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

Bladder cancer has been associated with chronic arsenic exposure. Monomethylarsonous acid [MMA(III)] is a metabolite of inorganic arsenic and has been shown to transform an immortalized urothelial cell line (UROtsa) at concentrations 20-fold less than arsenite. MMA(III) was used as a model arsenical to examine the mechanisms of arsenical-induced transformation of urothelium. A microarray analysis was performed to assess the transcriptional changes in UROtsa during the critical window of chronic 50nM MMA(III) exposure that leads to transformation at 3 months of exposure. The analysis revealed only minor changes in gene expression at 1 and 2 months of exposure, contrasting with substantial changes observed at 3 months of exposure. The gene expression changes at 3 months were analyzed showing distinct alterations in biological processes and pathways such as a response to oxidative stress, enhanced cell proliferation, anti-apoptosis, MAPK signaling, as well as inflammation. Twelve genes selected as markers of these particular biological processes were used to validate the microarray and these genes showed a time-dependent changes at 1 and 2 months of exposure, with the most substantial changes occurring at 3 months of exposure. These results indicate that there is a strong association between the acquired phenotypic changes that occur with chronic MMA(III) exposure and the observed gene expression patterns that are indicative of a malignant transformation. Although the substantial changes that occur at 3 months of exposure may be a consequence of transformation, there are common occurrences of altered biological processes between the first 2 months of exposure and the third, which may be pivotal in driving transformation.

Interdependent genotoxic mechanisms of monomethylarsonous acid: role of ROS-induced DNA damage and poly(ADP-ribose) polymerase-1 inhibition in the malignant transformation of urothelial cells.

Exposure of human bladder urothelial cells (UROtsa) to 50 nM of the arsenic metabolite, monomethylarsonous acid (MMA(III)), for 12 weeks results in irreversible malignant transformation. The ability of continuous, low-level MMA(III) exposure to cause an increase in genotoxic potential by inhibiting repair processes necessary to maintain genomic stability is unknown. Following genomic insult within cellular systems poly(ADP-ribose) polymerase-1 (PARP-1), a zinc finger protein, is rapidly activated and recruited to sites of DNA strand breaks. When UROtsa cells are continuously exposed to 50 nM MMA(III), PARP-1 activity does not increase despite the increase in MMA(III)-induced DNA single-strand breaks through 12 weeks of exposure. When UROtsa cells are removed from continuous MMA(III) exposure (2 weeks), PARP-1 activity increases coinciding with a subsequent decrease in DNA damage levels. Paradoxically, PARP-1 mRNA expression and protein levels are elevated in the presence of continuous MMA(III) indicating a possible mechanism to compensate for the inhibition of PARP-1 activity in the presence of MMA(III). The zinc finger domains of PARP-1 contain vicinal sulfhydryl groups which may act as a potential site for MMA(III) to bind, displace zinc ion, and render PARP-1 inactive. Mass spectrometry analysis demonstrates the ability of MMA(III) to bind a synthetic peptide representing the zinc-finger domain of PARP-1, and displace zinc from the peptide in a dose-dependent manner. In the presence of continuous MMA(III) exposure, continuous 4-week zinc supplementation restored PARP-1 activity levels and reduced the genotoxicity associated with MMA(III). Zinc supplementation did not produce an overall increase in PARP-1 protein levels, decrease the levels of MMA(III)-induced reactive oxygen species, or alter Cu-Zn superoxide dismutase levels. Overall, these results present two potential interdependent mechanisms in which MMA(III) may increase the susceptibility of UROtsa cells to genotoxic insult and/or malignant transformation: elevated levels of MMA(III)-induced DNA damage through the production of reactive oxygen species, and the direct MMA(III)-induced inhibition of PARP-1.

Monomethylarsonous acid produces irreversible events resulting in malignant transformation of a human bladder cell line following 12 weeks of low-level exposure.

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.

Arsenicals produce stable progressive changes in DNA methylation patterns that are linked to malignant transformation of immortalized urothelial cells.

Aberrant DNA methylation participates in carcinogenesis and is a molecular hallmark of a tumor cell. Tumor cells generally exhibit a redistribution of DNA methylation resulting in global hypomethylation with regional hypermethylation; however, the speed in which these changes emerge has not been fully elucidated and may depend on the temporal location of the cell in the path from normal, finite lifespan to malignant transformation. We used a model of arsenical-induced malignant transformation of immortalized human urothelial cells and DNA methylation microarrays to examine the extent and temporal nature of changes in DNA methylation that occur during the transition from immortal to malignantly transformed. Our data presented herein suggest that during arsenical-induced malignant transformation, aberrant DNA methylation occurs non-randomly, progresses gradually at hundreds of gene promoters, and alters expression of the associated gene, and these changes are coincident with the acquisition of malignant properties, such as anchorage independent growth and tumor formation in immunocompromised mice. The DNA methylation changes appear stable, since malignantly transformed cells removed from the transforming arsenical exhibited no reversion in DNA methylation levels, associated gene expression, or malignant phenotype. These data suggest that arsenicals act as epimutagens and directly link their ability to induce malignant transformation to their actions on the epigenome.