Regulation of mammary epithelial cell growth in mice and rats.

0163-769X/90/1104-0494$02.00/0 Endocrine Reviews Copyright © 1990 by The Endocrine Society

Vol. 11, No. 4 Printed in U.S.A.

Regulation of Mammary Epithelial Cell Growth in Mice and Rats* WALTER IMAGAWA, GAUTAM K. BANDYOPADHYAY, AND SATYABRATA NANDI Cancer Research Laboratory and Division of Cell and Developmental Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

I. Introduction

I. Introduction II. In Vivo "Classical" Studies A. Introduction B. Cellular composition of the mammary gland C. Stages of mammary epithelial cell growth in vitro D. Factors affecting mammary growth in vivo 1. Epithelial-stromal cell interactions 2. Endocrine effects on mammary growth E. Unanswered questions III. In Vitro Organ and Explant Cultures A. Introduction and overview B. Summary of in vitro findings C. Comparison of in vivo and in vitro findings and conclusions IV. In Vitro Cell Culture A. Introduction B. Cell culture on nonbiological substrates C. Cell culture on biological substrates 1. Growth in medium containing serum 2. Growth in serum-free medium 3. Cell matrix requirements for growth in vitro 4. Possible roles of the stromal/adipose cell compartments in the regulation of epithelial cell growth 5. Growth regulation by recently characterized growth factors D. Summary V. In Vivo Revisited A. Introduction B. Mammogenic hormones C. Growth factors D. Summary VI. Proliferation of Mammary Epithelial Cells and Intracellular Signalling Pathways A. Introduction B. Classification of in vitro growth stimulatory factors: groups 1, 2, 3 C. Group 1: peptides and steroids D. Group 2: cAMP, lithium, phorbol esters, phospholipids E. Group 3: linoleic acid and its eicosanoid products F. Multiple pathways regulate growth VII. Overall Summary and Conclusions

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HE mammary gland is a unique feature of mammals. Its chief biological function is to synthesize and secrete milk, which is essential for the nourishment of mammalian young. The parenchymal portion of the mammary gland is derived embryologically from ectoderm, while the surrounding stroma comes from mesoderm. Mammary gland growth takes place during both fetal and postnatal life; the gland is the site of milk production and secretion, and in several species (including humans) it is also a site of occurrence of preneoplastic and neoplastic lesions. Thus the mammary gland is an important organ of study by biologists from various disciplines, including endocrinologists, tumor biologists, and developmental biologists. During the last 30 yr, the primary interest of our laboratory has been the understanding of the neoplastic transformation process in mammary epithelial cells. One fundamental characteristic of tumor cells vis a vis their normal counterparts is deregulation of growth. Since cell proliferation is a prerequisite for neoplastic transformation in target tissues, we have tried to understand the regulation of mammary epithelial cell growth. Toward this end we have utilized a variety of in vivo techniques, in vitro organ and explant culture systems, and also cell culture methodologies. These tools have aided in the analysis of the roles of hormones, growth factors, lipids, metabolic products, and even extracellular matrix components in the control of growth in mouse and rat mammary epithelial cells. There are many excellent reviews dealing with various aspects of mammary biology. These cover especially well mammary gland architecture, its morphogenesis during fetal and postnatal life, and the endocrine requirements for its growth and for milk product synthesis and secretion (1-10). These topics will therefore not be covered in * This work was supported by Grants CA-40160, CA-09041, and CA05388 awarded by the National Institutes of Health, Department of Health and Human Services.

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MAMMARY EPITHELIAL CELL GROWTH

depth in the present review. Much new information is now being generated both from in vitro and from in vivo studies dealing with mammary epithelial cell proliferation. This work requires critical evaluation, particularly in terms of its relevance and mechanistic importance to the situation in vivo. The present review attempts to summarize chronologically our current knowledge of mammary epithelial cell growth regulation in mice and rats in vivo and in vitro, and to identify the problems and questions essential for the analysis of cellular and molecular events associated with cell proliferation in this organ. Section II of this article, with a few exceptions, deals chiefly with "classical" in vivo studies up through 1975 or so, as well as with the questions that were left unanswered by this relatively early work. Section HI summarizes in vitro explant and organ culture studies. In these studies, the effects on mammary epithelial cells of hormones, growth factors, and other agents could be examined in a system in which the parenchymal-stromal relationship had been maintained as close to that in vivo as possible. Section IV describes growth studies with isolated primary mammary epithelial cells, either alone or in conjunction with connective tissue components. Such experiments allow the examination of the interactions of these two components with various regulatory factors during mammary cell proliferation. Later (Section V) we analyze the impact of in vitro findings on recent in vivo studies and give our assessment of the relevance of this research to actual mammary epithelial cell growth in animals vis a vis the questions that were left unanswered after "classical" in vivo studies (Section II). Finally, in Section VI we attempt to summarize the current status of knowledge concerning intracellular pathways regulating mammary epithelial cell proliferation in mice and rats. II. In Vivo Classical Studies A. Introduction In vivo and in vitro (utilizing whole glands, pieces of glands, or isolated epithelial cells) approaches have been employed in the study of mammary growth and differentiation. Because of the complexity of the entire organism, the exact analysis of the roles of various regulatory factors is often extremely difficult. In vitro methods, with simpler and more controlled environments, are therefore frequently used to complement in vivo work. However, an adequate knowledge of in vivo findings is essential for the correct interpretation of in vitro results in determining the mechanisms of mammary cell proliferation, milk synthesis, and secretion in the whole animal. The aim of this section, therefore, is to summarize the

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classical in vivo work (along with a few contemporary studies) on mammary biology. These include studies of the nature of the cell types that comprise the mammary gland, the stages of mammary growth, and the stromal influences and hormones that have been implicated in the growth regulation of mammary epithelial cells. B. Cellular composition of the mammary gland The cellular composition of the mammary gland during embryonic, fetal, and postnatal stages in mice and rats has been described (3, 4, 6, 8, 11-16). Two components are involved: the parenchyma, which is the epithelial component, and the stroma, composed of connective tissue elements. The two compartments are separated by a basement membrane, which consists of extracellular matrix materials. The latter result from interactions among products derived from both the epithelial and the mesenchymal compartments (17). In postnatal life, the mammary tree (parenchyma) consists of lumina surrounded by single or multiple layers of epithelial cells with a basal layer of myoepithelium. The luminal epithelium is specialized for the synthesis and secretion of milk products and is organized into structures called ducts, end buds, alveoli, and lobules (18). Major ducts serve as channels and reservoirs for the milk during lactation. End buds are specialized groups of cells at the ends of ducts. These are the sites of the cell proliferation that results in ductal ramification in the growing gland. Alveolar cells, which are present in resting adult glands and during pregnancy, become the primary components for milk product synthesis during lactation. The clusters of alveoli usually developing in mid- to late pregnancy are called lobules. Myoepithelial cells form an almost continuous sheath around major ducts but become increasingly discontinuous around smaller ducts. The myoepithelium forms basket shapes around the terminal ducts and alveoli. During lactation, myoepithelial cells contract in response to oxytocin, thus forcing the alveoli to release milk into ductules. Milk passes from the latter into the major mammary ducts. During the fetal stage, the mesenchymal portion of the mammary gland consists of two separate entities (8, 17). One, the dense mesenchyme, is composed of several layers of fibroblasts, which are attached to the mammary anlage. The other is the fat pad precursor tissue, which develops separately and posterior to the mammary anlage. These fetal stromal elements both participate in mammary development (6, 8, 17-21). Heterogeneity among the luminal epithelial cells of mice and rats has been observed. In morphological studies, the Russos and their colleagues (22, 23) have described a number of different cell types in the mammary


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IMAGAWA, BANDYOPADHYAY, AND NANDI

glands of young adult rats. From labeling index experiments at various periods of the rat estrus cycle, as well as from immunocytochemical studies (24, 25), 10 different mammary cell types have been identified (these include the myoepithelial cells). Similar heterogeneity of various luminal epithelial cell populations from mouse mammary glands has been reported (4, 26, 27). At this time, the morphological as well as the functional significance of these various mammary epithelial cell types remains unknown. Recently heterogeneity has also been described among epithelial cells comprising the thyroid gland (28). Several observations suggest the presence of stem cell populations in the adult mammary gland (see Ref. 4). For example, all parts of the mammary tree of both male and female mice at various physiological states, when transplanted into parenchyma-free fat pads, are capable of regenerating into a fully functional mammary gland (excluding the nipple connection) (19, 29, 30). Williams and Daniel (31) demonstrated the occurrence of a monolayer of undifferentiated cells on the outer layers of growing mouse mammary end buds. They provide morphological evidence suggesting that these cells (called "cap cells") are a type of stem cell. Similar conclusions were drawn earlier (24, 25) from studies of rat mammary end buds. Recently, Smith and his colleagues (32, 33) have described another stem cell candidate. This cell is pale staining, contains specific keratin, and occurs in normal mammary glands of mice beginning at the 16th day of embryonic life. These same cells are also seen in large subpopulations of preneoplastic and neoplastic lesions. However, as carefully pointed out by Daniel and Silberstein (4), the identification of putative mammary gland stem cells must await their cloning and the demonstration of their capability to form a functional mammary tree upon transplantation into mammary glandfree fat pads. C. Stages of mammary epithelial cell growth in vivo A variety of methods have been employed in estimating the growth behavior of mammary epithelial cells during different developmental and physiological stages. These include whole mount preparation, measurements of volume, weight, area, DNA- and RNA-content of the mammary gland, determination of mitotic and labeling indices, and various qualitative and quantitative scoring methods (see Refs. 3 and 34). Broadly, five stages of mammary development can be distinguished: embryonic and fetal, prepubertal, pubertal, steady state cyclic adult growth, and growth during pregnancy and lactation. At the end of lactation, the mammary gland undergoes rapid involution, returning to a stage similar to the steady state with cyclic renewal. Excellent descriptions of mam-

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mary growth during these stages have already been made and reviewed (2, 4, 6-16, 35). The present discussion will only briefly summarize material pertinent to the growth of mammary epithelial cells. The pattern of fetal mammary development is essentially similar in mice and in rats (8,11-16, 35). Mammary buds are first evident in embryos at day 10 or 11. In mice, very little growth occurs between days 11 and 16, but from days 16 to 21 rapid proliferation of mammary epithelial cells occurs in females. This includes penetration of mammary epithelial cells into the mammary fat pad precursor tissue with accompanying morphogenesis, resulting in the typical mammary pattern composed of branching ducts. Male mice and rats lack nipples. In male mice, mesenchymal condensation around the mammary buds occurs between embryonic days 13 and 15, resulting in the rupture of the epithelial stalk which would connect the mammary rudiment with the epidermis. In most mouse strains, many of the mammary fat pads of males lack mammary anlage, and the glands remain rudimentary throughout life. The fate of the male mammary gland correlates with the secretion of androgens from the fetal testis and with the acquisition of androgen receptors in the mesenchyme (6, 36-39). In rats, however, most of the fat pads retain their mammary anlage. The latter undergo rapid proliferation, but the glands lack outlets to the exterior (9, 14). In females, considerable proliferation and branching of the mammary duct system occur between birth and puberty (12, 14, 16, 35). The mammary glands of newborns contain only rudimentary ducts with small club ends. The latter regress within a few days, but a second round of growth begins at about 3-4 weeks in mice or slightly earlier in rats. Large club-shaped end buds reappear and invade the mammary fat pad. This prepubertal growth is allometric and varies considerably among different strains of mice and rats. In mice (4, 40, 41), but not in rats (42, 43), this allometric growth appears to depend on the presence of the ovaries. At puberty (4-6 weeks of age), ductal arborization continues until the ducts reach the limits of the fat pad. In a series of elegant studies using [3H]thymidine labeling and autoradiography, Bresciani (44, 45) clearly showed that ductal growth in mice results from the division of end bud cells. The differentiation of an end bud cell into a duct cell is followed by the arrest of cell division. In C3H female mice, both body and mammary growth plateaus at 5-6 months of age. Bresciani (44, 45) observed that DNA synthesis then occurs only in alveolar cells, suggesting that the latter constitute the renewing cell population in adult animals. In prepubertal rats, ductal growth appears to be independent of the gonads, being essentially similar in males


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and females (42, 43, 46-50). After puberty, extensive mammary growth occurs in virgin female rats, resulting in a highly branched ductal system. Depending on the strain, a well developed alveolar system may or may not be present. In adult male rats, the mammary gland contains highly developed dense alveolar clumps (51). Variations in mammary epithelial cell proliferation occur during the estrus cycle of rats and mice. In general, the maximum [3H]thymidine labeling, greatest frequency of mitoses, and the most rapid DNA synthesis occur during diestrus when the glands are the least developed (44, 45, 52-54). (The glands are morphologically most developed at estrus.) The duration of DNA synthesis is longest during metestrus, while mitotic indices remain low throughout estrus and metestrus. Interductal spaces seen in virgin female mice are gradually filled during pregnancy by increased ductal branching and lobuloalveolar formation. Cell division occurs in both the alveolar and ductal cell populations, with the rate of mammary epithelial cell proliferation following a bimodal distribution. The highest rate of cell division occurs on day 4 of pregnancy, just before implantation, with a smaller peak on day 12. The proliferation of mammary epithelial cells continues during the early phase of lactation (34, 44, 45). D. Factors affecting mammary growth in vivo 1. Epithelial-stromal interactions. The in vivo studies done in the 1960s clearly indicated that multiple factors regulate the growth of mammary epithelial cells. In addition to hormones (discussed below), interactions between epithelial and stromal cells were implicated in this regulation. Pioneering studies of Emerman and Pitelka (55) made it obvious that the extracellular matrix, which includes the basement membrane, plays a pivotal role in mammary epithelial cell growth and differentiation. Several recent reviews (1-4, 6, 8, 9) have discussed this topic, so we will only touch upon it here. During rodent embryonic life, the stromal components (fibroblasts and adipocytes) that surround the mammary epithelial cells are involved with mammary growth and morphogenesis. We have already mentioned the fact that, under the influence of fetal androgens, the dense stroma destroys the epithelial stalk in male mice (6, 8). Interesting studies utilizing various chimera have demonstrated the specificity of the mesenchymal role in epithelial development. For example, chimera of salivary mesenchyme and mammary epithelium grown either in vitro (56) or in vivo (57) have resulted in salivary-like epithelial morphogenesis. Transplantation studies utilizing chimeras of mammary-specific mesenchymes with mammary epithelial cells have clearly demonstrated that fat

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pad precursor cells are essential for mammary-specific pattern formation (58). The precise contribution of adipocytes to mammary growth and morphogenesis remains an enigma, although it has been suggested that extracellular components derived from the adipocyte basement membrane may be involved (see Ref. 8). The essentiality of the mammary fat pad for epithelial cell growth, morphogenesis, and the limitation of the size of the mammary tree was first demonstrated by DeOme et al. (19). These workers devised a unique surgical procedure for the transplantation of mammary cells into parenchyma-free mouse mammary fat pads. Subsequent studies (20, 30, 59) revealed that only normal and precancerous mammary cells, and not mammary tumors, showed such dependence. White fat pads (and possibly brown fat as well) encourage the growth of normal mammary cells in endocrinologically intact female mice. Spleen, ovary, sc tissue, brain, uterus, and liver were incapable of supporting such growth. Other types of cell-cell interactions have been implicated in mammary growth regulation, although their significance remains unresolved. For example, interductal spacing in virgin mice may depend upon distant epithelial-epithelial interactions (20). Epithelialstromal/adipocyte interactions may also be involved in increasing the vascularity of the gland during mammary growth (60) and in the loss of fat from adipocytes during pregnancy and lactation (61). Clearly, much still remains to be explored in this area of cell-cell interaction, as well as in the involvement of the stroma and of growth factors (see Section HI) in the growth and functioning of the mammary gland. The better understood role of hormonal factors in mammary morphogenesis and proliferation is discussed below. 2. Endocrine effects on mammary growth Introduction: During the last 60 yr numerous efforts have been made to understand the effects of the various endocrine factors that may be involved in mammary epithelial cell proliferation in various physiological states (see Refs. 2, 3, 9, 35, and 62-65 for reviews). The present section discusses the "classical" studies, up through the early 1970s, before the effects of growth factors and autocrine-paracrine factors on the extracellular matrix and their importance in the control of mammary epithelial proliferation were completely understood. Ideally, to achieve a true understanding of the physiological control of growth regulation, it is necessary to correlate experimental results with the daily cycles and rhythmic variations in the endocrine environment. Additionally, an understanding of the dynamics of hormone receptors in mammary epithelial cells, as well as of cellcell interactions of both parenchyma and stroma, is needed to adequately interpret in vivo studies.


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Clearly none of the earlier (nor most of the more recent) studies have taken all these parameters into consideration, although a considerable amount of important information on the effects of the endocrine system on mammary epithelial cells has been generated. This information currently forms the basis for the analysis of the role of hormones and growth factors on growth and differentiation of mammary epithelial cells in cell culture. Early studies, summarized by Topper and Freeman (9), were based chiefly on three kinds of experimental design: effect of removal of endocrine gland(s), administration of hormones to intact animals, and hormone replacement therapy in endocrinectomized hosts. In all such experiments, attempts were made to induce mammary development resembling that occurring in various physiological states. The involvement of ovarian hormones in mammary development was evident from the different degrees of mammary growth in normal male vs. female mice and rats, in females in different physiological states, and from the effects of early transplantation or grafting of ovaries into male mice and rats (66, 67). The extensive mammary growth during pregnancy and pseudopregnancy of rodents, at stages during which the corpora lutea are highly active, also suggested a role of the ovaries in pregnancyrelated growth of this tissue (12, 68, 69). The possible direct involvement of anterior pituitary hormones was implicated first when Strieker and Grueter (70, 71) discovered that anterior pituitary extracts caused lactogenesis in rabbits. Subsequent availability of steroids (1930s) and of pituitary hormones (1940s and 1950s) resulted in extensive work evaluating the effects on mammary growth of hormones singly and in combination, using both intact and endocrinectomized hosts. These studies attempted to determine whether the active hormones affected mammary epithelial cells directly or through the stimulation of mammogenic hormone secretion from other organs, and also whether or not ovarian and pituitary hormones acted synergistically, additively, or independently. Some of these results were difficult to interpret. For example, estrogens were shown to stimulate mammary growth in intact but not in hypophysectomized mice (72). Investigators began using animals from which all the suspect endocrine organs were ablated either surgically or by other means. Commonly, the hormonal effects in intact animals were compared with those in ovariectomized + adrenalectomized, ovariectomized + hypophysectomized, or ovariectomized + adrenalectomized + hypophysectomized animals. In some cases thyroidectomy or the induction of diabetes with alloxan was also used (see Refs. 9 and 65). In our summary, below, we will describe selected data that we feel to be relatively un-

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ambiguous and that seem most relevant to the interpretation of the results using culture systems. Our description of the effects of the endocrine glands on mammary growth and morphogenesis in vivo will use the developmental stages discussed above (fetal, prepubertal, postpubertal, steady state growth in adult virgins, growth during pregnancy, and lactation). Fetal development: During fetal life, rapid mammary growth occurs only during the last few days of gestation. The male mammary growth pattern, as described above, appears dependent on androgen secretion from the fetal gonad. X-Ray destruction of the gonads in 13-day embryos results in female-like development of the mammary gland in males but produces no change in subsequent mammary development of female embryos (73, 74). Injection of testosterone proprionate into pregnant mothers or directly into the fetuses results in masculinization of mammary growth in the female young (75, 76). Similar injections of estrogen, on the other hand, cause malformation and retardation of mammary growth in the developing fetus (77, 78). Even now, almost nothing is known about the factors that may be involved in normal mammary growth during the last few days of fetal life. Hardy (79) cultured explants of body wall fragments containing the presumptive mammary region from 10-day-old mouse embryos. He used a medium containing a mixture of cock blood and chick embryo extract. Under these in vitro conditions, the primordia differentiated and developed to a level similar to that of a 7 day-old prepubertal mouse. Balinsky (80) obtained similar results but observed no additional effect of adding estrogens or mouse pituitary extracts to the culture medium. Although it is possible that mammogenesis during embryonic life is actually independent of hormones, these experiments are difficult to interpret. The likelihood of the occurrence of mammogenic growth factors in cock blood and/or chick embryo extracts cannot be ruled out. This is an important area which deserves further investigation. Mammary development has been enhanced in explant cultures of fetal mouse (81) and rat (82, 83) tissues by hormone additives that have been found to be mammogenic in adult mammary tissues. These hormones include insulin, adrenal corticoids, and PRL. Prepubertal development: The few club-shaped end buds that are seen in neonatal animals probably result from the residual effects of maternal and fetal hormones. In female mice and rats, mammary growth is isometric for 2 or 3 weeks, beginning at approximately day 7 of postnatal life. Growth becomes allometric at the end of this phase, and in mice, ovariectomy prevents further growth. This initial allometric mammary growth results in the development of large numbers of end buds, and in mice it appears to be dependent on ovarian estrogen (4,


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41). In female rats, on the other hand, mammary growth continues until puberty even after ovariectomy (42, 43). This result is probably due to the presence of sex steroids secreted by the adrenal cortex (see Ref. 3). Studies with intact and ovariectomized prepubertal mice have shown considerable ductal growth after treatment with ovarian estrogen. However, the conclusion that prepubertal ductal growth requires anterior pituitary hormones, in addition to those from the ovaries, became evident when ovarian hormones alone failed to induce ductal growth in hypophysectomized animals (35, 65, 72). In a number of subsequent studies with hypophysectomized + ovariectomized ± adrenalectomized mice and rats, it was observed that the prepubertal type of ductal development with large club-shaped end buds could be induced with hormone combinations containing estrogen + adrenal corticoid + GH (35, 65, 84, 85). Progesterone can substitute for adrenal hormones in these combinations, and elevated levels of progesterone have been reported in prepubertal females (see Ref. 3). Postpubertal development: In the postpubertal female, ductal growth continues until the ductal branches occupy the whole fat pad. The early pattern of postpubertal type of mammary gland development, as well as the steady state condition normally seen in 4- to 6-month-old rats and mice, shows much variation depending on the strain of origin. In some strains, the mammary tree remains primarily ductal, whereas others (for example, SpragueDawley rats or GR mice) show considerable alveolar development as well. Some strains fall within these two extremes. Such ductal growth is probably induced by the ductal mammogenic hormones described above. Alveolar growth in adult rats and mice also depends in part on ovarian hormones. Ovariectomy causes regression of both ducts and alveoli of the mammary gland. In ovariectomized mice, treatment with estrogen causes only ductal growth whereas estrogen + progesterone or progesterone (in large doses) alone causes both ductal branching and lobuloalveolar development (44, 45, 86, 87). In accordance with these morphological findings, estrogen was found to cause cell division only at the tips of the end buds while progesterone or estrogen + progesterone stimulated cell division in both the end buds and the surrounding ducts (44, 45). The role of estrogen may actually be limited to the induction of progesterone receptors on end bud cells, since progesterone alone can replace the action of estrogen. Estrogen is known to induce progesterone receptors in mammary tissues (see Ref. 5). Pregnancy: Studies with hypophysectomized + ovariectomized + adrenalectomized ("triply operated") rats (65) and mice (35) have shown that, in general, a minimum of estrogen + progesterone + PRL is necessary for lobuloalveolar development in all strains. In some strains

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of mice, GH can substitute for PRL. In all cases, a combination of all four hormones causes more lobuloalveolar growth than when only one of the pituitary hormones is included in the treatment. In a few studies, however, lobuloalveolar growth could be induced in ovariectomized + adrenalectomized (or even "triply operated") rats implanted with mammotropic pituitary tumors or injected with pituitary hormones alone, when given three times a day (88, 89). The physiological significance of this finding is still not known. During pregnancy, mammary growth involves enormous increases in branching of the ducts, as well as the development of large numbers of alveoli. Critical studies by Traurig (34) and Bresciani (44, 45) indicate that periods of maximum proliferation during pregnancy can be correlated with periods of high progesterone and PRL or placental lactogen production from ovaries, placenta, and anterior pituitary. Although it is well known that mammary growth continues during early lactation in mice and rats, the endocrine influences on growth during this latter period remain unknown (see Ref. 3). Male mammary glands: Much less attention has been paid to the mammary glands of males. As mentioned earlier, mammary glands are present only in some of the fat pads of male mice, and these remain rudimentary. In male rats, however, considerable ductal growth occurs during prepubertal life, and compact lobules of alveoli are visible even as early as the eighth week of life. Flux (85) studied mammary growth in hypophysectomized + castrated male CHI mice and found that the minimal requirements for ductal, alveolar, and lobuloalveolar development were similar to those reported for female mice of the C3H strain (35, 84). Castration of prepubertal male rats does not affect subsequent ductal growth, although fewer alveoli developed than occurred in controls (51). Ahren and Etienne (90) showed that gonadectomized or gonadectomized + adrenalectomized rats treated with testosterone developed lobules of alveoli. However, testosterone failed to induce such development in hypophysectomized + gonadectomized rats. These studies further showed that testosterone + GH, or estrogen + progesterone + PRL, can cause considerable lobuloalveolar development in gonadectomized + hypophysectomized rats. Estrogen + progesterone + GH did not produce this result (91). Conclusions: In conclusion, these studies have established the multihormonal requirements for mammary growth, and revealed much about the minimum hormonal requirements for the different stages of mammary development in both male and female mice and rats. However, this work does not necessarily accurately reflect the normal hormonal environment or the other factors that may be involved in mammogenesis in vivo during these stages. Surely placental hormones are involved in mam-



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mary growth during pregnancy (92). Other in vivo studies have shown that stromal components of the fat pads are also essential for hormones to successfully cause mammary proliferation. Additionally, knowledge about hormone receptors in mammary tissues at various stages of mammary development is still in its infancy. E. Unanswered questions The in vivo studies discussed above left many unanswered questions about the regulation of growth of the mammary gland during different physiological states. However, they set the stage for subsequent in vivo and in vitro studies designed to explore some of the questions presented below: 1. What, precisely, is the nature of the multiple hormonal requirements for growth and differentiation of mammary cell types and for their morphogenesis? a) How can hormonal synergisms, as well as the direct vs. indirect effects of hormones (systemic or local influences) be explained? Is there receptor modulation? b) Are still unknown factors being produced, locally and/or systemically? c) What is the precise role of the stromal and the adipose tissue compartments? 2. What intracellular mechanisms are involved in the hormonal regulation of growth? 3. What are the limitations of in vivo or in vitro studies in searching for answers to these questions? III. In Vitro Organ Culture Systems A. Introduction and overview In vivo experiments demonstrated that ovarian, adrenocortical, and pituitary hormones were required for the growth and morphogenesis of rat and mouse mammary glands. Because of the complexities inherent in intact physiological systems, it was apparent that an in vitro approach would be necessary to determine whether these hormones had direct effects on mammary tissues. Such in vitro model systems could be used for the systematic study of mammary tissue responses to a variety of hormonal and nonhormonal factors under controlled and defined conditions. Over the years two types of mammary gland organ culture have been used for investigating growth and lactogenesis in vitro. One of these, the explant culture system, utilizes approximately 1 mm3 pieces of mammary gland cultured on supporting grids. The other method uses whole mammary glands. Early explant culture studies by Elias (93, 94), using tissue from midpregnant mice, and Trowell (95), using tissue from postpubertal rats, showed that the histological architecture of mammary explants could be maintained in the presence of a synthetic medium containing only insulin. Rivera (96), using mammary explants from

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immature mice (3 to 5 weeks old) and an insulin-containing synthetic medium, observed epithelial proliferation in the primary duct but no lobuloalveolar development. Whole mammary gland cultures from prepubertal mice, in which the induction of alveolar growth was attempted in the presence of serum plus hormonal supplements (Refs. 97-100 and many others), initially had only limited success. In the mid-1960s, Ichinose and Nandi (101,102) demonstrated that in vivo pretreatment of immature mice with ovarian steroids, followed by culture of their whole mammary glands in synthetic medium supplemented with appropriate hormones, permitted full lobuloalveolar development in these glands in vitro. Banerjee (103) and Mukherjee et al. (104) confirmed the findings of Ichinose and Nandi and further developed a procedure in which regression of lobules that had developed in organ culture could be induced by hormone withdrawal. However, a second round of lobuloalveolar development could not be induced by the reintroduction of mammogenic hormones. Dilley and Nandi (105-107) were able to induce lobuloalveolar development in organ cultures of whole mammary glands from unprimed, immature rats. A significant finding by Tonelli and Sorof (108) showed that lobuloalveolar development could be influenced by the addition of a growth factor. Using whole organ culture of mouse mammary glands, they discovered that postregression, a second round of lobuloalveolar growth, could be induced when epidermal growth factor (EGF) was added to the hormonal "cocktail" capable of inducing the first round of growth. These and subsequent studies with explant and whole gland cultures in the laboratories of Lasfargues (81, 109), Topper (110-114), Sorof (108, 115), Elias (64), Turkington (116), Banerjee (117), Oka (118, 119), and Vonderhaar (120) confirmed many of the previous in vivo findings and extended our understanding of the hormonal requirements for mammary growth and differentiation. Excellent accounts of these developments are documented in reviews by Forsyth (121), Banerjee (103), Elias (64), Errick and KanoSueoka (122) and Borellini and Oka (2). B. Summary of in vitro findings The following conclusions about hormonal and other factors involved in mammary growth and morphogenesis in vitro can be derived from explant and cell culture studies: 1. Insulin appears to be essential for the maintenance but not mitogenesis of mammary cells in organ culture (104). This effect of insulin is thought to be related to its metabolic and housekeeping functions. In vivo evidence also indicates that insulin is not required for


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mitogenesis in mammary epithelial cells. In diabetic mice, lobuloalveolar development can occur although partially reduced, indicating that the cells are maintained in the presence of very low insulin concentrations and that insulin may not be required for growth (9). 2. The minimum hormonal requirement for DNA synthesis and mitosis in rat and mouse mammary epithelial cells in organ culture seems to be a combination of PRL and insulin (104, 106, 109, 112). 3. Both DNA synthesis and lobuloalveolar development can be induced in rat mammary glands in organ culture in the presence of insulin and PRL, although supplementation with ovarian steroids results in additional growth (106,107,123). Unlike rats, immature mice (3 to 5 weeks old) of the strains studied required in vivo pretreatment with estrogen and progesterone (priming) before organ culture in order for lobuloalveolar development to occur. Pretreatment did not change the morphology of the glands in vivo. The minimal hormonal requirement for lobuloalveolar development in vitro in mice appears to be insulin, PRL, plus a corticoid (101104,108). However, the response in vitro varied according to age and strain (99, 124-126). In contrast to immature mice, older mice of some strains may not need steroid pretreatment at all. 4. Tonelli and Soroff (108) found that two cycles of development and regression of the mouse mammary gland can be induced in vitro. The epithelial tree from the whole mammary gland of primed female mice grows and forms lobules when cultured in the presence of a combination of insulin, PRL, aldosterone, and hydrocortisone. However, regression of this tissue occurs when the hormonal cocktail is replaced by insulin alone. A second round of development requires the presence of EGF in the hormone mixture. One could speculate that the initial round of development was dependent upon endogenous EGF that remained associated with the tissue during the initial stage of organ culture. An effect of EGF on mammary development in vitro was also observed by Turkington (116). This finding of Tonelli and Soroff became the stimulus for much work examining the possible physiological role of EGF in mammary development (118, 120, 127). 5. Different parts of the mammary epithelial tree (e.g. ducts, end buds, alveoli, etc.) may respond differently to hormones depending on the maturity and the physiological state of the explanted glands (26, 112).

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differentiation only in synergism with one another and that PRL is a strongly mammogenic and lactogenic factor. Neither type of study has provided significant evidence concerning the precise role of ovarian steroids in mammary cell proliferation. In vitro studies showed that insulin is essential for the maintenance of mammary cells. The novel findings of Turkington (116) and of Tonelli and Sorof (108) suggested a role for EGF in mammary cell proliferation and lobuloalveolar differentiation. Significantly, in vitro studies demonstrate that different parts of the mammary tree have different hormone or growth factor requirements for growth and morphogenesis. Organ culture studies, especially those using synthetic media, avoid many of the interpretive complexities associated with in vivo results. Nevertheless, the former are fraught with at least two complications that preclude unequivocal interpretation of the results. First, there is still no clear explanation for differences between mouse and rat systems with regard to their requirements for in vivo pretreatment. Different degrees of carryover of hormones by the tissues during explantation, the possible induction of EGF receptors, and possible increased levels of circulating EGF have been suggested as causes. Until it is possible to induce mammary epithelial cell proliferation and lobuloalveolar differentiation in vitro, starting with glands from endocrinectomized animals (as is customarily done in vivo), these questions cannot be answered with certainty. Second, interpretation of the organ culture results is complicated by the difficulties in determining cellular targets of hormones and growth factors, e.g. whether stromal, epithelial, or both types of tissue are affected. In retrospect, organ culture studies have demonstrated new growth requirements for mammary tissues. However, information is still lacking concerning precise actions and interactions, at the mammary epithelial cell level, that result in proliferation and lobuloalveolar differentiation. The effects of hormones on stromal and epithelial cells still cannot be separated and thus the real targets and mechanisms of hormone action cannot be correctly interpreted. The use of cell cultures of epithelial and stromal cells, separately and together, may yet overcome some of the limitations now observed with organ culture systems. IV. In Vitro Cell Culture A. Introduction

C. Comparison of in vivo and in vitro findings and conclusions Comparison of in vitro findings with those in vivo reveals both similarities and differences. Both systems similarly show that hormones can induce growth and

Pioneering studies in vivo and in organ and explant culture revealed that mammogenic hormones and possibly EGF act in synergism to regulate the growth of the mammary gland. It was apparent that the elucidation of the nature of this synergism would require the evaluation


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of not only the availability of hormones and growth factors to the tissues but the responsiveness of the target cells to these regulatory factors alone and in combination. It was appreciated that the response of the target cell population might also be influenced directly by epithelial-stromal cell interactions and the presence of as yet unidentified paracrine, autocrine, or endocrine regulatory factors. These considerations stimulated efforts to develop cell culture systems in which the responses of isolated mammary epithelial cells to hormones or growth factors could be studied under controlled conditions. The first problem to overcome in establishing in vitro cell cultures was the separation of the mammary epithelium from the surrounding fat pad. This problem was solved by Lasfargues (128, 129) who found that enzymatic digestion of minced mouse mammary glands with a crude, bacterial collagenase prepared from Clostridium histolyticum yielded fragments of the mammary tree free of fat and stroma. Although the effect of hormones on proliferation was not examined, it was observed that the epithelial cells survived longer and underwent a morphological reorganization (forming projections) when cultured in association with fibroblasts as opposed to culture in their absence. Epithelial cells were able to accumulate lipid only when cultured with glandular stroma or in the presence of exogenously added lipid extracted from mammary adipose tissue. This prompted speculation that in vivo, the adipose tissue served as a lipid store for the epithelium. In a similar vein, a later study from the laboratory of F. J. A. Prop (130) examining collagenaseisolated mouse mammary epithelial cells in culture showed that these cells formed ridge-like structures when cultured in contact with fibroblasts and, furthermore, that this effect was dependent upon the presence of insulin and PRL. These very early studies, although rudimentary relative to current methodology, anticipated two important and interrelated themes that would become crucial for probing the regulation of the growth, morphogenesis, and differentiation of the mammary gland in vitro. These themes are those of the permissive role of the cell substrate and the importance of epithelialstromal cell/adipocyte interactions in directly regulating the growth of the epithelium or its response to growthpromoting factors. As these culture systems were developed further, the possible beneficial role of stromal interactions became lost when the epithelial cells were found to be quickly overcome by rapidly growing fibroblasts. In this section the evolution of mammary epithelial cell culture will be described. We will see that only through the development of more biological substrates and defined media could, once again, attention focus on these more physiological parameters.

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B. Cell culture on nonbiological substrates The collagenase dissociation technique was quickly adopted, and many attempts were made to establish both primary cultures and then cell lines. The discussion will focus on primary cell culture since they have proven to be the most useful in examining mammary epithelial growth in vitro. The major objectives in the pursuit of rodent mammary epithelial cell culture were the establishment of systems in which the cells would proliferate, remain viable for relatively long times, and retain their normal phenotypic and genotypic properties including most prominently, their hormonal responsiveness. In early cell culture systems for both normal and tumor rodent cells, the cells were plated onto plastic or glass substrates in culture medium containing serum. This procedure was not totally satisfactory for several reasons. Serum, which is a highly variable and complex cocktail of hormones, proteins, and nutritional factors, was required for attachment and growth of cells (128,131,132). Thus, the system contained a necessary yet undefined component. Since techniques for obtaining purified epithelial cell populations had not yet been developed, fibroblast contamination and eventual overgrowth of the cultures were continual problems (133, 134). Another serious potential problem was the appearance of polyploidy, and aneuploidy in normal and tumor mouse cells when seeded at low density (135). However, in spite of these difficulties, attempts were made to assess the effects of mammogenic hormones on proliferation by monitoring [3H]thymidine incorporation in short term cultures. Either no hormonal effect in mouse tumors (136) or some stimulation of DNA synthesis by combinations of insulin and mammogenic hormones were observed for normal mouse (137) or normal and tumor rat cells (133, 138, 139). These studies serve to illustrate the problems associated with demonstrating hormonal effects on the proliferation of mammary cells cultured on plastic substrates in undefined media. C. Cell culture on biological substrates 1. Growth in medium containing serum. Because of the availability and ease of use of sterilized plastic cultureware to which cells could attach and spread in serumcontaining media, its use became preeminent in cell culture. This was true for mammary epithelial primary cell culture until Emerman and associates (55, 140) cultured mouse mammary epithelial cells on the top of collagen gels. The cell culture of mouse mammary epithelial cells on the top of floating collagen gels permitted for the first time in vitro the induction and maintenance of cytodifferentiation and the hormonal regulation of casein synthesis and secretion (see Ref. 141 for a recent review of the effect of extracellular matrix on mammary


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epithelial cell differentiation). Type I collagen derived from rat tail tendon was used for these studies following the lead of Michalopoulos and Pitot (142). This effect on differentiation was thought to be mediated by changes in cell shape or nutrient/hormonal availability to the basal cell surface. The ability of collagen to maintain cytodifferentiation and hormonal sensitivity prompted Yang et al. (143,144) to embed normal or tumor mouse mammary epithelial cells within collagen gels in an attempt to promote their proliferation. When embedded within collagen gels these cells grew as three-dimensional colonies. (See Ref. 145 for a review of the use of collagen in cell culture). The collagenase dissociation procedure now included a Percoll gradient step after digestion to remove contaminating fibroblasts and the inclusion of cholera toxin which stimulated the growth of epithelial cells but inhibited the growth of fibroblasts. This culture method offered several advantages over previous methods: for the first time, normal cells in primary culture could undergo multifold increases in cell number while maintaining their normal phenotype, and growth occurred as duct-like colonies reminiscent of in vivo morphology. However, the culture medium required the presence of serum, and no effect of mammogenic hormones on growth could be detected. Instead only EGF, tissue extracts, and cholera toxin were found to be growth stimulatory (146). Using serumcontaining medium Richards et al. (147) compared the growth of mouse cells plated on plastic vs. on or inside collagen gels and showed that proliferation of cells on plastic was limited compared to that on or inside collagen gels. Cells inside collagen gels grew more than cells on top due in part to a larger proliferating population in the three-dimensional colonies. In another study examining the growth of mouse end buds inside collagen gels (148) exponential growth of individual colonies early in culture was noted as well as enhanced growth in medium containing mammogenic hormones and serum. The growth and differentiation of end buds from immature rats were also found to be enhanced by a cocktail of mammogenic hormones in the presence of serum (149). Pasco et al. (150) cultured alveolar cells from mature virgin rats inside collagen, again in serum-containing medium, and found that mammogenic hormones could stimulate growth. These studies utilizing the collagen gel culture system were able to show that mixtures of mammogenic hormones including ovarian steroids and PRL could stimulate proliferation in the presence of serum. However, the contributions of individual hormones to growth were not evaluated, and possible synergistic effects with unknown serum factors complicated interpretation of these responses. The development of serum-free media finally permitted a more

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detailed and comprehensive analysis of the regulation of proliferation by hormones and growth factors. Although the use of collagen gels proved to be a significant development in mammary epithelial cell culture it is clear that collagen gels per se are not required for sustained proliferation in vitro. An interesting coculture system was developed for mouse mammary epithelial cells in which they were cultured only in the presence of an irradiated rat mammary tumor epithelial cell line (151). The cells could be grown and passaged in this system. The nature of the "feeder" effect remains undetermined but could involve both soluble factors and a substrate effect (similar to collagen). The growth of the cells was inhibited by EGF, and no effect of mammogenic hormones on growth was reported. 2. Growth in serum-free medium. A serum-free medium developed for the culture of normal and tumor mouse mammary epithelial cells (152) originally contained insulin, EGF, and BSA as essential components. Later work showed that in a defined basal serum-free medium containing insulin and phospholipid, both progesterone and PRL (but not estrogen) alone and in synergism could stimulate the proliferation of cells from normal postpubertal virgin mice. The proliferation of cells from midpregnant mice was only slightly stimulated by mammogenic hormones probably because of their more differentiated phenotype (153). Cells from ovariectomized as well as ovariectomized and adrenalectomized animals can also undergo sustained growth in serum-free media in response to hormones and EGF followed by the induction of differentiated product synthesis (154). The cells from virgin mice contained progesterone receptors whose level was highest in the presence of PRL and either estrogen or progesterone (155). Only a low level of estrogen receptors was found; however, estrogen could elevate the level of progesterone receptors indicating that the estrogen receptors were functional. Inside collagen gels the cells from virgin mice formed colonies containing a lumen around which the cells became polarized with the apical surface forming characteristic microvilli. In the presence of PRL alone they developed a secretory morphology with the appearance of casein micelles (156). In media containing EGF or mammogenic hormones the cells were predominantly of a luminal epithelial morphology with only occasional myoepithelial cell types. Cells cultured in the presence of hormones or EGF formed normal ductal outgrowths (157) when transplanted into cleared fat pads in vivo. Cells from mature virgin rats were also successfully cultured in serum-free medium (158). Both a luminal and myoepithelial cell type were found to proliferate in an insulin-containing basal medium in response to, respectively, the combination of progesterone plus PRL,


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or EGF. Again, as was observed in the mouse, estrogen did not stimulate proliferation but could induce progesterone receptors (159). The possibility that phenol red in the culture medium might mask an effect of exogenously added estrogen (160) was eliminated when the lack of an effect of estrogen on the growth of mouse and rat cells was confirmed in phenol red-free medium (161). More recent experiments using this culture system (S. Nandi, unpublished observations) indicate that for the growth of the small, ductal/alveolar type of cell, PRL can be replaced optimally by the combination of fibroblast growth factor (FGF) and EGF. Progesterone is still absolutely required. Previous workers have shown that when rat cells are cultured in the presence of serum, several cell types are distinguishable (162). The observation of the selective proliferation of two different cell types in response to a growth factor or mammogenic hormones demonstrates how the growth of these cell types might be differentially regulated. In another type of collagen culture system, Ethier (163) found that rat cells from virgin rats could be cultured on type I collagencoated surfaces. In serum-free medium (164, 165) a variable response to progesterone and PRL was observed only in the presence of EGF and a low pH (7.1-7.2). A small effect of estrogen on proliferation was also reported in basal media containing EGF and cholera toxin. What was unclear in these studies was the nature of the cell types, luminal or myoepithelial, whose growth was stimulated in the presence of mammogenic hormones or EGF. 3. Cell matrix requirements for growth in vitro. A clue to the permissive effect of collagen on growth was provided by the observation that cells cultured on floating collagen gels (55) elaborated a visible basement membrane; cells cultured on plastic did not. This observation suggested that collagen might modify the ability of rodent mammary epithelial cells to synthesize basement membrane components and stabilize the assembly of a basal lamina. In a study using a mouse mammary epithelial cell line (166), type I collagen was found to inhibit the degradation of basal laminar heparan sulfate-rich proteoglycans suggesting a mechanism through which basal lamina formation may be promoted by collagen. Cells cultured on plastic degraded the proteoglycans releasing them into the culture medium. Primary cultures of mouse cells cultured on collagen also deposit glycosaminoglycans (GAGS) into an extracellular matrix while cells on plastic, although capable of synthesizing GAGS, release the majority of them into the medium (167). These studies suggested that the formation of a basal lamina or the deposition of basal laminar materials in the extracellular matrix might regulate proliferation. If so, then growth regulatory factors could conceivably affect growth by regulating the turnover of extracellular

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matrix components secreted by the cells. This was found when the growth response to EGF and hormones was compared in rat cells plated on plastic or different collagen substrata (168, 169). When cells were plated on type IV collagen (epithelial cell basement membrane collagen), EGF was not required for growth in serumfree medium containing fetuin. However, EGF was stimulatory to growth when cells were plated on plastic or on type I collagen-coated surfaces. This EGF requirement correlated with EGF's ability to stimulate type IV collagen synthesis, and it was hypothesized that EGF stimulated growth on type I collagen or plastic via its effect on type IV collagen synthesis. Cortisol was found to inhibit collagen degradation and stimulate growth. Newer findings indicate that transforming growth factored also can stimulate collagen synthesis in cultured mouse cells (170). In an earlier study of cultured rat cells, a hormonal cocktail including insulin, PRL, cortisol, progesterone, and estrogen inhibited the turnover of type IV collagen and the production of a type IV collagenase (171). If type IV collagen synthesis was inhibited by the proline analog, cis-hydroxyproline, then the growth of normal (172) or tumor (173) rat cells could be inhibited in vitro as well as in vivo (173, 174). Using collagen gel cultures, Richards (147) observed a similar growth inhibition by cis-hydroxyproline of normal and tumor mouse cells. The latter study was done in serum-containing medium, and a visible basal lamina was noted around the basal surface of the colonies. In growth-promoting, serum-free medium containing progesterone plus PRL, or EGF, no visible basal lamina could be seen although the presence of type IV collagen and laminin in and around the cells was verified using immunocytochemical methods (156). It is clear that hormones and growth factors can affect the turnover of extracellular matrix materials but further work is necessary to establish how this effect may be coupled to or coordinated with their activation of intracellular growth regulatory pathways. Examination of the turnover and deposition of basal lamina components demonstrated how the extracellular matrix could promote the growth of mammary epithelial cells and explain, in part, the requirements for hormones and growth factors. Following the example of liver cultures (175), Wicha et al. (176) isolated extracellular matrix from the mammary glands of pregnant rats and cultured rat cells on this matrix. Both growth and differentiation were achieved on this matrix with the maintenance of better functional differentiation compared to cells cultured on floating collagen gels. The effect was tissue specific since liver-derived matrix had no similar effect. Although illustrative of the importance of extracellular matrix, this system is difficult to use in practice since growth is difficult to quantitate and the matrix not easily obtained and used. (See Ref. 177 for a discussion


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of the relative merits of different in vitro cell and tissue culture systems used to study growth and differentiation.) 4. Possible roles of the stromal/adipose cell compartments in the regulation of epithelial cell growth. In this section we expand our discussion beyond the epithelium and associated basal lamina to the surrounding stromal matrix and fat cells. As we have seen, in vitro cell culture systems advanced quickly once techniques became available to reimpose the extracellular matrix or a facsimile thereof upon the epithelium. Implicit in culture systems using type I collagen is the notion that the collagenous substrate somehow mimics the in vivo stroma thus promoting the proper deposition and synthesis of the cells own unique extracellular matrix. We have previously discussed how in vivo, mammary epithelial cells require the adipose tissue matrix for proper growth, morphogenesis, and differentiation. The nature of the interactions between the parenchymal and extraparenchymal compartments has been unknown, but cell culture with collagen has suggested that at least the stromal compartment plays a permissive role in supporting epithelial cell growth. A more active role assumed by the fat cell compartment has been proposed, i.e. fat cells contributing lipid to the epithelium, but only relatively recently has this possibility been examined in vitro. This discussion will focus on studies exploring the possible contributions of the stromal and fat cell components of the mammary gland to epithelial growth. Coculture systems have been used to examine the role of extraparenchymal adipose tissue in the regulation of mammary epithelial cell growth. These systems have employed coculture with stromal fibroblasts or adipocytes as well as with pieces of mammary tissue and have revealed that both the substrate and soluble factors produced by fibroblasts or fat cells can stimulate mammary epithelial cell proliferation. The growth of mouse mammary epithelium was promoted when these cells were cultured in 5% serum on lethally irradiated preadipocytes (3T3-L1) or their differentiated adipocyte derivative (178, 179). A 3T3 cell line that did not undergo adipocyte conversion was much less effective in stimulating growth. This growth-promoting effect was attributed to two factors: the provision of the appropriate substrata since cell-free substrate attached material (principally GAGS and fibronectin) produced by the adipocytes had the same effect, and to the production of a soluble factor(s) as revealed by the use of preadipocyte or adipocyte-conditioned medium. Similar to the original observations of Lasfargues and Visser, when mammary epithelial cells were cultured on adipocytes (180), a ductlike morphogenesis occurred along with the formation of

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a basement membrane and biochemical and secretory differentiation upon exposure to lactogenic hormones. Mammary fibroblasts from normal glands also stimulate the proliferation of normal mouse mammary epithelial cells via a diffusible factor (181). This partially purified epithelial growth-stimulatory factor was a protein (not EGF) of about 100 kilodaltons recovered from fibroblast-conditioned medium. Similarly, a diffusible fibroblast factor(s) stimulated the growth of mouse mammary tumor cells (182). This activity was present only in cocultures in collagen gels, not on plastic. An effect on normal cells was not reported. The above studies did not address the effect of stromal or fat cells on the hormonal regulation of cell proliferation. Interestingly, however, the hormonal regulation of differentiated product synthesis was shown to be dependent on the presence of live cells not just an adipocyte extracellular matrix (179). Haslam (183, 184) examined the effect of mammary stromal fibroblasts on estrogen induction of progesterone receptors finding that this effect was present in epithelial cell-fibroblast cocultures but lost when epithelial cells were cultured on plastic. Again, as in the case of cell proliferation, this fibroblast effect was also due to the extracellular substrate since type I collagen could replace the fibroblasts and, as discussed earlier, this estrogen effect is seen in cells cultured inside collagen gels. An effect of fibroblasts on estrogen-stimulated DNA synthesis was claimed by McGrath (185). When normal mouse mammary epithelial cells plated on plastic physically touched fibroblasts, cells at the region of contact appeared to undergo enhanced DNA synthesis in the presence of estrogen. These results were difficult to interpret, however, but they do support the notion of a stromal influence on the hormonal sensitivity of the epithelium. A role for the lipid stores of the fat cells of the mammary gland in the regulation of mammary epithelial cell proliferation has been the subject of much interest over the years. Attempts to establish the validity of an epithelial-fat cell lipid axis first concentrated on examining the effect of exogenous fatty acids on the growth of both normal, preneoplastic, and tumor cells in vitro. In general, these studies, involving the addition of fatty acids alone (186-188) or entrapped in liposomes (189, 190), showed that unsaturated fatty acids, linoleic and oleic in particular, could stimulate the proliferation of mammary cells. Saturated fatty acids such as palmitate and stearate were inhibitory to growth. Studies by Bandyopadhyay et al. (186, 191) using serum-free medium showed that linoleic acid and its eicosanoid derivatives, arachidonic acid, prostaglandin E2, and hydroxy fatty acids, stimulated growth only in synergism with another growth-promoting factor such as EGF. That the fat pad could be the source of fatty acids (and phospholipids)


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that could stimulate epithelial proliferation was shown in vitro by using coculture with pieces of fat pad or fat pad-conditioned medium (192,193). Rudland (194) found that prostaglandin E2 (PGE2) could be released from stromal cells (preadipocytes) and stimulate the growth of fat mammary epithelial cells. A hormonal mechanism utilizing this fat cell-epithelial cell axis in growth stimulation was proposed by Kidwell et al. (169, 195). This hypothesis proposed that PRL, which can cause fatty acid release from the fat cells (possibly via the stimulation of histamine release from mast cells) and promote fatty acid uptake by the epithelium might promote mammary epithelial growth in part through this pathway. More recently phospholipids containing unsaturated fatty acids (196) have been shown to be mitogenic for mouse mammary epithelial cells in collagen gel culture. All these studies have demonstrated that the mammary epithelium can proliferate in response to lipids, but as yet no good evidence showing that these observations are of physiological relevance has been obtained. Nevertheless, these studies strongly suggest that the fat cells of the mammary gland are potentially capable of exerting a regulatory influence on the growth potential of the epithelium. A new finding was that mammary stromal fibroblasts and, perhaps, adipocytes, are a potential source of peptide growth factors for the epithelium. The possible role of lipid-dependent intracellular pathways involved in cell proliferation will be discussed later (Section IV). Overall, the in vitro systems utilized to examine the role of stromal cells and adipocytes in the regulation of the epithelial compartment have taken in vitro culture closer to the in vivo situation and support the concept that stromal influences, implied from collagen and extracellular matrix effects in vitro, depend upon interactions of the mammary epithelial cells with both the mammary stromal fibroblasts and fat cells. 5. Growth regulation by recently characterized growth factors. Much discussion has already focused on the effect of EGF on the growth of mammary epithelial, cells in vitro. EGF serves as an example of a possible growth regulatory factor for the mammary epithelium which was not detected or anticipated by in vivo studies. The effect of EGF on mammary epithelial cell proliferation was first detected in cell culture followed by organ culture. In the next section we will discuss efforts taken to establish that this growth factor is of physiological significance for the mammary gland. More recently the requirement for a high, superphysiological concentration of insulin in serum-free culture of rodent mammary epithelial cells has been attributed to its binding to the insulin-like growth factor I (IGF-I) receptor (197, 198). IGF-II was not growth stimulatory. IGF-I (somatomedin C) stimulated growth only

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in synergism with growth factors or mammogenic hormones. Since it is present in the blood in high concentrations it probably serves only a permissive role in the regulation of mammary gland development, but this speculation remains to be verified in vivo. Insulin at physiological concentrations (1 Mg/mO or IGF-I (50 ng/ml) is required to which one or more of the other growth-stimulatory factors listed in the table can be added. Lipids, excluding 18:2-PA and 18:2PS, require a factor from one other category in addition to insulin or IGF-I in order to stimulate growth. EET, Epoxy-eicosatrienoic acid; 18:2-PS, dilinoleoyl-phosphatidylserine.

become available from mouse mammary epithelial cell cultures, we shall dwell more on the mouse culture systems here. The proliferative responses to these nonhormonal factors have provided clues to the possible underlying mechanisms that may be involved. Understanding the effects of these factors on proliferation may lead to the elucidation of the intracellular mechanisms involved in the initiation of proliferation and help explain the synergistic growth-stimulatory relationships among hormones, growth factors, and lipids. B. Classification of in vitro growth stimulatory factors: Groups 1, 2, and 3 The growth-promoting factors shown in Table 1 can be reclassified according to their possible modes of action into three groups. One group consists of those factors that require specific receptors for their action, e.g. PRL, progesterone, corticoids, EGF, FGF, and stimulate growth in the presence of only insulin (group 1). The remaining two groups are those diffusible agents that may affect signal transduction pathways directly. These are further subdivided into those agents that can stimulate growth in the presence of insulin only e.g. cAMP lithium ion (Li+), 12-O-tetradecanoylphorbol-13-acetate (TPA), dilinoleoyl phosphatidic acid (18:2-PA) (group 2) or only in synergism with factors from groups 1 or 2 e.g. linoleic acid (18:2a;6), arachidonic acid (20:4a>6), dihydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs), PGE2 and PGEi (group 3). These group 3 agents may be thought of as comitogens that act via modifications of receptor or postreceptor functions of the mitogens. The response of mammary epithelial cell to the diffusible agents that are not hormones or peptide growth factors suggests the existence of intracellular pathways that may be separate from the receptormediated pathways. C. Group 1: peptides and steroids The hallmark of actions of peptide growth factors and hormones (such as the group 1 mitogens) that utilize

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plasma membrane-bound receptors is their tyrosine-kinase activity; a notable exception is the PRL receptor. Attempts have been made, using explant culture systems, to correlate the effect of PRL on mammary functions with phospholipase A2 and protein kinase C (PKC) activities, polyamine synthesis, PGE2 production, and phosphatidylinositol (PI)-metabolism (237-241). However, using explant cultures, it has been reported by other groups that the effects of prolactin on casein gene expression can not be mimicked by PGE2, cAMP, cyclic-3',5'GMP (cGMP), calcium fluxes, and polyamines (242, 243). It is probable that minimal levels of cAMP and polyamines have to be maintained in order to achieve growth and differentiation, but the modulation of these levels is not the critical growth-promoting function of PRL receptor. On the other hand, it is possible that PRL stimulates adipocytes of the explants to produce arachidonic acid (via phospholipase A2) and its metabolites which, in turn, act on mammary epithelial cells as proposed by Kidwell and Shaffer (169). In mouse mammary epithelial cells in collagen gel culture, PRL-stimulated proliferation can be potentiated by 18:2co6 or cAMP supplementation. Thus, it is possible that PRL may stimulate growth via extracellular and intracellular mechanisms. The availability of complementary DNA (cDNA) clones for the PRL receptor (244, 245) and their transfection into and expression in receptor-deficient cells may lead to a clearer understanding of the mechanism of action of PRL. The discovery of different forms of the PRL receptor (244-246) has also raised the possibility that the diverse actions of PRL may be mediated by different receptor subtypes. The receptors for insulin, EGF, FGF, and IGF-I and FGF receptor-related proteins have tyrosine kinase activity (247). Tyrosine phosphorylation plays an essential role in growth stimulation by EGF (248). A mutant EGF receptor, defective in tyrosine kinase activity, loses its mitogenic activity (249, 250). The recent observations that PI-3-kinase (251, 252), GTPase activator protein (GAP) for p21raA (253, 254), phospholipase C (PLC) (255, 256), and ra/-protein (257) are phosphorylated by tyrosine kinase receptor activity are of considerable significance because of their role in signalling pathways (247, 248). Although there are indications that some serine/ threonine kinases may relay tyrosine kinase-produced signals, how and what postreceptor messages reach the nucleus remain unknown. The steroid hormones of the group 1 mitogens function through either cytosolic or nuclear receptors. These receptors do not have any intrinsic kinase activity. Their main role is to bind to their respective DNA-responsive elements and function as transcriptional activators for specific sets of genes. How steroid-specific gene activation occurs and target cell specificity is maintained is


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currently under intense investigation. Recent reviews survey these areas (258-261). D. Group 2: cAMP, lithium, phorbol esters, phospholipids 1. cAMP. The group 2 mitogens (cAMP, 18:2-PA, lithium, TPA) for mammary epithelial cells, which do not need typical receptors for their action, suggest that some novel pathways operate in these cells. cAMP elevating agents (cholera toxin, PGE2, PGEl5 etc.) have been shown to have mitogenic effects on 3T3 cells (262, 263). Although it has been observed that cAMP levels in guinea pig (264), rat (265, 266), and mouse (267) mammary glands rise during the proliferative phase of pregnancy, it is not known what causes this increase and in which cellular compartment. It is interesting to note that cAMP-dependent protein kinase A (PKA) activity in the mammary gland also increases during pregnancy (268, 269, 270), but no specific substrate for PKA has been identified. Cell culture studies with mouse (234, 235, 271, 272), rat (165, 273), and human mammary epithelial cells (274) demonstrate the mitogenic activity of cAMP in the mammary gland without resolving the issue of physiological significance. However, cAMP or cholera toxin, when injected systemically (275) or released locally in the gland by slow release implants (276, 277), can stimulate ductal mammary growth in vivo. These findings show that mammary epithelial cells in vivo are capable of proliferating in response to cAMP and, therefore, contain a cAMP-responsive growth-regulatory mechanism. As yet not well understood is the nature of the stimulus that normally functions as the physiological agonist for adenylate cyclase in mammary epithelial cells. Possible candidates are linoleic acid and PGE2 or PGEi. It would be of interest to know whether PGE2 or PGEi, released in the gland by a minipump or implant, can cause mammary epithelial cell growth similar to cAMP or cholera toxin. However, from collagen gel cell culture studies we know that there is a difference between the effect of PGE2 and cAMP on growth (235). The response of cultured mammary epithelial cells to PGE2 (a comitogen, which potentiates the action of a mitogen) and a high concentration of exogenous cAMP (a mitogen) suggests that the surge in the intracellular cAMP level (which drops after few hours), induced by PGE2, may only modulate receptor pathways [possibly via PKA, cAMP response elements, and transcriptional activators (278, 279)]. In contrast, the maintenance of a high concentration of extracellular cAMP (100-200 Mg/ml) by exogenous supplementation may activate separate mitogenic pathways (280). The cAMP pathway in mammary epithelial cells can be stimulated by 18:2co6, arachidonic acid (20:4a>6), PGE2, PGEa, cholera toxin, and (Bu)2cAMP. The cAMP-

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mediated effects of all these agents can be inhibited by a very low dose (50-100 pg/ml) of pertussis toxin. Pertussis toxin which can ADP-ribosylate GTP-binding proteins (G-proteins), inhibits growth factor and hormone-stimulated mammary epithelial cell proliferation by about 25%, whereas, inhibition of the cAMP-mediated response was 70-75% (G. Bandyopadhyay, unpublished observation). These results suggest an important role for G-proteins in the cAMP-dependent pathway. Recently, a role for G-proteins in the proliferation of normal mammary epithelial cells from bovine mammary tissue has been reported (281). 2. Lithium. Lithium ion has long been known as an antidepressant. Surprisingly it also is mitogenic for cultured mouse mammary epithelial cells (282-284) as well as other cell types (285-287). In this regard, a functional role for PI metabolism in mammary epithelial cells becomes relevant because lithium is a strong inhibitor of inositol phosphate phosphatases and is known to cause intracellular accumulation of inositol phosphates (288290). In spite of much data showing a correlation between the stimulation of PI hydrolysis and cell proliferation (291), the role or the necessity for PI metabolism and PLC-mediated signalling in cell proliferation remains unresolved. For example, overexpression of PLC in transfected cells increased inositol trisphosphate levels (indicative of a stimulation of PI hydrolysis), but no stimulation of Ca2+ signalling and DNA synthesis occurred (292). This result reinforces the notion, expressed by some investigators, that PI metabolism may not be essential for mitogenesis (292-295). However, the involvement of PI metabolism in mitogenesis may depend upon the cell type and the particular mitogenic response being examined. For example, there is evidence that the specific blockade of PI metabolism with microinjection of antibodies to phosphatidylinositol-4,5-bisphosphate (296) or PLC (297) can inhibit the proliferative response to some but not all growth-stimulatory factors in NIH 3T3 cells. Lithium has also been found to alter cAMP synthesis (298, 299), Na+ (300-302) and Ca2+ transport (303-305), and G-protein function (306) in different cell types (for a general discussion, see Ref. 307). At present it is debatable whether or not the effects of lithium on cAMP, Na+, and Ca2+ metabolism are more relevant than PI metabolism for mitogenicity. 3. Phorbol esters. The phorbol ester, TPA, and the fatty acids, 18:2OJ6 and 20:4a;6, are activators of PKC (308312). Although TPA influences multiple biochemical pathways, its stimulatory effect on mammary epithelial cell proliferation also suggests a possible role for PKC (236, 313, 314). PKC activity in the mammary gland has been shown to remain high in the virgin and during early pregnancy before declining steadily during late preg-


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nancy and lactation (315). Both in vivo and in vitro results correlate PKC activity and mammary epithelial cell proliferation. Like PKA, true target proteins for PKC have not been identified. The proposed role of PKC in the activation of nuclear transcriptional regulators {e.g. AP-1, AP-2) has drawn much attention (316-318). 4. Phospholipids. Phosphatidic acid (PA) has been shown to be mitogenic for fibroblasts (319, 320) and mouse mammary epithelial cells (196). Unlike fibroblasts, mammary epithelial cells do not proliferate in the presence of PA containing saturated fatty acids; unsaturated fatty acids (UFA), preferably 18:2a>6 or 20:4co6, are essential. Although free UFAs themselves are not mitogenic, their presence as esterified fatty acids is required for the mitogenic effect of negatively charged phospholipids (PA, phosphatidylserine). In fibroblasts, lysophosphatidic acid (LPA), with or without UFA, has been shown to be mitogenic (321). Considering the effective concentration range for LPA mitogenesis in fibroblasts, the mitogenic effect of PA observed in mouse mammary epithelial cells is apparently not due to minor contamination with LPA. Although both PA and LPA can mobilize cellular calcium (321-324), their biochemical effects are likely to be different. Recently it has been found that PA can inhibit the activity of a cytoplasmic GAP which can stimulate the GTPase activity of wild type p21ros (325). It now seems that some UFAs can be just as or more effective than PA (326) in producing this effect. However, the fact that microinjection of anti-p21ras antibody can block PA-stimulated mitogenesis in NIH 3T3 cells suggests that cross-talk occurs between ras and PA signalling pathways (319). Unlike phosphatidylserine, PA is not a direct activator of PKC activity, but, since 18:2-PA can elevate cellular Ca2+ and diglyceride levels and generate free 18:2co6, its indirect role as intracellular activator of PKC activity can not be discounted. This issue needs careful examination. The expression of a number of oncogenes (ras, raf, fos, mos,jun, etc.) and activities of their products have been implicated to play important roles in signalling mechanisms (257, 278, 316, 327-333). Among these, the functions of wild type or mutated ras protein (p21ra') in cell proliferation and transformation have been subjected to rigorous scrutiny. For example, microinjection of a neutralizing antibody against p21ras into some cell lines causes dramatic inhibition of cell proliferation, even though the life of the microinjected antibody is short, 30-40 h (334, 335). On the other hand, cells expressing raf and mos (which are serine/threonine kinase) are resistant to such inhibition, suggesting that ras and the other two oncogene products follow different pathways. The same antibody can prevent the expression of src (which possesses tyrosine kinase activity) related func-

513

tions (for a general discussion on the effects of antip21ras antibody, see Refs. 327 and 328). Furthermore, overexpression of H-ras in Rat-1 fibroblast cells increases the potential for basal and mitogen-induced proliferation (336). It appears from the interactions between various kinases that the p-raf and p-raos serine/threonine kinases may act downstream to tyrosine kinases (247, 257, 332, 333). The possibilities that the control of p21ros activity by 18:2o;6 and 18:2-PA (through GAP inhibition) and the cAMP-pathway by PGE2 may constitute lipid-responsive pathways in mammary epithelial cells, should be examined. E. Group 3: linoleic acid and its eicosanoid products The group 3 compounds are lipidic in nature. The actions of these lipids and 18:2-PA of group 2 strengthen the notion that lipids are direct participants in mitogenic pathways in mammary epithelial cells. The effect of linoleic acid on growth is largely but not wholly dependent upon its eicosanoid derivatives. The eicosanoid-dependent effect can be demonstrated by blockade of the arachidonic acid cascade by specific inhibitors that also inhibit the synergistic action of this fatty acid on growth. This growth inhibition can be overcome by supplementation with lipids, such as PGE2 and HETEs, produced distally to the point of inhibition (186, 191). The source of these lipids for intracellular growth-regulatory mechanisms may be extracellular as previously described or generated intracellularly. It is likely that hormones and growth factors stimulate the intracellular production of lipids involved in signal transduction and, as such, these lipids act as messengers that can directly modulate or activate signal transduction pathways. F. Multiple pathways regulate growth Most of the significant results in the field of signal transduction have come from experimental systems using cells other than normal mammary epithelial cells. However, a number of mammary tumor cell lines, e.g. T47D, MCF-7, MDA-468, and some of their variants, have been extensively used for some of these studies. The results obtained with these cells corroborate those obtained in other systems. It should be cautioned, however, that many conclusions drawn from the experiments with mammary tumor cell lines may not hold for primary cultures of normal mammary epithelial cells. In the case of normal mammary epithelial cell proliferation, very little is known to describe a meaningful signalling pathway. However, aside from steroid hormone receptors, it appears that the transduction of signals from the plasma membrane involves lipid-responsive pathways. These lipid-responsive pathways may be initiated by the stimulation of phospholipid turnover by


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hormones and growth factors. We propose that investigating the following questions will help to clarify our understanding of the importance of these pathways: 1) Can these cells proliferate under inositol-deprived conditions? In other words, is PI metabolism essential for mitogenesis? 2) Does lithium act only by affecting PI metabolism or by modulating ion fluxes across the cells? 3) Why are UFA necessary for PA-stimulated mitogenesis in mammary epithelial cells? 4) Is the PA effect mediated by its role as substrate for increased PI synthesis, a regulator of Ca2+ metabolism, or as an activator of ras function through inhibition of GAP activity? 5) Does cAMP stimulate growth via multiple pathways? We are beginning to address some of these questions. From our experience, it appears that the persistence or maintenance of one or more activated signals (e.g. high Ca2+, cAMP, alkaline pH, activated p21ras and PKC) for a prolonged period, not their transient rise and fall, is necessary for growth stimulation. Our present conceptualization of the multiple pathways involved in growth regulation in the case of mouse mammary epithelial cells is depicted in Fig. 1. The relative importance and interactions of these intracellular pathways remain obscure, but we depict the array of initiating events in mammary epithelial cells. In Fig. 1, we have basically outlined five cascades of events that send specific messages to the nucleus: I, cAMP-PKA-related; II, p21ms-GAP-related; III, tyrosine kinase-p-ra//p-mos related; IV, PI cycle/ PKC-related; and V, pHi related. Based on the available information, it appears that lipid stimulation of mammary epithelial cell growth may be functioning through cascades I, II, and IV. Although a diverse variety of agents can influence mammary epithelial cell proliferation, it is possible that the actions of many of these agents may converge at the

level of transcriptional regulation. It has been observed that the expression of the protooncogenes c-fos and cjun are associated with growth stimulation by serum, EGF, and TPA (337-340). Two separate types of response elements containing promotors with AP-1 binding sites, serum response elements and TPA response elements, are involved in the action of growth factors and TPA. The products of jun and fos can up-regulate the transcription of promotors containing AP-1 binding sites (316, 331, 341-343). Also the binding protein for cAMP-response elements can activate AP-1 site-containing promotors (only after phosphorylation by PKA) (344, 345), and some of the hormone response elements also contain AP-1 binding sites (346). Another transcription factor, AP-2, has been shown to mediate activation of transcription by both TPA and cAMP (318). It appears, therefore, that the actions of most of the mitogenic signals may converge at sites for transcriptional activation by AP-1 and AP-2. The additive or synergistic effects of the various mitogens may relate to interactions or cooperation among their respective response elements. What specific gene products are involved and how they cause stimulation of DNA synthesis and cell division remain to be determined. VII. Overall Summary and Conclusions In this review we have attempted to show how our concepts about the regulation of the growth of the mammary epithelium have evolved, beginning from the initial in vivo studies to the present state of our knowledge. Our purpose has been to frame the in vitro observations within a physiological context since the intent of in vitro systems is to advance our understanding of the regulation of mammary epithelial cell growth in vivo. As hoped,

PGE2

18:2

FIG. 1. Proposed intracellular pathways for signal transduction in mammary epithelial cells: I, cAMP-PKA-related; II, p21ros/GAP-related; III, tyrosine kinase/ ra/-mos-kinase related; IV, PI cycle/ PKC-related; and V, pHrrelated.

Growth factors Hormones

Oncoproteins ser/threo-kinase (rat. mos )

Receptor Tyr-kinase

III * ?

Activation of nuclear DNAbinding and transcriptional factors, e.g., fos, jun, myc

AP-l.AP-2

IV

18:2-PA

PK-C Organelles e.g.. ER

Lithium Na*

Membrane

\ pHi

Cytosol

Nucleus


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increasingly sophisticated in vitro systems have succeeded in providing new insights into the complexity of mammary epithelial growth regulation in vivo much of which involves the consideration of new modes of action of hormones. The early in vivo studies identified a number of interacting factors that are important for growth, morphogenesis, and differentiation in the mammary gland. These factors are systemic factors (hormones) and local stromal influences of the gland, e.g. adipocyte environment. Among the systemic factors, a combination of estrogen, adrenal corticoids (or progesterone), and PRL was found to satisfy the minimum hormonal requirement for full lobuloalveolar development. The fact that epithelial organoids from any part of the mammary gland could undergo normal mammary development only when transplanted to a fat pad suggested that the adipose tissue in the gland provides the most permissive environment for the expression of mammary functions. These results might also imply that the epithelial organoids contained pluripotent stem cells capable of developing into a full gland. With appropriate systemic and local support, a developing mammary gland shows heterogeneity in cellular morphogenesis and hormonal responses at different parts of the mammary tree suggesting the existence of different cell types. This was further corroborated by labeling index studies and immunological marker analysis. Except for estrogen, in vitro studies generally supported the in vivo observations regarding the role of various factors in mammary epithelial cell proliferation. The basic contributions of the in vitro studies lie in the following observations: 1) the requirement of insulin for maintenance of cell or organ cultures, 2) the mitogenic role of peptide growth factors, 3) the mitogenic role of cAMP, 18:2-PA, and Li+, and 4) the regulatory role of the components of extracellular matrix. While all these in vitro observations have extended our knowledge of mammary biology, they have proven less useful for defining the cellular phenotypes present in vivo. In other words, we cannot be sure that the phenotype of the cells taken from glands is maintained when the epithelium is enzymatically dissociated and placed in cell culture. In later in vivo studies, mammary gland implants of elvax pellets containing diffusible agents provided evidence that local actions of estrogen, EGF, and cAMP were involved in mammary epithelial cell proliferation. With regard to the actions of ovarian steroids, it appears that while progesterone can be a direct mitogen for mammary epithelial cells, the estrogen effect on mammary epithelial cells in vivo may be mediated indirectly. The two known indirect effects of estrogen on mammary functions are stimulation of PRL (itself a mitogen for mammary epithelial cells) secretion and progesterone

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receptor induction. In this connection, the question of whether or not estrogen regulates the production of growth factors or extracellular matrix components locally by the stromal cells becomes pertinent for understanding the nature of local actions of estrogen. Another related question is whether or not progesterone, like estrogen, also acts locally. The answer to these questions will help clarify the nature of steroid action on mammary gland. Taken together, in vivo and in vitro studies have been of immense help in learning more about the factors, systemic and local, that regulate mammary epithelial cell proliferation. But, many key questions of biological importance remained unanswered. For example: 1) What is the basis of the synergism between the actions of various hormones on mammary epithelial cells? 2) If circulating growth factors are able to support mammary epithelial cell proliferation, why would ablation of endocrine glands cause suppression of mammary epithelial cell growth in vivoi Is there any role for growth-inhibitory factors in this type of suppression? Is it possible that there are some locally produced growth factors, regulated by hormones, which are more relevant than the circulating ones? In other words, what is the nature of interaction between hormones and growth factors? 3) Do the effects of cAMP, both in vivo and in vitro, indicate a possible physiological role for arachidonate metabolites, e.g., PGs, because unlike PGE2 and PGEi, known mitogenic hormones and growth factors do not elevate cellular cAMP levels. In this regard, the search for an in vivo response to 18:2-PA may be a worthwhile effort because eicosanoids and negatively charged phospholipids containing UFA may constitute a novel lipid-responsive pathway. 4) Do different molecules involved in intracellular signalling such as cAMP, phospholipases, phosphoinositides, protein kinases, GTP-binding proteins, calcium ions, GAP, and oncoproteins regulate growth by common pathways which might explain the response of mammary epithelial cells to a disparate variety of agents? 5) How many phenotypically different cell types are present in vivo in ductal or alveolar structures? Are phenotypes of different cells observed in vivo preserved in vitroi 6) Is there plasticity in the expression of different cellular phenotypes depending on the immediate microenvironment in the gland consisting of the stromal cells, extracellular matrix components, and soluble products secreted by both stromal and epithelial cells? Future research directed at answering all these questions seem to be vital to advance our understanding of growth, morphogenesis, and differentiation of mammary epithelial cells as well as the role various systemic and local factors play in the origin and growth of varieties of preneoplastic and neoplastic lesions in the mammary tissue of mice and rats. These studies should provide



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model systems for similar studies with human mammary epithelial cells.

Acknowledgments The authors thank Drs. Raphael Guzman, Shigeki Miyamoto, and James Richards for their critical review of the manuscript.

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Regulation of mammary epithelial cell homeostasis by lncRNAs.

Production and characterization of mammary-derived growth factor 1 in mammary epithelial cell lines.

Selenium retention and inhibition of cell growth in mouse mammary epithelial cell lines in vitro.

Regulation of vimentin expression in cultured human mammary epithelial cells.

Syndecan expression regulates cell morphology and growth of mouse mammary epithelial tumor cells.

Host Cxcr2-dependent regulation of mammary tumor growth and metastasis.

Regulation of mammary growth and function by TGF-beta.

Regulation of gene expression in gastric epithelial cell populations of fetal, neonatal, and adult transgenic mice.

c mice.

Mammary glands exhibit molecular laterality and undergo left-right asymmetric ductal epithelial growth in MMTV-cNeu mice.

Developmental regulation of mammary-derived growth inhibitor expression in bovine mammary tissue.

Differential regulation of GLUT1 and GLUT8 expression by hypoxia in mammary epithelial cells.

Regulation of bovine bronchial epithelial cell proliferation and proto-oncogene expression by growth factors.

GATA4 regulates epithelial cell proliferation to control intestinal growth and development in mice.

Regulation of transforming growth factor expression in rat intestinal epithelial cell lines.

Mutant p53 promotes epithelial-mesenchymal plasticity and enhances metastasis in mammary carcinomas of WAP-T mice.

Stromal regulation of embryonic and postnatal mammary epithelial development and differentiation.

Regeneration of Bovine Mammary Gland in Immunodeficient Mice by Transplantation of Bovine Mammary Epithelial Cells Mixed with Matrigel.

Bovine mammary epithelial cell cultures for the study of mammary gland functions.

Sustained growth and three-dimensional organization of primary mammary tumor epithelial cells embedded in collagen gels.

Regulation of growth of normal ovarian epithelial cells and ovarian cancer cell lines by transforming growth factor-beta.

TGF-beta regulation of epithelial cell proliferation.

Differential effects of the simian virus 40 early genes on mammary epithelial cell growth, morphology, and gene expression.

Runx1 stabilizes the mammary epithelial cell phenotype and prevents epithelial to mesenchymal transition.