INDUCIBLE OVEREXPRESSION OF HUMAN PROTEIN KINASE-C-ALPHA IN NIH 3T3 FIBROBLASTS RESULTS IN GROWTH ABNORMALITIES

We have stably overexpressed the human protein kinase C-alpha (hPKC-alpha) in NIH 3T3 fibroblasts under the control of the interferon (IFN) type I inducible murine Mx promoter. These cells showed a 10-fold increase in the transcription of hPKC-alpha-mRNA after induction with interferon-alpha. The increase in the amount and activity of protein kinase C (PKC)-protein in these cells was only about 3-fold after induction with interferon-alpha. Compared to control cells which were transfected with the vector only, the NIH 3T3 fibroblasts transfected with the hPKC-alpha-cDNA showed already a slightly increased PKC-activity and amount of PKC-protein in the absence of interferon-alpha. The hPKC-alpha-overexpressing cells had an altered, "transformed-like" morphology, which was reversed by staurosporine, an increased growth rate and a higher saturation density. The growth rate was further increased by treating the cells with interferon-alpha. The hPKC-alpha-overexpressing cells were able to grow in soft agarose after treatment with phorbol ester such as TPA (12-O-tetradecanoylphorbol 13-acetate). After phorbol ester and interferon treatment a stronger expression of the protooncogene c-jun was detectable in the hPKC-alpha-overexpressing cells, whereas expression of c-fos and c-myc was not affected. Since these cells show a specific response pattern due to induced PKC-alpha-expression they might be useful as an assay system for the development of PKC isozyme-specific inhibitors and activators.