RESUMEN
Bladder cancer is the fifth most common malignancy in the UK. Clinically, the most important process in determining prognosis is the development of invasion, initially of the lamina propria and then beyond as these transitional cell carcinomas (TCCs) progress from stage pT1 to stages T2+. Cadherins and catenins are the main mediators of cell-cell interactions in epithelial tissues, and loss of membranous E-cadherin immunoreactivity is strongly correlated with high grade, advanced stage and poor prognosis in bladder cancer and other malignancies. However, the role of P-cadherin is yet to be fully elucidated in bladder TCC. The objectives of this study were to establish how the expression of cadherins and catenins determines clinical and in vitro behaviour in bladder TCC. Utilizing immunohistochemistry, immunofluorescence and western blotting, we demonstrated a significant reduction in the expression of E-cadherin and beta-catenin as grade and stage of bladder TCC progress, accompanied by a significant increase in P-cadherin expression (all p < 0.05, Pearson's chi2 test). Increased P-cadherin expression was also associated with a significantly worse bladder cancer-specific survival (log rank p = 0.008), with Cox regression showing P-cadherin to be an independent prognostic factor. Utilizing a variety of tissue culture models in a range of functional studies, we demonstrated that P-cadherin mediates defective cell-cell adhesion and enhances anchorage-independent growth. The results provide evidence that increased P-cadherin expression promotes a more malignant and invasive phenotype of bladder cancer, and appears to have a novel role late in the disease.
Asunto(s)
Biomarcadores de Tumor/análisis , Cadherinas/metabolismo , Carcinoma de Células Transicionales/metabolismo , Cateninas/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Western Blotting/métodos , Cadherinas/análisis , Carcinoma de Células Transicionales/química , Carcinoma de Células Transicionales/patología , Cateninas/análisis , Humanos , Inmunohistoquímica , Pronóstico , Modelos de Riesgos Proporcionales , Análisis de Supervivencia , Neoplasias de la Vejiga Urinaria/química , Neoplasias de la Vejiga Urinaria/patología , beta Catenina/análisisRESUMEN
Lysates from the Jurkat T lymphocyte cell line were immunoblotted with anti-Pyk2, and two major forms of Pyk2 were identified. When lysates from the p56(Lck) negative (J.CaM1/Rep3) and CD45 negative Jurkat cell line derivatives were immunoblotted with anti-Pyk2, only the lower mobility form of Pyk2 was predominant. Transfection of J.CaM1 cells with p56(Lck) restored expression of the multiple forms of Pyk2. Using RT-PCR, we found that both species of the alternatively spliced mRNA for Pyk2 were present in all of the lines regardless of their ability to express CD45 or p56(Lck) protein. When p56(Lck) immunoprecipitates were immunoblotted with anti-Pyk2, only the higher mobility form of Pyk2 immunoprecipitated with p56(Lck). These data demonstrate that certain members of the Src family of kinases interact preferentially with the different isoforms of Pyk2 and may have a role in the regulation of the Pyk2 protein in lymphocytes.
Asunto(s)
Isoenzimas/metabolismo , Células Jurkat/enzimología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Quinasa 2 de Adhesión Focal , Humanos , Immunoblotting/métodos , Técnicas In Vitro , Isoenzimas/genética , Antígenos Comunes de Leucocito/inmunología , Antígenos Comunes de Leucocito/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Proteínas Tirosina Quinasas/genéticaRESUMEN
We previously demonstrated that stimulation of human T-lymphocytes with calcium ionophores induced the phosphorylation and enzymatic activation of ERK2. We now report on the mechanism by which calcium-ionophore-induced activation of ERK1 and 2 occurs in these cells. The activation of ERK1 and 2 by increases in intracellular calcium was inhibited by calmidazolium suggesting the involvement of calmodulin in this response. To further elucidate the mechanism by which calcium-induced ERK activation occurs, we used the CaM-kinase inhibitor KN-93 and an inactive analog of KN-93 (KN-92). KN-93, but not KN-92, blocked ionomycin-induced activation of ERK1 and 2 in human T lymphocytes. We previously demonstrated that stimulation of T lymphocytes with ionomycin or A23187 resulted in a CaM-kinase-dependent shift in the mobility of p56(Lck). To determine if p56(Lck) was involved in calcium-induced ERK activation, we stimulated the p56(Lck) negative Jurkat cell derivatives, J.CaM1.6 and J.CaM1/Rep3, with ionomycin. In these p56(Lck) negative cell lines, activation of ERK1 and 2 in response to ionomycin was only minimally detected. When J.CaM1 cells were reconstituted with p56(Lck), ionomycin induced ERK1 and 2 activation. Treatment of Jurkat cells with PP2, an inhibitor of p56(Lck), inhibited calcium-induced, but not PMA-induced, ERK1 and 2 activation. Treatment of Jurkat cells with the MEK inhibitor PD98059 blocked ionomycin-induced ERK activation, but not the shift in the mobility of p56(Lck). Our data suggests that increases in intracellular calcium induce the activation of ERK1 and 2 in human T lymphocytes via sequential activation of CaM-kinase and phosphorylation of p56(Lck).
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Calcio/fisiología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/fisiología , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Linfocitos T/enzimología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina , Línea Celular , Activación Enzimática , Humanos , Proteína Quinasa 3 Activada por MitógenosRESUMEN
We have previously shown that stimulation of B lymphocytes with calcium ionophores lead to the phosphorylation and enzymatic activation of ERK2. We have now determined that stimulation of human primary and Jurkat T lymphocytes with ionomycin also results in the activation of ERK1 and 2 as determined by; shifts in the mobility of this enzyme on SDS PAGE gels, the binding of an antibody that recognizes only the activated form of this enzyme, and increased ability to phosphorylate myelin basic protein (MBP). Another calcium ionophore, A23187, also induced activation of ERK1 and 2 in human primary and Jurkat T lymphocytes demonstrating that this is a general effect of calcium ionophores and is not limited to ionomycin. The activation of ERK1 and 2 by calcium ionophores was rapid, transient, and occurred in a dose-dependent manner. Activation of ERK1 and 2 by increases in intracellular calcium were blocked by the MEK inhibitor PD98059. These data point to a new role for calcium fluxes in T lymphocytes.