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Inhibition of Nuclear Factor κB Activation Attenuates Apoptosis Resistance in Lymphoid Cells
Abstract
Death-inducing ligands (DILs) such as tumor necrosis factor α (TNFα) or the cytotoxic drug doxorubicin have been shown to activate a nuclear factor κB (NFκB)-dependent program that may rescue cells from apoptosis induction. We demonstrate here that TRAIL (TNF-related apoptosis-inducing ligand), a recently identified DIL, also activates NFκB in lymphoid cell lines in a kinetic similar to TNFα. NFκB activity is independent from FADD, caspases, and apoptosis induction. To study the influence of NFκB activity on apoptosis mediated by TRAIL, CD95, TNFα, or doxorubicin, NFκB activation was inhibited using the proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal or transient overexpression of mutant IκBα. Sensitivity for induction of apoptosis was markedly increased by these treatments in apoptosis sensitive cell lines. Moreover, both in cell lines and in primary leukemia cells that are resistant towards induction of apoptosis by DILs and doxorubicin, antagonization of NFκB activity partially restored apoptosis sensitivity. These data suggest that inhibition of NFκB activation may provide a molecular approach to increase apoptosis sensitivity in anticancer treatment.
... Evidence for the importance of activation of NF-B in resistance to apoptosis came from studies on leukemia cells in which inhibition of NF-B was associated with increased sensitivity to apoptosis induced by TNF-␣ and activation of CD95 and TRAIL (21). TRAIL-induced apoptosis of keratinocyte lines was inhibited by IL-1, which was shown to activate NF-B (22). ...
... The proteasome inhibitor LLnL was shown in previous studies to inhibit the degradation of IB and thereby to prevent activation of NF-B (21,34). To determine whether LLnL inhibited activation of NF-B in melanoma cells, we used EMSA to measure NF-B activation in the melanoma lines before and after activation by TRAIL and the effect of LLnL on this activation. ...
... Another line, Mel-JG, was very sensitive to TRAIL-induced apoptosis alone, and LLnL could not further increase this sensitivity. These results suggest that NF-B activation is a significant factor in protection against TRAIL-induced apoptosis of melanoma, as reported by others in studies on lymphoid cells (21) and other cancers (24,25). In contrast to the increased sensitivity to TRAIL induced by LLnL, the latter did not increase the sensitivity of the melanoma lines to apoptosis induced by CD40L, TNF-␣, or FasL, the exception being small increases in sensitivity of one line to FasL. ...
Previous studies have shown that activation of NF-kB can inhibit apoptosis induced by a number of stimuli. It is also known that TNF-related apoptosis-inducing ligand (TRAIL) can activate NF-kB through the death receptors TRAIL-R1 and TRAIL-R2, and decoy receptor TRAIL-R4. In view of these findings, we have investigated the extent to which activation of NF-kB may account for the variable responses of melanoma lines to apoptosis induced by TRAIL and other TNF family members. Pretreatment of the melanoma lines with the proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (LLnL), which is known to inhibit activation of NF-kB, was shown to markedly increase apoptosis in 10 of 12 melanoma lines with death receptors for TRAIL. The specificity of results for inhibition of NF-kB activation was supported by an increase of TRAIL-induced apoptosis in melanoma cells transfected with a degradation-resistant IkBa. Furthermore, studies with NF-kB reporter constructs revealed that the resistance of melanoma lines to TRAIL-induced apoptosis was correlated to activation of NF-kB in response to TRAIL. TRAIL-resistant sublines that were generated by intermittent exposure to TRAIL were shown to have high levels of activated NF-kB, and resistance to TRAIL could be reversed by LLnL and by the superrepressor form of IkBa. Therefore, these results suggest that activation of NF-kB by TRAIL plays an important role in resistance of melanoma cells to TRAIL-induced apoptosis and further suggest that inhibitors of NF-kB may be useful adjuncts in clinical use of TRAIL against melanoma. The Journal of Immunology, 2001, 166: 5337-5345.
... TNF-related apoptosis-inducing ligand (TRAIL), also called Apo2 ligand, is a member of the TNF cytokine family with highest homology to CD95 ligand (Wiley et al., 1995;Pitti et al., 1996). Several reports have documented cytotoxic effects of TRAIL in cell lines originating from different types of human neoplasm, including breast cancer, lung cancer, bladder cancer, prostate cancer, Kaposi's sarcoma and leukemia in vitro (Wiley et al., 1995;Pitti et al., 1996;Jeremias et al., 1998;Ashkenazi et al., 1999;Bonavida et al., 1999;Keane et al., 1999;Mori et al., 1998) and in haematological tumours ex vivo (Snell et al., 1997). Unlike TNF-␣ and CD95 ligand or agonistic CD95 antibody, TRAIL is innocuous to normal cells and tissues, as demonstrated both in vitro and in vivo in non-human primates and mice (Wiley et al., 1995;Pitti et al., 1996;Ashkenazi et al., 1999;Walczak et al., 1999). ...
... Proteasomes have been implicated in conferring protection of tumour cells against apoptotic stimuli, including TRAIL (Jeremias et al., 1998). To examine whether proteasome inhibition would attenuate apoptosis resistance to TRAIL in ESFT cells, we evaluated the effect of MG132, a highly potent peptide aldehyde proteasome inhibitor (Lee and Goldberg, 1998). ...
... The susceptibility of some tumour cells to stimulus-induced apoptosis is believed to be dependent on the activity of the surviving nuclear transcription factor NF-B (reviewed by Barkett and Gilmore, 1999). Proteasome inhibition sensitises lymphoid cells to apoptosis by TRAIL, apparently through inactivation of TRAIL-activated NF-B (Jeremias et al., 1998). Thus, inhibition of NF-B activation by the proteasome inhibitor MG132 might enable apoptosis sensitivity for TRAIL in otherwise resistant ESFT cell lines. ...
We investigated the cytotoxic responsiveness of 40 cell lines derived from representatives of the Ewing's sarcoma family of tumours (ESFT), i.e., Ewing's sarcoma (ES), peripheral primitive neuroectodermal tumour (pPNET) and Askin tumour (AT), to tumour necrosis factor–related apoptosis-inducing ligand (TRAIL). Incubation with TRAIL at 100 ng/ml induced cell death at 24 hr in 19 of 26 ES, 11 of 12 pPNET and 2 of 2 AT cell lines. Half-maximal cell death concentrations (IC50 values) varied from 0.1 to 20 ng/ml. TRAIL displayed potent cytotoxic activity against freshly derived ESFT cell isolates. Cytotoxicity was associated with phosphatidylserine expression and internucleosomal DNA fragmentation, features characteristic of apoptosis. The apoptotic programme in the sensitive ESFT VH-64 cell line revealed TRAIL-induced activation of FLICE/MACH1 (caspase-8) and CPP32/Yama/apopain (caspase-3) and processing of the prototype caspase substrate poly(ADP-ribose) polymerase. In addition, TRAIL provoked a collapse of the mitochondrial transmembrane potential (ΔΨm), parallelled by a reduction in ATP levels and release of cytochrome c from mitochondria into the cytosol. Inhibition of caspase-8 and caspase-3 by zIETDfmk and zDEVDfmk, respectively, substantially prevented TRAIL-induced apoptosis. However, zIETDfmk, but not zDEVDfmk, reduced TRAIL-mediated ΔΨm dissipation, indicating that TRAIL causes mitochondrial dysfunction through caspase-8 acting upstream of mitochondria. While macromolecule synthesis inhibitors (actinomycin D, cycloheximide) augmented susceptibility to TRAIL in TRAIL-responsive cell lines, these agents did not render TRAIL-resistant cell lines susceptible to TRAIL. However, the proteasome inhibitor MG132 sensitised to TRAIL in resistant cell lines. Collectively, these results show that TRAIL initiates effective death in the vast majority (80%) of cell lines derived from ESFT. Since TRAIL provoked cell death in ESFT ex vivo, this cytokine may be a promising drug for the treatment of ESFT in vivo.Int. J. Cancer 88:252–259, 2000. © 2000 Wiley-Liss, Inc.
... Several groups have shown that the transcription factor NF-O B plays a role in protecting against death induced by TNF [11][12][13][14]. Cells that are unable to appropriately activate NF-O B are significantly more susceptible to TNF-mediated apoptosis. ...
... The data in this report address the issue of whether NF-O B can regulate apoptosis that is induced by Fas or TCR cross-linking. While abundant evidence has shown that NF-O B can decrease TNF-induced apoptosis [11][12][13][14], it has been suggested that NF-O B plays no role in the more common pathway for T cell apoptosis that is mediated by Fas [12]. A protective role for NF-O B has not been previously explored in TCR-induced death, the most stringent test for a possible role in T cell homeostasis, or in apoptosis induced in normal, non-transformed lymph node T cells. ...
The maintenance of lymphocyte homeostasis by apoptosis is a critical regulatory mechanism in the normal immune system. The transcription factor NF-κB has been shown to play a role in protecting cells against death mediated by TNF. We show here that NF-κB also has a role in regulating Fas / APO-1 / CD95-mediated death, a major pathway of peripheral T cell death. Transfection of Jurkat cells with the NF-κB subunits p50 and p65 confers resistance against Fas-mediated apoptosis. Reciprocally, inhibition of NF-κB activation by a soluble peptide inhibitor or a dominant form of the NF-κB inhibitor, IκB, makes the cells more susceptible to Fas-mediated apoptosis. Furthermore, inhibition of NF-κB activation by a soluble peptide inhibitor rendered a T cell hybridoma more susceptible to TCR-mediated apoptosis. Correspondingly, transfection of p50 and p65 provided considerable protection from TCR-mediated apoptosis. These observations were corroborated by studies on Fas-mediated death in primary T cells. Concanavalin A-activated cycling T cell blasts from mice that are transgenic for the dominant IκB molecule have increased sensitivity to Fas-mediated apoptosis, associated with a down-regulation of NF-κB complexes in the nucleus. In addition, blocking TNF, itself a positive regulator of NF-κB, with neutralizing antibodies renders the cells more susceptible to anti-Fas-mediated apoptosis. In summary, our results provide compelling evidence that NF-κB protects against Fas-mediated death and is likely to be an important regulator of T cell homeostasis and tolerance.
... Following activation, the IB becomes phosphorylated, ubiquitinated, and degraded by the 26 S proteasome (8), resulting in the release of NF-B complexes and their nuclear translocation (6,9). It was through the use of mutant IB␣ constructs and RelA-negative mice that the important role of NF-B in host immunity and antiapoptotic gene expression was established (8,10,11). ...
... The role of NF-B in T cell survival was confirmed by Kolenko et al. (21), who demonstrated that inhibition of NF-B with the SN50 peptide led to an apoptotic response. The functional importance of NF-B to T cell survival was further established in studies demonstrating a mutant IB␣ repressor, constitutively inhibiting NF-B, could sensitize Jurkat cells to TNF-␣-induced apoptosis (10). The ability of tumor-derived gangliosides to mediate both NF-B degradation and T cell apoptosis, on the one hand, and the finding that the RelA transgene can protect Jurkat cells from SK-RC-45 induced death, on the other, suggests that NF-B inhibition may be an immune escape mechanism used by RCC. ...
T cells from cancer patients are often functionally impaired, which imposes a barrier to effective immunotherapy. Most pronounced are the alterations characterizing tumor-infiltrating T cells, which in renal cell carcinomas includes defective NF-κB activation and a heightened sensitivity to apoptosis. Coculture experiments revealed that renal tumor cell lines induced a time-dependent decrease in RelA(p65) and p50 protein levels within both Jurkat T cells and peripheral blood T lymphocytes that coincided with the onset of apoptosis. The degradation of RelA/p50 is critical for SK-RC-45-induced apoptosis because overexpression of RelA in Jurkat cells protects against cell death. The loss of RelA/p50 coincided with a decrease in expression of the NF-κB regulated antiapoptotic protein Bcl-xL at both the protein and mRNA level. The disappearance of RelA/p50 protein was mediated by a caspase-dependent pathway because pretreatment of T lymphocytes with a pan caspase inhibitor before coculture with SK-RC-45 blocked RelA and p50 degradation. SK-RC-45 gangliosides appear to mediate this degradative pathway, as blocking ganglioside synthesis in SK-RC-45 cells with the glucosylceramide synthase inhibitor, PPPP, protected T cells from tumor cell-induced RelA degradation and apoptosis. The ability of the Bcl-2 transgene to protect Jurkat cells from RelA degradation, caspase activation, and apoptosis implicates the mitochondria in these SK-RC-45 ganglioside-mediated effects.
... This complex can promote caspase-8 activation, which is released to the cytosol and cleaves effector caspases-3 and -7, ultimately triggering apoptosis in the target cell [7]. However, TRAIL is also able to induce proliferation in cells that are resistant to cell death induction, since it can also activate NF-κB-dependent signaling pathways [8,9]. For instance, TRAIL enhances survival and proliferation in human vascular endothelial cells [10], erythroid differentiation [11], angiogenesis and neovascularization in microvascular endothelial cells [12] or promotes proliferation and survival [13]. ...
... Furthermore, curcumin upregulates the pro-apoptotic molecular Smac/Diablo to suppress NF-κB and IAPs (cIAP-1 and cIAP-2), which induces apoptosis [42]. Studies suggest a positive feedback system between NF-κB and IAPs, as IAPs can be upregulated by NF-κB and vice versa [146][147][148]. Hence, downregulation of both NF-κB and IAPs protein might further suppress GBM tumorigenesis. ...
Glioblastoma (GBM) is the most malignant brain tumor and accounts for most adult brain tumors. Current available treatment options for GBM are multimodal, which include surgical resection, radiation, and chemotherapy. Despite the significant advances in diagnostic and therapeutic approaches, GBM remains largely resistant to treatment, with a poor median survival rate between 12 and 18 months. With increasing drug resistance, the introduction of phytochemicals into current GBM treatment has become a potential strategy to combat GBM. Phytochemicals possess multifarious bioactivities with multitarget sites and comparatively marginal toxicity. Among them, curcumin is the most studied compound described as a potential anticancer agent due to its multi-targeted signaling/molecular pathways properties. Curcumin possesses the ability to modulate the core pathways involved in GBM cell proliferation, apoptosis, cell cycle arrest, autophagy, paraptosis, oxidative stress, and tumor cell motility. This review discusses curcumin’s anticancer mechanism through modulation of Rb, p53, MAPK, P13K/Akt, JAK/STAT, Shh, and NF-κB pathways, which are commonly involved and dysregulated in preclinical and clinical GBM models. In addition, limitation issues such as bioavailability, pharmacokinetics perspectives strategies, and clinical trials were discussed.
... 94 One critical factor in cell survival is NF-B, a transcription factor that is induced by a variety of signals including oncogenes, cytokines, and anticancer drugs. [95][96][97][98][99] Remarkably, inactivation of NF-B dramatically enhances apoptosis following treatment with TNF-and certain anticancer agents, 100 owing, at least partially, to NF-B's ability to modulate the expression of components of death receptor complexes. 17 Another survival signal cascade, the PI3K/Akt Fig. 1-DNA damage and oncogenes initiate pathways involved in apoptosis, cell-cycle arrest, or survival. ...
The dogma that antineoplastic treatments kill tumour cells by damaging essential biological functions has been countered by the notion that treatment itself initiates a programmed cellular response. This response often produces the morphological features of apoptosis and is determined by a network of proliferation and survival genes, some of which are differentially expressed in normal and malignant cells. Correspondingly, mutations that interfere with the initiation or execution of apoptosis may produce tumour‐cell drug resistance. Remarkably, many of the genes that modulate apoptosis in response to cytotoxic drugs also affect apoptosis during tumour development; hence, the process of apoptosis provides a conceptual framework for understanding how cancer genes can influence the outcome of cancer therapy. Although the relative contribution of apoptosis to radiation and drug‐induced cell death remains controversial, clinical studies have associated anti‐apoptotic mutations with treatment failure. While careful preclinical and clinical studies will be necessary to resolve this point, our current understanding of apoptosis should facilitate the design of rational new therapies. Copyright © 1999 John Wiley & Sons, Ltd.
... These inhibitors, which on their own, did not induce apoptosis under the experimental conditions used in this study, increased CD95-dependent DNA fragmentation in human chondrocytes. Thus, the results presented here show that in chondrocytes, CD95-induced NF-B may have protective effects, although in other cell systems, NF-B activation apparently does not interfere with apoptosis induction by CD95 (29,37). ...
Objective
To examine the role of nuclear factor κB (NF‐κB) and caspases 3, 8, and 9 in CD95‐mediated apoptosis of normal chondrocytes.
Methods
First‐passage chondrocytes from normal human knee cartilage were stimulated with CD95 antibody, and cell death was determined by annexin V binding and by an enzyme‐linked immunosorbent assay. Activation of caspases 3, 8, and 9 was measured by Western blotting, and their role in death signaling was evaluated using caspase‐specific small peptide inhibitors. The influence of NF‐κB was determined by electrophoretic mobility shift assay (EMSA) and proteasome inhibition–dependent blocking of the degradation of inhibitor of NF‐κB.
Results
Low levels of NF‐κB activity were detected by EMSA in unstimulated chondrocytes. NF‐κB activity was increased in response to agonistic CD95 antibody. CD95 antibody–induced apoptosis was potentiated by the proteasome inhibitors MG‐132 and PS1, and this was associated with a reduced nuclear translocation of NF‐κB. Proteasome inhibitors also caused the induction of DNA fragmentation by tumor necrosis factor α. Procaspase 3 processing was enhanced by the proteasome inhibitor MG‐132. Procaspase 8 was undetectable by immunoblotting in whole cell lysates of chondrocytes, but caspase 8 messenger RNA was detected by reverse transcription–polymerase chain reaction. Furthermore, apoptosis induced by CD95 stimulation and proteasome inhibitors was blocked by the caspase 8–specific inhibitor Ac‐IETD‐CHO. Processing of procaspase 9 was not observed, and inhibition of CD95‐dependent cell death by the caspase 9 inhibitor Ac‐LEHD‐CHO was not significant.
Conclusion
These results suggest that CD95‐dependent cell death is enhanced by NF‐κB inhibition at and/or downstream of caspase 8 activation and that caspase 9 activation is not involved in CD95‐mediated apoptosis in chondrocytes.
... The first pathway, the canonical, involves the IKKα/IĸBα pathway resulting in the formation of predominantly p50/p65 dimers, whereas the second non-canonical pathway which requires IKKαdependent processing of p100 NF-ĸB2 and the generation of p52/RelB heterodimers. A numbers of studies show that NF-ĸB can be activated by TRAIL (Jeremias et al., 1998) (Devin et al., 2000). In addition, activated NF-ĸB can upregulate the expression of anti-apoptotic genes like cFLIP that results in inhibition of TRAIL-induced apoptosis (Gonzalvez and Ashkenazi, 2010). ...
TRAIL is a member of TNF superfamily and binding of TRAIL to its receptors
induces cell death with apparent specificity for carcinoma cells compared with
normal epithelium. On this basis, a number of TRAIL-targeted therapies are
currently under investigation as anti-cancer agents. Recent studies in our lab
have shown that membrane bound forms of both FasL and CD40L are more
potent in cell death induction, suggesting that a membrane bound TRAIL could
be more effective in cell death induction compared to the wild-type or the
soluble counterparts. TRAIL gene therapies have demonstrated that ex vivo
infection of cancer cells with a recombinant adenovirus expressing TRAIL
(RAdTRAIL) enhances apoptosis and promotes tumour regression. In the
present study, we have generated two adenoviral vectors. The first one
expressing wild-type TRAIL (RAd wtTRAIL) that subject to cleavage from cell
membrane and the second adenoviral vector expressing fusion CD40LTRAIL
protein that is resistant to metalloproteinase cleavage (RAd CD40LTRAIL). The
direct effects of these viruses were examined on TRAIL receptor positive
carcinomas either alone or in combination with different chemotherapeutic
drug. The RAd CD40LTRAIL that expressed the membrane bound
CD40LTRAIL was found to exhibit more cell death than the RAd wtTRAIL.
This cell death was through the activation of caspase 3/7.
... In a multivariate Cox regression model, SP1 expression was a useful marker for predicting long-term prognosis in TNBC patients receiving doxorubicin treatment, and we thus suggest that SP1 expression could serve as a prognostic marker in these patients. . However, the DNA damage induced by doxorubicin activates the NF-κ B pathway, leading to doxorubicin resistance in cancer cell lines, including in breast cancer cells [15][16][17][18] . ...
We investigated gene expression profiles of the NF-κB pathway in patients with triple-negative breast cancer (TNBC) receiving adjuvant chemotherapy to determine the prognostic value of NF-κB pathway genes according to chemotherapeutic regimen. We used the nCounter expression assay to measure expression of 11 genes (NFKB1, NFKB2, RELA, RELB, REL, TP53, FOXC1, TBP, SP1, STAT3 and IRF1 genes) belonging to the NF-κB pathway using mRNA extracted from paraffin-embedded tumor tissues from 203 patients diagnosed with TNBC. Of the 203 patients, 116 were treated with a chemotherapeutic regimen containing doxorubicin. As revealed by the expression profiles of the 11 genes, increased expression of SP1 was associated with poor prognosis in TNBC patients treated with adjuvant doxorubicin chemotherapy (5-year distant recurrence-free survival [5Y DRFS], low vs. high expression [cut-off: median]: 92.3% vs. 71.6%, P = 0.001). In a multivariate Cox regression model, SP1 expression was a useful marker for predicting long-term prognosis in TNBC patients receiving doxorubicin treatment, and we thus suggest that SP1 expression could serve as a prognostic marker in these patients.
... NF-kB promotes cancer development (breast cancer and Hodgkin's disease) by increasing cell proliferation and by inducing various IAP family members (xiap, hiap1 and hiap2) (Bargou et al., 1997;Gilmore et al., 1996;Jeremias et al., 1998;LaCasse et al., 1998;Luque & Gelinas, 1997;Nakshatri et al., 1997;Stehlik et al., 1998). Therefore, induction of IAPs by NF-kB activation is partially responsible for certain tumor cell survival and progression to neoplastic disease (LaCasse et al., 1998). ...
Abstract Non-genotoxic carcinogens act by promoting the clonal expansion of preneoplastic cells by directly or indirectly stimulating cell division or inhibiting cell loss in the target organ. The specific mode-of-action (MoA) by which some non-genotoxic carcinogens ultimately cause cancer is not completely understood. To date, there are several proposed MoAs for non-genotoxic carcinogens and some of these propose inhibition of apoptosis as one of the Key Events. In general, inhibition of apoptosis is considered a necessary step for cell survival and in theory can occur in combination or in association with other key promotional events, such as cell proliferation, oxidative stress, and inhibition of intercellular communication to promote carcinogenesis. However, the evidence supporting the role of inhibition of apoptosis as a necessary step in promoting specific chemically-induced tumors is often debated. To address this evidence, we reviewed studies that utilized prototypical nuclear receptor- mediated hepatocarcinogens. Based on this review, it is proposed that the ability to determine the importance of inhibition of apoptosis as a Key Event in the MoA for tumor promotion is hampered by the limitations of the methods utilized for its detection. This review provides an assessment of the strengths and limitations of the current methodology used for detection of apoptosis and provide suggestions for improving its detection, thereby strengthening the weight of evidence supporting inhibition of apoptosis as a Key Event in a MoA for tumor promotion.
... Ved administration af NFκB-inhibitorer kan apoptose stimuleres in vitro i celler fra prostatatumorer [28] og in vivo i celler fra colontumorer [29]. Endvidere ophaeves resistensen mod apoptose i lymfoide celler ved inhibering af NFκB [30]. Det er endnu ikke helt klarlagt, hvordan morfin haemmer NFκB, men i et enkelt studie har man vist, at morfin direkte nedregulerer frit NFκB ved frisaetning af nitrogenoxid (NO) (se senere) [31]. ...
Resumé Eksogene og endogene opiate alkaloider som f.eks. morfin tilskri-ves en bred vifte af farmakologiske og fysiologiske funktioner. For-uden deres analgetiske effekt har opioider indflydelse på vaekstre-guleringen af normalt og neoplastisk vaev. I denne oversigtsartikel fokuseres der på morfins virkning på tumorvaekst med vaegt på im-munregulatoriske, proliferative og antiproliferative mekanismer. … Hvo som svaelger den saft, når den først i kummen er blandet, faelder ej en tåre på kind den dag fra morgen til aften ej engang om hans moder var død og hans fader tillige, ej om hans broder, og hans elskede søn ved hans side draebes af fjenden … Således beskrev Homer opiums berusende effekt i Odyséen. Opium udvindes fra opiumvalmuen, papaver somniferum og vi-des at vaere benyttet som rusmiddel siden sumerernes tid 3.400 år før vor tidsregning. I 1803 identificeredes morfin som vaerende den vigtigste aktive komponent i opium. Opmaerk-somheden har traditionelt vaeret rettet mod eksogent admini-streret morfin, som i vidt omfang anvendes i smertebehand-ling til bl.a. cancerpatienter. Det er mindre kendt, at morfin også produceres i kroppen. Det er således påvist, at endogent morfin syntetiseres i flere hvirveldyr [1, 2], herunder mennesket, hvor syntesen bl.a. sti-muleres i forbindelse med et kirurgisk stressrespons [3, 4]. Hos mennesket er endogent morfin foreløbig isoleret fra plasma [3, 4] og hjertemuskulatur [5]. Morfin tilhører gruppen af alkaloider, en kemisk gruppe i hvilken man også finder nikotin, kokain og koffein. Ud over morfin producerer kroppen også opioide peptider såsom endorfiner, enkefaliner, dynorfiner og endomorfiner. Både alkaloider og peptider fungerer som signalmolekyler og bin-des med forskellig affinitet til opioidreceptorer. Disse recep-torer inddeles i tre undertyper: µ-, κ-og δ-receptorer. Opioid-receptorerne er overvejende lokaliseret til centralnervesyste-met, men findes også i mave-tarm-systemet, endotelvaev m.m. [1]. Opioide alkaloider og peptider er involverede i en raekke farmakologiske og fysiologiske funktioner [6]. Immunologisk synes morfin at have antiinflammatorisk effekt, mens opioide peptider virker proinflammatorisk [1]. Således kan begge klas-ser af signalmolekyler potentielt modificere cancervaekst. I denne oversigt fokuseres der på eksogene og endogene opi-oiders virkning på tumorvaekst med vaegt på proliferative, antiproliferative og immunregulatoriske mekanismer. Artiklen er baseret på litteratur fundet ved søgning i MED-LINE med anvendelse af følgende søgeord: morphine, opioid peptides, neoplasms, opioid receptor og nitric oxide.
... 38 Inhibition of the NFkB signaling pathway and p53 induction have been linked to DR5 upregulation. [39][40][41] However, our results showed that the Medinduced upregulation of TRAIL receptor DR5 was independent of p53 and NFkB as it induced increased DR5 expression in p53-deficient K562 cells in agreement with earlier studies. 42,43 Consistent with this finding, an agonist antibody specific for DR5 also reduced cell viability in cells pretreated with Med. ...
Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent with cancer cell-selective cell death inducing effect. However, the major limitation in the usage of TRAIL as a chemotherapeutic agent is the development of TRAIL resistance in many cancer types including myeloid leukemia. In this study, we report for the first time that Medicarpin (Med), a naturally occurring phytoalexin sensitizes myeloid leukemia cells to TRAIL-induced apoptosis. Combination of Med and TRAIL induced significantly higher apoptosis compared with that of the individual treatments of either agent alone through activation of both the extrinsic and the intrinsic cell death pathways characterized by the activation of caspases 8, 9, 3, and 7. Med treatment downregulated antiapoptotic proteins (Survivin, Bcl2, Bcl-xL, XIAP, and c-FLIP), upregulated pro-apoptotic proteins (Bax, Cytochrome C, Smac/Diablo, Bid, truncated Bid (tBid), p-eIF2α, Bip, and CHOP (CCAAT-enhancer binding protein homologous protein)), induced G2/M cell-cycle arrest, and increased the expression of the functional TRAIL receptor DR5 through activation of the ROS-JNK-CHOP pathway. Gain and loss of function studies clearly indicated that DR5 expression was critical for Med-induced TRAIL sensitization. The Med-induced TRAIL sensitization did not involve the NFkB signaling pathway or redistribution of DR5 in lipid rafts. The concomitant treatment with Med and TRAIL showed robust apoptotic effects in primary myeloid leukemia cells but had no toxic effects in primary human peripheral blood mononuclear cells (PBMCs). In conclusion, our results suggest that Med sensitizes myeloid leukemia cells to TRAIL-induced apoptosis through the upregulation of DR5 through activation of the ROS-JNK-CHOP pathway.
... Well-tolerated in normal tissue, Bort is clinically used as an anti-cancer agent to treat myelomas and lung cancers, and is currently being tested as an adjuvant for a variety of other cancers [6,29,30]. Bort-induced toxicity in cancer cells arises from their need for NF-kB turnover to facilitate DNA transcription and rapid cell division [6,31]. Non-or slowlydividing cells in normal tissue are better able to tolerate Bort action. ...
Increasing numbers of female patients survive cancer, but succumb to primary ovarian insufficiency after chemotherapy. We tested the hypothesis that Bortezomib (Bort) protects ovaries from doxorubicin (DXR) chemotherapy by treating female mice with Bort 1 hour prior to DXR. By preventing DXR accumulation in the ovary, Bort attenuated DXR-induced DNA damage in all ovarian cell types, subsequent γH2AFX phosphorylation, and resulting apoptosis in preantral follicles. Bort pretreatment extended the number of litters per mouse, improved litter size and increased pup weight following DXR treatment, thus increasing the duration of post-chemotherapy fertility and improving pup health. As a promising prophylactic ovoprotective agent, Bort does not interfere with cancer treatment, and is currently used as a chemotherapy adjuvant. Bort-based chemoprotection may preserve ovarian function in a non-invasive manner that avoids surgical ovarian preservation, thus diminishing the health complications of premature menopause following cancer treatment.
... Over the past years, it has become increasingly evident that TRAIL-R1 and TRAIL-R2, besides death signaling pathways, can also activate multiple non-cell death signal transduction pathways like NF-kB (nuclear factor k-light-chain-enhancer of activated B cells), PKC (protein kinase C), PI3K (phosphatidylinositide 3-kinases), AKT/PKB, Src (Rous sarcoma virus oncogene cellular homolog) and TAK1 (transforming growth factor-b activated kinase 1), as well as different members of the MAP-kinase family like ERK1/ERK2 (extracellular regulated kinases), JNK (c-Jun N-terminal kinase) and p38 ( Figure 1). 14,[42][43][44][45][46][47][48][49][50][51] Regarding the molecular determinants for TRAIL-mediated activation of cell death or non-apoptotic pathways, the engagement of different protein complexes has been proposed. In this respect, non-apoptotic pathways have been suggested to be activated within a secondary signaling complexes formed downstream of the DISC. ...
Localized in the plasma membrane, death domain-containing TNF-related apoptosis-inducing ligand (TRAIL) receptors, TRAIL-R1 and TRAIL-R2, induce apoptosis and non-apoptotic signaling when crosslinked by the ligand TRAIL or by agonistic receptor-specific antibodies. Recently, an increasing body of evidence has accumulated that TRAIL receptors are additionally found in noncanonical intracellular locations in a wide range of cell types, preferentially cancer cells. Thus, besides their canonical locations in the plasma membrane and in intracellular membranes of the secretory pathway as well as endosomes and lysosomes, TRAIL receptors may also exist in autophagosomes, in nonmembraneous cytosolic compartment as well as in the nucleus. Such intracellular locations have been mainly regarded as hide-outs for these receptors representing a strategy for cancer cells to resist TRAIL-mediated apoptosis. Recently, a novel function of intracellular TRAIL-R2 has been revealed. When present in the nuclei of tumor cells, TRAIL-R2 inhibits the processing of the primary let-7 miRNA (pri-let-7) via interaction with accessory proteins of the Microprocessor complex. The nuclear TRAIL-R2-driven decrease in mature let-7 enhances the malignancy of cancer cells. This finding represents a new example of nuclear activity of typically plasma membrane-located cytokine and growth factor receptors. Furthermore, this extends the list of nucleic acid targets of the cell surface receptors by pri-miRNA in addition to DNA and mRNA. Here we review the diverse functions of TRAIL-R2 depending on its intracellular localization and we particularly discuss the nuclear TRAIL-R2 (nTRAIL-R2) function in the context of known nuclear activities of other normally plasma membrane-localized receptors.
... Previously, we showed that inhibition of NF-κB increases TRAIL sensitivity in breast cancer cell lines [18]. Similar results were reported in other cancer cell lines [18,[60][61][62][63]. Again, our findings in this article that IκBKB LOF leads to enhanced TRAIL-induced caspase activation provide support for further studies of NF-κB inhibitors in combination with TRAIL. ...
Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) binds to its receptors, TRAIL-receptor 1 (TRAIL-R1) and TRAIL-receptor 2 (TRAIL-R2), leading to apoptosis by activation of caspase-8 and the downstream executioner caspases, caspase-3 and caspase-7 (caspase-3/7). Triple-negative breast cancer (TNBC) cell lines with a mesenchymal phenotype are sensitive to TRAIL while other breast cancer cell lines are resistant. The underlying mechanisms that control TRAIL sensitivity in breast cancer cells are not well understood. Here, we performed small interfering RNA (siRNA) screens to identify molecular regulators of the TRAIL pathway in breast cancer cells.
We conducted siRNA screens of the human kinome (691 genes), phosphatome (320 genes), and [almost equal to]300 additional genes in the mesenchymal TNBC cell line MB231. Forty-eight hours post-transfection of siRNA, parallel screens measuring caspase-8 activity, caspase-3/7 activity, or cell viability were conducted in the absence or presence of TRAIL for each siRNA relative to a negative control siRNA (siNeg). A subset of genes was screened in cell lines representing epithelial TNBC (MB468), HER2-amplified breast cancer (SKBR3), and estrogen receptor positive breast cancer (T47D). Selected putative negative regulators of the TRAIL pathway were studied using small molecule inhibitors.
The primary screens in MB231 identified 150 genes, including 83 kinases, 4 phosphatases, and 63 non-kinases, as potential negative regulators of TRAIL. The identified genes are involved in many critical cell processes including apoptosis, growth factor receptor signaling, cell cycle regulation, transcriptional regulation, and DNA repair. Gene network analysis identified four genes (PDPK1, IKBKB, SRC, and BCL2L1) that formed key nodes within the interaction network of negative regulators. A secondary screen of a subset of the genes identified in additional cell lines representing different breast cancer subtypes and sensitivities to TRAIL validated and extended these findings. Further, we confirmed that small molecule inhibition of SRC or BCL2L1 in combination with TRAIL sensitizes breast cancer cells to TRAIL-induced apoptosis, including cell lines resistant to TRAIL-induced cytotoxicity.
These data identify novel molecular regulators of TRAIL-induced apoptosis in breast cancer cells and suggest strategies for the enhanced application of TRAIL as a therapy for breast cancer.
... To determine the other roles of NF-κBi nI κB-ζ expression, we used NF-κB signaling inhibitors. Both Bay 11-7082, an inhibitor of IκBα phosphorylation [48], and LLnL, a proteasomal inhibitor [49], inhibited the expression of IκB-ζ at the mRNA ( Figure 3I)a n dp r otein ( Figure 3J) levels and NF-κB binding to the IκB-ζκB2 and κB1 probes in HUT-102 cells ( Figure 3K ). Both inhibitors suppressed the Tax-induced expression of IκB-ζ mRNA in CdCl 2 -treated JPX-9 cells ( Figure 3L). ...
Human T cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T cell leukemia (ATL) and various inflammatory disorders including HTLV-I-associated myelopathy/tropical spastic paraparesis. HTLV-I oncoprotein Tax is known to cause permanent activation of many cellular transcription factors including nuclear factor-κB (NF-κB), cyclic adenosine 3',5'-monophosphate response element-binding protein, and activator protein 1 (AP-1). Here, we show that NF-κB-binding cofactor inhibitor of NF-κB-ζ (IκB-ζ) is constitutively expressed in HTLV-I-infected T cell lines and ATL cells, and Tax transactivates the IκB-ζ gene, mainly through NF-κB. Microarray analysis of IκB-ζ-expressing uninfected T cells demonstrated that IκB-ζ induced the expression of NF-κB. and interferon-regulatory genes such as B cell CLL/lymphoma 3 (Bcl3), guanylate-binding protein 1, and signal transducer and activator of transcription 1. The transcriptional activation domain, nuclear localization signal, and NF-κB-binding domain of IκB-ζ were required for Bcl3 induction, and IκB-ζ synergistically enhanced Tax-induced Bcl3 transactivation in an NF-κB-dependent manner. Interestingly, IκB-ζ inhibited Tax-induced NF-κB, AP-1 activation, and HTLV-I transcription. Furthermore, IκB-ζ interacted with Tax in vitro and this interaction was also observed in an HTLV-I-transformed T cell line. These results suggest that IκB-ζ modulates Tax-dependent and Tax-independent gene transcription in T cells. The function of IκB-ζ may be of significance in ATL genesis and pathogenesis of HTLV-I-associated diseases.
... These inhibitors, which on their own, did not induce apoptosis under the experimental conditions used in this study, increased CD95-dependent DNA fragmentation in human chondrocytes. Thus, the results presented here show that in chondrocytes, CD95-induced NF-B may have protective effects, although in other cell systems, NF-B activation apparently does not interfere with apoptosis induction by CD95 (29,37). ...
Objective
To examine the role of nuclear factor κB (NF-κB) and caspases 3, 8, and 9 in CD95-mediated apoptosis of normal chondrocytes.Methods
First-passage chondrocytes from normal human knee cartilage were stimulated with CD95 antibody, and cell death was determined by annexin V binding and by an enzyme-linked immunosorbent assay. Activation of caspases 3, 8, and 9 was measured by Western blotting, and their role in death signaling was evaluated using caspase-specific small peptide inhibitors. The influence of NF-κB was determined by electrophoretic mobility shift assay (EMSA) and proteasome inhibition–dependent blocking of the degradation of inhibitor of NF-κB.ResultsLow levels of NF-κB activity were detected by EMSA in unstimulated chondrocytes. NF-κB activity was increased in response to agonistic CD95 antibody. CD95 antibody–induced apoptosis was potentiated by the proteasome inhibitors MG-132 and PS1, and this was associated with a reduced nuclear translocation of NF-κB. Proteasome inhibitors also caused the induction of DNA fragmentation by tumor necrosis factor α. Procaspase 3 processing was enhanced by the proteasome inhibitor MG-132. Procaspase 8 was undetectable by immunoblotting in whole cell lysates of chondrocytes, but caspase 8 messenger RNA was detected by reverse transcription–polymerase chain reaction. Furthermore, apoptosis induced by CD95 stimulation and proteasome inhibitors was blocked by the caspase 8–specific inhibitor Ac-IETD-CHO. Processing of procaspase 9 was not observed, and inhibition of CD95-dependent cell death by the caspase 9 inhibitor Ac-LEHD-CHO was not significant.Conclusion
These results suggest that CD95-dependent cell death is enhanced by NF-κB inhibition at and/or downstream of caspase 8 activation and that caspase 9 activation is not involved in CD95-mediated apoptosis in chondrocytes.
... NOD2 protein, the product of CARD-15, induces activation of NF-jb, in response to microbial insults; this transcriptional activator is able to increase the production of pro-inflammatory cytokines, adhesion molecules and prevent the apoptosis of T lymphocytes. 33,34 In psoriatic lesions, there is an increase of particular mediators such as TNF-a, IL-1 and other molecules, activated by NF-jb; for this reason, it is possible to hypothesize that NOD2 plays an important role in the pathogenesis of psoriasis, despite the lack of studies demonstrating this correlation. This pathogenetic hypothesis has been shown in several studies [35][36][37] but different authors have demonstrated a lack of association between CARD15 ⁄ NOD2 SNP and psoriasis in different populations. ...
Psoriasis is a common, inflammatory, chronic, relapsing skin disease. New insight about the etiology of this disease shows the important role played by the epidermal barrier function, its integrity and pathogen responses in combination with microbial environmental factors. A pivotal role in the management of this balance is played by NLR genes, also known as NBD-LRR or CATERPILLER, that encode important mediators of innate immunity and provide the first line of defense against pathogens. The polymorphism of these genes is implicated in the pathogenesis of several immunological diseases and might be of importance in the pathogenesis of barrier organ disorders. Crohn's disease is considered archetypal of these kinds of disorders; similarities between Crohn's disease and psoriasis and their similar pathogenetic mechanisms may support the concept of psoriasis as a barrier organ disorder and common genetic ground lying behind these illnesses. Considering psoriasis as a "barrier organ disease" is not only a mere mental exercise; this consideration may, in fact, open new prospects in the treatment of these disorders just by preventing alterations of microbial flora or regulating the response of the host to infective diseases.
... These results emphasize the importance of signaling components involved in the activation of NF-κB in H. pylori-induced activation of the CD69 promoter. Because activation of the CD69 promoter by H. pylori infection requires the activation of NF-κB, we blocked NF-κB activation with Bay 11-7082, an inhibitor of IκBα phosphorylation [27] , or LLnL, a proteasome inhibitor [28] . The latter is known to inhibit the activation of NF-κB by blocking the degradation of the IκBα protein. ...
To investigate and elucidate the molecular mechanism that regulates inducible expression of CD69 by Helicobacter pylori (H. pylori) infection.
The expression levels of CD69 in a T-cell line, Jurkat, primary human peripheral blood mononuclear cells (PBMCs), and CD4+ T cells, were assessed by immunohistochemistry, reverse transcription polymerase chain reaction, and flow cytometry. Activation of CD69 promoter was detected by reporter gene. Nuclear factor (NF)-κB activation in Jurkat cells infected with H. pylori was evaluated by electrophoretic mobility shift assay. The role of NF-κB signaling in H. pylori-induced CD69 expression was analyzed using inhibitors of NF-κB and dominant-negative mutants. The isogenic mutants with disrupted cag pathogenicity island (cagPAI) and virD4 were used to elucidate the role of cagPAI-encoding type IV secretion system and CagA in CD69 expression.
CD69 staining was detected in mucosal lymphocytes and macrophages in specimens of patients with H. pylori-positive gastritis. Although cagPAI-positive H. pylori and an isogenic mutant of virD4 induced CD69 expression, an isogenic mutant of cagPAI failed to induce this in Jurkat cells. H. pylori also induced CD69 expression in PBMCs and CD4+ T cells. The activation of the CD69 promoter by H. pylori was mediated through NF-κB. Transfection of dominant-negative mutants of IκBs, IκB kinases, and NF-κB-inducing kinase inhibited H. pylori-induced CD69 activation. Inhibitors of NF-κB suppressed H. pylori-induced CD69 mRNA expression.
The results suggest that H. pylori induces CD69 expression through the activation of NF-κB. cagPAI might be relevant in the induction of CD69 expression in T cells. CD69 in T cells may play a role in H. pylori-induced gastritis.
... TRAIL can activate the transcription factor NF-jB, which can protect from TRAIL-induced apoptosis [12][13][14]. To investigate whether TRAIL-induced NF-jB activation requires TRADD, we treated WT and TRADD À/À MEFs with GST-TRAIL because we were able to increase the activity in MEFs compared to rmTRAIL (see Fig. 4A for cell death activity [11]). ...
One major obstacle in the clinical application of TRAIL as a cancer therapeutic agent is the acquisition of TRAIL resistance. We found that deficiency of TRADD sensitizes cells to TRAIL-induced apoptosis. Enhanced cell death in TRADD(-/-) MEFs is associated with defective NF-κB activation, indicating that the pro-survival function of TRADD in TRAIL signaling is mediated at least in part via NF-κB activation. Moreover, siRNA knock-down of TRADD in cancer cells sensitizes them to TRAIL-induced apoptosis. Thus, TRADD has a survival role in TRAIL signaling and may be one potential target for overcoming TRAIL resistance in cancer therapy.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
La surinfection par le Virus de l'Hépatite Delta (HDV) de patients chroniquement infectés par le virus de l'hépatite B (HBV) est la forme la plus agressive d'hépatite virale chronique avec une progression plus rapide vers la cirrhose ainsi qu’un risque accru d'insuffisance hépatique et de carcinome hépatocellulaire. Environs 15 à 20 millions de personnes sont co-infectés par ces deux virus ; il s’agit d’une des coinfections la plus répandue et difficile à combattre au monde. Les traitements disponibles pour HBV ne sont pas efficaces contre HDV. L'unique option thérapeutique pour les patients co-infectés par HBV et HDV repose sur l'utilisation d'interféron alpha pégylé avec de très nombreux effets secondaires et une efficacité très limitée. Très peu de molécules sont actuellement en R&D pour HDV et à priori aucune ne cible directement la réplication du virus. Il est donc urgent de développer de nouvelles stratégies antivirales efficaces pour traiter les patients co-infectés. Lors d’une infection virale classique, plusieurs cytokines sont produites. Plusieurs études ont montré une activation de la voie de signalisation de l’interféron par HDV in vitro et in vivo. Toutefois il n’y a aucune donnée sur l’activation de la voie « Nuclear factor κ B » (NFκB) par HDV, et même une absence d’activation de celui-ci a déjà été proposée par notre laboratoire. Notre objectif était de tester l'effet sur HDV d’immunomodulateurs activant cette voie. Après criblage de différents inducteurs des voies canoniques et non-canoniques de NFκB nous avons identifié un agoniste du récepteur "toll like" 1/2 (Pam3CSK4) et un agoniste du récepteur à la lymphotoxine β (BS1) diminuant les ARN et protéines d’HDV. Des études approfondies ont montré un effet anti-HDV dose-dépendant et stable malgré une augmentation de la quantité de particules virales utilisées pour infecter les cellules. Des expériences d'arrêt de traitement ont révélé un effet antiviral persistant et altérant l’infectivité des nouvelles particules d’HDV. Ceci suggère un effet irréversible sur la matrice de transcription et réplication d’HDV. Des analyses transcriptomiques sur des cellules infectées par HBV et HDV et traitées par Pam3CSK4, BS1 ont confirmé l’induction de cette voie NFκB et révélé l’activation de nombreux gènes impliqués dans une réponse inflammatoire. Une étude par Gene Ontology des fonctions moléculaires et processus biologiques significativement modulés lors des traitements par Pam3CSK4 et BS1 a permis d’identifier une liste de potentiels effecteurs ciblant les ARN et responsables du phénotype anti-HDV. L’identification mécanisme sous-jacent (i.e. dégradation des ARN ou régulation transcriptionnelle négative) reste toutefois à réaliser. Ce projet a montré que l’induction de la voie NFκB est une potentielle cible thérapeutique pour inhiber l’infection à HDV. Notre recherche du ou des effecteurs de l’effet anti-HDV nous permettra d’identifier à terme un nouveau facteur de restriction. Ainsi, nos efforts de recherche devraient ouvrir des pistes pour mener au développement de nouvelles stratégies antivirales pour éliminer HDV
Nonsteroidal antiinflammatory agents (NSAIA) have been shown to exert potent chemopreventive activity against colon, lung, and breast cancers. In this study, we show that at pharmacological concentrations (1 to 3 mmol/L) sodium salicylate (Na-Sal) can potently induce programmed cell death in several human myeloid leukemia cell lines, including TF-1, U937, CMK-1, HL-60, and Mo7e. TF-1 cells undergo rapid apoptosis on treatment with Na-Sal, as indicated by increased annexin V binding capacity, cpp-32 (caspase-3) activation, and cleavage of poly (ADP-ribose) polymerase (PARP) and gelsolin. In addition, the expression of MCL-1, an antiapoptotic member of the BCL-2 family, is downregulated during Na-Sal–induced cell death, whereas the expression of BCL-2, BAX, and BCL-XL is unchanged. Z-VAD, a potent caspase inhibitor, prevents the cleavage of PARP and gelsolin and rescues cells from Na-Sal–induced apoptosis. In addition, we show that Na-Sal accelerates growth factor withdrawal-induced apoptosis and synergizes with daunorubicin to induce apoptosis in TF-1 cells. Thus, our data provide a potential mechanism for the chemopreventive activity of NSAIA and suggest that salicylates may have therapeutic potential for the treatment of human leukemia.
We recently reported increased sensitivity of B-cell chronic lymphocytic leukemia (B-CLL) lymphocytes to apoptotic death activation by the proteasome-specific inhibitor lactacystin. Here, we show that only specific—not nonspecific—proteasomal inhibitors can discriminate between malignant and normal lymphocytes in inducing the apoptotic death response. Indeed, lactacystin and its active metaboliteclasto-lactacystin β-lactone induced apoptotic death in CLL but not in normal lymphocytes. This difference was completely abolished when tripeptide aldehydes such as MG132 or LLnL (which can also inhibit calpains) were used as less specific proteasomal inhibitors. Moreover, B-CLL cells exhibited a constitutive altered ubiquitin-proteasome system, including a threefold higher chymotrypsin-like proteasomal activity and high levels of nuclear ubiquitin-conjugated proteins compared with normal lymphocytes. Interestingly, B-CLL cells also displayed altered proteolytic regulation of wild-type p53, an apoptotic factor reported to be a substrate for the ubiquitin-proteasome system. Nuclear wild-type p53 accumulated after lactacystin treatment used at the discriminating concentration in malignant, but not in normal, lymphocytes. In contrast, p53 was stabilized by MG132 or LLnL in malignant and normal cells undergoing apoptosis, indicating that in normal lymphocytes p53 is regulated mainly by calpains and not by the ubiquitin-proteasome system. This work raises the possibility that two different proteolytic pathways controlling p53 stability may be pathologically imbalanced. This could result in modification of apoptosis control, since in CLL-lymphocytes a highly upregulated ubiquitin-proteasome system, which controls p53 stability among other apoptotic factors, was correlated with an increased propensity of these cells to apoptosis triggered by lactacystin.
6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) is a novel compound that represents the prototype of a new class of synthetic retinoids with apoptogenic properties in acute promyelocytic leukemia (APL) and other types of leukemia. In this article, using SCID mice xenografted with APL-derived NB4 cells, we demonstrate that CD437 has significant antileukemic activity in vivo. In addition, we report on the isolation and characterization of an APL cell line (NB4.437r) resistant to CD437. The cell line retains expression of PML-RAR and is approximately 33-fold more resistant than the parental counterpart to the apoptogenic effects of the retinoid. Resistance is relatively specific to CD437 and structural congeners because the NB4.437r cell line is still sensitive to various types of apoptogenic compounds. The CD437-resistant cell line maintains sensitivity to the antiproliferative and apoptotic action of all-trans-retinoic acid, AM580, and fenretinide, though it shows partial resistance to the cytodifferentiating effects of the first 2 compounds. Resistance to CD437 lays upstream of the CD437-induced release of cytochrome c from the mitochondria and the activation of caspase-3, -7, -8, and -9. Furthermore, NB4.437r cells are deficient in the CD437-dependent activation of nuclear NFkb and AP1-binding activities and in the phosphorylation of the protein kinase Akt. In the case of AP1, deficient assembly of the complex is not caused by the lack of activation of the Jun N-terminal kinase (JNK) family of kinases. The novel cell line will be useful in the elucidation of the molecular mechanisms underlying the apoptogenic action of CD437 and structurally related retinoids.
We recently reported increased sensitivity of B-cell chronic lymphocytic leukemia (B-CLL) lymphocytes to apoptotic death activation by the proteasome-specific inhibitor lactacystin. Here, we show that only specific—not nonspecific—proteasomal inhibitors can discriminate between malignant and normal lymphocytes in inducing the apoptotic death response. Indeed, lactacystin and its active metaboliteclasto-lactacystin β-lactone induced apoptotic death in CLL but not in normal lymphocytes. This difference was completely abolished when tripeptide aldehydes such as MG132 or LLnL (which can also inhibit calpains) were used as less specific proteasomal inhibitors. Moreover, B-CLL cells exhibited a constitutive altered ubiquitin-proteasome system, including a threefold higher chymotrypsin-like proteasomal activity and high levels of nuclear ubiquitin-conjugated proteins compared with normal lymphocytes. Interestingly, B-CLL cells also displayed altered proteolytic regulation of wild-type p53, an apoptotic factor reported to be a substrate for the ubiquitin-proteasome system. Nuclear wild-type p53 accumulated after lactacystin treatment used at the discriminating concentration in malignant, but not in normal, lymphocytes. In contrast, p53 was stabilized by MG132 or LLnL in malignant and normal cells undergoing apoptosis, indicating that in normal lymphocytes p53 is regulated mainly by calpains and not by the ubiquitin-proteasome system. This work raises the possibility that two different proteolytic pathways controlling p53 stability may be pathologically imbalanced. This could result in modification of apoptosis control, since in CLL-lymphocytes a highly upregulated ubiquitin-proteasome system, which controls p53 stability among other apoptotic factors, was correlated with an increased propensity of these cells to apoptosis triggered by lactacystin.
Apoptosis, the cell’s intrinsic death program, is a key regulator of tissue homeostasis, and an imbalance between cell death and proliferation may result in tumor formation. Also, killing of cancer cells by cytotoxic therapies such as chemotherapy, UPgamma-irradiation, or ligation of death receptors is predominantly mediated by triggering apoptosis in target cells. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that induces apoptosis upon binding to its receptors. TRAIL is of special interest for cancer therapy, as TRAIL has been shown to predominantly kill cancer cells, while sparing normal cells. Importantly, combined treatment with TRAIL together with chemotherapy or UPgamma-irradiation synergized to achieve antitumor activity in tumor cell lines and also in tumor models in vivo. However, failure to undergo apoptosis in response to TRAIL treatment may result in tumor resistance. Understanding the molecular events that regulate TRAIL-induced apoptosis and their deregulation in resistant forms of cancer may provide new opportunities for cancer therapy. Thus, novel strategies targeting tumor cell resistance will be based on further insights into the molecular mechanisms of cell death, for example, triggered by TRAIL.
Programmed cell death is an energy-dependent process of cellular elimination that is necessary in all stages of life, including development, tissue homeostasis, and aging in multicellular organisms. Work by Wyllie, Kerr, and Currie identified the large phenotypic changes in the morphology of cells undergoing programmed cell death such as nuclear condensation and the dismantling of cellular material into membrane-enclosed packages; they termed these changes apoptosis. In 1972 they published a seminal paper that correctly predicted the physiologic and pathophysiologic relevance of their findings (1), and Wyllie went on to explore some of the biochemical characteristics that are signs of apoptosis, describing the cleavage of DNA into discrete nucleosomal multimers (DNA ladders) (2). In the 1970s Horvitz and colleagues described the loss of 131 cells during the development of the nematode Caenorhabetitis elegans. Mutagenesis studies subsequently implicated three genes in this process (ced-3, ced-4, and egl-1) that when inactivated caused the retention of the normally eliminated cells (3,4). A fourth gene (ced-9) was identified that promoted cell survival, in that its loss resulted in the death of more cells than normal (5). These proapoptotic gene products, EGL-1, CED-3, and CED-4, along with their antiapoptotic counterpart, CED-9, proved to be components of an evolutionarily conserved, biochemical cascade responsible for cellular elimination.
Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a protein of the tumor necrosis factor (TNF) family that has excited much recent interest as a potentially novel anticancer agent. TRAIL induces apoptosis in a wide variety of human and murine cancer cells, with no apparent apoptotic activity on normal nontransformed cells. Furthermore, TRAIL has also shown therapeutic efficacy when administered both to mice bearing various murine tumors and to immunodeficient mice bearing human tumor xenografts. Although some tumor cells are exquisitely sensitive to the proapoptotic effects of TRAIL, many tumor cells are only moderately sensitive to TRAIL as a single agent, and some remain highly resistant. The proteasome inhibitor bortezomib has also shown much initial promise as an anitcancer agent in vitro and in vivo. Treatment of tumor cells with bortezomib results in multiple biologic effects including inhibition of cell cycle, changes in adherence, inhibition of nuclear factor-κB, and increases in levels of apoptosis. Combinations of bortezomib and TRAIL synergistically interact to promote apoptosis of some tumor cells. The molecular mechanism(s) whereby bortezomib sensitizes tumor cells to apoptosis remains unclear. Nonetheless, a combination of these two novel agents may hold some promise for cancer therapy in the future.
Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) mediates innate and adaptive immunity against the tumorigenesis and tumor progression. TRAIL binds its two death receptors, DR4 and DR5, which activate intracellular pathway of apoptosis for self destruction of tumor cells. To target this apoptotic pathway, recombinant human TRAIL and monoclonal antibodies to DR4 and DR5 have been generated as TRAIL agonists for clinical cancer therapies. A number of TRAIL agonists have passed drug safety evaluation in phase I trials; however, the data from phase II trials thus far are disappointing: TRAIL agonists either in monotherapy or combination have failed to show clinical antitumor activity. In this chapter, we will provide a historic review of the advances and the challenges in the development of TRAIL agonists for clinical treatment of human cancers.
Although approved for over a decade, the clinical utility of proteasome inhibitors (PIs) remains largely restricted to the treatment of patients with multiple myeloma (MM) and mantle cell lymphoma (MCL). This has fueled interest in understanding mechanisms of resistance to their antineoplastic actions, leading to the development of new and improved PIs (e.g., carfilzomib, ixazomib, marizomib) and rational combinations with other novel classes of targeted agents. With respect to the latter, histone deacetylase inhibitors (HDACIs) represent one of the most extensively studied classes of agents. PIs and HDACIs interact at multiple levels to trigger synergistic cell killing in a variety of tumor types through multiple mechanisms, including induction of oxidative stress and DNA damage, PI-mediated inhibition of the cytoprotective NF-κB pathway activated by HDACIs, and promotion of proteotoxic stress through simultaneous proteasome inhibition and disruption of aggresome formation and chaperone proteins, leading to the accumulation of misfolded proteins. Clinically, this combination may be closest to regulatory approval in MM, but represents a promising avenue of investigation in MCL, a relatively uncommon but challenging disease that has been the focus of much recent attention given Food and Drug Administration approvals in 2013 for the immunomodulatory drug lenalidomide and the first-in-class Bruton’s tyrosine kinase inhibitor, ibrutinib. In this chapter, we discuss the mechanisms of action of and interactions between PIs and HDACIs with an MCL focus and review the relevant preclinical and clinical data.
Although hepatic injury is reported in cases with dengue haemorrhagic fever and dengue shock syndrome, its mechanism remains poorly understood. Several findings suggest that dengue virus (DEN) induces apoptosis of hepatocytes in vivo . In this work, DEN type 2 (DEN-2) strain NGC was shown to induce apoptosis in the hepatic cell line HepG2, and infection of HepG2 cells was found to induce Apo2 ligand (Apo2L, also known as tumour necrosis factor-related apoptosis-inducing ligand or TRAIL) expression. Furthermore, Apo2L/TRAIL induced apoptosis in HepG2 cells, which expressed the Apo2L/TRAIL receptor DR5/TRAIL-R2 on their surface. Analysis of the Apo2L/TRAIL promoter revealed that this gene was activated by DEN-2 infection, whose responsive element was overlapping NF- κ B- and Sp1-binding sites located at nt −75 to −65. The proteasome inhibitor N -acetyl- l -leucinyl- l -leucinyl- l -norleucinal (LLnL) inhibited Apo2L/TRAIL mRNA expression, and LLnL and anti-Apo2L/TRAIL antibody inhibited DEN-2-induced apoptosis. It was proposed that DEN infection promotes apoptosis partly through the induction of Apo2L/TRAIL expression.
Hintergrund Doxorubicin ist ein wichtiges Chemotherapeutikum für die Behandlung krebskranker Kinder und fester Bestandteil der meisten Therapieprotokolle sowohl für akute Leukämien als auch für solide Tumoren. Der Einsatz wird dadurch eingeschränkt, dass Doxorubicin zu schweren Herzschädigungen führen kann, auch noch Jahre nach der erfolgreichen Heilung eines tumorkranken Kindes. Die Substanz Dexrazoxan (ICFR 187/Zinecard 7) vermindert die Herzschädigung von Doxorubicin über noch unbekannte Mechanismen. Diskussion Wir haben untersucht, ob das Apoptosesystem CD95/APO-1/FAS die protektive Wirkung von Dexrazoxan auf die Herzschädigung durch Doxorubicin vermittelt. Doxorubicin löst auf primären neonatalen Rattenkardiomyozyten Apoptose aus, was durch die Freisetzung des Todesliganden CD95L in den Zellkulturüberstand begleitet wird. Die zusätzliche Gabe von Dexrazoxan vermindert sowohl die Freisetzung von CD95L als auch den doxorubicininduzierten Zelltod von Herzmuskelzellen. Das molekulare Ausschalten des CD95-Apoptosesystems durch die stabile Transfektion von dysfunktionalem FADD hat jedoch keinen Einfluss auf die Herzschädigung durch Doxorubicin. CD95 ist somit nicht ursächlich an der protektiven Wirkung von Dexrazoxan auf den doxorubicininduzierten Herzschaden beteiligt.
It is not clear why on treatment with certain killer cytokines or chemotherapeutic agents, some cells undergo apoptosis while others do not. The delineation of sensitivity/resistance pathways should provide a more specific therapy for cancer and other hyperproliferative diseases. Most cells die either by apoptosis or by necrosis. The biochemical pathway that mediates these two modes of cell death has recently been described. The nuclear factor (NF)-κB and the genes regulated by this transcription factor have been shown to play a critical role in induction of resistance to killer agents. Thus, inhibitors of NF-κB activation have a potential in overcoming resistance to apoptosis induced by various agents. The evidence for and against such a notion is discussed.
These data show that 8-Cl-cAMP is cytotoxic to the lymphoblastic leukemia cell line CEM and its vinblastine selected multidrug resistant derivative, CEM/VLB100 although PKA was not involved in these effects. The cytotoxic effects of 8-Cl-cAMP was abrogated by cotreatment with either ADA or IBMX which indicated a degradation form of 8-Cl-cAMP was needed for this cytotoxicity. CEM and CEM/VLB100 cells displayed a notable sensitivity to 8-Cl-adenosine-induced growth inhibition and apoptosis. 8-Cl-adenosine increased the cytosolic levels of IκBα which prevented NF-κB nuclear translocation. 8-Cl-adenosine also prevented TNFα-induced IkB decay and NF-κB activation in CEM and CEM/VLB100 cells.
TRAIL (TNF-related apoptosis inducing ligand) is a newly identified member of the TNF gene family which appears to have tumor-selective cytotoxicity due to the distinct decoy receptor system. TRAIL has direct access to caspase machinery and induces apoptosis regardless of p53 phenotype. Therefore, TRAIL has a therapeutic potential in lung cancer which frequently harbors p53 mutation in more than 50% of cases. However, it was shown that TRAIL also could activates NF-κB in some cell lines which might inhibit TRAIL-induced apoptosis. This study was designed to investigate whether TRAIL can activate NF-κB in lung cancer cell lines relatively resistant to TRAIL-induced apoptosis and inhibition of NF-κB activation using proteasome inhibitor MG132 which blocks IκBα degradation can sensitize lung cancer cells to TRAIL-induced apoptosis.
Dendritic cell (DC)-based vaccines have received attention as a new therapeutic modality against cancer. However, increased STAT3 activity in the tumor microenvironment makes DCs tolerogenic and suppresses their antitumor activity. In this study, we explored the effects of a combination treatment consisting of a proteasome inhibitor, bortezomib, and an antigen specific STAT3-ablated (STAT3-/-) DC-based vaccine on the control of TC-1(P3) tumors, a p53-degraded immune resistant cancer cells. We found that E7-antigen expressing STAT3-/- DC (E7-DC-1STAT3-/-) vaccination enhanced generation of E7-specific CD8+ T cells, but was not enough to control TC-1(P3) cancer cells. Therefore, we investigated whether bortezomib could create a synergistic effect with E7-DC-1STAT3-/- vaccination. We found that apoptosis via down-regulation of STAT3 and NF-κB and up-regulation of Fas and death receptor 5 (DR5) expression in TC-1(P3) induced by bortezomib was independent of p53 status. We also observed that TC-1(P3) cells pretreated with bortezomib had markedly enhanced anti-tumor effects on E7-specific CD8+ T cells through a Fas/DR5-mediated mechanism. In addition, TC-1(P3) tumor-bearing mice treated with bortezomib prior to vaccination with E7-DC-1STAT3-/- demonstrated enhanced generation of E7-specific CD8+ T cells and prolonged survival compared to those treated with monotherapy. These results suggest that the anti-tumor effects against a p53-degraded immune resistant variant generated by antigen-expressing STAT3-ablated mature DCs may be enhanced by bortezomib via death receptor-mediated apoptosis.
Targeted degradation of key regulatory proteins is an essential element of cell-cycle control. The proteasome plays a pivotal role in the degradation of such proteins and has, therefore, become an important therapeutic target for diseases involving cell proliferation, notably malignant diseases. Several studies have demonstrated that proteasome inhibition induces apoptosis and sensitizes cancer cells to traditional tumoricidal agents (chemotherapy or irradiation), both in vitro and in vivo, by having a significant impact on the turnover of certain key cell cycle regulatory elements (p53, p21, p27, Bax, etc.) as well as central transcription factors (nuclear factor [NF]-κB). Inhibition of the latter can result in reversal of chemoresistance.Bortezomib, a potent and selective proteasome inhibitor, is particularly promising from a therapeutic perspective; it is the only such inhibitor that has progressed to clinical trials. Results from phase I/II studies indicate that the drug is well tolerated, and bortezomib has become an effective treatment for patients with hematologic malignancies (in particular multiple myeloma). This drug also seems to have significant activity in solid tumor malignancies. Phase I/II combination studies of bortezomib and other chemotherapeutics in hematologic and solid tumors are underway.
Besides inducing apoptosis, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) activates NF-κB. The apoptosis signaling pathway of TRAIL is well characterized involving TRAIL receptors, Fas-associated protein with death domain (FADD) and caspase-8. In contrast, the molecular mechanism of TRAIL signaling to NF-κB remains controversial. Here, we characterized the receptor-proximal mediators of NF-κB activation by TRAIL. Deletion of the DD of TRAIL receptors 1 and 2 revealed that it is essential in NF-κB signaling. Because FADD interacts with the TRAIL receptor DD, FADD was tested. RNAi-mediated knockdown of FADD or FADD deficiency in JURKAT T-cell leukemia cells decreased or disabled NF-κB signaling by TRAIL. In contrast, TRAIL-induced activation of NF-κB was maintained upon loss of receptor interacting protein 1 (RIP1) or knockdown of FLICE-like inhibitory protein (FLIP). Exogenous expression of FADD rescued TRAIL-induced NF-κB signaling. Loss-of-function mutations of FADD within the RHDLL motif of the death effector domain, which is required for TRAIL-induced apoptosis, abrogated FADD's ability to recruit caspase-8 and mediate NF-κB activation. Accordingly, deficiency of caspase-8 inhibited TRAIL-induced activation of NF-κB, which was rescued by wild-type caspase-8, but not by a catalytically inactive caspase-8 mutant. These data establish the mechanism of TRAIL-induced NF-κB activation involving the TRAIL receptor DD, FADD and caspase-8, but not RIP1 or FLIP. Our results show that signaling of TRAIL-induced apoptosis and NF-κB bifurcates downstream of caspase-8.
The maintenance of lymphocyte homeostasis by apoptosis is a critical regulatory mechanism in the normal immune system. The transcription factor NF-κB has been shown to play a role in protecting cells against death mediated by TNF. We show here that NF-κB also has a role in regulating Fas / APO-1 / CD95-mediated death, a major pathway of peripheral T cell death. Transfection of Jurkat cells with the NF-κB subunits p50 and p65 confers resistance against Fas-mediated apoptosis. Reciprocally, inhibition of NF-κB activation by a soluble peptide inhibitor or a dominant form of the NF-κB inhibitor, IκB, makes the cells more susceptible to Fas-mediated apoptosis. Furthermore, inhibition of NF-κB activation by a soluble peptide inhibitor rendered a T cell hybridoma more susceptible to TCR-mediated apoptosis. Correspondingly, transfection of p50 and p65 provided considerable protection from TCR-mediated apoptosis. These observations were corroborated by studies on Fas-mediated death in primary T cells. Concanavalin A-activated cycling T cell blasts from mice that are transgenic for the dominant IκB molecule have increased sensitivity to Fas-mediated apoptosis, associated with a down-regulation of NF-κB complexes in the nucleus. In addition, blocking TNF, itself a positive regulator of NF-κB, with neutralizing antibodies renders the cells more susceptible to anti-Fas-mediated apoptosis. In summary, our results provide compelling evidence that NF-κB protects against Fas-mediated death and is likely to be an important regulator of T cell homeostasis and tolerance.
Inhibition of the transcription factor NF-κB has been reported to increase cell sensitivity to TNF and some cytotoxic drugs. We investigated the effect of NK-κB inhibition on the susceptibility of tumor cells to freshly isolated, nonactivated, human NK cells and to a TCR / δ T cell clone displaying an MHC-unrestricted "NK-like" lysis. Using electrophoretic mobility shift assay, we first demonstrated that NF-κB / DNA binding activity was induced in target cells following coculture with NK cells or TCR / δ T cell clone. To investigate the effect of target cell NF-κB inhibition on NK-mediated lysis, we blocked NF-κB translocation by introducing a human cDNA coding for a mutated IκB-. Interestingly, our results indicated that inhibition of NF-κB did not induce any increase in either granzyme-dependent non-MHC-restricted cytotoxicity mediated by fresh non-stimulated NK cells and by TCR / δ T cell clone or in CD95-mediated lysis. These results emphasize that NF-κB expressed in target cells does not play a role in the molecular process related to the control of target cell susceptibility to NK-mediated lysis and suggest that the NF-κB pathway is not a general mechanism for controlling the cytotoxic response.
Crassocephalum crepidioides, a plant distributed in Okinawa Islands, is known in folk medicine; however, its anticancer activity has not been investigated. The aim of this study was to determine the in vitro and in vivo antitumor activities of C. crepidioides on murine Sarcoma 180 (S-180) and related molecular mechanisms.
The antitumor effect of C. crepidioides was evaluated in S-180-cell-bearing mice. Cell growth was assessed using a colorimetric assay. Nitrite and nitrate levels were measured by colorimetry. The expression levels of inducible NO synthase (iNOS) in murine RAW264.7 macrophages was assessed by reverse transcriptase-polymerase chain reaction. Activation of iNOS promoter was detected by reporter gene. Activation of nuclear factor-κB (NF-κB) was evaluated by electrophoretic mobility shift assay. The role of NF-κB signaling was analyzed using inhibitors of NF-κB and dominant-negative mutants, and Western blot analysis.
C. crepidioides extract delayed tumor growth in S-180-bearing mice. However, it did not inhibit S-180 cell growth in vitro. Supernatant of cultured C. crepidioides-stimulated RAW264.7 macrophages was cytotoxic to S-180 cells. This cytotoxicity was associated with nitric oxide (NO) production. NF-κB signaling pathway was crucial for the transcriptional activation of iNOS gene. Isochlorogenic acid, a component of C. crepidioides, induced NF-κB activation and iNOS expression.
The results highlight the oncolytic and immunopotentiation properties of C. crepidioides mediated through NF-κB-induced release of NO from macrophages.
Recent advances in cancer biology have generated novel cancer therapeutics that can activate apoptotic pathways in human cancers.
Among the apoptotic therapeutics, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received much attention
because it can selectively induce apoptosis in cancer cells. Preclinical studies in glioblastoma cell lines, primary cultures,
and xenografts have resulted in the development of TRAIL-based therapeutic modalities for the treatment of glioblastoma. These
include recombinant human TRAIL (rhTRAIL), agonistic antibodies against TRAIL death receptors, DR4 and DR5, TRAIL-producing
neural stem cells, TRAIL-expressing oncolytic vectors, and nanoparticles conjugated with TRAIL-expressing vectors. Although
rhTRAIL and DR4 and DR5 agonistic antibodies have entered clinical trials in patients with solid cancers and hematological
malignancies, many challenges remain before any of these therapeutic modalities can be successfully developed into clinical
treatments for glioblastoma. It remains to be seen whether and how the therapeutics can be effectively distributed through
glioblastoma that is diffusely infiltrating through the human brain. In addition, the vast majority of the tumors are resistant
to TRAIL and thus require combination treatments that can overcome the resistance. Finally, genetic analysis of human glioblastomas
is required to identify the genetic defects in apoptotic genes that could be used as biomarkers to predict the responsiveness
of the tumors to TRAIL-based therapies.
Recent research indicates that the proteasome is one of the non-caspase proteases involved in apoptotic signaling pathways. Nuclear factor-κB (NF-κB) activation, one of the key factors in apoptosis, can be prevented through abrogation of IκBα degradation by proteasome inhibition. We have investigated the effects of the proteasome inhibitors carbobenzoxyl-l-leucyl-l-leucyl-l-leucinal (MG132) and N-acetyl-l-leucinyl-l-leucinyl-l-norleucinal (LLnL) on apoptosis and NF-κB activation induced by etoposide, using a human leukemia cell line (U937) and leukemia blasts freshly isolated from patients with acute leukemia. Pretreatment of U937 cells with MG132 or LLnL inhibited etoposide-induced morphological apoptosis and caspase-3 activation. Furthermore, MG132 or LLnL prevented NF-κB activation and IκBα degradation, but not IκBα phosphorylation at Ser32. Other inhibitors of NF-κB activation, including pyrrrolidine dithiocarbamate (an antioxidant) and the peptide SN50 (an inhibitor of translocation of activated NF-κB into the nucleus), also attenuated etoposide-induced apoptosis. In leukemia blasts, although proteasome inhibitors suppressed NF-κB activation induced by etoposide, they were unable to prevent morphological apoptosis. Moreover, proteasome inhibitors by themselves caused apoptosis in leukemia blasts at the concentrations employed in this study. These results suggest that the role that NF-κB plays in apoptosis induced by etoposide in a human leukemia cell line may be different from the role it plays in freshly isolated leukemia blasts.
Large granular lymphocyte (LGL) leukemia is a rare disorder of mature cytotoxic T or natural killer cells. Large granular lymphocyte leukemia is characterized by the accumulation of cytotoxic cells in blood and infiltration in the bone marrow, liver, and spleen. Herein, we review clinical features of LGL leukemia. We focus our discussion on known survival signals believed to play a role in the pathogenesis of LGL leukemia and their potential therapeutic implications.
Apoptosis can be triggered by the engagement of cell surface receptors by their ligands. A growing number of receptors belonging to the TNF receptor family have been identified that contain a conserved cytoplasmic death domain. These include Fas, TNF-R1, lymphocyte-associated receptor of death (LARD), DR4, and TNF-related apoptosis-inducing ligand receptor inducer of cell killing-2 (TRICK2). The latter two are receptors for the cytotoxic ligand TNF-related apoptosis-inducing ligand (TRAIL), and one of the paradoxes raised by the cloning of these molecules was why do most cells not die upon contact with the widely expressed TRAIL molecule? This is a particular problem for lymphocytes that express DR4 and TRICK2 and are in constant circulation through TRAIL-expressing tissues. We have cloned LIT (lymphocyte inhibitor of TRAIL), which lacks a death domain. LIT is expressed predominantly on PBL, where it can competitively inhibit TRAIL-induced apoptosis through DR4/TRICK2, and may function to modulate lymphocyte sensitivity to TRAIL.
Taxol, a naturally occurring diterpene with antitumor activity, induces tubulin polymerization to generate abnormally stable and nonfunctional microtubules. Previously, we showed that taxol has lipopolysaccharide (LPS)-like effects on macrophages. As LPS is a potent inducer of macrophage cytokine production, we investigated whether a similar effect is exerted by taxol. In a dose-dependent manner, LPS-free taxol induced release of biologically active tumor necrosis factor alpha (TNF) by inflammatory murine macrophages. Taxol-induced production of TNF was inhibitable by interleukin-10. By Northern blot, taxol (10 and 1 microM) induced TNF mRNA expression to an extent similar to LPS. Induction of TNF mRNA by 10 microM taxol was detectable at 45 min of stimulation, maximal at 90 min, and evident for at least 8 h. The same low concentration of taxol also induced interleukin 1 (IL-1) alpha and beta mRNA expression. We conclude that taxol triggers macrophages for TNF and IL-1 production. These LPS-like effects of taxol might contribute to its antitumor activity.
The transcription factor NF-kappa B is sequestered in the cytoplasm by the inhibitor protein I kappa B alpha. Extracellular inducers of NF-kappa B activate signal transduction pathways that result in the phosphorylation and subsequent degradation of I kappa B alpha. At present, the link between phosphorylation of I kappa B alpha and its degradation is not understood. In this report we provide evidence that phosphorylation of serine residues 32 and 36 of I kappa B alpha targets the protein to the ubiquitin-proteasome pathway. I kappa B alpha is ubiquitinated in vivo and in vitro following phosphorylation, and mutations that abolish phosphorylation and degradation of I kappa B alpha in vivo prevent ubiquitination in vitro. Ubiquitinated I kappa B alpha remains associated with NF-kappa B, and the bound I kappa B alpha is degraded by the 26S proteasome. Thus, ubiquitination provides a mechanistic link between phosphorylation and degradation of I kappa B alpha.
Fas antigen/Apo-1 (Fas) and the p55 tumor necrosis factor receptor (TNF-R) are two related cell surface molecules that induce apoptosis in susceptible cells. With regard to their cytoplasmic homology region, we investigated whether Fas like the TNF-R activates nuclear factor kappa B (NF-kappa B), using human SV80 fibroblasts transfected with the cDNA encoding human Fas. In this cell line Fas mobilizes the p50/p65 heterodimeric form of NF-kappa B and induces interleukin-6 (IL-6) production. Compared to NF-kappa B activation via the TNF-R differences in kinetics and signal intensity were observed. Peak activation occurred 2 hr after Fas compared to 1 hr after TNF-R stimulation. Furthermore, when equitoxic concentrations of anti-Fas antibody and TNF were applied, TNF triggered a stronger NF-kappa B response. Studies using inhibitors of signal transduction suggest that both receptors mediate NF-kappa B activation via similar routes: D609, an inhibitor of the phospatidylcholine-specific phospholipase C, had an inhibitory effect, while the protein kinase C inhibitor staurosporine had an enhancing effect on both Fas and TNF-R induced NF-kappa B mobilization. Interestingly, D609 had no influence on Fas and TNF-R mediated cytotoxicity arguing against an involvement of NF-kappa B in the cell death pathway triggered by these receptors. This is the first indication that Fas may activate genes via NF-kappa B and may thus in addition to its role as a cell death inducing receptor serve a much broader range of biological functions.
Cytotoxic drugs used in chemotherapy of leukemias and solid tumors cause apoptosis in target cells. In lymphoid cells the CD95 (APO-1/Fas)/CD95 ligand (CD95-L) system is a key regulator of apoptosis. Here we describe that doxorbicin induces apoptosis via the CD95/CD95-L system in human leukemia T-cell lines. Doxorubicin-induced apoptosis was completely blocked by inhibition of gene expression and protein synthesis. Also, doxorbicin strongly stimulates CD95-L messenger RNA expression in vitro at concentrations relevant for therapy in vivo. CEM and jurkat cells resistant to CD95-mediated apoptosis were also resistant to doxorbicin-induced apoptosis . Furthermore, doxorbicin-induced apoptosis was inhibited by blocking F(ab')2 anti-APO-1 (anti-CD95) antibody fragments. Expression of CD95-L mRNA and protein in vitro was also stimulated by other cytotoxic drugs such as methotrexate. The finding that apoptosis caused by anticancer drugs may be mediated via the CD95 system provides a new molecular insight into resistance and sensitivity toward chemotherapy in malignancies.
In unstimulated cells, the transcription factor NF-kappaB is held in the cytoplasm in an inactive state by the inhibitor protein IkappaBalpha. Stimulation of cells results in rapid phosphorylation and degradation of IkappaBalpha, thus releasing NF-kappaB, which translocates to the nucleus and activates transcription of responsive genes. Here we demonstrate that in cells where proteasomal degradation is inhibited, signal induction by tumor necrosis factor alpha results in the rapid accumulation of higher molecular weight forms of IkappaBalpha that dissociate from NF-kappaB and are consistent with ubiquitin conjugation. Removal of the high molecular weight forms of IkappaBalpha by a recombinant ubiquitin carboxyl-terminal hydrolase and reactivity of the immunopurified material with a monoclonal antibody specific for ubiquitin indicated that IkappaBalpha was conjugated to multiple copies of ubiquitin. Western blot analysis of immunopurified IkappaBalpha from cells expressing epitope-tagged versions of IkappaBalpha and ubiquitin revealed the presence of multiple copies of covalently bound tagged ubiquitin. An S32A/S36A mutant of IkappaBalpha that is neither phosphorylated nor degraded in response to signal induction fails to undergo inducible ubiquitination in vivo. Thus signal-induced activation of NF-kappaB involves phosphorylation-dependent ubiquitination of IkappaBalpha, which targets the protein for rapid degradation by the proteasome and releases NF-kappaB for translocation to the nucleus.
Proteases are known to be involved in the apoptotic pathway. We report here that benzyloxycarbonyl (Z)-Leu-Leu-leucinal(ZLLLal), a leupeptin analogue, can induce apoptosis in MOLT-4 and L5178Y cells. ZLLLal is a cell-permeant inhibitor of proteasome. Among the protease inhibitors tested, only calpain inhibitor I (acetyl-Leu-Leu-norleucinal) and ZLLLal caused a marked induction of apoptosis in MOLT-4 cells. In contrast Z-Leu-leucinal, a specific inhibitor of calpain, did not induce apoptosis. When MOLT-4 cells were incubated in the presence of ZLLLal, p53 accumulated in the cells. These results strongly suggest that inhibition of proteasome induces p53-dependent apoptosis and that proteasome can protect cell from apoptosis.
Apoptosis of the WEHI 231 immature B cell lymphoma line following membrane interaction with an antibody against the surface IgM chains (anti-IgM) is preceded by dramatic changes in Nuclear Factor-kappaB (NF-kappaB)/ Rel binding activities. An early transient increase in NF-kappaB/Rel binding is followed by a significant decrease in intensity below basal levels. Here we have explored the role of these changes in Rel-related factors in B cell apoptosis. Treatment of WEH1 231 cells with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a protease inhibitor which prevents degradation of the inhibitor of NF-kappaB (IkappaB)-alpha, or with low doses of pyrrolidinedithiocarbamate (PDTC) selectively inhibited NF-kappaB/Rel factor binding and induced apoptosis. Bcl-XL expression protected WEHI 231 cells from apoptosis induced by these agents. Microinjection of WEHI 231 cells with either IkappaB-alpha-GST protein or a c-Rel affinity-purified antibody induced apoptosis. Ectopic c-Rel expression ablated apoptosis induced by TPCK or anti-IgM. Treatment of BALENLM 17 and A20 B lymphoma cells or normal murine splenic B lymphocytes with either TPCK or PDTC also resulted in apoptosis. These findings indicate that the drop in NF-kappaB/Rel binding following anti-IgM treatment activates apoptosis of WEHI 231 cells; furthermore, they implicate the NF-kappaB/Rel family in control of apoptosis of normal and transformed B cells.
Several members of the tumour-necrosis/nerve-growth factor (TNF/NGF) receptor family activate the transcription factor NF-kappaB through a common adaptor protein, Traf2 (refs 1-5), whereas the interleukin 1 type-I receptor activates NF-kappaB independently of Traf2 (ref. 4). We have now cloned a new protein kinase, NIK, which binds to Traf2 and stimulates NF-kappaB activity. This kinase shares sequence similarity with several MAPKK kinases. Expression in cells of kinase-deficient NIK mutants fails to stimulate NF-kappaB and blocks its induction by TNF, by either of the two TNF receptors or by the receptor CD95 (Fas/Apo-1), and by TRADD, RIP and MORT1/FADD, which are adaptor proteins that bind to these receptors. It also blocked NF-kappaB induction by interleukin-1. Our findings indicate that NIK participates in an NF-kappaB-inducing signalling cascade common to receptors of the TNF/NGF family and to the interleukin-1 type-I receptor.
TRAIL (also known as Apo-2L) is a member of the tumor necrosis factor (TNF) ligand family that rapidly induces apoptosis in
a variety of transformed cell lines. The human receptor for TRAIL was found to be an undescribed member of the TNF-receptor
family (designated death receptor-4, DR4) that contains a cytoplasmic “death domain” capable of engaging the cell suicide
apparatus but not the nuclear factor kappa B pathway in the system studied. Unlike Fas, TNFR-1, and DR3, DR4 could not use
FADD to transmit the death signal, suggesting the use of distinct proximal signaling machinery. Thus, the DR4-TRAIL axis defines
another receptor-ligand pair involved in regulating cell suicide and tissue homeostasis.
The anthracycline antibiotic, daunorubicin, can induce programmed cell death (apoptosis) in cells. Recent work suggests that this event is mediated by ceramide via enhanced ceramide synthase activity. Since the generation of ceramide has been directly linked with the activation of the transcription factor, NFkappaB, this was investigated as a novel target for the action of daunorubicin. Here we describe how treatment of HL-60 promyelocytes and Jurkat T lymphoma cells with daunorubicin results in the activation of the transcription factor NFkappaB. The effect of daunorubicin was evident following 1-2 h treatment, which was in contrast to the time course of activation obtained with the cytokine, tumor necrosis factor, where NFkappaB activation was detected within minutes of cellular stimulation. Activated complexes were shown to contain predominantly p50 and p65/RelA subunit components. Daunorubicin also induced IkappaB degradation and increased the expression of an NFkappaB-linked reporter gene. In addition, the drug was found to strongly potentiate the ability of tumor necrosis factor to induce an NFkappaB-linked reporter gene, suggesting a synergy between these two agents in this response. These events were sensitive to the iron chelator, deferoxamine mesylate (desferal), and the anti-oxidant and metal chelator pyrrolidine dithiocarbamate. A structurally related compound, mitoxantrone, which, unlike daunorubicin, is unable to undergo redox cycling in cells, also activated NFkappaB in a pyrrolidine dithiocarbamate-sensitive manner. A specific inhibitor of ceramide synthase, fumonisin B1, had no effect on daunorubicin induced NFkappaB activation at a range of concentrations previously reported to block apoptosis induced by this drug. However, this agent could inhibit increases in ceramide induced by daunorubicin, in addition to blocking ceramide synthase activity from HL-60 cells which was activated in response to daunorubicin treatment. These data therefore suggest that the effect of daunorubicin on NFkappaB is unlikely to involve ceramide, but may involve reactive oxygen species generated as a result of endogenous cellular processes rather than reductive metabolism of the drug. As NFkappaB may be involved in apoptosis, this effect may be an important aspect of the cellular responses to this agent.
Proteolysis by the ubiquitin/proteasome pathway controls the intracellular levels of a number of proteins that regulate cell proliferation and cell cycle progression. To determine whether this pathway of protein turnover was also linked to apoptosis, we treated Rat-1 and PC12 cells with specific proteasome inhibitors. The peptide aldehydes PSI and MG115, which specifically inhibit the chymotrypsin-like activity of the proteasome, induced apoptosis of both cell types. In contrast, apoptosis was not induced by inhibitors of lysosomal proteases or by an alcohol analog of PSI. The tumor suppressor p53 rapidly accumulated in cells treated with proteasome inhibitors, as did the p53-inducible gene products p21 and Mdm-2. In addition, apoptosis induced by proteasome inhibitors was inhibited by expression of dominant-negative p53, whereas overexpression of wild-type p53 was sufficient to induce apoptosis of Rat-1 cells in transient transfection assays. Although other molecules may also be involved, these results suggest that stabilization and accumulation of p53 plays a key role in apoptosis induced by proteasome inhibitors.
TRAIL (also called Apo2L) belongs to the tumor necrosis factor family, activates rapid apoptosis in tumor cells, and binds
to the death-signaling receptor DR4. Two additional TRAIL receptors were identified. The receptor designated death receptor
5 (DR5) contained a cytoplasmic death domain and induced apoptosis much like DR4. The receptor designated decoy receptor 1
(DcR1) displayed properties of a glycophospholipid-anchored cell surface protein. DcR1 acted as a decoy receptor that inhibited
TRAIL signaling. Thus, a cell surface mechanism exists for the regulation of cellular responsiveness to pro-apoptotic stimuli.
Anticancer agents have been shown to trigger apoptosis in chemosensitive tumors such as neuroblastomas. We previously identified activation of the CD95 system as one of the key mechanisms for doxorubicin-induced apoptosis in leukemic T cells. Here, we report that therapeutic concentrations of doxorubicin, cisplatinum, and VP-16 led to induction of CD95 receptor and CD95 ligand (CD95-L) that mediated cell death in chemosensitive neuroblastoma cells. Using F(ab')2 anti-CD95 antibody fragments to interfere with CD95-L-receptor interaction markedly reduced apoptosis induced by those drugs in vitro. Cyclosporin A inhibited induction of CD95 mRNA and CD95-L mRNA and blocked drug-mediated apoptosis. Drug-induced apoptosis involved activation of caspases (interleukin 1beta-converting enzyme/Ced-3-like proteases) and processing of the prototype caspase substrate PARP and was completely blocked by benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a peptide inhibitor of caspases. In addition, neuroblastoma cells that were resistant to CD95-triggered apoptosis also displayed cross-resistance to chemotherapeutic agents. These data provide new clues for understanding the molecular requirements for drug-induced apoptosis in chemosensitive neuroblastoma cells by demonstrating that cell death was mediated via the CD95-L-receptor system and may open new avenues for targeting drug resistance of neuroblastoma.
TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines and induces apoptosis in a wide variety of cells. Based on homology searching of a private database, a receptor for TRAIL (DR4 or TRAIL-R1) was recently identified. Here we report the identification of a distinct receptor for TRAIL, TRAIL-R2, by ligand-based affinity purification and subsequent molecular cloning. TRAIL-R2 was purified independently as the only receptor for TRAIL detectable on the surface of two different human cell lines that undergo apoptosis upon stimulation with TRAIL. TRAIL-R2 contains two extracellular cysteine-rich repeats, typical for TNF receptor (TNFR) family members, and a cytoplasmic death domain. TRAIL binds to recombinant cell-surface-expressed TRAIL-R2, and TRAIL-induced apoptosis is inhibited by a TRAIL-R2-Fc fusion protein. TRAIL-R2 mRNA is widely expressed and the gene encoding TRAIL-R2 is located on human chromosome 8p22-21. Like TRAIL-R1, TRAIL-R2 engages a caspase-dependent apoptotic pathway but, in contrast to TRAIL-R1, TRAIL-R2 mediates apoptosis via the intracellular adaptor molecule FADD/MORT1. The existence of two distinct receptors for the same ligand suggests an unexpected complexity to TRAIL biology, reminiscent of dual receptors for TNF, the canonical member of this family.
TRAIL-R3, a new member of the TRAIL receptor family, has been cloned and characterized. TRAIL-R3 encodes a 299 amino acid
protein with 58 and 54% overall identity to TRAIL-R1 and -R2, respectively. Transient expression and quantitative binding
studies show TRAIL-R3 to be a plasma membrane–bound protein capable of high affinity interaction with the TRAIL ligand.
The TRAIL-R3 gene maps to human chromosome 8p22-21, clustered with the genes encoding two other TRAIL receptors. In contrast
to TRAIL-R1 and -R2, this receptor shows restricted expression, with transcripts detectable only in peripheral blood lymphocytes
and spleen. The structure of TRAIL-R3 is unique when compared to the other TRAIL receptors in that it lacks a cytoplasmic
domain and appears to be glycosyl-phosphatidylinositol–linked. Moreover, unlike TRAIL-R1 and -R2, in a transient overexpression
system TRAIL-R3 does not induce apoptosis.
A human receptor for the cytotoxic ligand TRAIL (TRAIL receptor-1, designated DR4) was identified recently as a member of
the tumor necrosis factor receptor family. In this report we describe the identification of two additional human TRAIL receptors,
TRAIL receptor-2 and TRAIL receptor-3, that belong to the tumor necrosis factor receptor family. Interestingly, TRAIL receptor-2
but not TRAIL receptor-3 contains a cytoplasmic “death domain” necessary for induction of apoptosis and is hence designated
death receptor-5 (DR5). Like DR4, DR5 engages the apoptotic pathway independent of the adaptor molecule FADD/MORT1. Because
of its lack of a death domain, TRAIL receptor-3 is not capable of inducing apoptosis. However, by competing for TRAIL, it
is capable of inhibiting TRAIL-induced apoptosis. Thus, TRAIL receptor-3 may function as an antagonistic decoy receptor to
attenuate the cytotoxic effect of TRAIL in most tissues that are TRAIL+, DR4+, and DR5+.
Studies on mice deficient in nuclear factor kappa B (NF-κB) subunits have shown that this transcription factor is important for lymphocyte responses to antigens and cytokine-inducible gene expression. In particular, the RelA (p65) subunit is required for induction of tumor necrosis factor-α (TNF-α)-dependent genes. Treatment of RelA-deficient (RelA^(−/−)) mouse fibroblasts and macrophages with TNF-α resulted in a significant reduction in viability, whereas RelA^(+/+) cells were unaffected. Cytotoxicity to both cell types was mediated by TNF receptor 1. Reintroduction of RelA into RelA^(−/−) fibroblasts resulted in enhanced survival, demonstrating that the presence of RelA is required for protection from TNF-α. These results have implications for the treatment of inflammatory and proliferative diseases.
The cytotoxic effect of anticancer drugs has been shown to involve induction of apoptosis. We report here that tumor cells resistant to CD95 (APO-1/Fas) -mediated apoptosis were cross-resistant to apoptosis-induced by anticancer drugs. Apoptosis induced in tumor cells by cytarabine, doxorubicin, and methotrexate required the activation of ICE/Ced-3 proteases (caspases), similarly to the CD95 system. After drug treatment, a strong increase of caspase activity was found that preceded cell death. Drug-induced activation of caspases was also found in ex vivo-derived T-cell leukemia cells. Resistance to cell death was conferred by a peptide caspase inhibitor and CrmA, a poxvirus-derived serpin. The peptide inhibitor was effective even if added several hours after drug treatment, indicating a direct involvement of caspases in the execution and not in the trigger phase of drug action. Drug-induced apoptosis was also strongly inhibited by antisense approaches targeting caspase-1 and -3, indicating that several members of this protease family were involved. CD95-resistant cell lines that failed to activate caspases upon CD95 triggering were cross-resistant to drug-mediated apoptosis. Our data strongly support the concept that sensitivity for drug-induced cell death depends on intact apoptosis pathways leading to activation of caspases. The identification of defects in caspase activation may provide molecular targets to overcome drug resistance in tumor cells.
NF-κB, which consists of two polypeptides, p50 (M_r 50K) and p65/RelA (M_r 65K), is thought to be a key regulator of genes involved in responses to infection, inflammation and stress. Indeed, although developmentally normal, mice deficient in p50 display functional defects in immune responses. Here we describe the generation of mice deficient in the RelA subunit of NF-κB. Disruption of the relA locus leads to embryonic lethality at 15–16 days of gestation, concomitant with a massive degeneration of the liver by programmed cell death or apoptosis. Embryonic fibroblasts from RelA-deficient mice are defective in the tumour necrosis factor (TNF)-mediated induction of messenger RNAs for IκBα and granulocyte/macrophage colony stimulating factor (GM-CSF), although basal levels of these transcripts are unaltered. These results indicate that RelA controls inducible, but not basal, transcription in NF-κB-regulated pathways.
Cytokines in the tumor necrosis factor (TNF) family regulate development and function of the immune system. We have isolated a new member of this family, designated Apo-2 ligand (Apo-2L), via an expressed sequence tag. Apo-2L is a 281-amino acid protein, related most closely to Fas/Apo-1 ligand. Transfected Apo-2L is expressed at the cell surface with its C terminus exposed, indicating a type II transmembrane protein topology. Like Fas/Apo-1 ligand and TNF, the C-terminal extracellular region of Apo-2L (amino acids 114-281) exhibits a homotrimeric subunit structure. Soluble Apo-2L induces extensive apoptosis in lymphoid as well as non-lymphoid tumor cell lines. The effect of Apo-2L is not inhibited by soluble Fas/Apo-1 and TNF receptors; moreover, expression of human Fas/Apo-1 in mouse fibroblasts, which confers sensitivity to induction of apoptosis by agonistic anti-Fas/Apo-1 antibody, does not confer sensitivity to Apo-2L. Hence, Apo-2L acts via a receptor which is distinct from Fas/Apo-1 and TNF receptors. These results suggest that, along with other family members such as Fas/Apo-1 ligand and TNF, Apo-2L may serve as an extracellular signal that triggers programmed cell death.
TRAIL induces apoptosis through two closely related receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Here we show that TRAIL-R1 can associate with TRAIL-R2, suggesting that TRAIL may signal through heteroreceptor signaling complexes. Both TRAIL receptors bind the adaptor molecules FADD and TRADD, and both death signals are interrupted by a dominant negative form of FADD and by the FLICE-inhibitory protein FLIP. The recruitment of TRADD may explain the potent activation of NF-κB observed by TRAIL receptors. Thus, TRAIL receptors can signal both death and gene transcription, functions reminiscent of those of TNFR1 and TRAMP, two other members of the death receptor family.
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Almost exactly ten years following the first publication on NF-κB (Sen and Baltimore 1986), researchers working on transcriptional regulation by NF-κB/Rel and IκB proteins gathered for the third time to discuss recent developments in the field (Madrid, July 8-10, 1996). The first meeting of its kind was a Howard Hughes workshop at the NIH in November 1992 and the second one a Banbury Conference held at Cold Spring Harbor in October 1993. This year's meeting was organized by R. Bravo (Bristol-Myers Squibb, Princeton) and P. S. Lazo (Universidad de Oviedo) and held at the Juan March Foundation in Madrid, Spain.
TRAIL, also called Apo2L, is a cytotoxic protein that induces apoptosis of many transformed cell lines but not of normal tissues,
even though its death domain–containing receptor, DR4, is expressed on both cell types. An antagonist decoy receptor (designated
as TRID for TRAIL receptor without an intracellular domain) that may explain the resistant phenotype of normal tissues was
identified. TRID is a distinct gene product with an extracellular TRAIL-binding domain and a transmembrane domain but no intracellular
signaling domain. TRID transcripts were detected in many normal human tissues but not in most cancer cell lines examined.
Ectopic expression of TRID protected mammalian cells from TRAIL-induced apoptosis, which is consistent with a protective role.
Another death domain–containing receptor for TRAIL (designated as death receptor–5), which preferentially engaged a FLICE
(caspase-8)–related death protease, was also identified.
Paclitaxel can induce tumor necrosis factor (TNF) and interleukin-1 gene expression, similar to lipopolysaccharides. Since
lipopolysaccharide-induced expression of TNF is related to activation of NF-κB, we determined whether NF-κB could be activated
by paclitaxel. In the human lung adenocarcinoma cell line A549, paclitaxel activated NF-κB in a dose-dependent manner with
maximal activation after 2–4 h. Since paclitaxel could up-regulate TNF and interleukin-1 secretion and subsequent NF-κB activation
could be caused by these cytokines, the effect of two other groups of anticancer drugs including vinca alkaloids (vinblastine
and vincristine) and anthracyclines (daunomycin and doxorubicin), neither of which induce TNF or interleukin-1 gene expression,
were examined. Like paclitaxel, vinblastine, vincristine, daunomycin, and doxorubicin each caused activation of NF-κB. Therefore,
it is unlikely that activation of NF-κB caused by these agents or by paclitaxel is mediated via cytokine up-regulation. Furthermore,
actinomycin D and cycloheximide, inhibitors of transcription and translation, respectively, did not inhibit paclitaxel-induced
NF-κB activation. Several other transcription factors such as AP-1, AP-2, CREB, SP-1, or TFIID were not activated by antineoplastic
agents demonstrating specificity of NF-κB activation. The involvement of both subunits in the NF-κB DNA binding complex was
demonstrated by its abrogation by anti-p65 and by supershift by anti-p50 antibodies. Since protein phosphorylation is implicated
in the activation of NF-κB, the effect of anticancer drugs on protein kinase C activity was measured. Vincristine, daunomycin,
and paclitaxel significantly increased protein kinase C activity, and vinblastine and doxorubicin caused similar trends. Following
treatment with antineoplastics (1–4 h), cytoplasmic IκBα degradation occurred concomitantly with translocation of p65 to the
nucleus. Specific protein kinase C inhibitors (bisindolylmaleimide (GF109203X) and calphostin C) blocked the activation of
NF-κB by each compound. Hence, protein kinase C activation may contribute to NF-κB activation by antineoplastic agents.
T-cell cytotoxicity is primarily mediated by two cell surface proteins, Fas ligand (FasL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), and intracellular perforin and granzyme granules. FasL-deficient and perforin-deficient T lymphocytes maintain cytotoxicity but fail to induce graft-versus-host disease (GVHD) when transplanted into mice, suggesting that GVHD and graft-versus-tumour (GVT) effects can be dissociated, and that TRAIL is not involved in the pathogenesis of GVHD. Because TRAIL could mediate a favourable GVT effect it became important to study the spectrum of its activity and to investigate factors that can dissociate its expression from FasL. TRAIL induced apoptosis in 11/41 (27%) tumour specimens of haematological origin compared to 16/41 (39%) induced by FasL. Although eight specimens were sensitive to both FasL and TRAIL, no synergism was observed between these two ligands. TRAIL induced apoptosis in a dose and time dependent manner with an ED50 of 0.5 μg/ml and EDmax of 1 μg/ml. TRAIL activity was not reduced by the over-expression of the multidrug resistant (MDR) protein, and was not enhanced by 9-cis retinoic acid (RA), which can down-regulate bcl-2 protein. Both ligands were simultaneously up-regulated in normal peripheral blood lymphocytes in response to IL-2, IL-15 and anti-CD3 antibody, whereas IL-10 had no effect. Together, our data show that (1) TRAIL can mediate cell death in a variety of human haematological malignancies, (2) resistance to TRAIL is not mediated by MDR protein, (3) the lack of synergy between TRAIL and FasL suggests that either one is sufficient to mediate T-cell cytotoxicity, and (4) within the panel of cytokines tested, the expression of TRAIL and FasL could not be dissociated.
Members of the tumor necrosis factor (TNF) family such as CD95 (APO-1/Fas) ligand (L) trigger apoptosis in lymphoid cells. Recently, a new member of apoptosis-inducing ligands, TRAIL (TNF-related-apoptosis-inducing-ligand)/Apo-2 ligand, was identified that might act in a similar way. We compared TRAIL and CD95L-induced apoptosis in human lymphoid cells. Expression of TRAIL was found in CD4+ and CD8+ T cells following activation, suggesting that TRAIL participates in T cell-mediated induction of apoptosis. Similar to CD95L, TRAIL-induced apoptosis in target cells is mediated by activation of caspases (ICE/Ced-3 proteases). However, different human lymphoid cell lines and peripheral T cells differ in sensitivity towards induction of apoptosis by TRAIL and CD95L. In addition, T cells are highly sensitive towards CD95L-induced apoptosis after prolonged activation in vitro, but remain completely resistant to TRAIL-induced apoptosis. In contrast, T cells from HIV-1-infected patients previously shown to exhibit increased CD95 sensitivity are even more susceptible towards TRAIL-induced cell death. These data suggest that TRAIL might participate in CD95-independent apoptosis of lymphoid cells and might be involved in deregulated apoptosis in diseases such as leukemias and HIV-1 infection.
NF-kappa B is a ubiquitous transcription factor. Nevertheless, its properties seem to be most extensively exploited in cells of the immune system. Among these properties are NF-kappa B's rapid posttranslational activation in response to many pathogenic signals, its direct participation in cytoplasmic/nuclear signaling, and its potency to activate transcription of a great variety of genes encoding immunologically relevant proteins. In vertebrates, five distinct DNA binding subunits are currently known which might extensively heterodimerize, thereby forming complexes with distinct transcriptional activity, DNA sequence specificity, and cell type- and cell stage-specific distribution. The activity of DNA binding NF-kappa B dimers is tightly controlled by accessory proteins called I kappa B subunits of which there are also five different species currently known in vertebrates. I kappa B proteins inhibit DNA binding and prevent nuclear uptake of NF-kappa B complexes. An exception is the Bcl-3 protein which in addition can function as a transcription activating subunit in th nucleus. Other I kappa B proteins are rather involved in terminating NF-kappa B's activity in the nucleus. The intracellular events that lead to the inactivation of I kappa B, i.e. the activation of NF-kappa B, are complex. They involve phosphorylation and proteolytic reactions and seem to be controlled by the cells' redox status. Interference with the activation or activity of NF-kappa B may be beneficial in suppressing toxic/septic shock, graft-vs-host reactions, acute inflammatory reactions, acute phase response, and radiation damage. The inhibition of NF-kappa B activation by antioxidants and specific protease inhibitors may provide a pharmacological basis for interfering with these acute processes.
We demonstrate an essential role for the proteasome complex in two proteolytic processes required for activation of the transcription factor NF-kappa B. The p105 precursor of the p50 subunit of NF-kappa B is processed in vitro by an ATP-dependent process that requires proteasomes and ubiquitin conjugation. The C-terminal region of p105 is rapidly degraded, leaving the N-terminal p50 domain. p105 processing can be blocked in intact cells with inhibitors of the proteasome or in yeast with proteasome mutants. These inhibitors also block the activation of NF-kappa B and the rapid degradation of I kappa B alpha induced by tumor necrosis factor alpha. Thus, the ubiquitin-proteasome pathway functions not only in the complete degradation of polypeptides, but also in the regulated processing of precursors into active proteins.
Inducible gene expression in eukaryotes is mainly controlled by the activity of transcriptional activator proteins, such as NF-kappa B (refs 1-3), a factor activated upon treatment of cells with phorbol esters, lipopolysaccharide, interleukin-1 and tumour necrosis factor-alpha. Activation of NF-kappa B involves release of the inhibitory subunit I kappa B from a cytoplasmic complex with the DNA-binding subunits Rel-A (formerly p65) and p50 (refs 6, 7). Cell-free experiments have suggested that protein kinase C and other kinases transfer phosphoryl groups onto I kappa B causing release of I kappa B and subsequent activation of NF-kappa B. Here we report that I kappa B-alpha (formerly MAD-3) is degraded in cells after stimulation with phorbol ester, interleukin-1, lipopolysaccharide and tumour necrosis factor-alpha, an event coincident with the appearance of active NF-kappa B. Treatment of cells with various protease inhibitors or an antioxidant completely prevented the inducible decay of I kappa B-alpha as well as the activation of NF-kappa B. Our findings suggest that the activation of NF-kappa B relies on an inducible degradation of I kappa B-alpha through a cytoplasmic, chymotrypsin-like protease. In intact cells, phosphorylation of I kappa B-alpha is apparently not sufficient for activation of NF-kappa B.
Engagement of the CD95 (APO-1/Fas) receptor induces apoptosis in a variety of cell types. However, the nature of the cytotoxic signal and the intermediate messenger molecules remain to be elucidated. In an effort to understand CD95-mediated signaling, we assessed possible changes in the DNA binding activity of NF-kappaB as a result of CD95 engagement in various tumor cells. By performing electrophoresis mobility shift assays, we show that CD95 can stimulate the DNA binding activity of NF-kappaB in a variety of cells, irrespective of their sensitivity or resistance to CD95-mediated cytotoxicity. Moreover, deletion of 37 carboxyl-terminal residues from the cytoplasmic domain of CD95, which abrogates CD95-mediated apoptosis, only marginally affects NF-kappaB activation. Taken together, these observations indicate that CD95 has a function that involves activation of NF-kappaB and that appears to be unrelated to its role as an inducer of apoptotic cell death.
The transcription factor NF-kappa B has attracted widespread attention among researchers in many fields based on the following: its unusual and rapid regulation, the wide range of genes that it controls, its central role in immunological processes, the complexity of its subunits, and its apparent involvement in several diseases. A primary level of control for NF-kappa B is through interactions with an inhibitor protein called I kappa B. Recent evidence confirms the existence of multiple forms of I kappa B that appear to regulate NF-kappa B by distinct mechanisms. NF-kappa B can be activated by exposure of cells to LPS or inflammatory cytokines such as TNF or IL-1, viral infection or expression of certain viral gene products, UV irradiation, B or T cell activation, and by other physiological and nonphysiological stimuli. Activation of NF-kappa B to move into the nucleus is controlled by the targeted phosphorylation and subsequent degradation of I kappa B. Exciting new research has elaborated several important and unexpected findings that explain mechanisms involved in the activation of NF-kappa B. In the nucleus, NF-kappa B dimers bind to target DNA elements and activate transcription of genes encoding proteins involved with immune or inflammation responses and with cell growth control. Recent data provide evidence that NF-kappa B is constitutively active in several cell types, potentially playing unexpected roles in regulation of gene expression. In addition to advances in describing the mechanisms of NF-kappa B activation, excitement in NF-kappa B research has been generated by the first report of a crystal structure for one form of NF-kappa B, the first gene knockout studies for different forms of NF-kB and of I kappa B, and the implications for therapies of diseases thought to involve the inappropriate activation of NF-kappa B.
A new member of the tumor necrosis factor (TNF) cytokine family, designated Apo-2 ligand (Apo-21) [1] or TRAIL [2], has been shown recently to induce apoptosis in various tumor cell lines; however, its biological role is unknown. Here, we show that Apo-21, activated apoptosis in T-cell-enriched cultures of peripheral blood lymphocytes stimulated by interleukin-2 (IL-2), but not in unstimulated cells. This finding suggests that, like Fas/Apo-1 ligand and TNF [3-5], Apo-2L may play a role in regulating post-stimulation apoptosis of mature lymphocytes. Studies on the mechanism of Apo-2L action demonstrated marked membrane blebbing, a hallmark of apoptosis, within a few minutes of the addition of Apo-2L to tumor cells. Ectopic expression of a dominant negative mutant of FADD, a cytoplasmic protein that mediates death signalling by Fas/Apo-1 and by TNF receptor type 1 (TNFR1) [6-9], inhibited the induction of apoptosis by anti-Fas/Apo-1 antibody, but had little effect on Apo-2L function. In contrast, expression of CrmA, a cowpox virus-derived inhibitor of the Ced-2-like proteases ICE [10] and CPP32/Yama [11,12], blocked the induction of apoptosis by either Apo-2L or anti-Fas/Apo-1 antibody. These results suggest that Apo-2L activates a rapid, FADD-independent pathway to trigger a cell-death programme that requires the function of cysteine proteases such as ICE or CPP32/Yama.
Recent investigations indicate that proteolysis is an important event in generation of the apoptosis phenotype. Although various proteases have been suggested to be candidates for this proteolysis, the results from different laboratories are inconsistent. In the present studies, HL-60 cells were treated with cycloheximide to investigate proteases involved in apoptosis. The calpain inhibitors benzyloxycarbonyl-Leu-Leu-Tyr diazomethylketone and acetyl-Leu-Leu-Nle aldehyde were not capable of preventing apoptosis induced by cycloheximide. In the absence of cycloheximide, these two inhibitors could initiate apoptosis in HL-60 cells. The thiol protease inhibitor benzyloxycarbonyl-Leu-Val-Gly diazomethylketone neither prevented nor produced apoptosis. The serine protease inhibitors 3,4-dichloroisocoumarin (DCI) and tosyl-Phe chloromethylketone (TPCK) also induced apoptosis in the absence of cycloheximide. On the other hand, the latter two inhibitors decreased cycloheximide-induced apoptosis, assessed either by cell morphologic changes or DNA ladder generation. Benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone and iodoacetamide, inactivators of interleukin 1beta-converting enzyme (ICE)-like proteases, did not produce apoptosis and inhibited the induction of apoptosis by cycloheximide, calpain inhibitors, or serine protease inhibitors. These results are consistent with the ICE-like proteases having a central role in proteolysis during apoptosis, while calpain-like proteases and the serine proteases sensitive to DCI or TPCK are not required for generation of the apoptosis phenotype in HL-60 cells.
Tumor necrosis factor α (TNF-α) signaling gives rise to a number of events, including activation of transcription factor NF-κB
and programmed cell death (apoptosis). Previous studies of TNF-α signaling have suggested that these two events occur independently.
The sensitivity and kinetics of TNF-α-induced apoptosis are shown to be enhanced in a number of cell types expressing a dominant-negative
IκBα (IκBαM). These findings suggest that a negative feedback mechanism results from TNF-α signaling in which NF-κB activation
suppresses the signals for cell death.
Many cells are resistant to stimuli that can induce apoptosis, but the mechanisms involved are not fully understood. The activation
of the transcription factor nuclear factor-kappa B (NF-κB) by tumor necrosis factor (TNF), ionizing radiation, or daunorubicin
(a cancer chemotherapeutic compound), was found to protect from cell killing. Inhibition of NF-κB nuclear translocation enhanced
apoptotic killing by these reagents but not by apoptotic stimuli that do not activate NF-κB. These results provide a mechanism
of cellular resistance to killing by some apoptotic reagents, offer insight into a new role for NF-κB, and have potential
for improvement of the efficacy of cancer therapies.
Developments in supportive care, new regimens for induction of remission and consolidation therapy, and bone-marrow transplantation have improved the outlook for patients with acute leukaemia. The outlook, though, is influenced by age and factors related to the biology of the disease. In acute myeloid leukaemia age is such an important factor that it determines approach to therapy. Overcoming resistance to chemotherapy is an area that needs much attention.
A subset of the tumour necrosis factor (TNF) receptor family contain a conserved intracellular motif, the death domain. Engagement of these receptors by their respective ligands initiates a signalling cascade that rapidly leads to cell death by apoptosis. We have cloned a new member of this family, TRICK2, the TRAIL (TNF-related apoptosis-inducing ligand) receptor inducer of cell killing 2. TRICK2 is expressed in a number of cell types, and to particularly high levels in lymphocytes and spleen. Two isoforms of the TRICK2 mRNA are generated by alternative pre-mRNA splicing and differ by a 29 amino-acid extension to the extracellular domain. Overexpression of TRICK2 rapidly induced apoptosis in 293T cells; this induction was dependent upon the presence of the death domain of TRICK2. Using a soluble molecule containing the TRICK2 extracellular domain, we demonstrated that TRICK2, like DR4 [1], is a receptor for TRAIL/APO-2L [2,3] and could inhibit TRAIL-induced killing of lymphocyte lines, such as the Jurkat T-cell line. TRAIL is upregulated upon lymphocyte activation, as is the intensively studied ligand for Fas, FasL [4]. TRAIL and its receptors might therefore provide another system for the regulation of lymphocyte selection and proliferation, as well as providing an additional weapon in the armoury of cytotoxic lymphocytes.
Two receptors for TRAIL, designated TRAIL-R2 and TRAIL-R3, have been identified. Both are members of the tumor necrosis factor receptor family. TRAIL-R2 is structurally similar to the death-domain-containing receptor TRAIL-R1 (DR-4), and is capable of inducing apoptosis. In contrast, TRAIL-R3 does not promote cell death. TRAIL-R3 is highly glycosylated and is membrane bound via a putative phosphatidylinositol anchor. The extended structure of TRAIL-R3 is due to the presence of multiple threonine-, alanine-, proline- and glutamine-rich repeats (TAPE repeats). TRAIL-R2 shows a broad tissue distribution, whereas the expression of TRAIL-R3 is restricted to peripheral blood lymphocytes (PBLs) and skeletal muscle. All three TRAIL receptors bind TRAIL with similar affinity, suggesting a complex regulation of TRAIL-mediated signals.
Apo2 ligand (Apo2L [1], also called TRAIL for tumor necrosis factor (TNF)-related apoptosis-inducing ligand [2]) belongs to the TNF family and activates apoptosis in tumor cells. Three closely related receptors bind Apo2L: DR4 and DR5, which contain cytoplasmic death domains and signal apoptosis, and DcR1, a decoy receptor that lacks a cytoplasmic tail and inhibits Apo2L function [3-5]. By cross-hybridization with DcR1, we have identified a fourth Apo2L receptor, which contains a cytoplasmic region with a truncated death domain. We subsequently named this protein decoy receptor 2 (DcR2). The DcR2 gene mapped to human chromosome 8p21, as did the genes encoding DR4, DR5 and DcR1. A single DcR2 mRNA transcript showed a unique expression pattern in human tissues and was particularly abundant in fetal liver and adult testis. Upon overexpression, DcR2 did not activate apoptosis or nuclear factor-kappaB; however, it substantially reduced cellular sensitivity to Apo2L-induced apoptosis. These results suggest that DcR2 functions as an inhibitory Apo2L receptor.
Fas/Apo1 and other cytotoxic receptors of the tumor necrosis factor receptor (TNFR) family contain a cytoplasmic death domain (DD) [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] that activates the apoptotic process by interacting with the DD-containing adaptor proteins TNFR-associated DD protein (TRADD) [12] [13] and Fas-associated DD protein (FADD/MORT1) [14] [15], leading to the activation of cysteine proteases of the caspase family [16]. Stimulation of Fas/Apo1 leads to the formation of a receptor-bound death-inducing signaling complex (DISC), consisting of FADD and two different forms of caspase-8 [17] [18] [19]. Transient expression of a dominant-negative mutant of FADD impairs TNFR60-mediated and Fas/Apo1-mediated apoptosis [13] [20], but has no effect on TNF-related apoptosis-inducing ligand (TRAIL/Apo2L)-induced cell death [7] [8] [9] [10] [21]. To study the function of FADD in DD-receptor signaling in more detail, we established HeLa cells that stably expressed a green fluorescent protein (GFP)-tagged dominant-negative mutant of FADD, GFP-DeltaFADD. Interestingly, expression of this mutant inhibited cell death induced by TNFR60, Fas/Apo1 and TRAIL-R/Apo2. In addition, GFP-DeltaFADD did not interfere with TNF-mediated gene induction or with activation of NF-kappaB or Jun N-terminal kinase (JNK), demonstrating that FADD is part of the TNFR60-initiated apoptotic pathway but does not play a role in TNFR60-mediated gene induction. Fas/Apo1-mediated activation of JNK was unaffected by the expression of GFP-DeltaFADD, suggesting that in Fas/Apo1 signaling the apoptotic pathway and the activation of JNK diverge at a level proximal to the receptor, upstream of or parallel to FADD.
Death receptor 5, a new member of the TNFR family, and DR4 induce FADD-dependent apoptosis and activate the NF-κB pathway.
- Chaudhary