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Biomedical subjects

M M Gottesman

Publications and source records attributed to M M Gottesman.

At least 163 records · Page 9Linked to original sources
PubMed1991

Multidrug resistance.

Laboratory investigations indicate that cancer cells can become simultaneously resistant to many different chemotherapeutic drugs that are natural products via the expression of an energy-dependent drug efflux pump. This multidrug transporter is a plasma membrane glycoprotein encoded in the human by the MDR1 gene. Recent clinical studies indicate that expression of the multidrug transporter plays a major role in the intrinsic and acquired resistance to chemotherapy of many human cancers. Strategies aimed at inactivating this drug efflux pump may have significant impact on the treatment of human cancer.

Animals↗
PubMed1991

Human hepatocellular carcinoma cell lines exhibit multidrug resistance unrelated to MRD1 gene expression.

Multidrug resistance of human cancer cells may result from expression of P-glycoprotein, the product of the MRD1 gene, acting as an energy-dependent drug efflux pump. However, direct evidence that expression of the MDR1 gene contributes to the multidrug resistance of human liver carcinomas has not been established. In this study, we tested five cell lines derived from human hepatocellular carcinomas for sensitivity to a variety of drugs used widely as anticancer agents; these included vinblastine, doxorubicin, actinomycin D, mitomycin C, 5-fluorouracil, 6-mercaptopurine, melphalan, methotrexate, cis-platinum and etoposide (VP-16). All five hepatoma cell lines were resistant at different levels to these chemicals compared to human KB cells. Although it has been demonstrated that resistance to vinblastine, colchicine, doxorubicin and actinomycin D in human multidrug-resistant cells is associated with overexpression of P-glycoprotein, very little expression of P-glycoprotein was found in these human hepatoma cells. Neither verapamil nor quinidine, inhibitors of the drug efflux pump, were able to overcome multidrug resistance in hepatoma cells. These results indicate that the multidrug resistance phenotype in human hepatocellular carcinoma cells cannot be attributed to expression of the MDR1 gene, but that novel mechanisms may account for the resistance of these cancer cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Pseudomonas exotoxin fusion proteins are potent immunogens for raising antibodies against P-glycoprotein.

Antibodies to specific regions of human P-glycoprotein have been difficult to obtain. We developed a method to express in E. coli fusions between Pseudomonas exotoxin and specific regions of human P-glycoprotein. We used the polymerase chain reaction to amplify the desired regions of MDR1 cDNA and to introduce appropriate restriction sites. These fragments were cloned into the 3' end of the Pseudomonas exotoxin gene. With this system we produced large amounts of fusion proteins for immunizations, and we obtained positive rabbit antiserum against P-glycoprotein with most of these antigens. We now have a comprehensive panel of polyclonal antibodies against P-glycoprotein. This system should be generally useful to raise antibodies against other eukaryotic proteins that are difficult to prepare in large quantities.

ADP Ribose Transferases↗
PubMed1991

Fluorescent verapamil derivative for monitoring activity of the multidrug transporter.

Multidrug resistance to amphipathic natural product chemotherapeutic drugs is conferred on cancer cells by expression of the MDR1 gene, which encodes the 170-kDa multidrug transporter known as P glycoprotein. The P glycoprotein-mediated efflux of toxic chemotherapeutic drugs can be reversed by agents such as verapamil, which is a substrate for the multidrug transporter and appears to be a competitive inhibitor of the efflux pump. In this study, Bodipy-verapamil, a fluorescent derivative of verapamil, has been shown to be a substrate for the efflux pump activity of P glycoprotein. Single-cell fluorescence analysis reveals that Bodipy-verapamil accumulates in lysosomes of drug-sensitive NIH3T3 and KB cells but is rapidly effluxed from multidrug-resistant derivatives of these cell lines. Although Bodipy-verapamil is a substrate for the multidrug transporter, it is not an efficient inhibitor of the pump and does not reverse resistance to vinblastine and colchicine as effectively as does verapamil. This new derivative may be a useful tool for imaging of lysosomes in drug-sensitive cells and for rapid screening for the multidrug-resistant phenotype in other cell types.

3T3 Cells↗
PubMed1991

Downstream sequences mediate induction of the mouse cathepsin L promoter by phorbol esters.

The major excreted protein (MEP) of mouse fibroblasts is the precursor to a lysosomal acid protease (cathepsin L) whose synthesis is induced by malignant transformation, growth factors, tumor promoters, and cyclic AMP. We have previously cloned a functional gene for MEP from NIH 3T3 cells. When subcloned into chloramphenicol acetyl transferase (CAT) expression vectors, both 4-kilobase and 300 base pair fragments in the 5'-flanking region of the MEP gene confer CAT activity that is stimulated by cyclic AMP treatment but is not stimulated by phorbol ester treatment of NIH 3T3 cells. These fragments confer constitutive promoter activity that is comparable to that of the SV40 promoter. Primer extension, using RNA from cells transiently transfected with MEP-CAT fusion plasmids, demonstrates that phorbol ester treatment increases the amount of transcript from constructs containing both the promoter and sequences downstream of the transcription initiation site, including the first three introns, but not from constructs containing only the 5'-flanking region of the MEP gene. Nuclear run-off experiments confirm that the increase in endogenous MEP mRNA is mediated by increased transcription and not via relief of transcriptional attenuation. Since both the MEP promoter, which contains three potential binding sites for the AP-2 transcription factor, and the SV40 promoter, which contains both AP-1 and AP-2 binding sites, fail to respond to 12-O-tetradecanoylphorbol-13-acetate in NIH 3T3 cells, these upstream motifs are not sufficient to confer phorbol ester responsiveness in NIH 3T3 cells. These results suggest that the MEP gene is regulated in a complex manner by sequences both upstream and downstream of the transcription initiation site.

Animals↗
PubMed1990

Monoclonal antibodies to immunogenic lymphoma cell variants displaying impaired neoplastic properties: characterization and applications.

Immunogenic, nontumorigenic cell variants derived from the highly tumorigenic mouse lymphoma cell line S-49 were used to raise monoclonal antibodies (MAbs) in syngeneic BALB/c mice. MAbs of the following specifications were derived: (a) MAbs that interacted preferentially with the immunogenic variants, (b) MAbs that interacted with both immunogenic variants and parental tumorigenic cells, and (c) a MAb that interacted with both immunogenic and tumorigenic S-49 cells and the normal BALB/c splenocytes. Six MAbs raised in this way were found to recognize at least five different cell-surface epitopes. Functional analysis of the different MAbs suggested their potential usefulness in passive immunization against parental tumorigenic cells as well as in enrichment of immunogenic cells from a mixed population containing a preponderance of tumorigenic cells.

Animals↗
PubMed1990

Inhibitor studies indicate that active cathepsin L is probably essential to its own processing in cultured fibroblasts.

The lysosomal cysteine proteinase cathepsin L is synthesized in cultured mouse NIH 3T3 cells as a 39 kDa precursor and processed intracellularly into active 29 kDa and 20 kDa + 5 kDa lysosomal forms. Addition to culture media of the peptidyl aldehyde leupeptin, a non-covalent inhibitor of cathepsin L, results in the accumulation of the 20 kDa mature form of the enzyme, resulting in increased activity of cathepsin L as measured in an in vitro assay system in the absence of leupeptin. The more potent irreversible cathepsin L inhibitors benzyloxycarbonyl-Phe-Ala-diazomethane and L-transepoxysuccinyl-L-leucylamino-(4-guanidino)butane, when added to living cells at low concentrations, result in accumulation of all partially processed forms of cathepsin L, especially the 29 kDa form, suggesting that cathepsin L is responsible for its own processing. Exogenous procathepsin L introduced into CHO cells by endocytosis via the mannose 6-phosphate receptor is processed in a manner similar to endogenous procathepsin L. We conclude that the major intracellular pathway for processing of procathepsin L, either endogenous or exogenous, probably requires active cathepsin L.

Animals↗
PubMed1990

Transepithelial transport of vinblastine by kidney-derived cell lines. Application of a new kinetic model to estimate in situ Km of the pump.

We present a new transport model that may be useful for many kinds of transepithelial transport experiments. The model permits estimation of a pump Km and pump activity solely on the basis of transepithelial tracer fluxes. We apply the model to studies of a multidrug efflux pump, P-glycoprotein, which is normally located in the apical plasma membrane of certain transporting epithelia such as kidney proximal tubule cells. To determine the functional properties of this multidrug transporter in an epithelium, we studied the transepithelial transport of the chemotherapeutic drug, vinblastine, in epithelia formed by the kidney cell lines MDCK, LLC-PK1, and OK. We have previously shown that basal to apical flux of 100 nM vinblastine was about five times higher than apical to basal flux in MDCK epithelia, indicating that there is a net transepithelial transport of vinblastine across MDCK epithelia. Addition of unlabeled vinblastine reduced basal to apical flux of tracer and increased apical to basal flux of tracer in a concentration-dependent manner, a pattern expected if there is a saturable pump that extrudes vinblastine at the apical plasma membrane. The model permits estimation of a pump Km and pump activity solely on the basis of transepithelial tracer fluxes. According to the transport model the apical membrane pump has Michaelis-Menten kinetics with an apparent Km = 1.1 microM. Net basal to apical transport of vinblastine was also observed in LLC-PK1 cells and OK cells which are other kidney-derived cell lines. The order of potency of the transport is LLC-PK1 greater than MDCK greater than OK cells. The organic cation transporter is not involved in this vinblastine transport because vinblastine transport in MDCK cells was not affected by 3 mM tetramethyl- or tetraethylammonium. Inhibitors of vinblastine transport in MDCK cells was not affected by potency, were verapamil greater than vincristine greater than actinomycin D greater than daunomycin. The transport pattern we observed is that predicted to result from the function of the multidrug transporter in the apical plasma membrane.

Animals↗
PubMed1990

Expression of a human complementary DNA for the multidrug resistance gene in murine hematopoietic precursor cells with the use of retroviral gene transfer.

In multidrug resistance, cells become simultaneously resistant to anthracyclines, vinca alkaloids, epipodophyllotoxins, and certain other natural product cytotoxic drugs. Resistance results from synthesis of a multidrug transporter (P-glycoprotein) encoded by the MDR1 gene (also known as the PGY1 gene). In the present study, a retrovirus vector containing a complementary DNA for the human multidrug resistance gene HaMDR1/A was used to transfer the multidrug resistance phenotype to bone marrow cells of the DBA/2J mouse. A high proportion of transduced bone marrow cells showed resistance to both colchicine and vinblastine, as determined by in vitro colony formation of hematopoietic precursor cells. In addition, brief culturing of the cells in a cytotoxic drug following exposure to the retrovirus vector could be used to increase the proportion of bone marrow cell colonies that were resistant. These results may serve as a model for the generation and selection of bone marrow cells resistant to the toxic effects of chemotherapeutic agents in vivo.

Animals↗
PubMed1990

Cyclic AMP responses are suppressed in mammalian cells expressing the yeast low Km cAMP-phosphodiesterase gene.

A genomic DNA fragment from Saccharomyces cerevisiae which contains the SRA5 (=PDE2) gene, coding for a low Km cAMP-phosphodiesterase, was transfected into Chinese hamster ovary cells. Clones carring the cAMP-phosphodiesterase gene were capable of growth in the presence of cholera toxin, which slows the growth of untransfected cells by elevating their cAMP levels. The cholera toxin-resistant transfected cell lines expressed high levels of cAMP-phosphodiesterase mRNA and cAMP-phosphodiesterase activity. Basal intracellular cAMP levels were not significantly affected by the presence of the yeast cAMP-phosphodiesterase gene, but elevation of cAMP levels in response to cholera toxin or prostaglandin E1 was suppressed. Induction of the cAMP-responsive tyrosine aminotransferase promoter by cholera toxin was also blocked in cell lines carrying the yeast cAMP-phosphodiesterase gene. Cholera toxin-resistant transfected cell lines were sensitive to the growth inhibitory effects of N6,02'-dibutyryladenosine 3',5'-monophosphate, which can be used to bypass the effects of the yeast cAMP-phosphodiesterase.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Cellular and biochemical characterization of thioxanthenes for reversal of multidrug resistance in human and murine cell lines.

We have previously shown that phenothiazines sensitize multidrug resistant (MDR) cells to chemotherapeutic drugs in a manner related to specific structural features, and have identified structurally related thioxanthenes with increased anti-MDR activity. We have now studied the structure-activity relationships of 16 thioxanthenes in the human breast cancer line MCF-7 AdrR. trans-Thioxanthene stereoisomers were 2- to 7-fold more potent than cis-thioxanthenes for antagonizing MDR. The most potent thioxanthenes possessed a halogenated tricyclic ring connected by a 3-carbon alkyl bridge to a piperazinyl or piperadinyl side group. The chemosensitizing effects of the lead compound, trans-flupenthixol, its stereoisomer cis-flupenthixol, its phenothiazine homologue fluphenazine, and the calcium channel blocker verapamil, were further studied in a series of sensitive and MDR cell lines. trans-Flupenthixol caused a greater reversal of cellular resistance to doxorubicin, vinblastine, vincristine, and colchicine in MCF-7 AdrR, KB-V1, and P388/DOX MDR cells than the other chemosensitizers. In particular, trans-flupenthixol was 2- to 3-fold more potent for reversing MDR than equimolar concentrations of verapamil. Furthermore, trans-flupenthixol fully reversed resistance to doxorubicin, vincristine, and colchicine in MDR MCF-7 and NIH 3T3 cells transfected with the mdr1 gene. None of these agents altered MDR in a non-P-glycoprotein expressing MCF-7 cell line selected with mitoxantrone, nor in any of the parental cell lines. The stereoselective antagonism of the flupenthixol isomers on several putative cellular targets was studied to explore the mechanism of their chemosensitizing activity. cis- and trans-flupenthixol were equally active inhibitors of protein kinase C and calmodulin. Both cis- and trans-flupenthixol were also potent inhibitors of [3H]azidopine binding to P-glycoprotein. The apparent lack of clinical toxicity of trans-flupenthixol makes it an attractive drug for possible use in the modulation of tumor resistance in vivo if appropriate tissue concentrations can be achieved.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Expression of the human multidrug transporter in insect cells by a recombinant baculovirus.

The plasma membrane associated human multidrug resistance (MDR1) gene product, known as the 170-kDa P-glycoprotein or the multidrug transporter, acts as an ATP-dependent efflux pump for various cytotoxic agents. We expressed recombinant human multidrug transporter in a baculovirus expression system to obtain large quantities and further investigate its structure and mechanism of action. MDR1 cDNA was inserted into the genome of the Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter. Spodoptera frugiperda insect cells synthesized high levels of recombinant multidrug transporter 2-3 days after infection. The transporter was localized by immunocytochemical methods on the external surface of the plasma membranes, in the Golgi apparatus, and within the nuclear envelope. The human multidrug transporter expressed in insect cells is not susceptible to endoglycosidase F treatment and has a lower apparent molecular weight of 140,000, corresponding to the nonglycosylated precursor of its authentic counterpart expressed in multidrug-resistant cells. Labeling experiments showed that the recombinant multidrug transporter is phosphorylated and can be photoaffinity labeled by [3H]-azidopine, presumably at the same two sites as the native protein. Various drugs and reversing agents (e.g., daunomycin greater than verapamil greater than vinblastine approximately vincristine) compete with the [3H]azidopine binding reaction when added in excess, indicating that the recombinant human multidrug transporter expressed in insect cells is functionally similar to its authentic counterpart.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Photosensitized labeling of a functional multidrug transporter in living drug-resistant tumor cells.

A 170,000-Da glycoprotein (P170 multidrug transporter) becomes specifically labeled in multidrug-resistant human KB carcinoma cells by the photolabile lipophilic membrane probe 5-[125I]iodonaphthalene-1-azide ([125I]INA) when photoactivation of the probe is triggered by energy transfer from intracellular doxorubicin or rhodamine 123. In contrast, in drug-sensitive cells, drug-induced specific labeling of membrane proteins with [125I]INA was not observed. Instead, multiple membrane proteins became labeled in a nonspecific manner. This phenomenon of drug-induced specific labeling of P170 by [125I]INA is observed only in living cells, but not in purified membrane vesicles or lysed cells. It is generated by doxorubicin and rhodamine 123, drugs that are chromophores and to which the cells exhibit resistance; but it is not observed with other drugs or dyes. Verapamil, a calcium channel blocker which reverses resistance to doxorubicin, also abolishes doxorubicin-induced specific [125I]INA labeling of P170. These results reveal that a specific interaction between P170 and doxorubicin takes place in living cells and demonstrate that P170 is directly involved in the mechanism of drug resistance in vivo. They also provide a possible means to label functional domains in the multidrug transporter. The results demonstrate that photosensitized [125I]INA labeling is a technique which provides sufficient spatial and time resolution to detect specific intracellular interactions between chromophores and proteins in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Use of recombinant P-glycoprotein fragments to produce antibodies to the multidrug transporter.

Multidrug-resistance of human cancer cells may result from expression of a 170,000 dalton multidrug efflux pump called P-glycoprotein. To identify this multidrug transporter, and to study its structure and function, we have generated polyclonal rabbit antibodies against the amino-terminal and carboxy-terminal halves of the molecule using recombinant protein fragments produced in Escherichia coli. Two recombinant P-glycoprotein fragments, representing amino acids 140-228 and 919-1280, were overproduced in Escherichia coli by an inducible T7 expression system, gel-purified and injected into rabbits. Both antisera specifically immunoprecipitate 3H-azidopine and 35S-methionine labeled P-glycoprotein from multidrug-resistant cells and detect P-glycoprotein on Western blots with high sensitivity. Because these antisera were raised against epitopes in the amino- and carboxy-terminal halves of P-glycoprotein, they should be useful as research tools to define the function of these two halves of the molecule.

ATP Binding Cassette Transporter, Subfamily B, Mem↗