Effects of estradiol and medroxyprogesterone acetate on expression of the cell cycle proteins cyclin D1, p21 and p27 in cultured human breast tissues.

Estrogen and progesterone are key regulators of normal breast epithelial cell proliferation and differentiation. They are also involved in the initiation and progression of breast tumorigenesis. Several experimental studies have demonstrated that steroid hormones affect cell cycle proteins associated with tumor initiation and progression. Hormone replacement therapy (HT) is widely used to alleviate climacteric symptoms among postmenopausal women. Little is known, however, about cell cycle protein regulation during hormonal treatment of human breast tissue (HBT). In this study we aimed to evaluate the effects of 17beta-estradiol (E(2)) and medroxyprogesterone acetate (MPA) on cultured HBTs representing samples from reduction mammoplasty of premenopausal (pre-HBT) and postmenopausal (postm-HBT) women, and from peritumoral tissue (peritum-HBT) after breast tumor surgery among postmenopausal patients. Explants of HBT were cultured for 14 days in medium supplemented with E(2), MPA or E(2) + MPA. Expression of cyclin D1, p21 and p27 was assessed by immunohistochemical staining of explants cultured for 2 and 14 days. Further, Ki-67 staining was performed to evaluate correlation between proliferation and cell cycle regulatory protein expression. Our results showed that HBTs studied were positive for ERalpha, ERbeta and PR (> or =10% of the cells stained). The level of p21 was lower (p < 0.001) in pre-HBT than in postm-HBT, whereas p27 levels were higher (p < 0.05) in pre-HBT than in postm- and peritum-HBT. The level of Ki67 positive cells was higher in pre- HBT than in post-HBT. Interestingly, level of p21 positive cells showed an opposite pattern. Treatment with E(2) increased the relative number of cyclin D1-staining cells and decreased that of p27-staining cells in postm-HBT (p < 0.05), but not in pre-HBT. All hormone regimens (E(2), MPA, E(2) + MPA) increased the number of p21-positive cells in postm-HBTs at 14 days and E(2) even at 2 days. In pre-HBT p21 staining was increased (p < 0.05) in explants cultured with E(2) for 14 days but no response was observed in cyclin D1 and p27. The number of cyclin D1-staining cells was clearly higher (p < 0.05) in peritum-HBT than in non-tumorous pre- or postm-HBT, but the response cyclin D1 to all hormonal treatments in peritum-HBT was the same as in postm-HBT. Moreover, we found that E(2), MPA, E(2) + MPA in vitro increased numbers of Ki67 positive cells in post-HBTs at 14 days and E(2) also in pre-HBT. Stimulated proliferation rate was associated with increase of cyclin D1 and p21-positive cells and reduced numbers of p27, especially in post-HBTs. Taken together, our results suggest that cell cycle regulatory proteins are more sensitive to exogenous hormone treatment in postm-HBT than in pre-HBT.