Modulation of signal transduction pathways and global protein composition of macrophages by ionizing radiation

It is assumed that the exposure of cells to ionizing radiation modulates th eir signal transduction pathways, which then govern the early and late radi ation-induced alterations in gene expression. In this study we tested the e ffects of low doses of X-irradiation on the cell signaling and global prote in composition of an HL-60 human promyelocytic leukemia cell line different iated along a macrophage-like cell pathway by 4 beta-phorbol-12-myristate-1 3-acetate (PMA). Using sodium dodecyl sulfate-polyacrylamide gel electropho resis (SDS-PAGE) followed by immunoblotting of anti-phosphotyrosine immunop recipitates, we found radiation-induced changes in the level of phosphoryla tion of proteins with molecular masses of 45 and 48 kDa, but in the most in tensively stained area, ranging from 54 to 60 kDa, no alterations were obse rved. When two-dimensional electrophoresis (2-DE) immunoblotting was applie d, only proteins from this heavily stained region were visualized and in ad dition the evident differences in tyrosine phosphorylated protein patterns between nonirradiated and irradiated cells were found in this area. Further more, the immunostaining of extracellular signal-regulated kinase 2 (ERK2) which did not prove its tyrosine phosphorylation demonstrated the existence of several ERK2 charge isoforms showing differential expression after X-ir radiation. Comparing the whole protein profiles we found after the simultan eous quantitation of 1000 matched spots two proteins whose expression was r egulated in an opposite manner in nonirradiated and X-irradiated cells. The quantities of both spots showed increases or decreases by a factor of 2 or more between irradiated and nonirradiated samples and both these changes w ere statistically significant (P < 0.05).