Recurrent integration of human papillomaviruses 16, 45, and 67 near translocation breakpoints in new cervical cancer cell lines

Progressive chromosomal changes and integration of human papillomavirus (HP V) sequences mark the development of invasive cervical cancer. Chromosomal localization of HPV integration is essential to the study of genomic region s involved in HPV-induced pathogenesis. Yet, the available information abou t HPV integration loci is still limited, especially with respect to differe nt HPV types. We have established cell lines from five cervical cancers wit h HPV-16, HPV-45, and HPV-67. We have determined HPV integration sites and karyotype abnormalities by using the multicolor combined binary ratio-fluor escence in situ hybridization method (Tanke et at) with 24 chromosome-speci fic paints in combination with full-length HPV DNA probes. All cell lines were cytogenetically abnormal, and exhibited numerical and s tructural chromosomal deviations. HPV sequences were integrated at various (segments of) chromosomes. Duplicate integration sites were seen in all mul tiploid cell lines, suggesting that viral integration had preceded chromoso mal endoreduplication. HPV-16 was found near the t(3p14.1-14.3;14) breakpoi nt in cervical squamous cell carcinoma (CSCC)-7 and mainly in episomal form in CSCC-1. HPV-45 was integrated near 3q26-29 in cervical (adeno or adenos quamous) carcinoma (CC)-8 and near 1q21-23 as well as near the t(1q21;22q13 ) breakpoint in CC-10A and CC-10B variant lines. HPV-67 was Localized near the breakpoint of t(3p23-26;13q22-31) in CC-11. Southern blot analysis show ed that, except for CSCC-1, the physical state of HPV in the cell lines was the same as in the original tumor lesions. This set of six cervical cancer cell Lines included three lines with HPV-45 , a major non-Western high-risk HPV type, the first reported HPV-67-positiv e cell line, and two cell lines with integrated and episomal HPV-16 DNA, re spectively. The novel combined binary ratio-fluorescence in situ hybridizat ion technique enabled us to simultaneously map chromosomal rearrangements a nd HPV integration sites, thereby revealing recurrent integration near tran slocation junctions for all of these HPV types in the cell lines from three of the five primary tumors. The detection of multiple HPV integration site s at rearranged chromosomes at such high frequency in cervical cancer-deriv ed cells may reflect events that are relevant to the development of cervica l cancer.