Constitutive activation of a slowly migrating isoform of Stat3 in mycosis fungoides: Tyrphostin AG490 inhibits Stat3 activation and growth of mycosis fungoides tumor cell lines

Abstract

Mycosis fungoides (MF) is a low-grade cutaneous T cell lymphoma of unknown etiology. In this report, the Jak͞Stat (Janus kinase͞signal transducer and activator of transcription) signaling pathway was investigated in tumor cell lines established from skin biopsy specimens from a patient with MF. Jaks link cytokine receptors to Stats, and abnormal Jak͞Stat signaling has been observed in some hemopoietic cancers. In MF tumor cells, a slowly migrating isoform of Stat3, Stat3 sm , was found to be constitutively activated, i.e., (i) Stat3 sm was constitutively phosphorylated on tyrosine residues, and tyrosine phosphorylation was not enhanced by growth factor stimulation; (ii) band shift assays and immunoprecipitations of DNA͞Stat complexes showed constitutive DNA-binding properties of Stat3 sm ; and (iii) Stat3 sm was constitutively associated with Jak3. The abnormal activation of Stat3 sm was highly specific. Thus, neither the fast migrating isoform of Stat3 (Stat3 fm ) nor other Stats (Stat1, Stat2, and Stat4 through Stat6) were constitutively activated. The Jak kinase inhibitor, tyrphostin AG490, blocked the constitutive activation of Stat3 sm and inhibited spontaneous as well as interleukin 2-induced growth of MF tumor cells. In conclusion, we have provided evidence for an abnormal Jak͞ Stat signaling and growth regulation in tumor cells obtained from affected skin of an MF patient.

Characterization of the antiviral effects of interferon-α against a SARS-like coronoavirus infection in vitro

“…When L2 cells were treated with the Jak 1 inhibitor prior to IFN treatment, then infected with MHV-1, we observed a reduction in IFN-inducible protection from MHV-1 infection ( Figure 1B). As a negative control, we examined the effect of the inhibitor AG490, which specifically inhibits Jak2 at 100 µM [45]. As anticipated, treating cells with 100 µM AG490 did not influence the anti-MHV-1 activity of IFN-α4 ( Figure 1B).…”

“…Immunoprecipitations and immunoblotting using an ECL (enhanced chemiluminescence) method were performed as described previously [45]. In experiments in which pharmacological inhibitors were used, the cells were pre-treated for 60 min with indicated concentrations of the inhibitors and subsequently treated with IFN-α4 prior to lysis in phosphorylation lysis buffer.…”

Transcriptional regulatory effects of lymphoma-associated NFKB2/lyt10 protooncogenes

“…The HUT78 cell line also contains alterations in c-myc (Finger et al, 1988), c-cbl (Blake andLangdon, 1992), and p53 (Tolomeo et al, 1998). Furthermore, alterations in JAK/STAT signaling have also been strongly implicated in the pathogenesis of human CTCL (Nielsen et al, 1997;Zhang et al, 1996). Loss of the IkB-like activity of p100 NF-kB-2 could lead to enhanced anti-apoptotic activity in these cells as a result of constitutive NF-kB activity (Beg and Baltimore, 1996;Liu et al, 1996;Sonenshein, 1997;Van Antwerp et al, 1996;Wang et al, 1996).…”

Cutaneous T‐cell lymphoma: 2011 update on diagnosis, risk‐stratification, and management

“…Nuclear localization and DNA-binding of phosphorylated STAT3 has been convincingly demonstrated in CTCL [77,78]. Following nuclear translocation, STAT3 directly regulates a number of target genes in CTCL, including regulators of apoptosis (e.g., Bcl-2/Bax), cytokines (e.g., IL-5, IL-13) and suppressors of cytokine signaling (e.g., SOCS).…”

“…In addition, STAT3 indirectly regulates gene expression by inducing the expression of DNA methyltransferase 1 (DNMT1), which promotes the epigenetic silencing of tumor suppressor genes [79]. Not surprisingly then, pharmacologic inhibition of STAT3 promotes apoptosis in CTCL [77,[80][81][82]. Cytogenetic gains involving STAT5A and STAT5B or their activation in response to cytokines present within the tumor microenvironment suggests a pathogenic role for other STATs [83][84][85].…”