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1.
Nature ; 574(7777): 268-272, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31578521

RESUMEN

Liver cancer remains difficult to treat, owing to a paucity of drugs that target critical dependencies1,2; broad-spectrum kinase inhibitors such as sorafenib provide only a modest benefit to patients with hepatocellular carcinoma3. The induction of senescence may represent a strategy for the treatment of cancer, especially when combined with a second drug that selectively eliminates senescent cancer cells (senolysis)4,5. Here, using a kinome-focused genetic screen, we show that pharmacological inhibition of the DNA-replication kinase CDC7 induces senescence selectively in liver cancer cells with mutations in TP53. A follow-up chemical screen identified the antidepressant sertraline as an agent that kills hepatocellular carcinoma cells that have been rendered senescent by inhibition of CDC7. Sertraline suppressed mTOR signalling, and selective drugs that target this pathway were highly effective in causing the apoptotic cell death of hepatocellular carcinoma cells treated with a CDC7 inhibitor. The feedback reactivation of mTOR signalling after its inhibition6 is blocked in cells that have been treated with a CDC7 inhibitor, which leads to the sustained inhibition of mTOR and cell death. Using multiple in vivo mouse models of liver cancer, we show that treatment with combined inhibition of of CDC7 and mTOR results in a marked reduction of tumour growth. Our data indicate that exploiting an induced vulnerability could be an effective treatment for liver cancer.


Asunto(s)
Apoptosis/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Terapia Molecular Dirigida , Sertralina/farmacología , Animales , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Mutación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Sertralina/uso terapéutico , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genética
2.
Gastroenterology ; 156(6): 1788-1804.e13, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30641053

RESUMEN

BACKGROUND & AIMS: Patients with cirrhosis are at high risk for hepatocellular carcinoma (HCC) and often have increased serum levels of estrogen. It is not clear how estrogen promotes hepatic growth. We investigated the effects of estrogen on hepatocyte proliferation during zebrafish development, liver regeneration, and carcinogenesis. We also studied human hepatocytes and liver tissues. METHODS: Zebrafish were exposed to selective modifiers of estrogen signaling at larval and adult stages. Liver growth was assessed by gene expression, fluorescent imaging, and histologic analyses. We monitored liver regeneration after hepatocyte ablation and HCC development after administration of chemical carcinogens (dimethylbenzanthrazene). Proliferation of human hepatocytes was measured in a coculture system. We measured levels of G-protein-coupled estrogen receptor (GPER1) in HCC and nontumor liver tissues from 68 patients by immunohistochemistry. RESULTS: Exposure to 17ß-estradiol (E2) increased proliferation of hepatocytes and liver volume and mass in larval and adult zebrafish. Chemical genetic and epistasis experiments showed that GPER1 mediates the effects of E2 via the phosphoinositide 3-kinase-protein kinase B-mechanistic target of rapamycin pathway: gper1-knockout and mtor-knockout zebrafish did not increase liver growth in response to E2. HCC samples from patients had increased levels of GPER1 compared with nontumor tissue samples; estrogen promoted proliferation of human primary hepatocytes. Estrogen accelerated hepatocarcinogenesis specifically in male zebrafish. Chemical inhibition or genetic loss of GPER1 significantly reduced tumor development in the zebrafish. CONCLUSIONS: In an analysis of zebrafish and human liver cells and tissues, we found GPER1 to be a hepatic estrogen sensor that regulates liver growth during development, regeneration, and tumorigenesis. Inhibitors of GPER1 might be developed for liver cancer prevention or treatment. TRANSCRIPT PROFILING: The accession number in the Gene Expression Omnibus is GSE92544.


Asunto(s)
Carcinoma Hepatocelular/metabolismo , Estradiol/farmacología , Estrógenos/farmacología , Neoplasias Hepáticas/metabolismo , Hígado/crecimiento & desarrollo , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Pez Cebra/metabolismo , 9,10-Dimetil-1,2-benzantraceno , Animales , Carcinogénesis/efectos de los fármacos , Carcinoma Hepatocelular/patología , Proliferación Celular/efectos de los fármacos , Femenino , Expresión Génica/efectos de los fármacos , Hepatocitos , Humanos , Hígado/metabolismo , Cirrosis Hepática/metabolismo , Neoplasias Hepáticas/patología , Regeneración Hepática , Masculino , Tamaño de los Órganos/efectos de los fármacos , Fosfatidilinositol 3-Quinasa/metabolismo , Receptores Acoplados a Proteínas G/genética , Factores Sexuales , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Carga Tumoral/efectos de los fármacos , Pez Cebra , Proteínas de Pez Cebra/genética
3.
EMBO J ; 31(14): 3079-91, 2012 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-22692129

RESUMEN

Two types of stem cells are currently defined in small intestinal crypts: cycling crypt base columnar (CBC) cells and quiescent '+4' cells. Here, we combine transcriptomics with proteomics to define a definitive molecular signature for Lgr5(+) CBC cells. Transcriptional profiling of FACS-sorted Lgr5(+) stem cells and their daughters using two microarray platforms revealed an mRNA stem cell signature of 384 unique genes. Quantitative mass spectrometry on the same cell populations identified 278 proteins enriched in intestinal stem cells. The mRNA and protein data sets showed a high level of correlation and a combined signature of 510 stem cell-enriched genes was defined. Spatial expression patterns were further characterized by mRNA in-situ hybridization, revealing that approximately half of the genes were expressed in a gradient with highest levels at the crypt bottom, while the other half was expressed uniquely in Lgr5(+)stem cells. Lineage tracing using a newly established knock-in mouse for one of the signature genes, Smoc2, confirmed its stem cell specificity. Using this resource, we find-and confirm by independent approaches-that the proposed quiescent/'+4' stem cell markers Bmi1, Tert, Hopx and Lrig1 are robustly expressed in CBC cells.


Asunto(s)
Antígenos de Diferenciación/metabolismo , Regulación de la Expresión Génica , Mucosa Intestinal/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Madre/metabolismo , Animales , Antígenos de Diferenciación/genética , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Perfilación de la Expresión Génica , Intestinos/citología , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores Acoplados a Proteínas G/genética , Células Madre/citología
4.
EMBO Rep ; 15(1): 62-9, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24355609

RESUMEN

The concept of 'field cancerization' describes the clonal expansion of genetically altered, but morphologically normal cells that predisposes a tissue to cancer development. Here, we demonstrate that biased stem cell competition in the mouse small intestine can initiate the expansion of such clones. We quantitatively analyze how the activation of oncogenic K-ras in individual Lgr5(+) stem cells accelerates their cell division rate and creates a biased drift towards crypt clonality. K-ras mutant crypts then clonally expand within the epithelium through enhanced crypt fission, which distributes the existing Paneth cell niche over the two new crypts. Thus, an unequal competition between wild-type and mutant intestinal stem cells initiates a biased drift that leads to the clonal expansion of crypts carrying oncogenic mutations.


Asunto(s)
Células Madre Adultas/fisiología , Neoplasias Colorrectales/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptores Acoplados a Proteínas G/metabolismo , Animales , Ciclo Celular , Proliferación Celular , Transformación Celular Neoplásica , Neoplasias Colorrectales/genética , Mucosa Intestinal/patología , Intestino Delgado/patología , Ratones , Ratones Transgénicos , Mutación Missense , Oncogenes , Receptores Acoplados a Proteínas G/genética , Nicho de Células Madre
5.
Angew Chem Int Ed Engl ; 55(40): 12440-4, 2016 09 26.
Artículo en Inglés | MEDLINE | ID: mdl-27554600

RESUMEN

The ability to remotely trigger CRISPR/Cas9 activity would enable new strategies to study cellular events with greater precision and complexity. In this work, we have developed a method to photocage the activity of the guide RNA called "CRISPR-plus" (CRISPR-precise light-mediated unveiling of sgRNAs). The photoactivation capability of our CRISPR-plus method is compatible with the simultaneous targeting of multiple DNA sequences and supports numerous modifications that can enable guide RNA labeling for use in imaging and mechanistic investigations.


Asunto(s)
Sistemas CRISPR-Cas/genética , ARN Guía de Kinetoplastida/metabolismo , Secuencia de Bases , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Luz , Hibridación de Ácido Nucleico , Fotólisis/efectos de la radiación , ARN Guía de Kinetoplastida/química
6.
EMBO J ; 30(6): 1104-9, 2011 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-21297579

RESUMEN

Somatic cells have been proposed to be limited in the number of cell divisions they can undergo. This is thought to be a mechanism by which stem cells retain their integrity preventing disease. However, we have recently discovered intestinal crypt stem cells that persist for the lifetime of a mouse, yet divide every day. We now demonstrate biochemically that primary isolated Lgr5+ve stem cells contain significant telomerase activity. Telomerase activity rapidly decreases in the undifferentiated progeny of these stem cells and is entirely lost in differentiated villus cells. Conversely, asymmetric segregation of chromosomes has been proposed as a mechanism for stem cells to protect their genomes against damage. We determined the average cell cycle length of Lgr5+ve stem cells at 21.5 h and find that Lgr5+ve intestinal stem cells randomly segregate newly synthesized DNA strands, opposing the 'immortal strand' hypothesis.


Asunto(s)
Segregación Cromosómica , Mucosa Intestinal/citología , Mucosa Intestinal/enzimología , Nicho de Células Madre , Células Madre/enzimología , Células Madre/fisiología , Telomerasa/metabolismo , Animales , Ciclo Celular , Diferenciación Celular , Células Epiteliales/enzimología , Células Epiteliales/fisiología , Ratones , Factores de Tiempo
7.
Carcinogenesis ; 35(1): 237-46, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23996931

RESUMEN

Although Apc mutation is widely considered an initiating event in colorectal cancer, little is known about the earliest stages of tumorigenesis following sporadic Apc loss. Therefore, we have utilized a novel mouse model that facilitates the sporadic inactivation of Apc via frameshift reversion of Cre in single, isolated cells and subsequently tracks the fates of Apc-deficient intestinal cells. Our results suggest that consistent with Apc being a 'gatekeeper', loss of Apc early in life during intestinal growth leads to adenomas or increased crypt fission, manifested by fields of mutant but otherwise normal-appearing crypts. In contrast, Apc loss occurring later in life has minimal consequences, with mutant crypts being less prone to either increased crypt fission or adenoma formation. Using the stem cell-specific Lgr5-CreER mouse, we generated different sized fields of Apc-deficient crypts via independent recombination events and found that field size correlates with progression to adenoma. To evaluate this early stage prior to adenoma formation as a therapeutic target, we examined the chemopreventive effects of sulindac on Apc-deficient occult crypt fission. We found that sulindac treatment started early in life inhibits the morphologically occult spread of Apc-deficient crypts and thus reduces adenoma numbers. Taken together these results suggest that: (i) earlier Apc loss promotes increased crypt fission, (ii) a field of Apc-deficient crypts, which can form via occult crypt fission or independent neighboring events, is an important intermediate between loss of Apc and adenoma formation and (iii) normal-appearing Apc-deficient crypts are potential unappreciated targets for cancer screening and chemoprevention.


Asunto(s)
Focos de Criptas Aberrantes/prevención & control , Adenoma/genética , Genes APC , Neoplasias Intestinales/genética , Sulindac/farmacología , Focos de Criptas Aberrantes/tratamiento farmacológico , Adenoma/patología , Factores de Edad , Animales , Quimioprevención , Reparación del ADN/genética , Neoplasias Intestinales/patología , Intestinos/patología , Ratones , Ratones Transgénicos , Mutación , Células Madre/patología , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo
8.
Cell Rep Med ; 5(3): 101471, 2024 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-38508142

RESUMEN

Drug-tolerant persisters (DTPs) are a rare subpopulation of cells within a tumor that can survive therapy through nongenetic adaptive mechanisms to develop relapse and repopulate the tumor following drug withdrawal. Using a cancer cell line with an engineered suicide switch to kill proliferating cells, we perform both genetic screens and compound screens to identify the inhibition of bromodomain and extraterminal domain (BET) proteins as a selective vulnerability of DTPs. BET inhibitors are especially detrimental to DTPs that have reentered the cell cycle (DTEPs) in a broad spectrum of cancer types. Mechanistically, BET inhibition induces lethal levels of ROS through the suppression of redox-regulating genes highly expressed in DTPs, including GPX2, ALDH3A1, and MGST1. In vivo BET inhibitor treatment delays tumor relapse in both melanoma and lung cancer. Our study suggests that combining standard of care therapy with BET inhibitors to eliminate residual persister cells is a promising therapeutic strategy.


Asunto(s)
Neoplasias Pulmonares , Recurrencia Local de Neoplasia , Humanos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética
9.
PLoS One ; 17(9): e0273182, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36067171

RESUMEN

Inducing senescence in cancer cells is emerging as a new therapeutic strategy. In order to find ways to enhance senescence induction by palbociclib, a CDK4/6 inhibitor approved for treatment of metastatic breast cancer, we performed functional genetic screens in palbociclib-resistant cells. Using this approach, we found that loss of CDK2 results in strong senescence induction in palbociclib-treated cells. Treatment with the CDK2 inhibitor indisulam, which phenocopies genetic CDK2 inactivation, led to sustained senescence induction when combined with palbociclib in various cell lines and lung cancer xenografts. Treating cells with indisulam led to downregulation of cyclin H, which prevented CDK2 activation. Combined treatment with palbociclib and indisulam induced a senescence program and sensitized cells to senolytic therapy. Our data indicate that inhibition of CDK2 through indisulam treatment can enhance senescence induction by CDK4/6 inhibition.


Asunto(s)
Quinasa 6 Dependiente de la Ciclina , Inhibidores de Proteínas Quinasas , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Humanos , Piperazinas , Inhibidores de Proteínas Quinasas/farmacología , Piridinas , Sulfonamidas
10.
Nat Cancer ; 3(11): 1284-1299, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36414711

RESUMEN

Senolytics, drugs that kill senescent cells, have been proposed to improve the response to pro-senescence cancer therapies; however, this remains challenging due to a lack of broadly acting senolytic drugs. Using CRISPR/Cas9-based genetic screens in different senescent cancer cell models, we identify loss of the death receptor inhibitor cFLIP as a common vulnerability of senescent cancer cells. Senescent cells are primed for apoptotic death by NF-κB-mediated upregulation of death receptor 5 (DR5) and its ligand TRAIL, but are protected from death by increased cFLIP expression. Activation of DR5 signaling by agonistic antibody, which can be enhanced further by suppression of cFLIP by BRD2 inhibition, leads to efficient killing of a variety of senescent cancer cells. Moreover, senescent cells sensitize adjacent non-senescent cells to killing by DR5 agonist through a bystander effect mediated by secretion of cytokines. We validate this 'one-two punch' cancer therapy by combining pro-senescence therapy with DR5 activation in different animal models.


Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Neoplasias , Animales , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Apoptosis , FN-kappa B/metabolismo , Transducción de Señal , Neoplasias/tratamiento farmacológico
11.
Mol Cancer Res ; 19(10): 1613-1621, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34158393

RESUMEN

Pro-senescence therapies are increasingly being considered for the treatment of cancer. Identifying additional targets to induce senescence in cancer cells could further enable such therapies. However, screening for targets whose suppression induces senescence on a genome-wide scale is challenging, as senescent cells become growth arrested, and senescence-associated features can take 1 to 2 weeks to develop. For a screen with a whole-genome CRISPR library, this would result in billions of undesirable proliferating cells by the time the senescent features emerge in the growth arrested cells. Here, we present a suicide switch system that allows genome-wide CRISPR screening in growth-arrested subpopulations by eliminating the proliferating cells during the screen through activation of a suicide switch in proliferating cells. Using this system, we identify in a genome-scale CRISPR screen several autophagy-related proteins as targets for senescence induction. We show that inhibiting macroautophagy with a small molecule ULK1 inhibitor can induce senescence in cancer cell lines of different origin. Finally, we show that combining ULK1 inhibition with the senolytic drug ABT-263 leads to apoptosis in a panel of cancer cell lines. IMPLICATIONS: Our suicide switch approach allows for genome-scale identification of pro-senescence targets, and can be adapted to simplify other screens depending on the nature of the promoter used to drive the switch.


Asunto(s)
Proteínas Relacionadas con la Autofagia/genética , Autofagia/genética , Sistemas CRISPR-Cas/genética , Senescencia Celular/genética , Células A549 , Apoptosis/efectos de los fármacos , Apoptosis/genética , Autofagia/efectos de los fármacos , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Sistemas CRISPR-Cas/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Senescencia Celular/efectos de los fármacos , Células HEK293 , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Bibliotecas de Moléculas Pequeñas/farmacología
12.
ACS Chem Biol ; 16(9): 1770-1778, 2021 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-34427427

RESUMEN

The utility of in vitro human disease models is mainly dependent on the availability and functional maturity of tissue-specific cell types. We have previously screened for and identified small molecules that can enhance hepatocyte function in vitro. Here, we characterize the functional effects of one of the hits, FH1, on primary human hepatocytes in vitro, and also in vivo on primary hepatocytes in a zebrafish model. Furthermore, we conducted an analogue screen to establish the structure-activity relationship of FH1. We performed affinity-purification proteomics that identified NQO2 to be a potential binding target for this small molecule, revealing a possible link between inflammatory signaling and hepatocellular function in zebrafish and human hepatocyte model systems.


Asunto(s)
Biomarcadores/metabolismo , Inhibidores Enzimáticos/química , Hepatocitos/metabolismo , Quinona Reductasas/antagonistas & inhibidores , Animales , Inhibidores Enzimáticos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Interleucina-6/genética , Hígado , Simulación del Acoplamiento Molecular , Unión Proteica , Factor de Transcripción STAT3/genética , Transducción de Señal , Relación Estructura-Actividad , Factores de Necrosis Tumoral/genética , Pez Cebra
13.
Cell Rep ; 36(4): 109441, 2021 07 27.
Artículo en Inglés | MEDLINE | ID: mdl-34320349

RESUMEN

Cellular senescence is characterized as a stable proliferation arrest that can be triggered by multiple stresses. Most knowledge about senescent cells is obtained from studies in primary cells. However, senescence features may be different in cancer cells, since the pathways that are involved in senescence induction are often deregulated in cancer. We report here a comprehensive analysis of the transcriptome and senolytic responses in a panel of 13 cancer cell lines rendered senescent by two distinct compounds. We show that in cancer cells, the response to senolytic agents and the composition of the senescence-associated secretory phenotype are more influenced by the cell of origin than by the senescence trigger. Using machine learning, we establish the SENCAN gene expression classifier for the detection of senescence in cancer cell samples. The expression profiles and senescence classifier are available as an interactive online Cancer SENESCopedia.


Asunto(s)
Senescencia Celular , Neoplasias/patología , Compuestos de Anilina/farmacología , Azepinas/farmacología , Línea Celular Tumoral , Senescencia Celular/efectos de los fármacos , Etopósido/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias/genética , Pirimidinas/farmacología , Reproducibilidad de los Resultados , Fenotipo Secretor Asociado a la Senescencia/efectos de los fármacos , Fenotipo Secretor Asociado a la Senescencia/genética , Senoterapéuticos/farmacología , Sulfonamidas/farmacología
14.
Lab Chip ; 16(14): 2644-53, 2016 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-27296616

RESUMEN

In vitro models of human tissue are crucial to our ability to study human disease as well as develop safe and effective drug therapies. Models of single organs in static and microfluidic culture have been established and shown utility for modeling some aspects of health and disease; however, these systems lack multi-organ interactions that are critical to some aspects of drug metabolism and toxicity. Thus, as part of a consortium of researchers, we have developed a liver chip that meets the following criteria: (1) employs human iPS cells from a patient of interest, (2) cultures cells in perfusable 3D organoids, and (3) is robust to variations in perfusion rate so as to be compatible in series with other specialized tissue chips (e.g. heart, lung). In order to achieve this, we describe methods to form hepatocyte aggregates from primary and iPS-derived cells, alone and in co-culture with support cells. This necessitated a novel culture protocol for the interrupted differentiation of iPS cells that permits their removal from a plated surface and aggregation while maintaining phenotypic hepatic functions. In order to incorporate these 3D aggregates in a perfusable platform, we next encapsulated the cells in a PEG hydrogel to prevent aggregation and overgrowth once on chip. We adapted a C-trap chip architecture from the literature that enabled robust loading with encapsulated organoids and culture over a range of flow rates. Finally, we characterize the liver functions of this iHep organoid chip under perfusion and demonstrate a lifetime of at least 28 days. We envision that such this strategy can be generalized to other microfluidic tissue models and provides an opportunity to query patient-specific liver responses in vitro.


Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Hígado/citología , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodos , Albúminas/metabolismo , Diferenciación Celular , Técnicas de Cocultivo/instrumentación , Técnicas de Cocultivo/métodos , Sistema Enzimático del Citocromo P-450/metabolismo , Hepatocitos/citología , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Dispositivos Laboratorio en un Chip , Hígado/fisiología , Perfusión
15.
Cold Spring Harb Perspect Biol ; 4(4): a007989, 2012 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-22474007

RESUMEN

The Wnt signaling pathway was originally uncovered as one of the prototype developmental signaling cascades in invertebrates as well as in vertebrates. The first indication that Wnt signaling also plays a role in the adult animal came from the study of the intestine of Tcf-4 (Tcf7L2) knockout mice. The gastrointestinal epithelium continuously self-renews over the lifetime of an organism and is, in fact, the most rapidly self-renewing tissue of the mammalian body. Recent studies indicate that Wnt signaling plays a central role in the biology of gastrointestinal stem cells. Furthermore, mutational activation of the Wnt cascade is the principle cause of colon cancer.


Asunto(s)
Neoplasias Gastrointestinales/metabolismo , Transducción de Señal , Células Madre/metabolismo , Proteínas Wnt/metabolismo , Animales , Neoplasias Gastrointestinales/patología , Humanos
16.
Science ; 337(6095): 730-5, 2012 Aug 10.
Artículo en Inglés | MEDLINE | ID: mdl-22855427

RESUMEN

The concept that tumors are maintained by dedicated stem cells, the so-called cancer stem cell hypothesis, has attracted great interest but remains controversial. Studying mouse models, we provide direct, functional evidence for the presence of stem cell activity within primary intestinal adenomas, a precursor to intestinal cancer. By "lineage retracing" using the multicolor Cre-reporter R26R-Confetti, we demonstrate that the crypt stem cell marker Lgr5 (leucine-rich repeat-containing heterotrimeric guanine nucleotide-binding protein-coupled receptor 5) also marks a subpopulation of adenoma cells that fuel the growth of established intestinal adenomas. These Lgr5(+) cells, which represent about 5 to 10% of the cells in the adenomas, generate additional Lgr5(+) cells as well as all other adenoma cell types. The Lgr5(+) cells are intermingled with Paneth cells near the adenoma base, a pattern reminiscent of the architecture of the normal crypt niche.


Asunto(s)
Adenoma/patología , Neoplasias Intestinales/patología , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/fisiología , Receptores Acoplados a Proteínas G/análisis , Adenoma/genética , Adenoma/metabolismo , Animales , Biomarcadores/análisis , Linaje de la Célula , Transformación Celular Neoplásica , Perfilación de la Expresión Génica , Técnicas de Sustitución del Gen , Genes Reporteros , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Neoplasias Intestinales/genética , Ratones , Células Madre Multipotentes/patología , Células Madre Multipotentes/fisiología , Células de Paneth/patología , Nicho de Células Madre , Tamoxifeno/farmacología , Ensayo de Tumor de Célula Madre
17.
Nat Protoc ; 6(8): 1221-8, 2011 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-21799490

RESUMEN

In recent years, many mouse models have been developed to mark and trace the fate of adult cell populations using fluorescent proteins. High-resolution visualization of such fluorescent markers in their physiological setting is thus an important aspect of adult stem cell research. Here we describe a protocol to produce sections (150-200 µm) of near-native tissue with optimal tissue and cellular morphology by avoiding artifacts inherent in standard freezing or embedding procedures. The activity of genetically expressed fluorescent proteins is maintained, thereby enabling high-resolution three-dimensional (3D) reconstructions of fluorescent structures in virtually all types of tissues. The procedure allows immunofluorescence labeling of proteins to depths up to 50 µm, as well as a chemical 'Click-iT' reaction to detect DNA-intercalating analogs such as ethynyl deoxyuridine (EdU). Generation of near-native sections ready for imaging analysis takes approximately 2-3 h. Postsectioning processes, such as antibody labeling or EdU detection, take up to 10 h.


Asunto(s)
Proteínas Luminiscentes/análisis , Análisis de la Célula Individual/métodos , Procesamiento de Imagen Asistido por Computador , Imagenología Tridimensional , Microscopía Fluorescente/métodos , Microtomía
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