Retinoic acid is known to inhibit mammary carcinogenesis in rodents and to inhibit proliferation and steroid hormone receptor gene expression in human breast cancer cells. Since these effects are likely to be mediated by nuclear retinoic acid receptors (RARs) the present study was initiated to determine the expression and regulation of RARs in human breast cancer cell lines. Differential cellular gene expression of the RARs was determined by Northern blot analysis of total RNA prepared from 5 ER+ and 6 ER− cell lines. RARα was detected as mRNA species of 2.7 and 3.4 kilobases in all cell lines and the level of gene expression was greater in ER+ cell lines (P < 0.001). RARβ mRNA (3.7 kilobases) was detected in seven of the eleven lines tested and was expressed most commonly in ER− cell lines. RARγ mRNA was expressed in all cell lines as a transcript of 3.4 kilobases at levels that were similar in both ER+ and ER− cell lines. Retinoic acid failed to regulate the expression of the RARα and RARγ genes. The effect of steroid hormones on RARα and RARγ mRNA levels was also examined. In four PR+ cell lines (T-47D, BT 474, MCF-7M, and MDA-MB-361), progestins markedly decreased RARα mRNA levels. The progestin effect on RARα levels in T-47D cells was detectable at concentrations of 0.05 nm and was maximal at 1 nm 16α-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione ORG 2058, whereas dihydrotestosterone and dexamethasone were without effect. RARα and RARγ mRNA levels were rapidly decreased by progestin, and the effect was maximal 3–6 h after ORG 2058 treatment. However, the mRNA loss was transient, and recovery of RARα and RARγ mRNA levels was noted 12–24 h after retinoic acid treatment. Although RARγ mRNA returned to control levels by 24 h, the decrease in RARα mRNA was maintained at around 50% control until at least 48 h. In summary, RARα and RARγ were expressed in all human breast cancer cell lines and were regulated by progestins in the PR+ T-47D cell lines. The previously reported ability of retinoic acid to down-regulate PR mRNA and the present demonstration that progestins down-regulate RARα and RARγ mRNA suggest that mutual regulation may be a mechanism through which PR and the RARs interact in human breast cancer cells.
Supported by the National Health and Medical Research Council of Australia and MLC-Life, Ltd.