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 their signal transduction pathways, which then govern the early and late radiation-induced alterations in gene expression. In this study we tested the effects of low doses of X-irradiation on the cell signaling and global protein composition of an HL-60 human promyelocytic leukemia cell line differentiated along a macrophage-like cell pathway by 4beta-phorbol-12-myristate-13-acetate (PMA). Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting of anti-phosphotyrosine immunoprecipitates, we found radiation-induced changes in the level of phosphorylation of proteins with molecular masses of 45 and 48 kDa, but in the most intensively stained area, ranging from 54 to 60 kDa, no alterations were observed. When two-dimensional electrophoresis (2-DE) immunoblotting was applied, only proteins from this heavily stained region were visualized and in addition the evident differences in tyrosine phosphorylated protein patterns between nonirradiated and irradiated cells were found in this area. Furthermore, 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-irradiation. Comparing the whole protein profiles we found after the simultaneous quantitation of 1000 matched spots two proteins whose expression was regulated 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 were statistically significant (P<0.05).
Stulík
J
J
Purkyne Military Medical Academy, Institute of Radiobiology and Immunology, Hradec Králové, Czech Republic. jstulik@pmmfhk.cz
Koupilová
K
K
Hernychová
L
L
Macela
A
A
Bláha
V
V
Baaske
C
C
Kaffenberger
W
W
van Beuningen
D
D
D015180:Electrophoresis, Gel, Two-Dimensional; D018922:HL-60 Cells; D006801:Humans; D008264:Macrophages / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects*; D010766:Phosphorylation; D011506:Proteins / Q000032:analysis / Q000528:radiation effects*; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism
D011506:Proteins; D014443:Tyrosine