Viruses in cancer cell plasticity: the role of hepatitis C virus in hepatocellular carcinoma

International audienc

Detection and quantification of apoptosis in transiently transfected adherent cells.

Apoptosis is a highly conserved form of cell death present in all eukaryotic cell types and controlled by multiple genes. Several methods have been developed to quantify apoptosis, but none is adapted for all cell types. It is particularly difficult to reliably assay apoptosis of adherent cells. We describe a new, rapid and reliable flow cytometric method which can be used for quantifiying apoptosis in a sub-population of transiently transfected adherent cells. This technique is based on the detection of transfected cells and the apoptotic sub-population by immunofluorescence and Annexin-V labelling, respectively

Anti-apoptotic activity of low levels of wild-type p53.

Induction of apoptosis is a function of both an external stimulus and the physiology of the cell, which includes the expression of multiple oncogenes and tumor suppressors. Here we have studied the apoptotic response of immortalized mouse fibroblasts to serum withdrawal. We show that, in addition to the p53-independent apoptosis observed in p53- cells, overexpression of wild-type p53 tumor suppressor results in a high rate of programmed cell death. However, physiological range, low levels of the p53 protein protect fibroblasts from induction of apoptosis. Our results indicate that, as a function of its dose, the wild-type p53 can either protect from death or promote apoptosis. This new, anti-apoptotic, activity of p53 may have implications for the understanding of the role played by p53 in embryonic development as well as in initial stages of oncogenesis

Downregulation of Akt and FAK phosphorylation reduces invasion of glioblastoma cells by impairment of MT1-MMP shuttling to lamellipodia and downregulates MMPs expression

AbstractHuman malignant glioblastomas are highly invasive tumors. Increased cell motility and degradation of the surrounding extracellular matrix are essential for tumor invasion. PI3K/Akt signaling pathway emerges as a common pathway regulating cellular proliferation, migration and invasion; however, its contribution to particular process and downstream cascades remain poorly defined. We have previously demonstrated that Cyclosporin A (CsA) affects glioblastoma invasion in organotypic brain slices and tumorigenicity in mice. Here we show that CsA impairs migration and invasion of human glioblastoma cells by downregulation of Akt phosphorylation. Interference with PI-3K/Akt signaling was crucial for CsA effect on invasion, because overexpression of constitutively active myr-Akt antagonized drug action. Furthermore, the drug was not effective in T98G glioblastoma cells with constitutively high level of phosphorylated Akt. CsA, comparably to pharmacological inhibitors of PI3K/Akt signaling (LY294002, A443654), reduced motility of glioblastoma cells, diminished MMP-2 gelatinolytic activity and MMP-2 and MT1-MMP expression. The latter effect was mimicked by overexpression of dominant negative Akt mutants. We demonstrate that CsA and LY294002 reduced MMP transcription partly via modulation of IκB phosphorylation and NFκB transcriptional activity. Those effects were not mediated by inhibition of calcineurin, a classical CsA target. Additionally, CsA reduced phosphorylation and activity of focal adhesion kinase that was associated with rapid morphological alterations, rearrangement of lamellipodia and impairment of MT1-MMP translocation to membrane protrusions. Our results document novel, Akt-dependent mechanisms of interference with motility/invasion of human glioblastoma cells: through a rapid modulation of cell adhesion and MT1-MMP translocation to membrane protrusions and delayed, partly NFκB-dependent, downregulation of MMP-2 and MT1-MMP expression