Previous Article | Next Article 
Molecular and Cellular Biology, October 1999, p. 7061-7075, Vol. 19, No. 10
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Growth Retardation, DNA Repair Defects, and Lack of
Spermatogenesis in BRCA1-Deficient Mice
Victoria L.
Cressman,1
Dana C.
Backlund,2
Anna V.
Avrutskaya,3
Steven A.
Leadon,3
Virginia
Godfrey,4 and
Beverly
H.
Koller2,*
Curriculum in Genetics and Molecular
Biology,1 Department of
Medicine,2 Department of
Pathology,4 and Department of Radiation
Oncology,3 University of North Carolina at
Chapel Hill, Chapel Hill, North Carolina 27599
Received 2 February 1999/Returned for modification 31 March
1999/Accepted 22 June 1999
 |
ABSTRACT |
BRCA1 is a nuclear phosphoprotein expressed in a broad spectrum of
tissues during cell division. The inheritance of a mutant BRCA1 allele dramatically increases a woman's lifetime
risk for developing both breast and ovarian cancers. A number of mouse lines carrying mutations in the Brca1 gene have been
generated, and mice homozygous for these mutations generally die before
day 10 of embryonic development. We report here the survival of a small
number of mice homozygous for mutations in both the p53 and
Brca1 genes. The survival of these mice is likely due to
additional unknown mutations or epigenetic effects. Analysis of the
Brca1
/
p53
/
animals
indicates that BRCA1 is not required for the development of most organ
systems. However, these mice are growth retarded, males are infertile
due to meiotic failure, and the mammary gland of the female mouse is
underdeveloped. Growth deficiency due to loss of BRCA1 was more
thoroughly examined in an analysis of primary fibroblast lines obtained
from these animals. Like p53
/
fibroblasts,
Brca1
/
p53
/
cells
proliferate more rapidly than wild-type cells; however, a high level of
cellular death in these cultures results in reduced overall growth
rates in comparison to p53
/
fibroblasts.
Brca1
/
p53
/
fibroblasts are
also defective in transcription-coupled repair and display increased
sensitivity to DNA-damaging agents. We show, however, that after
continued culture, and perhaps accelerated by the loss of BRCA1 repair
functions, populations of Brca1
/
p53
/
fibroblasts with increased growth rates can
be isolated. The increased survival of BRCA1-deficient fibroblasts in
the absence of p53, and with the subsequent accumulation of additional
growth-promoting changes, may mimic the events that occur during
malignant transformation of BRCA1-deficient epithelia.
 |
INTRODUCTION |
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 |
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.
To confirm the decrease in cell viability, two additional assays were
performed. For the first assay, nonadherent cells from two
Brca1
/
p53
/
cell lines and
one Brca1+/
p53
/
cell line
were harvested as described above. The nonadherent cells were counted
and plated in 1 ml of medium in 24-well dishes. Adherent cells were
then trypsinized and plated at an equivalent density. Live adherent
cells (as determined by trypan blue exclusion) were counted from each
well 24 h later. The second assay utilized the MTT assay (Roche
Molecular Biochemicals, Indianapolis, Ind.) with the following
modifications. Nonadherent cells from two Brca1
/
p53
/
lines and one Brca1+/
p53
/
line were harvested as described above,
resuspended in <1 ml of medium, and counted. Adherent cells were
trypsinized and counted, and 5,000, 10,000, 20,000, and 40,000 cells
(nonadherent and adherent) were plated in duplicate. All cells were
immediately incubated with the MTT labeling reagent for 4 h and
then incubated with the solubilization solution for 24 h.
Conversion to formazan dye was detected at 595 nm.
To quantitate apoptosis in this dead cell population, the TUNEL assay
was performed on the nonadherent cells immobilized on a microscope
slide (as described above) as per the manufacturer's directions
(Trevigen). To determine the percentage of apoptotic cells, photographs
from random areas of each slide were taken.
For p21 protein analysis, asynchronous Brca1+/
p53
/
, Brca1
/
p53
/
, and wild-type fibroblasts were washed three
times with PBS and mechanically lysed for 15 min at 4°C in 1% CHAPS
{3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate} buffer (containing 20 mM Tris, 143 mM KCl, 5 mM EDTA, 10 mM
dithiothreitol, 20 mM NaCl) supplemented with a Complete Mini Protease
Inhibitor Cocktail Tablet (Roche Molecular Biochemicals, Indianapolis,
Ind.). Supernatants of the cell lysates were collected following
centrifugation (15,000 × g at 4°C for 15 min). Five
micrograms of protein was resolved on a sodium dodecyl sulfate-12%
polyacrylamide gel and then transferred to nitrocellulose. p21 was
detected by a monoclonal antibody (F-5, 1:1,000 dilution; Santa Cruz
Biotechnology, Inc., Santa Cruz, Calif.), followed by incubation with a
horseradish peroxidase-conjugated secondary antibody and visualized
with chemiluminescence regents (Pierce, Rockford, Ill.).
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.
Transcription-coupled repair analysis was performed as previously
described (29). For labeling of parental DNA, cells were grown for 3 days in medium containing 1 µCi of
[3H]thymidine (TdR; Amersham) per ml in the presence of 5 µg of unlabelled TdR per ml. The medium was removed, and the cells
were grown for an additional 2 days in nonradioactive medium. Prior to
irradiation, cells were incubated for 1 h in medium containing 10 µM BrdU and 1 µM fluorodeoxyuridine (FdUrd). Cultures were washed
with PBS and were irradiated with either a 60Co gamma
source at dose rates of 1.3 to 1.6 Gy/min or at 254 nm by using a
germicidal lamp at an incident dose rate of 0.63 J/m2/s.
After irradiation, the cultures were either harvested immediately or
incubated for various lengths of time in medium containing 10 µM BrdU
and 1 µM FdUrd. For measurements of thymine glycol production and
repair, cell cultures were exposed to 10 mM
H2O2 for 15 min at 37°C. The cells were then
incubated for various lengths of time in this medium. After incubation,
the cultures were washed twice with PBS and harvested by lysis in 10 mM
Tris-10 mM EDTA-0.5% sodium dodecyl sulfate.
Repair analysis of UV and ionizing-radiation-induced DNA damage was
carried out as previously described (28) by using a monoclonal antibody against BrUra. Briefly, purified DNA, digested with
BamHI, was centrifuged to equilibrium in a CsCl gradient to
separate parental density DNA (containing BrUra-substituted repair
patches) from hybrid density DNA (synthesized by semiconservative replication). Unreplicated, parental-density DNA was then reacted with
the monoclonal antibody against BrUra. The DNA bound by the antibody
was separated by centrifugation. Aliquots of the supernatant and pellet
were assayed for radioactivity by liquid scintillation counting to
determine the relative amount of DNA bound by the antibody.
Repair analysis of thymine glycols was carried out as previously
described (30) with a monoclonal antibody that recognizes thymine glycols in DNA. Heat-denatured DNA (50 to 100 µg) was incubated with the antibody (1:1,000 dilution) in PBS containing 0.1%
bovine serum albumin for 1 h at 37°C, followed by an overnight incubation at 4°C. An equal volume of ice-cold saturated
ammonium sulfate in PBS was added, and the mixture was incubated
at 4°C for 15 min. The DNA bound by the antibody was collected as a
pellet by centrifugation. Aliquots of the supernatant and pellet were assayed for radioactivity by liquid scintillation counting to determine
the relative amount of DNA bound by the antibody.
Equal amounts of DNA, based on the 3H prelabel, from the
supernatant and pellet were electrophoresed on 0.7% neutral agarose gels. After electrophoresis, the DNA was transferred to a GeneScreen Plus (NEN) membrane. RNA probes were prepared as previously described (29). After hybridization, the membranes were washed and
exposed to Kodak XAR-5 X-ray film. The intensity of hybridization to
the fragments of interest was measured by using a Bio-Rad
phosphorimager. The value for the density of each fragment was
multiplied by the amount of DNA in the bound or free fractions to
obtain the total amount of each gene in both fractions. The percentage
of the dihydrofolate reductase gene (DHFR) gene bound by the
antibody was then calculated from the total amount of the gene in the
bound fraction divided by the total amount of the gene in the
bound plus free fractions. For the studies on the time course of repair
of thymine glycols, the percentage of the genes containing thymine
glycols immediately after treatment was set at 100%.
 |
RESULTS |
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.
All three double-homozygous animals were severely growth retarded. The
female mouse reached a maximum weight of 14 g at 12 weeks of age
compared to the average weight of 21 g seen in the p53-deficient
females in our colony. Similarly, the two Brca1
/
p53
/
males weighed only 16.3 and 14.5 g
compared to an average weight of 27 g seen in
p53
/
males at this age.
Similar to the majority of the p53
/
mice,
the Brca1
/
p53
/
mice
developed tumors and were killed when moribund. All three mice died of
lymphomas, the most common tumor seen in the
p53
/
populations (14, 26).
Necropsy of one of the male mice also revealed the presence of a
hemangiosarcoma. Lymphomas and hemangiosarcomas are seen in 59 and
18%, respectively, of the p53
/
population
(23). While the tumor types observed in the
Brca1
/
p53
/
mice are
consistent with the tumors seen with p53 deficiency, the survival age
of the double-homozygous animals was substantially less than the
average 19 weeks of survival for p53
/
animals in our colony. All three Brca1
/
p53
/
mice died at between 10 and 12 weeks of age.
Alopecia (generalized loss of fur), as well as an unusually high
percentage of white hairs dispersed through their coats, was noted in
two of the three double-homozygous animals. On gross observation the
skin of all the double homozygous animals appeared thinner and more
transparent than that of the p53-deficient mice. Histological analysis
revealed atrophy of follicles and adnexa with a reduction in the size
of both the sebaceous glands and follicles. Follicles were also reduced
in number in comparison to p53
/
mice.
Loss of BRCA1 did not result in morphological or histological changes
in the kidney, liver, brain, lungs, pancreas, or gastrointestinal tract, other than those secondary to metastatic hematopoietic neoplasms. Histopathologic lesions were primarily confined to the
epithelia of the parotid, mammary, and prostate glands. Parotid salivary glands exhibited diffuse acinar atrophy that is usually observed only in geriatric animals. Mild and multifocal cytomegaly and
karyomegaly suggestive of polypoidy were seen in the acinar epithelium
(Fig. 2E and F). Parotid ductal cells were normal, as was the histology
of the submandibular and sublingual glands. While the coagulated and
vesicular glands of the male Brca1
/
p53
/
did not differ from control mice, a rare
benign granular cell tumor, usually found in older animals, was
observed in the prostate glands. Cytomegaly and karyomegaly were also
observed in the prostate glands.
BRCA1 is expressed in the mouse mammary gland during puberty,
pregnancy, and regression after discontinuation of lactation (34). The mammary gland in the female
Brca1
/
p53
/
mouse was
examined by whole mount. While the primary ducts were easily
identified, a substantial reduction in the branching is seen in
comparison to whole mounts prepared from an age-matched p53
/
virgin mouse (compare Fig. 1A and
B). In addition, the end buds of the
BRCA1-deficient female appeared underdeveloped (compare Fig. 1C and D),
and the fat pad in the region of the end buds contains finely dispersed
cellular material. To further examine the structure of the mammary
gland, the fat pad was embedded for histological analysis (Fig.
2A through D). Primary ducts in mammary glands from Brca1
/
p53
/
mice
were fewer and more dilated than those in the mammary gland of the
p53
/
mice. In the
Brca1
/
p53
/
mice (Fig. 2B
through D), the ducts were surrounded by loose, concentric aggregates
of individualized oval cells. These cells had variably sized round
nuclei and scant-to-modest eosinophilic cytoplasm. Some of the
periductal cells had pyknotic nuclei suggestive of cell death. These
cells may be myoepithelial cells or ductal cells that failed to migrate
and differentiate normally. We cannot, however, exclude the presence of
inflammatory cells in these cellular aggregates on the basis of
morphology.

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.
As expected, the seminiferous tubules of 10-week-old
p53
/
male mice contain cells in all stages
of spermatogenesis, including spermatogonia, spermatocytes and round
and fully elongated spermatids (Fig.
3A and C). The
spermatogonia, the stem cells of the testes, are located at the
periphery of a seminiferous tubule. The developing spermatocytes,
located just interior to the spermatogonia, undergo meiosis I and II to
form the haploid round spermatids, which are easily identified by their
light-staining nuclei. Fully elongated spermatids with tails extending
into the lumen form from these round spermatids after spermiogenesis.
The seminiferous tubules of the Brca1
/
p53
/
males appeared smaller in diameter in
comparison to those of an age-matched p53
/
male and contained fewer cells (Fig. 3B). While the spermatogonia appeared relatively normal in both Brca1
/
p53
/
males, spermatids and spermatozoa were not
observed (Fig. 3D). In addition, only two pachytene spermatocytes,
identified by their distinct chromatin structure, were observed on
survey of the entire section. To further define the defect in
spermatogenesis of the double-homozygous animals, sections were stained
with an antibody to HSP70-2 (13). This antibody is specific
for a heat shock protein whose expression is initiated as cells enter
into meiosis. As expected, the peripheral layer of cells, the
spermatogonia, of both the p53
/
(Fig. 3G)
and Brca1
/
p53
/
(Fig. 3H)
mice failed to stain with this antibody. Cells luminal to this single
layer of spermatogonia present in some of the seminiferous tubules of
the testes of the Brca1
/
p53
/
males stained brightly with this antibody,
indicating that these cells had likely entered into meiosis. Together
with the absence of pachytene spermatocytes, this suggests that meiotic
failure occurs during prophase I of meiosis in the
Brca1
/
p53
/
- mice.

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.
|
|
To determine whether meiotic failure observed in the
Brca1
/
p53
/
mice was
paralleled by increased levels of apoptosis of spermatocytes, tissue
sections obtained from the testes of both control
p53
/
mice and Brca1
/
p53
/
animals were analyzed by using the TUNEL
assay. Only a modest increase in the number of stained cells was
observed (Fig. 3E and F). In 25 randomly examined seminiferous tubules,
13 apoptotic cells were seen in the testes of the
p53
/
control mice compared to 37 apoptotic
cells per 25 tubules in the testes of the Brca1
/
p53
/
mouse. Examination of other tissues from the
double-homozygous animals also failed to reveal an increased level of apoptosis.
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).
Proliferation rates and saturation densities of fibroblast cultures
prepared from wild-type mice and from two
p53
/
and two Brca1
/
p53
/
mice were determined at both low and high
passage points (Fig. 4A and B,
respectively). Early-passage p53
/
fibroblasts grew more quickly and reached higher saturation densities than cells obtained from wild-type control animals. Both the growth rate and the saturation density of the early passage
Brca1
/
p53
/
fibroblasts
resembled more closely those of the wild-type cells than those observed
for the p53
/
cells.

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.
|
|
As an increase in the expression of p21 in BRCA1-deficient
embryos (embryonic days 6 to 8) was previously reported
(21), it was of interest to determine whether a similar
increase might underlie the decreased growth rate of the
Brca1
/
p53
/
cells. Because
these cells are deficient in p53, such an increase if observed would be
dependent on stimulation of p21 expression by p53-independent pathways
(1, 39, 55). Protein levels of p21 were determined by
Western blot analysis in asynchronous, proliferating
Brca1
/
p53
/
,
p53
/
, and wild-type fibroblasts. While high
levels of p21 were observed in the wild-type fibroblasts, a finding
consistent with the rapid senescence of these cells, only very low
levels of p21 were detected in the Brca1
/
p53
/
and p53
/
fibroblasts. These results fail to support a role of p21 in the decreased growth rate of the Brca1
/
p53
/
cells (Fig.
5A).

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.
|
|
We next wished to determine whether the growth differences between the
cell lines would be maintained after extended in vitro culture (passage
15 to 25). Senescence of the wild-type cells by passage 8 precluded
their inclusion in this experiment. While a small increase in the
growth rate and saturation density of one of the
p53
/
lines was observed on comparison of
early- and late-passage cell cultures, a dramatic increase in both
parameters was seen in both late passage Brca1
/
p53
/
lines (Fig. 4B).
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.
To determine whether the increased proliferation rate seen in the
late-passage Brca1
/
p53
/
cells was a result of an increased rate through the cell cycle, BrdU
and DNA content analyses were performed on asynchronous cells from a
p53
/
control line and two
Brca1
/
p53
/
lines. Again,
the percentage of cells in S phase was similar in all three lines
(p53
/
control, 58.2%;
Brca1
/
p53
/
line 1, 64.2%;
Brca1
/
p53
/
line 2, 57.6%).
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).
To confirm that this nonadherent population of cells was in fact dead
and did not simply reflect a change in anchorage dependence of the
Brca1
/
p53
/
fibroblasts
during various stages of the cell cycle, we examined the plating
efficiency of these cells. Brca1
/
p53
/
nonadherent cells were collected and plated
in 24-well plates at a density similar to that used in passage of
adherent cells. After 24 h, the numbers of adherent and
nonadherent cells were determined. A decrease in the total number of
cells present had occurred, and only 3% of the nonadherent cells
plated had attached. Analysis of these cells and the nonadherent
population indicated that only the attached cells excluded the dye. The
viability of the Brca1
/
p53
/
cells was further examined by using a colorimetric MTT-based assay
capable of reliably detecting small numbers of viable cells (37). Between 5 × 103 and 4 × 104 adherent or nonadherent cells were collected, and the
conversion of MTT to formazan dye was examined quantitatively. In two
Brca1
/
p53
/
fibroblast lines
and one p53
/
line, fewer than 15% of the
nonadherent cells examined were viable.
To determine whether cellular death was occurring through an apoptotic
pathway, the TUNEL assay was performed on the nonadherent cells of two
Brca1
/
p53
/
lines and two
Brca1+/
p53
/
lines. Although
nonadherent cells are not viable as determined by trypan blue
exclusion, fewer than 25% stained positively for apoptosis in the four
lines examined (data not shown).
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 ( |
|