Induction of Fanconi anemia cellular phenotype in human 293 cells by overexpression of a mutant FAC allele.
The polypeptide encoded by the Fanconi anemia (FA) complementation group C gene, FAC, binds to a group of cytoplasmic proteins in vitro and may form a multimeric complex. A known mutant allele of FAC resulting from the substitution of Pro for Leu at codon 554 fails to correct the sensitivity of FA group C cells to mitomycin C. We reasoned that overexpression of the mutant protein in a wild-type cellular background might induce the FA phenotype by competing with endogenous FAC for binding to the accessory proteins. After stable transfection of 293 cells with wild-type and a mutant FAC allele containing the L554P substitution, four independent clones that expressed four-to-fifteen fold higher levels of transcript from the mutant transgene relative to the endogenous FAC gene showed hypersensitivity to mitomycin C. By contrast, both parental and FAC-overexpressing cells maintained their relative resistance to mitomycin C. No differences in the biosynthesis, subcellular localization and protein interactions of the normal and mutant proteins were detected. The induction of the FA phenotype in this system is compatible with the competition hypothesis and provides support for a functional role of the FAC-binding proteins in vivo.
Youssoufian
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Department of Medicine, Hematology-Oncology Division, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Li
Y
Y
Martin
M E
ME
Buchwald
M
M
D000483:Alleles; D001483:Base Sequence; D002451:Cell Compartmentation; D018797:Cell Cycle Proteins; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins; D005199:Fanconi Anemia / Q000503:physiopathology*; D052218:Fanconi Anemia Complementation Group C Protein; D051856:Fanconi Anemia Complementation Group Proteins; D006801:Humans; D016685:Mitomycin / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D010641:Phenotype; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; C497651:FANCC protein, human; D052218:Fanconi Anemia Complementation Group C Protein; D051856:Fanconi Anemia Complementation Group Proteins; D009687:Nuclear Proteins; D011506:Proteins; D011994:Recombinant Proteins; D016685:Mitomycin