Expression of nuclear retinoic acid receptors in wild-type and mutant embryonal carcinoma PCC4.aza1R cells.
Retinoic acid (RA) induces differentiation of murine embryonal carcinoma PCC4.aza1R cells. In this study, the expression of nuclear retinoic acid receptors (RARs) in PCC4.aza1R cells is examined. Analyses of [3H]RA-labeled nuclear extracts prepared from PCC4.aza1R cells 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 acid-binding protein, the RARs bound RA analogues of the Ch-series very effectively. Northern blot analyses of total RNA with complementary DNA probes specific for RAR alpha, RAR beta, and RAR gamma showed that PCC4.aza1R cells contain predominantly transcripts encoding RAR alpha and RAR gamma; RAR beta transcripts were undetectable. Treatment of PCC4.aza1R cells with RA increased the levels of RAR beta mRNA in a dose- and time-dependent manner. The RA concentration for half-maximum induction of RAR beta mRNA was 1 nM. An increase in RAR beta 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 beta mRNA levels in PCC4.aza1R 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.(ABSTRACT TRUNCATED AT 250 WORDS)
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Cell Biology Group, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Vollberg
T M
TM
Grippo
J F
JF
Lucas
D A
DA
George
M D
MD
Sherman
M I
MI
Shudo
K
K
Jetten
A M
AM
D000818:Animals; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D015342:DNA Probes; D015870:Gene Expression / Q000187:drug effects; D066298:In Vitro Techniques; D051379:Mice; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics; D018168:Receptors, Retinoic Acid; D013724:Teratoma / Q000235:genetics*; D014212:Tretinoin / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D015342:DNA Probes; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; D014212:Tretinoin; D003513:Cycloheximide