Soluble epoxide hydrolase inhibitor promotes immunomodulation to inhibit bone resorption.

BACKGROUND AND OBJECTIVE: Soluble epoxide hydrolase (sEH) is an enzyme in the arachidonate cascade which converts epoxy fatty acids (EpFAs), such as epoxyeicosatrienoic acids (EETs) produced by cytochrome P450 enzymes, to dihydroxy-eicosatrienoic acids. In the last 20 years with the development of inhibitors to sEH it has been possible to increase the levels of EETs and other EpFAs in in vivo models. Recently, studies have shown that EETs play a key role in blocking inflammation in a bone resorption process, but the mechanism is not clear. In the current study we used the sEH inhibitor (1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea [TPPU]) to investigate the immunomodulatory effects in a mouse periodontitis model. MATERIAL AND METHODS: Mice were infected on days 0, 2, and 4 with Aggregatibacter actinomycetemcomitans and divided into groups (n = 6) that were treated orally, daily for 15 days, with 1 mg/kg of TPPU. Then, the mice were killed and their jaws were analyzed for bone resorption using morphometry. Immunoinflammatory markers in the gingival tissue were analyzed by microarray PCR or western blotting. RESULTS: Infected mice treated with TPPU showed lower bone resorption than infected mice without treatment. Interestingly, infected mice showed increased expression of sEH; however, mice treated with TPPU had a reduction in expression of sEH. Besides, several proinflammatory cytokines and molecular markers were downregulated in the gingival tissue in the group treated with 1 mg/kg of TPPU. CONCLUSION: The sEH inhibitor, TPPU, showed immunomodulatory effects, decreasing bone resorption and inflammatory responses in a bone resorption mouse model.

[Derivative of bleomycin generates less of ROS? Less of fibrosis? Alternative in the development of an efficacy anticancer therapy but less toxic]. / Dérivé de la bléomycine générant moins de ROS? Moins de fibrose? Une alternative dans le développement d'une thérapie anticancéreuse efficace mais moins toxique.

Deglycobleomycin (DBLM), the aglycon of the glycopeptide antitumor drug bleomycin (BLM), was first used since 1980 during comparative studies between BLM and DBLM in order to elucidate the role of the sugar component in the mechanism of action of BLM. In fact, the deglycosylation of BLM reduce the toxicity of this molecule and fails to produce reactive oxygen species, responsible for pulmonary fibrosis, and for anti-neoplastic activity of BLM. This causes toxic DNA lesions and ultimately leads to cell death. The therapeutic use of BLM is limited by a dose-dependent lung toxicity that eventually leads to fibrosis. Testing BLM-derivative molecules and defining their molecular mechanisms involved in tumor cell death may help to design new therapeutic approach with limited toxicity profile while maintaining anti-tumoral properties. The present review was undertaken to determine the effect of carbohydrate moiety in the mechanism of BLM induced cytotoxicity behind a comparative studies between BLM and DBLM.

Caspase-10 involvement in cytotoxic drug-induced apoptosis of tumor cells.

Anticancer drugs can induce tumor cell death by caspase-dependent apoptosis. The observation that procaspase-10 expression decreased in leukemic cells from acute myeloblastic leukemia patients at first relapse led us to explore the role of caspase-10 in cytotoxic drug-induced apoptosis. We show that caspase-10 is activated in etoposide-treated cells in a dose- and time-dependent manner. A caspase-10 peptide inhibitor, a caspase-10 dominant-negative mutant or a small interfering RNA (siRNA)-mediated downregulation of the enzyme negatively interfere with drug-induced cell death and caspase-2, -3, -8 and -9 activation. The extrinsic pathway to apoptosis is not involved in drug-induced caspase-10 activation that occurs downstream of Bax redistribution to mitochondria and cytochrome c release from this organelle. siRNA-mediated downregulation of Apaf-1 prevents etoposide-mediated activation of caspase-10. In a cell-free assay, cytochrome c and dATP treatment of cell extracts after immunodepletion of either caspase-3 or caspase-9 indicates that caspase-10 is activated downstream of caspase-9. Then, caspase-10 is involved in a feedback amplification loop that amplifies caspase-9 and -3 activities. Altogether, these data indicate an active role for caspase-10 in cytotoxic drug-induced tumor cell death, downstream of the mitochondria.

Role of protein kinase C zeta isoform in Fas resistance of immature myeloid KG1a leukemic cells.

Leukemic CD34(+) immature acute myeloid leukemia (AML) cells express Fas receptor but are frequently resistant to Fas agonistic reagents. Fas plays an important role in T-cell-mediated cytotoxicity, and recently it has been suggested that altered Fas signaling may contribute to drug resistance. Therefore, Fas resistance could be one of the mechanisms by which AML progenitors escape chemotherapy or T-cell-based immune intervention. However, the molecular mechanism of Fas resistance in AML cells has not been identified. Fas signaling can be interrupted at 3 mains levels: Fas clustering, alteration of death-inducing-signaling-complex (DISC) formation, and effector caspase inhibition of downstream caspase-8. This study shows that in the Fas-resistant CD34(+)CD38(-) KG1a cells, Fas agonists resulted in Fas aggregation but not in caspase-8 activation, related to a defect in DISC formation. However, pretreatment with chelerythrin, but not with calphostin C, resulted in the restoration of Fas-induced caspase-8 activation and cytotoxicity, suggesting that some atypical protein kinase C (PKC) isoforms contributed to the lack of DISC formation. Indeed, treatment with antisense oligonucleotides directed against PKC zeta and enforced expression of Par-4, a negative regulator of PKC zeta activity, restored Fas-induced caspase-8 activity and apoptosis. Moreover, it was found that PKC zeta interacts with FADD and that PKC zeta immunoextracts prepared from KG1a cells are able to phosphorylate FADD in vitro, whereas this phosphorylation is dramatically reduced in Par-4 transfectant cells. In conclusion, it is suggested that in AML cells, PKC zeta plays an important role in Fas resistance by inhibiting DISC formation, possibly by phosphorylating FADD.

Early increase in DcR2 expression and late activation of caspases in the platelet storage lesion.

Platelet transfusion is widely used to prevent bleeding in patients with severe thrombocytopenia. The maximal storage duration of platelet concentrates is usually 5 days, due to the platelet storage lesion that impairs their functions when stored for longer times. Some of the morphological and biochemical changes that characterize this storage lesion are reminiscent of cell death by apoptosis. The present study analyzed whether proteins involved in nucleated cell apoptosis could play a role in the platelet storage lesion. Storage of leukocyte-depleted platelets obtained by apheresis is associated with a late and limited activation of caspases, mainly caspase-3. This event correlates with an increased expression of the pro-apoptotic BH3-only protein Bim in the particulate fraction and a slight and late release of the pro-apoptotic mitochondrial protein Diablo/Smac in the cytosol. Platelets do not express the death receptors Fas, DR4 and DR5 on their plasma membrane, while the expression of the decoy receptor DcR2 increases progressively during platelet storage. Addition of low concentrations of the cryoprotector dimethylsulfoxide accelerates platelet caspase activation during storage, an effect that is partially prevented by the caspase inhibitor z-VAD-fmk. Altogether, DcR2 expression on the plasma membrane is an early event while caspase activation is a late event during platelet storage. These observations suggest that caspases are unlikely to account for the platelet storage lesion. As a consequence, addition of caspase inhibitors may not improve the quality of platelet concentrates stored in standard conditions.

Ceramide generation occurring during 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis is caspase independent and is not required to trigger cell death.

Biological activities of oxysterols seem tightly regulated. Therefore, the ability to induce cell death of structurally related oxysterols, such as those oxidized at C7(7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol), was investigated on U937 cells at different times of treatment in a concentration range of 5-80 microg/ml. Whereas all oxysterols accumulate inside the cells, strong inhibition of cell growth and increased permeability to propidium iodide were observed only with 7beta-hydroxycholesterol and 7-ketocholesterol, which trigger an apoptotic process characterized by the occurrence of cells with fragmented and/or condensed nuclei, and by various cellular dysfunctions: loss of mitochondrial transmembrane potential, cytosolic release of cytochrome c, activation of caspase-9 and -3 with subsequent enhanced activity of caspase-3, degradation of poly(ADP-ribose) polymerase, and increased accumulation of cellular C16 : 0 and C24 : 1 ceramide species. This ceramide generation is not attributed to caspase activation since inhibition of 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis by Z-VAD-fmk (100 microM), a broad spectrum caspase inhibitor, did not reduce C16 : 0 and C24 : 1 ceramide species accumulation. Conversely, when U937 cells were treated with 7beta-hydroxycholesterol and 7-ketocholesterol in the presence of fumonisin B1 (100 microM), a specific inhibitor of ceramide synthase, C16 : 0 and C24 : 1 ceramide species production was completely abrogated whereas apoptosis was not prevented. Noteworthy, 7alpha-hydroxycholesterol induced only a slight inhibition of cell growth. Collectively, these results are consistent with the notion that the alpha or beta hydroxyl radical position of oxysterols oxidized at C7 plays a key role in the induction of the apoptotic process. In addition, our findings demonstrate that 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis involve the mitochondrial signal transduction pathway and they suggest that C16 : 0 and C24 : 1 ceramide species generated through ceramide synthase play a minor role in the commitment of 7beta-hydroxycholesterol- and 7-ketocholesterol-induced cell death.

Role of Fas and granule exocytosis pathways in tumor-infiltrating T lymphocyte-induced apoptosis of autologous human lung-carcinoma cells.

We have isolated a cytotoxic T lymphocyte (CTL) clone, Heu161, that reacts specifically with the human autologous lung carcinoma cell line IGR-Heu. We first demonstrated that IGR-Heu lacked Fas-receptor expression and was resistant to CD95-induced apoptosis. To further elucidate the role of Fas in tumor immune surveillance, we have stably transfected IGR-Heu with a Fas-expression vector and isolated CD95-sensitive and -resistant clones. Our data indicated that the resistance of 2 selected Fas-transfected clones to CD95-mediated lysis correlated with down-regulation of caspase-8 or its lack of cleavage and subsequent activation. All Fas transfectants, either sensitive or resistant to anti-Fas agonistic antibody, were as efficiently lysed by the CTL clone as the parental cell line. In addition, neither anti-Fas-blocking antibody nor Fas-Fc molecule inhibited T-cell lysis of Fas-sensitive tumor clone. This cytotoxicity was extracellular Ca(2+)-dependent and abolished in the presence of EGTA, indicating that it was mainly granzyme-mediated. Interestingly, although the caspase inhibitor z-VAD-fmk had no effect on tumor-cell lysis, it efficiently blocked target DNA damage triggered by autologous CTLs via the granule exocytosis pathway, indicating that the latter event was caspase-dependent. The present results suggest that lung carcinoma-specific CTLs use mainly a granule exocytosis-dependent pathway to lyse autologous target cells and that these effectors are able to circumvent alteration of the Fas-triggered intracellular signalling pathway via activation of a caspase-independent cytoplasmic death mechanism.

Phosphatidylcholine-specific phospholipase C and phospholipase D are respectively implicated in mitogen-activated protein kinase and nuclear factor kappaB activation in tumour-necrosis-factor-alpha-treated immature acute-myeloid-leukaemia cells.

Tumour necrosis factor-alpha (TNFalpha) has been reported to induce potent growth inhibition of committed myeloid progenitor cells, whereas it is a potential growth stimulator of human CD34(+)CD38(-) multipotent haematopoietic cells. The present study was aimed at evaluating the respective role of two phospholipases, phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD) in the response of the CD34(+) CD38(-) KG1a cells to TNFalpha. In these cells TNFalpha triggered phosphoinositide 3-kinase (PI3K)-dependent PC hydrolysis within 4-8 min with concomitant production of both diacylglycerol (DAG) and phosphocholine (P-chol). DAG and P-chol production was accompanied by extracellular-signal-related protein kinase-1 ('ERK-1') activation and DNA-synthesis stimulation. PC-PLC stimulation was followed by PI3K-independent PLD activation with concomitant phosphatidic acid (PA) production followed by PA-derived DAG accumulation and sustained nuclear factor kappaB (NF-kappaB) activation. PLD/NF-kappaB signalling activation played no role in the TNFalpha proliferative effect and conferred no consistent protection of KG1a cells towards antileukaemic agents. Altogether these results suggest that, in KG1a cells, TNFalpha can stimulate in parallel PC-PLC and PLD, whose lipid products activate in turn mitogen-activated protein kinase (MAP kinase) and NF-kappaB signalling respectively. Finally, our study suggests that PC-PLC, but not PLD, plays a role in the TNFalpha proliferative effect in immature myeloid cells.

Positive and negative regulation of apoptotic pathways by cytotoxic agents in hematological malignancies.

Most chemotherapeutic drugs can induce tumor cell death by apoptosis. Analysis of the molecular mechanisms that regulate apoptosis has indicated that anticancer agents simultaneously activate several pathways that either positively or negatively regulate the death process. The main pathway from specific damage induced by the drug to apoptosis involves activation of caspases in the cytosol by pro-apoptotic molecules such as cytochrome c released from the mitochondrial intermembrane space. At least in some cell types, anticancer drugs also upregulate the expression of death receptors and sensitize tumor cells to their cognate ligands. The Fas-mediated pathway could contribute to the early steps of drug-induced apoptosis while sensitization to the cytokine TRAIL could be used to amplify the response to cytotoxic drugs. The Bcl-2 family of proteins, that includes anti- and pro-apoptotic molecules, regulates cell sensitivity mainly at the mitochondrial level. Anticancer drugs modulate their expression (eg through p53-dependent gene transcription), their activity (eg by phosphorylating Bcl-2) and their subcellular localization (eg by inducing the translocation of specific BH3-only pro-apoptotic proteins). Very early after interacting with tumor cells, anticancer drugs also activate lipid-dependent signaling pathways that either increase or decrease cell ability to die by apoptosis. In addition, cytotoxic agents can activate protective pathways that involve activation of NFkappaB transcription factor, accumulation of heat shock proteins such as Hsp27 and activation of proteins involved in cell cycle regulation. This review discusses how modulation of the balance between noxious and protective signals that regulate drug-induced apoptosis could be used to improve the efficacy of current therapeutic regimens in hematological malignancies.

Ligation of the CD44 adhesion molecule inhibits drug-induced apoptosis in human myeloid leukemia cells.

Adhesion molecules can improve hematopoietic cell survival; however, their role in leukemic cell resistance to drug-induced apoptosis is poorly documented. The CD44 adhesion molecule is strongly expressed on acute myeloid leukemia (AML) blasts. Using 2 myeloid cell lines, HL60 and NB4, evidence is presented that prior incubation with the CD44-specific monoclonal antibody (mAb) A3D8, reported to induce differentiation of AML blasts, significantly decreases apoptosis induced by 3 drugs used in AML chemotherapy: daunorubicin (DNR), mitoxantrone, and etoposide. In addition, in HL60 cells, CD44 ligation with A3D8 mAb fully abrogates the DNR-triggered generation of ceramide, a lipid second messenger involved in the DNR apoptotic signaling pathway. Moreover, results show that the A3D8 mAb and Bcl-2 additively inhibit DNR-induced apoptosis in HL60 cells overexpressing Bcl-2. These results suggest that, to eradicate AML blasts, the differentiation-inducing anti-CD44 mAb A3D8 should not be administered prior to apoptosis-inducing drugs.

Implication of radical oxygen species in ceramide generation, c-Jun N-terminal kinase activation and apoptosis induced by daunorubicin.

Anthracyclines such as daunorubicin (DNR) generate radical oxygen species (ROS), which account, at least in part, for their cytotoxic effect. We observed that early ceramide generation (within 6-10 min) through neutral sphingomyelinase stimulation was inhibitable by the antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate, which led to a decrease in apoptosis (>95% decrease in DNA fragmentation after 6 h). Furthermore, we observed that DNR triggers the c-Jun N-terminal kinase (JNK) and the transcription factor activated protein-1 through an antioxidant-inhibitable mechanism. Treatment of U937 cells with cell-permeant ceramides induced both an increase in ROS generation and JNK activation, and apoptosis, all of which were antioxidant-sensitive. In conclusion, DNR-triggered apoptosis implicates a ceramide-mediated, ROS-dependent JNK and activated protein-1 activation.

Involvement of de novo ceramide biosynthesis in lymphotoxin-induced oligodendrocyte death.

Both experimental and clinical studies suggest that lymphotoxin (LT) plays an important role in multiple sclerosis (MS) by inducing oligodendrocyte (OL) depletion. However, the mechanism of LT cytotoxicity is unknown. Because of the role of ceramide as a cell death mediator for a large variety of cytotoxic molecules, we have investigated the possible role of this second messenger in LT-induced cytotoxicity on SV40 immortalized new-born mice OL. Human recombinant LT exposure (50 ng/ml) resulted in intracellular ceramide accumulation which peaked at 48 h (approximately 170% increase) and paralleled LT-induced cytotoxicity. Moreover, fumonisin B1, a potent and specific ceramide synthase inhibitor, not only inhibited ceramide accumulation but also protected OL from LT cytotoxicity. These results suggest that LT-induced ceramide synthase stimulation and subsequent increased intracellular ceramide concentration are implicated in oligodendrocyte death.

The phosphoinositide 3-kinase/Akt pathway is activated by daunorubicin in human acute myeloid leukemia cell lines.

Daunorubicin induces apoptosis in myeloid leukemia cells by activation of neutral sphingomyelinase and ceramide generation occurring 4-10 min after daunorubicin addition. We show here that daunorubicin is able to increase the phosphoinositide 3-kinase activity and enhance intracellular phosphoinositide 3-kinase lipid products prior to ceramide generation. Daunorubicin activates Akt, a downstream phosphoinositide 3-kinase effector. Interestingly, the phosphoinositide 3-kinase inhibitors wortmannin and LY294002 accelerate daunorubicin-induced apoptosis in U937 cells. The phosphoinositide 3-kinase/Akt pathway has been involved in cell survival following serum deprivation, tumor necrosis factor alpha, anti-Fas and UV radiations. Our results suggest that anti-tumor agents such as daunorubicin may also activate anti-apoptotic signals that could contribute to drug resistance.

Methyltransferase inhibitor S-adenosyl-L-homocysteine sensitizes human breast carcinoma MCF7 cells and related TNF-resistant derivatives to TNF-mediated cytotoxicity via the ceramide-independent pathway.

In this study we investigated the signalling requirements for TNF-induced cytotoxicity modulated by the methyltransferase inhibitor S-adenosyl-L-homocysteine (AdoHcy) using the TNF-sensitive human breast carcinoma MCF7 cells and its established TNF-resistant clones (R-A1 and clone 1001). Our data indicate that inhibition of methylation reactions by adenosine plus homocysteine, which are known to condense within cells to AdoHcy, markedly potentiated TNF-induced cytotoxicity in MCF7 cells and rendered related TNF-resistant variants, TNF-sensitive by a mechanism independent from the ceramide pathway. We demonstrated that the dominant-negative derivative of FADD (FADD-DN) blocked methylation inhibition/TNF-induced cell death. Moreover, TNF-mediated cytotoxicity modulated by AdoHcy was blocked by the ICE-inhibiting peptide z-VAD-fmk, suggesting that an ICE-like protease is required for the methylation inhibition/TNF-inducible death pathway. In conclusion, these results suggest that the methyltransferase inhibitor AdoHcy potentiates TNF-induced cytotoxicity in MCF7 cells and renders TNF-resistant MCF7 clones, TNF-sensitive via the ceramide independent pathway and that FADD and the ICE-like protease are likely necessary components in transducing methylation inhibition/TNF signals for cell death.

Selective inhibition of apoptosis by TPA-induced differentiation of U937 leukemic cells.

U937 leukemic cells treated for 24 h with 16 nM 12-O-tetradecanoylphorbol 13-acetate (TPA), that induces their macrophagic terminal differentiation, become resistant to etoposide-induced apoptosis. Exposure of undifferentiated U937 cells to 50 microM etoposide for 6 h, that triggers apoptosis in 80% cells, activates procaspase-2L, -3 and -8, induces the mitochondrial release of cytochrome c and decreases Mcl-1 expression without modifying Bcl-2, Bcl-xL and Bax protein levels. All these events are inhibited in TPA-differentiated U937 cells that are also resistant to vinblastine-induced and Fas-mediated cell death. Interestingly, these cells are not inherently resistant to apoptosis induction. Exposure of TPA-differentiated U937 cells to 0.8 microg/ml cycloheximide for 24 h, that triggers apoptosis in 50% cells, activates procaspase-2L, -3 and -8, induces the mitochondrial release of cytochrome c and decreases Bcl-xL expression without modifying Bcl-2, Mcl-1 and Bax protein levels. All these events are not observed in undifferentiated cells treated in similar conditions. These results indicate that the apoptotic pathway that involves the release of cytochrome c from mitochondria and the cleavage of procaspases remains functional in TPA-differentiated cells.

Alteration of the daunorubicin-triggered sphingomyelin-ceramide pathway and apoptosis in MDR cells: influence of drug transport abnormalities.

We have previously shown that in myeloid leukemic cells, daunorubicin (DNR) induces apoptosis via the activation of the sphingomyelin-ceramide pathway. We have now investigated sphingomyelin (SM) hydrolysis, ceramide generation, and apoptosis in vincristine-selected multidrug resistant (MDR) HL-60 cells (HL-60/Vinc), compared with their parental counterparts. We show that DNR triggers the SM cycle (stimulation of neutral sphingomyelinase, SM hydrolysis, and ceramide generation) and apoptosis in both parental and MDR cells, when used at isotoxic doses (ie., 1 and 100 microM for HL-60 and HL-60/Vinc, respectively). However, in MDR cells treated with either 10 microM DNR or 1 microM DNR in association with the P-glycoprotein (P-gp) blocker verapamil (treatment conditions which yield an intracellular DNR concentration similar to that achieved with 1 microM in the parental cells), we were unable to detect SM hydrolysis, ceramide generation and apoptosis. This implies that inhibition of the DNR-induced SM cycle in MDR cells is not directly related to P-gp. We have also investigated the influence of intracellular drug localization on the DNR-induced SM-cycle in HL-60/Vinc cells. In these cells, DNR at 10 microM is mainly localized in cytoplasmic vesicles, while the drug is diffusely distributed when used at 100 microM. A diffuse distribution pattern was also observed when MDR cells were treated with 1 microM DNR in association with the cyclosporine derivative PSC-833, but not with verapamil. In parallel, PSC-833, but not verapamil, restored the induction of the SM cycle and the apoptotic potential of DNR, and markedly increased drug cytotoxicity in MDR cells. Our results suggest that altered intracellular drug transport plays an important role in limiting ceramide generation and cell death in MDR cells.

Daunorubicin- and mitoxantrone-triggered phosphatidylcholine hydrolysis: implication in drug-induced ceramide generation and apoptosis.

Several studies have suggested that diacylglycerol can affect the induction of apoptosis induced by toxicants and ceramide. The present study demonstrates that clinically relevant concentrations of the chemotherapeutic drugs daunorubicin and mitoxantrone (0.2-1 microM) transiently stimulated concurrently with sphingomyelin-derived ceramide generation and diacylglycerol and phosphorylcholine production within 4 to 10 min via phospholipase C hydrolysis of phosphatidylcholine. Pretreatment of cells with the xanthogenate compound D609, a potent inhibitor of phosphatidylcholine-phospholipase C, led to significant inhibition of drug triggered diacylglycerol and phosphorylcholine production and to a sustained increase in ceramide levels for a period up to 2 h. Moreover, D609 pretreatment induced both cell death and ceramide generation at daunorubicin and mitoxantrone concentrations previously shown to be ineffective (i.e., 0.1 microM). These results underline the importance of diacylglycerol in the regulation of programmed cell death and strongly argue for a balance between apoptotic (ceramide) and survival (diacylglycerol) signal transducers.

Analysis of human breast adenocarcinoma MCF7 resistance to tumor necrosis factor-induced cell death. Lack of correlation between JNK activation and ceramide pathway.

Considerable progress has been made in the understanding of tumor necrosis factor (TNF) signaling; however, the molecular and biochemical basis of tumor resistance to the cytotoxic action of TNF are still not definitively identified yet. Although a role of c-Jun N-terminal kinase (JNK) pathway has been suggested as an effector in TNF signaling, its exact relative contribution and its interaction with ceramide pathway and tumor resistance to TNF remain unknown. The relationship between JNK activation and human breast adenocarcinoma MCF7 resistance acquisition to the cytotoxic action of TNF was therefore investigated. We demonstrate that TNF triggers JNK activation in both TNF-sensitive MCF7 cells and its resistant derivative, RA1/1001. In addition, when MCF7 cells were stably transfected with mitogen-activated protein kinase kinase 4 (MKK4) dominant-negative cDNA or transiently transfected with a dominant-negative c-Jun mutant (TAM 67), their susceptibility to the cytotoxic action of TNF remains comparable with control cells. We also demonstrated that JNK activation does not require ceramide generation since in MCF7 cells transfected with a dominant-negative derivative of FADD (FADD-DN), which are resistant to the cytotoxic action of TNF, TNF induced JNK activation in the absence of ceramide generation. Furthermore, our data indicate that exogenous permeable synthetic ceramide C-6 induced the killing of MCF7 cells transfected with MKK4 dominant-negative cDNA. These results provide strong evidence indicating that tumor acquisition of resistance to the cytotoxic action of TNF may occur either independently or at a level downstream of JNK activation and suggest that JNK activation is not linked to ceramide pathway in TNF-mediated apoptosis.

Antitumor agent-induced apoptosis in myeloid leukemia cells: a controlled suicide.

Traditional antitumor research has generally believed that the cytotoxicity of antitumor agents was directly correlated with the amount of drug-induced cellular lesions. Accordingly, oncologists have tried to improve anticancer agent/target interactions by increasing the intracellular dose of active effectors. However, a growing body of evidence stemming from both clinical and experimental observations, strongly suggests that similar anticancer-induced lesions may result in different cellular responses, greatly influencing cytotoxicity. For example, it has been shown that in some but not all cellular models, antitumor agents trigger apoptosis, an irreversible process which leads to a rapid and complete elimination of tumor cells. Several of these studies also demonstrated that apoptosis induced by antitumor agents is highly regulated by multiple signaling pathways which are themselves influenced by oncogenes, protein kinase activities, external stimuli and the oxidative balance. Therefore, it appears that cell death commitment is controlled by both external and internal factors which interfere downstream of drug- or ionizing radiation-target interaction. The characterization of these mediators may provide novel strategies for modulating intracellular signaling pathways in order to promote apoptosis in drug-resistant tumor cells.