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G M Ringold

Publications and source records attributed to G M Ringold.

68 records · Page 4Linked to original sources
PubMed1981

Selection of rat hepatoma cells defective in hormone-regulated production of mouse mammary tumor virus RNA.

We have been studying the mechanism of glucocorticoid hormone action by using mouse mammary tumor virus (MMTV)-infected rat hepatoma cells as a model system. J2.17, a clonal cell line that contains one MMTV provirus, induces tyrosine aminotransferase (TyrATase; L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5), viral RNA, and the cell surface viral glycoprotein gp52 in response to dexamethasone. Using a fluorescence-activated cell sorter and a rabbit antiserum directed against gp52, we selected a cell population that displays a reduced hormone-mediated increase in cell surface gp52. Fourteen clones of this population were assayed for induction of viral gp52 and RNA and of cellular TyrATase. The results of these assays revealed that the clones display a variety of responses to hormone. One clone has retained wild-type responses of both TyrATase and gp52. Six clones exhibit coordinately reduced or abolished responses of both markers. Seven clones show normal induction of TyrATase but reduced or undetectable induction of gp52. These latter clones exhibit reduced production of MMTV RNA and thus may represent a unique class of variants defective in the regulation of MMTV gene expression.

Animals↗
PubMed1980

Isolation of glucocorticoid-unresponsive rat hepatoma cells by fluorescence-activated cell sorting.

We have used a mouse mammary tumor virus (MMTV)-infected rat hepatoma cell line as a model system for studying glucocorticoid action. These cells induce tyrosine aminotransferase and MMTV in response to the synthetic glucocorticoid, dexamethasone. The major viral antigen, a glycoprotein of 52,000 daltons (gp52), appears on the surface of infected cells in amounts which reflect the cytoplasmic content of viral RNA. Using an anti-gp52 antiserum and a fluorescence-activated cell sorter (FACS), we have selected variants which display low levels of pg52 in the presence of the hormone. Multiple cycles of enrichment for cells that fluoresce weakly in the presence of hormone have generated a population which fails to produce a detectable increase in cell surface gp52 in response to dexamethasone. This population of nonresponders and a number of independent clones derived from this population were analyzed for their ability to induce gp52 and TAT and for these presence of glucocorticoid receptors. All nonresponder clones exhibited little or no induction of either glucocorticoid-inducible marker. Two of the clones contained reduced levels of glucocrticoid receptor, while the remainder of the clones showed no detectable specific hormone binding. These results provide genetic evidence that a single class of glucocorticoid receptors is involved in the induction of both MMTV and TAT in HTC cells.

Animals↗
PubMed1979

Glucocorticoid regulation of mouse mammary tumor virus gene expression.

Glucocorticoid hormones act rapidly and specifically to stimulate the synthesis of mouse mammary tumor virus RNA in a variety of mouse mammary tumor cells and infected heterologous cells. The increase in viral RNA production appears to be mediated by receptor proteins and requires the presence of basal levels of viral RNA. Infection of heterologous cells with MMTV may alter host cell responses to glucocorticoids; in addition, production of unintegrated viral DNA in these cells has provided reagents required for studying the structure and function of the viral DNA itself. The advent of new techniques for genetic manipulation of eukaryotic cells and for isolation of large amounts of specific DNA sequences should now permit detailed analyses of steroid hormone action in this system.

Animals↗
PubMed1979

Integration and transcription of mouse mammary tumor virus DNA in rat hepatoma cells.

Rat hepatoma cells infected with mouse mammary tumor virus (MMTV) acquire viral DNA that becomes covalently linked to the cell DNA. Using restriction endonucleases and the DNA transfer procedure of Southern [Southern , E.M. (1975) J. Mol. Biol. 98, 503--517], we have studied the sites in cellular DNA into which MMTV DNA inserts. These experiments indicate that: (i) there are many sites in cell DNA into which MMTV DNA integrates; (ii) the junctions between viral and cellular DNA occur within a limited portion of the viral genome, (iii) clones that contain MMTV DNA do not necessarily produce viral RNA; and (iv) the extent of transcription and glucocorticoid responsiveness of MMTV proviruses may be dependent on the site(s) in cell DNA in which the viral DNA resides.

Animals↗
PubMed1978

Mapping of linear and circular forms of mouse mammary tumor virus DNA with restriction endonucleases: evidence for a large specific deletion occurring at high frequency during circularization.

Rat hepatoma cells infected with mouse mammary tumor virus contain multiple forms of unintegrated viral DNA when grown in the presence of glucocorticoids. Using the DNA transfer procedure of Southern, we have prepared restriction endonuclease fragment maps of these forms of viral DNA. The maps indicate that: (i) the major species of viral DNA is a linear molecule of 5.9 X 10(6) Mr located in the cytoplasm; (ii) the nuclei contain covalently closed circular viral DNA of two distinct sizes (5.1 X 10(6) and 5.9 X 10(6) Mr) in addition to linear molecules (5.9 X 10(6) Mr); (iii) the linear molecule has specific termini; (iv) there is extensive homology between regions at or near termini of the linear molecule; (v) the predominant form of circular DNA lacks 1.2 kilobase pairs present in both the larger circular molecule and the linear molecule; and (vi) the sequences deleted from the majority of the circular DNA molecules are located at the ends of the linear DNA that are joined during circularization.

Chromosome Mapping↗
PubMed1978

Production of unintegrated mouse mammary tumor virus DNA in infected rat hepatoma cells is a secondary action of dexamethasone.

Dexamethasone, a synthetic glucocorticoid, selectively increased the rate of synthesis of mouse mammary tumor virus (MTV) RNA in clonal isolates of chronically infected rat hepatoma tissue culture cells. This hormonal effect occurred extremely rapidly and appeared to be mediated directly by the glucocorticoid-specific receptor protein. In addition to the viral RNA, unintegrated MTV DNA was also detected in these cells. Several lines of evidence are consistent with the idea that the unintegrated viral DNA is synthesized by reverse transcription of MTV RNA. (i) Unintegrated viral DNA accumulated only in the presence of dexamethasone and was produced with a time course that closely paralleled the increased accumulation of viral RNA. (ii) Density labeling of the viral DNA revealed that both strands were newly synthesized, implying a non-semiconservative mode of replication. (iii) Inhibitors of viral RNA synthesis prevented the appearance of unintegrated viral DNA. These data suggest that the production of unintegrated MTV DNA after dexamethasone treatment occurs as a secondary consequence of the hormonal induction of synthesis of viral RNA. In contrast to infected rat hepatoma cells, no unintegrated MTV DNA was detected in mouse mammary tumor cells or mouse lymphoma cells, despite the presence of high levels of viral RNA.

Animals↗
PubMed1977

Infection of cultured rat hepatoma cells by mouse mammary tumor virus.

A continuous line of buffalo rat hepatoma (HTC) cells has been successfully infected with mouse mammary tumor virus (MMTV) produced by the GR mammary tumor cell line. Uniform infection required initial exposure of the HTC cells to greater than 10(5) MMTV particles per cell. The resultant chronically infected cell population was found to have stably acquired 20-30 copies of MMTV DNA. The infected cells contain viral RNA and express viral antigens; however, very few MMTV particles are released into the culture medium. In spite of the biochemical evidence for infection, we have not detected any alterations in the morphology or growth properties of the infected HTC cells. As is the case in mammary tumor cells, the intracellular concentration of viral RNA is strongly stimulated (50-150 fold) by the synthetic glucorcorticoid, dexamethasone. Thus it appears that the mechanisms by which glucorticoids regulate MMTV gene expression in mouse cells are maintained when this virus infects nonmurine cells.

Antigens, Viral↗
PubMed1977

Mouse mammary tumor virus DNA in infected rat cells: characterization of unintegrated forms.

Mouse mammary tumor virus (MMTV) DNA in chronically infected rat hepatoma cells is maintained in both the integrated and unintegrated state. Fractionation of DNA by the procedure of Hirt (1967) as well as by sedimentation through alkaline sucrose suggests that about two thirds of the viral DNA is associated with high molecular weight cell DNA. The remainder of the viral DNA is unintegrated and is present primarily as linear or open circular duplexes consisting of a genome-length strand complementary to the viral RNA ("minus" strand) and "plus" strands of subgenomic length. Approximately 20% of the unintegrated MMTV DNA is present as double-stranded, covently closed circles (form I) with a molecular weight of 6 X 10(6) daltons. Form I viral DNA is found primarily in the nucleus, whereas the open forms are both nuclear and cytoplasmic.

Cell Line↗
PubMed1977

Glucocorticoid-stimulated accumulation of mouse mammary tumor virus RNA: increased rate of synthesis of viral RNA.

Glucocorticoid hormones specifically increase the intracellular concentration of mouse mammary tumor virus (MMTV) RNA in a cultured cell line from a GR mouse mammary carcinoma (GR) and in an MMTV-infected rat hepatoma cell line (M1.19). In contrast, these steroids have no effect on the concentration of MMTV RNA in a lymphoma line, S49, from a Balb/c mouse. Using a molecular hybridization procedure to detect newly synthesized RNA, we have directly measured the effect of dexamethasone, a synthetic glucocorticoid, on the rate of MMTV RNA synthesis. In GR cells the hormone causes a 10-fold increase in the rate of synthesis of viral RNA without appreciably affecting the overall rate of cellular RNA synthesis. The transition from the basal to the maximally stimulated rate of MMTV RNA synthesis occurs within the earliest labeling period, 0-15 min after addition of the hormone. Thus, it appears that glucocorticoids regulate MMTV genes principally by this rapid and specific alteration of their rate of transcription. Similar results are obtained in M1.19 rat hepatoma cells. In contrast, dexamethasone does not affect the rate of viral RNA synthesis in S49 lymphoma cells.

Carcinoma, Hepatocellular↗
PubMed1975

Dexamethasone-mediated induction of mouse mammary tumor virus RNA: a system for studying glucocorticoid action.

We have investigated the mechanisms by which dexamethasone (a synthetic glucocorticoid) stimulates the production of mouse mammary tumor virus (MMTV) by cell cultures derived from mammary carcinomas of GR mice. Treatment of these cells with dexamethasone stimulates a rapid accumulation of intracellular virus-specific RNA which is dependent upon RNA synthesis but not upon DNA or protein synthesis. The effect of dexamethasone is probably mediated by a specific and saturable glucocorticoid receptor. We conclude that the accumulation of MMTV RNA is a primary response to dexamethasone and that the rate of synthesis of MMTV RNA is probably accelerated by treatment with dexamethasone.

Cell Line↗
PubMedDate not supplied

Mouse mammary tumor virus genes: regulation of expression by glucocorticoids and structural analysis with restriction endonucleases.

The production of mouse mammary tumor virus (MTV) is stimulated by treatment of mammary carcinoma cells with glucocorticoid hormones (e.g., dexamethasone). The reaction appears to be a primary and receptor-mediated phenomenon in which the rate of synthesis of MTV RNA is selectively increased; the maximally induced rate is reached within 15 min after addition of the hormone. Production of MTV RNA is also stimulated by dexamethasone in some but not all clonal isolates of nonmurine cells infected by the virus. Using restriction endonucleases, we have characterized both integrated and unintegrated viral DNA from clones of infected rat hepatoma (HTC) cells. We suggest that the site(s) in the rat cell genome into which MTV DNA integrates may be a determining factor for MTV RNA synthesis.

DNA Restriction Enzymes↗