Constitutive Activation Of NF-kappa Research Articles

Inhibition of I\u03baB\u03b1 phosphorylation potentiates regulated cell death induced by azidothymidine in HTLV-1 infected cells

Adult T cell leukemia/lymphoma (ATL) can be susceptible, at least transiently, to treatments with azidothymidine (AZT) plus IFNα and/or arsenic trioxide. However, the real role of AZT in this effect is still unclear. In fact, while reverse transcriptase (RT) inhibition could explain reduction of clonal expansion and of renewal of HTLV-1 infected cells during ATL progression, this effect alone seems insufficient to justify the evident and prompt decrease of the pro-viral load in treated patients. We have previously demonstrated that AZT is endowed with an intrinsic pro-apoptotic potential towards both peripheral blood mononuclear cells from healthy donors or some tumor cell lines, but this cytotoxic potential cannot be fully achieved unless IκBα phosphorylation is inhibited. Since the constitutive activation of NF-kappa B (NF-κB) appears a common biological basis of HTLV-1-infected cells, a pharmacological inhibition of IκBα phosphorylation seems a potential strategy for treating and preventing HTLV-1 related pathologies. In this study, we have demonstrated that a combination treatment with the IκBα phosphorylation inhibitor Bay 11-7085 and AZT induced increased levels of regulated cell death (RCD) by apoptosis compared to the single treatments in HTLV-1 infected cells of different origin. Importantly, levels of RCD were considerably higher in infected cells in comparison with the uninfected ones. Inhibition of NF-κB activation following the combined treatment was confirmed by analysis of both gel-shift and functional activity of the NF-κB complex proteins, p65/p52. Moreover, a transcriptional analysis revealed that the addition of Bay 11-7085 to AZT treatment in HTLV-1-infected cells modified their transcriptional profile, by inducing the upregulation of some pro-apoptotic genes together with the downregulation of some anti-apoptotic genes. Our data suggest that addition of adequate concentrations of IκBα phosphorylation inhibitor to therapeutic regimens including AZT could be a promising strategy in ATL.

Abstract 1300: The potential role of NF-κB activation in the immortalization of nasopharyngeal epithelial cells.

Abstract Nasopharyngeal carcinoma (NPC) is a common cancer among Southern Chinese. Many molecular and cellular events, particularly those occurring at the early stage of the NPC development, have not been well defined. Immortalization is a pre-requisite property of cancer cells and is regarded as an early event in human carcinogenesis. Defining and understanding these molecular and cellular events could provide us important insights to the early events involved in the carcinogenesis process. Our lab has established multiple immortalized nasopharyngeal epithelial (NP) cell lines using telomerase alone or in combination with other genetic elements including Bmi-1, cyclin D1 and knocking down p16. We observed that NF-Kappa B are commonly activated in immortalized cells compared to primary cells. Immunofluorescence staining and western blotting assay revealed activation of canonical NF-kappa B pathway in these immortalized nasopharyngeal epithelial cell lines with nuclear translocation of NF-kappa B p65 subunit. Interestingly, EGFR is also commonly expressed in our immortalized nasopharyngeal epithelial cells and may contribute to the constitutive activation of NF-kappa B in immortalized nasopharyngeal epithelial cells. Noteworthy, Epstein - Barr Virus (EBV) infection is closely associated with NPC development. There are studies reporting that activation of NF-kappa B and overexpression of EGFR are derived by EBV encoded LMP1 in NPC (Miller et al. Oncogene. 16, 1998). Our data also show LMP1 could induce NF-kappa B activation and EGFR overexpression in NPC cell line. Thus, the activation of NF-kappa B and overexpression of EGFR may be an important component of EBV infection in epithelial cells and contributing to the development of NPC. We further showed that activation of NF-kappa B signaling appears to be essential for the survival of immortalized nasopharyngeal epithelial cells as inhibition of NF-kappa B activity by a specific inhibitor (Nemo binding domain peptide), could selectively inhibit growth of the immortalized nasopharyngeal epithelial cells but not in primary nasopharyngeal epithelial cells. To study the functional interaction of NF-kappa B activation with EGFR, we have examined if EGFR overexpression may support immortalization of nasopharyngeal epithelial cells. We observed that functional EGFR could facilitate cell growth and protect cells from apoptosis. We are now exploring if activation of NF-kappa B and EGFR overexpression may be functionally related during immortalization of nasopharyngeal epithelial cells appears. Citation Format: Dandan Zhu, Yim Ling Yip, Wen Deng, Chi Man Tsang, Lai Man Cheung, Sai Wah Tsao. The potential role of NF-κB activation in the immortalization of nasopharyngeal epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1300. doi:10.1158/1538-7445.AM2013-1300 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

The ubiquitin‐proteasome pathway and enhanced activity of NF‐κB in gastric carcinoma

NF-kappa B, ubiquitin and proteasome have been shown be important factors in oncogenesis. The aim of this study was to determine whether NF-kappa B could be a sensitive biomarker for gastric carcinoma. Tumor and adjacent mucosal tissue specimens in 92 patients with gastric carcinoma were studied. The expression of NF-kappa B was evaluated by immunohistochemistry. The expression of I kappa B alpha, ubiquitin in cytoplasm and NF-kappa B in nucleoplasm was assayed by Western blot. DNA binding-activity of NF-kappa B was confirmed by electrophoretic mobility shift assay (EMSA). Fluorogenic technique was performed to measure the 26S proteasome activity. NF-kappa B positive expression in tumor tissues (82.4%) was significant higher than that in adjacent mucosal tissues (32.7%, P < 0.05). The increase of NF-kappa B activation was accompanied by the increases of ubiqutin, 26S proteasome activation and a degradation of I kappa B alpha but not the ubiquitin-conjugated I kappa B alpha/NF-kappa B complex in gastric carcinoma. NF-kappa B expression was significantly increased in patients with lymph node metastasis, TNM stage III/IV or with the habit of high intake of pickled vegetables. This study demonstrates that the constitutive activation of NF-kappa B is likely due to the activation of ubiquitin-proteasome pathway and NF-kappa B can be used as a prognostic biomarker.

The AKT/IκB kinase pathway promotes angiogenic/metastatic gene expression in colorectal cancer by activating nuclear factor-κB and β-catenin

Our laboratory has delineated that the phosphatidylinositol 3' kinase (PI3K)/AKT/I kappa B kinase (IKK) pathway positively regulates NF kappa B and beta-catenin, both important transcriptional regulators in colorectal cancer (CRC). Therefore, we investigated the effect of inhibiting the PI3K/AKT/IKK alpha pathway in regulating the inappropriate constitutive activation of NF kappa B and beta-catenin in CRC cell lines. SW480 and RKO CRC cell lines demonstrate constitutive activation of AKT as well as both NF kappa B- and beta-catenin-dependent transcription. The constitutive activation of NF kappa B- and beta-catenin-dependent transcription is inhibited by transiently transfecting either kinase dead (KD) IKK alpha, which blocks IKK alpha kinase activity, KD AKT, which blocks AKT activity, or wildtype (WT) PTEN, which inhibits PI3K and AKT activity. The ability of KD IKK alpha, KD AKT or WT PTEN to decrease beta-catenin-dependent transcription is independent of their effects on NF kappa B. Inducible expression of either KD IKK alpha or WT PTEN strongly inhibits both the constitutive NF kappa B- and beta-catenin-dependent promoter and endogenous gene activation. Targeted array-based gene expression analysis of this inducible system reveals that many of the genes downregulated upon inhibition of this pathway are involved in tumor angiogenesis and metastasis. The activation of this pathway and the expression of the three most repressed genes was further analysed in samples of CRC. These results indicate a role of this pathway in controlling gene expression important in tumor progression and metastasis.

PKCθ mediates pre-TCR signaling and contributes to Notch3-induced T-cell leukemia

Protein kinase (PK)C theta is a critical regulator of mature T-cell activation and proliferation, being implicated in TCR-triggered nuclear factor (NF)-kappa B activation and providing important survival signals to leukemic T cells. We previously showed that overexpression of pT alpha/pre-TCR and constitutive activation of NF-kappa B characterize the T-cell leukemia/lymphoma developing in Notch3-IC transgenic mice. We report here that PKC theta is a downstream target of Notch3 signaling and that its activation and membrane translocation require a functional pre-TCR in order to trigger NF-kappa B activation in thymocytes and lymphoma cells of transgenic mice. Furthermore, deletion of PKC theta in Notch3-IC transgenic mice reduces the incidence of leukemia, correlating with decreased NF-kappa B activation. This paper therefore suggests that PKC theta mediates the activation of NF-kappa B by pre-TCR in immature thymocytes and contributes to the development of Notch3-dependent T-cell lymphoma.

Monitoring NF-kappa B transactivation potential via real-time PCR quantification of I kappa B-alpha gene expression.

Nuclear factor-kappa B (NF-kappa B) is an important transcription factor involved in the regulation of immune responses as well as in cell proliferation and survival. An abnormal and constitutive activation of NF-kappa B is observed in many pathological states as diverse as inflammation, neurological diseases, and cancer. Termination of NF-kappa B transcription is mediated through the NF-kappa B-dependent synthesis of the I kappa B-alpha inhibitory subunit. To quantify NF-kappa B activation we measured by real-time PCR the expression of I kappa B-alpha mRNA. The PCR data perfectly matched the results obtained by Northern blot or gene reporter analysis when Jurkat leukemic T cells or HeLa carcinoma cells were stimulated with various activators of NF-kappa B, such as the cytokine tumor necrosis factor (TNF)-alpha or the phorbol ester PMA. Constitutive NF-kappa B activation in Hodgkin's lymphoma cell line could also be evaluated by this approach. Kinetic experiments in HeLa cells show that TNF stimulation first induced NF-kappa B DNA binding within 30 minutes, followed by I kappa B-alpha gene transcription 30 minutes later. Removal of TNF after stimulation resulted in a faster decrease in both NF-kappa B DNA binding activity and I kappa B-alpha mRNA levels. No accumulation or stabilization of I kappa B-alpha mRNA was detected that could bias interpretation of the results. The sensitivity of the method allowed the detection of NF-kappa B activation in stimulated normal peripheral blood lymphocytes. The real-time PCR measure of I kappa B-alpha mRNA levels is a rapid, sensitive, and powerful method to quantify the transcriptional power of NF-kappa B. It can be easily used for clinical evaluation of NF-kappa B status.

Analysis of expression of nuclear factor kappa B (NF-kappa B) in multiple myeloma: downregulation of NF-kappa B induces apoptosis.

Nuclear factor-kappa B (NF-kappa B) is an important transcription factor that regulates survival in many cells. Activated NF-kappa B has been shown to protect some haematopoietic neoplastic cells from apoptosis. In the present study, we analysed NF-kappa B status in 13 primary samples from patients with multiple myeloma (MM) and in four myeloma cell lines including U266, RPMI 8226, HS-Sultan and K620. Constitutive activation of NF-kappa B was evaluated by either immunohistochemistry or immunofluorescence using a monoclonal mouse anti-human p65 (Rel A) antibody, which recognizes the unbound, active form of p65 (Rel A). Constitutively active NF-kappa B was present in all MM patient samples as well as in all four myeloma cell lines. Inhibition of constitutively active NF-kappa B, by either proteasome inhibitors (MG132, gliotoxin) or inhibitors of I kappa B phosphorylation (Bay117082, and Bay117085), induced apoptosis as demonstrated by both flow cytometric analysis and light microscopic morphological evaluation. This chemically induced apoptosis was associated with decreased DNA binding of nuclear NF-kappa B as determined by the electrophoretic mobility shift assay. In addition, adenovirus vector with dominant negative I kappa B alpha (Ad5I kappa B) was used for inhibition of NF-kappa B in the U266 cell line. Compared with wild-type, super-repressor-treated cells showed an increased level of apoptosis. These results suggest that constitutive expression of NF-kappa B plays an important role in plasma cell survival in MM.

Expression of nuclear factor-kappa B, tumor necrosis factor receptor type 1, and c-Myc in human astrocytomas.

Tumor necrosis factor receptor type 1 (TNFR1) and c-Myc are important in signal transduction in tumor necrosis factor-alpha (TNF-alpha)-induced cytotoxicity, whereas activation of nuclear factor-kappa B (NF-kappa B) protects against TNF-alpha-induced apoptosis. This study investigated the expression of NF-kappa B, TNFR1, and c-Myc in human astrocytoma tissues by reverse transcriptase-polymerase chain reaction (PCR) and immunohistochemical analysis. TNFR1 messenger ribonucleic acid (mRNA) and c-Myc mRNA were frequently expressed in malignant astrocytomas, especially in glioblastomas, compared with low-grade astrocytomas by PCR analysis. TNFR1 and c-Myc mRNAs were barely detectable in normal brain tissues. NF-kappa B p50 and p65 subunit mRNAs were detected in various grades of astrocytomas, with frequent expression in malignant astrocytomas. The presence of activated NF-kappa B was confirmed by nuclear localization in neoplastic astrocytes as determined by immunohistochemistry. Both p50 and p65 subunits were inhomogeneously expressed in neoplastic astrocytes of glioblastoma, but only in a few scattered tumor cells in low-grade astrocytoma, and almost undetectable in normal brain tissues. These results indicate that TNFR1 and c-Myc are overexpressed in malignant astrocytomas, and this may increase the cellular sensitivity to the cytotoxic action of TNF-alpha. NF-kappa B p50 and p65 were simultaneously induced and activated in malignant astrocytomas. Our results suggest that the constitutive activation of NF-kappa B subunits in malignant astrocytoma, especially in glioblastoma, could be associated with the resistance to TNF-alpha immunotherapy, and indicates new therapeutic strategies for malignant astrocytomas.

Alteration of the cellular response to interleukin-1 beta by SV40 large T antigen in rheumatoid synovial fibroblasts.

The large T antigen of SV40 (LT) has been widely used to immortalize primary cells for various studies. In this study, synovial fibroblasts of a patient from rheumatoid arthritis (RA) were transformed with LT gene to analyze the effect of SV40-mediated transformation on the production of cytokines, such as IL-6, IL-8, and GM-CSF, that are under the control of interleukin-1 beta (IL-1 beta), a physiological inducer of nuclear factor kappa B (NF-kappa B). It was noted that the basal levels of GM-CSF and IL-8 were upregulated, whereas that of IL-6 was downregulated. Moreover, the extents of induction of these cytokines in response to IL-1 beta were markedly downregulated in synovial fibroblasts transformed by LT as compared from parental cells. Although IL-1 beta could translocate NF-kappa B to the nucleus in all cells, some of the transformed cells exhibited nuclear translocation of NF-kappa B even before the stimulation with IL-1 beta, suggesting that transformation of LT resulted in the constitutive activation of NF-kappa B, either directly or indirectly. In order to examine whether LT downregulate the kappa B-dependent gene expression, we performed the transient luciferase gene expression assay. We found that cotransfection of LT did not downregulate the kappa B-dependent gene expression that was stimulated with L-1 beta. These observations suggest that the apparent inhibitory effect of LT on the IL-1-induced expression of cytokines may not be through its direct action on the NF-kappa B transactivation.

Constitutive activation of NF-kappa B in an animal model of aging.

The pathophysiology of many disease states observed in aged individuals has been linked to the dysregulated production of several pleiotropic cytokines. We have demonstrated that NF-kappa B, a major transcriptional regulator of these aberrantly expressed cytokines, exists in a constitutively activated state in cells obtained from the major lymphoid organs of aged animals. Therapeutic treatment with dietary antioxidants or with agents capable of activating the peroxisome proliferator-activated receptor (PPAR)-alpha was able to correct the abnormal nuclear NF-kappa B activity, reduce lipid peroxide levels, and eliminate the dysregulated expression of cytokines and other genes under NF-kappa B control. These results suggest that abnormal activation of NF-kappa B in aging contributes to the dysregulated expression of certain pleiotropic cytokines. Effective therapeutic regimens for aging and other inflammatory disease states might benefit from the administration of antioxidants or other agents which specifically activate PPAR-alpha.

Homologous T and B cells immortalized in vitro by the Epstein-Barr virus exhibit differential genetical and functional features

After in vitro EBV infection of peripheral blood lymphocytes (PBL), we previously obtained IL-2-independent T-cell lines expressing EBNA1 and LMP1 viral latent genes. One tumorigenic clone, NC5, was further characterized for chromosomal abnormalities, rearrangement and expression of oncogenes, and constitutive or induced activation of cellular transduction pathways. NC5 as well as TC cells derived from an NC5-induced tumor exhibited the same few chromosomal abnormalities absent in normal PBL and B-cell lines (LCLs) from the same donor. No rearrangement or altered expression of C-MYC, BCL-2 and NF-KB2 oncogenes could be detected. In contrast, we found high levels of BCL-X and thioredoxin (TRX), as markers of EBV infection or T-cell activation/transformation status. No constitutive activation of NF-kappa B or STAT transcriptional complexes was observed in these cells. For NF-kappa B, this was in apparent contradiction with its reported inducibility mediated by LMP1, taking into account that NF-kappa B was still inducible by TNF alpha or PMA and ionomycin. Our results highlight independence of EBV protein-mediated transformation towards classical cellular pathways in T-lymphocytes.

Constitutive activation of NF-kappa B is essential for transformation of rat fibroblasts by the human T-cell leukemia virus type I Tax protein.

Human T-cell leukemia virus type I (HTLV-I) encodes a 40 kDa trans-acting protein, Tax, that regulates transcription of both the proviral and cellular genes, and can transform rat fibroblasts. To determine the functional importance of its trans-acting capacities in cell transformation, we have examined two representative pathways of transcriptional activation--HTLV-I long terminal repeat (LTR) mediated and NF-kappa B dependent--by mutational analysis of Tax. In contrast to a previous report, mutants lacking the ability to activate an NF-kappaB-dependent promoter failed to transform rat fibroblasts, whereas a mutation which abolishes the HTLV-I LTR-mediated trans-activation demonstrated a wild-type capacity for cell transformation. Stable expression of Tax competent for transformation caused enhanced DNA binding of NF-kappa B in rat fibroblasts. We also demonstrate that stable co-expression of the NFKB2 precursor, known as a member of the I kappa B proteins, with wild-type Tax blocked transformation as well as eliminated aberrant NF-kappaB activation by Tax without interference with the HTLV-I LTR-mediated trans-activation. Our results indicate that constitutive activation of NF-kappa B is essential for Tax-mediated transformation of rat fibroblasts.

Enhanced I kappa B alpha degradation is responsible for constitutive NF-kappa B activity in mature murine B-cell lines.

Nuclear factor kappa B (NF-kappa B) is a ubiquitous transcription factor which binds to decameric DNA sequences (kappa B sites) and regulates transcription of multiple genes. The activity of NF-kappa B is regulated by an inhibitor protein, I kappa B, which sequesters NF-kappa B in the cytoplasm. Release of I kappa B and subsequent nuclear translocation of NF-kappa B generally require activating signals. However, in mature murine B cells, the DNA-binding activity of NF-kappa B is constitutively nuclear and activates the Ig kappa gene, a marker for mature B cells. To understand the basis for the constitutive NF-kappa B activation, we examined the properties of NF-kappa B and I kappa B in both pre-B and mature B cells, the regulated and constitutive states, respectively. We found that expression of I kappa B alpha and p105, members of the I kappa B family, and Rel, a member of the NF-kappa B family, is augmented in mature B cells. Both I kappa B alpha and p 105 are associated with NF-kappa B proteins and sequester most of these proteins in the cytoplasm of mature B cells. However, rapid I kappa B alpha dissociation and degradation lead to continuous nuclear translocation of a small fraction of NF-kappa B proteins, which represent the constitutively active NF-kappa B in mature B cells. We estimate that the protease activity is at least 35-fold greater in mature B cells than in pre-B cells. Rapid degradation of I kappa B alpha is directly involved in constitutive NF-kappa B activation, because stabilization of I kappa B alpha by a protease inhibitor causes loss of NF-kappa B activity in mature B cells. These results provide evidence that continuous and rapid degradation of I kappa B alpha in the absence pf external stimuli is causally involved in the constitutive activation of NF-kappa B in mature murine B cells.

Enhanced I kappa B alpha degradation is responsible for constitutive NF-kappa B activity in mature murine B-cell lines.

Nuclear factor kappa B (NF-kappa B) is a ubiquitous transcription factor which binds to decameric DNA sequences (kappa B sites) and regulates transcription of multiple genes. The activity of NF-kappa B is regulated by an inhibitor protein, I kappa B, which sequesters NF-kappa B in the cytoplasm. Release of I kappa B and subsequent nuclear translocation of NF-kappa B generally require activating signals. However, in mature murine B cells, the DNA-binding activity of NF-kappa B is constitutively nuclear and activates the Ig kappa gene, a marker for mature B cells. To understand the basis for the constitutive NF-kappa B activation, we examined the properties of NF-kappa B and I kappa B in both pre-B and mature B cells, the regulated and constitutive states, respectively. We found that expression of I kappa B alpha and p105, members of the I kappa B family, and Rel, a member of the NF-kappa B family, is augmented in mature B cells. Both I kappa B alpha and p 105 are associated with NF-kappa B proteins and sequester most of these proteins in the cytoplasm of mature B cells. However, rapid I kappa B alpha dissociation and degradation lead to continuous nuclear translocation of a small fraction of NF-kappa B proteins, which represent the constitutively active NF-kappa B in mature B cells. We estimate that the protease activity is at least 35-fold greater in mature B cells than in pre-B cells. Rapid degradation of I kappa B alpha is directly involved in constitutive NF-kappa B activation, because stabilization of I kappa B alpha by a protease inhibitor causes loss of NF-kappa B activity in mature B cells. These results provide evidence that continuous and rapid degradation of I kappa B alpha in the absence pf external stimuli is causally involved in the constitutive activation of NF-kappa B in mature murine B cells.