The arginine decarboxylase (ADC) is a significant functional enzyme, synthesizes agmatine through arginine metabolism, and agmatine was reported to posses protective properties in various tissues. This study first optimized the conditions for efficient hexahistidine tagged human ADC (hisADC) gene delivery into mouse fibroblast cell line (NIH3T3) using retroviral vector (pLXSN). Later, the functionality of the delivered hisADC gene in synthesizing agmatine during H2O2 injury in NIH3T3 was also elucidated. Amplification of hisADC gene was performed using hisADC specific primers under specified conditions. The hisADC PCR product (1.4 kb) was ligated with pLXSN considering the restriction enzyme sites. The complete hisADC pLXSN clone was transfected into PT67 cell line following CalPhos Mammalian transfection method. RT-PCR and western blot results showed the specific and strong detection of hisADC genes in hisADC PT67 transfected cells compared with normal control and pLXSN transfected PT67 cells. The retrovirus containing hisADC gene (vhisADC) was infected into NIH3T3 (vhisADC NIH) using polybrene reagent. Immunocytochemical results showed hisADC expression in the cytoplasm of vhisADC NIH. HPLC analysis revealed high agmatine concentration in the vhisADC NIH, and the induced agmatine synthesized from the retroviral gene delivery prevented vhisADC NIH from H2O2 injury which is evident by the decrease in lactate dehydrogenase (P < 0.05) leakage into the medium and less number of propidium iodide positive cells during injury compared to control group. The obtained results provide compelling evidence that higher level of hisADC transgene expression completely triggered the endogenous agmatine synthesis during H2O2 injury thus protecting NIH3T3 cells against cytotoxicity.
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Acknowledgments This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. 2009-0052626), by a grant (#HMP-A080959-0902-0000100) of the 2008 Good Health R&D Project, Ministry of Health & Welfare, Korea. Special thanks to Dr. Regu-nathan for providing the ADC antibody to our research work.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Clinical Biochemistry
- Cell Biology