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S Bourgeois

Publications and source records attributed to S Bourgeois.

At least 55 records · Page 3Linked to original sources
PubMed1984

DNA binding properties of glucocorticosteroid receptors bound to the steroid antagonist RU-486.

RU-486 is an anti-fertility steroid which also has anti-glucocorticosteroid effects. RU-486 is shown to be a strong antagonist of the glucocorticosteroid-induced cytolytic response of the murine thymoma lines W7TB and T1M1b , and of the induction of mouse mammary tumor virus (MMTV) mRNA in T1M1b cells. The glucocorticosteroid receptor of W7 cells has high affinity for RU-486 (Kd = 3 X 10(-9) M) but the complex formed has low nuclear transfer capacity. Binding of RU-486, as compared with the glucocorticosteroid agonist triamcinolone acetonide, to mouse receptor results in a decreased affinity for DNA in general and a reduced specific recognition of a site in the promoter region of MMTV proviral DNA. The RU-486 complex formed with rat liver receptor exhibits the same behavior; in addition, it is shown that only a fraction of these complexes are activated by temperature and these form highly salt-sensitive interactions with DNA. These results indicate that the binding of RU-486 to glucocorticosteroid receptors mimics pharmacologically the properties of a class of receptor variants (nt-) which are non-functional and have reduced nuclear transfer and altered DNA binding capacity. These results substantiate the importance of DNA binding in receptor function.

Animals↗
PubMed1984

Nuclear interactions of wild type and variant glucocorticoid receptors.

Nuclease digestion of nuclei from glucocorticoid sensitive and resistant lymphoma cell lines was used to study the nuclear compartmentalization of wild type and variant glucocorticoid receptors. In comparison with wild type, the variant line (S49 143r) had an increased capacity to translocate to the nucleus (nti), but was more completely released from nuclei by nuclease digestion. Approximately 20% of the receptor in wild type nuclei was resistant to release by DNase I digestion, while only less than 5% of the receptor from nti nuclei was retained under the same conditions. Studies with wild type nuclei show that the nuclease resistant portion of receptors was also more resistant to release by increased ionic strength.

Animals↗
PubMed1984

Analysis of the cya locus of Escherichia coli.

A 9500-bp DNA segment containing the adenylate cyclase gene (cya) of Escherichia coli has been isolated and analyzed. Four large proteins are encoded within this fragment - the adenylate cyclase protein (92 kDal), two proteins of unknown function (37 and 32 kDal), and a part of the uvrD-coded protein. Various truncated adenylate cyclase proteins, made from cya genes having as much as 60% of their carboxy-terminal end deleted, are sufficient to complement cya- hosts. When these truncated cya genes are present on a multicopy plasmid in a cya- host, the synthesis of beta-galactosidase is still regulated by glucose. The "maxicell" technique was used to visualize the four proteins encoded by this region and some of the truncated adenylate cyclase proteins.

Adenylyl Cyclases↗
PubMed1984

A new murine model system for the in vitro development of thymoma cell heterogeneity.

We have established and characterized a continuous T-cell line derived from the bone marrow of an AKR mouse with disseminated lymphoma. The original tumor cell line is heterogeneous with respect to several markers of thymocyte differentiation. Clones from the line differ in the expression of ThB, Pgp-1, and H-2Kk surface antigens. These clones also differ in their sensitivity to glucocorticoid-induced cell lysis. The quantity, affinity, and nuclear translocation properties of the glucocorticoid receptor are similar in the hormone-sensitive and -resistant clones. Furthermore, dexamethasone-resistant T-cells can be selected in vitro from freshly cloned cells sensitive to hormone-induced lysis at high frequency and without mutagenesis. Of several randomly sampled, spontaneously arising, independently derived dexamethasone resistant clones, all show a coordinate reduction in cell surface Thy-1 and ThB expression with no detectable changes in glucocorticoid receptor properties. Following treatment with the DNA-demethylating agent 5-azacytidine, the original dexamethasone-resistant T-cell line as well as the dexamethasone-resistant derivatives obtained in vitro regain sensitivity to lysis. These results collectively suggest a role of DNA methylation in hormone resistance and are consistent with a model of thymocyte differentiation in which a glucocorticoid-sensitive cell is the progenitor of hormone-resistant T-cells.

Animals↗
PubMed1984

Membrane permeability as a determinant of dexamethasone resistance in murine thymoma cells.

The variant MS23 of the murine thymoma cell line W7 , selected for growth at low concentrations of dexamethasone (7.5 nM), is cross-resistant to various unrelated drugs, including colchicine. By stepwise selection in combinations of dexamethasone and colchicine at increasing concentrations, we have isolated a series of variants with increased resistance to dexamethasone and cross-resistance to puromycin, colchicine, daunomycin, gramicidin, and vincristine. Surprisingly, resistance to triamcinolone acetonide, a glucocorticoid structurally related to dexamethasone, did not develop. Assays for specific dexamethasone and triamcinolone acetonide binding sites in variant cell extracts reveal that the glucocorticoid receptors of these variants are unchanged as compared to the W7 parental line. However, whole cell binding assays yielded reduced apparent affinity for dexamethasone in the MS23 variant and drastically reduced dexamethasone binding after selection for increased resistance. We demonstrate that this is due to reduced drug uptake. Procaine, a membrane-active anesthetic, potentiates uptake of puromycin and dexamethasone in the variants. The variants are stable, and karyotypic analysis did not reveal double minute chromosomal structures. These results demonstrate that permeability can be a rate-limiting step in steroid hormone action and is the basis for dexamethasone resistance in these variants.

Animals↗
PubMed1983

Role of de novo DNA methylation in the glucocorticoid resistance of a T-lymphoid cell line.

A correlation has been shown between changes in the methylation pattern of cytosine residues in DNA and the expression of specific genes in differentiated tissues. The pattern of DNA methylation is conserved, through cell division, by a maintenance methylase but the mechanism by which a given pattern of methylation is established is unknown. De novo methylation of foreign DNA molecules has been shown to occur in several systems, and may serve as a signal to arrest gene expression. Conversely, treatment of cultured cell lines with 5-azacytidine results in DNA hypomethylation and leads to transcriptional activation of previously unexpressed genes. The results described here demonstrate spontaneous de novo methylation of DNA in a T-lymphoid cell line previously treated with 5-azacytidine to generate glucocorticoid sensitivity. This de novo methylation is accompanied by the acquisition of the glucocorticoid-resistant phenotype.

Animals↗
PubMed1983

Regulation of fibronectin biosynthesis by glucocorticoids in human fibrosarcoma cells and normal fibroblasts.

When treated with the synthetic glucocorticoid dexamethasone, HT1080 human fibrosarcoma cells show changes in morphology, adhesion, and the extracellular matrix. Dexamethasone treatment results in a tenfold increase in the rate of fibronectin biosynthesis in HT1080 cells and a twofold increase in untransformed, normal human fibroblasts. Maximal induction levels are attained within one cell generation, while decay of the response requires several cell cycles. Pulse-chase studies showed that most of the newly synthesized fibronectin is secreted into the medium. The glucocorticoid antagonist, RU-486, blocks the dexamethasone-induced changes but does not alter the basal rate of fibronectin production. Therefore, fibronectin biosynthesis appears to be controlled by two distinct mechanisms--one, regulating basal rates of fibronectin production, which is transformation-sensitive and glucocorticoid-independent; and another, which is mediated by the glucocorticoid receptor, resulting in elevated rates of fibronectin biosynthesis upon dexamethasone treatment both in normal fibroblasts and in HT1080 cells.

Cells, Cultured↗
PubMed1983

Demethylation and expression of murine mammary tumor proviruses in mouse thymoma cell lines.

Murine mammary tumor virus (MMTV) expression is analyzed in a T-lymphoid cell line (T1M1) sensitive to the killing effect of glucocorticoids and in two of its variants, one resistant (T1M1r) and one supersensitive (T1M1ss) to glucocorticoid-induced lymphocytolysis. In the T1M1 line, MMTV is expressed and induced approximately 10-fold by short treatment with dexamethasone. Southern blot analyses of restriction enzyme digests of DNA from T1M1 cells reveal three proviruses similar to those of normal C57BL mouse tissue. In the T1M1ss line, which has retained functional glucocorticoid receptors, MMTV mRNA is inducible by glucocorticoids, while induction is reduced in the T1M1r line defective in glucocorticoid receptors. Moreover, the T1M1r line expresses a strikingly elevated basal level of MMTV mRNA in the absence of hormone. No rearrangements or superinfection have occurred in the variants, but all the regions containing 5'-long terminal repeats are demethylated in the T1M1r variant although other sites of the provirus remain methylated. Because this variant was selected by prolonged treatment with dexamethasone, these observations raise the possibility that the continuous transcription of MMTV that occurred during this selection can result in glucocorticoid-induced demethylation of long-terminal-repeat sequences.

Animals↗
PubMed1983

A new determinant of glucocorticoid sensitivity in lymphoid cell lines.

The SAK cell line, derived from a spontaneous thymic lymphoma in an AKR mouse, is resistant to lysis by glucocorticoids in spite of the presence of functional glucocorticoid receptor. Receptor function was determined by hormone binding analyses, as well as characterization of hormonal effects on cell growth and on the accumulation of murine leukemia virus and metallothionein mRNAs. SAK cells were fused with a receptor-defective (and therefore resistant) variant of a well-characterized murine thymoma line, W7. The resulting hybrids are glucocorticoid sensitive, demonstrating complementation of the receptor defect in W7 cells by the functional glucocorticoid receptor of SAK. This fusion shows that SAK cells are resistant to the hormone due to the absence of another function designated "I" for lysis. SAK cells were also fused with glucocorticoid-sensitive W7 cells (containing wild-type receptor), generating glucocorticoid-sensitive hybrids, which demonstrate that the dexamethasone-resistant phenotype of the SAK cells is recessive. Resistant derivatives of this hybrid were found which still contain the full amount of receptor. Chromosome analysis revealed that, on the average, the resistant derivatives had lost two chromosomes, suggesting segregation of chromosomes carrying genetic material necessary for the "lysis" function. The drug 5-azacytidine (a known inhibitor of DNA methylation) has been shown to cause heritable changes in gene expression. Treatment of SAK cells with 5-azacytidine generated glucocorticoid-sensitive clones at high frequency, suggesting that the gene(s) involved in the "lysis" function are intact and have been inactivated through a process such as differentiation.

Animals↗
PubMed1981

Induction of glucocorticoid-resistant variants in a murine thymoma line by antitumor drugs.

Several antitumor drugs are shown to be mutagenic in murine thymoma lines: mitomycin C, bleomycin, streptonigrin, Colcemid, and BD40, an analog of ellipticine. Using conditions yielding 3 to 40% cell survival, all five drugs tested increase the frequency of glucocorticoid-resistant variants. Mitomycin C is as efficient as the classical alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine and ethyl methanesulfonate. The other drugs, previously untested for mutagenic activity on mammalian cells, are weak mutagens yielding variants at frequencies 1 to 2 orders of magnitude lower than the alkylating agents. All 152 variants obtained result from defects in the glucocorticoid receptor. Variants induced by mitomycin C, streptonigrin, Colcemid, and BD40 have very reduced receptor activity, as measured by dexamethasone binding. In contrast, bleomycin or the combination of mitomycin C and dexamethasone induce a majority of variants having dexamethasone-binding activity comparable to the parental line. However, assays of nuclear transfer capacity and genetic complementation show that these receptors are nonfunctional and may result from point mutations in the gene encoding the glucocorticoid receptor. This study suggests that, in combination therapies, antitumor drugs might induce glucocorticoid-resistant lymphoid cell variants that could be selected by the hormone.

Animals↗
PubMed1980

Glucocorticoid-induced lymphocytolysis: state of the genetic analysis.

The glucocorticoid-induced lysis of lymphoid cell lines offers a genetic approach to steroid hormone action because unresponsive variants can easily be selected as resistant to this lytic effect. The present state of analysis of lymphocytolysis in two murine cell lines, the S49 T-lymphoma and the W7 thymoma, is reviewed. All glucocorticoid-resistant variants isolated so far result from various defects in the glucocorticoid receptor. The absence of variants blocked at another step of the lytic mechanism is discussed. The observed hemizygosity of the glucocorticoid receptor locus in the S49 line and the instability of cell hybrids illustrate some of the potential problems encountered in somatic cell genetics.

Animals↗
PubMed1980

Analysis of steroid resistance in lymphoid cell hybrids.

In an attempt to obtain a more detailed understanding of the action of the glucocorticoid-receptor complex in mouse lymphoid cell lines, steroid sensitivity has been investigated in hybrids. Hybrids between dexamethasone (dex)-sensitive and dexamethasone-resistant (Dexr) variants, and hybrids between different Dexr variants were investigated. In the case of Dexr x Dexs hybrids, the possibility of negative complementation was tested; in the case of Dexr x Dexr hybrids, positive complementation was investigated. Neither positive nor negative complementation could be detected; dex sensitivity was always dominant over dex resistance. However, hybrids which contain positive receptor allele(s) segregated out Dexr clones at higher frequencies than expected from studies of pseudodiploid cell lines. This study suggests that different mechanisms give rise to the dex-resistant phenotype in pseudodiploid lymphoid cell lines and in pseudotetraploid hybrids of these cell lines.

Alleles↗
PubMed1979

Correlation between glucocorticoid receptor and cytolytic response of murine lymphoid cell lines.

By fusion between murine thymoma lines which are either homozygous (r+/r+ or r-/r-) or hemizygous (r+/r-) for the glucocorticoid receptor structural gene r, hybrid clones have been obtained which carry one, two three, or four copies of the r+ allele. These hybrids contain different amounts of normal glucocorticoid receptor as the result of a r+ gene dosage effect. Measurements of the cytolytic response of these hybrids to dexamethasone indicate a tight correlation between receptor content and sensitivity.

Animals↗
PubMed1978

Interaction of glucocorticoid receptors from lymphoid cell lines with their nuclear acceptor sites.

Procedures have been developed which provide simple means of determining binding constants of steroid receptors for glucocorticoids in mouse lymphoid cell lines and of characterizing the interaction of the steroid--receptor complex with the nucleus. An average of 70% of the steroid--receptor complexes is found associated with the nuclear fraction in three investigated cell lines, whereas 30% of the steroid--receptor complexes is found in the cytosol fraction. This distribution of the steroid-receptor complex within the cell is independent of whether steroid uptake of the cells is performed at low or at high steroid concentration. Part of the binding of the steroid receptor to the nuclear fraction is sensitive to high ionic strength and to high pH. A larger fraction of the steroid--receptor complex binding to the nuclear fraction is insensitive to high ionic strength and pH when the steroid uptake is performed at low steroid concentrations than when performed at high steroid concentrations. Steroid--receptor complex is released from the nuclear fraction by DNAase treatment but not by RNAase treatment. The possible correlation between the sensitivity to ionic strength and pH and the specificity of the binding is discussed.

Animals↗
PubMed1978

General features of steroid resistance on lymphoid cell lines.

Some general features of dexamethasone resistance in five murine lymphoid cell lines were investigated. To obtain large numbers of dexamethasone-resistant (Dexr) variants, a technique was developed by which mouse lymphoid cell lines can be grown with high efficiency on the surface of agar plates without a feeder layer. A total of 271 Dexr variants were investigated, and 90% of them turned out to lack detectable steroid receptor whereas 10% have receptor with, in most cases, a normal affinity for the steroid hormone. Most of this latter class of variants, however, have reduced amounts of receptor and the receptor of all of them displayed altered nuclear binding characteristics. None of the five investigated lymphoid cell lines yielded a Dexr variant with a normal receptor. These results confirm the idea that the high incidence of receptor variant may be due, at least in part, to the haploid state of a gene coding for the receptors. In cell fusion experiments it could be shown that Dexs is dominant over Dexr, but that a Dexr a-lele in a tetraploid cell can lead to an increased frequency of steroid resistance.

Animals↗