Identification and characterization of all-trans-retinoic acid receptor transcripts and receptor protein in human...

Retinoic acid (RA) induces the differentiation of tumor cells of neural origin and may do so by binding to one or more nuclear receptor proteins. We have identified transcripts and nuclear RA receptor (RAR) protein in a clonal line of human neuroblastoma cells that differentiate in response to RA. Prior to any exposure to RA, LA1-15n cells express two transcripts for RAR alpha (approximately 3.6 and approximately 2.7 kb) as well as low levels of transcript for RAR beta (approximately 3.4) and RAR gamma (approximately 2.8 kb). Exposure of LA1-15n cells to RA leads to the induction of a approximately 2.9-kb RAR beta mRNA, whereas the expression of transcripts for RARs alpha and gamma does not change appreciably. The 2.9-kb RAR beta transcript is increased by 4 h (8-fold) and continues to increase for 24-48 h (40- to 60-fold). The RA-associated increase in RAR beta mRNA in LA1-15n cells is not diminished by the addition of the protein synthesis inhibitor, cycloheximide, but is abolished by the addition of the RNA synthesis inhibitor, actinomycin D. In addition to RAR transcripts, LA1-15n cells contain a nuclear protein with the requisite characteristics of a RAR. The nuclear protein binds all-trans-[3H]RA with high affinity (Kd approximately 0.2 nM). The nuclear protein sediments at approximately 4S, which is consistent with the molecular mass deduced from RAR cDNAs (approximately 50,000 Da). The nuclear protein is clearly distinguishable from a all-trans-[3H]RA-binding protein found in the cytosolic fraction of LA1-15n cells. The cytosolic protein sediments at approximately 2S on sucrose density gradients, consistent with the expected molecular mass of the cellular retinoic acid-binding protein (approximately 16,000 Da). The nuclear [3H]RA-binding protein binds to DNA-cellulose and to the RAR beta response element. These results support the hypothesis that RARs are present in human neuroblastoma cells and may be involved in human neuroblastoma cell differentiation. They also demonstrate that RA markedly influences the expression of steady-state levels of mRNA for one of its own receptors, the RAR beta.