Cifuentes Eugenia, Croxen Richard, Menon Mani, Barrack Evelyn R, Reddy G Prem-Veer
Vattikuti Urology Institute, Henry Ford Health Sciences Center, Detroit, Michigan 48202, USA.
J Cell Physiol. 2003 Jun;195(3):337-45. doi: 10.1002/jcp.10317.
Androgen-ablation is a most commonly prescribed treatment for metastatic prostate cancer but it is not curative. Development of new strategies for treatment of prostate cancer is limited partly by a lack of full understanding of the mechanism by which androgen regulates prostate cancer cell proliferation. This is due, mainly, to the limitations in currently available experimental models to distinguish androgen/androgen receptor (AR)-induced events specific to proliferation from those that are required for cell viability. We have, therefore, developed an experimental model system in which both androgen-sensitive (LNCaP) and androgen-independent (DU145) prostate cancer cells can be reversibly blocked in G(0)/G(1) phase of cell cycle by isoleucine deprivation without affecting their viability. Pulse-labeling studies with (3)H-thymidine indicated that isoleucine-deprivation caused LNCaP and DU145 cells to arrest at a point in G(1) phase which is 12-15 and 6-8 h, respectively, before the start of S phase and that their progression into S phase was dependent on serum factors. Furthermore, LNCaP, but not DU145, cells required AR activity for progression from G(1) into S phase. Western blot analysis of the cell extracts prepared at regular intervals following release from isoleucine-block revealed remarkable differences in the expression of cyclin E, p21(Cip1), p27(Kip1), and Rb at the protein level between LNCaP and DU145 cells during progression from G(1) into S phase. However, in both cell types Cdk-2 activity associated with cyclin E and cyclin A showed an increase only when the cells transited from G(1) into S phase. These observations were further corroborated by studies using exponentially growing cells that were enriched in specific phases of the cell cycle by centrifugal elutriation. These studies demonstrate usefulness of the isoleucine-deprivation method for synchronization of androgen-sensitive and androgen-independent prostate cancer cells, and for examining the role of androgen and AR in progression of androgen-sensitive prostate cancer cells from G(1) into S phase.
雄激素剥夺疗法是转移性前列腺癌最常用的治疗方法，但并非根治性疗法。前列腺癌治疗新策略的发展在一定程度上受到限制，原因是对雄激素调节前列腺癌细胞增殖机制的全面理解不足。这主要归因于当前可用实验模型存在局限性，难以区分雄激素/雄激素受体（AR）诱导的特定于增殖的事件与细胞存活所需的事件。因此，我们开发了一种实验模型系统，在该系统中，通过异亮氨酸剥夺，雄激素敏感（LNCaP）和雄激素非依赖（DU145）前列腺癌细胞均可在细胞周期的G(0)/G(1)期被可逆性阻断，且不影响其活力。用(3)H-胸腺嘧啶进行的脉冲标记研究表明，异亮氨酸剥夺使LNCaP和DU145细胞分别在S期开始前12 - 15小时和6 - 8小时停滞在G(1)期的某一点，且它们进入S期依赖于血清因子。此外，LNCaP细胞（而非DU145细胞）从G(1)期进入S期需要AR活性。对从异亮氨酸阻断释放后定期制备的细胞提取物进行的蛋白质印迹分析显示，在从G(1)期进入S期的过程中，LNCaP和DU145细胞在细胞周期蛋白E、p21(Cip1)、p27(Kip1)和Rb的蛋白水平表达上存在显著差异。然而，在两种细胞类型中，与细胞周期蛋白E和细胞周期蛋白A相关的Cdk - 2活性仅在细胞从G(1)期过渡到S期时增加。使用通过离心淘析在细胞周期特定阶段富集的指数生长细胞进行的研究进一步证实了这些观察结果。这些研究证明了异亮氨酸剥夺方法在同步雄激素敏感和雄激素非依赖前列腺癌细胞以及研究雄激素和AR在雄激素敏感前列腺癌细胞从G(1)期进入S期过程中的作用方面的实用性。
