BRCA1 is required for common-fragile-site stability via its G2/M checkpoint function

Abstract

Common fragile sites are loci that form chromosome gaps or breaks when DNA synthesis is partially inhibited. Fragile sites are prone to deletions, translocations, and other rearrangements that can cause the inactivation of associated tumor suppressor genes in cancer cells. It was previously shown that ATR is critical to fragile-site stability and that ATR-deficient cells have greatly elevated fragile-site expression (A. M. Casper, P. Nghiem, M. F. Arlt, and T. W. Glover, Cell 111:779-789, 2002). Here we demonstrate that mouse and human cells deficient for BRCA1, due to mutation or knockdown by RNA interference, also have elevated fragile-site expression. We further show that BRCA1 functions in the induction of the G(2)/M checkpoint after aphidicolin-induced replication stalling and that this checkpoint function is involved in fragile-site stability. These data indicate that BRCA1 is important in fragile-site stability and that fragile sites are recognized by the G(2)/M checkpoint pathway, in which BRCA1 plays a key role. Furthermore, they suggest that mutations in BRCA1 or interacting proteins could lead to rearrangements at fragile sites in cancer cells.

FIG. 1.

Examples of expressed common-fragile-site detection by FISH. (A) Partial metaphase of HCC1937 cells treated with 0.3 μM aphidicolin for 24 h. Chromosomes are stained with DAPI and demonstrate multiple fragile-site breaks. White arrows indicate positions of gaps and breaks at FRA3B. (B) Examples of FISH results on the same partial metaphase. FRA3B was probed with YAC 850A6 (green). White arrows indicate colocalization of FISH signal and broken FRA3B sites. (C) Partial metaphase of HCC1937 cells treated with 0.3 μM aphidicolin for 24 h. Chromosomes are stained with DAPI and demonstrate multiple fragile-site breaks. White arrows indicate positions of gaps and breaks at FRA16D. (D) Examples of FISH results on the same partial metaphase. FRA16D was probed with BAC 264L1 (red). White arrows indicate colocalization of FISH signal and broken FRA3B sites.

FIG. 2.

HCC1937 cells lacking BRCA1 have elevated fragile-site expression after aphidicolin treatment. (A) Western blot of BRCA1 immunoprecipitation from clones of HCC1937 stably transfected with wild-type BRCA1, BRCA1 (S1423A), or BRCA1 (S1387A) expression constructs or an empty vector. (B) Average total chromosomal gaps and breaks per cell in HCC1937 cells stably transfected with the indicated BRCA1 expression constructs after 24 h in the presence (dark gray) or absence (light gray) of 0.3 μM aphidicolin; n = 100 metaphases for each data set. Error bars indicate the 95% confidence interval. (C) Frequency (%) of gaps and breaks at specific fragile sites FRA3B and FRA16D in HCC1937 cells stably transfected with the indicated BRCA1 expression constructs after 24 h of treatment with 0 μM (light gray) or 0.3 μM (dark gray) aphidicolin; n = 88 to 108 sites examined. Fragile sites were identified by FISH with probes specific to these sites. Frequency of fragile-site induction is presented as the percentage of chromosome 3 or 16 homologs with breaks at FRA3B or FRA16D, respectively.

FIG. 3.

Mouse cells lacking BRCA1 have elevated fragile-site expression after aphidicolin treatment. (A) Average total chromosomal gaps and breaks per cell in mouse cells with the indicated genotypes after 24 h in the presence (dark gray) or absence (light gray) of 0.3 μM aphidicolin; n = 100 metaphases for each data set. Error bars indicate the 95% confidence interval. (B) Frequency (%) of gaps and breaks at specific fragile site Fra14A2 in mouse cells after 24 h of treatment with 0 μM (light gray) or 0.3 μM (dark gray) aphidicolin; n = 89 to 100 sites examined. The genotype of each cell line is indicated. Fragile sites were identified by FISH with a probe specific to Fra14A2. Fragile-site induction frequency is presented as the percentage of chromosome 14 homologs with breaks at Fra14A2.

FIG. 4.

Human cells with BRCA1 expression reduced via RNAi have elevated fragile-site expression after aphidicolin treatment. (A) Western blot probed with αBRCA1 antibodies showing reduced BRCA1 expression in HCT116 cells 48 h after transfection with BRCA1 SMARTpool siRNA. Untransfected cells and cells transfected with control siRNA show no reduction in BRCA1 expression. (B) Average total chromosomal gaps and breaks per cell in HCT116 cells after transfection with BRCA1 SMARTpool siRNA, no siRNA, or control siRNA; n = 100 metaphases for each data set. Error bars indicate the 95% confidence interval. RNAi reduction of BRCA1 levels was achieved 48 h before harvest. Fragile-site induction was achieved by addition of 0.2 μM aphidicolin 24 h before harvest. (C) Frequency (%) of gaps and breaks at specific fragile sites FRA3B and FRA16D in HCT116 cells after transfection with BRCA1 SMARTpool siRNA, no siRNA, or control siRNA. Fragile-site expression was measured after 24 h of treatment with 0 μM (light gray) or 0.3 μM (dark gray) aphidicolin; n = 80 to 101 sites examined. Frequency of fragile-site induction is presented as the percentage of chromosome 3 or 16 homologs with breaks at FRA3B or FRA16D, respectively. (D) Western blot probed with αBRCA1 antibodies showing reduced BRCA1 expression in HeLa cells 48 h after transfection with BRCA1 SMARTpool siRNA. Untransfected cells and cells transfected with control siRNA show no reduction in BRCA1 expression. (E) Average total chromosomal gaps and breaks per cell in HeLa cells after transfection with BRCA1 SMARTpool siRNA, no siRNA, or control siRNA; n = 100 metaphases for each data set. Error bars indicate the 95% confidence interval. RNAi reduction of BRCA1 levels was achieved 48 h before harvest. Fragile-site induction was achieved by addition of 0.2 μM aphidicolin 24 h before harvest. (F) Frequency (%) of gaps and breaks at specific fragile sites FRA3B and FRA16D in HeLa cells after transfection with BRCA1 SMARTpool siRNA, no siRNA, or control siRNA. Fragile-site expression was measured after 24 h of treatment with 0 μM (light gray) or 0.3 μM (dark gray) aphidicolin; n = 100 to 102 sites examined. Frequency of fragile-site induction is presented as the percentage of chromosome 3 or 16 homologs with breaks at FRA3B or FRA16D, respectively.

FIG. 5.

BRCA1 serine 1423, but not serine 1387, is necessary for G₂/M checkpoint induction after aphidicolin treatment. (A) Flow cytometric profiles of cell cycle distribution of cells after 24 h in the presence of 0 and 0.5 μM aphidicolin (APH). Shown are HCC1937 cells stably transfected with constructs expressing wild-type BRCA1, BRCA1 containing serine-to-alanine mutations at serine 1423 or serine 1387, or vector alone. Cells were stained for DNA content with propidium iodide (y axis) and for histone H3 phosphorylation (x axis). The mitotic cell population is circled, and the percentage of total cells falling within that population is indicated. (B) Quantitation of flow cytometric profiles of cell cycle distribution following 24 h of exposure to 0.5 μM aphidicolin. Bars indicate the percentage of aphidicolin-treated cells that are in mitosis relative to untreated cells. Error bars indicate the standard deviation.

FIG. 6.

G₂/M checkpoint-deficient HCC1937 cells expressing a serine-to-alanine mutation at serine 1423 have increased fragile-site expression. (A) Average total chromosomal gaps and breaks per cell in HCC1937 cells stably transfected with the indicated BRCA1 expression constructs after 24 h in the presence (dark gray) or absence (light gray) of 0.3 μM aphidicolin; n = 100 metaphases for each data set. Error bars indicate the 95% confidence interval. (B) Frequency of fragile-site expression showing what percentage of specific fragile sites FRA3B and FRA16D were broken in HCC1937 cells stably transfected with the indicated BRCA1 expression constructs after 24 h of treatment with 0 μM (light gray) or 0.3 μM (dark gray) aphidicolin; n = 88 to 109 sites examined. Frequency of fragile-site induction is presented as the percentage of chromosome 3 or 16 homologs with breaks at FRA3B or FRA16D, respectively.