Abrogation of the Postmitotic Checkpoint Contributes to Polyploidization in Human Papillomavirus E7-Expressing Cells

High-risk types of human papillomavirus (HPV) are considered the major causative agents of cervical carcinoma. The transforming ability of HPV resides in the E6 and E7 oncogenes, yet the pathway to transformation is not well understood. Cells expressing the oncogene E7 from high-risk HPVs have a high incidence of polyploidy, which has been shown to occur as an early event in cervical carcinogenesis and predisposes the cells to aneuploidy. The mechanism through which E7 contributes to polyploidy is not known. It has been hypothesized that E7 induces polyploidy in response to mitotic stress by abrogating the mitotic spindle assembly checkpoint. It was also proposed that E7 may stimulate rereplication to induce polyploidy. We have tested these hypotheses by using human epithelial cells in which E7 expression induces a significant amount of polyploidy. We find that E7-expressing cells undergo normal mitoses with an intact spindle assembly checkpoint and that they are able to complete cytokinesis. Our results also exclude DNA rereplication as a major mechanism of polyploidization in E7-expressing cells upon microtubule disruption. Instead, we have shown that while normal cells arrest at the postmitotic checkpoint after adaptation to the spindle assembly checkpoint, E7-expressing cells replicate their DNA and propagate as polyploid cells. Thus, abrogation of the postmitotic checkpoint leads to polyploidy formation in E7-expressing human epithelial cells. Our results suggest that downregulation of pRb is important for E7 to induce polyploidy and abrogation of the postmitotic checkpoint.An important hallmark of human cancers is aneuploidy, the state in which a cell has extra or missing chromosomes (12, 25). Polyploidy is the state in which cells have more than two equal sets of chromosomes and is thought to be an early event in multistep carcinogenesis that can lead to aneuploidy (1, 24), as exemplified in Barrett''s esophagus (11). Polyploidy has recently been shown to occur as an early event in cervical carcinogenesis and to predispose the cells to aneuploidy (26). Other recent studies have shown that tetraploid but not diploid mouse or human cells induce tumor formation in mice (3, 9). These studies highlight the potential importance of polyploidy in carcinogenesis.The cellular mechanisms responsible for this polyploidy formation are as of yet undetermined, but several models have been proposed. First, abrogation of the spindle assembly checkpoint followed by cleavage failure may lead to polyploidy formation (36, 40). A second proposed model is rereplication, a process of multiple rounds of DNA replication without an intervening mitosis. Third, cells that adapt to the mitotic spindle checkpoint halt in a G₁-like state with 4C DNA content. Abrogation of this postmitotic checkpoint allows the cells to replicate their 4C DNA content, leading to polyploidy formation. This has been shown in cells that express the human papillomavirus type 16 (HPV-16) E6 oncogene that degrades p53 (21). Finally, cleavage failure, which yields binucleate cells with 4C DNA content, is also a potential mechanism for polyploidy formation (31).The postmitotic checkpoint becomes activated when cells with an intact spindle assembly checkpoint become arrested during mitosis for a prolonged period of time and eventually adapt to the checkpoint, exit mitosis without cleavage, and progress into a G₁-like state with 4C DNA content (19, 22). The cells are prevented from continuing through the cell cycle and replicating their DNA by a proposed p53- and pRb-dependent postmitotic checkpoint (18, 19).High-risk types of HPV (of which HPV-16 is the most prevalent) are commonly associated with lesions that can progress to cervical carcinoma, which is one of the leading causes of cancer death in women worldwide (42). The transforming properties of high-risk HPVs primarily reside in the E6 and E7 oncogenes (reviewed in reference 7). The ability of high-risk HPV E6 and E7 proteins to promote the degradation of p53 and pRb, respectively, has been suggested as a mechanism by which HPV induces cellular transformation (6, 30). Expression of the high-risk HPV E6 and E7 oncogenes in human keratinocytes leads to polyploidy, which is enhanced by DNA damage and by activation of the spindle checkpoint through microtubule disruption (15, 27, 37, 38).Previously, it was thought but not directly shown that high-risk E6 and E7 induce polyploidy in response to microtubule disruption by abrogating the spindle checkpoint and that degradation of the tumor suppressor p53 by E6 is the mechanism by which E6 accomplishes this polyploidy formation (27, 37, 38). Others have proposed that E7 may play a role in stimulating DNA rereplication that occurs prior to mitosis initiation and polyploidy formation (20). Our recent studies demonstrate that E6 does not affect the mitotic spindle checkpoint (21). Instead, E6 abrogates the postmitotic checkpoint to induce polyploidy after microtubule disruption. Interestingly, E6 mutant proteins defective in inducing p53 degradation also induce polyploidy (21). The mechanism by which HPV E7 induces polyploidy remains to be determined. In this study, we investigate these possible mechanisms through which HPV-16 E7 induces polyploidy formation.