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

The biologically active synthetic retinoid CD437 (6-[3-adamantyl-4-hydroxyp henyl]-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-2 31 HBC cell lines demonstrated a progressive increase in the 1.4 kb gadd45 transcript after exposure to 1 mu M CD437, Western blot analysis showed inc reased gadd45 protein levels in MDA-MB-468 HBC cells following exposure to CD437, CD437 increased gadd45 mRNA levels by similar to 20-fold in MDA-MB-4 68 cells, however, the transcriptional activity was increased similar to 2- 3-fold as demonstrated by the human gadd45 promoter-luciferase reporter con struct and nuclear run-off assays. Sublines of MDA-MB-468 HBC cells express ing stably integrated GADD45 cDNA fragments were obtained and CD437-depende nt induction of GADD45 analyzed. We report that similar to 300 nt located i n the 5'-untranslated region (5'-UTR) of gadd45 mRNA are involved in the CD 437-dependent 4-fold enhanced stability of gadd45 transcripts, MDA-MB-468 c ells were stably transfected with either a plasmid having a CMV promoter-dr iven rabbit beta-globin gene or plasmids having a CMV promoter-driven chime ric gadd45 5'-UTR-rabbit beta-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 beta-globin gene. CD437 was f ound to up-regulate expression of both the chimeric gadd45-rabbit beta-glob in transcripts, suggesting that cis element(s) involved in the CD437-depend ent enhanced stability of gadd45 mRNA are contained in the 45 nt of the 5'- UTR of the gadd45 mRNA.