부산대학교 도서관

OBJECTIVES: The main aim of this work is to study how E5, E6 and E7 oncogenes of human papillomaviruses type 18 could cooperate among each other to boost key cancer cell features such as uncontrolled cell proliferation, enhanced migration capacity, invasion, and how this relates with oxidative stress. METHODS: We generated three HaCaT cell lines, that are spontaneously immortalized, expressing the following combination of oncogenes: HaCaT E5–18, HaCaT E6/E7–18 and HaCaT E5/E6/E7–18 and non-transduced HaCaT cells as a control. Cell proliferation was assessed using a MTT assay. Cellular migration was studied using a wound healing assay by scratching the monolayer in order to generate a “wound”. Invasiveness potential of cells was studied using a transwell collagen invasion assay. Intracellular oxidants production in the four cell lines was measured using the fluorescent probe CM-H2DCFDA. Catalase activity was assayed spectrophotometrically, following the decomposition of 10 mM H2O2 by catalase contained in the samples at 240 nm. Total glutathione in cell lysates was quantified by HPLC, and PRX1 expression levels was assessed by Western Blot. RESULTS: MTT assay showed a statistically significant increment in cell proliferation in HaCaT E5/E6/E7 cell with respect to the other three cell lines. Similar results were obtained in cell migration assay, and in invasion assays. We measured levels of the three oncoproteins involved in ROS metabolism and observed that E5/E6/E7 diminished catalase activity but augmented significantly the levels of GSH and PRX1. CONCLUSIONS: This study demonstrates that cells with E5/E6/E7 together cooperate to augment the malignant transformation of HaCaT cells.