Ibtissam Youlyouz-Marfak

PK11195 potently sensitizes to apoptosis induction independently from the peripheral benzodiazepin receptor

1-(2-Chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195) is a prototypic ligand of the peripheral benzodiazepine receptor (PBR), a mitochondrial outer membrane protein. PK11195 can be used to chemosensitize tumor cells to a variety of chemotherapeutic agents, both in vitro and in vivo. PK11195 has been suggested to exert this effect via inhibition of the multiple drug resistance (MDR) pump and by direct mitochondrial effects which could be mediated by the PBR. Here, we established a model system in which PK11195 and another PBR ligand, 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro5-4864), sensitize to nutrient depletion-induced cell death. In this MDR-independent model, PK11195 and Ro5-4864 are fully active even when the PBR is knocked down by small interfering RNA. Cells that lack PBR possess low-affinity binding sites for PK11195 and Ro5-4864. The starvation-sensitizing effects of PK11195 are not due to a modulation of the adaptive response of starved cells, namely autophagy and NF-κB activation. Rather, it appears that the combination of PK11195 with autophagy or NF-κB inhibitors has a potent synergistic death-inducing effect. Starved cells treated with PK11195 exhibit characteristics of apoptosis, including loss of the mitochondrial transmembrane potential, mitochondrial cytochrome c release, caspase activation and chromatin condensation. Accordingly, stabilization of mitochondria by overexpression of Bcl-2 or expression of the viral mitochondrial inhibitor (vMIA) from cytomegalovirus inhibits cell death induced by PK11195 plus starvation. Thus, PK11195 potently sensitizes to apoptosis via a pathway that involves mitochondria, yet does not involve the PBR.

Latent Membrane Protein 1 Regulates STAT1 through NF-κB-Dependent Interferon Secretion in Epstein-Barr Virus-Immortalized B Cells

ABSTRACT Constitutive activation of signal transducer and activator of transcription 1 (STAT1) is a distinctive feature of Epstein-Barr virus (EBV)-immortalized B cells (lymphoblastoid cell lines [LCLs]). The expression of STAT1 in these cells is modulated by the latent membrane protein 1 (LMP1), but the mechanism of STAT1 activation has remained unclear. We demonstrate that the tyrosine phosphorylation of STAT1 in LCLs results from an indirect pathway encompassing an NF-κB-dependent secretion of interferons (IFNs). The cell culture supernatant of LCLs induced tyrosine phosphorylation of STAT1 in cells with no constitutively activated STAT1. Moreover, removal of supernatant from LCLs was sufficient to decrease the phosphorylation of STAT1. Inhibition of NF-κB activity by different pharmacological inhibitors (i.e., parthenolide, MG132 and BAY 11-7082) and by overexpressed mutated IκBα prevented the activation of STAT1. To identify the factors involved, we performed macroarray cDNA profiling with or without inhibition of NF-κB. The expression of several cytokines was NF-κB dependent among those alpha and gamma IFNs (IFN-α and IFN-γ), known activators of STAT1. By real-time PCR and enzyme-linked immunosorbent assay we show that IFN-α and IFN-γ are expressed and released by LCLs in an NF-κB-dependent manner. Finally, the blocking of the IFN-α and IFN-γ by neutralizing antibodies led to the complete inhibition of tyrosine phosphorylation of STAT1. Taken together, our results clearly show that LMP1-induced tyrosine phosphorylation of STAT1 is almost exclusively due to the NF-κB-dependent secretion of IFNs. Whether this response, which is usually considered to be antiviral, is in fact required for the persistence of the virus remains to be elucidated.

Molecular Basis of Cytotoxicity of Epstein-Barr Virus (EBV) Latent Membrane Protein 1 (LMP1) in EBV Latency III B Cells: LMP1 Induces Type II Ligand-Independent Autoactivation of CD95/Fas with Caspase 8-Mediated Apoptosis

ABSTRACT The Epstein-Barr virus (EBV) oncoprotein latent membrane protein 1 (LMP1) is thought to act as the major transforming protein in various cell types, by rerouting the tumor necrosis factor receptor family signaling pathway. Despite this implication in EBV-associated transformation of cells, LMP1 toxicity is a well-known but poorly studied feature, perhaps because it contradicts its role in transformation. We show that LMP1 physiological levels are very heterogeneous and that the highest levels of LMP1 correlate with Fas overexpression and spontaneous apoptosis in lymphoblastoid cell lines (LCLs). To understand the cytotoxic effect of LMP1 in LCLs, we cloned wild-type LMP1 into a doxycycline double-inducible episomal vector pRT-1, with a truncated version of NGFR as a surrogate marker of inducibility. We found that LMP1 overexpression induced apoptosis in LCL B cells, as shown by annexin V labeling, sub-G1 peak, and poly(ADP ribose) polymerase cleavage. Knocking down Fas expression by small interfering RNA abolished LMP1-induced apoptosis. The absence of detectable levels of Fas ligand mRNA suggested a ligand-independent activation of Fas. LMP1 induced Fas overexpression with its relocalization in lipid raft microdomains of the membrane. Fas immunoprecipitation detected FADD (Fas-associated death domain protein) and caspase 8, suggesting a Fas-dependent formation of the death-inducing signaling complex. Caspases 8, 9, 3, and 7 were activated by LMP1. Caspase 8 activation was associated with BID cleavage and truncated-BID mitochondrial relocalization, consistent with type II apoptosis. Therefore, our results are in agreement with a model where LMP1-dependent NF-κB activation induces Fas overexpression and autoactivation that could overwhelm the antiapoptotic effect of NF-κB, revealing an ambivalent function of LMP1 in cell survival and programmed cell death.

Identification of a novel p53-dependent activation pathway of STAT1 by antitumour genotoxic agents.

Chemotherapeutic drugs such as fludarabine*, doxorubicin or cisplatin are very potent activators of the anti-oncogene p53. Convergent studies suggest that p53 and STAT1 (signal transducer and activator of transcription 1) cooperate in the induction of cell death. We show that these drugs are also activators of STAT1 in p53-expressing cells, but not in p53-null cells. STAT1 activation was obtained in the presence of both the secretion inhibitor brefeldine A and the inhibitor of RNA synthesis, actinomycin D. p53-dependent STAT1 activation was reversed by overexpression of MDM2 and siRNAs against p53. Genetic analysis of p53 showed that expression of transcriptionally inactive p53 punctual mutants markedly increased Y701-STAT1 phosphorylation, and suggests that the p53 DNA-binding domain was alternatively involved in STAT1 activation or p53 multimerization. Immunoprecipitation experiments showed that ataxia telangiectasia mutated, p53, STAT1 and c-Abl1 (Abelson murine leukaemia viral oncogene homologue 1) were associated together. Treatment of cells with the c-Abl1 tyrosine kinase inhibitor STI571 decreased STAT1 activation by genotoxic drugs. Finally, genotoxic agents sensitized cells in response to very low doses of both interferon α and γ (IFNα and γ). These results show that genotoxic drugs induce STAT1 activation, an effect that depends on p53 protein but not on p53 transcriptional activity, and point to a novel pathway of STAT1 activation by genotoxic drugs, with involvement of c-Abl1 tyrosine kinase in sensitizing cells to IFN response.

B7-H1, Which Represses EBV-Immortalized B Cell Killing by Autologous T and NK Cells, Is Oppositely Regulated by c-Myc and EBV Latency III Program at Both mRNA and Secretory Lysosome Levels

EBV-immortalized B cells induce a complex immune response such that the virus persists as a clinically silent infection for the lifetime of the infected host. B7-H1, also called PD-L1, is a cosignaling molecule of the B7 family that can inhibit activated T cell effectors by interaction with its receptor PD-1. In this work, we have studied the dependence of B7-H1 on NF-κB and c-Myc, the two main transcription factors in EBV latency III proliferating B cells, on various lymphoblastoid and Burkitt lymphoma cell lines, some of them being inducible or not for the EBV latency III program and/or for c-Myc. We found that B7-H1 repressed killing of EBV-immortalized B cells by their autologous T and NK cells. At the mRNA level, NF-κB was a weak inducer whereas c-Myc was a strong repressor of B7-H1 expression, an effect mediated by STAT1 inhibition. At the protein level, B7-H1 molecules were stored in both degradative and unconventional secretory lysosomes. Surface membrane B7-H1 molecules were constitutively internalized and proteolyzed in lysosomes. The EBV latency III program increased the amounts of B7-H1–containing secretory lysosomes and their export to the surface membrane. By repressing actin polymerization, c-Myc blocked secretory lysosome migration and B7-H1 surface membrane export. In addition to B7-H1, various immunoregulatory molecules participating in the immunological synapse are stored in secretory lysosomes. By playing on actin polymerization, c-Myc could thus globally regulate the immunogenicity of transformed B cells, acting on export of secretory lysosomes to plasma membrane.

Novel function of STAT1β in B cells: induction of cell death by a mechanism different from that of STAT1α

Alternate splicing of STAT1 produces two isoforms: α, known as the active form, and β, previously shown to act as a dominant‐negative factor. Most studies have dealt with STAT1α, showing its involvement in cell growth control and cell death. To examine the specific function of either isoform in cell death, a naturally STAT1‐deficient human B cell line was transfected to express STAT1α or STAT1β. STAT1α, expressed alone, enhanced cell death, potentiated the fludarabine‐induced apoptosis, and enhanced the nuclear location, the phosphorylation, and the transcriptional activity of p53. Unexpectedly, STAT1β, expressed alone, induced cell death through a mechanism that was independent of the nuclear function of p53. Indeed, in STAT1β‐expressing B cells, p53 was stricktly cytoplasmic where it formed clusters, and there was no induction of the transcriptional activity of p53. These data reveal a novel role of STAT1β in programmed cell death, which is independent of p53.

T/B ratio does not reflect levels of ZAP70 expression in clonal CLL B-cells due to ZAP70 overexpression in patient T-cells†

Flow cytometry is the reference technique for assessing ZAP70 expression, a marker of poor prognosis in CLL. One of the most common methods is to assess ZAP70 levels in CLL cells by calculating the ratio between ZAP70 mean fluorescence intensities (MFIs) in residual T‐cells and CLL B‐cells (ZAP70 T/B ratio). In this study, we developed a new method for ZAP70 labeling. Cells were labeled with a combination of anti ZAP70 phycoerythrin‐conjugated SBZAP monoclonal antibody (mAb) and mAbs against CD45, CD19, and CD5. The latter three were used to specifically gate on different lymphocyte subsets. Staining was performed in absence (test) or in presence of excess unconjugated SBZAP mAb (isoclonic control). A so‐called ZAP70 isoclonic ratio between SBZAP MFIs in the test and isoclonic control was calculated. A series of 32 patients with CLL and 10 normal controls were studied. Prediction of IGHV mutation status by ZAP70 isoclonic and T/B ratios was similar. By using the ZAP70 isoclonic ratio, we showed that ZAP70 expression was increased in T‐cells from CLL patients. Nearly all cases with increased ZAP70 expression in CLL cells were associated with high ZAP70 expression in cognate T‐cells. Therefore, the ZAP70 isoclonic ratio was more likely to closely reflect the biology of ZAP70 dysregulation rather than the T/B ratio. These results also explained why ZAP70 T/B ratios were artefactually close to normal in cells from CLL patients with high levels of ZAP70.

RelA and RelB cross-talk and function in Epstein-Barr virus transformed B cells.

In this study, we determined the respective roles of RelA and RelB NF-κB subunits in Epstein–Barr virus (EBV)-transformed B cells. Using different EBV-immortalized B-cell models, we showed that only RelA activation increased both survival and cell growth. RelB activity was induced secondarily to RelA activation and repressed RelA DNA binding by trapping the p50 subunit. Reciprocally, RelA activation repressed RelB activity by increasing expression of its inhibitor p100. To search for such reciprocal inhibition at the transcriptional level, we studied gene expression profiles of our RelA and RelB regulatable cellular models. Ten RelA-induced genes and one RelB-regulated gene, ARNTL2, were repressed by RelB and RelA, respectively. Apart from this gene, RelB signature was included in that of RelA Functional groups of RelA-regulated genes were for control of energy metabolism, genetic instability, protection against apoptosis, cell cycle and immune response. Additional functions coregulated by RelA and/or RelB were autophagy and plasma cell differentiation. Altogether, these results demonstrate a cross-inhibition between RelA and RelB and suggest that, in fine, RelB was subordinated to RelA. In the view of future drug development, RelA appeared to be pivotal in both classical and alternative activation pathways, at least in EBV-transformed B cells.