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 microM 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 approximately 20-fold in MDA-MB-468 cells, however, the transcriptional activity was increased approximately 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 approximately 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 beta-globin gene or plasmids having a CMV promoter-driven chimeric 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 found to up-regulate expression of both the chimeric gadd45 -rabbit beta-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.
Rishi
A K
AK
Department of Internal Medicine and Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA, rishia@karmanos.org
Sun
R J
RJ
Gao
Y
Y
Hsu
C K
CK
Gerald
T M
TM
Sheikh
M S
MS
Dawson
M I
MI
Reichert
U
U
Shroot
B
B
Fornace
A J
AJ
Brewer
G
G
Fontana
J A
JA
D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D004249:DNA Damage / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D005914:Globins / Q000235:genetics / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012176:Retinoids / Q000494:pharmacology*; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects; D000097642:GADD45 Proteins
D020121:5' Untranslated Regions; D000970:Antineoplastic Agents; D005914:Globins; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012176:Retinoids; D000097642:GADD45 Proteins; C099555:CD 437