Distinctive role of integrin-mediated adhesion in TNF-induced PKB/Akt and NF-kappaB activation and endothelial cell survival.

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine exerting pleiotropic effects on endothelial cells. Depending on the vascular context it can induce endothelial cell activation and survival or death. The microenvironmental cues determining whether endothelial cells will survive or die, however, have remained elusive. Here we report that integrin ligation acts permissive for TNF-induced protein kinase B (PKB/Akt) but not nuclear factor (NF)-kappaB activation. Concomitant activation of PKB/Akt and NF-kappaB is essential for the survival of endothelial cells exposed to TNF. Active PKB/Akt strengthens integrin-dependent endothelial cell adhesion, whereas disruption of actin stress fibers abolishes the protective effect of PKB/Akt. Integrin-mediated adhesion also represses TNF-induced JNK activation, but JNK activity is not required for cell death. The alphaVbeta3/alphaVbeta5 integrin inhibitor EMD121974 sensitizes endothelial cells to TNF-dependent cytotoxicity and active PKB/Akt attenuates this effect. Interferon gamma synergistically enhanced TNF-induced endothelial cell death in all conditions tested. Taken together, these observations reveal a novel permissive role for integrins in TNF-induced PKB/Akt activation and prevention of TNF-induced death distinct of NF-kappaB, and implicate the actin cytoskeleton in PKB/Akt-mediated cell survival. The sensitizing effect of EMD121974 on TNF cytotoxicity may open new perspectives to the therapeutic use of TNF as anticancer agent.

A role for AP-1 in apoptosis: the case for and against.

The nuclear transcription factor AP-1, composed of dimers of Fos and Jun proteins, has been linked to a startling breadth of cellular events including cell transformation, proliferation, differentiation and apoptosis. AP-1 is often portrayed as a general, nuclear decision-maker that determines life or death cell fates in response to extracellular stimuli. However, it is increasingly clear that the cellular context is critical for determining the contribution of AP-1 to cellular fates, and the role of AP-1 in apoptosis should be considered within the context of a complex network of nuclear factors that respond simultaneously to a wide range of signal transduction pathways. We take a closer look at the evidence for and against a role for AP-1 in inducing apoptosis, drawing on examples of studies in neurons, lymphocytes and hepatocytes. Although AP-1 activation is associated with a large number of apoptotic scenarios, its role in ensuring cell survival seems equally important. It is, therefore, difficult to convict AP-1 as a killer without taking into account the cellular and extracellular context within which it is functioning. Defining the target genes regulated by AP-1 in these different contexts will help to decipher the contribution of AP-1 to cell fate decisions.

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.

Effect of nuclear factor kappaB inhibition on tumor cell sensitivity to natural killer-mediated cytolytic function.

Inhibition of the transcription factor NF-kappaB has been reported to increase cell sensitivity to TNF and some cytotoxic drugs. We investigated the effect of NK-kappaB inhibition on the susceptibility of tumor cells to freshly isolated, nonactivated, human NK cells and to a TCRgamma/delta T cell clone displaying an MHC-unrestricted "NK-like" lysis. Using electrophoretic mobility shift assay, we first demonstrated that NF-kappaB/DNA binding activity was induced in target cells following coculture with NK cells or TCRgamma/delta T cell clone. To investigate the effect of target cell NF-kappaB inhibition on NK-mediated lysis, we blocked NF-kappaB translocation by introducing a human cDNA coding for a mutated IkappaB-alpha. Interestingly, our results indicated that inhibition of NF-kappaB did not induce any increase in either granzyme-dependent non-MHC-restricted cytotoxicity mediated by fresh non-stimulated NK cells and by TCR gamma/delta T cell clone or in CD95-mediated lysis. These results emphasize that NF-kappaB 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-kappaB pathway is not a general mechanism for controlling the cytotoxic response.

Resistance to TNF-induced cytotoxicity correlates with an abnormal cleavage of cytosolic phospholipase A2.

To investigate the mechanism underlying the absence of arachidonic acid (AA) release by TNF in TNF-resistant cells, we first performed comparative analysis of phospholipid pools in both TNF-sensitive (MCF7) and their equivalent resistant cells (C1001). Quantification and incorporation studies of [(3)H]AA indicated that TNF-resistant cells were not depleted in AA. Furthermore, distribution of this fatty acid in different phospholipid pools was similar in both sensitive cells and their resistant counterparts, ruling out a defect in phospholipid pools. Since phospholipase A(2) (PLA(2)) are the main enzymes releasing free AA, we investigated their relative contribution in the acquisition of cell resistance to TNF-induced cell death and AA release. For this purpose, we used two PLA(2) inhibitors, methylarachidonyl fluorophosphate (MAFP) and bromoenol lactone (BEL), which selectively and irreversibly inhibit the cytosolic PLA(2) (cPLA(2)) and the Ca(2+)-independent PLA(2), respectively. Although a significant inhibitory effect of MAFP on both TNF-induced AA release and PLA(2) activity in MCF7 was observed, BEL had no effect. The inhibitory effect of MAFP on cPLA(2) activity correlated with an inhibition of TNF-induced cell death. Western blot analysis revealed that TNF induced a differential cleavage of cPLA(2) in TNF-sensitive vs TNF-resistant cells. Although the p70 (70-kDa) form of cPLA(2) was specifically increased in TNF-sensitive cells, a cleaved form, p50 (50 kDa), was selectively observed in TNF-resistant C1001 cells in the presence or absence of TNF. These findings suggest that the acquisition of cell resistance to this cytokine may involve an abnormal cPLA(2) cleavage.

Adenovirus-mediated wild-type-p53-gene expression sensitizes TNF-resistant tumor cells to TNF-induced cytotoxicity by altering the cellular redox state.

We have shown that the loss of p53 function contributed to resistance of tumor cells to TNF-induced cytotoxicity. In the present study, we evaluated the effect of wild-type p53 (wt-p53) expression on TNF sensitivity, by introducing wt-p53 into MCF7/Adr cells in which p53 was deleted, via a recombinant adenovirus encoding p53 (Ad-p53). Our results indicate that infection with Ad-p53 (50-100 viral particles per cell) resulted in pronounced cytotoxicity, whereas infection with 10 viral particles per cell, which was weakly toxic for the MCF7/Adr cells, sensitized these cells to TNF-induced cell death. Moreover, expression of wt-p53 in MCF7/Adr cells induced the production of reactive oxygen intermediates (ROIs) and caused glutathione (GSH) depletion, indicating disturbances in the cellular redox state. Additional treatment of cells with the anti-oxidant and glutathione (GSH) precursor N-acetylcysteine (NAC) resulted in inhibition of p53-induced ROIs production and in partial restoration of intracellular GSH levels, which was associated with the ability of NAC to inhibit p53-modulated TNF-induced cytotoxicity. Interestingly, Ad-p53 was able to inhibit TNF-induced MnSOD mRNA expression in MCF7/Adr cells, which might contribute to the sensitization of cells to the cytotoxic action of TNF. Taken together, our data strongly suggest that wt-p53 expression sensitizes TNF-resistant MCF7 cells with p53 deletion to TNF-induced cell death by a pathway that is dependent on ROIs production.

Adenovirus-mediated transfer of wild-type p53 gene sensitizes TNF resistant MCF7 derivatives to the cytotoxic effect of this cytokine: relationship with c-myc and Rb.

Tumor suppressor p53 is a nuclear transcription factor that blocks cell cycle progression and induces apoptosis. We have previously shown that the MCF7 resistance to the cytotoxic action of TNF correlates with p53 mutations. In the present study, we used a recombinant adenovirus carrying a wild-type p53 gene (Adwtp53) in order to investigate the effect of wt p53 transfer on modulation of cell resistance to the cytotoxic action of TNF. Our data indicate that infection of TNF resistant MCF7 cells (1001 and MCF7/Adr) with Adwtp53 resulted in the restoration of wt p53 expression and function as respectively revealed by the yeast assay and the induction of p53 inducible genes MDM2 and p21. Furthermore, the restoration of p53 function significantly sensitized TNF resistant cells to TNF cytotoxic action. This correlated with a significant down-regulation of c-myc in both TNF-resistant cell lines and a decrease of Retinoblastoma protein (Rb) in 1001 clone. In contrast, the effect of p53 seems to be independent from Bcl-2 and Bax protein level regulation. The present study suggests that the combination of TNF and Adwtp53 may be a potential strategy to sensitize mutant p53 TNF-resistant tumors to the cytotoxic action of this cytokine.

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.

Stable inhibition of nuclear factor kappaB in cancer cells does not increase sensitivity to cytotoxic drugs.

Several reports indicated that nuclear factor kappaB (NF-kappaB) activation by cytokines, cytotoxic drugs, or ionizing radiation protects cells against apoptosis. Therefore, we investigated the consequence of NF-kappaB inhibition on the efficiency of antineoplastic agents. HPB, HCT116, MCF7, and OVCAR-3 cells stably expressing a dominant negative IkappaBalpha inhibitor showed a decreased NF-kappaB activation following treatment with tumor necrosis factor a and various chemotherapeutic agents. However, there was no difference in survival between parental cells and cells expressing mutated IkappaBalpha. These studies suggest that, at least in these cell lines, stable NF-kappaB inhibition did not modify the response to cytotoxic drugs.

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.