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Springer, Molecular and Cellular Biochemistry, 1-2(279), p. 123-131, 2005

DOI: 10.1007/s11010-005-8284-2

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Sodium arsenite-induced inhibition of eukaryotic translation initiation factor 4E (eIF4E) results in cytotoxicity and cell death

Journal article published in 2005 by Sreekumar Othumpangat ORCID, Michael Kashon, Pius Joseph
This paper is available in a repository.
This paper is available in a repository.

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Abstract

Exposure to arsenic (As) is a risk factor for the development of diabetes, vascular diseases and cancer. Several theories have been proposed to account for the mechanisms potentially responsible for As toxicity and carcinogenesis. Currently, we have investigated whether the eukaryotic translation initiation factor 4E (eIF4E), the mRNA cap binding and rate limiting factor required for translation, is a target for As-induced cytotoxicity and cell death. We have also investigated the potential cellular mechanisms underlying the As-induced de-regulation of expression of eIF4E that are most likely responsible for the cytotoxicity and cell death induced by As. Exposure of four different human cell lines - HCT15 (colorectal adenocarcinoma), PLC/PR/5 (hepatocellular carcinoma), HeLa (cervical adenocarcinoma) and Chang (likely derived from HeLa cells) to sodium arsenite (NaAsO2) for time intervals up to 24 h resulted in a concentration-dependent cytotoxicity and cell death. All the NaAsO2-treated cells exhibited significant inhibition of eIF4E gene (protein). The potential involvement of eIF4E gene expression in the NaAsO2-induced cytotoxicity and cell death was investigated by silencing the cellular expression of the eIF4E gene by employing a small interfering RNA (SiRNA) specifically targeting the eIF4E gene's expression. The SiRNA-mediated silencing of eIF4E gene expression also resulted in significant cytotoxicity and cell death suggesting that the toxicity noticed among the NaAsO2-treated cells was probably due to the chemically induced inhibition of eIF4E gene expression. The potential involvement of inhibition of eIF4E gene expression in the NaAsO2-induced cytotoxicity and cell death was further investigated by employing transgenic cell lines overexpressing the eIF4E gene. Overexpression of the eIF4E gene in the Chinese hamster ovary cell line was protective against the NaAsO2-induced cytotoxicity and cell death. Additional studies conducted to understand the potential mechanisms responsible for NaAsO2-induced inhibition of eIF4E gene expression demonstrated that exposure to NaAsO2 resulted in transcriptional down-regulation of the eIF4E gene only in HCT-15 and HeLa cells, while in the NaAsO2-treated and PLC/PR/5 and Chang cells, the eIF4E mRNA expression level was comparable to those of the corresponding control cells. Cellular levels of ubiquitin and the process of ubiquitination were significantly higher in the NaAsO2-treated cells compared with the control cells. Immunoprecipitation of lysates obtained from the NaAsO2-treated cells and the subsequent western blot analysis of the immunoprecipitated protein(s) using the eIF4E antibody detected the presence of eIF4E protein in the immunoprecipitate suggesting possible ubiquitination of eIF4E protein in the NaAsO2-treated cells. Pre-exposure of the NaAsO2-treated cells to proteasome inhibitors blocked the inhibition of eIF4E gene expression as well as the resulting cytotoxicity and cell death. Furthermore, exposure of cells to NaAsO2 resulted in a significant inhibition of expression of the cell cycle and growth regulating gene, cyclin D1. Whether or not the inhibition of cyclin D1 in the NaAsO2-treated cells is mediated through the inhibition of eIF4E was tested by silencing the expression of eIF4E gene in the cells. Transfection of cells with SiRNA specifically targeting eIF4E gene expression resulted in a significant inhibition of cyclin D1 gene suggesting that the observed inhibition of cyclin D1 gene in the NaAsO2-treated cells is most likely mediated through inhibition of eIF4E gene. Taken together, our results indicate that the exposure of cells to NaAsO2 resulted in cytotoxicity and cell death, at least in part, due to the inhibition of eIF4E gene expression leading to diminished cellular levels of critical genes such as cyclin D1.