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M M Gottesman

Publications and source records attributed to M M Gottesman.

At least 109 records · Page 6Linked to original sources
PubMed1994

The role of the human genome project in disease prevention.

The Human Genome Project has made it easier to genetically map and clone mutant genes which predispose to a great many human diseases. In addition to diseases inherited in a simple Mendelian fashion, there are many degenerative and infectious diseases, and sensitivities to environmental insults, in which the genetic make-up of an individual contributes to the course of the disease. Once a gene or genes associated with disease has been cloned, it is possible to design DNA-based diagnostics to detect altered forms of the gene which predispose to disease. The ability to predict the development of disease makes possible early intervention to limit the severity of a disease or to use gene therapy to cure inherited disorders.

Disease Susceptibility↗
PubMed1994

Ketoconazole effectively reverses multidrug resistance in highly resistant KB cells.

The antifungal agent ketoconazole was found to overcome resistance to vinblastine and doxorubicin in multidrug resistant KB-V1 cells in vitro. These cells are several hundred-fold more resistant than the parental cell line KB-3-1. Ketoconazole had little or no effect on the parental KB-3-1 cells. The concentrations used to overcome drug resistance in vitro have already been safely used in vivo for treatment of fungal infections and in the monotherapy of hormone independent prostate carcinomas to block adrenal androgen production. Because of a possible beneficial effect of a combination of ketoconazole and a chemotherapeutic drug in multidrug resistant cancers, we examined a panel of 11 prostate carcinoma tissues for the expression of the MDR1 gene by an RNA-PCR assay. MDR1 expression was detectable, albeit at low levels, in 8 of the 11 tumors, suggesting a possible role of this gene in the drug resistance of prostate carcinomas. Our data suggest that ketoconazole might be useful in overcoming multidrug resistance in concentrations that are achievable in humans.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Efficient expression of drug-selectable genes in retroviral vectors under control of an internal ribosome entry site.

We describe a new retroviral vector system pSXLC/pHa that utilizes a putative internal ribosome entry site (IRES) from encephalomyocarditis virus downstream from a multicloning site to co-express drug-selectable markers with a second non-selectable cDNA in a eukaryotic expression vector. The positive drug-selectable marker, MDR1, and the positive-negative marker, herpes simplex virus thymidine kinase (HSV-TK), were successfully introduced and expressed in the pSXLC/pHa system. The pSXLC-MDR and pSXLC-TK vectors contain the drug-selectable genes under translational control of the IRES and multiple cloning sites upstream for insertion of second cDNAs which can be co-expressed in this system. The inserts of these pSXLC plasmids were designed for easy transfer to the pHa retrovirus vector which has a strong promoter from Harvey murine sarcoma virus. The IRES-MDR-carrying retroviral vector, pHa-MCS-IRES-MDR, conferred resistance to vincristine and adriamycin. The IRES-TK-containing vector, pHa-MCS-IRES-TK conferred HAT-resistance in TK-deficient cells and the transfectants showed hypersensitivity to ganciclovir. These "flexible" vectors should be useful for co-expression of genes for selectable gene transfer and for positive-negative (suicide) selections in vitro and in vivo.

3T3 Cells↗
PubMed1994

Interaction of bioactive hydrophobic peptides with the human multidrug transporter.

In this report we demonstrate that various biologically active hydrophobic peptide derivatives, e.g., proteinase inhibitors, chemoattractants, ionophores, enkephalins, and immunosuppressants, stimulate a membrane ATPase activity associated with the human multidrug transporter (MDR1). The stimulation of the MDR1-ATPase by these agents does not correlate with their known biochemical or pharmacological activities but rather with their hydrophobicity. The peptides that show high-affinity interaction with the MDR1-ATPase also interfere strongly with fluorescent dye extrusion catalyzed by the multidrug transporter in intact cells and some have been shown to reverse drug resistance in cultured cells. These data suggest that several hydrophobic peptides behave as substrates of the multidrug transporter and may be used to modulate the chemotherapy resistance of tumor cells.

3T3 Cells↗
PubMed1994

Kinetic evidence suggesting that the multidrug transporter differentially handles influx and efflux of its substrates.

A kinetic approach was used to analyze the mechanism by which a substitution of valine for glycine at position 185 in the multidrug transporter alters its substrate specificity so that colchicine and etoposide transport is increased, daunorubicin transport is unchanged, and vinblastine transport is decreased. Time courses for uptake and efflux of colchicine, vinblastine, etoposide, and daunorubicin for NIH/3T3 mouse cells transfected with wild-type (MDR1-G185) and mutant (MDR1-V185) strains of the human mdr1 gene were determined at room temperature in the presence and absence of an energy supply. The initial rate of vinblastine uptake was reduced approximately 5-fold by glucose feeding of ATP-depleted wild-type (MDR1-G185) cells but was only halved in MDR1-V185 transfectants. In contrast, glucose feeding decreased the initial rate of colchicine uptake approximately 4-fold in the MDR1-V185 (mutant) transfectant but not in the MDR1-G185 (wild-type) transfectant. Efflux of colchicine was accelerated > 5-fold in both the MDR1-V185 (mutant) and MDR1-G185 (wild-type) transfectants when glucose was given to raise ATP levels. The effects on initial rates of colchicine uptake accounted semiquantitatively for the increased colchicine resistance of MDR1-V185 (mutant) transfectants. Similar effects were found for etoposide in the MDR-V185 transfectants. Quinidine in the external medium greatly inhibited drug entry rates but had little effect on efflux, whereas verapamil inhibited both uptake and efflux. A possible interpretation of these data is that the multidrug transporter extracts drugs from the external and internal halves of the membrane bilayer by different paths, which are distinguishable by mutation and inhibitors.

3T3 Cells↗
PubMed1994

Localization of the forskolin labeling sites to both halves of P-glycoprotein: similarity of the sites labeled by forskolin and prazosin.

An iodinated derivative of forskolin, 6-O-[[2-[3-(4-azido-3-[125I] iodophenyl)propionamido]ethyl]carbamyl]forskolin ([125I]6-AIPP-Fsk), photolabels the multidrug efflux pump P-glycoprotein in membranes prepared from the multidrug-resistant cell lines KB-V1 and KB-C1. The labeling site for [125I]6-AIPP-Fsk was localized by immunoprecipitation of tryptic fragments of P-glycoprotein labeled in KB-C1 membranes. A 6-kDa, photolabeled, tryptic fragment was immunoprecipitated by antiserum raised against residues 348-419 of P-glycoprotein, PEPG9, but not by antisera raised against flanking regions PEPG7 and PEPG11. A peptide that corresponds to residues 343-359 of P-glycoprotein inhibited immunoprecipitation of the 6-kDa fragment by antiserum against PEPG9 but had no effect on the immunoprecipitation of photolabeled fragments by antiserum against PEPG7. A second peptide, corresponding to residues 360-376, had no effect on the immunoprecipitation by antiserum against PEPG9. [125I]6-AIPP-Fsk labels the carboxyl-terminal half of P-glycoprotein, because low molecular mass tryptic fragments were immunoprecipitated by three carboxyl-terminal antisera. Therefore, [125I]6-AIPP-Fsk labels both halves of P-glycoprotein, and labeling in the amino-terminal half can be localized to residues 291-359, which span proposed transmembrane regions 5 and 6. KB-V1 membranes photolabeled with [125I]6-AIPP-Fsk and [125I]iodoarylazidoprazosin were digested with either Staphylococcus aureus V8 protease or chymotrypsin and had similar digestion patterns, suggesting that the two drugs label the same sites on P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Transactivation of the human multidrug resistance (MDR1) gene promoter by p53 mutants.

Multidrug resistance in human cancer is associated with overexpression of the MDR1 gene, which encodes a plasma membrane energy-dependent efflux pump termed P-glycoprotein (or the multidrug transporter), which confers cross-resistance to multiple hydrophobic natural product cytotoxic drugs. We have previously shown in cotransfection experiments that activity of the human MDR1 gene promoter is modulated by Ras and p53, suggesting that expression of the MDR1 gene may be associated with the activation of oncogenes and/or functional loss of tumor suppressor genes during oncogenesis. To further characterize the effects of p53 on the MDR1 promoter, we have shown in the current study that the region of the promoter that is required for transactivation by p53 mutants overlaps with the region that is essential for basal promoter activity. In addition, we also have shown that several different p53 mutants transactivate the MDR1 promoter in several different cell types, including embryo fibroblasts derived from the p53-deficient (p53-l-) mice generated by gene targeting.

Animals↗
PubMed1993

Targeted disruption of the mouse mdr1b gene reveals that steroid hormones enhance mdr gene expression.

To evaluate the role of P-glycoprotein in steroid secretion in adrenal cells, we have used gene targeting to introduce a null mutation into one allele of the mdr1b gene in mouse Y1 adrenal cells. Characterization of both the wild-type and the mutant cell lines revealed the following. 1) The expression of mdr1b is enhanced by steroid hormones, in a feedback regulatory mechanism. Inhibition of steroid biosynthesis by 2-aminoglutethimide blocks the adrenocorticotropin (ACTH)-induced increase in mdr1b mRNA levels. 2) ACTH-stimulated steroid secretion is markedly decreased in the mutant cell line. This decreased steroid secretion in the mutant cells occurs despite an increase in the levels of mdr1b mRNA and P-glycoprotein. Kinetic analyses of vinblastine and daunomycin accumulation in both the wild-type and the mutant cell lines during ACTH-stimulated steroidogenesis show that in the mutant cells both drugs accumulated to higher levels than in Y1 cells, suggesting that the remaining mdr1b allele in the mutant cells is relatively inactive as an exporter of steroids, or that the targeted disruption of the mdr1b allele is associated with other changes in the mutant cells which block ACTH-stimulated steroid secretion.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

A human mitochondrial ATP-dependent protease that is highly homologous to bacterial Lon protease.

We have cloned a human ATP-dependent protease that is highly homologous to members of the bacterial Lon protease family. The cloned gene encodes a protein of 963 amino acids with a calculated molecular mass of 106 kDa, slightly higher than that observed by Western blotting the protein from human tissues and cell lines (100 kDa). A single species of mRNA was found for this Lon protease in all human tissues examined. The protease is encoded in the nucleus, and the amino-terminal portion of the protein sequence contains a potential mitochondrial targeting presequence. Immunofluorescence microscopy suggested a predominantly mitochondrial localization for the Lon protease in cultured human cells. A truncated LON gene, in which translation was initiated at Met118 of the coding sequence, was expressed in Escherichia coli and produced a protease that degraded alpha-casein in vitro in an ATP-dependent manner and had other properties similar to E. coli Lon protease.

ATP-Dependent Proteases↗
PubMed1993

Fluorescent cellular indicators are extruded by the multidrug resistance protein.

In this report we show that NIH-3T3 mouse fibroblasts stably expressing the human multidrug transporter (MDR1 or P-glycoprotein), in contrast to the control NIH-3T3 cells, actively extrude the hydrophobic acetoxymethyl ester (AM) derivatives used for cellular loading of various fluorescent calcium and pH indicators. This dye extrusion is blocked by competing substrates and inhibitors of the multidrug transporters, e.g. by verapamil, vincristine, sodium orthovanadate, oligomycin, and a monoclonal anti-MDR1 antibody. The hydrophilic free acid forms of the indicators are not exported by MDR1. We also demonstrate that in isolated cell membranes the MDR1-ATPase, similar to that by known substrates of the transporter, is stimulated by the AM derivatives of fluorescent dyes whereas the free acid forms of the dyes are without effect. Since (i) the AM derivatives of the fluorescent indicators rapidly permeate the cell membrane and are readily cleaved by high activity and large capacity cytoplasmic esterases and (ii) the free acid forms are not substrates for export by MDR1, the observations above suggest that dye extrusion by MDR1 may occur without a cytoplasmic appearance of the AM compounds. These data also call attention to the possible interaction of widely used hydrophobic fluorescent indicators with MDR1 and offer an efficient detection of MDR1-expressing tumor cells as well as a screening method for examining drug interactions with the multidrug transporter.

3T3 Cells↗
PubMed1993

Expression of Chinese hamster cAMP-dependent protein kinase in Escherichia coli results in growth inhibition of bacterial cells: a model system for the rapid screening of mutant type I regulatory subunits.

The regulatory and catalytic subunits of cAMP-dependent protein kinase (PKA) were coexpressed within the same bacterial cell using a polycistronic bacterial T7 expression vector encoding Chinese hamster cDNAs for the type I regulatory (RI) and catalytic alpha (C alpha) subunits of PKA. Basal expression of active RI/C alpha holoenzyme in the BL21(DE3) strain of Escherichia coli caused severe growth inhibition resulting in extremely small colony size. Several lines of evidence demonstrate that this growth inhibition requires active PKA subunits and cAMP: (i) this phenotype is dependent on cAMP since it is not seen in a strain lacking adenylyl cyclase activity, but the growth rate of these transformants is slower when exogenous cAMP is added; (ii) normal growth occurs when wild-type RI cDNA is replaced by a mutant RI cDNA encoding a RI protein with reduced cAMP binding; and (iii) the growth-inhibited phenotype of the transformed BL21(DE3) cells requires soluble, active C alpha protein. Holoenzyme expressed in bacteria is activated by cAMP, which stimulates phosphorylation of an endogenous 50-kDa protein that is missing in four host mutants selected for normal growth after transformation with PKA holoenzyme. A mutant RI cDNA library was generated by PCR random mutagenesis and screened by polycistronic expression in BL21(DE3) cells. The RI cDNA sequence from one revertant has base-pair substitutions creating two amino acid substitutions within the cAMP binding sites. The coexpression of the RI/C alpha subunits in BL21(DE3) bacterial cells provides a system for rapidly selecting mutations in the RI subunits of PKA.

Animals↗
PubMed1993

The effect of ion channel blockers, immunosuppressive agents, and other drugs on the activity of the multi-drug transporter.

The MDRI protein is an energy-dependent transport protein responsible for the multi-drug resistance seen in many tumors. A variety of drugs have been shown to inhibit the function of this pump, including compounds known to block various ion channels. The mouse lymphoma cell line L5178Y has been transduced with the human mdrI gene. Using this cell line, we have tested a number of compounds to determine whether there is a correlation between the ability to block a specific type of ion channel, or shift membrane potential, and the ability to act as an MDR-reversing agent using the fluorescent substrates Rhodamine 123 and daunorubicin as test compounds. Our results show no apparent correlation between the ability to block a specific ion channel and reversal of MDR transport ability. We have found active MDR inhibitors in compounds that affect K+, Na+, Ca++, H+, but not Cl- channels. Our data suggest that Cl- channel activity may be distinct from MDR activity. Several immunosuppressive compounds and analogs were also tested and found to be active reversing agents. Measurements suggest a significant difference in resting membrane potential between the L5178YvMDR line and the L5178Y parental cell line used in these experiments. No correlation was found between the ability of drugs to alter membrane potential and to inhibit MDR transport activity. Our results suggest that MDR transport function may be independent of the physiological movement of ions and show that a wide variety of compounds can inhibit MDR transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Cloning, genomic organization, and chromosomal localization of human cathepsin L.

Cathepsin L is a lysosomal cysteine protease whose expression and secretion is induced by malignant transformation, growth factors, and tumor promoters. Many human tumors express high levels of cathepsin L, which is a broad spectrum protease with potent elastase and collagenase activities. Two published human cathepsin L cDNA sequences differ only in their 5'-untranslated regions. In this study, we demonstrate the concurrent expression of two distinct human cathepsin L mRNAs (hCATL-A and hCATL-B) in adenocarcinoma, hepatoma, and renal cancer cell lines. Cloning of the human cathepsin L gene by polymerase chain reaction amplification of genomic DNA and subsequent sequencing reveals that hCATL-A and hCATL-B mRNAs are encoded by a single gene. The 3' end of the first intron contains the 5' portion of hCATL-B and is contiguous to the second exon of the gene. These data suggest either the possibility of alternative splicing or the presence of a second promoter within the first intron of the hCATL gene. We mapped the hCATL gene to chromosome 9q21-22. Sequencing of both the mouse and human cathepsin L genes demonstrates almost complete conservation of exon and intron position, but significant divergence in intron structure, possibly reflecting differences in regulation of expression of the mouse and human cathepsin L genes.

Amino Acid Sequence↗
PubMed1993

Characterization of an unusual mutant of human melanoma cells resistant to anticancer drugs that inhibit topoisomerase II.

The topoisomerase II inhibitor, VP-16 (etoposide), is an important component in many chemotherapeutic regimens. To characterize resistance to this drug, the human melanoma cell line, FEM-X, was selected in multiple steps with VP-16. To prevent the development of typical multidrug resistance, an inhibitor of P-glycoprotein, the tiapamil analog, RO-11-2933, was added to the selections. The resultant clone FVP3 is 56-fold resistant to VP-16 and cross-resistant to doxorubicin (Adriamycin) (9-fold) and VM-26 (27-fold). These cells are also two- to four-fold resistant to m-AMSA, daunorubicin, and mitoxantrone. FVP3 is not resistant to the P-glycoprotein substrates vinblastine, does not express the MDR1 gene at detectable levels, and does not show reduced 3H-VP-16 accumulation. Unlike other cell lines that exhibit resistance to inhibitors of topoisomerase II, FVP3 has the same level of topoisomerase II expression and activity as FEM-X. Using live cells treated with VP-16, band depletion assays and KCI/SDS precipitation assays show that topoisomerase II from FVP3 is much less susceptible to drug-induced cleavable complex formation than is that from FEM-X. This difference in sensitivity to VP-16 is also detected using lysates from disrupted cells, but not with isolated nuclei devoid of cytoplasmic and membrane components. In addition, the topoisomerase II present in nuclear extracts from FVP3 is not resistant to the effects of VP-16 as measured by: (1) inhibition of strand passing activity during decatenation of kinetoplast DNA, (2) drug-induced linearization of plasmid DNA, and (3) immunodepletion by VP-16. These results suggest that some component of the cytoplasm or cellular membranes, or a factor depleted from nuclei during their isolation, is responsible for the resistance to VP-16 in FVP3.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

P-glycoproteins: mediators of multidrug resistance.

Multidrug resistance represents a major obstacle to successful chemotherapy of metastatic disease. Elevated levels in cancer cells of the product of the multidrug resistance gene, P-glycoprotein or the multidrug transporter, have been associated with the development of simultaneous resistance to a great variety of amphiphilic cytotoxic drugs. P-glycoprotein is an integral plasma membrane protein which contains 12 putative transmembrane regions and two ATP binding sites. It confers multidrug resistance by functioning as an energy-dependent drug efflux pump. Here we describe recent studies on the biosynthesis, structure, function, and mechanism of action of P-glycoprotein which have provided insights into the complexity of this multifunctional transport system and revealed an additional chloride channel activity. The physiological role of P-glycoprotein, however, still remains to be elucidated.

ATP Binding Cassette Transporter, Subfamily B, Mem↗