IFN-γ Resistance Research Articles

Under Bcl-2 surveillance, cytokines, autophagy-dependently, induce p53-independent CD54 expression (161.4)

Abstract p53 is required for stress-induced CD54 expression; until now, its role has less investigated for IFN-γ- and TNF-α-induced such effects. However, IFN-γ caused CD54 expression in human non-small cell lung cancer A549 cells even with p53 silence, but controversially had no effects on H1299 and AS2 cells carried p53 mutation. To investigate the unexpected IFN-γ resistance, there was not as much of change on IFN-γ receptor expression among these cells; however, IFN-γ-activated Jak2/STAT1/IFN regulatory factor (IRF) 1 was decreased. Aberrant activation of Src homology domain-containing phosphatase (SHP) 2, but neither suppressor of cytokine signaling (SOCS) 1 nor SOCS3, was essential for IFN-γ resistance to decrease CD54. Notably, autophagy deficiency was shown in resistant cells and determined IFN-γ resistance accompanied by increasing mitochondria and reactive oxygen species. TNF-α also acted accordingly by autophagy to induce IκB degradation, NF-κB activation, and CD54 expression independent of p53. These results demonstrate that under Bcl-2 surveillance, IFN-γ and TNF-α induce p53-independent but autophagy-regulated CD54 expression.

Regulation of SHP2 by PTEN/AKT/GSK-3β signaling facilitates IFN-γ resistance in hyperproliferating gastric cancer

Oncogenic activation accompanied by escape from immune surveillance, such as IFN-γ resistance, is critical for cancer cell growth and survival. In this study, we investigated the crosstalk signaling between IFN-γ resistance and signaling of hyperproliferation in gastric cancer cells. IFN-γ inhibited the cell growth of MKN45 cells but not hyperproliferating AGS cells. AGS cells did not respond to IFN-γ because of a decrease in STAT1 but not due to dysfunctional IFN-γ receptors. Signaling of PI3K/AKT, as well as MEK/ERK, was required for the hyperproliferation; notably, PI3K/AKT alone mediated the IFN-γ resistance. Aberrant Src homology-2 domain-containing phosphatase (SHP) 2 determined IFN-γ resistance but unexpectedly had no effects on hyperproliferation or ERK activation. In the IFN-γ resistant cells, inactivation of glycogen synthase kinase (GSK)-3β by PI3K/AKT was important for SHP2 activation but not for hyperproliferation. An imbalance of AKT/GSK-3β/SHP2 caused by a reduction of PTEN was important for the crosstalk between IFN-γ resistance and hyperproliferation. PI3K is constitutively expressed in AGS cells and immunohistochemical staining showed a correlation between hyperproliferation and expression of SHP2 and STAT1 in gastric tumors. These results demonstrate the effects of PTEN/AKT/GSK-3β/SHP2 signaling on IFN-γ resistance in hyperproliferating gastric cancer cells.

Association of IFN-γ Signal Transduction Defects with Impaired HLA Class I Antigen Processing in Melanoma Cell Lines

Abnormalities in the constitutive and IFN-γ-inducible HLA class I surface antigen expression of tumor cells is often associated with an impaired expression of components of the antigen processing machinery (APM). Hence, we analyzed whether there exists a link between the IFN-γ signaling pathway, constitutive HLA class I APM component expression, and IFN-γ resistance. The basal and IFN-γ-inducible expression profiles of HLA class I APM and IFN-γ signal transduction cascade components were assessed in melanoma cells by real-time PCR (RT-PCR), Western blot analysis and/or flow cytometry, the integrity of the Janus activated kinase (JAK) 2 locus by comparative genomic hybridization. JAK2 was transiently overexpressed in JAK2(-) cells. The effect of IFN-γ on the cell growth was assessed by XTT [2,3-bis(2-methoxy-4-nitro-S-sulfophenynl)-H-tetrazolium-5-carboxanilide inner salt] assay. The analysis of 8 melanoma cell lines linked the IFN-γ unresponsiveness of Colo 857 cells determined by lack of inducibility of HLA class I surface expression on IFN-γ treatment to a deletion of JAK2 on chromosome 9, whereas other IFN-γ signaling pathway components were not affected. In addition, the constitutive HLA class I APM component expression levels were significantly reduced in JAK2(-) cells. Furthermore, JAK2-deficient cells were also resistant to the antiproliferative effect of IFN-γ. Transfection of wild-type JAK2 into JAK2(-) Colo 857 not only increased the basal APM expression but also restored their IFN-γ sensitivity. Impaired JAK2 expression in melanoma cells leads to reduced basal expression of MHC class I APM components and impairs their IFN-γ inducibility, suggesting that malfunctional IFN-γ signaling might cause HLA class I abnormalities.

Development of IFN-γ resistance is associated with attenuation of SOCS genes induction and constitutive expression of SOCS 3 in melanoma cells

The resistance to interferons (IFNs) limits their anticancer therapeutic efficacy. Here we studied the evolution of an IFN-resistant state in vitro using melanoma cell lines. We found that the cells became less sensitive to antiproliferative effect of IFN-γ after prolonged cultivation enabling us to isolate sensitive and resistant subclones of the parental line. We investigated transcription of signal transducer and activator of transcription (STAT) 1–6 and suppressor of cytokine signalling (SOCS) 1–3 genes, and phosphorylation of STAT 1 protein. The resistant subline (termed WM 1158R) differed from the sensitive subline (WM 1158S) by a constitutive expression of SOCS 3, lack or weak SOCS 1–3 activation following IFN-γ, and short duration of cytokine activatory signal. Similar correlations were observed in additional melanoma lines differing in IFN sensitivities. At the protein level, IFN-γ induced strong and prolonged STAT 1 activation at serine 727 (S727) in WM 1158R while in WM 1158S cells phosphorylation of this amino acid was much less pronounced. On the other hand, phosphorylation of tyrosine 701 (Y701) was stimulated regardless of the sensitivity phenotype. In conclusion, constitutive expression of SOCS 3 is correlated with attenuation of its induction following IFN treatment. These results suggest that progression of melanoma cells from IFN sensitivity to IFN insensitivity associates with changes in SOCS expression.

IFN-γ fails to antagonize fibrotic effect of TGF-β on keloid-derived dermal fibroblasts

Background: Interferon-γ (IFN-γ) has been noted as a potential therapeutic agent for various fibrotic disorders, in part, through its antagonistic effect on a fibrogenic cytokine, transforming growth factor-β (TGF-β). Keloid is a fibrotic skin disorder that results in an excessive deposition of extracellular matrix, which is associated with altered-expression of or -responses to TGF-β in dermal fibroblasts. Objective: We sought to determine whether IFN-γ antagonized TGF-β-mediated fibrotic response in keloid-derived dermal fibroblasts. Methods: Type I collagen production, fibroblast contractile activity, and α-smooth muscle actin (α-SMA) expression were assessed by using Western blotting, an in vitro type I collagen gel contraction assay, and immunofluorescence study in normal and keloid-derived human dermal fibroblasts in the presence or absence of IFN-γ and/or TGF-β. Results: In contrast to normal dermal fibroblasts, IFN-γ did not inhibit TGF-β-induced type I collagen production, contractile activity, and α-SMA expression in keloid-derived dermal fibroblasts. In addition, keloid-derived dermal fibroblasts constitutively expressed type I collagen and α-SMA with increased capacity to contract a collagen matrix. Conclusion: IFN-γ failed to antagonize TGF-β-mediated fibrotic response in keloid-derived dermal fibroblasts. Thus, IFN-γ may not be therapeutically useful for keloid and clarification of the molecular mechanisms underlying the IFN-γ resistance should be investigated for therapeutic application of IFN-γ for keloid.

Interferon-gamma-mediated activation and ubiquitin-proteasome-dependent degradation of PPARgamma in adipocytes.

Interferon-gamma (IFNgamma) treatment of adipocytes results in a down-regulation of the peroxisome proliferator-activated receptor gamma (PPARgamma). The decrease in PPARgamma expression is mediated by inhibition of PPARgamma synthesis and increased degradation of PPARgamma. In this study, we demonstrate that both PPARgamma1 and PPARgamma2 are targeted to the proteasome under basal conditions and that PPARgamma1 is more labile than PPARgamma2. The IFNgamma-induced increase in PPARgamma turnover is blocked by proteasome inhibition and is accompanied by an increase in PPARgamma-polyubiquitin conjugates. In addition, IFNgamma treatment results in the transcriptional activation of PPARgamma. Similar to ligand-dependent activation of PPARgamma, IFNgamma-induced activation was greater in the phosphorylation-deficient S112A form of PPARgamma when compared with wild-type PPARgamma. Moreover, the inhibition of ERKs 1 and 2 with a MEK inhibitor, U1026, lead to an inhibition in the decay of PPARgamma proteins, indicating that serine phosphorylation influences the degradation of PPARgamma in fat cells. Our results also demonstrate that the proteasome-dependent degradation of PPARgamma does not require nuclear export. Taken together, these results indicate that PPARgamma is targeted to the ubiquitin-proteasome pathway for degradation under basal conditions and that IFNgamma leads to an increased targeting of PPARgamma to the ubiquitin-proteasome system in a process that is affected by ERK-regulated serine phosphorylation of PPARgamma proteins.

Molecular pathogenesis of secondary myeloid leukemia: Resistance to inhibitory cytokines and autocrine vegf-dependence of the UOC-M1 cell line

Monosomy 7 (−7) is common in alkylating agent induced- or Fanconi anemia-related myelodysplastic syndrome and acute myeloid leukemia. Interested in using genetic complementation to discover tumor suppressor genes on chromosome 7 that may play a role in leukemogenesis, we are characterizing the phenotype of a factor-independent, megakaryoblastoid, CD34+ cell line (UoC-M1) with complex cytogenetics including (−7). UoC-M1 cells were resistant to 100ng/ml IFNγ and 5ng/ml TGFβ in colony forming unit assays. In contrast, TNFα inhibited growth and resulted in caspase 3 activation. UoC-M1 cells produced highly vascularized, solid tumors in NOD/SCID mice. The tumor cell karyotype was identical to UoC-M1. Supernatants from unstimulated UoC-M1 cells in vitro demonstrated VEGF production (240pg/ml/72 hours from 1×105 cells by ELISA) but no GM-CSF, FLT-3, SCF, IL-6, TPO, TNFα, or IL-1β. Normal human CD34+ cells yielded 4pg/ml VEGF/106 and 57pg/ml/106 when stimulated with 200U/ml rhGM-CSF for 3 days. UoC-M1 also expresses both VEGFR1 (FLT-1) and VEGFR2 (KDR). Because VEGF normally varies inversely with von Hippel-Lindau protein (VHL), we analyzed UoC-M1 mRNA for VHL and the VHL-interacting factors CUL2, HIF1α/β, elongin B/C, and SP1. All were constitutively expressed. We conclude that TGF and IFNγ resistance, VHL resistant-VEGF production, autocrine growth, and in vivo tumorigenesis will be of potential value in genetic complementation studies for the identification of tumor suppressor genes on chromosome 7.

Effects of interleukin-4 deprivation and treatment on resistance to Trypanosoma cruzi.

Trypanosoma cruzi (Y strain)-infected interleukin-4(-/-) (IL-4(-/-)) mice of strains 129/J, BALB/c, and C57BL/6 showed no significant difference in parasitemia levels or end point mortality rates compared to wild-type (WT) mice. Higher production of gamma interferon (IFN-gamma) by parasite antigen (Ag)-stimulated splenocytes was observed only for C57BL/6 IL-4(-/-) mice. Treatment of 129/J WT mice with recombinant IL-4 (rIL-4), rIL-10, anti-IL-4, and/or anti-IL-10 monoclonal antibodies (MAbs) did not modify parasitism. However, WT mice treated with rIL-4 and rIL-10 had markedly increased parasitism and suppressed IFN-gamma synthesis by spleen cells stimulated with parasite Ag, concanavalin A, or anti-CD3. Addition of anti-IL-4 MAbs to splenocyte cultures from infected WT 129/J, BALB/c, or C57BL/6 mice failed to modify IFN-gamma synthesis levels; in contrast, IL-10 neutralization increased IFN-gamma production and addition of rIL-4 and/or rIL-10 diminished IFN-gamma synthesis. We conclude that endogenous IL-4 is not a major determinant of susceptibility to Y strain T. cruzi infection but that IL-4 can, in association with IL-10, modulate IFN-gamma production and resistance.