The BMI1 polycomb protein represses cyclin G2-induced autophagy to support proliferation in chronic myeloid leukemia cells.

The BMI1 polycomb protein regulates self-renewal, proliferation and survival of cancer-initiating cells essentially through epigenetic repression of the CDKN2A tumor suppressor locus. We demonstrate here for the first time that BMI1 also prevents autophagy in chronic myeloid leukemia (CML) cell lines, to support their proliferation and clonogenic activity. Using chromatin immunoprecipitation, we identified CCNG2/cyclin G2 (CCNG2) as a direct BMI1 target. BMI1 downregulation in CD34+ CML cells by PTC-209 pharmacological treatment or shBMI1 transduction triggered CCNG2 expression and decreased clonogenic activity. Also, ectopic expression of CCNG2 in CD34+ CML cells strongly decreased their clonogenicity. CCNG2 was shown to act by disrupting the phosphatase 2A complex, which activates a PKCζ-AMPK-JNK-ERK pathway that engages autophagy. We observed that BMI1 and CCNG2 levels evolved inversely during the progression of CML towards an acute deadly phase, and therefore hypothesized that BMI1 could support acute transformation of CML through the silencing of a CCNG2-mediated tumor-suppressive autophagy response.

L-type amino-acid transporter 1 (LAT1): a therapeutic target supporting growth and survival of T-cell lymphoblastic lymphoma/T-cell acute lymphoblastic leukemia.

The altered metabolism of cancer cells is a treasure trove to discover new antitumoral strategies. The gene (SLC7A5) encoding system L amino-acid transporter 1 (LAT1) is overexpressed in murine lymphoma cells generated via T-cell deletion of the pten tumor suppressor, and also in human T-cell acute lymphoblastic leukemia (T-ALL)/lymphoma (T-LL) cells. We show here that a potent and LAT1 selective inhibitor (JPH203) decreased leukemic cell viability and proliferation, and induced transient autophagy followed by apoptosis. JPH203 could also alter the in vivo growth of luciferase-expressing-tPTEN-/- cells xenografted into nude mice. In contrast, JPH203 was nontoxic to normal murine thymocytes and human peripheral blood lymphocytes. JPH203 interfered with constitutive activation of mTORC1 and Akt, decreased expression of c-myc and triggered an unfolded protein response mediated by the C/EBP homologous protein (CHOP) transcription factor associated with cell death. A JPH203-resistant tPTEN-/-clone appeared CHOP induction deficient. We also demonstrate that targeting LAT1 may be an efficient broad spectrum adjuvant approach to treat deadly T-cell malignancies as the molecule synergized with rapamycin, dexamethasone, doxorubicin, velcade and l-asparaginase to alter leukemic cell viability.

Mast cells and cellularity of the colonic mucosa correlated with fatigue and depression in irritable bowel syndrome.

BACKGROUND: A subset of patients with irritable bowel syndrome (IBS) have an increased number of mast cells (MCs) in the colonic mucosa. Psychological factors are believed to contribute to the course of IBS. AIMS: To examine associations between fatigue, depression and MCs of the colonic mucosa in IBS. METHODS: Colonic biopsies were taken from 50 Rome II IBS patients, 21 healthy controls and 11 depressed/fatigued patients without IBS. The cellularity of the lamina propria was determined as the number of inflammatory cells per high power field (hpf) through a 400x microscope. The Fatigue Impact Scale (FIS) and the short form Beck Depression Inventory (BDI) evaluated the severity of fatigue and depression. RESULTS: IBS patients had a significant increase in the cellularity of the lamina propria compared with controls or with depressed patients (mean (SD) 94.5 (48-110) vs 68 (58-82) and 78 (87-90) cells per hpf, p = 0.005 and p = 0.05, respectively), in particular of MCs (9.3 (5.6-11.7) vs 4.0 (2.7-6.8) and 4.3 (2.8-7.8) cells per hpf, p = 0.001 and p = 0.005, respectively). Both the FIS and BDI scores were significantly higher in IBS or in depressed patients than in controls (p<0.001). In IBS, the FIS score correlated significantly with the cellularity of the lamina propria (r = 0.51, p<0.0001) and MCs (r = 0.64, p<0.0001). In IBS, the BDI score correlated significantly with MCs (r = 0.29, p = 0.03). CONCLUSIONS: Elevated MCs counts are a key feature of the low-grade inflammatory infiltrate in the caecal mucosa of IBS. Fatigue and depression are associated with mucosal cell counts, in particular MCs, suggesting that psychological factors are associated with the low-grade inflammatory infiltrate in IBS.

Pharmacological targeting of NF-kappaB potentiates the effect of the topoisomerase inhibitor CPT-11 on colon cancer cells.

NF-kappaB interferes with the effect of most anti-cancer drugs through induction of anti-apoptotic genes. Targeting NF-kappaB is therefore expected to potentiate conventional treatments in adjuvant strategies. Here we used a pharmacological inhibitor of the IKK2 kinase (AS602868) to block NF-kappaB activation. In human colon cancer cells, inhibition of NF-kappaB using 10 microM AS602868 induced a 30-50% growth inhibitory effect and strongly enhanced the action of SN-38, the topoisomerase I inhibitor and CPT-11 active metabolite. AS602868 also potentiated the cytotoxic effect of two other antineoplasic drugs: 5-fluorouracil and etoposide. In xenografts experiments, inhibition of NF-kappaB potentiated the antitumoural effect of CPT-11 in a dose-dependent manner. Eighty-five and 75% decreases in tumour size were observed when mice were treated with, respectively, 20 or 5 mg kg(-1) AS602868 associated with 30 mg kg(-1) CPT-11 compared to 47% with CPT-11 alone. Ex vivo tumour analyses as well as in vitro studies showed that AS602868 impaired CPT-11-induced NF-kappaB activation, and enhanced tumour cell cycle arrest and apoptosis. AS602868 also enhanced the apoptotic potential of TNFalpha on HT-29 cells. This study is the first demonstration that a pharmacological inhibitor of the IKK2 kinase can potentiate the therapeutic efficiency of antineoplasic drugs on solid tumours.

Inhibition of the NF-kappaB survival pathway via caspase-dependent cleavage of the IKK complex scaffold protein and NF-kappaB essential modulator NEMO.

Apoptosis is mediated by cysteine-dependent, aspartate-directed proteases of the caspase family that proteolyse strategic intracellular substrates to induce cell suicide. We describe here that engagement of apoptotic processes by Fas triggering or by staurosporine stimulation leads to the caspase-dependent inactivation of the nuclear factor kappa B (NF-kappaB) pathway after cleavage of IKK1 (IkappaB kinase 1) and NEMO (NF-kappaB essential modulator), which are needed to transduce NF-kappaB activation signals. In this study, we have analyzed in more detail, the role of NEMO cleavage, as NEMO, but not IKK1, is important for the pro-survival actions of NF-kappaB. We demonstrate that NEMO is cleaved after Asp355 to remove the last 64 C-terminal amino acids. This short form was unable to rescue NF-kappaB activation by tumor necrosis factor-alpha (TNF-alpha) when transfected in NEMO-deficient cells. Consequently, inactivation of NEMO resulted in an inhibition of the expression of antiapoptotic NF-kappaB-target genes coding for caspase inhibitors (cIAP-1, cIAP-2) or adaptors of the TNF receptor family. NEMO-deficient Jurkat cells transiently expressing a non-cleavable mutant of NEMO were less sensitive to TNF-alpha-induced apoptosis. Therefore, downmodulation of NF-kappaB activation via the proteolytic cleavage of NEMO could represent an amplification loop for apoptosis.

AS602868, a dual inhibitor of IKK2 and FLT3 to target AML cells.

Acute myeloid leukemia (AML) cells carry molecular defects that promote their leukemic proliferation, resistance to apoptosis and defect in differentiation. Pharmacological targeting of the nuclear factor kappaB (NF-kappaB) pathway has been shown to promote apoptosis of primary AML cells and to sensitize blasts to neoplastic drugs (Frelin, Blood 2005, 105, 804). The Fms-like tyrosine kinase 3 (FLT3), which sustains proliferation of normal hematopoietic progenitors is frequently overexpressed or mutated in AML patients. Using Ba/F3 murine pre-B cells transfected with various mutants of FLT3 (ITD, D835V, D835Y) and the MV4-11 human AML line, we show that normal or oncogenic stimulation of FLT3 led to activation of NF-kappaB. Pharmacological inhibition of either FLT3 with AG1296 or NF-kappaB with the small molecule inhibitor of IkappaB kinase-2 AS602868 reduced viability and triggered cell death. Moreover, AS602868 was also found to interfere directly with FLT3 kinase activation. AS602868 thus appears to target two different kinases that play a crucial role in the pathogenesis of AML, making it particularly attractive as a new therapeutical approach for AML.

[Biomodulation of transcriptional factor NF-kappa B by ionizing radiation]. / Biomodulation du facteur de transcription NF-kappaB par les radiations ionisantes.

NF-kappaB (Nuclear Factor-kappaB) was described for the first time in 1986 as a nuclear protein binding to the kappa immunoglobulin-light chain enhancer. Since then, NF-kappaB has emerged as an ubiquitous factor involved in the regulation of numerous important processes as diverse as immune and inflammatory responses, apoptosis and cell proliferation. These last two properties explain the implication of NF-kappaB in the tumorigenic process as well as the promise of a targeted therapeutic intervention. This review focuses on the current knowledge on NF-kappaB regulation and discusses the therapeutic potential of targeting NF-kappaB in cancer in particular during radiotherapy.

Activation of nuclear factor kappaB through the IKK complex by the topoisomerase poisons SN38 and doxorubicin: a brake to apoptosis in HeLa human carcinoma cells.

The transcription factor nuclear factor (NF) kappaB is involved in the regulation of cell survival. NFkappaB is activated in many malignant tumors and seems to play a role in the resistance to cytostatic treatments and escape from apoptosis. We have studied the effects on NFkappaB activation of two topoisomerase poisons and DNA damaging agents that are used in chemotherapy: SN38 (7-ethyl-10-hydroxycamptothecin), the active metabolite of CPT11, and doxorubicin. In HeLa cells, both drugs activate NFkappaB using a preexisting pathway that requires a functional IkappaB-specific kinase complex, IkappaB-specific kinase activation, IkappaB-alpha phosphorylation, and degradation. Blocking NFkappaB activation by stable expression of a mutant super-repressor IkappaB-alpha molecule sensitized HeLa cells to the apoptotic actions of drugs and tumor necrosis factor. RNase protection assay analysis demonstrate that NFkappaB is involved in the regulation of a complex pattern of gene activation and repression during the cellular response of HeLa cells to topoisomerase poisons. The blockade of NF-kappaB activation seems to shift the death/survival balance toward apoptosis.

Ikappa b-alpha, the NF-kappa B inhibitory subunit, interacts with ANT, the mitochondrial ATP/ADP translocator.

The transcription factor NF-kappaB regulates a wide set of genes involved in the establishment of many cellular processes that control cell activation, proliferation, and apoptosis. IkappaB inhibitory subunits integrate NF-kappaB activation signals through phosphorylation and ubiquitination of its N-terminal domain. Using the two-hybrid system in yeast, we searched for IkappaB-alpha N-terminal domain interactors and therefore potential NF-kappaB regulators. An interaction of IkappaB-alpha with the mitochondrial ATP/ADP translocator ANT was detected in yeast and confirmed in glutathione S-transferase pull-down assays and co-precipitation experiments in transfected cells. Subcellular cell fractionation, resistance to proteinase K treatment, and electron microscopy experiments demonstrated the presence of IkappaB-alpha and associated p65 NF-kappaB in the mitochondrial intermembrane space. IkappaB-alpha.NF-kappaB appeared to be released from mitochondria upon the induction of apoptosis by engagement of the Fas receptor. These data suggest that the mitochondrial IkappaB-alpha.NF-kappaB pool participates in the regulation of apoptosis.

Activation of nuclear factor kappaB and Bcl-x survival gene expression by nerve growth factor requires tyrosine phosphorylation of IkappaBalpha.

NGF has been shown to support neuron survival by activating the transcription factor nuclear factor-kappaB (NFkappaB). We investigated the effect of NGF on the expression of Bcl-xL, an anti-apoptotic Bcl-2 family protein. Treatment of rat pheochromocytoma PC12 cells, human neuroblastoma SH-SY5Y cells, or primary rat hippocampal neurons with NGF (0.1-10 ng/ml) increased the expression of bcl-xL mRNA and protein. Reporter gene analysis revealed a significant increase in NFkappaB activity after treatment with NGF that was associated with increased nuclear translocation of the active NFkappaB p65 subunit. NGF-induced NFkappaB activity and Bcl-xL expression were inhibited in cells overexpressing the NFkappaB inhibitor, IkappaBalpha. Unlike tumor necrosis factor-alpha (TNF-alpha), however, NGF-induced NFkappaB activation occurred without significant degradation of IkappaBs determined by Western blot analysis and time-lapse imaging of neurons expressing green fluorescent protein-tagged IkappaBalpha. Moreover, in contrast to TNF-alpha, NGF failed to phosphorylate IkappaBalpha at serine residue 32, but instead caused significant tyrosine phosphorylation. Overexpression of a Y42F mutant of IkappaBalpha potently suppressed NFG-, but not TNF-alpha-induced NFkappaB activation. Conversely, overexpression of a dominant negative mutant of TNF receptor-associated factor-6 blocked TNF-alpha-, but not NGF-induced NFkappaB activation. We conclude that NGF and TNF-alpha induce different signaling pathways in neurons to activate NFkappaB and bcl-x gene expression.

Tyrosine phosphorylation-dependent activation of NF-kappa B. Requirement for p56 LCK and ZAP-70 protein tyrosine kinases.

Phosphorylation of the N-terminal domain of I kappa B inhibitory subunits induces activation of the transcription factor NF-kappa B. Although serine phosphorylation has been shown to induce ubiquitination and subsequent proteasome-mediated degradation of I kappa B-alpha, little is known about the mechanisms that lead to release of active NF-kappa B in T cells as a consequence of tyrosine phosphorylation of I kappa B-alpha [Imbert, V., Rupec, R.A., Livolsi, A., Pahl, H.L., Traenckner, B.M., Mueller-Dieckmann, C., Farahifar, D., Rossi, B., Auberger, P., Baeuerle, P. & Peyron, J.F. (1996) Cell 86, 787--798]. The involvement of the tyrosine kinases p56(lck) and ZAP-70 in this reaction is demonstrated here using specific pharmacological inhibitors and Jurkat mutants unable to express these kinases. Although the inhibitors prevented both pervanadate-induced phosphorylation of I kappa B-alpha on Tyr42 and NF-kappa B activation, we observed that, in p56(lck)-deficient Jurkat mutants, NF-kappa B could still associate with I kappa B-alpha despite phosphorylation on Tyr42. Furthermore, the SH2 domain of p56(lck) appeared to be required for pervanadate-induced NF-kappa B activation but not for Tyr42 phosphorylation. These results show that p56(lck) and ZAP-70 are key components of the signaling pathway that leads to phosphotyrosine-dependent NF-kappa B activation in T cells and confirm that tyrosine kinases must control at least two different steps to induce activation of NF-kappa B. Finally, we show that H(2)O(2), which stimulates p56(lck) and ZAP-70 in T cells, is an activator of NF-kappa B through tyrosine phosphorylation of I kappa B-alpha.

Cleavage and relocation of the tyrosine kinase P59FYN during Fas-mediated apoptosis in T lymphocytes.

Ligation of Fas with its natural ligand or with anti-Fas antibodies induces an apoptotic program in Fas sensitive cells. We report here the identification of the tyrosine kinase p59Fyn as a substrate for CPP32-like proteinases and more particularly caspase 3 during Fas-mediated apoptosis in Jurkat T cells. Inhibition of CPP32-like proteinases by Ac-Asp-Glu-Val-Asp-aldehyde but not by Ac-Tyr-Val-Ala-Asp-aldehyde prevents CPP32, PARP and p59Fyn cleavage indicating that CPP32 or CPP32-like proteinases are responsible for the cleavage of p59Fyn. Cleavage occurs in the N-terminal domain of p59Fyn between Asp19 and Gly20 and is accompanied by relocation of an active p57Fyn kinase to cytoplasm of Fas-stimulated Jurkat cells as judged by both biochemical and confocal microscopy experiments. Thus, p59Fyn relocation and activity may play an important role during Fas-mediated cell death in human T lymphocytes.

Tumor necrosis factor alpha-mediated inhibition of melanogenesis is dependent on nuclear factor kappa B activation.

Melanogenesis is a physiological process resulting in the synthesis of melanin pigments which play a crucial protective role against skin photocarcinogenesis. In vivo, solar ultraviolet light triggers the secretion of numerous keratinocyte-derived factors that are implicated in the regulation of melanogenesis. Among these, tumor necrosis factor alpha (TNFalpha), a cytokine implicated in the pro-inflammatory response, down-regulates pigment synthesis in vitro. In this report, we aimed to determine the molecular mechanisms by which this cytokine inhibits melanogenesis in B16 melanoma cells. First, we show that TNFalpha inhibits the activity and protein expression of tyrosinase which is the key enzyme of melanogenesis. Further, we demonstrate that this effect is subsequent to a down-regulation of the tyrosinase promoter activity in both basal and cAMP-induced melanogenesis. Finally, we present evidence indicating that the inhibitory effect of TNFalpha on melanogenesis is dependent on nuclear factor kappa B (NFkappaB) activation. Indeed, overexpression of this transcription factor in B16 cells is sufficient to inhibit tyrosinase promoter activity. Furthermore, a mutant of inhibitory kappa B (IkappaB), that prevents NFkappaB activation, is able to revert the effect of TNFalpha on the tyrosinase promoter activity. Taken together, our results clarify the mechanisms by which TNFalpha inhibits pigmentation and point out the key role of NFkappaB in the regulation of melanogenesis.

Tumor necrosis factor-alpha activation of nuclear transcription factor-kappaB in marrow macrophages is mediated by c-Src tyrosine phosphorylation of Ikappa Balpha.

Tumor necrosis factor-alpha (TNF) exerts its transcriptional effects via activation of nuclear transcription factor-kappa B (NF-kappaB). NF-kappaB is sequestered in the cytosol by Ikappa Balpha and, in most cells, released upon serine phosphorylation of this inhibitory protein which then undergoes rapid, ubiquitin-dependent degradation. In contrast, we find TNF induction of NF-kappaB in murine bone marrow macrophages (BMMs), is mediated, by c-Src, in a cell, and cytokine specific manner. The non-receptor tyrosine kinase is rapidly mobilized and activated upon TNF exposure. Within the same time frame, TNF induced c-Src associates with Ikappa Balpha in a long lived complex. The proto-oncogene, when associated with Ikappa Balpha phosphorylates the inhibitory protein on tyrosine 42. Consistent with the pivotal role played by c-Src in TNF-induced Ikappa Balpha tyrosine phosphorylation, NF-kappaB activation, by the cytokine, is markedly delayed and reduced in c-src-/- BMMs. Underscoring the physiological significance of c-Src activation of NF-kappaB, TNF induction of IL-6, which is an NF-kappaB mediated event, is substantially diminished in c-src-/- BMMs.

Two distinct regions of the CD28 intracytoplasmic domain are involved in the tyrosine phosphorylation of Vav and GTPase activating protein-associated p62 protein.

The T cell-associated CD28 molecule plays a key role in T cell co-stimulation. Its ligation induces the tyrosine phosphorylation of numerous proteins including CD28 itself as well as a restricted set of substrates of 97 and 62-68 kDa which are poorly phosphorylated by the tyrosine kinases induced by CD3-TCR triggering. In this study, we identify these substrates as the product of the vav proto-oncogene and as a 62 kDa protein that could correspond at least in part to p62dok, the 62 kDa adaptor molecule associated to p120 Ras-GTPase activating protein. Both p97vav and p62 are tyrosine phosphorylated upon CD28 ligation by mAb or by its counter-receptor B7-1/CD80. Using CD28 mutants, we also show that Vav and p62 tyrosine phosphorylation is regulated by distinct domains within the CD28 cytoplasmic tail: residues 173-181 for Vav and residues 182-202 for p62. Finally, the phosphorylation of Vav and p62 does not require an intact binding site for Grb-2 or p85 SH2 domains. We thus demonstrate that the CD28 cytoplasmic domain contains at least three functionally independent regions involved in CD28-induced signal transduction, since in addition to the Grb-2 and p85 SH2 domain binding site (Tyr173), residues 173-181 and 182-202 are associated with Vav and p62 tyrosine phosphorylation respectively.

Endopeptidase 24.11 (CD10/NEP) is required for phorbol ester-induced growth arrest in Jurkat T cells.

Jurkat T cells express a functional endopeptidase 24.11 that is involved in the regulation of T cell activation. We have analyzed the effect of ectopic CD10 expression in mutant Jurkat cell clones that fail to express CD10 and, unlike wild-type cells, are resistant to the growth-inhibitory effects of the protein kinase C activator, PMA. No differences in the expression of the mRNA encoding the alpha, beta, gamma, delta, epsilon, and zeta isoforms of PKC were found in parental vs. PMA-resistant Jurkat cells, ruling out the possibility that the defect could be accounted for by an altered expression of one of these isoforms. Phorbol ester-induced growth arrest was not due to apoptosis since PMA failed to trigger DNA fragmentation in parental and mutant Jurkat T cells. CD10 mRNA expression and activity were abrogated in four independent PMA-resistant Jurkat T cell clones compared to parental cells, whereas the activities of several other peptidases were unaffected. Transfection of one mutant clone with a functional endopeptidase 24.11 restored in a significant manner PMA-induced growth arrest in all the clones selected and tested, whereas transfection of an inactive form of endopeptidase 24.11 had no effect, demonstrating that the enzymatic activity of CD10 is critical in the mediation of the PMA growth arrest. The data presented here demonstrate that a functional CD10 is required for PMA-induced growth arrest in Jurkat cells and provide further evidence for a role of endopeptidase 24.11 in the regulation of tumor cell proliferation.

The protein tyrosine kinase p56(lck) regulates the serine-base exchange activity in Jurkat T cells.

Different classes of protein kinase inhibitors for protein kinase C, cAMP-dependent protein kinase or protein tyrosine kinases have been studied for their effect on phospholipid metabolism. The results show that among the compounds studied, only 4'-aminohydroxyflavone (AHF), previously described as a specific inhibitor of the protein tyrosine kinase p56(lck), markedly increased phosphatidylserine synthesis in Jurkat T cells. The biosyntheses of phosphatidylcholine and phosphatidylethanolamine were not affected. Also, the synthesis of phospholipids from tritium-labeled fatty acid as precursor was left unchanged by the p56(lck) inhibitor. The decreased phosphatidylserine synthesis induced when triggering the CD3-TCR complex was impaired by AHF, suggesting that p56(lck) could be implicated in the regulation of the serine-base exchange enzyme system. Direct evidence for the participation of p56(lck) in the regulation of the serine-base exchange enzyme system was obtained by using p56(lck)-deficient Jurkat cells (J.CaM 1.6) in which the basal base exchange activity was markedly increased and on the other hand AHF had no effect. In addition, transfection of J.Cam 1.6 cells with p56(lck)-cDNA allowed recovery of the AHF activity.

CD10 (endopeptidase 24.11) is a thymic peptide-degrading enzyme possibly involved in the regulation of thymocyte functions.

Human immature thymocytes express significant levels of the CD10 (endopeptidase 24.11) cell surface antigen. We report here that IOB5, an anti-CD10 mAb, as well as the phorbol ester PMA down-regulate CD10 activity at the surface of human thymocytes. The kinetics of CD10 modulation were drastically different for both effectors, indicating different regulatory mechanisms. We also demonstrated that intact human thymocytes hydrolyze thymopentin and that CD10 significantly participates in this process. Finally, we found that thymopentin and to a lesser extent phosphoramidon, a specific endopeptidase 24.11 inhibitor, induced up-regulation of CD4 and CD8 molecules at the thymocyte cell surface. In view of these results, we suggest that down-regulation of endopeptidase 24.11 at the thymocyte cell surface might reduce its activity toward thymic factors possibly involved in the regulation of thymocyte functions.