The identification of sequence elements that mediate stabilization of the CHOP/Gadd153 mRNA by N-(4-hydroxyphenyl)retinamide (4HPR/fenretinide)

Abstract

The CHOP, or the growth-arrest and DNA-damage-inducible gene (Gadd153) encodes a C/EBP-homologous protein that was previously shown to express in cultured cancer cells in response to the anticancer agent, N-(4-hydroxyphenyl)retinamide (4HPR or fenretinide). The 4HPR-induced expression of Gadd153 involves the stabilization of Gadd153 mRNA through a mechanism that is dependent on the production of reactive oxygen species. In this communication, we demonstrated by base-substitution and deletion analyses of the full-length Gadd153 cDNA that an adenylate-uridylate rich (AU-rich) element present in the 3’UTR of Gadd153 mRNA is essential for the mRNA-stabilization effect of 4HPR. However, a chimeric mRNA consisting of the 3’UTR of Gadd153 mRNA joined to the 3’-end of the coding sequence of green-fluorescent protein (GFP) was not stabilized. On the contrary, a similar chimeric mRNA consisting of the GFP coding sequence and a longer Gadd153 mRNA-fragment (containing the last 358 nucleotides of the Gadd153 coding sequence and the entire 3’UTR) was stabilized by 4HPR. These observations suggest that the AU-rich element in the 3’UTR of Gadd153 mRNA alone is not sufficient for 4HPR-induced mRNA stability. Examination of stability of mRNA species transcribed from various deletion and base-substituted mutants of Gadd153 cDNA revealed the presence of a 14-nucleotide sequence element (lying 39 nucleotides upstream of the last codon in the reading frame) that acted as a co-requisite sequence element for 4HPR-induced stability of Gadd153 mRNA. The deletion of sequences in between the 14-nucleotide sequence element and the AU-rich element did not affect 4HPR-induced stability of the resultant mRNA species. Our data demonstrate for the first time that the 4HPR-induced stability of Gadd153 mRNA is co-mediated by the AU-rich element and a novel sequence element of Gadd153 mRNA. Such a mechanism may be required for the sustained expression of Gadd153.