NF-kappaB Activation and cIAP Expression in Radiation-induced Cell Death of A549 Lung Cancer Cells

Lee, Kye Young; Kwak, Shang June

Abstract

BACKGROUND: Activation of the transcription factor NF-kappaB has been shown to protect cells from tumor necrosis factor-alpha, chemotherapy, and radiation-induced apoptosis. NF-kappaB-dependent cIAP expression is a major antiapoptotic mechanism for that. NF-kappaB activation and cIAP expression in A549 lung cancer cells which is relatively resistant to radiation-induced cell death were investigated for the mechanism of radioresistance.
MATERIALS AND METHODS
We used A549 lung cancer cells and Clinac 1800C linear accelerator for radiation. Cell viability test was done by MTT assay. NF-kappaB activation was tested by luciferase reporter gene assay, Western blot for IkappaBalpha degradation, and electromobility shift assay. For blocking NF-kappaB, MG132 and transfection of IkappaBalpha-superrepressor plasmid construct were used. cIAP expression was analyzed by RT-PCR and cIAP2 promoter activity was performed using luciferase assay system.
RESULTS
MTT assay showed that cytotoxicity even 48 hr after radiation in A549 cells were less than 20%. Luciferas assay demonstrated weak NF-kappaB activation of 1.6+/-0.2 fold compared to PMA-induced 3.4+/-0.9 fold. Radiation-induced IkappaBalpha degradation was observed in Western blot and NF-kappaB DNA binding was confirmed by EMSA. However, blocking NF-kappaB using MG132 and IkappaBalpha-superrepressor transfection did not show any sensitizing effect for radiation-induced cell death. The result of RT-PCR for cIAP1 & 2 expression was negative induction while TNF-alpha showed strong expression for cIAP1 & 2. The cIAP2 promoter activity also did not show any change compared to positive control with TNF-alpha.
CONCLUSIONS
We conclude that activation of NF-kappaB does not determine the intrinsic radiosensitivity of cancer cells, at least for the cell lines tested in this study.