Dirks P B, Rutka J T, Hubbard S L, Mondal S, Hamel P A
Division of Neurosurgery, Hospital for Sick Children, University of Toronto, Ontario, Canada.
Oncogene. 1998 Aug 20;17(7):867-76. doi: 10.1038/sj.onc.1202008.
We previously demonstrated that P16Ink4a (p16) expression in p16-deficient U343 astrocytoma cells causes a G1 cell cycle arrest, profound changes in cytoskeletal proteins and alterations in expression and activity of the pRB and E2F family proteins. We examine here the effects of expressing wild type or mutant versions of the downstream targets of p16 in U343 astrocytomas. We first attempted to block proliferation of U343 cells using the dominant mutant of pRB, deltap34. Expression of this mutant in the human osteosarcoma, SAOS-2, potently blocked proliferation but did not affect the cell cycle of U343 cells. We next showed that expression of E2F-1, E2F-2, E2F-3 and E2F-4 are each able to overcome this p16-dependent cell cycle arrest but exhibit distinct biological activities. Adenoviral-mediated expression of E2F-1, E2F-2, E2F-3, or E2F-4 overcame the p16-dependent cell cycle block and induced alterations in cell morphology. E2F-5, only in conjunction with DP1, promoted cell cycle progression. For both E2F-1 and E2F-2, but not E2F-3 or E2F-5/DP1, cell cycle re-entry was associated with almost quantitative cell death. Only small numbers of dying cells were observed in E2F-4-expressing cultures. Expression of the different E2F's altered the expression of distinct sets of cell cycle regulatory proteins. E2F-1 induced endogenous E2F-4 expression and also caused an increase in pRB, p107 and cyclin E levels. Expression of E2F-4 caused a weak increase in E2F-1 levels but also strongly induced pRB, p107, p130 and cyclin E. However, E2F-1 and E2F-4 clearly regulate expression of distinct genes, demonstrated when E2F-4 caused a threefold increase in the levels of cdk2 whereas E2F-1 failed to increase in this cyclin dependent kinase. Similarly, expression of E2F-1 or E2F-2 were shown to have distinct effects on the expression of cdk2, cyclin E and pRB despite both of these closely related E2F-family members potently inducing cell death. Thus, E2F-1, E2F-2, E2F-3 and E2F-4 are able to overcome the p16-dependent proliferative block in U343 astrocytoma cells. While overcoming this cell cycle block, each of the E2F's uniquely affect the expression of a number of cell cycle regulatory proteins and have distinct abilities to promote cell death.
我们之前证明，在缺乏p16的U343星形细胞瘤细胞中表达P16Ink4a（p16）会导致G1期细胞周期停滞、细胞骨架蛋白发生深刻变化以及pRB和E2F家族蛋白的表达及活性改变。我们在此研究在U343星形细胞瘤中表达p16下游靶点的野生型或突变型的作用。我们首先尝试用pRB的显性突变体deltap34阻断U343细胞的增殖。该突变体在人骨肉瘤细胞SAOS-2中表达时能有效阻断增殖，但不影响U343细胞的细胞周期。接下来我们发现，E2F-1、E2F-2、E2F-3和E2F-4的表达均能克服这种p16依赖性细胞周期停滞，但表现出不同的生物学活性。腺病毒介导的E2F-1、E2F-2、E2F-3或E2F-4的表达克服了p16依赖性细胞周期阻滞并诱导细胞形态改变。E2F-5仅与DP1共同作用时能促进细胞周期进程。对于E2F-1和E2F-2而言，而非E2F-3或E2F-5/DP1，细胞周期重新进入与几乎定量的细胞死亡相关。在表达E2F-4的培养物中仅观察到少量死亡细胞。不同E2F的表达改变了不同组细胞周期调节蛋白的表达。E2F-1诱导内源性E2F-4表达，还导致pRB、p107和细胞周期蛋白E水平升高。E2F-4的表达使E2F-1水平略有升高，但也强烈诱导pRB、p107、p130和细胞周期蛋白E。然而，E2F-1和E2F-4明显调节不同基因的表达，当E2F-4使cdk2水平增加三倍而E2F-1未能使这种细胞周期蛋白依赖性激酶增加时得以证明。同样，尽管E2F-1和E2F-2这两个密切相关的E2F家族成员都能有效诱导细胞死亡，但它们对cdk2、细胞周期蛋白E和pRB的表达显示出不同的影响。因此，E2F-1、E2F-2、E2F-3和E2F-4能够克服U343星形细胞瘤细胞中的p16依赖性增殖阻滞。在克服这种细胞周期阻滞的同时，每个E2F都独特地影响多种细胞周期调节蛋白的表达，并具有不同的促进细胞死亡的能力。
