|Waterland, Robert - Rob|
|Waalkes, Michael p|
Submitted to: Journal of Toxicology and Applied Pharmacology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/16/2004
Publication Date: 1/18/2005
Citation: Benbrahim-Tallaa, L., Waterland, R.A., Styblo, M., Achanzar, W.E., Webber, M.M., Waalkes, M.P. 2005. Molecular events associated with arsenic-induced malignant transformation of human prostatic epithelial cells: aberrant genomic DNA methylation and K-ras oncogene activation. Toxicology and Applied Pharmacology. 206:288-298. Interpretive Summary: Arsenic exposure is linked to cancer in various tissues, including prostate. This study focused on the molecular events during the time of arsenic-induced malignant transformation in a prostate cancer cell line CAsE-PE. The oncogene K-ras was dramatically overexpressed in CAsE-PE cells, but we found that CpG methylation in the K-ras promoter region was not altered by arsenic exposure. Hence, up-regulation of K-ras occurred by some mechanism other than promoter region hypomethylation.
Technical Abstract: Numerous studies link arsenic exposure to human cancers in a variety of tissues, including the prostate. Our prior work showed that chronic arsenic exposure of the non-tumorigenic, human prostate epithelial cell line, RWPE-1, to low levels of (5 microM) sodium arsenite for 29 weeks resulted in malignant transformation and produced the tumorigenic CAsE-PE cell line. The present work focuses on the molecular events occurring during this arsenic-induced malignant transformation. Genomic DNA methylation was significantly reduced in CAsE-PE cells. A time course experiment showed that during malignant transformation DNA methyltransferase activity was markedly reduced by arsenic. However, DNA methyltransferase mRNA levels were not affected by arsenic exposure. Microarray screening showed that K-ras was highly overexpressed in CAsE-PE cells, a result further confirmed by Northern blot and Western blot analyses. Since ras activation is thought to be a critical event in prostate cancer progression, further detailed study was performed. Time course experiments also showed that increased K-ras expression preceded malignant transformation. Mutational analysis of codons 12, 13, and 61 indicated the absence of K-ras mutations. The K-ras gene can be activated by hypomethylation, but our study showed that CpG methylation in K-ras promoter region was not altered by arsenic exposure. Arsenic metabolism studies showed RWPE-1, CAsE-PE, and primary human prostate cells all had a very poor capacity for arsenic methylation. Thus, inorganic arsenic-induced transformation in human cells is associated with genomic DNA hypomethylation and K-ras overexpression. However, overexpression of K-ras occurred without mutations and through a mechanism other than promoter region hypomethylation.