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This is to certify that the

dissertation entitled

The role of ovarian hormones, age and mamnary
gland development in polyomavirus mamnary tumorigenesis

presented by

Rachel Mae Heindel

has been accepted towards fulfillment
of the requirements for

Ph . D. Microbiology

degree in

 

 

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Date February 21, 1994

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TO AVOID FINES return on or before date duo.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU I. An Affirmative Action/Emmi Opponunity Institution
Wan-m

-.__————___.

THE ROLE OF OVARIAN HORMONES, AGE AND MAMMARY GLAND
DEVELOPMENT IN POLYOMAVIRUS MAMMARY TUMORIGENESIS

BY

Rachel Mae Heindel

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Microbiology
Cell and Molecular Biology Program

1994

ABSTRACT

THE ROLE OF OVARIAN HORMONES, AGE AND MAMMARY GLAND
DEVELOPMENT IN POLYOMAVIRUS MAMMARY TUMORIGENESIS

BY

Rachel Mae Heindel

Polyomavirus (Py) infection of adult athymic female mice
causes a high incidence of mammary adenocarcinomas. In this
thesis, I have examined the role of ovarian hormones, age and
mammary gland development at the time of infection in Py
infection and tumorigenesis. Ovary intact mice were infected
at 3, 6, 10, 20 and 30 weeks of age with Py A2. In addition,
mice were ovariectomized (OVX) at 5, 9 and 19 weeks of age and
infected 1 week later. Mice were also OVX at 3 weeks, 1 week
or 48 hours before or 1 week after infection at 6 weeks of
age. To determine the effect of proliferation and
differentiation on tumor induction, eight or 10 week old mice
that were OVX and treated with 1 ug 17-beta estradiol (E) and
1 mg progesterone (P) for 3 or 1 weeks before infection and
late pregnant mice were also infected. OVX-saline treated

mice served as controls. All mice were palpated for 14 weeks

post infection (pi) for tumors or killed at 10 days pi to
assess the levels of viral genomes, early protein and middle
T kinase activity in their mammary glands. Mammary tumor
incidence and number were significantly decreased and mammary
tumor latency was significantly increased with increasing age
at the time of infection and by OVX at least 1 week before
infection. In addition, longer periods between OVX and
infection further decreased tumor induction. Late pregnant
mice with fully differentiated mammary glands remained
susceptible to Py transformation. ‘The severe effect of OVX on
tumorigenesis suggests a positive role for ovarian hormones in
this process and the target cell population may be influenced
by age and mammary development state. No significant
differences were observed in the levels of viral genomes (high
in all groups) or in viral early proteins and middle T kinase
activity (low in all groups) in mammary glands at 10 day pi.
However, the levels of all three were high in mammary tumors

and correlated with transformation.

To my parents

iv

ACKNOWLEDGMENTS

I would like to thank Drs. Michele M. Fluck and Sandra Z.
Haslam for their support and encouragement throughout this
thesis research. I also thank my guidance committee members,
Dr. Ronald Patterson, Dr. Richard Schwartz and Dr. Clifford
Welsch for their helpful discussions throughout the course of
this study; In addition, I would like to express my gratitude
to my lab co-workers, both in Dr. Fluck’s and Dr. Haslam's
labs for their acts of kindness and friendship. I would like
to thank Michael Tomaszewski for technical assistance.
Finally, I thank Michael Rondinelli for his love and complete
support of all my endeavors and for his assistance in

statistical analyses.

TABLE OF CONTENTS

List of Tables... ..................... . ...........

List of Figures ...................................

Chapter 1:

Chapter 2:

Chapter 3:

Literature Review .....................
List of References ....................

The role of ovarian hormones, age and mammar
gland development state in polyomavirus
mammary tumorigenesis.

Abstract ..............................
Introduction .........................
Materials and Methods .................
Results ...............................
Discussion ............................

List of References.. ..... . ............

Polyoma viral genome and early protein level
and middle T associated kinase activity in
mammary glands and mammary tumors of athymic
mice infected at different ages and ovarian
hormone responsiveness states.

Abstract ............... .... ...........
Introduction ..........................
Materials and Methods .................
Results ...............................
Discussion ............................

List of References ....................

vi

PAGE
vii

viii

Y

37
39
41
44
63
69

S

72
74
77
81
89
92

LIST OF TABLES

PAGE
Table 1. Skin and bone tumor induction by 14 weeks
post infection in athymic female mice
following polyomavirus A2 infection.. ........ 62

vii

Chapter 1:

Chapter 2:

Chapter 3:

Figure 1.
Figure 2.

Figure 3.

Figure 4.
Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure 11.

Figure 12.

Figure 13.

LIST OF FIGURES

PAGE

Physical map of the polyoma

virus genome.....................
Polyomavirus mediated signal
transduction.....................
Physical map of the enhancer
region of the polyomavirus A2
genome ....................... ....
Mouse mammary gland development..
Mammary gland wholemount analysis
of ovary intact and ovariectomized
mice at various ages .............
Effect of age on mammary tumor
induction................... .....
Effect of ovariectomy at various
ages on polyomavirus mammary tumor
induction ................ ........
Effect of ovariectomy at 6 weeks
of age ...................... .....
Mammary gland wholemount analysis
of ovariectomized, ovarian hormone
treated mice and late pregnant
mice ............. . ........... ....
Effect of mammary gland
proliferation and differentiation
on tumor induction ...............
Level of viral genomes in mammary
glands and mammary tumors ........
Level of viral early protein
expression in mammary glands and
mammary tumors. ...... ............
Level of middle T antigen
associated kinase activity in
mammary glands and mammary tumors.

viii

5

10

15
21

46

48

51

54

57

60

83

86

88

Chapter 1

LITERATURE REVI EW

Breast cancer is a major killer of North American women
and there has been an alarming and significant increase in
incidence for which there is no explanation (1). The National
Cancer Institute has predicted that one in nine women can
expect to develop breast cancer in her lifetime (2). While
the etiology of this disease has not been defined, there is
solid epidemiological evidence for an important role of
ovarian hormones in mammary cancer development.(3). A woman’s
total lifetime exposure to the ovarian hormones estrogen and
progesterone has been implicated in the etiology of breast
cancer (4). This hypothesis takes into account her age at
menarche, her age at first full-term pregnancy, her total
parity and her age at menopause. In addition to natural
exposure to ovarian hormones, women who have taken oral
contraceptives for long periods of time, starting at young
ages (<25 years) have an elevated risk for developing breast
cancer (5-7). A model that attempts to explain the risk
increases associated with total lifetime exposure to ovarian
hormones has been presented (5). This model emphasizes that
early menarche and older age at first full term pregnancy
result in higher levels of mammary proliferation due to longer
hormone.exposure and.correlate with.higher breast cancer risk.

It is this proliferative response of the mammary epithelium to

2

ovarian hormones that either creates a more susceptible gland
for transformation or induces a longer period of proliferation
that may act to expand a transformed cell population.
Therefore, it is apparent that this disease requires further
investigation into the mechanisms of neoplastic transformation
of the breast and the role of ovarian hormones in this
process.

Several experimental models have been investigated to
identify mechanisms of mammary neoplasia and the role of
ovarian hormones. A major model system involves the use of
chemical carcinogens to induce mammary tumors in rodents. N-
nitroso-n-methylurea (NMU) , 3-methylcholanthrene (3-MC or MCA)
and 7,12-dimethylbenz(a)anthracene (DMBA) have been used
extensively to induce mammary tumors in rodents since their
carcinogenic properties were first described (8-12). Pre-
cancerous ductal and alveolar dysplasias are induced by the
carcinogens and ovarian hormones are critically required for
tumor initiation and promotion (8, 9, 12-14).

In addition to chemical carcinogens, the mouse mammary
tumor virus (MMTV) has also been studied as a model of mammary
tumorigenesis (15). IMMTV infected 'mice develop :mammary
tumors, but only after undergoing several pregnancies,
suggesting that ovarian hormones are also critically required
for tumorigenesis in this model. Tumors are preceded by the
development of alveolar dysplasias and in contrast to
carcinogen induced tumors, MMTV induced tumors are ovarian

hormone independent for growth (16). Further research has

3

identified glucocorticoids (17, 18), progestins (19, 20),
prolactin (21) and androgens (20), as the positive regulating
molecules of the long terminal repeat (LTR) of MMTV. The LTR
controls gene expression of MTV and steroid hormones and
prolactin are therefore important in regulating the oncogenic
potential of MMTV. Thus in this model, hormones can interact
directly with the oncogenic agent.as well as act to expand the
transformed cell population.

Mouse polyomavirus (Py) is an important model system used
in the study of virally induced tumors in mice and cell
transformation in tissue culture. Py is a tumorigenic DNA
virus whose natural host is the mouse. Its genome is
comprised of 5292 base pairs with two coding regions, early
and late, and. a non-coding, enhancer-origin region. that
functions as the control region for viral transcription and
replication (see Figure 1). The early region codes for three
proteins, small, middle and large Tiantigens. Large Tiantigen
controls viral DNA replication and gene expression. Middle T
antigen also plays a role in viral gene expression and DNA
synthesis, as well as in capsid assembly. The function of
small T antigen, which is essentially a fragment of middle T
antigen, is poorly understood. The late region codes for the
three viral capsid proteins, VP1, VP2 and VP3.

Recently it has been reported that infection of adult
athymic, immuno-incompetent Balb/c mice with Py results in a
high incidence of mammary tumors with a short latency period

(23). Several aspects of the Py mammary tumor model are

Figure 1. Physical map of the polyomavirus genome. The outer
circles depict the two coding regions of the polyomavirus
genome, early (top half) and late (bottom half), each of which
produces.one transcript that is<differentially spliced (jagged
lines represent the introns) to produce three gene products.
The early region codes for three proteins, small T, middle T
and large T antigens. The late gene products are the capsid
proteins VP1, VP2 and VP3. The non-coding enhancer/origin
region is located between these two coding regions on the left
side of the map. The enhancer region is located between
nucleotides 5021 and 5262, on the late side of the origin.

This figure is taken from Soeda et a1. (22).

   
 
   
   

hssgf
BglI (87)

Barn H1(4631)

Figure 1.

6

intriguing and hold promise for providing some unique insights
into the mammary neoplastic process. Recently, the
histopathogenesis of Py-induced mammary tumors has been
characterized in 6 week old infected athymic female mice (24).
By 2 weeks post infection (pi), the mammary glands exhibit a
hyperplastic appearance, similar to that observed during early
to mid-pregnancy, characterized by moderate to extensive
ductal sidebranching. However, the mammary glands eventually
return to a normal state associated with the non-pregnant
condition. Ductal dysplasias are frequently observed and are
associated with the mammary adenocarcinomas, suggesting either
a precancerous nature of the ductal dysplasias or that they
are in fact, microscopic tumor foci. In addition, stromal
abnormalities are often observed in association with the
dysplasias and frank tumors. Similar morphological structures
occur in humans, such as hyperplasia and atypia of ducts and
lobules, and are associated with an increased relative risk
for breast cancer in humans (25) Therefore, the Py model
allows the study of early stages of tumor development that may
be similar to those occurring in human breast cancer.

Recent studies have revealed that ovarian hormones play
an important role in the induction of mammary tumors by Py
(26, 27). In this thesis, I have examined the effect of
ovariectomy at various times before and after Py infection and
I will present evidence that suggests that ovarian hormones
are important at the time of infection for Py mammary tumor

induction. These observations suggest that ovarian hormones

7

play an early role in the tumorigenic process. Thus, this
model system may provide unique insights into the role of
ovarian hormones in mammary carcinogenesis. Interestingly, Py
induced tumors appear to be ovarian hormone-independent for
continued growth (24, 26), similar to approximately 50% of
human breast cancers. Therefore, the Py tumor model also
provides an opportunity to investigate potential treatments of
hormonally unresponsive mammary cancers.

Many studies have examined the involvement of oncogenes
in human breast cancer and these findings have been recently
reviewed (28). The oncogenes that have been found to be
amplified or overexpressed in human breast tumors include myc,
myb, Ha-ras and neun ‘While these oncogenes are upregulated in
some human tumors, their involvement in the etiology of this
disease is unclear. To elucidate the role of oncogenes that
are implicated in human breast cancer, many lines of
transgenic mice have been created that overexpress these
oncogenes and induce tumors in their mammary glands (29-34).
To determine if any of these implicated oncogenes are involved
in the polyoma system, the expression of these various
oncogenes has also been recently investigated in infected
athymic female mice (24). This study found that with
increased time post infection, the level of c-myc gene
expression was slightly elevated. However, no increases were
observed in Ha-ras, c-neu, int-1 or int-2 gene expression,
indicating that transcriptional upregulation of these proto—

oncogenes does not appear to be a key step underlying Py

8
oncogenesis with the possible exception of c-myc.

The tumorigenic property of polyomavirus has been linked
to the action of middle T antigen (Figure 2). The mechanism
behind the neoplastic transformation induced by polyoma has
been suggested to be mediated by the interaction of middle T
antigen with pp60‘Hrc which activates c-src's tyrosine kinase
and phosphorylates middle T antigen (35). The activated
middle T/c-src complex can then associate with
phosphatidylinositol-3 (PI-3) kinase (36-41). Although the
metabolites of the PI-3 kinase are not cleaved by a known
phospholipase, this PI-3 kinase is suggested to play an
important role in growth control since middle T mutants that
are PI-3 kinase deficient have decreased transformation
capabilities (40, 42). In addition to its association with
pp60”", middle T antigen has also been reported to associate
with other protein kinases such as c-fyn and c-yes, both
tyrosine kinases, and can activate c-raf, a serine-threonine
kinase (43-46).

Middle T antigen has also been shown to activate the
protein kinase C (PKC) pathway (47, 48). This property of
middle T may be mediated by the interaction with phospholipase
C-gamma (49), activating the PI signal transduction pathway
downstream of PI kinase. Middle T antigen can also associate
with a protein phosphatase, pp2A (50). By interacting with
pp2A, middle T antigen may interfere with the regulation of
important proteins responsible for signal transduction and

cell cycle control. These perturbations caused by the

 

Figure 2. Polyomavirus mediated signal transduction.
PolyomaviruS'middle:Tiantigen (mT) has been shown to associate
with a variety of cellular proteins and the consequences of
these interactions may result in neoplastic transformation.
Py mT binds to c-src and activates c-src’s tyrosine kinase
activity which results in autophosphorylation of c-src and
phosphorylation of mT. The phosphorylated mT/c-src complex
can now associate with phosphatidylinositol-3 kinase (PIB-K).
PI3-K phosphorylates phosphatidylinositol (PI) on the 3
position, however, no known phospholipase can cleave this
metabolite. The mT/c-src complex has been shown to activate
phospholipase C-gamma (PLC) and.protein.kinase C (PKC). .Also,
the transcriptional activators.AP1 andic-ets, are activated by
middle TL In addition to c-src, mT can also associate in
complexeS‘with c-fyn, c-yes and.c-raf, other cellular kinases.
The consequences of these interactions have not been
determined. Middle T has also been shown to associate with a
cellular protein phosphatase, pp2A. Collectively or
individually, the perturbations that may be caused as a result
of mT interacting with these various cellular proteins may

lead to cellular transformation.

 

@633 assets 595 L 235 Egg

11

interaction of middle T antigen with cellular proteins may
play a role in Py transformation. It is also noteworthy to
mention that small T antigen, which is identical to the amino
terminus of middle T antigen except for 4 amino acids at its
carboxyl terminus can also associate with pp2A (50). The role
of small T in Py infection and transformation is not known but
it has been shown to cooperate with middle T in the
transformation of cells in culture by increasing foci growth
(51). Therefore, small T antigen may play a role in Py
transformation as well.

It has been shown that.polyomavirus replication, which is
bi-directional and mediated by large T antigen, is strongly
coupled to viral transcription (52). Studies by de Villiers
et al. (53) and Veldman et al. (54) demonstrated that polyoma
replication requires an enhancer. Indeed, the mutation of
individual nucleotides within the enhancer simultaneously
affected both viral DNA replication and transcription,
suggesting that these two molecular processes may share a
common mechanism (55). Due to this link, the study of the
polyoma enhancer region which is located between nucleotides
5020 and 5265 has been facilitated, since enhancer function
can be measured in terms of the amount of viral DNA that has
been replicated. The Py enhancer has been previously
implicated in the control of organ specific viral replication
in the mouse (56, 57).

Previous studies that examined vira1.DNA.replication in

different organs of both the neonate as well as the adult

12

mouse, revealed an organ specific pattern of polyoma
replication. After infection with Py, neonate mice exhibit
high levels of viral DNA replication throughout most of the
mouse with the exception of the brain and blood (56, 58, 59).
This stage of viremia, which peaks at 7-10 days post infection
(pi) is followed by almost complete viral clearance by the
host immune system. Infection of adult normal mice results in
very low levels of viral replication and clearance of the
virus by the immune system of the mouse. However, when adult
athymic, immuno-deficient mice are infected, moderate levels
of replication occur, resulting in the accumulation of high
copy numbers of viral genomes in long-term infections (59).
The three major sites of Py replication in the athymic adult
mouse are the mammary gland, skin and bone.

From these results, four broadly defined organ specific
patterns of Py replication have been identified in the mouse.
They include organs with 1) high levels of replication in
neonates and moderate levels in adults (i.e. mammary gland,
skin and bone), 2) high levels of replication in neonates and
very low levels in adults (i.e. kidney and to a lesser extent,
the liver and lung), 3) higher levels of replication in adult
females compared to adult males (i.e. mammary gland) and 4)
low levels of replication in both neonates and adults (i.e.
brain and blood) (59).

These age related, organ-specific viral replication
patterns observed in neonatally infected and adult infected

mice have led our laboratory to examine the effect of the non-

13

coding enhancer region within the polyoma genome as a possible
site controlling these patterns. The Py enhancer has been
segmented into domains and sub-domains, based primarily upon
restriction enzyme recognition sites. Very generally, it has
been split into two domains, A and B (Figure 3). The effects
of enhancer rearrangements on the pattern of replication of
the A22 genome in neonatally infected Balb/c mice were
previously investigated (56, 57). These studies demonstrated
that rearrangements within the enhancer, involving deletions
of the B domain and duplications of the A domain of Py,
severely decreased viral replication in the kidney, liver and
lung of the neonate mouse. However, these rearrangements did
not greatly affect replication in the mammary gland, skin and
bone of either neonate or athymic adult mice (57). These
studies suggested that the B domain is important for viral
replication in all organs at neonatal infection. In terms of
the mammary gland, these results suggest that either the B
domain has no mammary control element or that the duplications
within the A enhancer domain can compensate for the B
deletion. In either case, a major control of Py replication
in the mammary gland lies outside of the B enhancer domain and
it is the A domain that is implicated in this control.

The sites of polyoma tumorigenesis also show an age-,
organ- and sex-specific pattern. The oncogenicity of wild-
type strains of polyomaviruses was extensively studied in the
1960's and has been recently reviewed (60). Infection of

neonatal mice with the wild type strain A2 results in a

14

Figure 3. Physical map of the enhancer region of the
polyomavirus A2 genome. The polyomavirus enhancer has been
divided into two very general domains, A and B. PEAl, the
mouse homologue to AP1, and PEA3, the mouse homologue to c-
ets, both bind to multiple sites within the A and B domains.

This figure was designed by Larry G. Martin.

15

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16

variety of tumors of both epithelial and mesenchymal origin.
The major types include tumors of the mammary gland, skin,
bone, salivary glands and kidney (sarcomas). This review
described.over 30 different cell types that are susceptible to
infection and approximately 23 sites for tumorigenesis by
polyomavirus in neonatal mice. While mice that are neonatally
infected with Py are susceptible to the virus and develop
these tumors later in life, infection of normal adult mice
results in rapid immune clearance of the virus and no tumors
develop.

However, infection of adult athymic female mice with Py
results in the induction of mainly mammary adenocarcinomas and
osteosarcomas (23, 61). Mammary adenocarcinomas arise in a
very short period after infection (6 weeks) and at a very high
incidence (96%) in ovary intact athymic female Balb/c mice.
Infection of adult athymic male mice results mainly in
osteosarcomas; but no mammary tumors are induced (23). The
basis for the lack of mammary tumor development in males is
not known but it is intriguing that the virus does replicate
in the male mammary gland, albeit at lower levels (59).
Howeverq a threshold leve1.of organ specific viral replication
may not be all that is required for organ specific
tumorigenesis. Since Py does not cause tumors in all organs
in which it replicates, Py may require the cells to possess
additional characteristics for transformation. It may be that
these characteristics are present only in the adult female

mammary gland and are regulated by ovarian hormones. The

17
control of this mammary-tropic viral replication and sex-
specific mammary tumorigenesis remains to be elucidated.
It is possible that the organ-specificity as well as the
sex-specificity of Py replication and tumorigenesis are
mediated. by transcription factors that bind to enhancer

sequences. As discussed above, I vivo experiments with B

 

enhancer deletion mutants suggested that the major control of
Py replication in the adult mammary gland lies outside of the
B enhancer domain and implicated the A.domain (57). These age
dependent, organ-specific viral replication patterns have led
to the ongoing examination of the Py enhancer region for
specific sequences that control these patterns (62). Within
the A enhancer domain, there are known binding sites for the
transcriptional activators PEAI and PEA3 which are the mouse
homologues to API and c-ets, respectively (see Figure 3).
Therefore, the binding of these activators to the A enhancer
domain of Py may control viral replication and gene expression
in the mouse mammary gland“ The influence of ovarian hormones
on the activity of these factors may help to explain the sex-
specific pattern of Py mammary tumorigenesis.

Recent cell culture studies have shown that Py middle T
antigen activates PEAl (47) and PEA3 (63). This is taken as
evidence that middle T antigen induces a signal transduction
pathway which activates protein kinase C as described above.
PEA3 has recently been shown to cooperate with the PEAl
components, c-Fos and c-Jun, to activate transcription through

their binding sites in the Py enhancer (64). PEAl can exist

18

as a combination of protein complexes comprised of c-Fos, b-
Jun and/or c-Jun dimers (65). Both estrogen and progesterone
are able to increase expression of the human c-fos gene and
consequently, APl (66, 67). c-fos gene expression is induced
by estrogen in HeLa cells and by progesterone in T-47D human
breast cancer cells. If PEA1 is also induced in the mouse
mammary gland in response to ovarian hormones, this might
explain the ability of Py to replicate to higher levels in the
female mammary gland. However, this thesis will show that
ovariectomy does not decrease the level of Py genomes in the
mammary gland but clearly affects the transformation of the
gland. It remains plausible that an ovarian hormone regulated
factor(s) is required for Py transformation of the mammary
gland. It may be that an indirect factor (i.e. cellular
metabolic state, proliferative status, etc.) is upregulated in
the ovary intact gland and makes the gland immediately
susceptible to Py transformation which is reflected in short
latencies and high mammary tumor incidence.

Indeed, the activation of PEA1 and PEA3 by middle T
antigen in the mammary gland might play a major role in the
neoplastic cascade induced by the virus. A positive feedback
loop may be created by the middle T induced signal
transduction cascade, with PEA1 and PEA3 increasing the
expression of not only Py genes but also cellular genes that
may play a crucial role in transforming the mammary
epithelium. It may be that the ovary intact female mammary

gland may contain a factor(s) that mediates the organ-tropism

19
of viral replication and tumorigenesis. This factor may not
be present or may not function optimally in the male mammary
gland.

In.order to understand the possible basis for sex-related
patterns of tumorigenesis it is necessary to understand the
differences in the development of mammary glands of male and
female mice. These differences have been reviewed (68) and
are presented in Figure 4. During the first half of
gestation, mammary gland development is similar for both
sexes. At 13-15 days of gestation, androgen secretion begins
in the male embryo and the growth of the mammary epithelium is
halted and the primary duct detaches from the nipple region,
leaving only a rudimentary gland. The male gland does not
undergo any further development without hormonal manipulation.
By contrast the epithelial rudiment of the female continues to
grow'and branch; this embryonic growth is hormone independent.
The rudimentary gland remains quiescent and unresponsive to
estrogen and progesterone until puberty at 3-4 weeks of age
with the onset of ovarian cycles (68). At this time, the
epithelium proliferates in response to estrogen and extensive
ducts develop and elongate. It is also at this age that the
epithelium can respond to epidermal growth factor (EGF) which
induces proliferation and ductal elongation (69). Therefore,
at this age, the mammary gland is very proliferative in
response to estrogen and EGF, but is not yet responsive to
progesterone (70). The mammary epithelium continues to grow

until it reaches the boundaries of its stromal fat pad by 8-10

20

Figure 4. Mouse mammary gland development. In both sexes
during early gestation, the mammary epithelial bud is formed
and begins to grow. However, unlike the female primary duct
that continues to grow and branch until birth, the male
epithelial growth is halted at mid-gestation by the increase
in circulating androgens. The epithelium regresses and the
primary duct detaches from the nipple. At birth, the female
mammary gland remains rudimentary and very little growth
occurs until puberty at approximately 3 to 5 weeks of age. At
this time, the ductal epithelium elongates in response to
estrogen (E) and epidermal growth factor (EGF). At 7 to 8
weeks of age, the mammary gland now responds to E by inducing
epithelial progesterone :receptors (PgR) which, confer
responsiveness tijrogesterone (P). E’induces sidebranches at
this age. At 10 weeks of age, EGF receptors (EGFR) can be
induced by E and P and more extensive sidebranches are

induced. Increased sidebranching occurs as the mouse ages.

21

 

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22

weeks. At 10 weeks of age, the gland begins to develop
sidebranches as a result of the induction of progesterone
receptors.by estrogen and the acquisition of responsiveness to
progesterone (70-72). EGF receptors are inducible by estrogen
and progesterone at 10 weeks of age and confer a different
responsiveness to EGF which can now induce extensive
sidebranching to create a more differentiated gland (73). By
20 and 30 weeks of age, more extensive sidebranching occurs
(74). With the onset of pregnancy, in response to the
elevated levels of estrogen, progesterone, EGF and placental
hormones, extensive epithelial proliferation and morphogenesis
take place that produce a fully differentiated gland (74).
After parturition, with the onset of lactation, the mammary
gland becomes refractory to estrogen and progesterone (75).

Therefore, as the female mouse ages, the mammary gland
progresses through different morphological states that have
varying degrees of proliferation and differentiation. During
the early growth stages from puberty until about 10 weeks of
age, the gland is very proliferative, especially at the
terminal end buds (76). As the gland begins to develop
sidebranches at 10 weeks of age, it becomes more
differentiated, as these sidebranches are the structures from
which lobuloalveoli will form if the mouse becomes pregnant
(77). The proliferative end buds are present from puberty
until the epithelium reaches the boundaries of the stromal fat
pad between 8 and 10 weeks of age. It is at this time that

the end buds regress and the proliferative activity of the

I!!!

23
gland decreases (76, 78). At later ages, more sidebranching
and lobule development occurs creating a more differentiated
gland (74).

One specific difference between male and female mammary
glands that may determine sex-related differences in tumor
development may be differences in ovarian hormone and growth
factor receptor expression. The ontogeny of estrogen,
progesterone and EGF receptors and mammary gland
responsiveness to these factors has been described for the
female mouse (69-71, 79). Estrogen receptors are present in
the female mouse mammary gland at 3 days of age but the gland
does not respond to estrogen until at least 3-5 weeks of age.
At 3-5 weeks of age, the mouse mammary gland proliferates in
response to estrogen treatment, but it is not until 7 weeks of
age that.the:gland.produces progesterone receptors in response
to estrogen. The inability to induce progesterone receptors
before this age suggests a difference in the response to
estrogen or function of the estrogen receptors at different
developmental stages. Progesterone receptors are present in
low concentrations in the stromal cells of the mouse mammary
gland.at.5 weeks of age but are not responsive to progesterone
until 7 weeks of age. The responsiveness to progesterone has
been shown to correlate with the presence of the estrogen-
inducible progesterone receptors in the mammary epithelium
(70). EGF receptors are also present in immature animals (80)
but are not inducible by estrogen and progesterone until 10

weeks of age (73). Thus, the ability of the female mammary

 

24
gland to respond to ovarian hormones could account for the
difference in tumorigenesis between males and females. If a
sequential developmental process, such as the acquistion of
hormone receptors, that is influenced by ovarian hormones and
growth factors must occur to create a susceptible gland for Py
tumorigenesis, then the male mammary gland may not provide an
optimal target for Py transformation.

It is possible that the rudimentary development of the
mammary gland in male mice is not sufficient to create optimal
conditions for mammary tumorigenesis similar to that of the
females. One might expect that by increasing the number of
target epithelial cells, the chance for transformation by the
virus will also increase. In this regard, it is of interest
that Py infection of the neonate mouse is associated with high
levels of replication in the mammary gland (56, 59) and that
mammary tumors do appear in both sexes later in adulthood
(60). Thus, it is unlikely that the size of the gland alone
is a sufficient explanation for the lack of mammary tumors in
adult athymic male mice. A possible explanation for mammary
tumor induction in neonatally infected males may be that the
neonate mouse tissues which contain high numbers of stem
cells, may permit a sufficient level of replication and gene
expression for subsequent mammary tumorigenesis or have a
higher number/ratio of susceptible cells than adult male mice.
This could be explained by the presence of 1) a factor that is
ovarian hormone independent in the neonate and subsequently

hormonally regulated in the adult or 2) a unique

25

characteristic in the neonate male and female as compared to
the adult female mammary gland. Thus, by infecting the male
mouse neonatally, the virus may escape the sex specificity for
mammary tumor development that is present in the adult mouse.

Clearly, there is something about the adult female mouse
mammary gland that allows the production of tumors by Py.
Previous studies have examined the effect of age and
proliferative status on the induction of mammary tumors by
chemical carcinogens and have observed age dependencies for
initiation (11, 81-83). This age effect is inherent in the
tissue "age" and not the animal age or its accompanying
hormonal milieu as donor mammary glands from young rats are
equally susceptible to DMBA when transplanted into young or
old rats (1JJ. Whereas, older donor mammary glands remain
less susceptible to DMBA tumor induction, even when
transplanted into young animals. 11:has been shown that young
animals have more terminal end buds that are highly
proliferative (84) and.the presence of these proliferative end
buds in rats correlates with the ability of carcinogens to
induce mammary tumors, suggesting that they are the target
sites for transformation (82, 85-89). Sharkey and Bruce (76)
have demonstrated the targeting effect of ionizing radiation,
DMBA and NMU to the terminal end buds of 7 week old mice by
quantitating nuclear aberrations. Younger animals had higher
tumor incidence and shorter latency periods than.older animals
that do not have the end buds and have more differentiated

mammary glands that are generally much less proliferative.

.‘I

 

26

In this regard, high levels of cellular proliferation are
also observed in intralobular terminal ducts, the. human
equivalent to the mouse terminal end bud (90). This study
also identified increased growth fractions and longer S phases
in the terminal ducts of younger women. If rapidly
proliferating cells are the targets for carcinogenic agents,
then younger animals and women may be more susceptible to
initiation events. Indeed, increased breast cancer incidence
has been observed in young women (ages 10-20 years) who were
exposed to repeated fluoroscopic chest examinations and
ionizing radiation during the atomic bombing of.Japan compared
to their older counterparts (91, 92).

The age effect on carcinogenesis may also be due to the
process of differentiation that occurs as the animal ages (82,
86, 88, 89, 93). Several studies have shown protective
effects of full term. pregnancy, lactation and chorionic
gonadotropin, a placental hormone, on carcinogen induced
mammary tumors, suggesting that more differentiated cells are
less susceptible to initiation and/or promotion of neoplastic
foci (94-96). The target cell population for the carcinogen
is decreased in late pregnant and lactating mammary glands
that are fully differentiated and less proliferative (89).
However, when carcinogens are given before or at mid-
pregnancy, high mammary tumor incidence is induced, which may
be due to high proliferation rates at these stages of
development (97).

In this regard, I have infected ovary intact or

 

 

27
ovariectomized athymic mice with Py at various ages to
determine if the stage of mammary gland development and
hormonal responsiveness influences the level of Py genomes,
early viral protein and/or middle T associated kinase activity
and mammary tumorigenesis. If the cells in which Py can
replicate and produce middle T antigen differ due to the state
of the gland, one may expect different responses to Py at
various ages and developmental states. ‘This study may provide
clues about the nature of ovarian hormone- and age-related

influences on mammary tumorigenesis.

 

10.

11.

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nitroso-N-methylurea administration. J. Natl. Cancer
Inst., 1g:209-212, 1983.

Sinha, D. K., J. E. Pazik and T. L. Dao. Progression of
rat mammary development with age and its relationship to
carcinogenesis by a chemical carcinogen. Int. J. Cancer,
31:321-327, 1983.

Meranze, D. R., M. Gruenstein and M. B. Shimkin. Effect
of age and sex on the development of neoplasms in Wistar
rats receiving a single intragastric instillation of
7,12-dimethylbenz(a)anthracene. Int. J. Cancer, 4:480-
486, 1969.

Korfsmeier, K. H. Proliferation kinetics in the mammary
gland of the mouse during postnatal development. Anat.
Anz., 145:313-318, 1979.

Russo, J., J. Saby, W. M. Isenberg and I. H. Russo.
Pathogenesis of mammary carcinomas induced in rats by
7,12-dimethylbenz(a)anthracene. J. Natl. Cancer Inst.,
59:435-445, 1977.

Russo, J., G. Wilgus and I. H. Russo. Susceptibility of
the mammary gland to carcinogenesis. I. Differentiation
of the mammary gland as determinant of tumor incidence
and type of lesion. Am. J. Path., 96:721-733, 1979.

Haslam, S. Z. The effect of age on the histopathogenesis
of 7 , 12-dimethylbenz (a) -anthracene-induced mammary tumors
in the Lewis rat. Int. J. Cancer, 26:349-356, 1980.

88.

89.

90.

91.

92.

93.

94.

95.

96.

36

Russo, J. and I. H. Russo. DNA labeling index and
structure.of the rat mammary gland as determinants of its
susceptibility to carcinogenesis. J. Natl. Cancer Inst. ,
61:1451-1457. 1978.

Russo, J., L. K. Tay and I. H. Russo. Differentiation of
the mammary gland and susceptibility to carcinogenesis.
Breast Cancer Res. Treat., 2:5-73, 1982.

Russo, J., G. Calaf, L. Roi and I. H. Russo. Influence
of age and gland topography on cell kinetics of normal

human breast tissue. J. Natl. Cancer Inst., 18:413-418,
1987.

Boice, Jr., J. D. and R. R. Monson. Breast cancer in
women after repeated fluoroscopic examinations of the
chest. J. Natl. Cancer Inst., 52:823-832, 1977.

Tokunaga, M., J. E. Norman, Jr., M. Asano, S. Tokuoka, H.
Ezaki, I. Nishimori and Y. Tsuji. Malignant breast
tumors among atomic bomb survivors, Hiroshima and
Nagasaki, 1950-74. J. Natl. Cancer Inst., 6;:1347-1359,
1979.

Russo, I. H. and J. Russo. Developmental stage of the

rat mammary gland as determinant of its susceptibility to
7,12-dimethylbenz(a)anthracene. J. Natl. Cancer Inst.,
6;:1439-1445, 1978.

Dao, T. L., F. G. Bock and M. J. Greiner. Mammary
carcinogenesis by 3-methylcholanthrene. II. Inhibitory
effect of pregnancy and Lactation on Tumor Induction. .J.
Natl. Cancer Inst., 25:991-1002, 1960.

Grubbs, C. J., D. L. Hill, K. C. McDonough and J. C.
Peckham. N-nitroso-N-methylurea-induced mammary
carcinogenesis: Effect of Pregnancy on preneoplastic
cells. J. Natl. Cancer Inst., 11:625-628, 1983.

I. H. Russo, M. Koszalka, P. A. Gimotty and J. Russo.
Protective effects of chorionic gonadotropin on DMBA-
induced mammary carcinogenesis. Br. J. Cancer, 62:243-
247, 1990.

Chapter 2

The role of ovarian hormones, age and mammary gland

development state in polyomavirus mammary tumorigenesis.

ABSTRACT

Polyomavirus (Py) infection of adult athymic female mice
causes a high incidence of mammary adenocarcinomas. I have
examined the role of ovarian hormones, age and mammary gland
development at the time of infection in Py tumorigenesis.
Ovary intact mice were infected at 3, 6, 10, 20 and 30 weeks
of age with Py A2. In addition, mice were ovariectomized
(OVX) at 5, 9 and 19 weeks of age and were infected 1 week
later. To establish the time at which ovarian hormones are
required for tumorigenesis, mice were OVX at 3 weeks, 1 week
or 48 hours before or 1 week after infection at 6 weeks of
age. To determine the effect of proliferation and
differentiation on tumor induction, eight or 10 week old mice
that were OVX and treated with 1 ug 17-beta estradiol (E) and
1 mg progesterone (P) for 3 or 1 weeks before infection and
late pregnant mice were also infected. OVX-saline treated
mice served as controls. All mice were palpated for 14 weeks
post infection (pi). Mammary tumor incidence and number were
significantly decreased and mammary tumor latency was
significantly increased with increasing age at the time of

infection. In addition, OVX at older ages (10 and 20 weeks)

37

38
drastically reduced or eliminated tumor induction. Since OVX
at least 1 week before infection significantly reduced
tumorigenesis, ovarian hormones are important at the time of
infection. In addition, longer periods between OVX and
infection further decreased tumor induction. Late pregnant
mice with fully differentiated mammary glands remained
susceptible to Py transformation, suggesting that the decrease
in Py tumorigenicity in older animals is not simply due to
differentiation but may be due to the loss of proliferation or
certain target cell types that are present in young mice. 'The
drastic effect of OVX on tumorigenesis suggests a positive
role for ovarian hormones in the poloymavirus carcinogenic
process and it may be that the target cell population is

influenced by both age and mammary development state.

INTRODUCTION

Mouse polyomavirus (Py), a DNA tumor virus, is an
important model system used in the study of virally induced
tumors in mice and cell transformation in tissue culture.
Previous studies that examined viral DNA replication in
different organs of both the neonate as well as the adult
mouse revealed an age- and organ-specific pattern of Py
replication (1).

The sites of Py tumorigenesis in adult athymic mice also
show an age-, organ- and sex-specific pattern (2, 3).
Infection of adult athymic, immuno-incompetent Balb/c female
mice results in a high incidence of mammary tumors with a
short latency period (3, 4). .Athymic male mice do not develop
mammary tumors (3). However, the induction of other types of
tumors does occur but is somewhat less frequent than the
mammary tumors and the basis for this difference is not known.
Although it has been shown that ovarian.hormones are important
at or around the time of Py infection for mammary tumors to
develop in female mice (3, 4), the role of the ovarian
hormones in this process is not known. It is possible that an
appropriate state of mammary development and/or ovarian
hormone responsiveness of the gland may be required for Py
induction of tumors. Therefore, the purpose of the present
study was to define the effect of ovarian hormones on Py
mammary tumorigenesis and to investigate the transformation

susceptibility of the mouse mammary gland to Py at various

39

40
ages and different developmental states.

I report here that ovarian hormones are required at least
one week before Py infection for high levels of mammary tumor
induction. In addition, older animals (20 and 30 weeks of
age) are less susceptible to Py tumorigenesis than their 6 and
10 week old counterparts. Ovariectomy decreased tumor
incidence when performed one week before Py infection.at.6 and
10 weeks of age and eliminated tumor induction at 20 weeks of
age. This study has examined the ability of Py to induce
tumors in fully differentiated, late pregnant mammary glands
of athymic mice. These mice developed mammary tumors at a
high incidence, suggesting that the decrease in mammary tumor
incidence in older mice is not simply due to a more
differentiated mammary gland and may be related to the loss of
proliferative target cells or another type of

"differentiation" that occurs as the nulliparous mouse ages.

MATERIALS AND METHODS

Animals:

All athymic mice were obtained from Life Sciences, Inc.
and were housed in our Vivarium in a sterile laminar flow rack
and given sterile water and rodent chow (Purina) ad libitum.
Mice were infected subcutaneously with 10" plaque forming

units (pfu) of the wild type strain A2 of Py.

Treatments:

Female mice were infected with Py at 3, 6, 10, 20 and 30
weeks of age to assess the influence of different stages of
mammary gland development on mammary tumor induction. In
addition, 5, 9 and 19 week old mice were OVX before infection
at 6, 10 and 20 weeks of age, respectively. Two mice from
each group were killed on the day of infection by cervical
dislocation and the mammary glands were removed and
wholemounts were prepared and stained as previously described
(5) to assess the state of morphological development at the
time of infection. To examine the role of ovarian hormones at
the time.of Py infection, mice were bilaterally ovariectomized
(OVX) at 3 weeks, 1 week or 48 hours before or 1 week after
infection at 6 weeks of age. Ovary intact mice served as
controls.

To determine the effect of increasing differentiation on

41

42

Py mammary tumor induction, 8 or 10 week old mice were OVX and
given daily subcutaneous injections of 1 ug 17-beta estradiol
(Sigma) + 1 mg progesterone (Sigma) in 5% gum arabic (Sigma)
for 3 weeks or 1 week, respectively before infection. Ten
week old, ovary intact and age-matched and OVX-saline treated
mice served as controls. In addition, 8 week old mice were
mated with normal Balb/c male mice from our own colony for
timed pregnancies. All mice were infected with Py at late
pregnancy, 18-19 days gestation, and allowed to deliver and
nurse their progeny. All late pregnant mice were infected by
13 weeks of age. Two mice from each group were killed on the
day of infection by cervical dislocation and the mammary
glands were removed to determine the extent of morphological
development at the time of infection as described above.

All mice were palpated weekly for the appearance of
tumors. These mice were killed by cervical dislocation when
moribund or when tumor load exceeded more than 10% of their
body weight. Tumor incidence, number and latency' were
assessed as of 14 weeks post infection (pi). Mammary glands
and mammary tumors were removed at the time of death and were
analyzed for viral genome level, early protein production and
middle T associated kinase activity and these results are

presented in Chapter 3.

43

Parameter and statistical calculations:

Tumor incidence was determined by dividing the number of
mice that develOped tumors by the total number of mice in an
experimental group. Average mammary tumor number per mouse
was determined by dividing the total number of mammary tumors
that appeared in an experimental group by the number of mice
in the group. .Average mammary tumor latency was calculated by
assigning each tumor with the week pi that it was first
palpable and determining the mean for all tumors that appeared
within an experimental group.

All statistical analyses were performed using the SYSTAT
(SYSTAT, Inc.) statistical software package on a Packard Bell
386SX-II computer. Tumor incidence data were analyzed for
significance by Chi square analysis. Average mammary tumor
number per mouse and average mammary tumor latency data were
analyzed for significance:by Kruskal-Wallis tests, followed by
Mann-Whitney analyses with Bonferroni p value adjustments for

the number of comparisons.

RESULTS

Effect of age and hormonal responsiveness on tumor induction.

To examine the effect of various ages that are
characterized by various degrees of mammary epithelial
development and ovarian hormone responsiveness on the
induction of mammary tumors by Py, female mice were infected
at 3, 6, 10, 20 and 30 weeks of age. In addition, 5, 9, and
19 week old mice were OVX and then infected at 6, 10, or 20
weeks of age, respectively, to determine the effect of ovarian
hormone withdrawal at these ages on Py tumorigenesis.
Wholemount analysis of mammary glands taken from these mice on
the day of infection depict the expected morphology for that
age and ovarian status (Figure 5). These wholemounts
illustrate that each age is morphologically different. .As the
mouse ages or if it is OVX, the proliferative end buds are
lost. Increasing age creates a more differentiated gland with
respect to the amount of sidebranching that was present.

Mammary tumor incidence of the ovary intact mice groups
is shown in Figure 6a. This figure clearly demonstrates that
after 10 weeks of age, the susceptibility of the mouse mammary
gland to Py tumorigenesis is significantly decreased (55%;
p<0.05) as compared to that of younger mice (96%). This same
age influence was also observed in the average number of

mammary tumors that were induced (Figure 6b), with

44

45

Figure 5. Mammary gland wholemount analysis of ovary intact
and ovariectomized mice at various ages. Mammary glands were
removed from ovary intact mice at 3, 6, 10, 20 or 30 weeks of
age and from mice that were ovariectomized (OVX) for 1 week
before infection at 20 weeks of age. The bulbous,
proliferative end buds of the young glands (a and b) are lost
as the epithelium reaches the boundaries of the stromal fat
pad by 10 weeks of age (c). sidebranching begins at 10 weeks
of age and becomes more extensive with increasing age (d and
e). 1Pane&.f demonstrates the regression that occurs when mice
are OVX for 1 week. The ducts become thin and sidebranching

is diminished. LN, lymph node. Magnification x 42.

46

 

Figure 5.

47

Figure 6. Effect of age on mammary tumor induction. Ovary
intact mice at 3, 6, 10, 20 and 30 weeks of age were infected
with polyomavirus and followed for mammary tumor induction.
All statistical comparisons were made versus the 6 week old
mice, with a p value < 0.05 constituting significance (*).
(a). Mammary tumor incidence, n values represent the number
of mice in each group. (b). .Average mammary tumor number per
mouse, n values represent the number of mice. (c). Average

mammary tumor latency, n values represent the number of

mammary tumors.

48

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Figure 6.

49

significant decreases apparent in the 20 and 30 week age
groups (2 and 0.18, respectively) as compared to the 6 week
group (4.3; p<0.0125). Significantly longer average mammary
tumor latencies were observed in mice infected at 10 weeks of
age and older (1.9, 2.4 and 3.5 weeks, respectively;
p<0.0125) (Figure 6c). These results may reflect the changes
that are occurring developmentally and hormonally within the
mammary gland between these ages. The mammary gland is
vigorously growing at 6 weeks of age as the end buds
proliferate and penetrate the fat pad. However, by 10 weeks
of age, the end buds have reached the boundaries of the fat
pad, they have regressed and the gland is less proliferative
than that of younger mice. If tumor latency is determined by
the rate of target cell proliferation, then the decrease in
proliferation that occurs as the mouse ages (6) correlates
well with the increase in tumor latency and may determine the
lower tumor incidence and number that was observed in these
older groups as well.

The effect of OVX at these various ages on Py mammary
tumorigenesiS‘was also»determined. Figure 7a illustrates that
OVX 1 week before infection at 6, 10 or 20 weeks of age
significantly reduced (61% and 11%; p<0.05), and in the case
of the 20 week OVX group, eliminated mammary tumor induction
by Py as compared to the ovary intact, age-matched controls
(96%, 100% and 56%, respectively). Similarly, OVX 1 week

before Py infection at these various ages also significantly

50

Figure 7. Effect of ovariectomy at various ages on mammary
tumor induction. Mice were ovariectomized (OVX) at 5, 9 or 19
weeks of age before polyomavirus infection at 6, 10 and 20
weeks of age, respectively and were monitored for mammary
tumors. All statistical comparisons were made versus age-
matched, ovary intact controls, with a p value < 0.05
constituting significance (*) . (a) . Mammary tumor incidence,
n values represent the number of mice in each group. (b).
Average mammary tumor number, n values represent the number of
mice per group. (c). .Average mammary tumor latency, n values
represent the number of mammary tumors. Statistical analyses
were not performed on the 20 week OVX group because no tumors

were induced.

51

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Figure 7.

52

decreased (p<0.017) the average number of mammary tumors per
mouse by at least 4 fold (Figure 7b). It is intriguing that
between 6 and 10 weeks of age, tumor induction appeared to
become more sensitive to the withdrawal of ovarian hormones
than at the younger ages. However, OVX only had an effect on
average tumor latency when performed at 20 weeks of age

(Figure 7c). These results indicate a progression to strict
ovarian hormone dependence of Py for the induction of mammary

tumors in older aged mice.

Effect of ovariectomy at 6 weeks of age.

The next set of experiments was designed to examine the
role of ovarian hormones on the induction of mammary tumors.
Female mice were OVX at 3 weeks, 1 week or 48 hours before or
1 week after infection at 6 weeks of age. The animals were
palpated weekly to monitor the appearance of mammary tumors.
The results are shown in Figure 8a. In mice that were OVX at
3 weeks or 1 week before infection, the incidence of mammary
tumors*was significantly'decreased (25% and 61%, respectively)
as compared to the ovary intact controls (96%; p<0.05).
However, no difference was observed in mice that were OVX 48
hours before or 1 week after infection. .Average mammary tumor
number was also significantly decreased by 4 fold (p<0.017) in
mice that were OVX at least 1 week before infection (Figure

8b) while OVX at other times before or after infection had no

53

Figure 8. Effect of ovariectomy at 6 weeks of age. Mice were
ovariectomized (OVX) at 3 weeks, 1 week or 48 hours before or
at 1 week after infection with polyomavirus A2 at 6 weeks of
age. Ovary intact, 6 week old mice served as controls. All
mice ‘were followed for' mammary tumor incidence, average
mammary tumor number per mouse and average mammary tumor
latency. All statistical comparisons were made versus the

intact mice, with a p value < 0.05 constituting significance

(*). (a). Mammary tumor incidence, n values represent the
number of mice in each group. (b). Average mammary tumor
number per mouse. (c). Average mammary tumor latency, n

values represent the total number of mammary tumors.

54

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at 6 Weeks of Age

Figure 8.

55
effect. However, average mammary tumor latency was only
significantly increased when mice were OVX 3 weeks before
infection (Figure BC). This increased latency for tumor
appearance is reflected as well in tumor incidence and number
for this group. These results indicate that ovarian hormones
are required at the time of infection for Py mammary tumor

induction.

Effect of different proliferative and differentiated mammary

states on tumor induction.

To determine the effect of increasing differentiation on
Py induced mammary tumorigenesis, 8 or 10 week old mice were
OVX and given daily injections of 1 ug 17-beta estradiol (E)
«+ 1 mg progesterone (P) in 5% gum arabic for 3 weeks or 1
week, respectively before infection. One week of E + P is
expected to create an early pregnant-like, proliferative gland
while 3 weeks of E + P should create a late pregnant-like,
differentiated mammary gland. Ten week old, ovary intact and
age-matched and OVX-saline treated mice served as controls.
In addition, 8 week old mice were mated with normal male mice,
infected with Py at 18-19 days gestation and allowed to
deliver and nurse their progeny.

Mammary gland wholemounts taken from a subset of these
mice on the day of infection are shown in Figure 9 and

demonstrate the morphological changes that were induced by

56

Figure 9. Mammary gland wholemount analysis of
ovariectomized, ovarian hormone treated mice and late pregnant
mice. Mammary glands were removed on the day of infection
from a subset of mice that were ovariectomized (OVX) at 8 or
10 weeks of age and given daily injections of 1 ug 17-beta
estradiol (E) + 1 mg progesterone (P) for 3 weeks or 1 week,
respectively, before infection. The mammary glands of late
pregnant.mice were also removed on the day of virus infection.
Ten week old, ovary intact and OVX-NaCl treated mice served as
controls. Panels a and b demonstrate the increase in
sidebranching that occurs after 1 week and 3 weeks of E + P
injections, respectively. Panel b represents a slightly more
differentiated gland, similar to that created during early-
pregnancy. Panels c and d show the effect of the length of
OVX for 1 or 3 weeks, respectively. Note the greater extent
of ductal thinning and diminished sidebranching in the longer
OVX gland (d). Panel e demonstrates the extent of
lobuloalveolar development that occurs in the late pregnant
mouse mammary gland, conferring complete functional
differentiation. Panel f depicts the mammary gland of an
ovary intact 10 week old mouse. LN, lymph node.

Magnification x 42.

57

 

Figure 9.

58

OVX, length of E + P administration and pregnancy. In this
study, 3 weeks of E + P treatment induced only a mid-pregnant
like gland, suggesting that the response to exogenous hormones
is different in athymic mice than in normal mice. .Regardless,
the mammary glands of the groups examined are different from
each other in their extent of epithelial proliferation and
differentiation. With increased length of E + P treatment,
increased sidebranching occurs, creating a more differentiated
gland. In addition, a fully differentiated gland was achieved
in the late pregnant mice.

Tumor incidence was not significantly affected by
increasing amounts of epithelial differentiation when the 3
week E + P and pregnant groups were compared to the 10 week
old ovary intact controls (Figure 10a). In addition, these
results also demonstrated the effect of the length of OVX on
tumor incidence. Among the saline treated controls, OVX for
3 weeks before infection eliminated tumor induction by 14
weeks pi while mice that were OVX for 1 week before infection
remained susceptible to Py mammary tumorigenesis, albeit at
significantly lower levels (38%; p<0.05). ‘Tumor incidence was
significantly lower in the 1 week E + P treated group (50%:
p<0.05) which is interesting in that this length and dose of
treatment, which induces a highly proliferative gland in
normal mice, did not cause such a response in the athymic mice
(Figure 9), again suggesting that athymic mice have a less

efficient response to exogeneous hormone treatment. Thus,

59

Figure 10. Effect of mammary gland proliferation and
differentiation on tumor induction. Mice were ovariectomized
(OVX) at 8 or 10 weeks of age and given daily subcutaneous
injections of 1 ug 17-beta estradiol (E) + 1 mg progesterone
(P) for 3 weeks or 1‘week, respectively, before infection with
polyomavirus. Late pregnant mice were also infected with
polyomavirus” 'Ten‘week old, ovary intact and OVX-NaCl treated
mice served as controls. All mice were monitored for mammary
tumor induction. All statistical comparisons were made versus
the 10 week old ovary intact controls, with a p value < 0.05
constituting significance (*) . (a). Mammary tumor incidence,
n values represent the number of mice in each group. (b).
Average mammary tumor number per mouse, n values represent the
number of mice. Statistical analyses were not performed on
the 3 week OVX-NaCl treated group because no tumors were
induced. (c). Average mammary tumor latency, n values

represent the number of mammary tumors.

>

Mammary Tumor Intidence (X)

Average Mammary Tumor
Number per Mouse

n

Average Mammary Tumor LatenCy
(weeks)

 

60

    

 

 

 

 

 

 

    

 

 

 

 

 

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90 1r
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Differentiation
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Ovarian Hormone Status and Mammary Gland
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15 n=2 _
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NaCI E + P

Ovarian Hormone Status and Mammary Gland
Differentiation

Figure 10.

61
these glands are more like OVX mammary glands than hormone
treated glands.

Theiaverage number of mammary'tumors determined for these
experimental groups followed the same pattern as that seen in
mammary tumor incidence (Figure 10b) . Interestingly, average
mammary tumor latency (Figure 10c) was significantly shorter
by 1 week in the pregnant infected group than the ovary intact
nulliparous mice (p<0.01), while tumor latency was increased
in the OVX-saline treated and 1 week and 3 week E + P treated
animals (1.1, 5 weeks and 1.4 weeks respectively; p<0.01).
Collectively, these data indicate that a fully differentiated
mammary gland such as that created in late pregnancy is
equally susceptible to Py tumorigenesis as that of the ovary
intact, nulliparous controls, suggesting that increased
epithelial differentiation is not the reason for decreased Py

tumor induction in older aged mice.
Induction of other tumors.

Table 1 describes the incidence of skin and bone tumors.
Older and OVX mice that do not have a high incidence of
mammary tumors are able to live longer and will develop skin
and osteosarcomas at later stages following Py infection.
Therefore, these treatments that lower mammary tumor incidence
may be desirable to create a skin or bone tumor model using

the Py system.

 

62

Table 1. Skin and bone tumor induction by 14 weeks post
infection in athymic female mice following polyomavirus A2

infection.

 

 

Skin Tumors Bone Tumors
Ovary Intact Females:
3 weeks 75% (6/8) 0% (0/8)
6 weeks 44% (12/27) 7% (2/27)
10 weeks 64% (7/11) 0% (0/11)
20 weeks 44% (4/9) 0% (0/9)
30 weeks 18% (2/11) 9% (1/11)
Ovariectomized (OVX) Females:
ovx -3 weeks 100% (8/8) 50% (4/8)
} infected at 6
OVX -1 week weeks of age 67% (12/18) 11% (2/18)
10 week OVX 78% (7/9) 56% (5/9)

20 week ovx 71% (5/7) 14% (1/7)

DISCUSSION

If ovarian hormones play a role in Py mammary tumor
induction, then one might expect mice that are infected at
various ages that are characterized by different mammary
developmental and ovarian hormone and growth factor responsive
states will exhibit different susceptibilities to Py
transformation. This study has investigated the developmental
and ovarian hormone responsive states required by Py for
mammary'tumorigenesisu From the results presented here, there
is an age effect on Py mammary tumor induction, with
increasing age resulting in decreased susceptibility to Py
transformation. In addition, OVX at all ages examined
decreased mammary tumor induction, with the most pronounced
effects observed at 10 and 20 weeks of age. The effects of
ovarian hormones on tumor promotion at these older ages were
not examined, but the present findings suggest that Py is more
dependent upon ovarian hormones at older ages for mammary
tumor induction. The effect of ovarian hormones after the
time of infection at these older ages warrants further
investigation. Interestingly, when mammary tumor-bearing mice
that were infected with Py at 6 weeks of age are
ovariectomized, the tumors do not regress, indicating that
they are not ovarian hormone dependent for growth (4, 7).
Based on the arguments presented above, the ovarian hormone
dependency of Py tumors that are induced in older mice might

be different and should also be determined.

63

 

 

 

64

The drastic effects of OVX at older ages on Py mammary
tumor induction suggests that a transition occurs between 6
and 10 weeks of age that confers more ovarian hormone
dependency for Py tumorigenesis. This is interesting in
regard to the hormonal responsiveness and morphological
changes that occur in the mammary gland between these ages.
At approximately 7 weeks of age, the gland acquires the
ability’ to induce progesterone receptors in response to
estrogen which confers progesterone responsiveness and
consequently, induces sidebranches in response to progesterone
(8-10). Increased sidebranching represents a more
differentiated gland, as these are the structures from which
lobuloalveoli will develop (11). As the epithelial end buds
reach the end of the stromal fat pad at approximately 8 to 10
weeks of age, these highly proliferative structures regress
(6, 12). EGF has also been shown to induce proliferation in
the epithelium at 5 weeks of age, independently of estrogen
and may actually mediate estrogen effects at this age (13).
Therefore, young mouse mammary glands may remain more
proliferative due to EGF. Indeed, ovarian hormone independent
epithelial growth does occur in young mice to atgreater extent
than in older mice (14). In contrast, the older mice may have
mammary glands that are more ovarian hormone dependent for
proliferation and these differences may reflect their
increased sensitivity to ovarian hormone withdrawal and
decreased tumor susceptibilities to Py that.were identified in

this study.

“_fi

 

65

While this study demonstrated no effect of OVX at 48
hours before or 1 week after Py infection on mammary tumor
profiles, an effect was observed when mice were OVX at least
1 week before Py infection. The tumor incidence results
presented here may at first, appear to conflict with the
results of Berebbi et al. (4) which showed that in mice
ovariectomized at 3 weeks of age, the presence of estrogen was
critical at a stage between 10 to 21 days pi for the
subsequent development of mammary tumors. Indeed, the length
of time between ovariectomy and Py infection was found to
influence mammary tumor induction, which may explain the
requirement for estrogen in their study. By removing the
ovaries at such an early age, the development of mammary gland
hormonal responsiveness and/or development may have been
arrested in Berebbi et al.’s study (4). Thus a specific
treatment period with estrogen may have been required for the
acquisition of an appropriate hormonal responsiveness or state
of development in these mice. The lack of an effect of OVX at
48 hours before and 1 week after Py infection in the present
study suggests that ovarian hormones do not function as
promoting agents for Py tumorigenesis in mice that are
infected at 6 weeks of age.

The fact that fully differentiated mammary glands present
in the late pregnant mice infected in this study remained
susceptible to Py tumorigenesis indicates that the decrease in
tumorigenicity that was observed in older mice is not simply

due to increasing differentiation of the mammary gland. These

1‘.

66

results may suggest the loss of a certain cell type or
population that is present in the young mouse that is the
primary target for Py transformation. In addition, it may be
that this population or another target cell type is also
present in the late pregnant mouse mammary gland, conferring
the high tumorigenicity that was observed in this study.
While chemical carcinogen models have shown protective effects
of late pregnancy/lactation exposure on subsequent tumor
induction (15-17), the mice infected in this study may have
had a high level of proliferation remaining in the gland that
allowed tumor induction. Alternatively, the mechanism of Py
transformation may be such that a pregnant gland and its
functionally differentiated cell types remain susceptible to
tumor induction, while older mice have another type of "age"
acquired differentiation that is less susceptible to Py.

It is noteworthy that the wholemount analysis presented
here (Figure.9) of epithelial development following 3 weeks of
E + P injections to OVX mice was expected to produce a late
pregnant-like gland, however, only an early-pregnant state of
development was attained, suggesting a different response to
exogenously administered ovarian hormones. Decreased
effectiveness of exogenously administered ovarian hormones has
been previously observed in athymic mice (20).

131this study, skin tumors and osteosarcomas were induced
in the mice in addition to mammary adenocarcinomas. It is
curious that skin and bone tumors appear to occur less

frequently than mammary tumors. This may be due to the

 

67

observation that when 6 week old, ovary intact mice were
infected, they developed mammary tumors very rapidly after
infection and became moribund before the development of other
types of tumors which appear to have much longer latencies.
Some of the older mice groups that did not develop mammary
tumors, did develop skin and bone tumors. Therefore, this
study has defined conditions to induce primarily skin and bone
tumors, which may provide an interesting model in which to
study these types of tumors. It is worth mentioning, that
while this study observed both osteosarcomas and skin tumors,
those of Berebbi et al. (3, 4) and Demengeot et al. (18) found
only osteosarcomas, even though the same strains of mice and
virus were used. It is difficult to reconcile these
differences of organ tropic tumorigenesis but it may be that
these two A2 strains may be different in their tumorgenicity
of the skin, as both strains do replicate to high levels in
the skin (1, 18). Alternatively, there may be discrete
differences between the two types of Balb/c mice that were
used in these two separate studies.

It is clear that the stage of mammary gland development
and degree of hormonal responsiveness at the time of Py
infection affect mammary tumorigenesis in athymic female mice.
Previously, age effects have also been demonstrated for
chemical carcinogen mammary tumor initiation (21-24). These
age effects correlate with the levels of proliferation in
young glands and the loss of proliferation in terminal end

buds in older mice and rat mammary glands as they age and

‘ ._'...-M
'5 ' ' ._. q

68
differentiate (24-28). The mammary cells in which Py
replicates and transforms have not been identified, however,
only adenocarcinomas are induced following viral infection,
suggesting that it is the epithelium that is the target for Py
transformation. If proliferative cells are the targets for
Py, older mice would be less likely to develop mammary tumors.
While in late pregnancy, the gland is functionally
differentiated, the level of proliferation probably remains
high until lactation. This may explain the induction of
mammary tumors following Py infection of late pregnant mice.
However, it may be that the functional differentiation that
occurs during pregnancy and age induced differentiation may
result in very different cell type populations that have
different susceptibilities to Py transformation. These
possibilities cannot be discerned by the present study but it
is imperative to identify the target cells for Py in order to
understand. the differences in :mammary cell types, their
transformation potential and the role of ovarian hormones in

this process.

 

 

 

10.

11.

12.

LIST OF REFERENCES

Wirth, J. J., A. Amalfitano, R. Gross, M. B. A. Oldstone
and M. M. Fluck. Organ- and age-specific replication of
polyomavirus in the mouse. J Virol. , 6_6_:3278-3286, 1992.

Dawe, C. J., R. Freund, G. Mandel, K. Ballmer-Hofer, D.
A. Talmage and T. L. Benjamin. Variations in polyoma
virus genotype in relation to tumor induction in mice.
Am. J. Path., l;l:243-261, 1987.

Berebbi, M., L. Dandolo, J. Hassoun, A. M. Bernard and D.
Blangy. Specific tissue targeting of polyoma virus
oncogenicity in athymic nude mice. Oncogene, 2:149-
156, 1988.

Berebbi, M., P. M. Martin, Y. Berthois, A. M. Bernard and
D. Blangy. Estradiol dependence of the specific mammary
tissue targeting of polyoma virus oncogenicity in nude
mice. Oncogene, §:505-509, 1990.

Banerjee, M. R., B. G. Wood, F. K. Lin and L. R. Crump.
Organ culture of whole mammary glands of the mouse.
Tissue Culture Assoc. Manual, 2:457, 1976.

Sharkey, S. M. and W. R. Bruce. Quantitation of nuclear
aberrations as a screen for agents damaging to mammary
epithelium. Carcinogenesis, 1:1991-1995, 1986.

Haslam, S. 2., J. J. Wirth, L. J. Counterman and M. M.
Fluck. Characterization of the mammary hyperplasia,
dysplasia and neoplasia induced in athymic female
adult mice by polyomavirus. Oncogene, 1:1295-1303, 1992.

Haslam, S. Z. Progesterone effects on deoxyribonucleic
acid synthesis in normal mouse mammary glands.
Endocrinol., 122:464-470, 1988.

Haslam, S. Z. The ontogeny of mouse mammary gland
responsiveness to ovarian steroid hormones. Endocrinol. ,
125:2766-2772, 1989.

Wang, S., L. J. Counterman and S. Z. Haslam.
Progesterone action in normal mouse mammary gland.
Endocrinology, 127:2183-2189, 1990.

Vonderhaar, B. K. IHormones and growth factors in mammary
gland development. In; C. M. Veneziale (ed.) , Control of
cell growth and proliferation, pp. 11-33. New York: Van
Nostrand-Reinhold, 1985.

Bertalanffy, F. D. Comparison of mitotic rate in normal

69

-Am

 

 

 

 

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

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renewing and neoplastic cell populations. Can. Cancer
Conf., Ottawa, 1:65-83, 1967.

Haslam, S. 2., L. J. Counterman and K. A. Nummy. Effects
of epidermal growth factor, estrogen, and progestin on
DNA synthesis in mammary cells in vivo are determined by
the developmental state of the gland” .J. Cell. Physiol.,
.;§§:72-78, 1993.

Haslam, S. 2., personal communication.

Dao, T. L., F. G. Back and M. J. Greiner. Mammary
carcinogenesis by 3-methylcholanthrene. II. Inhibitory
effect of pregnancy and Lactation on Tumor Induction. .J.
Natl. Cancer Inst., ;§:991-1002, 1960.

I. H. Russo, M. Koszalka, P. A. Gimotty and J. Russo.
Protective effects of chorionic gonadotropin on DMBA-
induced mammary carcinogenesis. Br. J. Cancer, 62:243-
247, 1990.

Grubbs, C. J., D. L. Hill, K. C. McDonough and J. C.
Peckham. N-nitroso-N-methylurea-induced mammary
carcinogenesis: Effect of Pregnancy on preneoplastic

Demengeot, J., J. Jacquemier, M. Torrente, D. Blangy and
M. Berebbi. Pattern of polyomavirus replication from
infection until tumor formation in the organs of athymic
nulnu mice. J. Virol., 64:5633-5639, 1990.

Neri, C., D. Blangy, B. Schatz, K. Drieu, M. Berebbi and
P-M. Martin. Direct inhibiting effects of [D-
mef]Gonadotropin—releasing hormone on the estrogen-
sensitive progression of polyoma virus-induced mammary
tumors in athymic mice. Cancer Res. , fl:5892-5897, 1990.

Nagasawa, H. and R. Yanai. Mammary growth and function
and pituitary prolactin secretion in female nude mice.
Acta Endocrinologica, 86:794-802, 1977.

Dao, T. L. Mammary cancer induction by 7,12-
dimethylbenz(a)anthracene: relation to age. Science,
16;:810-811, 1969.

cells. J. Natl. Cancer Inst., 11:625-628, 1983.

Meranze, D. R., M. Gruenstein and M. B. Shimkin. Effect
of age and sex on the development of neoplasms in Wistar
rats receiving a single intragastric instillation of
7,12-dimethylbenz(a)anthracene. Int. J. Cancer, 4:480-
486, 1969.

Grubbs, C. J., J. C. Peckham and K. D. Cato. Mammary
carcinogenesis in rats in relation to age at time of N-

 

 

24.

25.

26.

27.

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71

nitroso-N-methylurea administration. J. Natl. Cancer
Inst., 1g:209-212, 1983.

Sinha, D. K., J. E. Pazik and T. L. Dao. Progression of
rat mammary development with age and its relationship to
carcinogenesis by a chemical carcinogen. Int. J. Cancer,
Q1:321-327, 1983.

Russo, J., J. Saby, W. M. Isenberg and I. H. Russo.
Pathogenesis of mammary carcinomas induced in rats by
7,12-dimethylbenz(a)anthracene. J. Natl. Cancer Inst.,
£2:435-445, 1977.

Russo, J., G. Wilgus and I. H. Russo. Susceptibility of
the mammary gland to carcinogenesis. I. Differentiation
of the mammary gland as determinant of tumor incidence

and type of lesion. Am. J. Path., 26:721-733, 1979.

Haslam, S. Z. The effect of age on the histopathogenesis
of 7 , 12-dimethylbenz (a) -anthracene-induced mammary tumors
in the Lewis rat. Int. J. Cancer, 26:349-356, 1980.

Russo, J., L. K. Tay and I. H. Russo. Differentiation of
the mammary gland and susceptibility to carcinogenesis.
Breast Cancer Res. Treat., 2:5-73, 1982.

mm

 

Chapter 3

Polyoma viral genome and early protein levels and middle T
antigen associated kinase activity in the mammary gland and
tumors of athymic mice infected at different ages and

mammary gland development states.

ABSTRACT

Polyomavirus (Py) infection of adult athymic female mice
causes a high incidence of mammary adenocarcinomas. I have
examined the role of ovarian hormones, age and mammary gland
development at the time of infection in Py infection. Ovary
intact mice were infected at 3, 6, 10, 20 and 30 weeks of age
with Py.A2. lktaddition, mice were ovariectomized (OVX) at 5,
9 and 19 weeks of age and were infected 1 week later. In
addition, mice were OVX at 3 weeks, 1 week or 48 hours before
or 1 week after infection at,6 weeks of age. To determine the
effect of proliferation and differentiation on infection,
eight or 10 week old mice that were OVX and treated with 1 ug
17-beta estradiol (E) and 1 mg progesterone (P) for 3 or 1
weeks before infection and late pregnant mice were also
infected. OVX-saline treated mice served as controls. Mice
were killed at 10 days post infection (pi) to assess the
levels of viral genomes, early viral proteins and middle T
kinase activity in their mammary'glandsu ‘While no significant

differences were observed in the levels of viral genomes,

72

 

73
which were high in all groups or in early viral protein and
middle leinase activity levels (low in all groups) in mammary
glands at 10 days pi, these levels were very high in mammary
tumors“ These results suggest that the level of viral genomes
in the mammary glands at 10 days pi is not indicative of the
tumorigenicity of Py. However, the levels of genomes, early
proteins and middle T kinase activity correlated with Py
transformation and may reflect a threshold level of gene

expression that is required for transformation.

 

 

 

I NTRODUCTI ON

Mouse polyomavirus (Py), a DNA tumor virus, is an
important model system used in the study of virally induced
tumors in mice and cell transformation in tissue culture.
Previous studies that examined viral DNA replication in
different organs of both the neonate as well as the adult
mouse revealed an age and organ specific pattern of Py
replication. After infection with Py, neonate mice exhibit
high levels of viral genomes throughout most of the mouse with
the exception of the brain and blood (1—3). This stage of
viremia, which peaks at 7-10 days post infection (pi) is
followed by almost complete viral clearance by the host immune
system to establish a persistent stage of Py infection, with
some mice developing tumors later in life (4). Py infection
of normal adult mice results in a quick viral clearance and no
tumors are induced. However, when adult athymic mice are
infected with Py, moderate levels of viral DNA replication
occur in three major sites, the mammary gland, skin and bone
(3).

The level of viral genomes was previously analyzed in the
mammary glands of Py infected athymic female mice and found to
be variable, though relatively high at all stages of the
infection (3). However, when viral gene transcription was
analyzed, a higher level of gene expression was found to
correlate with the presence of mammary tumors. This study

suggested that the levels of viral replication and gene

74

 

 

 

75
expression may determine the ability of Py to transform.

The oncogenicity of Py has been linked to the action of
middle T antigen and its activation of a signal transduction
cascade that is similar to that which is induced by platelet
derived growth factor (PDGF) (5). Middle T has also been
shown to activate protein kinase C (6, 7). These functions of
middle T may culminate in the activation of important
transcriptional activators PEA1 and PEA3 (6, 8), both of which
are able to bind the Py enhancer and increase viral
replication and gene expression. In addition, cellular genes
may be overexpressed in Py infected cells and this
overexpression as well as other, yet undefined, actions of
middle T may lead to neoplastic transformation.
Interestingly, when oncogenes that are suspected to be
involved in human breast cancer were analyzed for
overexpression in the Py model, a slight increase was only
observed in c-myc expression, indicating that activation of
other proto-oncogenes does not appear to be a key step in Py
transformation (9).

Similar to viral replication patterns, the sites of Py
tumorigenesis in adult athymic mice also show an organ- and
sex-specific pattern (3, 10-12). Recent studies have revealed
that ovarian hormones play a role in this oncogenic process of
the female mouse mammary gland (12-13). Although it has been
shown that ovarian hormones are required at or around the time
of Py infection (12) for mammary tumors to develop, the role

of the ovarian hormones in Py tumorigenesis is not known. It

I.
. ‘ '1
NW' '11:."

‘1“
‘4

 

 

76

is possible that an appropriate state of mammary development
and/or ovarian hormone responsiveness of the gland may be
required for Py to induce mammary tumors. Therefore, the
purpose of the present study was to define the effect of
ovarian hormones on viral replication, early protein
expression and middle T antigen associated kinase activity in
relation to Py mammary tumorigenesis. Mice that were infected
at various ages which are characterized by different
developmental and. ovarian Ihormone responsive states 'were
investigated.

While ovarian hormones, age and mammary gland development
state influence mammary tumor induction (12, 13 and this
thesis, Chapter 2), I report here that the levels of viral
genomes, early proteins and middle T kinase activity in the
mammary gland are not influenced by these factors, at least at
the levels that can be detected by the methods used herein.
However, in almost all cases tested, viral genome, early
protein and middle T kinase activity levels were very high in

Py induced mammary tumors.

 

 

MATERIALS AND METHODS
Animals:

All athymic mice were obtained from Life Sciences, Inc.
and were housed in our Vivarium in a sterile laminar flow rack
and given sterile water and rodent chow (Purina) ad libitum.
Mice were infected subcutaneously with 106 plaque forming

units (pfu) of the wild type strain A2 of Py.
Treatments:

Female mice were infected with Py at 3, 6, 10, 20 and 30
weeks of age to assess the influence of different degrees of
ovarian hormone responsiveness and mammary epithelial
development on the levels of Py genomes, early viral proteins
and middle T kinase activity. In addition, 5, 9 and 19 week
old mice were OVX before infection with Py at 6, 10 and 20
weeks of age, respectively. To examine the role of ovarian
hormones at the time of Py infection, mice were bilaterally
ovariectomized (OVX) at 3 weeks, 1 week or 48 hours before or
1 week after infection at 6 weeks of age. Six week old, ovary
intact mice served as controls.

To determine the effect of increasing differentiation on
the levels of Py genomes, early viral proteins and middle T
kinase activity in the mammary gland, 8 or 10 week old mice

were OVX and given daily subcutaneous injections of 1 ug 17-

77

 

 

78

beta estradiol (Sigma) + 1 mg progesterone (Sigma) in 5% gum
arabic (Sigma) for 3 weeks or 1 week, respectively before
infection. Ten week old, ovary intact and age-matched and
OVX-saline treated mice served as controls. In addition, 8
week old mice were mated with normal Balb/c male mice from our
own colony for timed pregnancies. .All mice were infected with
Py at late pregnancy, 18-19 days gestation and allowed to
deliver and nurse their progeny. All late pregnant mice were
infected by 13 weeks of age.

The effect of age, ovarian status and different
developmental and hormone responsive states of the mouse
mammary gland on viral genome and early protein levels and
middle T kinase activity were compared to the tumor incidence

profiles of these mice that were determined in Chapter 2.

Viral genomes, early proteins and middle T kinase activity in

polyoma induced mammary tumors.

Mammary tumors that were induced in the experimental
groups examined in this study in Chapter 2 were analyzed for
the levels of viral genomes, early proteins and middle T

kinase activity as described below.

79

Viral genome, early protein and middle T kinase assays.

Two mice from each group were killed at 10 days post
infection (pi) and their mammary glands were removed. In
addition, mammary tumors that were induced at later times pi
were also removed at the time of death. All mammary glands
and mammary tumors were analyzed for the levels of viral
genomes, early proteins and.middleeT antigen associated kinase
activity. All lymph nodes were removed from the mammary
glands prior to the extractions to reduce the amount of non-
mammary DNA and protein.

Total DNA was extracted as previously described (9) and
analyzed for the level of viral genomes by dot blot analysis
following standard protocols (14). Five ug of total RNase-
treated DNA was blotted onto a Hybond-N membrane (Amersham)
and hybridized at 65°C:with.a random-primed, 32P-labeled probe
that represented the entire polyoma A2 genome. A polyomavirus
transformed FR3T3 cell line which contains approximately 1
viral genome per host cell was used as a control. Following
autoradiography, the levels of viral genomes were quantitated
using a beta scanner (Ambis). Representative samples from
each group are shown.

The level of viral early proteins was assessed in mammary
gland and tumor protein extracts by Western blot analysis
following' standard. protocols (14). Mammary' glands from
uninfected mice and wildtype Py A2 and mutant Py 18-5 (small

and middle T minus) infected NIH3T3 cells served as controls.

 

 

 

8O

Fifty ug of total protein was denatured and electrophoresed on
a 10% SDS-polyacrylamide gel. The gel was blotted onto PVDF
membrane (Dupont) and small, middle and large T antigens were
detected using a polyclonal rat anti-polyoma T antigen ascites
serum, a goat anti-rat horseradish peroxidase labelled
secondary antibody (Kirkegaard.and.Perry Laboratories) and the
Lumiglo chemiluminescent detection system (Kirkegaard and
Perry Laboratories).

In addition, the associated kinase activity of middle T
antigen within mammary gland and tumor protein extracts was
analyzed as previously described (15, 16). The middle T
antigen/protein complexes were immunoprecipitated from 500 ug
of protein extract using a polyclonal rat anti-Py T antigen
ascites fluid that was prepared in Norwegian brown rats
(Harlan). The immunoprecipitated complexes were assayed for
kinase activity in vitro using [gamma-32P]ATP (New England
Nuclear). The radiolabeled complexes were denatured and
electrophoresed on a 10% SDS-polyacrylamide gel and
autoradiography of the dried gel was performed at -70° C.

Representative samples from each group are shown.

 

RESULTS

To examine the role of ovarian hormones in Py mammary
tumorigenesis, female mice were infected at different ages
with Py to assess the influence of different stages of mammary
gland development and different degrees of ovarian hormone
responsiveness on the levels of Py genomes, early viral
proteins and middle T kinase activity.

While mammary tumor induction by Py was influenced by age
and ovarian hormone status (Chapter 2), the level of viral
genomes was not. As shown in Figure 11, increased age at the
time of Py infection did not significantly influence the
ability of the virus to replicate to high levels in the
mammary glands of ovary intact mice. As compared to the Py
transformed FR3T3 cell line that contains approximately 1
viral genome copy per host cell genome, the mammary glands of
these mice that were taken at 10 days pi exhibited viral
genome levels that ranged from 100 to 400 copies per cell. In
addition, OVX at these various ages or for different lengths
before or after Py infection had no significant effect on the
level of viral genomes in the mammary gland (data not shown).
Mammary tumors from these mice also had very high levels of
viral genomes, ranging from 200 to over 1,000 copies per host
cell.

The expression of the viral early proteins, small, middle

and large T antigen was examined in mammary glands and mammary

81

 

 

82

Figure 11. Level of viral genomes in mammary glands and
mammary tumors. (a). Ovary intact mice at 3, 6, 10, 20 and
30 weeks of age were infected and their mammary glands were
removed at 10 days post infection (pi). In addition, mammary
tumors were removed from mice at later times post infection.
A polyomavirus transformed FR3T3 (Py-FR3T3) cell line which
contains approximately 1 viral genome per host cell genome was
used as a control. DNA was isolated from the cell line,
mammary glands and tumors and 5 ug was transferred to a
Hybond-N membrane. The blot was hybridized with a 32P—labeled
probe that represented the entire polyomavirus A2 genome and
autoradiographed to detect viral sequences. Net counts per
minute (cpm) were determined by beta-scanning and demonstrate
the high level of viral genomes per cell in both mammary

glands and mammary tumors.

net cpm a

* Py-FR3T3

.I

3

_fl-nn.

—.._... ...+ s .n—n .. .1- —_-'—t'_ -' '- *'_ .l' I ‘- '
. ‘ 7‘ - . .
I II I I
‘ u l r I .4 ‘fi ‘ 2'. ' -~ “ ‘2‘.“ .1 “km. r‘fl - Q |

9

83

MG ‘IO days pi

6

II

'10 20 30

3".-."r ,‘

37 20 43 55 21

Figure 11.

Tumors

27 77 46

 
 

"2

 

 

84
tumors from mice that were infected at various ages and at
late pregnancy. Western analysis of mammary protein extracts
at 10 days pi revealed that the levels of Py early viral
proteins were undetectable (Figure 12a). However, all mammary
tumors that were examined expressed high but variable levels
of T antigens (Figure 12a and b).

When the level of middle:T associated kinase activity was
determined in the mammary glands of mice at various ages and
ovarian status at 10 days pi, it was undetectable in all mice
(Figure 13). However, the level of middle T kinase activity

in mammary tumors was again high but variable.

 

85

Figure 12. iLevel of viral early protein expression in mammary
glands and mammary tumors. Ovary intact mice at 3, 6, 10, 20
and 30 weeks of age and late pregnant (P) mice were infected
and their mammary glands were removed at 10 days post
infection (pi). In addition, mammary tumors were removed at
later times post infection. Uninfected (U) mammary glands and
polyoma A2 (large T +, middle T + and small T +) and 18-5
(large T +) infected NIH3T3 cells served as controls.

Proteins were extracted and 50 ug of total protein was
denatured and loaded onto a 10% SDS-polyacrylamide gel and
electrophoresed, blotted onto PVDF membrane and detected using
a polyclonal rat anti-polyoma T antigen ascites serum, a goat
anti-rat horseradish peroxidase labeled secondary antibody and
Lumiglo chemiluminescent detection system. (a). Western
analysis of mammary glands at 10 days pi and mammary tumors.
(b). Western analysis of additional mammary tumors. The
viral early proteins, large, middle and small T antigen are

indicated by arrows.

86

lOdays pi
Mammary Glands Tumors
U 3 6 10 20 30 P

«‘9
<2

 

1 large T
. .. #4in ‘ «middle T
v Q“ a small T
in
U Lmors 2 9

 

.----.-... «largeT

Q“O~Q.O "

-o.-.‘.-—-- «middleT

-£3-“‘ . a small T

Figure 12.

87

Figure 13. Level of middle T antigen associated kinase
activity in mammary glands and mammary tumors. Mammary glands
of ovary intact mice that were infected at 3, 6, 10, 20 or 30
weeks of age or at late pregnancy (P) were removed at 10 days
post infection (pi). Iniaddition, mammary tumors were removed
from mice at later times post infection. Uninfected (Uninf)
mammary glands and polyoma A2 infected NIH3T3 cells (A2)
served as controls. Polyclonal rat anti-polyoma T antigen
ascites serum (+) was used to immunoprecipitate middle T
antigen protein complexes from 500 1K; of protein extract.
Preimmune rat serum (-) was used as a control for each
immunoprecipitation. The immunoprecipitated complexes were
assayed for kinase activity in vitro using [gamma-”P]ATP. 'The
radiolabeled complexes were denatured and electrophoresed on
a 10% SDS-polyacrylamide gel. The level of middle T
associated kinase activity was determined by autoradiography

of the dried gel. *, radiolabeled middle T antigen.

88

 

3
—

on ON 2 o n
2053. CUE—ca: a gut O— l wanna—0 EOE—=32
e s 0 s
S d 3 d l I) n T .
m m m m. m M MW W a m m MW m T mm

 

Figure 13.

DISCUSSION

This study has concentrated on the analysis of
polyomavirusigenome level, early protein.expression.and middle
T antigen.associated kinase activity in the mammary glands and
mammary tumors of mice that were infected at various ages that
differ in mammary epithelial development and their response to
ovarian hormones and growth factors. The purpose of this
study was to explore the link between viral replication/gene
expression and tumorigenesis.

Although Py mammary tumor induction has been shown to be
influenced by ovarian hormones, age and perhaps mammary gland
development stage, the level of viral genomes remained high in
the mammary glands. This finding suggests that viral genome
copy number at 10 days pi is not indicative of the mammary
gland's susceptibility to Py mammary tumor formation.
However, the possibility exists that a threshold level of
replication is required _i_n addition to another factor(s).
Indeed, organ-tropic replication of the virus may not be all
that is required for organ-tropic tumorigenesis. It is
possible that only cells with certain transcription factors
will replicate the virus but since polyoma does not cause
tumors in all tissues in which it replicates, the cells may
need to express other factors that are targeted by middle T
antigen. It is possible that this additional factor(s)
required for Py mammary tumorigenesis is present in high

amounts in young female mouse mammary glands and allows rapid

89

90
and high mammary tumor induction.

In contrast, the levels of Py early T antigen protein
expression and middle T kinase activity were undetectable in
whole mammary gland extracts at 10 days pi. However, as shown
for the first time in this study, high levels of viral
replication, early viral protein and middle T antigen
associated kinase activity correlate with mammary tumors.
This observation supports the model of Py tumorigenesis that
occurs through the continual activation of the signal
transduction pathway(s) by middle T, leading to increased
kinase activity and uncontrolled cell proliferation.

It may be that Py gene expression is affected by age,
ovarian hormones and/or mammary gland development state within
the virus’ target cell populations. More than likely, a
dilution problem exists within the assays used in this study.
At the time post infection that the infected mammary glands
were examined, the ratio of infected cells to uninfected cells
is probably fairly low. Therefore, any effects on kinase
activity/gene expression that may occur due to age or ovarian
hormone status in the target cells may be diluted out by the
high percentage of uninfected cells that are present when the
proteins are extracted. Indeed, a threshold level of
transgene expression was required in neu transgenic mice
mammary glands for mammary tumors to be induced (17). The
level of neu expression was high in the mammary tumors, but
was lower in the surrounding normal tissue. Therefore, T

antigen immunocytochemical analysis of Py infected mammary

91
glands from the various age and ovarian hormone status groups
that were analyzed in this present study might resolve this
issue.

Overall, this study has shown that the levels of viral
genomes, early proteins and middle T antigen kinase activity
were not significantly affected by age, ovarian hormone status
or mammary gland differentiation, at least as detected by the
assays used herein. However, high levels of viral genomes,
early proteins and middle T kinase activity were observed in
the mammary tumors that Py induced in these mice and these
levels may be reflective of the mechanism by which Py

transforms the mouse mammary gland.

10.

11.

12.

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