Strobeck M W, Fribourg A F, Puga A, Knudsen E S
Department of Cell Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio, OH 45267-0521, USA.
Oncogene. 2000 Apr 6;19(15):1857-67. doi: 10.1038/sj.onc.1203510.
Phosphorylation/inactivation of RB is typically required for cell cycle progression. However, we have identified a tumor cell line, C33A, which progresses through the cell cycle in the presence of an active allele of RB (PSM-RB). To determine how C33A cells evade RB-mediated arrest, we compared RB signaling to downstream effectors in this resistant cell line to that of the RB-sensitive SAOS-2 cell line. Although introduction of PSM-RB repressed E2F-mediated transcription in both C33A and SAOS-2 cells, PSM-RB failed to repress Cyclin A promoter activity in C33A. Ectopic expression of PSM-RB in SAOS-2 cells resulted in a decrease in both Cyclin A and Cdk2 protein levels without affecting Cyclin E or Cdk4. In contrast, over-expression of PSM-RB in C33A cells did not alter endogenous Cyclin A, Cyclin E, or Cdk2 protein levels or impact Cdk2 kinase activity, indicating that signaling from RB to down-stream targets is abrogated in this cell line. The importance of Cdk2 activity was demonstrated by p27Kip1, which attenuated Cdk2 activity and inhibited cell cycle progression in C33A cells. Since RB signaling to Cdk2 is disrupted in these tumor cells, we co-expressed two proteins that cooperate with RB in transcriptional repression, AHR and BRG-1, in an attempt to correct this signaling dysfunction. Co-expression of AHR/BRG-1 with PSM-RB attenuated Cyclin A and Cdk2 expression as well as Cdk2-associated kinase activity, resulting in cell cycle inhibition of C33A cells. Importantly, ectopic expression of Cyclin A was able to reverse the arrest mediated by co-expression of AHR/BRG-1 with PSM-RB. These results indicate that down-regulation of Cdk2 activity is requisite for RB-mediated cell cycle arrest. Thus, this study reveals a new mechanism through which tumor cells evade anti-proliferative signals, and provides insight into how RB-signaling is mediated.
细胞周期进程通常需要RB的磷酸化/失活。然而,我们鉴定出一种肿瘤细胞系C33A,它在存在RB活性等位基因(PSM-RB)的情况下仍能进行细胞周期进程。为了确定C33A细胞如何逃避RB介导的细胞周期停滞,我们将这种耐药细胞系中RB信号传导至下游效应器的情况与RB敏感的SAOS-2细胞系进行了比较。尽管引入PSM-RB在C33A和SAOS-2细胞中均抑制了E2F介导的转录,但PSM-RB未能抑制C33A中细胞周期蛋白A启动子的活性。在SAOS-2细胞中异位表达PSM-RB导致细胞周期蛋白A和Cdk2蛋白水平均下降,而不影响细胞周期蛋白E或Cdk4。相反,在C33A细胞中过表达PSM-RB并未改变内源性细胞周期蛋白A、细胞周期蛋白E或Cdk2蛋白水平,也未影响Cdk2激酶活性,这表明在该细胞系中从RB到下游靶点的信号传导被消除。p27Kip1证明了Cdk2活性的重要性,它减弱了Cdk2活性并抑制了C33A细胞的细胞周期进程。由于在这些肿瘤细胞中RB向Cdk2的信号传导被破坏,我们共表达了两种在转录抑制中与RB协同作用的蛋白AHR和BRG-1,试图纠正这种信号功能障碍。AHR/BRG-1与PSM-RB共表达减弱了细胞周期蛋白A和Cdk2的表达以及与Cdk2相关联的激酶活性,导致C33A细胞的细胞周期受到抑制。重要的是,细胞周期蛋白A的异位表达能够逆转由AHR/BRG-1与PSM-RB共表达介导的细胞周期停滞。这些结果表明Cdk2活性的下调是RB介导的细胞周期停滞所必需的。因此,本研究揭示了肿瘤细胞逃避抗增殖信号的一种新机制,并为RB信号传导的介导方式提供了见解。