Post-transcriptional regulation of the DNA damage-inducible gadd45 gene in human breast carcinoma cells exposed to a novel retinoid CD437

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Abstract
The biologically active synthetic retinoid CD437 (6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene, AHPN) and different human breast carcinoma (HBC) cell lines were used to examine the possible mechanism(s) of gadd45 induction. Northern blot analysis of mRNA isolated from MCF-7, MDA-MB-468 and MDA-MB-231 HBC cell lines demonstrated a progressive increase in the 1.4 kb gadd45 transcript after exposure to 1 μM CD437. Western blot analysis showed increased gadd45 protein levels in MDA-MB-468 HBC cells following exposure to CD437. CD437 increased gadd45 mRNA levels by ∼20-fold in MDA-MB-468 cells, however, the transcriptional activity was increased ∼2–3-fold as demonstrated by the human gadd45 promoter-luciferase reporter construct and nuclear run-off assays. Sublines of MDA-MB-468 HBC cells expressing stably integrated GADD45 cDNA fragments were obtained and CD437-dependent induction of GADD45 analyzed. We report that ∼300 nt located in the 5′-untranslated region (5′-UTR) of gadd45 mRNA are involved in the CD437-dependent 4-fold enhanced stability of gadd45 transcripts. MDA-MB-468 cells were stably transfected with either a plasmid having a CMV promoter-driven rabbit β-globin gene or plasmids having a CMV promoter-driven chimeric gadd45 5β-UTR—rabbit β-globin gene, where the entire gadd45 5′-UTR (from + 1 to + 298) or a 45 bp subfragment of the gadd45 5′-UTR (from + 10 to + 55) was positioned at the 5′-end of the rabbit β-globin gene. CD437 was found to up-regulate expression of both the chimeric gadd45—rabbit β-globin transcripts, suggesting that cis element(s) involved in the CD437-dependent enhanced stability of gadd45 mRNA are contained in the 45 nt of the 5′-UTR of the gadd45 mRNA.