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1.
Br J Cancer ; 105(9): 1313-21, 2011 Oct 25.
Artículo en Inglés | MEDLINE | ID: mdl-21952626

RESUMEN

BACKGROUND: Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) regulation of the Rho-like GTPase Cdc42 has a central role in epithelial polarised growth, but effects of this molecular network on apoptosis remain unclear. METHODS: To investigate the role of Cdc42 in PTEN-dependent cell death, we used flow cytometry, in vitro pull-down assays, poly(ADP ribose) polymerase (PARP) cleavage and other immunoblots in isogenic PTEN-expressing and -deficient colorectal cells (HCT116PTEN(+/+), HCT116PTEN(-/-), Caco2 and Caco2 ShPTEN cells) after transfection or treatment strategies. RESULTS: The PTEN knockout or suppression by short hairpin RNA or small interfering RNA (siRNA) inhibited Cdc42 activity, PARP cleavage and/or apoptosis in flow cytometry assays. Transfection of cells with wild-type or constitutively active Cdc42 enhanced PARP cleavage, whereas siRNA silencing of Cdc42 inhibited PARP cleavage and/or apoptosis. Pharmacological upregulation of PTEN by sodium butyrate (NaBt) treatment enhanced Cdc42 activity, PARP cleavage and apoptosis, whereas Cdc42 siRNA suppressed NaBt-induced PARP cleavage. Cdc42-dependent signals can suppress glycogen synthase kinase-ß (GSK3ß) activity. Pharmacological inhibition of GSK3ß by lithium chloride treatment mimicked effects of Cdc42 in promotion of PARP cleavage and/or apoptosis. CONCLUSION: Phosphatase and tensin homologue deleted on chromosome 10 may influence apoptosis in colorectal epithelium through Cdc42 signalling, thus providing a regulatory framework for both polarised growth and programmed cell death.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Fosfohidrolasa PTEN/metabolismo , Proteínas de Unión al ARN/metabolismo , Apoptosis , Células CACO-2 , Técnicas de Inactivación de Genes , Células HCT116 , Humanos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Factores de Empalme de ARN , ARN Interferente Pequeño/farmacología , Transducción de Señal
2.
Clin Exp Immunol ; 164(2): 202-10, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21361912

RESUMEN

Identification of immune modifiers of inherited cancer syndromes may provide a rationale for preventive therapy. Cowden disease (CD) is a genetically heterogeneous inherited cancer syndrome that arises predominantly from germline phosphatase and tensin homologue deleted on chromosome 10 (PTEN) mutation and increased phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) signalling. However, many patients with classic CD diagnostic features are mutation-negative for PTEN (PTEN M-Neg). Interferon (IFN)-γ can modulate the PI3K/mTOR pathway, but its association with PTEN M-Neg CD remains unclear. This study assessed IFN-γ secretion by multi-colour flow cytometry in a CD kindred that was mutation-negative for PTEN and other known susceptibility genes. Because IFN-γ responses may be regulated by killer cell immunoglobulin-like receptors (KIR) and respective human leucocyte antigen (HLA) ligands, KIR/HLA genotypes were also assessed. Activating treatments induced greater IFN-γ secretion in PTEN M-Neg CD peripheral blood lymphocytes versus healthy controls. Increased frequency of activating KIR genes, potentially activating KIR/HLA compound genotypes and reduced frequency of inhibitory genotypes, were found in the PTEN M-Neg CD kindred. Differences of IFN-γ secretion were observed among PTEN M-Neg CD patients with distinct KIR/HLA compound genotypes. Taken together, these findings show enhanced lymphocyte secretion of IFN-γ that may influence the PI3K/mTOR CD causal molecular pathway in a PTEN mutation-negative CD kindred.


Asunto(s)
Síndrome de Hamartoma Múltiple/metabolismo , Interferón gamma/metabolismo , Femenino , Citometría de Flujo , Genotipo , Antígenos HLA/biosíntesis , Síndrome de Hamartoma Múltiple/genética , Haplotipos/genética , Humanos , Ionomicina/farmacología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Masculino , Fosfohidrolasa PTEN/análisis , Linaje , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Reacción en Cadena de la Polimerasa , Receptores KIR/fisiología , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo
3.
Oncogene ; 32(10): 1305-15, 2013 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-22543585

RESUMEN

Disruption of glandular architecture associates with poor clinical outcome in high-grade colorectal cancer (CRC). Phosphatase and tensin homolog deleted on chromosome ten (PTEN) regulates morphogenic growth of benign MDCK (Madin Darby Canine Kidney) cells through effects on the Rho-like GTPase cdc42 (cell division cycle 42). This study investigates PTEN-dependent morphogenesis in a CRC model. Stable short hairpin RNA knockdown of PTEN in Caco-2 cells influenced expression or localization of cdc42 guanine nucleotide exchange factors and inhibited cdc42 activation. Parental Caco-2 cells formed regular hollow gland-like structures (glands) with a single central lumen, in three-dimensional (3D) cultures. Conversely, PTEN-deficient Caco-2 ShPTEN cells formed irregular glands with multiple abnormal lumens as well as intra- and/or intercellular vacuoles evocative of the high-grade CRC phenotype. Effects of targeted treatment were investigated. Phosphatidinylinositol 3-kinase (PI3K) modulating treatment did not affect gland morphogenesis but did influence gland number, gland size and/or cell size within glands. As PTEN may be regulated by the nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ), cultures were treated with the PPARγ ligand rosiglitazone. This treatment enhanced PTEN expression, cdc42 activation and rescued dysmorphogenesis by restoring single lumen formation in Caco-2 ShPTEN glands. Rosiglitazone effects on cdc42 activation and Caco-2 ShPTEN gland development were attenuated by cotreatment with GW9662, a PPARγ antagonist. Taken together, these studies show PTEN-cdc42 regulation of lumen formation in a 3D model of human CRC glandular morphogenesis. Treatment by the PPARγ ligand rosiglitazone, but not PI3K modulators, rescued colorectal glandular dysmorphogenesis of PTEN deficiency.


Asunto(s)
Anilidas/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , PPAR gamma/antagonistas & inhibidores , Fosfohidrolasa PTEN/deficiencia , Tiazolidinedionas/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Células CACO-2 , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/fisiología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Células HCT116 , Humanos , Ligandos , Células de Riñón Canino Madin Darby , Terapia Molecular Dirigida , PPAR gamma/genética , PPAR gamma/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Rosiglitazona , Transducción de Señal , Transfección , Proteína de Unión al GTP cdc42/metabolismo
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