HUMAN-PAPILLOMAVIRUS ONCOPROTEINS E6 AND E7 INDEPENDENTLY ABROGATE THE MITOTIC SPINDLE CHECKPOINT

The E6 and E7 genes of the high-risk human papillomavirus (HPV) types encode oncoproteins, and both act by interfering with the activity of cellular tumor suppressor proteins. E7 proteins act by associating wit h members of the retinoblastoma family, while E6 increases the turnove r of p53. p53 has been implicated as a regulator of both the G(1)/S ce ll cycle checkpoint and the mitotic spindle checkpoint. When fibroblas ts from p53 knockout mice are treated with the spindle inhibitor nocod azole, a rereplication of DNA occurs without transit through mitosis, We investigated whether E6 or E7 could induce a similar loss of mitoti c checkpoint activity in human keratinocytes. Recombinant retroviruses expressing high-risk E6 alone, E7 alone, and E6 in combination with E 7 were used to infect normal human foreskin keratinocytes (HFKs). Esta blished cell Lines were treated with nocodazole, stained with propidiu m iodide, and analyzed for DNA content by flow cytometry. Cells infect ed with high-risk E6 were found to continue to replicate DNA and accum ulated an octaploid (8N) population. Surprisingly, expression of E7 al one was also able to bypass this checkpoint. Cells expressing E7 alone exhibited increased levels of p53, while those expressing E6 had sign ificantly reduced levels. The p53 present in the E7 cells was active, as increased levels of p21 were observed. This suggested that E7 bypas sed the mitotic checkpoint by a p53-independent mechanism, The levels of MDM2, a cellular oncoprotein also implicated in control of the mito tic checkpoint, were significantly elevated in the E7 cells compared t o the normal HFKs. In E6-expressing cells, the levels of MDM2 were und etectable. It is possible that abrogation of Rb function by E7 or incr eased expression of MDM2 contributes to the loss of mitotic spindle ch eckpoint control in the E7 cells. These findings suggest mechanisms by which both HPV oncoproteins contribute to genomic instability at the mitotic checkpoint.