(1/2) EXPANDING THE CRITICAL PERIOD OF ARSENICAL-INCUDED BLADDER CARCINOGENESIS (2/2) EXAMINING THE ROLE OF PDGFRA AS A BIOMARKER IN ARSENICAL-INCUDED BLADDER CARCINOGENESIS

Daniel Troup , Medeiros, Matthew; Minh le, Tam; Gandolfi, A. Jay

(1/2) EXPANDING THE CRITICAL PERIOD OF ARSENICAL-INCUDED BLADDER CARCINOGENESIS (2/2) EXAMINING THE ROLE OF PDGFRA AS A BIOMARKER IN ARSENICAL-INCUDED BLADDER CARCINOGENESIS

(1/2) Continued exposure to arsenic has been associated with several types of cancer, including bladder cancer. Studies using monomethylarsonous acid [MMA(III)], a metabolite of inorganic arsenic, have been shown to induce a carcinogenic transformation in human urothelial cell lines (UROtsa). Through utilization of RT-PCR, we assessed gene expression changes in UROtsa following chronic 50 nM MMA(III) exposure at monthly time points until transformation was observed after 3 months of exposure. Examination of 29 genes (based upon previous microarray analysis) from 7 distinct pathways- mitogenic, PI3K/AKT, apoptosis, JAK/STAT, oxidative stress, DNA repair, and inflammation- was performed. Analysis revealed minor changes at one and two months of exposure, and significant changes occurring at 3 months of exposure. Incremental analysis was then performed to assess the gene expression changes between 2 and 3 months of exposure with the observation of progressive alterations in the expression of the genes: PDGFRA, COX2, and XAF1. The alterations in these genes are being correlated with expected phenotypic changes- hyperproliferation and colony formation in soft agar- in the transforming cells. Because short-term exposure (up to 2 months) has not been shown to induce transformation in UROtsa, while gene expression changes have been observed with exposure for 3 months and beyond, we believe a stress-threshold exists.

(2/2) Using monomethylarsonous acid [MMA(III)], a metabolite of inorganic arsenic, we have induced a carcinogenic transformation in human urothelial cell lines (UROtsa) by 3 months of chronic, 50nM exposure.  To determine the mechanisms of arsenical-induced bladder cancer, we examined the gene expressions of several biomarkers. Of these biomarkers, the tyrosine kinase receptor PDGFRA, has consistently shown to have significant increases in expression by 3 months of exposure.  We explored the role of PDGRA in the transformation of UROtsa through chemical inhibition, genomic intervention, and epigenetic analysis with correlated phenotypic changes. Chemical inhibition of PDGRA in transformed UROtsa with 3µM GLEEVEC reduced proliferation rates near that of untreated UROtsa.  Also, treatment of 3µM GLEEVEC with transformed UROtsa reduced the number of colonies grown in soft agar. We believe the manipulation of the MAPK pathway by MMA(III) through PDGFRA may be the initial biological alteration leading to the subsequent gene expression and phenotypic changes seen in the transformation of UROtsa. Currently, we are working to knockout the expression of the PDGRA gene in untreated UROtsa and short-term exposed UROtsa, and analyzing methylation patterns of the PDGRA gene at monthly time points in treated UROtsa.

These studies were supported by the Superfund Basic Research Program Grant (NIH grant ES04940) from National Institute of Environmental Health Sciences and the Trainee in Toxicology and Toxicogenomics (NIEHS grant ES007091). Additional support from NIH grant CA23074 and NIEHS grant ES06694.

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