The focus of this project is to better elucidate the possible involvement of transmembrane signal transmission systems in the regulation of cell growth and in malignant transformation. A new multiwell filtration assay was developed to determine protein kinase C (PKC) phosphotransferase and phorbol ester binding activities. This method is more rapid and is better suited for analyzing large numbers of samples than conventional methods. Exposure of cells to oxidant tumor promoters has been shown to directly modify PKC to convert it to an active form no longer dependent on Ca2+ and phospholipids. With this new method of assay, results indicate that short- term (15 to 30 min) pretreatment of cells with low (10 to 100 nM) concentrations of retinoic acid protects PKC from oxidants such as H2O2 and m-periodate. These results suggest that some of the anti-tumor promoter actions of retionoids may be through protection of PKC from oxidative modification. Phosphate ion (Pi) uptake into NIH 3T3 cells was found to be differentially regulated by activation of PKC and of cAMP-dependent protein kinase (PKA). Activation of PKC resulted in the rapid (within min) stimulation of short- term (2 min) sodium-dependent Pi uptake, while activation of PKA resulted in the rapid inhibition of Pi transport. These results suggest that stimulation of PKC and PKA through hormone-induced production of diacylglycerol and cAMP, respectively, likely serves to rapidly regulate the intracellular level of Pi. Raf-1 protein kinase (Raf-1 PK) levels and activity were found to be elevated in human radiation resistant squamous carcinoma cells. Treatment of these cells with antisense c-raf-oligodeoxyribonucleotides (ODNS) was found to decrease Raf-1 PK activity in a concentration (20-40 mM)-and time (12-36 hrs)-dependent manner. This experimental approach should be useful in determining the possible role of Raf-PK in radiation resistance. Previous studies have demonstrated a deficiency of PKA in fibroblasts and in erythrocytes isolated from patients with psoriasis. Retinoic acid treatment of psoriatic fibroblasts was shown to increase PKA to levels found in normal human fibroblasts. Results now indicate that the effects of retinoids on PKA are rapid. Treatment of isolated psoriatic erythrocytes with acitretin (a synthetic retinoid) for 15 min resulted in a significant increase in cAMP binding to the RI regulatory subunit of PKA. Oral administration of acitretin to psoriatic patients also resulted in a rapid (within 1 hr) increase in the ability of RI to bind cAMP. These data indicated that retinoids may act to rapidly modify PKA.
