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. The effects of HPV16 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 HPV16 E6 and E7.
Western Blotting and RT-qPCR were performed to characterize HPV16 E6 and E7 expression in selected doxycycline inducible HCT116 clones. Effects on centrosome numbers and spindle poles formation during mitosis were analyzed using gamma-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 number of aneuploid cells in response to HPV 16 E6 and E7 expression was determined by propidium iodide staining of the DNA and subsequent FACS analysis.
Induction of both oncogenes elevated the number of interphase cells showing abnormal centrosome numbers. Additionally, the percentage of cells with 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. As a result of the deregulated distribution of chromosomes during mitosis, E6- and E7-expressing cells showed increased rates of DNA damage and aneuploidy.
In conclusion, HPV16 E6 and E7 induce genomic instability in HCT116 cells as indicated by abnormal spindle pole formation and increased DNA damage rates. Subsequent analyses on gene copy number variations and differential methylation patterns will also help to understand how and how fast genomic stability is affected by the HPV oncogenes, deepening the insight into mechanisms and causes promoting malignant progression to cervical cancer.