The FANCG Fanconi anemia protein interacts with CYP2E1: possible role in protection against oxidative DNA damage.
Fanconi anemia (FA) is a genetic disorder that leads to aplastic anemia and birth defects and predisposes to cancer. FA cells exhibit characteristic hypersensitivity to DNA cross-linking agents such as mitomycin C (MMC), and FANCG is one of six known FA gene products. By immunocytochemical analysis of transfected cells, we discovered that although FANCG localized to both the nucleus and cytoplasm, there was an increase in cells with predominantly cytoplasmic staining after treatment with MMC. Concurrently, while searching by two-hybrid analysis for proteins that associate with FANCG, we identified a novel interaction between FANCG and cytochrome P450 2E1 (CYP2E1). A member of the P450 superfamily, CYP2E1 is associated with the production of reactive oxygen intermediates and the bioactivation of carcinogens. High constitutive levels of CYP2E1 were found in a FA-G lymphoblast cell line, whereas complementation of the FA-G line with wild-type FANCG was associated with decreased CYP2E1. These findings suggested that the interaction of FANCG with CYP2E1 might alter redox metabolism and increase DNA oxidation. Using a fluorescent assay, we found a dose-dependent increase in the oxidized DNA base, 8-oxoguanine (8-oxoG), after treatment of mutant FA-G cells with H(2)O(2) or MMC. Conversely, significantly lower levels of 8-oxoG were detected in FANCG-complemented FA-G cells. We conclude that the unknown function of FANCG involves at least transient interaction with cytoplasmic components, possibly including CYP2E1, and propose a role for FANCG in protection against oxidative DNA damage.
Futaki
Makoto
M
Hematology Branch, NHLBI, NIH, Building 10, Room 7C103, Bethesda, MD 20892, USA.
Igarashi
Takehito
T
Watanabe
Shinji
S
Kajigaya
Sachiko
S
Tatsuguchi
Atsushi
A
Wang
Jianxiang
J
Liu
Johnson M
JM
D015153:Blotting, Western; D002460:Cell Line; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D052241:Fanconi Anemia Complementation Group G Protein; D005434:Flow Cytometry; D005982:Glutathione Transferase / Q000378:metabolism; D006151:Guanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D018613:Microscopy, Confocal; D016685:Mitomycin / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D004268:DNA-Binding Proteins; C499909:FANCG protein, human; D052241:Fanconi Anemia Complementation Group G Protein; D012333:RNA, Messenger; D006151:Guanosine; C046215:8-hydroxyguanosine; D016685:Mitomycin; D006861:Hydrogen Peroxide; D019392:Cytochrome P-450 CYP2E1; D005982:Glutathione Transferase