Signal transducer and activator of transcription 1 (STAT1), a transcription factor known to participate in antiviral responses, acts as a tumor suppressor inhibiting cell growth and promoting apoptosis. To study the role of STAT1 in DNA damage-induced apoptosis in B lymphocytes, its active form, STAT1alpha, was specifically inhibited by the overexpression of STAT1beta, the STAT1alpha truncated inhibitory isoform. An episomal vector with a tetracycline-inducible bidirectional promoter was created to induce the expression of 2 proteins, STAT1beta and enhanced green fluorescence protein (EGFP). The same vector was used to overexpress STAT1alpha as a control. Expression of STAT1beta inhibited the phosphorylation, the DNA-binding activity, and the transcriptional activity of STAT1alpha, as well as the expression of STAT1alpha target genes such as p21WAF1/CIP1, TAP1, IRF1, and PKR. Inhibiting STAT1alpha by STAT1beta increased the growth rate of transfected cells and their resistance to fludarabine-induced apoptosis and cell cycle arrest. Overexpressing STAT1beta reversed the negative regulation of Mdm2 expression observed after treatment with interferon-gamma (IFN-gamma), which activates STAT1, or with fludarabine. Nuclear translocation of p53 after fludarabine treatment was decreased when STAT1beta was overexpressed, and it was increased when STAT1alpha was induced. Oligonucleotide pull-down experiments showed a physical STAT1/p53 interaction. Our results show that imbalance between the antiproliferative/proapoptotic isoform STAT1alpha and the proliferative isoform STAT1beta is likely to play a crucial role in the regulation of proliferation and apoptosis and that STAT1alpha may regulate p53 activity and sensitize B cells to fludarabine-induced apoptosis.
Epstein-Barr virus (EBV) induces IntroductionEpstein-Barr virus (EBV) was first reported to be associated with Burkitt lymphoma (BL). EBV is able to immortalize B cells both in vitro and in vivo. EBV is also the etiologic agent of infectious mononucleosis, a benign viral disease of young adults that regresses spontaneously. In most cases, primary EBV infection is asymptomatic. Epidemiologic and serologic studies showed that EBV is widely distributed in humans around the world and that more than 95% of adults have been infected by EBV in their past. The ability to grow EBV-infected B-cell lines from peripheral blood mononuclear cells (PBMCs) of EBV-seroconverted healthy subjects leads to the conclusion that this virus persists as a clinically silent infection for the lifetime of the infected host. 1 Nevertheless, it is of note that the virus remains continuously active in immunocompetent individuals even though at very low levels. 2 The EBV genome consists of a 180-kb double-stranded DNA that remains stable in the cell nucleus in an episomal form. In vitro immortalization of B cells by EBV is due to the expression of a restricted set of viral genes, the so-called latency III program, coding for latent membrane proteins (LMP1, LMP2a, LMP2b), Epstein-Barr nuclear antigens (EBNA1, 2, 3A, 3B, 3C, LP), and small noncoding RNAs (EBERs), and leads to the establishment of lymphoblastoid cell lines (LCLs). 3 Among these genes, EBNA1 is responsible for episomal maintenance of the EBV genome. 4 EBNA2 alone is responsible for the expression of the entire set of genes of the latency III program. 5 EBNA2 reroutes the Notch pathway by targeting RBP-jkappa. 6 EBNA2 is also the main viral transactivator of the BNLF1 gene coding for LMP1. LMP1 mimics a constitutively activated receptor of the TNF receptor family through aggregation of TRAF/TRADD molecules. 7,8 In vivo, the latency III program is expressed in newly infected B-blasts during primary EBV infection. 9 Most of these newly EBV-infected B-blasts are killed by cytotoxic T cells. However, some EBVinfected B-blasts are considered to follow the normal process of B-cell maturation until the B-cell memory stage. 10 The B-cell memory compartment constitutes the EBV reservoir in humans. EBV is almost silent in its reservoir. [10][11][12] The immune response against EBV is associated with a dramatic oligoclonal increase in EBV-specific CD8 cytotoxic T cells during the initial phase of primary EBV infection. [13][14][15] Cytotoxic T cells may prevent in vitro de novo infection of B cells. 16,17 In Purtilo syndrome, the defect of SH2D1A/SAP gene alters T-cell signaling in response to antigen. 18 The SAP signaling For personal use only. on May 10, 2018. by guest www.bloodjournal.org From pathway plays a key role in the cytolytic activity of T cells against EBV-positive targets, 19 and Purtilo syndrome is characterized by fatal infectious mononucleosis after primo-infection. 18 Emergence of EBV-associated lymphoma in patients with acquired T-cell immunodeficiencies such as HIV infecti...
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 a and c (IFNa and c). 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.
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