Program in Molecular Biology (K.L.S., S.M.A.) and Department of Pathology (M.M.R., S.M.A.) University of Colorado Health Sciences Center Denver, Colorado 80262
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ABSTRACT |
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INTRODUCTION |
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The use of mouse genetic models has been critical for gaining insight into the molecular processes of apoptosis during mammary gland involution. A conditional knockout of signal transducer and activator of transcription-3 (STAT3) in the mammary gland results in delayed involution and decreased epithelial cell apoptosis, suggesting that STAT3 is critical for the normal process of apoptosis during involution (9). Transgenic mice overexpressing insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein 3 (IGFBP-3) in the mammary gland also exhibit delayed involution and decreased apoptosis (10). IGF-I has been shown to be involved in both proliferation and suppression of apoptosis, and IGFBP-3 may be involved in facilitating the actions of IGF-I in the mammary gland. In addition, mice overexpressing the antiapoptotic protein Bcl-2 in the mammary gland show an inhibition of alveolar cell apoptosis, although the collapse of alveolar structures is not delayed (11). These observations suggest a role for these proteins in regulating mammary gland involution. Finally, the expression patterns of a number of apoptosis-related proteins, including Bcl-x, Bax, p53, transforming growth factor ß1 (TGF-ß1), and sulfated glycoprotein 2 (SGP-2), are regulated during involution (1, 12, 13). The further identification of proteins that regulate the apoptotic process during involution is important for understanding both the regulation of normal mammary gland development and the process of apoptosis in physiological systems.
Akt, also referred to as PKB (protein kinase B) and RAC (related to A
and C kinases), is a serine/threonine protein kinase that has been
shown to suppress apoptosis in a number of systems. c-Akt (PKB,
RAC
) was identified as the cellular homolog of the retroviral
oncogene, v-akt (14, 15). In addition to c-Akt (now known as Akt1), two
other members of the Akt family have been identified, Akt2 (PKBß,
RACß) and Akt3 (PKB
, RAC
) (15, 16). The three members of this
family are all serine/threonine protein kinases that share similar
structural features, including a pleckstrin homology (PH) domain in the
amino terminus and two regulatory phosphorylation sites, Thr308 and
Ser473 in Akt1, which are critical for activation (17). Numerous
studies have shown that Akt is activated in a phosphatidylinositol
3'-kinase (PI3K)-dependent manner (18, 19). The activation of PI3K by
growth factor receptors results in the generation of
3'-phosphoinositides, which recruit Akt to the plasma membrane by means
of its PH domain binding to these membrane-associated
phospholipids (20). Akt is then phosphorylated by
phosphatidylinositol-dependent kinase 1 (PDK1), which has been shown to
phosphorylate Thr308 (21, 22). Phosphorylation of Ser473 is also
required for catalytic activation of Akt, and it has been suggested
that this represents an autophosphorylation event (23), although other
kinases may also phosphorylate this site. Localization to the membrane
appears to be critical for Akt activation because modifications that
target Akt to the membrane, such as the gag sequence found in v-Akt
(24) or fusion of a myristoylation sequence from src-like kinases to
the amino terminus of Akt (25), result in the constitutive activation
of the kinase. Activation of Akt results in the suppression of
apoptosis induced by a number of stimuli including growth factor
withdrawal, detachment from extracellular matrix, UV irradiation, cell
cycle discordance, and activation of Fas (25, 26, 27, 28, 29, 30, 31). In addition,
expression of gag-Akt, a constitutively active form of Akt, in
thymocytes of transgenic mice prevents apoptosis in response to various
stimuli (32).
Akt activation can be induced by a number of growth factors, including epidermal growth factor (EGF) (30), IGF-I (17, 33), estrogen (34), and hepatocyte growth factor/scatter factor (35), which all regulate mammary gland development (36, 37, 38, 39, 40). The purpose of this study was to examine the role of Akt as a regulator of apoptosis during mammary gland involution. A constitutively active mutant of Akt, which contains a myristoylation sequence fused to the amino terminus of Akt (myr-Akt), was expressed in mammary glands of transgenic mice using the mouse mammary tumor virus (MMTV) promoter present in the long terminal repeat of MMTV. We demonstrate that the expression of constitutively active Akt in the mammary gland results in delayed involution and a delay in the onset of apoptosis, suggesting that Akt may be involved in regulating normal mammary gland involution.
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RESULTS |
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To determine localization of Akt in the mammary gland,
immunofluorescence was performed on paraffin-embedded sections from
lactating glands using an anti-Akt1 specific antibody. As shown in Fig. 1D, Akt1 is expressed in epithelial cells surrounding the luminal
spaces as visualized by the red staining. The luminal spaces
are visualized by Oregon-Green 488-conjugated wheat germ agglutinin
(WGA), which binds to mucins on the apical surfaces of epithelial cells
(44). Blue staining represents the
4,6-diamidino-2-phenylindole (DAPI)-stained nuclei. No staining is
observed in the negative control slides that received only the
secondary antibody (Fig. 1E
). These results indicate that Akt is
expressed in epithelial cells during lactation and is catalytically
activated during lactation, suggesting that the decrease of Akt in
these cells at the onset of involution may be necessary for apoptosis
to proceed.
Myr-Akt Is Expressed in the Mammary Glands of Transgenic Mice
Because endogenous Akt expression decreases during early
involution (Fig. 1, A and C), we hypothesized that the inappropriate
presence of activated Akt at this stage would result in a change in the
involution process. Therefore, we expressed a constitutively active
mutant of Akt in the mammary glands of mice under the control of the
MMTV promoter, which allows expression of the transgene in the
epithelial cells of the mammary gland. Although the MMTV promoter
allows expression during all stages of development, the promoter is
strongly expressed during lactation. Therefore, we can examine the
effect of Akt on the transition between lactation and involution in
these transgenic mice. The constitutively active mutant of Akt
(myr-Akt) used in these studies contains an amino-terminal
myristoylation sequence from c-src fused to the coding sequence of
Akt1. This targets the fusion protein to the plasma membrane where it
can be constitutively activated after phosphorylation of Thr308 and
Ser473 (25). The construct also contains a hemagglutinin (HA) epitope
tag at the carboxyl-terminal end of the protein to allow for detection
of transgene expression by immunoblotting.
Transgenic mice were produced that express the myr-Akt construct in the
mammary gland. Transgene-positive founder mice were identified by PCR
of tail DNA (Fig. 2A). Immunoblot
analysis of mammary gland protein for the HA epitope tag indicates that
two lines of mice (designated 1173 and 1176) express detectable levels
of the myr-Akt protein (Fig. 2B
). Expression of the transgene is
consistently higher in mice derived from the 1173 founder than
expression in mice from the 1176 founder (Fig. 2B
and data not shown).
These data show that two independently generated lines of MMTV-myr-Akt
transgenic mice, lines 1173 and 1176, have been produced that express
detectable levels of myr-Akt protein in the mammary gland.
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To localize expression of the transgenic myr-Akt in the mammary gland,
immunofluorescence was performed on mammary tissue at day 2 of
involution from normal (Fig. 2G) and transgenic (Fig. 2H
) mice using an
anti-Akt1 antibody. Data presented in Fig. 1B
indicate that the level
of endogenous Akt is low at this time point. Red staining
represents Akt1 protein and blue staining represents the
DAPI-stained nuclei. Some staining is observed in the epithelium of the
normal mammary gland (Fig. 2G
), which is to be expected because a small
amount of Akt protein is observed in normal mammary glands at this
stage (Fig. 1B
). However, expression of Akt1 in the mammary gland from
the transgenic mouse is much greater and is observed in cells
surrounding the luminal spaces (Fig. 2H
). Identical exposure times were
used for photographs in panels G and H of Fig. 2
. Sections
stained with secondary antibody alone show no staining (data not
shown). Therefore, it appears that the myr-Akt transgene is expressed
in mammary epithelial cells surrounding the alveolar lumina.
Involution Is Delayed in MMTV-myr-Akt Transgenic Mice
Mammary involution can be divided into an early phase (days 14)
and a late phase (days 58) (8). The early phase of involution is
characterized by apoptosis of the alveolar epithelial cells (2).
Because Akt can suppress apoptosis, we determined the effect of the
myr-Akt transgene upon involution. Involution was induced in both FVB
and MMTV-myr-Akt female mice using forced involution as described
above. The fourth inguinal mammary glands were removed at days 2, 4, 6,
8, 10, and 14 of involution, fixed, and embedded in paraffin.
Histological features of control and myr-Akt-expressing mammary
glands were examined in hematoxylin and eosin-stained sections (Fig. 3). At day 2 of involution, alveolar
structures, comprised of a single layer of epithelial cells surrounding
a lumen, are observed in mammary glands from both normal and transgenic
mice (Fig. 3
, A and B). No distinct morphological differences are
apparent between the two glands at this stage. At day 4 of involution,
the alveolar structures in mammary glands from normal mice have started
to collapse, and numerous apoptotic bodies are apparent in the ductal
lumens (Fig. 3C
). In addition, adipocytes have begun to reappear or,
alternatively, to reaccumulate lipid. The predominance of adipocytes is
typical of mammary glands in virgin mice and postinvolution mice. In
the mammary glands from transgenic mice, however, the alveoli have not
yet begun to collapse, and they appear distended (Fig. 3D
). In
addition, there is an accumulation of protein in the alveolar lumina,
which may represent inspissated milk typically observed during milk
stasis. At day 6 of involution, the alveoli in mammary glands from
normal mice have collapsed (Fig. 3E
) and the process of remodeling has
continued (8). In mammary glands from transgenic mice, the alveoli have
still not collapsed, although some apoptotic bodies are observed in the
luminal spaces and some adipocytes have begun to reappear (Fig. 3F
),
suggesting that the process of involution has begun. By day 8 of
involution, the normal mammary glands have been completely remodeled
and resemble that of a prepregnant gland (Fig. 3G
). In the mammary
glands from transgenic mice, the alveoli are beginning to collapse and
numerous apoptotic bodies are visible in the luminal spaces (Fig. 3H
),
similar to the morphology observed at day 4 of involution in the normal
gland (Fig. 3C
). Normal mammary glands at days 10 and 14 of involution
are similar to that seen at day 8, as the mammary gland has been
completely remodeled (Fig. 3
, I and K). In contrast, mammary glands
from transgenic mice are still undergoing the process of involution
through day 10 as indicated by the collapsing alveolar structures and
the disorganization of the gland (Fig. 3J
). By day 14, the mammary
glands from transgenic mice appear to have been remodeled, although
some alveolar structures still persist (Fig. 3L
).
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Myr-Akt Delays the Onset of Apoptosis during Involution
To determine whether the delayed involution observed in the
MMTV-myr-Akt mice was accompanied by a decrease in apoptosis, the
numbers of apoptotic cells were quantified. Figure 4A shows the results of quantifying
apoptotic bodies in hematoxylin and eosin-stained sections of mammary
glands from normal and myr-Akt transgenic mice. Consistent with
observations published previously (7), apoptosis peaked at day 4 of
involution in the mammary glands from normal mice. However, the peak of
apoptosis in the mammary glands from transgenic mice did not occur
until day 8 of involution, indicating a delay in the onset of apoptosis
in mammary glands from transgenic mice.
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To compare the epithelial content of the regressed mammary glands from
normal and transgenic mice, the amount of epithelium present in mammary
glands at day 21 of involution was quantified. As shown in Fig. 4C, epithelial content in mammary glands from transgenic mice was nearly
2-fold higher than that in mammary glands from normal mice. Figure 4D
shows representative pictures of hematoxylin and eosin-stained sections
from normal (upper panel) and transgenic (lower
panel) mammary glands at day 21 of involution. These results
suggest that less epithelial cells undergo apoptosis in the mammary
glands from transgenic mice and that these cells remain until the next
pregnancy.
ß-Casein and Whey Acidic Protein (WAP) Expression Are Sustained
in MMTV-myr-Akt Mice throughout Involution
One of the initial events after weaning is the rapid
decrease in the expression of milk proteins such as ß-casein and WAP
(1). These proteins are highly expressed during lactation, but diminish
during the first 5 days of involution (4). We hypothesized that the
observed delay in involution would be accompanied by sustained
production of ß-casein and WAP RNA. RNA was extracted from the fourth
inguinal mammary glands during lactation and involution, and the levels
of ß-casein, WAP, and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) RNA were examined by Northern blot analysis. Expression of
ß-casein and WAP are sustained in the mammary glands from transgenic
mice compared with mammary glands from normal mice (Fig. 5, A and B). ß-Casein expression
decreases dramatically in the normal gland after weaning, and levels
are undetectable by day 6 of involution (Fig. 5A
). WAP expression
similarly decreases after weaning, and levels are also undetectable by
day 6 (Fig. 5B
). In contrast, ß-casein expression in the mammary
glands from transgenic mice remains high and levels are detectable
until day 10 of involution, although they are beginning to decrease by
day 6 of involution (Fig. 5A
). WAP expression is also sustained in the
mammary glands from transgenic mice, but decreases by day 8 of
involution (Fig. 5B
). It appears that there is more WAP mRNA production
at day 2 of involution than during lactation (Fig. 5B
); however, there
is a corresponding increase in GAPDH (Fig. 5C
), indicating that this is
due to a loading artifact. Similar data were obtained in three
different studies using RNA isolated from mammary glands of different
mice. These data demonstrate that milk protein gene expression is
sustained in the mammary glands of transgenic mice compared with normal
mice.
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To examine STAT phosphorylation, protein was extracted from
mammary glands of both normal and MMTV-myr-Akt mice during lactation
and involution. The phosphorylated forms of STAT3 and STAT5a were
detected by immunoblot analysis using phospho-specific antibodies.
STAT3 phosphorylation is low during lactation, but increases by day 2
of involution in mammary glands from both normal and transgenic mice
(Fig. 6A). This increase is transient and
decreases by day 8 of involution in mammary glands from normal mice.
Phosphorylated STAT3 persists to day 8 in mammary glands from
transgenic mice, possibly reflecting the increased numbers of
epithelial cells remaining in the mammary glands from transgenic mice.
Overall levels of STAT3 are equivalent in mammary glands from both
normal and transgenic mice (Fig. 6B
).
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Increased and Prolonged TIMP-1 Expression in MMTV-myr-Akt
Glands
After apoptosis of the secretory epithelial cells during
involution, the mammary gland is remodeled back to a prepregnant state.
This process involves activation of MMPs, a family of enzymes that
degrades extracellular matrix, and the inactivation or decrease in the
concentration of their inhibitors, the TIMPs (4, 6). Expression
patterns of the MMP stromelysin-1 (MMP-3) and its inhibitor TIMP-1
during mammary gland involution have been well characterized (4, 50).
TIMP-1 is expressed at moderate levels during lactation, but increases
in early involution with a peak of expression at 4 days of involution
(50). MMP-3 is undetectable during lactation, but increases during
involution with a peak of expression at 46 days of involution (50).
The ratio of MMP to TIMP expression is important for the remodeling
process to occur, and disruption of this ratio has been shown to alter
involution. Implantation of TIMP-1 pellets in the mammary gland results
in delayed involution (4), and overexpression of MMP-3 in transgenic
mice results in precocious involution (51, 52).
To determine whether expression of MMP-3 and TIMP-1 are altered in the
MMTV-myr-Akt mice, the amount of TIMP-1 and MMP-3 RNAs was examined
using Northern blot analysis. TIMP-1 expression in mammary glands from
normal mice increases during early involution, peaking at days 24 and
decreasing by day 8 of involution (Fig. 7A). In mammary glands from transgenic
mice, TIMP-1 levels also increase during early involution but remain
high until day 10 of involution (Fig. 7A
). GAPDH levels are equivalent
in mammary glands from both normal and transgenic mice (Fig. 7B
). MMP-3
expression in mammary glands from normal mice is low during lactation
and increases by day 4 of involution, peaking at days 46, followed by
a subsequent decrease in expression by day 10 of involution (Fig. 7C
).
In transgenic mice, MMP-3 levels increase earlier than in normal mice
and are detectable on day 2 of involution, peaking at day 6, and are
followed by a decrease in expression at day 10 of involution. The
observed earlier increase of MMP-3 in mammary glands from transgenic
mice is not consistent with a delay in involution. However, since the
MMP to TIMP ratio is thought to be critical for MMP activity, the MMP-3
activity is likely being suppressed by the high levels of TIMP-1 at
this stage (Fig. 7A
). GAPDH levels are equivalent in mammary glands
from both normal and transgenic mice (Fig. 7D
). Similar results were
obtained in three different studies using tissue extracts prepared from
different mice. These data suggest that the increased and sustained
levels of TIMP-1 in the mammary glands of transgenic mice may be
involved in the delayed involution observed in the myr-Akt transgenic
mammary glands.
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DISCUSSION |
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We have generated transgenic mice that express a constitutively active Akt in the mammary gland using the MMTV promoter. These mice were shown to express the transgene during lactation as well as during the first 8 days of involution, with detectable levels remaining at day 14 of involution. Morphologically, the mammary glands of the transgenic mice show a delay in involution that corresponds with a delay in the onset of apoptosis. The mammary epithelial cells eventually undergo apoptosis, and the gland is remodeled suggesting that either transgene expression diminishes and can no longer suppress apoptosis, or that another apoptotic mechanism is present that can override the effects of the myr-Akt transgene.
Understanding the suppression of apoptosis is of great relevance to cancer, as overexpression of genes that suppress apoptosis lead to cancers such as leukemia (53). Although the mammary glands of the MMTV-myr-Akt mice eventually undergo involution, it appears that less epithelial cells are lost than in normal regressed mammary glands. This observation raises the possibility that the presence of the myr-Akt transgene in mammary glands may result in the retention of epithelial structures, particularly in uni- and multiparous mice, which could lead to the development of hyperplasia and/or tumors.
One of the initial characteristics of involution is the decrease in the
production of milk proteins in early involution (1). ß-Casein and WAP
mRNA expression were sustained in the mammary glands of the transgenic
mice compared with those of normal mice. In the histological sections
(Fig. 3), it is apparent that the alveolar structures are retained in
the glands from transgenic mice through day 6 of involution whereas the
alveolar structures in glands from normal mice regress after day 2 of
involution. It has been demonstrated that association of mammary
epithelial cells with extracellular matrix is important for maintaining
the differentiation status of the epithelial cells and that disruption
of this interaction results in the loss of ß-casein expression
(4). Therefore, it is possible that the epithelial cells that have not
undergone apoptosis in the mammary glands from transgenic mice are
still associated with the extracellular matrix, resulting in the
maintenance of their differentiation state as suggested by the
prolonged expression of milk protein genes.
STATs have been shown to be important regulators of mammary gland development. STAT5a knockout mice have impaired lobuloalveolar development during pregnancy and fail to lactate (54). STAT5a is activated during pregnancy and is involved in milk protein expression (48, 49, 55), but its activity decreases at the beginning of involution and has been suggested to be involved in the suppression of apoptosis of epithelial cells (9). As mentioned in the Introduction, STAT3 is required for normal involution as demonstrated by delayed involution in conditional knockouts (9). STAT3 activity is low during lactation and increases at the beginning of involution (9, 49). It has been suggested that STAT5 and STAT3 are reciprocally regulated and that this regulation may be important for the normal process of involution (9, 49). Because of the observed delay of involution in the myr-Akt mice, we examined the phosphorylation states of both STAT3 and STAT5a. Although phosphorylated STAT3 persists longer in mammary glands from transgenic mice, this may be due to higher epithelial cell content and the observed delay in involution. Both molecules were expressed equally at the protein level in mammary glands from both normal and transgenic mice. These data may suggest that either Akt functions independently of STAT3 to suppress apoptosis, or that Akt functions downstream of STAT3.
Studies have shown that STAT5a can induce the transcription of the milk proteins such as ß-casein (48, 56). Although ß-casein levels remain high after weaning in the myr-Akt mice, phosphorylated STAT5a levels decrease by day 2 of involution. Therefore, the increased level of ß-casein RNA observed in mammary glands from transgenic mice could reflect either continued transcription of the gene in the absence of phosphorylated STAT5a or an increase in the half-life of the mRNA.
MMPs and their inhibitors, the TIMPs, are critical for the process of remodeling of the gland after apoptosis (4, 6). TIMP-1 has been demonstrated to increase at the beginning of involution, presumably to inhibit the remodeling process until after apoptosis has occurred, and then decrease to allow the MMPs to begin the remodeling process (50). TIMP-1 expression increases during involution in the MMTV-myr-Akt mice, and it remains elevated until day 10 of involution. Interestingly, the expression pattern of MMP-3 in the MMTV-myr-Akt mammary glands is similar to that of the normal glands, although its expression is increased earlier in the mammary glands from transgenic mice when compared with control mice. It is possible that in response to the inability of the epithelial cells to undergo apoptosis, the mammary glands from transgenic mice are up-regulating pathways involved in inducing apoptosis and remodeling in an attempt to overcome this phenotype. It would be interesting to determine the expression patterns of other proteins known to be involved in inducing apoptosis in the mammary gland. Although MMP-3 is expressed earlier in the mammary glands of transgenic mice induced to undergo involution, the increased and sustained levels of TIMP-1 may inhibit MMP-3 activation. It would be interesting to determine whether the expression of other MMPs is altered in our transgenic mice and whether they are responsible for the remodeling that eventually occurs in the mammary glands of myr-Akt transgenic mice.
The coordination of apoptosis and tissue remodeling during involution is not completely understood. One explanation for the delayed involution observed in the mammary glands of transgenic mice is that myr-Akt suppresses apoptosis of the secretory epithelial cells; therefore, the signals required to initiate the remodeling process may not be present. An additional factor contributing to this phenotype may be the observed alteration in MMP to TIMP ratios. The ratio of TIMPs to MMPs is critical for normal involution. Talhouk et al. (4) used pellet implants to demonstrate that the presence of high levels of exogenous TIMP protein during involution resulted in delayed regression of alveolar structures and sustained expression of ß-casein, demonstrating that the TIMP to MMP ratio is important for normal involution and regulation of mammary epithelial function. Therefore, the altered ratio of TIMP to MMP might explain not only the delay in tissue remodeling, but also the sustained expression of ß-casein in the transgenic mammary glands. One possible mechanism for the increased levels of TIMP-1 is that Akt may induce TIMP-1 transcription or alter the molecules that regulate TIMP-1 gene expression. Another possibility is that activated Akt may be indirectly regulating TIMP-1 expression by modulating a signal from the apoptotic mammary epithelial cells that is normally required to down-regulate TIMP-1.
A recent study by Fata et al. (57) has implicated Akt activity in alveolar development during pregnancy. Impaired alveolar development in osteoprotegerin-ligand (OPGL) knockout mice correlated with loss of phosphorylated Akt. Expression of OPGL by pellet implants in pregnant mammary glands results in an increase in phosphorylated Akt and the restoration of alveolar development. The data presented in this paper focus on the antiapoptotic functions of Akt in the mammary gland and indicate that the loss of Akt at weaning is necessary for normal involution to occur. We have shown that expression of activated Akt in the mammary gland results in suppression of apoptosis and delayed involution. The delayed involution may be a result of increased TIMP-1 expression, and future studies will focus on the regulation of TIMP-1 expression in these mice. In addition, the identification of Akt targets will define other molecules involved in regulating both apoptosis and tissue remodeling. Clearly, the overexpression of signaling molecules in transgenic mice can reveal potential roles in normal developmental processes, but this analysis may be prone to artifacts. Confirmation that Akt is critical in regulating mammary gland involution will require analysis of knock-out mice that eliminate expression of Akt1, Akt2, or both.
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MATERIALS AND METHODS |
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Tissue Collection
Adult female mice were mated. Litters were normalized to eight
pups and were removed after 9 days of lactation. The females were
killed by cervical dislocation at day 9 of lactation, or days 2, 4, 6,
8, 10, and 14 of involution. The tissues were either snap frozen in dry
ice for RNA and protein extraction or were fixed in 10% neutral
buffered formalin and embedded in paraffin. The embedded tissues were
sectioned at 5 µM and stained with hematoxylin and eosin.
Mammary glands were removed from at least five mice per time point for
histological analysis, and all subsequent analyses were performed in
duplicate on tissue from at least three mice per time point.
Assessment of Epithelial Cell Apoptosis
Apoptotic cells were quantified using hematoxylin and
eosin-stained sections based on morphological criteria (59). The number
of apoptotic cells was calculated as a percentage of total cell counts.
TUNEL staining was performed on sections using the Apoptag kit
(Intergen, Purchase, NY) following the manufacturers
instructions. The number of apoptotic cells was calculated as a
percentage of total cell counts. Hematoxylin and eosin-stained sections
were used to calculate epithelial content of glands at day 21 of
involution. Epithelial content was quantified by overlaying a grid
consisting of 384 squares onto images of mammary glands taken at 100x
magnification. The numbers of squares containing epithelium were
counted and calculated as a percentage of the total number of squares.
The resulting percentage is a representation of the area of the gland
containing epithelium rather than a direct count of epithelial cells. A
total of 12 mammary glands at day 21 of involution, 6 glands from
normal mice and 6 glands from transgenic mice, were quantitated. The
study was performed blindly.
Northern Blot Analysis
RNA was extracted from frozen tissue using Trizol (Life Technologies, Gaithersburg, MD). The RNA was then denatured and
analyzed on a 1% agarose gel containing 6% formaldehyde and
transferred onto GeneScreen (NEN Life Science Products,
Boston, MA) according to manufacturers protocols. Probes for the
Northern blots were radiolabeled using random primers (Prime-It
II, Stratagene, San Diego, CA) and
[-32P]-dCTP (NEN Life Science Products). The ß-casein probe was obtained from Dr. Lothar
Hennighausen (NIH, Bethesda, MD). The WAP probe was obtained from Dr.
Jeffrey Rosen (Baylor College of Medicine, Houston, TX). The TIMP-1
probe was obtained from Dr. Zena Werb (University of California San
Francisco, San Francisco, CA). The stromelysin-1 (MMP-3) probe was
obtained from Dr. Lynn Matrisian (Vanderbilt College of Medicine,
Nashville, TN). The GAPDH probe was from the collection of this
laboratory.
Immunoblot Analysis
Protein was extracted from frozen tissue by homogenizing in RIPA
lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl,
2 mM EDTA, 50 mM NaF, 1% Triton X-100, 1%
DOC, 0.1% SDS, 1 mM dithiothreitol, 5 mM
sodium orthovanadate, 100 µg/ml phenylmethylsulfonyl fluoride, and a
protease inhibitor cocktail (no. P 8340, Sigma, St. Louis,
MO). The samples were then boiled for 10 min, chilled on ice, and
sonicated until homogeneous. Protein assays were performed using the
Pierce Coomassie Plus protein assay reagent (Pierce Chemical Co., Rockford, IL). Total protein (50 µg amounts) was
separated on 8% SDS-polyacrylamide gels, transferred to PVDF membrane
(Immobilon, Millipore Corp., Bedford, MA), and
immunoblotted with various antibodies as described previously (60, 61).
The anti-Akt, antiphospho-Akt, antiphospho GSK-3/ß,
antiphospho-STAT3, and anti-STAT3 rabbit polyclonal antibodies were
obtained from New England Biolabs, Inc. (Beverly, MA). The
antiactin and anti-GSK-3
antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antiphospho-STAT5
rabbit polyclonal antibody was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). The anti-STAT5 rabbit
polyclonal antibody was obtained from Transduction Laboratories, Inc. (Lexington, KY). The horseradish peroxidase-conjugated
anti-HA antibody (clone 3F10) was obtained from Roche Molecular Biochemicals (Indianapolis, IN). All antibodies were used at a
dilution of 1:1000, except the anti-HA antibody, which was used at a
dilution of 1:400. Bound antibodies were detected using horseradish
peroxidase-conjugated secondary antibody followed by detection with the
ECL system according to manufacturers recommendations (Amersham Pharmacia Biotech, Piscataway, NJ). Densitometric
analysis was performed on the Computing Densitometer, and images were
quantitated using ImageQuant software, both from Molecular Dynamics, Inc. (Sunnyvale, CA).
Akt Kinase Assay
Mammary glands were removed from animals at the indicated times
and snap frozen in liquid nitrogen. Lysates were prepared by
homogenizing the tissue in lysis buffer [150 mM NaCl, 50
mM HEPES, pH 7.4, 2 mM EGTA, pH 8.0, 1% Triton
X-100, 0.25% deoxycholate, and a protease inhibitor cocktail (no. P
8340, Sigma, St. Louis, MO)]. The samples were
centrifuged at 13,000 x g for 30 min and the
supernatants were used for immunoprecipitation. Protein assays were
performed using the Pierce Coomassie Plus protein assay reagent
(Pierce Chemical Co.). One milligram amounts of total
protein were used for each immunoprecipitation. The immunoprecipitation
and kinase assays were performed using the Akt Kinase Assay Kit (no.
9840, New England Biolabs, Inc.) following the
manufacturers instructions. The reactions were analyzed on a 12%
polyacrylamide gel and immunoblotted as described above using the
antiphospho-GSK-3/ß antibody.
Immunofluorescence
Mammary glands were removed from animals at the indicated times,
fixed in 10% neutral buffered formalin, and embedded in paraffin.
Sections were cut at 5 µm. After dehydration with graded alcohols,
microwave antigen retrieval was performed for 20 min in 10
mM sodium citrate, pH 6.0. The sections were permeabilized
with 0.2% glycine in PBS, blocked with 5% normal goat serum, and
incubated with the anti-Akt1 antibody kindly provided by Dr. Morrie
Birnbaum (University of Pennsylvania, Philadelphia, PA) at a dilution
of 1:200. The sections were then incubated with Cy3-conjugated
antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc.,
West Grove, PA), Oregon-Green 488-conjugated WGA (Molecular Probes, Inc., Eugene, OR), and 0.6 µg/ml DAPI
(Sigma). Images were collected using SlideBook
software (Intelligent Imaging Innovations, Inc., Denver, CO) on a
Diaphot TMD microscope (Nikon, Melville, NY) equipped for
fluorescence with a Xenon lamp and filter wheels (Sutter Instruments,
Novato, CA), fluorescent filters (Chroma, Brattleboro, VT), cooled CCD
camera (Cooke, Tonawanda, NY), and stepper motor (Intelligent Imaging
Innovations, Inc., Denver, CO).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This research was supported by a University of Colorado Cancer Center "Wines for Life" Seed Grant and NIH Grants CA-085736, DK-53858, and DK-48845. K.L.S. and M.M.R. are predoctoral and postdoctoral fellows supported by grants from the US Army Breast Cancer Research Program (BC980097 and BC990978, respectively). The University of Colorado Cancer Center Transgenic Mouse Core Facility is supported by a Cancer Center grant from the National Cancer Institute (CA-46934).
Received for publication January 8, 2001. Accepted for publication March 22, 2001.
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REFERENCES |
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