GNE Is Involved in the Early Development of Skeletal and Cardiac Muscle

UDP-N-acetylglucosamine 2 epimerase/N-acetylmannosamime kinase (GNE) is a bifunctional enzyme which catalyzes the two key sequential steps in the biosynthetic pathway of sialic acid, the most abundant terminal monosaccharide on glycoconjugates of eukaryotic cells. GNE knock out (GNE KO) mice are embryonically lethal at day E8.5. Although the role of GNE in the sialic pathway has been well established as well as the importance of sialylation in many diverse biological pathways, less is known about the involvement of GNE in muscle development. To address this issue we have studied the role of GNE during in vitro embryogenesis by comparing the developmental profile in culture of embryonic stem cells (ES) from wild type and from GNE KO E3.5 mice embryos, during 45 days. Neuronal cells appeared rarely in GNE KO ES cultures and did not reach an advanced differentiated stage. Although primary cardiac cells appeared at the same time in both normal and GNE KO ES cultures, GNE KO cardiac cells degraded very soon and their beating capacity decayed rapidly. Furthermore very rare skeletal muscle committed cells were detected in the GNE KO ES cultures at any stage of differentiation, as assessed by analysis of the expression of either Pax7, MyoD and MyHC markers. Beyond the supporting evidence that GNE plays an important role in neuronal cell and brain development, these results show that GNE is strongly involved in cardiac tissue and skeletal muscle early survival and organization. These findings could open new avenues in the understanding of muscle function mechanisms in health and in disease

TNFα Cooperates with IFN-γ to Repress Bcl-xL Expression to Sensitize Metastatic Colon Carcinoma Cells to TRAIL-mediated Apoptosis

BACKGROUND: TNF-related apoptosis-inducing ligand (TRAIL) is an immune effector molecule that functions as a selective anti-tumor agent. However, tumor cells, especially metastatic tumor cells often exhibit a TRAIL-resistant phenotype, which is currently a major impediment in TRAIL therapy. The aim of this study is to investigate the synergistic effect of TNFα and IFN-γ in sensitizing metastatic colon carcinoma cells to TRAIL-mediated apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: The efficacy and underlying molecular mechanism of cooperation between TNFα and IFN-γ in sensitizing metastatic colon carcinoma cells to TRAIL-mediated apoptosis were examined. The functional significance of TNFα- and IFN-γ-producing T lymphocyte immunotherapy in combination with TRAIL therapy in suppression of colon carcinoma metastasis was determined in an experimental metastasis mouse model. We observed that TNFα or IFN-γ alone exhibits minimal sensitization effects, but effectively sensitized metastatic colon carcinoma cells to TRAIL-induced apoptosis when used in combination. TNFα and IFN-γ cooperate to repress Bcl-xL expression, whereas TNFα represses Survivin expression in the metastatic colon carcinoma cells. Silencing Bcl-xL expression significantly increased the metastatic colon carcinoma cell sensitivity to TRAIL-induced apoptosis. Conversely, overexpression of Bcl-xL significantly decreased the tumor cell sensitivity to TRAIL-induced apoptosis. Furthermore, TNFα and IFN-γ also synergistically enhanced TRAIL-induced caspase-8 activation. TNFα and IFN-γ was up-regulated in activated primary and tumor-specific T cells. TRAIL was expressed in tumor-infiltrating immune cells in vivo, and in tumor-specific cytotoxic T lymphocytes (CTL) ex vivo. Consequently, TRAIL therapy in combination with TNFα/IFN-γ-producing CTL adoptive transfer immunotherapy effectively suppressed colon carcinoma metastasis in vivo. CONCLUSIONS/SIGNIFICANCE: TNFα and IFN-γ cooperate to overcome TRAIL resistance at least partially through enhancing caspase 8 activation and repressing Bcl-xL expression. Combined CTL immunotherapy and TRAIL therapy hold great promise for further development for the treatment of metastatic colorectal cancer