A Study on Apoptotic Signaling Pathway in HL-60 Cells Induced by Radiation

Kim, Hye Jung; Moon, Sung Keun; Lee, Jae Hoon; Moon, Sun Rock

Abstract

PURPOSE: The mechanical insights of death of cancer cells by ionizing radiation are not yet clearly defined. Recent evidences have demonstrated that radiation therapy may induce cell death via activation of signaling pathway for apoptosis in target cells. This study is designed whether ionizing radiation may activate the signaling cascades of apoptosis including caspase family cysteine proteases, Bcl2/Bax, cytochrome c and Fas/Fas-L in target cells.
MATERIALS AND METHODS
HL-60 cells were irradiated in vitro with 6 MV X-ray at dose ranges from 2 Gy to 32 Gy. The cell viability was tested by MTT assay and the extent of apoptosis was determined using agarose gel electrophoresis. The activities of caspase proteases were measured by proteolytic cleavages of substrates. Western blot analysis was used to monitor PARP, Caspase-3, Cytochrome-c, Bcl-2, Bax, Fas and Fas-L.
RESULTS
Ionizing radiation decreases the viability of HL-60 cells in a time and dose dependent manner. Ionizing radiation-induced death in HL-60 cells is an apoptotic death which is revealed as characteristic ladder-pattern fragmentation of genomic DNA over 16 Gy at 4 hours. Ionizing radiation induces the activation of caspase-2, 3, 6, 8 and 9 of HL-60 cells in a time-dependent manner. The activation of caspase-3 protease is also evidenced by the digestion of poly (ADP-ribose) polymerase and procaspase- 3 with 16Gy ionizing irradiation. Anti-apoptotic Bcl2 expression is decreased but apoptotic Bax expression is increased with mitochondrial cytochrome c release in a time- dependent manner. In additon, expression of Fas and Fas-L is also increased in a time dependent manner.
CONCLUSION
These data suggest that ionizing radiation-induced apoptosis is mediated by the activation of various signaling pathways including caspase family cysteine proteases, Bcl2/Bax, Fas and Fas-L in a time and dose dependent manner.