RESUMEN
Breast cancers are highly heterogeneous and their metastatic potential and response to therapeutic drugs is difficult to predict. A tool that could accurately gauge tumour invasiveness and drug response would provide a valuable addition to the oncologist's arsenal. We have developed a 3-dimensional (3D) culture model that recapitulates the stromal environment of breast cancers by generating anisotropic (directional) collagen scaffolds seeded with adipocytes and culturing tumour fragments therein. Analysis of tumour cell invasion in the presence of various therapeutic drugs, by immunofluorescence microscopy coupled with an optical clearing technique, demonstrated the utility of this approach in determining both the rate and capacity of tumour cells to migrate through the stroma while shedding light also on the mode of migration. Furthermore, the response of different murine mammary tumour types to chemotherapeutic drugs could be readily quantified.
Asunto(s)
Adipocitos/citología , Movimiento Celular/fisiología , Colágeno/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Células 3T3-L1 , Animales , Femenino , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Microscopía FluorescenteRESUMEN
While genetically engineered mice have made an enormous contribution towards the elucidation of human disease, it has hitherto not been possible to tune up or down the level of expression of any endogenous gene. Here we describe compound genetically modified mice in which expression of the endogenous E2f3 gene may be either reversibly elevated or repressed in adult animals by oral administration of tetracycline. This technology is, in principle, applicable to any endogenous gene, allowing direct determination of both elevated and reduced gene expression in physiological and pathological processes. Applying this switchable technology to the key cell cycle transcription factor E2F3, we demonstrate that elevated levels of E2F3 drive ectopic proliferation in multiple tissues. By contrast, E2F3 repression has minimal impact on tissue proliferation or homeostasis in the majority of contexts due to redundancy of adult function with E2F1 and E2F2. In the absence of E2F1 and E2F2, however, repression of E2F3 elicits profound reduction of proliferation in the hematopoietic compartments that is rapidly lethal in adult animals.
Asunto(s)
Factor de Transcripción E2F3/genética , Ingeniería Genética/métodos , Tetraciclina/administración & dosificación , Animales , Proliferación Celular , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Regiones Promotoras Genéticas , Tetraciclina/farmacología , Regulación hacia ArribaRESUMEN
The functional integrity of the intestinal epithelial barrier relies on tight coordination of cell proliferation and migration, with failure to regulate these processes resulting in disease. It is not known whether cell proliferation is sufficient to drive epithelial cell migration during homoeostatic turnover of the epithelium. Nor is it known precisely how villus cell migration is affected when proliferation is perturbed. Some reports suggest that proliferation and migration may not be related while other studies support a direct relationship. We used established cell-tracking methods based on thymine analog cell labeling and developed tailored mathematical models to quantify cell proliferation and migration under normal conditions and when proliferation is reduced and when it is temporarily halted. We found that epithelial cell migration velocities along the villi are coupled to cell proliferation rates within the crypts in all conditions. Furthermore, halting and resuming proliferation results in the synchronized response of cell migration on the villi. We conclude that cell proliferation within the crypt is the primary force that drives cell migration along the villus. This methodology can be applied to interrogate intestinal epithelial dynamics and characterize situations in which processes involved in cell turnover become uncoupled, including pharmacological treatments and disease models.-Parker, A., Maclaren, O. J., Fletcher, A. G., Muraro, D., Kreuzaler, P. A., Byrne, H. M., Maini, P. K., Watson, A. J. M., Pin, C. Cell proliferation within small intestinal crypts is the principal driving force for cell migration on villi.
Asunto(s)
Movimiento Celular/fisiología , Intestino Delgado/citología , Animales , Antimetabolitos Antineoplásicos/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular , Citarabina/farmacología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Involution of the mammary gland occurs at the end of every period of lactation and is an essential process to return the gland to a pre-pregnant state in readiness for the next pregnancy. Involution is a complex process of regulated alveolar cell death coupled with tissue remodeling and requires exquisite control of transcription and signaling. These processes can be investigated using a variety of molecular and morphological approaches.In this chapter we describe how to initiate involution and collect mammary glands, measure involution morphologically, and quantify lysosomal leakiness in mammary tissue and in cultured mammary epithelial cells. These procedures encompass a range of microscopy and molecular biology techniques.
Asunto(s)
Muerte Celular/fisiología , Glándulas Mamarias Animales/fisiología , Animales , Células Epiteliales/fisiología , Femenino , Lactancia/fisiología , Lisosomas/fisiología , Ratones , Embarazo , Transducción de Señal/fisiología , Transcripción Genética/fisiologíaRESUMEN
Bone (or body) morphogenetic proteins (BMPs) belong to the TGFß superfamily and are crucial for embryonic patterning and organogenesis as well as for adult tissue homeostasis and repair. Activation of BMP receptors by their ligands leads to induction of several signaling cascades. Using fluorescence recovery after photobleaching, FRET, and single particle tracking microscopy, we demonstrate that BMP receptor type I and II (BMPRI and BMPRII) have distinct lateral mobility properties within the plasma membrane, which is mandatory for their involvement in different signaling pathways. Before ligand binding, BMPRI and a subpopulation of BMPRII exhibit confined motion, reflecting preassembled heteromeric receptor complexes. A second free diffusing BMPRII population only becomes restricted after ligand addition. This paper visualizes time-resolved BMP receptor complex formation and demonstrates that the lateral mobility of BMPRI has a major impact in stabilizing heteromeric BMPRI-BMPRII receptor complexes to differentially stimulate SMAD versus non-SMAD signaling.
Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Membrana Celular/metabolismo , Complejos Multiproteicos/metabolismo , Transducción de Señal/fisiología , Proteínas Smad/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Membrana Celular/genética , Células HEK293 , Humanos , Complejos Multiproteicos/genética , Transporte de Proteínas/fisiología , Proteínas Smad/genéticaRESUMEN
The process of post-lactational regression, or involution, of the mammary gland is a complex event characterised by extensive death of the secretory epithelium coupled with remodelling of the extracellular matrix and adipogenesis to regenerate the fat pad. Associated with these events is an inflammatory cascade and acute phase response. The critical signalling pathways that regulated involution have been defined and a wide variety of genes have been shown to modulate the various processes involved, including cell death, phagocytosis, tissue remodelling and innate immune response.
Asunto(s)
Mama/crecimiento & desarrollo , Glándulas Mamarias Animales/crecimiento & desarrollo , Animales , Mama/citología , Mama/inmunología , Mama/fisiología , Muerte Celular/genética , Muerte Celular/fisiología , Femenino , Expresión Génica , Humanos , Lactancia/genética , Lactancia/fisiología , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/inmunología , Glándulas Mamarias Animales/fisiología , Modelos Biológicos , Embarazo , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
It is well established that lysosomes play an active role during the execution of cell death. A range of stimuli can lead to lysosomal membrane permeabilization (LMP), thus inducing programmed cell death without involvement of the classical apoptotic programme. However, these lysosomal pathways of cell death have mostly been described in vitro or under pathological conditions. Here we show that the physiological process of post-lactational regression of the mammary gland is accomplished through a non-classical, lysosomal-mediated pathway of cell death. We found that, during involution, lysosomes in the mammary epithelium undergo widespread LMP. Furthermore, although cell death through LMP is independent of executioner caspases 3, 6 and 7, it requires Stat3, which upregulates the expression of lysosomal proteases cathepsin B and L, while downregulating their endogenous inhibitor Spi2A (ref. 8). Our findings report a previously unknown, Stat3-regulated lysosomal-mediated pathway of cell death under physiological circumstances. We anticipate that these findings will be of major importance in the design of treatments for cancers such as breast, colon and liver, where cathepsins and Stat3 are commonly overexpressed and/or hyperactivated respectively.
Asunto(s)
Regulación de la Expresión Génica , Lisosomas/metabolismo , Neoplasias/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Catepsina B/metabolismo , Catepsina L/metabolismo , Catepsinas/metabolismo , Muerte Celular , Permeabilidad de la Membrana Celular , Cruzamientos Genéticos , Femenino , Inmunohistoquímica/métodos , Neoplasias Mamarias Animales/patología , Ratones , Ratones Endogámicos C57BLRESUMEN
Cysteine cathepsins are proteolytic enzymes that reside in endolysosomal vesicles. Some are expressed constitutively while others are transcriptionally regulated. However, the expression and subcellular localization of cathepsins changes during cancer progression and cathepsins have been shown to be causally involved in various aspects of tumorigenesis including metastasis. The use of mouse models of breast cancer genetically ablated for cathepsin B has shown that both the growth of the primary tumor and the extend of lung metastasis is reduced by the loss of cathepsin B. The role of cathepsins in involution of the mammary gland has received little attention although it is clear that cathepsins are involved in tissue remodeling in the second phase of involution. We discuss here the roles of cathepsins and their endogenous inhibitors in breast tumorigenesis and post-lactational involution.