Numerous studies link arsenic exposure to human cancers in a variety of tissues, including the liver. However, inorganic arsenic has never been unequivocally shown to be an animal carcinogen, and its carcinogenic mechanism remains undefined. Our previous studies indicate that chronic (> or =18 weeks), low-level (125 to 500 nM) exposure to arsenite induces malignant transformation in the normally nontumorigenic rat liver epithelial cell line (TRL 1215), and these chronic arsenic-exposed (CAsE) cells produce invasive and metastatic tumors upon inoculation into nude mice. In addition, a prior microarray screening analysis of aberrant gene expression showed several oncogenes were overexpressed in CAsE cells exposed to 500 nM arsenite, including a prominent overexpression of the protooncogene c-myc, as well as genes related to cell proliferation. Thus, to better understand the mechanism of arsenic carcinogenesis, we studied the role of c-myc overexpression in arsenite-induced cell transformation. The upregulation of c-myc was confirmed by RT-PCR at the transcription level and by Western blot analysis for the translation product. Further analysis showed that arsenite produced significant increases in the steady-state expression of c-myc in a time- and concentration-dependent manner during the malignant transformation process. The level of c-myc expression was highly correlated (r = 0.988) with tumor formation after inoculation of CAsE cells into nude mice and was also highly correlated (r = 0.997) with genomic DNA hypomethylation. CAsE cells showed a high cell proliferation rate in a fashion related to the level of arsenic exposure. The expression of c-myc was highly correlated with cellular hyperproliferation (r = 0.961). Consistent with the enhanced proliferation both proliferating cell nuclear antigen and cyclin D1 were overexpressed in CAsE cells. In summary, a prominent overexpression of c-myc, a gene frequently activated during hepatocarcinogenesis, is strongly correlated with several events possibly associated with arsenic-induced malignant transformation, including hyperproliferation, DNA hypomethylation and tumor formation upon inoculation into nude mice. These correlations provide convincing evidence c-myc overexpression is mechanistically important in arsenic-induced malignant transformation in this model system.