DNA from mammalian cells has been shown to contain significant amounts of 5-methyl cytosine resulting from enzymatic transfer of methyl groups from s-adenosylmethionine to cytosine residues in the DNA polymer. The function of this modification is not known . We have found that DNA synthesized during chemically induced differentiation of Friend erythroleukemia cells is hypomethylated, as measured by its ability to accept methyl groups transferred by homologous DNA methyltransferases in vitro. The extent of hypomethylation detected by this sensitive method is small, a decrease of < 1 .6% in 5-methylcytosine content .Hypomethylated DNA can be isolated from Friend erythroleukemia cells grown in the presence of dimethyl sulfoxide, butyrate, hexamethylene-bis-acetamide, pentamethylene-bis acetamide, and ethionine. However, hypomethylated DNA is found only under conditions where differentiation is actually induced . DNA isolated from cells of a dimethyl sulfoxideresistant subclone grown in the presence of that agent is not hypomethylated, although DNA of these cells becomes hypomethylated after growth in the presence of inducers that can trigger their differentiation. We also find that the DNA of Friend erythroleukemia cells does not become hypomethylated when the cells are exposed to inducing agents in the presence of substances that inhibit differentiation. These results suggest a close link between genome modification by methylation and differentiation of Friend erythroleukemia cells.

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There has been increasing evidence which suggests that abnormal expression of the estrogen receptor (ER) protein in nonmalignant breast tissue may be important in the carcinogenic process. To examine the effects of ER expression in immortalized nonmalignant mammary epithelial cells, an expression vector containing human ER cDNA was transfected into the ER negative human breast cells, MCF10A. Characterization of a clone stably expressing ER, 139B6, provided evidence for the regulated synthesis of a functional ER capable of binding estradiol-17 beta (E2) and undergoing processing. Expression of the ER gene did not enable E2 to stimulate endogenous genes [progesterone receptor (PgR), pS2, cathepsin D and TGF alpha] which normally respond to estrogens in breast cancer cells. The ER in 139B6 cells was, however, capable of inducing expression of an ERE-regulated reporter gene, indicating its ability to interact with transcriptional machinery. Furthermore, cultures in log growth displayed a slight increase in doubling time in the presence of E2. These results indicate that ER expression alone is not sufficient to induce a transformed phenotype. Thus, the 139B6 cell line should provide a new model for determining what additional changes lead to increased growth potential in response to E2 and for exploring how E2 itself may help bring about changes leading to progression of preneoplastic breast epithelial cells.

These experiments were designed to examine the effect of structural modifications to the estradiol-17 beta (E2) molecule on the estrogen response element (ERE) dependent activation of the thymidine kinase (tk) promoter. Estrogen receptor (ER) positive MCF-7 cells were transfected with plasmids containing one or two vitellogenin EREs inserted upstream of the tk promoter in p(-37)tk. Transient expression of the CAT gene in these constructs was measured after cells had been maintained for 36-42 h in the presence of E2 or an E2 analogue. E2 induced CAT expression at levels as low as 10(-13) M, with maximum induction at 10(-11) M. CAT activity decreased at higher concentrations of E2. Estratriene, which has low affinity for ER, was active only at micromolar concentrations. 3-Hydroxyestratriene displayed maximal activity at 10(-9) M, with higher levels being less active. Still higher concentrations (10(-7) M) of estratrien-17 beta-ol were required to induce maximum CAT activity. All positional and conformational alterations in the D-ring hydroxyl group of E2 yielded active ligands. Movement of the phenolic hydroxyl group of E2 to other positions on the A-ring produced dihydroxyestrogens with varied capacities to activate CAT (2-hydroxyestratrien-17 beta-ol produced maximum CAT activation at 10(-11) M; 1-hydroxyestratrien-17 beta-ol required a 10(-8) M concentration for maximum activity; 4-hydroxyestratrien-17 beta-ol gave maximum CAT activation at 10(-6) M). Only those androstanediols or 5-androstenediols with a 3 beta-hydroxyl group were capable of activating CAT expression.(ABSTRACT TRUNCATED AT 250 WORDS)

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