© 2000 by Oxford University Press
Journal of the National Cancer Institute Monographs, No. 27, 17-37,
2000
© 2000 Oxford University Press
Chapter 1: Developmental, Cellular, and Molecular Basis of Human Breast Cancer
Affiliation of authors: Breast Cancer Research Laboratory, Fox Chase Cancer Center, Philadelphia, PA
Correspondence to: Jose Russo, M.D., Breast Cancer Research Laboratory, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA (e-mail: J_russo{at}fccc.edu).
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ABSTRACT |
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 TopNotes Abstract IntroductionDevelopmental Pattern of the...Hormonal Influences on the...Architectural Pattern of the...Architectural Pattern of the...Breast Development, Hormones,...The In Vitro Model...Future PerspectivesReferences |
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Breast cancer, which is the most common neoplastic disease in females and accounts for up to one third of all new cases of women's cancer in North America, continues to rise in incidence. In addition, the mortality caused by this disease has remained almost unchanged for the past 5 decades, becoming only second to lung cancer as a cause of cancer-related death. The failure in eradicating this disease is largely due to the lack of identification of a specific etiologic agent, the precise time of initiation, and the molecular mechanisms responsible for cancer initiation and progression. Despite the numerous uncertainties surrounding the origin of cancer, there is substantial evidence that breast cancer risk relates to endocrinologic and reproductive factors. The development of breast cancer strongly depends on the ovary and on endocrine conditions modulated by ovarian function, such as early menarche, late menopause, and parity. However, the specific hormone or hormone combinations responsible for cancer initiation have not been identified, and their role as protective or risk factors is still incompletely understood. A highly significant female hormone is estrogen, which is involved in the development of a variety of cancers, but it is still unclear whether estrogens are carcinogenic to the human breast. An understanding of whether estrogens cause mutations, and, if so, whether they act through hormonal effects activated by receptor binding, cytochrome P450-mediated metabolic activation, or compromise the DNA repair system, is essential for determining whether this steroid hormone is involved in the initiation or progression of breast cancer. This knowledge has to be based on a multidisciplinary approach encompassing studies of the development of the breast, influence of hormones on the differentiation of individual structures, and their interrelations in the pathogenesis of breast cancer. The analysis of the mechanisms involved would require confirmation in the adequate in vitro models and determination of the role played by genomic alterations in both cancer initiation and progression.
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INTRODUCTION |
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TopNotesAbstractIntroductionDevelopmental Pattern of the...Hormonal Influences on the...Architectural Pattern of the...Architectural Pattern of the...Breast Development, Hormones,...The In Vitro Model...Future PerspectivesReferences |
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Breast cancer accounts for up to one third of all new cases of women's cancer in North America, representing the most common neoplastic disease in the female (1). The incidence rates of this disease have been approximately level during the past decade, and breast cancer mortality is declining in the United States and in certain industrialized areas. However, these favorable trends have not been observed in nations with lower breast cancer incidence, and societies traditionally known to have low breast cancer incidence are experiencing an increase in both incidence and mortality (1,2). Even though the U.S. mortality rates in females decreased an average of approximately 1.7% per year from 1990 through 1995, breast cancer retains the first place as a cause of cancer-related death in nonsmoking women (1,2).
The failure in eradicating this disease is largely due to the lack of identification of a specific etiologic agent, the precise time of initiation, and the molecular mechanisms responsible for cancer initiation and progression. Despite the numerous uncertainties surrounding the origin of cancer, intensive epidemiologic, clinical, and genetic studies have identified a number of biologic and social traits as risk factors associated with breast cancer (2–5). Principal among them are the evidence of BRCA1 and BRCA2 susceptibility genes; familial history of cancer in the breast, ovary, or endometrium; individual history of breast diseases; advanced age; higher socioeconomic status; excess ionizing radiation exposure; tallness in adult life; consumption of alcohol; and a variety of endocrinologic and reproductive factors (2–5). These latter factors include early onset of menstruation, nulliparity or delayed first childbirth, short duration of breast-feeding, late menopause, postmenopausal obesity, extended use of oral contraceptives, and prolonged estrogen replacement therapy (2–5).
Among the hormonal influences, a major role has been attributed to the unopposed exposure to elevated levels of estrogens (6), as has been indicated for a variety of female cancers, namely vaginal, hepatic, and cervical carcinomas (7–12). However, the mechanisms through which this phenomenon occurs have not been completely understood (6). In fact, it is still unclear whether estrogens are carcinogenic to the human breast. Most of the current understanding of the carcinogenicity of estrogens is based on clinical observations of a greater risk of endometrial hyperplasia and neoplasia associated with estrogen supplementation (10–12) and experimental data (13–15). At least three mechanisms are considered to be responsible for the carcinogenicity of estrogens; the most widely recognized is the receptor-mediated hormonal activity, which is generally related to stimulation of cellular proliferation, resulting in more opportunities for accumulation of genetic damage leading to carcinogenesis (16,17). The second mechanism is the cytochrome P450-mediated metabolic activation, which elicits direct genotoxic effects by increasing mutation rates (18–43), and a third mechanism is postulated to compromise the DNA repair system, resulting in the accumulation of lesions in the genome essential to estrogen-induced tumorigenesis (13).
Disappointingly, the molecular mechanisms underlying the development of breast cancer in general, and estrogen-associate breast carcinogenesis in particular, are not completely understood. It is generally believed that the initiation of breast cancer results from uncontrolled cellular proliferation and/or aberrant programmed cell death or apoptosis as a consequence of cumulative genetic damages that lead to genetic alterations that result in activation of proto-oncogenes and inactivation of tumor suppressor genes (44,45). Genetic alterations, in turn, can be inherited as germline mutations or acquired as somatic mutations. These latter ones might occur as a result of exposure to environmental carcinogens, either physical (e.g., excess ionizing radiation), chemical (e.g., polycyclic hydrocarbons or nitrosoureas), and/or biologic (e.g., viruses) (5). The classic two-stage animal model of chemical carcinogenesis has constituted the basis for the generally accepted conclusions that the altered genotype of an initiated cell is irreversible and that the expression of transformed phenotypes requires further genetic or epigenetic changes (46). Tumor progression, the second stage of carcinogenesis in this model, involves exclusively epigenetic changes that are considered to be reversible (46). It remains to be determined whether this is true in breast cancer. The mechanism through which endocrinologic factors, such as hormone replacement therapy, influence cancer initiation and progression in women has not been clarified as yet. The development of breast cancer entails multiple events; unfortunately, the two main factors elucidated in the experimental animal model, the causative agent and the time of initiation, are unknown in the human population (47,48). The elucidation of whether estrogens act as endogenous carcinogenic agents requires a better understanding of the normal development of the breast under the influence of physiologic conditions, which in turn is important for understanding the pathogenetic pathway leading to preneoplastic lesions and cancer. The analysis of pure populations of human breast epithelial cells (HBECs) at various stages en route to malignancy would be the direct approach to understanding the cellular and molecular processes of breast carcinogenesis (49). However, it has been extremely difficult to establish primary cultures of HBECs from breast lesions representing various stages of neoplastic progression, and no cell lines at the intermediate stages of neoplastic transformation are available for mechanistic studies (49). It is expected that an in vitro system of HBECs that can reproduce the main steps of the in vivo situation, such as cell immortalization and transformation, would constitute an adequate tool for determining what genomic changes are important in the initiation and progression of the neoplastic process (50–62). It is, therefore, predicted that an in vitro system of this nature will allow us to test whether estrogens are endogenous carcinogens and whether they play an essential role in the initiation and/or the promotional phase of breast cancer.
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DEVELOPMENTAL PATTERN OF THE HUMAN BREAST FROM ADOLESCENCE TO MATURITY |
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TopNotesAbstractIntroductionDevelopmental Pattern of the...Hormonal Influences on the...Architectural Pattern of the...Architectural Pattern of the...Breast Development, Hormones,...The In Vitro Model...Future PerspectivesReferences |
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The development of the breast, which is rigorously controlled by the ovary, can be defined by several parameters, such as its external appearance, total area, volume, degree of branching, number of structures present in the mammary gland, and degree of differentiation of individual structures, i.e., lobules and alveoli (63–68).
The breast undergoes changes that are progressive from birth to early childhood, becoming striking at puberty. The adolescent period begins with the first signs of sexual change at puberty, which in American females sets in between the ages of 10 and 12 years, and terminates with sexual maturity (64–66). Although puberty is often considered to be the point of initiation of ovarian function, the development of the ovary is a gradual process that depends on pituitary gonadotropins. Receptors for the pituitary luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are present in the ovary even during the infantile period, when they stimulate the secretion of androgens after binding to and activating their respective receptors (69). FSH and LH interact with growth hormone (GH) and prolactin in modulating ovarian steroidogenesis, a function that is also influenced by epinephrine, secreted by the adrenal medulla. The release of FSH is, in turn, modulated by inhibin and activin, glycoprotein hormones secreted by the ovary (69).
The mammary ductal tree undergoes progressive elongation and branching during childhood and puberty. These processes are positively regulated by GH, although its exact mechanism of action is unclear. GH directly stimulates ductal growth in hypophysectomized–ovariectomized rats, and it might act as well through its local mediator, the insulin-like growth factor I. Normal ductal development, however, requires the presence of estrogen and progesterone, the two ovarian steroid hormones that act on the mammary gland through their respective receptors. As puberty approaches, the rudimentary mamma begins to show growth activity both in the glandular tissue and in the surrounding stroma. Glandular increase is due to the growth and division of small bundles of primary ducts originated during intrauterine life from invaginations of the superficial ectoderm (63,65–67). The ducts grow and divide through a combination of dichotomous and sympodial branching, forming at the distal epithelial–stromal boundary a club-shaped terminal end bud. Each terminal end bud bifurcates into two smaller ductules or alveolar buds (67,70). The term alveolar bud applies to those structures that appear morphologically more developed than the terminal end bud. With further branching, alveolar buds become smaller and more numerous, and then they are called ductules. When an average of 11 alveolar buds/ductules cluster around a terminal duct, they form the lobule type 1 (Lob 1) or virginal lobule (Fig. 1
) (67). Terminal ducts and ductules are lined by a two-layered epithelium, whereas terminal end buds in the human fetus are lined by an epithelium composed of up to four layers of cells. Lobule formation in the female breast occurs within 1–2 years after onset of the first menstrual period. Afterward, the ulterior development of the gland varies greatly from woman to woman. Full differentiation of the mammary gland is a gradual process taking many years, and it can be assumed that in all women in whom pregnancy did not supervene, it was never attained (63).
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The normal breast tissue of adult women contains two other identifiable types of lobules in addition to the Lob 1 described above. These are designated lobule type 2 (Lob 2) and type 3 (Lob 3) (Figs. 2 and 3
). The transition from Lob 1 to Lob 2 and of these to Lob 3 is a gradual process of sprouting of new ductules, which increase in number from approximately 11 in Lob 1 to 47 and 80 in Lob 2 and Lob 3, respectively (Fig. 3, Table 1). As the number of ductules increases, so does the size of the lobules, even though individual ductules appear reduced in size (Table 1) (63,68).
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The breast of nulliparous women contains more undifferentiated structures, such as terminal ducts and Lob 1, although Lob 2 and Lob 3 are occasionally observed. These characteristics are not influenced by age or menopausal status (Fig. 4
). In parous premenopausal women, the predominant structure is the most differentiated Lob 3, whose number peaks during the early reproductive years. They start to decrease after the fourth decade of life, as the proportion of Lob 1 increases, and, when menopause sets in, they reach the same values observed in nulliparous women (Fig. 4). In the breast of nulliparous women, the Lob 2 is present in moderate numbers during the early years, sharply decreasing after age 23 years, whereas the number of Lob 1 remains significantly higher. This observation suggests that a certain percentage of Lob 1 might have progressed to Lob 2, but the number of Lob 2 progressing to Lob 3 is significantly lower in nulliparous women as compared with parous women. In the case of parous women, it is interesting to note that a history of parity between the ages of 14 and 20 years is associated with a significant increase in the number of Lob 3 that remains present as the predominant structure until the age of 40 years, the time at which a decrease in the number of Lob 3 occurs, probably because of their involution to predominantly Lob 1 (Fig. 4) (63,68).
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HORMONAL INFLUENCES ON THE DEVELOPMENT OF THE BREAST |
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TopNotesAbstractIntroductionDevelopmental Pattern of the...Hormonal Influences on the...Architectural Pattern of the...Architectural Pattern of the...Breast Development, Hormones,...The In Vitro Model...Future PerspectivesReferences |
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The breast is a hormone-responsive organ by excellence. Its development is influenced by a myriad of hormones and growth factors, responding selectively to given hormonal stimuli with either cell proliferation or differentiation. The type of response elicited is, in turn, modulated by specific topographic characteristics of the mammary parenchyma (70–78). In either case, the response of the mammary gland to these complex hormonal and metabolic interactions results in developmental changes that permanently modify both the architecture and the biologic characteristics of the gland (72,73). Among all of the complex hormonal influences, estrogens are considered to play a major role in promoting the proliferation of both the normal and the neoplastic breast epithelium (71,72). Estradiol acts locally on the mammary gland, stimulating DNA synthesis and promoting bud formation. Although the influence of estrogens on the proliferative activity of mammary epithelial cells has been traditionally considered to be mediated by at least three different mechanisms, a receptor-mediated (79–85), an autocrine/paracrine loop (85), and/or a negative feedback (86), it is generally accepted that the biologic activities of estrogens are mediated by the nuclear estrogen receptor (ER) that, on activation by cognate ligands, forms a homodimer with another ER-ligand complex and activates transcription of specific genes containing the estrogen response elements (87). According to this classic model, the biologic responses to estrogens are mediated by a well-characterized ER.
The recent cloning of a new type of ER, the ER
from the rat (88), mouse (89), and human (90) tissues, has required researchers to rename the traditional ER as ER
. The presence of ER in target tissue or cells is essential to their responsiveness to estrogen action. In fact, the expression levels of ER in a particular tissue have been used as an index of the degree of estrogen responsiveness (91). A vast majority of human breast carcinomas are initially positive for ER, and their growth can be stimulated by estrogens and inhibited by antiestrogens (92–94). ER and ER share high sequence homology, especially in the regions or domains responsible for specific binding to DNA and the ligands (88–90). ER can be activated by estrogen stimulation and blocked with antiestrogens (88,90,95). On activation, ER can form homodimers as well as heterodimers with ER (95–99). The existence of two ER subtypes and their ability to form DNA-binding heterodimers suggests three potential pathways of estrogen signaling: via the ER or ER subtype in tissues exclusively expressing each subtype and via the formation of heterodimers in tissues expressing both ER and ER (97). In addition, estrogens and antiestrogens can induce differential activation of ER and ER to control transcription of genes that are under the control of an AP1 element (100).
The importance of the integrity of ER in the mammary gland has been clearly elucidated by using the -ER knock-out (KO) mice (101). The mammary glands of these animals are poorly developed. Nonetheless, the -ERKO mammary gland appears to possess the intrinsic tissue components necessary for pubertal development and pregnancy-induced maturation, but it fails to develop because of the loss of multiple stimuli that are downstream of ER action (101). Unlike the dramatic underdevelopment observed in the mammary gland of the -ERKO mouse, no such phenotype is observed in adult -ERKO females (101). Although these studies are important for our understanding of the role of both ERs, extrapolation to the human breast needs to be carefully done because these genetic-engineered mice reflect only a portion of the functional complexity of the human situation.
Progesterone is another major, although controversial, player in mammary gland biology. This ovarian steroidal hormone also acts, in conjunction with estrogen, through its specific receptor PgR in the normal epithelium for regulating breast development. The role of these hormones on the proliferative activity of the breast, which is indispensable for its normal growth and development, has been for a long time, and still is, the subject of heated controversies. Although estrogen is known to stimulate cell proliferation, the breast epithelium of sexually mature and normally cycling women does not exhibit maximal proliferation during the follicular phase of the menstrual cycle (77,78,102–106), when estrogens reach peak levels of 200–300 pg/mL and progesterone is less than 1 ng/mL (107). Instead, the breast epithelium exhibits its maximal proliferative activity during the luteal phase, when progesterone levels reach 10–20 ng/mL and estrogen levels are twofold to threefold lower than those observed during the follicular phase (107). These observations are puzzling when analyzed to the light of in vitro and experimental data because estrogen stimulates the proliferation of cultured breast cells and breast tissues implanted in athymic nude mice. Progesterone, however, has no effect or even inhibits cell growth in the same models (105,106).
In addition to its response to circulating hormones, the proliferative activity of the mammary epithelium in both rodents and humans varies with the degree of differentiation of the mammary parenchyma (68,71–74,108,109). In humans, the highest level of cell proliferation is observed in the undifferentiated Lob 1 present in the breast of young nulliparous females (71–74). The progressive differentiation of Lob 1 into Lob 2 and Lob 3, occurring under the hormonal influences of the menstrual cycle, and the full differentiation into lobules type 4 (Lob4) as the result of pregnancy leads to a concomitant reduction in the proliferative activity of the mammary epithelium (68,71–74,108,109).
The relationship of lobular differentiation, cell proliferation, and hormone responsiveness of the mammary epithelium is just beginning to be unraveled. Of interest, the content of ER and PgR in the lobular structures of the breast is directly proportional to the rate of cell proliferation. These three parameters are maximal in the undifferentiated Lob 1, decreasing progressively in Lob 2, Lob 3, and Lob 4 (Fig. 5, Table 2). The determination of the rate of cell proliferation, expressed as the percentage of cells that stain positively with Ki67 antibody, has revealed that proliferating cells are predominantly found in the epithelium lining ducts and lobules and less frequently in the myoepithelium and in the intralobular and interlobular stroma. Ki67-positive cells are most frequently found in Lob 1 (Fig. 5, Table 2). The percentage of positive cells is reduced by threefold in Lob 2 and by more than 10-fold in Lob 3 (Figs. 5 and 6, Table 2) (73,110). ER- and PgR-positive cells are found exclusively in the epithelium; the myoepithelium and the stroma are totally devoid of steroid receptor-containing cells. The highest number of cells positive for both receptors is found in Lob 1, decreasing progressively in Lob 2 and Lob 3 (Fig. 5, Table 2) (110).
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To clarify the relationship between steroid receptor-positive cells and proliferating cells, we used a double-staining procedure, combining in the same tissue section anti-Ki67 and ER
, Ki67 and PgR, or ER and PgR antibodies. Each antibody was identified by its color reaction, brown with 3,3'-diaminobenzidine-HCl (DAB) or red with the alkaline phosphatase-vector red (110). This procedure allowed us to quantitatively determine the spatial relationship between those cells that are proliferating and those that react with either ER or PgR antibodies. It was found that a higher percentage of cells reacted simultaneously with both ER and PgR, appearing purple red in color (Fig. 6
), whereas the number of cells positive for both ER and Ki67 or PgR and Ki67 was very low (Table 2). The highest percentage of ER-, PgR-, and Ki67-positive cells was observed in Lob 1 (Fig. 6, Table 2). The percentages of Ki67-, ER-, and PgR-positive cells was reduced to 1.6%, 3.8%, and 0.7% in Lob 2, respectively. Their percentages became negligible in Lob 3 (Table 2).
Of interest was the observation that even though there were similarities in the relative percentages of Ki67-, ER- and PgR-positive cells and in the progressive reduction in the percentage of positive cells as the lobular differentiation progressed, those cells positive for Ki67 were not the same that reacted positively for ER or PgR (Fig. 6) (110). Very few cells, less than 0.5% in Lob 1 and even fewer in Lob 2 and Lob 3, were positive for both Ki67 and ER (Ki67+ER) or Ki67 and PgR (Ki67+PgR) (Table 2). Despite their low percentage, still double-labeled (Ki67+ER) cells were more numerous in Lob 1, decreasing gradually in Lob 2 and Lob 3. The percentage of cells exhibiting double labeling with Ki67 and PgR, however, were more numerous in Lob 2 than in Lob 1 but decreased to the same levels observed for ER in Lob 3 (Table 2).
The simultaneous immunocytochemical detection of proliferating cells and of those containing ER and PgR in normal breast tissue led us to conclude that their number varies with the degree of lobular development of the organ and that steroid receptor content is linearly related to the rate of cell proliferation. The use of a double-labeling immunocytochemical technique has allowed us to demonstrate that the expression of the receptors occurs in cells other than the proliferating cells, confirming results reported by others (104). The findings that proliferating cells are different from those that are ER positive and PgR positive support data that indicate that estrogen controls cell proliferation by an indirect mechanism. This phenomenon has been demonstrated with the use of a supernatant of estrogen-treated ER-positive cells that stimulates the growth of ER-negative cell lines in culture. The same phenomenon has been shown in vivo in nude mice bearing ER-negative breast tumor xenografts (111,112). ER-positive cells treated with antiestrogens secrete tumor growth factor- that inhibits the proliferation of ER-negative cells (113). The fact that the highest proliferative activity and the highest percentage of ER- and PgR-positive cells are present in Lob 1 provides a mechanistic explanation for the higher susceptibility of these structures to be transformed by chemical carcinogens in vitro (47,114), supporting as well the observations that Lob 1 is the site of origin of ductal carcinomas (115).
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ARCHITECTURAL PATTERN OF THE NORMAL BREAST AT MENOPAUSE |
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TopNotesAbstractIntroductionDevelopmental Pattern of the...Hormonal Influences on the...Architectural Pattern of the...Architectural Pattern of the...Breast Development, Hormones,...The In Vitro Model...Future PerspectivesReferences |
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Menopause supervenes as the consequence of the atresia of more than 99% of the 400 000 follicles that are present in the ovaries of a female fetus of a gestational age of 5 months (69). Gonadotropin-releasing hormone secretion is also implicated in this phenomenon, indicating that a hypothalamic process is involved in the development of menopause. The most characteristic sign of menopause is amenorrhea, which is the result of the almost complete cessation of ovarian estrogen and progesterone production. The years leading to the final menstrual period, until menopause sets in, generally at around the age of 51 years, constitute the perimenopause. During this period, many women ovulate irregularly, either because the rise in estrogen during the follicular phase is insufficient to trigger a LH surge or because the remaining follicles are resistant to the ovulatory stimulus (69). The increase in human longevity occurring in our society has caused a considerable increment in the number of women that will live one third or more of their lives after menopause, a period characterized by profound ovarian hormone deprivation.
After menopause the breast undergoes regression in both nulliparous and parous women. This regression is manifested as an increase in the number of Lob 1 and a concomitant decline in the number of Lob 2 and Lob 3. At the end of the fifth decade of life, the breast of both nulliparous and parous women is composed predominantly of Lob 1 (Fig. 4) (68). These observations have led us to conclude that the understanding of breast development requires a horizontal study in which all different phases of growth are taken into consideration. For example, the analysis of breast structures at a single given point, i.e., age 50 years, would lead one to conclude that the breasts of both nulliparous and parous women are identical. However, the phenomena occurring in prior years might have imprinted permanent changes in the breast, which affect its susceptibility to carcinogenesis but are no longer morphologically observable. Thus, from a quantitative point of view, the regressive phenomenon occurring in the breast at menopause differs between nulliparous and parous women. In the breast of nulliparous women, the most predominant structure is the Lob 1, which comprises 65%–80% of the total lobule type components and their relative percentage is independent of age. Second in frequency is the Lob 2, and the least frequent structure is the Lob 3, which represent 10%–35% and 0%–5% of the total lobular population, respectively. In the breast of premenopausal parous women, however, the predominant lobular structure is the Lob 3, which comprises 70%–90% of the total lobular component. Only after menopause the number of Lob 3 declines and the relative proportion of the three lobular types approaches that observed in nulliparous women. Full lobular differentiation only occurs in the parous women, especially in those completing full-term pregnancy at a young age, but lobular differentiation in nulliparous women seldom reaches the Lob 3 and never the Lob 4 stages (68). These differences in the pattern of breast development between nulliparous and parous women greatly explain the protective effect induced by pregnancy from breast cancer development. They also highlight the need to determine whether the undifferentiated Lob 1 of nulliparous women differ from those of the parous postmenopausal woman in their ability to metabolize estrogens or in the ability of the cells to repair genotoxic damage (116,117).
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ARCHITECTURAL PATTERN OF THE BREAST WITH PROLIFERATIVE DISEASE |
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TopNotesAbstractIntroductionDevelopmental Pattern of the...Hormonal Influences on the...Architectural Pattern of the...Architectural Pattern of the...Breast Development, Hormones,...The In Vitro Model...Future PerspectivesReferences |
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Our studies of the pattern of breast development in tissues devoid of mammary pathology, such as those obtained from reduction mammoplasties, led us to establish certain criteria of normality specific for a given age and parity status of the donors. In these tissues, we identified parenchymal structures exhibiting variations in the degree of differentiation, rate of cell proliferation, and content of ER
and PgR (118). To answer the question of whether breast lesions of either benign, premalignant, or malignant nature develop as a reflection of the stage of development of the breast, we compared parenchymal structures present in 33 reduction mammoplasty specimens with those found in 45 breast biopsies performed because of mammographic abnormalities or clinically suspicious breast masses (Table 3). Because the initiation of the neoplastic process is inversely related to the degree of differentiation of the breast, which in turn is a function of reproductive history, the patient populations were subdivided according to parity status (Table 3). In this study, we confirmed previous observations that in the reduction mammoplasty specimens (RM) the breast of nulliparous women of all ages was composed predominantly of Lob 1, whereas the breast of parous premenopausal women contained a higher concentration of Lob 3 (Table 3) (118). Those breast tissues obtained from biopsies had an architectural pattern different from that obtained from RM for women of comparable parity status because parous women that had a breast biopsy contained a higher percentage of Lob 1 and a lower percentage of Lob 3 than the parous population of the RM group (Table 3) (118).
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The patient population that had breast biopsies was also subdivided into subgroups, based on the histopathologic diagnosis of their lesions. One group of 21 patients had no pathology present (normal breast or control group), one group of 15 patients had ductal hyperplasia (DH group), four patients had blunt duct adenosis (BDA group), and five patients had sclerosing adenosis (SAD group) (Table 4
). Tissue sections from all these groups were analyzed for lobular architecture, type of pathologic lesions, and proliferative activity of the breast (Figs. 7 and 8) (118). The breast tissues of the groups classified as normal breast (control) and DH had a significantly higher percentage of Lob 1 than Lob 2 and Lob 3 (P<.0008 and P<.0001, respectively) (Fig. 7). The breast tissues containing BDA were also characterized by having a higher percentage of Lob 1, whereas the SAD group had a higher percentage of Lob 2 (Table 4, Fig. 7) (118). In all of the groups, the percentage of Lob 3 was significantly lower than that of Lob 1, although the relative percentage of Lob 3 was significantly higher in breast biopsies with BDA and SAD than in the normal breast biopsies or in those diagnosed with DH (P<.05) (Fig. 7) (118).
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The number of proliferating epithelial cells, determined by immunostaining of the Ki67 nuclear antigen, was higher in Lob 1 than in Lob 2 and Lob 3 (P<.001), with a similar pattern in the normal breast, DH, and SAD groups, although the differences were not statistically significant between Lob 1 and Lob 2 in the SAD group (Fig. 8
). In the BDA group, however, the rate of cell proliferation was higher in Lob 2 (P<.01) than in Lob 1 and Lob 3 (Fig. 8) (118). Although the proliferative activity was on an average higher in Lob 1 than in Lob 3, the differences were not statistically significant (Fig. 8) (118). These data allowed us to conclude that breast tissues obtained from biopsies performed because of mammographic or clinical abnormalities, even in the absence of cancer, have architectural and cell kinetic patterns different from the normal breast tissues obtained from reduction mammoplasties. More important is the observation that even in those cases in which no pathology or only benign lesions were diagnosed, the pattern of breast development in biopsies was more similar to that of the cancer-bearing breast than it is to the population not requiring a biopsy. Our findings that in DH-containing biopsies Lob 1 are the most frequent structures present and have the highest rate of cell proliferation support our postulate that DH originates from Lob 1 (115). Lob 2 and Lob 3, which are the sites of origin of more differentiated lesions, such as BDA and SAD, are more prominently represented and are more proliferative in those biopsies containing these types of pathologic lesions. It is of importance to clarify that parity does not seem to influence the pattern of development in DH-containing breast tissues. A similar observation has been made in cancer-bearing breasts or in breasts of nulliparous women in terms of lobular composition. |
BREAST DEVELOPMENT, HORMONES, AND THE PATHOGENESIS OF BREAST CANCER |
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TopNotesAbstractIntroductionDevelopmental Pattern of the...Hormonal Influences on the...Architectural Pattern of the...Architectural Pattern of the...Breast Development, Hormones,...The In Vitro Model...Future PerspectivesReferences |
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From our studies associating normal breast development and the pathogenesis of both experimental and spontaneous mammary carcinogenesis emerged an important concept: that the Lob 1, the most undifferentiated structure found in the breast of young nulliparous women, is equivalent to the terminal ductal lobular unit, a structure originally identified by Wellings et al. (119) as the site of origin of ductal carcinomas (Fig. 9
) (63,70). This observation was supported by comparative studies of normal and cancer-bearing breasts obtained at autopsy. We observed that the nontumoral parenchyma of those breasts that had developed a malignancy contain a significantly higher number of hyperplastic terminal ducts, atypical Lob 1, and ductal carcinomas in situ originating from Lob 1 than those breasts free of malignancies. These findings indicate that the Lob 1 is affected by both preneoplastic and neoplastic processes (115). More differentiated lobular structures have been found to be affected by neoplastic lesions as well, although they originate tumors whose histologic type and malignancy are in an inverse relationship with the degree of differentiation of the parent structure (52,115,120,121). The finding that the most undifferentiated structures originate the most aggressive neoplasms is clinically important because these structures are more numerous in the breasts of nulliparous women who are, in turn, at a higher risk of developing breast cancer (68).
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The analysis of the nontumoral breast tissues from cancer-bearing lumpectomy or mastectomy specimens reveals that the breasts in nulliparous women have an architecture dominated by Lob 1, being their overall architecture similar to that of nulliparous female free of mammary pathology (120,122). Although the breast tissues of parous women from the general population contain predominantly Lob 3 and a very low percentage of Lob 1, in those parous women who have developed breast cancer, their breast tissues have also the Lob 1 as the predominant structure, appearing in this sense similar to those of nulliparous women. It is of interest that all of the parous women in our studies who had developed breast cancer had a history of late first full-term pregnancy or a family history of breast cancer. The analysis of these samples allowed us to conclude that the architecture of the breast of parous women with breast cancer differs from that of parous women without cancer. The similarities found between the architecture of the breast of nulliparous women and that of parous women with cancer support our hypothesis that the degree of breast development is of importance in the susceptibility to carcinogenesis and, furthermore, that parous women who develop breast cancer might exhibit a defective response to the differentiating influence of the hormones of pregnancy (52,115,120–122).
Breast cancer is a hormone-dependent malignancy. The risk of developing breast cancer has been traditionally linked to exposure to estrogen, mainly because a majority of breast cancers contain receptors for this hormone. The ER content of a tumor is considered to be a parameter of prognostic significance (123,124). There is no information available as yet of the prognostic significance of the ER content of a tumor. The presence of ER-positive and ER-negative cells and of proliferating cells, regardless of the receptor status of the normal breast, may help to elucidate the genesis of ER-positive and ER-negative breast cancers (125,126). It has been suggested that ER-negative breast cancers result from either the loss of the ability of the cells to synthesize ER during the clinical evolution of ER-positive cancers or that ER-positive and ER-negative cancers are different entities (125,127).
We have observed that Lob 1 contains at least three cell types: ER-positive cells that do not proliferate, ER-negative cells that are capable of proliferating, and a small proportion of ER-positive cells that can proliferate as well (Fig. 10) (110). Therefore, estrogen might stimulate ER-positive cells to produce a growth factor that might, in turn, stimulate neighboring ER-negative cells to proliferate (Fig. 10) (110). In the same fashion, the small proportion of cells that are ER-positive and can proliferate could be the source of ER-positive tumors. The possibility exists, as well, that the ER-negative cells convert to ER-positive cells. The newly discovered ER opens the possibility that those cells traditionally considered to be negative for ER might be positive for ER (88–90,128–131). It has recently been found that ER is expressed during the immortalization and transformation of ER-negative human breast epithelial cells (Fig. 11) (131), supporting the hypothesis of a conversion from negative to positive receptor cell. The functional role of ER-mediated estrogen signaling pathways in the pathogenesis of malignant diseases is essentially unknown. ER-mediated mechanisms have been implicated in the increased PgR expression in the dysplastic acini of the dorsolateral rat prostate in response to treatment with testosterone and estradiol-17 (132). ER has been detected either alone or co-expressed with ER in both normal and cancerous human breast tissues and breast cell lines (129–131). These observations suggest the possibility that ER and ER proteins interact with each other and discriminate between target sequences leading to differential responsiveness to estrogens. In addition, estrogen responses mediated by ER and ER may vary with different composition of their co-activators that transmit the effect of ER-ligand complex to the transcription complex at the promoter of target genes (133).
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Although the receptor-mediated mode of estrogen action is the most widely studied and accepted, evidence is mounting that a membrane receptor, coupled to alternative second messenger signaling mechanisms, is operational and may stimulate the cascade of events leading to cell proliferation (134,135). This knowledge suggests that ER
-negative cells found in the human breast may respond to estrogens through this one pathway or other known or as yet undiscovered pathways. Definitively more studies need to be done in this direction, especially when taking into consideration that, in the normal breast, the proliferating and steroid hormone receptor-positive cells are not the same. This finding has opened new possibilities for clarifying the mechanisms through which estrogens stimulate cell proliferation for initiating the cascade of events leading to cancer.
There is evidence as well that estrogen may not need to activate its nuclear receptors to initiate or promote breast carcinogenesis. The metabolic activation of estrogens can be mediated by various cytochrome P450 (CYP) complexes, generating through this pathway reactive intermediates that elicit direct genotoxic effects by increasing mutation rates [(18–43), Chapters 4 and 5]. The two major endogenous estrogens, estradiol-17 (E2) and estrone (E1), are continuously interconverted by 17-hydroxylase. They are generally metabolized via two major pathways: hydroxylation at C-16 position and at the C-2 or C-4 positions (16,20,21). The carbon position of the estrogen molecules to be hydroxylated differs among various tissues, and each reaction is probably catalyzed by various CYP isoforms (136). For example, in MCF-7 human breast cancer cells, which produce catechol estrogens (CE) in culture (23,34), CYP IA1 catalyzes hydroxylation of E2 at C-2, C-15, and C-16, CYP IA2 predominantly at C-2 (22), and a member of the CYP IB subfamily at C-4 (24–26). The hydroxylated estrogens are CE that have a very short half-life in vivo because of rapid inactivation via monomethylation at the 2-, 3- or 4-hydroxy (OH) groups catalyzed by blood-borne catechol-O-methyltransferase (COMT) [(20,27,28), Chapter 6]. Steady-state concentrations of CE are determined by the CYP-mediated hydroxylations of estrogens and COMT-catalyzed methylation of catechols (22). An increase in CE because of either elevated rates of synthesis or reduced rates of monomethylation will easily lead to their autoxidation to semiquinones and subsequently quinones. These two compounds are electrophiles capable of covalently binding to nucleophilic groups on DNA via a Michael addition and, thus, serve as the ultimate carcinogenic reactive intermediates in the peroxidatic activation of CE (29). This pathway still needs to be demonstrated in normal breast epithelial cells.
Collectively, the alternative pathways described above offer new paradigms for determining the role of estrogens as endogenous carcinogens and for clarifying whether they act as initiators or promoters of the neoplastic process. Many questions remain to be answered, such as are there differences in the levels of aromatases, sulfotransferases, CYP IA1, or CYP IB1 present in the differentiated breast of parous women and those found in the poorly differentiated gland of young nulliparous women? By the same token, are the oxidative adduct byproducts of the metabolism of estrogens to CEs, semiquinones, and quinones different in various types of lesions found in the breast? These are important questions that need to be answered to fully understand the role of estrogens in breast carcinogenesis.
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THE IN VITRO MODEL OF BREAST CANCER |
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Culture of Human Breast Epithelial Cells In Vitro
Under in vitro conditions, HBECs have a life span comparable to that of adult human fibroblasts (30–64 doublings) when cultured in a medium supplemented with bovine pituitary extract and a standard concentration of calcium (1.05 mM) (53,54). However, when the concentration of calcium (Ca2+) in the medium is reduced to 0.04 mM (low Ca2+), the growth and population longevity of the cells are profoundly affected (56–59). HBECs cultured under low Ca2+ conditions divide for periods of more than 1000 days and for more than 50 generations of linear growth, without expressing terminal differentiation. Similar effects have been observed in other cell types, such as WI-38 (137), mouse epidermal (138–140), and rat esophageal epithelial cells (141). In addition, under these conditions HBECs maintain a normal diploid karyotype and human breast epithelial characteristics, including morphologic and ultrastructural features, as well as formation of domes and duct-like structures in collagen. They also express specific keratin filaments and milk fat globule membrane antigen (50,53,57). The behavior of HBECs in vitro, however, is in great part modulated by the biologic conditions of the breast from which they were obtained. Cells obtained from lobules varying in their degree of differentiation show variations in their in vitro growth properties (53,57,58,114). The number of doublings is higher in those HBECs derived from less differentiated breast tissues, i.e., the Lob 1 of young nulliparous women, which also exhibit a greater rate of cell proliferation (47,114). Furthermore, these HBECs are more susceptible to be transformed in vitro by etiologically important environmental chemical carcinogens, a phenomenon that does not occur in cells obtained from the more differentiated Lob 3 of older and parous women (47,114). These observations indicate that both the in vitro growth characteristics of HBECs and their susceptibility to be transformed by chemical carcinogens are profoundly influenced by the degree of lobular differentiation and the rate of cell proliferation of the breast epithelium in vivo.
Immortalization of Human Breast Epithelial Cells
As previously indicated, normal HBECs maintained in vitro senesce after 10–20 passages. Very few immortal breast epithelial cell lines of nonmalignant origin have been established (50,142–145). Among them, MCF-10, a HBEC line derived from a primary culture of sample S#130, designated as MCF-10M, became immortalized after culture in low Ca2+ medium for more than 2 years. Two immortal cell lines arose spontaneously, MCF-10A and MCF-10F (50,145). The immortalization of these cells was characterized by their continuous growth in conventional, 1.05 mM Ca2+ (also called high Ca2+ medium) without entering into senescence after more than 20 passages in vitro. Although the growth curves of MCF-10M, MCF-10A, and MCF-10F cells were similar when grown in low- and high-Ca++ media, MCF-10M cells were unable to continue growing in high-Ca++ medium after the 20th passage, whereas the immortal MCF-10A and MCF-10F cells grew indefinitely under high Ca++ conditions. An important difference between MCF-10M and its immortalized derivatives was their survival efficiencies in soft agar (Table 5), even though there were no significant differences in survival efficiencies among these cell types when they were seeded in agar-methocel (Table 5). None of the three cell types formed colonies in soft agar or in agar-methocel. Instead, the neoplastic breast epithelial cell line MCF-7 had a significantly higher survival efficiency, and they formed colonies in both culture conditions (Table 5).
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The immortalized cells MCF-10A and MCF-10F are bona fide HBECs, expressing epithelial sialomucins and keratins usually reported in human breast (50,145). Ultrastructurally, the cells appear low cuboidal, with their lateral borders joined by numerous desmosomes and the free surfaces are lined by short microvilli. When plated on plastic surfaces, they grow in monolayers forming domes. In a collagen matrix and under the control of hormones and growth factors the cells form ductular structures. They lack anchorage-independent growth and are not tumorigenic in severe compromised immune deficient mice. Cytogenetic analysis of MCF-10M cells showed a normal diploid karyotype, whereas the immortalized MCF-10A and MCF-10F cells had a near diploid karyotype. Genotypically, these cells were demonstrated to be of human origin by DNA hybridization with probes for highly polymorphic sequences, such as the hypervariable single-copy gene PUM. The relationship of MCF-10A and MCF-10F cells to a specific donor was demonstrated by hybridization of identical size HaeIII fragments with a M13 probe that detects multiple hypervariable minisatellites (145). The immortal MCF-10 cells do not have amplification of c-erbB2/HER-2-neu, erbA-1, int-2, int-1 or mutated c-Ha-ras-1 gene and do not contain SV40 antigen. These characteristics make this cell line the most near to a normal HBEC line available. Undoubtedly, the establishment of the immortal cell line MCF-10F that arose spontaneously from the mortal diploid HBECs without viral or chemical intervention provides an important tool for understanding how chemical carcinogens are able to induce transformation phenotypes. At the same time, it also provides the basis for understanding the differences in the processes that control senescence, immortalization, and malignancy.
Neoplastic Transformation of Human Breast Epithelial Cells With Chemical Carcinogens
Exposure of primary cultures of HBECs to chemicals such as benzo[a]pyrene (BP), N-methyl-N-nitrosourea (NMU), and 7,12-dimethylbenz[a]anthracene (DMBA), all known to be carcinogenic in animals (115), reveals that their susceptibility to be transformed is modulated by host factors and by specific characteristics of the cells. Treatment of MCF-10F cells with BP and DMBA has given rise to a series of chemical-carcinogen-initiated cell lines, each one expressing a well-defined transformed phenotype. The transformed cells express anchorage independence, loss of ductule-like formation in collagen gel, and increase in chemotactic and invasive properties, tumorigenicity in heterologous hosts (e.g., BP1E tumor cell lines) (47,49,146). Thus, the highest susceptibility of HBECs to be transformed has been found to be associated with a high rate of cell proliferation and undifferentiated condition of the donor organ in vivo (114), immortalization prior to exposure to the carcinogens (47), and inherited predisposition to breast cancer, as revealed by our studies of HBECs obtained from prophylactic mastectomies performed in women with familial history of breast cancer and genetic predisposition, as evidenced by linkage analysis (147).
Primary cultures established from outgrowths of organoids obtained from prophylactic mastectomies received two 24-hour treatments with BP or DMBA in a week. After 30 days, the cells were plated in agar-methocel for evaluation of anchorage-independent growth. By day 21 postplating, viable cells and colonies, ranging in size from 50 to 250 mm in diameter, were counted. Results were expressed as survival efficiency, colony efficiency, and colony size. Survival efficiency (Fig. 12, A) and colony efficiency (Fig. 12, B) in agar-methocel were calculated as number of cells that survived per 100 cells plated and as number of colonies formed per 100 cells plated, respectively. The average size of the colonies was measured with a micrometer disc under an inverted phase contrast microscope (Fig. 13).
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BP and DMBA did not affect survival efficiency but significantly increased colony efficiency of treated cells. The colonies formed by treated cells showed considerable anchorage-independent growth during the 21-day assay period (Fig. 12
, A and B) (147). Because the formation of colonies in agar is generally construed as an indication of anchorage-independent growth, a hallmark of neoplastic cells, our results clearly indicated that HBECs from women with familial history of breast cancer manifested phenotypic changes indicative of initial stages of neoplastic transformation in response to the treatment with carcinogens (147). In contrast, the same treatments when applied to HBECs of women without familial history of breast cancer induced phenotypic alterations indicative of partial transformation only, such as increased survival efficiency in agar methocel (Fig. 12, A and B), which is perceived to precede the acquisition of anchorage independence. Our observations led us to conclude that genetic predisposition in women with familial history of breast cancer confers inherited susceptibility to environmental chemical carcinogens (147). However, induction of the full spectrum of transformed phenotypes by chemical carcinogens requires immortalization of the cells prior to exposure to the carcinogens. It is not known how estrogens or other endocrine disrupters influence the susceptibility of the epithelial cells to undergo neoplastic transformation, or whether these cells will express transformation phenotypes when estrogen is metabolized or interacts with specific nuclear receptors.
Molecular Basis of Cell Immortalization and Neoplastic Transformation
Genomic Alterations
Genomic analysis of MCF-10F cells, using single-strand conformational polymorphism technique revealed the presence of a variant band in one of the heterozygous alleles of TP53. On DNA sequencing, a point mutation (TAG 
TTA) of codon 254 in exon 7 of this gene was detected (Table 6) (148). There was a coexistence of a mutation of TP53 and instability of microsatellite DNA in the intragenic TP53 in these cells, manifested by additional bands with slower mobility. We used polymerase chain reaction (PCR) amplification of microsatellite DNA length polymorphism to detect allelic loss as well as microsatellite instability (MSI). These microsatellites are highly polymorphic, flanked by unique sequences that can serve as primers for PCR amplification. They have been proven to be useful markers for investigating multiple areas of MSI and loss of heterozygosity (LOH) and should be applicable to allelotyping as well as regional mapping of deletions in specific chromosomal regions. We have studied 466 markers that represent approximately 4.6% of the 10 000 microsatellite markers identified. Microsatellite PCR analysis of MCF-10F cells did not reveal LOH with any of the markers analyzed in this study when compared with their parental MCF-10M cells. However, MCF-10F cells did show MSI in chromosome 11 in the locus D11S392 and in chromosome 17 in the loci represented by markers D17S849, TP53, D17S786, and D17S520 (149).
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Neoplastic transformation of MCF-10F cells with chemical carcinogens (e.g., BP1 and BP1E cells) is associated with genetic instability on chromosomes 11 and 13, in addition to that observed on chromosome 17, which has been detected in association with immortalization (150–153). MSI was found on chromosome 11 by using marker D11S912 and expressed as an allelic expansion in the BP1 and BP1E cells, representing an additional location affected in the early stage of transformation of HBECs (Fig. 14
) (151). On chromosome 13, MSI was found in both BP1 and BP1E cells by using markers D13S260 and D13S289 at 13ql2–13 (flanking the BRCA2 locus) (Fig. 14) (150). In addition, we have also observed other genomic alterations on chromosomes 9 and 16 (154).
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Activation of Telomerase in the Immortalized MCF-10F Cells There is evidence that the repetitive TTAGGG sequences located at the ends of human chromosomes (i.e., telomeres) may act as a molecular mitotic clock (155). It is generally believed that each successive genomic replication is accompanied by gradual shortening of 50–200 base pairs (bp) because of incomplete replication of the 3' ends, and cellular senescence occurs when telomeres reach a critically short length that replication of the genome cannot be maintained (156). The stabilization of the telomeric sequences at the ends of chromosomes, which is required for the continuous proliferation of immortal cells, involves the activation of the enzyme telomerase, which adds TTAGGG repeats to the 3' ends of chromosomes (157,158). The genetic nature of cellular senescence implicates activation of telomerase as a key element of cell immortalization (144,158). Elevated levels of telomerase activities have been detected in a number of immortal cell lines and human tumor tissues (159,160). We have observed telomerase activity in immortal MCF-10F but not in the mortal MCF-10M cells (161), suggesting that telomerase activation may play a role in the spontaneous immortalization of MCF-10F cells.
Increase of H-Ferritin in MCF-10F Cells
In efforts to identify genes underlying the process of immortalization, we have performed subtractive hybridization and differential display analysis between immortal MCF-10F and its parental mortal MCF-10M cells. With the use of a 10F(+)/10M(-) subtractive complementary DNA (cDNA) library, we isolated more than 15 clones, one of which contains sequences identical to H-ferritin (162). We observed marked increases in messenger RNA (mRNA) levels of ferritin H in immortal MCF-10F cell lines (particularly in late passages) (Fig. 15) and in tissues exhibiting an increase in growth rate, such as ductal hyperplasia, carcinoma in situ, and invasive carcinoma (Fig. 16) (162). An increase in transcript signal was also confirmed by in situ hybridization of breast tissues containing lesions representative of progressive stages of neoplastic evolution (Fig. 17). The levels of expression were undetectable in normal tissues; they increased progressively from moderately elevated in DH, a stage of cell progression from normal to neoplasia that may be a histopathologic parallel of cell immortalization, greater expression in carcinoma in situ, with the highest transcript levels being detected in infiltrating ductal carcinoma (Fig. 17).
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Ferritin is a large protein found in most cell types of vertebrates, as well as of invertebrates, plants, and bacteria (163). The main function of ferritin is iron storage. This function, in turn, can be subdivided into iron storage for other cells (specialized-cell ferritin), iron storage for intracellular needs (normal housekeeping ferritin), and iron storage for intracellular protection from iron overload (stress housekeeping ferritin) (163). Iron is required for DNA synthesis necessary for cell growth and multiplication (164–166). It is also required for electron transport and for oxygen metabolism, generating harmful activated oxygen species capable of damaging DNA, lipids, and proteins (167). The iron-catalyzed conversion of H202 is a major route to the synthesis of highly reactive OH radicals that inflict damage on the nucleotide bases of DNA, inducing mutations and increasing the risk of cancer (42,168).
Normally, the concentration of iron capable of catalyzing these reactions is tightly controlled and regulated, and a critical homeostatic balance is achieved by the synthesis of ferritin (163,169). Many compounds, such as flavins and xanthine oxidase, are capable of reductively releasing iron from ferritin (170,171). Once released from ferritin, iron in the ferrous (Fe++) state is capable of participating in free-radical reactions (Fenton reactions) leading to oxidative damage (Fig. 18). Thus, a disruption in normal iron homeostasis may lead to an increase in the level of reactive iron and to a corresponding increase in oxygen free-radical generation and DNA damage (172). Oxidative stress has also been implicated in metastasis, because it results in loss of cell adhesion, a prerequisite for cell detachment and subsequent host tissue invasion (173,174).
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Progression of invasive breast cancer to the metastatic state is linked to OH radical-induced DNA damage (175–177). Thus, ferritin-dependent oxidative damage to DNA may be one of the mechanisms contributing to immortalization of HBECs. An increase in ferritin H chain levels may provide iron necessary for the clonal selection and uncontrolled growth of cells. It has been shown that iron and its binding proteins participate in a variety of reactions required for cell proliferation (56,178) and are critical for the activity of the enzyme ribonucleotide reductase, a rate-limiting enzyme in DNA synthesis (165,179). Ferritin has been shown to have an immunosuppressive effect on host immune response in cancer patients (180,181). Placental isoferritin, an acidic form of ferritin, and its p43 super heavy chain have been reported to be synthesized by breast cancer cells but are absent in normal breast epithelium (182). Breast cancer-associated p43 induces alterations in the expression of cell-surface molecules in neoplastic cells, which in turn could have an effect on the modulation of the cells' adhesive interactions (183). Cytokines, such as tumor necrosis factor, interleukin-1
, and the NF-
B family of transcription factors, specifically induce synthesis of ferritin H by selectively increasing ferritin H transcription (Fig. 18) (183–185).
Our observations that the immortalization of HBECs was associated with an increased ferritin H chain gene transcription led us to postulate that this increase might have contributed to the immortalization of HBEC; probably through one or more of the following mechanisms: a) providing a source of iron required by rapidly dividing cells for clonal expansion, b) providing iron capable of participating in free-radical reactions leading to oxidative DNA damage and mutation, or c) affecting immune surface antigens and thus providing immortal cells a growth advantage by allowing them to escape immune surveillance (Fig. 18). However, the possibility that ferritin H chain gene induction may be a consequence of the immortalized condition of the cells, rather than its cause, cannot be ruled out. In either instance, it may prove to be a valuable marker of cell immortalization or an early indicator of malignant transformation.
Reversion of Immortalized and Transformed Phenotypes
To investigate the functional role of genomic changes on chromosomes 11 and 17 that were detected in the immortalized and transformed cells, single normal human fibroblast A9-derived chromosome 11 or 17, tagged with a neomycin-resistant gene, was transferred into 6 x 106 transformed BP1E cells. Surviving cells or clones of microcell hybrids from chromosome 11 or 17 were designated BP1E-11neo or BP1E-17neo cells. A total of 16 colonies was isolated from BP1E-11neo and BP1E-17neo cells each. The transfer efficiency in BP1E cells was approximately 2.6 x 10-6 cells (149). During a selection period of up to 6 months, BP1E-17neo cells, and to a lesser degree BP1E-11neo cells, exhibited altered cellular morphology and growth pattern, such as contact inhibition and cellular senescence (149). In addition to the acquired ability to survive in the G-418 selection medium that indicates the active function of the neomycin-resistance gene tagged on the donor chromosome 11 or 17, the physical presence of these chromosomes was further confirmed by dual-color fluorescence in situ hybridization (FISH) analysis. As shown in Fig. 19, A and B, the metaphases of the BPlE-11neo#145 cells were confirmed to contain an extra chromosome 11 that had a stronger signal with the painting probe (red). Anchorage-independent growth in agar-methocel gel was reduced from 17% in control BP1E cells to 7% in the BP1E-11neo#145 cells, whereas BP1E-17neo D100 cells failed to form any colony (100% reduction), reflecting a more potent suppression of the BP1E cells by chromosome 17 than that by chromosome 11. These data indicated that the introduced chromosome 11 caused a partial growth inhibition, whereas chromosome 17 produced a nearly complete growth suppression of the BP1E cells (149). Another phenotypic reversion induced by the chromosome transfer was the recovery of the ability of cells to form ductule-like structures in collagen gel, a property exhibited by BP1E-17neo D100 cells, similar to that of MCF-10F cells, whereas BP1E cells grew in loosely arranged clusters or as isolated cells. These observations confirmed the reversion of the transformed phenotype by chromosome 17 transfer.
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Microsatellite analysis showed that the preexisting instability in the parental BP1E cells at loci D17S849 (17pl3.3), TP53 (17p13.1), D17S786 (17pl3.1), and D17S520 (17pl2.0) was reverted in BP1E-17neo D100 cells, which acquired an allelic pattern similar to that of the mortal MCF-10M cells. In contrast, the instability of these markers was not restored in the BPlE-11neo #145 cells. These data indicated a specific effect associated with transfer of chromosome 17. Surprisingly, none of the corresponding donor alleles observed in the A9–17neo cells could be detected in the BPlE-17neo D100 cells, suggesting that other untested regions of the donor chromosome 17 might be the responsible ones for the phenotypic reversion and the restored microsatellite stability.
In summary, our data provide supportive evidence for the hypothesis that MSI within or near genes can confer instability to these genes and alter their expression or functions. However, further investigation is required for determining what is their functional role in the initiation and progression of neoplasia. TP53 has been considered as the guardian of the genome by allowing cells to undergo DNA repair prior to entering a new cell cycle (186–188). The observation that introduction of an unaffected chromosome 17 can correct instability on the corresponding chromosome, including that of marker TP53, in addition to the reversion of transformed phenotypes in the transformed BP1E cells, suggests that other important genes on chromosome 17 may control this process.
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FUTURE PERSPECTIVES |
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TopNotesAbstractIntroductionDevelopmental Pattern of the...Hormonal Influences on the...Architectural Pattern of the...Architectural Pattern of the...Breast Development, Hormones,...The In Vitro Model...Future PerspectivesReferences |
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In the paradigm described above, it is clear that if estrogens play a role in the early stages of cell immortalization and transformation, this experimental system will allow us to demonstrate such phenomena. The demonstration of the ability of the mammary epithelial cells to metabolize estradiol and/or to accumulate "genotoxic" metabolites could profoundly influence our understanding of the neoplastic transformation of the mammary epithelium (189). Metabolic biotransformation of estradiol occurs in human mammary explant cultures composed of a mixture of epithelial and stromal cells (190,191). Treatment of normal mouse mammary epithelial cells with the mutagenic polycyclic hydrocarbon DMBA results in production of 16
-hydroxyestrone. This predominant metabolite of estrogen increases unscheduled DNA synthesis, cellular proliferation, and anchorage-independent growth, all phenomena indicative of preneoplastic transformation (192). Because normal HBECs are susceptible to be transformed by environmental carcinogens that require metabolic activation (116,117) and many of the enzymes (e.g., CYP IA1) that catalyze the oxidation of drugs, alkaloids, and environmental pollutants also catalyze the hydroxylation of estrogens (136,193,194), we hypothesize that HBECs, regardless of their ER status, are capable of metabolic activation of estrogen and, thus, susceptible to estrogen-induced carcinogenesis. It is possible that the rates of metabolic activation of estrogen might vary among HBECs with different carcinogenic susceptibility. We postulate that the susceptibility of cells to be transformed by estrogens would depend on their rate of proliferation, genetic predisposition, and mortal status of the cells, rather than their ER contents, similar to what has been observed with chemical carcinogens (47,114,146,147). If these assumptions are true, the efficiency and extent of estrogen-induced neoplastic transformation will be high in immortalized MCF-10F cells, moderate in those HBECs derived from breasts of women with family history of breast cancer, and low in cells derived from the breast of parous women and of those women with no family history of breast cancer. The independence of ER contents in estrogen-induced carcinogenesis would support the postulate that metabolic activation of estrogen is involved in the neoplastic transformation of susceptible HBECs. Alternatively, estrogen or its metabolites may not initiate neoplastic transformation, but they may act by promoting neoplastic progression in chemically transformed HBECs by increasing the genomic instability of the cells. In essence, estrogen, on metabolic activation, might serve as an initiator and/or as a promoter of carcinogenesis in the human breast. However, the metabolism of estrogen in normal HBECs and the carcinogenic potential of estrogen and its metabolites in the human breast are virtually unknown.
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Supported by Public Health Services grants R01CA6728 (National Cancer Institute) and ES07280 (National Institute of Environmental Health Sciences), National Institutes of Health, Department of Health and Human Services.
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REFERENCES |
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