Mol Pharmacol, 1989 Oct, 36(4), 543 - 6 Identification of the multidrug resistance-related P-glycoprotein as a cyclosporine binding protein; Foxwell BM et al.; The immunosuppressive agent cyclosporine A has been shown to reverse multidrug resistance (MDR) in malignant cells . In the present study, a 3H-cyclosporine diazirine analogue was used to photolabel viable MDR Chinese hamster ovary cells . The 170-kDa membrane P-glycoprotein, which functions as a drug efflux pump, was strongly labeled . The binding of 3H-cyclosporine diazirine analogue to P-glycoprotein was competable by excess cyclosporine A and by the nonimmunosuppressive cyclosporine H . These results suggest that cyclosporine reverses the MDR phenotype by binding directly to P-glycoprotein and that this binding is not dependent on the immunosuppressive potential of the cyclosporine derivative . The identification of P-glycoprotein as a cyclosporine binding protein has obvious implications for cancer chemotherapy. Cancer Res, 1989 Oct 1, 49(19), 5281 - 7 Atypical multidrug resistance in a therapy-induced drug-resistant human leukemia cell line (LALW-2): resistance to Vinca alkaloids independent of P-glycoprotein; Haber M et al.; Near diploid leukemic T-cells (LALW-2), exposed to cytotoxic drugs only as a consequence of therapy administered to the donor patient, have been maintained by serial xenograft in nude mice . In comparison with the leukemic line CCRF-CEM, using a growth inhibition assay, LALW-2 cells were resistant to Vinca alkaloids and actinomycin D (relative resistance, 200-fold or more), were slightly resistant to Adriamycin (relative resistance, 4-fold), and showed no resistance to daunorubicin or teniposide . By comparison, a vincristine-resistant CEM subline developed in our laboratory (CEM/VCR R) was resistant to all these agents by at least 30-fold . The VCR R subline served as a positive control, confirming the previously reported correlation between multidrug resistance and amplification of the P-glycoprotein gene . Comparison of CEM, CEM/VCR R, and LALW-2 cells establish that the P-glycoprotein gene was not amplified or overexpressed in the LALW-2 cells; neither could the gene product be detected by immunoblotting in extracts from these cells . The LALW-2 cells were further distinguished from CEM/VCR R cells due to the lack of increased vincristine efflux by the xenografted cells, an effect readily demonstrable in the CEM/VCR R cells . However, although LALW-2 cells efflux vincristine at the same rate as CCRF-CEM cells, the xenografted cells exhibited a reduced rate of vincristine accumulation . Uptake of daunorubicin by LALW-2 cells was not distinguished from that by CEM cells, consistent with similar 50% inhibitory dose levels for this drug in both cell populations, and differentiating both from CEM/VCR R cells . Thus, clinical resistance in this case appears to be an "atypical" form of multidrug resistance specifically distinguished by resistance to Vinca alkaloids and actinomycin D occurring in the absence of increased amounts of P-glycoprotein and manifesting decreased drug uptake. J Biol Chem, 1989 Sep 25, 264(27), 16261 - 7 Membrane protein changes in an L1210 leukemia cell line with a translocation defect in the methotrexate-tetrahydrofolate cofactor transport carrier; Schuetz JD et al.; We report on membrane protein changes in an L1210 leukemia cell line with a highly specific defect in the function of the methotrexate (MTX)-tetrahydrofolate cofactor transport carrier . This clonal line, MTXrA, made 100-fold resistant to MTX, was derived in a single step and exhibited stable resistance over 120 generations in the absence of drug . The transport defect was associated with a 10-fold decrease in influx Vmax without a change in influx Km . There was no difference between the MTXrA and parent lines in the levels or affinities of specific cell surface binders for MTX nor in the labeling of the 44-kDa membrane protein upon treatment with the specific affinity label, N-hydroxysuccinimide ester of tritiated MTX . Consistent with impaired carrier function was the observation that trans-stimulation of MTX influx by intracellular 5-formyltetrahydrofolate observed in the parent line was not demonstrated in the MTXrA line . The transport defect was highly specific for the MTX-tetrahydrofolate cofactor transport carrier . Initial uptake rates for 5-fluoro-2'-deoxyuridine and 2-deoxyglucose were unchanged and influx and net transport of alpha-aminoisobutyric acid were, in fact, increased . There was no cross-resistance of this line to phenylalanine mustard or cytosine arabinoside, agents that utilize specific amino acid and nucleoside transport carriers, respectively . SDS-polyacrylamide gel electrophoresis of purified plasma membrane preparations stained with Coomassie Blue revealed several protein differences between the parental and MTXrA lines . Most prominent is a band at approximately 190 kDa which ran with slightly greater mobility than a lesser staining band in the parent line . {3H}Borohydride labeling of cells also identified a distinct protein peak in the MTXrA line at approximately 190 kDa eliminated by prior treatment of cells with neuraminidase . Absence of expression of protein or mRNA related to the multidrug resistance gene as well as lack of cross-resistance to daunorubicin or trimetrexate indicate that this mechanism of resistance to MTX is completely unrelated to the multidrug resistance phenomenon observed with high molecular weight heterocyclic compounds . These data represent the first demonstration of membrane protein differences in a highly resistant L1210 murine leukemia cell line with a marked unique defect in MTX transport which appears to be related to impaired mobility of the tetrahydrofolate-cofactor carrier . Further studies are now required to elucidate the possible role of one or more of these proteins in the transport defect. J Biol Chem, 1989 Sep 25, 264(27), 16054 - 8 Alternate overexpression of two P-glycoprotein {corrected} genes is associated with changes in multidrug resistance in a J774.2 cell line; Lothstein L et al.; In multidrug-resistant murine J774.2 cells, the mdr1a and mdr1b genes encode the 120- and 125-kDa P-glycoprotein precursors, respectively (Hsu, S . I., Lothstein, L., and Horwitz, S.B . (1989) J . Biol . Chem . 264, 12053-12062) . It is shown here that a J774.2 cell line selected for vinblastine resistance (J7.V3) switched from the 125- to 120-kDa precursor when cells that were maintained in 20 nM vinblastine were grown in 40 nM vinblastine for 20 months . The rate of switching was accelerated by growing cells in higher levels of vinblastine . These findings suggest that cells which express mdr1a have a selective growth advantage compared to cells which express mdr1b . Consistent with this hypothesis, the switching event that occurs in cells maintained at 40 nM vinblastine was correlated with 3.5-5-fold higher levels of resistance to vinblastine, taxol, and doxorubicin in the absence of any detectable increase in the amount of immunoreactive P-glycoprotein . These findings suggest that P-glycoproteins derived from mdr1a and mdr1b are functionally distinct. J Natl Cancer Inst, 1989 Sep 20, 81(18), 1401 - 5 Correlation of MDR1 gene expression with chemotherapy in neuroblastoma; Bourhis J et al.; Forty-one neuroblastoma tumor specimens have been analyzed by Northern and slot blot hybridization techniques with human MDR1 gene probes . Only one of 15 (6%) tumors from patients who had not received chemotherapy exhibited high levels of MDR1 transcripts, while 11 of 26 (42%) treated tumors showed high levels of MDR1 expression (Fisher exact test: P = .03) . The results indicate that the level of MDR1 mRNA expression is associated with previous chemotherapy, including drugs that select the multidrug resistance phenotype in vitro regardless of neuroblastoma tissue origin or N-myc content in the genome . For the 26 treated neuroblastomas, the number of nonresponsive tumors was found to be significantly higher among those with high levels of MDR1 mRNA. Cancer Res, 1989 Sep 15, 49(18), 5062 - 5 Expression of a human multidrug resistance gene in ovarian carcinomas; Bourhis J et al.; Expression of the human MDR1 gene has been shown to confer the multidrug resistance (MDR) phenotype to sensitive cells . To investigate the possible contribution of the MDR phenotype to chemoresistance in ovarian carcinoma, we have analyzed MDR1 gene expression in fresh carcinoma specimens from 50 patients . Fifteen received chemotherapy before surgery and were judged as poor responders . Thirty-five patients did not receive any drug before surgery . Control tissues were lymphocytes from 7 patients . Total RNAs were analyzed by Northern and slot blot hybridization techniques using human MDR1 complementary DNA and human gamma-actin complementary DNA probes sequentially as qualitative and quantitative controls . MDR1 transcripts (4.5 kilobases) were observed in the RNA preparations obtained from 3 of 10 patients who were treated with doxorubicin or vincristine, 2 drugs known to select the MDR phenotype in vitro . In 40 other RNA preparations obtained from 35 untreated patients and 5 patients treated exclusively with cyclophosphamide and cis-platinum, no transcript could be detected . Using the exact Fisher test, the difference between the 2 groups was found to be significant (P less than 0.01) . The three tumors with elevated MDR1 expression did not show MDR1 DNA amplification . Our study suggests that, in spite of the weak occurrence of the MDR process in patients with ovarian cancers, MDR1 expression can be related to previous treatment with doxorubicin or vincristine . These results favor the expression of the MDR1 gene as one of the determinants involved in the acquired chemoresistance of ovarian cancers. Biochim Biophys Acta, 1989 Sep 15, 992(3), 307 - 14 Biosynthesis, processing and half-life of P-glycoprotein in a human multidrug-resistant KB cell; Yoshimura A et al.; The biosynthesis, processing, and half-life of the drug efflux pump, P-glycoprotein, were studied in human multidrug-resistant KB (KB-C2) cells selected for resistance to colchicine . An antibody directed against a synthetic oligopeptide corresponding to the amino-acid sequence (Glu-393-Lys-408) of P-glycoprotein from human mdr1 cDNA was prepared in rabbits . With immunoblotting and immunoprecipitation, we detected a 140-170 kDa protein in KB-C2 cells but not in parental sensitive KB cells . KB-C2 cells made a 125 kDa precursor that was slowly processed (t1/2 = 45 min) to the mature form of 140-150 kDa . The processing rate of P-glycoprotein was slower than that of low-density lipoprotein receptor . We detected another 160-180 kDa smear band, which might be a completely denatured form of P-glycoprotein . With immunoblotting, a minor band of high molecular mass (greater than 500 kDa) was also detected and this form increased after the cells were treated with chemical cross-linker, 1,5-difluoro-2,4-dinitrobenzene . The half-life of P-glycoprotein was long; no significant loss of P-glycoprotein was observed within 24 h after synthesis . Cells treated with tunicamycin produced a 120 kDa form of P-glycoprotein which was no longer processed but showed stability similar to that of the mature 140-150 kDa form . Agents that reverse multidrug resistance, phorbol ester and transport substrate did not affect the stability of P-glycoprotein. Cancer Res, 1989 Sep 15, 49(18), 5002 - 6 Reversal mechanism of multidrug resistance by verapamil: direct binding of verapamil to P-glycoprotein on specific sites and transport of verapamil outward across the plasma membrane of K562/ADM cells; Yusa K et al.; The calcium channel blocker verapamil has been shown to reverse multidrug resistance (T . Tsuruo et al., Cancer Res . 41: 1967-1972, 1981), but the mechanism of action of this agent has not been fully elucidated . A radioactive photoactive analogue of verapamil, N-{benzoyl-3,5-3H-(+/-)-5-{(3,4-dimethoxyphenetyl)methylamino}-2- (3,4-dimethoxyphenyl)-2-isopropyl-N-p-azidobenzoylpentylamine, was used to label the plasma membranes of a human myelogenous leukemia cell line (K562), a multidrug-resistant subline selected for resistance to Adriamycin (K562/ADM) and its revertant cell (R1-3) . Sodium dodecyl sulfate-polyacrylamide gel electrophoretic fluorograms revealed the presence of an intensely radiolabeled Mr 170,000-180,000 protein in the membranes from K562/ADM but not from the drug-sensitive parental K562 and revertant R1-3 cells . The Mr 170,000-180,000 verapamil acceptor was immunoprecipitated by monoclonal antibody MRK16 specific for P-glycoprotein associated with multidrug resistance, indicating that P-glycoprotein in the plasma membrane is a major target of verapamil in K562/ADM cells . The photolabeling of P-glycoprotein with N-{benzoyl-3,5-3H}-(+/-)-5-{(3,4-dimethoxyphenetyl)methylamino}-2- (3,4-dimethoxyphenyl)-2-isopropyl-N-p-azidobenzoylphentylamine was significantly blocked by other calcium channel blockers, nicardipine and diltiazem, that have been shown to overcome multidrug resistance . In addition, the photolabeling was partially blocked by Adriamycin, vincristine, and colchicine, suggesting that the specific binding sites for verapamil on P-glycoprotein are closely related to the binding sites for these calcium channel blockers and antitumor agents . To determine whether verapamil could be a substrate for P-glycoprotein, the cellular accumulation of {3H}verapamil into K562 and K562/ADM was evaluated . The accumulation of {3H}verapamil in the multidrug-resistant cells was 30% of K562 cells and increased when K562/ADM cells were treated with vincristine and nicardipine at 5 microM, indicating that the P-glycoprotein transports verapamil as well as other antitumor agents in the multidrug-resistant cells . These results suggest that verapamil enhances antitumor agent retention through competition for closely related binding sites on P-glycoprotein. J Biol Chem, 1989 Sep 15, 264(26), 15483 - 8 Two different regions of P-glycoprotein {corrected} are photoaffinity-labeled by azidopine; Bruggemann EP et al.; Cells that express P-glycoprotein are resistant to many unrelated anticancer drugs . All evidence suggests that P-glycoprotein is a plasma membrane protein that confers multidrug resistance by actively transporting these cytotoxic drugs out of cells . The objective of our work is to locate drug binding sites on P-glycoprotein . Azidopine is a photoaffinity drug analog that specifically labels P-glycoprotein . To determine the region of P-glycoprotein that binds azidopine, we labeled P-glycoprotein with azidopine and digested the labeled protein into fragments . We then identified the labeled fragments with specific antibodies . We have determined that azidopine labels two different regions of P-glycoprotein: one region is in the amino half of P-glycoprotein, and the other is in the carboxyl half of the protein . Our results suggest that P-glycoprotein contains either two binding sites for azidopine or a single site formed by the two homologous halves of the protein. J Biol Chem, 1989 Sep 5, 264(25), 15094 - 103 Multiple drug resistance and conservative amplification of the H region in Leishmania major; Ellenberger TE et al.; Amplification of the H region has been previously observed in methotrexate (MTX)-resistant strains of Leishmania major and in unselected laboratory stocks of L . tarentolae . We now show that selection of L . major with the structurally unrelated drugs primaquine or terbinafine generated resistant lines exhibiting H region amplification and 23- and 12-fold cross-resistance to MTX, respectively . These and other drug-resistant lines bearing H region amplification also exhibited weak cross-resistance to primaquine and terbinafine, associating the amplified H region with pleiotropic resistance to MTX and other drugs . In contrast, lines selected for chloroquine or pentamidine resistance did not show H region amplification or this pattern of drug cross-resistance . The primaquine- and terbinafine-selected lines exhibited wild-type levels of dihydrofolate reductase-thymidylate synthase and normal uptake and accumulation of MTX, and the MTX resistance of these lines was not reversed by verapamil . These data suggest that the mechanism of MTX cross-resistance associated with H region amplification is novel and distinct from that mediated by overexpression of MDR genes in multidrug-resistant mammalian cells . Structural studies indicated that the amplified H region DNA in these L . major lines was largely (possibly exclusively) extra-chromosomal and consisted of circular inverted repeats joined at two DNA rearrangement junctions . Southern blot analyses showed that these rearrangement junctions were identical in four independent cell lines, suggesting that these sites are "hotspots" for DNA rearrangement . H region amplification in all of these lines was conservative, defined as retention of the chromosomal H region locus without structural alteration or reduction in copy number . This finding is consistent with an over-replication/recombination model for amplification of the H region. J Biol Chem, 1989 Sep 5, 264(25), 14880 - 4 Transepithelial transport of drugs by the multidrug transporter in cultured Madin-Darby canine kidney cell epithelia; Horio M et al.; We studied transepithelial transport of 3H-labeled hydrophobic cationic drugs in epithelia formed by wild-type and by drug-resistant Madin-Darby canine kidney (MDCk) cells that had been infected with a retrovirus carrying the multidrug-resistance (MDR1) cDNA which encodes the P-glycoprotein . P-glycoprotein is an ATP consuming plasma membrane multidrug transporter responsible for the efflux of cytotoxic chemotherapeutic drugs from resistant cancer cells . Wild-type MDCK cells have small amounts of P-glycoprotein detected by immunoprecipitation . Net transepithelial transport across wild-type MDCK epithelia was demonstrated . Basal to apical flux of 100 nM vinblastine was about six times higher than apical to basal flux . Addition of unlabeled vinblastine reduced basal to apical flux of tracer and increased apical to basal flux of tracer, a pattern expected if there is a saturable pump that extrudes vinblastine at the apical plasma membrane . Daunomycin, vincristine, and actinomycin D were also actively transported and at 20 microM these agents inhibited transport of vinblastine, suggesting that wild-type MDCK cells have a common transporter for all these drugs . Vinblastine transport was also inhibited by 20 microM verapamil, which inhibits the multidrug transporter and reverses multidrug-resistance in non-polarized cells . Net transepithelial transport of all these cytotoxic drugs and of verapamil was much higher in epithelia formed by MDCK cells infected with a human MDR1 virus (MDR-MDCK) which is expressed on the apical surface of MDR-MDCK monolayers . Because the transport of these cytotoxic drugs and verapamil is increased in MDR-MDCK epithelia compared to wild-type MDCK epithelia, transport in both these cell populations can be attributed to P-glycoprotein . These results are consistent with a role for P-glycoprotein in multidrug secretory transport across the epithelium of the proximal tubule since P-glycoprotein is normally expressed on the apical membrane of proximal tubule cells. Eur J Cancer Clin Oncol, 1989 Sep, 25(9), 1287 - 93 Localization of a nonintercalative DNA binding antitumour drug in mitochondria: relationship to multidrug resistance; Liley DT et al.; The bis-(n-butyl) quaternary salt of N,N'-bis-(6-quinolyl)terephthalamide (QBQ), a fluorescent antitumour compound in the phthalanilide series which is thought to bind to the minor groove of the DNA double helix, has been investigated with respect to its in vitro activity and subcellular localization . Cultured MCF-7 human breast carcinoma cells concentrated QBQ in mitochondria by a time-dependent process which was inhibited by the ionophore valinomycin, suggesting a possible mode of antitumour action of QBQ through mitochondrial poisoning . Growth of cultured P388 murine leukaemia cells was inhibited 50% in the presence of 0.52 microM QBQ and multidrug-resistant P388 sublines developed for resistance to actinomycin D, vincristine, Adriamycin and the phthalanilide NSC 38280 were cross-resistant to the drug . Cross-resistance was reduced in all lines by the presence of 11 microM verapamil, suggesting that a transport resistance mechanism operates on QBQ . The actinomycin D-resistant P388 cell line was found to be cross-resistant to the aromatic cations rhodamine 123, which binds to proteins, and ethidium and pyronin Y, which bind intercalatively to DNA . Thus mitochondrion-specific drugs with different macromolecular binding properties all appear to be excluded by multidrug-resistant cells. Blood, 1989 Sep, 74(4), 1388 - 95 Expression of the mdr-1/P-170 gene in patients with acute lymphoblastic leukemia; Rothenberg ML et al.; Increased expression of the multidrug resistance gene (mdr-1/P-170) and the dihydrofolate reductase (DHFR) gene have been implicated in the development of in vitro drug resistance . Overexpression, with or without gene amplification, is seen in the development of drug resistance in culture and it has been postulated that genetic modulation of mdr-1/P-170 and DHFR may also be involved in the development of clinical drug resistance . We screened lymphoblasts from 28 patients with acute lymphoblastic leukemia (ALL) for evidence of overexpression of mdr-1/P-170 using RNAse protection, RNA in situ hybridization and immunohistochemistry . Overexpression of mdr-1/P-170 without gene amplification was detected in samples from four patients (three after multiple relapses, one at presentation) . Overexpression of mdr-1/P-170 was heterogeneous within the population of malignant lymphoblasts as demonstrated by RNA in situ hybridization, immunohistochemistry, and drug uptake using daunomycin autofluorescence analysis . There was no evidence of overexpression of DHFR in any of the eight patient samples tested by RNAse protection nor was there any evidence of gene amplification in 11 patient samples on Southern blot analysis . From these observations it appears that overexpression without gene amplification of mdr-1/P-170 may be one mechanism of clinical drug resistance in ALL. Br J Cancer, 1989 Sep, 60(3), 339 - 42 Amplification and expression of mdr1 gene in a multidrug resistant variant of small cell lung cancer cell line NCI-H69; Reeve JG et al.; Amplification and expression of the mdr1 gene encoding P-glycoprotein have been studied in H69/LX4 a multidrug resistant variant (MDR) of small cell lung cancer (SCLC) cell line NCI-H69 . Recently a second independently derived MDR variant of this cell line designated H69/AR was found by others not to show amplification, rearrangement or over-expression of the mdr1 gene . The present study reports that in marked contrast to H69/AR, H69/LX4 shows amplification and expression of the P-glycoprotein gene and raises the possibility that P-glycoprotein hyperexpression may be a clinically relevant component of MDR in some SCLC tumours. Mol Cell Biol, 1989 Sep, 9(9), 3808 - 20 Structure and expression of the human MDR (P-glycoprotein) gene family; Chin JE et al.; The human MDR (P-glycoprotein) gene family is known to include two members, MDR1 and MDR2 . The product of the MDR1 gene, which is responsible for resistance to different cytotoxic drugs (multidrug resistance), appears to serve as an energy-dependent efflux pump for various lipophilic compounds . The function of the MDR2 gene remains unknown . We have examined the structure of the human MDR gene family by Southern hybridization of DNA from different multidrug-resistant cell lines with subfragments of MDR1 cDNA and by cloning and sequencing of genomic fragments . We have found no evidence for any other cross-hybridizing MDR genes . The sequence of two exons of the MDR2 gene was determined from genomic clones . Hybridization with single-exon probes showed that the human MDR1 gene is closely related to two genes in mouse and hamster DNA, whereas MDR2 corresponds to one rodent gene . The human MDR locus was mapped by field-inversion gel electrophoresis, and both MDR genes were found to be linked within 330 kilobases . The expression patterns of the human MDR genes were examined by enzymatic amplification of cDNA . In multidrug-resistant cell lines, increased expression of MDR1 mRNA was paralleled by a smaller increase in the levels of MDR2 mRNA . In normal human tissues, MDR2 was coexpressed with MDR1 in the liver, kidney, adrenal gland, and spleen . MDR1 expression was also detected in colon, lung, stomach, esophagus, muscle, breast, and bladder. Proc Natl Acad Sci U S A, 1989 Sep, 86(17), 6488 - 92 Mammalian multidrug-resistance gene: correlation of exon organization with structural domains and duplication of an ancestral gene; Raymond M et al.; Analysis of the nucleotide and deduced amino acid sequences of the biologically active mouse mdr1 cDNA clone indicates that the protein is formed by two highly homologous halves, each containing six putative transmembrane domains and a nucleotide-binding site . The duplicated unit shows high sequence homology to the proposed energy-coupling subunit of bacterial periplasmic transport proteins . We have cloned and characterized the mouse mdr1 gene and have analyzed the genomic organization of the two homologous halves forming the mdr1 protein . The gene spans 68 kilobases, is split into 28 exons, and the two homologous halves are encoded by 14 and 13 exons . The transcriptional initiation site of the gene has been mapped and putative TATA and consensus CAAT sequences have been found at positions -27 and -83, respectively . Discrete structural domains of the mdr1 protein are encoded by separate exons: Ten of the 12 putative transmembrane domains are encoded by individual exons and the two nucleotide-binding sites are each encoded by three exons . The exon/intron organization of the gene is conserved in the two highly homologous regions encoding the nucleotide-binding sites . The conservation of certain pairs of introns, together with the high degree of sequence homology, indicate that the mouse mdr1 gene originated from the duplication of an intron-containing ancestral gene. Cancer Res, 1989 Sep 1, 49(17), 4829 - 34 Elimination of chemoresistant multiple myeloma clonogenic colony-forming cells by combined treatment with a plasma cell-reactive monoclonal antibody and a P-glycoprotein-reactive monoclonal antibody; Tong AW et al.; Patients with multiple myeloma (MM) commonly become refractory to chemotherapy despite a favorable response to induction treatment . We examined the effectiveness of a previously characterized plasma cell-reactive monoclonal antibody, MM4, in eliminating MM clonogenic colony-forming cells (CCC) with a multidrug-resistant (MDR) phenotype . Experiments were performed using MM cell lines that exhibit 6 (RPMI 8226/DOX6)- and 40 (RPMI 8226/DOX40)-fold resistance to doxorubicin (DOX) . Both lines were selected from the chemosensitive MM line RPMI 8226/S and were cross-resistant to mitoxantrone, acronycine, etoposide, and vincristine . Surface marker analysis conducted in this study showed that DOX6 and DOX40 overexpressed the MDR1 gene product p170 . Both MDR lines remained reactive to the plasma cell-reactive monoclonal antibodies MM4 and PCA-1 and expressed the relevant cytoplasmic immunoglobulin light chain . Treatment with MM4 and rabbit complement (C') was equally cytotoxic to RPMI 8226/S {80 +/- 5.6% (SD)}, DOX6 {74 +/- 8.5}, and DOX40 cells {75 +/- 11.3%}, based on short-term chromium release studies . Furthermore, MM4 + C' deleted up to 3 logs of CCC colonies from chemosensitive and MDR lines (RPMI 8226/S, 99.87 +/- 0.11%; DOX6, 99.91 +/- 0.08%; DOX40, 99.55 +/- 0.44%) . By comparison, the P-glycoprotein-reactive monoclonal antibody MRK-16 and C' inhibited tumor colony formation of MDR cells (8226/DOX6, 95.71 +/- 2.51%; 8226/DOX40, 99.61 +/- 0.43%) but affected that of chemosensitive cells only slightly (8.9 +/- 17.8%) . In an attempt to optimize the depletion of myeloma CCC, MM4 was used together with MRK-16 . This approach resulted in uniform depletion of myeloma clonogenic colony-forming cells from the chemosensitive (98.32 +/- 1.53%, n = 4) and MDR lines (8226/DOX6, 98.83 +/- 0.08%, n = 4; 8226/DOX40 99.29 +/- 0.62, n = 7) but did not result in enhanced CCC depletion . When DOX40 cells were mixed with normal bone marrow (BM) in the ratio of 90:10 (BM:MM), either MM4 or MRK-16 and C' depleted MM colonies (98.8 +/- 0.71% and 98.10 +/- 1.0%, respectively) without affecting the majority of BM progenitor cells . These observations suggest that either MM4 or MRK-16 is useful for depleting MDR myeloma clonogenic colony-forming cells. J Clin Oncol, 1989 Sep, 7(9), 1359 - 64 Toremifene: pharmacologic and pharmacokinetic basis of reversing multidrug resistance; DeGregorio MW et al.; Triphenylethylene compounds, such as tamoxifen, have shown chemosensitizing activity independent of estrogen receptor status in doxorubicin-resistant cells . We examined the chemosensitizing activity of a new triphenylethylene, toremifene, and its major metabolites in a doxorubicin-resistant human breast cell line, MCF-7/DOX . In addition, we examined the chemosensitizing activity of unbound plasma toremifene and its metabolites isolated from patients treated with toremifene doses of 20 to 400 mg/d . MCF-7/DOX cells were exposed to ultrafiltrate plasma specimens in the absence and presence of doxorubicin . These latter studies were single-blinded . Toremifene and its major metabolites were capable of sensitizing multidrug-resistant cells to doxorubicin . The degree of chemosensitizing activity in vitro correlated with the plasma concentrations of toremifene and its metabolites (P less than .05) . Plasma samples isolated from patients receiving high-dose toremifene (400 mg/d) had the greatest chemosensitizing activity . We present evidence that toremifene and its metabolites can sensitize resistant MCF-7/DOX cells to doxorubicin, that this effect is concentration-dependent, and that sensitizing activity can be detected at clinically achieved concentrations. J Biol Chem, 1989 Aug 25, 264(24), 14376 - 81 Deletion and insertion mutants of the multidrug transporter; Currier SJ et al.; The multidrug transporter is a 170,000-dalton membrane glycoprotein which confers multidrug resistance through its activity as an ATP-dependent efflux pump for hydrophobic, cytotoxic drugs . To determine the essential structural components of this complex membrane transporter we have altered an MDR1 cDNA in an expression vector by deletion and insertion mutations . The structure of the transporter deduced from its amino acid sequence suggests that it consists of two homologous, perhaps functionally autonomous, halves each with six transmembrane segments and a cytoplasmic ATP-binding domain . However, several carboxyl-terminal deletions, one involving 53 amino acids, the second removing 253 amino acids, and an internal deletion within the carboxyl-terminal half of the molecule, totally eliminate the ability of the mutant transporter to confer drug resistance . An internal deletion of the amino-terminal half, which removed residues 140-229, is also nonfunctional . Small carboxylterminal deletions of up to 23 amino acids leave a functional transporter, although the removal of 23 COOH-terminal amino acids reduces its ability to confer colchicine resistance . Insertions of 4 amino acids in a transmembrane domain, and in one of the two ATP-binding regions, have no effect on activity . These studies define some of the limits of allowable deletions and insertions in the MDR1 gene, and demonstrate the requirement for two intact halves of the molecule for a functional multidrug transporter. J Natl Cancer Inst, 1989 Aug 16, 81(16), 1250 - 4 Resistance to drugs associated with the multidrug resistance phenotype following selection with high-concentration methotrexate; Haber M et al.; To study patterns of resistance at extreme but nevertheless clinically relevant drug concentrations, we developed a series of methotrexate-selected CCRF-CEM sublines, all of which were highly resistant to this antifolate (relative resistance, 10(2)- to greater than 10(5)-fold) . The least methotrexate-resistant subline was completely sensitive to drugs associated with the multidrug resistance phenotype . However, more highly methotrexate-resistant sublines were significantly cross-resistant to vincristine, vinblastine, and dactinomycin (maximum relative resistance, 40-fold) . These sublines were not cross-resistant to doxorubicin, daunorubicin, and teniposide . Regression analysis indicated that relative resistance to methotrexate was correlated with relative resistance to vincristine (r = 0.96) and vinblastine (r = 0.99) . Such cross-resistance in highly methotrexate-resistant cells may have important clinical implications. Biochem Biophys Res Commun, 1989 Aug 15, 162(3), 1402 - 8 Photoaffinity labeling of P-glycoprotein in multidrug resistant cells with photoactive analogs of colchicine; Safa AR et al.; Two photoactive radiolabeled analogs of colchicine, N-(p-azido{3,5-{3H}benzoyl)aminohexanoyldeacetylcolchicine ({3H}NABC}) and N-(p-azido-{3-125I}salicyl)aminohexanoyldeacetylcolchicine ({125I}NASC) were synthesized and used to identify colchicine-specific acceptor(s) in membrane vesicles from multidrug resistant (MDR) variant DC-3F/VCRd-5L Chinese hamster lung cells . Both {3H}NABC and {125I}NASC specifically photolabeled a prominent 150-180 kDa polypeptide in membrane vesicles from DC-3F/VCRd-5L cells . The photolabeled polypeptide was immunoprecipitated by monoclonal antibody C219 specific for the MDR-related P-glycoprotein (P-gp) indicating the identity of this protein with P-gp . Colchicine at 1000 microM reduced {3H}NABC photolabeling of P-gp by 72% . Furthermore, 100 microM of colchicine, vincristine, vinblastine, doxorubicin and actinomycin D inhibited {125I}NASC photolabeling by 45, 88.8, 91.1, 61.5, and 51% respectively . However, methotrexate did not affect the {125I}NASC photolabeling of P-gp, indicating the multidrug specificity of the P-gp colchicine acceptor for drugs to which these cells are resistant. Blood, 1989 Aug 15, 74(3), 913 - 7 P-glycoprotein expression in plasma-cell myeloma is associated with resistance to VAD; Epstein J et al.; Tumor cell-associated expression of multidrug resistance (MDR) was quantitated in 22 patients with DNA-aneuploid myeloma using 2-parameter flow cytometry with monoclonal antibody (MoAb) C-219 for the detection of cytoplasmic p-170 and propidium iodide for nuclear DNA content . The proportion of cells expressing p-170 and the intensity of p-170-related fluorescence were determined for each patient . Among the 14 patients treated with vincristine-adriamycin-dexamethasone (VAD), the proportion of p-170-positive cells distinguished sensitive from resistant disease (P less than .01) . Among a subgroup of seven patients with MDR analysis available prior to VAD therapy, two subsequent nonresponders had high proportions of C-219-reactive cells . The presence de novo of high proportions of p-170-expressing cells in another still untreated patient and in a further individual with resistance to dexamethasone and interferon (not associated with MDR) warrants systematic analysis of p-170 expression prior to therapy to determine its clinical implications for response to MDR-associated drugs as combined in the VAD regimen . Concurrent MDR expression by aneuploid tumor cells and cells in the diploid subcompartment may represent involvement of diploid cells in the myeloma disease process. Cancer Res, 1989 Aug 15, 49(16), 4542 - 9 Multidrug resistance in mitoxantrone-selected HL-60 leukemia cells in the absence of P-glycoprotein overexpression; Harker WG et al.; A multidrug-resistant variant of the human HL-60 promyelocytic leukemia cell line (HL-60/MX2) has been isolated in vitro by subculturing these cells in progressively increasing concentrations of mitoxantrone . The MX2 cells are cross-resistant to etoposide, teniposide, bisantrene, dactinomycin, 4'-(9-acridinylamino)methanesulfon-m-anisidide, and the anthracyclines daunorubicin and doxorubicin but retain sensitivity to the Vinca alkaloids melphalan and mitomycin C . In addition, the MX2 cells display slight collateral sensitivity to bleomycin . Despite being 30-35-fold less sensitive to mitoxantrone, net {14C}mitoxantrone accumulation at 60 min was reduced by only 10% in the mitoxantrone-resistant cells compared to the parental line . Furthermore, at later time points, e.g., 120 and 180 min, mitoxantrone accumulation in the MX2 cells exceeded that in HL-60 cells by 8.5 and 6.4%, respectively . No significant differences were observed between the sensitive and resistant cell lines in the initial (first 60 s) accumulation of mitoxantrone, and only minor (3-6%) enhancement of mitoxantrone efflux was detected in the resistant cell type . Monoclonal antibodies to P-glycoprotein had no detectable reactivity with membrane vesicles from either the sensitive or resistant cell types as determined by standard immunoblotting techniques . The mitoxantrone-resistant cells displayed a reciprocal translocation {rcpt(1;3)-(q21;p23)} not found in the sensitive parent, but there were no demonstrable double minute chromosomes or homogeneous staining regions in cells from either line . Thus, these mitoxantrone-resistant human leukemia cells display many features which are atypical for the "classic" multidrug resistance phenotype and should provide a useful model for the study of multidrug resistance which is not mediated by P-glycoprotein. Cancer Res, 1989 Aug 15, 49(16), 4499 - 503 Characterization of a K562 multidrug-resistant cell line; Yanovich S et al.; A daunorubicin-resistant variant of the K562 human leukemia cell line (K562-R), which demonstrates cross-resistance to other anthracycline antibiotics and Vinca alkaloids, has been developed in vitro by continuous exposure to daunorubicin . Cross-resistance to anthracyclines and Vinca alkaloids is reversed when cells are exposed to drugs in the presence of verapamil, a calcium channel blocker . The K562-R cell line overexpresses a 4.5-kilobase mRNA, which is thought to code for the Mr 170,000 membrane glycoprotein associated with multidrug resistance . Transport studies indicate reduced intracellular accumulation and retention of daunorubicin in the K562-R cells as compared to the parent cell line . These studies further suggest the presence of distinct cellular pools composed of both rapidly and slowly exchanging drug, with the rapidly exchanging pool being more pronounced in the resistant line . The development of multidrug resistance in the K562-R cell line is also associated with the overexpression of five different cell surface membrane proteins ranging in molecular weight between 50,000 and 210,000, whose function remains to be defined. Nature, 1989 Aug 3, 340(6232), 400 - 4 The yeast STE6 gene encodes a homologue of the mammalian multidrug resistance P-glycoprotein; McGrath JP et al.; Mammalian tumours displaying multidrug resistance overexpress a plasma membrane protein (P-glycoprotein), which is encoded by the MDR1 gene and apparently functions as an energy-dependent drug efflux pump . Tissue-specific expression of MDR1 and other members of the MDR gene family has been observed in normal cells, suggesting a role for P-glycoproteins in secretion . We have isolated a gene from the yeast Saccharomyces cerevisiae that encodes a protein very similar to mammalian P-glycoproteins . Deletion of this gene resulted in sterility of MATa, but not of MAT alpha cells . Subsequent analysis revealed that the yeast P-glycoprotein is the product of the STE6 gene, a locus previously shown to be required in MATa cells for production of a-factor pheromone . Our findings suggest that the STE6 protein functions to export the hydrophobic a-factor lipopeptide in a manner analogous to the efflux of hydrophobic cytotoxic drugs catalysed by the related mammalian P-glycoprotein . Thus, the evolutionarily conserved family of MDR-like genes, including the hlyB gene of Escherichia coli and the STE6 gene of S . cerevisiae, encodes components of secretory pathways distinct from the classical, signal sequence-dependent protein translocation system. J Natl Cancer Inst, 1989 Aug 2, 81(15), 1144 - 50 MDR1 gene expression in lung cancer; Lai SL et al.; The MDR1 gene (also known as PGY1) is frequently overexpressed in multidrug-resistant cell lines . We investigated the role of MDR1 gene expression in lung cancer by performing RNA slot blot analysis in samples from a panel of 24 lung cancers, 10 corresponding nontumorous lung tissues, and 67 tumor cell lines of several histologic types . Almost all of the tumors, nontumorous lung tissues, and cell lines expressed low levels of MDR1 RNA . Relatively higher levels were found in only one type of lung cancer, a subgroup of non-small cell lung cancers expressing neuroendocrine markers . No evidence of MDR1 gene amplification or rearrangements was detected . We found no correlation between MDR1 gene expression in cell lines and (a) in vitro chemosensitivity of the cells, (b) prior therapy status of the patients, or (c) clinical response to therapy . We conclude that the clinical multidrug resistance of many lung cancers cannot be explained solely on the basis of expression of the MDR1 gene. Arzneimittelforschung, 1989 Aug, 39(8), 828 - 31 Acquired drug resistance in human lung carcinoma xenografts; Volm M et al.; The development of resistance to vincristine and dactinomycin has been investigated in a human epidermoid lung xenograft line grown in nude mice . With 1 mg/kg BW vincristine and 0.5 mg/kg BW dactinomycin per passage, resistance of the solid tumors was visible already at the second transplantation . The vincristine-resistant subline showed cross-resistance to dactinomycin and doxorubicin, and the dactinomycin-resistant subline only to vincristine . To determine whether multidrug resistance genes were overexpressed in the resistant sublines, slot blots were performed using the cDNA probe pcDR 1.5 . Slightly elevated RNA levels could be detected in the vincristine-resistant subline and in the dactinomycin-resistant subline. Pharm Res, 1989 Aug, 6(8), 690 - 6 Saturable process involved in active efflux of vincristine as a mechanism of multidrug resistance in P388 leukemia cells; Watanabe T et al.; Kinetic analysis of vincristine (VCR) efflux in multidrug-resistant and parental P388 leukemia cells was performed to investigate the difference in activity between the two cell lines . Efflux velocities of VCR were directly determined from the slope of the initial release of drug induced by resuspending the preloaded cells in VCR-free medium, representing unidirectional efflux from intracellular free or loosely bound drug pools . Further, the equilibrium binding of VCR to whole-cell homogenates was analyzed by ultrafiltration to estimate intracellular unbound drug concentrations . A two-site binding model was found to fit the data best for both cell lines, and depletion of ATP by the addition of apyrase decreased binding . The binding parameters were similar between the two cell lines . A Hofstee plot of efflux demonstrated the existence of both linear and saturable transport of VCR in both cell lines . The greater maximum velocity observed with VCR efflux in the resistant cells suggests that an increased number of transporters causes greater activity of this process in the resistant cells. Anticancer Drug Des, 1989 Aug, 4(2), 125 - 35 Potentiation of vincristine and actinomycin D by a new synthetic imidazole anti-tumor agent YM534 active against human cancer cells and multidrug-resistant cells; Sato S et al.; Ethyl 6-p-5-(l-imidazolyl) pentyloxyphenoxy-2, 2-dimethylhexanoate hydrochloride (YM534) is a new synthetic anti-tumor compound . Combinations of YM534 with other anti-cancer agents were examined to ascertain whether YM534 potentiated other anti-cancer agents against the KB cell line and its multidrug-resistant counterpart, VJ-300 . YM534 potentiated the cytotoxic action of vincristine and actinomycin D about 2-fold against KB cells, but not those of daunomycin and adriamycin . By contrast, YM534 only slightly reversed drug-resistance to adriamycin and daunomycin in VJ-300 while it reversed 5-fold vincristine resistance and 60-fold actinomycin D resistance in VJ-300 . The reversal effect of YM534 on actinomycin D and vincristine-resistance in VJ-300 cells appeared to be due to enhanced accumulation of {3H} actinomycin D and {3H} vincristine in VJ-300 cells by YM534 . YM534 inhibited efflux of actinomycin D and vincristine from VJ-300 cells, and it also enhanced cellular uptake of these anti-cancer agents . YM534 enhanced cellular accumulation of both actinomycin D and vincristine in the sensitive KB cells . YM534 is thus a unique anti-cancer agent since combinations of other anti-cancer agents with YM534 are expected to augment anti-tumor activity of them . By contrast, YM212, a carboxy analog of YM534, had much less activity to potentiate vincristine and actinomycin D) . YM534 at 100-1000 microM almost completely inhibited the photoaffinity labeling of {3H} azidopine to the 170-kD P-glycoprotein of VJ-300 cell membranes, but YM212 showed much less inhibitory action on the photoaffinity labeling . YM534 could also inhibit the photoaffinity labeling of deglycosylated P-glycoprotein. Semin Oncol, 1989 Aug, 16(4 Suppl 6), 58 - 65 Chemotherapy in ovarian carcinoma: present role and future prospects; Thigpen JT et al.; Epithelial carcinoma of the ovary is characterized by presentation at an advanced stage, spread primarily by an intraperitoneal (IP) route, and relative sensitivity to chemotherapy . An initial surgical approach is essential to proper staging of the disease process and to aggressive cytoreduction, which in turn improves response to chemotherapy and survival . The use of chemotherapy is the mainstay of definitive therapy after completion of the initial surgery . A large number of drugs have activity against the disease, with the most important single category of agents being the platinum compounds . Studies by the Gynecologic Oncology Group (GOG) document the superiority of cisplatin-based combination chemotherapy over single alkylating agents and combinations that do not include cisplatin . The current regimen of choice is a two-drug combination of cisplatin plus cyclophosphamide . Efforts to improve results further focus on enhancing dose intensity of the drug combination through either escalating intravenous (IV) doses or administering cisplatin via an IP route . Also offering an opportunity for further improvement of therapeutic results are three drugs identified as having activity in patients no longer candidates for cisplatin: carboplatin, ifosfamide, and taxol . Biologic agents, such as alpha-interferon, also have potential roles in future combination therapy . The management of patients with limited (stage I or II) disease is based on studies of the GOG and the Ovarian Cancer Study Group, which indicate that this population can be divided by prognostic factors into a group at low risk for recurrence and a group at high risk . Those at low risk require only surgery, whereas those at high risk should receive either IP radioactive chromic phosphate or systemic chemotherapy following surgery . The future prospects for additional improvement of results in all patients appear bright on the basis not only of studies of dose intensity and IP therapy but also of efforts directed at overcoming multidrug resistance and at devising noninvasive means of assessing disease status. Rinsho Ketsueki, 1989 Aug, 30(8), 1128 - 32 {Drug resistance and implication for therapy}; Tsuruo T; One of the major problems in cancer chemotherapy is the development of drug resistance during treatment . The nature of drug resistance in cancer patients is complex . Recently, it has been found that tumor cells can acquire resistance to anticancer drugs . It is now generally accepted that drug resistance at the cellular level (cellular resistance) is also an important mechanism of drug resistance in patients . The elucidation of the resistance mechanisms has progressed well recently owing to the application of cellular and molecular techniques in addition to the usual biological and biochemical techniques . In this article, I describe the mechanisms of cellular resistance, especially those of multidrug resistance at the molecular level, and I also discuss possible approaches to overcoming drug resistance. Rinsho Byori, 1989 Aug, 37(8), 899 - 904 {Analysis of P-glycoprotein in patients with acute leukemias by flow cytometry}; Funato T et al.; The identification of a P-glycoprotein product of multidrug-resistant gene (mdr 1) was reported recently . To examine the expression of the P-glycoprotein in acute leukemias of various types, we have prepared leukemic blast cells from patients and measured their positivity of P-glycoprotein using monoclonal anti-P-glycoprotein antibody (C219) by flow cytometry . P-glycoprotein is expressed in 8 out of 44 cases including leukemic blast cells but not lymphocytes and monocytes . In these cases showed drug resistance was shown clinically . In addition, the expression of the P-glycoprotein was not observed in the drug-sensitive cases and at the time of initial chemotherapy . Our results suggest that measurement of P-glycoprotein in acute leukemias by flow cytometry may prove to be a valuable tool for the design of chemotherapy protocols. J Protein Chem, 1989 Aug, 8(4), 563 - 73 Conformational effects of amino acid substitutions in the P-glycoprotein of the mdr 1 gene; Brandt-Rauf PW et al.; The P-glycoprotein of the mdr 1 gene is responsible for the phenomenon of multidrug resistance in human cells . The presumed drug-binding site of the wild-type P-glycoprotein contains a glycine at position 185 . A mutant P-glycoprotein which contains valine at this position causes cells to retain resistance to colchicine, but to lose cross-resistance to other drugs such as the chemotherapeutic agents vinblastine and Adriamycin . This has been hypothesized to be due to a conformational change in the protein induced by the amino acid substitution . Using conformational energy analysis, we have determined the allowed three-dimensional structures for the wild-type and mutant proteins in the region of position 185 . The results indicate that the wild-type protein adopts a unique left-handed conformation at position 185 which is energically unfavorable for the protein with L-amino acids (including valine) at this position . This conformational change induced by amino acid substitutions for Gly 185 could explain the differences in binding to the P-glycoprotein of various drugs and, hence, the differences in drug resistance exhibited by various cell lines expressing these proteins. Genomics, 1989 Aug, 5(2), 371 - 4 Application of natural partial digests to pulsed-field gel analysis of the amplified MDR locus; Meese E et al.; The analysis by pulsed-field gel electrophoresis of partial digestion products visualized by probes for the human multidrug resistance (MDR) locus has been used to further establish the restriction map of this region . Results place the MDR1 and MDR2 genes on a single SfiI fragment, with partial digestion products establishing the distance between these genes to be 230-250 kb . The feasibility and potential advantages of using "natural" partials to generate detailed restriction maps within an amplified DNA domain are discussed. Cancer Res, 1989 Aug 1, 49(15), 4175 - 8 Resistance mechanisms in three human small cell lung cancer cell lines established from one patient during clinical follow-up; de Vries EG et al.; Mechanisms for resistance were studied in three classic type, human small cell lung cancer cell lines, GLC14, GLC16, and GLC19, that were established from one patient during clinical follow-up . Clinically the tumor changed from sensitive (GLC14) to completely resistant to (chemo)therapy (GLC19) during this period . The stain with JSB-1 antibody, detecting the Mr 170,000 multidrug resistance associated glycoprotein, was most pronounced in GLC16 and absent in GLC19 . Intracellular Adriamycin (Adr) concentrations were decreased in GLC16 and GLC19 versus GLC14 . Glutathione levels were 12.9, 15.5, and 16.6 micrograms/mg protein; total sulfhydryl groups were 36.5, 45.7, and 48.8 micrograms/mg protein; and glutathione S-transferase activity was 13, 29, and 43 nmol I-chloro-2,4-dinitrobenzene/min/mg protein for GLC14, GLC16, and GLC19, respectively . Incubation with DL-buthionine-S,R-sulfoximine increased Adr and cisplatin induced cytotoxicity, whereas X-ray induced cytotoxicity remained the same . Catalase activity increased from 0.88 to 1.73 to 3.83 mumol H2O2/min/mg protein in, respectively, GLC14, GLC16, and GLC19 . Compared to GLC14 and GLC16, Adr induced a higher amount of DNA strand breaks in GLC19 . In none of the three cell lines could Adr induced DNA strand breaks be repaired . X-ray induced a comparable amount of DNA strand breaks in all three cell lines but all cell lines were capable of repairing the X-ray induced DNA strand breaks within 90 min . It is concluded that a number of different mechanisms are operative and that some but not all of the observed changes in mechanisms for drug resistance in these lines correlate with the clinical data. Cancer Res, 1989 Aug 1, 49(15), 4098 - 102 In vitro evaluation of the new anticancer agents KT6149, MX-2, SM5887, menogaril, and liblomycin using cisplatin- or adriamycin-resistant human cancer cell lines; Ohe Y et al.; A new model to predict antitumor activity of new analogues was developed, and the cross-resistance against cisplatin (CDDP) and Adriamycin (ADM) was examined . A preclinical evaluation of various new analogues using this new model was performed . The antitumor activities of KT6149, MX-2 (KRN8602), SM5887, menogaril (TUT-7), and liblomycin (NK313) were evaluated against four non-small cell lung cancer cell lines, PC-7, -9, -13, and -14; two small cell lung cancer cell lines, H69 and N231; four CDDP-resistant cell lines, PC-7/1.0, PC-9/0.5, PC-14/1.5, and H69/0.4; a human myelogenous leukemia cell line, K562; and its ADM-resistant subline, K562/ADM by clonogenic assay . The relative antitumor activities of these new analogues were compared with those of parental agents, mitomycin C, ADM, bleomycin, and several anticancer drugs, CDDP, daunomycin, vindesine, and etoposide . KT6149 was more active than mitomycin C against all lung cancer cell lines and the human myelogenous leukemia cell line . Menogaril showed greater activity than ADM, and MX-2 showed activity similar to ADM . However, the antitumor activity of SM5887 was lower than that of ADM . SM5887 and menogaril showed cross-resistance to K562/ADM . Nevertheless, the antitumor activity against K562/ADM of MX-2 was similar to that of the parental cell lines . The activity of liblomycin was similar to that of bleomycin . Thus, KT6149 appears to be the best analogue for use in a clinical trial against lung cancer . MX-2 was active even against ADM-resistant cancer cells . The values of relative resistance to CDDP or ADM were 4.7, 8.1, 7.5, 20.0, and 13.6 for PC-7/1.0, PC-9/0.5, PC-14/1.5, H69/0.4, and K562/ADM, respectively . CDDP-resistant cell lines showed no cross-resistance with other drugs in this study . K562/ADM showed cross-resistance against daunomycin, etoposide, and vindesine . In contrast, mitomycin C and bleomycin had nearly equal activity against K562 and K562/ADM . However, K562/ADM was 2.4-fold more sensitive to CDDP than its parental cell line, K562 (P less than 0.001) . These results suggested that the mechanism of CDDP resistance is different from that of multidrug resistance. Cancer Res, 1989 Jul 15, 49(14), 3867 - 71 Enhanced efflux of {3H}vinblastine from Chinese hamster ovary cells transfected with a full-length complementary DNA clone for the mdr1 gene; Hammond JR et al.; Multidrug-resistant Chinese hamster ovary cell clones stably transfected with, and overexpressing, the mouse mdr1 complementary DNA clone along with drug-sensitive Chinese hamster ovary control cells were characterized for their capacities to accumulate and retain {3H}vinblastine . Multidrug-resistant mdr1 transfectants show a 3-4-fold decrease in {3H}vinblastine accumulation, compared to their drug-sensitive counterparts . After ATP depletion, this difference in {3H}vinblastine accumulation between mdr1 transfectants and control cells effectively disappears . This ATP-dependent decreased drug accumulation is paralleled in mdr1 transfectants by an enhanced capacity of these cells to extrude the drug in an ATP-dependent manner . In medium containing glucose and glutamine, the mdr1 transfectants release preloaded drug at a rate five times that of control, drug-sensitive cells . In ATP-depleted control and mdr1-transfected cells, there is little difference in the rate or extent of {3H}vinblastine release . The observation that the mdr1 transfectants show a decreased {3H}vinblastine accumulation and an increased vinblastine release, both of which are abolished when cellular ATP levels are reduced, provides a direct demonstration that the product of the transfected mdr1 gene is responsible for a mechanism controlling cellular drug levels in an ATP-dependent manner . However, attempts to establish competition for {3H}vinblastine transport by vincristine, daunomycin, and actinomycin D were only partly successful in mdr1 transfectants. Eur J Biochem, 1989 Jul 15, 183(1), 189 - 97 Equilibrium, kinetic and photoaffinity labeling studies of daunomycin binding to P-glycoprotein-containing membranes of multidrug-resistant Chinese hamster ovary cells; Busche R et al.; The binding of daunomycin and its Bolton-Hunter derivative iodomycin to plasma membranes isolated from multidrug-resistant Chinese hamster ovary cells (CHO B30) and their drug-sensitive parents (B1) was investigated . The thermodynamics and kinetics of equilibrium binding monitored by fluorescence titrations and temperature-jump relaxation spectrometry were compared with the specificity of covalent photolabeling with {3H}daunomycin and {125I}iodomycin . The facts that the uptake of anthracycline from aqueous solution into the CHO membranes was not accompanied by any substantial increase of fluorescence anisotropy nor by any spectral shift of the fluorescence emission spectrum and that the partition ratio into the membrane was 20-30-fold higher when compared to a lecithin bilayer, provided evidence that the non-covalent drug binding sites are constituted by polar protein domains without any substantial contribution from the surrounding lipids . Photoaffinity labeling with nanomolar concentrations of anthracycline and equilibrium binding curves independently showed that a 150-170-kDa plasma membrane glycoprotein (P-glycoprotein), whose overexpression is the major difference between B1 and B30 membranes, provides the binding sites of highest affinity for daunomycin and iodomycin (K approximately equal to 4 x 10(7) M-1) . Comparison of photolabeling and equilibrium data suggested that the same binding sites on P-glycoprotein were most probably being monitored . The photolabeling of P-glycoprotein by iodomycin was inhibited in a dose-dependent manner by other compounds to which multi-drug-resistant cells are either resistant or collaterally sensitive with the following orders of effectiveness: vinblastine greater than verapamil greater than nitrendipine greater than daunomycin much greater than colchicine . Temperature-jump experiments covering the time range of 1 microseconds to 1 s revealed a single concentration-dependent relaxation time of 10-30 microseconds . The association of daunomycin with its binding sites in the membranes was found to be a diffusion-controlled process with kon rates of 2-4 X 10(9) M-1 s-1 . Therefore, the selectivity of drug binding was entirely reflected in the dissociation rates. J Biol Chem, 1989 Jul 15, 264(20), 11693 - 8 The function of Gp170, the multidrug resistance gene product, in rat liver canalicular membrane vesicles; Kamimoto Y et al.; Gp170 (also known as P-glycoprotein) is a transmembrane glycoprotein which is overexpressed in multidrug-resistant tumor cells and is also found in the apical plasma membrane domain of several normal human and animal tissues . Gp170 has been postulated to function as an energy-dependent efflux pump for cytotoxic drugs . In rat liver, Gp170 is restricted to the bile canalicular domain of the plasma membrane . Canalicular membrane vesicles (CMV), but not sinusoidal membrane vesicles, contained a approximately 160-kDa protein which reacts with anti-Gp170 monoclonal antibody and manifest ATP-dependent {3H}daunomycin transport which is temperature dependent, osmotically sensitive, and saturable . Among several nucleotides, ATP was a potent stimulator of transport whereas non- or slowly hydrolyzable analogues (adenosin-5-O-(3-thiotriphosphate, adenyl-5-yl-imidodiphosphate) were ineffective . ATP-dependent daunomycin transport was inhibited by cytotoxic drugs (vinblastine, vincristine, and adriamycin) and other drugs, such as verapamil and quinidine, which restore anti-cancer drug sensitivity in resistant cells . Inside-out CMV were separated from right side-out CMV by antibody-induced affinity density perturbation . Only inside-out CMV manifested ATP-dependent daunomycin transport . These results suggest that Gp170 is an ATP-dependent efflux pump which is responsible for the undirectional, energy-dependent transport of daunomycin and other drugs by rat liver into the bile. Int J Cancer, 1989 Jul 15, 44(1), 149 - 54 Reversal of drug resistance by erythromycin: erythromycin increases the accumulation of actinomycin D and doxorubicin in multidrug-resistant cells; Hofsli E et al.; Development of resistance to one type of lipophilic chemotherapeutic drug often leads to resistance to other, structurally unrelated, lipophilic drugs . This suggests that non-toxic lipophilic agents may interfere with and reverse drug resistance by saturating the pathway through which multidrug-resistant (MDR) cells protect themselves against cytotoxic drugs . The lipophilic antibiotic, erythromycin, can significantly reverse the resistance of MDR WEHI 164 murine fibrosarcoma cells to the chemotherapeutic drugs, doxorubicin and actinomycin-D . The MDR cells showed an approximately 10-fold higher expression of the P-glycoprotein than the drug-sensitive parental cells from which the resistant cells were derived . The accumulation of actinomycin-D and doxorubicin was much lower in the drug-resistant cells than in the sensitive parental cells . The concentrations of erythromycin which reversed the drug resistance of the MDR cells increased the accumulation of actinomycin-D and doxorubicin in these cells to a level comparable to that observed in the sensitive parental cells . Our data suggest that erythromycin reverses drug resistance by saturating the drug-binding sites on the P-glycoprotein, thereby reducing the capacity of this protein to pump drugs out of resistant cells . Some of our MDR cells have also become more resistant to tumour necrosis factor (TNF) . However, erythromycin did not reverse TNF resistance, suggesting that the mechanisms of multi-drug and TNF resistance are different . TNF did not influence drug accumulation in MDR cells. J Biol Chem, 1989 Jul 15, 264(20), 12053 - 62 Differential overexpression of three mdr gene family members in multidrug-resistant J774.2 mouse cells . Evidence that distinct P-glycoprotein precursors are encoded by unique mdr genes; Hsu SI et al.; A hallmark of the multidrug-resistant phenotype is the overproduction of a family of 130-180-kDa integral membrane phosphoglycoproteins collectively called P-glycoprotein . Gene-specific hybridization probes were derived from three classes of mouse P-glycoprotein cDNAs . These probes revealed the differential amplification and/or transcriptional activation of three distinct but closely related mdr genes (mdr1a, mdr1b, and mdr2) in independently selected multidrug-resistant J774.2 mouse cell lines . Overexpression of mdr1a and mdr1b was found to correlate, in general, with the differential overproduction of either a 120- or 125-kDa P-glycoprotein precursor, respectively . This same correlation was observed in a single cell line during the course of stepwise selection for resistance to vinblastine in which a switch in gene expression from mdr1b to mdr1a resulted in a switch from the 125- to 120-kDa P-glycoprotein precursor . These findings suggest that differential overexpression of distinct mdr genes which encode unique P-glycoprotein isoforms is a possible mechanism for generating diversity in the multidrug-resistant phenotype. Biochem Biophys Res Commun, 1989 Jul 14, 162(1), 244 - 52 Identification of P-glycoprotein in renal brush border membranes; Lieberman DM et al.; A monoclonal antibody (C219) that recognizes the P-glycoprotein (Mr = 170,000) in plasma membranes of multidrug-resistant Chinese hamster ovary (CHO) cell lines was used to assay renal brush border membrane (BBM) and basolateral membrane (BLM) fractions for the presence of a cross-reactive polypeptide . The C219 antibody bound to a 155,000 dalton protein in immunoblots of rat BBM but not BLM proteins resolved by sodium dodecyl sulfate gel electrophoresis . The corresponding human kidney BBM and dog kidney BBM proteins had molecular weights of 170,000 and 160,000 respectively . The glycoprotein nature of the renal protein was shown by its sensitivity to N-glycanase treatment which reduced the apparent molecular weight of the dog protein to 120,000 . In addition, dog P-glycoprotein could be bound to and eluted from immobilized wheat germ agglutinin . The molecular weight, antibody crossreactivity, glycosidase sensitivity and lectin binding show that this protein is a normal kidney analogue of the P-glycoprotein induced in multidrug resistant cell lines. Biochem Biophys Res Commun, 1989 Jul 14, 162(1), 224 - 31 P-glycoprotein gene (MDR1) cDNA from human adrenal: normal P-glycoprotein carries Gly185 with an altered pattern of multidrug resistance; Kioka N et al.; We isolated a full-length MDR1 cDNA from human adrenal where P-glycoprotein is expressed at high level . The deduced amino acid sequence shows two amino acid differences from the sequence of P-glycoprotein obtained from colchicine-selected multidrug resistant cultured cells . The amino acid substitution Gly----Val at codon 185 in P-glycoprotein from colchicine resistant cells occurred during selection of cells in colchicine . As previously reported, cells transfected with the MDR1 cDNA carrying Val185 acquire increased resistance to colchicine compared to other drugs . The other amino acid substitution Ser----Ala at codon 893 probably reflects genetic polymorphism . The MDR1 gene, the major member of the P-glycoprotein gene family expressed in human adrenal, is sufficient to confer multidrug-resistance on culture cells. Cytometry, 1989 Jul, 10(4), 463 - 8 A rapid and sensitive flow cytometric method for the detection of multidrug-resistant cells; Herweijer H et al.; Multidrug-resistant (MDR) cells are characterized by a defect in drug accumulation caused by activity of an energy-dependent rapid drug efflux pump . The action of this drug pump can be inhibited by specific agents, referred to as membrane transport modulating agents (MTMAs), resulting in a restoration of the intracellular drug accumulation . This paper presents a flow cytometric assay for the detection of MDR cells, which is based on the ability of these cells to respond to MTMAs . Daunorubicin net-uptake kinetics were measured of anthracycline-sensitive (A2780/S) and -resistant (A2780/R) human ovarian carcinoma cells in vitro . A2780/R cells accumulated significantly less (about a factor of 5) daunorubicin as compared to A2780/S cells . Addition of verapamil or cyclosporin A to A2780/R cells at steady-state daunorubicin uptake led to a dose-dependent increase in cellular daunorubicin accumulation . The sensitivity of the assay was determined by testing mixtures of A2780/S and A2780/R cells . Analysis of A2780/S cells contaminated with A2780/R cells showed that as few as 2.5% MDR cells could readily be detected in the mixture . In conclusion, this functional assay enables the detection of MDR cells in a heterogeneous cell suspension and is ideally suited for the study of the occurrence of typical MDR in human cancer. Rinsho Byori, 1989 Jul, 37(7), 779 - 83 {Expression of P-glycoprotein (multidrug-resistance gene product) in haematological tumors}; Funato T et al.; The fact that cancer cell acquires multidrug resistance to carcinostatics at cancer treatment is a very important subject clinically . The mode of multidrug-resistance is complicated, but the gene associated with multidrug resistance (MDR 1) has been isolated . It has become evident that MDR 1 gene carries membrane glycoprotein (P-glycoprotein) which occurs in the cell acquired drug-resistance . Assessment has been made this time regarding the occurrence of P-glycoprotein in the tumorous cells and tissues by the use of monoclonal antibody (C 219) to P-glycoprotein . Occurrence of P-glycoprotein in malignant lymphoma exhibited positivity in 9 cases out of 36 immunohistologically . 170 KD P-glycoprotein was detected in 4 cases out of 10 at Western blotting analysis of the protein isolated from the nuclear cell in the peripheral blood in the patients with leukemia . Further, P-glycoprotein positive cases were all progressive cases clinically and showed resistance to treatment . From these results, it has been clarified that occurrence of P-glycoprotein in haematological tumors is related to multidrug resistance. Jpn J Cancer Res, 1989 Jul, 80(7), 627 - 31 Inhibition of multidrug-resistant human tumor growth in athymic mice by anti-P-glycoprotein monoclonal antibodies; Tsuruo T et al.; In an effort to devise an effective treatment for human drug-resistant cancers, we have developed monoclonal antibodies, MRK16 and 17, reactive to the multidrug transporter protein, P-glycoprotein . The monoclonal antibodies given intravenously effectively prevented tumor development in athymic mice inoculated subcutaneously with drug-resistant human ovarian cancer cells 2780AD . Treatment with MRK16 induced rapid regression of established subcutaneous tumors and apparent cures of some animals . Complement-dependent cytotoxicity (MRK16) and antibody-dependent cell-mediated cytolysis (MRK16 and 17) were observed with these antibodies . These monoclonal antibodies may have potential as treatment tools against multidrug resistant human tumors possessing the P-glycoprotein. J Clin Pathol, 1989 Jul, 42(7), 719 - 22 Comparison of western blot analysis and immunocytochemical detection of P-glycoprotein in multidrug resistant cells; Friedlander ML et al.; A sensitive immunocytochemical technique was developed to detect a 170,000 dalton cell membrane glycoprotein (P-gp) in cell lines resistant to vincristine and vinblastine with varying degrees of resistance . P-gp was shown very clearly using the C219 monoclonal antibody and immunocytochemical detection with either antialkaline phosphate or peroxidase-antiperoxidase with silver gold intensification . There was good correlation between the results obtained with immunocytochemical detection of P-gp in single cells and Western blot analysis . The technique is easily performed and can detect P-gp in relatively small numbers of cells that Western blot analysis could miss and is suitable for clinical application. Proc Natl Acad Sci U S A, 1989 Jul, 86(13), 5128 - 32 Essential features of the P-glycoprotein pharmacophore as defined by a series of reserpine analogs that modulate multidrug resistance; Pearce HL et al.; We have shown previously that reserpine is an effective "modulator" of P-glycoprotein-associated multidrug resistance (MDR) . In addition to enhancing drug cytotoxicity in our multidrug-resistant human leukemia cell line, CEM/VLB100, reserpine strongly competes with a photoactivatible analog of vinblastine, N-(p-azido-3-{125I}iodosalicyl)-N'-(beta-aminoethyl)vindesine, for binding to P-glycoprotein . We also demonstrated previously that there are three substructural domains present in many compounds that modulate P-glycoprotein-associated MDR: a basic nitrogen atom and two planar aromatic rings . In the present study, we wished to test more rigorously the hypothesis that not only are these domains necessary for modulators of MDR but also they must exist in an appropriate conformation . Reserpine is a modulator of MDR in which these domains are present in a well-defined conformation . Accordingly, we prepared eight compounds that vary the spatial orientation of these domains, using either naturally occurring reserpine or yohimbine as chemical templates . When tested for their ability to enhance the cytotoxic activity of natural product antitumor drugs in CEM/VLB100 cells, five compounds that retained the pendant benzoyl function in an appropriate spatial orientation all modulated MDR . By contrast, compounds lacking this moiety failed to do so . These active modulators competed strongly with the 125I-labeled vinblastine analog for binding to P-glycoprotein in plasma membrane vesicles prepared from these cells . Conformational analysis using molecular mechanics revealed the structural similarities of the active modulators . Our results support the hypothesis that the relative disposition of aromatic rings and basic nitrogen atom is important for modulators of P-glycoprotein-associated MDR, and they suggest a ligand-receptor relationship for these agents . These results also provide direction for the definition of an MDR "pharmacophore." J Histochem Cytochem, 1989 Jul, 37(7), 1141 - 5 The multidrug transporter: rapid modulation of efflux activity monitored in single cells by the morphologic effects of vinblastine and daunomycin; Konen PL et al.; Double-label fluorescence microscopy was used to demonstrate the efflux activity of the multidrug transporter in single cultured cells . NIH3T3 cells expressing a transfected MDR1 gene (NIH3T3-MDR) were treated with vinblastine or daunomycin . The accumulation of vinblastine was monitored by examining the morphology of tubulin in cells, using immunofluorescence . Overnight treatment of drug-sensitive cells caused disassembly of microtubules and formation of paracrystals; the absence of vinblastine effects was evident by the presence of intact microtubules . Daunomycin accumulation was detected in nuclei using the inherent fluorescence of the drug with rhodamine epifluorescence microscopy . Drug efflux in multidrug-resistant cells was inhibited with verapamil . When multidrug-resistant cells were treated overnight in vinblastine, an effect of 0.5 microM vinblastine on microtubules was seen only in the presence of verapamil . Similarly, when cells were treated with daunomycin, this drug accumulated in nuclei only when verapamil was present . When cells incubated with vinblastine and verapamil were washed free of drugs, they did not accumulate daunomycin in a subsequent incubation, indicating that the multidrug transporter was still active; this occurred even though the morphologic effects of vinblastine persisted . Cells incubated with vinblastine alone showed an immediate inhibition of efflux activity when verapamil was subsequently added with daunomycin . These results show that the efflux activity of the multidrug transporter can be rapidly manipulated by agents such as verapamil, despite a prior history of drug treatment, and that the effects of inhibition of the transporter are rapidly reversible. FEMS Microbiol Lett, 1989 Jul 1, 51(1), 31 - 6 Serotypes and antibiotic resistance of verotoxigenic (VTEC) and necrotizing (NTEC) Escherichia coli strains isolated from calves with diarrhoea; Gonzalez EA et al.; Serotypes and antibiotic resistance of 51 Verotoxigenic (VTEC) and 33 Necrotizing (NTEC) bovine Escherichia coli strains were determined and compared with those shown by 205 non-VTEC non-NTEC strains isolated from the same batch of calves . E . coli untypable for O-antigen represented 47% of the VTEC, 12% of the NTEC and 8.8% of the non-VTEC non-NTEC . Typable VTEC belonged to serotypes 02:K?, 0103:K-, 0104:K?, 0128:K?, 0153:K- and O157:K-:H7, whereas typable NTEC were of serotypes 08:K87, 015:K14, 015:K-, 054:K?, 076:K-, 078:K(80), 088:K?, 0123:K-, 0139:K- and 0153:K- . Non-VTEC non-NTEC showed a wide variety of serotypes which were generally unrelated to those found in VTEC and NTEC . VTEC were resistant to antibiotics at higher rates than NTEC and non-VTEC non-NTEC, and showed also the highest multidrug-resistant pattern . Our results show that bovine VTEC strains belonged to O-groups usually found in human VTEC causing sporadic diarrhoea, haemorrhagic colitis and/or haemolytic uraemic syndrome, such as 02, 0103, 0104, 0153 and especially 0128 and O157 . In contrast, bovine NTEC strains belonged to serotypes different from those previously found in necrotizing E . coli strains of human origin. Cell, 1989 Jun 16, 57(6), 921 - 30 Amplification of the multidrug resistance gene in some chloroquine-resistant isolates of P . falciparum; Foote SJ et al.; Resistance of Plasmodium falciparum to chloroquine shares features with the multidrug resistance (MDR) phenotype of mammalian tumor cells . We report here the sequence of pfmdr, the P . falciparum homolog of mdr . We show that pfmdr is amplified in some chloroquine-resistant parasites but not in any of the sensitive isolates examined and that pfmdr transcript levels are increased . The gene is located on chromosome 5, and in one chloroquine-resistant line with an amplified pfmdr gene, chromosome 5 is greatly enlarged . The chromosome heterogeneity is due to varying copy numbers of different-sized pfmdr-containing amplicons . The existence of an mdr gene in P . falciparum and its amplification in some chloroquine-resistant lines greatly adds to the circumstantial evidence that pfmdr mediates chloroquine resistance in these lines. Cancer Res, 1989 Jun 15, 49(12), 3209 - 14 Characterization of monoclonal antibodies recognizing a Mr 180,000 P-glycoprotein: differential expression of the Mr 180,000 and Mr 170,000 P-glycoproteins in multidrug-resistant human tumor cells; Meyers MB et al.; P-glycoprotein is a plasma membrane protein believed to mediate resistance to natural product drugs such as vincristine, Adriamycin, and actinomycin D . To facilitate the study of human P-glycoprotein, monoclonal antibodies (designated HYB-612, HYB-241, and HYB-195) were raised against vincristine-resistant human neuroblastoma (SH-SY5Y/VCR) cells . The antibodies recognize a Mr 180,000 plasma membrane phosphoglycoprotein produced in increased amounts in SH-SY5Y/VCR as well as in vincristine-resistant human neuroepithelioma (MC-IXC/VCR), vinblastine-resistant human leukemia (CEM/VLB100), and actinomycin D- or vincristine-resistant Chinese hamster (DC-3F/AD X and DC-3F/VCRd-5L) cells, as compared to control cells . Radioimmunoprecipitation of proteins in cells metabolically labeled with {35S}methionine, 32Pi, or {3H}glucosamine and Western transfer procedures were used for these studies . Characterization of the HYB-612 or HYB-241 antigen by destructive degradation produced a pattern of results typical of a conformation-dependent protein epitope . HYB-612 recognizes complexes of the Mr 180,000 antigen with an iodinated photoaffinity analogue of vinblastine or with tritiated azidopine . Furthermore, pretreatment of MC-IXC and MC-IXC/VCR cells with HYB-612 or HYB-241 before measurement of tritium-labeled actinomycin D or vincristine uptake increases the amount of drug accumulation in resistant, but not in sensitive, cells . Of importance is the fact that the Mr 180,000 protein is expressed in cells which also contain a Mr 170,000 P-glycoprotein . The relative amounts of the Mr 180,000 and 170,000 species vary from one drug-resistant cell line to another . Evidence that the Mr 180,000 protein is a P-glycoprotein and that there is a conserved complex pattern of resistance-related surface proteins in multidrug-resistant cells is presented in this report. Cancer Res, 1989 Jun 15, 49(12), 3190 - 5 Correlation between reversing of multidrug resistance and inhibiting of {3H}azidopine photolabeling of P-glycoprotein by newly synthesized dihydropyridine analogues in a human cell line; Kamiwatari M et al.; Ten synthetic dihydropyridine analogues were investigated for their ability to reverse drug resistance in a multidrug-resistant human carcinoma cell line, KB-Cl . Four dihydropyridine analogues completely reversed the resistance, three lowered the resistance, and three had little effect . The radioactive photoactive dihydropyridine calcium channel blocker, {3H}azidopine, photolabels P-glycoprotein in membrane vesicles from KB-Cl cells . This photolabeling was almost completely inhibited by excess dihydropyridine analogues that reversed or lowered drug resistance . In contrast, the labeling was not significantly inhibited by analogues that do not reverse resistance . Among other reversing agents, cepharanthine and reserpine inhibited the {3H}azidopine photolabeling, but thioridazine did not . N-Solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine slightly inhibited the labeling at 100 microM . An anticancer agent, vinblastine, also inhibited the labeling . The correlation between the reversing of the drug resistance and the inhibition of the {3H}azidopine photolabeling of P-glycoprotein by dihydropyridine analogues suggests a role for P-glycoprotein in multidrug resistance and also the reversing of the resistance by dihydropyridine analogues. J Natl Cancer Inst, 1989 Jun 7, 81(11), 844 - 9 MDR1 RNA levels in human renal cell carcinomas: correlation with grade and prediction of reversal of doxorubicin resistance by quinidine in tumor explants; Kanamaru H et al.; We examined the distribution of RNA levels expressed by the multidrug-resistance gene (MDR1, also known as PGY1) in 42 renal cell carcinoma (RCC) samples (38 primary and four metastatic lesions) . The median MDR1 RNA level for the 38 primary lesions, expressed relative to the level for KB-3-1 cells, was approximately one-half of the level in multidrug-resistant KB-8-5 cells . Elevated MDR1 RNA levels were also observed in three of the four metastatic lesions . The mean MDR1 RNA level was higher in well-differentiated RCCs than in those that were poorly differentiated, suggesting that the increased expression of the MDR1 gene in RCCs originates from the increased expression in renal proximal tubule cells . To clarify the association of the MDR1 protein product P-glycoprotein with natural resistance to doxorubicin (ADR) in RCCs, we evaluated the effects of quinidine on in vitro sensitivity to ADR in 16 RCC samples, using a {3H}thymidine incorporation assay . The enhancing effect of quinidine (7.5 micrograms/mL) on sensitivity to ADR was statistically significant only in the group with high MDR1 RNA levels . Similar enhancement by quinidine of sensitivity to ADR was also observed in the established RCC cell lines in which MDR1 RNA levels were high . These results suggest that P-glycoprotein is active in the natural resistance of RCCs to ADR. Antimicrob Agents Chemother, 1989 Jun, 33(6), 881 - 5 Feeder layer-free in vitro assay for screening antitrypanosomal compounds against Trypanosoma brucei brucei and T . b . evansi; Kaminsky R et al.; A drug-susceptible Trypanosoma brucei brucei stock, a multidrug-resistant T . b . brucei stock, and a T . b . evansi stock resistant to two commercial trypanocides were adapted to a feeder layer-free culture system . Bloodstream forms were grown continuously in a liquid medium at 37 degrees C in 4% CO2 in air . Samples of trypanosome populations in the logarithmic growth phase were incubated with various concentrations of commercial and experimental compounds . Growth inhibition was monitored after a 24-h incubation and quantified by comparing the number of generations between control and drug-treated cultures . Some of the experimental compounds {taxol, formicin B, thioridazine, Ro 15-0216, and DL-alpha-(difluoromethyl)ornithine hydrochloride monohydrate} showed activity against both drug-susceptible and drug-resistant trypanosomes . Other compounds {sinefungin, 1,3,5-triacetylbenzene tris(guanylhydrazone)trimethanesulfonate hydrate, and 9-deazainosine} which inhibited the growth of drug-susceptible trypanosomes showed little or no effect upon drug-resistant parasites . Gossypol, however, had no antitrypanosomal effect on either trypanosome stock . The results obtained in this study correlate with observations obtained from drug screening in mice . The main advantages of the described in vitro screening assay are as follows: (i) lower amounts of drugs are required, (ii) results are obtained more rapidly, (iii) animals are not necessary, and (iv) the method is less labor intensive . These advantages result in an economical and rapid assay for primary drug screening. Cancer, 1989 Jun 1, 63(11), 2103 - 10 Therapy-induced drug resistance in a human leukemia line (LALW-2) . A clinically relevant model; White L et al.; A human leukemic T-cell line, LALW-2, established by xenografting in nude mice, has been maintained through 14 serial passages . The cells display consistent morphologic features, immunophenotype, and karyotypic aberrations (including an 11;14 translocation) and exhibit rearrangement of the T-cell receptor beta-chain gene . The growth rate of LALW-2 xenografts was differentially affected by drugs administered to host mice, the cells being resistant to cytotoxic agents (particularly methotrexate and doxorubicin) used in treatment of the donor patient . In short-term in vitro culture, LALW-2 cells exhibited extreme resistance to methotrexate and were also resistant to vincristine, vinblastine, dactinomycin, and doxorubicin . The findings differ from those obtained with laboratory-derived methotrexate or multidrug-resistant cell lines . The response of LALW-2 cells, in both the nude mouse model and in vitro, is consistent with acquisition of drug-resistance as a result of clinical treatment. Am J Anat, 1989 Jun-Jul, 185(2-3), 109 - 27 Use of colloidal gold cytochemistry in the study of the basic cell biology of cancer; Willingham MC; We are currently investigating the morphologic aspects of two areas of the basic cell biology of cancer: tumor-specific surface antigens as targets for immunotoxins, and the phenomenon of multidrug resistance in chemotherapy of human tumors . Colloidal gold cytochemistry has provided a useful method for the electron-microscopic cytochemical detection of materials endocytosed by cells in culture . This technique has been used to study the internalization pathway of ligands bound to the surface of cancer cells, particularly antibodies for use as immunologic targeting reagents for the construction of immunotoxins . These colloidal gold conjugates with monoclonal antibodies have demonstrated the internalization of these immunologic reagents through coated pits and receptosomes, which is a necessary step in the delivery of immunotoxins into the cell where they can mediate their cell-killing functions . Morphologic methods have been employed for the screening and selection of monoclonal antibodies reactive with the surface of human ovarian cancer cells for use as immunotoxins and have demonstrated the in vivo activity of immunotoxins made with these antibodies and Pseudomonas exotoxin in a nude mouse model system . In other studies, we have employed such reagents for the immunocytochemical detection of the surface expression of P170, the cell-surface efflux pump protein responsible for the phenotype of multidrug resistance in tumor cells, and to investigate the distribution of this protein by using immunocytochemistry in normal human tissues . These results have suggested a role for P170 in normal cell membrane transport of metabolites in various organ systems. Cancer Res, 1989 Jun 1, 49(11), 2988 - 93 Correlation of multidrug resistance with decreased drug accumulation, altered subcellular drug distribution, and increased P-glycoprotein expression in cultured SW-1573 human lung tumor cells; Keizer HG et al.; Four multidrug-resistant variants of the human squamous lung cancer cell line SW-1573 with levels of doxorubicin resistance ranging from 10- to 2000-fold were characterized with respect to drug accumulation and efflux, subcellular drug distribution pattern, antioxidant defenses, and P-glycoprotein expression . For all these parameters except the antioxidant defenses a correlation was observed with the level of doxorubicin resistance; with increasing drug resistance cellular drug accumulation capacity (as measured for doxorubicin) progressively decreased, initial drug efflux rates (as measured for daunorubicin) progressively increased, while the subcellular doxorubicin distribution (as measured by fluorescence microscopy) gradually shifted from a "mainly nuclear" to a "mainly cytoplasmic" pattern . Our data suggest that in the present set of cell lines the same mechanism of resistance is operating at all levels of doxorubicin resistance. Cancer Res, 1989 May 15, 49(10), 2661 - 7 Modulation of doxorubicin resistance by valinomycin (NSC 122023) and liposomal valinomycin in Chinese hamster ovary cells; Daoud SS et al.; Recently, we have reported that the toxicity of the membrane-active agent valinomycin (VM) can be reduced with maintenance and/or enhancement of its antitumor activity by incorporation in liposomes (S . S . Daoud and Juliano, Cancer Res., 46:5518-5525, 1986) . Since the underlying defect(s) in multidrug resistance reside mainly in the cell membrane, it seems reasonable to attempt to overcome multidrug resistance with membrane-active drugs . Here, we report on the in vitro restoration of Adriamycin (ADR) sensitivity in a resistant Chinese hamster ovary cell line (CHRC5) by treatment with nontoxic doses of valinomycin or of liposomal valinomycin . During a 1-h drug exposure, the sensitivity of CHRC5 to ADR was enhanced 21- to 28-fold when 20 or 40 nM VM was present, doses which are not toxic to CHRC5 cells . At the same time, modest synergistic toxicity could be seen in the parent drug-sensitive cell line (AUX B1) . At 100 nM VM, the sensitivity of CHRC5 to ADR was restored to almost that of the sensitive AUX B1 cells . The effects of liposomal VM on ADR sensitivity were similar to the effects produced by free VM . At nontoxic doses and with continuous exposure of the drug, valinomycin was highly active in restoring ADR sensitivity in CHRC5 cells . In cells treated for 72 h, valinomycin enhanced the sensitivity to ADR 208- to 250-fold in CHRC5 and 3- to 5-fold in AUX B1 cells . Measurements of ADR uptake and efflux indicate that, unlike other multidrug resistance modifiers, valinomycin exerts its actions in modulating ADR resistance by mechanism(s) other than increasing intracellular accumulation of Adriamycin . The possible mechanisms of the restoration of ADR sensitivity by valinomycin are discussed. Cancer Res, 1989 May 15, 49(10), 2790 - 6 P-glycoprotein expression in multidrug-resistant human ovarian carcinoma cell lines; Bradley G et al.; Multiple selections with either vinblastine or vincristine in the human ovarian carcinoma cell line SKOV3 resulted in variants with increasing degrees of multidrug resistance . SKOV3 derivatives that span a wide range in resistance (4- to 2000-fold) were obtained and analyzed for P-glycoprotein expression . In general, we observed a progressive increase in P-glycoprotein level (detected by Western blot) that paralleled the increase in multidrug resistance . However, a more detailed analysis of the P-glycoprotein mRNA and gene level indicated that the amount of P-glycoprotein expressed may be under complex control . At low levels of resistance, only an increase in P-glycoprotein mRNA and protein was observed . At intermediate to high levels of resistance P-glycoprotein gene amplification became evident . At the high level of resistance, an example was observed where only the amount of P-glycoprotein was increased without a concomitant increase in mRNA or gene copy . The mechanisms through which the content of P-glycoprotein in the plasma membrane is mediated are not understood; it is possible that the resistant variants identified here represent perturbations at different levels of regulation. Cancer Res, 1989 May 15, 49(10), 2729 - 33 Characterization of the multidrug resistance protein expressed in cell clones stably transfected with the mouse mdr1 cDNA; Schurr E et al.; Structural features of the multidrug resistance protein encoded by the mouse mdr1 gene were studied in multidrug-resistant cell clones stably transfected with a biologically active cDNA clone . Independently derived transfectant cell clones, initially selected in Adriamycin, were shown to be cross-resistant to several drugs, including actinomycin D, amsacrine, mitoxantrone, VP-16, and vinblastine but remained sensitive to cis-platinum, 5-fluorouracil, arabinocytosine, and bleomycin . In drug-resistant transfectants the mdr1 gene product was greatly overexpressed as a polypeptide of apparent molecular weight 160,000-170,000 . This protein was present in membrane enriched fractions and could be metabolically labeled with {3H )glucosamine, confirming that the transfected mdr1 gene encodes a membrane glycoprotein . The protein was found phosphorylated on serine residues and was shown to be photolabeled by both the calcium antagonist azidopine and the ATP analogue 8-azido ATP . Tryptic mapping of the ATP-photoaffinity labeled protein indicated that ATP crosslinking was site-specific and limited to two discrete peptide fragments of the protein, suggesting that the overexpressed mdr protein is capable of direct and specific ATP binding. J Natl Cancer Inst, 1989 May 10, 81(10), 798 - 803 Reversal of the multidrug-resistant phenotype of Chinese hamster ovary cells by L-histidinol; Warrington RC et al.; The amino acid analogue L-histidinol reverses the multidrug-resistance (MDR) attribute of the colchicine-resistant (CHR) variant CHRC5, a Chinese hamster ovary cell line that overexpresses a plasma membrane-associated glycoprotein and is resistant to colchicine (CH), daunorubicin, and vinblastine sulfate (VS) . The level of cell kill achieved in CHRC5 cells by combinations of L-histidinol and either daunorubicin or CH approached that achieved in AUXB1 parent cells by these two drugs, whereas L-histidinol-VS combinations killed even more CHRC5 cells than VS in the parental line . The capacity of L-histidinol to reverse the MDR phenotype of the CHRC5 line was time and dose dependent and was eliminated by the addition of a twofold molar excess of L-histidine . The reversal of the MDR trait by L-histidinol appears to be independent of the drug uptake mechanism. J Biol Chem, 1989 May 5, 264(13), 7418 - 24 Expression of a multidrug resistance-adenosine deaminase fusion gene; Germann UA et al.; A novel fusion gene has been created in which the expression of a dominant selectable marker, the human multidrug resistance gene, is directly linked to the expression of human adenosine deaminase cDNA . The chimeric gene was inserted between the long terminal repeats of a Harvey murine sarcoma virus expression vector and used to transfect drug-sensitive human KB carcinoma cells . Transfectants were selected in increasing concentrations of colchicine and found to contain multiple copies of the intact fusion gene, which is stably and efficiently expressed . A membrane-associated 210-kDa human P-glycoprotein-adenosine deaminase fusion protein is synthesized which retains function of the multidrug transporter and also exhibits adenosine deaminase activity . The data indicate that the human multidrug resistance gene may be used as a dominant selectable marker to introduce other genes in the form of gene fusions into cultured cells. J Natl Cancer Inst, 1989 May 3, 81(9), 706 - 9 Increased cytosolic pH in multidrug-resistant human lung tumor cells: effect of verapamil; Keizer HG et al.; In a set of four increasingly multidrug-resistant variants of SW-1573 human lung tumor cells, the pHi (i.e., steady-state cytosolic pH) increased up to 0.44 U as a function of the level of doxorubicin resistance . The elevated pHi in the most resistant (2,000-fold) variant dropped to the control level upon addition of verapamil, a known inhibitor of P-glycoprotein activity . These data suggest that, in the absence of xenobiotic substrates, P-glycoprotein activity can affect cellular pHi . This finding may be important for the elucidation of the physiological function of this protein. J Natl Cancer Inst, 1989 May 3, 81(9), 696 - 701 Prediction of doxorubicin resistance in vitro in myeloma, lymphoma, and breast cancer by P-glycoprotein staining; Salmon SE et al.; Prior studies have shown that the P-glycoprotein is a cell membrane efflux pump that is quantitatively increased in expression in multidrug-resistant tumor cell lines . In this study, fresh tumor tissues from patients with multiple myeloma, malignant lymphoma, or metastatic breast cancer were studied immunohistochemically for P-glycoprotein expression and for in vitro sensitivity to doxorubicin . Twenty-six patients who were either previously untreated or in relapse after chemotherapy had tumor specimens submitted that could be evaluated in both assays . The testing was done independently and blindly in separate laboratories instead of our being provided relevant clinical data on the patients . Tumor cells from 12 of the 26 patients (46%) stained positively for P-glycoprotein . Fifteen of the 26 specimens (58%) exhibited drug resistance in vitro . Although only three (21%) of the 14 P-glycoprotein-negative tumors exhibited in vitro resistance to doxorubicin, all 12 fresh tumors that stained positively for P-glycoprotein were resistant to doxorubicin . The difference in frequency of intrinsic doxorubicin resistance between P-glycoprotein-negative and -positive tumors was highly significant (P less than .001) . Similar trends were observed in each of the individual tumor categories and were statistically significant in myeloma and breast cancer . Four of the biopsy specimens that stained positively for P-glycoprotein and exhibited doxorubicin resistance were from patients who had not received prior cytotoxic chemotherapy . Similar conclusions were reached when results of drug sensitivity tests were ranked in relation to the median infective dose rather than by criteria based on correlations with clinical drug resistance . Our findings indicate that positive staining for P-glycoprotein associated with multidrug resistance predicts intrinsic cellular resistance of human cancers to doxorubicin . We anticipate that immunohistochemical staining for P-glycoprotein will prove useful in clinical oncology. Br J Haematol, 1989 May, 72(1), 40 - 4 Multidrug resistance in haemopoietic cell lines, myelodysplastic syndromes and acute myeloblastic leukaemia; Holmes J et al.; Resistance to cytotoxic agents is a common clinical problem encountered in the treatment of human myelodysplastic syndromes (MDS) and acute myeloblastic leukaemia (AML) . Cellular acquisition of the multidrug resistance (MDR) phenotype confers loss of sensitivity to a wide range of structurally dissimilar anti-neoplastic agents . This state can arise through increased expression of the mdrl (P-glycoprotein) gene . We have used the mdrl gene probe to investigate adriamycin resistant (HL60/AR) and vinblastine resistant (CEM/VLB100) human leukaemic cell lines . In addition, peripheral blood or bone marrow cells from 66 patients with MDS and AML have been screened for gene amplification and 40 cases for increased mRNA expression . P-glycoprotein gene amplification was observed only in the (CEM/VLB100) and not in the HL60/AR on any other leukaemic cell line . Gene amplification was not found in any patient's cells . Eighteen out of 40 patients showed an increase (2----20) of mdrl mRNA expression . These results are not only of significance in understanding the biology of human drug resistance but have practical importance in the design of anti-leukaemic therapy. Trans R Soc Trop Med Hyg, 1989 May-Jun, 83(3), 313 - 5 The effectiveness of chemoprophylaxis against malaria for non-immune migrant workers in eastern Thailand; Kamolratanakul P et al.; A randomized, double-blind field trial was carried out to compare the effectiveness of mefloquine plus sulfadoxine-pyrimethamine (MSP) with that of sulfadoxine-pyrimethamine (SP) in chemoprophylaxis against malaria . The study was conducted in 193 migrant workers in the eastern rural areas of Thailand which are known to be highly endemic for multidrug-resistant Plasmodium falciparum infection . MSP was found to be more effective than SP in the suppression of both P . falciparum and P . vivax parasitaemias, when administered weekly for 12 weeks (P = 0.0014) . Complete suppression of P . falciparum was achieved by MSP while 8 subjects receiving SP developed parasitaemia . One subject in the MSP group developed P . vivax parasitaemia, compared with 4 in the SP group . However, in view of the reported complications associated with the use of long-acting sulphonamides, some of which can be life threatening, prophylactic regimens containing sulfadoxine, though proved efficacious, must be used with extreme caution. Anticancer Res, 1989 May-Jun, 9(3), 575 - 8 Expression of the P-glycoprotein gene in multidrug-resistant Chinese hamster ovary cells; Mukhopadhyay T et al.; A series of multidrug-resistant (MDR) Chinese hamster ovary (CHO) cells was established to survive in stepwise increasing concentrations of vincristine . These MDR cells contained amplified P-glycoprotein (mdr) genes . Using monoclonal antibody against the P-glycoprotein, we present here results showing that the levels of P-glycoprotein and its phosphorylation and glycosylation did not correlate with those of drug resistance . Our results suggest that overproduction of the P-glycoprotein cannot account for the differences in drug resistance in these MDR cells, and that multiple modalities are involved in multiple drug resistance in mammalian cells. Jpn J Cancer Res, 1989 May, 80(5), 475 - 81 Potentiation of some anticancer agents by dipyridamole against drug-sensitive and drug-resistant cancer cell lines; Asoh K et al.; In this study, we have used two different vincristine (VCR)-resistant variants, VJ-300 and HC-7-5/VCR . VJ-300 was isolated from a human cancer KB cell line and HC-7-5/VCR from a human cancer HC-7-5 cell line . VJ-300 and HC-7-5/VCR are both multidrug-resistant (MDR) variants, showing resistance to multiple anticancer drugs such as VCR, adriamycin, actinomycin D and daunomycin . Dipyridamole, a specific inhibitor of nucleoside transport, potentiated these anticancer drugs about 2- to 10-fold against KB and VJ-300 . Dipyridamole almost completely reversed drug resistance to actinomycin D in VJ-300 cells with about a 70-fold higher resistance to actinomycin D . Dipyridamole inhibited the efflux of actinomycin D and VCR from VJ-300 cells . Dipyridamole enhanced the uptake of VCR but not that of actinomycin D in VJ-300 and KB . Dipyridamole at 10-100 microM inhibited photoaffinity labeling with {3H}azidopine of the cell-surface protein P-glycoprotein in VJ-300 cells . Dipyridamole potentiated 5-fluorouracil and hexylcarbamoyl-5-fluorouracil in cultured KB and VJ-300, but it annihilated the cytotoxic action of 5-fluorouridine . Potentiation of 5-fluorouracil by dipyridamole against HC-7-5 and HC-7-5/VCR was also observed, but appeared to be less than in VJ-300 and KB cells . Dipyridamole almost completely inhibited the cellular accumulation of 5-fluorouridine, but not that of 5-fluorouracil . Thus, dipyridamole appeared to potentiate anticancer agents through pleiotropic action sites, one of which is inhibition of enhanced efflux of MDR cell lines and the other of which is inhibition of nucleoside transport . Dipyridamole might be a useful and potent agent to potentiate anticancer agents and reverse drug-resistance. Jpn J Cancer Res, 1989 May, 80(5), 469 - 74 Immunocytochemical identification and localization of the Mr 22,000 calcium-binding protein (sorcin) in an adriamycin-resistant myelogenous leukemia cell line; Sugawara I et al.; Monoclonal antibody against the Mr 22,000 calcium-binding protein (sorcin) from an adriamycin-resistant myelogenous leukemia cell line K562 (K562/ADM) was prepared and used as a probe to study the localization of sorcin in K562/ADM cells and the parental cell line, K562 . Analysis of extracts from K562/ADM cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorescence image analysis showed that K562/ADM cells possessed abundant sorcin in the cytoplasm which was almost entirely absent from the drug-sensitive parental cell line, K562 . Furthermore, immuno-electron microscopic studies revealed that sorcin was closely associated with free ribosomes, rough endoplasmic reticulum, mitochondria, microfilament bundles and perinuclear membranes . These observations provide the first clue that the Ca-binding protein, sorcin, may play an important role in the development of the multidrug resistance phenomenon, although the relationship between sorcin and P-glycoprotein is still unknown. Br J Cancer, 1989 May, 59(5), 682 - 5 P-glycoprotein gene amplification and expression in multidrug-resistant murine P388 and B16 cell lines; Capranico G et al.; P-glycoprotein gene (mdrl) amplification and expression were examined in murine leukaemia P388/DX and melanoma B16VDXR cell lines, which exhibit a high level of resistance to a selecting agent, doxorubicin, and express a multidrug-resistant phenotype because they are cross-resistant to multiple cytotoxic drugs . The multidrug-resistant phenotype was obtained in different conditions of selection (in vivo and in vitro for P388/DX and B16VDXR, respectively) . In both multidrug-resistant cell lines, an increased expression of P-glycoprotein gene (5 kb transcript detected in Northern blots) was observed and the level of P-glycoprotein mRNA correlated with the degree of resistance . In addition, high molecular weight mRNAs homologous to mdrl gene sequence were consistently detected only in P388/DX cells . Overexpression was associated with a high level of gene amplification only in resistant melanoma cells, whereas it occurred in P388/DX cells with a marginal increase in gene copy number . These results, suggesting that different genetic mechanisms could be responsible for P-glycoprotein overexpression, emphasise the complexity of genetic regulation that may affect tumour cell sensitivity to cytotoxic agents. Cancer Res, 1989 May 1, 49(9), 2422 - 6 Genetic characterization of the multidrug-resistant phenotype of VM-26-resistant human leukemic cells; Wolverton JS et al.; Our human T-cell leukemia line, CEM/VM-1, selected for resistance to VM-26 (teniposide), is cross-resistant to several drugs that interact with topoisomerase II, including VP-16 (etoposide), 4'-(9-acridinylamino)methanesulphon-m-anisidide, daunorubicin, and mitoxantrone . However, in contrast to cell lines exhibiting multidrug resistance (MDR) associated with overexpression of P-glycoprotein, this line is not cross-resistant to the Vinca alkaloids, is not impaired in drug accumulation, and does not overexpress the mdrl gene (Cancer Res., 47: 1297, 5455, 1987) . More recently we found that nuclear extracts of these cells exhibit decreased topoisomerase II catalytic and cleavage activity, compared to the drug-sensitive line (Biochemistry, 1988) . These results suggest that an alteration in topoisomerase II or a modulator of this enzyme may be responsible for this altered topoisomerase II-form of multidrug resistance (at-MDR) . In the present work, we studied the somatic cell genetics of at-MDR . We produced hybrid cell lines by polyethylene glycol-mediated fusion of the CEM/VM-1 line with a hypoxanthine-guanine phosphoribosyl transferase-deficient, ouabain-resistant CEM line (CEM.AG1.OU1.5) that exhibits VM-26 sensitivity . Ten of the hybrid lines that grew in selective medium were randomly chosen for expansion and four were analyzed for both DNA content by flow cytometry and VM-26 sensitivity in a 72-h growth inhibition assay . The hybrid lines all contained approximately 2x DNA compared to unfused controls, indicating that the fusions were successful . The IC50 for VM-26 in 3 of the 4 lines was the same as that of the sensitive controls, ranging from 4.7 to 7.4 x 10(-8) M, and another was 76 x 10(-8) M . These data indicate that drug sensitivity was reconstituted by the hybridization procedure . By comparison, the VM-26 IC50 values in the CEM/VM-1 cells and CEM/VM-1 x CEM/VM-1 control "fusions" were 360 and 750 x 10(-8) M, respectively . To determine whether a topoisomerase II-mediated function was reconstituted in the hybrids, we measured drug-stimulated DNA cleavage ("cleavable complex formation") . Using 32P-labeled pBR322 DNA as substrate with nuclear extracts from drug sensitive cells, 100 microM VM-26 maximally stimulated DNA cleavage by approximately 11-fold compared to no-drug controls.(ABSTRACT TRUNCATED AT 400 WORDS) Anticancer Res, 1989 May-Jun, 9(3), 637 - 41 Sensitivity of multidrug resistant KB-C1 cells to an antibody-dextran-adriamycin conjugate; Sheldon K et al.; Resistance to adriamycin is an important limitation to the use of the drug in cancer therapy . This resistance is often a manifestation of the multidrug resistance phenotype . Studies with multidrug resistant cell lines in vitro may be useful to design approaches for overcoming the drug resistance encountered clinically . We investigated the possibility of overcoming adriamycin resistance in vitro in a multidrug resistant subline (KB-C1) of human epidermal carcinoma (KB-3-1) cells using antibody-mediated drug targeting . Adriamycin was conjugated through a dextran bridge to a monoclonal antibody (mAb), 10B, which bound to KB-3-1 cells with a Ka of 4 x 10(8) M-1 . The conjugate retained immunoreactivity with the target cells . Adriamycin (0.2 micrograms/ml) caused a 50% inhibition of DNA synthesis in KB-3-1 cells, but failed to achieve this degree of inhibition in KB-C1 cells at levels as high as 10 micrograms/ml . In contrast, the 10B-dextran-adriamycin conjugate produced 50% inhibition of DNA synthesis in KB-C1 cells at a concentration of 2.5 micrograms/ml . This was significantly more cytotoxic than adriamycin conjugated to control mAb or bovine serum albumin (BSA) . Similarly, a 10B-recombinant ricin A (rRA) immunotoxin was more cytotoxic to KB-C1 cells than free rRA . These results indicate that adriamycin resistance in KB-C1 cells in vitro can be partially overcome by specifically targeting adriamycin to the cells using an 10B-dextran-adriamycin conjugate . This approach may be useful in overcoming adriamycin resistance encountered during the course of cancer therapy. FEBS Lett, 1989 Apr 24, 247(2), 405 - 10 Glycolysis in P-glycoprotein-overexpressing human tumor cell lines . Effects of resistance-modifying agents; Broxterman HJ et al.; We show that drugs, such as verapamil, which reverse multidrug resistance (MDR), in P-glycoprotein-overexpressing tumor cells, increased the rate of lactate production in four human MDR cell lines, but not in the parent, sensitive cell lines . The effect on glycolytic rate was maximal at a medium concentration of 2 microM verapamil . The glycolytic rate in sensitive (A2780) and MDR 2780AD) cells showed the same pH dependence, but the effect of verapamil was seen only in 2780AD cells at all pH values investigated (6.6, 7.4 and 8.2) . A series of drugs such as nigericin, oligomycin, amiloride and monensin had similar effects in the two cells . Phorbol myristate acetate increased lactate formation in neither cell line . Verapamil induced an extra amount of ATP consumption in P-glycoprotein-expressing 2780AD cells of approx . 25 pmol/s per 10(6) cells, which was estimated to be about 10% of cellular energy turnover. Cancer, 1989 Apr 15, 63(8), 1534 - 8 Increased P-glycoprotein expression and multidrug-resistant gene (mdr1) amplification are infrequently found in fresh acute leukemia cells . Sequential analysis of 15 cases at initial presentation and relapsed stage; Ito Y et al.; Using a DNA probe of mdr1 and an anti-P-glycoprotein monoclonal antibody (MRK16), the authors investigated 19 cases of adult acute leukemia patients (one M1, six M2, three M3, one M4, three M5, two L1, and three L2), comparing leukemia cells at the initial presentation (I) with those at the relapsed stage (R) . By Southern hybridization analysis mdr1 DNA levels were not amplified in 32 samples from 19 patients (I: 14, R: 18) . By Northern hybridization analysis mdr1 mRNA levels were not expressed in ten samples from seven patients (I: 4, R: 6) . By indirect immunofluorescent assay with MRK16 antibody P-glycoprotein was not detected in 30 samples from 18 patients (I: 13, R: 17) . Thus, P-glycoprotein expression and mdr1 gene amplification occurred in