Evaluation of kinase inhibitors as potential therapeutics for flavivirus infections.

The recent introduction of Zika virus (ZIKV), the recurrence of dengue virus (DENV), and the lethality of yellow fever virus (YFV) have had a significant impact on Brazilian society and public health. Here, we targeted two cellular kinases implicated in cell proliferation and cancer that are also important for viral replication: mitogen-activated protein kinase kinase (MEK) and Src. We used two MEK inhibitors - trametinib and selumetinib - and two Src inhibitors - saracatinib and bosutinib - to inhibit ZIKV, DENV, and YFV replication in cell culture. The cytotoxicity of the four inhibitors was determined by the observation of abnormal morphology and quantification of adherent cells by crystal violet staining. The antiviral activity of these drugs was assessed based on the reduction of plaque-forming units in cell culture as evidence of the inhibition of the replication of the selected flaviviruses. All four inhibitors showed antiviral activity, but among them, trametinib was the safest and most efficacious against all of the viruses, inhibiting the replication of ZIKV and YFV by 1000-fold, and DENV2/3 by nearly 100-fold. This pan-antiviral effect shows that trametinib could be repurposed for the treatment of flaviviral infections.

The small molecule AZD6244 inhibits dengue virus replication in vitro and protects against lethal challenge in a mouse model.

Dengue virus (DENV) is the most common mosquito-borne viral disease. The World Health Organization estimates that 400 million new cases of dengue fever occur every year. Approximately 500,000 individuals develop severe and life-threatening complications from dengue fever, such as dengue shock syndrome (DSS) and dengue hemorrhagic fever (DHF), which cause 22,000 deaths yearly. Currently, there are no specific licensed therapeutics to treat DENV illness. We have previously shown that the MEK/ERK inhibitor U0126 inhibits the replication of the flavivirus yellow fever virus. In this study, we demonstrate that the MEK/ERK inhibitor AZD6244 has potent antiviral efficacy in vitro against DENV-2, DENV-3, and Saint Louis encephalitis virus (SLEV). We also show that it is able to protect AG129 mice from a lethal challenge with DENV-2 (D2S20). The molecule is currently undergoing phase III clinical trials for the treatment of non-small-cell lung cancer. The effect of AZD6244 on the DENV life cycle was attributed to a blockade of morphogenesis. Treatment of AG129 mice twice daily with oral doses of AZD6244 (100 mg/kg/day) prevented the animals from contracting dengue hemorrhagic fever (DHF)-like lethal disease upon intravenous infection with 1 × 105 PFU of D2S20. The effectiveness of AZD6244 was observed even when the treatment of infected animals was initiated 1-2 days postinfection. This was also followed by a reduction in viral copy number in both the serum and the spleen. There was also an increase in IL-1ß and TNF-α levels in mice that were infected with D2S20 and treated with AZD6244 in comparison to infected mice that were treated with the vehicle only. These data demonstrate the potential of AZD6244 as a new therapeutic agent to treat DENV infection and possibly other flavivirus diseases.

c-Jun integrates signals from both MEK/ERK and MKK/JNK pathways upon vaccinia virus infection.

Usurpation of the host's signalling pathways is a common strategy employed by viruses to promote their successful replication. Here we show that infection with the orthopoxvirus vaccinia virus (VACV) leads to sustained stimulation of c-Jun activity during the entire infective cycle. This stimulation is temporally regulated through MEK/ERK or MKK/JNK pathways, i.e. during the early/mid phase (1 to 6 hpi) and in the late phase (9 to 24 hpi) of the infective cycle, respectively. As a transcriptional regulator, upon infection with VACV, c-Jun is translocated from the cytoplasm to the nucleus, where it binds to the AP-1 DNA sequence found at the promoter region of its target genes. To investigate the role played by c-Jun during VACV replication cycle, we generated cell lines that stably express a c-Jun-dominant negative (DNc-Jun) mutation. Our data revealed that c-Jun is required during early infection to assist with viral DNA replication, as demonstrated by the decreased amount of viral DNA found in the DNc-Jun cells. We also demonstrated that c-Jun regulates the expression of the early growth response gene (egr-1), a gene previously shown to affect VACV replication mediated by MEK/ERK signalling. VACV-induced stimulation of the MKK/JNK/JUN pathway impacts viral dissemination, as we observed a significant reduction in both viral yield, during late stages of infection, and virus plaque size. Collectively, our data suggest that, by modulating the host's signalling pathways through a common target such as c-Jun, VACV temporally regulates its infective cycle in order to successfully replicate and subsequently spread.

MEK2 controls the activation of MKK3/MKK6-p38 axis involved in the MDA-MB-231 breast cancer cell survival: Correlation with cyclin D1 expression.

The Ras-Raf-MEK-ERK1/2 signaling pathway regulates fundamental processes in malignant cells. However, the exact contributions of MEK1 and MEK2 to the development of cancer remain to be established. We studied the effects of MEK small-molecule inhibitors (PD98059 and U0126) and MEK1 and MEK2 knock-down on cell proliferation, apoptosis and MAPK activation. We showed a diminution of cell viability that was associated with a downregulation of cyclin D1 expression and an increase of apoptosis marker in MEK2 silenced cells; by contrast, a slight increase of cell survival was observed in the absence of MEK1 that correlated with an augment of cyclin D1 expression. These data indicate that MEK2 but not MEK1 is essential for MDA-MB-231 cell survival. Importantly, the role of MEK2 in cell survival appeared independent on ERK1/2 phosphorylation since its absence did not alter the level of activated ERK1/2. Indeed, we have reported an unrevealed link between MEK2 and MKK3/MKK6-p38 MAPK axis where MEK2 was essential for the phosphorylation of MKK3/MKK6 and p38 MAPK that directly impacted on cyclin D1 expression. Importantly, the MEK1 inhibitor PD98059, like MEK1 silencing, induced an augment of cyclin D1 expression that correlated with an increase of MDA-MB-231 cell proliferation suggesting that MEK1 may play a regulatory role in these cells. In sum, the crucial role of MEK2 in MDA-MB-231 cell viability and the unknown relationship between MEK2 and MKK3/MKK6-p38 axis here revealed may open new therapeutic strategies for aggressive breast cancer.

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.

Mimivirus circulation among wild and domestic mammals, Amazon Region, Brazil.

To investigate circulation of mimiviruses in the Amazon Region of Brazil, we surveyed 513 serum samples from domestic and wild mammals. Neutralizing antibodies were detected in 15 sample pools, and mimivirus DNA was detected in 9 pools of serum from capuchin monkeys and in 16 pools of serum from cattle.

The interplay between Araçatuba virus and host signaling pathways: role of PI3K/Akt in viral replication.

In this study, we describe the interaction between Araçatuba virus (ARAV), a naturally occurring Brazilian vaccinia virus isolated from an outbreak at a dairy farm, and the host cell's signal transduction pathways. Even though ARAV infection led to phosphorylation of MAPKs MEK/ERK, JNK, and p38MAPK, genetic or pharmacological inhibition of these pathways had no impact on viral replication. We also provide evidence that ARAV stimulated the phosphorylation of Akt (PKB) at serine 473 (S473-P), a signaling event that is required for full activation of Akt during the infectious cycle. Furthermore, pharmacological inhibition of PI3K (LY294002) abrogated ARAV-induced Akt activation (S473-P) and affected early and late viral gene expression, which was followed by a decrease in virus yield (~1 log). Taken together, our data shed some light onto the biological differences between ARAV and vaccinia virus strain WR (VACV-WR), which could contribute, at least in part, to the low-virulence phenotype displayed by ARAV. Thus, while the requirement for the PI3K/Akt pathway for successful ARAV replication is also shared with VACV-WR and cowpox virus strain BR (CPXV-BR), ARAV showed a lower replicative capacity, as well as a smaller plaque-size phenotype after infection of A31 cells when compared to VACV-WR.

The dengue virus nonstructural protein 1 (NS1) increases NF-κB transcriptional activity in HepG2 cells.

Dengue virus nonstructural protein 1 (NS1) is a glycoprotein involved in viral RNA replication. NS1 associates with host cell proteins and can be found in lipid raft domains on the host cell surface, suggesting an involvement in signal transduction events. In this work, we observed that NS1 expression in HepG2 cells increases nuclear translocation of NF-κB p65 protein, which was paralleled by DNA-protein complex formation. Luciferase assays showed an increase in NF-κB transcriptional activities in NS1-expressing cells when compared to parental cells. NS1 may enhance NF-κB function in host cells and contribute to the pathogenesis of dengue.

Vaccinia virus regulates expression of p21WAF1/Cip1 in A431 cells.

In this paper, we provide evidence that both the mRNA and protein levels of the cyclin-dependent kinase (CDK) inhibitor p21WAF1/CDK-interacting protein 1 (Cip1) increase upon infection of A431 cells with Vaccinia virus (VACV). In addition, the VACV growth factor (VGF) seems to be required for the gene expression because infection carried out with the mutant virus VACV-VGF- revealed that this strain was unable to stimulate its transcription. Our findings are also consistent with the notion that the VGF-mediated change in p21WAF1/Cip1 expression is dependent on tyrosine kinase pathway(s) and is partially dependent on mitogen-activated protein kinase/extracellular-signal regulated kinase 1/2. We believe that these pathways are biologically significant because VACV replication and dissemination was drastically affected when the infection was carried out in the presence of the relevant pharmacological inhibitors.

Vaccinia virus infection in monkeys, Brazilian Amazon.

To detect orthopoxvirus in the Brazilian Amazon, we conducted a serosurvey of 344 wild animals. Neutralizing antibodies against orthopoxvirus were detected by plaque-reduction neutralizing tests in 84 serum samples. Amplicons from 6 monkey samples were sequenced. These amplicons identified vaccinia virus genetically similar to strains from bovine vaccinia outbreaks in Brazil.

Vaccinia virus regulates expression of p21WAF1/Cip1 in A431 cells

In this paper, we provide evidence that both the mRNA and protein levels of the cyclin-dependent kinase (CDK) inhibitor p21WAF1/CDK-interacting protein 1 (Cip1) increase upon infection of A431 cells with Vaccinia virus (VACV). In addition, the VACV growth factor (VGF) seems to be required for the gene expression because infection carried out with the mutant virus VACV-VGF- revealed that this strain was unable to stimulate its transcription. Our findings are also consistent with the notion that the VGF-mediated change in p21WAF1/Cip1 expression is dependent on tyrosine kinase pathway(s) and is partially dependent on mitogen-activated protein kinase/extracellular-signal regulated kinase 1/2. We believe that these pathways are biologically significant because VACV replication and dissemination was drastically affected when the infection was carried out in the presence of the relevant pharmacological inhibitors.

PDE4 inhibition drives resolution of neutrophilic inflammation by inducing apoptosis in a PKA-PI3K/Akt-dependent and NF-kappaB-independent manner.

PDE4 inhibitors are effective anti-inflammatory drugs whose effects and putative mechanisms on resolution of inflammation and neutrophil apoptosis in vivo are still unclear. Here, we examined the effects of specific PDE4 inhibition on the resolution of neutrophilic inflammation in the pleural cavity of LPS-challenged mice. LPS induced neutrophil recruitment that was increased at 4 h, peaked at 8-24 h, and declined thereafter. Such an event in the pleural cavity was preceded by increased levels of KC and MIP-2 at 1 and 2 h. Treatment with the PDE4 inhibitor rolipram, at 4 h after LPS administration, decreased the number of neutrophils and increased the percentage of apoptotic cells in the pleural cavity in a PKA-dependent manner. Conversely, delayed treatment with a CXCR2 antagonist failed to prevent neutrophil recruitment. Forskolin and db-cAMP also decreased the number of neutrophils and increased apoptosis in the pleural cavity. The proapoptotic effect of rolipram was associated with decreased levels of the prosurvival protein Mcl-1 and increased caspase-3 cleavage. The pan-caspase inhibitor zVAD-fmk prevented rolipram-induced resolution of inflammation. LPS resulted in a time-dependent activation of Akt, which was blocked by treatment with rolipram or PI3K and Akt inhibitors, and PI3K and Akt inhibitors also enhanced apoptosis and promoted neutrophil clearance. Although LPS induced NF-kappaB activation, which was blocked by rolipram, NF-kappaB inhibitors did not promote resolution of neutrophil accumulation in this model. In conclusion, our data show that PDE4 inhibition resolves neutrophilic inflammation by promoting caspase-dependent apoptosis of inflammatory cells by targeting a PKA/PI3K/Akt-dependent survival pathway.

Cyclic AMP enhances resolution of allergic pleurisy by promoting inflammatory cell apoptosis via inhibition of PI3K/Akt and NF-kappaB.

Selective and timely induction of apoptosis is an effective means of resolving inflammation. The effects and putative mechanisms by which cyclic AMP (cAMP) modulates leukocyte apoptosis in vivo are still unclear. The present study aims at identifying intracellular pathways underlying the ability of cAMP elevating agents to resolve eosinophilic inflammation in a model of allergic pleurisy in mice. Ovalbumin (OVA) challenge of immunized mice induced eosinophil recruitment that peaked at 24h and persisted till 48h. Treatment with the PDE4 inhibitor rolipram, cAMP mimetic db-cAMP or adenylate cyclase activator forskolin, at 24h after antigen-challenge resulted in profound resolution of eosinophilic inflammation, without a decrease of mononuclear cell numbers. There was a concomitant increase in number of apoptotic cells in the pleural cavity. The effects of rolipram and db-cAMP were inhibited by the PKA inhibitor H89. Inhibition of PI3K/Akt or NF-kappaB induced resolution of inflammation that was associated with increased apoptosis. OVA-challenge resulted in a time-dependent activation of Akt and NF-kappaB, which was blocked by treatment with rolipram or PI3K/Akt pathway inhibitors. Thus, cAMP elevating agents resolve established eosinophilic inflammation by inducing leukocyte apoptosis. Mechanistically, the actions of cAMP are dependent on PKA and target a PI3K/Akt-dependent NF-kappaB survival pathway.

Activation of the PI3K/Akt pathway early during vaccinia and cowpox virus infections is required for both host survival and viral replication.

Viral manipulation of the transduction pathways associated with key cellular functions such as actin remodeling, microtubule stabilization, and survival may favor a productive viral infection. Here we show that consistent with the vaccinia virus (VACV) and cowpox virus (CPXV) requirement for cytoskeleton alterations early during the infection cycle, PBK/Akt was phosphorylated at S473 [Akt(S473-P)], a modification associated with the mammalian target of rapamycin complex 2 (mTORC2), which was paralleled by phosphorylation at T308 [Akt(T308-P)] by PI3K/PDK1, which is required for host survival. Notably, while VACV stimulated Akt(S473-P/T308-P) at early (1 h postinfection [p.i.]) and late (24 h p.i.) times during the infective cycle, CPXV stimulated Akt at early times only. Pharmacological and genetic inhibition of PI3K (LY294002) or Akt (Akt-X and a dominant-negative form of Akt-K179M) resulted in a significant decline in virus yield (from 80% to >/=90%). This decline was secondary to the inhibition of late viral gene expression, which in turn led to an arrest of virion morphogenesis at the immature-virion stage of the viral growth cycle. Furthermore, the cleavage of both caspase-3 and poly(ADP-ribose) polymerase and terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end labeling assays confirmed that permissive, spontaneously immortalized cells such as A31 cells and mouse embryonic fibroblasts (MEFs) underwent apoptosis upon orthopoxvirus infection plus LY294002 treatment. Thus, in A31 cells and MEFs, early viral receptor-mediated signals transmitted via the PI3K/Akt pathway are required and precede the expression of viral antiapoptotic genes. Additionally, the inhibition of these signals resulted in the apoptosis of the infected cells and a significant decline in viral titers.

Differential sensitivity of C57BL/6 (M-1) and BALB/c (M-2) macrophages to the stimuli of IFN-gamma/LPS for the production of NO: correlation with iNOS mRNA and protein expression.

C57BL/6 and BALB/c mice are prototype hosts for the study of resistance and susceptibility to several infectious diseases. In many cases, resistance of C57BL/6 is due to the microbicidal effect of nitric oxide (NO) produced by macrophages in response to interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), mainly secreted by Th1 cells and macrophages, respectively. BALB/c, usually unable to give rise to Th1 lymphocytes, does not control certain infections. However, we and others have previously observed that regardless of the adaptive immune response, C57BL/6 (M-1) macrophages are far more sensitive to the stimulus of IFN-gamma-plus lipopolysaccharide (LPS) for the production of NO than are BALB/c (M-2) cells, a feature that might also account for resistance. Here, we report that the differential production of NO by M-1 and M-2 macrophages correlates with the accumulation of inducible nitric oxide synthase (iNOS) mRNA and protein, which shows that expression of iNOS is differentially regulated in M-1 and M-2 cells. The higher accumulation of iNOS mRNA in M-1 cells is independent of its stability, and, thus, it is possible that transcription of the iNOS gene in these cells may be more efficient than in M-2 cells. A remarkable finding is that the level of iNOS protein is much higher in M-1 macrophages than in M-2 cells, as compared with the mRNA levels, which makes us speculate that differential translational or posttranslational controls of iNOS gene are operative.

The ATP-sensitive potassium channel blocker glibenclamide prevents renal ischemia/reperfusion injury in rats.

BACKGROUND: Renal ischemia/reperfusion (I/R) is a complex neutrophil-mediated syndrome. Adenosine-triphosphate (ATP)-sensitive potassium (K(ATP)) channels are involved in neutrophil migration in vivo. In the present study, we have investigated the effects of glibenclamide, a K(ATP) channel blocker, in renal I/R injury in rats. METHODS: The left kidney of the rats was excised through a flank incision and ischemia was performed in the contralateral kidney by total interruption of renal artery flow for 45 minutes. Renal perfusion was reestablished, and the kidney and lungs were removed for analysis of vascular permeability, neutrophil accumulation, and content of cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-10] 4 and 24 hours later. Renal function was assessed by measuring creatinine, Na(+), and K(+) levels in the plasma and by determination of creatinine clearance. Drugs were administered subcutaneously after the onset of ischemia. RESULTS: Reperfusion of the ischemic kidney induced local (kidney) and remote (lung) inflammatory injury and marked renal dysfunction. Glibenclamide (20 mg/kg) significantly inhibited the reperfusion-associated increase in vascular permeability, neutrophil accumulation, increase in TNF-alpha levels and nuclear factor-kappaB (NF-kappaB) translocation. These inhibitory effects were noticed in the kidney and lungs. Moreover, glibenclamide markedly ameliorated the renal dysfunction at 4 and 24 hours. CONCLUSION: Treatment with glibenclamide is associated with inhibition of neutrophil recruitment and amelioration of renal dysfunction following renal I/R. Glibenclamide may have a therapeutic role in the treatment of renal I/R injury, such as after renal transplantation.

Plasminogen/plasmin regulates c-fos and egr-1 expression via the MEK/ERK pathway.

In this study, we showed that plasminogen (Plg) and plasmin (Pla) bind to lysine-binding sites on cell surface and trigger a signaling pathway that activates the mitogen-activated protein kinase (MAPK) MEK and ERK1/2, which in turn leads to the expression of the primary response genes c-fos and early growth response gene egr-1. Our data show that the Plg/Pla-stimulated steady-state mRNA levels of both genes reached a maximum by 30 min and then returned to basal levels by 1h. The gene induction was sensitive to both pharmacological and genetic inhibition of MEK. Leupeptin, a serine protease inhibitor, suppressed Pla but not Plg-induced c-fos and egr-1 expression, emphasizing the role played by the serine protease activity associated with Pla. Pre-incubation with cholera toxin completely blocked the Plg/Pla-induced gene expression, suggesting that another signaling pathway, which recruits G protein-coupled receptors, may also be involved. Furthermore, Plg/Pla also stimulated AP-1 and EGR-1 DNA-binding activities, which were abrogated by pharmacological inhibition of MEK. Altogether, these results suggest that Plg/Pla stimulates c-fos and egr-1 expression via activation of the MEK/ERK pathway.

The vaccinia virus-stimulated mitogen-activated protein kinase (MAPK) pathway is required for virus multiplication.

Early events play a decisive role in virus multiplication. We have shown previously that activation of MAPK/ERK1/2 (mitogen-activated protein kinase/extracellular-signal-regulated kinase 1/2) and protein kinase A are pivotal for vaccinia virus (VV) multiplication [de Magalhães, Andrade, Silva, Sousa, Ropert, Ferreira, Kroon, Gazzinelli and Bonjardim (2001) J. Biol. Chem. 276, 38353-38360]. In the present study, we show that VV infection provoked a sustained activation of both ERK1/2 and RSK2 (ribosomal S6 kinase 2). Our results also provide evidence that this pattern of kinase activation depends on virus multiplication and ongoing protein synthesis and is maintained independently of virus DNA synthesis. It is noteworthy that the VGF (VV growth factor), although involved, is not essential for prolonged ERK1/2 activation. Furthermore, our findings suggest that the VV-stimulated ERK1/2 activation also seems to require actin dynamics, microtubule polymerization and tyrosine kinase phosphorylation. The VV-stimulated pathway MEK/ERK1/2/RSK2 (where MEK stands for MAPK/ERK kinase) leads to phosphorylation of the ternary complex factor Elk-1 and expression of the early growth response (egr-1) gene, which kinetically paralleled the kinase activation. The recruitment of this pathway is biologically relevant, since its disruption caused a profound effect on viral thymidine kinase gene expression, viral DNA replication and VV multiplication. This pattern of sustained kinase activation after VV infection is unique. In addition, by connecting upstream signals generated at the cytoskeleton and by tyrosine kinase, the MEK/ERK1/2/RSK2 cascade seems to play a decisive role not only at early stages of the infection, i.e. post-penetration, but is also crucial to define the fate of virus progeny.

Inhibition of ERK1/2 and CREB phosphorylation by caspase-dependent mechanism enhances apoptosis in a fibrosarcoma cell line treated with butyrate.

We evaluated the role of MAPKs on apoptosis induced by butyrate in cells derived from a human fibrosarcoma (2C4). Culture of 2C4 cells in 5% of fetal bovine serum (FBS) induced ERK1/2 and CREB phosphorylation and delayed apoptosis induced by butyrate. Butyrate inhibited phosphorylation of ERK1/2 and CREB. Furthermore, the use of specific inhibitors PD98059 (MEK) and H89 (PKA), which block ERK1/2 and CREB phosphorylation, accelerated butyrate induced cell death in 2C4 cells. The butyrate effect was shown to be dependent on caspase activation, once caspase inhibitors restored phosphorylation of ERK1/2 and CREB in 2C4 cells. However, the proteolytic effect of caspases was not directly on ERK1/2 and CREB proteins. In conclusion, butyrate induced apoptosis in 2C4 cells is regulated by the levels of ERK1/2 and CREB phosphorylation in a caspase dependent mechanism.

Apoptosis induced by butyrate is independent of Jak/STAT signaling in a fibrosarcoma cell line.

The aim of this study was to evaluate the participation of the Jak-1 and STAT-1 proteins in sodium butyrate-induced apoptosis in 2C4 cells derived from human fibrosarcoma. Making use of Jak-1 or STAT-1 deficient cell lines, we demonstrated that the apoptotic process induced by butyrate is independent of the presence of these proteins. In addition, this work showed that, although the constitutive expression of pro-caspases-2 and -3 is reduced in STAT-1 cells, the activity of caspase-3 is preserved in both Jak-1 and STAT-1 deficient cells and is similar to that seen in 2C4 parental cells. In conclusion, we demonstrated that the absence of functionally active Jak-1 or STAT-1 protein directly affects the TNF-alpha-induced apoptosis, but does not alter the sodium butyrate-induced apoptosis in cells derived from human fibrosarcoma.