Stable transfection of Chinese hamster ovary cells with glutamate-cysteine ligase catalytic subunit cDNA confers increased resistance to tert-butyl hydroperoxide toxicity.
Glutathione (GSH) plays vital roles in antioxidant defense mechanisms. To determine whether gene transfection strategies could be used to enhance GSH synthetic capacities and protect mammalian cells against oxidant stresses, we used liposome-mediated transfer of the cDNA for rat glutamate-cysteine ligase (GLCL) catalytic subunit (GLCLC) to transfect Chinese hamster ovary (CHO) cells. CHO cell lines (CHOhi) with stably enhanced GLCL activities (14.61+/-0.82 mU/mg protein) and greater GSH contents (45.7+/-1.37 nmol/mg protein) than observed in wild-type CHO K1 cells (0.26+/-0.01 mU/mg protein and 20.7+/-1.15 nmol/mg protein, respectively) were developed and were confirmed to have integrated the GLCLC cDNA into their genomic DNA and to exhibit increased GLCLC mRNA levels, by Southern and northern analyses, respectively. Similarly treated and selected CHO cell lines that showed no increases in GLCL activities (CHOun) were studied as controls for the effects of GLCLC transgene expression. CHOhi cells showed significantly greater resistance to oxidant stress caused by exposure to tert-butyl hydroperoxide (tBuOOH) than did CHO or CHOun cells. Twenty-four hours after exposure to 400 or 800 microM tBuOOH, wild-type CHO cells had released more cellular lactate dehydrogenase (67.3+/-14.5% and 94.4+/-2%) than had CHOhi cells (5.11+/-0.5% and 46.0+/-5.4%, n=4, P<0.05). The present data demonstrate improved resistance to oxidant injury of CHO cells stably transfected with the GLCLC cDNA. Although additional enhancements in GLCL activities are possible by transfection with cDNAs for both catalytic and regulatory GLCL subunits, our results demonstrate that the increases in GLCL activities that can be attained by transfection of the GLCLC cDNA alone can enhance cellular antioxidant defense function.
Fernandes
Caraciolo J
CJ
Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA. cfernandes@neo.bcm.tmc.edu
Rong
Liang
L
Tamura
Toshiya
T
Stewart
Kerry D
KD
Rogers
Lynette K
LK
McMicken
Harilyn W
HW
Elliston
Jonathan F
JF
Hansen
Thomas N
TN
Smith
Charles V
CV
D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics*; D005721:Glutamate-Cysteine Ligase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015151:Immunoblotting; D018384:Oxidative Stress / Q000187:drug effects; D021122:Protein Subunits; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D013997:Time Factors; D014162:Transfection; D020122:tert-Butylhydroperoxide / Q000633:toxicity*
D000975:Antioxidants; D018076:DNA, Complementary; D021122:Protein Subunits; D012333:RNA, Messenger; D020122:tert-Butylhydroperoxide; D005721:Glutamate-Cysteine Ligase