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Biomedical subjects

M M Gottesman

Publications and source records attributed to M M Gottesman.

At least 217 records · Page 12Linked to original sources
PubMed1988

In situ hybridization analysis of acquisition and loss of the human multidrug-resistance gene.

The extent of multidrug-resistance of human KB carcinoma cell lines has been shown to be proportional to the level of expression of the MDR1 gene. Using an in situ hybridization analysis with 35S-labeled RNA probes, we have found that there is some heterogeneity in expression of the MDR1 gene from cell to cell, but that the average level of expression is proportional to the resistance of the cell line. In the absence of selective pressure, a colchicine-selected multidrug-resistant population with a highly amplified MDR1 gene loses its resistance in parallel with the loss of the amplified gene. Loss of resistance also parallels a decrease in MDR1 RNA expression in the whole cell population. Loss of MDR1 expression in this population is highly heterogeneous, with small clusters of cells maintaining expression even after the population as a whole has become relatively sensitive. This heterogenous loss of expression of the MDR1 gene is consistent with random segregation of amplified DNA segments in the selected cells. The analysis of MDR1 RNA expression by in situ hybridization which is validated by this study should be useful in the study of normal human tissue and tumor samples expressing the MDR1 gene.

Carcinoma↗
PubMed1988

Isolation and sequence of a cDNA for human pro-(cathepsin L).

The major excreted protein (MEP) of malignantly transformed mouse fibroblasts is the precursor to an acid proteinase with enzymic specificity similar to that of human cathepsin L. By cross-hybridization with a mouse MEP sequence, cDNA clones of the human form of MEP in an SV40 expression vector were isolated. A 1.6 kb cDNA showed 70% deduced amino acid sequence identity with mouse MEP. The deduced amino acid sequence of the cloned human MEP was the same, except for two amino acids, as the N-terminal sequence of mature human cathepsin L, thereby establishing that human MEP is human pro-(cathepsin L). Use of this human pro-(cathepsin L) cDNA clone allowed the detection of a 1.6-1.8 kb pro-(cathepsin L) mRNA in human cells which was not detected with a mouse pro-(cathepsin L) probe.

Amino Acid Sequence↗
PubMed1988

Cloning and expression of the gene for the major excreted protein of transformed mouse fibroblasts. A secreted lysosomal protease regulated by transformation.

The major excreted protein (MEP) of mouse fibroblast cells is the 39,000 Mr precursor to a lysosomal acid protease (cathepsin L) induced by malignant transformation, growth factors, and tumor promoters. We have cloned and characterized the gene for MEP from NIH-3T3 cells. This cosmid clone (pcosMMEP), containing the unique 12,000-base pair mouse MEP gene, has been transfected into monkey kidney (CV-1) cells and human epidermoid carcinoma (A431) cells. The stable A4MEP transfectants produce mouse MEP that is an active cathepsin which is secreted, glycosylated, and processed intracellularly to lower molecular weight forms as in the wild-type NIH-3T3 cells. The CVMEP cells (nontransformed phenotype) produce quantities of mouse MEP similar to that found in NIH-3T3 cells, whereas the A4MEP cells (transformed phenotype) produce greater amounts of MEP similar to the levels seen in Kirsten virus-transformed NIH-3T3 cells. The MEP mRNAs from both mouse cells and stably transfected human cells are the same size and have the same single major site for initiation of transcription, indicating that the cloned mouse MEP promoter is active in transfected cells.

Animals↗
PubMed1988

Epidermal growth factor-dependent growth of human KB cells in a defined medium and altered growth factor requirements of KB mutants resistant to EGF-Pseudomonas exotoxin conjugates.

A serum-free culture system was established for human KB carcinoma (HeLa) cells that consisted of a chemically defined medium and several growth factors including epidermal growth factor (EGF), insulin, transferrin, hydrocortisone, and ethanolamine. EGF and insulin showed the greatest effects on the growth rate of KB cells. Insulin-like growth factor I (IGF-I) at the same concentration as insulin stimulated cell growth less than insulin. Transferrin, hydrocortisone, or ethanolamine had no growth-stimulatory effects alone but were stimulatory when combined with EGF and/or insulin. Transforming growth factor-beta inhibited growth and triiodothyronine stimulated growth. The growth factor requirements were established for several KB mutants with low EGF receptor levels that had been selected for resistance to a conjugate of EGF with Pseudomonas exotoxin (EGF-PE). Three of five KB mutants did not respond to EGF; two other mutants responded to a lesser extent than the parental KB cells. Four mutants had a reduced response to insulin and responded to T3; one mutant (ET-30) responded to neither. These results indicate that KB cells selected for EGF-PE resistance have lost their growth response to EGF and illustrate the usefulness of serum-free medium for studying the growth factor requirements of mutants with altered receptor levels.

ADP Ribose Transferases↗
PubMed1988

Genetic characterization of human KB cell lines resistant to epidermal growth factor: Pseudomonas exotoxin conjugates.

Pleiotropic human KB cell mutants, selected for resistance to a conjugate of epidermal growth factor with Pseudomonas exotoxin (PE-EGF), were characterized genetically. These mutants have a pleiotropic phenotype, which includes reduced number of EGF receptors and reduced growth rate. Hybrid cells between HeLa D98 and four out of five of these resistant cell lines were more resistant to PE-EGF than hybrids formed between HeLa D98 and parental KB cells. This result indicates that the phenotype of PE-EGF resistance is incompletely dominant in four out of five cases and recessive in one out of five variants. In three separate experiments, transfection of DNA from two of the dominant resistant cell lines resulted in transformation of wild-type KB cells to PE-EGF resistance, confirming the dominant nature of these mutations, which affect levels of EGF receptor in KB cells.

ADP Ribose Transferases↗
PubMed1988

Measurement of multidrug-resistance messenger RNA in urogenital cancers; elevated expression in renal cell carcinoma is associated with intrinsic drug resistance.

We measured the levels of messenger RNA of the human multidrug-resistance gene (MDR1) in 25 urogenital tumors before chemotherapy. Many of the renal cell carcinomas continued to express MDR1 gene at high levels, reflecting the increased expression of MDR1 RNA in normal kidneys. In other urogenital tumors, the MDR1 RNA levels were low reflecting low MDR1 RNA levels in normal bladder, prostate and testis. For comparative purposes, we performed in vitro chemosensitivity testing on many tumor samples using soft agar culture techniques. Vinblastine sensitivity in vitro inversely correlated with MDR1 RNA levels (p less than 0.01). Moreover, mean sensitivity of seven renal cell carcinomas to vinblastin was significantly lower than that of the other seven cancers (p less than 0.05). As for doxorubicin, mean sensitivity of six renal cell carcinomas was lower than the others (p less than 0.1). These results suggest that the high MDR1 RNA levels in renal cell carcinomas are associated with intrinsic multidrug-resistance.

Carcinoma, Renal Cell↗
PubMed1988

ATP-dependent transport of vinblastine in vesicles from human multidrug-resistant cells.

Resistance of human cancer cells to multiple cytotoxic hydrophobic agents (multidrug resistance) is due to overexpression of the "MDR1" gene, whose product is the plasma membrane P-glycoprotein. Plasma membrane vesicles partially purified from multidrug-resistant human KB carcinoma cells, but not from drug-sensitive cells, accumulate [3H]vinblastine in an ATP-dependent manner. This transport is osmotically sensitive, with an apparent Km of 38 microM for ATP and of approximately equal to 2 microM for vinblastine. The nonhydrolyzable analog adenosine 5'-[beta, gamma-imido]triphosphate does not substitute for ATP but is a competitive inhibitor of ATP for the transport process. Vanadate, an ATPase inhibitor, is a potent noncompetitive inhibitor of transport. These results indicate that hydrolysis of ATP is probably required for active transport of vinblastine. Several other drugs to which multidrug-resistant cell lines are resistant inhibit transport, with relative potencies as follows: vincristine greater than actinomycin D greater than daunomycin greater than colchicine = puromycin. Verapamil and quinidine, which reverse the multidrug-resistance phenotype, are good inhibitors of the transport process. These results confirm that multidrug-resistant cells express an energy-dependent plasma membrane transporter for hydrophobic drugs, and establish a system for the detailed biochemical analysis of this transport process.

Adenosine Triphosphate↗
PubMed1988

A retrovirus carrying an MDR1 cDNA confers multidrug resistance and polarized expression of P-glycoprotein in MDCK cells.

A full-length cDNA for the human multidrug resistance gene 1 (MDR1) has been inserted into a retroviral vector containing a murine Harvey sarcoma virus from which the viral oncogene was deleted. Ecotropic and amphotropic virus was produced after transfection of this vector into psi-2 and PA-12 packaging cell lines. This virus conferred the full phenotype of multidrug resistance on mouse and human cell lines. Viral titers of up to 2 X 10(5) drug-resistant colonies per ml were observed. Infected cells became resistant to colchicine, vinblastine, doxorubicin, VP16 (etoposide), and puromycin, but not cisplatin, indicating that the presence of the human MDR1 gene is sufficient to cause multidrug resistance. When the dog kidney cell line MDCK was infected with the MDR1 virus, P-glycoprotein was expressed in a polarized manner on the upper surface of the cells, showing that the cloned cDNA also encodes information for polarized expression of P-glycoprotein. The MDR1 virus should be useful for introducing this drug resistance gene into a variety of cell types for biological experiments in vitro and in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

An acid protease secreted by transformed cells interferes with antigen processing.

The major excreted protein of malignantly transformed mouse fibroblasts (MEP), which is the precursor to lysosomal cathepsin L, was used to study the effect of exogenous acid proteases on antigen processing. When MEP and native pigeon cytochrome c were added to Chinese hamster ovary (CHO) cells expressing transfected major histocompatability complex class II gene products, the antigen-specific T-cell hybridoma 2B4 did not respond to the antigen. MEP appears to destroy the antigen in an acid compartment of the presenting cell because: (a) MEP is only active as a protease under acid conditions; (b) mannose 6-phosphate inhibited the internalization of MEP and blocked its effect on antigen processing; (c) the destruction required the simultaneous entry of the antigen and MEP into the cells; and (d) cytochrome c fragment 66-104 which does not need to be processed stimulated 2B4 in the presence of MEP. These results support the hypothesis that antigen processing requires internalization of the antigen into an acidic compartment, and they provide a new model for the investigation of the contribution of acid proteases to the reduced immunocompetence of tumor-bearing animals.

Animals↗
PubMed1988

Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease.

Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L). To evaluate the possible role of this protease in the transformed phenotype, we transfected cloned genes for mouse or human MEP into mouse NIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene. The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine. The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells. The MEP synthesized by cells transfected with the cloned mouse and human MEP genes was also secreted. Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.

Animals↗
PubMed1988

Most drugs that reverse multidrug resistance also inhibit photoaffinity labeling of P-glycoprotein by a vinblastine analog.

Multidrug-resistant human KB carcinoma cells express a 170,000-dalton membrane glycoprotein (P-glycoprotein) that can be photoaffinity labeled with the vinblastine analog N-(p-azido-[3-125I]salicyl]-N'-(beta-aminoethyl)vindesine. Several agents that suppress the multidrug-resistant phenotype, including N-solanesyl-N,N'-bis(3,4-dimethylbenzyl)ethylenediamine, cepharanthine, quinidine, and reserpine, were found to inhibit photolabeling of P-glycoprotein at doses comparable to those that reverse multidrug resistance. However, the phenothiazines chlorpromazine and trifluoperazine, which also effectively reverse multidrug resistance, were poor inhibitors of the photoaffinity labeling of P-glycoprotein. Chloroquine, propranolol, or atropine, which only partially reversed the drug resistance, also did not inhibit photolabeling. Naphthalene sulfonamide calmodulin inhibitors, W7 and W5, as well as many other drugs that did not circumvent multidrug resistance, did not inhibit photolabeling. These studies suggest that most, but not all, agents that phenotypically suppress multidrug resistance also inhibit drug binding to a site on P-glycoprotein with which a photoaffinity analog of vinblastine interacts.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1987

Isolation and sequence of the promoter region of the human multidrug-resistance (P-glycoprotein) gene.

Intrinsic and acquired multidrug resistance is an important problem in cancer therapy. Multidrug resistance results from overexpression of the MDR 1 gene, which encodes a drug-efflux pump called P-glycoprotein. We have isolated a 1-kilobase genomic fragment containing the major transcription initiation sites for the human MDR 1 gene. Ribonuclease protection experiments using this fragment indicate that normal human adrenal, colon, and liver cells, the human hepatoma cell line HepG2, and vinblastine-selected human KB multidrug-resistant cells initiate transcription of the MDR 1 gene at the same site within this fragment. The 0.43-kilobase region upstream from the major transcription initiation site linked to the chloramphenicol acetyltransferase gene showed promoter activity in CV-1 monkey kidney cells and in human KB cells. The putative promoter region has a consensus CAAT box and two GC box-like sequences, but no TATA sequence. This identification and isolation of promoter sequences for the MDR 1 gene will permit studies on how expression of this gene is regulated in normal human tissues and cancers.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1987

The identification of the major excreted protein (MEP) from a transformed mouse fibroblast cell line as a catalytically active precursor form of cathepsin L.

The major excreted protein (MEP) purified from Kirsten-virus-transformed 3T3 fibroblasts and mature human cathepsin L were compared in respect to a number of catalytic criteria and found to be similar. The Mr of MEP is 39,000, whereas that of mature human cathepsin L is 30,000. Sequence data suggested that MEP could be a pro-form of mouse cathepsin L. Both enzymes acted on the synthetic substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide with similar catalytic constants and acted optimally at pH 5.5. Both were rapidly inactivated by the active-site-directed inhibitors benzyloxycarbonyl-Phe-Phe-diazomethane and L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane, and furthermore, 3H-labelled L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-acetamid o)butane, which binds covalently to the heavy chain of mature cathepsin L, also bound to MEP. MEP autolyses rapidly at pH 3.0 to give lower-Mr (35,000 and 30,000) forms, but all forms react with the radiolabelled inhibitor. No autolysis occurred above pH 5.0. MEP hydrolysed azocasein at pH 5.0, demonstrating that it is capable of hydrolysing protein substrates without autolytic activation. Unlike mature forms of cathepsin L, MEP is stable, but not active, at neutral pH. The present work shows that cathepsin L can be secreted as a higher-Mr precursor that is stable in extracellular fluids but only active where local pH values fall below 6.0. These results suggest that the extra N-terminal peptide on MEP is not an activation peptide, but is a regulatory peptide affecting the pH-stability and activity of mouse cathepsin L.

Binding Sites↗
PubMed1987

Sequence and expression of the cDNA for MEP (major excreted protein), a transformation-regulated secreted cathepsin.

The major excreted protein (MEP) of malignantly transformed mouse fibroblasts is a secreted thiol proteinase. Sequencing of the MEP cDNA shows the coding region for the protein to be identical with the sequence for a mouse cysteine proteinase isolated from macrophages, but the MEP cDNA is polyadenylated at a different site in the 3' non-coding region. Strong homology of MEP with human cathepsin L suggests that MEP is the mouse analogue of cathepsin L. Amino acid sequencing of the N-terminus of the secreted form of MEP indicates that, during secretion, the polypeptide is cleaved between amino acids 17 and 18. We have placed the MEP cDNA in a eukaryotic expression vector and demonstrated the production of the 39 kDa polypeptide form of mouse MEP in monkey CV-1 cells.

Amino Acid Sequence↗
PubMed1987

Isolation of human KB cell lines resistant to epidermal growth factor-Pseudomonas exotoxin conjugates.

Mutants of the human KB carcinoma cell line resistant to a cytotoxic conjugate of epidermal growth factor (EGF) and Pseudomonas exotoxin (PE) were selected. EGF-PE and the drug verapamil, which enhanced EGF-PE cytotoxicity, were used in the selection process. These mutants also showed some cross-resistance to PE. All of the EGF-PE resistant variants displayed lower levels of 125I-EGF binding, 20-50% of parental KB levels, without altered affinity for EGF and grew at a slower rate than the parental cell line KB-3-1. These results indicate that EGF-PE resistant KB cells have a complex phenotype which includes a reduction in the number of EGF receptors and reduced sensitivity to unconjugated PE. Resistance to toxin-conjugates, although pleiotropic, is specific and does not lead to resistance to multiple other anticancer drugs, nor are independently selected multidrug resistant KB lines resistant to PE. These results argue that protocols for cancer treatment could effectively use specifically designed cytotoxic toxin conjugates as an adjunct to conventional chemotherapy.

Bacterial Toxins↗