Differential roles of STAT1  and STAT1  in fludarabine-induced cell cycle arrest and apoptosis in human B cells

Abstract

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.

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

“…The results suggest a model according which p53-dependent STAT1 activation depends on p53 DNA-binding domain. The fact that constitutive engagement of p53 DNA-binding domain in p53 tetramerization prevents STAT1 activation and that both p53 and STAT1 interact physically with each other (Townsend et al, 3 Baran-Marszak et al 4 and our results) strongly suggests that STAT1 interacts directly with the DNA-binding domain of p53.…”

“…We previously showed that inhibition of STAT1 activation blocks p53-dependent cell cycle arrest by fludarabine*, 4 and Townsend et al 3,16 showed that STAT1 and p53 cooperate together in induction of apoptosis. These results led us to raise the question of STAT1 activation by genotoxic agents.…”

“…Since STAT1 and p53 may interact with each other, 3,4 we wanted to assess the role of p53 protein in STAT1 activation by genotoxic agents. Figure 3a shows that Dx did not induce Y701-STAT1 phosphorylation in HL60 or in Jurkat cells, two p53-null cell lines.…”

“…In addition to its transcriptional activity, p53 interacts directly or indirectly with other transcription factors such as c-myc, the nuclear factor kappa B (NF-kB) subunit RelA 2 or STAT1 (signal transducer and activator of transcription 1). 3,4 It is noteworthy that both STAT1 and p53 share various target genes involved in cell cycle arrest or in apoptosis such as p21 5 and CD95. 6 The cytotoxic activity of p53 inducers such as fludarabine* is increased by STAT1 transcriptional activation.…”

“…6 The cytotoxic activity of p53 inducers such as fludarabine* is increased by STAT1 transcriptional activation. 4 Physical interaction between STAT1 and p53 is associated with a negative regulation of MDM2 expression and with an increase in p53 activation. 3 STAT1 is essential for signalling by interferons (IFNs).…”