Affiliations of authors: R. G. Mehta, M. E. Hawthorne, R. R. Mehta, K. Christov (Department of Surgical Oncology, College of Medicine), K. P. L. Bhat, J. M. Pezzuto (Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy), University of Illinois at Chicago; L. Kopelovich, G. J. Kelloff, V. E. Steele, Chemopreventive Agent Development Group, Division of Cancer Prevention and Control, National Cancer Institute, Bethesda, MD.
Correspondence to: John M. Pezzuto, Ph.D., Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy (M/C 877), University of Illinois at Chicago, 833 South Wood St., Chicago, IL 60612 (e-mail: jpezzuto{at}uic.edu).
Mammary glands undergo morphologic and biochemical changes during various physiologic stages of life, specifically during the transition from virgin to pregnancy, lactation, and involution (13). The complete cycle of structural and functional differentiation depends on the coordinated action of prolactin, insulin, adrenal corticoids, and ovarian hormones (4,5). Atypical ductal hyperplasia is an abnormal ductal epithelial cell proliferative condition that does not invade the periductal stroma (6). In women, atypical ductal hyperplasia, which may become more aggressive and ultimately fill the lumen of the duct, is considered to be a physiologic precursor to the development of ductal carcinoma in situ (DCIS).
Although the histopathology of DCIS subtypes is well defined, there are few experimental models to evaluate the molecular mechanisms underlying DCIS formation or to evaluate cancer chemopreventive agents. One model is the mouse mammary gland organ culture (MMOC) (7,8), in which the entire cycle of mammary gland morphology and physiology can be simulated with appropriate hormonal supplementation of a chemically defined medium. We have shown previously that the MMOC model is useful for evaluating the underlying molecular mechanisms of lesion formation because mammary glands exposed to 7,12-dimethylbenz[a]anthracene (DMBA) develop hyperplastic mammary alveolar lesions in the presence of the nonovarian steroid hormones aldosterone and hydrocortisone (9). These lesions do not regress to the nonproliferating state comprised mainly of ducts and a few end-buds after the removal of growth-promoting hormones. Moreover, cells isolated from these lesions form adenocarcinomas when transplanted into syngeneic mice (10). In addition, the model is useful for testing the efficacy of chemopreventive agents to inhibit DMBA-induced lesions (11,12), although tamoxifen did not reduce lesion formation. Because of the proposed role of ovarian hormones in breast carcinogenesis, we evaluated the effects of estrogen and progesterone on DMBA-induced lesions in the MMOC model.
Ductal lesions were induced by incubating mouse mammary glands in serum-free Waymouth MB752/1 medium (7,8), in which aldosterone and hydrocortisone were replaced with estradiol-17 (0.001 µg/mL) and progesterone (1 µg/mL). The glands were treated with DMBA (2 µg/mL) for 24 hours on the third day of culture. After 24 days, glands were fixed in 10% formalin, and histopathologic sections were evaluated for ductal lesions. For progesterone receptor analyses, mammary glands were incubated with growth-promoting hormones either alone or in the presence of estradiol-17
(1 nM) and/or tamoxifen (1 µM) for 6 days. On day 6, the glands were fixed in 10% formalin, and histologic sections were prepared and immunostained with antibodies to the progesterone receptor (Neomarkers, Fremont, CA) (13). Sections were evaluated semiquantitatively for the expression of progesterone receptor according to the intensity of staining (14). The difference between means of percent incidence (of atypical ductal hyperplasia) and percent induction (of progesterone receptor expression) of control and treated groups was analyzed by use of Student's t test for independent samples (SPSS® statistical software; version 6.1.3; Chicago, IL). All statistical tests were two-sided.
Histologically, ovarian hormone-dependent lesions induced by DMBA in MMOC were predominantly of ductal rather than alveolar origin, similar to human breast hyperplastic and premalignant lesions. The ducts in control glands (i.e., not treated with DMBA) were largely lumina lined with one or two layers of epithelial cells (Fig. 1, A). The ducts in DMBA-treated glands were thickened and lined with five to six layers of hyperplastic cells (defined by a thickness of more than three cell layers) (Fig. 1
, C). The lumina of some ducts were occluded completely by intraductal outgrowths, and the epithelial cells often formed alveolar and papillary structures (Fig. 1
, E). Transverse sections through the lesions showed a combination of proliferating epithelial cells and areas of necrosis (Fig. 1
, F). Close examination of the intraductal lesions showed that aggressive lesions (i.e., those completely occluded with ductal epithelial cells) were composed of atypical cells (variable in size and form), nuclei with intense chromatin staining, reduced intracellular spaces, and a reduced number of mitotic figures (Fig. 1
, G).
|
We next evaluated the effect of tamoxifen and other modulators of estrogen function on DMBA-induced atypical ductal hyperplasia. Vehicle (dimethyl sulfoxide)-treated control glands had normal morphology. Glands treated with tamoxifen (1 µM) during the first 10 days of growth, in the presence of estrogen and progesterone but in the absence of DMBA, had thin ducts and small normal end-buds. DMBA-treated glands had few alveoli, thickened, enlarged end-buds, and dense hyperplastic ducts (Fig. 1, C and E). The number of hyperplastic ductal lesions was greatly reduced in glands treated with DMBA in the presence of tamoxifen (Fig. 1
, H). Additional antiestrogens and modulators of estrogen metabolism were also found to inhibit DMBA-induced ductal lesions (IC50 [i.e., concentration of test substance required to reduce ductal hyperplasia by 50%] values are summarized in Table 1
).
|
One of the initial effects of estradiol-17 in mammary cell differentiation is the induction of estrogen-inducible genes, including the progesterone receptor (15). We found that the expression of progesterone receptors was localized in the epithelial cell nuclei of glands incubated with insulin, prolactin, and estradiol-17
and that treatment of glands with tamoxifen for 6 days decreased estrogen-inducible progesterone receptor expression. Consistent with our studies, a recent report (16) showed that progesterone receptor knockout mice developed fewer DMBA-induced tumors than isogeneic wild-type mice. These data indirectly suggest a regulatory role for progesterone and the progesterone receptor in carcinogenesis. Although the complete role of the progesterone receptor in the development of breast cancer remains unclear, the induction of DMBA-induced lesions will be a valuable tool for studying the mechanism of progesterone action in mammary carcinogenesis.
In this study, we show for the first time that DMBA can induce estrogen- and progesterone-dependent mammary atypical ductal hyperplastic lesions in MMOC with high frequency. Although histologically distinct from human DCIS, these estrogen-dependent lesions can be considered analogous to atypical ductal hyperplasia observed in women, which are distinct and often a prerequisite to the development of breast can-cer (6). Therefore, our MMOC model could serve as a tool for identifying and studying the alterations in molecular markers during the development of atypical ductal lesions and DCIS. Moreover, because tamoxifen and other modulators of estrogen action suppressed the formation of estrogen-dependent ductal lesions (Table 1), this MMOC model is useful for determining the potential of cancer chemopreventive agents to inhibit the development of transformed atypical hyperplastic ductal lesions.
NOTES
Supported by Public Health Service contracts CN55135 and CN65114 and by grant P01CA48112 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services.
We thank Drs. Henry Thompson and Meenakshi Singh, AMC Cancer Research Center, Denver, CO, for valuable suggestions during the initiation of the project and critical evaluation of the manuscript.
REFERENCES
1 Medina D. The mammary gland: a unique organ for the study of development and tumorigenesis. J Mammary Gland Biol Neoplasia 1996;1:519.[Medline]
2
Nandi S, Guzman RC, Yang J. Hormones and mammary carcinogenesis in mice, rats and humans: a unifying hypothesis. Proc Natl Acad Sci U S A 1995;92:36507.
3
Guzman RC, Yang J, Raukumar L, Thordarson G, Chen X, Nandi S. Hormonal prevention of breast cancer: mimicking the protective effect of pregnancy. Proc Natl Acad Sci U S A 1999;96:25205.
4 Imagawa W, Yang J, Guzman R, Nandi S. Control of mammary gland development. In: Knobil E, Neill JD, editors. The physiology of reproduction. New York (NY): Raven Press; 1994. p. 103365.
5 Russo J, Gusterson BA, Rogers AE, Russo IH, Wellings SR, van Zwieten MJ. Comparative study of human and rat mammary tumorigenesis. Lab Invest 1990;62:24478.[Medline]
6 Bellamy CO, McDonald C, Salter DM, Chetty U, Anderson TJ. Noninvasive ductal carcinoma of the breast: the relevance of histologic categorization. Hum Pathol 1993;24:1623.[Medline]
7 Mehta RG, Banerjee MR. Action of growth-promoting hormones on macromolecular biosynthesis during lobulo-alveolar development of the entire mammary gland in organ culture. Acta Endocrinol (Copenh) 1975;80:50116.[Medline]
8 Ganguly R, Mehta NM, Ganguly N, Banerjee MR. Glucocorticoid modulation of casein gene transcription in mouse mammary gland. Proc Natl Acad Sci U S A 1979;76:646670.[Abstract]
9 Mehta RG, Hawthorne ME, Steele VE. Induction and prevention of carcinogen induced precancerous lesions in mouse mammary gland organ culture. Meth Cell Sci 1997;19:1924.
10 Telang NT, Banerjee MR, Iyer AP, Kundu AB. Neoplastic transformation of epithelial cells in whole mammary gland in vitro. Proc Natl Acad Sci U S A 1979;76:588690.[Abstract]
11
Jang M, Cai L, Udeani GO, Slowing KV, Thomas CF, Beecher CW, et al. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 1997;275:21820.
12
Mehta RG, Moriarty RM, Mehta RR, Penmasta R, Lazzaro G, Constantinou A, et al. Prevention of preneoplastic mammary lesion development by a novel vitamin D analogue 1-hydroxyvitamin D5. J Natl Cancer Inst 1997;89:2128.
13
Mehta RR, Bratescu L, Graves JM, Green A, Mehta RG. Differentiation of human breast carcinoma cells by a novel vitamin D analog: 1-hydroxyvitamin D5. Int J Oncol 2000;16:6573.[Medline]
14 Hata H, Kuramoto H. Immunocytochemical determination of estrogen and progesterone receptors in human endometrial adenocarcinoma cells (Ishikawa cells). J Steroid Biochem Mol Biol 1992;42:20110.[Medline]
15 Cuzick J. Chemoprevention of breast cancer with tamoxifen. IARC Sci Publ 1996;136:95109.[Medline]
16
Lydon JP, Ge G, Kittrell FS, Medina D, O'Malley BW. Murine mammary gland carcinogenesis is critically dependent on progesterone receptor function. Cancer Res 1999;59:427684.
Manuscript received November 20, 2000; revised April 24, 2001; accepted May 4, 2001.
This article has been cited by other articles in HighWire Press-hosted journals:
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |