Integration of human papillomavirus type 16 into the human genome correlates with a selective growth advantage of cells

Integration of human papillomavirus type 16 (HPV-16) DNA into a host chromosome has been hypothesized to result in altered expression of two viral transforming genes, E6 and E7, in cervical cancers. In order to investigate the role that changes in viral genomic state and gene expression play in cervical carcinogenesis, we have derived clonal populations of human cervical epithelial cells which harbor multiple copies of either extrachromosomal or integrated viral DNA. The clonal populations harboring extrachromosomal HPV-16 DNA stably maintained approximately 1,000 viral copies for at least 15 passages (approximately 100 cell doublings), which contrasted with the unstable HPV-16 replicons in the parental counterpart. In the clonal populations harboring integrated viral DNA, 3 to 60 copies of HPV-16 DNA were found integrated in either of two forms: type 1, in which all the copies of HPV-16 DNA were disrupted in the E2 open reading frame upon integration, and type 2, in which intact viral copies were flanked by disrupted viral copies and cellular sequences. Despite the lower HPV-16 DNA copy number, the clonal populations with integrated viral DNA had levels of E7 protein that were in most cases higher than those found in the clonal populations harboring extrachromosomal viral DNA. Irrespective of viral genomic state, the clonal populations were capable of undergoing terminal differentiation and unable to form colonies in soft agar, which is indicative of the nontumorigenic nature of these cells. Importantly, a cell population with integrated viral DNA was found to outgrow another with extrachromosomal DNA when these cells were cocultured over a period of time. Thus, integration of human papillomaviral DNA correlates with increased viral gene expression and cellular growth advantage. These observations are consistent with the hypothesis that integration provides a selective advantage to cervical epithelial precursors of cervical carcinoma.