Radiation-induced Apoptotic Signaling Pathway in HL - 60 Cells

Ahn, Sung Ja; Park, Rae Kil; Lee, Sang Rock; Chung, Woong Ki; Nah, Byung Sik; Nam, Taek Keun; Chung, Hun Taeg; Moon, Sun Rock; Kang, Heoung Keun; Park, Seung Jin

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 was designed whether ionizing radiation may activate the signaling cascades of apoptosis including caspase family cystein proteases, mitogen-activated protein (MAP) kinases, and transcriptional activation factors in target cells eventually leading to death.
MATERIALS AND METHODS
HL-60 cell line in the log phase was used in this study and the culture media was RPMI 1640. The irradiation was done using the linear accelarator and the radiation does was 10 Gy, 20 Gy, and 30 Gy, respectively. The cell viability was tested by MTT assay and apoptosis was identified by the DNA fragmentation assay. JNK1 (cJun N-terminal kinase) and ERK (extracellular-signal regulated protein kinase) activity was analyzed by the in vitro Ig complex kinase assay. NF- kB (Nuclear Factor- kB) and AP-1 (activator protein-1) activity was assayed by the electrophoretic mobility sbift assay.
RESULTS
Ionizing radiation decreased the viability of HL-60 cells in a time and dose dependent manner. Ionizing radiation-induced cell death of HL-60 cells may be an apo- ptotic death which was evidenced as apoptotic characteristic ladder pattern fragmentation of DNA over 20 Gy at 4 hours. Ionizing radiation specifically induced the activation of CPP32-like cystein protease rather than ICE-like protease of HL-60 cells in a time and dose dependent manner. The activation of CPP32-like cystein protease was also evidenced by the digestion of poly (ADP-ribose) polymerase with 30 Gy ionizing irradiation at 2 hours. The activity of JNK1 was transiently increased up to 3.6 fold by 30 Gy ionizing radiation at 2 hours. Ionizing radiation also rapidly activated the transcriptional activation factors including AP-1 and NF- kB at 10 or 30 min.
CONCLUSION
These data suggested that ionizing radiation-induced apoptosis was mediated by the activation of CPP32-like cystein protease, JNK1, and transcriptional activation factors