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Eppley Institute for Research in Cancer and Allied Diseases, Department of Biochemistry and Molecular Biology, and Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARYREFERENCES |
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KEY WORDS: • estrogen • rats • pituitary tumor • mammary cancer • dietary energy restriction • prolactin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARYREFERENCES |
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, Hunter and Willett 1993
,
Slattery et al. 1997
, Willett 1997
).
Chronic treatment with estrogen induces development of prolactin
(PRL)6-producing pituitary tumors in a variety of inbred rat strains,
including Fischer 344 (F344) (Lloyd 1983
, Wiklund et al. 1981b
), ACI (Holtzman et al. 1979
,
Shull et al. 1997b
) and Copenhagen (Spady et al. 1998a
). Estrogens also induce development of mammary carcinoma
in certain inbred rat strains, with the ACI strain probably the most
notable (Dunning et al. 1947
, Shull et al. 1997b
). We hypothesize that dietary energy consumption may
modulate the ability of estrogens to regulate cell proliferation and/or
survival, either of which could affect tumorigenesis in
estrogen-responsive tissues. To test this hypothesis, we are examining
the effects of dietary energy restriction on the induction, by
administered estrogen, of PRL-producing pituitary tumors and mammary
carcinomas in different inbred rat strains. Data presented herein
indicate that dietary energy restriction modulates the responsiveness
of specific cell populations to estrogenic hormones and thereby
inhibits estrogen-induced tumorigenesis in a manner specific to both
rat strain and tissue. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARYREFERENCES |
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and 1988
,
Spady et al. 1998a and 1998b
). Briefly,
F344 and ACI rats were housed in an accredited animal facility under
controlled conditions. Upon receipt at our facility, the rats were
caged singly and fed a control (Cont) diet that was formulated in
accordance with recommendations of the American Society of Nutritional
Science (AIN 1977
). One week later, the rats were
assigned randomly to groups fed either the Cont diet or an
energy-restricted (EnRes) diet that was formulated through reductions
in consumption of carbohydrate and fat in such a manner that rats that
consumed 0.64 g of this diet for each gram of diet consumed by the
Cont diet-fed rats consumed 40% less energy than rats fed the Cont
diet, while consuming equivalent protein, fiber, vitamins and minerals.
Treatment with either the synthetic estrogen, diethylstilbestrol (DES),
or the naturally occurring estrogen, 17ß-estradiol (E2), was
initiated 1 wk later. Food consumption and body weights were monitored
throughout each experiment. The rats were killed at various times and
pituitary tumor and/or mammary carcinoma development was assessed.
Trunk blood sera were collected and retained for measurement of
circulating hormones by RIA. Statistical significance was assessed by
ANOVA. Values of P 
0.05 were considered significant. |
RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARYREFERENCES |
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A 40% restriction of energy consumption virtually abolished
development of PRL-producing pituitary tumors in F344 rats treated with
estrogen (Shull et al. 1998
, Spady et al. 1998b
). Markedly enlarged, deformed and congested pituitary
glands were observed in the majority of male F344 rats that were fed
the Cont diet and treated with DES for 8 wk. We (Shull et al. 1998
, Spady et al. 1998b
) and others
(Lloyd 1983
) have defined these estrogen-induced
pituitary tumors as benign masses that display diffuse lactotroph
hyperplasia and hypertrophy but lack distinct adenomatous foci. In
contrast, slightly enlarged glands of normal shape and color were
observed in DES-treated rats fed the EnRes diet. Because pituitary
tumor mass correlates with cell number and DNA content per gland, mass
is often used as a quantitative indicator of pituitary tumor
development (Shull et al. 1998
, Wiklund et al. 1981b
). The marked inhibitory (P 0.05)
effect of energy restriction on pituitary tumor development in male
F344 rats treated with DES for 8 wk was apparent when either pituitary
weight (data not shown) or pituitary weight to body weight ratio
(Fig. 1A
)was measured. A 40% restriction of energy consumption similarly
inhibited (P 0.05) pituitary tumor development in
ovariectomized female F344 rats treated with E2 for 10 wk (Fig. 1
B), whereas a 25% restriction was without significant
effect (Spady et al. 1998b
). In the latter experiment,
circulating levels of E2 in the treated rats were slightly
hyperphysiologic, but were observed to be equivalent in treated rats
fed the Cont, 25% EnRes or 40% EnRes diets (Spady et al. 1998b
). A marked inhibitory effect of a 40% restriction of
energy consumption on estrogen-induced pituitary tumor development was
also observed in experiments in which male F344 rats were treated with
E2 for either 10 or 26 wk (data not shown). In contrast to that
observed in the F344 rat strain, dietary energy restriction did not
inhibit estrogen-induced pituitary tumor development in ovariectomized
female ACI rats, as evidenced by measures of pituitary mass and
pituitary to body mass ratio (Fig. 1
C) (unpublished data).
|
). A similar inhibitory effect of energy restriction
on induction of circulating PRL was observed in male F344 rats treated
with E2 for either 10 or 26 wk (unpublished data). In contrast to these
observations in the F344 rat strain, no inhibition of E2 induction of
circulating PRL by dietary energy restriction was observed in the ACI
rat strain. In ovariectomized female ACI rats treated with E2 for 20
wk, circulating PRL was increased 282-fold, from 8.1 ± 1.9 to
2285 ± 310 ng/mL, in rats fed the Cont diet, and 485-fold, from
4.5 ± 2.0 to 2183 ± 368 ng/mL, in rats fed the 40% EnRes
diet (unpublished data). These data provide additional support to our
conclusion that a 40% restriction of energy consumption is a potent
inhibitor of estrogen-induced pituitary tumor development in the F344
rat strain, but not the ACI rat strain. This lack of an inhibitory
effect of dietary energy restriction on estrogen-induced pituitary
tumor development in the ACI rat strain is most interesting. The
propensity of the F344 rat strain to develop pituitary tumors when
treated with estrogens is conferred through the actions of multiple
genes (Wendell and Gorski 1997
, Wiklund et al. 1981a
). Data from our laboratory indicate that the genetic
etiology of estrogen-induced pituitary tumor development in the ACI
strain differs from that of the F344 strain (Spady et al. 1997
, unpublished data). Genetic studies in progress will
enhance our understanding of the mechanisms underlying the rat
strain–specific antitumorigenic actions of dietary energy restriction
in the anterior pituitary gland. Dietary energy restriction modulates estrogen regulation of pituitary cell survival, not cell proliferation.
Data from the laboratory of Jack Gorski (Wiklund and Gorski 1982
) and our laboratory (Shull et al. 1998
)
indicate that the proliferative response to estrogen within the
anterior pituitary gland of the F344 rat is aberrantly robust, relative
to that observed in the pituitary gland of the Holtzman rat, an outbred
strain that does not develop PRL-producing pituitary tumors in response
to chronic estrogen treatment. Therefore, we have examined the
interactions between dietary energy consumption and administered
estrogen in the regulation of cell proliferation in the F344 anterior
pituitary gland. Pituitary cell proliferation was examined by two
methods as follows: 1) measurement of
[3H]thymidine incorporation as an indicator of ongoing
DNA synthesis; and 2) identification of lactotrophs and
nonlactotrophs in the S phase of the cell cycle by coupled
immunohistochemistry using antibodies to PRL and
5-bromo-2'-deoxyuridine. Data from these studies clearly indicate that
dietary energy restriction does not inhibit the ability of administered
DES or E2 to stimulate proliferation within either the lactotroph or
nonlactotroph pituitary cell populations (Shull et al. 1998
, Spady et al. 1998b
). From these data, we
conclude that the inhibitory effect of dietary energy consumption on
estrogen-induced pituitary tumor development is exerted at a step
subsequent to induction of cell proliferation by administered hormone.
We have also examined interactions between dietary energy consumption
and administered estrogen in the regulation of pituitary cell survival.
Using an in situ immunohistochemistry method (Gavrieli et al. 1992
), we have determined that cells exhibiting DNA
fragmentation, a hallmark of apoptotic cells, are rare in the anterior
pituitary gland, probably reflecting rapid phagocytosis of apoptotic
cells after DNA fragmentation. Therefore, we have quantified expression
of TRPM-2 (Testosterone Repressed
Prostatic Message) mRNA as a surrogate indicator
of apoptosis. Numerous studies indicate that expression of
TRPM-2 mRNA is up-regulated in cells and tissues in which
apoptosis is occurring (Buttyan et al. 1989
,
Kyprianou et al. 1991
, Leger et al. 1987
). In ovariectomized female F344 rats fed the Cont diet,
the level of TRPM-2 mRNA was reduced by >75% (P 0.05) in response to 10 wk of E2 treatment, suggesting that E2
inhibits apoptosis in the anterior pituitary gland of the F344 rat and
that this contributes to development of estrogen-induced pituitary
tumors in this rat strain. In contrast, no significant reduction in
TRPM-2 mRNA was observed in response to E2 in animals fed
the 40% EnRes diet, suggesting that dietary energy restriction
inhibits the ability of this estrogen to enhance pituitary cell
survival and thereby inhibits pituitary tumor development in the F344
rat strain (Spady et al. 1998b
). When expression of
TRPM-2 mRNA was examined in the pituitary gland of the
ovariectomized female ACI rat, E2 was observed to reduce the level of
this mRNA by 70% (P 0.05); this reduction was observed
in rats fed the Cont or 40% EnRes diets (unpublished data). Together,
these data illustrate a correlation between the ability of dietary
energy restriction to modulate regulation of pituitary cell survival by
E2 and its ability to inhibit estrogen-induced pituitary tumor
development in these two rat strains.
The inhibitory effects of dietary energy restriction on estrogen-induced tumorigenesis are tissue specific.
We have demonstrated that near physiologic levels of E2 rapidly induced
development of mammary carcinoma in the female ACI rat (Shull et al. 1997b
). Unpublished data from our laboratory indicate that
the genetic etiology of E2-induced mammary cancer development in the
ACI rat strain is distinct from the genetic etiology of carcinogen- and
radiation-induced mammary cancers. Data summarized above indicate that
a 40% restriction of energy consumption does not inhibit development
of estrogen-induced pituitary tumors and gross hyperprolactinemia in
the ovariectomized female ACI rat. Because of these data and published
observations that energy restriction markedly inhibits development of
mammary cancers in rats treated with the chemical carcinogen
7,12-dimethylbenz(a)anthracene (Klurfeld et al. 1989
, Kritchevsky et al. 1989
), it was of
interest to determine whether energy restriction would inhibit
induction of mammary cancers in ACI rats treated with E2. Ovary intact
female ACI rats, fed either the Cont or EnRes diet, were treated with
E2 beginning at the age of 9 wk. Mammary cancers and PRL-producing
pituitary tumors developed in rats fed the Cont diet in a manner
similar to that reported by us previously (Shull et al. 1997b
). A 40% restriction of energy consumption markedly
reduced (P 0.05) the incidence of E2-induced mammary
cancers, but not development of E2-induced pituitary tumors, in ovary
intact ACI rats (unpublished data). These data clearly indicate that
the inhibitory effects of dietary energy restriction on E2-induced
tumorigenesis are tissue specific. Moreover, in conjunction with
previously published data from our laboratory (Shull et al. 1997b
), these data clearly indicate that pituitary
tumor–associated hyperprolactinemia is insufficient for development of
mammary carcinoma in the ACI rat. We are presently working toward
identification of the genes that confer upon the ACI rat strain its
unique susceptibility to estrogen-induced mammary cancers (Shull et al. 1997a
). The ACI rat would then provide a genetically
defined and physiologically relevant animal model in which to study
diet/hormone interactions in mammary cancer development.
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SUMMARY |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARYREFERENCES |
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,
Spady et al. 1998b
) and unpublished data described
herein indicate for the first time that dietary energy restriction
inhibits estrogen-induced tumorigenesis in a manner specific to both
rat strain and tissue. Energy restriction virtually abolishes the
ability of administered estrogens to promote development of pituitary
tumors and associated hyperprolactinemia in the F344, but not ACI rat
strain. Moreover, inhibition of pituitary tumorigenesis in F344 rats
occurs at a step subsequent to induction of lactotroph proliferation,
most probably at the level of apoptosis. Although estrogen-induced
pituitary tumor development in ACI rats was not inhibited by energy
restriction, development of estrogen-induced mammary carcinoma was
markedly inhibited.
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FOOTNOTES |
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1 Presented at the symposium "Steroid Hormone
Receptor and Nutrient Interactions: Implications for Cancer
Prevention" as part of Experimental Biology 98, April 18–22, 1998,
San Francisco, CA. The symposium was sponsored by the American Society
for Nutritional Sciences and was supported in part by educational
grants from Loders Croklaan, Inc. and Slimfast Nutrition Institute.
Published as a supplement to The Journal of Nutrition. Guest
editors for the symposium publication were Diane F. Birt, Iowa State
University and Martha Belury, Purdue University.
2 This research in the Shull laboratory was
supported by grants R01CA68529 and R01CA77876 from the National
Institutes of Health and grant 97A146 from the American Institute for
Cancer Research. NIH grant P30CA36727 to the UNMC/Eppley Cancer Center
and Special Institutional Grant SIG-16 from the American Cancer Society
to the Eppley Cancer Institute also supported this research. T.J.S. was
supported by a fellowship from the Graduate College of the University
of Nebraska. D.M.E.H. and A.L.-W. were supported in part by NIH grant
T32CA09476 to the Cancer Research Training Program of the Eppley Cancer
Institute.
3 T.J.S. and D.M.E.H. contributed equally to the
research described in this manuscript and should be considered as joint
first authors.
4 Current address: Department of Food Sciences and
Human Nutrition, Iowa State University, 2312 Food Sciences Building,
Ames, IA 50011–1061.
5 Abbreviations used: Cont, control; DES,
diethylstilbestrol; E2, 17ß-estradiol; EnRes, energy restricted;
F344, Fischer 344; PRL, prolactin; TRPM, testosterone
repressed prostatic message.
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