Antiapoptotic action of prolactin is associated with up-regulation of Bcl-2 and down-regulation of Bax in HC11 mouse mammary epithelial cells

The effect of prolactin on apoptosis and the expression of bcl-2 and bax in HC11 mouse mammary epithelial cells were investigated. Flow cytometric ana lysis of Bcl-2 level (FITC-conjugated monoclonal anti-Bcl-2 antibody and FI TC-conjugated monoclonal anti-IgG(1) antibody as a negative control), numbe r of apoptotic cells and cell cycle phases (DNA stained with DAPI) was perf ormed. Bax transcript was measured using the RT-PCR method with GAPDH servi ng as a reference gene. Administration of prolactin (5 mu g/ml) in the pres ence of insulin stimulated differentiation of mammary epithelial cells, whi ch manifested in stopping cells at G(0)/G(1) phase, cell swelling and incre ase of cell number with enhanced protein content. Moreover, prolactin highl y significantly reduced the extent of apoptosis of HC11 cells during 48 h o f incubation. Nevertheless, the apoptotic cell number rose with increased t ime length of cell culture, probably due to the resulting high cell density and EGF withdrawal from the incubation medium. The antiapoptotic effect of prolactin was associated with up-regulation of bcl-2 expression, shown as an increase in cell numbers expressing this protooncogene and elevated Bcl- 2 content in these cells. A negative relationship (r = -0.87, p less than o r equal to 0.001) between the number of apoptotic cells and those expressin g bcl-2 was also found. Prolactin administration lowered Bax transcript by 68.8% and 70.7% after 3 and 6 h, respectively. In conclusion, the results p resented indicate that stimulation of bcl-2 expression with simultaneous su ppression of bax may be key events in the mechanism of antiapoptotic action of prolactin in HC11 mammary epithelial cells.