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Biomedical subjects

M M Gottesman

Publications and source records attributed to M M Gottesman.

At least 289 records · Page 16Linked to original sources
PubMed1982

Genetic evidence that a phorbol ester tumor promoter stimulates ornithine decarboxylase activity by a pathway that is independent of cyclic AMP-dependent protein kinases in CHO cells.

Ornithine decarboxylase (ODC) inductions by cholera toxin and by the phorbol ester tumor promoter, TPA, were compared in wild-type Chinese hamster ovary (CHO) cells and in mutant cells having altered cyclic AMP-dependent protein kinase activity. The aim of these studies was to determine whether cyclic AMP-dependent protein kinase is involved in these inductions. The time course and the magnitude of ODC inductions by either 100 ng/ml cholera toxin or 100 ng/ml TPA were similar in wild-type cells with a maximum at 3-4 hours after treatment and a return to unstimulated levels by 8 hours. Induction of ODC by cholera toxin was suppressed more than 80% in the four protein kinase mutants studied (10215, 10248, 10260, and 10265), strongly implicating a cyclic AMP-dependent kinase step in the mechanism of induction. Similar results were found with the cyclic AMP analog 8-Br-cyclic AMP and the phosphodiesterase inhibitor, methyl-isobutylxanthine. The induction of ODC by TPA, on the other hand, was only partially inhibited (approximately 50%) in three of four mutants. Lower ODC activity in two mutants stimulated by cholera toxin or TPA whose kinetics were studied in more detail could not be ascribed to a reduced affinity (Km) of ornithine for the enzyme, but appeared to be due to reduced catalytic activity (Vmax) in the extracts. These results suggest that the induction of ODC by TPA proceeds by a mechanism which is only partially dependent on an intact cyclic AMP-dependent protein kinase activity.

Animals↗
PubMed1982

Transfer of genes to Chinese hamster ovary cells by DNA-mediated transformation.

We have transferred DNa to Chinese hamster ovary (CHO) cells by DNA-mediated transformation. CHO tk- cells were transformed with the clones gene for herpes simplex virus thymidine kinase (HSV-tk) and were found to have a 50-fold lower frequency of transformation than mouse Ltk- cells at the same DNA dosage. By altering the amount of tk gene and carrier DNA present, frequencies of up to 5 x 10(-5) were obtained. CHO HSV-tk+ transformants were very stable, and in several clones the HSV-tk gene copies integrated in higher-molecular-weight DNA. These cells also exhibited cotransformation for unselected markers. CHO lines were also transformed at a frequency of 10(-4) with the bacterial gene Ecogpt in a SV40-pBR322 vector. CHO tk-cells could be transformed at a frequency of 10(-7) with cellular DNA isolated from CHO tk+ cells. CHO cells offer a well-defined genetic system within which to transfer either cloned or whole cellular DNAs.

Animals↗
PubMed1982

Revertants of a Chinese hamster ovary cell mutant with an altered beta-tubulin: evidence that the altered tubulin confers both colcemid resistance and temperature sensitivity on the cell.

We recently described the isolation of a mutant Chinese hamster ovary cell (Cmd 4) resistant to the cytotoxic effects of colcemid (Cabral et al., Cell 20:29-36, 1980). This mutant carries an altered beta-tubulin but still grows normally at 37 degrees C. In the present study we found that Cmd 4 is temperature sensitive for growth at 40.3 degrees C. A class of revertants selected for temperature resistance had simultaneously lost colcemid resistance and the altered beta-tubulin. In addition, we isolated a temperature-resistant revertant which carries a further alteration in the mutant beta-tubulin polypeptide. This second alteration appears to make the mutant beta-tubulin incompetent to assemble into microtubules, resulting in a strain which is again colcemid sensitive. These revertant cell lines provide strong evidence that a mutation in beta-tubulin can confer both colcemid resistance and temperature sensitivity on a mammalian cell line. Cellular microtubules studied by indirect immunofluorescence in both mutant and revertant cell lines had an apparently normal distribution at permissive and nonpermissive temperatures, yet mitosis appears to be abnormal in the mutant cell line. We conclude from these studies that incorporation of the altered beta-tubulin into microtubules does not affect their distribution but may affect their function during mitosis.

Animals↗
PubMed1981

Glycogen synthase activity in Chinese hamster ovary cells. Studies with wild-type and mutant cells defective in cyclic AMP-dependent protein kinase.

Glycogen synthase (EC 2.4.1.11) activity was studied in cell extracts from wild-type Chinese hamster ovary (CHO) cells and three mutants resistant to cyclic AMP effects on cell shape and cell growth. Based on the capacity of crude extracts to phosphorylate exogenous histone, two of the mutants appeared to have altered cyclic AMP-dependent protein kinase (EC 2.7.1.37) and one of them had apparently normal amounts of kinase activity. Glycogen synthase activity was present in comparable amounts in wild-type and all three mutant strains in a presumably inactive phosphorylated form since activity was virtually completely dependent upon the presence of glucose 6-phosphate. The enzyme could be partially dephosphorylated by endogenous phosphatases and rephosphorylated by exogenous cyclic AMP-dependent protein kinase. Attempts to find culture conditions (e.g. glucose starvation) or cell treatment (e.g. insulin) which might activate glycogen synthase in intact cells were unsuccessful. since glycogen synthase activity present in CHO cells was independent of the level of cyclic AMP-dependent kinase, we conclude that cyclic AMP-dependent protein kinase does not play a critical role in regulating the state of phosphorylation of the synthase.

Animals↗
PubMed1981

Purification and characterization of a transformation-dependent protein secreted by cultured murine fibroblasts.

The major excreted protein (MEP) of transformed mouse fibroblasts has been purified, and monospecific antisera against it have been prepared. Synthesis and secretion of this protein have previously been shown to be stimulated by transformation or treatment with tumor-promoting phorbol esters, but its function is still not known [Gottesman, M. M. (1978) Proc, Natl. Acad. Sci. U.S.A. 75, 2767--2771; Gottesman, M. M., & Sobel, M. E. (1980) Cell (Cambridge, Mass.) 19, 449--455]. The purified protein shows charge heterogeneity by two-dimensional gel electrophoresis; the major intracellular and extracellular species have a molecular weight of 35 000 and a pI of 6.8--7.3. The purified secreted protein contains approximately 5--10% neutral sugar by weight and binds specifically to a concanavalin A--Sepharose affinity column. Translation of messenger ribonucleic acid (mRNA) from cells actively synthesizing MEP in cell-free reticulocyte or wheat germ systems, which are reported to be unable to glycosylate translated proteins, results in a product of Mr 33 000 which is presumably devoid of neutral sugar. However, on two-dimensional electrophoresis, the MEP mRNA translation products continue to show charge heterogeneity similar to that seen in intact cells, suggesting that there may be multiple coordinately controlled mRNAs for MEP or a single mRNA species which can be translated in a variety of ways.

Animals↗
PubMed1981

Intermediate filaments from Chinese hamster ovary cells contain a single protein. Comparison with more complex systems from baby hamster kidney and mouse epidermal cells.

Intermediate filaments (IF) were prepared under identical conditions from Chinese hamster ovary (CHO), baby hamster kidney (BHK), and primary mouse epidermal cells. CHO and BHK cells each have a major IF protein (Mr = 55,000); these proteins comigrate on two-dimensional gels. BHK cells have an additional protein constituent (Mr = 54,000) which is absent from CHO. The IF preparation from the epidermal cells was considerably more complex, containing at least five proteins with molecular weights between 50,000 and 70,000, none of which has a mobility similar to the CHO protein. If assembled in vitro from crude CHO IF preparations were morphologically indistinguishable from filaments assembled from other cell types. Analysis of these in vitro polymerized filaments from CHO cells using circular dichroism and wide angle x-ray diffraction revealed that the CHO filaments were structurally similar to filaments prepared from BHK or epidermal cells. The Mr = 55,000 proteins from CHO and BHK cells were found to have identical one- and two-dimensional peptide maps, demonstrating that their amino acid sequences must be very similar. These studies indicate that CHO cells possess the simplest system thus far described for studying the assembly of intermediate filaments.

Animals↗
PubMed1981

Characterization of cyclic AMP-resistant Chinese hamster ovary cell mutants lacking type I protein kinase.

A group of three mutants of Chinese hamster ovary cells (10260, 10265, and 10223) which are resistant to cyclic AMP (Gottesman, M. M., LeCam, A., Bukowski, M., and Pastan I. (1980) Somatic Cell Genet. 6, 45-61) have been characterized in this work. By genetic analysis, these mutants are all recessive and fall into two complementation groups. Cycl AMP-stimulated protein kinase activity in crude extracts of these mutants using histone as a substrate is decreased to 10 and 7% (complementation group I), and 31% (complementation group II), respectively, of the activity found in wild type extracts. The binding of cyclic [3H]AMP by extracts of all of these mutants is decreased to 30 to 50% of the binding found in wild type extracts. We have used the photoaffinity label 8-azidoadenosine 3':5'-[32P]monophosphate to label the regulatory subunits of type I and type II protein kinase in wild type and mutant extracts analyzed by DEAE-cellulose and Sephadex chromatography. We find that all three mutants lack type I cyclic AMP-dependent protein kinase and have reduced amounts of type II kinase activity. The regulatory subunits of type I and type II kinase are present in both complementation groups. We conclude that type I protein kinase is not needed for normal growth of Chinese hamster ovary cells. The defect in both classes of mutants appears to be in the failure of the catalytic subunit to associate normally with its regulatory subunits.

Affinity Labels↗
PubMed1981

In vitro assembly of homopolymer and copolymer filaments from intermediate filament subunits of muscle and fibroblastic cells.

This paper presents evidence that the intermediate filament (IF) subunits of muscle cells (skeletin or desmin) and fibroblastic cells (decamin or vimentin) separately form homopolymer IF in vitro and, when mixed, prefer to form copolymer IF in vitro. Because they coexist in cells, they may also form copolymers in vivo. The IFs of baby hamster kidney fibroblasts (BHK-21) consist of a major subunit, decamin, and two minor subunits which, on the basis of two-dimensional gel and peptide mapping criteria, are identical to the alpha and beta subunits of smooth muscle desmin. The subunits differ only in their degrees of phosphorylation: alpha desmin contained 2 mol/mol of O-phosphoserine whereas beta desmin contained none. The decamin and desmin subunits assembled into homopolymer IF in vitro in high yield from purified denatured subunits under identical conditions of pH and ionic strength. However, homopolymer decamin IF disassembled into soluble protofilaments in solutions of ionic strength less than 0.05 mol/liter whereas homopolymer desmin IF disassembled at ionic strength less than 0.03 mol/liter. When decamin and desmin were mixed together as denatured subunits or as soluble protofilaments, the IF assembled in vitro had solubility properties intermediate between those of the homopolymer IFs, indicating that the two subunits had formed copolymer IF. The stoichiometry of copolymerization as determined in mixtures in which one subunit was present in excess was suggestive of the formation of three-chain units. The possibility of nonspecific aggregation was eliminated by isolation of stable three-chain alpha-helix-enriched particles from such IF. When tracer amounts of [35S]methionine-labeled decamin were mixed with desmin, labeled IFs were obtained under conditions in which homopolymer decamin IFs were soluble. These in vitro findings may be of physiological significance because native BHK-21 IF also had solubility properties similar to those of the copolymer IF.

Animals↗
PubMed1981

Isolation of a taxol-resistant Chinese hamster ovary cell mutant that has an alteration in alpha-tubulin.

Taxol is a plant alkaloid that has antimitotic activity and appears to stabilize microtubules [Schiff, P. B., Fant, J. & Horwitz, S. B. (1979) Nature (London) 277, 665-667]. Taxol-resistant cells were selected from a population of UV-mutagen-treated Chinese hamster ovary cells by a single-step procedure. These mutants have normal morphologies and growth rates but are 2- to 3-fold more resistant to the toxic effects of the drug than the wild-type parent. One out of 20 mutants screened by two-dimensional electrophoresis for chemical alterations in tubulin had an "extra" spot with a more acidic isoelectric point that alpha-tubulin. This extra spot was shown to be an electrophoretic variant alpha-tubulin by its copurification with tubulin in crude microtubule-containing preparations and by one-dimensional peptide mapping. The alpha-tubulin mutant was found to be temperature sensitive for growth, and this property was used as the basis for the selection of revertants. Seventeen temperature-resistant revertants of the alpha-tubulin mutant were selected for their ability to grow at 40 degrees C and three of these revertants were found to have simultaneously lost their taxol resistance and the electrophoretic variant alpha-tubulin. These results provide evidence that an alteration in alpha-tubulin can confer taxon resistance on a mammalian cell line and suggest that alpha-tubulin is essential for cell viability.

Alkaloids↗
PubMed1981

Tumor promoters induce the synthesis of a secreted glycoprotein in mouse skin and cultured primary mouse epidermal cells.

The effect of tumor promoters and anti-promoters on the synthesis and secretion of a 35,000 mol wt transformation-dependent secreted glycoprotein (MEP) by primary mouse epidermal cells has been investigated. MEP is synthesized and secreted at low levels by cultured epidermal basal cells. The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) induces a 3-fold increase in the synthesis of MEP as determined by quantitative immunoprecipitation from pulse-labeled cell extracts. This increase is maximal 6 h after treatment and parallels an increase in secretion of MEP measured by appearance of radiolabeled protein in the medium or by radioimmunoassay of culture supernatants. An increase in MEP synthesis is also observed in the skin of mice exposed to TPA. The phorbol ester antagonists retinoic acid, lidocaine and fluocinolone acetonide were examined to determine their effect on the synthesis and secretion of MEP. Of these agents, only fluocinolone acetonide affects MEP levels by suppressing the basal level of MEP synthesis but not its relative induction by TPA. These data suggest that the modifying effects of these three agents on tumor promotion are not mediated via an alteration in MEP synthesis per se is not sufficient for promotion.

Animals↗
PubMed1981

Induction of specific protein synthesis by phorbol esters in mouse epidermal cell culture.

The induction of specific protein synthesis by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), was studied in cultured mouse epidermal cells by two-dimensional gel electrophoresis after pulse-labeling with [35S]methionine. While overall protein synthesis was moderately inhibited by TPA, the synthesis of five specific proteins was increased. Three of these proteins (M.W. 25,000, M.W. 55,000, and M.W. 70,000) were either not synthesized or synthesized at low rates in untreated cells, while two proteins (M.W. 35,000 and M.W. 50,000) were also synthesized in controls but to a lesser extent than in TPA-treated cells. Increased synthesis of the M.W. 50,000 protein could be seen as early as 1 hr after TPA exposure, while maximum induction of all proteins was observed at 6 hr. By 24 hr, synthesis rates of these proteins had returned to near basal levels even if TPA exposure was continued. The amino acid analog canavanine (at levels which inhibited protein synthesis to the same extent as does TPA) or nonpromoting analogs of TPA did not induce these proteins. Pulse-chase studies indicated that these proteins were not degradation products which may have resulted from TPA exposure and that the M.W. 25,000 and M.W. 35,000 protein appear to be rapidly turned over. 32PO4 labeling indicated that only the M.W. 55,000 protein was significantly phosphorylated and that TPA did not induce a qualitative change in the pattern of phosphorylation of epidermal proteins. Definitive identification of these proteins has not been made, but the specific stimulation of their synthesis supports a model of tumor promoter action in which promoters induce a specific program of changes in macromolecular synthesis in the epidermis.

Animals↗
PubMed1980

Mechanism of cyclic AMP effect on nutrient transport in Chinese hamster ovary cells. A genetic approach.

The effect of 8-bromo cyclic adenosine 3':5'-monophosphate (8-Br-cAMP) on sugar and amino acid transport was investigated in wild-type Chinese hamster ovary (CHO) cells and in mutants selected for resistance to cAMP inhibition of cell growth. In wild type cells, both 3-O-methyl-D-glucose and alpha-aminoisobutyric acid transport were decreased in cells treated for 24 h with 8-Br-cAMP; kinetic analysis indicated that a decrease in Vmax, without a significant change in Km, accounted for the lower transport capacity of 8-Br-cAMP treated cells. Among the different transport systems contributing to amino acid entry, "alanine" preferring transport system (system A) appeared to be specifically affected. The sensitivity of transport processes to 8-Br-cAMP was tested in three cAMP-resistant cell lines. When tested for their capacity to phosphorylate histones in crude extracts, one strain had apparently normal amounts of protein kinase activity, one strain had a decreased enzyme sensitivity to cAMP, and one strain had little or no enzyme activity. In all three mutants, no effect of 8-Br-cAMP on 3-O-methyl glucose and alpha-aminoisobutyric acid transport could be observed, regardless of the level of cAMP-dependent protein kinase activity. These data do not indicate whether the effect of cAMP on nutrient transport in CHO cells is the cause or consequence of growth inhibition. However, they support the conclusion that, in CHO cells, the presence of a normally functioning cAMP-dependent protein kinase appears to be necessary but may not be sufficient to observe the effects of cAMP on nutrient transport as well as cell shape and cell growth.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1980

Regulation of transglutaminase activity in Chinese hamster ovary cells.

We have investigated the regulation of transglutaminase activity (epsilon-(gamma-glutamyl)lysine crosslinking enzyme) in Chinese hamster ovary cells in culture. We report that transglutaminase activity increases several-fold in CHO cells at maximum density in suspension culture. This increase cannot be explained by the presence of soluble regulators of the enzyme activity or the appearance of a new enzyme activity with a different affinity for substrate, but appears to be due to an increase in total enzyme activity. Treatment of CHO cells at low cell density with 8-bromo cyclic AMP results in a small increase (20--70%) in transglutaminase activity. By studying CHO mutants which have altered or absent cyclic-AMP-dependent protein kinases, we have demonstrated that the effect of cyclic AMP on transglutaminase activity at low cell density is mediated by cyclic-AMP-dependent protein kinase. However, the protein kinase mutants show normal increases in transglutaminase activity at high cell density, indicating that cyclic AMP-dependent protein kinase does not mediate density-dependent changes in transglutaminase activity.

Animals↗
PubMed1980

Sodium butyrate affects expression of fibronectin on CHO cells: specific increase in antibody-complement-mediated cytotoxicity.

Cellular fibronectin is expressed only at very low levels on the surface of the spontaneously transformed Chinese Hamster Ovary (CHO) cell line, as detected by immunofluorescence studies and confirmed by resistance of CHO cells to complement-mediated lysis in the presence of anti-fibronectin antibody. Treatment of CHO cells with sodium butyrate results in a marked susceptibility to anti-fibronectin complement-mediated killing. Using selected complement-containing sera from rabbits, it is possible to demonstrate that killing of butyrate-treated CHO cells is absolutely and specifically dependent on the presence of antibody to fibronectin. The increased susceptibility of butyrate-treated cells to complement-mediated killing correlates with an increase in cell surface fibronectin detected by immunofluorescence studies. This increased antigen expression should allow the isolation of mutants with altered regulation of fibronectin expression in a cell line of proven value for somatic cell genetic studies.

Animals↗
PubMed1980

Isolation of multiple classes of mutants of CHO cells resistant to cyclic AMP.

From mutagenized Chinese hamster ovary (CHO) cells we have isolated, in a single step, 11 independent mutants resistant to the growth-inhibitory effects of 8-Br-cyclic AMP, cholera toxin, and methylisobutylxanthine. Two major classes and several subclasses of mutants were obtained. Mutants from all classes have a normal doubling time. None of the mutants respond to cyclic AMP treatment with increased flattening and elongation as do the parental cells. Members of the first class have an altered protein kinase activity which has either an increased Ka for cyclic AMP or an absent response to cyclic AMP. Most of those mutations which result in a protein kinase with increased Ka for cyclic AMP (6/11) are dominant in somatic cell hybrids. Those mutations which result in a protein kinase with little or no response to cyclic AMP (3/11) are recessive. Members of the second major class (2/11) have normal levels of basal and cyclic AMP-dependent protein kinase activity. One is recessive and one is dominant by genetic tests. The basis for the defect in this second class of mutants has not been determined.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1980

CHO mutants resistant to colchicine, colcemid or griseofulvin have an altered beta-tubulin.

Single-step mutants of Chinese hamster ovary (CHO) cells have been isolated which are resistant to killing by the anti-mitotic drugs colchicine, colcemid or griseofulvin. Two-dimensional gel analysis showed that two mutants resistant to griseofulvin, one resistant to colcemid and one resistant to colchicine carry an alteration in the beta-tubulin subunit. Most of the remaining isolates are believed to be permeability mutants on the basis of their cross resistance to drugs which do not interefere with microtubular polymerization or function (Ling and Thompson, 1974; Bech-Hansen, Till and Ling, 1976). A reduced amount of the wild-type beta-tubulin protein remained in each of the beta-tubulin mutants, but a beta-tubulin protein with a more basic isoelectric point also appeared. Messenger RNAs coding for both wild-type and variant beta-tubulins were found in at least one mutant as assayed by in vitro translation in a reticulocyte lysate. This indicates that the altered tubulin does not arise as the result of a posttranslational modification.

Animals↗