Regulated expression of the retinoblastoma gene in differentiating embryonal carcinoma cells.

The expression of the retinoblastoma susceptibility (RB) gene was investigated in P19 embryonal carcinoma cells and in these cells induced to differentiate with retinoic acid (RA) or with dimethyl sulfoxide (DMSO). In undifferentiated cells very low levels of RB mRNA and protein were present. DMSO-treated P19 cell cultures develop into mesodermal and endodermal cells and RB expression increased only slightly in these differentiating cells. RA-treated P19 cells develop into neuroectoderm and this differentiation was accompanied by a marked increase in RB expression with mRNA levels increasing 15 fold by 4-6 days following initiation of RA treatment. No such increase occurred in mutant cells that fail to respond to RA. The RB promoter did not appear to be directly activated by RA. Nevertheless, the increase in RB expression in RA-treated cells appeared to be due to enhanced initiation of transcription because cells transfected with a reporter gene driven by the RB promoter expressed the reporter gene with kinetics similar to that of the RB gene. Thus the activation of the RB gene appears to be achieved indirectly by RA-induced factor(s) in differentiating neuroectodermal cells. The post-mitotic neurons that developed in RA-treated cultures contained only the hypophosphorylated form of the RB protein. Recent studies (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992) have shown that mice lacking the RB gene have abnormalities in early brain development suggesting that the rapid rise in RB expression and the hypophosphorylation of the protein are essential for neuronal cell differentiation.