Kokontis J M, Hay N, Liao S
Ben May Institute for Cancer Research, University of Chicago, Illinois 60637, USA.
Mol Endocrinol. 1998 Jul;12(7):941-53. doi: 10.1210/mend.12.7.0136.
The molecular mechanism of androgen-independent growth of prostate cancer after androgen ablation was explored in LNCaP cells. An androgen-dependent clonal subline of the LNCaP human prostate carcinoma cell line, LNCaP 104-S, progressed to a slow growing stage (104-R1) and then to 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 proliferation of 104-R1 and 104-R2 cells is unaffected by Casodex. This indicates that proliferation of 104-R1 and 104-R2 cells is not supported by low 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 androgen receptor (AR), and proliferation of these two cell lines is paradoxically 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-R2 sublines was further evaluated by examining the role of critical regulatory 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 p21waf1/cip1 in 104-R1 cells, while expression of the cdk inhibitor p27Kip1 was persistently induced by androgen in both 104-R1 and 104-R2 cells. Induced expression of murine p27Kip1 in 104-R2 cells resulted in G1 arrest. Specific immunoprecipitates of Cdk2 but not Cdk4 from androgen-treated 104-R1 cells contained both p21waf1/cip1 and p27Kip1. This observation was confirmed by in vitro assay of histone H1 and Rb (retinoblastoma protein) phosphorylation by the proteins associated with the immune complex. Furthermore, inhibition of Cdk2 activity correlated with the accumulation of p27Kip1 and not p21waf1/cip1. From these results we conclude that androgenic repression of LNCaP 104-R1 and 104-R2 cell proliferation is due to the induction of p27Kip1, which in turn inhibits Cdk2, a factor critical for cell cycle progression and proliferation.
在LNCaP细胞中探索了雄激素去除后前列腺癌雄激素非依赖性生长的分子机制。LNCaP人前列腺癌细胞系的雄激素依赖性克隆亚系LNCaP 104-S，在无雄激素的情况下连续培养超过2年的过程中，进入生长缓慢阶段（104-R1），然后进入生长较快阶段（104-R2）。抗雄激素药物康士得可抑制雄激素诱导的104-S细胞增殖，而104-R1和104-R2细胞的增殖不受康士得影响。这表明104-R1和104-R2细胞的增殖不受培养基中低水平雄激素的支持。与LNCaP 104-S细胞相比，104-R1和104-R2细胞均表达更高基础水平的雄激素受体（AR），并且这两种细胞系的增殖反而受到雄激素的抑制。在含雄激素的培养基中连续传代后，104-R1细胞恢复为雄激素依赖性表型。通过研究参与细胞周期进程调控的关键调节因子的作用，进一步评估了104-R1和104-R2亚系雄激素抑制的机制。在抑制生长的浓度下，雄激素可短暂诱导104-R1细胞中细胞周期蛋白依赖性激酶（cdk）抑制剂p21waf1/cip1的表达，而在104-R1和104-R2细胞中，雄激素持续诱导cdk抑制剂p27Kip1的表达。在104-R2细胞中诱导小鼠p27Kip1的表达导致G1期停滞。来自雄激素处理的104-R1细胞的Cdk2而非Cdk4的特异性免疫沉淀复合物中同时含有p21waf1/cip1和p27Kip1。通过与免疫复合物相关的蛋白质对组蛋白H1和视网膜母细胞瘤蛋白（Rb）磷酸化的体外测定证实了这一观察结果。此外，Cdk2活性的抑制与p27Kip1而非p21waf1/cip1的积累相关。从这些结果我们得出结论，LNCaP 104-R1和104-R2细胞增殖的雄激素抑制是由于p27Kip1的诱导，其反过来抑制Cdk2，而Cdk2是细胞周期进程和增殖的关键因子。
