INTRODUCTION

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Germ line mutations in BRCA1 account for 45% of all hereditary cases of breast cancer (35). Inheritance of one defective copy of BRCA1 confers an estimated 80 to 90% overall lifetime risk for breast or ovarian cancer (15). Analysis of the primary structure of BRCA1 identified a number of potential functional motifs, including a RING finger domain in the N terminus and two putative nuclear localization sequences (5, 50, 52). In addition, two BRCT domains, characteristic of proteins involved in DNA repair, were identified in the C terminus (3). Expression of BRCA1 is detected early in embryonic development and continues to be found in a broad spectrum of tissues in the adult animal (27, 34). It has now been established that BRCA1 is a nuclear phosphoprotein expressed in dividing cells in a cell-cycle-specific manner, with maximal expression of BRCA1 occurring in the S phase (6, 42). Phosphorylation levels of BRCA1 also change throughout the cell cycle, peaking in late S phase (6, 42).

Despite extensive study, the function(s) of BRCA1 has not yet been clearly defined. The expression and phosphorylation of BRCA1 in a cell-cycle-specific manner suggest that this protein may be involved in the regulation of cell cycle transition. BRCA1 has been shown to induce expression of p21 and, more recently, BRCA1 has been shown to act as a p53 coactivator (38, 48, 54). In addition, a potential role as a transcriptional activator has been shown by the fusion of the C terminus of BRCA1 with a GAL4 DNA-binding domain (4, 36).

The hyperphosphorylation and relocalization of BRCA1 with RAD51 to PCNA-containing foci after exposure to DNA-damaging agents suggest an additional or alternate function for this protein (43). RAD51 has been implicated in DNA repair, as demonstrated by the high sensitivity to DNA-damaging agents of Saccharomyces cerevisiae rad51 mutants (17, 46). This increased sensitivity is thought to be due to a defect in recombinational repair of double-strand breaks. The recent demonstration that embryonic stem (ES) cells deficient in BRCA1 are more sensitive to some DNA-damaging agents provides direct evidence supporting a role for BRCA1 in maintaining genomic integrity (19). These BRCA1-deficient cells are unable to carry out transcription-coupled repair (TCR) after exposure to DNA-damaging agents, demonstrating the participation of this protein in at least one cellular repair pathway. This repair system, which preferentially repairs the transcribed strand of active genes, is important for the removal of lesions for which the global repair process is too slow.

Several mouse lines carrying mutations in the Brca1 gene have been generated (18, 21, 32, 33). Unlike humans, mice carrying a mutant Brca1 allele do not display an increased risk for tumor formation. This observation and the early embryonic lethality of mice homozygous for the mutant allele have to date limited the contribution of these models to understanding the role of BRCA1 in tumorigenesis. However, we have recently reported the generation of five mammary tumors from mice heterozygous for both a mutant Brca1 allele and p53 allele after exposure to ionizing radiation (9). Furthermore, loss of heterozygosity of both Brca1 and p53 could be demonstrated in tumor tissue obtained from three of these tumors. This suggests that exposure of Brca1^+/ mice to specific environmental risk factors may be necessary for the development of mammary tumors during the short lifespan of the mouse. It also suggests that mutations in genes, such as p53, which confer a growth advantage and in particular allow the continued growth of cells having incurred DNA damage may be critical to the development of these tumors.

Evidence for a relationship between p53 and BRCA1 is further suggested by the demonstration that survival of BRCA1-deficient embryos is extended in the absence of p53 expression (22, 33). However, the Brca1^/ p53^/ embryos still die early in embryogenesis, precluding examination of the loss of BRCA1 in later stages of development and in tumorigenesis. The early lethality of the Brca1^/ p53^/ embryos did not permit the generation of Brca1^/ p53^/ embryonic fibroblast cell lines.

We report here the survival and characterization of a small number of Brca1^/ p53^/ mice after extensive breeding of Brca1^+/ p53^/ animals. Although growth retarded, the development of the somatic tissues of these mice is largely normal. Male mice are infertile due to a defect in spermatogenesis, thus defining a role for BRCA1 in meiosis. Brca1^/ p53^/ animals die of tumors typical of p53^/ mice; however, tumor latency is reduced compared to that observed in the p53^/ population in our colony. Fibroblast lines were generated from the Brca1^/ p53^/ mice and compared to the fibroblast lines from both wild-type and p53^/ animals. While, as expected, p53^/ fibroblasts grow rapidly, the growth of early passage Brca1^/ p53^/ fibroblasts is similar to that of wild-type cells. This decreased growth rate is largely the result of a decrease in the survival of the BRCA1-deficient cells. We also show here that the Brca1^/ p53^/ cells are more sensitive than p53^/ cells to DNA-damaging agents and that the ability of the cells to carry out transcription-coupled repair in response to DNA damage is compromised. These observations support the hypothesis that BRCA1 plays an important role in DNA repair pathways. In summary, our results suggest that loss of heterozygosity of BRCA1 is not an initiating event in tumorigenesis. Cells must instead attain a growth advantage, by mechanisms such as loss of cell cycle checkpoint control or responsiveness to trophic factors, for loss of BRCA1 function to predispose towards tumorigenesis.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Animal husbandry. p53^+/ mice were obtained from Jackson Labs and bred to Brca1^+/ mice generated in our colony (18, 26). Genomic DNA was recovered from tail biopsy, and genotypes were determined by PCR amplification, as described elsewhere (18, 26). All three Brca1^/ p53^/ mice were generated from matings of Brca1^+/ p53^/ females with a Brca1^+/ p53^/ male. The Brca1 genotype was verified by Southern blotting analysis. Genomic DNA was digested with EcoRV and analyzed by Southern blot with a probe containing a portion of intron 9 and exon 10 of the Brca1 gene. Each mouse was euthanized when moribund and then necropsied. Tissues were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Sections were cut (5 µm) and stained with hematoxylin and eosin. Testes sections were also stained with toluidine blue. The TUNEL (terminal deoxynucleotidytransferase-mediated dUTP-biotin nick end labeling) assay was performed on testes sections to determine the level of apoptosis, as per the manufacturer's instructions (Trevigen, Gaithersburg, Md.). Additional testes sections were immunostained with a rabbit polyclonal antibody for HSP70-2 (13) and staining was detected by a Peroxidase Elite ABC Kit (Vector Laboratories, Burlingame, Calif.) as per the manufacturer's instructions. Whole-mount preparations were done as described elsewhere (37a). Briefly, mammary epithelium was fixed in 10% phosphate-buffered neutral formalin and then rehydrated. The mammary glands were stained overnight with carmine alum, dehydrated, cleared in xylene, and mounted in Permount.

Generation of cell lines. Primary fibroblasts were obtained from the skin and ears of all three Brca1^/ p53^/ mice. These tissues were washed repeatedly with phosphate-buffered saline (PBS), finely minced, and digested overnight with collagenase (Life Technologies, Grand Island, N.Y.). The fibroblasts released were cultured in Dulbecco modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and supplemented with L-glutamine, penicillin, streptomycin, and gentamicin at 37°C with 5% CO₂. Cells were passaged before they reached 100% confluence. Fibroblast lines were also generated from two Brca1^+/ p53^/ mice and a Brca1^+/+ p53^+/+ mouse.

Analysis of cellular growth. Determination of cellular growth was performed as described elsewhere (12). Briefly, asynchronous fibroblasts from each cell line were plated onto a series of 35-mm dishes. Each of the Brca1^/ p53^/ lines was plated at a density of 4 × 10⁴ cells per well, while 2 × 10⁴ cells of each of the Brca1^+/ p53^/ lines per well were plated. These values were determined based on the slower growth of the Brca1^/ p53^/ cells. Cells from two wells per line were counted daily by using trypan blue exclusion. The medium was changed daily for the remaining cells. For cell cycle analysis, asynchronous fibroblasts from each cell line were harvested after a 4-h incubation with 10 µM bromodeoxyuridine (BrdU; Boehringer Mannheim, Indianapolis, Ind.) and then fixed in cold 70% ethanol. Fixed cells were stored at 4°C until analysis. Nuclei were released by treatment with 0.08% pepsin in 0.1 N HCl at 37°C for 20 min. Nuclei were then treated with 2 N HCl at 37°C for 20 min, followed by neutralization with 0.1 M sodium borate. Cells were then incubated with the fluorescein isothiocyanate-conjugated anti-BrdU antibody (Becton Dickinson, San Jose, Calif.) and counterstained with propidium iodide (50 µg/ml) containing RNase (5 µg/ml) overnight at 4°C. Cell cycle analysis was performed on a FACScan cell sorter (San Jose, Calif.) with Cytomation data acquisition software (Fort Collins, Colo.). WinMDI software was used to determine the percentage of cells in each cell cycle phase.

Analysis of cellular death. To determine the ratio of dead to live cells, asynchronous cells from each line were plated onto six 100-mm dishes and grown to 70 to 80% confluence. The medium was removed, and the cells were incubated with 8 ml of fresh medium for 7 h. For quantitation of live cells, each plate was trypsinized, and cells were counted by trypan blue exclusion with a hemocytometer. For the dead cell count, the medium from each plate was centrifuged in separate tubes. Pelleted cells were resuspended in 10% FBS in PBS and cytospun onto a microscope slide. The dead cells were stained with Diff-Quik stain (Dade Diagnostics of P.R., Inc., Miami, Fla.), and photographs from random areas of each slide were taken to facilitate counting. Only cells containing nuclei were counted.

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DNA damage analysis. The survival of the fibroblasts following treatment with DNA-damaging agents was determined as described elsewhere (40). In brief, to determine survival after ionizing radiation, asynchronous cells were irradiated in suspension at room temperature by using a ¹³⁷Cs source. The medium was changed immediately after irradiation. Brca1^/ p53^/ cells were then plated at a density of 4 × 10⁴ cells per 35-mm dish and Brca1^+/ p53^/ cells were plated at a density of 2 × 10⁴ cells per dish. The surviving cells were counted 4 days later by trypan blue exclusion and expressed as a percentage, by using untreated, nonconfluent cells as the 100% level. To measure survival after hydrogen peroxide treatment, Brca1^/ p53^/ cells were plated at a density of 4 × 10⁴ cells per 35-mm dish, and Brca1^+/ p53^/ cells were plated at a density of 2 × 10⁴ cells per dish. When cells were adherent 10 h later, cells were incubated with hydrogen peroxide for 15 min at 37°C. Cells were then washed twice with medium. Quantitation of cells was done 4 days later by trypan blue exclusion, and this is expressed as a percentage, using untreated, nonconfluent cells as the 100% value. To determine survival after UV irradiation, cells were plated as described above for the hydrogen peroxide treatment. The cells were incubated for 10 h to allow adherence. The medium was then aspirated, and cells were irradiated by using a Stratalinker (Stratagene, La Jolla, Calif.). Medium was then immediately added. Quantitation was done 4 days later by trypan blue exclusion and expressed as a percentage, by using untreated, nonconfluent cells as the 100% level. Each of these experiments was conducted in duplicate, and in each replicate, the cell survival was determined four times at each experimental point.

	RESULTS

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Identification of Brca1^/ p53^/ mice. Mice heterozygous for the Brca1 mutation, Brca1^223-763, were intercrossed with mice carrying a mutant p53 allele to obtain animals heterozygous for both mutations (18, 26). While litters obtained from the intercrossing of the derived double-heterozygous animals yielded p53^/ animals at reported frequencies, no Brca1^/ animals were identified among either the p53^/ or p53^+/ offspring. Brca1^+/ p53^/ mice were further intercrossed and again no double-homozygous animals were obtained on examination of 56 offspring obtained from nine different litters (9). Further expansion of this mouse population, however, led to the identification of three Brca1^/ mice, two males and a single female, all also homozygous for the p53 mutant allele. Southern blot analysis revealed that only the targeted Brca1 allele was present (data not shown). The absence of the normal Brca1 mRNA transcript was further verified by Northern blot analysis (data not shown). Interestingly, two of these animals not only shared one parent but were further related in that the dam of the second Brca1^/ p53^/ mouse was the sibling of the first Brca1^/ p53^/ animal identified.

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View larger version (116K): [in this window] [in a new window]   FIG. 1.   Abnormal end bud formation and decreased ductal branching in BRCA1-deficient mice. (A) Mammary epithelium of a 12-week-old p53/ female mouse displays extensive branching and numerous end buds. (B) In contrast, very few branches from the primary ducts are seen in the mammary gland of the Brca1/ p53/ female mouse. At a higher magnification, many end buds can be visualized in the p53/ female (C), while only a few underdeveloped end buds are seen in the Brca1/ p53/ mouse (D). Magnification bars: A and B, 500 µm; C and D, 200 µm.

View larger version (170K): [in this window] [in a new window]   FIG. 2.   Cellular abnormalities within the mammary and salivary glands of the Brca1/ p53/ mice. (A) Histological analysis of the mammary gland ducts from a p53/ mouse shows a duct lined by one to two layers of low cuboidal epithelia. Mammary ducts of the Brca1/ p53/ female are dilated and lined by a single layer of flattened-to-low cuboidal cells. (B to D) In some regions, the ducts are surrounded by an array of individual cells. While acinar cells in the p53/ salivary gland appear to be uniform (E), cytomegaly and karyomegaly (arrows) are seen sporadically throughout the acinar units of the salivary glands of the Brca1/ p53/ animals (F). Staining was done with hematoxylin and eosin. Magnification bar, 50 µm.

Spermatogenesis is disrupted in the Brca1^/ p53^/ mice. Male Brca1^/ p53^/ mice failed to impregnate female mice. This deficiency was not the result of a failure of the mice to copulate, since copulation plugs were observed in females following mating with a Brca1^/ p53^/ male. On gross examination, the testes of both of the double-homozygous males were smaller in size than those of wild-type males of similar weight. We next compared the histological development of the seminiferous tubules of these testes to those from p53^/ control animals of similar age. Sertoli cells, the supporting cells within the tubules, were seen throughout the testes of the Brca1^/ p53^/ males, suggesting that spermatogenesis was not aborted due to loss of this cell population.

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View larger version (123K): [in this window] [in a new window]   FIG. 3.   Absence of spermatids and spermiogenesis in a BRCA1-deficient male. The testes and seminiferous tubules of a Brca1/ p53/ male (B) are smaller than those of an age-matched p53/ control male (A). While the seminiferous tubules of the p53/ control male mouse (C) contained all of the cells involved in spermatogenesis (spermatogonia, thin arrows; spermatocyte, wide arrow; spermatid, large arrowhead; spermatozoa, small arrowhead), only spermatogonia (solid arrows) can be seen in the testes of the Brca1/ p53/ male (D). Sertoli cells (double arrow) were seen in abundance in the seminiferous tubules of the Brca1/ p53/ males and the p53/ males (C and D). (E) Apoptosis as determined by the TUNEL assay (apoptotic cells, arrow) occurs rarely in spermatogenesis in p53/ males. Apoptotic cells (arrows in panels E and F) appear to be more numerous in the testes of the Brca1/ p53/ male. However, to correct for the decreased size of the seminiferous tubules of the Brca1/ p53/ male in a given area, we counted the number of apoptotic cells present in 25 randomly chosen seminiferous tubules from the two animals. (F) TUNEL-positive cells were only increased approximately threefold in testes of the Brca1/ p53/ male. (G) Spermatocytes (arrows), spermatids, and spermatozoa can be detected in the testes of a p53/ control male stained with HSP70-2 antibody. Incubation with the HSP70-2 antibody revealed the presence of spermatocytes (arrows) in the testes of a Brca1/ p53/ male. Staining: A and B, hematoxylin and eosin; C and D, toluidine blue; E and F, diaminobenzidine and methyl green; G and H, diaminobenzidine. Magnification bars: A and B, 200 µm; C and D, 20 µm; E and F, 100 µm; G and H, 67 µm.

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Growth properties of primary skin fibroblasts. Skin fibroblasts were prepared from Brca1^/ p53^/ mice, Brca1^+/ p53^/ mice, and wild-type mice, and the growth properties of these primary cultures were examined. Brca1^+/ p53^/ fibroblasts had growth characteristics similar to Brca1^+/+ p53^/ lines. For simplicity we will refer to the Brca1^+/ p53^/ lines as p53^/ lines throughout the peper. Before these experiments were begun, the plating efficiency of the fibroblasts of each genotype was established. This was done by plating known numbers of cells, allowing the cells to adhere, and then determining the number of viable surviving cells 7 h later. At this time point, cell numbers reflect the plating efficiency of the cells rather than differences in the growth rates of the various lines. No differences between the plating efficiencies of the Brca1^/ p53^/, p53^/, and wild-type lines were observed (data not shown).

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View larger version (30K): [in this window] [in a new window]   FIG. 4.   Growth rate of Brca1/ p53/ fibroblasts. (A) At early passages, Brca1/ p53/ and p53/ control lines and a wild-type fibroblast line were plated at a low density and were counted daily. Brca1/ p53/ fibroblasts have a growth rate similar to that of the wild-type fibroblasts. In contrast, the Brca1+/ p53/ control fibroblast lines have a higher growth rate, and contact inhibition occurs at a higher density. (B) However, at later passages, the growth rate of the Brca1/ p53/ fibroblasts has increased, and contact inhibition occurs at a higher cell density. (C) Brca1/ p53/ fibroblasts were incubated with BrdU and stained with anti-BrdU antibody and propidium iodide. Quantitation of the percentage of cells in each cell cycle stage was performed by flow cytometry. One Brca1+/ p53/ fibroblast line and one wild-type fibroblast line were used as controls. Cells at passage numbers 3 and 4 were considered early-passage cells, while cells at passage numbers 10 through 14 were considered late-passage cells.

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View larger version (21K): [in this window] [in a new window]   FIG. 5.   A higher rate of cellular death occurs in Brca1/ p53/ primary fibroblast cultures. (A) p21 protein levels are not elevated in the Brca1/ p53/ fibroblasts (lanes a and b) or the Brca1+/ p53/ fibroblasts (lane c). In contrast, high levels of p21 were detected in wild-type fibroblasts (lanes d and e). (B) Nonadherent Brca1/ p53/ fibroblasts were collected and counted. This number was normalized to the number of adherent live cells in each fibroblast population. Nonadherent and adherent counts were obtained from six plates for each of the cell lines. The bars represent the standard errors.

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Cell cycle kinetics of Brca1^/ p53^/ fibroblasts. Previous studies of p53^/ and wild-type embryonic fibroblasts indicated that the increased proliferation rate of the p53^/ cells correlated with an increase in the percentage of cells in S phase and a decrease in the number of cells in G₀/G₁ (24). These alterations are believed to stem largely from a loss of the G₁/S checkpoint in the p53^/ lines. To determine whether further alterations in the cell cycle kinetics underlie the decreased growth rate of the Brca1^/ p53^/ lines, asynchronous early-passage fibroblasts of each of the three genotypes were labeled with BrdU, stained with propidium iodide, and subjected to flow cytometry analysis (Fig. 4C). As reported previously and consistent with observed growth rates, a higher percentage of p53^/ cells are observed in S phase than seen in similarly labeled wild-type cells (24). In addition, loss of the spindle checkpoint due to p53 deficiency results in an increased percentage of tetraploid cells (11). Surprisingly, in light of the growth properties of the Brca1^/ p53^/ cells, the percentage of these cells in S phase was similar to that observed in the p53^/ fibroblast cultures. Thus, while at low passage the growth rate of the Brca1^/ p53^/ lines is similar to wild-type lines, the percentage of cells in S phase is approximately two to three times greater than seen in this control population.

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Decreased growth rate of Brca1^/ p53^/ cells is due to an increased rate of cellular death. In order to resolve the discrepancy between the observed growth rate of the Brca1^/ p53^/ cells and the percentage of the cells in S phase, we next determined the rate of cellular death in the cultures of Brca1^/ p53^/, p53^/, and wild-type fibroblasts. Dead cells can be collected and enumerated easily in these cultures because after cellular death, the fibroblasts become nonadherent, lift off from the surface of the culture dishes, and can be harvested by aspiration and centrifugation of the tissue culture medium. Collection of these cells from cultures of all three genotypes and analysis with trypan blue exclusion dye confirmed that virtually all of the cells harvested in this manner were dead and that the collection procedure had not resulted in disturbance of the loosely attached mitotic cells (Fig. 5B).

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Brca1^/ p53^/ fibroblasts show increased sensitivity to DNA-damaging agents. A number of lines of evidence support a role for BRCA1 in the maintenance of genome integrity (19, 43). If increased cellular death in the Brca1^/ p53^/ lines is directly related to the inability of these cells to repair DNA damage, it would be expected that the growth of these cells would be further compromised by exposure to DNA-damaging agents. We therefore determined the survival rate of the two Brca1^/ p53^/ lines and two p53^/ lines after treatment with ionizing radiation, hydrogen peroxide, and UV light. A dose-dependent decrease in cellular survival was seen in cells of both genotypes after exposure to 2.5, 5.0, and 7.5 Gy of ionizing radiation (Fig. 6A). However, the decrease in survival after treatment was significantly greater in the double-homozygous lines. A similar increase in sensitivity to DNA-damaging agents was observed after exposure of the cells to hydrogen peroxide (Fig. 6B). Again, while hydrogen peroxide treatment decreased cellular survival of both Brca1^/ p53^/ and p53^/ fibroblasts, the impairment of survival of the double-homozygous cells was significantly greater. The difference between the Brca1^/ p53^/ and p53^/ cells was most pronounced after exposure to 500 µM hydrogen peroxide. Although differences in the survival of the Brca1^/ p53^/ and p53^/ cells were not as dramatic after exposure of these lines to ultraviolet light (Fig. 6C), survival impairment of the double-homozygous lines was greater than that observed in the p53^/ cultures. These differences achieved statistical significance in two of the UV radiation doses examined.

View larger version (17K): [in this window] [in a new window]   FIG. 6.   Brca1/ p53/ fibroblasts are sensitive to gamma radiation, hydrogen peroxide, and UV radiation. Fibroblasts from two Brca1/ p53/ cell lines and two Brca1+/ p53/ cell lines were exposed to 0, 2.5, 5.0, or 7.5 Gy of gamma radiation (A); 0, 250, 500, or 750 µM hydrogen peroxide (B); or 0, 5, 10, or 30 J of UV radiation (C). Survival was determined in each cell line by normalizing surviving cells following DNA damage to the number of cells which were untreated. An asterisk indicates statistical significance (P < 0.05).

Transcription-coupled repair in p53^/ and Brca1^/ p53^/ cells. We have previously demonstrated an increased sensitivity to ionizing radiation and hydrogen peroxide in an ES cell line that is BRCA1 deficient and that this increased sensitivity paralleled a defect in TCR (19). However, as this BRCA1-deficient ES cell line was a single isolate and as no additional lines could be identified in numerous additional experiments, it is possible that this phenotype was not directly related to the loss of BRCA1 but rather to the accumulation of other mutations in the cell line. To determine if TCR is dependent on normal BRCA1 function, a Brca1^/ p53^/ fibroblast line and a Brca1^+/ p53^/ control line were exposed to ionizing radiation, UV light, or hydrogen peroxide, and the rate of repair on the DHFR gene was measured. After exposure to 10 Gy of ionizing radiation, a rapid repair of the transcribed strand of the DHFR gene was found in the Brca1^+/ p53^/ cells (Fig. 7A). In contrast, the rate of repair on the transcribed strand of the DHFR gene was similar to that of the nontranscribed strand and the genome overall in the Brca1^/ p53^/ cells, similar to what was observed in the BRCA1-deficient ES cells. Similarly, when cells were exposed to 10 mM H₂O₂ and the TCR of thymine glycols was examined, a deficiency in the rapid removal of this oxidized base from the transcribed strand of the DHFR gene was also observed in Brca1^/ p53^/ cells (Fig. 7B). However, when cells were exposed to 10 J of UV per m², no deficiency in TCR was observed in the Brca1^/ p53^/ cells compared to the Brca1^+/ p53^/ control line (Fig. 7C). These results indicate that TCR of oxidative DNA damage is absent in cells defective in BRCA1.

View larger version (21K): [in this window] [in a new window]   FIG. 7.   TCR is defective in the DHFR gene of Brca1/ p53/ fibroblasts after exposure to ionizing radiation and hydrogen peroxide but not after exposure to UV light. Cells were exposed to 10 Gy of gamma rays (A) or 10 J of UV radiation per m2 (C) and allowed to repair in the presence of 10 µM BrdU. Genomic DNA, digested with BamHI, was reacted with an antibody to BrdU. DNAs from the bound and free fractions were electrophoresed and transferred to a GeneScreen Plus membrane. The percentage of total DNA (