Recombinant hexahistidine arginine decarboxylase (hisADC) induced endogenous agmatine synthesis during stress.
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 H(2)O(2) 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 H(2)O(2) 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 H(2)O(2) injury thus protecting NIH3T3 cells against cytotoxicity.
Moon
Sung-Ung
SU
Department of Anatomy, Yonsei University College of Medicine, 134 Shinchon-dong Seodaemun-gu, Seoul, 120-752, Korea.
Kwon
Ki-Hyo
KH
Kim
Jae-Hwan
JH
Bokara
Kiran Kumar
KK
Park
Kyung Ah
KA
Lee
Won Taek
WT
Lee
Jong-Eun
JE
D016475:3T3 Cells; D000376:Agmatine / Q000378:metabolism*; D000818:Animals; D002262:Carboxy-Lyases / Q000008:administration & dosage / Q000235:genetics* / Q000494:pharmacology; D006639:Histidine / Q000235:genetics*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D051379:Mice; D009842:Oligopeptides / Q000235:genetics*; D018384:Oxidative Stress / Q000187:drug effects*; D011994:Recombinant Proteins; D015533:Transcriptional Activation; D014162:Transfection / Q000379:methods
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine; D000376:Agmatine; D006861:Hydrogen Peroxide; D002262:Carboxy-Lyases; C019722:arginine decarboxylase