Immortalised breast epithelia survive prolonged DNA replication stress and return to cycle from a senescent-like state.

Mammalian cells have mechanisms to counteract the effects of metabolic and exogenous stresses, many of that would be mutagenic if ignored. Damage arising during DNA replication is a major source of mutagenesis. The extent of damage dictates whether cells undergo transient cell cycle arrest and damage repair, senescence or apoptosis. Existing dogma defines these alternative fates as distinct choices. Here we show that immortalised breast epithelial cells are able to survive prolonged S phase arrest and subsequently re-enter cycle after many days of being in an arrested, senescence-like state. Prolonged cell cycle inhibition in fibroblasts induced DNA damage response and cell death. However, in immortalised breast epithelia, efficient S phase arrest minimised chromosome damage and protected sufficient chromatin-bound replication licensing complexes to allow cell cycle re-entry. We propose that our observation could have implications for the design of drug therapies for breast cancer.

Apoptosis regulation in the mammary gland.

Epithelial apoptosis has a key role in the development and function of the mammary gland. It is involved with the formation of ducts during puberty and is required to remove excess epithelial cells after lactation so that the gland can be prepared for future pregnancies. Deregulated apoptosis contributes to malignant progression in the genesis of breast cancer. Since epithelial cell apoptosis in the lactating mammary gland can be synchronised by forced weaning, it has been possible to undertake biochemical analysis of the pathways involved. Together with the targeted overexpression or deletion of candidate genes, these approaches have provided a unique insight into the complex mechanisms of apoptosis regulation in vivo. This review explores what is currently known about the triggers for apoptosis in the normal mammary gland, and how they link with the intrinsic apoptotic machinery.

The insecticidal toxin Makes caterpillars floppy (Mcf) promotes apoptosis in mammalian cells.

Photorhabdus bacteria produce a number of toxins to kill their insect hosts. The expression of one of these, Makes caterpillars floppy (Mcf), is sufficient to allow Escherichia coli to persist within and kill caterpillars. Mcf causes shedding of the insect midgut epithelium and destructive blebbing of haemocytes suggesting it may trigger apoptosis. To investigate this hypothesis, here we examine the effects of E. coli-expressed Mcf on the mammalian cell lines COS-7, NIH 3T3 and HeLa cells. Cells treated with Mcf show apoptotic nuclear morphology, active caspase-3, DNA laddering after 6 h, and the presence of cleaved PARP after 16 h. These effects are prevented by the apoptosis inhibitor zVAD-fmk. Transfection of cells with constructs expressing only the NH2-terminal 1280 amino acids of Mcf, as a fusion with Myc, also triggered cell destruction. The expressed fusion protein was concentrated into the Golgi apparatus before cell death. These results confirm that the novel insecticidal toxin Mcf induces apoptosis but the precise intracellular pathway remains obscure.

Autocrine signalling through erbB receptors promotes constitutive activation of protein kinase B/Akt in breast cancer cell lines.

The protein kinase PKB/Akt plays a pivotal role in promoting cell survival and proliferation. This study investigated the regulation of PKB/Akt activity in breast cancer cells. In primary invasive breast cancers PKB/Akt exhibited elevated phosphorylation at regulatory site Ser473 in 80% of cases, using immunohistochemistry. The degree of phospho-PKB/Akt immunoreactivity was positively correlated with the extent of its nuclear accumulation. Moderate/strong staining was seen in 31% of the samples but was absent in tumour-associated normal breast epithelia. To examine the mechanisms of PKB/Akt activation, we studied its phosphorylation in a panel of breast cancer cell lines. PKB/Akt was constitutively phosphorylated on both regulatory sites (Thr308 and Ser473) in the absence of serum growth factors in 7 of 8 lines but not in two cell lines derived from normal breast epithelia. Further analysis revealed that constitutive PKB/Akt phosphorylation was associated with loss of PTEN phosphatase expression (CAL51, MDA-MB-468, BT549 cells) and constitutive activation of erbB2 (SKBR3, BT474 cells). In two further breast cancer lines (T47D and HS578T) PKB/Akt phosphorylation was dependent upon autocrine factors acting primary through the epidermal growth factor receptor (EGFR) and erbB2. Conditioned medium from HS578T cells stimulated EGFR-dependent PKB/Akt phosphorylation in normal breast cells. These results demonstrate that PKB/Akt is frequently activated in breast cancer through diverse mechanisms, including autocrine signalling via erbB receptors.

Assessment of apoptosis in human breast tissue using an antibody against the active form of caspase 3: relation to tumour histopathological characteristics.

Apoptosis is of important significance in the pathogenesis of cancer. Many methods are available for the measurement of apoptosis but the 'gold standard' is to identify apoptotic cells by their morphological features using microscopy. Caspase 3 is a cytosolic enzyme that is activated only in cells committed to undergo apoptosis. The activation of caspase 3 precedes the development of the classical morphological features of apoptosis. Using immunohistochemistry with an antibody against the active form of caspase 3, the apoptotic index (AI) was measured in 116 samples of human breast tissue (22 normal/benign and 94 invasive carcinomas). The AI obtained by measuring caspase activation has a strong correlation with the AI derived by morphological assessment (r = 0.736, P < 0.01). The AI is higher in the invasive group than in the benign group (P = 0.008), and in invasive cancer high AI is associated with high tumour grade (P = 0.013), positive node status (P < 0.001) and negative steroid receptor status (P = 0.001 for ER; P = 0.004 for PR). No significant association is observed between AI and tumour size. Measurement of apoptosis by immunohistochemistry using an antibody against the active form of caspase 3 is therefore reliable and correlates strongly with morphological assessment.

Desmosomal adhesion regulates epithelial morphogenesis and cell positioning.

Desmosomes are intercellular junctions of epithelia and are of widespread importance in the maintenance of tissue architecture. We provide evidence that desmosomal adhesion has a function in epithelial morphogenesis and cell-type-specific positioning. Blocking peptides corresponding to the cell adhesion recognition (CAR) sites of desmosomal cadherins block alveolar morphogenesis by epithelial cells from mammary lumen. Desmosomal CAR-site peptides also disrupt positional sorting of luminal and myoepithelial cells in aggregates formed by the reassociation of isolated cells. We demonstrate that desmosomal cadherins and E-cadherin are comparably involved in epithelial morphoregulation. The results indicate a wider role for desmosomal adhesion in morphogenesis than has previously been considered.

Epithelial development and differentiation in the mammary gland is not dependent on alpha 3 or alpha 6 integrin subunits.

In the mammary gland, both laminin and integrins have been shown to be required for normal ductal morphogenesis during development in vivo, and for functional differentiation in culture models. Major integrin receptors for laminins in the mammary gland are alpha 3 beta 1, alpha 6 beta 1, and alpha 6 beta 4. However, the specific subunits that contribute to laminin-mediated mammary cell function and development have not been identified. In this study, we use a genetic approach to test the hypothesis that laminin-binding integrins are required for the function of the mammary gland in vivo. Rudiments of embryonic mammary gland were shown to develop in the absence of these integrin subunits. Postnatal development of the mammary gland was studied in integrin null tissue that had been transplanted into the mammary fat pads of syngeneic hosts. In mammary epithelium lacking alpha 6 integrin, the beta 4 subunit was not apparent and hemidesmosome formation was only rudimentary. However, despite this deficiency, normal ductal morphogenesis and branching of the mammary gland occurred and myoepithelial cells were distributed normally with respect to luminal cells. Mammary alveoli devoid of alpha 3 or alpha 6 integrin formed in pregnancy and were histologically and functionally identical to those in wild-type mammary gland. The tissue underwent full morphological differentiation, and the epithelial cells retained the ability to synthesize beta-casein. This work demonstrates that mammary tissue genetically lacking major laminin-binding integrin receptors is still able to develop and function.

Quantitative image analysis of laminin immunoreactivity in skin basement membrane irradiated with 1 GeV/nucleon iron particles.

We previously reported that laminin immunoreactivity in mouse mammary epithelium is altered shortly after whole-body irradiation with 0.8 Gy from 600 MeV/nucleon iron ions but is unaffected after exposure to sparsely ionizing radiation. This observation led us to propose that the effect could be due to protein damage from the high ionization density of the ion tracks. If so, we predicted that it would be evident soon after radiation exposure in basement membranes of other tissues and would depend on ion fluence. To test this hypothesis, we used immunofluorescence, confocal laser scanning microscopy, and image segmentation techniques to quantify changes in the basement membrane of mouse skin epidermis. At 1 h after exposure to 1 GeV/nucleon iron ions with doses from 0.03 to 1.6 Gy, neither the visual appearance nor the mean pixel intensity of laminin in the basement membrane of mouse dorsal skin epidermis was altered compared to sham-irradiated tissue. This result does not support the hypothesis that particle traversal directly affects laminin protein integrity. However, the mean pixel intensity of laminin immunoreactivity was significantly decreased in epidermal basement membrane at 48 and 96 h after exposure to 0.8 Gy 1 GeV/nucleon iron ions. We confirmed this effect with two additional antibodies raised against affinity-purified laminin 1 and the E3 fragment of the long-arm of laminin 1. In contrast, collagen type IV, another component of the basement membrane, was unaffected. Our studies demonstrate quantitatively that densely ionizing radiation elicits changes in skin microenvironments distinct from those induced by sparsely ionizing radiation. Such effects may might contribute to the carcinogenic potential of densely ionizing radiation by altering cellular signaling cascades mediated by cell-extracellular matrix interactions.

Integrin-mediated survival signals regulate the apoptotic function of Bax through its conformation and subcellular localization.

Most normal cells require adhesion to extracellular matrix for survival, but the molecular mechanisms that link cell surface adhesion events to the intracellular apoptotic machinery are not understood. Bcl-2 family proteins regulate apoptosis induced by a variety of cellular insults through acting on internal membranes. A pro-apoptotic Bcl-2 family protein, Bax, is largely present in the cytosol of many cells, but redistributes to mitochondria after treatment with apoptosis-inducing drugs. Using mammary epithelial cells as a model for adhesion-regulated survival, we show that detachment from extracellular matrix induced a rapid translocation of Bax to mitochondria concurrent with a conformational change resulting in the exposure of its BH3 domain. Bax translocation and BH3 epitope exposure were reversible and occurred before caspase activation and apoptosis. Pp125FAK regulated the conformation of the Bax BH3 epitope, and PI 3-kinase and pp60src prevented apoptosis induced by defective pp125FAK signaling. Our results provide a mechanistic connection between integrin-mediated adhesion and apoptosis, through the kinase-regulated subcellular distribution of Bax.

Laminin and beta1 integrins are crucial for normal mammary gland development in the mouse.

We have examined the role of integrin-extracellular matrix interactions in the morphogenesis of ductal structures in vivo using the developing mouse mammary gland as a model. At puberty, ductal growth from terminal end buds results in an arborescent network that eventually fills the gland, whereupon the buds shrink in size and become mitotically inactive. End buds are surrounded by a basement membrane, which we show contains laminin-1 and collagen IV. To address the role of cell-matrix interactions in gland development, pellets containing function-perturbing anti-beta1 integrin, anti-alpha6 integrin, and anti-laminin antibodies respectively were implanted into mammary glands at puberty. Blocking beta1 integrins dramatically reduced both the number of end buds per gland and the extent of the mammary ductal network, compared with controls. These effects were specific to the end buds since the rest of the gland architecture remained intact. Reduced development was still apparent after 6 days, but end buds subsequently reappeared, indicating that the inhibition of beta1 integrins was reversible. Similar results were obtained with anti-laminin antibodies. In contrast, no effect on morphogenesis in vivo was seen with anti-alpha6 integrin antibody, suggesting that alpha6 is not the important partner for beta1 in this system. The studies with beta1 integrin were confirmed in a culture model of ductal morphogenesis, where we show that hepatocyte growth factor (HGF)-induced tubulogenesis is dependent on functional beta1 integrins. Thus integrins and HGF cooperate to regulate ductal morphogenesis. We propose that both laminin and beta1 integrins are required to permit cellular traction through the stromal matrix and are therefore essential for maintaining end bud structure and function in normal pubertal mammary gland development.

Extracellular matrix remodelling and cellular differentiation.

The extracellular matrix is not merely a passive structure. In the past few years, it has emerged that the matrix is a dynamic action zone that functions to instruct cellular phenotype. Extracellular matrix proteins interact directly with cell surface receptors to initiate signal transduction pathways and to modulate those triggered by differentiation and growth factors. The extracellular matrix also controls the activity and presentation of a wide range of growth factors. Thus modulation of the extracellular matrix, by remodelling its structure and activity, has profound effects on its function and the consequent behaviour of cells residing on or within it.

Type IV collagen and laminin regulate glomerular mesangial cell susceptibility to apoptosis via beta(1) integrin-mediated survival signals.

Postinflammatory scarring is characterized by changes in extracellular matrix (ECM) composition and progressive loss of normal resident cells. In glomerular inflammation there is now evidence that unscheduled apoptosis (programmed cell death) of mesangial and other resident cells may mediate progression to irreversible glomerulosclerosis. In the current study we examined the hypothesis that ECM components may differ in their capacity to support mesangial cell survival by suppression of apoptosis. Using a well-established in vitro model of mesangial cell apoptosis, we found that collagen IV and laminin, components of normal mesangial ECM, protected rat mesangial cells from apoptosis induced by serum starvation and DNA damage, by a beta(1) integrin-mediated, but arg-gly-asp (RGD)-independent mechanism. In contrast, collagen I, fibronectin, and osteonectin/SPARC, which are overexpressed in diseased glomeruli, failed to promote rat mesangial cell survival. However, the survival-promoting effect of collagen IV and laminin was not associated with changes in cellular levels of apoptosis regulatory proteins of the Bcl-2 family. These experiments demonstrate that glomerular mesangial cell survival is dependent on interactions with ECM and provide insights into potential mechanisms by which resident cell loss may occur during acute inflammation and postinflammatory scarring of the kidney and other organs.

Adhesion-mediated signaling in the regulation of mammary epithelial cell survival.

Tissue architecture in multicellular organisms is maintained through adhesive interactions between cells and their neighbors, and between cells and the underlying extracellular matrix. These interactions are important in the dynamic regulation of tissue organization as well as the control of cell proliferation, differentiation and apoptosis. The ultimate goal of this regulation is to promote cell growth and differentiation only when the cell is in the correct location, and to delete cells that have become displaced from their proper environment. It therefore plays an important role in development and tissue remodeling. In this review we consider the molecular mechanisms by which cell-matrix interactions contribute to cell survival, and discuss their role in mammary gland development and function.

Extracellular matrix selectively modulates the response of mammary epithelial cells to different soluble signaling ligands.

In adherent cells, cell-substratum interactions are essential for the propagation of some growth factor signaling events. However, it has not been resolved to what extent different types of extracellular matrix regulate the signals elicited by different soluble ligands. Our previous work has shown that prolactin signaling in mammary epithelium requires a specific cell interaction with the basement membrane and does not occur in cells plated on collagen I. We have now investigated whether the proximal signaling pathways triggered by insulin, epidermal growth factor (EGF), and interferon-gamma are differentially regulated in primary mammary epithelial cell cultures established on basement membrane and collagen I. Two distinct signaling pathways triggered by insulin exhibited a differential requirement for cell-matrix interactions. Activation of insulin receptor substrate (IRS) and phosphatidylinositol 3-kinase was restricted to cells contacting basement membrane, whereas the phosphorylation of Erk occurred equally in cells on both substrata. The amplitude and duration of insulin-triggered IRS-1 phosphorylation and its association with phosphatidylinositol 3-kinase were strongly enhanced by cell-basement membrane interactions. The mechanism for inhibition of IRS-1 phosphorylation in cells cultured on collagen I may in part be mediated by protein-tyrosine phosphatase activity since vanadate treatment somewhat alleviated this effect. In contrast to the results with insulin, cell adhesion to collagen I conferred greater response to EGF, leading to higher levels of tyrosine phosphorylation of the EGF receptor and Erk. The mechanism for increased EGF signaling in cells adhering to collagen I was partly through an increase in EGF receptor expression. The interferon-gamma-activated tyrosine phosphorylation of Jak2 and Stat3 was independent of the extracellular matrix. It is well recognized that the cellular environment determines cell phenotype. We now suggest that this may occur through a selective modulation of growth factor signal transduction resulting from different cell-matrix interactions.

Developmental regulation of Bcl-2 family protein expression in the involuting mammary gland.

Epithelial cells within the mammary gland undergo developmental programmes of proliferation and apoptosis during the pregnancy cycle. After weaning, secretory epithelial cells are removed by apoptosis. To determine whether members of the Bcl-2 gene family could be involved in regulating this process, we have examined whether changes in their expression occur during this developmental apoptotic program in vivo. Bax and Bcl-x were evenly expressed throughout development. However, expression of Bak and Bad was increased during late pregnancy and lactation, and the proteins were present during the time of maximal apoptotic involution. Thereafter, their levels declined. In contrast, Bcl-w was expressed in pregnancy and lactation but was downregulated at the onset of apoptosis. Bcl-2 was not detected in lactating or early involuting mammary gland. Thus, the pro-apoptotic proteins Bax, Bak and Bad, as well as the death-suppressors Bcl-x, Bcl-2 and Bcl-w, are synthesised in mouse mammary gland, and dynamic changes in the expression profiles of these proteins occurs during development. To determine if changes in Bak and Bcl-w expression could regulate mammary apoptosis, their effect on cultured mouse mammary epithelial cells was examined in transient transfection assays. Enforced expression of Bak induced rapid mammary apoptosis, which could be suppressed by coexpression of Bcl-w. In extracts of mammary tissue in vivo, Bak heterodimerized with Bcl-x whereas Bax associated with Bcl-w, but Bak/Bcl-w heterodimers were not detected. Thus, Bak and Bcl-w may regulate cell death through independent pathways. These results support a model in which mammary epithelial cells are primed for apoptosis during the transition from pregnancy to lactation by de novo expression of the death effectors Bak and Bad. It is suggested that these proteins are prevented from triggering apoptosis by anti-apoptotic Bcl-2 family proteins until involution, when the levels of Bcl-w decline. Our study provides evidence that regulated changes in the expression of cell death genes may contribute to the developmental control of mammary apoptosis.

Extracellular matrix regulates apoptosis in mammary epithelium through a control on insulin signaling.

Adherent epithelial cells require interactions with the extracellular matrix for their survival, though the mechanism is ill-defined. In long term cultures of primary mammary epithelial cells, a laminin-rich basement membrane (BM) but not collagen I suppresses apoptosis, indicating that adhesion survival signals are specific in their response (. J. Cell Sci. 109:631-642). We now demonstrate that the signal from BM is mediated by integrins and requires both the alpha6 and beta1 subunits. In addition, a hormonal signal from insulin or insulin-like growth factors, but not hydrocortisone or prolactin, is necessary to suppress mammary cell apoptosis, indicating that BM and soluble factors cooperate in survival signaling. Insulin induced autophosphorylation of its receptor whether mammary cells were cultured on collagen I or BM substrata. However, both the tyrosine phosphorylation of insulin receptor substrate-1 and its association with phosphatidylinositol 3-kinase were enhanced in cells cultured on BM, as was the phosphorylation of the phosphatidylinositol 3-kinase effector, protein kinase B. These results suggest a novel extracellular matrix-dependent restriction point in insulin signaling in mammary epithelial cells. The proximal signal transduction event of insulin receptor phosphorylation is not dependent on extracellular matrix, but the activation of downstream effectors requires adhesion to BM. Since phosphatidylinositol 3-kinase was required for mammary epithelial cell survival, we propose that a possible mechanism for BM-mediated suppression of apoptosis is through its facilitative effects on insulin signaling.

Leydig cell apoptosis in the rat testes after administration of the cytotoxin ethane dimethanesulphonate: role of the Bcl-2 family members.

Ethane dimethanesulphonate (EDS) is cytotoxic to Leydig cells in the adult rat. To investigate the role and regulation of apoptosis in the Leydig cell, EDS (100 mg/kg i.p.) was administered to adult male rats and the testes examined 6, 12, 18, 24, 48 and 72 h later. Numbers of Leydig cells, identified by 3 beta-hydroxysteroid dehydrogenase immuno-histochemistry started to fall by 12 h after EDS injection and were almost undetectable by 72 h. Apoptotic cells in the interstitium, visualised by in situ end labelling of DNA, increased in number to reach a maximum 24 h after injection of EDS, and were undetectable by 72 h. In many tissues the apoptosis-related gene products act in cohort: Bcl-2 and Bcl-xl promoting survival of a cell, whilst Bax promotes cell death often positively regulated by the tumour-suppressor gene p53. Western blot analysis showed that: (1) Bcl-2 and p53 were absent from interstitial Leydig cells but were expressed in the seminiferous tubules. (2) Bax protein although expressed in the interstitium was not present in the Leydig cells. (3) Bcl-xl in Leydig cells was transiently increased after EDS. In conclusion, EDS kills Leydig cells by apoptosis; however the control of Leydig cell death does not involve p53 or the Bcl-2 family members but may require other gene products yet to be identified.