RESUMEN
Interferon-gamma, a known inhibitor of tumor cell growth, has been used in several protocols for the treatment of melanoma. We have studied the molecular events underlying interferon-gamma-induced G0/G1 arrest in four metastatic melanoma cell lines with different responsiveness to interferon-gamma. The growth arrest did not result from enhanced expression of cyclin-dependent kinase inhibitors p21 and p27. Instead, it correlated with downregulation of cyclin E and cyclin A and inhibition of their associated kinase activities. We show that interferon-gamma-induced growth inhibition could be abrogated by overexpression of dominant negative STAT1 (signal transducer and activator of transcription 1) in the melanoma cell line A375, suggesting that STAT1 plays a crucial part for the anti-proliferative effect. Erythropoietin stimulation of a chimeric receptor led to a concentration-dependent STAT1 activation and concomitant growth arrest when it contained the STAT recruitment motif Y440 of the interferon-gamma receptor 1. In contrast, dose-response studies for interferon-gamma revealed a discrepancy between levels of STAT1 activation and the extent of growth inhibition; whereas STAT1 was activated by low doses of interferon-gamma (10 U per mL), growth inhibitory effects were only visible with 100-fold higher concentrations. Our results suggest the presence of additional signals emanating from the interferon-gamma receptor, which may counteract the anti-proliferative function of STAT1.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Unión al ADN/metabolismo , Interferón gamma/farmacología , Melanoma , Neoplasias Cutáneas , Transactivadores/metabolismo , Línea Celular Tumoral/citología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Antineoplásicos , Fase G1/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Factor de Transcripción STAT1 , Transducción de Señal/efectos de los fármacos , Transactivadores/genéticaRESUMEN
Dysregulated cytokine production contributes to inflammatory and proliferative diseases. Therefore, inhibition of proinflammatory mediators such as TNF, IL-1, and IL-6 is of great clinical relevance. Actual strategies are aimed at preventing receptor activation through sequestration of the ligand. Here we describe the development of an inhibitor of murine IL-6 based on fused receptor fragments. Molecular modeling-guided analysis of the murine IL-6Ralpha revealed that mutations in the Ig-like domain D1 severely affect protein function, although D1 is not directly involved in the ligand-binding interface. The resulting single chain IL-6 inhibitor (mIL-6-RFP) consisting of domains D1-D3 of mgp130, a flexible linker, and domains D1-D3 of mIL-6Ralpha is a highly potent and specific IL-6 inhibitor. mIL-6-RFP will permit further characterization of the role of IL-6 in various disease models and could ultimately lead to anti-IL-6 therapy.