Prem S. Subramaniam

A SOCS-1 peptide mimetic inhibits both constitutive and IL-6 induced activation of STAT3 in prostate cancer cells

Prostate cancer is the second highest cause of cancer-related deaths of men in the US. Signal transducers and activators of transcription (STATs) proteins are a small family of latent cytoplasmic transcription factors that act downstream of Janus kinase (JAK) activation and mediate intracellular signaling from a wide variety of cytokines, growth factors, and hormones. Aberrant activation of STAT3 has been implicated in the progression of many human carcinomas, including prostate cancer. Previously, we have characterized a novel tyrosine kinase inhibitor peptide, Tkip, that is a mimetic of suppressor of cytokine signaling 1 (SOCS-1). Similar to SOCS-1, Tkip binds to the autophosphorylation site of JAK2 and inhibits phosphorylation of STAT1α. In this study, we determined the inhibitory effects of Tkip on the human prostate cancer cell lines DU145 and LNCaP. Tkip inhibited cellular proliferation of both DU145 and LNCaP cells, with a slightly greater antiproliferative effect on DU145 cells. Cell cycle analysis using flow cytometry showed Tkip blockage of progression into the S phase of the cell cycle. Tkip also inhibited constitutive (DU145) and IL-6-induced (LNCaP) activation of STAT3, consistent with the fact that STAT3 activation is mediated by JAK2. Tkip also slightly reduced the levels of cyclin D1, an important regulator of cell cycle progression into S phase, in DU145 and LNCaP cancer cell lines. These data describe a potentially important therapeutic that targets both constitutive and IL-6-induced STAT3 activation in human prostate cancer cell lines.

IFNα induces the expression of the cyclin-dependent kinase inhibitor p21 in human prostate cancer cells

Prostate cancer, like other types of cancer, is associated with the loss of cell cycle control, resulting in unregulated growth of cells. We report here on the inhibitory effects of interferon α (IFNα) on the cell cycle of prostate cancer cells, using the human prostate carcinoma cell line DU145 that has mutations in the tumor suppressor genes pRB, p53 and KAI1. IFNα inhibited growth and colony formation of DU145 cells and analysis by flow cytometry suggests that IFNα inhibited the progression of these cancer cells from the G1 through S phase of the cell cycle. IFNα treatment of DU145 cells reduced cyclin dependent kinase 2 (cdk2) activity. In particular, cyclin E dependent cdk2 activity was inhibited by IFNα treatment. IFNα treatment, however, did not affect the amount of cdk2 bound to cyclin E. Consistent with this data, IFNα was able to induce expression of the kinase inhibitor p21 in DU145 cells. Furthermore, IFN treatment increased the amounts of p21 complexed with cdk2 in these cells. These data support a role for p21 in mediating the antiproliferative action of IFNα. The induction of p21 and its growth inhibitory effects in DU145 cells appears independent of p53, pRB and KAI1 status.

Characterization of a Peptide Inhibitor of Janus Kinase 2 That Mimics Suppressor of Cytokine Signaling 1 Function

Positive and negative regulation of cytokines such as IFN-γ are key to normal homeostatic function. Negative regulation of IFN-γ in cells occurs via proteins called suppressors of cytokine signaling (SOCS)1 and -3. SOCS-1 inhibits IFN-γ function by binding to the autophosphorylation site of the tyrosine kinase Janus kinase (JAK)2. We have developed a short 12-mer peptide, WLVFFVIFYFFR, that binds to the autophosphorylation site of JAK2, resulting in inhibition of its autophosphorylation as well as its phosphorylation of IFN-γ receptor subunit IFNGR-1. The JAK2 tyrosine kinase inhibitor peptide (Tkip) did not bind to or inhibit tyrosine autophosphorylation of vascular endothelial growth factor receptor or phosphorylation of a substrate peptide by the protooncogene tyrosine kinase c-src. Tkip also inhibited epidermal growth factor receptor autophosphorylation, consistent with the fact that epidermal growth factor receptor is regulated by SOCS-1 and SOCS-3, similar to JAK2. Although Tkip binds to unphosphorylated JAK2 autophosphorylation site peptide, it binds significantly better to tyrosine-1007 phosphorylated JAK2 autophosphorylation site peptide. SOCS-1 only recognizes the JAK2 site in its phosphorylated state. Thus, Tkip recognizes the JAK2 autophosphorylation site similar to SOCS-1, but not precisely the same way. Consistent with inhibition of JAK2, Tkip inhibited the ability of IFN-γ to induce an antiviral state as well as up-regulate MHC class I molecules on cells at a concentration of ∼10 μM. This is similar to the Kd of SOCS-3 for the erythropoietin receptor. These data represent a proof-of-concept demonstration of a peptide mimetic of SOCS-1 that regulates JAK2 tyrosine kinase function.

IFNγ inhibition of cell growth in glioblastomas correlates with increased levels of the cyclin dependent kinase inhibitor p21 WAF1/CIP1

Glioblastoma is a highly aggressive form of brain cancer characterized by uncontrolled cell growth resulting from a loss of cell cycle regulation. In this study we determined the antiproliferative effects of interferon gamma (IFNγ) on the glioblastoma cell lines T98G, SNB-19 and U-373, focusing on the ability of IFNγ to increase levels of p21WAF1/CIP1, an important negative regulator of cell cycle events. IFNγ was found to inhibit the growth of all cell lines, with inhibition ranging from 82.2% to 45.4%. Flow cytometry analysis showed that IFNγ treatment caused a cell cycle delay in the G1 or S phases. The strength of this delay varied, correlating with the degree by which IFNγ inhibited proliferation of each cell line. IFNγ treatment increased the production of the cyclin dependent kinase inhibitor (CKI) p21WAF1/CIP1 in all cell lines, the level and kinetics of production of which correlated with the degree and stage of inhibition of cellular proliferation. Further, immunoprecipitation of p21WAF1/CIP1 in complexes of p21WAF1/CIP1/cyclin-dependent kinase 2 (cdk2)/cyclin showed that the amount of p21WAF1/CIP1 in the complexes and the inhibition of cdk2-cyclin kinase activity correlated with the level of p21WAF1/CIP1 produced in the cells by IFNγ. These results show that IFNγ has significant antiproliferative effects on the glioblastoma cell lines and suggest that p21WAF1/CIP1 plays a role in mediating these effects.

The Carboxyl Terminus of Interferon-γ Contains a Functional Polybasic Nuclear Localization Sequence

Cytokines such as interferon-gamma (IFN-γ), which utilize the well studied JAK/STAT pathway for nuclear signal transduction, are themselves translocated to the nucleus. The exact mechanism for the nuclear import of IFN-γ or the functional role of the nuclear translocation of ligand in signal transduction is unknown. We show in this study that nuclear localization of IFN-γ is driven by a simple polybasic nuclear localization sequence (NLS) in its COOH terminus, as verified by its ability to specify nuclear import of a heterologous protein allophycocyanin (APC) in standard import assays in digitonin-permeabilized cells. Similar to other nuclear import signals, we show that a peptide representing amino acids 95–132 of IFN-γ (IFN-γ(95–132)) containing the polybasic sequence126RKRKRSR132 was capable of specifying nuclear uptake of the autofluorescent protein, APC, in an energy-dependent fashion that required both ATP and GTP. Nuclear import was abolished when the above polybasic sequence was deleted. Moreover, deletions immediately NH2-terminal of this sequence did not affect the nuclear import. Thus, the sequence126RKRKRSR132 is necessary and sufficient for nuclear localization. Furthermore, nuclear import was strongly blocked by competition with the cognate peptide IFN-γ(95–132) but not the peptide IFN-γ(95–125), which is deleted in the polybasic sequence, further confirming that the NLS properties were contained in this sequence. A peptide containing the prototypical polybasic NLS sequence of the SV40 large T-antigen was also able to inhibit the nuclear import mediated by IFN-γ(95–132). This observation suggests that the NLS in IFN-γ may function through the components of the Ran/importin pathway utilized by the SV40 T-NLS. Finally, we show that intact IFN-γ, when coupled to APC, was also able to mediate its nuclear import. Again, nuclear import was blocked by the peptide IFN-γ(95–132) and the SV40 T-NLS peptide, suggesting that intact IFN-γ was also transported into the nucleus through the Ran/importin pathway. Previous studies have suggested a direct intracellular role for IFN-γ in the induction of its biological activities. Based on our data in this study, we suggest that a key intracellular site of interaction of IFN-γ is the one with the nuclear transport mechanism that occurs via the NLS in the COOH terminus of IFN-γ.

Lipid Microdomains Are Required Sites for the Selective Endocytosis and Nuclear Translocation of IFN-γ, Its Receptor Chain IFN-γ Receptor-1, and the Phosphorylation and Nuclear Translocation of STAT1α

IFN-γ contains a nuclear localization sequence that may play a role in the nuclear transport of activated STAT1α via a complex of IFN-γ/IFN-γ receptor (IFNGR)-1/STAT1α with the nuclear importer nucleoprotein interactor 1. In this study, we examine the mechanism of endocytosis of IFNGR-1 and the relationship of its nuclear translocation to that of STAT1α. In untreated WISH cells, both IFNGR-1 and IFNGR-2 were constitutively localized within caveolae-like microdomains isolated from plasma membrane. However, treatment of cells with IFN-γ resulted in rapid migration of IFNGR-1, but not IFNGR-2, from these microdomains. Filipin pretreatment, which specifically inhibits endocytosis from caveolae-like microdomains, inhibited the nuclear translocation of IFN-γ and IFNGR-1 as well as the tyrosine phosphorylation and nuclear translocation of STAT1α, but did not affect the binding of IFN-γ to these cells. In the Jurkat T lymphocyte cell line, which does not express caveolin-1, nuclear translocation of IFNGR-1 and STAT1α were similarly inhibited by filipin pretreatment. Isolation of lipid microdomains from Jurkat cells showed that both IFNGR-1 and IFNGR-2 were associated with lipid microdomains only after stimulation with IFN-γ, suggesting that the IFNGR subunits are recruited to lipid microdomains by IFN-γ binding in lymphocytes (Jurkat) in contrast to their constitutive presence in epithelial (WISH) cells. In contrast, treatments that block clathrin-dependent endocytosis did not inhibit either activation or nuclear translocation of STAT1α or the nuclear translocation of IFN-γ or IFNGR-1. Thus, membrane lipid microdomains play an important role in IFN-γ-initiated endocytic events involving IFNGR-1, and the nuclear translocation of IFN-γ, IFNGR-1, and STAT1α.

Differential Nuclear Localization of the IFNGR-1 and IFNGR-2 Subunits of the IFN-γ Receptor Complex Following Activation by IFN-γ

We have recently identified a nuclear localization sequence (NLS) in the C-terminus of murine type II interferon (IFN), IFN-γ, that is responsible for the internalization and nuclear translocation of extracellularly added IFN-γ. Because the uptake of IFN-γ is a receptor-mediated endocytotic process, we examined in this study the fate of both the receptor subunits (IFNGR-1 and IFNGR-2) of the heterodimeric IFN-γ receptor complex. Human IFN-γ (HuIFN-γ) was also found to contain a polybasic NLS in a conserved C-terminal region capable of directing its nuclear translocation. Like the ligand, the IFNGR-1 subunit of the receptor complex on WISH cells was found to be translocated to the nucleus on treatment with HuIFN-γ. Using a combination of immunoprecipitation and immunofluorescence techniques, we found the nuclear accumulation of IFNGR-1 to be ligand dependent, and it was evident within 10-20 min after ligand stimulation. IFNGR-1 was found to colocalize, in a time-dependent and dose-dependent fashion, with t...

Bovine Interferon-τ Stimulates the Janus Kinase-Signal Transducer and Activator of Transcription Pathway in Bovine Endometrial Epithelial Cells

Abstract Trophoblastic bovine interferon-tau (bIFN-τ) suppresses luteolytic pulses of endometrial prostaglandin F2α (PGF2α) at the time of maternal recognition of pregnancy. This results in maintenance of the corpus luteum in cattle. The hypothesis that effects of bIFN-τ in the endometrium were through activation of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway of signal transduction was tested. Whole cell, cytosolic, and nuclear extracts from bovine endometrial cells treated with bIFN-τ were analyzed by immunoprecipitation, immunoblotting, and electrophoretic mobility shift assays in a series of dose- and time-dependency experiments. Bovine IFN-τ stimulated tyrosine phosphorylation, homo- and heterodimer formation, nuclear translocation, and DNA binding of STAT proteins 1, 2, and 3. Moreover, bIFN-τ induced synthesis of interferon-regulatory factor. In conclusion, bIFN-τ stimulates the JAK-STAT pathway in the bovine endometrium. It is proposed that activation of the JAK-STAT pathway is involved in regulating the antiluteolytic effects of bIFN-τ.

Type I interferon induction of the Cdk-inhibitor p21WAF1 is accompanied by ordered G1 arrest, differentiation and apoptosis of the Daudi B-cell line.

We show, in this study, that type I IFN induction of the cyclin-dependent kinase (cdk) inhibitor p21WAF1 in the human Burkitt lymphoma B cell-line Daudi and ensuing cell cycle arrest correlate with the terminal differentiation of these cells, and is ultimately followed by apoptosis and cell death. The expression of p21WAF1 paralleled the onset of G1 arrest and the reduction of surface IgM expression which was used as a marker of the differentiation response, and the IFN treated cells acquired a typical plasma cell-like morphology. The type II IFN IFNγ, which does not inhibit the growth of Daudi cells, did not induce the expression of p21WAF1, nor affect the expression of surface IgM. The induction of p21WAF1 which paralleled the inhibition of the phosphorylation of the retinoblastoma protein, pRB, was preceded by the strong reduction in c-myc levels. We propose that the coupled down-regulation of c-myc and induction of p21WAF1 may be crucial to the induction of differentiation and G1 arrest in Daudi cells by type I IFN. Growth arrest and differentiation was followed by apoptosis and cell death, and was accompanied by the induction of the activity of the apoptotic ICE-family protease CPP32. G1 arrest and differentiation followed by apoptotic cell death are characteristics of terminal differentiation. Thus, our data suggest that the induction of p21WAF1 and G1 arrest mediated by type I IFN in Daudi cells is part of terminal differentiation response in these cells, highlighting a role for type I IFN as B cell terminal differentiation factors.

Oral feeding of interferon τ can prevent the acute and chronic relapsing forms of experimental allergic encephalomyelitis

IFN tau is a member of the type I IFN family but unlike IFN alpha and IFN beta, IFN tau lacks toxicity at high concentrations. Recently, ovine IFN tau was shown to prevent acute induction and superantigen reactivation of experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). In this report, we examined the ability of IFN tau when administered by oral feeding to block development of EAE. Oral feeding of INF tau prevented paralysis in the acute form of EAE in NZW mice and chronic-relapsing EAE in SJL/J mice. In addition, oral feeding of IFN tau at 10(5) U/dose was as effective as intraperitoneal (i.p.) injection in preventing chronic-relapsing EAE, and both forms of IFN tau administration resulted in IL10 production. Histological examination revealed no inflammatory lymphocytic infiltration to the CNS in IFN tau treated animals as compared to controls. Prolonged treatment of IFN tau was shown to be necessary for chronic-relapsing EAE since removal of IFN tau treatment by either oral feeding or i.p. injection resulted in onset of disease. Lastly, sera from SJL/J mice which received prolonged IFN tau treatment by oral feeding exhibited little to no development of anti-IFN tau antibodies. Thus, oral feeding of ovine IFN tau may be a successful form of IFN tau administration for treatment of autoimmune diseases such as MS and may circumvent potentially debilitative antibody responses.