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p53-dependent G1 arrest involves pRB-related proteins and is disrupted by the human papillomavirus 16 E7 oncoprotein
R J Slebos1, M H Lee, B S Plunkett
1Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Abstract:
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.
Insights
High-risk human papillomaviruses (HPVs) inactivate tumor suppressors p53 and pRB, crucial for cell cycle arrest after DNA damage. HPV oncoproteins disrupt this process, potentially driving cancer development by preventing genetic stability.
Area of Science:
- Molecular Biology
- Virology
- Cell Cycle Regulation
Background:
- Tumor suppressor proteins p53 and pRB are critical for cell cycle control.
- High-risk human papillomaviruses (HPVs) inactivate p53 and pRB using E6 and E7 oncoproteins, respectively.
- The p53 pathway mediates G1 cell cycle arrest following DNA damage, but its precise mechanism involving pRB is unclear.
Purpose of the Study:
- To investigate the role of pRB in the p53-mediated DNA damage response.
- To determine how HPV oncoproteins E6 and E7 interfere with cell cycle checkpoints.
Main Methods:
- Gamma-irradiation of cells with varying p53 and HPV oncoprotein status.
- Analysis of G1 arrest, WAF1/CIP1 mRNA levels, and pRB phosphorylation status.
- Assessment of cells with disrupted RB genes.
Main Results:
- Wild-type p53 induced G1 arrest, increased WAF1/CIP1 mRNA, and hypophosphorylated pRB after irradiation.
- Cells with inactivated p53 or HPV16 E6/E7 failed to arrest in G1 and showed altered pRB phosphorylation.
- HPV16 E7 expression prevented G1 arrest despite normal p53 and WAF1/CIP1 induction.
- RB gene disruption alone did not fully abolish G1 arrest.
Conclusions:
- p53 indirectly regulates pRB phosphorylation, a key step in DNA damage-induced G1 arrest.
- pRB and/or pRB-like molecules interacting with HPV16 E7 are essential for this G1 arrest signal.
- HPV E6 and E7 oncoproteins likely subvert cell cycle checkpoints, promoting genetic instability and tumorigenesis.