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M M Gottesman

Publications and source records attributed to M M Gottesman.

At least 181 records · Page 10Linked to original sources
PubMed1990

Heat shock and arsenite increase expression of the multidrug resistance (MDR1) gene in human renal carcinoma cells.

The multidrug transporter, initially identified as a multidrug efflux pump responsible for resistance of cultured cells to natural product cytotoxic drugs, is normally expressed on the apical membranes of excretory epithelial cells in the liver, kidney, and intestine. This localization suggests that the multidrug transporter may have a normal physiological role in transporting cytotoxic compounds or metabolites. In the liver, hepatectomy or treatment with chemical carcinogens increases expression of the MDR1 gene which encodes the multidrug transporter. To evaluate conditions which increase MDR1 gene expression, we have investigated the induction of the MDR1 gene by physical and chemical environmental insults in the renal adenocarcinoma cell line HTB-46. There are two strong heat shock consensus elements in the major MDR1 gene promoter. Exposure of HTB-46 cells to heat shock, sodium arsenite, or cadmium chloride led to a 7- to 8-fold increase in MDR1 mRNA levels. MDR1 RNA levels did not change following glucose starvation or treatment with 2-deoxyglucose and the calcium ionophore A23187, conditions which are known to activate the expression of another family of stress proteins, the glucose-regulated proteins. The levels of the multidrug transporter, P-glycoprotein, as measured by immunoprecipitation, were also increased after heat shock and sodium arsenite treatment. This increase in the level of the multidrug transporter in HTB-46 cells correlated with a transient increase in resistance to vinblastine following heat shock and arsenite treatment. These results suggest that the MDR1 gene is regulatable by environmental stress.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Genomic organization of the human multidrug resistance (MDR1) gene and origin of P-glycoproteins.

The MDR1 gene, responsible for multidrug resistance in human cells, encodes a broad specificity efflux pump (P-glycoprotein). P-glycoprotein consists of two similar halves, each half including a hydrophobic transmembrane region and a nucleotide-binding domain. On the basis of sequence homology between the N-terminal and C-terminal halves of P-glycoprotein, we have previously suggested that this gene arose by duplication of a primordial gene. We have now determined the complete intron/exon structure of the MDR1 gene by direct sequencing of cosmid clones and enzymatic amplification of genomic DNA segments. The MDR1 gene includes 28 introns, 26 of which interrupt the protein-coding sequence. Although both halves of the protein-coding sequence are composed of approximately the same number of exons, only two intron pairs, both within the nucleotide-binding domains, are located at conserved positions in the two halves of the protein. The other introns occur at different locations in the two halves of the protein and in most cases interrupt the coding sequence at different positions relative to the open reading frame. These results suggest that the P-glycoprotein arose by fusion of genes for two related but independently evolved proteins rather than by internal duplication.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Genetic basis of multidrug resistance of tumor cells.

Multidrug resistance in animal cells is defined as the simultaneous resistance to a variety of compounds which appear to be structurally and mechanistically unrelated. One type of multidrug resistance is characterized by the decreased accumulation of hydrophobic natural product drugs, a phenotype which is mediated by an ATP-dependent integral membrane multidrug transporter termed P-glycoprotein or P170. The gene coding for P170 is called MDR. The nucleotide-binding domain of P-glycoprotein shares sequence homology with a family of bacterial permease ATP-binding components. In addition, P170 as a whole is structurally very similar to a number of prokaryotic and eukaryotic proteins believed to be involved in transport activities. This review summarizes our current knowledge of the molecular biology and clinical significance of MDR expression and P-glycoprotein transport activity, as well as some theories about the function of this protein in normal cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Transfection of a mutant regulatory subunit gene of cAMP-dependent protein kinase causes increased drug sensitivity and decreased expression of P-glycoprotein.

Wild-type Chinese hamster ovary (CHO) cells were transfected with a DNA clone (MT-REV, site A) carrying a mouse gene for a dominant mutant regulatory subunit (RI) gene of cAMP-dependent protein kinase (PKA) from S49 cells along with a marker for G418 resistance. G418-resistant transfectant clone R-2D1 was resistant to 8-Br-cAMP-induced growth inhibition and morphological changes. The cells also did not phosphorylate a 50-kDa protein after cAMP stimulation and had decreased PKA activity, both characteristics of PKA mutants. Northern blot analysis indicated that clone R-2D1 was actively transcribing the MT-REV (site A)-specific RNA. We also tested clone R-2D1 for sensitivity to certain natural product hydrophobic drugs and found increased sensitivity to several drugs including adriamycin. Hypersensitivity to these drugs has previously been shown by us to be a characteristic of a CHO PKA mutant cell line. Expression of the mutant RI gene is also associated with a decrease in expression of the multidrug resistance associated P-glycoprotein (gp170) mRNA and protein. These results show that the PKA mutant RI gene from S49 cells acts as a dominant mutation to reduce the total PKA activity in the CHO transfectants as it does in mouse S49 cells. This study also confirms that reduced PKA activity modulates the basal multidrug resistance of these cells, apparently by causing decreased expression of the mdr gene at the protein and mRNA level.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Expression of the multidrug resistance gene in myeloid leukemias.

The human multidrug-resistance gene (MDR1) encodes an energy-dependent multidrug efflux protein responsible for the cross-resistance of cultured cells to natural product chemotherapeutic agents such as the anthracyclines and vinca alkaloids. RNA transcript levels were measured in leukemia cells obtained from 15 adult acute nonlymphocytic leukemia (ANLL) cases and 15 cases of chronic myelogenous leukemia (CML). Expression of MDR1 RNA was common in ANLL, and appears to be most frequent in leukemic cells of patients with the poorest response to chemotherapy. Expression of the MDR1 gene was not detectable in the peripheral white blood cells of any of the CML cases during the chronic phase, but was detectable in the immature cells present during this phase of the disease. The cells of the three blastic crisis patients contained detectable levels of MDR1 RNA. These studies support the idea that expression of the MDR1 gene contributes to drug resistance in ANLL, and may play a role in some instances in the drug-resistance of CML in blastic crisis. In contrast, studies of the level of expression of anionic glutathione transferase and DNA polymerase B failed to show any relationship between the RNA transcript levels of these enzymes and responsiveness to chemotherapy.

Adult↗
PubMed1990

Retroviral transfer of a chimeric multidrug resistance-adenosine deaminase gene.

A fusion between a selectable multidrug resistance (MDR1) cDNA and an adenosine deaminase (ADA) cDNA concomitantly confers multidrug resistance and ADA activity on transfected cells. We have produced a Harvey murine sarcoma virus-derived, replication-defective, recombinant retrovirus to transduce this chimeric MDR-ADA gene efficiently into a great variety of cells. Infection with the MDR-ADA retrovirus conferred the multidrug resistance phenotype on drug-sensitive cells, therefore allowing selection in the presence of colchicine. Colchicine-resistant cells synthesized large amounts of a membrane-associated 210-kDa MDR-ADA fusion protein that preserved both MDR and ADA functional activities. To monitor expression of the chimeric gene in vivo, Kirsten virus-transformed NIH cells were infected with the MDR-ADA retrovirus, and after drug-selection, injected into athymic nude mice. Tumors developed that contained the bifunctionally active MDR-ADA fusion protein. When these mouse tumor cells were placed in tissue culture without the selecting drug, they did not lose the bifunctionally active MDR-ADA fusion protein. The replication-defective, recombinant MDR-ADA retrovirus should be useful to stably introduce the chimeric MDR-ADA gene into a variety of cell types for biological experiments in vitro and in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

In situ localization of the human multidrug-resistance gene mRNA using thymine-thymine dimerized single-stranded cDNA.

In order to detect the mRNA transcribed from the multidrug-resistance gene (MDR1), thymine-thymine (T-T) dimerized single-stranded DNA probes have been utilized for hybridization with mRNA either on nitrocellulose filters or in cells and tissues. S1 nuclease digestion rather than sonication was used to obtain short T-T dimerized single-stranded DNA (300-400 bases) so that they could penetrate well into the cytoplasm. The hybridized T-T DNA was detected immunohistochemically using rabbit anti-T-T DNA antibody (Ab) and peroxidase-labeled goat anti-rabbit IgG Ab. Employing this system, MDR1 mRNA could be localized clearly in the human multidrug-resistant cell lines K562/ADM, CEM/VLB, 2780AD, and KBC4 cells as well as in human fetal kidney and gastric carcinoma. Furthermore, our system successfully detected the expression of MDR1 mRNA in cell lines of increasing resistance. These results paralleled results obtained at the protein level by immunohistochemistry. The analysis of MDR1 RNA expression by this in situ hybridization technique should be useful in the study of normal human tissues and tumor samples expressing the MDR1 gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Activity of the multidrug transporter results in alkalinization of the cytosol: measurement of cytosolic pH by microinjection of a pH-sensitive dye.

Multidrug-resistant cells contain a plasma membrane efflux pump, the multidrug transporter, which actively expels certain hydrophobic drugs from the cytosol to the cell exterior. These drugs are usually positively charged at physiological pH. Because one might predict that this efflux of positively charged molecules might deplete the cytosol of protons, raising the cytosolic pH, we examined the cytosolic pH of multidrug-resistant cells directly using a pH-sensitive dye coupled to a membrane-impermeable molecule. The dye (SNARF), covalently coupled to 10,000 MW dextran, was mechanically microinjected into the cytosol of cultured multidrug-resistant mouse NIH3T3 cells which express the human multidrug transporter. The fluorescence emission of the dye in living cells was measured using epifluorescence microscopy at different wavelengths to provide a measure of the pH of the cytosolic environment. Multidrug-resistant cells had a higher cytosolic pH than drug-sensitive normal parental cells. As the pH of the culture medium was increased, normal cells maintained their cytosolic pH below 7.0, whereas the cytosolic pH of multidrug resistant cells rose. The difference in cytosolic pH between the two cell types was more than 0.2 pH units at an external culture medium pH of 8.2. Treatment with agents that inhibit multidrug transporter-mediated efflux, such as verapamil and vinblastine, essentially eliminated the elevation of cytosolic pH, presumably because they are good substrates for the pump which overwhelm its capacity to pump other materials. These results suggest that the multidrug transporter is indirectly a proton pump, and that cells may contain an endogenous substrate or substrates for this transporter in the absence of added drugs.

Alkalies↗
PubMed1990

Expression of the multidrug resistance, MDR1, gene in neuroblastomas.

Metastatic neuroblastoma is a childhood malignancy that is frequently responsive to chemotherapy with doxorubicin, vincristine, and teniposide (VM26), among other drugs, but in the majority of treated patients, the tumor recurs during or after chemotherapy. In this work, we have examined the hypothesis that the development of resistance to chemotherapy in neuroblastoma might be related to the expression of the human MDR1 gene, which encodes a multidrug transporter that functions as an energy-dependent drug efflux pump. RNA samples from 49 neuroblastomas were analyzed, including 31 from untreated and 18 from treated patients. MDR1 RNA was detectable in the majority of treated and untreated tumors using a sensitive, semiquantitative slot blot assay. Of the samples from treated patients, five of 18 were found to have high MDR1 RNA levels, whereas only three of 31 from untreated patients had high MDR1 levels, a statistically significant difference (P less than .01). These results show that high levels of MDR1 RNA are often associated with resistance to chemotherapy in neuroblastoma and suggest that they may contribute to this resistance. Many of the neuroblastoma samples were also evaluated for N-myc amplification but there was no correlation between N-myc copy number and the level of MDR1 mRNA expression.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1990

Regulation of mdr RNA levels in response to cytotoxic drugs in rodent cells.

The mdr gene, which encodes an energy-dependent multidrug efflux pump termed P-glycoprotein, is expressed in some normal human and rodent tissues, including the adrenal gland, kidney, liver, colon, small intestine, and brain and testis capillary endothelial cells. Because of the important role played by the multidrug transporter in determining sensitivity of normal tissues and resistance of cancers to chemotherapeutic drugs, we and others have been determining the environmental factors which regulate expression of the mdr gene. In previous studies, expression of the human MDR1 gene has been shown to be regulated by heat shock, arsenite, and cadmium in a kidney carcinoma cell line, and mdr RNA is dramatically elevated in rat liver after partial hepatectomy or treatment of the animals with cytotoxic agents. We have now investigated the genetic response of the mdr gene to acute cytotoxic insults in rodent and human tissue culture cells. Following exposure to several drugs, most of which are known to be substrates for the multidrug transporter, mdr RNA levels were found to increase substantially in the rodent cells, but not the human cells. Furthermore, RNA levels for topoisomerase II, an intracellular target for these drugs, decreased in the rodent cells. These results suggest a complex pattern of regulation of mdr RNA levels, depending on animal species and cell type, and possible coordinate regulation with topoisomerase II RNA levels.

Animals↗
PubMed1990

Regulation of the multidrug resistance gene in regenerating rat liver.

The MDR1 gene encodes an Mr 170,000 energy-dependent drug efflux pump (P-glycoprotein) which transports hydrophobic agents such as Adriamycin, colchicine, the Vinca alkaloids, and actinomycin D out of cells. Increased expression of the mdr gene has been observed in preneoplastic and neoplastic carcinogen-induced rat liver nodules as well as in regenerating rat liver, suggesting that the mdr gene is regulated in response to liver injury. To determine whether the increased levels of mdr mRNA seen in regenerating liver are the result of an increased rate of transcription or a posttranscriptional event, nuclear run-on assays were performed on nuclei isolated from regenerating rat livers 4-72 h after partial hepatectomy. Whereas Northern blot analysis of regenerating rat liver demonstrated a greater than 20-fold increase in mdr mRNA levels, there was little or no increase in mdr gene transcription as measured by nuclear run-on analyses. beta-Actin and metallothionein gene transcription levels, known to increase transiently in regenerating liver, both showed increased nuclear run-on activity 4 h posthepatectomy, indicating that the nuclei were functional. Failure to demonstrate a substantial increase in mdr gene transcription suggests that the observed increase in mdr mRNA levels may result from a posttranscriptional event such as message stabilization. The sequence of the 3' noncoding region of the MDR1 gene shares strong homology with other unstable mRNAs, suggesting that RNA stabilization could account for the rise of mdr mRNA after partial hepatectomy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

The role of cathepsin L in malignant transformation.

Procathepsin L is the major excreted protein of malignantly transformed mouse fibroblasts and it is one of the major acid cysteine proteases in mammalian cells. The broad general protease activity of cathepsin L against a variety of extracellular matrix proteins as well as other intracellular protein substrates, and its regulation by factors which stimulate growth, suggest that it contributes to the transformed phenotype. Possible functions in antigen processing, invasiveness, metastasis, and turnover of intracellular and secreted proteins involved in growth regulations have been proposed.

Amino Acid Sequence↗
PubMed1989

A new vector using the human multidrug resistance gene as a selectable marker enables overexpression of foreign genes in eukaryotic cells.

A new vector, pSK1.MDR, has been constructed for expressing nonselectable genes in eukaryotic cells. The vector uses the human multidrug resistance gene, MDR1, as a dominant selectable marker and contains an additional transcription unit plus a unique SalI cloning site for inserting nucleotide sequences to be expressed. To test this expression system, a cDNA (IL2R) for the 55-kDa interleukin-2 receptor was inserted into the SalI site, and the resulting plasmid was transfected into NIH3T3 cells. Cells which acquired the MDR1 gene were selected with colchicine, and cells with high levels of MDR1 expression were selected by growth in increasing concentrations of the drug. Drug resistant cells also expressed the cotransferred, nonselected IL2R gene, and its expression was increased to 740,000 receptors per cell by growing cells in high concentrations of colchicine. The MDR1 system represents a very efficient method for synthesizing large amounts of protein in a wide variety of eukaryotic cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Activity and deletion analysis of recombinant human cathepsin L expressed in Escherichia coli.

A cDNA clone encoding the human cysteine protease cathepsin L was expressed at high levels in Escherichia coli in a T7 expression system. The insoluble recombinant enzyme was solubilized in urea and refolded at alkaline pH. 38-kDa procathepsin L was purified by gel filtration at pH 8.0, and a 29-kDa form of the enzyme was purified by gel filtration after autoprocessing of the proenzyme at pH 6.5. The kinetic properties of the 29-kDa species of recombinant cathepsin L were similar to those published for the human liver enzyme (Mason, R. W., Green, G. D. J., and Barrett, A.J. (1985) Biochem. J. 226, 233-241), using benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide as substrate. However, the stability of the recombinant enzyme, and its pH optimum for this substrate was shifted to a higher pH. Structure-function studies of cathepsin L were performed by constructing mutations in either the propeptide portion or the carboxyl-terminal light chain portion of the protein. These constructions were expressed in the E. coli system, and enzymatic activities were assayed following solubilization, renaturation, and gel filtration chromatography of the mutated proteins. Deletions of increasing size in the propeptide resulted in large proportional losses of activity, indicating that the propeptide is essential for proper enzyme folding and/or processing in this renaturation system. Deletion of part of the light chain containing a disulfide-forming cysteine residue or a single amino acid substitution of alanine for this cysteine residue resulted in almost complete loss of activity. These data suggest that the disulfide bond joining the heavy and light chains of cathepsin L is essential for enzymatic activity.

Amino Acid Sequence↗
PubMed1989

Glycosylation of procathepsin L does not account for species molecular-mass differences and is not required for proteolytic activity.

Cathepsin L is a major lysosomal cysteine proteinase in mouse and human cells. Despite similar predicted molecular masses, procathepsin L in these two species migrates on SDS/polyacrylamide gels with apparent molecular masses of 39 kDa and 42 kDa respectively. To determine if glycosylation differences account for this discrepancy, and to ascertain whether glycosylation is essential for enzymic activity, mouse and human procathepsins L were expressed at high concentrations in mouse NIH 3T3 cells or in human A431 cells after DNA-mediated transfection of cloned DNAs for these enzymes. In pulse-chase studies, human procathepsin L transfectants synthesized and secreted large amounts of enzymically active 42 kDa proenzyme and processed it into 34 kDa and 26 kDa intracellular peptides, a pattern of secretion and processing similar to that seen with endogenous or transfected mouse procathepsin L. Both translation of cloned procathepsin L cDNAs in vitro and Endoglycosidase H treatment of 39 kDa mouse and 42 kDa human procathepsin L resulted in non-glycosylated proteins 2 kDa lower in molecular mass than the untreated proteins for both species. This suggests that glycosylation differences are not responsible for the molecular-mass disparity between the two species. Moreover, Endoglycosidase H-treated mouse enzyme retained full proteolytic activity, indicating that glycosylation of cathepsin L is not essential for enzymic function.

Acetylglucosaminidase↗
PubMed1989

Two different regions of P-glycoprotein [corrected] are photoaffinity-labeled by azidopine.

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Expression of a human multidrug resistance gene in ovarian carcinomas.

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.

Cell Line↗