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Biomedical subjects

M M Gottesman

Publications and source records attributed to M M Gottesman.

At least 127 records · Page 7Linked to original sources
PubMed1993

Pseudomonas exotoxin conjugated to monoclonal antibody MRK16 specifically kills multidrug resistant cells in cultured renal carcinomas and in MDR-transgenic mice.

Using renal carcinoma and prostate carcinoma cell lines, we investigated the concept of targeting and killing multidrug resistant cells in urogenital cancers. Renal carcinoma lines HTB44, 45, 46, and 47 expressed a relatively low, but detectable level of multidrug resistance (MDR)1 mRNA as indicated by Northern blot analysis, whereas prostate lines LNCaP and DU145 were found to be MDR1-negative. Anti-P-glycoprotein monoclonal antibody MRK16 was conjugated to Pseudomonas exotoxin (PE) by a stable thioether bond. Treatment with MRK16-PE resulted in a dose-dependent killing of multidrug resistant renal carcinoma cells, while non-MDR expressing prostate carcinoma cells were not affected. Addition of excess MRK16 blocked the effect of MRK16-PE. Furthermore, MOPC-PE, a non-MDR associated monoclonal antibody control conjugate, did not target and kill multidrug resistant renal carcinoma cells. Having established that MRK16-PE was active against and specific for multidrug resistant cells in culture, we also tested bioactivity in MDR-transgenic mice, whose bone marrow cells express the human MDR1 gene at a level approximately equal to that found in many human cancers. Again, MRK16-PE killed multidrug resistant bone marrow cells with high efficiency in an intact animal, and killing was blocked by unconjugated MRK16.

ADP Ribose Transferases↗
PubMed1993

Function and regulation of the human multidrug resistance gene.

The discovery of an energy-dependent pump system for natural product anticancer drugs has important implications for the biology of related energy-dependent transport systems as well as for the treatment of human cancer. To fully realize the therapeutic potential associated with manipulation of the multidrug transporter, it will be necessary to understand the mechanisms of action of the transporter and its mode of regulation. This review has summarized recent developments in these areas which suggest that both the activity of the pump and its genetic regulation are potential targets for new anticancer therapies.

Clinical Trials as Topic↗
PubMed1992

Identification of residues in the first cytoplasmic loop of P-glycoprotein involved in the function of chimeric human MDR1-MDR2 transporters.

The human MDR1 gene encodes the multidrug transporter (P-glycoprotein), a multidrug efflux pump. The highly homologous MDR2 gene product does not appear to be a functional multidrug pump. We have constructed a chimeric protein in which the first intracytoplasmic loop and the third and fourth transmembrane domains of the MDR1 protein were replaced by the analogous region of MDR2. Substitution of the MDR2 sequences encompassing amino acid residues 140 to 229 resulted in 17 amino acid changes, 10 in the intracytoplasmic loop (amino acids 141-188) and 7 in the transmembrane regions. This chimeric protein was expressed on the surface of NIH 3T3 cells where it bound [3H]azidopine but did not confer drug resistance. When only 4 residues, 165, 166, 168, and 169, were changed back to MDR1 amino acids, a functional drug transporter was recovered. When residues 165, 166, 168, and 169 from MDR2 were substituted into a functional MDR1 cDNA, the resulting construction was not able to confer drug resistance. These results indicate that the major functional differences between MDR1 and MDR2 in this region of P-glycoprotein reside in a small segment of the first intracytoplasmic loop. We also independently analyzed the effect of replacing Asn183 of MDR1 with Ser which occurs in MDR2. Substitution of Ser at position 183 in combination with Val at position 185 in P-glycoprotein resulted in a relative increase in resistance to actinomycin D, vinblastine, and doxorubicin in transfected NIH 3T3 cells. These results emphasize the importance of the first intracytoplasmic loop in P-glycoprotein in determining function and relative drug specificity of the transporter.

3T3 Cells↗
PubMed1992

Construction of a new universal vector for insertional mutagenesis by homologous recombination.

We describe here the construction of a vector (pSSC-9) which can be used for the insertional mutagenesis of any gene for which genomic sequences have been cloned. This vector contains a neomycin-resistance-encoding gene (neoR) which is driven by a modified thymidine kinase (tk) promoter for positive selection. Flanking neoR are two tk genes driven by their own promoters for negative selection of nonhomologous insertions. The neoR and tk cassettes are separated by four unique cloning sites on the right-hand side of the neoR cassette and three unique sites on the left-hand side. The vector also includes two SfiI sites, one on each side of the tk cassettes, for the excision of the cloned genomic DNA fragments along with the selectable markers. Electroporation of pSSC-9 into mouse embryonic stem (ES) cells and cultured diploid mouse adrenal Y-1 cells conferred resistance to G418 and sensitivity to ganciclovir in both cell lines. These results illustrate the expression of the positive and negative selectable markers in two different cell lines and thus suggest that the vector could be used in ES cells, as well as in cultured somatic cells.

Animals↗
PubMed1992

Characterization of the azidopine and vinblastine binding site of P-glycoprotein.

To determine the number of drug binding sites that exist on the multidrug transporter, P-glycoprotein, we used azidopine, a dihydropyridine photoaffinity compound that reverses multidrug resistance and labels P-glycoprotein. Azidopine labels P-glycoprotein in two distinct locations: one labeled site is within the amino half of P-glycoprotein between amino acid residues 198 and 440, and the other site is within the carboxy half of the protein. Vinblastine is a cytotoxic drug that is used in cancer chemotherapy and is a substrate for transport by P-glycoprotein. We found that vinblastine inhibits azidopine labeling to approximately the same extent at each labeled site on P-glycoprotein. Because several studies have shown that amino acid residue 185 of P-glycoprotein plays a critical role in some aspects of drug binding and transport, we also studied the effect that amino acid residue 185 has on azidopine labeling. These studies show that azidopine labels both sites equivalently in both wild-type (G185) and mutant (V185) P-glycoproteins. We conclude from our results that the two halves of P-glycoprotein approach each other to form a single binding site for these drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Effect of P-glycoprotein expression on sensitivity to hormones in MCF-7 human breast cancer cells.

BACKGROUND: Data obtained from studies of primary human breast cancers and established cell lines indicate that overexpression of the MDR1 gene (also known as PGY1) is associated with decreased expression of steroid hormone receptors and increased expression of epidermal growth factor (EGF) receptors. Other study results indicate that both progestins and triphenylethylene antiestrogens may be substrates for P-glycoprotein, the product of the MDR1 gene. These findings together suggest an association between overexpression of the MDR1 gene and cross-resistance to progestin and antiestrogen therapies. PURPOSE: This study was designed to determine (a) the ability of MDR1 expression to alter tumor sensitivity to hormone therapy and (b) the role of MDR1 expression in expression of functional hormone receptors in human breast cancer. METHODS: We transduced MCF-7 cells with MDR1 complementary DNA, using a retroviral vector directing the constitutive expression of the MDR1 gene. Transduced cells (MCF-7MDR1) were examined for ability to produce P-glycoprotein, expression of steroid hormone receptors, and responsivity to antiestrogens. For comparison, we used MCF-7ADR human breast cancer cells, which overexpress MDR1 and have also lost the requirement for 17 beta-estradiol supplementation to form tumors in nude mice. We also investigated the level of EGF-R mRNA expression by using a sensitive RNase protection analysis. RESULTS: MCF-7MDR1 cells retained both estrogen receptor and progesterone receptor expression as well as sensitivity to 4-hydroxytamoxifen. Expression of the estrogen-inducible pS2 and EGF receptor genes was similar in parental MCF-7 and transduced MCF-7MDR1 cells. EGF receptor expression was increased, and pS2 expression was lost (undetectable) in MCF-7ADR cells. CONCLUSIONS: The data indicate that overexpression of the MDR1 gene alone confers a multidrug-resistant phenotype, but it does not directly result in either cross-resistance to antiestrogens or a loss of steroid hormone receptor expression. IMPLICATIONS: MCF-7MDR1 cells provide an important model for study of the interactions of cytotoxic drugs, hormones, and the MDR1 glycoprotein in human hormone-responsive breast cancer cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Partial purification and reconstitution of the human multidrug-resistance pump: characterization of the drug-stimulatable ATP hydrolysis.

Multidrug-resistant human tumor cells overexpress the MDR1 gene product P-glycoprotein, which is believed to function as an ATP-dependent efflux pump. In this study we demonstrate that the partially purified P-glycoprotein, when reconstituted in an artificial membrane, catalyzes drug-stimulated ATP hydrolysis. Plasma membrane proteins of a human multidrug-resistant cell line, KB-V1, were solubilized with 1.4% (wt/vol) octyl beta-D-glucopyranoside in the presence of 0.4% phospholipid and 20% (vol/vol) glycerol, and the crude detergent extract was chromatographed on DEAE-Sepharose CL-6B. The 0.1 M NaCl fraction, enriched in P-glycoprotein but devoid of Na,K-ATPase, was reconstituted by the detergent-dilution method. P-glycoprotein constituted 25-30% of the reconstituted protein in proteoliposomes. ATP hydrolysis by proteoliposomes was stimulated 3.5-fold by the addition of vinblastine but was unaffected by the hydrophobic antitumor agent camptothecin, which is not transported by P-glycoprotein. The stimulatory effect of vinblastine was observed only if the protein was reconstituted in proteoliposomes, suggesting that either the substrate binding site(s) was masked by detergent or that the conformation of the soluble P-glycoprotein might not be suitable for substrate-induced activation. Several other drugs that are known to be transported by P-glycoprotein enhanced the ATPase activity in a dose-dependent manner with relative potencies as follows: doxorubicin = vinblastine greater than daunomycin greater than actinomycin D greater than verapamil greater than colchicine. The basal and vinblastine-stimulated ATPase activities were inhibited by vanadate (50% inhibition observed at 7-10 microM) but were not affected by agents that inhibit other ATPases and phosphatases. These data indicate that the P-glycoprotein, similar to other ion-transporting ATPases, exhibits a high level of ATP hydrolysis (5-12 mumol per min per mg of protein).

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Monoclonal antibody MRK16 reverses the multidrug resistance of multidrug-resistant transgenic mice.

Using multidrug-resistant (MDR)-transgenic mice, whose bone marrow cells express the human MDR1 gene at a level approximately equal to that found in many human cancers, we determined the efficacy of human-specific anti-P-glycoprotein monoclonal antibody MRK16 in overcoming multidrug resistance in an intact animal. MRK16 alone (2 mg) did not significantly affect the WBC counts of the MDR-transgenic mice, but MRK16, as well as the F(ab')2 fragments of MRK16, led to a dose-dependent circumvention of bone marrow resistance against daunomycin, doxorubicin, vincristine, vinblastine, etoposide, and taxol. This sensitizing effect could not be enhanced by combining MRK16 with low molecular weight chemosensitizing agents such as verapamil, quinine, quinidine, or cyclosporin A. We also investigated the concept of specifically targeting and killing multidrug-resistant cells by using MRK16 coupled to Pseudomonas exotoxin (PE). MRK16-PE resulted in a dose-dependent killing of bone marrow cells in MDR-transgenic mice, whereas no bone marrow toxicity was observed in normal control mice. Administration of excess MRK16 prior to injection of MRK16-PE successfully blocked the effect of MRK16-PE. MOPC-PE, a non-MDR-related control monoclonal antibody conjugate, did not target and kill multidrug-resistant bone marrow cells in MDR-transgenic mice. Thus, these immunological approaches to reversing multidrug resistance appear to be both specific and effective.

Alkaloids↗
PubMed1992

Transplantation of bone marrow cells from transgenic mice expressing the human MDR1 gene results in long-term protection against the myelosuppressive effect of chemotherapy in mice.

Many human cancers that are initially responsive to chemotherapy eventually fail to respond to treatment. For some drugs, dose escalation that may be required for a cure cannot be achieved because sensitive tissues such as bone marrow (BM) limit cytotoxic therapy. Approaches to prevent or circumvent BM toxicity are therefore a high priority of research on dose escalation protocols. In this study, we have transplanted BM cells from transgenic mice that constitutively express physiologic amounts of a functional human multidrug resistance (MDR1) cDNA to lethally irradiated C57BL/6 x SJL F1 mice (n = 36). From 6 weeks to 10 months after the transplant, all animals contained MDR1 DNA in spleen and BM specimens as indicated by Southern blot analysis, and expressed MDR1 messenger RNA in BM samples as detected by slot blot analysis. In addition, these animals were resistant to the myelosuppressive effect of doxorubicin, daunomycin, taxol, vinblastine, vincristine, etoposide, and actinomycin D, whereas control animals that were reconstituted with normal BM were drug sensitive. Finally, the chemoprotection afforded by the MDR1 gene could readily be reversed by adding chemosensitizers such as cyclosporin A and R-verapamil to chemotherapy. Hence, it appears that BM cells expressing the human MDR1 gene maintain this function after transplantation to host animals for a minimum of 10 months, and confer multidrug resistance to these BM recipients. This selective advantage conferred by expression of the MDR1 cDNA suggests a strategy for the use of MDR1 gene therapy in cancer chemotherapy and for the introduction of otherwise nonselectable genes into BM.

Animals↗
PubMed1992

Modulation of activity of the promoter of the human MDR1 gene by Ras and p53.

Drug resistance in human cancer is associated with overexpression of the multidrug resistance (MDR1) gene, which confers cross-resistance to hydrophobic natural product cytotoxic drugs. Expression of the MDR1 gene can occur de novo in human cancers in the absence of drug treatment. The promoter of the human MDR1 gene was shown to be a target for the c-Ha-Ras-1 oncogene and the p53 tumor suppressor gene products, both of which are associated with tumor progression. The stimulatory effect of c-Ha-Ras-1 was not specific for the MDR1 promoter alone, whereas a mutant p53 specifically stimulated the MDR1 promoter and wild-type p53 exerted specific repression. These results imply that the MDR1 gene could be activated during tumor progression associated with mutations in Ras and p53.

3T3 Cells↗
PubMed1992

Reduced mRNA levels for the multidrug-resistance genes in cAMP-dependent protein kinase mutant cell lines.

We have previously shown that in Chinese hamster ovary (CHO) cells, a mutant cell line with a defective regulatory subunit (RI) for the cAMP-dependent protein kinase (Abraham et al: Mol. Cell. Biol., 7:3098-3106, 1987), and a transfectant cell line expressing the same mutant kinase, showed increased sensitivity to a number of drugs that are known to be substrates for the multidrug transporter (P-glycoprotein). In the current study we have investigated the mechanism by which cAMP-dependent protein kinase controls drug resistance. We report here that the sensitivity of the kinase defective CHO cell lines to multiple drugs results from decreased RNA levels for the multidrug-resistance gene. Similar results were obtained with mouse Y1 adrenal cells. Wild-type Y1 cells had high levels of P-glycoprotein due to expression of both the mdr1b and mdr2 genes, whereas the cAMP-dependent protein kinase mutant Kin 8 cells had decreased RNA levels for these genes. A Kin 8 transfectant with restored cAMP-dependent protein kinase activity recovered mdr expression, indicating a cause and effect relationship between the protein kinase mutations and mdr expression. No changes in nuclear run-off assays could be detected, suggesting a non-transcriptional mechanism of regulation. Wild-type Y1 cells are more drug sensitive despite having higher levels of P-glycoprotein than the mutant cells. This paradoxical result may be explained by the higher rate of synthesis of steroids by the wild-type Y1 cells, which appear to be inhibitors of P-glycoprotein transport activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Ablation of stimulation of a cAMP-responsive promoter in CHO cell lines defective in their cAMP-dependent protein kinase system.

We have studied the requirement for an intact cAMP-dependent protein kinase (PKA) system to regulate cAMP-mediated gene transcription in Chinese hamster ovary (CHO) cells. Wild-type CHO cells and mutant CHO cell lines selected for their resistance to the growth inhibitory effect of 8-Br-cAMP and defective in their PKA system were transiently transfected with reporter plasmids containing 2.5 and 3.0 kb of the 5'-flanking sequence of the rat tyrosine aminotransferase (TAT) gene promoter. This segment of DNA contains no CRE-like sequences, yet wild-type transfectants exhibited a specific increase in TAT promoter activity following growth in medium containing 8-Br-cAMP. In CHO cell lines defective in their PKA, the transfected TAT promoter failed to respond to cAMP treatment. We conclude that an intact PKA system is necessary for the cAMP-mediated increase in TAT promoter activity in CHO cells and that there is no requirement for a CRE to see this effect.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Is the multidrug transporter a flippase?

The phenomenon of multidrug resistance is correlated with the presence of a membrane protein, P-glycoprotein, which pumps a wide variety of drugs out of cells thus reducing their toxicity. However, the mechanism of this pumping action remains unclear. In this article, we suggest that several properties of the multidrug transporter may be explained if it acts as a 'flippase' to transport drugs from the inner leaflet of the lipid bilayer to the outer or to the external medium.

ATP Binding Cassette Transporter, Subfamily B, Mem↗