Differential regulation of retinoblastoma protein by hormonal and antihormonal agents in T47D breast cancer cells

Phosphorylation of the rumor suppressor protein, retinoblastoma (pRb), regu lates the progression of the cell cycle. Previous work from this laboratory had shown that estradiol (E-2) regulates tumor suppressor proteins, p53 an d retinoblastoma in breast cancer cells. In the present study, we have exam ined the phosphorylation of pRB in T47D breast cancer cells following treat ments with R5020 and antiprogestins. In growth medium containing serum depl eted of endogenous steroids by charcoal treatment, pRb appeared mainly in i ts hypophosphorylated form. Addition of 10 nM R5020 to the culture medium c aused hyperphosphorylation of pRb within 24 h, but the hypophosphorylated f orm of pRb began to accumulate after 72 h. Upon prolonged R5020 treatment ( 72-96 h), pRb was detected exclusively in its hypophosphorylated form. Whil e treatment of cells with R5020 caused a transient increase in the level of cyclin D1. E-2 addition caused a sustained increase in the level of cyclin D1 consistent with its role in stimulating pRb phosphorylation. Antagonist s of both estrogen receptor (ER) and progesterone receptor (PR) blocked the E-2 and R5020-induced pRb phosphorylation, respectively. These results sug gest that R5020 induces pRb phosphorylation via a transient increased expre ssion of cyclin D1, whereas E-2 treatment results in sustained expression o f cyclin D1 and increased pRb phosphorylation. Furthermore, R5020 effects o n pRb phosphorylation appear PR-mediated as no cross-antagonism of pRb phos phorylation was observed: the R5020 effects were blocked by RU486 and ZK982 99, but not by the pure ER antagonist. ICI 182, 780 (ICI). (C) 2001 Elsevie r Science Ltd. All rights reserved.