Relationship between IκBα constitutive expression, TNFα synthesis, and apoptosis in EBV-infected lymphoblastoid cells

Abstract

In order to understand the role of NF-kB in EBV transformation we have established stably transfected IkBa into lymphoblastoid cells. Two clones were obtained in which the loss of NF-kB binding activity correlated with the constitutive expression of the transgenic IkBa. Protein latency expression was determined by immunocytochemistry. Expression of surface markers, intracytoplasmic content of cytokines cell cycle analysis after BrdU incorporation and DNA staining with propidium iodide were studied by¯ow cytometry. Percentage of apoptotic cells was determined by in-situ labelling of DNA strand breaks. No signi®cative changes in EBV latency nor in cell surface marker expression was found. In contrast, intracytoplasmic TNFa levels were strongly reduced in transfected clones. Furthermore, 30% of IkBa transfected cells were apoptotic after 8 h of TNFa treatment. This correlated with a strong reduction of BrdU incorporation after 24 h of TNFa treatment. No e ect was seen with non transfected cells or with cells transfected with a control plasmid. Our results suggest that the TNFa gene could be one of the targets of NF-kB in EBV infected cells and that NF-kB protects EBV-infected cells from apoptosis induced by TNFa, which may favour the proliferative e ect of this cytokine.

Nuclear Factor-κB Activation: A Question of Life or Death

“…A large number of reports have demonstrated the anti-apoptotic effect of NF-κB in a wide variety of cell types. The protective role of NF-κB is shown in a large variety of cell types, including the human breast carcinoma (Liu et al, 1996) , T cells (Van Antwerp et al, 1996;Chu et al, 1997;Khoshnan et al, 2000), fibroblasts and macrophages (Beg and Baltimore, 1996), endothelial cells (Stehlik et al, 1998), EBV-infected lymphoblastoid cells (Asso-bonnet et al, 1998), non-small lung cancer cells , glomerular mesangial cells (Sugiyama et al, 1999), human ovarian cancer cells (Shao et al, 1997), human pancreatic cancer cell lines (Kajino et al, 2000), Ewing sarcoma cells (Javelaud and Besancon, 2001), cardiomyocytes (Bergmann et al, 2001), mouse embryos , and HT1080 fibrosarcoma . Treatment of RelA-deficient (the transcriptionally active subunit of NF-κB) mouse fibroblasts and macrophages with TNF significantly reduced cell viability, whereas RelA +/+ cells were unaffected.…”

Gene Array Identification of Epstein Barr Virus-Regulated Cellular Genes in EBV-Converted Burkitt Lymphoma Cell Lines

“…EBV infection of B-cells induces TNF␣ secretion (Williamson et al, 1983) and TNF␣ gene is a NF-〉 target gene Shakhov et al, 1990). Loss of NF-〉 in LCLs may be associated with a decrease of TNF synthesis and a synergy between LMP1 and TNF␣ has been suggested regarding both NF-〉 activation and B-cell proliferation (Asso Bonnet et al, 1998). …”

“…Doubling time was measured during the exponential growth phase. Cell cycle analysis and detection of apoptosis was performed by flow-cytometry as described (Asso Bonnet et al, 1998) on the basis of BrdU incorporation into DNA and staining with propidium iodide. Fluorescence analysis of chromatin condensation and fragmentation was performed after cytocentrifugation of 50,000 cells and DNA staining with the Hoechst 33258 diluted at 20 g/ml in PBS.…”

The contribution of NF-κB activity to spontaneous proliferation and resistance to apoptosis in human T-cell leukemia virus type 1 Tax-induced tumors

“…For instance, NF-B activity was shown to protect Epstein-Barr virus (EBV)-infected cells from apoptosis induced by TNF-␣, contributing to increased proliferation of B lymphoblastoid cells. 17 TNF-␣ coactivates the NF-B pathway, which is then able to limit apoptosis, presumably through up-regulation of anti-apoptotic gene transcription. 18 However, in the absence of new protein synthesis, TNF-␣-induced apoptosis proceeds through caspase activation.…”