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In this paper we reported a detailed analysis of the physical state of the HPV16 genome in the 140/7 cervical carcinoma-derived cell line. We found that HPV16 DNA in 140/7 cells exists as monomeric episomes of 10 kb in length as well as bands of identical electrophoretic mobilities in either one-dimensional or two-dimensional gels were generated after digestion with three no-cut restriction enzymes. Fluorescent in situ hybridization analysis of over 5000 140/7 cells with a HPV16 probe revealed no specific pattern of hybridization on any chromosome. Taken together, these results indicate that the 140/7 cell line contains exclusively extrachromosomal HPV DNA. Previous studies of established cell lines have found that episomal copies of HPV are lost or integrate into cellular DNA With time in culture. The episomal HPV16 DNA sequences were rapidly lost from the parental W12 line, but could be maintained as stable episomes in two clonal derivatives of this line for at least 15 passages. In 140/7 cells, HPV16 DNA remained extrachromosomal for at least 50 passages in culture and after growth for 3 weeks in nude mice (Kusumoto-Matsuo, 2010).

These results indicate that the 140/7 cervical carcinoma cell line supports stable replication of episomal HPV16 in vivo and in vitro. multimeric copies of increasing size. Discrete Human papillomaviruses (HPVs) belong to a family of small DNA viruses that usually produce benign lesions on cutaneous and mucosal surfaces. Of the more than 100 HPV types described to date, only subsets are consistently found in human cancer. HPV DNA, including that from types 16, 18, 31, 33 and 35, has been detected in over 90% of cervical cancers. HPV type 16 (HPV16) is the most common HPV type found in cervical cancers, detected in over half of malignant lesions. The etiologic role of HPV in cervical neoplasia has been firmly established, although the precise mechanisms of transformation have yet to be elucidated. Initial studies of cervical biopsies reported that premalignant lesions contained extrachromosomal viral DNA whereas HPV DNA was integrated in cancer tissue. Integration usually disrupts the E1 and/or the E2 open reading frames (ORFs), two genes whose products are important in regulation of virus replication and transcription. It has been reported that integration may also disrupt a potential mRNA instability element in the 3' untranslated region of E6 and E7 mRNA, resulting in increased stability and steady state levels of E6 and E7 transcripts). Thus, integration of viral DNA with subsequent disruption of regulatory DNA sequences provides a plausible mechanism for the progression of HPVinduced malignancy.

Several investigators have shown that a significant proportion of HPV16-positive primary cervical cancers contain both episomal as well as integrated HPV sequences and that, in approximately 20-30% of tumors, HPV16 DNA is found exclusively in an extra chromosomal form. This suggests that, without the integration-mediated disruption of virus regulatory elements, other changes in the viral genome may be necessary for transformation to occur. Although the mechanisms by which extra chromosomal HPV contributes to transformation are not known, studies suggest that mutations ...
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