Vol. 7, 3-11,
January
1996
Cell Growth
& Differentiation
Expression of a Viral Oncoprotein during Mammary Gland Development Alters Cell Fate and Function: Induction of p53-independent Apoptosis Is Followed by Impaired Milk Protein Production in Surviving Cells1
Minglin Li, Jiadi Hu, Kathnn Heermeier, Lothar Hennighausen, and Priscilla A. Furth2 Division
of Infectious
Maryland
Medical
Center,
Diseases, School
Baltimore, Maryland of Biochemistry
Laboratory Diabetes,
20892
Digestive
and
Department
and
the
Baltimore
of Medicine, Veterans
Introduction
University Affairs
21201 [M. L, J. H., P. A. F.], and and Metabolism, National Institutes
Kidney
Diseases,
NIH,
Bethesda,
of
Medical
of
Maryland
[K. H., L. H.]
Abstract The disruption of cell cycle regulation is associated with developmental abnormalities and tumorigenesis. The SV4O large T antigen (Tag) interferes with cell cycle control by interacting with the pRb family and p53. Mice carrying a transgene composed of the whey acidic protein (WAP) gene promoter and the Tag coding sequence express Tag during pregnancy and are unable to nurse their young. Tag expression induced apoptosis in mammary epithelial cells during late pregnancy. At least 5% of mammary epithelial cells were undergoing apoptosis at any one time. In contrast, less than 0.2% of mammary epithelial cells in nontransgenic littermates was undergoing apoptosis. Apoptosis in Tag mice was associated with increased steady-state RNA levels of bax and bcl-xL+S, with a relative increase in bcI-x expression. Since p53 was sequestered by Tag, it is likely that p53-independent mechanisms precipitated apoptosis. The Tag-expressing mammary alveolar cells that did not undergo apoptosis continued to differentiate through late pregnancy, as measured by the sequential activation of milk protein gene expression. However, milk protein production, processing, and secretion was impaired, resulting in lactation failure.
Received
6/19/95;
revised
The costs of publication payment of page charges. advertisement in accordance cate this fact.
10/3/95;
accepted
10/5/95.
of this article were defrayed in part by the This article must therefore be hereby marked with
1 8 U.S.C.
Section
1 734
solely
to mdi-
The pRb3 family and p53 have critical functions in regulating the cell cycle. In transgenic mice, expression of the SV4O large Tag and the papillomavirus E6 and E7 proteins can lead to developmental defects and cancer by disrupting cell cycle control through interactions with pRb and p53 (1-4). We used mice carrying a transgene composed of the WAP promoter and the Tag coding sequence (5) to identify cellular pathways disrupted through the expression of this viral oncoprotein in the developing mammary gland. Mammary gland development consists of well characterized steps that culminate in lactation (6-9). Developmental abnormalities at specific stages can be recognized through the examination of mammary gland structure and the evaluation of cellular differentiation. The function of the gland can be assessed by the presence or absence of a successful lactation. In virgin mice, the mammary parenchyma is composed of an organized system of ducts within the mammary fat pad. The major sites of growth are at the terminal end buds. With each estrous cycle, the lateral end buds differentiate and subdivide progressively. Extensive development mediated by lactogenic hormones begins with pregnancy and is completed at the onset of lactation. A rapid increase in the number and size of alveoli occurring during the second half of pregnancy results in development of fully differentiated and functional secretory lobules. Differentiation of mammary epithelial cells and formation of alveolar structures during pregnancy leads to the sequential expression of milk protein genes (7), followed by milk protein secretion and lactation. Lactation is maintained as long as the dams are suckled. After weaning, the mammaty gland involutes, and the entire lobulo-alveolar compartment collapses through PCD. This results in a ductal network resembling that of a mature virgin. The mammary gland can serve as a paradigm to investigate how loss of cell cycle regulation, mediated by a viral oncoprotein, affects both organ development and function. Female mice carrying a WAP-Tag transgene specifically express Tag in mammary tissue beginning around day 13 of pregnancy (5), but tumor development does not occur until after three to five pregnancies. These mice are unable to nurse their young, starting with the first pregnancy. This suggests that expression of a viral oncoprotein leads to functional defects in mammary epithelial cells prior to malignant transformation.
I This work was supported in part by a research contract from Galagen (Arden Hills, MN; to P. A. F.) and Grant 01-067482532 from the Veterans
Administration Research Advisory Group (to P. A. F.). 2 To whom requests for reprints should be addressed, at Department of Medicine, Room 5D-136, Baltimore VA Medical Center, University of Maryland Medical School, 10 North Greene Street, Baltimore, MD 21201. Phone: (410) 605-7181; Fax: (410) 605-7914; E-mail:
[email protected].
The abbreviations used are: pRb, retinoblastoma protein; Tag, SV4O large T antigen; WAP, whey acidic protein; PCD, programmed cell death; nt, nucleotide; TGF31 , transforming growth factor 31 ; MDGI, mammaryderived growth inhibitor; RT-PCR, reverse transcriptase PCR. 3
3
4
5V40 Tag Alters Mammary
Cell Fate and Function
Fig. 1. Mammary glands from Tag mice exhibit decreased alveolar density as compared to control mice. Whole-mount analysis of mammary glands from Tag (A and C) and control (B and D) mice. The inguinal glands of day 16 pregnant mice were mounted on a glass slide, fixed, and stained. Similar results were seen in glands from late preg-
nancy and on the day of parturition. A and B show the entire gland; C and D show the same glands at increased magnification. LN, lymph node.
In this study, differentiating
we demonstrate mammary
that the presence
epithelial
cells
altered
both
of Tag in cell fate
and function. Tag synthesis induced p53-independent apoptosis in mammary epithelial cells during late pregnancy. This was probably mediated by changes in bax and bcl-xL#{247}S expression. However, only a subset of Tag-expressing cells underwent
apoptosis,
and
alveolar
structures
were
not de-
stroyed. Tag-expressing cells that did not undergo apoptosis continued to differentiate throughout late pregnancy and expressed major milk protein RNAs. However, milk protein production and secretion were impaired in these cells, resuiting in lactation failure.
Results Tag Mice Cannot Nurse Their Offspring. Tag was detected in alveolar cells at approximately day 1 3 of the first pregnancy. Although nontransgenic females nursed all of their litters, Tag mice were unable to feed their offspring, beginning with the first litter. Pups delivered to transgenic females were healthy initially and attempted to suckle. However, no milk was observed in their stomachs, and 100% of the offspring died within 48 h. In cross-fostering experiments, 76% of offspring born to Tag mice could be rescued by fostering them to a nontransgenic female. Conversely, 1 00% of offspring born to nontransgenic mice died after being fostered to Tag mice (data not shown). To investigate why Tag mice cannot lactate at the time of delivery, studies were focused on the developmental state of the gland just prior to delivery. In normal mice, milk production and secretion into the alveoli begins a few days prior to delivery. Gestation in the Tag mice and controls was 20 days. Mice were evaluated during the first pregnancy to identify events that occur shortly after Tag expression.
Tag-induced, p53-independent Apoptosis Was Associated with Increased Steady-State Levels of bax and bcl-xL+s RNA. Whole-mount analyses of mammary tissue from
Tag
mice
revealed
decreased
alveolar
density,
starting
at day 16 of pregnancy through parturition (Fig. 1). Histologcal analyses demonstrated that the average alveolar circumference of Tag mice was smaller than that of control mice (Fig. 2). In situ
detection
of apoptotic
cells
demonstrated
that
ap-
proximately 5% of mammary epithelial cells were actively undergoing apoptosis in Tag mice at day 18 to day 19 of pregnancy (Table 1 ; Fig. 2, G and H). In contrast, less than 0.2% of cells underwent apoptosis in mammary glands of nontransgenic pregnant mice. Tag expression was detected in the majority of mammary epithelial cells (Fig. 2, D and E). Immunohistochemical staining localized p53 to the nucleus of epithelial cells of transgenic mice but was undetected in nontransgenic mice (data not shown; Ref. 5). These results suggest that the apoptosis induced by Tag expression during pregnancy was mediated by p53-independent mechanisms. To identify which apoptosis pathway genes were transcriptionally
formed
activated
following
Tag expression,
we per-
Northern
blot analyses of steady-state levels of bax, bcl-xL±s, p53, and bcl-2 on RNA extracted from mammary glands of late pregnant mice. Steady-state levels of bax (Fig. 3C) and bcl-xL#{247}s (Fig. 3A) RNA were increased approximately 5- and 2-fold, respectively, as compared to nontransgenic controls. To distinguish between bcl-xL and bcl-x RNA, a RT-PCR analysis was performed with primers which simultaneously
amplified
both
forms,
ization with oligonucleotides that two forms. Both bcl-xL and bcl-x in Tag
transgenic
mice,
bcl-x
RNA levels
was observed
but
followed
distinguished RNA levels
a relatively
by a hybridbetween the were increased
greater
(Fig. 3B).
Bcl-xL
increase
in
RNA
re-
Cell Growth & Differentiation
5
Fig. 2. Tag-induced apoptosis in mammary epithelial cells during pregnancy. Histological analysis of mammary tissue from day 18 pregnant Tag (A, B, D, E, G, and H) and control (C, F, and I) mice. Tissue was sectioned and stained with H&E (A, B, and C). Fat pad, alveoli, lumen, and secretions are indicated. A: arrow, an apoptotic cell. lmmunohistochemical analysis for the presence of Tag was performed (0, E, and F). D, solid arrow, a Tag-expressing cell. Note: the majority of nuclei in the Tag mice are positive for Tag. In situ detection of apoptosis was performed (G, H, and I). G: arrows, apoptotic cells. Cells undergoing apoptosis appear brown.
Table
1
Percentage
Each mouse
of cells
no. represents
undergoing ma mmary
apoptosis tissue
in sections
taken
of mammary
from a different
mouse.
gland
tissue
A poptotic
from
control
(C) and Tag (1) mice
cells we re identified
usin g the Apotag
Control Mouse
a
Mouse
Apoptotic cells
flO.a
mice blot
Mouse
no.
Total cells counted
% apoptotic
2
1035
0.19
5
48
1064
4.5
1040
0.19
6
43
1079
4.0
3 4
2
1039
0.19
7
60
1110
5.4
2
1042
0.19
8
64
1046
6.1
Mean
2
1039
0.19
Mean
54
1075
(P) Day (D) 17. Mouse
was
at least
form
in both
transgenic
5- to
1 0-fold
more
No p53 or bcl-2 expression (data
not shown).
RNA in the Tag mice translated (Fig.
2: C, PD 18. Mouse
3: C, PD 18. Mouse
4: C, PD 19. Mouse
cells
5.0
5: T, PD 17. Mouse
6: T, PD 18. Mouse
7:
8: T, PD 19.
the predominant
analyses
protein
cells
2
and
bcl-x.
#{176}“#{176} apoptotic
Apoptotic cells
1
1: C, Pregnancy
MD).
Tag
Total cells counted
2
T, PD 18. Mouse
mained
kit (0 ncor, Gaithersburg,
and control abundant
was detected The
increased
into increased
than
by Northern levels
of bax
levels of bax
3D).
Expression of bcl-xL±S and bax was mammary gland tumors from Tag mice.
performed pregnancy patterns tumor
also This
evaluated analysis
in was
of apoptosis
pathway
progression
detected The
to determine if the changes observed during persisted and because changes in expression
in tumor
bcl-xL:bcl-xS
mammary indicate
(10).
glands that
Both
specimens
genes
with
bax RNA were (Fig. 3 and data not shown).
RNA
ratio
from
late pregnant
changes
can be associated
bcl-xL±S was
in the ratio
and
similar
to that
Tag mice. of bcl-xL
found These
to bcl-x
in the results expres-
6
SV4O Tag Alters
Mammary
Cell Fate and Function
A
Control
WAP-Tag
Tumor
5 678
910
WAP-Tag
Tumor
I 2 3 4
c
Control
WAP-Tag
#{149}
bax#{149}
bcl-xl 2
---
12
-.----
Control
B
12345678
910
1
bcI-x
34
Control
bax
WAP-Tag
Con
Tumor
.
1234567
MI
bcI-x
Fig. 3. Bax and bcl-xL±S expression increased in Tag mice coincident with the appearance of PCD. A, Northern blot analysis of steady-state RNA levels of bcl-xL+S at late pregnancy. RNA from control mice, Tag (WAP-Tag) mice, and two Tag-induced mammary gland tumors (Tumor, Lanes 9 and 10) were analyzed. Each lane represents a mammary gland or tumor taken from a different mouse. Lane 1, day 17 of pregnancy; Lane 2, day 18; Lane 3, day 18; Lane 4, day 19; Lane 5, day 17; Lane 6, day 18; Lane 7, day 18; Lane 8, day 19. Ethidium bromide staining of 285 and 185 RNA was used as a loading standard. B, Southem blot analysis of bcl-xL- and bcl-x-specific PCR products following RT-PCR. The same samples analyzed in A were used in the RT-PCR assay. C, Northem blot analysis of steady-state bax RNA levels at late pregnancy. A subset of the same samples analyzed in A were examined. Lane 1, day 1 8 of pregnancy; Lane 2, day 19; Lane 3, day 18; Lane 4, day 19. 0, Western blot analysis of steady-state levels of bax protein at late pregnancy and in tumor tissue. Each lane represents a mammary gland or tumor specimen (Lane 7) taken from a different mouse. Lane 1, day 17 of pregnancy; Lane 2, day 18; Lane 3, day 18; Lane 4, day 19; Lane 5, day 18; Lane 6, day 18. Coomassie blue staining of the protein gel demonstrated equal loading of all
samples.
sion were correlated with Tag expression. Elevated concentrations of bax proteins were also detected in tumor specimens (Fig. 3D). Epithelial Cells Expressing Tag Differentiate Through Late Pregnancy as Measured by the Sequential Activation of Milk Protein Genes, but Milk Protein Production Was Impaired. Histological examination of mammary tissue from day 16 of pregnancy through parturition demonstrated a dramatic reduction in the number of fat globules present in the
alveolar
cells.
To assess
the
differentiation
state
of the
Tag-expressing cells, Northern blot analyses were used to evaluate expression of the differentiation-specific milk protein genes. The milk protein genes f3-casein, WAP, and a-lactalbumin were expressed from late pregnancy through the day
of delivery
(Fig.
4; Ref.
11). Milk
protein
production
was evaluated from day 17 of pregnancy through parturition. Mammary glands obtained from Tag and control mice during pregnancy or within 24 h of delivery revealed differences in milk accumulation upon visual examination. When sectioned in half,
milk readily
but not Tag
mice.
leaked The
from
presence
glands
of nontransgenic
of milk
proteins
was
Both
5E).
mammary
WAP
precursor
tissue
of late
and mature pregnant
WAP were
control
mice.
found
in
In contrast,
Tag mice demonstrated only a single WAP precursor band consistent with failure of cleavage of the signal peptide (12). Differential Expression of the Tumor Marker Gene WDNMI in Tag Mice. Northern blot analyses were used to determine
if there
was
evidence
of differential
expression
of
other developmentally regulated genes. Three additional genes, the expression of which normally increases during pregnancy, were examined: WDNM1, TGFj31, and MDGI. WDNM1 is a developmentally regulated gene the expression of which is reduced in c-myc and neu but not ras and mt transformed mammary
cells lines (1 3). TGFI31
expression
normally
increases
during
late pregnancy and falls at the time of delivery (14). Expression of MDGI normally increases during differentiation of
mammary
epithelial
cells (15). Only expression
of WDNM1
reduced in mammary tissue of Tag transgenic first pregnancy (Fig. 6 and data not shown).
was
mice during the
mice evalu-
ated by immunohistochemistry and Western blot analysis using antibodies directed against either mouse WAP or total mouse milk proteins. Milk proteins were abundant in both mammary epithelial cells and the alveolar lumens of control mice (Fig. 5, C and D). In contrast, very little milk protein was detected in either epithelial cells or alveolar lumens in Tag mice using immunohistochemistry (Fig. 5, A and B). Western blot analysis demonstrated decreased levels of WAP protein in Tag mice and reduced posttranslational processing (Fig.
Discussion This
study
protein
demonstrates
in developing
that
mammary
expression
gland
of a viral
alters
function during
(Fig. 7). Shortly after Tag synthesis pregnancy, mammary epithelial cells
dergo
PCD. At the same
but
did
not
undergo
time,
apoptosis
cells were
which
onco-
cell fate and commenced began to un-
expressed
unable
Tag
to produce
and secrete milk proteins. This occurred despite the presence of sufficient steady-state levels of the corresponding RNAs.
Control
WAP-Tag
Cell Growth
life, while
during
1234
following
5678
the mammary
each
lactation.
that
there
and
p53-dependent
are distinct
morigenesis. ing pregnancy play
The marked infers that
a protective
plexus
roles
role
through
raise
the
for p53-independent during
PCD
apoptosis
mammary
gland
tu-
induction of apoptosis by Tag durp53-independent apoptosis could
in early
(4), it is possible
steps
that
of Tag-induced
development
apoptosis
of breast
7
possibility
tumori-
since resistance to p53-dependent with tumor progression in the
p53-independent gression
involutes
results
apoptosis
genesis. However, tosis is associated
WAP
gland
These
& Differentiation
of resistance
pathways
cancer
in these
apopchoroid
contributes
to
to
pro-
mice.
bax and bcl-x family of genes mediate by forming homo- and heterodimers (1 8). The
Members
of the
apoptosis relative
levels
termine
whether
of each
Steady-state
or
levels
(20), and
protein
of both
bcl-xL±S
in an individual
a cell
not
RNA
will
bax,
which
were
cell
undergo
may
de-
apoptosis
promotes
increased
in the
(19).
cell
death
Tag
mice
coincident with the appearance of PCD. Bcl-x can be transcribed and processed into two different RNA forms with
Tag
opposing
functions
form
inhibits
and
and accelerates
Because
(21
22). Bcl-xL
,
PCD.
Bcl-x
cell death
is the most
is expressed
in transfection
abundant
at lower
studies
levels
in vitro
(21).
inducing
of its lower expression levels, the role of bcl-x PCD in vivo has not been obvious (22). Similar
previous
studies,
we found
that
bcl-xL
was
in to
the predominant
RNA form expressed. However, a relative increase in bcl-x expression as compared to bcl-xL expression was detected coincident
Fig. 4. Milk protein RNA expression in control and Tag mice. Northern blot analysis of steady-state RNA levels of 3-casein, WAP, a-lactalbumin, and Tag at late pregnancy in control and Tag mice. Each lane represents a mammary gland taken from a different mouse. Lane 1, day 1 7 of pregnancy; Lane 2, day 1 8; Lane 3, day 18; Lane 4, day 1 9; Lane 5, day 17; Lane 6, day 18; Lane 7, day 18; Lane 8, day 19. Ethidium bromide staining of 28S and 185 RNA was used as a loading standard.
with
optotic
cells in the developing
distinctly
abnormal.
expressed manly
domains
coincident tosis was gland.
of Tag
is important study
Different
related
pRb-binding
ofTag portion
plexus
(1-4),
Since
both
expressed
with the concept in normal
In support
Expression
pRb-
p53-
cell
functional types
gland
hypothesis,
that mammary
gland
a
involution
to developmental
needs.
cellular tion.
For instance,
Milk
conditions
have different capacities to activate either p53-depenor p53-independent pathways (1 6, 1 7). This could be the
Although
Pregnancy cells
in impaired sufficient Intraluminal
(23), these p53.
Led to Impaired
of Lactation.
did not destroy
milk protein steady-state milk
in the presence
the
protein
of Tag.
Tag
alveolar
production amounts of secretion
Western
blot
was anal-
yses indicated that the milk protein present was not processed and secreted properly. Some WAP precursor was on Western
blots
either
within tissue that
but not on immunocytochemistry,
function
prior
proteins
protein direct
WAP, which and lumen,
sections.
expression whether
is not secreted is not efficiently
These results
of a viral
to evidence
It is not known
of nonmilk physiological
resulted despite
to
gland.
of the bax gene does not require
epithelial
mRNAs.
reduced
recognized
p53 (11).
or different
protein
onstrated
development
we performed
in-
contribute
mammary
and Failure
suggesting that unprocessed into the endoplasmic reticulum
that p53-independent
form
in the
in mammary
detected
tissue
short
Production
structures but and secretion,
of Tag and
the
of Tag during
Protein
milk
is
toward
of apoptosis
p53 can activate transcription studies suggest that activation
sharply
pri-
a similar
may
efficiency
mice was
it induces
in mammary
mammary
of this
that demonstrated
does not require may dent
were
gland
with PCD, we suggest that p53-independent apopactivated by expression of Tag in the mammary
involution.
related
the
apoptosis.
This is consistent
apoptosis and
When
in the lens or choroid
p53-dependent
binding
mammary
Cells
Since
ing
appearance
synthesis of ap-
apoptosis.
described during nor(11), increased splic-
Milk Tag-induced Apoptosis in Mammary Epithelial during Pregnancy. The appearance of large numbers
Tag-induced
crease in bcl-x expression has been mal involution of the mammary gland
oncoprotein
of malignant
the production
further
dem-
impaired transforma-
and
processing
were also affected.
production or indirect
in Tag mechanisms.
mice
may
It does
be inhibited not appear
by that
lens and
choroid plexus develop just once, while mammary gland development repeats with each pregnancy. The lens and choroid plexus do not normally undergo extensive apoptosis
K. Heermeier and L Hennighausen. Bax and bcl-x onset of apoptosis in involuting mammary epithelial 4
publication.
are induced at the cells, submitted for
8
SV4O Tag Alters
Mammary
Cell Fate and Function
A.
,
,
‘
.
T+
c\ ;*
“
p,
‘A
Fig. 5. lmmunoNstochemic (4-0) and Western blot (E) analyses of WAP and total milk proteins in Tag (A and B) and control (C and D) mice. Immunohistochemical analyses for the presence of WAP (A and C) and total milk protein (B and D) was performed. E, Western blot analysis of steady-state levels of WAP protein at late pregnancy in control (Lanes 1 and 2) and Tag (Lanes 3-5) mice. Each lane represents a mammary gland taken from a different mouse. Lane 1, day 1 8 of pregnancy; Lane 2, day 18; Lane 3, day 18; Lane 4, day 19; Lane 5, day 18. The upper band (open arrow) points to the WAP precursor, and the lower band (solid arrow) points to mature WAP. Coomassie Blue staining of the protein gel revealed equal loading of all samples.
.,,
,
A..’
4, .k
‘
1
.
‘
‘*.‘
,
4P
r
1
S
‘n.4”
:9’
‘‘.
.-
.
..
.
-.
.
‘4,
.,
‘b
E pre WAP
I’ll’.
WAP’
12345 Control
production. Tag synthesis might affect milk protein production by association with pRb family members and dysregulation of cell cycle control (1-4). In muscle cells, functional pRb is required for terminal differentiation, which is coupled
WAP-Tag
5678
1234
with
cessation
protein
w D N Ml
of cell
production
epithelial
cells
ofWDNM1 RNA in Tag mice. Northern blot analysis of steady-state RNA levels of WDNM1 and TGFf31 at late pregnancy in control (Lanes 1-4) and Tag (Lanes 5-8) mice. Each lane represents a mammary gland taken from a different mouse. Lane 1, day 1 7 of pregnancy; Lane 2, day 18; Lane 3, day 18; Lane 4, day 19; Lane 5, day 17; Lane 6, day 18; Lane 7, day 18; Lane 8, day 19. Ethidium bromide staining of 285 and 185 RNA was used as a loading control and is illustrated in Fig. 4.
direct apoptotic alveolar
structures
expression
destruction and
of the gland milk
protein
is responsible mRNAs
Tag
with
the ability
could
the cell cycle
and
mammary
tissue,
In normal
inhibit
milk
of mammary undergo
termi-
epithelial
cell
proliferation falls during late pregnancy, coincident with the onset of milk protein synthesis. pRb or a related family member could be required for terminal differentiation and exit
TGF1 Differential
(24).
to exit from
nal differentiation.
Fig. 6.
divisions
by interfering
are
since present
throughout late pregnancy and parturition. In Tag mice, mammary gland involution and lobulo-alveolar collapse with reduced levels of milk protein mRNAs does not occur until after parturition and lactation failure (1 1). There are several mechanisms through which Tag could act to inhibit milk
from
the cell cycle
of mammary
epithelial
cells.
Exit from
the
cell cycle could be required for proper and abundant milk protein synthesis. This hypothesis can be tested by generating
transgenic
tant Tag constructs sion
using
the
mice
that
carry
either
(2, 4). Conditional tet-responsive
system
truncated
control could
Tag
or mu-
of Tag expresbe
used
to
establish if the effect of Tag on lactation was reversible (25, 26). It appears less likely that interruption of p53 function by Tag was responsible for the inability to produce milk proteins since p53 -Imice lactate normally. It is, however, possible that these mice lactate in the absence of p53 due to cornpensation from another gene product selected for during embryonic development. The presence of Tag in mammary epithelial cells could block the required growth-suppressive effects of TGFI3 pro-
Cell Growth & Differentiation
I WAP-Tagmice Fig.
7.
effect
Mammary
gland
to alveolar
I Controlmice
ccIII::i:::I:::
Model illustrating the of disrupting the cell cy-
stemcella
cle by expression of Tag during mammary gland development.
rise
virgin
stem cells give structures
dur-
ing pregnancy. In Tag mice, expression of the viral oncoprotein
lowed
during
pregnancy
by induction
independent
PCD
cells.
Surviving
normal
amounts
is fol-
in
cells
late pregnancy
\
I-tlumen----1
of p53alveolar
o#{232}J
express
of milk protein
RNA but do not produce milk proteins. Expression of the tumor marker gene WDNM1 is down-regulated. In control mice, synthesis and secretion of milk proteins begins in late pregnancy. When pups begin to nurse, lactation starts, and the diameter of the alveolar lumen increases. In Tag mice, no milk protein production occurs, lactation cannot be established, and the lumen diameter remains small.
parturition
pups
4, begin
teins during pregnancy. TGF1 and TGFI33 are synthesized at high levels in the mammary gland during pregnancy and decrease at parturition (1 4). Their growth-inhibitory properties may be necessary for alveolar cells to proceed into terminal differentiation at late pregnancy. TGFI3 family members block cell proliferation, in part, by maintaining pRb in an active hypophosphorylated form. Tag binding to pRb would interrupt this effect on mammary epithelial cells (27, 28). In addition, the association of Tag with pRb would result in release of E2F. Elevated levels of E2F1 expression can block growth suppressive effects of TGFf31 in cell culture (29). It is possible that Tag synthesis affected fat-related gene expression or synthesis since the mammary epithelial cells of Tag mice exhibit a marked reduction in the number of fat globules. Impairment of fat production similar to that seen for milk protein production would contribute to poor milk production. Finally, since Tag can interact with other cellular proteins than pRb and p53, it is possible that one of these interactions also contributed to the impairment of milk production (30-34). In summary, expression of Tag in mammary epithelial cells during pregnancy altered mammary epithelial cell fate and function. The induction of apoptosis in the presence of Tag illustrates the important role of p53-independent apoptosis pathways in the mammary gland. The failure of milk protein production following Tag expression indicates that factors beyond transcriptional control of milk protein gene expression are required for efficient milk production and lactation.
0
milk
N
WENM1
and Methods
Mice, Analysis of NursIng Behavior, Cross-Fostering, Mammary Gland BIopsies, and Tumor Specimens. Transgenic mice carrying a WAP-Tag hybrid gene were a gift from Adolf Graessmann (Freie
protein
RNA
protein
Universit#{228}t, Berlin, Germany; Ref. 5). The WAP-Tag hybrid gene consists of a 1600-bp WAPgene promoter(BgllI-Kpnl fragment) linked to the 5V40 early coding region (Bglll-BamHI fragment) containing the coding sequence
for both
large
Tag
and small Tag. Progeny
mice containing
the
WAP-Tag transgene
were screened using the PCR. The transgene was identified using primers corresponding to the WAP promoter from nudeotide nt -88 to nt -68 (5’ TAG AGC TGT GCC AGC CTC TTC 3’) and Tag sequences from nt -4950 to nt -4931 (5’ CAG MG CCT CCA AAG TCA
GG 3’). Both transgenic
and nontransgenic
littermates
were observed
and
analyzed during pregnancy and after parturition. Pups bom to these mice were observed after delivery and assessed for suckling behavior and the presence of milk in the stomachs. To cross-foster pups, matings of transgenic and nontransgenic females were synchronized, and the offspring of transgenic and nontransgenic mice were exchanged within 24 h of delivery. Mammary gland biopsies were performed between days 16 and 19 of
the first pregnancy.
Mice were anesthetized
using 0.7 ml of 0.175%
avertin
i.p. Under sterile conditions, the inguinal mammary gland from either the right or left side was exposed and removed. The mice recovered from the anesthesia uneventfully and went on to deliver at the normal time. Tumor specimens used in the present study were obtained from two different mice harvested by biopsy of the affected mammary gland. These
tumors first appeared
after three pregnancies.
Mammary Gland Whole-Mount Preparations. Each whole mammary gland specimen was spread on a glass slide and fixed in Camoy’s solution (100% ethanol:chloroform:glacial acetic acid, 6:3:1) for 60 mm at room temperature. Following fixation, the glands were washed with 70% ethanol for 15 mm, followed by a wash with distilled water for 5 mm. The staining of the glands was performed in carmine alum (1 g carmine; Sigma Chemical Co., St. Louis, MO) and 2.5 g aluminum potassium sulfate (Sigma) in 500 ml water) at 4#{176}C ovemlght. The tissues were then dehy-
drated and mounted on glass slides using routine methods. HIstologIcal Examination and lmmunohlstochemlstry. gland
Transgenic
to nurse
74 milk
Materials
9
specimens
were fixed
In 1 0% neutral
formalin
solution
Mammary and embed-
ded in paraffin using routine methods. Five-p.m tissue sections were prepared using routine methods for hematoxylin and eosin staining and for the detection was
detected
technology,
of Tag protein, WAP, and total milk protein. Tag protein using the monoclonal antibody Pab 1 01 (Santa Cruz BioInc., Santa Cruz, CA). Tissue sections were inffially treated
10
SV4O
Tag Afters
wfth pepsin
(2-10
Cell Fate and
Mammary
g/ml
in 0.01
N
Function
HCI buffer)
for 15 mm at room
temper-
ature and quenched
with 0.03% H202 in PBS for 30 mm at room temperature. After treatment with normal horse serum for 30 mm at room temperature, the specimens were incubated for 1 h with a 1 :1000 dilution of PablOl, followed by an incubation for 1 h with biotinylated horse antimouse lgG at a I :400-500 dilution (Vectastain ABC kft; Vector, L.aboratories, Inc., Burlingame, CA). The color reaction was performed with 0.05% 3,3’-dimethylaminoazobenzene (Sigma) and 0.01 % H202. Sections were counterstained with hematoxylin. WAP was detected using an anti-WAP polyclonal antibody raised in rabbits (35). Tissue sections were treated with pepsin, followed by quenching as described above. After blocking with normal goat serum for 1 h at room temperature, the spec-
imens were incubated for 1 h with a 1 :200-400
dilution
Tween 20] for 1 h at room temperature,
the membranes
Acknowledgments We thank Gertraud Robinson, Gilbert Smith, David Kerr, and Robert for helpful discussion and Albert Lewis for technical help.
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were exposed to
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