NF-kappaB Activation and cIAP Expression in Radiation-induced Cell Death of A549 Lung Cancer Cells.
10.4046/trd.2003.55.5.488
- Author:
Kye Young LEE
1
;
Shang June KWAK
Author Information
1. Department of Internal Medicine, College of Medicine, Dankook University, Cheonan, Korea.
- Publication Type:Original Article
- Keywords:
Radiation;
Cell Death;
NF-kappaB;
cIAP1 & 2
- MeSH:
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
- From:Tuberculosis and Respiratory Diseases
2003;55(5):488-498
- CountryRepublic of Korea
- Language:Korean
-
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.
