A comparative study of protein kinase C activation in γ-irradiated proliferating and confluent human lung fibroblast cells

Abstract

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 γ-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-βII, PKC-α/β, PKC-θ) and non-phosphorylated PKC-α 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-βII, PKC-α/β and PKC-θ with the radiation dose. However, in the nuclear fraction, PKC-βII and PKC-θ showed higher activity than the PKC-α/β. In the plateau phase cells of the cytosolic fraction, PKC-βII showed higher activity than the PKC-α/β and PKC-θ isoforms. Furthermore, in the nuclear fraction PKC-βII and PKC-α/β isoforms showed higher activity than the PKC-θ. In total cellular protein of the log phase cells, a dose dependent increase in PKC-βII activity followed by PKC-α/β was observed and in the plateau phase of cells, PKC-βII showed higher activity than the PKC-α/β. 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.