Slebos R J, Lee M H, Plunkett B S, Kessis T D, Williams B O, Jacks T, Hedrick L, Kastan M B, Cho K R
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5320-4. doi: 10.1073/pnas.91.12.5320.
The cell cycle regulatory tumor suppressor proteins p53 and pRB are targeted for inactivation by several tumor viruses, including the high-risk types of human papillomaviruses (HPVs) via interactions of the HPV E6 and E7 oncoproteins with p53 and pRB, respectively. p53 plays a central role in a signal transduction pathway that mediates G1 arrest after DNA damage, though the mechanism by which G1 arrest occurs has not been elucidated. The cyclin-associated protein p21waf1/cip1 has recently been shown to be induced by p53 and to inhibit cyclin complex-mediated phosphorylation of pRB in vitro. Thus, we investigated a possible role for pRB in the p53-mediated DNA damage response. After gamma-irradiation, cells expressing wild-type p53 arrested in G1, contained increased levels of WAF1/CIP1 mRNA, and demonstrated accumulation of hypophosphorylated pRB. In contrast, cell lines with abnormal p53 genes or with p53 functionally inactivated by the E6 oncoprotein of HPV16 (a high-risk HPV) failed to arrest in G1, did not elevate WAF1/CIP1 mRNA, and did not accumulate hypophosphorylated pRB. Despite apparently normal elevation of p53 protein and WAF1/CIP1 mRNA after irradiation, cells expressing HPV16 E7 also failed to arrest in G1 and did not accumulate hypophosphorylated pRB. Disruption of RB genes alone did not totally abrogate this G1 arrest. Our results suggest that p53 indirectly regulates phosphorylation of pRB and that pRB and/or other pRB-like molecules that bind to HPV16 E7 participate in the DNA damage-mediated G1 arrest signal. In the process of HPV infection, the HPV E6 and E7 oncoproteins may undermine this cell cycle checkpoint, contributing to the accumulation of genetic alterations during tumorigenesis.
细胞周期调节性肿瘤抑制蛋白p53和pRB是几种肿瘤病毒的作用靶点，会被其灭活，其中包括高危型人乳头瘤病毒（HPV），HPV的E6和E7癌蛋白分别与p53和pRB相互作用来实现这一目的。p53在介导DNA损伤后G1期停滞的信号转导通路中起核心作用，不过G1期停滞发生的机制尚未阐明。细胞周期蛋白相关蛋白p21waf1/cip1最近被证明可由p53诱导产生，并在体外抑制细胞周期蛋白复合物介导的pRB磷酸化。因此，我们研究了pRB在p53介导的DNA损伤反应中可能发挥的作用。γ射线照射后，表达野生型p53的细胞停滞在G1期，WAF1/CIP1 mRNA水平升高，并出现低磷酸化pRB的积累。相比之下，p53基因异常或被HPV16（一种高危HPV）的E6癌蛋白功能失活的细胞系无法停滞在G1期，WAF1/CIP1 mRNA未升高，也没有积累低磷酸化pRB。尽管照射后p53蛋白和WAF1/CIP1 mRNA明显正常升高，但表达HPV16 E7的细胞也无法停滞在G1期，且没有积累低磷酸化pRB。单独破坏RB基因并没有完全消除这种G1期停滞。我们的结果表明，p53间接调节pRB的磷酸化，并且pRB和/或其他与HPV16 E7结合的pRB样分子参与了DNA损伤介导的G1期停滞信号。在HPV感染过程中，HPV的E6和E7癌蛋白可能会破坏这个细胞周期检查点，导致肿瘤发生过程中基因改变的积累。
