Monomethylarsonous Acid Produces Irreversible Events Resulting in Malignant Transformation of a Human Bladder Cell Line Following 12 Weeks of Low-Level Exposure

Abstract

Arsenic is a known human bladder carcinogen; however, the mechanisms underlying arsenical-induced bladder carcinogenesis are not understood. Previous research has demonstrated that exposure of a nontumorigenic human urothelial cell line, UROtsa, to 50nM monomethylarsonous acid (MMA^III) for 52 weeks resulted in malignant transformation. To focus research on the early mechanistic events leading to MMA^III-induced malignancy, the goal of this research was to resolve the critical period in which continuous MMA^III exposure (50nM) induces the irreversible malignant transformation of UROtsa cells. An increased growth rate of UROtsa cells results after 12 weeks of MMA^III exposure. Anchorage-independent growth occurred after 12 weeks with a continued increase in colony formation when 12-week exposed cells were cultured for an additional 12 or 24 weeks without MMA^III exposure. UROtsa cells as early as 12 weeks MMA^III exposure were tumorigenic in severe combined immunodeficiency mice with tumorigenicity increasing when 12-week exposed cells were cultured for an additional 12 or 24 weeks in the absence of MMA^III exposure. To assess potential underlying mechanisms associated with the early changes that occur during MMA^III-induced malignancy, DNA methylation was assessed in known target gene promoter regions. Although DNA methylation remains relatively unchanged after 12 weeks of exposure, aberrant DNA methylation begins to emerge after an additional 12 weeks in culture and continues to increase through 24 weeks in culture without MMA^III exposure, coincident with the progression of a tumorigenic phenotype. Overall, these data demonstrate that 50nM MMA^III is capable of causing irreversible malignant transformation in UROtsa cells after 12 weeks of exposure. Having resolved an earlier timeline in which MMA^III-induced malignant transformation occurs in UROtsa cells will allow for mechanistic studies focused on the critical biological changes taking place within these cells prior to 12 weeks of exposure, providing further evidence about potential mechanisms of MMA^III-induced carcinogenesis.