Reactive oxygen species stimulated human hepatoma cell proliferation via cross-talk between PI3-K/PKB and JNK signaling pathways
Section snippets
Materials and methods
Materials. RPMI 1640 medium and TRIzol reagent were purchased from Gibco/BRL. Rabbit phospho-Akt (Thr308), phospho-SAPK/JNK (Thr183/Tyr185) antibody, phospho-p38 MAPK (Thr180/Tyr182) antibody, phospho-ERK (Thr202/Tyr204) antibody, sheep MnSOD antibody, horseradish peroxide-conjugated rat anti-sheep antibody, horseradish peroxide-conjugated goat anti-rabbit antibody, neomycin, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), penicillin, streptomycin, recombinant human insulin, PD98059, and
Results
Characterization of MnSOD expression in transfectants. The expression of MnSOD in transfected 7721 cells was determined using RT-PCR and compared with the control cells transfected with the vector alone. It was found that the intensity of the 255-bp band was obviously increased in the samples from pHβA-SOD(+)-transfected 7721 cells (MnSOD-7721), and markedly decreased in the samples from pHβA-SOD(−)-transfected 7721 cells (MnSOD-AS7721) (Fig. 1A). These results indicated that MnSOD mRNA
Discussion
Tumor formation can be initiated by many kinds of factors. ROS, which are important factors in the initiation of carcinogenesis, react with and modify DNA, resulting in alterations in DNA structure and leading to oncogene or antioncogene mutation. However, ROS can also act as intracellular signaling molecules to regulate protein kinase activity, transcription factor activity, early response gene expression, cell growth, apoptosis, differentiation, and transformation. The deficiency of an
Acknowledgements
We are grateful to Chen Hui-li for generously providing the PKB-7721 and PKB-AS7721 hepatoma cells. We also grateful to Kunitaka Hirose for providing pHβA-SOD(+)/pHβA-SOD(−) constructs. This work was supported by the National Natural Science Foundation Key Program Grant 30130100 and Shanghai National Science Foundation Key Program Grant O1JC1Y017 Shanghai Educational Committee Key Program Grant B990805.
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