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The role of human cytomegalovirus in transformation and in the development of cervical intraepithelial neoplasia

The aim of this study was to investigate the role of human cytomegalovirus (HCMV) in transformation and its possible involvement in the development of cervical intraepithelial neoplasia (CIN). Although cell transformation by HCMV is well documented in the literature, the mode of transformation has not yet been elucidated. Nelson et al. (1982) identified a DNA sequence within the HindIII E fragment of the HCMV AD169 genome which when transfected into NIH 3T3 cells, can initiate colony formation in methylcellulose and tumourigenicity in nude mice. It was intended to extend these experiments to investigate the molecular basis for HCMV cell transformation. Repeated attempts to transform 3T3, human embryo lung (Helu) and rat embryo (RE) cells with the HCMV AD169 HindIII E fragment were unsuccessful. However, transformation of RE cells was repeatedly observed after infection of the cells with UV-irradiated HCMV AD169 virus. Southern blot analysis could not detect HCMV DNA in HCMV transformed cell and derived tumour cell lines. This suggested that the retention of viral DNA in the transformed cells may be transient and only necessary to initiate the transformation event. One mechanism by which HCMV DNA could initiate transformation is by activating a cellular proto-oncogene. Experiments were carried out to see if the DNA from a rat tumour induced after inoculation with HCMV transformed cells contained a transforming gene. The rat tumour DNA was found to contain a transforming gene that could be transfected into 3T3 cells and could initiate tumourigenesis when the transfected cells were injected into nude mice. As viral DNA could not be detected in the original HCMV transformed cells, the transforming gene must have been of cellular origin. Preliminary experiments suggested that HCMV may have activated a ras oncogene in the transformed cells. Past seroepidemiological studies have implicated HCMV as an oncogenic agent in the development of CIN and cervical carcinoma. The second aim of this study was to provide molecular evidence for an association between HCMV and CIN. The DNA from biopsies of 43 CIN patients in the West of Scotland was examined for the presence of HCMV DNA sequences by Southern blot analysis. Two biopsies, C2 and C17 were found to contain DNA sequences that hybridized to the HCMV AD169 HindIII E fragment. In C17 the hybridizable DNA sequences were only present at about 0.1 copy/cell and therefore in an amount too low to permit detailed sequence analysis. The hybridizable DNA sequences in C2 were present at about 20 copies/cell and were found to contain BamHI restriction fragments that comigrated with the BamHI P, W, c and e fragments of HCMV AD169. The hybridizable CIN DNA sequences may represent HCMV sequences that were retained in the tissue after HCMV infection and may have instigated the development of CIN. Two other BamHI fragments of C2 DNA were detected using the HCMV AD169 HindIII E fragment as a probe and these may represent rearranged sequences in the retained HCMV DNA. Rearrangements could have occurred when the viral DNA became integrated into the cellular DNA of this patient. As only a small amount of DNA was available from the C2 biopsy, further analysis of the viral sequences retained and the nature of the rearrangements was only possible if the relevant sequences could be cloned. A l library of the C2 DNA was constructed using the vector EMBL3 and three clones were isolated that contained C2 sequences that hybridized to the cloned HCMV AD169 HindIII E fragment. One of the clones was found to contain C2 sequences that hybridized to pAT vector sequences present in the probe. The other two clones contained C2 sequences that hybridized specifically to the HCMV AD169 HindIII E fragment but were unfortunately lost during the purification procedure after only 2 or 3 rounds of replication. Since HSV-2 and HPV have also been implicated in the development of CIN and cervical carcinoma, the same 43 CIN biopsies were analysed for the presence of HSV-2 and HPVII DNA sequences. Authentic HPVII DNA was detected in two CIN biopsies (C13 and C19) but no conclusive evidence of HSV-2 specific DNA could be found, although sequences that hybridized to vector pBR322 were detected. (Abstract shortened by ProQuest.).