Potentiation of oncogenic N‐ras‐induced neurite outgrowth and ornithine decarboxylase activity by phorbol dibutyrate and protein kinase inhibitor H‐8

A recombinant N‐ras oncogene, under the transcriptional control of a corticosteroid‐inducible mouse mammary tumor virus (MMTV) promoter, has been stably transfected into a PC12 rat pheochromocytoma subline. This cell line, designated UR61, undergoes N‐ras‐induced neurite outgrowth and cessation of division when treated with dexamethasone (Guerrero et al.: Biochemical and Biophysical Research Communications 150:1185–1192, 1988). We have employed the UR61 cell line as a model for ras oncogene‐induced neuronal differentiation. In UR61 cells, dexamethasone‐induced expression of the recombinant N‐ras gene resulted in time‐dependent expression of ornithine decarboxylase enzyme (ODC) activity. Prompted by recent reports of possible functional (Lacal et al.: Molecular and Cellular Biology 7:4146–4149, 1987; Wolfman and Macara: Nature 325: 359–361, 1987) and direct (Jeng et al.: Biochemical and Biophysical Research Communications 145:782–788, 1987) interactions between oncogene ras‐coded p21 and protein kinase C (PK‐C; Ca^{+ +}/phospholipid‐dependent protein kinase), we employed the protein kinase inhibitor H‐8 (N‐[2‐(methylamino)ethyl]‐5‐isoquinoline sulfonamide dihydrochloride) and phorbol 12, 13‐dibutyrate (PDBu) to investigate this putative interaction in the UR61 cells, where ODC activity and neurite outgrowth were used as indicators of oncogenic N‐ras action. Treatment of UR61 cells with PDBu depleted cells of PK‐C and failed to promote neurite outgrowth but enhanced N‐ras‐induced neurite outgrowth and ODC activity. H‐8, which suppressed ODC induction by forskolin and phorbol myristate acetate, enhanced both N‐ras‐induced ODC activity and neurite outgrowth. Inhibition of ODC activity by difluoromethylornithine (DFMO) did not suppress oncogenic ras‐induced neurite outgrowth, suggesting that these two ras‐triggered events are mechanistically independent. These findings suggest that certain actions of N‐ras can occur in cells depleted of PK‐C, and thus, the role of PK‐C in ras‐induced differentiation differs from its role in ras‐induced mitogenesis and transformation.