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.
Apoptosis ; Blotting, Western ; Cell Death* ; Cell Line ; Cell Survival ; DNA ; Drug Therapy ; Genes, Reporter ; Luciferases ; Lung Neoplasms* ; Lung* ; NF-kappa B* ; Particle Accelerators ; Plasmids ; Radiation Tolerance ; Transcription Factors ; Transfection ; Tumor Necrosis Factor-alpha