Effects of retinoic acid and staurosporine on the protein kinase C activity and the morphology of two related human neuroblastoma cell lines

Abstract

Studies on the involvement of protein kinase C in retinoic acid-induced differentiation of human neuroblastoma were carried out with two variants of the SK-N-SH cell line namely the SH-F subline, which differentiates to give a fibroblast-like phenotype, and the SH-N subline, which develops into the typical neuronal phenotype. In SH-F, a substantial increase in protein kinase C activity accompanied morphological differentiation. Accordingly, after 7 days of retinoic acid treatment, EDTA-extracted, cytosolic protein kinase C activity increased by slightly more than 2-fold over vehicle-treated controls. Again, detergent-extracted activity, representing membrane-bound or total protein kinase C, showed a similar 2.6- to 5.1-fold increase in treated cells. A time-course study revealed an earliest increase in total activity after two days of retinoic acid treatment which continued linearly for the first 6 to 8 days, and then levelled off. A study of the effect of retinoic acid on the protein kinase C in vitro with SH-F cell extracts showed only a slight increase in activity (of 25%) at the relatively high concentration of 10(-4) M; however, no significant differences were observed at lower concentrations. In contrast, the SH-N cell line responded to retinoic acid by a 45% decrease in EDTA-extractable, and a 63% decrease in detergent-extractable protein kinase C activity. Added to SH-F cell cultures, 15 nM staurosporine was found to inhibit protein kinase C in vivo and to a lesser extent, the protein kinase A. Present together with retinoic acid, staurosporine not only prevented the augmentation but caused a marked decrease of protein kinase C activity in this cell line. Morphological studies indicated that when SH-N cells are treated with staurosporine, or staurosporine and retinoic acid together, a neuronal phenotype similar to that produced by retinoic acid alone is observed. In contrast, when the SH-F cell line is treated with staurosporine or staurosporine and retinoic acid together, the flattened fibroblast-like cell type normally induced by retinoic acids is not observed. Instead, these cells display much smaller cell bodies and elaborate extensions resembling the neuronal phenotype produced by retinoic acid induced differentiation of the SH-N variant. These results suggest that changes in the protein kinase C activity may be involved in regulating the expression of the phenotype during cell differentiation.