A Comparative Study of Protein Kinase C Activation in γ-irradiated Proliferating and Confluent Human Lung Fibroblast Cells

Exposure to low doses of radiation has been recently proven to be much more mutagenic and carcinogenic than previously thought. Since radiation sensitivity varies with different phases of cell cycle, we have investigated the activation of protein kinase C (PKC) after low doses (0.10-1 Gy) of gamma-irradiation on proliferating (log) and non-proliferating (confluent/plateau) human normal lung fibroblast (MRC-5) cells. PKC isoforms have been shown to play key roles in the regulation of proliferation, differentiation, migration and survival. In this study, we have examined the activation of phosphorylated forms of PKC isoforms (PKC-betaII, PKC-alpha/beta, PKC-theta) and non-phosphorylated PKC-alpha in an attempt to understand its kinases in total and subcellular (cytosolic and nuclear) fractions. Cytosolic fraction of the log phase cells showed an increase in activity of PKC-betaII, PKC-alpha/beta and PKC-theta with the radiation dose. However, in the nuclear fraction, PKC-betaII and PKC-theta showed higher activity than the PKC-alpha/beta. In the plateau phase cells of the cytosolic fraction, PKC-betaII showed higher activity than the PKC-alpha/beta and PKC-theta isoforms. Furthermore, in the nuclear fraction PKC-betaII and PKC-alpha/beta isoforms showed higher activity than the PKC-theta. In total cellular protein of the log phase cells, a dose dependent increase in PKC-betaII activity followed by PKC-alpha/ beta was observed and in the plateau phase of cells, PKC-betaII showed higher activity than the PKC-alpha/ beta. The specific activation of PKC isoforms in the plateau phase cells, as demonstrated for the first time, may help us to understand the radiation induced initiation of cellular transformation like hyper-proliferative phenotype, if any.