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      • Mutational and Functional Analysis of HPV-16 URR Derived from Korean Cervical Neoplasia

        Park, Jong-Sub,Hwang, Eun-Seong,Lee, Chan-Jae,Kim, Chan-Joo,Rha, Jong-Gu,Kim, Weung-Jo,Namkoong, 0Sung-Eun,Um, Soo-Jong 가톨릭대학교 2000 Bulletin of The Catholic Research Institutes of Me Vol.28 No.-

        Objective : The YY1 mutation has been suggested as one of the indicators that explains development of cervical neoplasia by episomal-type HPV. To extend this hypothesis, we examined whether a mutations(s) in the YY1 site is functionally related to the invasiveness of cervical neoplasia and the physical status of HPV DNA. Methods : The URR sequences were obtained by PCR amplification of HPV-16 genome from CIN and invasive cancer patients and cloned into pUC18 for sequencing and into pBLCAT8+ for functional CAT assay. Results : Our previous data classified HPV-infected patients into three groups : 3 cancer cases carrying episomal HPV DNA ; 12 cancer cases carrying integrated HPV DNA; 12 CIN cases carrying episomal HPV DNA. The specific variants in HPV-16 URR were found in Korean women : G→A transition at nt 7520 (200%, 27/27), A→C transition at nt 7729 (70% ; 21/27). Selective mutations were observed at the YY1 binding 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). Additionally, C→T transition at nt 7785(YY1-binding site 3 ; from 7781 to 7790) was transition at nt 7785(YY1-binding site 3 ; from 7781 to 7790) was found in 2 of 3 patients. No YY1 site mutations were detected in the 12 CIN patients and in the HPV-integrated invasive cancer patients. To determine 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 episomal 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 CIN, where no mutation of the YY1 binding site was detected, showed similar levels of promoter activity to that of the URR prototype. Conclusions : Our results support the hypothesis that the mutation the YY1 binding site is functionally related to the development of cervical neoplasia 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 progression of cervical cancer. (Gynecologic Oncology 74:23-29, 1999)

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