RESUMEN
The intestinal epithelium is highly regenerative. Rapidly proliferating LGR5+ crypt base columnar (CBC) cells are responsible for epithelial turnover needed to maintain intestinal homeostasis. Upon tissue damage, loss of LGR5+ CBCs can be compensated by activation of quiescent +4 intestinal stem cells (ISCs) or early progenitor cells to restore intestinal regeneration. LGR5+ CBC self-renewal and ISC conversion to LGR5+ cells are regulated by external signals originating from the ISC niche. In contrast, little is known about intrinsic regulatory mechanisms critical for maintenance of LGR5+ CBC homeostasis. We found that LGR5 expression in intestinal crypt cells is controlled by the circadian core clock gene BMAL1 and the BMAL1-regulated RNA-binding protein MEX3A. BMAL1 directly activated transcription of Mex3a. MEX3A in turn bound to and stabilized Lgr5 mRNA. Bmal1 depletion reduced Mex3a and Lgr5 expression and led to increased ferroptosis, which consequently decreased LGR5+ CBC numbers and increased the number of crypt cells expressing +4 ISC marker BMI1. Together, these findings reveal a BMAL1-centered intrinsic regulatory pathway that maintains LGR5 expression in the crypt cells and suggest a potential mechanism contributing to ISC homeostasis.
Asunto(s)
Factores de Transcripción ARNTL , Intestinos , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Mucosa Intestinal/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Epithelial ovarian cancer is a highly heterogeneous and malignant female cancer with an overall low survival rate. Mutations in p53 are prevalent in the major ovarian cancer histotype, high-grade serous ovarian carcinoma (HGSOC), while p53 mutations are much less frequent in other ovarian cancer subtypes, particularly in ovarian clear cell carcinoma (OCCC). Advanced stage OCCC with wild-type (WT) p53 has a worse prognosis and increased drug resistance, metastasis, and recurrence than HGSOC. The mechanisms responsible for driving the aggressiveness of WT p53-expressing ovarian cancer remain poorly understood. Here, we found that upregulation of MEX3A, a dual-function protein containing a RING finger domain and an RNA-binding domain, was critical for tumorigenesis in WT p53-expressing ovarian cancer. MEX3A overexpression enhanced the growth and clonogenicity of OCCC cell lines. In contrast, depletion of MEX3A in OCCC cells, as well as ovarian teratocarcinoma cells, reduced cell survival and proliferative ability. MEX3A depletion also inhibited tumor growth and prolonged survival in orthotopic xenograft models. MEX3A depletion did not alter p53 mRNA level but did increase p53 protein stability. MEX3A-mediated p53 protein degradation was crucial to suppress ferroptosis and enhance tumorigenesis. Consistently, p53 knockdown reversed the effects of MEX3A depletion. Together, our observations identified MEX3A as an important oncogenic factor promoting tumorigenesis in ovarian cancer cells expressing WT p53. SIGNIFICANCE: Degradation of p53 mediated by MEX3A drives ovarian cancer growth by circumventing p53 tumor suppressive functions, suggesting targeting MEX3A as a potential strategy for treating of ovarian cancer expressing WT p53.
Asunto(s)
Adenocarcinoma de Células Claras , Ferroptosis , Neoplasias Ováricas , Proteínas de Unión al ARN , Proteína p53 Supresora de Tumor , Femenino , Humanos , Adenocarcinoma de Células Claras/tratamiento farmacológico , Carcinogénesis/genética , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Ferroptosis/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Glutathione (GSH), the major non-enzymatic antioxidant, plays a critical role in cellular reactive oxygen species (ROS) neutralization. Moreover, GSH is required for the self-renewal maintenance of human embryonic stem cells (hESCs), and is highly accumulated in undifferentiated cells. Among 8 GSH biosynthesis-related enzymes, we found CHAC2 is highly enriched in undifferentiated hESCs. CHAC2 downregulation in hESCs efficiently decreased the levels of GSH and blocked self-renewal. The self-renewal of sh-CHAC2 cells can be rescued by GSH supplement. CHAC2 downregulation promoted mesoderm differentiation and hampered both teratoma formation and the expression of Nrf2 and glutamate-cysteine ligase (GCL). Notably, CHAC1 knockdown restored the self-renewability of CHAC2-downregulated cells. Although both CHAC1 and CHAC2 purified protein alone showed the catalytic activities to GSH, our data extraordinarily revealed that CHAC2 prevented CHAC1-mediated GSH degradation, which suggests that CHAC2 competes with CHAC1 to maintain GSH homeostasis. This is the first report to demonstrate that CHAC2 is critical for GSH maintenance and the novel roles of the CHAC family in hESC renewal.
Asunto(s)
Glutamato-Cisteína Ligasa/genética , Glutatión/biosíntesis , Células Madre Embrionarias Humanas/enzimología , Factor 2 Relacionado con NF-E2/genética , gamma-Glutamilciclotransferasa/genética , Animales , Bioensayo , Línea Celular , Proliferación Celular , Células Nutrientes/citología , Fibroblastos/citología , Regulación de la Expresión Génica , Glutamato-Cisteína Ligasa/metabolismo , Glutatión/genética , Células Madre Embrionarias Humanas/citología , Humanos , Ratones , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Teratoma/enzimología , Teratoma/genética , Teratoma/patología , gamma-Glutamilciclotransferasa/antagonistas & inhibidores , gamma-Glutamilciclotransferasa/metabolismoRESUMEN
An important safety concern in the use of human pluripotent stem cells (hPSCs) is tumorigenic risk, because these cells can form teratomas after an in vivo injection at ectopic sites. Several thousands of undifferentiated hPSCs are sufficient to induce teratomas in a mouse model. Thus, it is critical to remove all residue-undifferentiated hPSCs that have teratoma potential before the clinical application of hPSC-derived cells. In this study, our data demonstrated the cytotoxic effects of cardiac glycosides, such as digoxin, lanatoside C, bufalin, and proscillaridin A, in human embryonic stem cells (hESCs). This phenomenon was not observed in human bone marrow mesenchymal stem cells (hBMMSCs). Most importantly, digoxin and lanatoside C did not affect the stem cells' differentiation ability. Consistently, the viability of the hESC-derived MSCs, neurons, and endothelium cells was not affected by the digoxin and lanatoside C treatment. Furthermore, the in vivo experiments demonstrated that digoxin and lanatoside C prevented teratoma formation. To the best of our knowledge, this study is the first to describe the cytotoxicity and tumor prevention effects of cardiac glycosides in hESCs. Digoxin and lanatoside C are also the first FDA-approved drugs that demonstrated cytotoxicity in undifferentiated hESCs.
Asunto(s)
Adipogénesis/efectos de los fármacos , Glicósidos Cardíacos/farmacología , Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias Humanas/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Teratoma/prevención & control , Animales , Técnicas de Cultivo de Célula , Células Cultivadas , Células Madre Embrionarias Humanas/patología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Teratoma/metabolismo , Teratoma/patologíaRESUMEN
Patients with type 1 diabetes mellitus are associated with impairment in vitamin A metabolism. This study evaluated whether treatment with retinoic acid, the biologically active metabolite of vitamin A, can ameliorate diabetes. All-trans retinoic acid (atRA) was used to treat streptozotocin (STZ)-induced diabetic mice which revealed atRA administration ameliorated blood glucose levels of diabetic mice. This hyperglycemic amelioration was accompanied by an increase in the amount of ß cells co-expressed Pdx1 and insulin and by restoration of the vascular laminin expression. The atRA-induced production of vascular endothelial growth factor-A from the pancreatic islets was possibly the key factor that mediated the restoration of islet vascularity and recovery of ß-cell mass. Furthermore, the combination of islet transplantation and atRA administration significantly rescued hyperglycemia in diabetic mice. These findings suggest that vitamin A derivatives can potentially be used as a supplementary treatment to improve diabetes management and glycemic control.
Asunto(s)
Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Islotes Pancreáticos/metabolismo , Tretinoina/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Glucemia/metabolismo , Relación Dosis-Respuesta a Droga , Hipoglucemiantes/administración & dosificación , Insulina/sangre , Islotes Pancreáticos/efectos de los fármacos , Masculino , Ratones , Estreptozocina , Resultado del TratamientoRESUMEN
Islet transplantation has been proven to be an effective treatment for patients with type 1 diabetes, but a lack of islet donors limits the use of transplantation therapies. It has been previously demonstrated that hepatocytes can be converted into insulin-producing ß-like cells by introducing pancreatic transcription factors, indicating that direct hepatocyte reprogramming holds potential as a treatment for diabetes. However, the efficiency at which functional ß-cells can be derived from hepatocyte reprogramming remains low. Here we demonstrated that the combination of Pdx1 and Ngn3 can trigger reprogramming of mouse and human liver cells to insulin-producing cells that exhibit the characteristics of pancreatic ß-cells. Treatment with PDGF-AA was found to facilitate Pdx1 and Ngn3-induced reprogramming of hepatocytes to ß-like cells with the ability to secrete insulin in response to glucose stimulus. Importantly, this reprogramming strategy could be applied to adult mouse primary hepatocytes, and the transplantation of ß-like cells derived from primary hepatocyte reprogramming could ameliorate hyperglycemia in diabetic mice. These findings support the possibility of developing transplantation therapies for type 1 diabetes through the use of ß-like cells derived from autologous hepatocyte reprogramming.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Reprogramación Celular/efectos de los fármacos , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Transactivadores/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Glucemia/análisis , Transdiferenciación Celular/efectos de los fármacos , Células Cultivadas , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/terapia , Exenatida , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Péptido 1 Similar al Glucagón/farmacología , Receptor del Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Glucosa/farmacología , Hepatocitos/citología , Hepatocitos/metabolismo , Proteínas de Homeodominio/genética , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/trasplante , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Transgénicos , Microscopía Fluorescente , Proteínas del Tejido Nervioso/genética , Péptidos/farmacología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transactivadores/genética , Ponzoñas/farmacologíaRESUMEN
A short G1 phase is a characteristic feature of the cell cycle structure of pluripotent cells, and is reestablished during Yamanaka factor-mediated pluripotent reprogramming. How cell cycle control is adjusted to meet the requirements of pluripotent cell fate commitment during reprogramming is less well understood. Elevated levels of cyclin D1 were initially found to impair pluripotency maintenance. The current work further identified Cyclin D1 to be capable of transcriptionally upregulating Pax6, which promoted reprogramming cells to commit to a neural progenitor fate rather than a pluripotent cell fate. These findings explain the importance of reestablishment of G1-phase restriction in pluripotent reprogramming.
Asunto(s)
Diferenciación Celular , Reprogramación Celular , Ciclina D1/metabolismo , Células-Madre Neurales/citología , Células Madre Pluripotentes/citología , Animales , Biomarcadores/metabolismo , Ciclina D1/biosíntesis , Proteínas del Ojo/genética , Fibroblastos/citología , Fase G1 , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Ratones , Proteína Homeótica Nanog , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Células Madre Pluripotentes/metabolismo , Proteínas Represoras/genética , Transcripción GenéticaRESUMEN
Targeted inhibition of multidrug ABCG2 transporter is believed to improve cancer therapeutics. However, the consequences of ABCG2 inhibition have not been systematically evaluated since ABCG2 is expressed in several organs including the liver. Here, we demonstrate that ABCG2-deficient hepatocytes have increased amounts of fragmental mitochondria accompanied by disruption of mitochondrial dynamics and functions. This disruption was due to ABCG2 knockout elevating intracellular protoporphyrin IX, which led to upregulation of DRP-1-mediated mitochondrial fission. The finding that ABCG2 deficiency can generate dysfunctional mitochondria in hepatocytes raises concerns regarding the systematic use of ABCG2 inhibitor in cancer patients.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Hepatocitos/metabolismo , Mitocondrias Hepáticas/metabolismo , Dinámicas Mitocondriales , Protoporfirinas/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Animales , Células Cultivadas , Glucógeno/metabolismo , Ratones , Ratones Noqueados , Mitocondrias Hepáticas/fisiologíaRESUMEN
BACKGROUND & AIMS: The appearance of hepatic foci in pancreas has been well-documented in animal experiments and in patients with pancreatic cancer. We previously demonstrated that transdifferentiation of pancreatic exocrine cells to hepatocytes required members of the CCAAT enhancer binding protein family. Although the molecular basis of hepatic transdifferentiation is understood, the early cellular events remain to be defined. METHODS: Dexamethasone and oncostatin M were used to induce transdifferentiation of primary cultures of mouse acinar cells and exocrine cell lines into hepatocytes. Fluorescent-activated cell sorting was used to identify intermediate cell types and side-population characteristics. Cre-loxP-based lineage tracing was used to investigate whether acinar cells contribute directly to hepatocytes via intermediates that express adenosine triphosphate-binding cassette subfamily G member 2 (ABCG2). RESULTS: Lineage tracing studies showed that hepatocytes were derived directly from pancreatic cells via ABCG2-expressing intermediates. Exposure of cells to insulin increased Akt phosphorylation, ABCG2 expression, and hepatic transdifferentiation. Inhibition of the phosphoinositide 3-kinase pathway, through addition of LY294002 or overexpression of a dominant-negative form of Akt, was sufficient to prevent transdifferentiation. When ABCG2-expressing cells were incubated with glucagon-like-peptide 1 or epidermal growth factor, the intermediate cells could differentiate into insulin-producing beta-like cells. CONCLUSIONS: The phosphoinositide 3-kinase pathway is important in the transdifferentiation of acinar cells to hepatocytes and those hepatocytes arise from acinar cells via ABCG2-expressing intermediates. Furthermore, ABCG2-expressing cells are multipotent and able to differentiate into hepatocytes and insulin-producing beta cells.
Asunto(s)
Linaje de la Célula , Hepatocitos/citología , Páncreas Exocrino/citología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/fisiología , Animales , Proteína beta Potenciadora de Unión a CCAAT/fisiología , Diferenciación Celular , Células Cultivadas , Dexametasona/farmacología , Factor de Crecimiento Epidérmico/farmacología , Insulina/farmacología , Ratones , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Ratas , Transducción de SeñalRESUMEN
Although JC virus (JCV), a human polyomavirus, has been detected in colon cancers, the association between JCV and colon cancer remains controversial. In Taiwan, the prevalence of JCV infection in colon cancer patients has not been reported. Thus, the purpose of this study was to investigate JCV infection in colon cancers in Taiwan. Formalin-fixed, paraffin-embedded tissues from 22 colon cancer patients were examined in this study. Nested PCR was performed to detect viral genomic DNA. The product of the nested PCR flanking the JCV regulatory region was sequenced further. Viral large tumor protein, LT, and late capsid protein, VP1, were examined by immunohistochemistry (IHC). Nested PCR revealed JCV genomic DNA in 86.4% (19/22) of the colon cancer tissue samples. Both rearranged and archetypal genotypes of JCV were identified. Expression of JCV LT was positive in 63.6% (14/22) of the examined colon cancer tissue samples but not in any adjacent normal region. Expression of viral capsid protein VP1 was not detected in any of the tissues examined. The current study demonstrates that JCV genomic DNA was present in the examined colon cancer tissues. The genotypes of JCV in colon cancer tissues were also identified. Expression of viral early protein but not structural capsid protein was detected in the examined colon cancer tissues. Furthermore, a high prevalence of JCV infection in colon cancer tissues in Taiwan was also demonstrated.