This study explores the mechanism underlying the generation of polyploidy in HPV E7-expressing human epithelial cells. We tested the conflicting possibilities, and our data indicate that E7 does not have an effect on the spindle assembly checkpoint or on the length of time cells spent in mitosis. Our data also suggest that DNA rereplication is not a significant cause of polyploidy in E7-expressing human epithelial cells in response to microtubule disruption. Rather, we have shown that wild-type epithelial cells treated with low doses of nocodazole adapt to the spindle assembly checkpoint and exit mitosis. They then proceed into G1 with 4C DNA content, where they halt at the postmitotic checkpoint. Our results then demonstrate that E7-expressing cells are able to proceed through this checkpoint, replicate their DNA, and become polyploid.
Our results also demonstrate that Rb downregulation does not have a major effect on the spindle checkpoint. This is in contrast to the work of Hernando et al., which suggests that Rb downregulation and the subsequent effects on the Rb pathway, including upregulation of Mad2, result in a hyperactive spindle checkpoint (14
) and may predispose cells to additional genomic instability. We believe that the discrepancy may be due to our interpretation of the time-lapse videomicroscopy results, which differs from theirs. They measured the length of mitosis from prometaphase to late anaphase by examining chromosome condensation and cell division (14
). On the other hand, we measured mitosis from nuclear envelope breakdown to nuclear envelope reformation, which are more distinctly defined and easily seen parameters. Another difference between our experiments and those of Hernando et al. is the types of cells used. We have used primary MEFs and the immortalized RPE1 cells, while Hernando et al. used the established mouse NIH 3T3 cells as well as the human colon carcinoma line HCT116. A more recent study with Mad2-inducible upregulation demonstrates an increase in polyploid cells and a link to chromosomal instability upon Mad2 overexpression (33
). However, cells overexpressing Mad2 do not appear to be equivalent to Rb-downregulated cells, as Mad2 overexpression led to a partial mitotic block of proliferating cells, a phenomenon never seen in Rb−/−
As previously suggested in studies with fibroblasts and as our data indicate, Rb plays an important role in postmitotic checkpoint control (19
). However, our results do not rule out the role of inactivation of p107 or p130 in E7-induced polyploidy, nor can we explain how the E7 zinc finger domain mutants that are defective for Rb derepression induce polyploidy (27
). Notably, Patel et al. tested the E7 mutants in the presence of E6, which by themselves could induce polyploidy under our experimental conditions. In addition, we examined the role of Rb by using MEFs, which might be different from or less stringent than using human keratinocytes in regulation of the postmitotic checkpoint. The conclusions from our study and those by Patel et al. suggest that E7 possesses both Rb-dependent and Rb-independent activities in the abrogation of the postmitotic checkpoint.
p53 appears to play a key role in mediating the postmitotic checkpoint (1
), and p21 is responsible for at least part of this p53-mediated postmitotic arrest (18
). The cdk inhibitor p21 binds to and inactivates cyclin/cdk complexes, including cyclinE1/cdk2 and cyclinA2/cdk2, resulting in pRb hypophosphorylation that inhibits E2F activity and arrests the cell cycle at the G1
/S transition (35
). Consistent with observations made by others, we have previously shown that p53 levels were upregulated in E7-expressing PHKs (21
). E7 may abrogate the transcriptional activity of p53 (23
). E7 has also been shown to inactivate the Cdk inhibitory activity of p21 (10
). These functions may contribute to E7-induced polyploidy.
This study has important implications for cancer in general, and for cervical cancer in particular, because of our results with the HPV oncogene E7. HPV E6 and E7 are the oncogenes necessary for transformation in high-risk HPVs, which are associated with cervical lesions that eventually may lead to cervical carcinomas. Although E6 and E7 are able to immortalize primary human epithelial cells, they are not sufficient to induce the transformation of human cells (7
). Instead, it is believed that genomic instability caused by E6 and E7 predisposes the cells to accumulate additional genomic aberrations necessary for malignant transformation (4
). As shown here and in other studies, HPV E7-expressing cells are able to become polyploid (27
), and Olaharski et al. concluded that polyploidy and chromosomal instability are related events that predispose cells to aneuploidy and subsequently to malignancy (26
). Our finding that E7 expression results in polyploidy formation through abrogation of the postmitotic checkpoint has important implications for cancer prevention and/or treatment. Understanding this mechanism of polyploidy formation will be useful in developing therapies against its accumulation in E7-expressing, untransformed cells in order to prevent the accumulation of additional genomic aberrations that could lead to carcinogenesis.