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

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 5'-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.