[The expression of Recombinant adenovirus IkappaBalphaM in human hepatocarcinoma HepG2 and it's inhibitive effect to the activity of NF-kappaB].
OBJECTIVE
To investigate the expression of recombinant IkappaBalphaM (AdIkappaBalphaM) in human hepatocarcinoma cell line HepG(2) and the inhibiting effect to NF-kappaB.


METHODS
To test the virus titer in 293 cells and the infective efficiency virus titer in HepG(2) cells with GFP and limited dilution method, then to assay the IkappaBalphaM expression of recombinant adenovirus and the alteration after induction of TNF-alpha in 293 cells and HepG(2) by Western blotting, furthermore, to observe NF-kappaB activity in HepG(2) before and after treatment of TNF-alpha by EMSA.


RESULTS
The titer of AdIkappaBalphaM is 2 x 10(8) pfu/L, MOI equals to 20. AdIkappaBalphaM could be expressed stably and efficiently in HepG(2) and will not degrade by induction of TNF-alpha; but IkappaBalpha in the uninfection cell as well as AdIkappaBalpha control was increased at first and then decreased. EMSA demonstrated that the infected cells showed no activation of NF-kappaB before and after the treatment of TNF-alpha, but the cells of uninfected and infected with AdIkappaBalpha appeared excessive activation of NF-kappaB.


CONCLUSIONS
AdIkappaBalphaM could be amplified in 293 cell and effectively infect to target cells HepG(2), and could be expressed stably in cells, and wouldn't be degrade with treatment of TNF-alpha, also it can effectually inhibit the excessive activation of NF-kappaB in HepG(2). The result of our research indicates the theoretical value of IkappaBalphaM as a super inhibitor, inhibition activity of NF-kappaB with IkappaBalphaM super-suppressor aided with routine anti-tumor therapy would become an effective method.
Xiang
Ming-Que
MQ
Institute for Viral Hepatitis, Chongqing University of Medical sciences, Chongqing 400010, China.
Huang
Ai-Long
AL
Tang
Ni
N
Xiao
Yu-Jun
YJ
Yan
Ge
G
He
Tong-Chuan
TC
D000256:Adenoviridae / Q000235:genetics; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D020672:I-kappa B Proteins; D008164:Luminescent Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D049452:Green Fluorescent Proteins