Genomic instability develops at early stages of HPV-infected neoplasias and is associated with deregulated expression of the oncogenes E6 and E7, which were both shown to induce centrosome abnormalities, multipolar mitosis and aneuploidy [1, 2]. The effects of HPV 16 E6 and E7 on genomic integrity have been described in primary keratinocytes and in cervical cancer cell lines, which are either critical for long-term culturing or already chromosomally instable. To analyze the effects of the HPV oncogenes on genomic stability in a time dependent manner we intended to use chromosomally stable HCT116 colon carcinoma cells for the generation of clones that allow doxycycline inducible expression of HPV 16 E6 and E7.
Western Blotting and RT-qPCR were performed to characterize HPV 16 E6 and E7 expression in selected doxycycline inducible HCT116 clones. Effects on centrosome numbers and spindle pole formation during mitosis were analyzed using γ-tubulin immunostainings. DNA damage in HCT116 clones induced for E6 and E7 expression was evaluated by staining of the phosphorylated histone component γH2AX, a marker for DNA double strand breaks. The induction of DNA single and double strand breaks were further analyzed by performing Comet Assay.
Induction of both oncogenes elevated the number of interphase cells showing abnormal centrosome numbers. Additionally, the percentage of cells containing abnormal spindle poles during mitosis was significantly increased. Both effects could already be observed after 48 hours of oncogene induction and were found to be elevated after longer induction phases. Induction of E6 and E7 expression also affected DNA integrity of HCT116 cells. Moderate increases in γH2AX phosphorylation indicating DNA double strand break formation could be observed in response to E6 and E7 expression.
The results of the present study suggest that expression of both HPV 16 oncogenes affects the genomic stability in HCT116 cells. Induction of abnormal centrosome numbers may result in aberrant spindle pole formation during mitosis increasing the number of DNA damaged daughter cells. Despite the fact that severely DNA damaged cells potentially undergo apoptosis, the effects of E6 and E7 on mitotic progression might increase the genomic variability promoting the outgrowth of selected cells. As these effects could already be observed 48 hours after inducing the expression of the HPV 16 oncogenes, they may represent a very early event during HPV-mediated transformation.
1. Duensing S, Lee LY, Duensing A, Basile J, Piboonniyom S, Gonzalez S, Crum CP and Munger K. The human papillomavirus type 16 E6 and E7 oncoproteins cooperate to induce mitotic defects and genomic instability by uncoupling centrosome duplication from the cell division cycle. Proc Natl Acad Sci U S A. 2000; 97(18):10002-10007.
2. Duensing S and Munger K. The human papillomavirus type 16 E6 and E7 oncoproteins independently induce numerical and structural chromosome instability. Cancer Research. 2002; 62(23):7075-7082.