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* Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA;
Department of Radiation Therapy, University of Texas Medical Branch, Galveston, Texas 77550, USA;
Department of Veterinary Medicine and Surgery, University of Texas, M.D. Anderson Cancer Center, Houston, Texas 77030, USA
1Correspondence: Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, MS 112A, Houston, TX 77030, USA. E-mail: dmedina{at}bcm.tmc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: estrogen • aneuploidy • centrosomes • steroid hormone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
, at the gene level with
both gain and loss of gene expression (1
, 3
, 4)
, and at
the chromosomal level as the gain or loss of chromosomes (i.e.,
aneuploidy) by cytogenetic or comparative genomic hybridization (CGH)
methods (5
6
7)
. DNA content changes, i.e., loss of
heterozygosity (LOH) and aneuploidy, can be detected at early stages of
morphological atypia, supporting the hypothesis that aneuploidy is a
critical event driving neoplastic development and progression (8
, 9)
. In contrast to human breast cancers, most mammary cancers
induced in traditional models of mice and rats appear to be diploid
and/or show very low frequency of LOH (10)
.
Aneuploidy is the gain or loss of chromosomes; it is a dynamic,
progressive, and accumulative event that is almost universal in solid
tumors and occurs early in neoplastic development
(11
12
13
14)
. The chromosome imbalance occurring in aneuploid
cell populations results in altered gene dosage, gene balance, and loss
of heterozygosity, common events associated with tumorigenesis.
Consequences of these events include alterations in cellular metabolic
control (15)
, transcriptional regulation, and signal
transduction (reviewed in ref 14
). The extensive array of
altered gene expression observed in tumors and the numerous altered
chromosomes detected by CGH (5
6
7
8)
provide striking
evidence that aneuploidy can totally disrupt cell homeostatic control.
The question of whether aneuploidy is a consequence of neoplastic
development or a cause of neoplastic development is currently being
debated (14
15
16
17)
. It is likely that this question will be
unresolved until the mechanisms generating aneuploidy are understood at
the molecular level. One mechanism that has been proposed for
generating aneuploidy is failure to appropriately segregate chromosomes
(14
, 16
, 17)
. The processes of chromosome segregation and
movement are well defined at a cellular level, but molecular events
that interfere with appropriate chromosome segregation and resulting in
aneuploidy are not well understood and are likely to involve multiple
targets. For instance, one can conceive of interference with mitotic
spindle dynamics, abnormal centrosome duplication, altered chromosome
condensation and cohesion, defective centromeres, and loss of mitotic
checkpoints as being events generating aneuploidy (reviewed in ref
14
).
One well-known and important regulator of the cell cycle is the tumor
suppressor gene p53 (18
, 19)
. The loss or mutation of p53
results in tumorigenesis, aneuploidy, and centrosome amplification
(20
21
22
23)
. Centrosome anomalies have been reported for a
variety of solid tumors and in tumor-derived cell lines
(22
23
24
25
26
27
28)
. Defects affect spindle polarity, microtubule
nucleation, abnormal centrosome duplication, and centrosome separation
during mitosis. Functional consequences of centrosome defects may play
a role during neoplastic transformation and tumor progression,
increasing the incidence of multipolar mitoses that lead to chromosomal
segregation abnormalities and aneuploidy.
Recently, a p53 null mammary epithelial cell model has been developed
(29
, 30)
. The absence of p53 expression does not alter
normal mammary gland development but does result in a markedly
increased incidence of tumorigenesis. The mammary tumors are frequently
aneuploid and the tumorigenic process is markedly increased by chronic
hormone stimulation (30)
.
Both estrogen and progesterone are critical hormones in mammary
development in rodents and humans (31)
. While estrogen is
essential for ductal outgrowth (31
, 32)
, progesterone is
essential for tertiary branching and alveolar morphogenesis (33
, 34)
. The importance of progesterone as a mitogen, as well as a
morphogen, for mammary development has been reemphasized recently with
the demonstration that progesterone stimulates DNA synthesis and
mammary morphogenesis in ovariectomized mice (35)
.
Antiprogestins also inhibit mammary epithelial cell growth
(36)
. In chemical carcinogen-induced mouse mammary
tumorigenesis, progesterone is the critical hormone required for
tumorigenesis (37
, 38)
. In human breast cancer, there is
recent compelling evidence that progesterone, in addition to estrogen,
promotes breast tumorigenesis. In some human breast cell lines, there
is a correlation between progesterone receptors and cell growth
(39
, 40)
. Women on hormone replacement therapy that
includes progestins exhibit a 7- to 13-fold increased
mammographic-defined parenchymal density compared to conjugated
estrogens alone (41)
and a 4-fold increase in breast
cancer risk compared to estrogen alone (42
, 43)
. The
biological and molecular mechanisms responsible for the progesterone
effects are unknown and likely to be complex.
The experiments described here examine the occurrence of aneuploidy in normal and tumorigenic mammary epithelial cells that are null for the tumor suppressor gene p53 and the role of ovarian steroid hormones in facilitating the frequency of aneuploidy. The results suggest that progesterone, not estrogen, stimulates aneuploidy in p53 null mammary cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Donor mammary ducts were dissected from 7 to 10 wk old Balb/c p53 null
female mice or p53 wild-type female mice and transplanted as 
1
mm3 fragments into the cleared contralateral
mammary fat pads of 3 wk old BALB/c p53 wild-type mice
(44)
. Two weeks after transplantation, some of the mice
were exposed to chronic hormone treatment either via a pituitary
isograft (45)
or silastic tubing (46)
containing estradiol 17B (50 µg), progesterone (20 mg), or both
hormones or remained untreated. After 4–5 wk of hormone stimulation,
the mammary glands were collected and evaluated for development in
whole mount preparations (44)
or prepared for primary cell
culture (47)
. Some of the mice were left undisturbed to be
evaluated for proliferative/apoptotic indices at 10 wk or to develop
tumors for subsequent chromosomal analysis. Each individual treatment
group contained at least six fat pads. Experiments were performed
either twice (estrogen, progesterone studies) or five times (pituitary
isograft studies) and always included untreated p53 null mammary
transplants.
The protocol for hormone exposure was based on the results of Guzman et
al. (46)
, who demonstrated that silastic tubing implants
containing estrogen and progesterone deliver sustainable blood levels
of these hormones equivalent to that found in midpregnancy. Previous
studies have shown that a pituitary isograft under the kidney capsule
generates elevated blood levels of prolactin and progesterone, but only
slightly elevated blood levels of estrogen (45)
.
The mammary tumors examined in this study were derived from mice
bearing p53 null mammary cells that had been left untreated or were
treated with a pituitary isograft or pituitary isograft and
7,12-dimethylbenzanthracene (DMBA) (30)
.
Immunohistochemistry
For BrdU analysis of cell proliferation, mice were injected
intraperitoneal (i.p.) with BrdU (70 mg/kg BW) and killed 2 h
later. The no. 4 inguinal gland was fixed in Methacarn, processed, and
evaluated for BrdU labeling index as described in ref 48
.
There were 3–4 mice per group, and 500 cells from each gland were
counted.
For the apoptosis index, standard hemotoxylin and eosin-stained 4 µm
sections, adjacent to sections used for BrdU staining, were evaluated
for apoptotic cells by morphological criteria as described and
illustrated (49)
. The apoptotic index is based on scoring
500 nuclei per gland.
Estrogen and progesterone receptors were evaluated as described in ref
50
. The rabbit anti-estrogen receptor IgG, MC-20, and
rabbit anti-progesterone receptor IgG, C-19 (Santa Cruz
Biotechnology, Inc., Santa Cruz, Calif.) were used at 1:200 dilution.
Chromosome analysis
Mice were anesthetized (Nembutal, injected i.p. at 80 mg/kg) and
the no. 4 mammary glands were removed as described previously
(51)
. Epithelial cells suspensions were prepared using the
protocol of Ethier et al. (52)
with some modification.
Briefly, glands were minced with scalpels and suspended in medium 199
containing type III collagenase (200 U/ml) and Dispase (1 mg/ml). The
suspension was incubated at 37°C for 3 h with gentle agitation.
The cells were then washed extensively with medium 199 and plated in
2% fetal bovine serum for 90 min to allow unwanted fibroblasts to
attach. The supernatant containing epithelial cells was then collected
and cells were counted as described previously (53)
. After
isolation, cells were seeded onto collagen-coated 60 mm dishes at
densities of 1 x 105 cells/dish. Cells were
grown under 10% CO2 in JRH Ham’s F-12 medium
supplemented with insulin, hydrocortisone, transferrin, epidermal
growth factor, Fungizone, gentamicin, and 5% fetal bovine serum. The
population doubling time for these cells was estimated to be 30 h.
Cell populations were maintained in culture for 5 days, which was
approximately four population doublings. Blind-coded chromosome
preparations of the various populations were made as described
previously (53)
. Briefly, on the fifth day of culture,
cell cultures were incubated in 0.1 µg/ml Colcemid for 3 h.
Cells were collected and metaphase spreads were made, stained with
Giemsa after treatment with 0.1 M HCl at 55°C for 1 h, and
chromosome numbers were recorded. A minimum of 50 metaphases were
scored from each preparation.
Immunocytochemistry
For immunocytochemical staining, freshly isolated epithelial
cells were grown on coverslips for 48 h. Cells were fixed in 4%
paraformaldehyde in PEM buffer (80 mM Pipes, pH 7.0, 1 mM EGTA, 1 mM
MgCl2) for 15 min at room temperature. After
fixation, cells were permeabilized in 0.5% Triton/PEM buffer for 5
min, washed for 15 min in PEM buffer, and incubated in blocking
solution (PEM buffer with 5% goat serum) for 1 h at 37°C.
Incubation of primary antibodies diluted in blocking buffer was carried
out for 1 h at 37°C. We used a human autoantibody (a gift from
J. B. Rattner) for centrosome staining (1:1000) and an 
-tubulin
specific antibody for microtubule staining (1:20). Coverslips were
washed three times in PEM buffer. The cells were then incubated with an
Alexa 488-conjugated goat-anti-mouse (1:800) (Molecular Probes, Eugene,
Oreg.) and a Texas red-conjugated goat anti-human antiserum (1:500)
(Pierce, Rockford, Ill.). Nuclei were stained for 5 min in DAPI (10
µg/ml) (Sigma, St. Louis, Mo.). Immunofluorescence microscopy was
performed using a Deconvolution microscope (Applied Precision,
Issaquah, Wash.) to collect images of single mitotic cells.
Confocal imaging and scoring of centrosomes
Mammary tumor tissue was embedded in cryo-gel and sectioned in
50 µm using a minitome cryostat (23)
. The thick frozen
sections were mounted onto positively charged slides, fixed [4%
formaldehyde in phosphate-buffered saline (PBS)] for 20 min, and
permeabilized (0.05% Triton X-100 in PBS) for 5 min. Incubation with
the primary antibody was performed overnight at 4°C in 1% bovine
serum albumin in PBS. Subsequently, the preparations were washed for
1 h in PBS at RT and incubated with the secondary antibody at
37°C for 3 h. Tissue was stained with PI for 5 min, washed, and
slides were mounted using Vecta shield as mounting media. Centrosomes
were detected with a human anticentrosome autoantibody (1:1000) and
nuclei were stained with propidium iodide (2 µg/ml).
Immunofluorescence microscopy was performed with a confocal microscope
(Molecular Dynamics, Sunnyvale, Calif.; SARASTRO 2010). Scoring was
accomplished by counting the number of stained nuclei and the
corresponding number of centrosomes in sections of the tumor tissue.
The number of centrosomes and nuclei were counted in adjacent 5 µm
optical sections in order to avoid double counting of individual cells.
Counts were made on cells in the terminal ducts, alveolar buds, and the
stroma. A minimum of 300 nuclei/sample were counted and plotted as
number of centrosomes vs. percentage of cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Of six primary tumors arising 21–31 wk after transplantation of p53
null normal mammary cells into the cleared fat pads of wild-type Balb/c
mice, all were markedly aneuploid (mean percent aneuploid cells=72,
mean chromosome number=56), whereas normal mammary cells from p53
wild-type mice were diploid. An example of the chromosome distribution
of one tumor arising in a pituitary isografted mouse is shown in
Fig. 1A
.
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To determine the ploidy status of p53 null normal mammary cells,
mammary epithelial cells were collected at 6–7 wk after
transplantation. The original donor mammary epithelium was from 7–10
wk old p53 null BALB/c female mice. As shown in Table 1
and Fig. 1B
, the p53 null mammary epithelial cells taken from young
mice were not aneuploid with respect to either percent of aneuploid
cells or mean chromosome number. These two measurements were the same
as measured in p53 wild-type mammary epithelial cells (Table 1
, Fig. 1C
). In contrast, the same mammary epithelial cells
transplanted into wild-type mice and subsequently hormonally stimulated
for 4 or 5 wk with a pituitary isograft developed a striking increase
in aneuploidy as measured by either mean percent aneuploid cells =
51 or mean chromosome number = 53 (Table 1
, Fig. 1D
).
The comparison of the p53 null untreated cells vs. the p53 null,
hormone-stimulated cells was performed in five independent experiments,
all of which yielded similar results.
Aneuploidy in p53 null normal mammary cells in untreated mice was found to increase with age. In two cases we serially transplanted p53 mammary cells for two consecutive transplant generations, so the chronological age of the mammary epithelial cells was 24–26 wk when we collected and examined them for chromosome distribution. In these two cases, aneuploid cells comprised 20 and 30% of all cells with modal chromosome numbers of 49 and 40, respectively.
To ascertain any major morphological or biological alterations in p53
null mammary cells experiencing chronic hormone stimulation, we
examined selected sets of developmental characteristics by whole mount
preparations and immunohistochemical staining. Figure 2
demonstrates that the subgross morphology of the mammary gland in the
virgin gland and the hormone-stimulated gland was identical for the p53
null and p53 wild-type glands under both developmental states.
Table 2
demonstrates that differences in frequency of estrogen receptor and
progesterone receptor-positive cells in the BrdU-labeling index and in
apoptotic index were not detected between the p53 null and p53
wild-type cells in hormonally stimulated mice after 6–10 wk of hormone
stimulation.
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Steroid hormone-induced aneuploidy
Pituitary isografted mice display markedly elevated blood levels
of prolactin and progesterone and slightly elevated levels of estrogen
(45)
. To determine whether the steroid hormones were
responsible for the increase in aneuploidy detected in pituitary
isografted mice, we examined the effect of steroid hormones
administered singly or combined on aneuploidy. Figure 3
illustrates the results of two experiments. p53 null mammary cells, but
not wild-type cells, exposed to estrogen and progesterone for 4–1/2 wk
exhibited the same marked increase in aneuploidy as observed in p53
null cells exposed to hormones induced by a pituitary isograft. For
hormones administered singly, progesterone but not estrogen markedly
increased aneuploidy and to the same extent as the cells exposed to
both hormones. Mammary cells exposed to progesterone and estrogen or
just progesterone contained 38–40% aneuploid cells, respectively. The
progesterone exposed mammary cells demonstrated a large increase in
tertiary ductal branching not observed in the estrogen-exposed cells
(Fig. 4
).
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Aneuploidy need not involve centrosome amplification
Primary tumors and cultured normal p53 null cells were examined
for centrosomal amplification. Unlike results observed with fibroblasts
from p53 null mice (22
, 54)
, the majority of the samples
from the p53 null mammary epithelial cells from primary tumors
(Fig. 5
, Fig. 6
), untreated, or estrogen-progesterone-treated normal mammary gland did
not exhibit an increase in centrosome amplification. Using confocal
microscopy, we collected optical sections of p53 null tumors arising in
untreated mice, p53 null tumors arising in hormonal stimulated mice
(pituitary isografting) or after DMBA treatment, or the combination of
both (hormone + DMBA). Figure 5
shows the centrosome numbers in five
primary tumors arising in p53 null mammary epithelium. Two tumors
arising in pituitary isografted mice contained normal centrosome
numbers (Fig. 6C
). One of
these tumors had a DNA index of 1.6, with 32% of the cells being
aneuploid. One tumor arising in mice treated with both a pituitary
isograft and DMBA contained 12% of the cells, with more than two
centrosomes (Fig. 6D
). This tumor had a diploid DNA content
by flow cytometry. One of two tumors (Fig. 5)
arising in untreated p53
null mice demonstrated 15% of the cells, with more than two
centrosomes in cells surrounding a definable lumen [i.e., cells were
organized as ducts (Fig. 6B
)], but only 2% of the cells
exhibited abnormal centrosome number in cells that were organized as
sheets (Fig. 6A
). This tumor was also diploid by flow
cytometry analysis.
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After 2 days in culture, normal mammary epithelial cells show a typical
pattern of small islands. Cells were fixed and stained for centrosomes,
microtubules, and DNA and images were collected using a Deconvolution
microscope. Centrosomes were scored in two different sets of cultures.
In the control group (p53 null transplant without hormonal
stimuli), the distribution of centrosomes represents a population
of normal growing cells: 67% of cells have one centrosome, 31% have
two, and 2% have three centrosomes/cell (Fig. 7A
). The primary cell culture from the
estrogen/progesterone-treated gland was similar to that of the
untreated gland with respect to cell morphology and centrosome number,
indicating that aneuploidy in the hormone-treated p53 null mammary
cells was not mediated by centrosome amplification (Fig. 7B
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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32
33
34
35)
. Progesterone is primarily
important for tertiary side branching whereas estrogen is important for
duct elongation. If progesterone-driven epithelial cell proliferation
per se is the stimulus for the occurrence and accumulation
of aneuploidy, then it is difficult to understand the different levels
of aneuploidy found in cells experiencing estrogen and progesterone
alone. Both are mitogenic hormones for the gland and stimulate
proliferation over and above the basal growth rate occurring in
transplanted epithelial cells. Three possible explanations are
immediately apparent. First, the frequency of proliferation in
progesterone-exposed cells must be significantly greater than in
estrogen-exposed cells, resulting in an accumulation of aneuploid cells
generated by chromosome missegregation occurring in mitosis. At this
time we do not have results on the frequency of proliferation in
progesterone and/or estrogen exposed cells at 1, 2, and 3 wk. Second,
the mammary subpopulation responsive to progesterone might be different
and more susceptible to chromosome missegregation than the cells
responsive to estrogen. Some authors have suggested the presence of
cells that localize to lateral branch points and are uniquely
stimulated to proliferate by progesterone (34)
. Third, an
alternative hypothesis would state that progesterone alters the
expression and/or function of critical proteins that regulate
chromosome segregation. The possible candidates are numerous
(14)
, but the effect of progesterone on any of the
possible candidates is unknown. Although it is known that estrogen can
affect microtubules (11)
, to our knowledge the effect of
progesterone has not been reported. Whatever the mechanism, the data
suggest a unique interaction between progesterone stimulation of
mammary cells and the absence of p53. The absence of p53 per
se does not alter the overall cell proliferation kinetics,
apoptosis, or steroid receptor positivity of the mammary cells
experiencing prolonged hormonal stimulation. This is in accordance with
the results of Jerry et al. (29)
that normal mammary gland
development in the postpubescent or the pregnant mouse is not altered
by the absence of p53.
The result that progesterone alone can facilitate aneuploidy may have
greater importance with the recent publications that demonstrate women
on hormone replacement treatments that include progesterone have
increased mammographic breast density and increased breast cancer risk
than women taking only estrogen (41
42
43)
. The studies of
aneuploidy in mouse cells and the human epidemiological studies suggest
that progesterone has unanticipated deleterious effects on mammary
epithelial cells. Although we have reported that prolonged hormonal
exposure (i.e., progesterone and prolactin) provided by a pituitary
isograft strongly enhances tumorigenesis in the p53 null mammary cells,
similar experiments with progesterone alone remain to be performed. In
summary, the accumulated recent results warrant a better understanding
of progesterone on mammary cell biology and function.
Second, the presence of a normal chromosome distribution in less than
16 wk old p53 null mammary epithelial cells indicates that aneuploidy
or chromosomal instability is not rampant in all somatic cells that
have lost p53. This result is in contrast to the studies of Fukasawa et
al. (22
, 54)
, who demonstrated that fibroblasts and
hematopoietic cells obtained from p53 null mice demonstrated a high
frequency of chromosome instability. The experiments of Fukasawa et al.
(22
, 54)
differ from the experiments reported here in at
least two fundamental ways. Most important, the cell types are
dissimilar. The biology of epithelial cells differs in multiple ways
from mesenchymal cells and it is likely that extrinsic and intrinsic
regulatory pathways differ for mitogenesis. It is well established that
in vitro assays for transformation of fibroblast cells are
not always applicable to mammary epithelial cells (55
, 56)
, suggesting that the two cell types differ in factors and/or
pathways that regulate growth control and cell–cell and
cell–substrate interactions. Furthermore, the major cell type
neoplastically transformed in p53 null mice is of mesenchymal origin
(20)
, not ectodermal or endodermal, again suggesting that
cells of mesenchymal origin are at increased risk.
It is also worthwhile to note that the cell culture conditions used in
experiments by Fukasawa et al. (22)
were different from
the short-term primary cultures of the mammary cells. Passage 2 cells
were used in their original studies (22)
. This is
important because mouse fibroblasts in culture are genetically unstable
and aneuploidy can arise within a few passages (57)
. In
the more recent experiment (54)
, passage 0 cells were
used; however, the protocol required a period of growth followed by
60 h without growth factors, then 15 h of medium with growth
factors to restimulate growth before metaphase cells were collected.
Finally, it should be noted that Tsukada et al. (58)
reported that cloned fibroblasts from p53 null mice were highly diploid
and had a normal karyotype. They concluded that chromosomal changes
were not essential for an enhanced proliferative potential. Tsukada et
al. (58)
also examined mammary epithelial cells that
exhibited an enhanced proliferative potential in vitro and
retained differentiated morphogenic potential in 3-dimensional
cultures. The authors did not report on ploidy status of the mammary
cells.
These results do not imply that chromosomal instability is absent in
p53 null mammary cells. Several results suggest aneuploidy in mammary
epithelial cells is a function of age and/or proliferation. Aneuploid
mammary epithelial cells were observed in two cases where the donor
cells were of chronological age equal to 24–26 wk, as they had been
transplanted in the mammary fat pad twice serially. The number of cell
population doublings would have been even greater than that occurring
in 24 wk old mammary gland that had not been transplanted, as each
transplantation results in a round of extensive proliferation.
Furthermore, hormones that induce proliferation enhance aneuploidy.
Finally, mammary tumors were aneuploid. These results suggest that
aneuploidy, as judged by chromosome gain or loss, is a function of age
and/or proliferation history. It is feasible that age is a surrogate
for proliferation history and that the mitotic machinery is the source
of the genetic instability. Lengauer and co-workers suggest that the
chromosomal missegregation rate is 10-2 per
chromosome per generation in the colorectal tumors exhibiting
chromosomal instability (59)
. Cells containing chromosomal
missegregation would accumulate in p53 null cells undergoing extensive
proliferation.
A third result of interest is the role of centrosome amplification as
one mechanistic basis for aneuploidy. The precedents for this
mechanistic defect are the observations that p53 null fibroblasts
exhibit a high frequency of centrosome abnormalities coincident with
aneuploidy (22
, 54)
and the frequent occurrence of
centrosome abnormalities in tumor cells (25
26
27
28)
. The
results presented here suggest that centrosome amplification need not
be coincident with aneuploidy as neither p53 null normal diploid cells
nor p53 null hormone-stimulated cells (estrogen plus progesterone)
showed detectable amplification. These results are in accord with
recent observations that centrosome amplification is not a frequent
event in a p53 null mouse mammary epithelial cell line (Murphy and
Rosen, personal communication). These results do not rule out the
possibility that centrosome abnormalities in mammary epithelial cells
may contribute to tumorigenesis in some instances. Centrosome numbers
were amplified in some but not all mammary tumors. Amplified
centrosomes were correlated with the formation of structurally and
functionally aberrant mitotic spindles (Goepfert and Medina,
unpublished observation).
In summary, the results of these experiments suggest that specific hormone combinations will enhance aneuploidy and subsequently tumorigenesis in normal mammary epithelial cells that have lost p53 gene function. In view of the recent reports indicating the increased risk for breast cancer in women taking progesterone in hormone replacement therapy, the mechanisms of progesterone action in normal mammary cell growth and function deserve more intense investigation.
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ACKNOWLEDGMENTS |
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Received for publication March 27, 2000.
Revision received May 1, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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L. Sivaraman, O. M. Conneely, D. Medina, and B. W. O'Malley p53 is a potential mediator of pregnancy and hormone-induced resistance to mammary carcinogenesis PNAS, October 12, 2001; (2001) 221459098. [Abstract] [Full Text] [PDF]
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L. Sivaraman, O. M. Conneely, D. Medina, and B. W. O'Malley p53 is a potential mediator of pregnancy and hormone-induced resistance to mammary carcinogenesis PNAS, October 23, 2001; 98(22): 12379 - 12384. [Abstract] [Full Text] [PDF]
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