Expression of nuclear retinoic acid receptors in wild-type and mutant embryonal carcinoma PCC4.aza1R cells.

Retinoic ack (RA) induces differentiation of murine embryonal :arcinoma PCC4.azalR cells. In this study, the expres ion of nuclear retinoic acid receptors (RARs) in ‘CC4.iza1R cells is examined. Analyses of [3H]RAlabeled nuclear extracts prepared from PCC4.azalR ells by size-exclusion high-performance liquid chromatography demonstrated the presence of a specific RA-binding activity that migrated with a molecular weight of approximately 50,000. More than 95% of this binding activity was associated with the nuclear fraction. In contrast to cytosolic retinoic acidbinding protein, the RARs bound RA analogues of the Ch-series very effectively. Northern blot analyses of total RNA with complementary DNA probes specific for RARor, RARfl, and RAR#{176}y showed that PCC4.azalR cells contain predominantly transcripts encoding RARcr and RAR”y; RARfI transcripts were undetectable. Treatment of PCC4.azalR cells with RA increased the levels of RARI9 mRNA in a doseand time-dependent manner. The RA concentration for half-maximum induction of RARfl mRNA was 1 n i. An increase in RAR$ mRNA was detectable as early as 2 h after the addition of RA. This increase was not abrogated by cycloheximide, suggesting that protein synthesis is not required for this response. The ability of several retinoids to increase RAR 9 mRNA levels in PCC4.azalR cells correlated well with their binding affinity to the RARs but not with their binding affinity to cytosolic retinoic acid-binding protein. Two mutant cell lines, PCC4(RA)-1 and (RA)-2, which do not undergo differentiation after RA treatment, contained levels of RAR-binding activity very similar to those of the parental cells. PCC4(RA)-1 and PCC4(RA)-2 cells expressed RARa and RAR”y transcripts identical in size to that of PCC4.azalR cells. However, PCC4(RA)-1 cells expressed only one RAR”y transcript of 3.1 kilobases. The loss of the expression of the 3.3-kilobase RAR’y transcript in PCC4(RA)-1 cells may be causally related to their inability to differentiate in response to RA. RA treatment increased the levels of RAR$ mRNA in both PCC4(RA)-1 and (RA)-2 cells, indicating that these mutant cell lines have not totally lost their responsiveness to RA.