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Expression and purification of TAT-fused carbonic anhydrase III and its effect on C2C12 cell apoptosis induced by hypoxia/reoxygenation.

ABSTRACT: INTRODUCTION:Carbonic anhydrase III (CAIII) is remarkably abundant in slow skeletal muscles. It has multiple biological activities which could dissipate or resist some fatigue-related substances. In this study, we purified trans-activating transcriptional activator (TAT) fused CAIII protein and investigated its effect on C2C12 cell apoptosis induced by hypoxia/reoxygenation. MATERIAL AND METHODS:The CAIII and TAT-CAIII genes were constructed, cloned into plasmid pET28a and expressed in Escherichia coli BL21 (DE3). The fusion proteins were purified with a nickel-nitrilotriacetic acid affinity chromatography column and then verified by Western blot and phosphatase activity staining subsequently. The C2C12 cells were treated respectively with serum-free medium containing 1 ?M TAT-CAIII or 1 ?M CAIII for 1 h and the intracellular distributions of fusion proteins were observed by indirect immunofluorescence. The effect of TAT-CAIII on C2C12 cell apoptosis induced by hypoxia/reoxygenation was detected by flow cytometry. RESULTS:The CAIII and TAT-CAIII fusion proteins were expressed and purified successfully. After being cultured for 1 h, green fluorescence was visible in TAT-CAIII group cells under the fluorescence microscope, while no fluorescence was found in the CAIII group. Compared with the oxygen-glucose deprivation group, the apoptosis rate of C2C12 cells induced by hypoxia/reoxygenation in the TAT-CAIII group decreased significantly (p < 0.001). CONCLUSIONS:The purified TAT-CAIII could be transferred into cells efficiently and clearly decreased the apoptosis rate of C2C12 cells induced by hypoxia/reoxygenation, which indicated that it had antioxidative activity. This study lays an experimental basis for future research on the relationship between CAIII and muscle fatigue.

SUBMITTER: Shang X

PROVIDER: S-EPMC3460508 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Expression and purification of TAT-fused carbonic anhydrase III and its effect on C2C12 cell apoptosis induced by hypoxia/reoxygenation.

Shang Xiliang X Bao Yuanyuan Y Chen Shiyi S Ren Huimin H Huang He H Li Yunxia Y

Archives of medical science : AMS 20120901 4

<h4>Introduction</h4>Carbonic anhydrase III (CAIII) is remarkably abundant in slow skeletal muscles. It has multiple biological activities which could dissipate or resist some fatigue-related substances. In this study, we purified trans-activating transcriptional activator (TAT) fused CAIII protein and investigated its effect on C2C12 cell apoptosis induced by hypoxia/reoxygenation.<h4>Material and methods</h4>The CAIII and TAT-CAIII genes were constructed, cloned into plasmid pET28a and express ...[more]

PMID: 23056085

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Project description:AIM:To study the expression of carbonic anhydrase (CA) 9 in human hepatocellular carcinoma (HCC) cells. METHODS:We studied CA9 protein, CA9 mRNA and hypoxia-inducible factor-1 alpha (HIF-1?) protein levels in Hep3B cells exposed in different parallel approaches. In one of these approaches, HCC cells were exposed to extreme in vitro hypoxia (24 h 0.1% O(2)) without or with interleukin (IL)-1, IL-6, tumor necrosis factor-alpha (TNF-?) and transforming growth factor-beta (TGF-?) stimulation for the same hypoxic exposure time or exposed to normoxic oxygenation conditions without or with cytokine stimulation. RESULTS:The tumour cell line analysed showed a strong hypoxic CA9 mRNA expression pattern in response to prolonged severe hypoxia with cell-line specific patterns and a marked induction of CA9 protein in response to severe hypoxia. These results were paralleled by the results for HIF-1? protein under identical oxygenation conditions with a similar expression tendency to that displayed during the CA9 protein expression experimental series. Continuous stimulation with the cytokines, IL-1, IL-6, TNF-? and TGF-?, under normoxic conditions significantly increased the carbonic anhydrase 9 expression level at both the protein and mRNA level, almost doubling the CA9 mRNA and CA9 and HIF-1? protein expression levels found under hypoxia. The findings from these experiments indicated that hypoxia is a positive regulator of CA9 expression in HCC, and the four signal transduction pathways, IL-1, IL-6, TNF-? and TGF-?, positively influence CA9 expression under both normoxic and hypoxic conditions. CONCLUSION:These findings may potentially be considered in the design of anti- cancer therapeutic approaches involving hypoxia-induced or cytokine stimulatory effects on expression. In addition, they provide evidence of the stimulatory role of the examined cytokine families resulting in an increase in CA9 expression under different oxygenation conditions in human cancer, especially HCC, and on the role of the CA9 gene as a positive disease regulator in human cancer.

Dataset Information

Expression and purification of TAT-fused carbonic anhydrase III and its effect on C2C12 cell apoptosis induced by hypoxia/reoxygenation.

Publications

Expression and purification of TAT-fused carbonic anhydrase III and its effect on C2C12 cell apoptosis induced by hypoxia/reoxygenation.

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