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Incorporation of an Internal Ribosome Entry Site–Dependent Mechanism in Arsenic-Induced GADD45 Expression

¹ The Health Effects Laboratory Division, National Institute for Occupational Safety and Health; ² Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, West Virginia; and ³ Institute for Nutritional Sciences, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

Requests for reprints: Fei Chen, Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, 1095 Willowdale Road, Morgantown, WV 26505. Phone: 304-285-6021; E-mail: LFD3{at}cdc.gov.

We have previously shown that trivalent arsenic (arsenite, As³⁺) is able to induce GADD45 expression in human bronchial epithelial cells through activation of c-Jun NH₂-terminal kinase and nucleolin-dependent mRNA stabilization. In the present report, we show that As³⁺ is capable of inducing translation of the GADD45 protein through a cap-independent, or rather, an internal ribosome entry site (IRES)–dependent mechanism. In growth-arrested cells, As³⁺ elevated the GADD45 protein level in a dose- and time-dependent manner which did not correlate with the GADD45 mRNA expression. Pretreatment of the cells with rapamycin, an inhibitor for the cap-dependent translation machinery through the suppression of mTOR and p70S6 kinase, failed to affect the induction of the GADD45 protein induced by As³⁺. Sequence analysis revealed a potential IRES element in the 5'-untranslated region of the GADD45 mRNA. This IRES element in the 5'-untranslated region of the GADD45 mRNA is functional in mediating As³⁺-induced translation of the GADD45 protein in a dicistronic reporter gene activity assay. Immunoprecipitation and proteomic studies suggest that As³⁺ impairs the assembly of the cap-dependent initiating complex for general protein translation but increases the association of human elongation factor 2 and human heterogeneous nuclear ribonucleoprotin with this complex. Thus, these results suggest that in growth-arrested cells, As³⁺ is still capable of inducing GADD45 expression through an IRES-dependent translational regulation. [Cancer Res 2007;67(13):6146–54]