Primary fibroblasts from BRCA1 heterozygotes display an abnormal G1/S cell cycle checkpoint following UVA irradiation but show normal levels of micronuclei following oxidative stress or mitomycin C treatment.
OBJECTIVE
There is evidence to suggest that the breast cancer predisposing gene, BRCA1, is involved in cell cycle control and the response to damage but mouse brca1+/- heterozygotes have no distinctive phenotype. Here the response to the three forms of cellular stress was examined in primary human fibroblasts from individuals with a +/+ or +/- genotype for BRCA1.


METHODS
Fibroblasts from individuals carrying mutations in the BRCA1 gene were compared with those from those wild-type for BRCA1 in their response to long wavelength uv (UVA), hydrogen peroxide, and mitomycin C (MMC). Cell cycle progression and micronucleus formation (MN) were used as end points.


RESULTS
After UVA treatment there was no difference between +/- and +/+ cells in the initial fall in DNA synthetic activity (G(1) arrest) but the reentry into S-phase was restored at a faster rate in the BRCA1+/- cells after UVA exposure. Thus, for three normal (+/+) cell lines irradiated in monolayer, S-phase values averaged 15 +/- 3.7% 14 h post-UVA (1 x 10(5) J/m(2)), as compared with 35.7 +/- 1.9 (range) for two BRCA1(+/-) strains. Because a defective G(1)/S checkpoint in BRCA1 heterozygotes could lead to a greater proportion of S-phase cells with unrepaired DNA damage (strand breaks) and a resultant increase in chromosomal instability, the frequency of micronuclei induced by UVA was examined. Three normal (+/+) and three mutant (+/-) strains (two of which were used in the cell cycle experiments) produced mean micronuclei frequencies of 0.077 +/- 0.016 and 0.094 +/- 0.04/binucleate cell respectively (not statistically significant), 48 h after UVA exposure. No differences were found between BRCA1+/+ and +/- cells in MN formation after treatment with MMC or hydrogen peroxide.


CONCLUSIONS
Our data suggest a defective G(1)/S checkpoint in cells from BRCA1 heterozygotes in response to UVA although this is not reflected in genomic instability as measured by micronuclei induction after oxidative stress or MMC treatment.
Shorrocks
Julie
J
Department of Biological Sciences, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster LA1 4YQ, UK.
Tobi
Simon E
SE
Latham
Harry
H
Peacock
John H
JH
Eeles
Ros
R
Eccles
Diana
D
McMillan
Trevor J
TJ
D000477:Alkylating Agents / Q000494:pharmacology; D001943:Breast Neoplasms / Q000235:genetics; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D004249:DNA Damage; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects*; D016193:G1 Phase / Q000235:genetics*; D019398:Genes, BRCA1 / Q000502:physiology*; D020022:Genetic Predisposition to Disease; D006579:Heterozygote; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D048629:Micronuclei, Chromosome-Defective; D015162:Micronucleus Tests; D016685:Mitomycin / Q000494:pharmacology; D009154:Mutation; D018384:Oxidative Stress; D016196:S Phase / Q000235:genetics*; D014466:Ultraviolet Rays
D000477:Alkylating Agents; D016685:Mitomycin; D006861:Hydrogen Peroxide