Molecular Pharmacology Group, Departament de Medicina Experimental, IRBLLEIDA-Universitat de Lleida, C/Montserrat Roig 2, 25008, Lleida, Catalunya, Spain.
Apoptosis. 2011 Jul;16(7):660-70. doi: 10.1007/s10495-011-0603-3.
Chemical inhibitors of cyclin-dependent kinase (CDK), like roscovitine, are promising drugs in the context of new cancer therapies. Roscovitine and related compounds, like seliciclib and olomoucine, are effective inducers of apoptosis in many proliferating cells in culture. These compounds are known to activate the intrinsic or mitochondrial pathway of apoptosis. In order to better characterize this intrinsic pathway, a transcriptional analysis was performed using the reverse transcriptase-multiplex ligation-dependent probe amplification procedure (RT-MLPA). In five cell lines, we detected an early and marked reduction of most transcripts, which is consistent with the disruption of transcription that results from the inhibition of CDK7 and CDK9. However, the mRNA of p53-upregulated modulator of apoptosis (PUMA) gene escaped from this transcription inhibition in neuroblastoma cells with a functional p53 protein. The increase of PUMA mRNA was not found in roscovitine-treated cell lines defective in p53, which underwent apoptosis like their p53 proficient counterparts. In addition, in SH-SY5Y cells, sublethal and lethal concentrations of roscovitine produced equivalent increases of PUMA mRNA and protein. In conclusion, the increased expression of PUMA was not associated with apoptosis induction. On the contrary, mRNA and protein depletion of MCL-1 gene correlated the best with cell demise. Moreover, NOXA protein suffered a far minor decrease than MCL-1. Because of the selective neutralization of NOXA by MCL-1, we hypothesize that the disruption of this balance is a critical event in apoptosis induction by roscovitine and related compounds.
细胞周期蛋白依赖性激酶（CDK）的化学抑制剂，如罗沙司汀，在新的癌症治疗中是很有前途的药物。罗沙司汀和相关化合物，如塞利昔布和olomoucine，在培养中的许多增殖细胞中是有效的凋亡诱导剂。这些化合物已知能激活凋亡的内在或线粒体途径。为了更好地描述这种内在途径，使用逆转录-多重连接依赖的探针扩增程序（RT-MLPA）进行了转录分析。在五种细胞系中，我们检测到大多数转录本的早期和显著减少，这与 CDK7 和 CDK9 抑制导致的转录中断一致。然而，在具有功能性 p53 蛋白的神经母细胞瘤细胞中，p53 上调凋亡调节剂（PUMA）基因的 mRNA 逃避了这种转录抑制。在 p53 缺陷的罗沙司汀处理细胞系中未发现 PUMA mRNA 的增加，这些细胞像其 p53 功能正常的细胞一样发生凋亡。此外，在 SH-SY5Y 细胞中，亚致死和致死浓度的罗沙司汀产生了相当程度的 PUMA mRNA 和蛋白的增加。总之，PUMA 的表达增加与凋亡诱导无关。相反，MCL-1 基因的 mRNA 和蛋白耗竭与细胞死亡最相关。此外，NOXA 蛋白的减少远小于 MCL-1。由于 MCL-1 对 NOXA 的选择性中和，我们假设这种平衡的破坏是罗沙司汀和相关化合物诱导凋亡的一个关键事件。
