Mutational and functional analysis of HPV-16URR derived from Korean cervical neoplasia

Objective. The YY1 mutation has been suggested as one of the indicators tha t explains development of cervical neoplasia by episomal-type HPV. To exten d this hypothesis, we examined whether a mutation(s) in the YY1 site is fun ctionally related to the invasiveness of cervical neoplasia and the physica l status of HPV DNA, Methods. The URR sequences were obtained by PCR amplification of HPV-16 gen ome from CIN and invasive cancer patients and cloned into pUC18 for sequenc ing and into pBLCAT8+ for functional CAT assay. Results. Our previous data classified HPV-infected patients into three grou ps: 3 cancer cases carrying episomal HPV DNA; 12 cancer cases carrying inte grated HPV DNA; 12 CIN cases carrying episomal HPV DNA, The specific varian ts in HPV-16 URR were found in Korean women: G-->A transition at nt 7520 (1 00%, 27/27), A-->C transition at nt 7729 (70%; 19/27), and G-->A transition at nt 7841 (78%; 21/27), Selective mutations were observed at the YY1 bind ing sites of HPV-16 URR in the 3 patients with invasive cervical cancer who have the episomal forms of HPV-16 DNA: A-->C transition at nt 7484 and G-- >A transition at nt 7488 (YY1-binding site 2; from 7481 to 7489), Additiona lly, C-->T transition at nt 7785 (YY1-binding site 3; from 7781 to 7790) wa s found in 2 of 3 patients. No YY1 site mutations were detected in the 12 C IN patients and in the HPV-integrated invasive cancer patients. To determin e whether these mutations have effects on the expression of HPV E6/E7 genes driven by URR, the transient transfection assay was employed using URR-CAT reporter plasmid. The relative activities of three URR mutants from episom al HPV-16 DNA of cervical cancers were two- to fourfold higher than that of the HPV-16 URR prototype. In contrast, the URRs from integrated HPV-16 DNA in cervical cancer and from episomal HPV-16 DNA in GIN, where no mutation of the YY1 binding site was detected, showed similar levels of promoter act ivity to that of the URR prototype. Conclusions. Our results support the hypothesis that the mutation at the YY 1 binding site is functionally related to the development of cervical neopl asia caused by episomal HPV-16 DNA in Korean cervical cancer patients. Thus , mutation in the YY1 site of episomal HPV-16 URR may play a corresponding role of HPV integration in the progression of cervical cancer. (C) 1999 Aca demic Press.