Santoni-Rugiu E, Falck J, Mailand N, Bartek J, Lukas J
Institute of Cancer Biology, Danish Cancer Society, DK-2100 Copenhagen O., Denmark.
Mol Cell Biol. 2000 May;20(10):3497-509. doi: 10.1128/MCB.20.10.3497-3509.2000.
The retinoblastoma protein (pRb)/E2F pathway regulates commitment of mammalian cells to replicate DNA. On the other hand, mitogen-stimulated cells deprived of E2F activity can still maintain physiologically relevant levels of cyclin E-dependent kinase activity and gradually enter S phase, suggesting the existence of a DNA synthesis-inducing mechanism parallel to the pRb/E2F axis. Here we show that regulatable ectopic expression of cyclin E or transcriptionally active Myc can rapidly induce DNA synthesis in U2OS-derived cell lines whose E2F activity is blocked by a constitutively active pRb (pRbDeltacdk) mutant. The effect of Myc is associated with Cdc25A phosphatase and cyclin E-CDK2 kinase activation and abolished by antagonizing Myc activity with the dominant-negative (dn) MadMyc chimera. Moreover, while abrogation of either endogenous E2F or Myc activity only delays and lowers DNA synthesis in synchronized U2OS cells or rat diploid fibroblasts, concomitant neutralization of both abolishes it. Whereas ectopic Myc and E2F1 rescue the G(1)/S delay caused by pRbDeltacdk (or dnDP1) and MadMyc, respectively, cyclin E or Cdc25A can restore DNA replication even in cells concomitantly exposed to pRbDeltacdk and MadMyc. However, coexpression of dnCDK2 neutralizes all of these rescuing effects. Finally, proper transcription of cyclin E and Cdc25A at the G(1)/S transition requires both Myc and E2F activities, and subthreshold levels of ectopic cyclin E and Cdc25A synergistically restore DNA synthesis in cells with silenced Myc and E2F activities. These results suggest that Myc controls a G(1)/S-promoting mechanism regulating cyclin E-CDK2 in parallel to the "classical" pRb/E2F pathway.
视网膜母细胞瘤蛋白（pRb）/E2F通路调节哺乳动物细胞进行DNA复制的进程。另一方面，缺乏E2F活性的有丝分裂原刺激细胞仍能维持细胞周期蛋白E依赖性激酶活性的生理相关水平，并逐渐进入S期，这表明存在一种与pRb/E2F轴平行的DNA合成诱导机制。在此我们表明，细胞周期蛋白E的可调节异位表达或转录活性Myc能在U2OS衍生细胞系中快速诱导DNA合成，这些细胞系的E2F活性被组成型活性pRb（pRbDeltacdk）突变体阻断。Myc的作用与Cdc25A磷酸酶和细胞周期蛋白E-CDK2激酶的激活相关，并可通过用显性负性（dn）MadMyc嵌合体拮抗Myc活性而消除。此外，虽然内源性E2F或Myc活性的缺失仅会延迟并降低同步化的U2OS细胞或大鼠二倍体成纤维细胞中的DNA合成，但两者同时失活则会使其完全消除。异位表达的Myc和E2F1分别挽救了由pRbDeltacdk（或dnDP1）和MadMyc导致的G(1)/S期延迟，而细胞周期蛋白E或Cdc25A即使在同时暴露于pRbDeltacdk和MadMyc的细胞中也能恢复DNA复制。然而，共表达dnCDK2会中和所有这些挽救作用。最后，细胞周期蛋白E和Cdc25A在G(1)/S期转换时的正常转录需要Myc和E2F活性，并且异位表达的细胞周期蛋白E和Cdc25A的亚阈值水平能协同恢复Myc和E2F活性沉默细胞中的DNA合成。这些结果表明，Myc控制着一种与“经典”pRb/E2F通路平行的、促进G(1)/S期进程并调节细胞周期蛋白E-CDK2的机制。
