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

Abstract

Murine mammary tumor virus (MMTV) expression is analyzed in a T-lymphoid cell line (TIMI) sensitive to the killing effect of glucocorticoids and in two of its variants, one resistant (TIMir) and one supersensitive (T MI"5) to glucocorticoidinduced lymphocytolysis. In the TIM, line, MMTV is expressed and induced approximately 10-fold by short treatment with dexamethasone. Southern blot analyses of restriction enzyme digests of DNA Glucocorticoids induce in a-variety of tissues specific responses by mechanisms that appear to be similar to those ofother steroid hormones. Glucocorticoids induce cytolysis ofsome murine cell lines derived from malignant lymphoid T cells (1, 2). This response is mediated by the glucocorticoid receptor, which interacts with chromatin, but the mechanism ofcell lysis remains unknown. Lymphoid cells allow a genetic approach to study of the mechanism of glucocorticoid hormone action because variants resistant to killing can be selected easily. A number of resistant variants have been isolated and characterized from S49 and W7 cell lines (1-3). More recently, variants supersensitive to glucocorticoid-induced killing have been isolated from W7 and TIM, cell lines (4).Glucocorticoids also regulate the transcription of integrated murine mammary tumor virus (MMTV) proviral DNA in various cell types (5, 6) including lymphoid cell lines (7). The availability of lymphoid cell variants having different sensitivities to the lytic effect ofglucocorticoids allows the study of MMTV expression as a function of glucocorticoid sensitivity. Therefore, we have analyzed the control of MMTV RNA concentration by dexamethasone in wild-type, resistant, and supersensitive TIM, cell lines by using cloned MMTV sequences.There is evidence that modification of DNA such as methylation may be correlated with changes in gene expression (8), in particular, in MMTV proviral DNA of mouse tissue (9). By using methylation-sensitive restriction enzymes to detect specific methylation of.MMTV sequences, we have compared the methylation of sites within or flanking the long terminal repeat (LTR) (10) with that of a site within the MMMTV envelope gene.In this paper, we describe the regulation of MMTV RNA synthesis and the methylation ofthe MMTV LTR.and envelope sequences in sensitive, supersensitive, and resistant thymoma cell lines. We present evidence that the expression of endogenous proviruses is different in the various cell lines and appears to correlate with methylation of the LTR. t To whom reprint requests should be addressed. MATERIALS AND METHODS Cell 110The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Chronic exposure to dexamethasone induces hypomethylation of ornithine decarboxylase genes in a human myeloma cell line

“…Even more relevant to the present results are the findings of Mermod et al [19] who found that the selection of a dexamethasone-resistant mouse lymphoid cell line (in the presence of 10#M dexamethasone) was specifically associated with demethylation of the 5'-long terminal repeats of MMTV in the absence of any MMTV genome rearrangements or overall genome demethylation.…”

Genomic sequencing reveals a positive correlation between the kinetics of strand-specific DNA demethylation of the overlapping estradiol/glucocorticoid-receptor binding sites and the rate of avian vitellogenin mRNA synthesis.

“…Since role in eukaryotic cell metabolism in general and in liver in particular (44), a gradual demethylation of the CpG b may take-place during development. A similar effect of glucocorticoids on the demethylation of mouse mammary tumor virus DNA has also been reported in some mutants of myeloma cells (45). Whether glucocorticoids and estradiol trigger the same mechanism of demethylation remains unknown.…”

Control of gene expression by glucocorticoid hormones

“…No change in DNA methylation, however, could be detected during tyrosine aminotransferase induction by glucocorticoids in hepatoma cells, a short-term event (Lapeyre et al, 1980). Yet, experiments with MTV-infected lymphoid cells suggest that long-term treatment with glucocorticoids could lead to demethylation of the induced gene, and this might play a role in cell differentiation events induced by these hormones (Mermod et al, 1983). In any case, glucocorticoids induce the non-methylated transfected forms of MTV, but not their methylated endogenous forms (Groner et al, 1983b (Tymoczko et al, 1983;Rossini & Barbiroli, 1983).…”