suggest that broad cross-resistance among structurally unrelated drugs, i.e., multidrug resistance, is associated with an efflux-oriented transport carrier with broad affinity for these lipophilic drugs with a polycyclic structure.

Cancer Genet Cytogenet, 1987 Mar, 25(1), 141 - 8
Chromosomal location of human P-glycoprotein gene sequences; Bell DR et al.; Nonresponse to chemotherapy may result from the acquisition of multidrug resistance by malignant cells . Overexpression of the 170,000 dalton cell surface P-glycoprotein is associated with this phenotype and this appears to result from amplification of a multigene family coding for this protein . A cDNA encoding a conserved portion of P-glycoprotein has been cloned from hamster cells, and this was used in the present study to localize human P-glycoprotein gene sequences to chromosome 7q36.

Science, 1987 Feb 20, 235(4791), 899 - 901
Reversal of chloroquine resistance in Plasmodium falciparum by verapamil; Martin SK et al.; The parasite Plasmodium falciparum, like neoplastic cells, develops resistance to multiple structurally unrelated drugs . If the mechanisms by which P . falciparum and neoplastic cells become resistant are similar, then it may be possible to reverse the resistance in the two types of cells by the same pharmacological agents . Verapamil, a calcium channel blocker, completely reversed chloroquine resistance in two chloroquine-resistant P . falciparum clones from Southeast Asia and Brazil . Verapamil reversed chloroquine resistance at the same concentration (1 X 10(-6)M) as that at which it reversed resistance in multidrug-resistant cultured neoplastic cells . This same concentration of verapamil had no effect on chloroquine-sensitive parasites . Hence, chloroquine resistance in P . falciparum may fit the criteria for the multidrug-resistant phenotype.

J Biol Chem, 1987 Feb 15, 262(5), 2166 - 70
Certain calcium channel blockers bind specifically to multidrug-resistant human KB carcinoma membrane vesicles and inhibit drug binding to P-glycoprotein; Cornwell MM et al.; The calcium channel blockers verapamil and diltiazem have been shown to reverse multidrug resistance, but the mechanism of action of these agents is still unknown . We measured {3H}verapamil, {3H}desmethoxyverapamil, {3H}diltiazem, and {3H}nitrendipine binding to membrane vesicles made from drug-sensitive (KB-3-1), multidrug-resistant (KB-C4 and KB-V1), and revertant (KB-V1-R2) cells . Membrane vesicles from KB-V1 cells bound 10-20-fold more {3H}verapamil and {3H}diltiazem and about 30-fold more {3H}desmethoxyverapamil than did vesicles from the parental KB-3-1 or revertant KB-V1-R2 cell lines . These drugs reverse the multidrug resistance phenotype by increasing accumulation of drugs in the resistant cells . No difference in binding of {3H}nitrendipine, which did not reverse drug resistance, was observed . The binding of vinblastine, desmethoxyverapamil, and diltiazem to KB-V1 vesicles was specific and saturable and was inhibited by desmethoxyverapamil and quinidine greater than vinblastine and diltiazem much greater than daunomycin . In addition, verapamil and diltiazem inhibited the vinblastine photoaffinity labeling of P170, the protein previously shown to be a marker of multidrug resistance.

Anal Biochem, 1987 Feb 1, 160(2), 483 - 8
Determination of membrane antigens by a covalent crosslinking method with monoclonal antibodies; Hamada H et al.; Monoclonal antibodies that recognize cell surface proteins may serve as very useful tools for the study of the biological functions of membrane proteins . However, solubilization of the antigens with detergents may lead to major conformational changes of the protein, making their determination with monoclonal antibodies by immune blot or ordinary immunoprecipitation methods difficult . This is especially evident when the monoclonal antibodies recognize tertiary structures of the proteins in the membrane . We have generated two monoclonal antibodies which are specific for the cell surface antigens of multidrug-resistant human cell lines . However, the antigens of both monoclonal antibodies were difficult to detect by either immune blot or ordinary immunoprecipitation methods . We used a cleavable crosslinking reagent dithiobis(succinimidyl propionate) to covalently link the monoclonal antibody with its antigenic determinant in the membrane of intact cells . By this method, we were able to detect the antigens for these two monoclonal antibodies following solubilization, immunoprecipitation, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . This method should have wide applicability in determination of membrane antigens recognized by monoclonal antibodies when immune blot or ordinary immunoprecipitation methods are not successful.

Mol Cell Biol, 1987 Feb, 7(2), 718 - 24
Expression of hamster P-glycoprotein and multidrug resistance in DNA-mediated transformants of mouse LTA cells; Deuchars KL et al.; The overexpression of a plasma membrane glycoprotein, P-glycoprotein, is strongly correlated with the expression of multidrug resistance . This phenotype (frequently observed in cell lines selected for resistance to a single drug) is characterized by cross resistance to many drugs, some of which are used in cancer chemotherapy . In the present study we showed that DNA-mediated transformants of mouse LTA cells with DNA from multidrug-resistant hamster cells acquired the multidrug resistance phenotype, that the transformants contained hamster P-glycoprotein DNA sequences, that these sequences were amplified whereas the recipient mouse P-glycoprotein sequences remained at wild-type levels, and that the overexpressed P-glycoprotein in these cells was of hamster origin . Furthermore, we showed that the hamster P-glycoprotein sequences were transfected independently of a group of genes that were originally coamplified and linked within a 1-megabase-pair region in the donor hamster genome . These data indicate that the high expression of P-glycoprotein is the only alteration required to mediate multidrug resistance.

Lancet, 1987 Jan 17, 1(8525), 135 - 7
Detection of a multidrug resistant phenotype in acute non-lymphoblastic leukaemia; Ma DD et al.; Most adult patients with acute non-lymphoblastic leukaemia (ANLL) relapse with drug resistance . Overexpression of a plasma membrane protein, P-glycoprotein, correlates with multidrug resistance in human and animal cell lines . We have detected a multidrug resistance phenotype in two patients with drug resistant ANLL by an immunocytochemical assay using a monoclonal antibody to P-glycoprotein . Sequential analysis of peripheral blood samples from both patients showed a progressive increase in both the intensity of staining and the proportion of leukaemic cells that bound antibody as the disease progressed . The assay is simple, and may have prognostic and therapeutic implications.

J Biol Chem, 1987 Jan 15, 262(2), 505 - 8
The human multidrug resistance (mdr1) gene . cDNA cloning and transcription initiation; Ueda K et al.; Multidrug resistance in human KB carcinoma cells selected for resistance to colchicine, vinblastine, or adriamycin results from overexpression, and frequently amplification, of a specific gene (mdr1) . Overlapping cDNA clones representing a complete 4.7-kilobase mdr1 transcript have been obtained from multidrug-resistant KB cells . Primer extension and S1 nuclease protection experiments show that two transcripts initiate 136 and 140 bases upstream from the first ATG codon in all human multidrug-resistant cell lines . The mdr1 gene is expressed in human normal kidney cells and HepG2 liver cells as a poly(A)+ RNA which starts from the same sites . Less prominent transcripts were found to initiate 155-180 bases upstream from the first ATG codon in vinblastine- or adriamycin-selected cell lines and 480-630 bases upstream in colchicine-selected cell lines . Southern hybridization analyses with different portions of a full-length cDNA indicate that the human mdr1 gene encompasses at least 70 kilobases of DNA amplified in all highly multidrug-resistant cell lines.

Eksp Onkol, 1987, 9(4), 42 - 7
{Characteristics of anthracycline-resistant strains of P388 leukemia}; Goncharova SA et al.; Two strains of P388 murine leukemia with acquired resistance to rubomycin (P388/rm) and its nitroxyl derivative ruboxyl (P388/rx) . The rubomycin resistance has been developed by the 14th generation and ruboxyl one-by the 8th generation . The growth kinetic patterns and the cell cycle time of the parent and resistant strains were similar . An increased tumourogenicity of both resistant strains cells was found . The resistance development was accompanied by the appearance of the additional chromosome materials, namely of homogeneously staining region (P388/rx) and of double chromatin bodies (P388/rm) . The partial recovery of sensitivity to rubomycin occurred during 36 generations (1 year) . Simultaneously the genetic markers have been lost . The recovery of sensitivity to ruboxyl in this period was not observed . The obtained resistant strains possessed the multidrug resistance: the cross resistance of P388/rm and P388/rx to actinomycin D, Vinca alkaloids and colchicine was shown.

Chromosoma, 1987, 95(2), 117 - 25
Specific gene amplification associated with consistent chromosomal abnormality in independently established multidrug-resistant Chinese hamster ovary cells; Sen S et al.; Multidrug-resistant (MDR) Chinese hamster ovary (CHO) cell lines were established by selection for resistance to the toxicities of vinblastine (VB) and Adriamycin (AD) in progressively increasing drug concentrations . These cell lines have amplified the DNA sequence that has previously been shown to be amplified in another MDR CHO cell line which was selected with vincristine (VC) . An overproduced 4.5 kb mRNA was detected in these MDR cell lines . We report here that the levels of DNA amplification and the 4.5 kb transcript do not correlate with the levels of drug resistance, suggesting that either translational control for the expression of the amplified gene is involved or multiple genes are participating in conferring drug resistance in these cell lines . The amplified DNA sequence was used as a probe and localized by in situ hybridization to chromosome 1q 26-28 (middle portion of the long arm) in the drug-sensitive CHO line, but proximal to the telomere of chromosome 1q in both VB- and AD-selected MDR cell lines . This is consistent with results that have been previously reported for the VC-selected MDR cell lines . Cytogenetic analyses revealed abnormal chromosomal banding patterns or homogeneously staining regions (HSR) between 1q 26-28 and the 1q ter in these independently established MDR lines . These results, taken together, suggest that chromosomal rearrangements leading to gene translocation have consistently accompanied gene amplification in these MDR cell lines . The mechanisms of translocation and its implication in multidrug resistance in these cell lines are discussed.

Anticancer Res, 1987 Jan-Feb, 7(1), 65 - 9
Characterization of multidrug resistance in SEWA mouse tumor cells: increased glutathione transferase activity and reversal of resistance with verapamil; Dahllof B et al.; The glutathione transferase (EC 2.5.1.18) activity of multidrug-resistant and drug-sensitive SEWA mouse tumor cells was determined . Increase in activity was found in all multidrug-resistant sublines . Electrophoretic separation of cytosolic proteins and immunodetection, using antisera against Class Alpha, Class Mu and Class Pi glutathione transferase, revealed a two-fold increase of the Class Pi transferase in the resistant cells . The role of glutathione transferase of Class Pi in the multidrug resistance and its distinction from the over-produced 21,000 dalton protein p21 is discussed . Reversal of the resistance, using the calcium antagonist verapamil, and different sensitivity to verapamil between different sublines are also reported.

Cytogenet Cell Genet, 1987, 45(2), 99 - 101
Localization of the multidrug resistance-associated 170 kDa P-glycoprotein gene to mouse chromosome 5 and to homogeneously staining regions in multidrug-resistant mouse cells by in situ hybridization; Martinsson T et al.; This report describes the localization of the 170 kDa P-glycoprotein gene(s) to mouse chromosome 5, subbands A2 or A3 . Overproduction of P-glycoprotein is associated with multidrug resistance (MDR) . MDR cell lines derived from the SEWA mouse tumor carry multiple copies of the P-glycoprotein gene . These were found to reside in homogeneously staining regions, situated in different locations in different sublines.

Clin Physiol Biochem, 1987, 5(3-4), 140 - 51
Molecular mechanism of multidrug resistance in tumor cells; Roninson IB; The ability of tumor cells to develop simultaneous resistance to multiple lipophilic cytotoxic compounds represents a major problem in cancer chemotherapy . This review describes recent molecular biological studies which resulted in the identification and cloning of the gene responsible for multidrug resistance in human tumor cells . This gene, designated mdr1, is overexpressed in all and amplified in many of the multidrug-resistant cell lines analyzed . Gene transfer and expression assays have indicated that the mdr1 gene is both necessary and sufficient for multidrug resistance . The product of the mdr1 gene is P-glycoprotein, a transmembrane protein which shares homology with several bacterial proteins involved in active membrane transport . P-glycoprotein appears to function as an energy-dependent efflux pump responsible for the removal of drugs from multidrug-resistant cells . The functions of the mdr system in normal cells and its potential clinical implications are discussed.

Proc Natl Acad Sci U S A, 1987 Jan, 84(1), 265 - 9
Expression of a multidrug-resistance gene in human tumors and tissues; Fojo AT et al.; The identification and cloning of a segment of a human multidrug resistance gene (mdr1) was reported recently . To examine the molecular basis of one type of multidrug resistance, we have prepared RNA from human tumors and normal tissues and measured their content of mdr1 RNA . We find that the mdr1 gene is expressed at a very high level in the adrenal gland; at a high level in the kidney; at intermediate levels in the lung, liver, lower jejunum, colon, and rectum; and at low levels in many other tissues . The mdr1 gene is also expressed in several human tumors, including many but not all tumors derived from the adrenal gland and the colon . In addition, increased expression was detected in a few tumors at the time of relapse following initial chemotherapy . Although controlled clinical studies will be required, our results suggest that measurement of mdr1 RNA may prove to be a valuable tool in the design of chemotherapy protocols.

Biochem Biophys Res Commun, 1986 Dec 30, 141(3), 956 - 62
The mdr1 gene, responsible for multidrug-resistance, codes for P-glycoprotein; Ueda K et al.; The development of simultaneous resistance to multiple drugs in cultured cells occurs after selection for resistance to single agents . This multidrug-resistance phenotype is thought to mimic multidrug-resistance in human tumors treated with chemotherapy . Both the expression of a membrane protein, termed P170 or P-glycoprotein, and the expression of a cloned DNA fragment, termed mdr1, have been shown independently to be associated with multidrug-resistance in cultured cells . In this work, we show that human KB carcinoma cells which express the mdr1 gene also express P-glycoprotein, and that cDNAs encoding P-glycoprotein cross-hybridize with mdr1 cDNAs . Thus, the mdr1 gene codes for P-glycoprotein.

Nature, 1986 Dec 4-10, 324(6096), 485 - 9
Homology between P-glycoprotein and a bacterial haemolysin transport protein suggests a model for multidrug resistance; Gerlach JH et al.; Increased expression of P-glycoprotein, a plasma membrane glycoprotein of relative molecular mass (Mr) 170,000 (170K), occurs in a wide variety of cell lines that exhibit pleiotropic resistance to unrelated drugs . The presence of P-glycoprotein in human cancers refractory to chemotherapy suggests that tumour cells with multidrug resistance can arise during malignant progression . We have discovered striking homology between P-glycoprotein and the HlyB protein, a 66K Escherichia coli membrane protein required for the export of haemolysin (protein of Mr 107K) . P-glycoprotein can be viewed as a tandem duplication of the HlyB protein . The hydropathy profiles of the two proteins are similar and reveal an extensive transmembrane region resembling those found in pore-forming plasma membrane proteins . The C-terminal region of P-glycoprotein and the HlyB protein contain sequences homologous to the nucleotide-binding domains of a group of closely related bacterial ATP-binding proteins . We propose a model for multidrug resistance in which P-glycoprotein functions as an energy-dependent export pump to reduce intracellular levels of anticancer drugs.

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