PROGRESSION OF LNCAP PROSTATE TUMOR-CELLS DURING ANDROGEN DEPRIVATION- HORMONE-INDEPENDENT GROWTH, REPRESSION OF PROLIFERATION BY ANDROGEN, AND ROLE FOR P27(KIP1) IN ANDROGEN-INDUCED CELL-CYCLE ARREST

The molecular mechanism of androgen-independent growth of prostate can cer after androgen ablation was explored in LNCaP cells. An androgen-d ependent clonal subline of the LNCaP human prostate carcinoma cell lin e, LNCaP 104-S, progressed to a slow growing stage (104-R1) and then t o a faster growing stage (104-R2) during more than 2 yr of continuous culture in the absence of androgen. Androgen-induced proliferation of 104-S cells is inhibited by the antiandrogen Casodex, while proliferat ion of 104-R1 and 104-R2 cells is unaffected by Casodex. This indicate s that proliferation of 104-R1 and 104-R2 cells is not supported by lo w levels of androgen in the culture medium. Compared with LNCaP 104-S cells, both 104-R1 and 104-R2 cells express higher basal levels of and rogen receptor (AR), and proliferation of these two cell lines is para doxically repressed by androgen. After continuous passage in androgen- containing medium, 104-R1 cells reverted back to an androgen-dependent phenotype. The mechanism of androgenic repression of 104-R1 and 104-R 2 sublines was further evaluated by examining the role of critical reg ulatory factors involved in the control of cell cycle progression. At concentrations that repressed growth, androgen transiently induced the expression of the cyclin-dependent kinase (cdk) inhibitor p21(waf1/ci p1) in 104-R1 cells, while expression of the cdk inhibitor p27(Kip1) w as persistently induced by androgen in both 104-R1 and 104-R2 cells. I nduced expression of murine p27(Kip1) in 104-R2 cells resulted in G1 a rrest. Specific immunoprecipitates of Cdk2 but not Cdk4 from androgen- treated 104-R1 cells contained both p21(waf1/cip1) and p27(Kip1). This observation was confirmed by in vitro assay of histone H1 and Rb (ret inoblastoma protein) phosphorylation by the proteins associated with t he immune complex. Furthermore, inhibition of Cdk2 activity correlated with the accumulation of p27(Kip1) and not p21(waf1/cip1). From these results we conclude that androgenic repression of LNCaP 104-R1 and 10 4-R2 cell proliferation is due to the induction of p27(Kip1) which in turn inhibits Cdk2, a factor critical for cell cycle progression and p roliferation.