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Biomedical subjects

G M Ringold

Publications and source records attributed to G M Ringold.

At least 55 records · Page 3Linked to original sources
PubMed1986

Hormonal control of adipogenesis.

The concept that hormonal substances can alter the expression of entire developmental programs is in itself not particularly new. The ability to define conditions under which a specific hormone can precociously activate the differentiation of a well-defined population of cells and under which another hormone can both block and reverse such a developmental progression, however, provides a major step forward toward unraveling the biochemical events that define the transition from a committed precursor to a fully differentiated cell. Further analysis of the molecular events initiated by glucocorticoids and TNF should provide insights into the control of adipogenesis and may generate a foundation for understanding the mechanisms by which other cells enter a particular differentiative lineage. In a more applied sense, such knowledge may also provide a rational approach to controlling metabolic disease syndromes related to adipogenesis gone awry such as obesity-associated diabetes and cachexia.

Adipose Tissue↗
PubMed1985

A macrophage factor inhibits adipocyte gene expression: an in vitro model of cachexia.

Certain infections and malignancies in mammals cause the development of a condition known as cachexia in which the animal continues to lose weight, often while consuming an adequate diet. When macrophages are stimulated with an endotoxin, they produce a factor or factors, termed cachectin, that inhibits the activity of fat-producing (lipogenic) enzymes in cultured adipocytes. This effect may reflect one of the physiological bases for cachexia. In the present study, clones of complementary DNA from genes whose expression is increased during the differentiation of adipocytes were used to study the molecular basis of cachectin's actions. In the presence of cachectin, the expression of the corresponding genes was reversibly and specifically inhibited. Furthermore, when mature adipocytes were exposed to cachectin, the messenger RNA's of those genes diminished and rapidly approached the levels present before differentiation.

Actins↗
PubMed1985

Characterization of wild type and mutant glucocorticoid receptors from rat hepatoma and mouse lymphoma cells.

Using a combination of immunological blotting techniques and hormone affinity labeling, we have characterized the glucocorticoid receptors present in wild type and mutant rat hepatoma (HTC) and mouse thymoma (S49 and WEHI7) cells. Mutant HTC and WEHI7 cells of the receptorless phenotype, which contain greatly reduced amounts of glucocorticoid hormone binding activity, show parallel decreases in immunoreactive material using a monoclonal antibody raised against the rat liver glucocorticoid receptor. This indicates that these receptorless mutant cells harbor defects in either the production or accumulation of receptor protein. Quantitation of immunoreactivity and hormone binding activity present in wild type and mutant S49 cells indicates that these cells contain significantly more immunoreactive material than hormone binding activity. We conclude that S49 cells produce, in addition to their well characterized wild type or mutant receptors, a mutant receptor from a second allele which is of wild type size, is immunologically reactive, but is unable to bind hormone. The S49 mutant cell line nti (nuclear transfer increase) contains a glucocorticoid receptor which has a molecular weight of 40,000, while the wild type receptor has a molecular weight of 94,000. Affinity labeling of glucocorticoid receptors in nti cells with [3H]dexamethasone mesylate indicates that nti cells do not contain wild type sized precursor molecules which bind hormone, nor do they contain immunoreactive fragments of a molecular mass smaller than 94 kDa. It is proposed that the 40-kDa nti receptor is produced as a truncated protein most likely resulting from a nonsense mutation or from a truncated messenger RNA.

Affinity Labels↗
PubMed1985

Glucocorticoid regulation of adipocyte differentiation: hormonal triggering of the developmental program and induction of a differentiation-dependent gene.

We have analyzed the hormonal basis for the acceleration of differentiation by dexamethasone and insulin in the stable adipogenic cell line TA1. These cells, which were derived from 5-azacytidine-treated 10T1/2 mouse embryo fibroblasts, undergo differentiation in culture after reaching confluence. The ensuing morphological changes are accompanied by widespread alterations in the pattern of protein synthesis and the increased accumulation of specific mRNAs. Using cDNA clones corresponding to mRNAs that are induced during adipogenesis, we find that dexamethasone elicits the precocious accumulation of differentiation-specific gene products. This effect appears to be mediated by the glucocorticoid receptor, yet unlike standard steroid inductions, most of the RNAs reach the same maximal levels in the absence of dexamethasone. Glucocorticoids thus may increase the expression of a regulatory factor required for activating the entire set of differentiation-dependent genes. We also describe a gene whose transcription is not only activated during adipogenesis but is also specifically inducible by dexamethasone in the mature adipocyte. Moreover, the glucocorticoid responsiveness of this gene in differentiated cells appears to be dependent on its prior developmental activation.

Adipose Tissue↗
PubMed1984

Analysis of gene expression during differentiation of adipogenic cells in culture and hormonal control of the developmental program.

Treatment of 10T1/2 mouse embryo fibroblasts with 5-azacytidine, an inhibitor of mammalian DNA methylation, leads to the appearance of several new cell types, including adipocytes. We have isolated several such adipogenic cell lines and characterized two of them, TA1 and TA2. When subconfluent these cells resemble fibroblasts. After growth is arrested at high density, both clones express a functional adipose phenotype characterized by accumulation of lipid droplets. This in vitro differentiation is accompanied by a greater than 100-fold increase in glycerol phosphate dehydrogenase activity, an enzyme characteristic of mature adipocytes. Consistent with these morphologic and enzymatic changes, differentiated TA1 cells show a widespread alteration in protein composition as well as a substantial change in the pattern of secreted proteins. We have constructed a cDNA library of TA1 adipocytes and have isolated 12 different cDNA clones corresponding to mRNAs that are induced during adipogenesis. Among these RNAs, some are not expressed prior to initiating differentiation whereas others are expressed in 10T1/2 cells and TA1 preadipocytes. Treatment of TA1 cells with insulin and the synthetic glucocorticoid dexamethasone leads to an acceleration of the phenotypic changes observed during adipogenesis. We have found that hormone treatment leads to a precocious accumulation of specific RNA for all of the clones studied. Analysis of the temporal control of RNA accumulation during differentiation indicates that there are different categories of RNAs, some of which accumulate by day 1 after treatment while others are not apparent until day 3.

Adipose Tissue↗
PubMed1984

Functional analysis of the steroid hormone control region of mouse mammary tumor virus.

Gene fusions between the mouse mammary tumor virus long terminal repeat and the E. coli lacZ gene have been shown to exhibit hormone dependent expression of beta-galactosidase activity. These constructions were used in transient expression experiments to assess the effects of specific modifications introduced into the region upstream of the transcription initiation site. 5' deletions demonstrate that sequences sufficient for wild-type promoter function are contained downstream of residue -64 relative to the initiation site. Other deletions define a region of approximately 80 base pairs between -220 and -140 which contains sequences essential for hormonal control. Between this control region and the promoter lie sequences dispensable for both functions.

Animals↗
PubMed1984

Inhibition of protein synthesis stimulates the transcription of human beta-interferon genes in Chinese hamster ovary cells.

Using Chinese hamster ovary (CHO) cells transfected with a plasmid carrying the human beta-interferon gene, we find that inhibitors of protein synthesis, in the absence of any other inducer, stimulate the production of interferon RNA; this effect is maintained in cells in which the plasmid sequences have been amplified 25- to 50-fold. Nuclear transcription assays show that a major effect of cycloheximide is to increase the rate of transcription of the interferon gene. This contradicts the generally accepted explanation that inhibitors of protein synthesis augment interferon production by stabilizing interferon mRNA. In addition, we have studied the effects of double stranded RNA [poly(rI) X poly(rC)] on the induction of interferon RNA in the presence and absence of cycloheximide. Our results indicate that poly(rI) X poly(rC) by itself causes a transient increase in interferon RNA; however, in the presence of cycloheximide this effect is prolonged. We do not, however, find an increase in transcription of the interferon gene(s) as an early response to poly(rI) X poly(rC). Finally, we have found that cells treated with cycloheximide or infected with Newcastle disease virus induce large amounts of a secreted 11-kDa protein. This cellular protein is not inducible by poly(rI) X poly(rC). We propose that both interferon and this 11-kDa protein belong to a family of proteins in which production is regulated in a coordinate fashion during viral inhibition of cellular protein synthesis.

Animals↗
PubMed1984

Glucocorticoid-mediated induction of alpha 1-acid glycoprotein: evidence for hormone-regulated RNA processing.

We have studied the glucocorticoid-mediated accumulation of alpha 1-acid glycoprotein (AGP) in mRNA in HTC rat hepatoma cells. In contrast to the well-characterized primary response of mouse mammary tumor virus, in vitro transcription assays in isolated nuclei show that the rate of transcription of the AGP gene is high even in the absence of hormone. Despite the constitutive transcription of the AGP gene, no detectable AGP RNA can be found in either the cytoplasm or the nuclei of untreated cells. Previous experiments have shown that the glucocorticoid induction of AGP RNA requires ongoing protein synthesis. In conjunction with the present study, our data suggest that glucocorticoids stimulate accumulation of AGP RNA by inducing an RNA processing factor that allows production of stable transcripts.

Animals↗
PubMed1984

Use of the mouse mammary tumor virus long terminal repeat to promote steroid-inducible expression of v-mos.

We used the mouse mammary tumor virus long terminal repeat to promote dexamethasone-regulated expression of the Moloney murine sarcoma virus (M-MSV) transforming gene, v-mos. A recombinant DNA vector containing the mouse mammary tumor virus long terminal repeat fused to the M-MSV 124 v-mos gene was cotransfected with a plasmid containing the herpes simplex virus thymidine kinase gene (tk) into 3T3TK- cells. Individual clones of cells which grew in hypoxanthine-aminopterin-thymidine medium were tested for dexamethasone-regulated expression of p37mos as well as several transformation-specific phenotypic parameters. In the absence of dexamethasone, the v-mos transfectants appeared morphologically similar to the control cells despite low basal levels of p37mos expression. Upon hormone treatment, the levels of p37mos increased 5- to 10-fold, coincident with morphological changes typical of M-MSV transformation of 3T3 cells. The ability to form foci in monolayers also correlated with p37mos induction. The extent of morphological changes varied in individual clones of cells with similar levels of induced p37mos. Although the induced levels of p37mos were comparable to those seen in stable M-MSV 124 virus-transformed NIH 3T3 cells, the transfectants were unable to grow in soft agar under conditions which support growth of the virus-transformed cells. Acute infection of the transfectants with M-MSV 124 virus, a situation which resulted in elevated levels of p37mos, allowed these cells to grow in soft agar. The results described in this paper suggest that different threshold levels of p37mos may be necessary for the expression of various parameters of the transformed phenotype and also that continued expression of p37mos is necessary for maintenance of the transformed state. However, it also appears that the sensitivity to given levels of p37mos varies among clonal cell lines.

Animals↗
PubMed1983

Mouse 3T6 cells that overproduce glutamine synthetase.

A mouse 3T6 subline that grows in glutamine-free medium has been cloned and exposed to a regimen of increasing concentrations of the glutamine synthetase inhibitor, methionine sulfoxime. Cells selected for resistance to 700 microM methionine sulfoxime show a 75-fold increase in glutamine synthetase activity relative to the original subclone. Immune precipitation of extracts prepared from cells pulse-labeled with L-[35S] methionine indicates that the increase in enzyme activity reflects an increase in biosynthesis of glutamine synthetase. Results obtained from in vitro translation followed by immune precipitation suggests that the methionine sulfoxime-resistant cells are highly enriched in mRNA encoding glutamine synthetase. The increase in enzyme activity is lost upon culture of the cells in nonselective medium--a finding consistent with the observation of double minute chromosomes in only the drug-resistant cells. These data strongly support the notion that methionine sulfoxime treatment has resulted in selection of cells that have amplified the gene encoding glutamine synthetase.

Animals↗
PubMed1983

Induction of the acute-phase reactant, alpha 1-acid glycoprotein, by glucocorticoids in rat hepatoma cells.

alpha 1-acid glycoprotein (alpha 1-AGP), or orosomucoid, is shown to be inducible by glucocorticoids in HTC rat hepatoma cells. Immunoprecipitation of [35S]methionine pulse-labeled proteins from these cells reveals secreted proteins of Mr = 35,000-48,000 (alpha 1-AGP) and Mr greater than 180,000, both of which are greatly enhanced by glucocorticoid treatment. The amount of alpha 1-AGP-specific mRNA in HTC cells is greatly increased (at least 100-fold) in response to glucocorticoids. The new steady-state level of RNA is approached with a t 1/2 of about 8 hr and the RNA consists of a single species of approximately 850 bases. The response is specific for glucocorticoids since: (i) the EC50 for dexamethasone is 30 nM; (ii) the glucocorticoid antagonist, progesterone, inhibits the induction by dexamethasone; and (iii) a glucocorticoid receptor-deficient cell line is incapable of alpha 1-AGP mRNA induction. This is a secondary hormonal response since inhibition of protein synthesis blocks the induction of alpha 1-AGP mRNA by dexamethasone whereas the induction of mouse mammary tumor virus (MMTV) RNA is unaffected.

Animals↗
PubMed1983

Amplification and hormone-regulated expression of a mouse mammary tumor virus-Eco gpt fusion plasmid in mouse 3T6 cells.

Mouse 3T6 cells were transformed with a chimeric DNA plasmid, pSVMgpt, in which the mouse mammary tumor virus (MMTV) promoter was fused to the Escherichia coli gene encoding xanthine-guanine phosphoribosyl transferase (Eco gpt). The transformants exhibited glucocorticoid-inducible expression of Eco gpt. With limiting xanthine concentrations, conditions were established in which cell growth became hormone dependent. Cells selected for their ability to grow in limiting concentrations of both xanthine and glucocorticoids contained amplified levels of Eco gpt DNA, and expression of Eco gpt remained glucocorticoid inducible in these amplified cells. Thus, amplification of the MMTV promoter region in itself did not abolish hormonal responsiveness of a gene. In addition to increased levels of Eco gpt DNA, some of the selected cells also exhibited increased levels (two- to threefold) of glucocorticoid receptors. Lastly, we found that excessive expression of Eco gpt is toxic to 3T6 cells; by maintaining low hormone levels and, therefore, low levels of expression, we were able to select cells with amplified Eco gpt. Thus, the MMTV promoter may be of general utility in expressing genes whose products may be lethal if they are produced in excessive quantities.

Animals↗
PubMed1983

Analysis of glucocorticoid-inducible genes in wild-type and variant rat hepatoma cells.

We present pharmacological and genetic evidence that regulation of different genes by glucocorticoid hormones in the rat hepatoma cell line, HTC, occurs in a coordinate manner. We have analyzed the responses of four different glucocorticoid-inducible proteins, tyrosine aminotransferase [L-tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5)], glutamine synthetase [L-glutamate:ammonia ligase (EC 6.3.1.2)], a secreted glycoprotein Belt I, and the mouse mammary tumor virus (MMTV)-encoded protein (gp52) in these cells. The concentration of dexamethasone necessary for half-maximal induction of each of these proteins is 10-20 nM, the same concentration necessary to half-saturate glucocorticoid receptors. Furthermore, glucocorticoids of varying potency elicit parallel inductions of these markers. MSN5.3, a glucocorticoid receptor-defective cell line selected for its inability to induce gp52, is also unable to induce the other three cellular gene products. In contrast, another class of variants incapable of gp52 induction retains inducibility of the other three markers. We show here by "superinfection" with MMTV that these cells harbor a defect in the original integrated provirus itself and not in the cellular induction machinery. The results presented here suggest that the induction of glucocorticoid-responsive genes in these cells is mediated by a single glucocorticoid induction pathway.

Animals↗
PubMed1983

Expression and regulation of Escherichia coli lacZ gene fusions in mammalian cells.

Gene fusions between the Escherichia coli lacZ gene and DNA segments containing the simian virus 40 early promoter or the mouse mammary tumor virus (MMTV) promoter direct the synthesis of functional beta-galactosidase in Cos 7 monkey cells and mouse Ltk-cells. Enzymatic activity was measured either 72 h after transfection or in stable transformants. The sensitive beta-galactosidase assay was used to measure gene expression and to optimize the efficiency of DNA-mediated transfection. Glucocorticoids stimulated the production of beta-galactosidase when lacZ was fused to the hormonally regulated MMTV promoter.

DNA, Recombinant↗