Chinese hamster cell mutant, V-C8, a model for analysis of Brca2 function.
The previously described Chinese hamster cell mutant V-C8 that is defective in Brca2 shows a very complex phenotype, including increased sensitivity towards a wide variety of DNA damaging agents, chromosomal instability, abnormal centrosomes and impaired formation of Rad51 foci in response to DNA damage. Here, we demonstrate that V-C8 cells display biallelic nonsense mutations in Brca2, one in exon 15 and the other in exon 16, both resulting in truncated Brca2 proteins. We generated several independent mitomycin C (MMC)-resistant clones from V-C8 cells that had acquired an additional mutation leading to the restoration of the open reading frame of one of the Brca2 alleles. In two of these revertants, V-C8-Rev 1 and V-C8-Rev 6, the reversions lead to the wild-type Brca2 sequence. The V-C8 revertants did not gain the entire wild-type phenotype and still show a 2.5-fold increased sensitivity to mitomycin C (MMC), higher levels of spontaneous and MMC-induced chromosomal aberrations, as well as abnormal centrosomes when compared to wild-type cells. Our results suggest that Brca2 heterozygosity in hamster cells primarily gives rise to sensitivity to DNA cross-linking agents, especially chromosomal instability, a feature that might also be displayed in BRCA2 heterozygous mutation carriers.
Wiegant
Wouter W
WW
Department of Toxicogenetics, Leiden University Medical Center, Einthovenweg 20, Postzone S-6-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
Overmeer
René M
RM
Godthelp
Barbara C
BC
van Buul
Paul P W
PP
Zdzienicka
Małgorzata Z
MZ
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D024682:BRCA2 Protein / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D018385:Centrosome / Q000378:metabolism; D043171:Chromosomal Instability; D002869:Chromosome Aberrations / Q000187:drug effects; D018389:Codon, Nonsense; D018388:Codon, Terminator; D006224:Cricetinae; D003412:Cricetulus / Q000235:genetics*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D005260:Female; D024522:Genes, BRCA2; D006579:Heterozygote; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010641:Phenotype; D051135:Rad51 Recombinase / Q000378:metabolism; D012854:Sister Chromatid Exchange
D024682:BRCA2 Protein; D018389:Codon, Nonsense; D018388:Codon, Terminator; D003432:Cross-Linking Reagents; D051135:Rad51 Recombinase