Oestrogenic activity of tamoxifen and its metabolites on gene regulation and cell proliferation in MCF-7 breast cancer cells

Abstract

Summary The effects of tamoxifen, three of its in vivo metabolites and 3-hydroxytamoxifen on cellular proliferation and the induction of four oestrogen-regulated RNAs (pNR-1, pNR-2, pNR-25 and cathepsin D) have been measured in MCF-7 breast cancer cells in phenol red-free culture medium. Tamoxifen and 3-hydroxytamoxifen acted as partial oestrogens to stimulate cell growth and the levels of the pNR-2 and pNR-25 RNAs. They were full oestrogens for the induction of cathepsin D RNA and induced the pNR-l RNA above the level found in oestrogen-treated cells. N-Desmethyltamoxifen and 4-hydroxytamoxifen behaved like tamoxifen except that N-desmethyltamoxifen did not induce the pNR-2 RNA and was only a partial oestrogen for the induction of cathepsin D RNA, and 4-hydroxytamoxifen did not induce the pNR-2 or pNR-25 RNAs. In the presence of oestradiol, the four anti-oestrogens prevented the stimulation of growth and reduced (pNR-2 and pNR-25) or increased (pNR-1) the RNA levels to those present in MCF-7 cells treated with the anti-oestrogen alone. In contrast, for cathepsin D RNA levels there was a synergistic effect of the anti-oestrogens and oestradiol. The concentration at which each anti-oestrogen was effective was related to its affinity for the oestrogen receptor. Metabolite E was a full oestrogen for the induction of cell proliferation and the oestrogen-regulated RNAs. pNR-25 and pNR-2 RNA levels correlated most closely with effects on cell proliferation. These RNAs are therefore potentially the most useful for predicting the response of breast cancer patients to tamoxifen therapy.

Modulation of the proliferative response of breast cancer cells to growth factors by oestrogen

“…This involves the use of phenol-red free medium supplemented with stripped serum and a withdrawal period which involves frequent changes of medium to remove the influences of steroids present in normal growth medium. These culture conditions have been used to identify oestrogen regulated mRNAs in breast cancer cells (May & Westley, 1988) and to study the effects of antioestrogens on the expression of oestrogen regulated genes (Johnson et al, 1989).…”

“…Overall, bFGF had the greatest effect on cell proliferation although this was less marked than the previously described effect of the IGFs and insulin. (Lippman et al, 1976;Johnson et al, 1989). They therefore provide useful systems for studying the interactions between various growth factors and for understanding the mechanisms involved in the stimulation of proliferation by oestrogens and the interactions between growth factors and steroids.…”

“…Cells (10,000) were plated in 16-mm diameter wells in 0.5 ml and allowed to attach over 2 days. Prior to treatment, the cells were progressively withdrawn from the oestrogens present in the normal growth medium as described by Johnson et al (1989). This involved culturing the cells in phenol red-free modified Eagle's medium supplemented with charcoal-treated newborn calf serum (10%) and insulin (1 ,tg ml-1) for 4 days.…”

“…DNA assay DNA was measured using bisbenzimidazole (Hoechst 33258) as described previously (Johnson et al, 1989). FGF binding FGF binding was measured essentially as described by Kan et al (1988).…”

“…Growth experiments were performed as described previously (Johnson et al, 1989). Cells (10,000) were plated in 16-mm diameter wells in 0.5 ml and allowed to attach over 2 days.…”