Nervi C, Vollberg T M, Grippo J F, Lucas D A, George M D, Sherman M I, Shudo K, Jetten A M
Cell Biology Group, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Cell Growth Differ. 1990 Nov;1(11):535-42.
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)
维甲酸（RA）可诱导小鼠胚胎癌细胞PCC4.aza1R细胞分化。在本研究中，检测了PCC4.aza1R细胞中核维甲酸受体（RARs）的表达。通过尺寸排阻高效液相色谱法对从PCC4.aza1R细胞制备的[3H]RA标记核提取物进行分析，结果表明存在一种特异性RA结合活性，其分子量约为50,000。这种结合活性的95%以上与核部分相关。与胞质维甲酸结合蛋白不同，RARs能非常有效地结合Ch系列的RA类似物。用针对RARα、RARβ和RARγ的互补DNA探针对总RNA进行Northern印迹分析表明，PCC4.aza1R细胞主要含有编码RARα和RARγ的转录本；未检测到RARβ转录本。用RA处理PCC4.aza1R细胞可使RARβmRNA水平呈剂量和时间依赖性增加。RARβmRNA半最大诱导的RA浓度为1 nM。早在添加RA后2小时即可检测到RARβmRNA增加。这种增加未被放线菌酮消除，表明该反应不需要蛋白质合成。几种类维生素A增加PCC4.aza1R细胞中RARβmRNA水平的能力与其对RARs的结合亲和力密切相关，但与其对胞质维甲酸结合蛋白的结合亲和力无关。两种突变细胞系PCC4(RA)-1和(RA)-2在RA处理后不发生分化，其RAR结合活性水平与亲本细胞非常相似。（摘要截短于250字）
