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 50 nM 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 (50 nM) 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 50 nM 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.

The Redox Basis of Epigenetic Modifications: From Mechanisms to Functional Consequences

“…The Futscher group has been active in this field through a series of studies investigating exposure to arseniccontaining compounds. Their findings illustrate that arsenic promotes a broad, gradual, and lasting perturbation in DNA methylation patterns that contributes to arsenic-mediated carcinogenesis in urothelial cells (135,136,329). Collectively, these results strongly suggest that the direct perturbation of the iron loaded into epigenetic enzymes is a strong stimulus for the modification of cellular epigenetic status.…”

Global gene expression changes in human urothelial cells exposed to low-level monomethylarsonous acid

“…The work by Wnek and colleagues (2010) suggested that the observed phenotypic changes should coincide with perturbations in the transcriptional activity of the cells. Hyper-proliferation, morphological changes, acquisition of anchorage-independent growth in semi-solid media, and the formation of tumors in immuno-compromised mice all coincide with alterations in pathways associated with cancer progression (from Gene Ontology and SPIA-based analysis).…”

“…However, Wnek et al (2010) were able to observe these changes as early as 12 weeks of exposure, and this acquired phenotype was sustained even after withdrawal of MMA(III). Therefore, the documented biochemical effects from MMA(III) exposure in UROtsa, such as the induction of ROS, DNA damage, production of pro-inflammatory cytokines, activation of mitogenic signals, and the increase in proliferation rate, exemplifies the utility of the model in the study of arsenic carcinogenesis and the unique effects of MMA(III) on the biology of the urothelium (Eblin et al, 2008b; Wnek et al, 2009; Escudero-Lourdes et al, 2010).…”

Chronic inorganic arsenic exposure in vitro induces a cancer cell phenotype in human peripheral lung epithelial cells

“…Furthermore, human skin keratinocyte (HaCaT) cells have been malignantly transformed following chronic low-level arsenic exposure (Pi et al, 2008) and have aided in the study of the mechanisms of co-carcinogenic effects of arsenic and ultraviolet irradiation (Sun et al, 2011). Along similar lines, other groups have established and used target cell line relevant models of arsenical carcinogenesis such as transformed human urinary bladder cells (Eblin et al, 2007; Wnek et al, 2010; Escudero-Lourdes et al, 2012; Garrett et al, 2014) or other cell lines including bronchial epithelial cell lines (Stuecke et al, 2012; Xu, et al, 2013) to help better define carcinogenic mechanisms. These have all been a great aid in advancing our knowledge of the remarkable diversity of the toxic and carcinogenic actions of this metalloid.…”