Multidrug resistant

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 infrequently not only in leukemia cells at the initial presentation but also in those at the relapsed cases and may not be a major cause of refractoriness to antileukemia drugs in adult acute leukemia.

Cancer Res, 1989 Apr 1, 49(7), 1722 - 6
In vivo circumvention of vincristine resistance in mice with P388 leukemia using a novel compound, AHC-52; Shinoda H et al.; A novel compound partially analogous to nifedipine, AHC-52, was found to sensitize multidrug-resistant tumor cells . AHC-52 at 0.5 microgram/ml completely reversed the in vitro resistance to vincristine (VCR) in VCR-resistant P388 cells (P388/VCR) . Of various regimens examined for the in vivo treatment of P388/VCR-bearing mice, the combination of 0.05 mg/kg of VCR with 100 mg/kg twice a day of AHC-52 daily demonstrated the best result with a 206% increase in life prolongation . This result was comparable with that observed in parental P388-bearing mice treated with the optimal dose of VCR alone, indicating almost complete circumvention of resistance by combination VCR-AHC-52 therapy . In addition, the combination of both agents exhibited therapeutic effects in the treatment of P388-bearing mice with some long term survivors . This result was presumably due to the elimination of heterogeneity of VCR sensitivity in this cell population . These results suggest that combination chemotherapy using a sensitizing agent such as AHC-52 will be effective in not only circumvention of multidrug resistance but also retardation of its occurrence.

Gan To Kagaku Ryoho, 1989 Apr, 16(4 Pt 2-1), 1280 - 2
{Drug resistance and a strategy to eradicate resistant malignant cells}; Shimoyama M; Monoclonal antibody to detect multidrug resistant cell was interesting and useful for clinical application . Other type of drug resistance is also important, but it has not been elucidated in human cancer . Strategy to eradicate resistant cells is discussed.

Gan To Kagaku Ryoho, 1989 Apr, 16(4 Pt 2-1), 1273 - 9
{Cross and non-cross resistant anticancer agents to multidrug-resistant cultured cells from hematologic malignancies}; Shimoyama M et al.; We report here that typical and atypical multidrug resistant (MDR) cells can be identified by monoclonal antibodies, MRK16 and MRK20, respectively . Typical MDR cells were cross-resistant to vinca alkaloids, anthracyclines, mitoxantrone (MXT), etoposide (VP-16) and actinomycin-D (ACT-D), and reactive to MRK16 . Atypical MDR cells were cross-resistant to anthracyclines, MXT, VP-16, and bleomycin but sensitive to vinca alkaloids and ACT-D . They were reactive to MRK20, but not to MRK16 . Among anthracyclines, aclacinomycin A was not cross-resistant to either typical or atypical MDR cells . Methotrexate was not cross-resistant to doxorubicin, vincristine, Ara-C and cisplatin . Cisplatin was not cross resistant to 5-FU, either . Identification of cross and non-cross resistant anticancer agents may be useful to plan a cure-oriented combination chemotherapy for hematologic malignancies.

Gan To Kagaku Ryoho, 1989 Apr, 16(4 Pt 2-1), 1266 - 72
{A molecular basis for multidrug resistance and reversal of the resistance}; Akiyama S et al.; Multidrug-resistance is frequently characterized by enhanced drug efflux resulting from a membrane glycoprotein of 170,000 daltons (P-glycoprotein) . Analysis of cloned cDNAs for the human MDR 1 gene, whose product is the P-glycoprotein, indicates that P-glycoprotein is an energy-dependent drug-efflux system for cytotoxic hydrophobic anticancer drugs . We have demonstrated that a photoanalog of a reversing agent, SDB-ethylenediamine, specifically binds to P-glycoprotein . The binding site on P-glycoprotein seems to be identical with that of anticancer agents and other reversing agents . On the other hand, the radioactive photoactive dihydropyridine calcium channel blocker, {3H} azidopine, photolabels P-glycoprotein in membrane vesicles from multidrug-resistant cells . This photolabeling is almost completely inhibited by excess dihydropyridine analogs that reverse or lower drug-resistance . In contrast, the labeling is not significantly inhibited by analogs that do not reverse resistance . These results suggest that it may be possible to quickly screen for dihydropyridine analogs that reverse multidrug resistance by measuring the inhibition of {3H} azidopine labeling of P-glycoprotein.

Eur J Cancer Clin Oncol, 1989 Apr, 25(4), 743 - 9
Detection of the multidrug resistant phenotype in human tumours by monoclonal antibodies and the streptavidin-biotinylated phycoerythrin complex method; Volm M et al.; The aim of this study was to find out whether the membrane glycoprotein P-170 can be detected in human tumours with both acquired and intrinsic resistance to chemotherapeutic agents using monoclonal antibodies (265/F4 and C219) and the streptavidin-biotinylated phycoerythrin complex method . Pretreated leukaemia cells and untreated lung and ovarian carcinomas were analysed . Two plasmacytomas and one leukaemia expressed high levels of P-glycoprotein, whereas two leukaemias showed moderate, and three leukaemias no expression of this protein . The intrinsic resistance was analysed with a panel of four human epidermoid lung cancer xenografts grown in nude mice . The expression of P-glycoprotein could be correlated with the degree of resistance . In addition, one out of five ovarian carcinomas revealed a high level of P-glycoprotein.

Blut, 1989 Apr, 58(4), 215 - 7
Detection of P-glycoprotein in human leukemias using monoclonal antibodies; Mattern J et al.; Overexpression of a Mr 170,000 membrane glycoprotein (P-glycoprotein) is consistently associated with multidrug resistance in cell lines . Two monoclonal antibodies (Mab) against P-glycoprotein (265/F4 and C 219) were used to examine tumour samples from patients with leukemias for evidence of P-glycoprotein overexpression . High levels of P-glycoprotein (greater than 5% positive cells) were detected with both antibodies in samples from 3 out of 18 patients suggesting that a multidrug resistant phenotype may also occur in human leukemias.

Mol Pharmacol, 1989 Apr, 35(4), 414 - 21
Preparation and utility of a radioiodinated analogue of daunomycin in the study of multidrug resistance; Busche R et al.; Anthracyclines are an important class of cytotoxic drugs that are frequently used in cancer chemotherapy, especially in acute leukemia . The pharmacokinetics and disposition of these compounds in whole animals and in cells have been studied employing 3H-labeled forms . However, their usefulness is limited by their low specific activities and the low energy of 3H . Therefore, we have labeled daunomycin using 125I-Bolton-Hunter reagent . The resultant anthracycline analogue, iodomycin, has a specific activity of approximately 2000 Ci/mmol . Although this compound was 10-fold less toxic to normal cells than daunomycin, multidrug-resistant cells were cross-resistant to it . Like other drugs to which these cells are cross-resistant, its accumulation by them was greatly reduced, compared with drug-sensitive cells . We have also utilized this compound in photoaffinity labeling experiments to identify its target in multidrug-resistant cells . We observed the specific binding of iodomycin to P-glycoprotein in membrane vesicles as well as in intact cells, thereby directly demonstrating that this protein specifically binds anthracyclines as well as Vinca alkaloids.

Jpn J Cancer Res, 1989 Apr, 80(4), 373 - 9
Altered expression of epidermal growth factor receptor gene in a classical multidrug-resistant variant of a human cancer cell line, KB; Takano H et al.; A variant clone resistant to high doses of colchicine (KB-C1) derived from human cancer KB cell line is resistant to various anticancer agents . The KB-C1 cells were much more resistant to epidermal growth factor and a chimeric toxin, EGF-Pseudomonas exotoxin (PE), than the parental KB cells . KB-C1 cells have decreased numbers of EGF-receptors, though the affinity of the receptors is similar to that in the parental KB cells . A drug-sensitive revertant (C1-R2) partially recovered its EGF-receptor activity . Northern blot analysis showed a decreased level of EGF-receptor mRNA in KB-C1 cells, while the multidrug-resistance gene, mdr-1, was expressed at very high levels in KB-C1 cells, but not in KB or C1-R2 cells . The drug-resistant cells were less tumorigenic than the parental cells when injected into nude mice . A decreased expression of EGF-receptor in these cells may be one of the pleiotropic properties of multidrug-resistant cells and may perhaps represent the basis for their reduced tumorigenicity.

Int J Cancer, 1989 Mar 15, 43(3), 520 - 5
Effect of erythromycin and tumour necrosis factor on the drug resistance of multidrug-resistant cells: reversal of drug resistance by erythromycin; Hofsli E et al.; WEHI 164 murine fibrosarcoma cells were rendered multidrug-resistant (MDR) by culture in the presence of actinomycin D . In addition to resistance to actinomycin D, the cells acquired resistance to doxorubicin, mitomycin, vincristine and cycloheximide . The fact that development of resistance to one type of lipophilic chemotherapeutic drug also results in resistance to other structurally unrelated lipophilic drugs suggests that non-toxic lipophilic agents may interfere with drug resistance by saturating the pathway by which MDR-cells inhibit drug cytotoxicity . We show that the antibiotic erythromycin significantly reverses the resistance of MDR WEHI 164 cells to doxorubicin and actinomycin D . In addition to cross-resistance to chemotherapeutic drugs, 3 out of 4 actinomycin D-resistant WEHI 164 cell lines also showed higher resistance to tumour necrosis factor (TNF) than the parental WEHI 164 cells . However, whereas verapamil, a calcium antagonist known to reverse multidrug-resistance, rendered resistant cells more sensitive to chemotherapeutic drugs, it protected the cells from killing by TNF, suggesting that drug resistance and TNF resistance may not be directly connected . A synergistic cytotoxic effect of TNF and actinomycin D was obtained on both the parental and the MDR cells . However, higher concentrations of TNF and actinomycin D were required to obtain a cytotoxic effect in the MDR cells, reflecting actinomycin D and TNF resistance in these cells.

Int J Cancer, 1989 Mar 15, 43(3), 487 - 91
Circumvention of adriamycin resistance by dipyridamole analogues: a structure-activity relationship study; Ramu N et al.; Dipyridamole restores sensitivity to Adriamycin (ADR) in drug-resistant cells . In an effort to elucidate the relationship between activity and chemical structure of dipyridamole, the ability to enhance the growth inhibitory effect of ADR, in multidrug-resistant (MDR) P388 murine leukemia cells, was determined for 43 derivatives and related compounds . Since both substituted pyrimidopyrimidines and pteridines enhanced the growth-inhibitory effect of ADR in drug resistant cells, the core skeleton may not be directly involved and rather serve as a carrier for the substituents connected with this activity . The exact positions of the active substituents on the core skeleton did not seem to be critical for exertion of the activity . Activity was dependent on the presence of 3 tertiary amine groups . However, not all tertiary amines showed the same potency which might be related to the degree of basicity and/or the spatial structure of these groups . The most active derivatives carried piperidine and pyrrolidine groups while derivatives with thiomorpholine, 3-hydroxypiperidine or dimethylamine groups had low activity . Activity was also dependent on the presence of a substituent with partial electronegative charges as found in a diethanolamine group . However, this function could be carried out, with even higher efficiency, by a substituent containing 6 pi electrons.

Cancer Res, 1989 Mar 15, 49(6), 1452 - 5
Competitive inhibition by verapamil of ATP-dependent high affinity vincristine binding to the plasma membrane of multidrug-resistant K562 cells without calcium ion involvement; Naito M et al.; Verapamil, a calcium channel blocker, can inhibit the efflux of antitumor agents from multidrug-resistant cells and reverse drug resistance . We have recently reported that the plasma membrane prepared from an adriamycin (ADM)-resistant variant (K562/ADM) of human myelogenous leukemia K562 cells showed ATP/Mg2+-dependent high affinity binding of vincristine (VCR), which is closely related to the drug transport mechanism in this cell line . To clarify how calcium channel blockers inhibit the transport of antitumor agents from the resistant cells, we analyzed the effect of calcium channel blockers and Ca2+ ion on the VCR binding to K562/ADM plasma membrane . The ATP-dependent VCR binding was inhibited by calcium channel blockers (verapamil, nicardipine, and diltiazem), which are known to inhibit drug efflux from the resistant cells . Addition of {ethylenebis(oxyethylenenitrilo)}tetraacetic acid or high concentration of Ca2+ decreased the amount of VCR binding to some extent; however, a substantial amount of VCR still could bind to K562/ADM plasma membrane . The inhibitory effect of verapamil on the VCR binding was observed regardless of the Ca2+ concentration . Klotz plot analysis revealed that the inhibition of the VCR binding to K562/ADM plasma membrane by verapamil was competitive . Dissociation constant (Kd) of VCR and apparent inhibitory constant (Kiapp) of verapamil were calculated to be 0.1 +/- 0.1 microM (SD) and 1 +/- 1 microM, respectively . These results indicate that Ca2+ ion is not required for the VCR binding and that verapamil competitively inhibits the VCR binding without concerning Ca2+ ion . Antitumor agents (vinblastine, actinomycin D, ADM, and colchicine) and other agents known to reverse multidrug resistance (nicardipine, diltiazem, cyclosporin A, quinidine, and trifluoperazine) also inhibited the VCR binding competitively.

Cancer Res, 1989 Mar 15, 49(6), 1422 - 8
Expression of anionic glutathione-S-transferase and P-glycoprotein genes in human tissues and tumors; Moscow JA et al.; The development of multidrug resistance in MCF-7 human breast cancer cells and the acquisition of broad resistance to xenobiotics in rat hyperplastic nodules are both associated with increased P-glycoprotein (mdr) gene expression as well as changes in activities of intracellular detoxication enzymes; among these changes is a significant increase in the activity of the anionic isozyme of glutathione-S-transferase (GST) . We have isolated a cDNA encoding the human anionic glutathione-S-transferase, GST pi-1, from a cDNA library constructed from multidrug-resistant MCF-7 cells . The deduced amino acid sequence of GST pi-1 shows that while the human anionic GST displays 85% nucleotide and amino acid sequence homology to the rat anionic isozyme, it is markedly less related to human basic GST isozymes . We have examined the expression of GST pi and P-glycoprotein in 170 specimens of human tissues and tumors . P-Glycoprotein RNA expression was positive in eight of 23 lymphomas and two of 12 colon tumors; however, many other normal and malignant tissues, including lung, bladder, and breast tumors, had low or undetectable levels of P-glycoprotein RNA expression . In contrast, GST pi was readily detected in a wide variety of normal and malignant tissues . The level of GST pi mRNA expression in normal tissues was heterogeneous, with lowest levels found in liver and the highest levels found in lung, esophagus, and placenta . GST pi was also variably expressed in human tumors, with the lowest relative levels occurring in lymphoma and breast cancer and the highest levels found in lung cancer and head and neck tumors . In addition, comparison of paired specimens from the same patient indicated that GST pi expression was increased in many tumors relative to matched normal tissue.

Biochem Pharmacol, 1989 Mar 1, 38(5), 787 - 93
Role of glutathione and its associated enzymes in multidrug-resistant human myeloma cells; Bellamy WT et al.; Multidrug resistance (MDR) is a phenomenon associated with the emergence of simultaneous cross-resistance to the cytotoxic action of a wide variety of structurally and functionally unrelated antineoplastic agents . The present study was undertaken to determine if 8226 human myeloma cells possessing the MDR phenotype had an increased ability to resist the intercalating drug doxorubicin (DOX) via glutathione-based detoxification systems . Glutathione S-transferase (GST) was isolated by affinity chromatography, and the enzyme activity was assessed using 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) as substrates . There was no difference in overall GST activity between the sensitive and resistant cells . Using a cDNA probe (pGTSS1-2) for the human placental, anionic GST isoenzyme, no overexpression of mRNA for this isoenzyme was noted in the resistant line . When glutathione peroxidase activity (GSH-px) was assessed using either H2O2 or cumene hydroperoxide as substrate, again there was no difference in enzyme activity . Non-protein sulfhydryl (NPSH) levels were found to be elevated significantly in the resistant 8226/DOX40 subline (19.2 +/- 0.1 nmol NPSH/10(6) cells) as compared to the drug-sensitive parental subline 8226/S (11.6 +/- 1.9 nmol NPSH/10(6) cells) (P less than 0.001) . In addition, when the 8226/DOX40 cells were cultured in medium without doxorubicin, there was a consistent decline in NPSH values reaching a steady state identical to that of the 8226/S cells . However, the decrease in NPSH level was not accompanied by a change in the level of doxorubicin resistance as assessed by colony-forming assays . Depletion of glutathione by D,L-buthionine-S,R-sulfoximine had no effect on doxorubicin sensitivity in either subline . Thus, it appears that GSH-based detoxification systems are not causally involved in maintaining the MDR phenotype in 8226 human myeloma cells; rather they appear to comprise an epiphenomenon associated with the resistance selection procedure.

Anticancer Res, 1989 Mar-Apr, 9(2), 469 - 74
Chemosensitivity and multidrug resistance to antineoplastic drugs in retinoblastoma cell lines; Chan HS et al.; Chemosensivity testing in 11 retinoblastoma (RB) cell lines identified five tumors showing cross-resistance to plant alkaloids, alkylating agents, anthracycline antibiotics, and antimetabolites, suggestive of the multidrug-resistant (MDR) phenotype described in other tumors . Only two tumors had been clinically exposed to chemotherapy, and both showed MDR . All RB tumors were resistant to clinically achievable concentrations of methotrexate and hydrocortisone, and only sensitive to cytosine arabinoside . These results provide a rational basis on which to develop new chemotherapeutic strategies for the treatment of RB.

Anticancer Res, 1989 Mar-Apr, 9(2), 367 - 71
Effect of the tiapamil analog RO11-2933 on cellular sensitivity to antitumor drugs in sensitive and multidrug resistant human ovarian cancer cells; Mazzoni A et al.; The ability of the Tiapamil analog N-(3,4 Dimethoxyphenyl)-N-methyl-2-(naphthyl)-m-dithiane-2-propylamine hydrocloride (RO11-2933) to influence the cytotoxic activity of Doxorubicin (DX) and seven other antitumor agents was evaluated in sensitive and treatment-induced multidrug resistant human ovarian cancer cells . In vitro treatment of A2780 sensitive cells with 1 microM RO11-2933 for 72hr potentiated by less than 3-fold the cytotoxic effects of each antitumor drug tested, with the exception of Cisplatin and Fluorouracil, which were potentiated by 7.2- and 5.0-fold, respectively . In A2780-DX3 resistant cells, RO11-2933 increased by a mean of 50-fold the cytotoxic effects of the anthracyclines and the Vinka alkaloids analyzed . A potentiation of Cisplatin activity was also observed (6.5-fold), whereas the Tiapamil analog did not significantly modify the antiproliferative activity of Bleomycin, Fluorouracil or Ara-C . Analysis of DX uptake and retention showed that in resistant cells RO11-2933 restored the intracellular DX content to levels comparable to those of sensitive A2780 cells, suggesting that the cytoplasmic membrane might represent a possible site of action of this compound . The results obtained indicate that RO11-2933 might represent an effective agent in combination with several anticancer drugs for the treatment of drug resistant ovarian carcinomas.

Cytometry, 1989 Mar, 10(2), 185 - 91
Improvement of flow-cytometric detection of multidrug-resistant cells by cell-volume normalization of intracellular daunorubicin content; Ross DD et al.; To improve the ability of flow cytometry to detect multidrug-resistant cells, we studied the extent to which cell volume heterogeneity accounts for the variance of intracellular daunorubicin (DNR) content . For P388 murine or HL-60 human leukemia cells exposed to DNR (1 micrograms/ml, 60 min), log intracellular DNR content varied in direct proportion to log cell volume measured by flow cytometry, with a correlation coefficient of .9 . This relationship was confirmed by cell sorting based on intracellular DNR content with subsequent volume determination of the sorted cells . Normalization of intracellular DNR content for cell volume (thus obtaining intracellular DNR concentration) was accomplished by subtracting log cell volume from log intracellular DNR content for each cell . This resulted in a 34% decrease (range 23-58%) in standard deviation compared to DNR content measurements without volume normalization for all cell types tested . Following exposure to DNR (as above), intracellular DNR content of drug-sensitive P388 or HL-60 cells measured by flow cytometry was 12- and 8-fold greater than that of the multidrug-resistant sublines P388/ADR and HL-60/AR, respectively . However, because of the variance of intracellular DNR content, the predictive value of flow-cytometric determination of intracellular DNR content as a discriminant assay for detecting the frequency of drug-resistant cells in a mixed population was acceptable only when the frequency of resistant cells in the population exceeded 10% . In contrast, volume normalization of intracellular DNR content enhanced the ability of the flow-cytometric assay to discriminate resistant cells by 10-fold for P388 cells and 100-fold for HL-60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Anticancer Res, 1989 Mar-Apr, 9(2), 267 - 71
Chromatin bodies in multidrug resistant hybrid cells have centromeres and originate from homogeneously staining regions; Jakobsson AH et al.; Antikinetochore antibodies from patients with the CREST syndrome of scleroderma were used as probes to study homogeneously staining regions (HSRs) in multidrug resistant (MDR) mouse tumor cells, and chromatin bodies (CBs) in MDR mouse-hamster hybrid cells . In one mouse tumor line the C-band positive HSR showed antigenic properties and displayed many weekly fluorescent spots, i.e . it contained kinetochore proteins . The immunofluorescence pattern of the HSR could also be observed in interphase nuclei . The C-band positive CBs of the hybrid cells had active centromeres, as shown by double kinetochore spots . These results support our hypothesis that CBs originate from C-band positive HSRs.

Gan To Kagaku Ryoho, 1989 Mar, 16(3 Pt 2), 592 - 8
{Anti-cancer drug resistance and glutathione S-transferases}; Sato K et al.; Glutathione S-transferase (GST) is a family of multimolecular forms with multi-functions for detoxication of drugs, and certain GST forms have been reported to concern multidrug resistance (MDR) mechanisms of neoplastic cells to anticancer drugs . In this paper, recent studies of GSTs concerning MDR are briefly reviewed, and the problems to be clarified are discussed . The reduced glutathione (GSH) is known to play important roles in the inactivation (detoxication) of the anticancer drugs . Most of them, especially alkylating agents, are conjugated with GSH by GSTs and detoxified, and the peroxides from drugs such as adriamycin are also reduced with GSH and detoxified by the GSH peroxidase activity of certain GST forms . Rat GST-P (GST 7-7) and human GST-pi, both of which belong to Class pi in the species-independent classification of GST, have been known as a marker enzyme for rat and human (pre) neoplastic lesions, respectively . GST-P is increased in rat hepatic preneoplastic foci resistant to cytotoxic agents . GST-pi is also increased not only in cancer cells such as colon carcinoma and non-small cell lung carcinoma, which exhibit "natural resistance" to anticancer drugs, but also increased in breast, ovarian and other tissue carcinomas with increased "acquired resistance" to certain drugs . A few research groups have attempted to confirm by transfection of a vector expressing a GST form such as GST-pi into non-resistant cell lines whether there is a direct relationship between the expression of a specific GST form and the appearance of MDR . However, MDR did not always appear . Recently, it was found in our laboratory that GST-P, GST-pi and even mouse GST II in Class pi, all are very strongly inactivated by SH-modifiers and active oxygens, indicating that these properties may be useful for overcoming MDR, if the forms really are involved with MDR.

Rinsho Ketsueki, 1989 Mar, 30(3), 275 - 81
{Multidrug resistance in acute leukemia}; Nara N; Despite substantial recent advances in leukemia therapy, most leukemia patients eventually relapse and die of recurrent or refractory leukemia . Important issues are how to treat the patients in relapse of leukemia and how to overcome drug-resistant leukemic cells . Multidrug resistance of tumor cells has been vigorously studied in terms of P-glycoprotein, which may play important roles in the transport of antitumor drugs through cell membrane . In the present article, the author has reviewed the recent advances in understanding the pathophysiology of drug-resistance in leukemic cells and discussed the clinical approaches to drug-resistant leukemia.

Mol Cell Biol, 1989 Mar, 9(3), 1224 - 32
Identification of members of the P-glycoprotein multigene family; Ng WF et al.; Overproduction of P-glycoprotein is intimately associated with multidrug resistance . This protein appears to be encoded by a multigene family . Thus, differential expression of different members of this family may contribute to the complexity of the multidrug resistance phenotype . Three lambda genomic clones isolated from a hamster genomic library represent different members of the hamster P-glycoprotein gene family . Using a highly conserved exon probe, we found that the hamster P-glycoprotein gene family consists of three genes . We also found that the P-glycoprotein gene family consists of three genes in mice but has only two genes in humans and rhesus monkeys . The hamster P-glycoprotein genes have similar exon-intron organizations within the 3' region encoding the cytoplasmic domains . We propose that the hamster P-glycoprotein gene family arose from gene duplication . The hamster pgp1 and pgp2 genes appear to be more closely related to each other than either gene is to the pgp3 gene . We speculate that the hamster pgp1 and pgp2 genes arose from a recent gene duplication event and that primates did not undergo this duplication and therefore contain only two P-glycoprotein genes.

Gan To Kagaku Ryoho, 1989 Mar, 16(3 Pt 2), 611 - 6
{Detection of multidrug resistant cells in human malignant diseases by monoclonal antibodies and strategy to eradicate resistant malignant cells}; Shimoyama M et al.; Two monoclonal antibodies of F (ab')2 form, MRK 16 and MRK 20 that recognize P-glycoprotein and P85 kD protein respectively, were useful to detect multidrug resistant cells in human lymphoma, leukemia and gastrointestinal cancer cell lines . They were classified into 4 groups: Group I (4 cell lines) was insensitive to vinca alkaloids, anthracyclines, etoposide (VP-16) and actinomycin-D (ACT-D), and reactive to MRK 16 and MRL 20 . Group II (2 cell lines) was insensitive to vincristine (VCR), but not reactive to both antibodies . Group III (3 cell lines) was insensitive to anthracyclines and VP-16, but sensitive to vinca alkaloids and ACT-D, and reactive to MRK 20 but not to MRK 16 . Group IV (all other cell lines) was sensitive to these drugs, and not reactive to both antibodies . MRK 16 detects P-glycoprotein-associated multidrug resistance (MDR), while MRK 20 detects P 85kd-associated novel MDR . These monoclonal antibodies were useful for detection of MDR cells in clinical samples.

Gan To Kagaku Ryoho, 1989 Mar, 16(3 Pt 2), 585 - 91
{Expression and function of proteins associated with multidrug resistance}; Hamada H et al.; We have identified the proteins specifically expressed in multidrug-resistant tumor cells and studied the functions of these proteins . The 170-to 180-kDa membrane glycoprotein (P-glycoprotein) is an ATPase which works as a pump molecule transporting chemotherapeutic drugs outside the resistant cells . The 22-kDa soluble protein (sorcin) is a calcium binding protein of unknown function . The 85-kDa membrane protein is specifically expressed in adriamycin-resistant cells and induced by treatment with adriamycin, suggesting a mechanism unique for adriamycin resistance . Our monoclonal antibodies to these proteins may well become useful tools for the diagnosis of clinical drug resistance.

Sci Am, 1989 Mar, 260(3), 44 - 51
Multidrug resistance in cancer; Kartner N et al.; Chemotherapy often fails because a tumor develops resistance to an array of different drugs . A single glycoprotein turns out to be responsible: it proliferates in some cells and pumps out the drugs . Now that the protein pump has been identified it may be possible to interfere with its action or to make it the target for drugs that destroy the cancer cell.

Mol Pharmacol, 1989 Mar, 35(3), 271 - 8
Role of differential drug uptake, efflux, and binding of etoposide in sensitive and resistant human tumor cell lines: implications for the mechanisms of drug resistance; Politi PM et al.; In order to study the mechanism of etoposide (VP-16) resistance in human tumor cells and to assess the role of P-170 glycoprotein in VP-16 accumulation, we have examined the uptake and efflux of VP-16 in both sensitive and multidrug-resistant MCF-7 human breast and HL60 human promyelocytic leukemia cells . The drug-resistant cells, MCF-7/ADR and HL60/ADR, were selected for resistance to adriamycin and were 200- to 250-fold resistant to VP-16 . Whereas MCF-7/ADR cells overexpress the P-170 glycoprotein and show the multidrug-resistant phenotype, HL60/ADR cells do not overexpress the P-170 glycoprotein . Although there was a 2-fold decrease in accumulation of VP-16 in MCF-7/ADR cells, this decrease did not correlate with a 250-fold resistance to the drug . VP-16 efflux was rapid and almost complete from MCF-7 cell lines and it was decreased at 4 degrees . Further, there was a significant increase in VP-16 accumulation in the MCF-7/ADR cells in the presence of glucose-free medium supplemented with sodium azide . However, no change in the pattern of VP-16 efflux was observed . Under these conditions, addition of glucose caused release of VP-16 from MCF-7/ADR cells, suggesting energy-dependent modifications in the drug binding . Coincubation of vincristine with VP-16 also increased the drug accumulation and decreased the rate of efflux of VP-16 in both sensitive and resistant MCF-7 cells, suggesting that vincristine and VP-16 may compete for similar binding and efflux mechanisms in these cell lines . In contrast, daunorubicin increased VP-16 accumulation only in the sensitive MCF-7 cell line, whereas the efflux rate of VP-16 was not significantly changed in either cell line . HL60 sensitive cells accumulated 4- to 5-fold more VP-16 than the resistant subline . Both sensitive and resistant cells showed an important noneffluxable pool of the drug, 3-fold larger for sensitive cells (79 +/- 12 versus 25 +/- 2 pmol of VP-16/mg of protein, for sensitive and resistant cells, respectively) . The efflux of VP-16 was temperature dependent only in sensitive cells . VP-16 accumulation in HL60/ADR cells was increased in glucose-free medium supplemented with sodium azide; however, the noneffluxable pool of VP-16 was not significantly changed . In contrast, although these conditions had no effect on the drug accumulation in the parental line, they caused a decrease in the noneffluxable pool of VP-16, suggesting an energy-dependent binding and retention of VP-16.(ABSTRACT TRUNCATED AT 400 WORDS)

Cancer Res, 1989 Mar 1, 49(5), 1148 - 53
Phenotypic instability of drug sensitivity in a human colon carcinoma cell line; Ferguson PJ et al.; Colon cancer is one of the tumors most refractory to treatment by chemotherapy, and this may be due to inherent phenotypic instability of such tumor cells with respect to the biochemical determinants of drug sensitivity . To test this hypothesis, a clonal human colon carcinoma cell line, clone A, was passaged in culture in the absence of selection conditions or mutagens . During this time, sensitivity to several drugs was examined, and was found to decrease 4-fold during 30 weeks of culture . Five randomly selected subclones, having never been exposed to drug or mutagen, displayed a range of sensitivities to etoposide (50% inhibitory concentrations ranging from 1.5 to 4.9 microM) and to vincristine (9-fold range), but all had the same sensitivity to methotrexate . With time these sensitivities also changed, and subsequent subclones were chosen from the lines with highest and lowest drug sensitivity . Again a wide range of phenotypes was observed . Sensitivity to vincristine ranged 14-fold and to doxorubicin 3-fold . Several biochemical determinants of drug sensitivity had a broad range of expression between cell lines . Cellular accumulation of {3H}vincristine, as well as expression of multidrug resistance protein P170 and glutathione transferase activity all varied significantly between subclonal lines . This suggests that some human colon tumors may be phenotypically unstable with respect to drug sensitivity, and this could contribute to clinical resistance to chemotherapeutic compounds.

Mol Cell Biol, 1989 Mar, 9(3), 1346 - 50
The three mouse multidrug resistance (mdr) genes are expressed in a tissue-specific manner in normal mouse tissues; Croop JM et al.; The gene responsible for multidrug resistance (mdr), which encodes the P-glycoprotein, is a member of a multigene family . We have identified distinct mdr gene transcripts encoded by three separate mdr genes in the mouse . Expression levels of each mdr gene are dramatically different in various mouse tissues . Specific mdr RNA transcripts of approximately 4.5, 5, and 6 kilobases have been detected . Each of the mdr genes has a specific RNA transcript pattern . These results should be considered in relation to understanding the normal physiological function of the mdr multigene family.

Cancer, 1989 Feb 15, 63(4), 675 - 81
Establishment of tumor cell lines from a patient with head and neck cancer and their different sensitivities to anti-cancer agents; Komiyama S et al.; The authors established five cell lines from a human head and neck tumor . The five cell lines (HC-2, HC-3, HC-4, HC-7, and HC-9) exhibited different sensitivities to Adriamycin, cisplatin, bleomycin, 5-fluorouracil, vincristine, and daunomycin . The D50 was 200 ng/ml Adriamycin (doxorubicin) for HC-7 and 45 ng/ml for HC-2 . At the inception of long-term culture (11 months) in the absence of any drug, the sensitivity to Adriamycin of HC-7-5 (subcloned from HC-7) was 3.4 times greater than that of HC-2-6 (subcloned from HC-2); by 11 months, it decreased to 1.6 times that of HC-2-6 . The cytocidal action of Adriamycin on HC-2-6 and HC-7-5 was potentiated when Adriamycin was combined with verapamil or cepharanthine . Cepharanthine also potentiated daunomycin and vincristine (VCR) against HC-2-6 and HC-7-5 cells, and it almost completely overcame drug-resistance to daunomycin and vincristine in HC-7-5/VCR, a multidrug-resistant variant isolated after long exposure to vincristine of HC-7-5 cells in culture . The cellular accumulation of {3H}-daunomycin by HC-7-5 cells was about 70% that of HC-2-6 cells . By Northern blot analysis, using a multidrug-resistance gene (mdr-1) probe, neither HC-2-6 nor HC-7-5 expressed the mdr-1 gene, but HC-7-5/VCR or other multidrug-resistant variants showed active expression of the mdr-1 gene . Differential sensitivities among the five cell lines to 5-fluorouracil, cisplatinum, and bleomycin appear to be mediated through other mechanism beside the mdr-1 gene.

Biochem Biophys Res Commun, 1989 Feb 15, 158(3), 1066 - 71
Steroid hormones inhibit binding of Vinca alkaloid to multidrug resistance related P-glycoprotein; Naito M et al.; Multidrug-resistant cells are characterized by the presence of P-glycoprotein on the plasma membrane, which binds and probably transports antitumor agents outside the cells . P-glycoprotein is also present in various normal tissues such as the adrenal gland . To investigate the physiological function of P-glycoprotein, we examined possible endogenous materials which inhibit the binding of vincristine to the resistant cell membrane . The binding was inhibited by steroid hormones, most efficiently by progesterone . Progesterone also reduced the photoaffinity labeling of P-glycoprotein by a photoactive analogue of vindesine . These results suggest that P-glycoprotein in the adrenal gland could have a role in the secretion of steroid hormones.

Blood, 1989 Feb 15, 73(3), 747 - 52
Immunohistochemical detection and quantitation of P-glycoprotein in multiple drug-resistant human myeloma cells: association with level of drug resistance and drug accumulation; Dalton WS et al.; Using several multiple drug-resistant human myeloma cell lines as standards, we developed an immunohistochemical staining technique and means of quantitating P-glycoprotein in individual myeloma cells . The level of staining intensity for P-glycoprotein in individual myeloma cells was quantitated by measuring the average optical density of each cell with a microscopic computerized cell analysis system . Using this system, we observed that the level of P-glycoprotein for individual cells within a cell population of known drug sensitivity was very homogeneous (coefficient of variation less than or equal to 13%) . Analysis of cell lines with gradually increasing levels of multidrug resistance (8226/S, 8226/Dox6 and 8226/Dox40) demonstrated a close association between the level of resistance to doxorubicin, defined by the mean lethal dose (D0) and the amount of P-glycoprotein on individual cells determined by the optical density (r = 0.82, P less than 0.0005) . Intracellular doxorubicin (DOX) accumulation in the individual cell lines was inversely related to the level of drug resistance expressed as D0 . P-glycoprotein was also detected in the marrow-derived myeloma cells of patients with drug refractory disease using immunohistochemical staining . The amount of P-glycoprotein in the cells of one patient was directly compared to the amount found in the simultaneously stained standard cell lines (8226/Dox6 and 8226/Dox40) by comparing the optical densities for individual cells . Using this immunohistochemical technique to detect and quantitate P-glycoprotein in patient myeloma cells and comparing it to standard multidrug resistant myeloma cell lines may be of value in determining the contribution of P-glycoprotein to clinical drug resistance in patients with multiple myeloma.

J Natl Cancer Inst, 1989 Feb 15, 81(4), 290 - 4
Cross-resistance to the lipid-soluble antifolate trimetrexate in human carcinoma cells with the multidrug-resistant phenotype; Assaraf YG et al.; We are studying mechanisms of resistance to hydrophilic and lipophilic antifolates in cultured mammalian cells . We determined the cytotoxicity of methotrexate and the lipid-soluble antifolate trimetrexate to various human carcinoma cells and their doxorubicin-resistant sublines . These multidrug-resistant cells were 17-fold to 26-fold more resistant to trimetrexate but as sensitive to methotrexate as their parental cells . Verapamil and quinidine, which are known to modulate the degree of pleiotropic drug resistance, reversed the cross-resistance to trimetrexate but did not alter methotrexate toxicity in multidrug-resistant cells . By flow cytometry, we show that multidrug-resistant Chinese hamster ovary cells retained eightfold more fluorescein-methotrexate bound to dihydrofolate reductase upon competition with trimetrexate than the drug-sensitive cells did . However, methotrexate displaced fluorescein-methotrexate equally well in sensitive and multidrug-resistant cells . Furthermore, verapamil produced dose-dependent displacement of fluorescein-methotrexate from the multidrug-resistant cells in the presence of low concentrations of trimetrexate but had no significant effect on displacement of fluorescein-methotrexate from sensitive cells . Hamster cells that overproduce dihydrofolate reductase by 93-fold were 145-fold and 96-fold more resistant to methotrexate and trimetrexate, respectively, than their sensitive parental cells . This form of antifolate resistance was not altered by verapamil or quinidine . We conclude that the cross-resistance to trimetrexate in cells that do not overproduce dihydrofolate reductase is associated with the multidrug-resistant phenotype . Possible implications of trimetrexate resistance in cancer chemotherapy are discussed.

Cancer Res, 1989 Feb 1, 49(3), 677 - 80
Cyclosporin A and verapamil enhancement of daunorubicin-produced nucleolar protein B23 translocation in daunorubicin-resistant and -sensitive human and murine tumor cells; Sweet P et al.; It has recently been shown that anthracycline antibiotic-resistant tumor cells are less responsive to daunorubicin-stimulated B23 nucleolar phosphoprotein translocation than drug-sensitive cells . Since cyclosporin A and verapamil reverse primary acquired and secondary cross-resistance to daunorubicin, we investigated the effect of these agents on nucleolar B23 translocation in sensitive and resistant tumors . We compared modified to baseline B23 phosphoprotein distribution between predominantly nucleolar, mixed nucleolar-nuclear, or nuclear immunofluorescence using a monoclonal anti-B23 antibody in parental drug-sensitive and multidrug-resistant acute lymphatic leukemia and in daunorubicin-sensitive and -resistant murine hepatoma . Our experiments show that cyclosporin A and verapamil alone have no effect on B23 phosphoprotein translocation, but that the addition of either agent to sensitive parental or resistant tumor sublines markedly enhances daunorubicin-stimulated translocation . This effect correlates with the correction of impaired daunorubicin inhibition of RNA synthesis by cyclosporin A and verapamil in the resistant sublines . Our observations suggest that nucleolar B23 phosphoprotein is an important site in the modulation of anthracycline antibiotic antitumor activity.

FEMS Microbiol Lett, 1989 Feb, 48(3), 275 - 8
Loss of plasmid linked antibiotic resistance in Escherichia coli on treatment with some phenolic compounds; Lakshmi VV et al.; Treatment of E . coli 46R641 cells carrying multidrug resistant TP181 plasmid with phenolic compounds namely bharangin, gossypetin, gossypin and quercetin lead to the concurrent loss of all the six plasmid linked antibiotic resistance markers . Among these test compounds, bharangin exhibited higher efficiency in curing of plasmids belonging to IncF1, H1 and X groups . However, multicopy plasmids with ColE1 origin of replication were totally refractory to these curing agents under similar conditions . The curing activity of the test compounds was much higher as compared to some of the known curing agents.

Biochem Pharmacol, 1989 Feb 1, 38(3), 519 - 27
Analysis of structural features of dihydropyridine analogs needed to reverse multidrug resistance and to inhibit photoaffinity labeling of P-glycoprotein; Nogae I et al.; Synthetic dihydropyridine analogs were screened to determine whether they would reverse multidrug resistance of a multidrug-resistant human KB carcinoma cell line, KB-C1 . Among twenty-four dihydropyridine analogs examined, thirteen almost completely overcame drug resistance (group A), nine partially overcame resistance (group B) and two did not reverse resistance (group C) . The twenty-two compounds that reversed drug-resistance (groups A and B) were hydrophobic dihydropyridine derivatives . Three compounds that reversed resistance, NK-113, NK-138 and NK-194, increased the accumulation of {3H}vincristine in the resistant KB-C1 cells, but not in the parental KB cells, nor in a revertant cell line, KB-C1-R2 . NK-101 (group C), which did not reverse resistance, had no effect on drug accumulation . Enhanced efflux of vincristine from the resistant cells was inhibited completely by NK-194, but NK-194 did not affect vincristine influx . Nine of the twenty-four compounds were screened to determine whether they inhibited photoaffinity labeling of the cell surface protein gp170 (P-glycoprotein) in KB-C1 cells by N-(p-azido-{3-125I}-salicyl)-N'-beta-aminoethylvindesine {( 125I}NASV) . All five compounds of group A, NK-138, NK-194, NK-200, NK-203 and NK-220, inhibited the photoaffinity labeling of gp170 at less than 10-100 microM, whereas NK-113 and NK-196 of group B inhibited the labeling at 100-200 microM . By contrast, NK-101 and NK-102 of group C did not inhibit labeling even at 2000 microM . These studies confirm the relationship among reversal of multidrug resistance, decreased efflux of vincristine, and inhibition of {125I}NASV labeling of P-glycoprotein.

Cancer Res, 1989 Feb 1, 49(3), 511 - 5
Potentiation of doxorubicin cytotoxicity by buthionine sulfoximine in multidrug-resistant human breast tumor cells; Dusre L et al.; Resistance to antineoplastic drugs is a major problem in the clinical management of cancer . Previous studies have demonstrated that the cytotoxicity of certain anticancer drugs is increased by lowering the glutathione (GSH) levels with buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase . In this study we report that the resistance to doxorubicin, an anthracycline antibiotic and the most active agent in the treatment of breast cancer, can be partially reversed by exposing MCF-7 doxorubicin-resistant breast tumor cells (MCF-7/ADRR) to minimally cytotoxic doses of BSO . We found that the BSO treatment (50 microM, 48 h) of MCF-7/ADRR cells resulted in 80 to 90% depletion in total GSH concentrations . The toxicity of doxorubicin, as determined by growth inhibition and clonogenic assays, was significantly potentiated in the BSO-treated GSH-depleted cells relative to control breast tumor cells, and a dose-modifying factor of 5 to 7 was observed . Since the cytotoxicity of doxorubicin has been associated with its ability to undergo enzymatic activation and to form hydroxyl (OH) radicals in this cell line, we also quantitated the OH formation in the BSO-treated and untreated MCF-7/ADRR cells using electron spin resonance spintrapping techniques . These results show that doxorubicin stimulated at least 2-fold more OH formation in the tumor cells after GSH levels were decreased by 90% . These results indicate that GSH plays an important role in modulating doxorubicin-induced OH formation via the scavenging of hydrogen peroxide by glutathione peroxidase and thus partially protects MCF-7/ADRR cells from the cytotoxic effect of doxorubicin.

J Histochem Cytochem, 1989 Feb, 37(2), 159 - 64
Immunohistochemical localization in normal tissues of different epitopes in the multidrug transport protein P170: evidence for localization in brain capillaries and crossreactivity of one antibody with a muscle protein; Thiebaut F et al.; Using peroxidase immunohistochemistry, we examined the distribution of P170, a multidrug transport protein, in normal tissues by use of two different monoclonal antibodies (MAb) . MAb MRK16 is a MAb that has been shown to react with an epitope in P170 located on the external face of the plasma membrane of multidrug-resistant human cells . MAb C219 has been shown to react with P170 in many mammalian species, and detects an epitope located on the cytoplasmic face of the plasma membrane . Using MRK16, we have previously described the localization of P170 on the bile canalicular face of hepatocytes, the apical surface of proximal tubular cells in kidney, and the surface epithelium in the lower GI tract in normal human tissues . In this work, we report that MRK16 also detects P170 in the capillaries of some human brain samples . A similar pattern was found using MAb C219 in rat tissues . in addition, MAb C219 showed intense localization in selected skeletal muscle fibers and all cardiac muscle fibers in rat and human tissues . ATPase cytochemistry showed that these reactive skeletal muscle fibers were of the type I (slow-twitch) class . Other additional sites of C219 reactivity in rat tissues were found in pancreatic acini, seminal vesicle, and testis . Electrophoretic gel immunoblotting showed two protein bands reactive with MAb C219 . In liver, MAb C219 reacted with a approximately 170 KD band . In skeletal and cardiac muscle, MAb C219 reacted with a approximately 200 KD band which migrated in the same position as myosin . This band also reacted with an antibody to skeletal muscle myosin . This result suggests that C219 may crossreact with the heavy chain of muscle myosin in cardiac and skeletal muscle . Because MAb C219 reacts with proteins other than P170, it should be used with caution in studies of multidrug resistance.

J Natl Cancer Inst, 1989 Jan 18, 81(2), 116 - 24
Expression of a multidrug resistance gene in human cancers; Goldstein LJ et al.; Many cancers have been cured by chemotherapeutic agents . However, other cancers are intrinsically drug resistant, and some acquire resistance following chemotherapy . Cloning of the cDNA for the human MDR1 gene (also known as PGY1), which encodes the multidrug efflux protein P-glycoprotein, has made it possible to measure levels of MDR1 RNA in human cancers . We report the levels of MDR1 RNA in greater than 400 human cancers . MDR1 RNA levels were usually elevated in untreated, intrinsically drug-resistant tumors, including those derived from the colon, kidney, adrenal gland, liver, and pancreas, as well as in carcinoid tumors, chronic myelogenous leukemia in blast crisis, and cell lines of non-small cell carcinoma of the lung (NSCLC) with neuroendocrine properties . MDR1 RNA levels were occasionally elevated in other untreated cancers, including neuroblastoma, acute lymphocytic leukemia (ALL) in adults, acute nonlymphocytic leukemia (ANLL) in adults, and indolent non-Hodgkin's lymphoma . MDR1 RNA levels were also increased in some cancers at relapse after chemotherapy, including ALL, ANLL, breast cancer, neuroblastoma, pheochromocytoma, and nodular, poorly differentiated lymphoma . Many types of drug-sensitive and drug-resistant tumors, including NSCLC and melanoma, contained undetectable or low levels of MDR1 RNA . The consistent association of MDR1 expression with several intrinsically resistant cancers and the increased expression of the MDR1 gene in certain cancers with acquired drug resistance indicate that the MDR1 gene contributes to multidrug resistance in many human cancers . Thus, evaluation of MDR1 gene expression may prove to be a valuable tool in the identification of individuals whose cancers are resistant to specific agents . The information may be useful in designing or altering chemotherapeutic protocols in these patients.

Cancer Res, 1989 Jan 15, 49(2), 340 - 4
Antitumor activity and murine pharmacokinetics of parenteral acronycine; Dorr RT et al.; The lipophilic antitumor alkaloid acronycine (ACRO) was solubilized in the cosolvent system used for etoposide . ACRO in this etoposide diluent (VPD) was found to be cytotoxic (less than or equal to 50% colony formation in soft agar) in fresh human tumors from patients with renal cell cancer, ovarian cancer, uterine cancer, and metastatic tumors of unknown primary . In P-glycoprotein-positive, multidrug-resistant (MDR) cell lines, ACRO in VPD was active in MDR Chinese hamster ovary cells but not against MDR L1210 murine leukemia cells, 8226 human myeloma cells, or human CCRF-CEM lymphoblasts . In mice, ACRO in VPD was active in two solid tumor models and an i.p . MOPC-315 plasmacytoma model . ACRO i.p . in 10% VPD (v/v%) produced significant tumor growth delays in (a) nude mice bearing human MCF-7 breast cancer xenografts and (b) C57BL mice bearing colon 38 tumor . In MOPC-315-bearing mice, a single i.p . ACRO dose of 25 mg/kg was as effective as melphalan (15 mg/kg) at prolonging life span . Finally, ACRO pharmacokinetics was evaluated in mice given single 25-mg/kg doses i.p . or p.o . The oral bioavailability of an ACRO solution in VPD was only 50% but both i.p . and p.o . regimens achieved plasma levels greater than 1.0 micrograms/ml . The plasma half-life was just under 2 h . These results show that parenteral ACRO in VPD comprises a cytotoxic antitumor agent with improved bioavailability over p.o . administration . ACRO is active in vitro against several human solid tumors but is cross-resistant in 3 of 4 MDR tumor cell lines . The prior clinical activity of p.o . ACRO in myeloma and the new results in MOPC-315 plasmacytomas in mice suggest that ACRO in VPD could have activity against human multiple myeloma.

J Biol Chem, 1989 Jan 15, 264(2), 782 - 8
Progesterone interacts with P-glycoprotein in multidrug-resistant cells and in the endometrium of gravid uterus; Yang CP et al.; P-Glycoprotein (P-GP) plays a pivotal role in maintaining the multidrug-resistant (MDR) phenotype . This membrane glycoprotein is overproduced in MDR cells and the endometrium of the mouse gravid uterus (Arceci, R.J., Croop, J.M., Horwitz, S.B., and Housman, D . (1988) Proc . Natl . Acad . Sci . U.S.A . 85, 4350-4354) . This latter observation and an interest in endogenous substrates for P-GP led to a study of the interaction of steroids with P-GP found in the endometrium of the mouse gravid uterus and in MDR cells derived from the murine macrophage-like cell J774.2 . {3H}Azidopine labeling of P-GP from these two sources was inhibited by various steroids, particularly progesterone . Progesterone also markedly inhibited {3H}vinblastine binding to membrane vesicles prepared from MDR cells, enhanced vinblastine accumulation in MDR cells, and increased the sensitivity of MDR cells to vinblastine . In addition, we have demonstrated that the hydrophobicity of a steroid is important in determining its effect on inhibition of drug binding to P-GP . It is concluded that progesterone, a relatively nontoxic endogenous steroid, interacts with P-GP and is capable of reversing drug resistance in MDR cells.

Cancer Chemother Pharmacol, 1989, 23(3), 140 - 4
Comparative effectiveness of mitoxantrone and doxorubicin in overcoming experimentally induced drug resistance in murine and human tumour cell lines in vitro; Hill BT et al.; Using a range of cell lines of murine and human tumour origin in which relatively modest levels (2- to 17-fold) of drug resistance have been selected in vitro by exposure to a range of standard antitumour drugs, we compared the cytotoxic effects of doxorubicin (DOX) and mitoxantrone (MITO) . In general, significantly lower concentrations of MITO than of DOX were required to achieve comparable cytotoxicity, confirming previously published data . MITO appears more generally effective against the murine L5178Y drug-resistant sublines than DOX, although there was no expression of collateral sensitivity to this newer agent . In the various human tumour lines there was a lack of cross-resistance to both DOX and MITO in two 5-fluorouracil (FU)-resistant lines and one of two cisplatin (CDDP)-resistant cells, but cross-resistance was expressed in one subline resistant to vincristine (VCR) and two etoposide (VP-16)-resistant sublines . One murine and two human DOX-resistant sublines were effectively killed by MITO, whilst DOX proved effective against the human MITO-resistant subline . This apparent lack of cross-resistance between DOX and MITO in these resistant sublines expressing low levels of resistance in vitro therefore appears to contrast with previous reports involving highly multidrug-resistant DOX-selected sublines . However, since the latter lines generally exhibited profound cross-resistance to VCR and definite cross-resistance to VP-16, this may at least in part dictate their responses to MITO . Therefore, attempts to use experimentally derived drug-resistant sublines for preclinical drug screening should be approached with caution, since patterns of drug response appear to be influenced by the level of drug resistance expressed . The need remains to determine which type of model system provides the most relevant clinical information.

Cancer Chemother Pharmacol, 1989, 23(1), 19 - 25
Association of sorcin with drug resistance in L1210 cells; Roberts D et al.; L1210 sublines independently selected for resistance to teniposide (VM-26), etoposide (VP-16), doxorubicin (DOX), dactinomycin (DACT), or vincristine (VCR) express an anionic, 22-kDa protein that is not observed in extracts of parental L1210 cells . Antibody raised against sorcin, an acidic calcium-binding protein overproduced in many other cells resistant to these agents, cross-reacts with the 22-kDa polypeptide . The levels of the 22-kDa protein (sorcin) increase with the relative levels of drug resistance of the L1210 sublines . The appearance of sorcin in these various sublines further supports the notion that the overproduction of this protein is related to the general phenomenon of multidrug resistance rather than to specific drug resistance and that selection for resistance to teniposide produces L1210 sublines with multidrug resistance.

Cancer Chemother Pharmacol, 1989, 24(6), 367 - 70
Reversal of multidrug resistance by new dihydropyridines with lower calcium antagonistic activity; Yoshinari T et al.; BS compounds, a series of new dihydropyridines, successfully overcame multidrug resistance in P388/ADR cells in vitro . These agents synergistically potentiated the cytotoxicity of Adriamycin to P388/ADR cells at a concentration of 1-2 microM, whereas they showed hardly any synergistic effect in the parental cell line (P388/S) at the same concentration . They inhibited the active drug efflux in P388/ADR cells as well as the binding of {G-3H}-vinblastine to membrane vesicles from P388/ADR, which was increased in resistant P388 cells as compared with parental cells . Besides, unlike the activity of clinically used calcium antagonists, the calcium antagonistic activity associated with BS compounds was very weak: their arterial relaxation activity was less than 21% of that of verapamil . These data suggest that BS compounds specifically overcome multidrug resistance without the serious hypotensive side effects that accompany the use of verapamil or other calcium antagonists.

Pediatr Pathol, 1989, 9(2), 117 - 30
Immunophenotyping to detect and characterize acute lymphocytic leukemia in testicular biopsies; Verdi CJ et al.; This study establishes the utility of immunophenotyping testicular biopsy specimens in patients with acute lymphoid leukemia . The value of immunophenotyping in detecting or excluding leukemic testicular infiltration is demonstrated in six children with acute lymphocytic leukemia . A panel of monoclonal antibodies was employed on snap-frozen testicular biopsies, allowing both detection and immunologic characterization of four neoplastic lymphocytic infiltrates . Two samples were proven both histologically and phenotypically negative for leukemic infiltration . One of the four leukemic cases was clinically silent and might have escaped detection except for phenotyping . One leukemic infiltrate was also suspected to possess a multidrug-resistant phenotype (p-glycoprotein +); the latter possibility was excluded by an absence of reactivity with anti-p-glycoprotein monoclonal antibody . Thus, three clinically useful applications are demonstrated: (1) confirmation of testicular leukemic relapse, gaining assertion in histologically uncertain cases; (2) exclusion of clinically suspected disease relevant to cessation of therapy, and (3) detection/exclusion of drug-resistant phenotypes . Unexpectedly, we found expression of plasma cell-associated antigen in testicular germ cells, which may prove to be diagnostically useful in the future evaluation of germ cell tumors.

Eksp Onkol, 1989, 11(4), 70 - 3
{Phosphorus-containing metabolites in the cells of anthracycline-resistant strains of leukemia P388}; Shiriaeva OA et al.; The method of 31P nuclear magnetic resonance has been used to study in vivo the level of phosphorus-containing metabolites in cells of two strains of murine leukemia P388 with the phenotype of the multidrug resistance and in cells of the parent strain . Cells of both resistant strains showed a depressed level of phosphomonoesters in comparison with the parent one . The influence of rubomycin and emoksil on the level of phosphorus-containing metabolites of drug-resistant and -sensitive strains has been evaluated . The drugs were established not to affect practically the pool of these metabolites of the resistant strains . Both drugs significantly increased the pool of phosphomonoesters in the parent strain cells.

Cancer Chemother Pharmacol, 1989, 24(4), 219 - 24
Activity of the pyrazoloacridines against multidrug-resistant tumor cells; Sebolt J et al.; A series of 2-aminoalkyl-5-nitropyrazolo {3,4,5-kl}acridines (pyrazoloacridines) were tested in vitro against a panel of multidrug-resistant cell lines comprising Adriamycin-resistant P388 leukemia, B16 melanoma, and mammary adenocarcinoma 16c . This new class of anticancer agents, particularly the 9-substituted methoxy derivatives, exhibited significant activity against all of the lines tested . The degree of cross-resistance to these compounds ranged from zero to 8-fold in the 138-fold Adriamycin-resistant P388/ADR line and was greatly diminished in the B16/ADR and 16c/ADR lines . Selected pyrazoloacridines were subsequently tested in vivo against B16 and B16/ADR cells established as solid tumors from the tissue culture line and shown to retain a significant degree of Adriamycin resistance . Whereas the B16/ADR line exhibited 2 logs less net tumor-cell kill than the B16 parent in response to Adriamycin treatment, the resistant tumor was completely sensitive to the pyrazoloacridines tested and proved in some experiments to be collaterally sensitive . The favorable activity of the pyrazoloacridines against these Adriamycin-resistant tumor lines points to the potential efficacy of these compounds against multidrug-resistant tumors encountered clinically.

Cancer Chemother Pharmacol, 1989, 23(5), 296 - 300
Effect of cyclosporin A on daunorubicin accumulation in multidrug-resistant P388 leukemia cells measured by real-time flow cytometry; Nooter K et al.; We investigated the mode of action of cyclosporin A (Cy-A) as a modifier of multidrug resistance in P388 mouse leukemia cells . A fluorescence-activated flow cytometer (FCM) was modified with a flow-through cuvette to allow continuous on-line monitoring of daunorubicin uptake in vitro . The addition of Cy-A to multidrug-resistant P388/R cells at steady-state daunorubicin uptake, led to a dose-dependent increase in cellular daunorubicin accumulation, as measured by FCM and high-performance liquid chromatography (HPLC) . A linear relationship was found between the daunorubicin concentration in the incubation medium and the Cy-A concentration required for optimal stimulation of cellular anthracycline accumulation . The results of a cytotoxicity assay indicated that Cy-A completely restored the chemosensitivity of the P388/R cells . Intracellular Cy-A measurements in P388/S and P388/R cells showed that P388/R cells accumulated significantly less Cy-A than P388/S cells . Relatively high daunorubicin concentrations could not restore that accumulation defect . These results suggest that Cy-A promotes cellular anthracycline accumulation by competing for an outward drug-transport system that operates in multidrug-resistant cells.

Cancer Commun, 1989, 1(6), 359 - 65
Differential increases in glutathione S-transferase activities in a range of multidrug-resistant human tumor cell lines; Whelan RD et al.; Modified glutathione S-transferase (GST) activity was detected in MCF-7 cell lines selected in vitro for modest levels of resistance (2.6- to 13.7-fold) not only to Adriamycin but also to Novantrone, vincristine (VCR), and etoposide (VP-16) . Western blot analysis revealed a qualitative increase in the expression of the pi class GST isozyme and some apparent down regulation of the mu class in these resistant lines relative to the parental line . Overexpression of GST pi protein was also observed after in vitro exposure of MCF-7 cells to fractionated x-irradiation, which resulted in a subline that exhibited 5-fold resistance to VCR and 3-fold resistance to VP-16 . Northern blot analysis provided qualitative evidence that these changes in GST pi and mu appeared to be at the transcriptional level in these VCR- and VP-16-resistant MCF-7 sublines . In contrast, similarly selected VCR-resistant, VP-16-resistant, and x-ray-pretreated-resistant sublines, derived from the SuSa cell line established from a human testicular teratoma, did not exhibit significant alterations in GST isozyme profiles compared with their parental cell lines . All parental and resistant SuSa lines that were studied expressed the GST pi isozyme constitutively . Total glutathione content and glutathione peroxidase activities were not modified in parallel with the GST alterations in these human tumor drug resistant sublines . These data suggest that the increased cellular GST activity was associated with selection of resistance to many of the drugs associated with the multidrug resistant phenotype in MCF-7 sublines but that this was not the case for the other human tumor cell line (SuSa) tested.

Acta Oncol, 1989, 28(6), 877 - 81
Anthracycline resistance; Friche E et al.; The major limitation of the usefulness of anthracyclines is the development of drug resistance . Most of the data related to anthracycline resistance has been obtained in experimental animal tumors or in cultured human cell lines . An important characteristic obtained in these models is the common finding of cross-resistance to a series of drugs structurally unrelated to the anthracyclines and with a mechanism of action which apparently differ from that of the anthracyclines . This phenomenon is defined as pleiotropic drug resistance or multidrug resistance (MDR) . The cross-resistance demonstrated includes the vinca alkaloids, actinomycin D, colchicin and the epipodophyllotoxins . The best documented mechanism of resistance and mechanism of cross-resistance is decreased cellular drug accumulation . Several findings indicate that the underlying factor of the altered net uptake is an active drug extrusion in the resistant cells . Another important characteristic is the demonstration of a cell surface protein with a molecular weight of about 170,000 (GP 170) . Genetic and biochemical evidence indicate that the MDR phenotype in both animal and human cells result from overexpression of the MDR-1 gene which encodes GP 170 . Some findings suggest that GP 170 is a transport protein for cytotoxic hydrophobic drugs like the anthracyclines . Other findings suggest that this gene is also associated with intrinsic (natural) anthracycline resistance.

Arch Gynecol Obstet, 1989, 244(2), 123 - 8
Detection of drug resistance in human ovarian carcinoma; Volm M et al.; Drug-resistant cancer cells with the multidrug-resistance phenotype show overexpression of P-glycoprotein, and we therefore tested carcinoma tissue from five patients with stage III or IV ovarian cancer for P-glycoprotein using 265/F4 and C 219 monoclonal antibodies, prepared against membrane glycoproteins in colchicine-resistant CHO cells . Using immunofluorescence and immunoblotting techniques, one of the tumors showed a positive reaction . Using the pcDR 1.5 clone we found that the same cancer tissue had elevated expression of the genes responsible for multidrug resistance . The demonstration of elevated P-glycoprotein in ovarian carcinomas indicates that P-glycoprotein overexpression is not limited to experimental tumor models.

Mol Cell Biol, 1989 Jan, 9(1), 109 - 15
Autonomously replicating episomes contain mdr1 genes in a multidrug-resistant human cell line; Ruiz JC et al.; Gene amplification in human tumor cells is frequently mediated by extrachromosomal elements (e.g., double minute chromosomes {DMs}) . Recent experiments have shown that DMs can be formed from smaller, submicroscopic circular precursors referred to as episomes (S . M . Carroll, M . L . DeRose, P . Gaudray, C . M . Moore, D . R . Needham-Vandevanter, D . D . Von Hoff and G . M . Wahl, Mol . Biol . 8:1525-1533, 1988) . To investigate whether episomes are generally involved as intermediates in gene amplification, we determined whether they mediate the amplification of the mdr1 gene, which when overexpressed engenders cross resistance to multiple lipophilic drugs . A variety of methods including electrophoresis of undigested DNAs in high-voltage gradients, NotI digestion, and production of double-strand breaks by gamma irradiation were used to distinguish between mdr1 sequences amplified on submicroscopic circular molecules and those amplified within DMs or chromosomal DNA . The gamma-irradiation procedure provides a new method for detecting and determining the size of circular molecules from 50 kilobases (kb) to greater than 1,000 kb . These methods revealed that some of the amplified mdr1 genes in vinblastine-resistant KB-V1 cells are contained in supercoiled circular molecules of approximately 600 and approximately 750 kb . Analysis of the replication of these molecules by a Meselson-Stahl density shift experiment demonstrated that they replicate approximately once in a cell cycle . The data lend further support to a model for gene amplification in which DMs are generally formed from smaller, autonomously replicating precursors.

Cancer Commun, 1989, 1(1), 35 - 43
Activity of cyclosporin A and a non-immunosuppressive cyclosporin against multidrug resistant leukemic cell lines; Hait WN et al.; Cyclosporin A (CsA) has been shown to increase the sensitivity of multidrug resistant (MDR) cells to chemotherapeutic agents . Although the concentration of drug required to produce this effect is clinically achievable, the use of this drug would be hampered by significant immunosuppression . We report a comparison of the effects of 11-methyl-leucine cyclosporin (11-met-leu CsA), a non-immunosuppressive homolog to the parent drug, on MDR cell lines . Both cyclosporins sensitized resistant cell lines to doxorubicin, including P388 murine leukemia and GM 3639 human T-cell leukemia . The action of the cyclosporins was more pronounced with resistant cells than with sensitive ones . 11-Met-leu CsA was less potent than, but equally effective as, the parent drug . Both agents increased the intracellular accumulation and retention of doxorubicin in MDR cells . The sensitization caused by the cyclosporins was independent of their effects on cyclophilin, calmodulin, and protein kinase C . Furthermore, there were no differences in the binding of labelled CsA to MDR cells compared to the binding to sensitive cells, suggesting that P-glycoprotein was also not the molecular site of action . These studies demonstrate that a non-immunosuppressive cyclosporin can modulate multidrug resistance and suggest its further evaluation for use in clinical trials.

Cancer Commun, 1989, 1(1), 21 - 7
Further characterization of drug-sensitivity and cross-resistance profiles of cloned cell lines of Adriamycin-sensitive and -resistant P388 leukemia; Seneviratne C et al.; The drug-sensitivity and cross-resistance profiles of cloned cell lines of Adriamycin-sensitive and -resistant P388 murine leukemia have been further characterized . A range of drug sensitivity that was more than 50,000-fold was observed for Adriamycin-sensitive cells; the most potent cytotoxic agent was the Adriamycin analog, 3'-(3-cyano-4-morpholinyl)-3'-deamino adriamycin, and the least active compound was vinblastine . Adriamycin-resistant cells, which express the multidrug resistance phenotype, were cross-resistant to the DNA topoisomerase II interactive drugs: actinomycin D, daunorubicin, mitoxantrone, etoposide, and 4'-(9-acridinyl-amino)methanesulfon-m-anisidide, to the vinca alkaloids: vincristine and vinblastine, and to colchicine but not to the Adriamycin analog, 3'-(3-cyano-4-morpholinyl)-3'-deamino adriamycin or the alkylating agent, melphalan . These findings are consistent with other studies suggesting that 3'-(3-cyano-4-morpholinyl)-3'-deamino adriamycin acts as an alkylating agent . Studies with DNA topoisomerase II interactive agents, including mitoxantrone, the DNA intercalator, and etoposide, the epipodophyllotoxin, showed that, as with Adriamycin, cytotoxicity correlated closely with the formation of DNA double-strand breaks.

Cancer Commun, 1989, 1(3), 145 - 9
Exploring multidrug resistance using rhodamine 123; Kessel D; Accumulation of the dye rhodamine 123 was characterized by fluorescence in murine leukemia P388 cells and the Adriamycin-resistant subline, P388/ADR . Dye uptake by P388 cells was a slow, temperature-sensitive process, and involved binding at relatively hydrophobic and heterogeneous loci . Dye uptake by P388/ADR cells was temperature-insensitive, a steady state was quickly achieved, and the fluorescence emission spectrum suggested a relatively hydrophilic dye-binding site . Treatment of P388/ADR cells with verapamil resulted in enhanced accumulation of dye at hydrophobic loci . The fluorescence studies suggest that this dye may not reach the cytoplasm of P388/ADR cells, a result which may have implications with regard to the mechanism of multidrug resistance.

Cancer Commun, 1989, 1(2), 141 - 4
Expression of a multidrug resistance gene in blast crisis of chronic myelogenous leukemia; Pirker R et al.; Three of four patients with chronic myelogenous leukemia in blast crisis were found to express the multidrug resistance (MDR1) gene in their blast cells . Expression of the MDR1 gene may contribute to the poor response of these patients to chemotherapy.

Cancer Commun, 1989, 1(2), 133 - 9
Glutathione, glutathione S-transferases, and related redox enzymes in Adriamycin-resistant cell lines with a multidrug resistant phenotype; Schisselbauer JC et al.; Friend erythroleukemia cells (FLC) selected by exposure to Adriamycin (doxorubicin) express an approximate 2.5-fold (ARN1) or 13-fold (ARN2) resistance to the drug with various degrees of cross-resistance to other anthracyclines, vinca alkaloids, and epipodophyllotoxins . Because the redox cycling of the quinone moiety of Adriamycin is known to produce oxidative stress, however, an analysis of glutathione (GSH) and related enzyme systems was undertaken in the wild-type and selected resistant cells . In ARN1 and ARN2, superoxide dismutase (SOD) and catalase activities were slightly decreased, intracellular GSH and GSH reductase were essentially unchanged, and total GSH peroxidase, glutathione S-transferase (GST), and DT-diaphorase activities were slightly elevated . In each case there was no stoichiometric relationship between degree of resistance and level of activity . GST isozymes were purified from each cell line by HPLC GSH affinity column chromatography . Two-dimensional gel electrophoresis and western blot immunoreactivity against a battery of GST isozyme polyclonal antibodies determined that both the resistant and sensitive cells expressed isozymes of the alpha, pi, and mu classes (alternative murine nomenclature: M1, M2, M3) . Of significance, both ARN1 and ARN2 cell lines expressed a unique alpha subunit which was absent from the parent FLC cell line . This isozyme presumably accounted for the increased GSH peroxidase activity (cumene hydroperoxide as substrate) found in ARN1 and ARN2 and may play a role in the small incremental resistance to melphalan found for both resistant lines . Expression of the isozyme was not stoichiometric with respect to degree of resistance . The presence of this isozyme may contribute to the resistant phenotype or may be the consequence of a more general cellular response to oxidative stress.

Adv Enzyme Regul, 1989, 29, 231 - 45
Resistance to methotrexate and multidrug resistance in childhood malignancies; Niethammer D et al.; Resistance to drugs, either primary or acquired, is a main problem in cancer chemotherapy . The paper summarizes our results in regard to resistance to methotrexate and multiple drug resistance in human cell lines of pediatric malignancies and in children with resistant cancer . In cell lines as well as in children we could demonstrate amplification of the gene coding for dihydrofolate reductase as a cause for resistance to MTX . Procedures to overcome drug resistance such as treatment with high dose MTX and leucovorin rescue are discussed . The increased expression of the mdrl gene coding for the P-glycoprotein is related to multidrug resistance . This could be shown in cell lines and in children . The expression decreased when the drug, used for induction of resistance, was omitted for a few weeks from the cell culture medium . Readdition of the drug caused a rapid increase of expression . For the first time data in children are presented which demonstrate the amplification of the gene coding for dihydrofolate reductase or increased expression of the mdrl gene as cause of drug resistance . The clinical implications of these findings are discussed.

Prog Clin Biol Res, 1989, 316B, 171 - 81
Induced differentiation of murine erythroleukemia cells (MELC) by polar compounds: marked increased sensitivity of vincristine resistant MELC; Marks PA et al.; Hexamethylene bisacetamide (HMBA) is a most effective compound as an inducer of MELC differentiation . HMBA-mediated terminal differentiation of MELC is a multistep process . There is a latent period during which a number of changes occur including the appearance of Ca2+ and phospholipid independent PKC activity in the cytosol, and modulation in expression of several genes, including c-myc, c-myb, c-fos and the p53 genes . During this latent period there is neither detectable commitment to terminal differentiation (including terminal cell division) or increased transcription of the globin genes . HMBA-mediated commitment to terminal differentiation is first detected at about 12 hr and increases in a stochastic fashion, until over 95% of the population has been recruited to terminal differentiation by 48 to 60 hr . Commitment is associated with persistent HMBA-mediated suppression of c-myb gene expression . By 36 to 48 hr, transcription of the globin genes has increased by 10 to 30 fold, whereas transcription of rRNA genes is suppressed . The steroid, dexamethasone, and the tumor promotor, phorbol-12-myristate-13-acetate, suppress HMBA-induced MEL cell terminal differentiation . The evidence indicates that these agents act at a late step during the latent period . Recently, we showed that MELC variants selected for resistance to vincristine have a marked increased sensitivity to HMBA . Compared to the parental MELC strains, vincristine resistant MELC are: A) responsive to 1/5 to 1/10 the concentration of HMBA; B) induced to terminal differentiation without a latent period and C) resistant to inhibition of HMBA induced terminal differentiation by dexamethasone or tumor promotor . The vincristine resistant MELC have characteristics of the multidrug resistant phenotype . A number of independently derived vincristine resistant MELC lines show similar altered response to HMBA . These findings suggest that vincristine resistance leads to a constitutive expression of a factor or factors induced by HMBA in vincristine sensitive (wild type) MELC during the latent period and which are essential to the transition to terminal differentiation.

Cancer Chemother Pharmacol, 1989, 25(2), 77 - 83
Relationship between cytotoxicity, drug accumulation, DNA damage and repair of human ovarian cancer cells treated with doxorubicin: modulation by the tiapamil analog RO11-2933; Alaoui Jamali MA et al.; The effect of N-(3,4-dimethoxyphenyl) N-methyl-2-(naphthyl)-m-dithiane-2-propylamine hydrochloride (RO11-2933), an analog of the calcium channel blocker tiapamil, on doxorubicin (DOX)-induced cytotoxicity and DNA damage in human ovarian cancer cells sensitive and resistant to DOX was investigated . A2780-DX2, A2780-DX3, and A2780-DX6 cell sublines were characterized by 7-, 26-, and 48-fold resistance after 2 h DOX exposure and 30-, 50-, and 500-fold resistance after 72 h DOX exposure, respectively . Increased drug efflux resulting in a lower intracellular drug accumulation, decreased DOX-induced DNA single-strand breaks (DNA SSBs), and rapid DNA repair correlated with the degree of resistance . In addition, DNA SSBs were rapidly repaired within 8 h in A2780-DX3 cells, whereas no significant repair of DNA SSBs was observed in sensitive cells . In comparison with verapamil, RO11-2933 was found to reverse DOX resistance at lower and nontoxic concentrations (2 microM as compared with 10 microM verapamil) . This reversion was complete in cells with a low degree of resistance (A2780-DX1 and A2780-DX2) but partial in highly resistant cells (A2780-DX3 and A2780-DX6), and continuous exposure to RO11-2933 was essential for optimal reversal of drug resistance . Interestingly, RO11-2933 was found to inhibit the repair of DNA SSBs induced by DOX but not those induced by X-ray . These results suggest that the potentiation of DNA SSBs and the specific inhibition of DNA repair by RO11-2933 in multidrug-resistant cells could be of particular value in overcoming MDR in the clinic.

Cancer Commun, 1989, 1(5), 311 - 6
Modulation of multidrug resistance in Chinese hamster cells by liposome-encapsulated doxorubicin; Thierry AR et al.; A Chinese hamster cell line (LZ), selected for multidrug resistance (MDR), exhibits a 3,000-fold resistance to doxorubicin, compared to parental V-79 cells . These drug resistant cells have amplified MDR genes, overexpress P-glycoprotein, and in the presence of doxorubicin show reduced intracellular drug accumulation . Using liposome-encapsulated doxorubicin (Rahman et al . Cancer Res . 45:796-803; 1985), we observed partial reversal of the resistance of LZ cells to this drug and a higher intracellular drug accumulation, compared to free drug . Parental V-79 cells, however, did not exhibit differences in survival or in drug accumulation when treated with encapsulated or free doxorubicin . Comparison of the effect of liposome-encapsulated doxorubicin with that of verapamil in reversing drug resistance showed that the liposomal preparation was as effective as verapamil used at its maximum clinically relevant concentration (1.5 microM) . These results suggest that the use of liposomes as carriers of anticancer drugs may offer a strategy for overcoming MDR in tumor cells.

Cancer Commun, 1989, 1(5), 285 - 92
Human multidrug resistant KB cells overexpress protein kinase C: involvement in drug resistance; Posada JA et al.; Among the many phenotypic characteristics of multidrug resistance (MDR), the presence of P-glycoprotein is nearly always observed, and it appears that the plasma membrane of the multidrug resistant cell is integrally involved in controlling drug resistance . Another membrane-associated protein kinase, protein kinase C (PKC), has been shown to regulate the flow of information to the cell interior and to control the efflux of a number of different compounds . We therefore initiated a study of PKC and MDR . We found that multidrug resistant sublines from both mouse sarcoma 180 and human KB lines exhibited 80-90% increases in basal PKC activity . The mechanism of the increase appears to be quite different in the two cell lines . The human KB cells overexpress the alpha isozyme of PKC, commensurate with the increase in alpha-PKC protein, whereas the mouse cells do not overexpress alpha-mRNA but increase alpha-PKC protein . Furthermore, it appears that PKC activity plays a functional role in drug resistance, since inhibition of endogenous PKC activity by staurosporine resulted in decreased resistance to Adriamycin . We also found that phosphorylation of MDR cell membrane vesicles by purified PKC, followed by immunoprecipitation of P-glycoprotein with monoclonal antibody C219, resulted in a level of phosphorylation of P-glycoprotein that was greater than the endogenous phosphorylation level . The data presented indicate that MDR cells of diverse species exhibited enhanced PKC activity but that the mechanisms were different . The increased kinase activity may have biological relevance to MDR since PKC appears to be coupled to P-glycoprotein function.

Cancer Treat Res, 1989, 48, 37 - 53
Structure and function of P-glycoprotein; Gerlach JH; P-glycoprotein remains the best understood molecule that has been implicated in the multidrug resistance phenomenon . As I have attempted to show, our understanding of this molecule is still in its infancy, with many questions as yet unanswered . It is anticipated that the answers to these questions will contribute to our understanding of drug resistance, as it relates to cancer, and also to our understanding of what appears to be a fundamental transport system.

Cancer Commun, 1989, 1(4), 233 - 41
Protein phosphorylation in multidrug resistant Chinese hamster cells; Meyers MB; Protein phosphorylation is altered in multidrug resistant, reverse transformed Chinese hamster cells selected for resistance to vincristine (DC-3F/VCRd-5L) or actinomycin D (DC-3F/AD X), as compared to drug-sensitive parental DC-3F cells . Evidence for this was obtained by gel electrophoretic analysis of proteins phosphorylated in vitro in the presence of {gamma -32P}ATP . In general, the level of incorporation of 32P into resistant cell proteins was higher than into proteins of sensitive cells, when reactions were carried out in either the presence or absence of exogenous protein kinase modulators . Phosphorylation of P-glycoprotein a multidrug resistance-related protein, and of sorcin, a 22 kDa calcium-binding protein overproduced in many multidrug resistant cells including DC-3F/VCRd-5L, was demonstrated . Analysis of proteins metabolically labeled with {32P}-orthophosphate suggests that protein phosphorylation differences in cell-free extracts are representative of events in the intact cells . Data support the probability that a variety of kinase and/or phosphatase activities were altered in the multidrug resistant cells . These may be associated with resistance development, P-glycoprotein function, reverse transformation, state of differentiation, inhibition of cellular proliferation, or all of these components.

Cancer Commun, 1989, 1(4), 217 - 24
Progressive resistance to doxorubicin in mouse leukemia L1210 cells with multidrug resistance phenotype: reductions in drug-induced topoisomerase II-mediated DNA cleavage; Ganapathi R et al.; Cells selected for resistance to doxorubicin (DOX) express the multidrug resistance (MDR) phenotype, and resistance has been suggested to be due primarily to enhanced cellular efflux of drug . A progressively DOX-resistant (10- and 40-fold) L1210 mouse leukemia model system, which does not exhibit enhanced DOX efflux as a primary mechanism of resistance, was found to display the MDR phenotype, based on overexpression of P-glycoprotein in western blots and cross-resistance to vinca alkaloids . Cross-resistance to another topoisomerase II inhibitor, etoposide (VP-16), was similar to that of DOX (10- and 40-fold), whereas resistance to N-{4-(9-acridinylamino)-3-methoxyphenyl}methanesulfonamide (m-AMSA) was 5-fold lower . In contrast, no cross-resistance to camptothecin, an inhibitor of topoisomerase I, was observed . Topoisomerase II decatenation activity in nuclear extracts from 10- and 40-fold DOX-resistant cells was 2- and 4-fold lower, respectively, when compared to sensitive cells . In these cells, however, marked reductions in m-AMSA- and VP-16-induced topoisomerase II mediated DNA cleavage were found to exceed decreases in the catalytic activity of the enzyme . Results from this study demonstrated that, in progressively DOX-resistant L1210 mouse leukemia cells with the MDR phenotype, a better relation existed between the degree of resistance and reduced VP-16- and m-AMSA-induced topoisomerase II mediated DNA cleavage, than between increases in P-glycoprotein and concomitant reduction in DOX accumulation.

Cancer Commun, 1989, 1(3), 181 - 90
In vitro multidrug resistance of P388 murine leukemia selected for resistance to diaziquone; Gutierrez PL et al.; P388 leukemia sublines were isolated from leukemia-cell-bearing CD2F1 mice that had been treated in vivo with increasing amounts of diaziquone (AZQ) . The sublines isolated for in vitro studies were AZQ19 and AZQ30 which corresponded to the 19th and 30th in vivo passages, respectively . The AZQ19 subline displayed a very low degree of resistance to AZQ (1.5-fold), whereas the AZQ30 subline was sensitive . Both sublines, however, had much higher degrees of resistance to Adriamycin than to AZQ (24-fold for AZQ30 cells and 10-fold for AZQ19 cells) . Both cell lines were also more resistant to actinomycin D, colchicine, and vincristine than to AZQ . The AZQ19 line was resistant to the alkylator thio-TEPA to the same degree that it was to AZQ, but the AZQ30 line was sensitive to thio-TEPA . On the other hand, AZQ30 cells were resistant to hydrogen peroxide with a very low degree of resistance (1.27-fold, P less than 0.05), whereas the AZQ19 line was sensitive . Drug accumulation experiments indicated that AZQ-resistant cells differed from the parental line in that they did not accumulate Adriamycin or vinblastine . In the case of AZQ, however, resistant and parental lines accumulated the same amounts of exchangeable AZQ . Using the immunoblotting technique, no P-glycoprotein was found in resistant cells . The resistant lines consumed oxygen at greater rates than the parental line . Oxygen consumption (Mean +/- SD) in sensitive cells was 2.0 +/- 0.4% O2 consumed/min, whereas in resistant cells it was nearly 3.1 +/- 0.6% O2 consumed/min . The increase in oxygen consumption with drug resistance was statistically significant (P less than 0.01) . The kinetics of production of hydroxyl free radicals and of AZQ free radicals were faster in the resistant lines reflecting, in essence, their increased oxygen consumption . It appears that the two sublines analyzed here show resistance mechanisms that may have been elicited by the two distinct chemical constituents of AZQ . Therefore, in the AZQ19-resistant line, the alkylating aspect of AZQ was emphasized, whereas in the AZQ30 line, the quinone and, thus, free radical aspect was emphasized . This is consistent with AZQ30 cells being sensitive to the alkylator thio-TEPA and resistant to hydrogen peroxide, and the AZQ19 line being resistant to thio-TEPA and sensitive to hydrogen peroxide . In addition, the AZQ30 cell line was relatively more resistant than the AZQ19 line to Adriamycin.

Hematol Pathol, 1989, 3(2), 73 - 8
Detection of cells with multidrug-resistant phenotype in myeloproliferative disorders before therapy; Kemnitz J et al.; The expression of the multidrug resistance (mdr) phenotype is connected with the overexpression of the P-glycoprotein . By applying the immunocytochemical assay, we have demonstrated that in myeloproliferative diseases (AML, ALL, MDS, CGL) in single cases in smear preparations from the peripheral blood as well as from the bone marrow P-glycoprotein-positive cells, respectively, cells with mdr-positive phenotype can be detected in the material obtained from patients before therapy and without clinically and anamnestically known exposure to cytotoxic or immunosuppressive drugs . In the control group of probands without hematologic disorders and also without clinically or anamnestically confirmed contact with cytotoxic or immunosuppressive drugs, we have found P-glycoprotein-positive subpopulations of cells with positive mdr phenotype in a few cases as well . The uniqueness of our results lies in the fact that this finding demonstrates the presence of subpopulations of mdr-positive cells in leukemias and myelodysplastic syndromes before therapy, and furthermore makes evident that a positive mdr phenotype is not necessarily associated with a malignant phenotype or a malignant cell transformation.

Br J Cancer, 1989 Jan, 59(1), 42 - 6
Effect of calcium antagonists on the chemosensitivity of two multidrug-resistant human tumour cell lines which do not overexpress P-glycoprotein; Cole SP et al.; We have examined the ability of eight compounds to enhance adriamycin (ADM) sensitivity of two human tumour cell lines (a small cell lung cancer cell line, NCI-H69, and a fibrosarcoma cell line, HT1080) and their multidrug-resistant variants . The resistant cell lines (H69AR and HT1080/DR4) do not overexpress P-glycoprotein . Verapamil, nicardipine, perhexiline maleate, chloroquine, tamoxifen, clomiphene, prenylamine and trifluoperazine were tested alone and in combination with ADM for their cytotoxic effects . No major differences in sensitivity between the parent and resistant cell lines were noted when these agents were tested alone, except for HT1080/DR4 cells which exhibited a slight collateral sensitivity to nicardipine and H69AR cells which showed cross-resistance to chloroquine and clomiphene . When the chemosensitisers were combined with ADM no enhanced cytotoxicity of either parent cell line was observed . In HT1080/DR4 cells, verapamil showed only a modest dose-dependent chemosensitising effect while the other compounds had no effect . Verapamil and nicardipine enhanced ADM cytotoxicity in H69AR cells slightly but these effects were not dose-dependent . These results demonstrate that the reversal of drug resistance by verapamil and other calcium antagonists in a dose-dependent fashion is not an invariable property of multidrug-resistant tumour cells.

Virchows Arch B Cell Pathol Incl Mol Pathol, 1989, 56(5), 327 - 35
Freeze-fracture study of plasma membranes in wild type and daunorubicin-resistant Ehrlich ascites tumor and P388 leukemia cells; Sehested M et al.; The plasma membrane is considered to play a major role in the development and maintenance of the multidrug resistance (MDR) phenotype, a role which may in part be mediated by an inducible 170 kD transmembrane protein (P-170) . The present freeze-fracture study of plasma membranes of daunorubicin-resistant Ehrlich ascites and P388 leukemia cells demonstrated a significant increase in the density of intramembrane particles (IMP) in the P-face, but not the E-face, of resistant sublines compared with wild type cells . Furthermore, a three-dimensional histogram plot of the diameters of P-face IMPs in Ehrlich ascites tumor cells showed the emergence of a subpopulation of 9 X 11 nm IMPs not found in wild type cells . The size of these IMPs would be consistent with a MW of approximately 340 kD, thus indicating that P-170, shown to be present in both resistant cell lines by Western blot analysis and immunohistochemical staining, exists as a dimer in the plasma membrane . Incubation with the calcium channel blocker verapamil, in concentrations known to inhibit daunorubicin efflux in resistant cells, showed evidence of membrane disturbance in the form of IMP clustering in both wild type and resistant Ehrlich ascites tumor cells . However, incubation with daunorubicin itself did not alter the freeze-fracture morphology of the plasma membranes.

J Cancer Res Clin Oncol, 1989, 115(1), 17 - 24
Induced multidrug resistance in murine leukemia L1210 and associated changes in a surface-membrane glycoprotein; Volm M et al.; The aim of this study was to find out whether only resistant cells of the "multidrug-resistant" phenotype show the described changes of plasma membrane glycoprotein (170 kDa) or whether resistant cells that do not express this phenotype reveal corresponding results . Doxorubicin-resistant (L1210dox) and daunorubicin-resistant L1210 ascites tumor cells (L1210dnr) (multidrug-resistant tumor cells) were therefore compared with cytosine-arabino-side-resistant (L1210AraC) and cyclophosphamide-resistant L1210 ascites tumor cells (L1210ctx) (not multi-drug-resistant tumor cells) . The resistant cell lines were generated in vivo in tumor-bearing mice and the resistance to cytostatic agents was evaluated in vivo and in vitro . Using the accumulation assay with rhodamine-123, the multidrug resistance can be detected . In order to determine alterations in the plasma membranes we used the monoclonal antibodies 265/F4 and C219, which were prepared against the membrane glycoprotein P170 (170 kDa) in colchicin-resistant Chinese hamster ovary cells . The results demonstrate that L1210dox and L1210dnr tumor cells show an intense immunostaining by the streptavidin/biotinylated-peroxidase-complex method and by the streptavidin/biotin/phycoerythrin immunofluorescence method . In contrast no specific immunostaining was observed in parental (sensitive) and L1210AraC or L1210ctx tumor lines . The results were confirmed by immunoblotting . To determine whether multidrug-resistant DNA sequences were expressed in the multidrug-resistant tumor cells, Northern blots with RNA od sensitive and resistant cells were performed using the clone pcDR1.5 . Elevated RNA levels were detected only in resistant cells with the multidrug-resistant phenotype . Thus, the results of this study demonstrate that only resistant cells with the multidrug-resistant phenotype show an increased expression of the membrane 170-kDa glycoprotein.

Mol Pharmacol, 1989 Jan, 35(1), 105 - 15
Structural features determining activity of phenothiazines and related drugs for inhibition of cell growth and reversal of multidrug resistance; Ford JM et al.; Phenothiazines and structurally related compounds inhibit cellular proliferation and sensitize multidrug-resistant (MDR) cells to chemotherapeutic agents . To identify more potent pharmaceuticals, we studied the structure-activity relationships of 30 phenothiazines and related compounds on cellular proliferation and MDR in sensitive MCF-7 and resistant MCF-7/DOX human breast cancer cells . Substitutions on the phenothiazine ring that increased hydrophobicity increased antiproliferative and anti-MDR activities . For example, -Cl and -CF3 groups increased whereas -OH groups decreased potency . Modifying the length of the alkyl bridge and the type of amino side chain also influenced potency . Compounds with increased activity against cellular proliferation and MDR possessed a four-carbon bridge rather than a three- or two-carbon bridge and a piperazinyl amine rather than a noncyclic amino group . Compounds with tertiary amines were better anti-MDR agents than those with secondary or primary amines but were equipotent antiproliferative agents . The effects of these substituents were unrelated to hydrophobicity . The structure-activity relationships suggest that an ideal phenothiazine structure for reversing MDR has a hydrophobic nucleus with a -CF3 ring substitution at position 2, connected by a four-carbon alkyl bridge to a para-methyl-substituted piperazinyl amine . We subsequently studied related compounds having certain of these properties . Substitution of a carbon for a nitrogen at position 10 of the tricyclic ring, with a double bond to the side chain (thioxanthene), further increased activity against MDR . For example, (trans)-flupenthixol, the most potent of these compounds, increased the potency of doxorubicin against MDR cells by 15-fold, as compared with its stereoisomer (cis)-flupenthixol (5-fold) or its phenothiazine homolog fluphenazine (3-fold) . (cis)- and (trans)-flupenthixol were equipotent antiproliferative agents . (trans)-flupenthixol was not accumulated more than (cis)-flupenthixol in MDR cells, implying that their stereospecific anti-MDR effects were not the result of selective differences in the access of the drugs to intracellular targets . Both drugs increased the accumulation of doxorubicin in MDR cells, but not in sensitive cells, suggesting that they modulate MDR by interacting with a uniquely overexpressed cellular target in these resistant cells . The apparent lack of clinical toxicity of (trans)-flupenthixol makes it an attractive drug for further investigation.

Proc Natl Acad Sci U S A, 1989 Jan, 86(2), 695 - 8
Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites; Cordon-Cardo C et al.; Endothelial cells of human capillary blood vessels at the blood-brain and other blood-tissue barrier sites express P-glycoprotein as detected by mouse monoclonal antibodies against the human multidrug-resistance gene product . This pattern of endothelial cell expression may indicate a physiological role for P-glycoprotein in regulating the entry of certain molecules into the central nervous system and other anatomic compartments, such as the testes . These tissues, which limit the access of systemic drugs, are known pharmacologic sanctuaries for metastatic cancer . P-glycoprotein expression in capillary endothelium of brain and testes and not other tissues (i.e., kidney and placenta) may in part explain this phenomenon and could have important implications in cancer chemotherapy.

Adv Enzyme Regul, 1989, 29, 61 - 72
Mediation of cellular anion detoxification in leukemic cells by unidirectional efflux pumps; Henderson GB; Methotrexate influx in L1210 cells is mediated almost entirely by a single system, whereas efflux occurs via three routes, the influx carrier functioning in reverse and two additional systems which are operationally unidirectional . The two unidirectional routes show considerable similarities in energy-dependence and inhibitor sensitivities but can be separated by their differential inhibition by bromosulfophthalein (BSP), probenecid, and vincristine . The predominant route is inhibited by BSP and vincristine, whereas the other route is sensitive to probenecid . A search for additional anion substrates for the unidirectional efflux systems for methotrexate led to the finding that cholate exits L1210 cells via the same two unidirectional efflux systems as methotrexate . Cholate efflux is carrier-mediated, energy-dependent, and unidirectional, and it can be inhibited by several compounds which inhibit the efflux of methotrexate . Moreover, the same concentration of each compound which produced a half-maximal inhibition of methotrexate efflux also inhibited the efflux of cholate by 50% . Cholate efflux occurred predominantly (85%) via the BSP-sensitive route . The observation that methotrexate and cholate share the same efflux systems in L1210 cells suggest that perhaps other large anions are also accommodated by these systems since methotrexate and cholate differ in net negative charge and have few common structural features . The hypothesis advanced from these studies has been that the function of the unidirectional efflux systems is to extrude various organic anion catabolites which might otherwise become toxic if allowed to accumulate within cells . Possible intracellular anions in this latter category include sulfonated or carboxylated steroids, bilirubin, and products of vitamin D and prostaglandin catabolism . Unidirectional efflux pumps have been identified for other anionic compounds including cyclic AMP and oxidized glutathione, and in both cases similarities were apparent with the efflux of methotrexate and cholate . The most compelling comparison was with prostaglandin A1 which inhibited the efflux of cyclic AMP, methotrexate, and cholate half-maximally at the same low concentration of 0.1 microM . Energy-dependent unidirectional efflux pumps for large neutral or cationic drugs have also been identified in cells with acquired multidrug resistance . The latter efflux activity is thought to be mediated by a membrane glycoprotein (p170) which is also sensitive to several of the various inhibitors (reserpine, verapamil, and quinidine) which reduce the efflux of methotrexate and cholate.

Bull Mem Acad R Med Belg, 1989, 144(3-4), 213 - 21; discussion 222-3
{Clinical and biological aspects of intensive chemotherapy with autologous bone marrow grafts in small-cell bronchial cancer}; Symann M et al.; Small cell lung cancer (SCLC) is a frequent and aggressive tumor characterized by its major sensitivity to chemotherapy since 80% of the patients respond dramatically to the treatment . Nevertheless, the majority of them eventually die from their cancer . We have addressed the effect of late intensification with autologous bone marrow transplantation on SCLC through a randomized clinical trial . This study included 101 patients, 45 of whom could be randomized to either conventional post-induction therapy or late intensification . Of the 32 patients with limited disease, patients who did not receive intensification relapsed on a median of 10 weeks compared with 35 weeks for patients who received intensification (p = 0.001) . An important observation was that there was no patient disease free at 1 year in the group that did not receive intensification whereas there were patients who were free of disease up to 271 weeks in the group receiving late intensification . To improve upon these results we have developed a panel of anti-lung cancer monoclonal antibodies that we use for the detection of occult cancerous disease in the bone marrow and for the in vitro elimination of clonogenic tumor cells contaminating the marrow graft . In addition we are monitoring the immune defect induced by ABMT and its compensation by an in vitro recombinant human IL-2 perfusion . Finally we have devised a new induction and intensification chemotherapy regimens with different drugs in order to prevent multidrug resistance . These new tools are currently under investigation in the clinical setting.

Cancer Chemother Pharmacol, 1989, 24(5), 284 - 90
Identification of anthracyclines and related agents that retain preferential activity over adriamycin in multidrug-resistant cell lines, and further resistance modification by verapamil and cyclosporin A; Coley HM et al.; A range of anthracyclines and related compounds were evaluated for activity against murine and human cell lines exhibiting multidrug resistance (MDR) . Cell lines used were the NCI-H69 human small-cell lung cancer line and the EMT6 murine mammary tumour line, together with their multidrug-resistant counterparts produced by in vitro exposure to Adriamycin (ADM) . Chemosensitivity testing was carried out using the tetrazolium (MTT) dye assay . Results were expressed as the ratio of the ID50 for the resistant line to that obtained in the parent, i.e . the resistance factor (RF) . Compounds exhibiting much lower RF values than ADM in both resistant cell lines were identified as those anthracyclines with 9-alkyl substituents and those with certain changes to the amino sugar residue at position 3' and 4', together with the anthracenedione mitoxantrone (MIT) . In a further attempt to overcome resistance, we used four of these compounds, Ro 31-1215, 4'-deoxy-4'-iodo-ADM (iodo-ADM), aclacinomycin A (ACL) and MIT (all yielding low RF values), in combination with the resistance modifiers verapamil (VRP) and cyclosporin A (CYA) . Additional enhancement of chemosensitivity was achieved in the ADM-resistant sublines, as shown by the further decrease in RF values . At the concentrations used, the largest effects were generally seen with CYA, and the combination of this modifier with ACL and MIT was particularly effective . For the H69/LX4 resistant line, the latter combinations gave RF values approaching unity . These findings point to the use of analogues with the 9-alkyl substituent and/or specific changes to the sugar residue in combination with resistance modifiers as a therapeutic strategy for circumvention of the MDR phenotype.

Cancer Chemother Pharmacol, 1989, 23(3), 161 - 8
Reduced formation of lesions in the DNA of a multidrug-resistant L1210 subline selected for teniposide resistance; Roberts DW et al.; Earlier studies have suggested that higher cellular levels of teniposide (VM-26) are required for the inhibition of growth in L1210/VM-26 sublines than in parental L1210 cells . On the basis of this observation, we hypothesized that resistance to VM-26, which is partly attributed to multidrug resistance, also resulted in reduced formation of DNA lesions by the drug . In confirmation of this hypothesis, equitoxic concentrations of VM-26 produced fewer breaks in the DNA of LIa5 microM cells, the prototype L1210/VM-26 subline, than in that of L1210 cells . Previously, potassium cyanide (KCN) and verapamil were shown to increase the levels of VM-26 in LIa5 microM but not L1210 cells . These agents also selectively increased the formation of breaks in the DNA of LIa5 microM but not L1210 cells . The DNA unknotting assay with phage P4 DNA indicated equivalent DNA type II topoisomerase activity in nuclear extracts of LIa5 microM and L1210 cells . The factor that reduced the formation of breaks in cellular LIa5 microM DNA by VM-26 provided less protection against equitoxic levels of doxorubicin, to which LIa5 microM cells are cross-resistant.

Eur J Cancer Clin Oncol, 1989 Jan, 25(1), 133 - 7
Development of tumour cell resistance to tumour necrosis factor does not confer resistance to cytotoxic drugs; Neale ML et al.; Tumour necrosis factor (TNF) is a protein product of macrophages with potential anti-cancer activity . As with other anti-cancer agents, tumour cells can develop resistance to the cytotoxic effects of TNF . The aim of his study was to see whether development of resistance to TNF resulted in a concomitant resistance to other anti-cancer agents, in particular those associated with multidrug resistance . Three TNF-susceptible tumour cell lines (L929, U937 and RK13) and their TNF-resistant sublines were compared for susceptibility in vitro to several cytotoxic drugs . The TNF-resistant sublines were not significantly more resistant to these drugs . In addition, an L929 subline selected for resistance to actinomycin D retained its susceptibility to TNF . These observations show that tumour cell resistance to TNF develops independently of resistance to cytotoxic drugs.

Tumour Biol, 1989, 10(5), 252 - 7
Immunohistochemical analysis of pulmonary and pleural tumors with the monoclonal antibody HYB-612 directed against the multidrug resistance (MDR-1) gene product, P-glycoprotein; Radosevich JA et al.; In this study, 212 untreated primary pulmonary and pleural neoplasms were studied immunohistochemically with the monoclonal antibody HYB-612 which detects the multidrug resistance (MDR)-related P-glycoprotein (gp180) . A tumor was considered positive for the expression of the MDR phenotype, even if a single rare positive cell was detected . Using this criterion, all of the various histologic subtypes were found to express MDR to varying degrees . The frequency of expression of this phenotype was found to be notably higher in non-small-cell carcinomas than in small-cell carcinomas . These findings are consistent with the known clinical responses of these neoplasms . The detection of gp180 in untreated lung neoplasms may be predictive of the responsiveness of neoplasms to chemotherapeutic agents . In addition, its presence or absence might be useful in determining the appropriate treatment protocol for given patients.

Mol Endocrinol, 1989 Jan, 3(1), 157 - 64
Relation between cytochrome P450IA1 expression and estrogen receptor content of human breast cancer cells; Vickers PJ et al.; Multidrug resistance (MDR) in an MCF-7 human breast cancer cell line (MCF7/Adr) is associated with decreased drug accumulation and overexpression of P-glycoprotein as well as alterations in the levels of specific drug-metabolizing enzymes, including decreased activity of the phase I drug-metabolizing enzyme aryl hydrocarbon hydroxylase (AHH) and increased expression of the anionic form of the phase II drug-metabolizing enzyme glutathione S-transferase . Since the development of MDR in this MCF-7 cell line is also associated with a loss of estrogen receptors (ER), we have examined the expression of cytochrome P450IA 1, the gene encoding AHH activity, in other breast cancer cell lines not selected for drug resistance but expressing various levels of ER . These studies show that a relationship exists between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible AHH activity and the ER content in a series of breast cancer cell lines . In these cell lines expression of AHH activity is regulated, at least in part, at the level of P450IA 1 RNA . While TCDD-specific binding proteins (Ah receptors) were found in each of the breast cancer cell lines, there was no apparent relation between the level of nuclear TCDD-binding proteins and the level of TCDD-inducible P450IA 1 expression . Previous studies from our laboratory have described an inverse relationship between levels of the anionic form of glutathione S-transferase and ER in breast cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1988 Dec 15, 263(35), 19119 - 25
Altered regulation of P-450IA1 expression in a multidrug-resistant MCF-7 human breast cancer cell line; Ivy SP et al.; MCF-7 human breast cancer cells, selected for resistance to adriamycin (AdrR), exhibit the phenotype of multidrug resistance (MDR) . Previous studies have shown that resistance in AdrR MCF-7 cells is associated with several biochemical changes that are similar to those induced in rat hyperplastic nodules, preneoplastic liver lesions which display broad spectrum resistance to carcinogens and hepatotoxins . In this report, we show that these changes in the AdrR MCF-7 cells are also associated with the development of cross-resistance to the procarcinogen benzo(a)pyrene (BP) and are associated with a marked defect in the conversion of BP to its cytotoxic, carcinogenic metabolites by AdrR cells . Since aryl hydrocarbon hydroxylase is the principle enzyme activity which converts benzo(a)pyrene to toxic hydroxylated forms, the regulation of cytochrome P-450IA1 expression, the gene encoding this enzyme activity in MCF-7 cells, was examined . Incubation with 100 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 h results in a marked increase in aryl hydrocarbon hydroxylase activity in wild type (WT) but not AdrR MCF-7 cells . The alteration in aryl hydrocarbon hydroxylase expression in the AdrR cells is not overcome by incubation either with higher concentrations of TCDD (1 microM) or for longer periods of time (4 days) . Northern blot analysis indicates that this defect in AdrR MCF-7 cells involves a regulatory defect at the level of P-450IA1 RNA . Following transfection of a construct containing the normal mouse P-450IA1 promoter fused to a reporter gene (bacterial chloramphenicol acetyltransferase) into WT and AdrR MCF-7 cells, TCDD induced chloramphenicol acetyltransferase activity in WT MCF-7 cells only . Furthermore, TCDD also induces both DT-diaphorase and UDP-glucuronyltransferase activities in WT, but not AdrR cells . These data suggest that the defect in the AdrR MCF-7 cells is not due to a structural P-450IA1 gene mutation, but rather involves a product regulating the polycyclic hydrocarbon-inducible expression of several drug-metabolizing enzyme activities . This defect in the AdrR MCF-7 cells is also associated with the development of resistance to ellipticine, an anticancer agent which is converted to more toxic hydroxylated species by aryl hydrocarbon hydroxylase or a similar mixed function oxidase . The WT and AdrR MCF-7 cells represent a useful model to study the regulation of the P-450IA1 gene in human cells.

Cancer Res, 1988 Dec 15, 48(24 Pt 1), 6986 - 91
Characterization of trimetrexate transport in human lymphoblastoid cells and development of impaired influx as a mechanism of resistance to lipophilic antifolates; Fry DW et al.; The purpose of this study was to characterize the transport properties of trimetrexate in WI-L2 human lymphoblastoid cells and determine the mode of resistance that had developed in a subline, WI-L2/TMQ, that was grown in increasing concentrations of trimetrexate . WI-L2/TMQ cells were 62-fold resistant to trimetrexate and 68- and 96-fold cross-resistant to the other lipophilic antifolates metoprine and piritrexim (BW 301U) . No cross-resistance was observed with vincristine or doxorubicin, and sensitivity was not increased with 5 micrograms/ml of verapamil, indicating that it was not a typical multidrug resistance phenotype . WI-L2/TMQ exhibited a 2-fold increase in dihydrofolate reductase; however, this did not contribute significantly to the observed resistance, since these cells retained full sensitivity to methotrexate . Nor were there any kinetic alterations in dihydrofolate reductase toward trimetrexate or differences in the levels of thymidylate synthase . The major difference between the sensitive and resistant cell line was a 50% decrease in the influx rate of WI-L2/TMQ cells which produced a corresponding decrease in cellular trimetrexate at the steady state . No difference in efflux rates was detected nor were there any differences in intracellular water or metabolism of trimetrexate . Additional characterization of trimetrexate transport in WI-L2 showed that influx was nonsaturable up to 5 mM extracellular trimetrexate, relatively insensitive to sodium azide, and exhibited a Q10 of 2.7 . Influx was, however, inhibited in a dose-dependent manner by concentrations of p-chloromercuribenzylsulfonate above 10 microM . Efflux studies revealed a large nonexchangeable fraction of trimetrexate that was well above the dihydrofolate reductase binding capacity and varied depending on the initial level of cell-associated drug . The intracellular exchangeable trimetrexate concentration at the steady state was always several-fold higher than the extracellular concentration . Retention of trimetrexate appeared to be coupled to some component of energy metabolism, since the presence of sodium azide stimulated this process by 2- to 3-fold . The data suggest that trimetrexate enters cells by passive diffusion but then is distributed and concentrated within the cell through more complex mechanisms which may involve energy coupling, compartmentation, or binding to macromolecules or organelles, although some type of carrier-mediated process cannot be ruled out.(ABSTRACT TRUNCATED AT 250 WORDS)

Cancer Res, 1988 Dec 15, 48(24 Pt 1), 7082 - 7
Mr 85,000 membrane protein specifically expressed in adriamycin-resistant human tumor cells; Hamada H et al.; For the characterization of membrane changes related to Adriamycin resistance in tumor cells, we have developed monoclonal antibodies against Adriamycin-resistant human myelogenous leukemia K562 (K562/ADM) . In addition to the monoclonal antibodies which recognize P-glycoprotein, we have obtained two monoclonal antibodies (designated MRK4 and MRK20) which recognize an Mr 85,000 membrane protein . Using MRK20 as a probe, we have studied the expression of the Mr 85,000 protein in various human multidrug-resistant and -sensitive cell lines . The Mr 85,000 protein was overexpressed in K562/ADM and in a human ovarian cancer cell line resistant to Adriamycin, 2780AD . The protein, if any, was not detected in other drug-resistant human cell lines such as colchicine-resistant KB cells (KB-C4), vinblastine-resistant CEM cells (CEM/VLB100), and vincristine-resistant K562 cells (K562/VCR) . We have isolated subclones of K562/ADM cells which express different amounts of the Mr 85,000 protein . The expression of the Mr 85,000 protein diminished when the cells were not kept in Adriamycin, and increased when the clones were kept in the presence of Adriamycin . In contrast, the expression of P-glycoprotein remained constant whether in the presence or absence of Adriamycin during these experiments . These findings suggest that the Mr 85,000 membrane protein is closely related to the resistant mechanism specific to Adriamycin resistance, which is different from that of the pleiotropic drug resistance.

Arzneimittelforschung, 1988 Dec, 38(12), 1771 - 4
Rapid detection assays for multidrug resistance; Efferth T et al.; In attempts to develop simple and rapid assays for detection of multidrug resistance (MDR) doxorubicin-resistant S180 (S180DOX), colchicine-resistant CHO (CHOCOL) and cytarabine (cytosine-arabinoside)-resistant L1210 cell lines (L1210AraC) were investigated . S180DOX and CHOCOL cells express the MDR-phenotype while L1210AraC does not . Using a previously described short-term assay firstly, inhibition of incorporation of radioactive nucleic acid precursors into tumour cells after addition of doxorubicin was measured . Secondly, the accumulation of the fluorescent dye rhodamine 123 (R123) in the different cell lines were analyzed . It could be observed that the resistant S180DOX and CHOCOL cells needed significantly more time to accumulate R123 than their sensitive parental cell lines or L1210AraC cells . Therefore, both assays are adequate tools for the rapid detection of MDR.

Cancer Res, 1988 Dec 1, 48(23), 6653 - 7
MX2, a morpholino anthracycline, as a new antitumor agent against drug-sensitive and multidrug-resistant human and murine tumor cells; Watanabe M et al.; MX2, a new morpholino anthracycline, showed similar or superior chemotherapeutic effects to Adriamycin (ADM) against several experimental murine tumors . i.v . administration of MX2 against L1210-bearing mice induced a prolongation of life-span by twice or more compared to ADM . MX2 was equally or slightly more effective against Lewis lung carcinoma and colon adenocarcinomas 26 and 38 than ADM when either drug was given i.v . The antitumor activity of MX2 against human tumor xenografts was similar to that of ADM, and the compound was effective against three out of four gastric adenocarcinomas, one out of two non-small-cell lung carcinomas, and two out of two mammary adenocarcinomas . In particular, this compound exhibited a marked effect against MX-1, a human mammary adenocarcinoma . MX2, in contrast to ADM, was effective against sublines of P388 leukemia resistant to ADM or aclacinomycin A in vivo as well as in vitro . A maximum percentage increase in life-span of about 90% was obtained in mice bearing these resistant tumors . MX2 is a unique anthracycline antibiotic effective on drug-sensitive as well as multidrug-resistant murine and human cells.

Mol Cell Biol, 1988 Dec, 8(12), 5206 - 15
The brown protein of Drosophila melanogaster is similar to the white protein and to components of active transport complexes; Dreesen TD et al.; The brown gene of Drosophila melanogaster is required for deposition of pteridine pigments in the compound eye and other tissues . We isolated a ca . 150-kilobase region including brown by microdissection and chromosome walking using cosmids . Among the cDNAs identified by hybridization to the cosmids, one class hybridized to a genomic region that is interrupted in two brown mutants, bw and In(2LR)CK, and to 2.8- and 3.0-kilobase poly(A)+ RNAs which are altered in the mutants . Nucleotide sequencing of these cDNAs revealed that the two transcripts differ as a consequence of alternative poly(A) addition and that both encode the same predicted protein of 675 amino acids . Searches of available databases for amino acid sequence similarities detected a striking overall similarity of this predicted protein to that of the D . melanogaster white gene . The N-terminal portion aligned with the HisP family of membrane-associated ATP-binding proteins, most of which are subunits of active transport complexes in bacteria, and to two regions of the multidrug resistance P-glycoprotein . The C-terminal portion showed a structural similarity to integral membrane components of the same complexes . Taken together with earlier biochemical evidence that brown and white gene products are necessary for uptake of a pteridine precursor and genetic evidence that brown and white proteins interact, our results are consistent with suggestions that these proteins are subunits of a pteridine precursor permease.

Lab Invest, 1988 Dec, 59(6), 870 - 5
A sensitive method for immunocytochemical detection of P-glycoprotein in multidrug-resistant human ovarian carcinoma cell lines; Chan HS et al.; P-glycoprotein, a molecular weight 170 kilodalton membrane component can be accurately detected in a series of human ovarian carcinoma cells with increasing degrees of multidrug resistance by using a modified immunoperoxidase "sandwich" method . Drug-resistant derivatives were selected from a drug-sensitive parent ovarian carcinoma cell line, SKOV3, by continuous exposure to increasing concentrations of the cytotoxic drug vincristine . These cells had corresponding overexpression of P-glycoprotein demonstrable at both protein and mRNA levels . Monoclonal antibodies against P-glycoprotein localized staining for P-glycoprotein to the plasma membrane and the Golgi region in individual drug-resistant cells, in proportion to their P-glycoprotein expression . P-glycoprotein was not demonstrable in drug-sensitive SKOV3 cells by either immunoblotting or immunocytochemical staining methods . The immunocytochemical staining method allowed detection of P-glycoprotein in the least drug-resistant cell line with as low as 8-fold relative resistance to vincristine . This method is as sensitive as Northern blot, and more sensitive than standard Western blot in detection of P-glycoprotein . We conclude that this highly sensitive immunocytochemical staining method for P-glycoprotein can be suitable for determination of P-glycoprotein expression in biopsy samples of tumors, and it can be a powerful diagnostic and prognostic tool in the study of the natural history of drug resistance . This may have important applications in the clinical management of cancer chemotherapy.

Carcinogenesis, 1988 Dec, 9(12), 2329 - 32
Transformation of rat liver epithelial cells with v-H-ras or v-raf causes expression of MDR-1, glutathione-S-transferase-P and increased resistance to cytotoxic chemicals; Burt RK et al.; We have examined the relationship between transformation and multidrug resistance by employing the v-H-ras or v-raf oncogenes to transform rat liver epithelial (RLE) cells in vitro . The data indicate that transformation of RLE cells with v-H-ras or v-raf results in increased resistance to the cytotoxins adriamycin, vinblastine and 2-acetylaminofluorene . This multidrug resistance is accompanied by increasing expression of P-glycoprotein (MDR-1) and glutathione-S-transferase P (GST-P) . Thus, neoplastic transformation of RLE cells with v-raf or v-H-ras, independently of chemical exposure, results in multidrug resistance.

Cancer Res, 1988 Nov 15, 48(22), 6365 - 70
Verapamil reversal of doxorubicin resistance in multidrug-resistant human myeloma cells and association with drug accumulation and DNA damage; Bellamy WT et al.; Verapamil reversed resistance to doxorubicin in a human multiple myeloma cell line selected for multiple drug resistance . The drug-resistant cell line 8226/DOX40 is known to have reduced intracellular drug accumulation associated with the overexpression of P-glycoprotein when compared to the sensitive parent cell line 8226/S . Verapamil alone was minimally cytotoxic in both cell lines, but reversed doxorubicin resistance in a dose-related manner in 8226/DOX40 . A similar dose-response relationship was observed for verapamil in increasing net intracellular doxorubicin accumulation . This increased net accumulation was secondary to block of enhanced doxorubicin efflux by verapamil from resistant cells . In contrast, verapamil did not alter initial doxorubicin accumulation over the first 60 s when incubated with resistant cells . Addition of verapamil to the 8226/DOX40 cells enhanced the formation of doxorubicin-induced DNA single strand breaks, double strand breaks, and DNA-protein cross-links . Verapamil had no effect on these lesions in the drug-sensitive cells . In addition, verapamil did not affect chemotherapeutic cytotoxicity or transport in the drug-sensitive cell line . Verapamil appears to reverse doxorubicin resistance in this human myeloma cell line by blocking enhanced drug efflux, leading to increased drug accumulation and enhanced DNA damage.

Cancer Res, 1988 Nov 15, 48(22), 6360 - 4
Direct relation of DNA lesions in multidrug-resistant human myeloma cells to intracellular doxorubicin concentration; Bellamy WT et al.; Using a human myeloma cell line selected for resistance to doxorubicin (8226/DOX), which expresses the multidrug resistance phenotype, we studied the effects of drug accumulation on DNA damage and cytotoxicity in multidrug-resistant cells . The resistant 8226 subline showed a decrease in {14C}doxorubicin accumulation as compared to the sensitive (8226/S) subline at all time points investigated . DNA alkaline elution techniques were used to examine the formation of single and double strand breaks and DNA-protein cross-links following exposure to doxorubicin in both sensitive and resistant sublines . When 8226/S and 8226/DOX40 cells were exposed to the same extracellular concentration of doxorubicin there was a difference in the quantity of DNA lesions occurring, with the 8226/DOX40 line exhibiting less DNA damage . However, when the extracellular concentration of drug was adjusted to yield equivalent intracellular concentrations these differences were removed and both lines exhibited the same degree of damage for all three DNA lesions . The same DNA lesions were also studied in isolated nuclei from sensitive and resistant cells . Such a model removes any confounding factors due to drug accumulation such as altered cytosolic distribution and/or metabolism of drug . We observed no difference in the formation of single or double strand breaks, or DNA-protein cross-links when the nuclei were exposed to the same concentration of doxorubicin . Results from colony-forming assays have shown that when resistant and sensitive 8226 cells were exposed to high concentrations of doxorubicin, there was a good correlation between DNA damage, drug accumulation, and cytotoxicity . This relationship did not hold for lower concentrations of doxorubicin, for which there was a lack of correlation between drug accumulation and cytotoxicity . Such findings may possibly be due to a prolonged retention of drug by the sensitive cell line as compared to the resistant cells . Further studies are under way to examine this possibility.

Cancer Res, 1988 Nov 15, 48(22), 6348 - 53
Increased mdr gene expression and decreased drug accumulation in multidrug-resistant human melanoma cells; Lemontt JF et al.; Multidrug-resistant clones of a drug-sensitive human malignant melanoma cell line were isolated by single-step selection in culture medium containing either vincristine (4.5 ng/ml or 7.5 ng/ml), vinblastine (3 ng/ml), or colchicine (8 ng/ml) . This protocol yielded primary colonies showing relatively low (4- to 24-fold) levels of drug resistance . These clones exhibit the classical multidrug resistance (MDR) phenotype, being cross-resistant to Vinca alkaloids, anthracyclines, colchicine, and actinomycin D . The appearance of an MDR phenotype in these cells was linked to a decreased accumulation and increased efflux of the drug {3H}vinblastine when compared to the drug-sensitive melanoma cell line . This increased drug efflux was dependent on the presence of cellular ATP and could be reduced by treatment of the cells with rotenone and deoxyglucose . A partial human mdr complementary DNA clone was used to monitor the degree of amplification and the level of transcription of this gene in the cloned lines . All 5 MDR sublines expressed increased levels of the specific 4.5-kilobase mdr mRNA, but did not show mdr gene amplification . Our results indicate that relatively low levels of drug resistance, similar to those observed clinically and in experimental xenografts, can be achieved by single-step drug selection and result from increased expression of at least one member of the mdr gene family.

J Natl Cancer Inst, 1988 Nov 2, 80(17), 1383 - 6
Coinduction of MDR-1 multidrug-resistance and cytochrome P-450 genes in rat liver by xenobiotics; Burt RK et al.; The levels of mRNA for multidrug-resistance (MDR-1) and selective cytochrome P-450 genes were determined in adult rat liver following administration of various natural and synthetic xenobiotics . MDR-1 (also known as PGY1) was induced following administration of aflatoxin B1, 2-(acetylamino)fluorene (AAF), N-hydroxy-2-(acetylamino)fluorene, isosafrole, phenothiazine, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but not after phenobarbital or 7-hydroxy-2-(acetylamino)fluorene treatment . Cytochrome P-450 isoform d was induced by TCDD, isosafrole, phenothiazine, and AAF, while cytochrome P-450 isoform b was induced by phenobarbital, and to a lesser extent by isosafrole . These observations suggest that both MDR and cytochrome P-450 gene families are evolutionarily selected by the capacity of various xenobiotics to induce their own detoxification either through metabolism to hydrophilic derivatives by the cytochrome P-450 system or direct excretion from the cell by the MDR gene family . Furthermore, the data indicate that induction of selective members of the MDR and the cytochrome P-450 gene families may depend on overlapping regulatory elements.

Anticancer Res, 1988 Nov-Dec, 8(6), 1169 - 78
Induced multidrug-resistance in murine sarcoma 180 cells grown in vitro and in vivo and associated changes in expression of multidrug-resistance DNA-sequences and membrane glycoproteins; Volm M et al.; The aim of this investigation was to analyze the resistance to doxorubicin and daunorubicin of murine sarcoma 180 cells grown in vitro (monolayer) and in vivo (ascites form) . The colchicine-resistant CHO cells were used as controls . A multidrug-resistant phenotype was found in all investigated cell lines . Multidrug resistant cells grown in tissue culture or as ascites tumor cells needed more time to accumulate rhodamine 123 than their sensitive parental cells . In order to evaluate whether the resistant cells show alterations in the plasma membranes, different methods were applied (immunocytochemistry, immunofluorescence, immunoblotting) using the monoclonal antibodies 265/F4 and C 219 . These methods all revealed an increased expression of the glycoprotein Mr 170 kd in the multidrug-resistant cell lines . To determine whether multidrug DNA sequences were expressed in the resistant cell lines, slot blots and Northern blots with RNA of sensitive and resistant cells were performed using the clones pDR 7.8 and pcDR 1.5 . Elevated RNA levels were detected in all resistant cell lines.

Jpn J Cancer Res, 1988 Nov, 79(11), 1238 - 46
Vincristine-resistant human cancer KB cell line and increased expression of multidrug-resistance gene; Kohno K et al.; A multidrug-resistant clone of human cancer KB cells was isolated by stepwise selection on exposure to increasing doses of vincristine . The final clone, VJ-300, obtained after ethylmethane sulfonate mutagenesis showed 400-fold higher resistance to vincristine than did KB cells . Cellular accumulation of vincristine in VJ-300 was decreased to less than one-tenth of that in KB . The cells were also cross-resistant to daunomycin, adriamycin, actinomycin D, colchicine and VP-16 . During continuous culturing in the absence of any drug for several months, a different colchicine-resistant and multidrug-resistant clone, KB-C1, reverted almost completely to drug sensitivity, whereas drug resistance in VJ-300 was stably maintained . Amplification of the multidrug-resistance-1 (mdr-1) gene was more than 20-fold in KB-C1, but less than 2-fold in VJ-300 . mdr-1 mRNA was, however, expressed in VJ-300 at a rate comparable to KB-C1 . Acquisition of high multidrug resistance in VJ-300 might be correlated with both activated transcription of mdr-1 gene and amplification.

Cancer Res, 1988 Nov 1, 48(21), 5927 - 32
Genes amplified and overexpressed in human multidrug-resistant cell lines; Van der Bliek AM et al.; Multidrug resistance (MDR) is associated with overproduction of Mr 170,000 membrane proteins (P-glycoproteins) caused by either gene amplification, transcriptional activation, or both . In rodents the amplified domain comprises genes that encode P-glycoproteins and at least five unrelated genes, one of which encodes the calcium-binding protein sorcin . The amplification and increased expression of these genes always includes one P-glycoprotein-encoding gene (pgp1 in hamsters, homologous to mdr1 in humans) . In human MDR cells only elevated mdr1 expression has been shown thusfar, although another P-glycoprotein encoding gene (mdr3, homologous to hamster pgp3) is closely linked . Here we show that the human homolog of the hamster sorcin gene resides on chromosome 7 like the P-glycoprotein-encoding genes . Furthermore, gene classes designated 4, 5, and 6 are coamplified with mdr1 and mdr3 in the human ovarian carcinoma cell line 2780AD, which strongly suggests that the overall structure of the human MDR domain is the same as in rodents . Class 6 was moderately and mdr1 was highly overexpressed in this cell line . Four other human MDR cell lines also have much higher mdr1 overexpression than expected from the relatively low levels (2- to 30-fold) of gene amplification . This contrasts with the results of previous work with rodent MDR cells, in which the increase in P-glycoprotein mRNA levels usually parallels the increase in gene copy number . Although four of the five human MDR cell lines have coamplified mdr3, its expression was undetectable . Our results confirm the central role of the mdr1 (pgp1) gene in MDR and suggest that different cross-resistance patterns are not due to differential expression of different P-glycoprotein genes.

J Natl Cancer Inst, 1988 Oct 19, 80(16), 1294 - 8
DNA cross-linking and cytotoxicity of the alkylating cyanomorpholino derivative of doxorubicin in multidrug-resistant cells; Scudder SA et al.; The cyanomorpholino derivative of doxorubicin (MRA-CN) is an anthracycline that is extremely potent and non-cross-resistant with doxorubicin (DOX) in multidrug-resistant cells . MRA-CN binds to and cross-links DNA and thus has been proposed to act as a targeted alkylating agent . In our study, the number of DNA interstrand and DNA-protein cross-links produced by MRA-CN was identical in multidrug-resistant Dx5 and parental MES-SA cells, as shown by alkaline elution analysis . The amount of cross-linking was directly proportional to drug concentration at concentrations from 10(-11) to 10(-7) M MRA-CN . Extensive DNA cross-linking was evident within 30 minutes of drug exposure . After 1 hour of drug exposure, the number of DNA cross-links increased for 90 minutes, reached a plateau, and then began to decrease after 120 minutes . Loss of cell viability was also observed as early as 3 hours after exposure to MRA-CN . The finding of the same number of DNA cross-links in MES-SA and Dx5 cells indicates that similar amounts of MRA-CN are likely to enter the nuclei of multidrug-resistant and sensitive cells . Other anthracyclines have major differences in nuclear distribution in sensitive and resistant cells . Several factors may contribute to the non-cross-resistance of MRA-CN in multidrug-resistant cells . (a) The lipophilicity of MRA-CN facilitates cell entry . (b) The substitution and loss of basicity at the amino nitrogen may reduce the affinity of the drug for the P-glycoprotein efflux pump, compared with that of DOX . (c) The detoxification function of P-glycoprotein may be less effective for drugs that produce rapid and irreversible cell damage, such as the DNA-targeted alkylation caused by MRA-CN.

Biochemistry, 1988 Oct 4, 27(20), 7607 - 13
Stability and covalent modification of P-glycoprotein in multidrug-resistant KB cells; Richert ND et al.; An antipeptide antibody (P7) to P-glycoprotein has been produced by immunizing rabbits with a synthetic peptide . Antibody P7 is directed against the amino-terminal region of P170 (residues 28-35) . The antibody immunoprecipitates a 170-kDa P-glycoprotein from extracts of drug-resistant KB-V1 cells that is not present in the drug-sensitive cell line KB-3-1 . Antibody P7 was used to quantitate the amount of P-glycoprotein present in drug-resistant KB lines at various levels of resistance and to demonstrate the presence of P-glycoprotein in NIH 3T3 cells transfected with a cloned MDR1 cDNA or human genomic DNA encoding MDR1 . Pulse-chase labeling experiments demonstrated that P-glycoprotein is synthesized as a 140-kDa precursor which is slowly converted over 2-4 h to a 170-kDa glycoprotein . Tunicamycin treatment blocks the conversion of the precursor to the mature form, and removal of N-linked oligosaccharides with Endo F reduces the relative molecular weight of P-glycoprotein to 140K . The mobility of mature P-glycoprotein is unaffected by treatment with neuraminidase and Endo H . These data indicate that P-glycoprotein is N-glycosylated and contains little or no neuraminic acid . P-Glycoprotein is also phosphorylated, and the extent of phosphate incorporated is proportional to the amount of protein present in drug-resistant cells.

Br J Cancer, 1988 Oct, 58(4), 441 - 7
Cross resistance pattern towards anticancer drugs of a human carcinoma multidrug-resistant cell line; Gupta RS et al.; Puromycin-resistant (PurR) mutants/variants of a human carcinoma cell line (HeLa), which show greatly reduced cellular uptake of 3H-puromycin and 3H-daunomycin have been isolated after one- and two-step selections in presence of the drug . The cross-resistance pattern of these mutant cell lines towards numerous anticancer drugs and other inhibitors has been examined . Both the first- and the second-step mutants exhibited increased resistance to a number of antimitotic drugs (viz . vinblastine, vincristine, colchicine, taxol and maytansine), several protein synthesis inhibitors (viz . chalcomycin, bruceantin, harringtonine, homoharringtonine), a large number of DNA interactive compounds (viz . aclacinomycin A, actinomycin D, adriamycin, m-AMSA, chromomycin A3, coralyne sulphoacetate, daunomycin, ellipticine, mithramycin, mitoxantrone, 5-methoxysterigmatocystin, rubidazone, variamycin, VM26 and VP16-213) and a number of other drugs acting via other mechanisms (viz . Baker's antifol, nitidine chloride and rhodamine 123) . Whereas the first-step mutants showed stable resistance to these drugs, the second-step lines partially reverted upon growth in non-selective medium . Further, treatment of these mutant lines with non-cytotoxic doses of the calcium channel blocker verapamil reverted or abolished their resistance to the above drugs in a dose-dependent manner . In contrast to the above compounds, the PurR mutants showed no significant cross-resistance to a large number of other drugs which included asaley, AT-125, 5-azacytidine, azaserine, cyclocytidine, cis-platin, cytosine arabinoside, chlorambucil, chlorpromazine, alpha-difluoromethyl ornithine, 5-fluorouracil, ftorafur, gallium nitrate, hydroxyurea, ICRF-159, ICRF-187, imipramine, methotraxate, 6-methylmercaptopurine riboside, mycophenolic acid, melphalan, mitomycin C, methyl GAG, nafoxidine, reumycin, 6-selenoguanosine, 6-thioguanine, tiazofurin, tamoxifen, thalicarpine, tiapamil and verapamil) . These cross-resistance data should prove useful in developing suitable drug combinations to which cellular resistance would not develop readily.

Mol Endocrinol, 1988 Oct, 2(10), 886 - 92
A multidrug-resistant MCF-7 human breast cancer cell line which exhibits cross-resistance to antiestrogens and hormone-independent tumor growth in vivo; Vickers PJ et al.; MCF-7 human breast cancer cells provide a useful in vitro model system to study hormone-responsive breast cancer as they contain receptors for estrogen and progesterone, and estrogen both induces the synthesis of specific proteins in these cells and increases their rate of proliferation . An MCF-7 cell line which was selected for resistance to adriamycin (MCF-7/AdrR) exhibits the phenotype of multidrug resistance (MDR), and displays multiple biochemical changes . MDR in MCF-7/AdrR is also associated with a loss of mitogenic response to estrogen and the development of cross-resistance to the antiestrogen 4-hydroxytamoxifen . In addition, while the parental MCF-7 cell line responds to estrogen with increased levels of progesterone receptors and the secretion of specific proteins, these estrogen responses are lost in MCF-7/AdrR . Furthermore, while the formation of tumors in nude mice by wild-type MCF-7 cells is dependent upon the presence of estrogen, MCF-7/AdrR cells form tumors in the absence of exogenous estrogen administration . These changes in hormonal sensitivity and estrogen-independent tumorigenicity of the multidrug-resistant MCF-7 cell line are associated with a loss of the estrogen receptor and a concomitant increase in the level of receptors for epidermal growth factor . Thus, in MCF-7/AdrR cells, the development of MDR is associated with alterations in the expression of both cytosolic and membrane receptors, resulting in resistance to hormonal agents and the expression of hormone-independent tumor formation.

Int J Radiat Oncol Biol Phys, 1988 Oct, 15(4), 931 - 6
Radiation resistance in a multidrug resistant human T-cell leukemia line; Shimm DS et al.; In clinical practice, cancers refractory to chemotherapy commonly appear to be comparatively radioresistant . One mechanism by which cancer cells become resistant to chemotherapy is pleiotropic multidrug resistance, characterized by cross resistance to a number of otherwise unrelated heterocyclic antineoplastic agents, including vinca alkaloids, anthracyclines, dactinomycin, and others . We have studied a drug sensitive human leukemia cell line, CEM; a pleiotropic multidrug resistant subline of CEM, CEM/VLB100; VLB-1, a drug sensitive revertant subline arising during in vivo passage of CEM/VLB100; and a methotrexate resistant subline of CEM, CEM-MTX . Using soft-agar colony formation after graded doses of X rays as an endpoint, we found that CEM, CEM-MTX, and CEM/VLB100 had similar terminal slopes (D0 = 0.66 Gy) . However, the CEM/VLB100 survival curve had a broader initial shoulder (n = 3.0, Dq = 0.75 Gy) than did CEM (n = 1.6, Dq = 0.25 Gy) or CEM/MTX (n = 1.0, Dq = 0 Gy), suggesting that CEM/VLB100 has an increased capacity to repair radiation-induced DNA damage . This was tested by comparing the cell lines' abilities to accumulate sublethal damage . In split dose recovery experiments, CEM/VLB100 demonstrated increased ability to repair sublethal radiation damage following fractionated irradiation compared with the CEM parental line . Although it no longer demonstrated multidrug resistance, VLB-1 still displayed diminished radiation sensitivity . On the basis of these and other investigators' results, we suggest that diminished radiation sensitivity is separate from, but can be closely associated with, the multidrug-resistant phenotype.

Exp Cell Res, 1988 Oct, 178(2), 513 - 7
The membrane transport system responsible for multidrug resistance is operating in nonresistant cells; Neyfakh AA et al.; Cultured hamster fibroblasts of the DM-15 cell line stained by rhodamine 123 gradually release the dye when placed in dye-free medium . Here we demonstrate that reserpine, verapamil, and trifluoperazine are capable of blocking this release . We also show that reserpine can inhibit the efflux of another dye, phosphine 3R, from DM-15 cells and the release of rhodamine 123 from mouse embryo fibroblasts, four mouse cell lines, and MDCK cells . The three substances that block the release of the dyes are potent inhibitors of the membrane transport system implicated in the phenomenon of multidrug resistance (MDR) . By using this system MDR cells can pump many structurally unrelated drugs and dyes, including rhodamine 123 and phosphine 3R, from the cytoplasm to the outer medium . It appears from our results that the membrane transport system responsible for MDR operates slowly in nonresistant cells and can play a role in normal cell physiology.

Oncology (Huntingt), 1988 Oct, 2(10), 55 - 63, 66-8
Mechanisms and clinical significance of multidrug resistance; Morrow CS et al.; Tumor cells often become refractory to diverse drugs with different mechanisms of cytotoxic action . This paper reviews the current state of our knowledge of multidrug resistance, the limitations of present concepts to fully explain the diversity of the MDR phenotypes, and the clinical relevance of these studies derived largely from cell culture systems . The authors discuss the use of markers associated with the multi-drug resistance phenotype to identify potentially drug-resistant tumors and outline strategies that might be used to overcome the resistance phenomenon.

Jpn J Cancer Res, 1988 Oct, 79(10), 1101 - 10
Cellular and tissue distribution of MRK20 murine monoclonal antibody-defined 85-kDa protein in adriamycin-resistant cancer cell lines; Sugawara I et al.; A murine monoclonal antibody (MAb) specific to adriamycin-resistant K-562 (K-562/ADM) cells, MRK20, was found to react strongly with an 85-kDa protein present in K-562/ADM and adriamycin-resistant ovarian cancer (2780AD) cells . This protein was present at only very low levels in parental cells (K-562 and A2780), methotrexate-resistant K-562 cells (K-562/MTX3, K-562/MTX4 and K-562/MTX5) and cisplatin-resistant ovarian cells (KFr) . Immunoelectron microscopically, the protein was found to be located on the cell membrane of K-562/ADM and 2780AD cells . Furthermore, the presence of the protein in various cell lines, normal tissues and surgical materials from patients given no anti-cancer agents was examined by immunocytochemistry and flow cytometry . MRK20 reacted with granulocytes, monocytes and endothelial cells in various tissues, but did not react with tissue macrophages . This 85-kDa protein recognized by MRK20 seems to be the second multidrug-resistance gene-encoded product appearing in adriamycin-resistant cancer cells, following the characterization of 170-180-kDa glycoprotein, and may be important for elucidating the multidrug-resistance mechanism relevant to adrimycin and Vinca alkaloids.

Gan To Kagaku Ryoho, 1988 Oct, 15(10), 2863 - 70
{Detection of multidrug resistant phenotype in leukemia and lymphoma by monoclonal antibodies}; Shimoyama M et al.; Two monoclonal antibodies, MRK16 and MRK20 that recognize P-glycoprotein and P-85 kd protein on the surface of adriamycin (ADM) resistant cells, respectively, were tested for the reactivity with 40 cultured leukemia/lymphoma cell lines . F(ab')2 form is essential to avoid false reaction through Fc gamma-R . Drug sensitivity of 19 representative cell lines were also examined in vitro . From this study, it was found that these cell lines were classified into 4 groups . Group 1 (4 cell lines) was insensitive to ADM, mitoxantron (MXT), etoposide (VP-16) and vincristine (VCR), and reactive to MRK16 and MRL20 . Group II (1 cell line) was insensitive to the 4 drugs, but not reactive to both antibodies . Group III (3 cell lines) was insensitive to ADM, MXT and VP-16, but sensitive to VCR, and reactive to MRK20, but not to MRK16 . Group IV (all other cell lines) was sensitive to these drugs, and not reactive to both antibodies . From these results, MRK16 detects P-glycoprotein-associated multidrug resistance (MDR), while MRK20 does P 85-kd-associated another type MDR (cross resistance to ADM, MXT and VP-16, but not to VCR) . MRK20 reacted with monocytes, but MRK16 did not with any WBC type . One hundred and ninety eight clinical samples obtained from blood cancer were tested for the reactivity with MRK16 . MRK16 did not react with any of 98 samples obtained before treatment, but did with 9 of 100 obtained at relapse or refractory stage after chemotherapy . The results indicate that MRK16 is useful to detect drug resistance phenotype of leukemia and lymphoma.

Gan To Kagaku Ryoho, 1988 Oct, 15(10), 2858 - 62
{The multidrug-resistance gene MDR1}; Ueda K et al.; MDR1 gene encodes a membrane glycoprotein (P-glycoprotein) that acts as a energy-dependent pump to transport antitumor drugs out of the cells . P-glycoprotein, 1280 amino acids long, consists of two homologous parts of approximately equal length . The protein has binding sites for ATP, antitumor drugs and calcium channel blockers . MDR1 gene is expressed tissue-specific in human normal adrenal, kidney, liver and colon . The normal function and transcriptional regulation of this gene are also discussed.

Gan To Kagaku Ryoho, 1988 Oct, 15(10), 2848 - 52
{Therapeutic approaches for multidrug resistance}; Tsuruo T; One of the major problems in cancer chemotherapy is the development of drug resistance during treatment . In the treatment of tumors with antitumor agents, tumor cells can acquire resistance to the drugs . This type of resistance is called as acquired drug resistance and the acquired drug resistances is a major factor for limiting the clinical use of antitumor agents . 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, the mechanisms of cellular resistance, especially those of multidrug resistance at a molecular level, and possible approaches to overcoming drug resistance are described and discussed.

Proc Natl Acad Sci U S A, 1988 Oct, 85(19), 7187 - 91
Photoaffinity labeling of the multidrug-resistance-related P-glycoprotein with photoactive analogs of verapamil; Safa AR; Verapamil, a phenylalkylamine calcium channel blocker, has been shown to reverse multidrug resistance in tumor cells, possibly by increasing drug retention through interaction with an outward drug transporter of the resistant cells . In this study two photoactive radioactive analogs of verapamil, N-(p-azido{3,5-3H}benzoyl)aminomethyl verapamil and N-(p-azido{3-125I}salicyl)aminomethyl verapamil, were synthesized and used to identify the possible biochemical target(s) for verapamil in multidrug-resistant DC-3F/VCRd-5L Chinese hamster lung cells selected for resistance to vincristine . The results show that a specifically labeled 150- to 180-kDa membrane protein in resistant cells was immunoprecipitated with a monoclonal antibody specific for P-glycoprotein . Phenylalkylamine binding specificity was established by competitive blocking of specific photolabeling with the nonradioactive photoactive analogs as well as with verapamil . Photoaffinity labeling was also inhibited by 50 microM concentrations of the calcium channel blockers nimodipine, nifedipine, nicardipine, azidopine, bepridil, and diltiazem and partially by prenylamine . Bay K8644, a calcium channel agonist, also inhibited P-glycoprotein photolabeling . Moreover, P-glycoprotein labeling was inhibited in a dose-dependent manner by vinblastine with half-maximal inhibition at 0.2 microM compared to that by verapamil at 8 microM . Photolabeling was also partially inhibited by two of the drugs to which these cells are cross-resistant, doxorubicin and actinomycin D, at 100 microM, but not by colchicine . These data provide direct evidence that P-glycoprotein has broad drug recognition capacity and that it serves as a molecular target for calcium channel blocker action in reversing multidrug resistance.

J Cell Biochem, 1988 Oct, 38(2), 87 - 97
Increased epidermal growth factor receptor in multidrug-resistant human neuroblastoma cells; Meyers MB et al.; Multidrug-resistant human neuroblastoma cell lines obtained by selection with vincristine or actinomycin D from two independent clonal lines, SH-SY5Y and MC-IXC, have 3- to 30-fold more cell surface epidermal growth factor (EGF) receptors than the drug-sensitive parental cells as indicated by EGF binding assays and immunoprecipitation, affinity-labeling, and phosphorylation studies . Reversion to drug sensitivity in one line was accompanied by a return to the parental level of EGF receptor . SH-EP cells, a clone derived from the same neuroblastoma cell line as SH-SY5Y but which displays melanocyte rather than neuronal lineage markers, also express significantly more EGF receptor than SH-SY5Y cells . By nucleic acid hybridization analysis with a molecularly cloned probe, increased receptor level in multidrug-resistant cells was shown to be the result of higher levels of EGF receptor mRNA in drug-resistant than in drug-sensitive cells . The increased steady state amount of specific RNA did not result from amplification of receptor-encoding genes . A small difference was observed in the electrophoretic mobility under denaturing conditions of EGF receptor immunoprecipitated from drug-resistant and drug-sensitive cells . Quantitative and qualitative modulation of the EGF receptor might reflect alterations in the transformation and/or differentiation phenotype of the resistant cells or might result from unknown selective pressures associated with the development of multidrug resistance.

Biochem Biophys Res Commun, 1988 Sep 15, 155(2), 754 - 60
Expression of a P-glycoprotein gene is inducible in a multidrug-resistant human leukemia cell line; Gekeler V et al.; A human T lymphoblastoid CCRF-CEM cell line exhibiting cross resistance to a variety of drugs was selected with increasing doses of actinomycin D . A subline, designated CCRF ACTD400+, was permanently cultured in the presence of 400 ng/ml Actinomycin D for several months . Using a fragment of the human mdr1 cDNA we found high expression of a 5 kb mRNA species which was not detectable in the sensitive parental CCRF-CEM cell line . The extent of the mdr-mRNA expression in resistant cells, however, depended on the presence or absence of actinomycin D in the culture medium: when the inhibitor was omitted, the expression decreased to about 60% after one month . In reverse, the steady state level of the P-glycoprotein mRNA increased about 2.5-fold within 72 h after the original dose of the drug was added again . In further experiments we recorded the actinomycin D or adriamycin dose response curves of the variously treated sublines by evaluation of {3H}uridine or {3H}thymidine incorporation, respectively, into acid insoluble material . Consistently, the drug sensitivity of the respective macromolecular synthesis was found to decrease with increasing mdr-mRNA levels.

Cancer Res, 1988 Sep 15, 48(18), 5096 - 100
DNA strand breaks produced by etoposide (VP-16,213) in sensitive and resistant human breast tumor cells: implications for the mechanism of action; Sinha BK et al.; Pleotropic resistant human breast cancer cells (MCF-7), selected for resistance to Adriamycin, were used to study the production of DNA strand breaks by etoposide (VP-16) and its relationship to drug cytotoxicity . It was shown that the resistant MCF-7 cell line was cross-resistant to VP-16, and the degree of resistance was found to be 125-200-fold . Alkaline elution studies indicated that the parental cell line was very sensitive to VP-16 which caused extensive DNA strand breakage . In contrast, little DNA strand breakage was detected in the resistant MCF-7 cells, even at very high drug concentrations, indicating a good agreement between strand breaks and cytotoxicity . Further studies indicated that the nuclei isolated from the parental cell line were more resistant to VP-16-induced DNA strand breaks than the intact cells, while the opposite was found in the resistant cell line . In addition, the alkaline elution studies in isolated nuclei showed only a 2-fold reduction of VP-16-induced DNA breaks in nuclei from the resistant cells . In agreement with this result, it was found that nuclear extract from the resistant cells produced 2-3-fold less VP-16-induced DNA breaks than that from the sensitive cells in 32P-end-labeled SV40 DNA . VP-16 uptake and efflux studies indicated that there was a 2-3-fold decrease in net cellular accumulation of VP-16 in the resistant cells . Although the reduced uptake of VP-16 and decreased drug sensitivity of topoisomerase II appear to contribute to the mechanism of action and the development of resistance to VP-16, they do not completely explain the degree of resistance to VP-16 in this multidrug-resistant MCF-7 cell line indicating that other biochemical factors, such as activation of VP-16, are also involved in drug resistance and suggesting that the resistance is multifactorial.

FASEB J, 1988 Sep, 2(12), 2791 - 6
Resistance of multidrug-resistant lines to natural killer-like cell-mediated cytotoxicity; Woods G et al.; Multidrug resistance (MDR) refers to a complex phenotype that describes a number of features characterized primarily by resistance to a wide range of structurally unrelated drugs . In this paper we investigated the relationship between drug resistance and resistance to NK-mediated cytotoxicity . Studies with two independently selected multidrug-resistant cell lines indicated that increased drug resistance was associated with both an increased resistance to NK-mediated cytotoxicity and increased levels of membrane P-glycoprotein expression . This resistance to cytotoxicity appears to result partly from an alteration in the membrane structure of the target cells inasmuch as there was a reduction in effector:target cell recognition . Resistance to NK-mediated cytotoxicity should be included with the numerous pleiotropic changes associated with the multidrug resistance phenotype.

Isr J Med Sci, 1988 Sep-Oct, 24(9-10), 477 - 82
Mechanism of acquired resistance to methotrexate in P388 murine leukemia cells and in their doxorubicin-resistant subline; Ramu N et al.; The mechanisms of acquired resistance to MTX were studied in P388 murine leukemia cell lines that were sensitive or resistant to ADR . The rate of MTX accumulation in ADR-sensitive cells that have acquired resistance to MTX was found to be lower than that measured in cells that were sensitive to both drugs . Furthermore, in contrast to drug-sensitive cells, in the ADR-sensitive MTX-resistant cells, most of the intracellular MTX (86.2%) was bound and MTX polyglutamation was not detected . The initial rate of MTX accumulation in cells that were resistant to both drugs was comparable to that measured in cells that were sensitive to both drugs or that were resistant only to ADR . However, in the cells that were resistant to both drugs, the rate of MTX accumulation was maintained at its initial level for a period that was considerably longer than that found in the other cell lines . After 3 h of exposure to MTX, the accumulation of MTX in cells that were resistant to both drugs was fourfold higher than that measured in cells that were sensitive to both drugs . Furthermore, while 65 to 70% of the intracellular MTX was free, in cells sensitive to both drugs, or resistant only to ADR, the corresponding value in cells that were resistant to both drugs was less than 1.5%, and a much lower proportion of the MTX was polyglutamated . The sensitivity to TMQ of ADR-sensitive, MTX-resistant cells was similar to that found in cells that were sensitive to ADR and MTX . However, ADR-resistant cells, sensitive or resistant to MTX, were markedly resistant to TMQ . The sensitivity of ADR-resistant MTX-sensitive cells to TMQ was restored by the presence of 10 microM verapamil . Such an effect was not observed in cells resistant to both drugs . It is suggested that P388 cells that have previously acquired resistance to ADR, when now selected by MTX, retain the MTX-transport system (in contrast to ADR-sensitive, MTX-resistant cells) and become resistant to MTX by increasing the activity of DHFR . The results obtained in ADR-resistant cells also suggested that resistance to TMQ was part of the multidrug resistance phenomenon.

Cancer Res, 1988 Sep 1, 48(17), 4926 - 32
Characterization of the ATPase activity of the Mr 170,000 to 180,000 membrane glycoprotein (P-glycoprotein) associated with multidrug resistance in K562/ADM cells; Hamada H et al.; The Mr 170,000 to 180,000 membrane glycoprotein associated with multidrug resistance (P-glycoprotein) is involved in drug transport mechanisms across the plasma membrane of multidrug-resistant cells . We have recently reported the purification of P-glycoprotein . The purified P-glycoprotein was found to have an ATPase activity, which might be coupled with the active efflux of anticancer drugs . In the present study, we have further studied the properties of the P-glycoprotein ATPase activity by an immobilized enzyme assay procedure using a P-glycoprotein-antibody-Protein A-Sepharose complex . GTP was also hydrolyzed by the P-glycoprotein, although less efficiently than ATP . The ATPase activity of P-glycoprotein had an optimal pH range around neutrality (pH 6.5-7.4) . The detergent concentration of 3-{(3-cholamidopropyl)dimethyl-ammonio}-1-propane sulfonate used for protein solubilization was essential for enzyme recovery . Maximum activity was obtained when 0.1-0.2% 3-{(3-cholamidopropyl)dimethyl-ammonio}-propane sulfonate was used, while higher concentrations markedly inhibited the ATPase activity . The ATPase activity was dependent on Mg2+; maximum activity was obtained at 2-10 mM . Manganese and cobalt could substitute for magnesium as ionic cofactors . Divalent cations such as Ca2+, Zn2+, Ni2+, Cd2+, and Cu2+ inhibited the Mg2+-catalyzed ATP hydrolysis . N-Ethylmaleimide and vanadate inhibited the ATPase activity, while sodium azide or ouabain had no effect . Anticancer agents such as vincristine and Adriamycin did not affect the enzyme activity . In contrast, verapamil and trifluoperazine, agents which inhibit active drug efflux and restore drug sensitivity in resistant cells, caused an increase in the P-glycoprotein ATPase activity suggesting that P-glycoprotein might be the target molecule of these agents.

Proc Natl Acad Sci U S A, 1988 Sep, 85(17), 6518 - 22
Isolation of the human anionic glutathione S-transferase cDNA and the relation of its gene expression to estrogen-receptor content in primary breast cancer; Moscow JA et al.; The development of multidrug resistance in MCF7 human breast cancer cells is associated with overexpression of P-glycoprotein, changes in activities of several detoxication enzymes, and loss of hormone sensitivity and estrogen receptors (ERs) . We have cloned the cDNA for one of the drug-detoxifying enzymes overexpressed in multidrug-resistant MCF7 cells (AdrR MCF7), the anionic isozyme of glutathione S-transferase (GST pi) . Hybridization with this GST pi cDNA, GST pi-1, demonstrated that increased GST pi activity in AdrR MCF7 cells is associated with overexpression but not with amplification of the gene . We mapped the GST pi gene to human chromosome 11q13 by in situ hybridization . Since multidrug resistance and GST pi overexpression are associated with the loss of ERs in AdrR MCF7 cells, we examined several other breast cancer cell lines that were not selected for drug resistance . In each of these cell lines we found an inverse association between GST pi expression and ER content . We also examined RNA from 21 primary breast cancers and found a similar association between GST pi expression and ER content in vivo . GST pi mRNA content in 11 ER-positive tumors (less than or equal to 10 fmol/mg of protein) was significantly different from the GST pi content of 10 ER-negative tumors (P = 0.002; Mann-Whitney Wilcoxon test for two independent samples) . The finding of similar patterns of expression of a drug-detoxifying enzyme and of ERs in vitro as well as in vivo suggests that ER-negative breast cancer cells may have greater protection against antineoplastic agents conferred by GST pi than ER-positive tumors.

Mol Immunol, 1988 Sep, 25(9), 913 - 5
A simple metabolic system for selection of hybrid hybridomas (tetradomas) producing bispecific monoclonal antibodies; Chervonsky AV et al.; A metabolic selective system has been proposed for the selection of hybrid hybridomas (tetradomas) based on the introduction in one of the parental cell lines of two traits simultaneously--a recessive one (resistance to 8-azaguanine) and a dominant one (multidrug resistance) . Tetradomas were selected in the presence of two selective agents: aminopterin and actinomycin D . Using this approach we produced tetradomas secreting bispecific MAbs to horseradish peroxidase and human alpha-fetoprotein.

Biochem Biophys Res Commun, 1988 Aug 30, 155(1), 476 - 81
Reversal of chloroquine resistance in falciparum malaria independent of calcium channels; Ye ZG et al.; Racemic verapamil and close structural derivatives gallopamil and devapamil completely reverse chloroquine-resistance in falciparum malaria at 1-2 micromolar concentrations . If the R-(+) isomers of these calcium channel inhibitors are used, chloroquine-resistance is again completely reversed at similar doses . However, these R-(+) isomers do not bind to cardiovascular calcium channels which are stereospecific for the S-(-) isomer of the drugs . Further since calcium channel inhibition is not involved, toxicity associated with this activity can be avoided . Therefore it is possible that a series of R-(+) isomers could be found that alter the resistant state without possessing significant toxicity . It is postulated that these lipophilic drugs are interacting with the mechanism of resistance, possibly a multidrug resistance glycoprotein pump.

J Biol Chem, 1988 Aug 25, 263(24), 11887 - 91
ATP/Mg2+-dependent binding of vincristine to the plasma membrane of multidrug-resistant K562 cells; Naito M et al.; To study the mechanism of active drug efflux in multidrug-resistant cells, the interaction between {3H} vincristine (VCR) and plasma membrane prepared from an adriamycin (ADM)-resistant variant (K562/ADM) of human myelogenous leukemia K562 cells was examined by filtration method . {3H}VCR bound to the plasma membrane prepared from K562/ADM cells, but not from parental K562 cells, depending on the concentrations of ATP and Mg2+ . Adenosine 5'-O-(3-thio)triphosphate was not effective in the binding of {3H}VCR, indicating that ATP hydrolysis is required for this binding . Dissociation constant (Kd) of VCR binding was 0.24 +/- 0.04 microM in the presence of 3 mM ATP . In the absence of ATP, specific binding of VCR to K562/ADM membrane was also observed; however, the affinity (Kd = 9.7 +/- 3.1 microM) was 40 times lower than that observed in the presence of ATP . The high affinity VCR binding to K562/ADM membrane was dependent on temperature . The bound {3H}VCR molecules were rapidly released by unlabeled VCR added to the reaction mixture at 25 degrees C . The high affinity binding of {3H}VCR to K562/ADM membrane was inhibited by VCR, vinblastine, actinomycin D, and ADM, to which K562/ADM cells exhibit cross-resistance, whereas 5-fluorouracil and camptothecin, to which K562/ADM cells are equally sensitive as K562 cells, did not inhibit the {3H}VCR binding . Furthermore, verapamil and other agents, which are known to circumvent drug resistance by inhibiting the active efflux of antitumor agents from resistant cells, could also inhibit the high affinity {3H}VCR binding . These results indicate that ATP/Mg2+-dependent high affinity VCR binding to the membrane of resistant cells closely correlates with the active drug efflux of this resistant cell line.

Cancer Res, 1988 Aug 15, 48(16), 4477 - 83
Properties of verapamil-hypersensitive multidrug-resistant Chinese hamster ovary cells; Warr JR et al.; Two vincristine-resistant Chinese hamster ovary cell lines have been shown previously to be hypersensitive to the calcium channel blocker, verapamil . They are now shown to be hypersensitive to the membrane-active agent quinidine sulfate and to the calcium channel blockers diltiazem and nicardipine . Hypersensitivity to quinidine sulfate implies that calcium channels are not the primary target for these drug effects on these cell lines and is consistent with our previous observation that their calcium accumulation is normal in the presence and absence of verapamil . The two cell lines have elevated levels of membrane P-glycoprotein and of two cytosolic proteins, Mr 27,000 and pI 6.0 and 6.4 . Revertants have normal levels of these cytosolic proteins, suggesting that these proteins may play a role in conferring resistance . {3H}Verapamil accumulation by the two cell lines is lower than in controls . One of the cell lines has been hybridized to normal cells and the vincristine resistance and verapamil sensitivity of three hybrid clones has been determined . Vincristine resistance is semidominant but verapamil hypersensitivity is completely recessive.

Cancer Lett, 1988 Aug 15, 41(2), 169 - 77
The role of oxygen-derived free radicals in the cytotoxicity of doxorubicin in multidrug resistant and sensitive human ovarian cancer cells; Cervantes A et al.; The role of oxygen-derived free radicals in the cytotoxicity of doxorubicin (Dox) was studied in a Dox sensitive human ovarian cancer cell line (A2780) and its multidrug resistant counterpart (2780AD) using reactive oxygen scavengers . In both cell lines, a significant inhibition of Dox toxicity was found after treatment with the hydroxyl radical scavengers, N-acetylcysteine, sodium benzoate and dimethyl sulfoxide, but not with mannitol . The protection was similar in sensitive and resistant cells: 13-39% less growth inhibition was found at Dox concentrations of 0.2 and 0.5 microM for A2780 as well as at 20 and 50 microM for 2780AD . This protection was not due to effects of the scavengers on Dox accumulation, as shown by uptake experiments with radio-labelled Dox . The superoxide anion free radical scavenger ascorbic acid or the enzyme superoxide dismutase as well as the hydrogen peroxide scavenger catalase did not protect cells against Dox-induced cell growth inhibition . Preloading the cells with the enzymes, a treatment which resulted in a two to nine-fold increase in their cellular contents, was not effective either . It is concluded that hydroxyl radicals, but not superoxide anion or hydrogen peroxide likely play a role in the antitumor activity of Dox in sensitive and resistant human ovarian cancer cells.

Science, 1988 Aug 5, 241(4866), 694 - 7
Role of the glutathione redox cycle in acquired and de novo multidrug resistance; Kramer RA et al.; Drug resistance represents a major obstacle to successful cancer chemotherapy . However, the specific biochemical mechanisms responsible for clinical drug resistance are unknown . In these studies resistance to the antitumor agent adriamycin was found to involve two mechanisms, one that decreased drug accumulation by the P170 mechanism and another that altered the glutathione redox cycle, an important pathway in the detoxification of reactive oxygen . This dual mechanism of drug resistance was demonstrated in cell lines that had acquired the multidrug-resistant phenotype and in human colorectal cancer cells with de novo resistance . These studies support a model of acquired and de novo multidrug resistance that includes alterations in both drug accumulation and the glutathione redox cycle.

Cancer Res, 1988 Aug 1, 48(15), 4334 - 9
In situ hybridization analysis of acquisition and loss of the human multidrug-resistance gene; Shen DW et al.; The extent of multidrug-resistance of human KB carcinoma cell lines has been shown to be proportional to the level of expression of the MDR1 gene . Using an in situ hybridization analysis with 35S-labeled RNA probes, we have found that there is some heterogeneity in expression of the MDR1 gene from cell to cell, but that the average level of expression is proportional to the resistance of the cell line . In the absence of selective pressure, a colchicine-selected multidrug-resistant population with a highly amplified MDR1 gene loses its resistance in parallel with the loss of the amplified gene . Loss of resistance also parallels a decrease in MDR1 RNA expression in the whole cell population . Loss of MDR1 expression in this population is highly heterogeneous, with small clusters of cells maintaining expression even after the population as a whole has become relatively sensitive . This heterogenous loss of expression of the MDR1 gene is consistent with random segregation of amplified DNA segments in the selected cells . The analysis of MDR1 RNA expression by in situ hybridization which is validated by this study should be useful in the study of normal human tissue and tumor samples expressing the MDR1 gene.

Arzneimittelforschung, 1988 Aug, 38(8), 1189 - 93
Intrinsic drug resistance in a human lung carcinoma xenograft is associated with overexpression of multidrug-resistance DNA-sequences and of plasma membrane glycoproteins; Volm M et al.; The purpose of this study was to examine whether multidrug resistance can be detected in human lung tumors not previously treated with chemotherapy . Therefore, the intrinsic sensitivity or resistance of 8 human epidermoid lung cancer xenografts grown in nude mice has been examined . The tumor lines responded differently to vincristine and the other cytostatic agents . When the effects of vincristine on the 8 tumor lines are correlated with the effect of dactinomycin (actinomycin D), a close relationship could be found (r = 0.96) . These results demonstrate that xenografts derived from human lung tumors not previously treated with chemotherapy exhibited a similar pattern of cross-resistance as is observed in sublines of murine and human tumors which were resistant to various drugs following repeated exposure of the cells to a sublethal concentration of these drugs . The resistant lung tumor HXL 54 also shows an overexpression of the P170-membrane glycoprotein (detected by Mab 265/F4) . To determine whether multidrug genes were expressed in resistant lung tumors, slot blots and Northern blots were performed using the probes pDR 7.8 and pcDR 1.5 . The level of expression can be correlated with the degree of drug resistance.

J Cell Physiol, 1988 Aug, 136(2), 341 - 7
Altered molecular properties of tubulin in a multidrug-resistant variant of Chinese hamster cells selected for resistance to vinca alkaloids; Pain J et al.; The basis for the markedly altered intracellular binding of {3H}vincristine in a multidrug-resistant variant (DC-3F/VCRd-5L) of Chinese hamster lung cells (DC-3F) was investigated . Binding of {3H}vincristine by protein in cytosol derived from each cell type exhibited a differing requirement for GTP in MgCl2 containing buffer of low-ionic strength . Binding of {3H}vincristine occurred to cytosolic protein derived from both variant and parental DC-3F cells, but after removal of GTP, binding only occurred to cytosolic protein from parental cells regardless of the presence of added GTP . Although binding by cytosolic protein from parental DC-3F cells did not require GTP, the addition of 0.1 mM GTP increased by two-fold the rate and extent of binding . When cytosol from variant and parental DC-3F cells was incubated with low concentrations of {3H}vincristine in high-ionic strength buffer and analyzed by molecular-sieve HPLC, most of the protein binding {3H}vincristine in parentally derived cytosol eluted as Mr 110,000-115,000 daltons, corresponding to that for dimeric tubulin . The same binding species was not detected in cytosol derived from variant cells . However, these same fractions derived with both parental and variant cytosols contained tubulin as shown by SDS-PAGE and immunoblotting . A smaller peak of {3H}vincristine binding and an amount of tubulin equal to that found in later fractions were found in the void volume during the same HPLC elution runs with cytosol from both variant and parental DC-3F cells . Evidence was also obtained for differences between parental and variant DC-3F cells in beta-tubulin isoforms following isoelectric focusing and immunoblotting . Parental-cell cytosol contains a single isoform of beta-tubulin . However, in variant cell cytosol the same isoform and, in addition, three more basic isoforms were found . These alterations in {3H}vincristine binding and in isoform compositions of beta-tubulin in variant versus parental DC-3F cells may have importance in regard to vincristine resistance in DC-3F cells.

Mol Cell Biol, 1988 Aug, 8(8), 3316 - 21
Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease; Kane SE et al.; Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L) . To evaluate the possible role of this protease in the transformed phenotype, we transfected cloned genes for mouse or human MEP into mouse NIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene . The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine . The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells . The MEP synthesized by cells transfected with the cloned mouse and human MEP genes was also secreted . Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.

Cancer Res, 1988 Jul 15, 48(14), 3959 - 63
Mechanisms of multidrug resistance in HL60 cells: evidence that a surface membrane protein distinct from P-glycoprotein contributes to reduced cellular accumulation of drug; McGrath T et al.; HL60 cells exhibiting a 140-fold increase in resistance to vincristine contain three surface membrane proteins with molecular weights of 210,000 (P210), 180,000 (P180), and 150,000 (P150) which are highly phosphorylated in vivo and in an in vitro system in the presence of Mn2+ and {gamma-32P}ATP . These phosphorylated proteins are either absent or present in very low levels in membranes of drug-sensitive cells . Growth of the vincristine-resistant isolate in the absence of drug results in a decrease in the level of resistance and a major reduction in the phosphorylation of P210 and P180 . The phosphorylation of P150 is not altered in the revertant which still exhibits substantial levels of resistance . Further studies show that P210 and P180 are highly reactive with a monoclonal antibody against P-glycoprotein . These two proteins are present in only very low levels in revertant cells . The monoclonal antibody exhibits no reactivity with P150 . In HL60 cells isolated for a 25-fold increase in vincristine resistance proteins reactive with P-glycoprotein monoclonal antibody are essentially absent . P150 is however highly phosphorylated in these cells . Additional experiments using lectin binding of 32P-labeled proteins demonstrates that P150 has properties distinct from P210 and P180 . Analysis of drug uptake patterns in the vincristine-resistant isolates and the revertant shows that resistance is related to a reduced intracellular accumulation of drug . Reduced accumulation of vincristine is also found in HL60 cells isolated for resistance to Adriamycin . These cells are devoid of P-glycoprotein but contain phosphorylated P150 . These results suggest that proteins P150, P180, and P210 may contribute to multidrug resistance in HL60 cells through a mechanism which involves reduced cellular accumulation of drug . P180 and P210 are structurally related whereas P150 is distinct from these two proteins.

Mol Cell Biol, 1988 Jul, 8(7), 2770 - 8
Cloning and characterization of a second member of the mouse mdr gene family; Gros P et al.; The mammalian mdr gene family comprises a small number of closely related genes . Previously, we have shown that one member, mdr1, has the capacity to convey multidrug resistance to drug-sensitive recipient cells in a gene transfer protocol . However, the functional characteristics of other members of this gene family have not been examined . In this report, we characterize a second member of the mdr gene family which we designated mdr2 . We determined the nucleotide sequence corresponding to the complete coding region of this mdr2 transcript . The predicted amino acid sequence of this protein (1,276 amino acids) showed that it is a membrane glycoprotein highly homologous to mdr1 (85%), strongly suggesting that both genes originate from a common ancestor . Regions of divergence between mdr1 and mdr2 proteins are concentrated in two discrete segments of the predicted polypeptides, each approximately 100 residues in length . The mdr2 protein appears to be formed by the duplication of a structural unit which encodes three putative transmembrane loops and a predicted nucleotide-binding fold and is highly homologous to bacterial transport proteins such as hlyB . This strong homology suggests that mdr2 also participates in an energy-dependent membrane transport process . However, the direct relationship, if any, of this new member of the mdr family to multidrug resistance remains to be established . Knowledge of the complete nucleotide sequence and predicted amino acid sequence of the mdr2 gene product will enable the preparation of gene-specific probes and antibodies necessary to study the functional role of this gene in multidrug resistance and normal physiological processes.

Cytometry, 1988 Jul, 9(4), 359 - 67
Isolation of highly multidrug-resistant P388 cells from drug-sensitive P388/S cells by flow cytometric cell sorting; Ross DD et al.; To investigate the spontaneous frequency of occurrence of stable multidrug-resistant cells in a population of drug-sensitive cells, we exposed drug sensitive P388/S cells to daunorubicin (dnr) for 1 h, then used fluorescence-activated cell sorting based on intracellular dnr fluorescence to isolate cells within P388/S having different intracellular content of drug . One of the sort windows chosen (low dnr content sort window) isolated only P388/S cells with intracellular drug content equal to or less than that of the known multidrug-resistant subline P388/adr . This sort window constituted approximately 3% of P388/S cells with lowest dnr content . By such a procedure we were able, on one of seven attempts, to isolate and cultivate stable, highly multidrug-resistant cells (comparable to that of P388/adr) from the P388/S cells obtained from the low dnr-content sort window . Net growth of cells in culture was observed 15-20 days after sorting, indicating that of the P388/S cells collected from the low dnr-content sort window, very few were actually highly drug-resistant . On no occasion could resistant cells be cultivated from cells sorted from P388/S with higher dnr content, as would be expected if mutation to a multidrug-resistant phenotype had occurred as a result of exposure to drug . The resistant cells isolated from P388/S by sorting (called P388/LoSort) displayed low intracellular accumulation of dnr that was enhanced by verapamil, were cross-resistant to vincristine and actinomycin-D, and distinct from P388/S, possessed a 150- to 160-kD membrane species identified by Vinca alkaloid photoaffinity labeling.(ABSTRACT TRUNCATED AT 250 WORDS)

Clin Pharmacokinet, 1988 Jul, 15(1), 15 - 31
Clinical pharmacokinetics of doxorubicin; Speth PA et al.; Doxorubicin (adriamycin) has a very wide antitumour spectrum, compared with other anticancer drugs; however, except for Hodgkin's disease, it is not associated with curative chemotherapy . Doxorubicin has been in clinical use for more than 2 decades, and only recently has it been recognised that the cytotoxic effect is produced at the cellular level by multiple mechanisms which have not yet been conclusively identified . Key factors are a combination of doxorubicin-induced free radical formation due to metabolic activation, deleterious actions at the level of the membrane, and drug-intercalation into DNA . Multiple aspects of the clinical pharmacokinetics of this drug have been described . Wide interpatient variations in plasma pharmacokinetics have been noted, but without firm relation to clinical outcome . An apparent volume of distribution of approximately 25 L/kg points to extensive uptake by tissues . Up to several weeks after administration, significant concentrations of doxorubicin have been found in haematopoietic cells and in several other tissues . The maximum cellular doxorubicin concentrations reached in vivo remain significantly below those at which all clonogenic leukaemic cells are killed in vitro . Doxorubicin has been administered as frequent (weekly) low doses, single high doses, and as a continuous infusion . The optimal schedule with respect to tumour cytotoxicity and dose-limiting side effects such as myelosuppression or cardiotoxicity, has never been investigated in a prospective, randomised manner . Clinical trials large enough to study optimal, and possibly individualised, doxorubicin chemotherapy need to be performed . This review summarises pharmacological and pharmacodynamic data of doxorubicin, and discusses these in relation to possible improvement of its therapeutic index . Furthermore, drug interactions, dose-response relationships, mechanisms of action, multidrug resistance, and treatment scheduling are discussed in the perspective of the development of novel treatment strategies.

Leukemia, 1988 Jul, 2(7), 453 - 8
Molecular/cytogenetic alterations accompanying the development of multidrug resistance in the J774.2 murine cell line; Slovak ML et al.; Mouse macrophage-like J774.2 cells were selected for resistance to colchicine and examined by molecular/cytogenetic analysis to determine whether the acquisition of the multidrug resistant (mdr) phenotype was associated with specific chromosomal rearrangements . Cytogenetic studies of the J774.2 parental and two colchicine-resistant (CLCR) sublines--J7.Cl-30 (770-fold CLCR) and J7.Cl-100 (2500-fold CLCR)--demonstrated specific numeric and structural karyotypic alterations accompanying the emergence of mdr . The parental cells demonstrated a modal chromosome number of 63, while the modal number of the J7.Cl-30 subline was 53 . The most striking difference between the parental and J7.Cl-30 subline was the presence of an average of 60 double minutes (DMs) per cell in the CLCR cells . The 2500-fold resistant J7.Cl-100 subline displayed a modal number of 50, which included structural rearrangements involving chromosomes 2 and 7 and concomitant replacement of DMs by a homogeneously staining region (HSR) . Southern blotting analysis demonstrated a approximately 35-fold amplification of P-glycoprotein homologous sequences in the J7.Cl-30 subline and approximately 70-fold amplification in the J7.Cl-100 subline . Chromosomal in situ hybridization localized the amplified P-glycoprotein sequences to DMs (J7.Cl-30) and the HSR (J7.Cl-100) in these CLCR sublines . Our results suggest that CLCR in J774.2 cells results from overexpression of P-glycoprotein via gene amplification which was accompanied by chromosomal evolution from DMs to an HSR.

Cancer Res, 1988 Jul 1, 48(13), 3595 - 602
Multifactorial resistance to adriamycin: relationship of DNA repair, glutathione transferase activity, drug efflux, and P-glycoprotein in cloned cell lines of adriamycin-sensitive and -resistant P388 leukemia; Deffie AM et al.; Cloned lines of Adriamycin (ADR)-sensitive and -resistant P388 leukemia have been established, including P388/ADR/3 and P388/ADR/7 that are 5- and 10-fold more resistant than the cloned sensitive cell line P388/4 (Cancer Res., 46: 2978, 1986) . A time course of ADR-induced DNA double-strand breaks revealed that in sensitive P388/4 cells, evidence of DNA repair was noted 4 h after removal of drug, whereas in resistant clone 3 and 7 cells repair was observed 1 h after drug removal . The earlier onset of DNA repair was statistically significant (p = 0.0154 for clone 3 cells, and p = 0.0009 for clone 7 cells) . By contrast, once the repair process was initiated, the rate of repair was similar for all three cell lines . The level of glutathione transferase activity was determined in whole cell extracts . Enzyme activity (mean +/- SE) in sensitive cells was 9.49 +/- 1.00 nmol/min/mg protein, that in resistant clone 3 cells was 13.36 +/- 1.03 nmol/min/mg, and that in clone 7 cells was 13.96 +/- 1.44 nmol/min/mg; the 1.44-fold increase in enzyme activity in resistant cells was statistically significant (p = 0.01) . Further evidence of induction of glutathione transferase was provided by Northern blot analysis using a 32P-labeled cDNA for an anionic glutathione transferase, which demonstrated approximately a twofold increase in mRNA in resistant clone 7 cells . Western blot analysis with a polyvalent antibody against anionic glutathione transferase also revealed a proportionate increase in gene product in resistant cells . Dose-survival studies showed that ADR-resistant cells were cross-resistant to actinomycin D, daunorubicin, mitoxantrone, colchicine, and etoposide, but not to the alkylating agent melphalan; this finding provided evidence that these cells are multidrug resistant . Using a cDNA probe for P-glycoprotein, a phenotypic marker for multidrug resistance, Northern blot analysis showed an increase in the steady state level of mRNA of approximately twofold in resistant clone 3 and 7 cells . Southern analysis with the same cDNA probe showed no evidence of gene amplification or rearrangement . Western blot analysis with monoclonal C219 antibody demonstrated a distinct increase in P-glycoprotein in resistant cells . Efflux of Adriamycin as measured by the efflux rate constant was identical in all three cell lines . Furthermore, the metabolic inhibitors azide and dinitrophenol did not augment drug uptake in either sensitive or resistant cells . These findings suggest that despite the increase in P-glycoprotein, an active extrusion pump was not operational in these cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochem Biophys Res Commun, 1988 Jun 30, 153(3), 959 - 66
Effects of indole alkaloids on multidrug resistance and labeling of P-glycoprotein by a photoaffinity analog of vinblastine; Beck WT et al.; Multidrug resistant cells are characterized by decreased drug accumulation and retention, thought to be mediated by a high molecular weight glycoprotein, P-glycoprotein (P-gp) . Agents such as verapamil have been shown to increase anticancer drug cytotoxicity and increase the amount of drug accumulated and retained by such cells . We show here that in addition to verapamil, reserpine, chloroquine, quinine, quinacrine, yohimbine, vindoline, and catharanthine also enhance the cytotoxicity of vinblastine (VLB) in a multidrug resistant, human leukemic cell line, CEM/VLB1K, described here for the first time . These cells express P-gp as a doublet that is photoaffinity labeled by the analog of VLB, N(p-azido-{3-125I}salicyl)-N'-beta-aminoethylvindesine ({125I}NASV) . Both reserpine and, to a lesser extent, verapamil, compete with {125I}NASV for binding to P-gp . We also found that chloroquine, quinacrine, vindoline, and catharanthine, each of which enhanced VLB cytotoxicity in CEM/VLB1K cells by 10- to 15-fold, similarly inhibited {125I}NASV labeling of P-gp . However, neither quinine nor yohimbine inhibited this labeling, and the inhibition produced by catharanthine and vindoline was the greatest or exclusively on the lower band of the P-gp doublet . Our results suggest a complex relationship between the ability of a compound to modulate MDR and its ability to compete for binding to P-gp.

Biochem Biophys Res Commun, 1988 Jun 16, 153(2), 598 - 605
Intrinsic multidrug resistance phenotype of Chinese hamster (rodent) cells in comparison to human cells; Gupta RS; In comparison to human cells, cell lines of Chinese hamster and mouse origin exhibit between 10-50-fold resistance to a number of different drugs (viz . actinomycin D, daunomycin, chromomycin A3, colchicine, maytansine, mithramycin, puromycin, rhodamine 123, vinblastine and taxol) . Studies with a representative Chinese hamster line (CHO) and a single-step multidrug resistant (MDR) mutant of human (HeLa) cells show that: (i) In comparison to the sensitive human cells, both cell lines show a comparable degree of resistance to the above mentioned drugs; (ii) In the presence of non-toxic dosage of verapamil, the drug-resistance phenotype of both cell lines is completely reversed; (iii) Both these cell lines showed greatly reduced uptake/intracellular levels of 3H-daunomycin, 3H-puromycin and 3H-vinblastine, which was restored to sensitive human cell's level in the presence of non-toxic doses of verapamil . The striking similarity in the behaviour of the naturally resistant Chinese hamster cells and a human MDR cell line with regard to the above characteristics, strongly suggests that the species-related differences in sensitivity to the above drugs result from a similar mechanism as that responsible for the MDR phenotype.

Cancer Res, 1988 Jun 15, 48(12), 3355 - 9
cis-diamminedichloroplatinum(II)-resistant sublines derived from two human ovarian tumor cell lines; Kuppen PJ et al.; In two human ovarian tumor cell lines, resistance to cis-diamminedichloroplatinum(II) (CDDP) was induced by continuous exposure of the parental lines to an increasing CDDP concentration in the culture medium . In contrast, a six times repeated pulse exposure of 6.7 microM or 16.7 microM CDDP for 1 h did not result in a cell line that showed a higher survival in CDDP-containing medium . The ID50 value for CDDP was seven to eight times higher for the resistant lines and those lines were able to grow at 3.3 microM CDDP . The induced resistance was stable during at least 25 doubling times . The CDDP-resistant sublines showed cross-resistance to the CDDP-analogues cis-diammine-1,1-cyclobutane-dicarboxylateplatinum(II) and cis-dichlorobis(isopropylamine)-trans-dihydroxyplatinum(IV) indicating that resistance to the different platinum compounds is generated by a common mechanism . The resistant sublines were also cross-resistant to mitomycin C and melphalan . The degree of cross-resistance for the tested drugs varied widely between the two cell lines . Resistance to CDDP was clearly correlated to decreased amounts of platinum in the resistant cells as compared to the sensitive cells . The amplification and expression of genes encoding proteins that had been shown to be involved in multidrug resistance, e.g., the Mr 170,000 P-glycoprotein was also studied . No amplification or overexpression of these genes could be shown in the resistant cell lines.

Proc Natl Acad Sci U S A, 1988 Jun, 85(12), 4350 - 4
The gene encoding multidrug resistance is induced and expressed at high levels during pregnancy in the secretory epithelium of the uterus; Arceci RJ et al.; A survey of the expression of the multidrug-resistance gene (mdr) in mouse tissues revealed that a mdr mRNA species is expressed at extremely high levels in the gravid uterus . mdr mRNA expression levels increase dramatically during pregnancy compared to the relatively low levels of expression observed in the nongravid uterus . In situ hybridization experiments revealed that the increased expression of the mdr mRNA is specifically localized to the secretory epithelial cells of the endometrium . Immunocytochemistry studies with a mdr glycoprotein-specific antiserum demonstrate that the mdr glycoprotein is predominantly localized to the luminal surface of the secretory epithelial cells . These results indicate that the mdr gene expression in the uterus is controlled by the physiologic changes associated with pregnancy . Our data are consistent with a potential role for the mdr glycoprotein in the transport of substrate across the secretory epithelium of the gravid uterus.

Cancer Res, 1988 Jun 1, 48(11), 3179 - 87
Chromosomal organization of amplified genes in multidrug-resistant Chinese hamster cells; Biedler JL et al.; Six independently derived multidrug-resistant Chinese hamster cell lines selected with vincristine, daunorubicin, actinomycin D, or colchicine were probed by in situ hybridization techniques with the cloned cDNA, p5L-18, to chromosomally localize known or presumed amplified P-glycoprotein genes . One or two clusters of amplified genes were demonstrable in all of the highly resistant sublines and were localized to homogeneously staining regions and/or abnormally banding regions, gene amplification-associated cytogenetic abnormalities, on eight different chromosomes . Analysis of trypsin-Giemsa banded karyotypes revealed additional, multiple chromosomal rearrangements that were apparently nonspecific . Mapping studies localized the native P-glycoprotein gene(s) to the region q23 to 31 (most probably band 26) on the long arm of chromosome 1 of normal Chinese hamster bone marrow fibroblasts and normal chromosome 1 homologues in resistant cells . Southern blot analysis of restriction endonuclease fragments indicated the amplification of one or both of (at least) two wild-type nonallelic genes in four of the lines and the presence in one line (DC-3F/DMM XX) of a unique 5.0-kilobase BamHI fragment resulting from a recombinational event during amplification . Comparison with the cytogenetic data indicated no correlation between restriction patterns generated by EcoRI, HindIII, PstI, or BamHI and chromosomal location of amplified genes . However, the only sublines in which the homogeneously staining region or abnormally banding region is positioned at 1q26 (at or near the site of the native gene) exhibit either alterations in gene structure (DC-3F/DM XX) or in regulation of gene expression (DC-3F/AD X), suggesting a process more complex than simply amplification of the gene in loco.

Cancer Res, 1988 Jun 1, 48(11), 3173 - 8
Purification of the Mr 22,000 calcium-binding protein (sorcin) associated with multidrug resistance and its detection with monoclonal antibodies; Hamada H et al.; A low molecular weight cytoplasmic protein (Mr 19,000-22,000) has been reported to be overexpressed in some multidrug-resistant cells . We have found that a cytoplasmic protein with a molecular weight of 22,000 is highly expressed in the human myelogenous leukemia K562 cells resistant to Adriamycin (K562/ADM) . The Mr 22,000 protein was shown to be one of the major calcium-binding proteins in the cytoplasmic extract from K562/ADM cells . The protein was purified to apparent homogeneity from K562/ADM cells using a four-step procedure including ammonium sulfate fractionation, anion-exchange chromatography, and gel filtration . 1.5 mg of the Mr 22,000 protein was purified from 3.0 x 10(9) of K562/ADM cells . The protein was acidic (pI 5.3) and exists as a homodimer (Mr 44,000) as revealed by gel filtration and sucrose density-gradient centrifugation . The purified protein appeared as a single band (Mr 22,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence or absence of reducing agents, suggesting that the homodimer was generated by noncovalent linkage . Monoclonal antibodies specific to the Mr 22,000 protein were raised by in vitro immunization with purified protein or by in vivo immunization with the crude membrane fraction of K562/ADM . These antibodies were used as probes for the detection of the protein . We have surveyed the expression of the Mr 22,000 protein in various multidrug-resistant and -sensitive cell lines, and found that the overexpression of the protein is not a sufficient nor a necessary condition for the acquisition of the multidrug-resistant phenotype.

Proc Natl Acad Sci U S A, 1988 Jun, 85(12), 4486 - 90
A retrovirus carrying an MDR1 cDNA confers multidrug resistance and polarized expression of P-glycoprotein in MDCK cells; Pastan I et al.; A full-length cDNA for the human multidrug resistance gene 1 (MDR1) has been inserted into a retroviral vector containing a murine Harvey sarcoma virus from which the viral oncogene was deleted . Ecotropic and amphotropic virus was produced after transfection of this vector into psi-2 and PA-12 packaging cell lines . This virus conferred the full phenotype of multidrug resistance on mouse and human cell lines . Viral titers of up to 2 X 10(5) drug-resistant colonies per ml were observed . Infected cells became resistant to colchicine, vinblastine, doxorubicin, VP16 (etoposide), and puromycin, but not cisplatin, indicating that the presence of the human MDR1 gene is sufficient to cause multidrug resistance . When the dog kidney cell line MDCK was infected with the MDR1 virus, P-glycoprotein was expressed in a polarized manner on the upper surface of the cells, showing that the cloned cDNA also encodes information for polarized expression of P-glycoprotein . The MDR1 virus should be useful for introducing this drug resistance gene into a variety of cell types for biological experiments in vitro and in vivo.

Proc Natl Acad Sci U S A, 1988 Jun, 85(11), 3762 - 6
Distinct P-glycoprotein precursors are overproduced in independently isolated drug-resistant cell lines; Greenberger LM et al.; A family of P-glycoproteins are overproduced in multidrug-resistant cells derived from the murine macrophage-like line J774.2 . To determine whether individual family members are overproduced in response to different drugs, the P-glycoprotein precursors in several independently isolated cell lines, which were selected for resistance to vinblastine or taxol, were compared . Individual cell lines selected with vinblastine overproduced P-glycoprotein precursors of either 120 or 125 kDa . Taxol-selected cell lines overproduced either the 125-kDa precursor or both precursors simultaneously . Two similar but distinct peptide maps for the mature P-glycoproteins were observed . These maps corresponded to each precursor regardless of the drug used for selection . One vinblastine-resistant cell line switched from the 125- to the 120-kDa precursor when grown in increasing concentrations of drug . This change coincided with the overexpression of a distinct subset of mRNA species that code for P-glycoprotein . It is concluded that precursor expression is not drug-specific . These data suggest that individual overproduced P-glycoprotein family members are translated as distinct polypeptides . The results may help to explain the diversity in the multidrug-resistant phenotype.

J Natl Cancer Inst, 1988 Jun 1, 80(7), 506 - 10
Electrophoretic analysis of P-glycoproteins produced by mouse J774.2 and Chinese hamster ovary multidrug-resistant cells; Greenberger LM et al.; P-glycoprotein (P-gp) plays a fundamental role in multidrug resistance . The quantity of P-gp relates to the degree of drug resistance . A comparison was made between P-gps in mouse and hamster cell lines in both Laemmli and modified Fairbanks gel systems . Both proteins are derived from precursors of similar size that undergo differential N-linked glycosylation . The electrophoretic mobility and the amount of P-gp are remarkably dependent on the conditions of analysis . Notably, boiling P-gp before Laemmli gel electrophoresis decreases its mobility by an amount that is equivalent to approximately equal to 15 kDa and results in an apparent diminution in the amount of protein . The latter effect can give a false impression concerning the quantity of P-gp in cells.

Biull Eksp Biol Med, 1988 Jun, 105(6), 684 - 6
{Identification of cells with lowered sensitivity to cytostatic agents by the use of fluorescent dyes}; Neifakh AA et al.; With a help of stepwise increase of vincristine concentrations in culture medium several lines of mouse myeloma X63 Ag 8.863 cells resistant to low concentrations of vincristine (6-35-fold) were selected . Rhodamine 123 stained resistant cells and wild-type cells with an equal intensity . However, resistant cells differ significantly from the sensitive ones by the rate of rhodamine efflux . The rate of the efflux was in proportion to the degree of resistance . The efflux of the dye could be blocked by the addition to reserpine, the inhibitor of multidrug resistance . Thus, fluorescent dyes can be used for the detection of cells with low levels of multidrug resistance.

Cell, 1988 May 20, 53(4), 519 - 29
An altered pattern of cross-resistance in multidrug-resistant human cells results from spontaneous mutations in the mdr1 (P-glycoprotein) gene; Choi KH et al.; Multidrug resistance in human cells results from increased expression of the mdr1 (P-glycoprotein) gene . Although the same gene is activated in cells selected with different drugs, multidrug-resistant cell lines can be preferentially resistant to their selecting agent . The mdr1 cDNA sequence from vinblastine-selected KB cells, which are uniformly resistant to different lipophilic drugs, was compared with the corresponding sequence from colchicine-selected KB cells preferentially resistant to colchicine . These sequences differ at three positions, resulting in a single amino acid change in P-glycoprotein . These differences result from mutations that occurred during colchicine selection . The appearance of these mutations coincides with the emergence of preferential resistance to colchicine . We have constructed biologically active mdr1 cDNA clones that express either wild-type or mutant P-glycoprotein . Multi-drug-resistant transfectants obtained with the mutant sequence were characterized by increased relative resistance to colchicine compared with transfectants obtained with wild-type sequence . mdr1 mutations are therefore responsible for preferential resistance to colchicine in multidrug-resistant KB cells.

J Natl Cancer Inst, 1988 May 4, 80(5), 361 - 5
Doxorubicin and the alkylating anthracycline 3'-deamino-3'-(3-cyano-4-morpholinyl) doxorubicin: comparative in vitro potency against leukemia and bone marrow cells; Beckman RA et al.; The new anthracycline analogue 3'-deamino-3'-(3-cyano-4-morpholinyl) doxorubicin (MRA-CN) is an intensely potent compound that has been shown to be 100-1,000 times more potent than doxorubicin (DOX) in vivo and in vitro . In addition, MRA-CN has been non-cross-resistant with DOX in DOX-selected models of multidrug resistance . We now report the effect of MRA-CN (and DOX) on leukemia cell lines established from patients with common, T-cell, and B-cell acute lymphoblastic leukemia, as well as with monoblastic leukemia . The effect of MRA-CN on the leukemia cells was compared to its toxicity on normal myeloid progenitors (therapeutic ratio) and to the effect of DOX on the leukemia and normal cells . MRA-CN was found to be 100 times more potent than DOX against normal myeloid progenitors--colony-forming units, granulocyte-macrophage (CFU-GM)--and 40-240 times more potent than DOX against leukemia cell lines . In addition, the therapeutic ratio was uniformly greater than 1, indicating that each leukemia cell line tested was more sensitive than CFU-GM to MRA-CN in vitro . There was a lack of correlation between MRA-CN and DOX at a drug concentration at which the colony formation is inhibited by 50% in the leukemia cell lines (correlation coefficient = 0.38), which supported the previous reports of non-cross-resistance between these two agents . The favorable therapeutic ratio, the non-cross-resistance with DOX, and the previously described lack of cardiac toxicity all make MRA-CN an attractive candidate for clinical trials in patients with acute leukemia.

Anticancer Res, 1988 May-Jun, 8(3), 307 - 12
The semistability of centric chromatin bodies during long-term passage of multidrug resistant mouse-Chinese hamster cell hybrids; Jakobsson AH; In a cell fusion experiment with multidrug resistant (MDR) mouse SEWA tumor cells and sensitive Chinese Hamster CHO cells, the resistant hybrid cells were completely without recognizable mouse chromosomes . Instead, numerous chromatin bodies (CB) were found that contained copies of a high molecular weight P-glycoprotein gene (PGY1) associated with the MDR condition . The present paper reports on the CB under long-term selective and nonselective growth . The CB were of a stability intermediate between that of double minutes (DM) and homogeneously staining regions (HSR) . The stability of the CB was different in the two hybrid lines studied.

Proc Natl Acad Sci U S A, 1988 May, 85(10), 3580 - 4
ATP-dependent transport of vinblastine in vesicles from human multidrug-resistant cells; Horio M et al.; Resistance of human cancer cells to multiple cytotoxic hydrophobic agents (multidrug resistance) is due to overexpression of the "MDR1" gene, whose product is the plasma membrane P-glycoprotein . Plasma membrane vesicles partially purified from multidrug-resistant human KB carcinoma cells, but not from drug-sensitive cells, accumulate {3H}vinblastine in an ATP-dependent manner . This transport is osmotically sensitive, with an apparent Km of 38 microM for ATP and of approximately equal to 2 microM for vinblastine . The nonhydrolyzable analog adenosine 5'-{beta, gamma-imido}triphosphate does not substitute for ATP but is a competitive inhibitor of ATP for the transport process . Vanadate, an ATPase inhibitor, is a potent noncompetitive inhibitor of transport . These results indicate that hydrolysis of ATP is probably required for active transport of vinblastine . Several other drugs to which multidrug-resistant cell lines are resistant inhibit transport, with relative potencies as follows: vincristine greater than actinomycin D greater than daunomycin greater than colchicine = puromycin . Verapamil and quinidine, which reverse the multidrug-resistance phenotype, are good inhibitors of the transport process . These results confirm that multidrug-resistant cells express an energy-dependent plasma membrane transporter for hydrophobic drugs, and establish a system for the detailed biochemical analysis of this transport process.

Biochem Biophys Res Commun, 1988 Apr 29, 152(2), 552 - 8
Reduced cyclosporin accumulation in multidrug-resistant cells; Goldberg H et al.; Cyclosporin accumulation was reduced by 50% or more in multidrug- resistant CHRC5 CHO cells with high levels of P-glycoprotein expression compared to drug sensitive AuxB1 CHO cells . This difference could be overcome by verapamil which is known to interact with P-glycoprotein and reverse multidrug resistance . The difference in cyclosporin accumulation between sensitive and resistant cells decreased with increasing cyclosporin concentrations suggesting that cyclosporine itself regulated its own accumulation through interaction with P-glycoprotein . Indeed, cyclosporin also reversed differences in vinblastine accumulation between resistant and sensitive cell lines . Since P-glycoprotein is highly expressed in the kidney which is also a target for cyclosporin toxicity, the effects of verapamil on cyclosporin accumulation were studied in two renal cell lines, rat mesangial cells and LLCPK1, cells . Verapamil increased cyclosporin accumulation by approximately 70% . These results suggest that cellular cyclosporine accumulation is regulated at least in part by its interaction with P-glycoprotein.

J Natl Cancer Inst, 1988 Apr 20, 80(4), 269 - 75
Expression of the mdr (P-glycoprotein) gene in Chinese hamster digestive tracts; Mukhopadhyay T et al.; P-glycoprotein has been shown to be responsible for multidrug resistance in mammalian cells . However, its physiological roles in normal cells are not known . The gene encoding this protein has been shown to express at a relatively high level in human digestive tracts . In the present study, in situ hybridizations were employed to determine the expression of this gene in gastrointestinal tissues . Epithelial cells in the villi of small intestine, colon, and stomach were rich in the P-glycoprotein gene transcript . Observations were consistent with the idea that the P-glycoprotein plays a role in detoxification by pumping potentially harmful compounds into the lumen of digestive tracts in animals.

Br J Cancer, 1988 Apr, 57(4), 348 - 52
Verapamil sensitizes normal and neoplastic rodent intestinal tissues to the stathmokinetic effect of vincristine in vivo; Ince P et al.; A morphological method has been developed allowing measurement of the effect on intestinal epithelia of vincristine . In routinely prepared tissue sections the proportion of mitotic events progressing beyond metaphase is counted by microscopy . When estimated over a range of doses of vincristine this post-metaphase index (PMI) can be used to compare the sensitivity of differing intact tissues . Intestinal tumours were induced in rats by chemical carcinogenesis . Administration of vincristine in the presence or absence of verapamil was performed in these tumour-bearing animals . Sections were prepared from colonic and small-bowel tumours and from normal mucosa . The results show that verapamil increases the sensitivity of the tissues studied to vincristine . A dose dependent effect of verapamil on vincristine sensitisation was demonstrated in colonic tissues . These findings indicate a shared pharmacological property between the resistance of primary tumour tissue and the multidrug-resistance phenotype.

J Urol, 1988 Apr, 139(4), 862 - 5
Measurement of multidrug-resistance messenger RNA in urogenital cancers; elevated expression in renal cell carcinoma is associated with intrinsic drug resistance; Kakehi Y et al.; We measured the levels of messenger RNA of the human multidrug-resistance gene (MDR1) in 25 urogenital tumors before chemotherapy . Many of the renal cell carcinomas continued to express MDR1 gene at high levels, reflecting the increased expression of MDR1 RNA in normal kidneys . In other urogenital tumors, the MDR1 RNA levels were low reflecting low MDR1 RNA levels in normal bladder, prostate and testis . For comparative purposes, we performed in vitro chemosensitivity testing on many tumor samples using soft agar culture techniques . Vinblastine sensitivity in vitro inversely correlated with MDR1 RNA levels (p less than 0.01) . Moreover, mean sensitivity of seven renal cell carcinomas to vinblastin was significantly lower than that of the other seven cancers (p less than 0.05) . As for doxorubicin, mean sensitivity of six renal cell carcinomas was lower than the others (p less than 0.1) . These results suggest that the high MDR1 RNA levels in renal cell carcinomas are associated with intrinsic multidrug-resistance.

FASEB J, 1988 Apr, 2(7), 2278 - 82
Induction by verapamil of a rapid increase in ATP consumption in multidrug-resistant tumor cells; Broxterman HJ et al.; A marked increase in cellular ATP consumption was induced by verapamil in the multidrug-resistant (MDR) cell line 2780AD, but not in the drug-sensitive parental cell line A2780 . A group of structurally unrelated drugs in concentrations known to reverse MDR, but not the verapamil analog tiapamil, a weak modulator of MDR, had similar effects . This effect was saturated at verapamil concentrations of about 1 microM . These data demonstrate that verapamil concentrations in MDR cells are maintained at a low level at the expense of ATP hydrolysis, and provide a first indication of the amount of metabolic energy used in this process.

Mol Pharmacol, 1988 Apr, 33(4), 454 - 62
Physical-chemical properties shared by compounds that modulate multidrug resistance in human leukemic cells; Zamora JM et al.; Multidrug resistance (MDR), typified by resistance to Vinca alkaloids and anthracyclines, is a well characterized experimental phenomenon that may have some clinical correlates . Verapamil, chloroquine, and related drugs have been shown previously to be capable of enhancing anticancer drug cytotoxicity in multi-drug-resistant cells, but the mechanism(s) by which these agents do this is(are) unclear . Since these agents did not seem to have common features, we studied these and other compounds for their ability to "modulate" Vinca alkaloid resistance in order to determine whether they possessed any common chemical or physical features . In addition to verapamil, 24 compounds, consisting of indole alkaloids, lysosomotropic agents, and amines, were tested for their ability to enhance the cytotoxicity of vinblastine and/or vincristine in our human leukemic multidrug-resistant cell line, CEM/VLB100 . Seventeen compounds that enhance the cytotoxicity of the Vinca alkaloids by more than 5-fold have been identified . These include quinolines (chloroquine, quinine, chinchonidine, and primaquine), acridines (acridine, acridine orange, and quinacrine), and indole alkaloids (yohimbine, corynanthine, reserpine, physostigmine, and the vindoline and catharanthine moieties of the Vinca alkaloids), as well as other alkaloids and amines (chlorpromazine, propranolol, atropine, and tryptamine) . Vindoline, catharanthine, and quinacrine also enhanced the cytotoxicity of doxorubicin and teniposide in these cells, indicating that this "modulation" was not limited to Vinca alkaloids . We examined some well known lysosomotropic compounds (methylamine, epinephrine, suramin, and trypan blue) and found that they were not able to enhance the cytotoxicity of vincristine in the CEM/VLB100 cells, indicating that lysosomotropic activity per se is not required for modulator activity . Three-dimensional computer modeling permitted molecular comparisons of conformationally related congeners of vinblastine, vindoline, and verapamil and revealed three regions of structural homology . We measured the hydrophobicity (by oil/water partitioning) and calculated the molar refractivity (by the additive substituent constant method) of active and inactive compounds . We found that those cationic agents--verapamil, quinacrine, indole alkaloids, and quinolines--that were lipid soluble at physiologic pH and had similar molar refractivities were best able to enhance the cytotoxicity of the Vinca alkaloids in our multidrug-resistant cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Gan To Kagaku Ryoho, 1988 Apr, 15(4 Pt 2-1), 1026 - 30
{Therapeutic approach targeting the proteins involved in mechanisms of multidrug resistance}; Tsuruo T; Recently, the mechanisms of multidrug resistance have been partly elucidated . Based on these resistant mechanisms, therapeutic approaches targeting the proteins involved in the mechanisms of multidrug resistance are under investigation . These include therapies with monoclonal antibodies, drug resistant genes and agents interacting with the proteins of resistant tumor cells.

Cancer Res, 1988 Apr 1, 48(7), 1926 - 9
Tissue distribution of P-glycoprotein encoded by a multidrug-resistant gene as revealed by a monoclonal antibody, MRK 16; Sugawara I et al.; A monoclonal antibody, MRK 16, specific to a human myelogenous leukemia cell line, K-562, and resistant to Adriamycin, was used to determine the localization of the antigen molecules (P-glycoprotein) recognized by the monoclonal antibody . P-glycoprotein was found to be expressed very strongly in the adrenal cortex and medulla of adults and strongly in the renal tubules of the kidney and the placenta . Interestingly, P-glycoprotein was not distributed in fetal and neonatal adrenals, and thus may be closely related to adrenal maturation . A high level of P-glycoprotein expression was also seen in one case each of untreated lung cancer (one of ten) and breast cancer (one of nine) . Immunoelectron microscopically, the P-glycoprotein was distributed evenly on the membranes of K-562/ADM and 2780 cells . These results imply that the presence of the glycoprotein may be useful as a marker for in vitro studies of multidrug resistance in various malignancies and as an indicator of therapeutic efficacy of ex vivo eradication of multidrug-resistant cancer cells, although other mechanisms of drug resistance may exist, and there is a possibility that this MRK 16 monoclonal antibody may not recognize all P-glycoprotein.

Cancer Res, 1988 Apr 1, 48(7), 1882 - 8
Cytogenetic and phenotypic analysis of a human colon carcinoma cell line resistant to mitoxantrone; Dalton WS et al.; A human colon carcinoma cell line selected for a 21-fold resistance to mitoxantrone was cross-resistant to the anthracycline, doxorubicin, but not to the anthracene, bisantrene . A 2-fold resistance was observed with vinblastine, another drug associated with multidrug resistance . Net intracellular mitoxantrone and doxorubicin accumulation were decreased at 1 h for all dose levels in the resistant cell line compared to the sensitive cell line . Although the resistant cells were more resistant to mitoxantrone than doxorubicin, the net accumulation of mitoxantrone was only 19% less than the sensitive cell line; whereas doxorubicin accumulation was decreased by 49% . No significant difference between the sensitive and resistant cell lines was observed in the initial accumulation of mitoxantrone; however, the efflux of mitoxantrone was increased in the resistant cell line . Verapamil did not overcome the resistance to mitoxantrone and did not increase the net accumulation of drug . No alterations in the electrophoretic mobility of membrane proteins were observed . Using immunoblotting techniques, the resistant cell line did not express P-glycoprotein which is frequently observed for cells resistant to anthracycline antibiotics . Cytogenetic analysis showed a putative homogenously staining region on the short arm of chromosome 7 in the resistant cell line . The limited cross-resistant phenotype, lack of verapamil reversal, nondetection of P-glycoprotein, and cytogenetic evidence of gene amplification suggests the involvement of a novel drug-resistant gene associated with resistance to mitoxantrone.

FEBS Lett, 1988 Mar 14, 229(2), 329 - 32
The tissue dependent expression of hamster P-glycoprotein genes; Baas F et al.; Using a sensitive RNase protection assay, we have measured the levels of P-glycoprotein mRNA in fourteen different Chinese hamster tissues and in three cell lines to determine whether a relationship exists between the level of P-glycoprotein expression and the occurrence of primary and acquired multidrug resistance (MDR) in cancer . P-Glycoprotein mRNA was detected in all tissues, suggesting that the protein is a normal constituent of the cell . High levels of P-glycoprotein mRNA were found in oesophagus, testis and uterus . Intermediate levels in brain, lung and ovary, and a very low level in the adrenal gland, bladder, bone marrow, heart, kidney, liver and spleen . There is no obvious correlation between this expression pattern and the occurrence of primary or acquired MDR.

J Natl Cancer Inst, 1988 Mar 2, 80(1), 14 - 20
Multidrug resistance; Moscow JA et al.; The ability of malignant cells to develop resistance to cytotoxic drugs poses a major obstacle to the ultimate success of cancer therapy . While some mechanisms of resistance allow cells to survive exposure to a single agent, the phenomenon of multidrug resistance (MDR) confers upon cells the ability to withstand exposure to lethal doses of many structurally unrelated antineoplastic agents . MDR has been strongly linked to the overexpression of a membrane-associated glycoprotein, P-glycoprotein, which appears to play a role in drug efflux . However, several lines of evidence suggest that other mechanisms of resistance are involved in MDR; biochemical similarities observed in a human breast cancer cell line after the acquisition of MDR and in carcinogen-induced rat preneoplastic hepatic nodules indicate that changes in regulation of phase I and phase II drug-metabolizing enzymes may also play a role in MDR . An atypical pattern of MDR has been characterized and related to altered topoisomerase activity . Improvement in current cancer chemotherapy may be achieved by interfering with the regulation and expression of mechanisms of MDR.

Proc Natl Acad Sci U S A, 1988 Mar, 85(5), 1595 - 9
Retroviral transfer of a murine cDNA for multidrug resistance confers pleiotropic drug resistance to cells without prior drug selection; Guild BC et al.; We have constructed a retrovirus expression vector that carries the murine mdr cDNA transcribed under the control of the human H4 histone promoter to examine the feasibility of efficiently transferring a multidrug resistance phenotype to cells without requiring drug selection . This approach will facilitate the transfer of mdr cDNA to hematopoietic progenitor cells for the study of multidrug resistance in vivo . The retrovirus vector pHmdr has been used for transmission and expression of the mdr cDNA in initially drug-sensitive NIH 3T3 fibroblasts . Selection of pHmdr infectants in the cytotoxic agents colchicine or doxorubicin gave rise to highly multidrug-resistant colonies containing a single gene copy of the vector . Moreover, in the analysis of 12 cloned unselected NIH 3T3 cell infectants, a multidrug resistance phenotype was conferred by as few as two copies of the pHmdr vector . Overexpression of the mdr cDNA in drug-selected and unselected pHmdr infectants was directly related to cell survival in three cytotoxic agents tested . These results hold significant implications for the study of multidrug resistance in vivo.

Eur J Cancer Clin Oncol, 1988 Mar, 24(3), 449 - 54
Identification of a multidrug resistance associated antigen (P-glycoprotein) in normal human tissues; Hitchins RN et al.; The multidrug resistance (NDR) phenotype describes a pattern of cross-resistance to unrelated compounds observed in mammalian cell lines selected in vitro for resistance to a single agent . Overexpression of a 170,000 dalton cell membrane glycoprotein (P-glycoprotein) is associated consistently with this phenotype in these cell lines . Recently, several human tumours have been shown to contain P-glycoprotein and expression was greatest in tumours exhibiting clinical drug resistance . To explore further the significance of P-glycoprotein, we examined normal human tissues obtained at autopsy by polyacrylamide gel electrophoresis and immunoblotting using a monoclonal antibody directed against P-glycoprotein . We showed expression of P-glycoprotein in normal liver and small bowel mucosa but not in other organs examined . This suggests there may be significant expression of P-glycoprotein in certain normal human tissues and any plan to exploit P-glycoprotein clinically must take these findings into account.

Br J Cancer, 1988 Mar, 57(3), 254 - 8
Modification of cytotoxic drug resistance by non-immuno-suppressive cyclosporins; Twentyman PR; We have examined the ability of a series of non- or minimally-immunosuppressive analogues of cyclosporin A to modify cytotoxic drug resistance in vitro . The series includes both cyclosporins derived from naturally-occurring compounds and synthetic cyclosporins . In contrast to our previous findings, we now report that several of these analogues are highly effective modifiers of resistance to adriamycin and vincristine in a multidrug resistant subline of the human small cell lung cancer cell line NCI-H69 . Two of the analogues (W8-032 and B3-243) maintain considerable activity in the dose range 1-2 micrograms ml-1 whereas little activity remains for cyclosporin A when the dose is reduced to this level . B3-243, however, in contrast to cyclosporin A and W8-032, does itself show growth inhibitory effects in this dose range . Possible clinical trial of these cyclosporins as resistance modifiers will depend upon their in vivo toxicology and pharmacokinetic properties.

Proc Natl Acad Sci U S A, 1988 Mar, 85(6), 2019 - 23
Comparison of ion channels in multidrug-resistant and -sensitive human leukemic cells; Lee SC et al.; Tumor cell lines selected to grow in the presence of one "natural product" antineoplastic drug often develop cross-resistance to others . This multidrug resistance (MDR) is believed to be a major problem in cancer therapy . Organic Ca2+-channel blockers, such as verapamil, can reverse this resistance and render MDR cells in culture nearly as sensitive to the antineoplastic drugs as the drug-sensitive cells from which they were derived . It has therefore been suggested that Ca2+ channels may play a role in MDR . To determine directly whether there are electrophysiological correlates of MDR, we used whole-cell and single-channel patch-clamp techniques to survey the ion channels in a drug-sensitive human T-cell leukemia line, CCRF-CEM, and a MDR variant, CEM/VLB100 . We found no evidence for a voltage-gated Ca2+ channel . However, we did identify three other current/channel types: a voltage-gated tetrodotoxin-sensitive inward current carried by Na+, a voltage-gated labile outward current carried by K+, and a nonselective cation channel reversing at 0 mV . Drug-sensitive and -resistant cells were the same with respect to the level of expression of these channels.

Biochim Biophys Acta, 1988 Feb 18, 938(2), 315 - 21
Altered plasma membrane ultrastructure in multidrug-resistant cells; Arsenault AL et al.; Multidrug resistance is mediated by P-glycoprotein, an integral plasma membrane component which is thought to function as a drug export pump . This model can explain drug resistance, but fails to account for the broader pleiotropy of the multidrug resistance phenotype . We report here a freeze-fracture study revealing increases in the densities of protoplasmic face intramembrane particles in multidrug-resistant Chinese hamster ovary (CHO) and human leukemic cells . The intramembrane particle density in a CHO cell revertant which had lost the characteristics of the multidrug resistance phenotype was indistinguishable from that of the drug-sensitive parental cell line . This demonstration of a global multidrug resistance-linked change in plasma membrane architecture may have significant implications for understanding the variety of concurrent membrane-related changes which are not easily explained by the current model for multidrug resistance.

Biochem Pharmacol, 1988 Feb 15, 37(4), 613 - 9
Effects of verapamil on the cellular accumulations and toxicity of several antitumor drugs in 9-hydroxy-ellipticine-resistant cells; Delaporte C et al.; 9-OH-Ellipticine (9-OH-E)-resistant cells are not only resistant to the DNA topoisomerase II inhibitors, but also to some other antitumor agents, such as actinomycin D (AD), adriamycin (ADM), daunorubicin and vincristine . It was previously shown that a decreased uptake accounts for the cross-resistance of these cells to AD and ADM which then suggested that the 9-OH-E-resistant cells might display some of the properties usually associated with the multidrug resistance phenotype . In this work, we have examined the effects of verapamil, a drug which is known to overcome the multidrug resistance, on the toxicity and the cellular accumulation of four cytotoxic agents: 9-OH-E, 2N-methyl-9-hydroxy-ellipticinium (NMHE), AD and ADM, either on 9-OH-E resistant cells or on a multidrug resistant subline derived from the same sensitive parental cells . Verapamil inhibited the cellular accumulation of the ellipticine derivatives in the sensitive DC-3F cells, and the toxicity of these drugs on these cells was correspondingly decreased . On either one of the resistant cell lines, verapamil had no effect on the toxicity and the cellular accumulation of 9-OH-E . In contrast, in the presence of verapamil, the cellular accumulation of NMHE by the 9-OH-E and the multidrug resistant cells was about 50% and 300% increased, respectively . The increased NMHE cellular concentration in the multidrug resistant cells was associated with an 8-fold increased toxicity . The major structural characteristics which might account for this difference between the sensitivities of both ellipticine derivatives to the effects of verapamil on the multidrug resistant cells is the presence of a positive charge on the nitrogen in position 2 of the 6H-pyridocarbazole molecule . Finally, verapamil circumvented partially the cross-resistance of DC-3F/9-OH-E cells to AD and ADM by increasing the accumulation of these drugs inside the cells.

Eur J Cancer Clin Oncol, 1988 Feb, 24(2), 205 - 10
Relationship between the structure of analogues of amsacrine and their degree of cross-resistance to adriamycin-resistant P388 leukaemia cells; Baguley BC et al.; A series of derivatives of 9-anilinoacridine related to the anti-leukaemia agent amsacrine have been tested in continuous exposure growth inhibition assays to determine the degree of cross-resistance in the Adriamycin-resistant P/ADR murine leukaemia line . Measured IC50 values for the two cell lines were only poorly correlated (r = 0.51), and cross-resistance as measured by the ratio of IC50 values varied from 2-fold and 272-fold . A high degree of resistance was found to be associated with the presence of amino or substituted amino groups on the acridine ring system . Logarithmic IC50 values were determined for other cell lines (L1210 leukaemia, Lewis lung carcinoma and HCT-8 human colon carcinoma) and were compared with those for the P388 lines to determine the degree of linear correlation . HCT-8 values were strongly correlated with P/ADR values (r = 0.84) while L1210 values correlated strongly with those of the sensitive P388 line (r = 0.98) . Values for Lewis lung cells showed an intermediate pattern and correlated with a linear combination of values for both P388 lines (r = 0.88) . Examination of available IC50 values for a number of rodent and human cell lines indicates that their sensitivity patterns are either P388-like or else intermediate between P388 and P/ADR . The series of amsacrine derivatives may be useful in characterizing the nature and degree of multidrug-resistance in cultured cell lines.

Mol Pharmacol, 1988 Feb, 33(2), 144 - 7
Most drugs that reverse multidrug resistance also inhibit photoaffinity labeling of P-glycoprotein by a vinblastine analog; Akiyama S et al.; Multidrug-resistant human KB carcinoma cells express a 170,000-dalton membrane glycoprotein (P-glycoprotein) that can be photoaffinity labeled with the vinblastine analog N-(p-azido-{3-125I}salicyl}-N'-(beta-aminoethyl)vindesine . Several agents that suppress the multidrug-resistant phenotype, including N-solanesyl-N,N'-bis(3,4-dimethylbenzyl)ethylenediamine, cepharanthine, quinidine, and reserpine, were found to inhibit photolabeling of P-glycoprotein at doses comparable to those that reverse multidrug resistance . However, the phenothiazines chlorpromazine and trifluoperazine, which also effectively reverse multidrug resistance, were poor inhibitors of the photoaffinity labeling of P-glycoprotein . Chloroquine, propranolol, or atropine, which only partially reversed the drug resistance, also did not inhibit photolabeling . Naphthalene sulfonamide calmodulin inhibitors, W7 and W5, as well as many other drugs that did not circumvent multidrug resistance, did not inhibit photolabeling . These studies suggest that most, but not all, agents that phenotypically suppress multidrug resistance also inhibit drug binding to a site on P-glycoprotein with which a photoaffinity analog of vinblastine interacts.

Cancer Res, 1988 Feb 1, 48(3), 539 - 43
Partial reversal of doxorubicin resistance by forskolin and 1,9-dideoxyforskolin in murine sarcoma S180 variants; Wadler S et al.; Acquired resistance to chemotherapeutic agents is an important clinical problem . One preclinical model, termed multidrug resistance (MDR), is characterized by a complex phenotype of cross-resistance to biochemically unrelated antineoplastic agents, the presence of a high-molecular-weight membrane glycoprotein, and impaired accumulation of drug . To determine whether MDR is mediated in part by altered cyclic 3',5'-adenosine monophosphate (cAMP) levels, the effect of incubation with the adenylate cyclase agonist, forskolin, was investigated in the murine sarcoma S180 cell line and two MDR variants (A5-.8, A5-2.5) . Basal cAMP levels in sensitive and MDR lines were not significantly different (range, 0.15 +/- 0.05 to 0.31 +/- 0.09 pmol/mg protein); however, 1-h incubation with forskolin, 10 microM, elevated intracellular cAMP 2-fold in the parent line and 43- and 35-fold in the variants . The adenylate cyclase agonists, prostaglandin E2 and cholera toxin, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine had no significant effect on cAMP levels . To determine the effect of forskolin on doxorubicin-induced cell lethality, S180 and MDR lines were incubated with doxorubicin plus forskolin for 1 h and cloned in soft agar . Coincubation with forskolin partially reversed doxorubicin resistance in the MDR lines in a dose-dependent fashion . To determine whether this effect was mediated solely by elevation of intracellular cAMP, the inactive 1,9-dideoxy analogue of forskolin (DF) was used . Incubation with DF resulted in no elevation of cAMP levels in the sensitive or resistant cell lines; however, DF also partially reversed doxorubicin resistance in the MDR variants . Furthermore, coincubation of the A5-2.5 cell line with doxorubicin and 8-bromo cAMP, 1 mM, did not result in reversal of resistance to doxorubicin . To determine whether the reversal of resistance by the diterpenes was associated with alteration of doxorubicin transport, uptake and efflux of {14C}doxorubicin were measured . Coincubation with both forskolin and DF, 10 microM, enhanced {14C}doxorubicin uptake in the resistant cells, while drug efflux was significantly affected only in the cell line exhibiting intermediate resistance . Since both forskolin and its inactive analogue are effective in partially reversing resistance to doxorubicin and augmenting anthracycline uptake, a mechanism other than elevation of cAMP is most likely responsible.

Presse Med, 1988 Jan 30, 17(3), 99 - 102
{Efficacy of halofantrine in Plasmodium falciparum or Plasmodium vivax malaria in a resistance area (French Guiana)}; Maisonneuve H et al.; Halofantrine (WR 171.669) is a phenanthrene methanol derivative effective against the multidrug resistant strains of Plasmodium falciparum . One hundred and one patients, 48 men and 53 women, 53 adults and 48 children (less than or equal to 12 years old) aged from 1.5 to 57 years were treated . Fifty-one patients received a single 16 mg/kg dose and 50 patients received 24 mg/kg/day in 3 doses at 6-hour intervals . Parasite counts with examination of both thin and thick smears were performed twice daily for 5 to 6 days following treatment, or until smears were negative for parasites for 24 hours, and then weekly for 4 weeks . Thirteen patients reported clinical side effects . Six treated patients had no parasites . One patient had mixed parasitemia . Eighty three patients had P . falciparum malaria, with mean parasitemias between 26,850 +/- 36,679 and 35,412 +/- 50,527 per cubic millimeter . Halofantrine was very effective in the two doses tested from 87.5 to 100 p . 100 . Eleven patients had in vivo resistant strains; ten in vitro tests were successful and nine were resistant to chloroquine . Thirteen patients with P . vivax and a mean parasitemia of 13,858 +/- 10,835 per cubic millimeter were cured but 3 had a relapse 3 to 4 weeks after treatment . At the 2 dosage levels tested halofantrine proved highly effective in the treatment of malaria caused by resistant and sensitive strains to P . falciparum.

J Biol Chem, 1988 Jan 25, 263(3), 1454 - 8
Purification of the 170- to 180-kilodalton membrane glycoprotein associated with multidrug resistance . 170- to 180-kilodalton membrane glycoprotein is an ATPase; Hamada H et al.; 170-180-kDa membrane glycoprotein (P-glycoprotein) associated with multidrug resistance is involved in drug transport mechanisms across the plasma membrane of resistant cells . From sequence analysis of cDNAs of the P-glycoprotein gene, it is postulated that the active drug-efflux pump function may be attributable to the protein . However, purification of the P-glycoprotein while preserving its enzymatic activity has not been reported . In this study, we have purified the P-glycoprotein from the human myelogenous leukemia K562 cell line resistant to adriamycin (K562/ADM) by means of one-step immunoaffinity chromatography using a monoclonal antibody against P-glycoprotein . The procedure was simple and efficiently yielded an electrophoretically homogeneous P-glycoprotein sample . By solubilization with 3-{(3-cholamidopropyl)dimethylammonio}-1-propanesulfonate, the purified P-glycoprotein was found to have ATPase activity . This ATP hydrolysis may be coupled with the active efflux of anticancer drugs across the plasma membrane of multidrug-resistant cells.

Cancer Res, 1988 Jan 15, 48(2), 393 - 8
Cytogenetic and molecular characterization of tumors in nude mice derived from a multidrug-resistant human leukemia cell line; Hill AB et al.; We have evaluated whether selection of a human tumor leukemic line for resistance to vinblastine (Velban; VLB) alters its tumorigenicity . To address this question, CEM and CEM/VLB100 cells {which express the multiple drug-resistant (MDR) phenotype via amplification of the P-glycoprotein gene} were characterized by several techniques including chromosome banding, in situ hybridization, Southern blotting, RNA dot blotting, in vitro drug sensitivity, and tumorigenicity in nude mice . Analysis of the chromosome banding patterns of both drug-sensitive CEM cells and the MDR CEM/VLB100 cells revealed that the two lines differed primarily by the presence of a large metacentric marker chromosome associated with the acquisition of VLB resistance . In situ hybridization of a P-glycoprotein complementary DNA to metaphase chromosomes showed that the amplified P-glycoprotein genes in the CEM/VLB100 cell line were localized to this large marker . Tumorigenicity of both the CEM and CEM/VLB100 cell lines was measured after injection of 10(7) cells/nude mouse . The results showed that 4 of 4 drug-sensitive and 4 of 5 drug-resistant cell lines formed tumors in 5-10 wk . By comparison with the parental line, three of the four tumors arising from the CEM/VLB100 line retained their drug-resistance properties as measured by vinblastine resistance in vitro and elevated P-glycoprotein mRNA expression associated with P-glycoprotein gene amplification . In addition, tumors retaining the MDR phenotype also retained the large metacentric marker chromosome . One tumor arising from CEM/VLB100 reverted to the drug-sensitive phenotype, with a resultant decrease in P-glycoprotein mRNA expression and loss of P-glycoprotein gene amplification . This revertant was also missing the large metacentric marker present in all cells from the CEM/VLB100 parent . Our experiments show that the acquisition of the MDR phenotype resulting from overexpression of P-glycoprotein in the plasma membrane does not effect the tumorigenicity of human CEM cells.

Proc Natl Acad Sci U S A, 1988 Jan, 85(2), 582 - 6
Phorbol esters induce multidrug resistance in human breast cancer cells; Fine RL et al.; Mechanisms responsible for broad-based resistance to antitumor drugs derived from natural products (multidrug resistance) are incompletely understood . Agents known to reverse the multidrug-resistant phenotype (verapamil and trifluoperazine) can also inhibit the activity of protein kinase C . When we assayed human breast cancer cell lines for protein kinase C activity, we found that enzyme activity was 7-fold higher in the multidrug-resistant cancer cells compared with the control, sensitive parent cells . Exposure of drug-sensitive cells to the phorbol ester phorbol 12,13-dibutyrate {P(BtO)2} led to an increase in protein kinase C activity and induced a drug-resistance phenotype, whereas exposure of drug-resistant cells to P(BtO)2 further increased drug resistance . In sensitive cells, this increased resistance was accompanied by a 3.5-fold increased phosphorylation of a 20-kDa particulate protein and a 35-40% decreased intracellular accumulation of doxorubicin and vincristine . P(BtO)2 induced resistance to agents involved in the multidrug-resistant phenotype (doxorubicin and vincristine) but did not affect sensitivity to an unrelated alkylating agent (melphalan) . The increased resistance was partially or fully reversible by the calcium channel blocker verapamil and by the calmodulin-antagonist trifluoperazine . These data suggest that stimulation of protein kinase C plays a role in the drug-transport changes in multidrug-resistant cells . This may occur through modulation of an efflux pump by protein phosphorylation.

Cancer Chemother Pharmacol, 1988, 22(1), 17 - 20
Comparison of glutathione S-transferase activity between drug-resistant and -sensitive human tumor cells: is glutathione S-transferase associated with multidrug resistance?
Yusa K, Hamada H, Tsuruo T.
We have studied the levels of glutathione S-transferase in drug-resistant and -sensitive human tumor cell lines to examine a possible involvement of glutathione S-transferase (GST) in multidrug resistance mechanisms . No increase in the activity of glutathione S-transferase was detected in myelogenous leukemia K562 resistant to adriamycin (K562/ADM), ovarian carcinoma cell line A2780 resistant to adriamycin (2780AD), or acute lymphoblastic leukemia cell line CCRF-CEM resistant to vinblastine (CEM-VLB100), compared with the drug-sensitive parent tumor cells . The human breast cancer cell lines Hattori and MCF-7 had a 12- to 63-fold lower level of glutathione S-transferase activity than K562, A2780, CCRF-CEM, and their drug-resistant sublines . Induction of ADM resistance in Hattori did not increase the activity of glutathione S-transferase . However, induction of colchicine resistance in MCF-7 resulted in a 70-fold increase in the activity of glutathione S-transferase . A revertant of the colchicine-resistant MCF-7 contained a level of glutathione S-transferase activity similar to that of the resistant subline . The increase of glutathione S-transferase activity did not alter the sensitivity of the cell to cytotoxic drugs . The increased activity was due to the appearance of glutathione S-transferase pi, as shown by enzyme inhibition using anti-glutathione S-transferase pi antibody . Our findings indicate that increased cellular glutathione S-transferase activity is not associated with the development of multidrug resistance.

NCI Monogr, 1988, (6), 187 - 91
Chinese hamster pleiotropic multidrug-resistant cells are not radioresistant; Mitchell JB et al.; The inherent cellular radiosensitivity of a Chinese hamster ovary pleiotropic cell line that is multidrug resistant (CHRC5) was compared to that of its parental cell line (AuxB1) . Radiation survival curve parameters n and D0 were 4.5 and 1.1 Gy, respectively, for the CHRC5 line and 5.0 and 1.2 Gy, respectively, for the parental line . Thus, the inherent radiosensitivity of the two lines was similar even though key intracellular free radical scavenging and detoxifying systems employing glutathione, glutathione transferase, and catalase produced enzyme levels that were 2.0-, 1.9-, and 1.9-fold higher, respectively, in the drug-resistant cell line . Glutathione depletion by buthionine sulfoximine resulted in the same extent of aerobic radiosensitization in both lines (approximately 10%) . Incorporation of iododeoxyuridine into cellular DNA sensitized both cell lines to radiation . These studies indicate that pleiotropic drug resistance does not necessarily confer radiation resistance.

Cancer Chemother Pharmacol, 1988, 21(1), 14 - 8
Membrane transport changes in an adriamycin-resistant murine leukemia cell line and in its sensitive parental cell line; Bose R et al.; Multidrug resistance in cancer chemotherapy occurs when cells develop resistance towards structurally and functionally unrelated drugs . It is speculated that alteration of some fundamental process(es) in the cells leads to the development of multidrug resistance . The sodium pump activity of murine leukemia cell lines P388/S (sensitive) and P388/ADR (resistant) was measured and found to be different in the two cell lines . The rate of sodium pumping, i.e., the ouabain-sensitive rubidium uptake, was consistently lower in the resistant cells compared to their parental controls . Uptake of adriamycin was lower in the resistant cells . Depolarizing the cells with potassium chloride or by inhibiting the pump with ouabain increased the adriamycin uptake in the sensitive cells but not in the resistant cells . Adriamycin did not have any acute effects on the sodium pump activity . It is concluded that the development of drug resistance in cell line P388 is associated with a decrease in sodium pump activity and a lack of depolarization-induced adriamycin uptake; these processes may be causally linked via alterations in cytosolic calcium concentration.

Exp Cell Res, 1988 Jan, 174(1), 168 - 76
Use of fluorescent dyes as molecular probes for the study of multidrug resistance; Neyfakh AA; Fluorescence microscopy has shown that 18 different fluorescent dyes, staining various intracellular structures in transformed hamster fibroblasts (DM-15), did not stain or stained weakly multidrug-resistant cells selected from DM-15 by colchicine . Reduced staining by fluorescent dyes was characteristic also of five other tested multidrug-resistant cell lines of hamster and mouse origin, selected by actinomycin D, colcemid, rubomycin, and ruboxyl . The intensity of staining of two revertant cell lines was similar to that of parental sensitive cells . All tested inhibitors of multidrug resistance, including weak detergent, metabolic inhibitors, calcium channel blockers, calmodulin inhibitors, and reserpine, restored normal staining of multidrug-resistant cells . The dyes accumulated in resistant cells in presence of these inhibitors left the cells several minutes after the removal of the inhibitor from the incubation medium . Sensitive cells retained the dyes for several hours . The efflux of the dyes from resistant cells is an active process since it occurred even in the presence of the dyes in the incubation medium . The efflux could be blocked by all tested inhibitors of multidrug resistance and it is possibly a basic mechanism of the reduced staining of resistant cells . These data support the idea that multidrug resistance is based on active nonspecific efflux of the drugs and indicate that the simple procedure of cell staining can be used for the detection of resistant cells and further study of the phenomenon of multidrug resistance.

Am J Trop Med Hyg, 1988 Jan, 38(1), 24 - 9
In vitro activity of pyronaridine against field isolates and reference clones of Plasmodium falciparum; Childs GE et al.; Pyronaridine, a 9-substituted 1-aza-acridine, was assayed for in vitro activity against clinical and field isolates as well as characterized clones of Plasmodium falciparum . The in vitro antimalarial activity of pyronaridine was compared to activities of standard antimalarials against multidrug-resistant isolates of P . falciparum from eastern and northern Thailand using an assay based on the inhibition of schizont maturation . Isolates from eastern Thailand (n = 30) were susceptible to pyronaridine (IC50 8.40 nM), mefloquine (IC50 6.97 nM), and amodiaquine (IC50 12.7 nM) and resistant to chloroquine (IC50 361 nM), quinine (IC50 388 nM), and pyrimethamine (IC50 11,800 nM) . The isolates from northern Thailand (n = 7) showed no statistical difference in susceptibility to pyronaridine (IC50 10.1 nM), amodiaquine (IC50 7.29 nM), and mefloquine (IC50 5.48 nM); however, isolates were significantly more susceptible to chloroquine (IC50 167 nM), quinine (IC50 248 nM), and pyrimethamine (IC50 1,980 nM) . These data suggest a lack of cross-resistance between pyronaridine and either chloroquine, quinine, or pyrimethamine . Using the same assay system the in vitro activity of pyronaridine was evaluated against isolates from treatment failures of mefloquine or enpiroline from eastern Thailand . The IC50 values for mefloquine against five recrudescent isolates were significantly higher (IC50 16.4 nM) than the field isolates collected from the same region (IC50 6.97 nM); however, there was no significant difference in the pyronaridine susceptibility between the isolates from the field study (IC50 8.89 nM) and the isolates from the treatment failures (IC50 8.40 nM) . These observations suggest a lack of cross-resistance to mefloquine following treatment failure with either mefloquine or enpiroline.(ABSTRACT TRUNCATED AT 250 WORDS)

Bull World Health Organ, 1988, 66(6), 763 - 7
Effect of oral contraceptive steroids on the clinical course of malaria infection and on the pharmacokinetics of mefloquine in Thai women; Karbwang J et al.; PIP: 6 healthy Thai women volunteers who regularly used oral contraceptives (OCs) and 12 Thai women patients with falciparum malaria, 6 of whom also were using OCs, participated in this study designed to evaluate the results of an investigation of the pharmacokinetics of mefloquine, an effective treatment for multidrug-resistant malaria, and the clinical course of malaria infection in OC users . The healthy volunteers received 3 tablets of mefloquine, each containing 250 mg mefloquine base and 1 tablet daily of a combined OC containing 0.5 mg norgestrel and 0.05 mg ethinylestradiol . Group 1 patients (non-OC users with falciparum malaria) received 3 tablets of mefloquine; group 2 patients (OC users with falciparum malaria) received 3 tablets of mefloquine plus 1 tablet daily of the OC . The level of mefloquine was determined by high-performance liquid chromatography . The area under the plasma mefloquine concentration-time curve (AUC) was determined using the trapezoidal rule . The 6 healthy volunteers who used OCs tolerated 750 mg mefloquine well . The main side effects were transient nausea and vomiting (3 subjects) that required no treatment . All patients with malaria infection had a history of fever that lasted 1-3 days and all but 1 was febrile . The geometric mean of the parasite counts was greater and the mean erythrocyte volume fraction was higher among non-OC users . There was no statistically significant differences between OC users and nonusers in exhibited fever and parasite clearance times . There was considerable interindividual variation in the peak plasma concentration of mefloquine, particularly among the OC users . No significant differences were found between the pharmacokinetic parameters for the OC users and nonOC users . There were significant differences between the parameters for the OC user volunteers and those for the OC user patients: the healthy volunteers had a significantly longer and mean retention time than patients . The study findings fail to indicate that OCs had any major deleterious effect on the course of human falciparum malaria .

Cancer Chemother Biol Response Modif, 1988, 10, 1 - 22
Antimetabolites; Allegra CJ et al.; The mechanisms of action of MTX and 5-FU have been further elucidated . Such studies will be important for the design of drug combinations and for the development of novel antifolate and fluoropyrimidine analogs . A greater understanding of MTX and ara-C transport and drug levels required to optimize transport may also aid in these endeavors . Pharmacokinetic parameters have been found to be predictors of relapse in children with acute leukemia, particularly with respect to MTX, 6-MP and ara-C . The intracellular terminal half-life of ara-C was correlated with remission duration in AML . Assay systems aimed at uncovering response predictors through biochemical analysis of patient tumor samples are being developed, including an interesting use of NMR spectroscopy to study the pharmacokinetics of fluorine-19-labeled 5-FU in vivo . Such an approach may yield valuable information on 5-FU anabolism in tumors in situ . A high frequency of resistance to MTX apparently may be generated within a single cell cycle by transient exposures to DNA synthesis inhibitors . The resistance may be based on either target enzyme amplification or altered membrane transport . These important studies provided bases for the rapid emergence of clinical resistance . Further, the multidrug-resistant phenotype appears to be a much broader based phenomenon as MTX resistance was found to be a frequent event in cells selected for multidrug resistance . A variety of novel approaches have been proposed to overcome antimetabolite resistance and to improve the selectivity of these agents, including the use of guanosine nucleotides, leucovorin and allopurines as biochemical modulators of 5-FU . Efficient techniques for the transfection of resistant DHFR into tissues using retroviruses have been reported . These studies serve as starting point for the ultimate development of more effective strategies for the treatment of human malignancies.

Anticancer Res, 1988 Jan-Feb, 8(1), 137 - 43
Chlorozotocin-induced selection of autocrine and multidrug resistant variants from a rat rhabdomyosarcoma; Pauwels-Vergely C et al.; The ability of tumor cells to develop simultaneous resistance to structurally different cytotoxic drugs constitutes a major problem in cancer chemotherapy . We previously described that a nitrosourea, chlorozotocin (CZT), increased in vivo the pulmonary metastatic dissemination of a rat rhabdomyosarcoma and enhanced in vitro the cloning efficiency (CE) of the same tumor cell line . The selection procedure for chlorozotocin-selected cell lines (R1 to R9 lines), by in vitro CZT-treatment and cloning assay, indicated that 2.5% of the parental cell line was a CZT-resistant subpopulation . This subset acquired, in the course of the selection steps in semi solid medium, a multidrug-resistant phenotype . In a stem cell assay, the resistance of the R9 line was increased 10-fold for adriamycin (ADR) and up to 2 fold for cisplatinum . In comparison, C8 control line sensitivity to ADR did not change significantly . This resistance did not affect the non clonogenic population, as shown in a monolayer proliferation assay . Re-exposure of R (R1-R9) lines to CZT or initial CZT contact with C lines transiently but consistently increased the CE; however, clonogenicity remained stable, and this was also observed when deticene, another alkylating drug, was used instead of CZT . Moreover, the CZT-selected R9 line easily proliferated in serum-free medium in the presence of transferrin and insulin, whereas serum was necessary to maintain the growth of the parental cell line or C lines . Finally, we show that in vitro selection of variants, by both resistance to CZT and cloning, led to the isolation of a multi-drug resistant subpopulation, serum independent for its proliferation - two properties associated with progressive malignancy.

Cancer Chemother Biol Response Modif, 1988, 10, 73 - 84
Multidrug resistance; Fine RL; The understanding of the mechanism(s) of multidrug resistance has increased at a rapid rate over the past year . Many new inhibitors, new agents equitoxic to MDR and sensitive cells, techniques for screening, and application to therapies will no doubt be within the grasp of the clinical oncologist over the next few years . However, whether in-vitro MDR is synonymous with clinical resistance is still an unanswered question, and other forms of resistance may be co-existing in patient tumors.

J Biol Chem, 1987 Dec 25, 262(36), 17432 - 6
Isolation and sequence of the promoter region of the human multidrug-resistance (P-glycoprotein) gene; Ueda K et al.; Intrinsic and acquired multidrug resistance is an important problem in cancer therapy . Multidrug resistance results from overexpression of the MDR 1 gene, which encodes a drug-efflux pump called P-glycoprotein . We have isolated a 1-kilobase genomic fragment containing the major transcription initiation sites for the human MDR 1 gene . Ribonuclease protection experiments using this fragment indicate that normal human adrenal, colon, and liver cells, the human hepatoma cell line HepG2, and vinblastine-selected human KB multidrug-resistant cells initiate transcription of the MDR 1 gene at the same site within this fragment . The 0.43-kilobase region upstream from the major transcription initiation site linked to the chloramphenicol acetyltransferase gene showed promoter activity in CV-1 monkey kidney cells and in human KB cells . The putative promoter region has a consensus CAAT box and two GC box-like sequences, but no TATA sequence . This identification and isolation of promoter sequences for the MDR 1 gene will permit studies on how expression of this gene is regulated in normal human tissues and cancers.

J Biol Chem, 1987 Dec 15, 262(35), 16871 - 9
The multidrug resistance gene PDR1 from Saccharomyces cerevisiae; Balzi E et al.; The Saccharomyces cerevisiae gene PDR1, responsible for pleiotropic drug resistance, was isolated from a genomic DNA cosmid library by hybridization to the flanking LEU1 gene, followed by subcloning the drug-sensitive phenotype into the transformed pdr1-1, pdr1-2, and pdr1-3 drug-resistant mutants . A RNA molecule of 3.5 kilobases was identified as the PDR1 transcript . The nucleotide sequence of the complementing DNA fragment contained a 3192-nucleotide open reading frame . Disruption of the pdr1 and PDR1 genes restored or increased drug sensitivity . Analysis of the PDR1 deduced amino acid sequence revealed several homologies to four different regulatory proteins involved in the control of gene expression, including a cysteine-rich motif suggested to be a metal-binding domain for DNA recognition . A model is proposed of a general transcriptional control by PDR1 of several target genes encoding proteins from plasma, mitochondria, and possibly other permeability barriers.

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