UP-REGULATION OF CYTOKERATIN-8 AND CYTOKERATIN-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 expre ssing cytokeratins (K) 8, 18 and 19, and basal/myoepithelial cells exp ressing cytokeratins 5 and 14. Human breast cancer T47D cells have a l uminal phenotype and are growth-inhibited by retinoids, a class of com pounds known to regulate cytokeratin expression. To extend our knowled ge of retinoid action in breast cancer, we have studied the retinoid r egulation of cytokeratin expression in the T47D model. We found that r etinoid inhibition of T47D cell growth was accompanied by increases in K8, K18 and K19 mRNA steady-state levels (Northern blot analysis). Th e effect on K8 was studied in greater detail. This effect was seen wit h as low as 1 nM all-trans retinoic acid (tRA) and was maximal (up to 7 fold over control) with 1 mu M tRA (the highest dose tested). Time-c ourse studies revealed a detect able effect at 1 h and a maximal effec t at 8-24 h. Non-retinoidal growth inhibitors (tamoxifen, BrcAMP and g enistein) did not modulate K8 expression, demonstrating that the effec t of tRA was specific. K8 mRNA upregulation was blocked by actinomycin D and cycloheximide, suggesting, in accordance with other studies, th at tRA exerted a transcriptional effect that was secondary to de novo protein synthesis. Five retinoids known to activate retinoic acid rece ptor (RAR) and/or retinoid X receptor (RXR) - tRA; 9-cis-retinoic acid , 9cRA; 13-cis RA, 13cRA; retinyl acetate; and N-(4-hydroxyphenyl) ret in-amide 4HPR - inhibited T47D cell growth and increased K8 expression , whereas an arotinoid (Re-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 blocke d by the RAR alpha-selective antagonist Re-41-5253. Analogous results were obtained throughout when blots were reprobed with K18 cDNA. Weste rn blot and immunocytochemistry experiments demonstrated that protein levels of K8 and K18 increased by 2 days of treatment with 1 mu M tRA. These results show that retinoids enhance the expression of cognate c ytokeratin markers of luminal differentiation in T47D breast cancer ce lls.