Gene transfection of H25A mutant heme oxygenase-1 protects cells against hydroperoxide-induced cytotoxicity.
Heme oxygenase (HO)-1 is a stress-inducible enzyme protecting cells against oxidative stress, and mechanisms have been considered to depend exclusively on its enzyme activity. This study aimed to examine if the protein lacking catalytic activities could also display such resistance against oxidative stress. Stable transfectants of rat wild type HO-1 cDNA (HO-1-U937) and those of its H25A mutant gene (mHO-1-U937) were established using human monoblastic lymphoma cell U937. HO-1-U937 and mHO-1-U937 used in the study exhibited similar levels of the protein expression, while only the former increased enzyme activities. HO-1- and mHO-1 U937 cells became more and less sensitive to H(2)O(2) than mock transfectants, respectively; such distinct susceptibility between the cells was ascribable to differences in the capacity to scavenge H(2)O(2) through catalase and to execute iron-mediated oxidant propagation. On the other hand, both cell lines exhibited greater resistance to tert-butyl hydroperoxide than mock transfectants. The resistance of HO-1-U937 to hydroperoxides appeared to result from antioxidant properties of bilirubin, an HO-derived product, while that of mHO-1-U937 was ascribable to increased contents of catalase and glutathione. These results provided evidence that gene transfection of the activity-lacking mutant HO-1 protects cells against oxidative stress through multiple mechanisms involving up-regulation of catalase and glutathione contents.
Hori
Rio
R
Department of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, Tokyo 160-8582, Japan.
Kashiba
Misato
M
Toma
Tomoko
T
Yachie
Akihiro
A
Goda
Nobuhito
N
Makino
Nobuya
N
Soejima
Akinori
A
Nagasawa
Toshihiko
T
Nakabayashi
Kimimasa
K
Suematsu
Makoto
M
D000818:Animals; D002374:Catalase / Q000096:biosynthesis; D016923:Cell Death; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000096:biosynthesis / Q000378:metabolism; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics* / Q000378:metabolism*; D051547:Heme Oxygenase-1; D006427:Hemin / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007501:Iron / Q000378:metabolism; D008565:Membrane Proteins; D009154:Mutation; D016877:Oxidants / Q000494:pharmacology*; D018384:Oxidative Stress; D012313:RNA / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D020298:U937 Cells; D015854:Up-Regulation
D018076:DNA, Complementary; D008565:Membrane Proteins; D016877:Oxidants; D012313:RNA; D006427:Hemin; D006861:Hydrogen Peroxide; D007501:Iron; D002374:Catalase; C496541:HMOX1 protein, human; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; D005978:Glutathione