[Enhancement of HepG2 cell radiosensitivity by mutant IkappaBalpha gene transfection].
OBJECTIVE
To investigate the efficacy of transfection with a mutant IkappaBalpha gene (mIkappaBalpha) in enhancing the radiosensitivity of hepatocellular carcinoma cells.


METHODS
The hepatocellular carcinoma Hep G2 cells were divided into 3 group and transfected with the adenovirus containing mIkappaBalpha vector (Ad-mIkappaBalpha group), Ad vector (Adv group), or without treatment (parental control cells). Before and after irradiation of the cells with 6 Gy high-energy X ray, Western blotting was performed to measure the expression level of IkappaBalpha in the cytoplasm, and electrophoresis mobility shift assay (EMSA) carried out to evaluate the nuclear factor-kappaB (NF- kappa;B) activity in the cell nuclei, with the cell apoptosis detected using TUNEL assay. The radiosensitivity of the HepG2 cells were determined by comparison between the 3 groups in term of the surviving cell fractions (SF2) after 2-Gy X-ray exposure, and of the Do and Dq values obtained using the single-hit multi target model.


RESULTS
Before the X-ray exposure, the Hep G2 cells in Adv group and the control group showed low levels of IkappaBalpha absorbance in the cytoplasm, which were further decreased after the exposure. The NF-kappaB activity in the nuclei of the cells in the two groups was positive (+) before irradiation, and substantially enhanced (++) after the exposure and maintained the stably activated state. The apoptotic index of the cells in the two groups was 1.4 and 1.6 before irradiation, and increased to 8.9 and 11.7 after the irradiation, respectively. The cells in the Ad-mIkappaBalpha group, however, exhibited high levels of IkappaBalpha absorbance either before or after the irradiation, which were approximately 3 times that of the Adv group, but the NF-kappaB activity remained negative irrespective of the irradiation. The apoptotic index of the cells in the Ad-mIkappaBalpha group was 18.2 before irradiation, was increased to 88.3 after the irradiation. Among the 3 groups, Ad-mIkappaBalpha group had the smallest SF(2) value of 0.301 but the highest sensitivity enhancement ratio (SER) of2.99, with the lowest Do and Dq values (1.468 and 0.709, respectively).


CONCLUSIONS
mIkappaBalpha gene transfection into HepG2 cells inhibits the anti-apoptotic activity of NF-kappaB to enhance the radiosensitivity of the cells.
Jin
Wu-dong
WD
Department of Radiation Oncology, Nangfang Hospital, Southern Medical University, Guangzhou 510515, China. jwd18@21cn.com
Chen
Long-hua
LH
Mu
Feng
F
D000256:Adenoviridae / Q000235:genetics; D017209:Apoptosis / Q000235:genetics / Q000502:physiology / Q000528:radiation effects*; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D005822:Genetic Vectors; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D014162:Transfection
D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; D000072000:NF-KappaB Inhibitor alpha