Extracellular matrix (ECM) is an intricate network composed of an array of macromolecules, the importance of which is becoming increasingly apparent. The ECM is an integral part of the machinery that regulates cell function; its role in cell differentiation and tissue-specific gene expression, although essential, is not yet understood. It can act as a positive as well as a negative regulator of functional differentiation depending on the cell type and the genes studied. It also acts in a hierarchical fashion, exacting higher and higher degrees of stringency to achieve full functional differentiation. Regulation by ECM is closely interrelated with the action of other regulators of cellular function, such as growth factors and hormones. But ECM may exert its regulation of gene expression by mechanisms distinct from those known for soluble transcription factors. In this short review, we describe three systems in which ECM has been shown to play a crucial role in functional differentiation, but we emphasize mainly the work from our own laboratory to provide a more in-depth analysis of one system. The three systems are: mouse mammary epithelial cells, rat hepatocytes, and human keratinocytes. The crucial role of ECM in normal cell differentiation implies that its alteration may have serious consequences in malignancies and other diseases. The current functional cell culture models could provide powerful tools not only for understanding regulation of normal cell function but also for the studies of tumorigenesis and possibly cancer therapy.

Mammary epithelial cells undergo changes in growth, invasion, and differentiation throughout much of adulthood, and most strikingly during pregnancy, lactation, and involution. Although the pathways of milk protein expression are being elucidated, little is known, at a molecular level, about control of mammary epithelial cell phenotypes during normal tissue morphogenesis and evolution of aggressive breast cancer. We developed a murine mammary epithelial cell line, SCp2, that arrests growth and functionally differentiates in response to a basement membrane and lactogenic hormones. In these cells, expression of Id-1, an inhibitor of basic helix-loop-helix transcription factors, declines prior to differentiation, and constitutive Id-1 expression blocks differentiation. Here, we show that SCp2 cells that constitutively express Id-1 slowly invade the basement membrane but remain anchorage dependent for growth and do not form tumors in nude mice. Cells expressing Id-1 secreted a ϳ120-kDa gelatinase. From inhibitor studies, this gelatinase appeared to be a metalloproteinase, and it was the only metalloproteinase detectable in conditioned medium from these cells. A nontoxic inhibitor diminished the activity of this metalloproteinase in vitro and repressed the invasive phenotype of Id-1-expressing cells in culture. The implications of these findings for normal mammary-gland development and human breast cancer were investigated. A gelatinase of ϳ120 kDa was expressed by the mammary gland during involution, a time when Id-1 expression is high and there is extensive tissue remodeling. Moreover, high levels of Id-1 expression and the activity of a ϳ120-kDa gelatinase correlated with a less-differentiated and more-aggressive phenotype in human breast cancer cells. We suggest that Id-1 controls invasion by normal and neoplastic mammary epithelial cells, primarily through induction of a ϳ120-kDa gelatinase. This Id-1-regulated invasive phenotype could contribute to involution of the mammary gland and possibly to the development of invasive breast cancer.The epithelial cells of the mammary gland undergo coordinate changes in growth, differentiation, and invasion of the surrounding ECM during embryonic development and puberty, and throughout much of adulthood during each menstrual cycle. Particularly striking changes occur during pregnancy, lactation, and involution. The molecular mechanisms that control the growth and functional differentiation of mammary epithelial cells are slowly being elucidated, but far less is known about the transient invasive behavior of normal breast epithelial cells.Normal breast epithelial cells proliferate and invade the surrounding ECM during the fetal and postnatal development of the gland, and then more vigorously at puberty as the branches of the mammary epithelial tree are formed. After puberty, there are minor waves of mammary epithelial-cell proliferation during each estrous cycle (16,46). The most striking activity of mammary epithelial-cell proliferation and invasion occurs during pregnancy,...

Mammary epithelial cells proliferate, invade the stroma, differentiate, and die in adult mammals by mechanisms that are poorly understood. We found that Id-1, an inhibitor of basic helix-loop-helix transcription factors, regulates mammary epithelial cell growth, differentiation, and invasion in culture. Here, we show that Id-1 is expressed highly during mammary development in virgin mice and during early pregnancy, when proliferation and invasion are high. During mid-pregnancy, Id-1 expression declined to undetectable levels as the epithelium differentiated fully. Surprisingly, Id-1 increased during involution, when the epithelium undergoes extensive apoptosis. To determine whether Id-1 regulates both proliferation and apoptosis, we constitutively expressed Id-1 in mammary epithelial cell cultures. Id-1 stimulated proliferation in sparse cultures but induced apoptosis in dense cultures, which reflect epithelial cell density during early pregnancy and involution, respectively. To understand how Id-1 acts, we screened a yeast two-hybrid library from differentiating mammary epithelial cells and identified ITF-2, a basic helix-loop-helix transcription factor, as an Id-1-interacting protein. Overexpression of ITF-2 significantly reduced Id-1-stimulated proliferation and apoptosis. We show further that, in contrast to Id-1, Id-2 was expressed highly in differentiated mammary epithelial cells in vivo and in culture. In culture, Id-2 antisense transcripts blocked differentiation. Our results suggest that Id-1, ITF-2, and Id-2 comprise a network of interacting molecular switches that govern mammary epithelial cell phenotypes.The mammary gland undergoes striking changes in morphology and function during development and puberty and adult life. During each menstrual cycle, and particularly during pregnancy, mammary epithelial cells undergo cycles of proliferation, invasion, differentiation, and apoptotic cell death. During pregnancy, mammary epithelial cells proliferate, invade the surrounding stromal extracellular matrix (ECM), 1 and form lobulo-alveolar structures that prepare the gland for lactation (1-3). At late pregnancy, prior to parturition, breast epithelial cells cease proliferation and invasion and functionally differentiate into cells that express and secrete milk proteins. Throughout lactation, epithelial cells remain quiescent but continue to express milk proteins (4). After weaning, the mammary gland undergoes involution, a phase of extensive remodeling characterized by degradation of ECM and epithelial cell death by apoptosis.The molecular mechanisms that regulate and coordinate the changes in mammary epithelial cell phenotypes are poorly understood. Genes that are up-or down-regulated at different developmental stages have been identified, but little is known about how they are regulated and coordinated. The identification of tissue-specific transcriptional regulators in mammary epithelial cells is crucial, not only for deciphering the regulatory mechanisms of normal growth and differentiation but also for ...

Abstract. Whey acidic protein (WAP) is an abundant rodent milk protein. Its expression in mouse mammary epithelial cell cultures was previously found to require the formation of an extracellular matrix (ECM)-induced three-dimensional alveolar structure. In the absence of such structures, cells were shown to secrete diffusible factors leading to suppression of WAP expression. We demonstrate here that (a) TGF-c~ production and secretion by mammary cells is downregulated by the basement membrane-dependent alveolar structure, and (b) compared with/3-casein, WAP expression is preferentially inhibited both in culture and in transgenic mice when TGF-ot is added or overexpressed. Thus, (c) the enhanced TGF-a production when cells are not in three-dimensional structures largely accounts for the WAP-inhibitory activity found in the conditioned medium. Since this activity can be abolished by incubating the conditioned medium with a function blocking antibody to TGF-tx. The data suggest that ECM upregulates WAP by downregulating TGF-ct production. We also propose that changes in TGF-ct activity during mouse gestation and lactation could contribute to the pattern of temporal expression of WAP in the gland. These results provide a clear example of cooperation among lactogenic hormones, ECM, and locally acting growth factors in regulation of tissue-specific gene expression.

Mammary epithelial cells undergo cycles of proliferation, invasion, differentiation and apoptotic cell-death throughout adult life. The molecular mechanisms that regulate these complex and co-ordinated developmental programs are poorly understood. We have identified Id-1 protein, a negative regulator of basic helix-loop-helix transcription factors, as a critical regulator of these normal mammary epithelial cell phenotypes. We also found that Id-1 is an important regulator of the aggressive and invasive phenotype, as well as mediator of the effects of sex steroid hormones, in human breast cancer cells.

B-CLL cells, like many solid and hematologic malignancies, are characterized by short telomeres, suggesting that they would be acutely susceptible to telomerase inhibition. We and others have documented that CLL patients in poorer prognosis subsets, i.e., those without IgVH mutations, had shorter mean telomere lengths and higher telomerase levels than patients with IgVH mutations (Damle et al, 2004; Keating et al, 2003; Bechter et al 1998, Hultdin et al, 2003). Treatment with the telomerase inhibitor GRN163L, a lipid-conjugated 13-mer thio-phosphoramidate oligonucleotide (Geron Corporation), inhibits the growth of human hepatoma (Djojosubroto et al, 2005), ovarian carcinoma (Ertem et al, 2004, 2005), and multiple myeloma (CAG, MM.1S) cell lines in vitro and in vivo (AACR 2004 and 2005 Annual Meetings). Although no validated human B-CLL xenograft models exist, preliminary data indicate effective inhibition of telomerase in freshly thawed B-CLL cells upon exposure to GRN163L. We will present data demonstrating robust uptake of GRN163L into primary B-CLL patient cells, along with the subcellular distribution of the oligonucleotide into cytoplasmic and nuclear compartments. Comparison of the effect of GRN163L and a mismatch oligonucleotide control on telomerase inhibition will be described. Geron initiated a Phase I/II trial with GRN163L in chronic lymphocytic leukemia in July 2005.