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Molecular Biology and Genetics |
Department of Medicine [M. E. M.] and Cell Biology Program [T. Y. C., M. J.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One type of damage to which mouse and human BRCA1-deficient cells respond abnormally is the DNA DSB.4 Chromosome DSBs arise from both endogenous processes and exogenous agents such as IR, causing cell death or genomic alterations if not repaired appropriately. BRCA1 mutant human cells (i.e., HCC1937 cancer cells) are hypersensitive to IR, and this sensitivity is partially relieved (2–3x) by expression of wild-type BRCA1. Of note, significant IR sensitivity remains in the BRCA1-complemented HCC1937 cells (3) . Brca1 mutant mouse cell lines, such as the 236.44 ES cell line, are also sensitive to IR, as are Brca1 mutant embryos (1 , 11) . In mammalian cells two major repair pathways, homologous recombination and NHEJ, prevent deleterious outcomes after treatment of cells with agents that cause DSBs. The association of Rad51, the eukaryotic homologue of the Escherichia coli RecA protein, with BRCA1 in immunoprecipitable complexes, as well as the observed subnuclear localization of BRCA1 with Rad51 in IR-induced foci (12 , 13) , is highly suggestive of a role for BRCA1 in homology-directed repair (HDR). However, after IR treatment of cells, BRCA1 also colocalizes with the Rad50 complex, which, in yeast, has a role in NHEJ as well as in HDR, raising the possibility of a role for BRCA1 in this DSB repair pathway (14) .
We have recently examined DSB repair at the molecular level in the Brca1-/- 236.44 ES cell line to determine the nature of the repair defect. This cell line was constructed through consecutive rounds of gene targeting such that both Brca1 alleles are mutated by deletion of the 5' end of exon 11 (15) . The targeted alleles are hypomorphs, inasmuch as they express a truncated product arising from an alternatively spliced transcript that skips exon 11 (16) . In addition to being hypersensitive to IR, the 236.44 cell line is hypersensitive to cisplatin, has reduced Rad51 focus formation after cisplatin treatment, and is defective in transcription-coupled repair of oxidative damage (1 , 17) . By examining repair of an endonuclease-generated DSB in this cell line, we demonstrated that HDR is reduced, though NHEJ is not impaired but, rather, slightly elevated (2) . Because DSB repair mutants often exhibit spontaneous chromosome instability, there was a concern that additional genomic changes had occurred in the Brca1-/- 236.44 cells, such that the observed repair defects are not fully attributable to loss of wild-type Brca1. Partial complementation of these cells by the reintroduction of wild-type Brca1 has, in fact, not demonstrated a clear restoration of homologous recombination (16) .
In the current study, we have examined repair phenotypes of the Brca1 mutant 236.44 cell line as well as derivative cell lines in which wild-type Brca1 is reexpressed as a result of either complementation from a Brca1 transgene or correction of one of the Brca1 hypomorphic alleles. Using a recombination reporter assay recently developed for the analysis of Brca2 mutant cells, we show that the 236.44 cell line has impaired HDR of an induced chromosome break and reduced gene targeting, and we confirm that wild-type Brca1 expression in this cell line restores levels of both types of homologous recombination. We also demonstrate that Brca1 deficiency results in chromosome instability in ES cells, although not the dramatic instability detected in MEFs. Moreover, Brca1-deficient cells exhibit exquisite sensitivity to the interstrand cross-linking agent mitomycin-C, suggesting that this or related DNA-damaging agents may have a highly favorable therapeutic ratio. Notably, restoration of Brca1 by transgene complementation was less effective at reversing repair defects than correction of a defective Brca1 allele by gene targeting, presumably because of the difficulty in expressing a physiologically relevant amount of Brca1.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Transfections and Nucleic Acid Analysis.
For stable transfection of pBBxAx, Brca1-/- ES cells were cotransfected by electroporation at 800 V/3 µF with 75 µg of the linear fragment from pBBxAx and 25 µg of a phosphoglycerate kinase-puromycin expression plasmid with subsequent selection after 24 h in 1.0 µg/ml puromycin. purR clones were picked and expanded 12–16 days later. Southern blots of genomic DNA from these clones were hybridized with a Brca1 probe. For gene targeting, ES cells were electroporated with 75 µg of linear targeting fragment from p59xDR-GFP6 with subsequent selection after 48 h in hygromycin 110 µg/ml and puromycin 1.0 µg/ml. hygR and purR clones were picked and expanded 10–12 days later. Southern blots of genomic DNA from these clones were digested with HincII and hybridized with a radiolabeled pim1 probe. For Brca1 correction by gene targeting, Brca1-/- ES cells were electroporated with 75 µg of linear pBrca1+S1Rhyg targeting fragment with subsequent selection after 24 h in hygromycin 200 µg/ml. HygR clones were picked and expanded 10–12 days later. Southern blots of genomic DNA from these clones were digested with EcoRV and hybridized with a 3' 1.2-kb Brca1 probe. For DSB repair assays, actively growing cells were electroporated at 250 V/960 µF with 30–50 µg of pCßASce (20)
, mock DNA, or pNZE-CAG (21)
and plated in nonselective media. Cells were trypsinized at 45–48 h and analyzed by flow cytometry. Data were analyzed with Lysis software. For Brca1 expression, total mRNA was extracted from actively growing cells by RNAzol (Biotecx Laboratories) treatment. Northern analysis was performed by standard techniques.
Mitomycin-C Clonogenic Survival Assays.
For mitomycin-C survival assays, cells were exposed to various mitomycin-C doses for 4 h and then rinsed three times in PBS. The cells were replated and allowed to grow undisturbed for 8–10 days. The colonies were stained and counted. Survival experiments were performed in triplicate.
Metaphase Spreads and Karotype Analysis.
For karotype analysis, cells were cultured with 0.05 µg/ml colcemid (Life Technologies, Inc.) for 1.5 h, then trypsinized and resuspended in hypotonic saline (0.075 M KCl) at 37°C for 10 min. The cells were fixed in a 3:1 mix of methanol and acetic acid. Fixed cell suspensions were transferred to glass slides and allowed to air-dry. Metaphase spreads were stained with 4% Giemsa in PBS for 20 min.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Ref. 18
). Gene targeting with the p59x vector is highly efficient, because the selectable hygromycin resistance gene (hygR) is fused in-frame to pim1 coding sequences such that expression of hygR is dependent on either targeting to the pim1 locus on chromosome 17 or a fortuitous nonhomologous integration adjacent to promoter sequences of another locus (23)
. The p59xDR-GFP6 vector additionally contains an intact puromycin resistance gene (purR), which can also be used to select for vector integration events.
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10–20% normal Brca1 levels (Ref. 16
and data not shown). Doubly resistant hygR/purR colonies were selected, and genomic DNA from individual colonies was analyzed by Southern blotting using a pim1 probe located outside the targeting fragment (Fig. 1A)
. Efficient gene targeting was observed in the Brca1+/- cells, with 94% of hygR/purR clones correctly targeted (Fig. 1B
; Table 1
). However, homologous integrations were rarely detected in the Brca1-/- cells, with only 6% of the hygR/purR clones correctly targeted, which is a 16-fold lower frequency of gene targeting as compared with Brca1+/- cells. This diminished ability to gene target is comparable with the 23-fold lower frequency we originally reported at the pim1 locus in the Brca1-/- cells. The small improvement in gene targeting may be related to the more stringent combined hygromycin and puromycin selection used in the targeting of the p59xDR-GFP6 fragment, because we observed that background colonies observed during hygromycin selection of Brca-/- cells were significantly reduced with the combined selection. The addition of puromycin to the hygromycin selective media for Brca1+/+ and Brca1+/- cells did not alter or improve upon the gene targeting frequencies obtained with hygromycin alone (data not shown). The addition of puromycin to the Brca1-/-,Tg+ would not effect gene targeting, because these cells were selected for purR during the integration of the Brca1 transgene (16)
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and Table 1
). This is a 10-fold improvement in gene targeting as compared with the Brca1-/- mutant cells. However, when compared with the Brca1+/- cells, a 1.6-fold decreased targeting efficiency remained in the transgene corrected cell line, suggesting only partial complementation of the gene targeting defect by expression from the Brca1 transgene. In this experiment we also examined gene targeting in parental Brca1+/+ cells, because loss of a single BRCA1 allele in human cell lines has been implicated in sensitivity to DNA-damaging agents (24) . Similar to the Brca1+/- cells, 97% of hygR/purR clones were correctly targeted. Thus, mouse cells heterozygous for a Brca1 mutation have no detectable defect in gene targeting as compared with Brca1+/+ ES cells. In addition, these results imply that the Brca1 exon 10–12 splice variant that is expressed in the Brca1+/- cells has no detectable dominant-negative effect on gene targeting.
HDR of a Chromosomal DSB in Brca1-/- ES Cells Is Also Partially Restored by Brca1 Transgene Expression.
The DR-GFP reporter substrate (21)
, which was incorporated at the pim1 locus of cell lines during targeting of the p59xDR-GFP6 vector, measures HDR of an endonuclease-induced DSB by a gene conversion mechanism. It is composed of two differentially mutated GFP genes oriented as direct repeats and separated by the purR gene (Fig. 2A)
. The SceGFP gene is a GFP gene that is mutated by 11 bp substitutions to contain the 18 bp recognition sequence for the I-Sce I endonuclease. Downstream of SceGFP is the 0.8-kb GFP fragment iGFP, which is a wild-type GFP gene truncated at both its 5' and 3' ends. Expression of I-Sce I in cells that have the DR-GFP substrate integrated into their genome results in a DSB in the chromosome at the position of the I-Sce I site. Repair of the induced DSB in SceGFP by a non-crossover gene conversion with iGFP reconstructs a GFP+gene, expression of which can be scored by cellular fluorescence. Although other DSB repair events at the I-Sce I site are possible, they are not detected, because the 11-bp substitutions in the SceGFP gene cannot be restored to the wild-type GFP sequence except through a templated gene conversion event. Molecular analysis of the DR-GFP substrate in sorted GFP-positive cells after I-Sce I expression has verified that cellular green fluorescence, as measured by flow cytometry, results from repair by gene conversion (18
, 21)
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. As with gene targeting, therefore, HDR of an induced chromosomal break is not diminished in ES cells containing only one wild-type Brca1 allele. Proficient HDR in the Brca1+/- cells also indicates that the exon 10–12 splice variant arising from the mutated Brca1 allele does not exhibit a dominant-negative effect on the wild-type Brca1 allele.
The Brca1-/- cells exhibited many fewer GFP-positive cells after I-Sce I expression, such that there was a 6-fold decrease in HDR relative to the Brca1+/+ and Brca1+/- cell lines (Fig. 2B)
. This decrease in gene conversion measured by GFP expression is comparable with the previously reported 5–6-fold decrease in gene conversion measured by a PCR-based assay with a neo substrate (2)
. The transgene-complemented Brca1-/-,Tg+ cells, however, showed a 2-fold increase in HDR over the Brca1-/- cells. Thus, HDR was increased by wild-type Brca1 expression, although, as with gene targeting, it was not completely restored.
Electroporations were also performed with a GFP expression vector to verify that the different cell lines had a similar transfection efficiency. The GFP expression vector that was electroporated, pCAG-NZE, uses the same control elements to express GFP as the pCßASce vector uses to express I-Sce I. Electroporated cells were examined by flow cytometry. No difference in GFP expression, which ranged from 44–58%, was detected between any of the Brca1+/+, Brca1+/-, Brca1-/-, and Brca1-/-,Tg+ targeted cell clones (data not shown), indicating that the transfection efficiency was not appreciably different for these lines.
Brca1-deficient Cells Are Hypersensitive to the DNA Interstrand Cross-linking Agent Mitomycin-C.
The first recognized mammalian HDR mutants, the irs1 and irs1SF hamster cell lines deficient for the Rad51-related proteins XRCC2 and XRCC3, respectively, show moderate sensitivity to IR, as do Brca1- deficient cells, but an extreme sensitivity to mitomycin-C (25)
. Mitomycin-C produces several types of DNA damage, one of which is an interstrand DNA cross-link. This lesion could potentiate DSBs through replication fork blockage, producing an intermediate that is much more dependent for repair on HDR than on NHEJ, although the exact mode of repair is unknown (26)
. To investigate whether Brca1 deficiency also results in mitomycin-C sensitivity, clonogenic survival assays were performed in the Brca1+/+, Brca1+/-, Brca1-/-, and Brca1-/-,Tg+ cell lines after exposure to increasing doses of mitomycin-C.
Hypersensitivity was seen at all mitomycin-C doses in the Brca1-/- cells as compared with the wild-type and heterozygous Brca1 cell lines (Fig. 3)
. The extent of sensitivity to mitomycin-C was dose dependent. At a mitomycin-C concentration of 0.5 µM, which was approximately the LD50 for wild-type cells, the Brca1-/- cells exhibited a >100-fold increased sensitivity (Fig. 3)
. No difference in sensitivity to mitomycin-C in the Brca1+/- and Brca1+/+ cell lines was observed at this or other doses. Consistent with the homologous recombination assays, the Brca1-/-,Tg+ cell lines revealed an intermediate sensitivity to mitomycin-C.
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Correction of the Brca1Exon 11 Deletion through Gene Targeting.
The incomplete restoration of homologous recombination and mitomycin-C resistance in the Brca1 transgene-complemented cell lines could be attributable to inadequate expression of Brca1 or undetected alterations in the Brca1 transgene upon integration. It is also formally possible that genomic changes occurred in the Brca1-/- cell line that contributed to the observed repair defects. This was a particular concern, given the dramatic chromosomal instability observed in MEFs harboring a similar Brca1 mutation.
To better address the dependence of the repair defects on Brca1-deficiency, a gene-targeting fragment was constructed to correct the exon 11 deletion mutation in the Brca1-/- 236.44 cell line (Fig. 4A)
. The Brca1 alleles in this line are disrupted by replacement of the 3' end of intron 10 and 1.5 kb from the 5' end of exon 11 with neo and hprt selectable marker genes. To correct one of these alleles, a targeting fragment was constructed in which the hygR gene was inserted into intron 9 of wild-type murine Brca1 genomic sequences. Gene targeting of this fragment, termed S1Rhyg, at either Brca1 allele would therefore restore an intact exon 11, giving rise to a Brca1+gt allele. In wild-type ES cells, we found that the S1Rhyg fragment yielded a high targeting efficiency, such that 75–90% of the hygR clones had homologously integrated this fragment at the Brca1 locus (data not shown). Thus, although gene targeting is substantially reduced in the Brca1-/- cell line, the high targeting efficiency of this fragment suggested that we would be able obtain homologous integrations with sufficient screening of hygR clones.
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. Two of 159 hygR clones yielded EcoRV fragments indicative of correction of the neo allele (Fig. 4B)
. Whereas most of the clones gave two distinct fragments for the hprt and neo alleles (12.3 and 5.9 kb, respectively), as in the parental Brca1-/- cell line, in the two targeted clones, E4 and H7, the neo fragment was replaced with a fragment expected for a targeted Brca1+gt hyg allele (11.3 kb). Because of the similarity in size of the hprt and hyg alleles, the blots were reprobed with an hprt fragment to determine whether the hprt allele was gene-targeted. In each of the clones, the hprt allele was still present, indicating that none of the other 157 clones was targeted (data not shown).
Because insertions into the intron of a gene can sometimes disrupt its expression, we determined whether Brca1 mRNA was restored in the targeted clones by performing Northern blot analysis. Brca1+/- cells were found to express the wild-type 7.2-kb Brca1 mRNA (Fig. 5)
, which was not present in the Brca1-/- cells (data not shown). Rather, a 3.9-kb transcript was seen in the mutant cells that resulted from the exon 10–12 splice. In the E4 and H7 Brca1-/+gt clones, the 7.2-kb mRNA was observed, indicating restoration of Brca1 expression (Fig. 5)
. Quantitation of Brca1 expression in these clones indicated that mRNA levels were similar to the Brca1+/- cells. A third hygR clone, H10, that was derived from the S1Rhyg targeting experiment but which had randomly integrated the fragment, was also examined for Brca1 expression. As expected, only the 3.9-kb exon 10–12 variant mRNA was observed in the H10 clone, similar to the parental Brca1-/- cells (Fig. 5)
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. This functional restoration suggests that the repair defects observed in the Brca1-/- 236.44 cell line are attributable specifically to the Brca1 mutation rather than to other genetic alterations.
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. The control cell lines expressing wild-type Brca1, i.e., Brca1+/+, Brca1+/-, and corrected Brca1-/+gt cell lines, each had a similar low frequency of observed chromosome aberrations, 3–4% of metaphases. By contrast, the Brca1 mutant cells had a substantially increased number, with 20% of metaphases containing aberrations. Spontaneous chromosomal instability is significantly higher (P = 0.0079) in Brca1-/- cells as compared with Brca1+/+, Brca1+/- and Brca1-/+gt cells. The increase was attributable to a number of different chromosomal abnormalities, including chromatid breaks and exchanges and chromosome breaks, deletions, and translocations. As seen with other assays, the Brca1-/-Tg+ cell lines fell between mutant and wild-type cells. They had 2-fold fewer aberrations than Brca1-/- cells (P = 0.043), but 2–3-fold more aberrations than wild-type cells, in accordance with partial correction of the Brca1 defect in these cells. This contrasted with the gene-targeted E4 and H7 clones that were fully corrected.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mammalian cell lines identified to have substantially impaired HDR defects, i.e., the XRCC2 and XRCC3 mutants, were originally characterized on the basis of their weak IR sensitivity but substantial mitomycin-C sensitivity and a high frequency of both spontaneous and induced chromosomal aberrations (25 , 29) . The repair phenotypes arising from Brca1 mutation described in this report are similar to those found in classical HDR mutants, and recent results suggest that similar phenotypes arise from Brca2 mutation (30) . These phenotypes may define the paradigm for HDR mutants and provide a contrast to NHEJ mutants, for which severe IR sensitivity and little or no cross-linking agent sensitivity is observed (26 , 31) . Induced chromosomal aberrations are also frequent in NHEJ mutants, although spontaneous instability is not uniformly observed in cells with reduced NHEJ (32, 33, 34, 35) . An additional emerging characteristic that may be attributable to defects in HDR is that of centrosome abnormalities, which have been observed in XRCC2, XRCC3, BRCA1, and BRCA2 mutant cells, in contrast with the lack of abnormalities in cells defective for NHEJ (27 , 34 , 36 , 37) . Thus, a proportion of gross chromosome abnormalities may result from chromosome missegregation. Although aneuploidy was not evident in Brca1-deficient ES cells, it is commonly observed in other HDR mutant cells.
Gross chromosomal instability is observed in MEFs and tumor cells derived from mice harboring Brca1 hypomorphic alleles (11 , 27 , 28) . However, as shown here, ES cells harboring similar hypomorphic Brca1 alleles have chromosome instability that is much less pronounced, suggesting that MEFs (as well as tumors) may be inherently prone to accumulate damage. The ES cell ultimately is required to generate all tissues of the adult organism and therefore may be exquisitely sensitive to a very low threshold of genetic change. As the cell cycle distribution of the mutant ES cells is normal, increased cell death may eliminate cells with unrepaired damage. Recently it has been shown that although p53 accumulates during stalled DNA replication, it is functionally impaired, suggesting repression of the p53 response during S-phase arrest (38) . One possibility for the lack of aneuploidy is that p53 function is less tightly regulated during replication blocks in ES cells, resulting in the elimination of cells when unrepaired chromosome breaks are encountered.
We found that correction of a mutant Brca1 allele led to complete rescue of the repair phenotypes, yet transgene expression of Brca1, even from a Brca1 promoter, led to only partial complementation. It remains uncertain why transgene rescue was incomplete; however, these results point to the difficulty of ectopic expression and suggest that a larger genomic region is necessary for proper regulation of Brca1 gene expression. Previously, transgene expression was shown to be insufficient for even partial restoration of normal gene-targeting efficiency in the Brca1 mutant cell line (16) . It is possible that targeting at loci tested in this previous study had a more stringent requirement for normal Brca1 levels than the pim1 locus. Nevertheless, Brca1 transgene expression led to a robust 10-fold improvement in pim1 targeting, which was confirmed by molecular analysis.
We expect that HDR of a chromosomal DSB is more physiologically relevant than gene targeting. In HDR assays, Brca1 transgene expression led to increased recombination, improving it to an intermediate level as seen for the other repair phenotypes. DSBs arising from exogenous sources like I-Sce I are potent inducers of recombination between sister-chromatids, which are templates for repair after DNA replication (39) . DSBs may also arise during normal S-phase progression in mammalian cells from replication fork disruption, such that HDR is required to restart the fork for a proper completion of replication as in E. coli (40) . Consistent with a similar role in mammalian cells is the colocalization of BRCA1 with PCNA after hydroxyurea treatment of cells (13) .
Mice and cells harboring a Brca1 mutation that deletes exon 11 are considered to be hypomorphic but not null for Brca1, because null alleles cause early embryonic lethality, whereas mice with an exon 11 deletion can survive at low frequency on a p53 background (41) . These mice have underdeveloped mammary glands, as do mice with an exon 11 deletion specifically targeted to breast tissue, consistent with impaired cellular proliferation (28 , 41) . It is possible that extended viability results from residual HDR, because, even if diminished, we find that HDR is not completely abrogated by the exon 11 deletion. Although the role of nuclear foci is uncertain, Rad51 foci are also not completely abrogated in this cell line, consistent with residual HDR (17) .
Spliced isoforms of BRCA1 found in vivo include BRCA1
11b and BRCA1672–4095 (42
, 43)
. Some lack the nuclear localization signals that are present in the 5' end of exon 11 and are predominantly located in the cytoplasm (42
, 43)
. The cytoplasmic localization of these products would seem to be predictive of a HDR defect, as improper cellular localization of BRCA2 and, surprisingly, Rad51 has been implicated as the mechanism for defective HDR observed in cells with a BRCA2 mutation (44)
. The exon 11 splice variants would still be expected to interact with other proteins, such as BARD1 or other BRCA1 RING-interacting proteins and the BRCT interacting proteins, as these interactions involve NH2- and COOH-terminal sequences, respectively, rather than the central exon 11 encoded sequences (45)
. Nevertheless, functions arising from these interactions may be compromised through sequestration of these interacting proteins in the cytoplasm. The recently described BRCA1 nuclear export sequence suggests that this protein does not reside solely in the nucleus but may shuttle between the cytoplasm and nucleus (46)
. However, a direct functional analysis of these endogenous spliced products has not been performed.
Heterozygosity for a BRCA1 mutation in humans results in a predisposition to early-onset breast and ovarian cancer. This predisposition is incurred when the remaining wild-type allele is lost or mutated, as is the case for classical tumor suppressor genes. A phenotype for the heterozygote state has been inferred for some tumor suppressor gene mutations through a dominant- negative phenotype (47 , 48) . Evidence for loss of tumor suppression solely from haploinsufficiency is less well documented (49 , 50) . A heterozygote phenotype for viability or tumorigenesis has not been reported in mice with targeted Brca1 alleles, and thus far human tumors derived from BRCA1 mutation carriers consistently reveal a loss of the wild-type allele. However, subtle changes in ovary and breast morphology have been described in mice heterozygous for either Brca1 or Brca2 mutations (51) . Furthermore, it has been reported that cells from BRCA1 and BRCA2 mutation carriers are more radiosensitive than cells from wild-type individuals (24) . In our analysis of the Brca1+/- cells, there was no decrease in the ability to gene target, repair a DSB by gene conversion, or maintain genetic integrity or mitomycin-C resistance. However, only partial complementation of repair defects was found with low transgene expression, which measured 10- 20% of wild-type Brca1 expression. Therefore, the possibility remains that a significant reduction in the expression of BRCA1 or the presence of dominant-negative truncated alleles may impart functional consequences that predispose to tumorigenesis, especially under conditions of increased DNA damage or increased proliferation.
We have found that Brca1-/- cells exhibit nearly 100-fold sensitivity as compared with the Brca1+/+ cells after treatment with mitomycin-C at the LD50 for wild-type cells. Recently, it was shown that in Saccharomyces cerevisiae, the pathways used in the repair of cisplatin-induced interstrand cross-links were cell cycle dependent. The intermediate in the repair of the interstrand cross-links in dividing cells is a DSB that is repaired by homologous recombination (52) . Agents that produce DNA interstrand cross-links are some of the most effective antitumor agents in clinical use, although their use is often limited by the toxicity incurred in normal cells. If the preference for HDR in cycling mammalian cells is preserved, it suggests that the exquisite sensitivity of Brca1-deficient cells to interstrand cross-linking agents may provide an extremely favorable therapeutic ratio in the treatment of tumors derived from BRCA1 mutation. This hypothesis can be explored in the recently derived Brca1-deficient mouse mammary tumors (28) .
The predominant function of BRCA1 that suppresses tumorigenesis has been difficult to determine, likely because of the tightly coordinated functions of DNA damage signaling, repair, and transcriptional regulation of cellular damage response genes. Genes involved in limiting chromosome aberrations would be anticipated to act cooperatively with both cell checkpoints and transcription machinery. An expectation supported by this study is that cells with defective DNA DSB repair develop chromosome instability that leads to a selective advantage either in cell growth or in the suppression of cell death and, ultimately, to tumorigenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by DAMD17-98-1-8334 from the Department of the Army, 1P50CA68425 from the NIH Specialized Program of Research Excellence in Breast Cancer, and the Breast Cancer Research Fund. 
2 To whom requests for reprints should be addressed, at Department of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue New York, NY 10021. Phone: (212) 639-2168; Fax: (212) 717-3821; E-mail: moynaham{at}mskcc.org
3 Current address: Department of Physiology, University of Michigan, Ann Arbor, MI 48109.
4 The abbreviations used are: DSB, double-strand break; IR, ionizing radiation; ES, embryonic stem; NHEJ, nonhomologous end-joining; HDR, homology-directed repair; MEF, mouse embryonic fibroblasts; Tg, transgene; pBS, pBluescript; purR, puromycin-resistant; hygR, hygromycin-resistant; DR-GFP, direct repeat-green fluorescent protein; neo, neomycin phosphoribosyl transferase; hprt, hypoxanthine phosphoribosyl transferase; XRCC, X-ray cross-complementing repair.
Received 1/22/01. Accepted 5/ 2/01.
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M. Laufer, S. V. Nandula, A. P. Modi, S. Wang, M. Jasin, V. V. V. S. Murty, T. Ludwig, and R. Baer Structural Requirements for the BARD1 Tumor Suppressor in Chromosomal Stability and Homology-directed DNA Repair J. Biol. Chem., November 23, 2007; 282(47): 34325 - 34333. [Abstract] [Full Text] [PDF]
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 M. A. Dimopoulos, V. L. Souliotis, A. Anagnostopoulos, C. Bamia, A. Pouli, I. Baltadakis, E. Terpos, S. A. Kyrtopoulos, and P. P. Sfikakis Melphalan-induced DNA damage in vitro as a predictor for clinical outcome in multiple myeloma Haematologica, November 1, 2007; 92(11): 1505 - 1512. [Abstract] [Full Text] [PDF]
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 A. D. Treszezamsky, L. A. Kachnic, Z. Feng, J. Zhang, C. Tokadjian, and S. N. Powell BRCA1- and BRCA2-Deficient Cells Are Sensitive to Etoposide-Induced DNA Double-Strand Breaks via Topoisomerase II Cancer Res., August 1, 2007; 67(15): 7078 - 7081. [Abstract] [Full Text] [PDF]
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 C. R. James, J. E. Quinn, P. B. Mullan, P. G. Johnston, and D. P. Harkin BRCA1, a Potential Predictive Biomarker in the Treatment of Breast Cancer Oncologist, February 1, 2007; 12(2): 142 - 150. [Abstract] [Full Text] [PDF]
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 S. Shanley, K. McReynolds, A. Ardern-Jones, R. Ahern, I. Fernando, J. Yarnold, G. Evans, D. Eccles, S. Hodgson, S. Ashley, et al. Acute Chemotherapy-Related Toxicity Is Not Increased in BRCA1 and BRCA2 Mutation Carriers Treated for Breast Cancer in the United Kingdom Clin. Cancer Res., December 1, 2006; 12(23): 7033 - 7038. [Abstract] [Full Text] [PDF]
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 S. V. Diaz-Perez, D. O. Ferguson, C. Wang, G. Csankovszki, C. Wang, S.-C. Tsai, D. Dutta, V. Perez, S. Kim, C. D. Eller, et al. A Deletion at the Mouse Xist Gene Exposes Trans-effects That Alter the Heterochromatin of the Inactive X Chromosome and the Replication Time and DNA Stability of Both X Chromosomes Genetics, November 1, 2006; 174(3): 1115 - 1133. [Abstract] [Full Text] [PDF]
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 N. S. Sivarajasingham, L. Cawkwell, R. P. Baker, S. L. O'Kane, E. F. Smyth, J. V. T. Tilsed, M. B. Watson, J. Greenman, and J. R. T. Monson Implication of the BRCA2 and Putative ''BRCA3'' Genes in Dukes' Stage C, Replication Error-Negative Colon Cancer Ann. Surg. Oncol., June 1, 2006; 13(6): 881 - 886. [Abstract] [Full Text] [PDF]
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R. I. Yarden and M. Z. Papa BRCA1 at the crossroad of multiple cellular pathways: approaches for therapeutic interventions. Mol. Cancer Ther., June 1, 2006; 5(6): 1396 - 1404. [Abstract] [Full Text] [PDF]
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A. M. Simons, A. A. Horwitz, L. M. Starita, K. Griffin, R. S. Williams, J.N. M. Glover, and J. D. Parvin BRCA1 DNA-Binding Activity Is Stimulated by BARD1 Cancer Res., February 15, 2006; 66(4): 2012 - 2018. [Abstract] [Full Text] [PDF]
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I. Cousineau, C. Abaji, and A. Belmaaza BRCA1 Regulates RAD51 Function in Response to DNA Damage and Suppresses Spontaneous Sister Chromatid Replication Slippage: Implications for Sister Chromatid Cohesion, Genome Stability, and Carcinogenesis Cancer Res., December 15, 2005; 65(24): 11384 - 11391. [Abstract] [Full Text] [PDF]
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 R. D. Kennedy and A. D. D'Andrea The Fanconi Anemia/BRCA pathway: new faces in the crowd Genes & Dev., December 15, 2005; 19(24): 2925 - 2940. [Abstract] [Full Text] [PDF]
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 J. Zhang and S. N. Powell The Role of the BRCA1 Tumor Suppressor in DNA Double-Strand Break Repair Mol. Cancer Res., October 1, 2005; 3(10): 531 - 539. [Abstract] [Full Text] [PDF]
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A. D. Choudhury, H. Xu, A. P. Modi, W. Zhang, T. Ludwig, and R. Baer Hyperphosphorylation of the BARD1 Tumor Suppressor in Mitotic Cells J. Biol. Chem., July 1, 2005; 280(26): 24669 - 24679. [Abstract] [Full Text] [PDF]
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S. A. Narod and K. Offit Prevention and Management of Hereditary Breast Cancer J. Clin. Oncol., March 10, 2005; 23(8): 1656 - 1663. [Full Text] [PDF]
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K. Trenz, P. Schutz, and G. Speit Radiosensitivity of lymphoblastoid cell lines with a heterozygous BRCA1 mutation is not detected by the comet assay and pulsed field gel electrophoresis Mutagenesis, March 1, 2005; 20(2): 131 - 137. [Abstract] [Full Text] [PDF]
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K. Nakanishi, Y.-G. Yang, A. J. Pierce, T. Taniguchi, M. Digweed, A. D. D'Andrea, Z.-Q. Wang, and M. Jasin Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair PNAS, January 25, 2005; 102(4): 1110 - 1115. [Abstract] [Full Text] [PDF]
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 A.N.J. TUTT, C.J. LORD, N. MCCABE, H. FARMER, N. TURNER, N.M. MARTIN, S.P. JACKSON, G.C.M. SMITH, and A. ASHWORTH Exploiting the DNA Repair Defect in BRCA Mutant Cells in the Design of New Therapeutic Strategies for Cancer Cold Spring Harb Symp Quant Biol, January 1, 2005; 70(0): 139 - 148. [Abstract] [PDF]
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 R. D. Kennedy, J. E. Quinn, P. B. Mullan, P. G. Johnston, and D. P. Harkin The Role of BRCA1 in the Cellular Response to Chemotherapy J Natl Cancer Inst, November 17, 2004; 96(22): 1659 - 1668. [Abstract] [Full Text] [PDF]
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 J. M. Stark, A. J. Pierce, J. Oh, A. Pastink, and M. Jasin Genetic Steps of Mammalian Homologous Repair with Distinct Mutagenic Consequences Mol. Cell. Biol., November 1, 2004; 24(21): 9305 - 9316. [Abstract] [Full Text] [PDF]
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 Y. Canitrot, J.-P. Capp, N. Puget, A. Bieth, B. Lopez, J.-S. Hoffmann, and C. Cazaux DNA polymerase {beta} overexpression stimulates the Rad51-dependent homologous recombination in mammalian cells Nucleic Acids Res., September 27, 2004; 32(17): 5104 - 5112. [Abstract] [Full Text] [PDF]
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A. D. Choudhury, H. Xu, and R. Baer Ubiquitination and Proteasomal Degradation of the BRCA1 Tumor Suppressor Is Regulated during Cell Cycle Progression J. Biol. Chem., August 6, 2004; 279(32): 33909 - 33918. [Abstract] [Full Text] [PDF]
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 M. S. Van der Heijden, J. R. Brody, E. Gallmeier, S. C. Cunningham, D. A. Dezentje, D. Shen, R. H. Hruban, and S. E. Kern Functional Defects in the Fanconi Anemia Pathway in Pancreatic Cancer Cells Am. J. Pathol., August 1, 2004; 165(2): 651 - 657. [Abstract] [Full Text] [PDF]
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 G. S. Boehden, A. Restle, R. Marschalek, C. Stocking, and L. Wiesmuller Recombination at chromosomal sequences involved in leukaemogenic rearrangements is differentially regulated by p53 Carcinogenesis, August 1, 2004; 25(8): 1305 - 1313. [Abstract] [Full Text] [PDF]
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P. Morales-Ramirez, T. Vallarino-Kelly, V. L. Cruz-Vallejo, R. Lopez-Iturbe, and H. Alvaro-Delgadillo In vivo kinetics of micronuclei induction by bifunctional alkylating antineoplastics Mutagenesis, May 1, 2004; 19(3): 207 - 213. [Abstract] [Full Text] [PDF]
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J. Zhang, H. Willers, Z. Feng, J. C. Ghosh, S. Kim, D. T. Weaver, J. H. Chung, S. N. Powell, and F. Xia Chk2 Phosphorylation of BRCA1 Regulates DNA Double-Strand Break Repair Mol. Cell. Biol., January 15, 2004; 24(2): 708 - 718. [Abstract] [Full Text] [PDF]
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A. Rothfuss and M. Grompe Repair Kinetics of Genomic Interstrand DNA Cross-Links: Evidence for DNA Double-Strand Break-Dependent Activation of the Fanconi Anemia/BRCA Pathway Mol. Cell. Biol., January 1, 2004; 24(1): 123 - 134. [Abstract] [Full Text] [PDF]
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S. Seal, R. Barfoot, H. Jayatilake, P. Smith, A. Renwick, L. Bascombe, L. McGuffog, D. G. Evans, D. Eccles, D. F. Easton, et al. Evaluation of Fanconi Anemia Genes in Familial Breast Cancer Predisposition Cancer Res., December 15, 2003; 63(24): 8596 - 8599. [Abstract] [Full Text] [PDF]
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B. Deans, C. S. Griffin, P. O'Regan, M. Jasin, and J. Thacker Homologous Recombination Deficiency Leads to Profound Genetic Instability in Cells Derived from Xrcc2-Knockout Mice Cancer Res., December 1, 2003; 63(23): 8181 - 8187. [Abstract] [Full Text] [PDF]
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U. K. Westermark, M. Reyngold, A. B. Olshen, R. Baer, M. Jasin, and M. E. Moynahan BARD1 Participates with BRCA1 in Homology-Directed Repair of Chromosome Breaks Mol. Cell. Biol., November 1, 2003; 23(21): 7926 - 7936. [Abstract] [Full Text] [PDF]
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J. E. Quinn, R. D. Kennedy, P. B. Mullan, P. M. Gilmore, M. Carty, P. G. Johnston, D. P. Harkin, U. Benatti, L. C. Boffa, and M. Ferrarini BRCA1 Functions as a Differential Modulator of Chemotherapy-induced Apoptosis Cancer Res., October 1, 2003; 63(19): 6221 - 6228. [Abstract] [Full Text] [PDF]
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S. Houghtaling, C. Timmers, M. Noll, M. J. Finegold, S. N. Jones, M. S. Meyn, and M. Grompe Epithelial cancer in Fanconi anemia complementation group D2 (Fancd2) knockout mice Genes & Dev., August 15, 2003; 17(16): 2021 - 2035. [Abstract] [Full Text] [PDF]
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S. L. Donahue, R. Lundberg, R. Saplis, and C. Campbell Deficient Regulation of DNA Double-strand Break Repair in Fanconi Anemia Fibroblasts J. Biol. Chem., August 8, 2003; 278(32): 29487 - 29495. [Abstract] [Full Text] [PDF]
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E. E. McCarthy, J. T. Celebi, R. Baer, and T. Ludwig Loss of Bard1, the Heterodimeric Partner of the Brca1 Tumor Suppressor, Results in Early Embryonic Lethality and Chromosomal Instability Mol. Cell. Biol., July 15, 2003; 23(14): 5056 - 5063. [Abstract] [Full Text] [PDF]
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M. S. van der Heijden, C. J. Yeo, R. H. Hruban, and S. E. Kern Fanconi Anemia Gene Mutations in Young-onset Pancreatic Cancer Cancer Res., May 15, 2003; 63(10): 2585 - 2588. [Abstract] [Full Text] [PDF]
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 X. Xu, O. Aprelikova, P. Moens, C.-X. Deng, and P. A. Furth Impaired meiotic DNA-damage repair and lack of crossing-over during spermatogenesis in BRCA1 full-length isoform deficient mice Development, May 1, 2003; 130(9): 2001 - 2012. [Abstract] [Full Text] [PDF]
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 C.-X. Deng and R.-H. Wang Roles of BRCA1 in DNA damage repair: a link between development and cancer Hum. Mol. Genet., April 2, 2003; 12(90001): R113 - 123. [Abstract] [Full Text] [PDF]
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J. M. Stark and M. Jasin Extensive Loss of Heterozygosity Is Suppressed during Homologous Repair of Chromosomal Breaks Mol. Cell. Biol., January 15, 2003; 23(2): 733 - 743. [Abstract] [Full Text] [PDF]
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H. Zheng, X. Wang, A. J. Warren, R. J. Legerski, R. S. Nairn, J. W. Hamilton, and L. Li Nucleotide Excision Repair- and Polymerase {eta}-Mediated Error-Prone Removal of Mitomycin C Interstrand Cross-Links Mol. Cell. Biol., January 15, 2003; 23(2): 754 - 761. [Abstract] [Full Text] [PDF]
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S. L. Donahue and C. Campbell A DNA Double Strand Break Repair Defect in Fanconi Anemia Fibroblasts J. Biol. Chem., November 22, 2002; 277(48): 46243 - 46247. [Abstract] [Full Text] [PDF]
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 T. Taniguchi, I. Garcia-Higuera, P. R. Andreassen, R. C. Gregory, M. Grompe, and A. D. D'Andrea S-phase-specific interaction of the Fanconi anemia protein, FANCD2, with BRCA1 and RAD51 Blood, September 18, 2002; 100(7): 2414 - 2420. [Abstract] [Full Text] [PDF]
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T. Taniguchi and A. D. D'Andrea The Fanconi anemia protein, FANCE, promotes the nuclear accumulation of FANCC Blood, September 18, 2002; 100(7): 2457 - 2462. [Abstract] [Full Text] [PDF]
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 N. G. Howlett, T. Taniguchi, S. Olson, B. Cox, Q. Waisfisz, C. de Die-Smulders, N. Persky, M. Grompe, H. Joenje, G. Pals, et al. Biallelic Inactivation of BRCA2 in Fanconi Anemia Science, July 26, 2002; 297(5581): 606 - 609. [Abstract] [Full Text] [PDF]
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J. M. Stark, P. Hu, A. J. Pierce, M. E. Moynahan, N. Ellis, and M. Jasin ATP Hydrolysis by Mammalian RAD51 Has a Key Role during Homology-directed DNA Repair J. Biol. Chem., May 31, 2002; 277(23): 20185 - 20194. [Abstract] [Full Text] [PDF]
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W. S. Joo, P. D. Jeffrey, S. B. Cantor, M. S. Finnin, D. M. Livingston, and N. P. Pavletich Structure of the 53BP1 BRCT region bound to p53 and its comparison to the Brca1 BRCT structure Genes & Dev., March 1, 2002; 16(5): 583 - 593. [Abstract] [Full Text] [PDF]
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M. Kraakman-van der Zwet, W. J. I. Overkamp, R. E. E. van Lange, J. Essers, A. van Duijn-Goedhart, I. Wiggers, S. Swaminathan, P. P. W. van Buul, A. Errami, R. T. L. Tan, et al. Brca2 (XRCC11) Deficiency Results in Radioresistant DNA Synthesis and a Higher Frequency of Spontaneous Deletions Mol. Cell. Biol., January 15, 2002; 22(2): 669 - 679. [Abstract] [Full Text] [PDF]
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M. Grompe and A. D'Andrea Fanconi anemia and DNA repair Hum. Mol. Genet., October 1, 2001; 10(20): 2253 - 2259. [Abstract] [Full Text] [PDF]
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, the percentage of GFP-positive cells subsequent to transfection of the I-Sce I expression vector normalized to the wild-type Brca1+/+ cells. I-Sce I expression strongly induces the number of GFP-positive Brca1+/+ and Brca1+/- cells, indicating robust DSB repair by gene conversion; however, Brca1-/- cells have 6-fold fewer GFP-positive cells, indicating impaired HDR of a DSB by gene conversion (P < 0.0001). Brca1 transgene expression in the Brca1-/-, Tg+ cells exhibited a 2-fold improvement in HDR of an induced DSB as compared with the Brca1-/- cells (P = 0.032). This graphical depiction includes quadruplicate experiments wherein three independent clones from each different genotype were analyzed. The number of GFP-positive cells for the three independent clones from each genotype was averaged for each experiment. The percentage of GFP-positive wild-type cells was normalized to one, and the relative frequencies of the other Brca1 genotypes were calculated; +/+, Brca1+/+ cells; +/-, Brca1+/-cells; -/-, Brca1-/- cells; and -/-, Tg+, Brca1-/-, Tg+ cells targeted with the p59xDR-GFP6 vector. Bars, SE.
