Menopausal hormone replacement therapy has been widely used to alleviate the symptoms of menopause and to decrease the detrimental effects of ovarian hormone loss on bone density and cardiovascular health. Multiple studies of colorectal cancer epidemiology also support a role for hormone replacement therapy in prevention of colorectal cancer. We studied the effect of ovariectomy and estrogen replacement on tumor formation in C57BL/6J-Min/+ (Min/+) mice, animals that bear a germ-line mutation in murine Apc. These mice develop multiple intestinal tumors that show loss of wild-type Apc protein. After ovariectomy, intestinal adenomas in Min/+ mice increased by 77% (P = 0.0004). Ovariectomized Min/+ mice that were treated with a replacement dose of 17β-estradiol had the same number of tumors as Min/+ mice that were neither castrated nor treated with estrogen replacement (P = 0.85). Examination of estrogen receptor (ER) levels in intestinal tissue by immunoblot showed changes in relative expression levels of ERα and ERβ, with highest ERα and lowest ERβ expression in the normal-appearing intestine of Min/+ mice, and lowest ERα and highest ERβ expression in the enterocytes of animals that received 17β-estradiol. These results suggest that endogenous estrogens protect against Apc-associated tumor formation and that tumor prevention by 17β-estradiol is associated with an increase in ERβ and a decrease in ERα expression in the target tissue.

Although males and females develop CRC3 with approximately the same frequency, women in developed countries who are normally at high risk for CRC have recently experienced a substantial decline in mortality from this disease (1, 2). This decline has been attributed to the use of menopausal HRT, a practice that began in the 1970s (3). Numerous epidemiological studies of CRC incidence show a protective effect of menopausal HRT use. A recent meta-analysis of studies published up to December 1996 suggests as much as a 25% reduction with HRT use, with a summary relative risk of 0.85 (95% CI, 0.73–0.99). Studies identifying the duration of HRT use also suggest a dose response, with a relative risk among current or recent HRT users of 0.69 (95% CI, 0.52–0.91) compared to 0.88 (95% CI, 0.64–1.21) for short-term users (4).

The mechanism responsible for HRT-associated prevention of CRC is unclear. Estrogens undergo metabolism to a variety of compounds that have different half-lives and receptor affinities, and they produce varying effects on cell growth. For example, the endogenous estrogen 17β-estradiol is metabolized by cytochrome P450 enzymes to form monohydroxy metabolites, and some of these metabolites may play important roles in the induction of estrogen-induced cancers (5). In addition, some of these metabolites (2-hydroxyestradiol and 4-hydroxyestradiol) can be further metabolized by catechol-O-methyltransferase to methylated estrogens. One of these methylated metabolites (2-methosyestradiol) may protect against tumor formation or inhibit tumor growth (5, 6, 7). A tissue-specific distribution of estrogen-metabolizing enzymes influences the effect of a systemically administered estrogen, such as those used for HRT, on cell proliferation in target tissues.

The activity of estrogens and antiestrogens in both males and females is mediated through binding of these compounds to ERs, which are ligand-activated transcription factors. In the absence of ligand, ER resides in the nucleus, where it is bound to an inhibitory heat shock protein complex (8). Upon ligand binding, the ER forms a stable dimer that then interacts with specific estrogen response elements to initiate the transcription of target genes. There are at least two major ERs, known as ERα and ERβ. These two ER isoforms appear to have a differential tissue distribution: ERα is the predominant isoform in breast and uterine tissue (9, 10); and ERβ is expressed in significant quantities in the urogenital tract, the central nervous system, and endothelial cells (10, 11, 12). The two ER isoforms also exhibit differences in binding affinity, potency, and efficacy after interaction with various estrogenic compounds (13, 14). To add yet another level of complexity, multiple ERβ isoforms have been identified (15, 16).

Several studies have examined the relative expression of ERα and ERβ in the colon. ERα is present at very low levels in the colon, with no difference in mRNA or protein expression between normal colon, adenomas, and colon cancers and no differences between males and females (17, 18). ERβ protein is the predominant isoform present in normal colon, and expression of ERβ protein may be decreased in colon cancers (18), although ERβ mRNA levels remain unchanged, suggesting that this decrease is due to a posttranscriptional mechanism. The ERα:ERβ protein ratio observed in normal colon is the reverse of that found in human endometrium (18).

The Min/+ mouse bears a germ-line mutation in the murine Apc gene and is therefore a genetic model for both familial adenomatous polyposis and sporadic human CRC. Loss of APC protein function produces increased intracellular levels of the oncoprotein β-catenin (19). In the nucleus, β-catenin combines with the protein Tcf-4, providing a DNA-binding domain that allows transcription of genes modulating cell proliferation and apoptosis (20). Min/+ mice develop multiple intestinal adenomas and frequently succumb to adenoma-related anemia or intestinal obstruction by 18–20 weeks of age (21). The development of adenomas in Min/+ mice is sensitive to a variety of compounds administered in the diet, making these animals an excellent screen for promising chemopreventive agents. Adenoma number in Min/+ mice is decreased by a variety of agents including aspirin (22), sulindac (23), piroxicam (24), and plant phenolics (25).

We investigated the role of estrogens in intestinal tumorigenesis in Min/+ mice by examining the effect of ovariectomy with and without estrogen replacement on tumor number and intestinal ER expression. The results suggest that endogenous estrogens protect against Apc-associated tumor formation and that tumor prevention by 17β-estradiol is associated with a relative increase in ERβ and a decrease in ERα in the target tissue.

Materials.

Min/+ mice and their wild-type littermates (+/+) were purchased from The Jackson Laboratory (Bar Harbor, ME). AIN-76A pelleted diet was obtained from Research Diets (New Brunswick, NJ). Controlled release 90-day pellets containing 17β-estradiol (NE-121) or control vehicle were obtained from Innovative Research of America (Sarasota, FL). Primary antibodies and protein standards were obtained from Santa Cruz Biotechnology (Santa Cruz, CA; ERα, MC-20 rabbit polyclonal antibody sc-542) and Affinity Bioreagents (Golden, CO; ERα standard, rp-310; ERβ, PA1–310A rabbit polyclonal antibody; ERβ standard, RP-311). Western blot analyses used Optitran nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Electrotransfer of proteins used the electroblot buffers of Owl Separation Systems (Woburn, MA). Western blotting used the enhanced chemiluminescence detection reagents of Amersham Pharmacia Biotech (Piscataway, NJ).

Animal Treatments and Tissue Harvesting.

Female Min/+ mice and their wild-type littermates (+/+) were obtained at 5 weeks of age. Ovariectomy was performed at The Jackson Laboratory on Min/+ mice at 4 weeks of age. Animals receiving estrogen replacement underwent implantation of a 90-day controlled release pellet containing 1.7 mg/pellet 17β-estradiol at 5–6 weeks of age. Control animals underwent placement of an inert pellet containing identical substances but lacking the hormone. Beginning at 5–6 weeks of age, all mice were fed AIN-76A chow diet. The animals were checked daily for signs of distress or anemia, and animals and their food were weighed weekly. During the course of the experiment, there were no differences in body weight or food consumption among the various study groups. Hence, the typical approximate intake of the animals included 2.5 grams/day AIN-76A and 3.0 ml/day water. At 15 weeks of age, all mice were euthanized by CO2 inhalation, and their intestinal tracts were removed from esophagus to distal rectum, opened, flushed with saline, and examined under ×3 magnification to obtain tumor counts. The tumors were counted by an individual blinded to the animal’s genetic status and treatment. Successful ovariectomy was documented by histological comparison of uterine mucosa from ovariectomized animals, animals treated with estrogen replacement, and control animals. Analyses of the different animal tissues were performed by an observer blinded to the animal’s genetic status and treatment group.

Enterocyte Preparation and Western Blotting.

Several tumors from all regions of the small intestine (duodenum, jejunum, and ileum) were excised from each animal, as well as segments of tumor-free duodenum, small bowel, and colon. These tissues were snap frozen and stored separately in liquid nitrogen. Enterocyte samples for Western blotting were obtained by mechanical dissociation using the edge of a glass microscope slide (26) and washed twice in cold PBS before storage at −70°C. For consistency, all samples of enterocytes were collected from 4-cm segments of the proximal small intestines. These were deemed macroscopically “normal” because they were collected after removal of all adenomas seen under ×3 magnification. Samples from two mice of the same treatment group were pooled to prepare the protein extracts. Procedures and buffers for cell lysis, protein determination, and Western blot analyses were performed exactly as described previously (27). Western blots used 50 μg protein/lane (500 ng for standards), 10% SDS-PAGE, and the following primary antibodies: (a) ERα, MC-20 rabbit polyclonal antibody sc-542; (b) ERα standard, rp-310; (c) ERβ, PA1–310A rabbit polyclonal antibody; and (d) ERβ standard, RP-311. All antibodies were used at a concentration of 1:1000. The Western blot determinations were repeated separately at least two times.

Ovariectomy Increased Tumor Production in Min/+ Mice.

To simulate the hormonal environment of menopause, ovariectomy was performed on healthy female Min/+ mice at 4–5 weeks of age. At this age, Min/+ mice have not yet developed tumors measurable by our counting method. Both ovariectomized Min/+ mice and control Min/+ mice with intact ovarian function were fed a standard AIN-76A diet. At 15 weeks of age, the animals were euthanized, and the entire intestinal tract was examined under ×3 magnification.

The average tumor number in the control Min/+ animals was 23.9 ± 11.9, a value consistent with that seen in previous studies using the tumor counting technique used here (Fig. 1). Ovariectomized Min/+ mice contained an average of 42.3 ± 13.9 tumors, a 77% increase in total tumor number compared to the control animals (P = 0.0004). Consistent with previous studies, 93% of the tumors were found in the small intestine, with 5% in the duodenum, and approximately 2% in the colon (Table 1). The distribution of tumors throughout the intestinal tract was not altered by ovariectomy (Table 1).

Administration of 17β-Estradiol Decreased Intestinal Tumor Number in Ovariectomized Min/+ mice.

To simulate the effect of postmenopausal HRT, 17β-estradiol was given to ovariectomized Min/+ mice in the form of controlled release pellets implanted in the s.c. tissue of 5–6-week-old animals. Control Min/+ mice were given a sham operation, with implantation of a pellet containing the only inert vehicle and lacking the hormone. As in the previous experiment, the mice were allowed to reach 15 weeks of age before tumor counts were obtained. The Min/+ control mice that were implanted with an inert pellet each contained 28.3 ± 7.5 tumors, an amount unchanged from that seen in previous Min/+ control animals (P = 0.19). The ovariectomized animals that also received estrogen replacement, however, contained 29.2 ± 12.4 tumors each and were therefore equal in tumor number to the sham vehicle-treated Min/+ controls (P = 0.85). When the distribution of tumors throughout the gastrointestinal tract was examined, the number of small intestinal tumors in the animals receiving 17β-estradiol was no different from that of animals receiving a sham operation (P = 0.36; Table 2). In the duodenum, treatment with 17β-estradiol decreased the number of tumors per mouse from 6.7 ± 1.2 to 3.7 ± 3.2 (P = 0.04). In the colon, the opposite effect was noted, with an increase in tumor number from 0.22 ± 0.44 tumor/animal in the sham operation group to 0.77 ± 0.66 tumor/animal in the 17β-estradiol-treated mice (P = 0.05). Although statistically significant, these differences are unlikely to be important because of the very small numbers of tumors found in the duodenum and colon of Min/+ mice.

Ovariectomy and Estrogen Replacement Altered the Relative Expression of ERα and ERβ in Enterocytes from Min/+ Mice.

ERs effect the biological responses to estrogens at the tissue level. We therefore performed Western blotting to determine the relative expression of ERα and ERβ in enterocytes isolated from Min/+ mice and their wild-type littermates, as well as in pooled preparations of adenomas arising in the intestines of Min/+ mice.

The small intestine of the Min/+ mouse is the region of the gastrointestinal tract most prone to tumor formation. By immunoblot, the tumor-prone small intestine of Min/+ mice showed elevated ERα and lowered ERβ levels when compared to comparable tissues from wild-type animals (Fig. 2). The intestinal mucosa that was used for the Min/+ small intestine immunoblots appeared normal under ×3 magnification, although it is possible that a small fraction of this preparation contained adenoma cells. When pooled samples of adenomas from Min/+ small intestine were examined, however, Western blots showed slightly more ERβ and slightly less ERα than the normal-appearing Min/+ mucosa. Thus, potential adenoma contamination is unlikely to explain the different ERα:ERβ ratios observed between Min/+ and wild-type animals.

To assess the consequences of hormone deficiency resulting from ovariectomy, as well as hormone replacement, Western blotting for ERα and ERβ was performed on enterocyte lysates prepared as described above. When compared to the control Min/+ animals, ovariectomized mice had lower ERα expression and higher ERβ expression in the tumor-prone small intestine. When ovariectomized animals were given 17β-estradiol, ERα expression was reduced further, in conjunction with a significant rise in ERβ levels.

Similar analyses were performed using enterocytes isolated from the colon (Fig. 2). In the colonic mucosa, there is apparently little difference in ER expression between wild-type, Min/+, and ovariectomized Min/+ mice. The overall expression of ERα appeared higher than that of ERβ in these three tissue types, although this experiment was not performed to quantitate exact protein levels. Just as in the small intestine, however, supplementation of ovariectomized Min/+ mice with 17β-estradiol produced a relative decrease in ERα and an increase in ERβ expression. These qualitative results also suggest that augmented ERβ expression relative to ERα correlates with suppression of tumors in Min/+ intestine.

Although it is clear that estrogens modulate tumor initiation in hormone-sensitive tissues such as the breast (5, 6, 7, 28), the role of estrogens in colorectal carcinogenesis is unclear. Data from observational studies suggest that some women may have a reduced CRC incidence because HRT replacement and multiparity may be protective (2). Despite these data, there are no clear gender differences in CRC incidence. The peak incidence of CRC for both men and women comes approximately 10 years after the onset of age-related decline in hormone production, an appropriate time span for completion of carcinogenesis once a protective effect has been removed (29, 30). Male Min/+ mice do not have a higher tumor number than female Min/+ mice (21). Despite this, our data show that tumor formation is increased in female Min/+ mice after ovarian hormone withdrawal. Taken together, these observations suggest that both male and female hormones may protect against intestinal tumors. Studies of the effect of castration on tumor formation in Min/+ mice are in progress and will help resolve this question.

Several studies suggest that ERα and ERβ are differentially regulated in estrogen-sensitive tissues. In blood vessels, removal of the endothelial cell layer to simulate vascular injury causes a dramatic increase in ERβ expression in the underlying vascular smooth muscle, with little change in the levels of ERα (31). This study suggests that estrogens act through ERβ to protect against arteriosclerotic vascular lesions by inhibiting smooth muscle proliferation. Another example of reciprocal regulation of ERα and ERβ is seen after neonatal estrogenization of male rats (32). This treatment produces increased susceptibility to estrogen-induced carcinogenesis of the urogenital tract and is associated with increased expression of ERα and decreased expression of ERβ in the prostate (32).

The ERα:ERβ ratio may be a determinant of the susceptibility of a tissue to estrogen-induced carcinogenesis. Estrogens induce gene transcription both by binding to the classic estrogen response element and by signaling through an AP1 enhancer element requiring the products of c-fos and c-jun(33). ERα and ERβ may have similar effects on gene transcription mediated via the estrogen response element but opposite effects on promoters containing AP1 (34). Estradiol activates AP1-mediated gene transcription when bound to ERα but inhibits promoter activity when bound to ERβ (34). The converse is true for the antiestrogens, such as tamoxifen, raloxifene, and ICI 164384, which are AP1 transcriptional suppressors via ERα and activators via ERβ (34). In addition, tumor-prone tissues responsive to hormones, such as breast and endometrium, have overall higher expression levels of ERα than ERβ (9, 10).

These observations led to the theory that binding of estrogens to ERα induces a cancer-promoting response, whereas ERβ binding is protective. The results of our study support a reciprocal role for ERα and ERβ expression in the promotion of intestinal tumor formation in vivo. In animals with intact ovaries, the tumor-prone Min/+ small intestine showed increased ERα and decreased ERβ when compared to wild-type tissue. The effect of estradiol replacement after ovariectomy was also informative. The tumor-reducing effect of estradiol replacement in the ovariectomized mice was accompanied by increased ERβ and decreased ERα in the target tissue. It is not known precisely what metabolites of estrogen are present in the intestinal mucosa, nor it is known whether ERα and ERβ compete for ligands in this tissue. The apparent tumor-promoting effects of increased ERα expression in Min/+ enterocytes are unlikely to be due to differential ligand binding, however, because the affinity of 17β-estradiol for ERα and ERβ is essentially equivalent (12, 35, 36).

By in situ hybridization, ERβ is expressed in the mucosa of the human duodenum, colon, and rectum (10). In the colonic tissue, expression of ERβ protein is significantly higher than that of ERα (18). We found the opposite result for the colonic tissue of both wild-type and Min/+ mice, although site-specific comparisons between human and mouse intestinal mucosa tend not to be useful. For example, the phenotype of Min/+ mice is different from that of humans with APC-associated tumors because most Min/+ tumors are located in the small intestine, with very few found in the mouse colon. When we examined noninvasive adenomas in Min/+ mice, we found less ERα and slightly more ERβ than in the normal-appearing Min/+ mucosa. This result is again opposite that observed in humans because Western blot of human CRCs showed a marked decrease in ERβ compared to normal tissue, whereas ERα levels remained the same (18). It is possible that a shift from ERβ to ERα predominance occurs when a tumor becomes invasive, although this is not supported by previous studies of ER expression in human colonic mucosa, adenomas, and carcinomas (17, 18). Unfortunately, invasive cancers cannot be studied in our model because Min/+ mice die from adenoma-related anemia or bowel obstruction before developing a significant number of invasive tumors. Studies of ER distribution and activity in human CRC cell lines show variable results. ERα is present in HT29 colon cancer cells (37), although these cells did not show increased proliferation in response to in vitro culture with estradiol. Additional studies showed that ERβ mRNA and protein were present in HCT8, HCT116, DLD-1, and LoVo cells (38), whereas ERα mRNA and protein were absent. Binding studies determined that all four of these cell lines contained a high affinity receptor for 17β-estradiol. Cell proliferation studies, however, yielded mixed results because 17β-estradiol induced the growth of HCT8 cells but inhibited the growth of HCT116, DLD-1, and LoVo cultures (38).

The effect of ovariectomy on ER expression in Min/+ intestine was interesting. The withdrawal of ligand generally produces up-regulation of its receptor. In this case, loss of endogenous estrogen production by ovariectomy increased ERβ and decreased ERα. This supports a role for ERβ as the natural receptor in the intestinal mucosa. This result also suggests that the response to ovariectomy may be due to more than just loss of estradiol stimulation of the intestinal mucosa. ER undergoes a conformational change after ligand binding, releasing inhibitory proteins and leading to dimerization followed by DNA binding and transcriptional activation (39, 40). ERα and ERβ can either homo- or heterodimerize. ERα and ERβ differ significantly in their NH2-terminal domains and are therefore likely to interact with different sets of proteins. The various ER ligands may also produce distinct conformational changes in these receptors (41). Subtle changes in the ERα:ERβ ratio, such as those produced here by ovariectomy, may therefore result in significant changes in the transcriptional activity and/or gene promoter specificity of these receptors.

ER ligands other than endogenous hormones may alter intestinal tumor formation. Diet is known to influence the development of CRC, with high consumption of fruits and vegetables conferring a protective effect. These food categories contain a variety of phytoestrogens that are capable of modulating ER activity. Phytoestrogens appear to have a greater affinity for ERβ than they do for ERα (42). In the brain, where estrogen may play a protective role against the progression of neurodegenerative conditions such as Alzheimer’s disease, ERβ mRNA expression in the paraventricular nucleus of ovariectomized rats is decreased by 17β-estradiol and up-regulated by the phytoestrogen coumestrol, a substance found in legumes (43). Our results, which show that ovariectomy followed by estrogen replacement markedly increased expression of ERβ in a susceptible tissue, suggest that phytoestrogens may also decrease tumor formation when endogenous hormones are lacking.

One of the possible mechanisms of estrogen-induced carcinogenesis is induction of peroxisome proliferation. Estrogen-induced peroxisome proliferation may be mediated through regulation of members of the PPAR family of nuclear transcription factors (44, 45, 46). PPAR activation is associated with carcinogenesis in the liver and may also be important in the development of APC-associated colorectal tumors (47). Interestingly, ligands of PPARγ, an isoform expressed in human colonic mucosa (48), stimulate tumorigenesis in Min/+ mice (49) as well as diet-induced carcinogenesis in mice (50), although they can induce differentiation and suppress the growth of certain human colon cancer cell lines (51). Thus, estrogens may modify tumorigenesis under conditions of APC deficiency via the regulation of PPAR activity. The possible effects of differential ERα and ERβ expression on PPAR activity in the intestinal tissue remain to be tested.

In conclusion, hormone-associated carcinogenesis is a complex process with species- and tissue-specific differences in receptor expression, receptor isoform distribution, and ligand metabolism as well as significant cross-talk between the different signaling pathways that govern cell fate. This study demonstrates a role for estrogen in modulating Apc-associated intestinal tumors and indicates that tumorigenesis is associated with reciprocal modulation of ER isoform expression in adult female Min/+ mice. Further study is needed to address how ER-estrogen interactions affect enterocyte proliferation and/or differentiation in vivo.

Fig. 1.

Ovariectomy increases tumors in Min/+ mice. Female Min/+ mice underwent ovariectomy at 4–5 weeks of age. Mice receiving estrogen replacement underwent s.c. implantation of a controlled release estrogen pellet at 5–6 weeks of age. Tumor counts from the entire intestine were obtained at 15 weeks of age. Values represent total tumor number/mouse ± SD, where n = 32 (Min/+, Experiment A), n = 9 (Min/+ sham, Experiment B), n = 15 (Min/+ ovariectomy, Experiment A), and n = 9, Min/+ E2 + ovariectomy, Experiment B). ∗, P = 0.0004 compared to Min/+; ∗∗, P = 0.85 compared to Min/+ animals treated with sham implant.

Fig. 1.

Ovariectomy increases tumors in Min/+ mice. Female Min/+ mice underwent ovariectomy at 4–5 weeks of age. Mice receiving estrogen replacement underwent s.c. implantation of a controlled release estrogen pellet at 5–6 weeks of age. Tumor counts from the entire intestine were obtained at 15 weeks of age. Values represent total tumor number/mouse ± SD, where n = 32 (Min/+, Experiment A), n = 9 (Min/+ sham, Experiment B), n = 15 (Min/+ ovariectomy, Experiment A), and n = 9, Min/+ E2 + ovariectomy, Experiment B). ∗, P = 0.0004 compared to Min/+; ∗∗, P = 0.85 compared to Min/+ animals treated with sham implant.

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Fig. 2.

ERα and ERβ are differentially expressed in Min/+ intestine. Samples of intestinal mucosa from wild-type (WT), control Min/+ (Min/+), Min/+ mice after ovariectomy (Min/+/OVX), and ovariectomized Min/+ mice receiving 17β-estradiol replacement (Min/+/OVX/17βE2) and pooled adenomas from control Min/+ mice (Min/+ adenoma) were examined by immunoblotting using antibodies to ERα and ERβ. The blots shown are representative of two or more using separately prepared cell lysates and show 50 μg protein/lane and 500 ng of protein for the standards.

Fig. 2.

ERα and ERβ are differentially expressed in Min/+ intestine. Samples of intestinal mucosa from wild-type (WT), control Min/+ (Min/+), Min/+ mice after ovariectomy (Min/+/OVX), and ovariectomized Min/+ mice receiving 17β-estradiol replacement (Min/+/OVX/17βE2) and pooled adenomas from control Min/+ mice (Min/+ adenoma) were examined by immunoblotting using antibodies to ERα and ERβ. The blots shown are representative of two or more using separately prepared cell lysates and show 50 μg protein/lane and 500 ng of protein for the standards.

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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by National Cancer Institute Grant NCI-1R29CA74162 (to M. M. B.), NIH Surgical Oncology Research Training Grant T32-CA-68971 (to M. J. W.), and the Irving Weinstein Foundation (A. M. C.).

3

The abbreviations used are: CRC, colorectal cancer; HRT, hormone replacement therapy; ER, estrogen receptor; Min/+, C57BL/6J-Min/+; CI, confidence interval; PPAR, peroxisome proliferator-activated receptor.

Table 1

Distribution of intestinal tumors

Tumor numbers were counted for each intestinal segment indicated. Numbers reported are tumors/segment/animal ± SD. An unpaired t test was used to compare results at each location. Tumor distribution and total tumor counts were measured for 32 Min/+ and 15 Min/+ ovariectomized animals.
DuodenumSmall intestineColonTotal
Min/+ mice 1.2 ± 1.1 22.3 ± 11.5 0.47 ± 0.62 23.9 ± 11.9 
Min/+ ovariectomized mice 2.6 ± 2.1 39.2 ± 14.4 0.47 ± 0.74 42.3 ± 13.9 
P 0.02 0.0006 0.993 0.0004 
Tumor numbers were counted for each intestinal segment indicated. Numbers reported are tumors/segment/animal ± SD. An unpaired t test was used to compare results at each location. Tumor distribution and total tumor counts were measured for 32 Min/+ and 15 Min/+ ovariectomized animals.
DuodenumSmall intestineColonTotal
Min/+ mice 1.2 ± 1.1 22.3 ± 11.5 0.47 ± 0.62 23.9 ± 11.9 
Min/+ ovariectomized mice 2.6 ± 2.1 39.2 ± 14.4 0.47 ± 0.74 42.3 ± 13.9 
P 0.02 0.0006 0.993 0.0004 
Table 2

Effect of 17β-estradiol supplementation on tumor distribution

Tumor numbers were counted for each intestinal segment indicated. Numbers reported are tumors/segment/animal ± SD. A t test was used to compare results at each location. Tumor distribution and total tumor counts were measured for nine Min/+ mice treated with sham implant and nine Min/+ mice treated with ovariectomy + 17β-estradiol implant.
 Duodenum Small intestine Colon Total 
Min/+ treated with sham implant 6.7 ± 1.2 21.4 ± 6.6 0.22 ± 0.44 28.3 ± 7.5 
Min/+ treated with ovariectomy + 17β-estradiol 3.7 ± 3.2 24.9 ± 9.3 0.77 ± 0.66 29.2 ± 12.4 
P 0.03 0.36 0.05 0.85 
Tumor numbers were counted for each intestinal segment indicated. Numbers reported are tumors/segment/animal ± SD. A t test was used to compare results at each location. Tumor distribution and total tumor counts were measured for nine Min/+ mice treated with sham implant and nine Min/+ mice treated with ovariectomy + 17β-estradiol implant.
 Duodenum Small intestine Colon Total 
Min/+ treated with sham implant 6.7 ± 1.2 21.4 ± 6.6 0.22 ± 0.44 28.3 ± 7.5 
Min/+ treated with ovariectomy + 17β-estradiol 3.7 ± 3.2 24.9 ± 9.3 0.77 ± 0.66 29.2 ± 12.4 
P 0.03 0.36 0.05 0.85 
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