Upregulation of cytokeratins 8 and 18 in human breast cancer T47D cells is retinoid-specific and retinoic acid receptor-dependent.
The mamary gland is chiefly composed of luminal epithelial cells expressing cytokeratins (K) 8, 18 and 19, and basal/myoepithelial cells expressing cytokeratins 5 and 14. Human breast cancer T47D cells have a luminal phenotype and are growth-inhibited by retinoids, a class of compounds known to regulate cytokeratin expression. To extend our knowledge of retinoid action in breast cancer, we have studied the retinoid regulation of cytokeratin expression in the T47D model. We found that retinoid inhibition of T47D cell growth was accompanied by increases in K8, K18 and K19 mRNA steady-state levels (Northern blot analysis). The effect on K8 was studied in greater detail. This effect was seen with as low as 1 nM all-trans retinoic acid (tRA) and was maximal (up to 7 fold over control) with 1 microM tRA (the highest dose tested). Time-course studies revealed a detectable effect at 1 h and a maximal effect at 8-24 h. Non-retinoidal growth inhibitors (tamoxifen, BrcAMP and genistein) did not modulate K8 expression, demonstrating that the effect of tRA was specific, K8 mRNA upregulation was blocked by actinomycin D and cycloheximide, suggesting, in accordance with other studies, that tRA exerted a transcriptional effect that was secondary to de novo protein synthesis. Five retinoids known to activate retinoic acid receptor (RAR) and/or retinoid X receptor (RXR) - tRA; 9-cis-retinoic acid, 9cRA; 13-cis RA, 13cRA; retinyl acetate; and N-(4-hydroxyphenyl) retinamide 4HPR - inhibited T47D cell growth and increased K8 expression, whereas an arotinoid (Ro-40-8757) that is not a RAR activator caused growth inhibition but did not upregulate K8. Activation of RAR alpha contributed to K8 upregulation, since this effect was partially blocked by the RAR alpha-selective antagonist Ro-41-5253. Analogous results were obtained throughout when blots were reprobed with K18 cDNA. Western blot and immunocytochemistry experiments demonstrated that protein levels of K8 and K18 increased by 2 days of treatment with 1 microM tRA. These results show that retinoids enhance the expression of cognate cytokeratin markers of luminal differentiation in T47D breast cancer cells.
Jing
Y
Y
Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.
Zhang
J
J
Waxman
S
S
Mira-y-Lopez
R
R
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D001943:Breast Neoplasms / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007633:Keratins / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D018168:Receptors, Retinoic Acid / Q000502:physiology*; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000502:physiology*; D014212:Tretinoin / Q000494:pharmacology*; D015854:Up-Regulation
D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D003609:Dactinomycin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D014212:Tretinoin; D007633:Keratins