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

Abstract

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 G 1 -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 yiel...

Human Papillomavirus E7 Induces Rereplication in Response to DNA Damage

“…To test this possibility, we added nocodazole to trap cells that may have bypassed the G 2 checkpoint in mitosis. Our previous studies have demonstrated that HPV E6 and E7 do not have much effect on the spindle checkpoint and that cells expressing E7 arrest in mitosis for an extended period of time (25). As shown in Fig.…”

“…Although no significant spontaneous polyploidy was seen in early-passage RPE1 cells expressing E7 (Fig. 1B), over an extended period of culture, a small but statistically significant increase in the percentage of polyploidy in these cells has been found (25). Bleomycin treatment enhanced the extent of DNA damage (Fig.…”

“…We have recently demonstrated that in response to microtubule disruption, E6 and E7 induced polyploidy through endoreduplication, a process including cell arrest at the spindle checkpoint, mitotic slippage, and abrogation of the postmitotic checkpoint (25,26). Induction of polyploidy by HPV E7 in mouse NIH 3T3 cells treated with doxorubicin was proposed by Polager and Ginsberg to be a result of endoreduplication, though evidence was not provided (22).…”

Human Papillomavirus: Biology and Pathogenesis

“…It is reported that expression of either HPV E6 or E7 alone is sufficient to deregulate cytokinesis and consequently produce the tetraploid cell (Heilman et al, 2009). However, it was demonstrated that the formation of these cells is primarily attributed to E5-induced cell fusion, rather than E6, E7, and cytokinesis failure (Ho et al, 2009).…”

Development of New Human Papillomavirus Vaccines

“…First, either HR-HPV E6 or E7 alone can also contribute to tetraploid cell formation by inducing cytokinesis failure (Incassati et al, 2006;Heilman et al, 2009;Duensing et al, 2001a). Second, it is widely accepted that increasingly deregulated expression of E6 and E7 has been identified as the major transforming factor in the pathogenesis of cervical dysplasia and derived cancers.…”