Expression of human papilloma virus E7 protein causes apoptosis and inhibits DNA synthesis in primary hepatocytes via increased expression of p21Cip-1/WAF1/MDA6

The impact of human papilloma virus (HPV16) E7 proteins and retinoblastoma (RB) antisense oligonucleotides upon mitogen-activated protein kinase (MAPK)-mediated inhibition of DNA synthesis via p21(Cip-1/WAF1/MDA6) (p21) was determined in primary hepatocytes. Prolonged activation of the MAPK pathway in p21(+/+) or p21(-/-) hepatocytes caused a large decrease and increase, respectively, in DNA synthesis. Either transfection with RE antisense oligonucleotides, expression of wild type E7, or RE binding mutant E7 (C24S) proteins increased p21 levels and reduced DNA synthesis in p21(+/+) hepatocytes. RE antisense oligonucleotides and E7 proteins increased apoptosis in p21(+/+), but not p21(-/-), hepatocytes. Expression of wild type E7 increased DNA synthesis above control levels in p21(-/-) cells, which was additive with prolonged MAPK activation. In contrast, expression of mutant E7 did not alter DNA synthesis above control levels in p21(-/-) cells and was supra-additive with prolonged MAPK activation. Antisense ablation of RE in p21(-/-) hepatocytes had a weak stimulatory effect upon DNA synthesis itself but enhanced the capacity of mutant E7 protein to stimulate DNA synthesis to the same level observed using wild type E7. The ability of prolonged MAPK activation to stimulate DNA synthesis in the presence of mutant E7 and antisense RE was additive. Collectively, the present data demonstrate that loss of RE function together with loss of pal function plays an important role in the E7- and MAPK-dependent modulation of apoptosis and DNA synthesis in primary hepatocytes.