Cell cycle basis for the onset and progression of c-Myc-induced, TGFalpha-enhanced mouse mammary gland carcinogenesis.

Using single and double transgenic mouse models, we investigated how c-Myc modulates the mammary epithelial cell cycle to induce cancer and how TGFalpha enhanced the process. In c-myc transgenic mice, c-myc expression was high in the hyperplastic mammary epithelium and in the majority of tumor areas. However, the tumors displayed focal areas of low expression of c-myc but high rates of proliferation. In contrast to E2F1 and cyclin A2, which were induced and co-localized with c-myc expression, induction of cyclins D1 and E occurred only in these tumor foci. Overexpression of cyclin D1 also occurred in the hyperplastic epithelium of tgfalpha-single and tgfalpha/c-myc-double transgenic mice. In tgfalpha/c-myc tumors, cells positive for cyclins D1 and E were randomly spread, without showing a reciprocal relationship to c-myc expression. In contrast to c-myc tumors, most tgfalpha/c-myc tumors showed undetectable levels of retinoblastoma protein (pRB), and the loss of pRB occurred in some cases at the mRNA level. These results suggest that E2F1 and cyclin A2 may be induced by c-Myc to mediate the onset of mammary cancer, whereas overexpression of cyclins D1 and E may occur later to facilitate tumor progression. TGFalpha may play its synergistic role, at least in part, by inducing cyclin D1 and facilitating the loss of pRB.

Decreased turnover of phosphatidylinositol accompanies in vitro differentiation of embryonic chicken lens epithelial cells into lens fibers.

The in vivo differentiation of embryonic chicken lens epithelial cells into lens fibers is accompanied by a marked decrease in the rate of degradation of phosphatidylinositol. The present experiments were undertaken to determine whether a similar change in phosphatidylinositol metabolism occurs during in vitro lens fiber formation in cultured explants of embryonic chicken lens epithelia. Lens epithelial cells in the explants differentiate into lens fibers following the addition of fetal calf serum, insulin or chicken vitreous humor to the culture medium. The results show that phosphatidylinositol is degraded with a half-life of 3-6 h in cultured lens epithelia that are not stimulated to differentiate. In contrast, no degradation occurs for at least 6 h in lens epithelia stimulated to form lens fibers. The stabilization of phosphatidylinositol is apparent within 4 h after the onset of fiber cell formation, and thus represents an early event in differentiation. The rapid degradation of phosphatidylinositol in lens epithelia is accompanied by comparably rapid synthesis. During this metabolic turnover only the phosphorylinositol portion of the molecule is renewed, as expected if hydrolysis occurs by the action of a phospholipase C, such as phosphatidylinositol phosphodiesterase. Thus, these data suggest that agents which produce in vitro differentiation of embryonic chicken lens epithelial cells into lens fibers lead to a reduction in either the amount or the activity of phospholipase C.

Differential alteration of stem and other cell populations in ducts and lobules of TGFalpha and c-Myc transgenic mouse mammary epithelium.

Genes associated with proliferation are active in stem and progenitor cells, and their over-expression can promote cancer. Two such genes, c-Myc and TGFalpha, promote morphologically dissimilar mammary tumors in transgenic mice. We investigated whether their over-expression affects population size and cell cycle activity in stem and other cell populations in non-neoplastic mammary epithelia. Results indicated that both cell population and cell cycle regulation are cell type- and microenvironment-specific. To create a tool for identifying and categorizing the five cellular phenotypes by light microscopy, we adapted previously established ultrastructural criteria. Using nulliparous MMTV-c-myc or MT-tgfalpha mice, we determined and compared the relative sizes the putative stem, progenitor and differentiated cell populations. PCNA staining was used to compare the portion of each cell population in the cell cycle. Cell population sizes were analyzed relative to: (1) their location in ducts versus lobules (microenvironment), (2) genotype, and (3) cell type. Population sizes differed significantly by genotype, depending on microenvironment (p=0.0008), by genotype, depending on cell type (p<0.0001), and by microenvironment, depending on cell type (p=0.03). The number of cycling cells was also affected by all three factors, confirming that the interplay of cell type, gene expression and three-dimensional organization are very important in tissue morphogenesis and function. We describe a structure in mammary epithelium consistent with that of a stem cell niche, and show that it is altered in MMTV-c-myc and likely altered in MT TGFalpha transgenic epithelia.

Progesterone induces side-branching of the ductal epithelium in the mammary glands of peripubertal mice.

Development of the functional secretory epithelium in the mammary gland of the female mouse requires the elongation of the anlage through the mammary fat pad to form the primary/secondary ductal network from which tertiary ductal side-branches and lobuloalveoli develop. In this study we examined the hormonal requirements for the spatial development of the primary/secondary epithelial network and tertiary side-branches by quantifying ductal growth and epithelial cell proliferation in normal and hormone-treated BALB/c mice between 21 and 39 days of age. In normal mice, an allometric increase in ductal length commenced at 31 days of age and resulted in completion of the primary/secondary ductal network by 39 days of age. Concurrent with this allometric growth was a significant increase in cellular proliferation in the terminal end-buds (TEBs) of the ductal epithelium from 29 days of age, as determined by 5-bromo-2'-deoxyuridine (BrdU) incorporation. A level of cellular proliferation similar to that in the TEBs of 33-day-old control mice could be induced in the TEBs of 25-day-old mice following treatment for 1 day with estrogen (E), or progesterone (P) or both (E/P), indicating that both E and P were mitogenic for epithelial cells of the peripubertal TEBs. However, the period of allometric ductal growth in untreated mice did not correspond to an increase in serum E or P (which might have been expected during the estrous cycle). In addition, epithelial growth was not observed in mammary glands from 24-day-old mice that were cultured in vitro with E, P or E/P. In contrast to treatment with E, treatment with P promoted a dramatic increase, relative to control mice, in the number of tertiary branch points upon the primary/secondary ductal network. BrdU labeling of mammary glands from 24- 33-day-old mice pelleted with cholesterol (C), E, P or E/P confirmed the greater mitogenicity of P on the epithelial cells of the secondary/tertiary ducts as compared with C or E. Concurrent with these changes, localized progesterone receptor (PR) expression in clusters of cells in the ductal epithelium was associated with structures that histologically resembled early branch points from ductules. In conclusion, our results suggest that additional endocrine growth factor(s) other than E and P contribute to the development of the primary/secondary ductal network, and that P is responsible for the formation of tertiary side-branches in the mammary glands of mice during puberty.

Mammary epithelial stem cells.

It has recently been shown that the progeny from a single cell may comprise the epithelial population of a fully developed lactating mammary outgrowth in mice. Serial transplantation of epithelial fragments from this clonally derived gland demonstrates that the subsequently generated outgrowths are also comprised of progeny from the original antecedent. All epithelial cell types were found to be present within these clonal normal populations including luminal, myoepithelial, ductal, and lobule-committed epithelial progenitors and fully competent mammary epithelial stem cells. These observations demonstrate the presence of multipotent tissue-specific epithelial stem cells among the parenchyma of the murine mammary gland. Similarly, genetic analysis of contiguous portions of individual human mammary ducts within the same breast indicates their clonal derivation. Here, we discuss the properties, location, division-potential, senescence, and plasticity associated with mammary epithelial stem cells and present the developing evidence for their presence in human breast and their important role in the risk for breast cancer development. Further, we review the present morphologic and genetic evidence for the characterization of specific stem cell markers and lineage-limited progenitor cells in human and rodent mammary epithelium. Microsc. Res. Tech. 52:190-203, 2001. Published 2001 Wiley-Liss, Inc.

Three division-competent, structurally-distinct cell populations contribute to murine mammary epithelial renewal.

Markers for the division-competent cells in mammary gland epithelium are important to the understanding of normal and neoplastic mammary epithelial growth and architectural maintenance, but have been difficult to reveal. Using the presence of condensed chromosomes as an indicator of division competence, we have described morphological markers in the nucleus and cytoplasm that reliably characterize two sets of structurally-distinct, division-competent cells in murine (i.e. mouse and rat) mammary epithelium. The markers, based, in part, on cell size, nuclear and cytoplasmic staining characteristics, and distinctive morphological features of the nucleus and cytoplasm allow classification of the division-competent cells into two categories: 'large light cells' and 'small light cells'. Based on the degree of cytoplasmic organelle differentiation, the small light cells are the least differentiated cells in the epithelium, and the large light cells appear, structurally and functionally, to be in the early stages of secretory differentiation. We demonstrate, using statistical analysis of cell counts (per unit area of epithelium) from four stages of mammary epithelial growth, that there are, in fact, three division-competent cell populations in the rat mammary epithelium, and that the large light cell is a direct precursor to terminally differentiated cells, both secretory and myoepithelial. Using our results we synthesize a morphological model of cell mitosis and the progression of epithelial differentiation in murine mammary gland from a stem cell through two progenitors.

Ultrastructure of the putative stem cell niche in rat mammary epithelium.

There is now strong evidence that the stem cells of many tissues reside in specialized structures termed niches. The stem cell niche functions to house and regulate symmetric and asymmetric mitosis of stem cells in mammalian skin, mouse and human bone marrow, mouse brain, gut, and hair follicle, and Drosophila ovary and testis. This regulation is effected through the action of various signaling pathways such as Notch, Hedgehog, Wnt and others. The hormones of the estrous cycle, pregnancy and lactation that initiate growth in mouse mammary epithelium appear to act at a paracrine level to regulate mitosis through Notch receptors. Previous work has established that the putative stem cells of the mammary epithelium in several animal species reside near the basement membrane and never make contact with the ductal lumen. We show that these putative stem cells are found in anatomically specialized places created by the cytoplasmic extensions and modifications of neighboring differentiated cells. Such specializations may help to regulate stem cell activity by modulating molecular traffic to putative stem cells and contact with signaling molecules in the basement membrane. The histological characteristics of these putative niches vary as to the kinds of relationships the cells can have with the basement membrane and neighboring cells and as to how many stem or progenitor cells they may contain. This suggests a plasticity that may be relevant to the response of niches to tissue demands, such as wound healing, the periodic growth and regression of mammary epithelium, the process of mammary tumorigenesis therapeutic strategies for breast cancer.

Mammary epithelial stem cells: our current understanding.

It has recently been shown that the progeny from a single cell may comprise the epithelial population of a fully developed lactating mammary outgrowth in mice. Serial transplantation of epithelial fragments from this clonally derived gland demonstrates that the subsequently generated outgrowths are also comprised of progeny from the original antecedent. Similarly, genetic analysis of contiguous portions of individual human mammary ducts within the same breast indicates their clonal derivation. These observations support the concept that multipotent tissue-specific epithelial stem cells are present among the parenchymal cells of the mammary gland. Here, we present the developing evidence for the presence of stem cells in virtually every renewing mammalian tissue as well as some classically considered to consist only of differentiated cells. Further, we review the present morphologic and biologic evidence for stem cells and lineage-limited progenitor cells in human and rodent mammary epithelium. Although a number of selective markers are known for various lineage-limited hematopoietic cells and their progeny, our understanding of the biology of the precursor cells for mammary epithelium is just beginning. Our purpose here is to develop further interest in the clarification of these issues in the biology of the mammary gland.