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First published online 12 January 2005
doi: 10.1242/dev.01609
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Laboratory of Mammary Gland Biology and Tumorigenesis, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA
e-mail: gs4d{at}nih.gov
Accepted 2 December 2004
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SUMMARY |
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 TOP SUMMARY IntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Key words: Mammary, Stem cell, Asymmetric division, Autoradiography
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Introduction |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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;
Cotsarelis et al., 1990;
Morris et al., 1985). In 1975,
Cairns (Cairns, 1975), and
subsequently Potten (Potten et al.,
1978), suggested that one of the reasons for this property is that
somatic stem cells selectively segregate their template DNA strands to
themselves and pass the newly synthesized chromatids to their daughters during
asymmetric divisions. In a recent paper, Potten and his co-workers
(Potten et al., 2002) have
convincingly demonstrated that stem cells in the crypt of the small intestine
do indeed retain their template DNA (3HTdR) and pass the newly
synthesized strands marked with 5-bromo-deoxyuridine (5BrdU) to their progeny.
This property is claimed to effectively protect long-lived stem cells from
mutagenesis related to errors occurring during DNA replication and
subsequently explains, in part, why stem cells in the small intestine rarely
give rise to intestinal cancers (Potten et
al., 2002).
Long label retaining cells (LREC) have been reported among the epithelium
of the murine mammary gland using both 3HTdR and 5BrdU
(Welm et al., 2002;
Zeps et al., 1998;
Zeps et al., 1996). It has
been reported that as many as 50% of mammary epithelial cells are labeled with
3HTdR after three consecutive injections and much of this label is
lost after 2 weeks, consistent with the loss of label by semi-conservative
exponential cell divisions. Some cells retained label following this 2-week
period and had autoradiographical grain counts similar to cells immediately
following 3HTdR injection. A greater number of these cells were
obtained when 3HTdR injection was made just at estrus or met-estrus
during the estrus cycle (Zeps et al.,
1998; Zeps et al.,
1996). These authors chased the label for just two weeks and used
adult females 9-16 weeks of age. In preliminary studies they determined that
no heavily labeled cells were present after 5 weeks. In a very different
approach, Welm et al. (Welm et al.,
2002) labeled mice with 5BrdU delivered from an implanted Alzet
pump for 14 days beginning at 3 weeks of age. Subsequently the pump was
removed and the number and location of labeled mammary cells was analyzed at
weekly periods for 9 more weeks. These investigators found that the number of
labeled epithelial cells decreased quite rapidly reaching <5% by 9 weeks.
These label-retaining cells remaining at 9 weeks were variously determined to
be expressing progesterone receptor (PR), 
1.5% and keratin K14 or K18,
myoepithelial and luminal epithelial cell markers respectively. In addition,
these authors found that the LREC epithelial population at 9 weeks was more
prevalent in side population (SP) cells after fluorescence-activated-cell
sorting (FACS), suggesting that they may represent mammary epithelial stem
cells.
To develop LREC among the mammary epithelium, mammary implants in the cleared mammary fat pads of syngeneic or immune-compromised recipients and the intact host mammary glands were labeled with 3HTdR for a 5-day period in the fifth week of life. Implants, host mammary glands and small intestine were removed on the third day following the last 3HTdR administration to determine the efficiency of labeling. At the 10th week of life, samples were again taken to assess for the presence of 3HTdR-positive LREC. In the 11th week, 5BrdU was administered to determine if mammary LRECs could be labeled simultaneously with 3H and 5BrdU. Subsequently, the mice were treated with various hormonal combinations in an attempt to chase 5BrdU from doubly labeled LREC. Our results indicate that a very large percentage of LREC were doubly labeled with 3HTdR and 5BrdU and that this number dropped precipitously following a 6 day chase providing evidence that a large proportion of mammary LREC are actively traversing the cell cycle and are capable of retaining their template strands during asymmetric cell divisions.
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Materials and methods |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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)
contralateral #4 and #9 fat pads (Fig.
1). This procedure was repeated in a second experiment with
transgenic mammary implants from parous
WAP-Cre/Rosa26-flox-Stop-flox-lacZ females
(Wagner et al., 2002). Sixteen
to twenty mice were used for each experiment. Wound clips were removed after
10 days. On the same day, the hosts received injections of 1.0 µg of
estradiol, intraperitoneal, daily at 4:00 PM followed by an intraperitoneal
injection of 3H-thymidine of 25 µCi at 6:00 PM. This was
continued for 5 consecutive days. Two animals were removed for tissue analysis
on the Monday following the final 3HTdR injection to determine the
number of mammary cells that were labeled. The #3 and #8 host mammary glands
were collected and the implanted #4 and #9 mammary fat pads. The small
intestine from each animal was excised and bundled to provide a positively
labeled control for autoradiography and for 5BrdU incorporation and as an
indicator of successful incorporation of the nuclear labels. Subsequently,
estradiol (1.0 µg) was given every other day for 3 weeks to promote mammary
growth. Upon cessation of estradiol treatment (the 8th week of life), the
animals were held for 2 weeks; at the end of the 10th week of life, tissues
were removed from two animals to determine the number and location of
long-label-retaining mammary cells. The remaining mice were placed in three
groups of at least four and treated as follows: group I (1.0 µg estradiol,
i.p. at 4:00 PM followed by an i.p. injection of 5BrdU, 1.0 mg in 0.1 ml
saline); group II (1.0 µg estradiol and 1.0 mg progesterone i.p. at 4:00 PM
and the same dose of 5BrdU; and group III (5BrdU, estradiol, progesterone as
per Groups I and II, plus 0.5 µg prolactin per gram body weight twice a
day). 5BrdU was given Monday and Tuesday of the 11th week of life. One animal
from each group was removed for tissue analysis Wednesday morning. All hormone
treatments were maintained for 5 consecutive days. The remaining animals were
analyzed 3 days (Monday) following the final hormone treatment.
|
Autoradiography and immunochemistry
All immunohistochemistry was performed after autoradiographical exposure.
The sections were deparaffinized and rehydrated and the endogenous peroxidase
was inactivated with 1% hydogen peroxide in methanol for 30 minutes.
Antibodies used were anti-5BrdU, 1:500 (DAKO-0744, clone BU20); anti-smooth
muscle actin 1:150 (Sigma A2547, clone 1A4); anti-progesterone receptor 1:75
(DAKO A009B, lot 126) and anti-estrogen receptor 1:50 [Santa Cruz
Biotech.-Era(MC-20) sc-542, lot 171]. Antigen retrieval was accomplished
according to the direction of the manufacturer. Negative tissue controls were
included in all immunocytochemical analyses. Sections were counterstained with
Hematoxylin or Nuclear Fast Red after immunostaining.
For autoradiography, 5-6 µm sections were cut placed upon slides, dewaxed, rehydrated through ethanol and subsequently dipped in Kodak NTB-2 liquid emulsion diluted 1:1 with distilled water. After drying, the slides were stored in lightproof slide boxes at constant humidity and temperature for 20 and 30 days. After exposure, the slides were developed in Kodak D-19, washed in distilled water and fixed in Kodak rapid fixer diluted 1:1 with distilled water. After staining and mounting, the slides were observed and evaluated for autoradiographical grains and for immunostaining under oil with a 63 x or 100 x objective. Images were recorded with a Kodak digital microscopy documentation system 290.
Determination of autoradiographical grain counts in LREC was made by counting the grains over at least 100 label-retaining epithelial cells in sections from each of the four mammary glands taken from each experimental mouse (2) in each experiment (2). These numbers were compared with the average number of grains found over labeled cells (within the ducts) in the four glands taken from each of two mice (in each experiment), 3 days after the last 3TdR injection was delivered. At least 500 labeled cells were counted in each of these sections (8). These determinations were made upon slides that had been equivalently treated for 5BrdU antigen retrieval, detection of 5BrdU by immunocytochemistry and autoradiography so that any loss of grains caused by these manipulations would be taken into account. In each experiment, the frequency of LREC was determined on the same slides comparing mammary tissues (8) from animals sacrificed following the 3TdR chase with those stained for 5BrdU after introduction of that label (12 glands) and its subsequent chase (36 glands). The frequency of LREC remained essentially unchanged (2.1±0.1%) among all of these tissues. At least 3000 nuclei were examined in each slide. Examination of autoradiographical slides from these tissues that were stained for PR, ER and SMA disclosed similar numbers of autoradiographical grains over LREC nuclei.
X-Gal and immunostaining of mammary gland whole mounts
To identify lacZ-positive progeny in
WAP-Cre/Rosa26-flox-Stop-flox-lacZ mammary outgrowths, whole mounts
of the entire implanted gland were fixed and stained as described earlier
(Wagner et al., 2002).
Briefly, the gland was spread on a glass slide, fixed in paraformaldehyde
(4.0%) for 1-2 hours, permeabilized in 0.01% NP-40 in phosphate buffered
saline (PBS) overnight at 4°C and subsequently processed for X-Gal as
described (Wagner et al.,
1997). Stained glands were repeatedly rinsed in PBS, then
post-fixed in Carnoy's fixative, cleared in 100% ethanol and the placed in
xylene before whole-mount analysis. For histological examination X-Gal-stained
whole mounts were embedded in paraffin wax, sectioned at 6 µm and
counterstained with nuclear Fast Red.
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Results |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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; Kordon and Smith,
1998). Consequently, all regions of mouse mammary glands contain
mammary epithelial stem cells (Smith and
Chepko, 2001). In addition, all parts of the regenerated gland are
likewise capable of recapitulating a new mammary tree upon transplantation to
a second round of cleared mammary fat pads. Therefore, mammary stem cells must
be self-renewed through expansive symmetric cell divisions during this
regenerative process. To access this property of mammary stem cells,
3HTdR was injected into 5-week-old females bearing autochthonous
implants of mammary tissue (Fig.
1). At this age, both the intact and implanted mammary tissues
would be in the midst of allometric growth when 3HTdR was
administered. Complete growth of the mammary ducts in intact glands is
attained between 10-12 weeks of age. During active growth, most, if not all,
of the dividing epithelial cells are present within or at the outermost
boundary of the terminal end buds of the growing ducts. Thus, during the 5-day
pulse (a daily injection of 3H-TdR, 24 hours apart) both renewing
stem cells and dividing epithelial (transit) cells destined to differentiate
along the various epithelial cellular lineages in the gland will be labeled.
Thereafter, cells that continue to divide will partition their labeled DNA
among daughters in a semi-conservative manner and become progressively free of
label. Only cells that immediately go out of cycle, possess very long cell
cycles or divide asymmetrically retaining their template strands will maintain
significant levels of the label. Zeps et al.
(Zeps et al., 1996) have
reported that mammary epithelial cell labeling efficiency was greatest during
estrus and metestrus in cycling mature virgin female mice. Therefore, we
injected the mice with 1.0 µg of estradiol every other weekday during the
chase period to mimic estrus and promote epithelial cell proliferation and
duct morphogenesis. Estradiol treatment was discontinued after the eighth week
of life (3 weeks after 3HTdR injection). On the 5th Friday after
3TdR injection, when the mice were 10 weeks old, tissues from two
mice (in each experiment) were harvested to determine the frequency and
location of LRECs (Fig. 1). On
Monday, in the 6th week post 3HTdR pulse, the remaining mice (12)
were placed into three groups and were given 1.0 mg 5BrdU for 2 days; on the
3rd day, tissue was harvested from one mouse in each group to ascertain the
level of 5BrdU incorporation. Group I received 1.0 µg estradiol, group II
received 1.0 µg estradiol plus 1.0 mg progesterone and group III received
estradiol, progesterone and 0.5 µg prolactin. These treatments were given
Monday to Friday, and were intended to promote epithelial cell proliferation
and duct side branch development. In addition, the degree of mammary
epithelial cell proliferation in the fully developed gland varies
significantly through the estrus cycle
(Zeps et al., 1999). The
hormone treatments provide a constant stimulus to epithelial proliferation and
therefore reduce variation in the proliferation index among the experimental
animals because of the estrus cycle. On the following Monday, the nine animals
(three in each group) remaining were sacrificed and the mammary implants, host
glands and small intestines were collected and prepared for autoradiography
and immunohistochemistry.
Assessment of labeling efficiency and the number of LREC
Sections were cut from the tissues taken following the initial pulse of
3HTdR and prepared for immunostaining and autoradiography. Slides
were prepared for staining with anti-smooth muscle actin (SMA), anti-estrogen
receptor (ER), anti-progesterone receptor (PR) and anti-5BrdU. Several
thousand cells were counted from each and the percent of labeled cells in the
mammary glands was calculated to be greater than 50%. Mammary cells associated
with growing terminal end buds were nearly 70% labeled (not shown). The high
frequency of labeled cells was anticipated in the mammary tissues sampled only
3 days following the last thymidine injection. The distribution of labeled
cells in the growing ducts was similar to that described by others
(Zeps et al., 1998). Mammary
epithelial cells in the terminal end buds that were positively labeled with
3H-TdR were the cap cells, body cells and cells in the subtending
duct. In addition to the epithelium, periductal cells in the stroma also
incorporated label. Subsequent to the 5-week chase period, tissue slides were
similarly prepared for staining and autoradiography, and the number of LREC
remaining was determined by counting several thousand (3000-4000) cells from
each sample (eight mammary glands in two experiments). The average number of
LREC in the mammary tissues after the chase was 2.1±0.1%. This
frequency of LREC was not significantly altered in samples taken subsequent to
5BrdU labeling and chase. The average number of grains per labeled nucleus in
3HTdR post pulse samples that had been prepared for 5BrdU antigen
retrieval and stained with anti-5BrdU was determined and found to be
4.75±1.15. These slides also served as negative controls for 5BrdU
immunostaining. The LREC in the tissue slides (12 mammary glands) evaluated
for the combined estimation of nuclear labeling with tritium and 5BrdU
following the 5BrdU pulse had an average of 4.12±0.88 grains and those
present in the mammary tissues (36 glands) following the 5BrdU chase period
had 4.11±0.95 grains per nucleus. At least 80-100 LREC nuclei
(5000 total cells) per experimental animal (n=9) were assessed
in the chased mammary tissues to obtain these grain counts. Glands taken the
day following the final 5BrdU injection contained 7.3±0.9%
5BrdU-labeled cells; this number increased during the chase period to
11.2±1.2% because of cell division and distribution of the label into
the daughter cells (Fig.
2).
|
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; Zeps et al.,
1999). The association of ER and PR staining in LREC has been
previously reported (Zeps et al.,
1998). Under our conditions of labeling, we did not encounter the
label-retaining myoepithelial cells reported by Zeps et al.
(Zeps et al., 1998) following
the chase period. However, similar to his report, the presence of stromal LRC
closely associated with epithelial structures was quite evident (not
shown).
|
;
Wagner et al., 1997). These
cells survive the massive cell death during remodeling of the gland following
the cessation of lactation and originally represent 7% of the surviving
mammary epithelium, although they increase in frequency upon successive
pregnancies. The PI-MEC were shown to be capable of self-renewal upon
transplantation and to contribute to the population of mammary epithelium
found in the resulting mammary outgrowth
(Wagner et al., 2002). These
cells also acted as secretory lobule-specific progenitors upon subsequent
pregnancies. The PI-MEC and their progeny are lineally marked by the
constitutive expression of lacZ and therefore can be detected by
X-gal staining in transplanted mammary outgrowths. To determine if the PI-MEC
might become LREC during self-renewal and contribution to mammary transplants
in mammary fat pads, mammary fragments containing lacZ-positive
PI-MEC were implanted in three-week-old Nu/Nu hosts. Labeling with
3HTdR and 5BrdU was conducted as described above. Examination of
doubly labeled mammary outgrowths from PI-MEC implants revealed the presence
of lacZ-positive, 3HTdR and 5BrdU-positive PI-MEC progeny
among the epithelium (Fig. 5), demonstrating that certain of the progeny of self-renewing PI-MEC become LREC
scattered among other lacZ-positive epithelial cells during the
process of mammary duct morphogenesis. The observation that the PI-MEC progeny
that retain 3HTdR also incorporate 5BrdU following its introduction
into the mice suggests that these cells are actively cycling and equivalent to
the LREC described above in intact glands. In addition, PI-MEC have been shown
to be pluripotent and self-renewing, both in situ and upon transplantation
(Boulanger et al., 2004),
suggesting that LREC in mammary epithelium represent cells that have the
capacity to produce progeny of several epithelial lineages and to possess
extensive self-renewal capacity.
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|
Discussion |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). When
contralateral glands were taken from the same mouse 2 weeks later, only 1/50
to 1/1000 cells retained label at the level seen immediately after the
3H pulse. These observations indicate that a considerable amount of
cellular turnover occurs in the adult gland. Further study by these authors
demonstrated that introducing 3HTdR at estrus and metestrus in
cycling virgins provided the greatest number of label retaining cells 2 weeks
later. Despite this, mammary glands labeled in this way contained no
detectable LREC when examined after 5 weeks
(Zeps et al., 1996). This
result implies that all the cells labeled by this method were exponentially
cycling and distributed the labeled DNA in a semi-conservative manner. It is
therefore unlikely that this method detects asymmetrically dividing cells
because expansive stem cell renewal is not occurring during maintenance of the
fully developed mammary ductal system. Conversely, when glands were labeled
for 14 consecutive days during the 3rd to the 5th week of life (during active
expansion of mammary stem cells in the allometrically growing ducts), LREC
were observed even 9 weeks after the cessation of labeling
(Welm et al., 2002). This
method, and the one employed here, tags stem cells (sic LREC) at their
inception and label is retained in these cells through asymmetric divisions,
even though they may often traverse the cell cycle during the weeks subsequent
to their derivation.
The experiment was designed to determine whether LREC in mouse mammary
gland selectively segregate their template DNA strands to themselves while
traversing the cell cycle. The frequency of LREC (1/50) detectable among
the mammary epithelium in these experiments agrees well with the numbers
reported by other investigators (Welm et
al., 2002; Zeps et al.,
1998; Zeps et al.,
1999; Zeps et al.,
1996). Our data show that >8/10 of mammary LREC become doubly
labeled upon the introduction of a secondary DNA synthesis marker (5BrdU).
This strongly supports the conclusion that mammary LREC are traversing the
cell cycle and are neither out of cycle nor cycling very slowly. In addition,
over a chase period of 5-6 days, a large proportion of the doubly labeled LREC
become 5BrdU-negative, while retaining the 3HTdR marker. This
demonstrates that the preponderance of doubly labeled LREC is actively
dividing and selectively segregating the old (3HTdR) DNA to
themselves and partitioning the newly labeled (5BrdU) DNA into their daughter
cells.
This unexpected result raises several questions regarding the principal
functions of LREC in mammary glands and how these may relate to putative stem
cell properties. One prospect is that the LREC represent a specific epithelial
cell subpopulation whose function is to divide asymmetrically to produce
committed transiently amplifying daughters to replace naturally occurring cell
loss among the mammary epithelium. Asymmetric cell division is a property of
stem cells and particularly of stem cells functioning within a tissue-specific
stem cell niche, reviewed by Lin (Lin,
2002). But are LREC multipotent stem cells or simply giving rise
to epithelial cells committed to a single epithelial cell lineage? In the
current study, it was not possible to determine whether LREC daughters
represented epithelial cells committed to one epithelial lineage or to
several. In either case, LREC are shown to be self-renewing by retention of
the 3HTdR-labeled DNA. This is apparently accomplished by
asymmetric distribution of the old and new DNA strands. Therefore mammary LREC
possess at least one property commonly ascribed to somatic stem cells. A
second property is the ability to divide symmetrically to produce an expanded
population of stem cells. To approach this issue, implants of mammary
fragments bearing parity-induced mammary epithelial cells (PI-MEC) were
examined after the double labeling procedure. PI-MEC marked by constitutive
lacZ expression expansively self-renew in outgrowths from mammary
fragments. We have estimated that each PI-MEC must undergo at least eight
doublings during the generation of a complete mammary outgrowth if all are
equivalently capable of self-renewal
(Wagner et al., 2002). PI-MEC,
lacZ-positive progeny became LREC in mammary outgrowths and were
doubly labeled with 3HTdR and 5BrdU. Therefore, mammary cells
(PI-MEC) that are pluripotent and capable of self-renewal and expansion during
the allometric growth of mammary ducts can become actively dividing LREC
(Boulanger et al., 2004). This
observation suggests that certain self-renewing mammary cells might occupy
specific micro-environmental locales in the fully developed gland and adopt
asymmetric cell division kinetics as defined by retention of a template DNA
strand during mitosis.
The observation of long label retaining mammary stromal cells was not
reported in the earlier papers, broaching the subject of LREC in the rodent
mammary gland (Welm et al.,
2002; Zeps et al.,
1996). However, in 1983, a paper was published
(Berger and Daniel, 1983)
describing the stimulation of DNA synthesis in the proximate mammary stroma
associated with actively growing terminal end buds. Here, we also observed DNA
synthesis in the mammary stroma surrounding the growing end buds and
subsequently the appearance of label-retaining stromal cells following the 5-6
week chase of the 3HTDr. These 3HTdR-labeled cells
appeared both in the periductal stroma and in the fat pad stroma. None of
these cells incorporated 5BrdU during the 2-day pulse. This result suggests
that the label-retaining stromal cells are not cycling or are cycling very
slowly in contrast to the LREC.
The significance of strand retention in asymmetrically dividing cells has
been implicated in the protection of such cells from mutations resulting from
errors during DNA duplication (Cairns,
2002) and thus from cancer risk. The relatively constant turnover
of mammary epithelial cells in the cycling female mouse was demonstrated by
the very large percentage (>50%) of epithelial cells labeled with
3HTdR in a 24-hour period (Zeps
et al., 1996). The rapidity with which this label is diluted
through cell divisions in 2 weeks (roughly three estrus cycles) suggests that
this strategy would be of selective advantage in preventing the accumulation
of mutations in proliferatively competent mammary cells that survive for
extended periods. An early pregnancy confers a twofold lifelong protection
from mammary cancer risk in rodents and humans. The observation that PI-MEC
appear to adopt the strategy of template strand retention during their
expansion and self-renewal in mammary transplants offers one possible
explanation for this pregnancy-induced refractoriness to carcinogenesis.
However, additional studies regarding the susceptibility of PI-MEC to various
carcinogenic agents and their capacity to adopt and maintain asymmetric cell
kinetics in situ are needed to address this possibility.
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ACKNOWLEDGMENTS |
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