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M M Gottesman

Publications and source records attributed to M M Gottesman.

At least 73 records · Page 4Linked to original sources
PubMed1996

In vitro and in vivo liposome-mediated gene transfer leads to human MDR1 expression in mouse bone marrow progenitor cells.

The ability to select bone marrow cells (BMC) expressing a selectable gene that confers resistance to anticancer drugs would be useful to protect bone marrow during chemotherapy. The human multidrug resistance (MDR1) gene encodes a 170-kD glycoprotein (P-gp), an ATP-dependent transmembrane efflux pump for many different cytotoxic drugs. In this work, we demonstrate efficient expression of the human MDR1 gene in mouse BMC after transfection with a liposomal delivery system (DLS-liposomes). The human MDR1 cDNA expression plasmid (pHaMDR1/A) was encapsulated in DLS-liposomes and delivered to mouse BMC using two approaches: (i) in vitro transfection of BMC followed by bone marrow transplantation and (ii) in vivo direct systemic gene transfer. After both the in vitro and the in vivo approaches, polymerase chain reaction (PCR) analysis confirmed that the human MDR1 gene was successfully transfected to bone marrow, spleen, and peripheral blood (PB) cells, with the human MDR1 gene detected in BMC for up to 30 days after bone marrow transplantation and 28 days after direct systemic administration. Efflux studies using rhodamine-123 demonstrated function of the MDR1 gene product in the in vitro-transfected BMC. Flow cytometry studies using the human MDR1-specific MRK16 monoclonal antibody confirmed the presence of P-gp in BMC after in vitro transfection, as well as in BMC from reconstituted or in vivo-transfected mice. Transgene expression in both lymphoid and myeloid subpopulations of BMC was demonstrated. Colony-forming units (CFU-Mix) were obtained after exposure of BMC to lethal doses of vincristine, demonstrating functional expression of the MDR1 gene in hematopoietic progenitor cells for up to 1 month.

3T3 Cells↗
PubMed1996

Gene-specific DNA repair and steady state transcription of the MDR1 gene in human tumor cell lines.

We have explored the relationship between DNA repair and transcription in vivo. A gene-specific repair assay has been employed to study removal of ultraviolet light-induced cyclobutane pyrimidine dimers in the MDR1 gene at different levels of MDR1 mRNA expression. The parental human adenocarcinoma cell line, KB-3-1, has very low levels of MDR1 mRNA expression, but its multidrug resistant derivatives KB-8-5 and KB-C1 have 42-fold and 3800-fold increases in MDR1 mRNA expression, respectively. In the KB-3-1 cell line that has a low level of MDR1 mRNA expression, we find a low level of MDR1 gene-specific repair and inefficient repair of the transcribed strand of the gene. In the KB-8-5 cell line that has a modest increase in MDR1 mRNA expression, we find only a minor increase in dimer repair in the MDR1 gene. Here, the repair in the transcribed strand is not significantly higher than that in the KB-3-1 cell line. However, in the KB-C1 derivative, where there is a 3800-fold increase in the level of MDR1 mRNA expression, we find a substantial increase in the level of dimer repair in the MDR1 gene. In addition, the MDR1 transcribed strand repair is markedly more efficient than the repair in the nontranscribed strand. Our data suggest that the rate of transcription in the MDR1 gene must be substantially increased before there is any measurable effect on DNA repair. Repair in the housekeeping gene, dihydrofolate reductase (DHFR), was similar in all three tumor cell lines. Repair in its transcribed strand was markedly lower than previously reported in normal human fibroblasts. We suspect that these human HeLa-derived tumor cell lines have deficient gene-specific DNA repair. This may be an important aspect of their malignant phenotype.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1996

Altered drug-stimulated ATPase activity in mutants of the human multidrug resistance protein.

The characteristics of P-glycoprotein (MDR1), an ATP-dependent drug extrusion pump responsible for the multidrug resistance of human cancer, were investigated in an in vitro expression system. The wild-type and several mutants of the human MDR1 cDNA were engineered into recombinant baculoviruses and the mutant proteins were expressed in Sf9 insect cells. In isolated cell membrane preparations of the virus-infected cells the MDR1-dependent drug-stimulated ATPase activity, and 8-azido-ATP binding to the MDR1 protein were studied. We found that when lysines 433 and/or 1076 were replaced by methionines in the ATP-binding domains, all these mutations abolished drug-stimulated ATPase activity independent of the MgATP concentrations applied. Photoaffinity labeling with 8-azido-ATP showed that the double lysine mutant had a decreased ATP-binding affinity. In the MDR1 mutant containing a Gly185 to Val replacement we found no significant alteration in the maximum activity of the MDR1-ATPase or in its activation by verapamil and vinblastine, and this mutation did not modify the MgATP affinity or the 8-azido-ATP binding of the transporter either. However, the Gly185 to Val mutation significantly increased the stimulation of the MDR1-ATPase by colchicine and etoposide, while slightly decreasing its stimulation by vincristine. These shifts closely correspond to the effects of this mutation on the drug-resistance profile, as observed in tumor cells. These data indicate that the Sf9-baculovirus expression system for MDR1 provides an efficient tool for examining structure-function relationships and molecular characteristics of this clinically important enzyme.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Characterization of phosphorylation-defective mutants of human P-glycoprotein expressed in mammalian cells.

To assess the role of phosphorylation of the human multidrug resistance MDR1 gene product P-glycoprotein for its drug transport activity, phosphorylation sites within its linker region were subjected to mutational analysis. We constructed a 5A mutant, in which serines at positions 661, 667, 671, 675, and 683 were replaced by nonphosphorylatable alanine residues, and a 5D mutant carrying aspartic acid residues at the respective positions to mimic permanently phosphorylated serine residues. Transfection studies revealed that both mutants were targeted properly to the cell surface and conferred multidrug resistance by diminishing drug accumulation. In contrast to wild-type P-glycoprotein, the overexpressed 5A and the 5D mutants exhibited no detectable levels of phosphorylation, either in vivo following metabolic labeling of cells with [32P]orthophosphate or in vitro in phosphorylation assays with protein kinase C, cAMP-dependent protein kinase, or a P-glyco-protein-specific protein kinase purified from multidrug-resistant KB-V1 cells. These results reconfirm that the major P-glycoprotein phosphorylation sites are located within the linker region. Furthermore, the first direct evidence is provided that phosphorylation/dephosphorylation mechanisms do not play an essential role in the establishment of the multidrug resistance phenotype mediated by human P-glycoprotein.

3T3 Cells↗
PubMed1996

A bicistronic retrovirus vector containing a picornavirus internal ribosome entry site allows for correction of X-linked CGD by selection for MDR1 expression.

Chronic granulomatous disease (CGD) is an inherited hematologic disorder involving failure of phagocytic cell oxidase to produce superoxide (O2-.), resulting in recurrent infections. The success of retrovirus gene therapy for hematopoietic diseases will be limited both by the efficiency of ex vivo transduction of target cells and by the ability of corrected cells to replace uncorrected cells in vivo. Using MFG-based retrovirus vectors containing oxidase genes, we have previously demonstrated in vitro correction of CGD, but transduction rates were low. In the present study we explore a strategy for providing a selective growth advantage to transduced cells, while retaining the single promoter feature of MFG responsible for high virus titer and enhanced protein production. We constructed a bicistronic retrovirus producing a single mRNA encoding both the therapeutic gene for the X-linked form of CGD (X-CGD), gp91phox, and the selectable human multidrug resistance gene, MDR1 linked together by the encephalomyocarditis virus internal ribosome entry site (IRES). As a control we constructed a bicistronic vector with the polio virus IRES element and using the bacterial neomycin resistance gene (neor) as the selective element. In Epstein-Barr virus transformed B (EBV-B) cells from an X-CGD patient, a tissue culture model of CGD, we show correction of the CGD defect and complete normalization of the cell population using either of these vectors and appropriate selection (vincristine for MDR1 and G418 for neor). Using a chemiluminescence assay of O2-. production, populations of cells transduced with either vector demonstrated initial correction levels of from less than 0.1% up to 2.7% of normal EBV-B cell oxidase activity. With either construct, cell growth under appropriate selection enriched the population of transduced cells, resulting in correction of X-CGD EBV-B cells to a level of O2-. production equalling or exceeding that of normal EBV-B cells. These studies show that a therapeutic gene can be linked to a resistance gene by an IRES element, allowing for selective enrichment of cells expressing the therapeutic gene. Furthermore, the use of MDR1 as a selective element in our studies validates an important approach to gene therapy that could allow in vivo selection and is generalizable to a number of therapeutic settings.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

The multidrug-resistance gene in gene therapy of cancer and hematopoietic disorders.

Chemoresistance genes have been identified as an impediment to anticancer drug treatment. In particular, P-glycoprotein, the product of the multidrug-resistance (MDR1) gene, plays a major role in clinical treatment failure. Conversely, expression of an MDR1 cDNA in bone marrow of transgenic animals renders hematopoietic cells chemoresistant. Efficient transfer of drug-resistance genes to normal hematopoietic progenitor cells has been achieved with the use of retroviral vectors. In this article we review approaches which use the multidrug-resistance gene to protect bone marrow from myelosuppression following chemotherapy and as a selectable markerin vivo to increase the expression of nonselectable genes which correct hereditary diseases of the hematopoietic system.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

P-glycoprotein and multidrug resistance.

Although the phenomenon of simultaneous resistance to multiple cytotoxic drugs (multidrug resistance) in cancer cells has been discussed for more than two decades, and the human and mouse genes encoding an energy-dependent transporter (the multidrug transporter or P-glycoprotein) responsible for multidrug resistance were cloned 10 years ago, there is still considerable controversy about the mechanism of action of this efflux pump and its true biological function. This review summarizes the current research on the mechanism of action of the multidrug transporter, including the hydrophobic cleaner and altered partitioning models, the possible function of P-glycoprotein as a chloride and/or ATP channel, the role of phosphorylation in its function and fact and speculation about its physiological role.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

MDA435/LCC6 and MDA435/LCC6MDR1: ascites models of human breast cancer.

We have established a novel ascites tumour model (MDA435/LCC6) from the oestrogen receptor-negative, invasive and metastatic MDA-MB-435 human breast cancer cell line. MDA435/LCC6 cells grow as both malignant ascites and solid tumours in vivo in nude mice and nude rats, with a tumour incidence of approximately 100%. Untreated mice develop ascites following i.p. inoculation of 1 x 10(6) cells and have a reproducible life span of approximately 30 days, with all animals dying within a 48 h period. The in vivo response of MDA435/LCC6 ascites to several cytotoxic drugs, including doxorubicin, etoposide (VP-16), BCNU and mitomycin C, closely reflects the activity of these single agents in previously untreated breast cancer patients. MDA435/LCC6 cells also retain the anchorage-dependent and anchorage-independent in vitro growth properties of the parental MDA-MB-435 cells, and can be used in standard in vitro drug screening assays. The drug resistance pattern of the MDA435/LCC6 cells suggests that they may have few active endogenous drug resistance mechanisms. To generate a model for the screening of MDR1-reversing agents, MDA435/LCC6 were transduced with a retroviral vector directing the constitutive expression of the MDR1 cDNA, producing a cell line with a classical MDR1 resistance pattern (MDA435/LCC6MDR1). THese ascites models may be a viable alternative to the murine leukaemia ascites (L1210, P388) and, in conjunction with other breast cancer cell lines, facilitate the in vitro and in vivo screening of new cytotoxic drugs and drug combinations.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Functional characterization of a glycine 185-to-valine substitution in human P-glycoprotein by using a vaccinia-based transient expression system.

Human P-glycoprotein (Pgp) is a 170-kDa plasma membrane protein that confers multidrug resistance to otherwise sensitive cells. A mutation in Pgp, G185-->V, originally identified as a spontaneous mutation, was shown previously to alter the drug resistance profiles in cell lines that are stably transfected with the mutant MDR1 cDNA and selected with cytotoxic agents. To understand the mechanism by which the V185 mutation leads to an altered drug resistance profile, we used a transient expression system that eliminates the need for drug selection to attain high expression levels and allows for the rapid characterization of many aspects of Pgp function and biosynthesis. The mutant and wild-type proteins were expressed at similar levels after 24-48 h in human osteosarcoma (HOS) cells by infection with a recombinant vaccinia virus encoding T7 RNA polymerase and simultaneous transfection with a plasmid containing MDR1 cDNA controlled by the T7 promoter. For both mutant and wild-type proteins, photolabeling with [3H]azidopine and [125I]iodoarylazidoprazosin, drug-stimulated ATPase activity, efflux of rhodamine 123, and accumulation of radiolabeled vinblastine and colchicine were evaluated. In crude membrane preparations from HOS cells, a higher level of basal Pgp-ATPase activity was observed for the V185 variant than for the wild-type, suggesting partial uncoupling of drug-dependent ATP hydrolysis by the mutant. Several compounds, including verapamil, nicardipine, tetraphenylphosphonium, and prazosin, stimulated ATPase activities of both the wild-type and mutant similarly, whereas cyclosporin A inhibited the ATPase activity of the mutant more efficiently than that of the wild-type. This latter observation explains the enhanced potency of cyclosporin A as an inhibitor of the mutant Pgp. No differences were seen in verapamil-inhibited rhodamine 123 efflux, but the rate of accumulation was slower for colchicine and faster for vinblastine in cells expressing the mutant protein, as compared with those expressing wild-type Pgp. We conclude that the G185-->V mutation confers pleiotropic alterations on Pgp, including an altered basal ATPase activity and altered interaction with substrates and the inhibitor cyclosporin A.

3T3 Cells↗
PubMed1996

MDR1/P-glycoprotein function. I. Effect of hypotonicity and inhibitors on rhodamine 123 exclusion.

The MDR1 protein (P-glycoprotein) is a membrane ATPase whose expression results in resistance to several anti-tumor drugs. It has been proposed that the MDR1 protein, in addition to its pumplike properties, can function as (Gill et al. Cell 71: 23-32, 1992; Altenberg et al. Cancer Res. 54:618-622, 1994) or mediate the activity of (Hardy et al. EMBO J. 14: 68-75, 1995) a hypotonic stress-induced Cl- current. In addition, one study found that drug transport and Cl- channel-associated functions of MRD1 were separable and mutually exclusive and that, when cells were swelled, the MDR1 protein could not transport substrate. This hypothesis was tested in four pairs of isogenic cell lines with MDR1 transfectants expression 8,000-55,000 MDR1 antibody binding sites per cell. Cytoplasmic exclusion of rhodamine 123 was used as an indicator of MDR1 function to measure the effect of hypotonic stress, MDR1 inhibitors, and Cl- channel blockers on MRD1 transport function. It was found that MDR1 activity and its inhibition by cyclosporine A or flufenamic acid were unaffected by hypotonicity alone or in combination with Cl- channel blockers.

3T3 Cells↗
PubMed1996

Construction and characterization of a selectable multidrug resistance-glucocerebrosidase fusion gene.

Gene fusions can be employed to ensure concomitant expression of two different proteins under the same transcriptional control elements. We have synthesized a retroviral expression vector (pHaMG1) containing a human multidrug resistance (MDR1)-glucocerebrosidase (GC) chimeric gene inserted between the long terminal repeats of the Harvey murine sarcoma virus. When introduced into psi-CRE mouse fibroblasts, pHaMG1 conferred the drug-selectable multidrug resistance phenotype, and drug-resistant clones produced active human GC of about 60 kDa. Percoll gradient fractionation of homogenates prepared from transfectants confirmed correct targeting of P-glycoprotein to the plasma membrane and of GC to lysosomes. Although this construction was designed as a translational fusion of the MDR1 gene product, P-glycoprotein, and human GC, no evidence for a fusion protein was found in transfected cells, and an analysis of the RNAs transcribed from the integrated pHaMG1 retroviral vector suggests that either P-glycoprotein and GC are translated from one mRNA and rapidly processed into two proteins or they are translated separately from different mRNAs. These results reveal the feasibility of using fusion genes, which are smaller than alternative constructions with two promoters or with an internal ribosome entry site, for coexpression of selectable and nonselectable cDNAs in retroviral vectors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Retroviral transfer of the human MDR1 gene confers resistance to bisantrene-specific hematotoxicity.

In this work, we demonstrate a protective effect conferred by the human multidrug resistance gene (MDR1) to populations of the murine hematopoietic system against the toxic effects of bisantrene, a novel intercalating cytotoxic agent under investigation as an anticancer agent. In vitro, MDR1-expressing cell lines are highly cross-resistant to bisantrene, and low levels of P-glycoprotein (the MDR1 gene product cell surface protein) confer resistance to the drug. MDR1-positive mice were generated after transplantation of bone marrow cells (BMC) transduced in vitro with a MDR1 retrovirus. Control mice were transplanted with BMC transduced with the neomycin resistance gene. Administration of a single i.v. dose of 50 mg/kg of bisantrene resulted in a decrease of the total WBC count of approximately 40%. In contrast, a decrease of the total WBC count of only 17% was observed in mice transplanted with MDR1-transduced BMC. Immunofluorescence studies with cell lineage-specific monoclonal antibodies showed that bisantrene was specifically toxic for B lymphocytes and macrophages. Double-staining with MRK16 (a monoclonal antibody specific for P-glycoprotein) demonstrated that a single dose of bisantrene increased the relative number of MDR1-transduced positive B cells, macrophages, and (to some extent) granulocytes when compared to the number found in MDR1-untreated mice or the bisantrene-treated neomycin-transduced control mice. These results provide in vivo evidence that bisantrene is a hematotoxic drug capable of selecting for MDR1-transduced hematopoietic cells. Bisantrene might be useful for gene therapy as an in vivo selective agent for cells transduced with MDR1 vectors that also coexpress therapeutic genes of interest.

3T3 Cells↗
PubMed1995

Bacterial expression of the linker region of human MDR1 P-glycoprotein and mutational analysis of phosphorylation sites.

Phosphorylation may play a role in modulating multidrug resistance by P-glycoprotein (P-gp). The linker region between the two homologous halves of human P-gp harbors several serine residues which are phosphorylated by protein kinase C (PKC) in vitro. We used the glutathione S-transferase gene fusion system to express and purify a series of fusion proteins containing the relevant portion (residues 644-689) of the linker region of the human MDR1 gene product. The fusion proteins were subjected to in vitro phosphorylation and phosphopeptide mapping analysis to identify specific phosphorylation sites. On the basis of a mutational strategy in which individual serine residues were systematically replaced with nonphosphorylatable alanine residues, Ser-661 and Ser-667 were identified as major PKC sites and Ser-683 was identified as a minor PKC site. Ser-661 and Ser-667 were also found to be the primary sites of phosphorylation for a novel membrane-associated P-gp specific kinase isolated from the multidrug-resistant KB-V1 cell line. Individual phosphorylation sites were recognized independently of each other. These data show that the linker region of P-gp represents a target for multisite phosphorylation not only for PKC but also for the P-gp specific V1 kinase. Specific serine phosphorylation sites are identified, and evidence is presented that the V1 kinase has a specificity which overlaps, but is more restricted than, that of PKC. In addition, these studies also suggest that the use of GST fusion peptides may be applicable for the analysis of multisite and ordered protein phosphorylation in other systems.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Behavior of N-acylated daunorubicins in MDR1 gene transfected and parental cells.

The substrate specificity of the P-glycoprotein (P-170), a multidrug transporter, was studied using N-acylated daunorubicin derivatives and four MDR1 cDNA transfected cell lines. Results showed that N-acetyl-daunorubicin is a substrate, but the longer fatty acid derivatives, N-octanoyl and N-dodecanoyl daunorubicins, are not. This conclusion was reached by flow cytometric drug uptake assay, cell proliferation assays, and confocal microscopy. It was concluded that the longer fatty acid derivatives interact with plasma membranes in a way that affected P-glycoprotein function.

3T3 Cells↗
PubMed1995

Efficient expression of functional human MDR1 gene in murine bone marrow after retroviral transduction of purified hematopoietic stem cells.

A procedure for efficient transfer of the human MDR1 (multi-drug resistance) gene into murine hematopoietic stem cells was developed. Cells expressing Sca-1 but no lineage-specific or major histocompatibility complex (MHC) class II antigens (Lin-MHC II-Sca-1+) were enriched from 5-fluorouracil-pretreated bone marrow by Ficoll density-gradient and immunomagnetic sorting. Purified cells were cocultured with growth factors and fibroblasts producing replication-deficient retroviruses containing human MDR1 cDNA. Fluorescence-activated cell sorter analysis and rhodamine-123 efflux experiments showed that greater than 60% of cocultured hematopoietic cells expressed functional human P-glycoprotein. After 6 to 8 days, hematopoietic cells were injected intravenously into sublethally irradiated SCID mice. Stem cell properties of the isolated population were confirmed by sustained expression of MDR1 marker cDNA for greater than 4 to 6 months after transplantation, multilineage engraftment, and presence of MDR1 cDNA in bone marrow of secondary recipient mice after retransplantation. Reconstitution of H-2K-mismatched SCID mice showed high engraftment capacity of Lin-MHC II-Sca-1+ cells. MDR1 cDNA was detected in blood of 78% of recipients. P-glycoprotein was expressed in bone marrow of 71% of mice, in both lymphocytes and myelomonocytoid progenitors. P-glycoprotein function in host marrow was confirmed by rhodamine-123 efflux. Transduction of P-glycoprotein may be useful for gene therapy in two ways: to protect bone marrow from myelosuppression after chemotherapy and as a selectable marker in vivo for the introduction of otherwise nonselectable genes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Differential effects of P-glycoprotein inhibitors on NIH3T3 cells transfected with wild-type (G185) or mutant (V185) multidrug transporters.

Multidrug resistance (MDR) may be associated with the expression of the MDR1 gene which encodes the 170-kDa cell surface P-glycoprotein (PGP) acting as an energy-dependent multidrug efflux pump. This pump can be inhibited by a variety of drugs including cyclosporin A, quinidine, and verapamil. Substrate specificity of the MDR1 gene product can be altered by a point mutation at amino acid residue 185 in which valine is substituted for glycine, but the effect of this mutation on inhibition of PGP is unknown. Multidrug-resistant NIH3T3 cells transfected with the MDR1 retroviral vector pHaMDR-1/A (G185) or pHaMDR1/A (V185) expressing comparable levels of PGP were compared for patterns of drug resistance and inhibition of drug resistance by MDR reversing agents. The NIH-MDR-G185 transfectants were somewhat preferentially resistant to daunorubicin, taxol, and vinblastine. The mutant (V185) conferred increased resistance to colchicine. This MDR phenotype in both NIH-MDR-G185- and NIH-MDR-V185-transfected NIH3T3 cells was overcome by the addition of cyclosporin A, quinidine, or verapamil. Verapamil was the most potent of the three agents affecting wild-type PGP. However, specific inhibitors showed different potency with wild-type or mutant transporters, depending on the cytotoxic drug whose resistance was being reversed. For example, cyclosporin A at a concentration of 1 microgram/ml, was a powerful reverser of taxol and colchicine resistance for the mutant drug transporter, but was much less effective for the wild-type transporter. In contrast, verapamil reversed resistance to vinblastine more efficiently for the wild-type transporter than for the mutant transporter. These results suggest that the sensitivity of a multidrug transporter to a reversing agent will depend on the reversing agent, the cytotoxic drug, and the presence or absence of mutations which alter substrate specificity.

3T3 Cells↗