Department of Genetics, Case Western Reserve University, Cleveland, OH 44106, USA.
Cancer Lett. 2010 Oct 28;296(2):225-32. doi: 10.1016/j.canlet.2010.04.012. Epub 2010 May 4.
The tumor suppressor protein p53 plays a key role in regulation of negative cellular growth in response to EGCG. To further explore the role of p53 signaling and elucidate the molecular mechanism, we employed colon cancer HCT116 cell line and its derivatives in which a specific transcriptional target of p53 is knocked down by homologous recombination. Cells expressing p53 and p21 accumulate in G1 upon treatment with EGCG. In contrast, same cells lacking p21 traverse through the cell cycle and eventually undergo apoptosis as revealed by TUNEL staining. Treatment with EGCG leads to induction of p53, p21 and PUMA in p21 wild-type, and p53 and PUMA in p21(-/-) cells. Ablation of p53 by RNAi protects p21(-/-) cells, thus indicating a p53-dependent apoptosis by EGCG. Furthermore, analysis of cells lacking PUMA or Bax with or without p21 but with p53 reveals that all the cells expressing p53 and p21 survived after EGCG treatment. More interestingly, cells lacking both PUMA and p21 survived ECGC treatment whereas those lacking p21 and Bax did not. Taken together, our results present a novel concept wherein p21-dependent growth arrest pre-empts and protects cells from otherwise, in its absence, apoptosis which is mediated by activation of pro-apoptotic protein PUMA. Furthermore, we find that p53-dependent activation of PUMA in response to EGCG directly leads to apoptosis with out requiring Bax as is the case in response to agents that induce DNA damage. p21, thus can be used as a molecular switch for therapeutic intervention of colon cancer.
抑癌蛋白 p53 在调节 EGCG 诱导的细胞负性生长中发挥关键作用。为了进一步探讨 p53 信号通路的作用并阐明其分子机制，我们采用结肠癌细胞系 HCT116 及其衍生细胞系，通过同源重组敲除 p53 的特定转录靶基因。用 EGCG 处理表达 p53 和 p21 的细胞后，细胞在 G1 期积累。相比之下，同样缺乏 p21 的细胞会穿过细胞周期，最终通过 TUNEL 染色显示发生凋亡。用 EGCG 处理可诱导 p53、p21 和 PUMA 在 p21 野生型细胞中表达，以及 p53 和 PUMA 在 p21(-/-)细胞中表达。用 RNAi 敲除 p53 可保护 p21(-/-)细胞，表明 EGCG 通过 p53 诱导细胞凋亡。此外，分析缺乏 PUMA 或 Bax 的细胞，无论是否有 p21 但有 p53 的情况下，所有表达 p53 和 p21 的细胞在用 EGCG 处理后都存活下来。更有趣的是，缺乏 PUMA 和 p21 的细胞在用 ECGC 处理后存活下来，而缺乏 p21 和 Bax 的细胞则没有。总之，我们的研究结果提出了一个新的概念，即在缺乏 p21 的情况下，p21 依赖性的生长抑制会预先阻止细胞凋亡，而凋亡是由促凋亡蛋白 PUMA 的激活介导的。此外，我们发现，EGCG 诱导的 p53 依赖性 PUMA 激活直接导致凋亡，而不需要 Bax，这与诱导 DNA 损伤的药物的情况不同。因此，p21 可以作为结肠癌治疗干预的分子开关。
