Retinoid receptor antisense DNAs inhibit alkaline phosphatase induction and clonogenicity in malignant keratinocytes

Abstract

Antisense oligodeoxynucleotides [oligo(dN)s] corresponding to human cellular retinol-binding protein I (cRBP) and human nuclear retinoic acid receptor alpha (hnRAR) were synthesized. Exposure of human malignant keratinocytes to these oligo(dN)s significantly attenuated the level of cytoplasmic cRBP and hnRAR in a concentration- and time-dependent manner. Further, the induction of alkaline phosphatase by retinol in these cells was blocked by treatment with 30 microM antisense oligo(dN) to cRBP or hnRAR but not by 30 microM of sense oligo(dN) to cRBP. Antisense oligo(dN) treatments concomitantly induced cell rounding, loss of cell-cell attachment, and cell adhesion to the substratum. By contrast, treatment of cells with an anticytokinetic agent, cytochalasin B, or with a cytostatic concentration of sodium azide failed to reduce cytoplasmic cRBP or hnRAR from nuclear extracts, even though antisense oligo(dN)-like changes in cell morphology were observed. Treatment of the cells for greater than 2.75 hr with 20-40 microM of either antisense oligo(dN) also led to the loss of clonogenic potential. These results show that both cytoplasmic and nuclear receptors for retinoids are important in the transduction of a retinoid signal response critical to cellular growth and differentiation. Our findings also suggest that defined genes, which are specified by retinoids and their receptors, may account for the pleiotropic effect of vitamin A compounds.