Progression to steroid autonomy in S115 mouse mammary tumor cells: role of DNA methylation.

Abstract

Although monoclonal in origin, mammary tumors acquire a marked heterogeneity of cell phenotypes, including a mixture of steroid hormone-sensitive cells and insensitive cells. We describe here long-term studies on the effects of androgen withdrawal on cloned androgen-responsive $115 mouse mammary tumor cells as a model system to investigate mechanisms by which tumor cells lose their steroid sensitivity. In the prolonged absence of androgen, the cells lost hormone-sensitive parameters reproducibly, including loss of proliferative response, saturation density response, cell morphology response, and mouse mammary tumor virus long terminal repeat (MMTV-LTR)-related RNA. These experiments have demonstrated that when deprived of hormone in the long term, a clone of responsive cells gives rise reproducibly to a population of unresponsive cells in an ordered series of phenotypic changes. At the time when the cells lost all androgen response in terms of cell biology and MMTV-LTR-RNA, increased methylation of MMTV-LTR sequences in the DNA was detected. Thereafter recovery of androgen sensitivity has not been achieved in any of these parameters. The possible role of de novo DNA methylation in the progression to androgen autonomy of $115 cells is discussed.Manipulation of the steroid environment affects breast tumor growth in many species (26, 29) but only on a temporary basis, and even in rodents, where effects are greatest, it never eliminates the tumor completely (19). In man, 30% of breast cancers regress after endocrine therapy, and a recent approach has been to select likely hormone-sensitive tumors on the basis of estrogen and progesterone receptor levels (9). However, possession of receptors is not the sole criterion for response to therapy and in any case regression is at best temporary, to be followed by hormone-independent tumors and metastatic disease. This progression to hormone insensitivity is a major clinical problem.Tumors are composed of mixed populations of cells that have a wide range of phenotypes. Cells obtained from individual rodent mammary tumors differ not only in hormone receptors (35) and hormone-dependent growth (8, 39) but also in immunological properties (24), transplantability (36), metastatic capability (18), tumorigenicity (38), drug resistance (23), growth rate (11), karyotype (11,22), mouse mammary tumor virus (MMTV) 1 expression (35), and MMTV proviral Abbreviations used in this paper. +A and -A, androgen responsive and unresponsive, respectively; DC, dextran-charcoal; DME, Dulbeccopies (32). It is now accepted that the origin of mammary tumors is monoclonal (10), but how such diversity arises is still unknown. The mechanism could involve either genotypic or phenotypic change. Evidence for a genotypic mechanism, involving mutation followed by cell selection, has been implied from karyotype studies (11,22,25) and from changes in exogenous integrated MMTV (32). A phenotypic mechanism would involve stable alteration in the program of gene expression and could result from either sele...

Steroid 5.ALPHA. Reductase mRNA Type I is Differentially Regulated by Androgens and Glucocorticoids in the Rat Liver

“…It has been previously demonstrated [20] that in vitro methylation of ARE occurs as a consequence of hormone removal from MMTV-LTR transfected mammary tumor cells [20]. This hypermethylation has been shown to be accompanied by irreversible loss of sensitivity to androgen actions [32,[36][37][38]. Our data do not address the question of whether eight days of androgen deprivation, in vivo, is sufficient to cause irreversible loss of sensitivity to androgen action on steroid 5aR type I and Andro-UDPGT gene expression.…”

“…First, differential effects of these two steroids on several gene products and cellular functions have been demonstrated, such as androgen receptor concentrations [24,31], mouse mammary tumor virus (MMTV)-LTR related transcripts [21,[32][33][34] and growth rates of tumor cell lines [31][32][33][34]. Secondly, there is substantial homology between sequences mediating androgen effects and DNA sequences known to be essential for glucocorticoid responses as in the case of MMTV-LTR [21,[22][23][24][25][26][27][28][29][30][31][32][33][34][35]. Finally, defective formation of 5a-reduced androgens and glucocorticoids has been demonstrated in patients with a genetic deficiency of steroid 5aR [23,24] suggesting that both steroids can serve as substrates for a single enzyme.…”

Factors controlling the expression of mouse mammary tumour virus

“…This suggests that methylation of proviral DNA may prevent its expression in hepatocytes. Conversely, the transition of mammary cells that express MMTV from a hormone-responsive to a hormone-unresponsive state is accompanied by a loss of MMTV expression and an increase in methylation (Darbre et al, 1983;Darbre & King, 1984). However, hypomethylation alone appears not to be sufficient for expression since at least one provirus (Mtv-20) is hypomethylated in a number of mouse organs and yet there is no evidence for the expression of this provirus .…”

Androgen regulation by the long terminal repeat of mouse mammary tumor virus.

“…We have been studying the way in which steroids regulate cell growth and MMTV-related RNA in the S115 mouse mammary tumor cell line (4,6,7). Our data indicate that both androgens and glucocorticoids regulate MMTV RNA, whereas previous work showed that only glucocorticoids were active (35).…”