Apoptosis induced by interferon-alpha and antagonized by EGF is regulated by caspase-3-mediated cleavage of gelsolin in human epidermoid cancer cells

We have previously reported that interferon-alpha (IFNalpha) induces apoptosis and EGF can antagonize this effect in human epidermoid cancer KB cells. Since apoptosis occurs together with cytoskeleton reorganization we have evaluated if IFNalpha and EGF could modulate cell remodeling in our experimental conditions. We have found that 48 h 1,000 IU/ml IFNalpha induced structural reorganization of stress fibers and membrane delocalization and partial capping of the actin severing protein gelsolin. The transfection of KB cells with both a wild type (WT) or a C-terminal truncated form of gelsolin caused overexpression of the protein and an increase of both the spontaneous and IFNalpha-induced apoptosis and cell cytoskeletal modifications. In fact, after 48 h of treatment IFNalpha induced 45% of apoptotic cell death in parental cells while an approximately 80% of cell population was apoptotic in transfected cells. These effects occurred together with an increase of the expression and consequent degradation of gelsolin. Again the addition of EGF to IFNalpha-treated transfected cells caused a recovery of the apoptosis. Notably, IFNalpha and EGF did not modify the expression of other molecules associated to cytoskeleton such as focal adhesion kinase and vinculin. In the same experimental conditions IFNalpha induced also gelsolin cleavage that occurred together with caspase-3 activation and release of cytochrome c. All these effects were antagonized by the exposure of IFNalpha-treated KB to 10 nM EGF for the last 12 h. Moreover, the specific inhibition of caspase-3 with 20 microM DEVD completely abrogated apoptosis and gelsolin cleavage induced by IFNalpha. In conclusion, our data are the first demonstration that IFNalpha can induce morphological cell changes that are peculiar of apoptosis onset through the caspase-3-mediated cleavage of gelsolin. Furthermore, we have demonstrated that EGF is able to antagonize these effects through the inhibition of caspase-3 activation.     

EGF activates an inducible survival response via the RAS-> Erk-1/2 pathway to counteract interferon-alpha-mediated apoptosis in epidermoid cancer cells

The mechanisms of tumor cell resistance to interferon-alpha (IFNalpha) are at present mostly unsolved. We have previously demonstrated that IFNalpha induces apoptosis on epidermoid cancer cells and EGF antagonizes this effect. We have also found that IFNalpha-induced apoptosis depends upon activation of the NH(2)-terminal Jun kinase-1 (Jnk-1) and p(38) mitogen-activated protein kinase, and that these effects are also antagonized by EGF. At the same time, IFNalpha increases the expression and function of the epidermal growth factor receptor (EGF-R). Here we report that the apoptosis induced by IFNalpha occurs together with activation of caspases 3, 6 and 8 and that EGF also antagonizes this effect. On the basis of these results, we have hypothesized that the increased EGF-R expression and function could represent an inducible survival response that might protect tumor cells from apoptosis caused by IFNalpha via extracellular signal regulated kinase 1 and 2 (Erk-1/2) cascades. We have found an increased activity of Ras and Raf-1 in IFNalpha-treated cells. Moreover, IFNalpha induces a 50% increase of the phosphorylated isoforms and enzymatic activity of Erk-1/2. We have also demonstrated that the inhibition of Ras activity induced by the transfection of the dominant negative Ras plasmid RASN17 and the inhibition of Mek-1 with PD098059 strongly potentiates the apoptosis induced by IFNalpha. Moreover, the selective inhibition of this pathway abrogates the counteracting effect of EGF on the IFNalpha-induced apoptosis. All these findings suggest that epidermoid tumor cells counteract the IFNalpha-induced apoptosis through a survival pathway that involves the hyperactivation of the EGF-dependent Ras->Erk signalling. The selective targeting of this pathway appears to be a promising approach in order to enhance the antitumor activity of IFNalpha.     

The eukaryotic initiation factor 5A is involved in the regulation of proliferation and apoptosis induced by interferon-alpha and EGF in human cancer cells

Interferon-alpha (IFNalpha) can induce apoptosis, a process regulated by a complex network of cell factors. Among these, eukaryotic initiation factor-5A (eIF-5A) is peculiar because its activity is modulated by the post-translational formation of the amino acid hypusine. Here we report the effects of IFNalpha and epidermal growth factor (EGF) on apoptosis and eIF-5A activity in human epidermoid oropharyngeal KB and lung H1355 cancer cells. We found that 48-h exposure to 1000 and 2000 IU/ml IFNalpha induced about 50% growth inhibition and apoptosis in H1355 and KB cells, respectively, and the addition of EGF completely antagonized this effect. When IFNalpha induced apoptosis, a hyperactivation of MEK-1 and ERK signalling and a decrease of the hypusine-containing form and, thus, of eIF-5A activity were recorded. The latter effect was again antagonized by the addition of EGF to IFNalpha-pretreated cells, probably through the activation of the EGF-->ERK-dependent pathway, since the addition of the specific MEK-1 inhibitor PD098059 abrogated the recovery of intracellular hypusine content induced by EGF in IFNalpha-pretreated cancer cells. Subsequently, we evaluated if the hypusine synthesis inhibitor (and eIF-5A inactivator) N1-guanyl-1,7-diaminoheptane (GC7) synergized with IFNalpha in the induction of cell growth inhibition and apoptosis. The analysis of the isobologram of IFNalpha and GC7 demonstrated a strong synergism between the two drugs in inducing cell growth inhibition. We also found that GC7 and IFNalpha had a synergistic effect on apoptosis. These data suggest that the apoptosis induced by IFNalpha could be regulated by eIF-5A that, therefore, could represent a useful target for the potentiation of IFNalpha antitumor activity.     

Bovine serum amine oxidase and spm potentiate docetaxel and interferon-alpha effects in inducing apoptosis on human cancer cells through the generation of oxidative stress

It was previously demonstrated that bovine serum amine-oxidase (BSAO) and SPM (SPM) addition to cancer cells induces cell growth inhibition and over-run the multi-drug resistance (MDR) phenotype through the oxidative stress caused by polyamine metabolites. In this study, it is reported that BSAO/SPM enzymatic system antagonizes the survival pathway induced by either docetaxel (DTX) or interferon alpha (IFNalpha) in human epidermoid cancer KB cells. The combination of BSAO/SPM with either DTX or IFNalpha had a synergistic effect on cell growth inhibition through apoptosis in both human epidermoid KB and breast cancer MCF-7 cell lines. The effects of the BSAO/SPM-DTX combination on apoptosis were caspase 3 and 9-dependent and were paralleled by the enhancement of intracellular O(2-), nitric oxide levels and of lipo-oxidation. The scavenger moiety N-acetyl-cysteine antagonized the effects on apoptosis and cell growth inhibition induced by the combination suggesting a role of the oxidative products of SPM. These effects occurred together with a decrease of the physiological scavenger MnSOD and an increase of both p38 kinase activity and DNA damage. The results suggest that DTX and IFNalpha could sensitize tumour cells to the oxidative stress and apoptosis induced by BSAO/SPM through the induction of a survival ras-dependent pathway and the consequent elevation of the intracellular polyamine pool. These data allow the design of new therapeutic strategy based on the use of this combination in human neoplasms.     

C-Raf antagonizes apoptosis induced by IFN-alpha in human lung cancer cells by phosphorylation and increase of the intracellular content of elongation factor 1A

Interferon alpha (IFNalpha) induces both apoptosis and a counteracting epidermal growth factor Erk-dependent survival response in cancer cells. In this report, IFNalpha increased eukaryotic elongation factor 1A (eEF-1A) protein expression by inhibition of eEF-1A degradation via a proteasome-dependent pathway. The reduction of the expression level of eEF-1A by RNA interference enhanced the apoptosis induced by IFNalpha on the same cells. Moreover, IFNalpha induced the phosphorylation of both serine and threonine in eEF-1A. These effects were paralleled by an increased co-immunoprecipitation and colocalization of eEF-1A with C-Raf. The suppression of C-Raf kinase activity with the inhibitor BAY 43-9006 completely antagonized the increase of both eEF-1A phosphorylation and expression and of C-Raf/eEF-1A colocalization induced by IFNalpha and enhanced apoptosis and eEF-1A ubiquitination. Cell transfection with the mutated K48R ubiquitin increased EF-1A expression and desensitized tumor cells to the modulating effects of IFNalpha. The dynamic simulation of 3Dstructure of eEF-1A identified putative serine and threonine phosphorylation sites. In conclusion, the interaction between eEF-1A and C-Raf increases eEF-1A stability and induces a survival activity.     

Atorvastatin suppresses oxidized LDL-induced dendritic cell-like differentiation of RAW264.7 cells regulated by the p38 MAPK pathway

The molecular signaling events leading to protection from oxidative stress-induced apoptosis upon contact inhibition have not been fully investigated. Previous research has indicated a role for mitogen-activated protein kinases (MAPKs) in the regulation of contact inhibition, and these proteins have also been associated with cell cycle regulation and stress-induced apoptosis. The potential role of the MAPK JNK-1 in the stress-response of actively proliferating and contact-inhibited cells was investigated. Actively proliferating normal fibroblasts (BJ) and fibrosarcoma cells (HT-1080) were stressed with H2O2, and levels of activated JNK-1 and cleaved PARP were ascertained. Similarly, these results were compared with levels of activated JNK-1 and cleaved PARP detected in H2O2-stressed confluent fibrosarcoma or contact-inhibited fibroblast cells. Contact-inhibited fibroblasts were protected from apoptosis in comparison to subconfluent fibroblasts, concurrent with decreased JNK-1 activation. Increased culture density of fibrosarcoma cells was not protective against apoptosis, and these cells did not demonstrate density-dependent alterations in the JNK-1 stress response. This decreased activation of JNK-1 in stressed, contact-inhibited cells did not appear to be dependent upon increased expression of MKP-1; however, over-expression of MKP-1 was sufficient to result in a slight decrease in H2O2-stimulated PARP cleavage. Increasing the antioxidant capacity of fibroblasts through NAC-treatment not only lessened H2O2-stimulated JNK-1 activation, but also did not influence the expression of MKP-1. Taken together, these results suggest that regulation of negative regulation of JNK-1 upon contact inhibition is protective against apoptosis, and that this regulation is independent of MKP-1.     

Interferon alpha-induced apoptosis in tumor cells is mediated through the phosphoinositide 3-kinase/mammalian target of rapamycin signaling pathway

Interferon (IFN) alpha induces a caspase-dependent apoptosis that is associated with activation of the proapoptotic Bak and Bax, loss of mitochondrial membrane potential, and release of cytochrome c. In addition to the onset of the classical Jak-STAT pathway, IFNalpha also induced phosphoinositide 3-kinase (PI3K) activity. Pharmacological inhibition of PI3K activity by Ly294002 disrupted IFN-induced apoptosis upstream of mitochondria. Inhibition of mTOR by rapamycin or by overexpression of a kinase dead mutant of mTOR, efficiently blocked IFNalpha-induced apoptosis. A PI3K and mTOR-dependent phosphorylation of p70S6 kinase and 4E-BP1 repressor was induced by IFNalpha treatment of cells and was strongly inhibited by Ly294002 or rapamycin. The activation of Jak-STAT signaling upon IFNalpha stimulation was not affected by abrogating PI3K/mTOR pathway. Neither was the expression of several IFNalpha target genes affected, nor the ability of IFNalpha to protect against virus-induced cell death affected by inhibition of the PI3K/mTOR pathway. These data demonstrate that an intact PI3K/mTOR pathway is necessary for the ability of IFNalpha to induce apoptosis, whereas activation of the Jak-STAT pathway alone appears to be insufficient for this specific IFNalpha-induced effect.     

Epidermal growth factor protects epithelial cells against Fas-induced apoptosis. Requirement for Akt activation.

Chemotherapeutic drugs that damage DNA kill tumor cells, in part, by inducing the expression of a death receptor such as Fas or its ligand, FasL. Here, we demonstrate that epidermal growth factor (EGF) stimulation of T47D breast adenocarcinoma and embryonic kidney epithelial (HEK293) cells protects these cells from Fas-induced apoptosis. EGF stimulation of epithelial cells also inhibited Fas-induced caspase activation and the proteolysis of signaling proteins downstream of the EGF receptor, Cbl and Akt/protein kinase B (Akt). EGF stimulation of Akt kinase activity blocked Fas-induced apoptosis. Expression of activated Akt in MCF-7 breast adenocarcinoma cells was sufficient to block Fas-mediated apoptosis. Inhibition of EGF-stimulated extracellular signal-regulated kinase (ERK) activity did not affect EGF protection from Fas-mediated apoptosis. The findings indicate that EGF receptor stimulation of epithelial cells has a significant survival function against death receptor-induced apoptosis mediated by Akt.     

Type I interferons activate apoptosis in a Jurkat cell variant by caspase-dependent and independent mechanisms

Although the antiviral actions of interferons (IFNs) are observed in most types of cells, the antiproliferative effects of IFNalpha/beta are variable as are the mechanisms of growth inhibition that may or may not be due to the induction of apoptosis. To understand more about the mechanisms that are responsible for IFNalpha/beta-stimulated apoptosis, we have characterized a new human Jurkat T cell variant named H123 where IFNalpha activates programmed cell death (PCD). No differences in IFNalpha-stimulated, Stat-dependent gene expression were detected between H123 cells and the parental Jurkat cells, which are growth inhibited, but do not undergo apoptosis with IFNalpha. Although IFNalpha stimulates the activity of both caspase 3 and 9 in H123 cells, the general caspase inhibitor Z-VAD only partially reverses the apoptotic actions of IFNalpha. Induction of apoptosis by IFNalpha occurs through a mitochondrial-dependent pathway in H123 cells, as demonstrated by the release of cytochrome C from the mitochondria. Furthermore, IFNalpha treatment of H123 cells stimulates the release of the serine protease HtrA2/Omi from the mitochondria, suggesting that it plays a role in the apoptotic actions of this cytokine. These results provide evidence for a novel type 1 IFN-mediated pathway that regulates apoptosis of T cells through a mitochondrial-dependent and caspase-dependent and independent pathway.     

Epidermal Growth Factor-dependent Activation of the Extracellular Signal-regulated Kinase Pathway by DJ-1 Protein through Its Direct Binding to c-Raf Protein

DJ-1 is an oncogene and also a causative gene for familial Parkinson disease. DJ-1 has various functions, and the oxidative status of cysteine at position 106 (Cys-106) is crucial for determination of the activation level of DJ-1. Although DJ-1 requires activated Ras for its oncogenic activity and although it activates the extracellular signal-regulated kinase (ERK) pathway, a cell growth pathway downstream of Ras, the precise mechanism underlying activation of the ERK pathway by DJ-1 is still not known. In this study, we found that DJ-1 directly bound to the kinase domain of c-Raf but not to Ras and that Cys-106 mutant DJ-1 bound to c-Raf more weakly than did wild-type DJ-1. Co-localization of DJ-1 with c-Raf in the cytoplasm was enhanced in epidermal growth factor (EGF)-treated cells. Knockdown of DJ-1 expression attenuated the phosphorylation level of c-Raf in EGF-treated cells, resulting in reduced activation of MEK and ERK1/2. Although EGF-treated DJ-1 knock-out cells also showed attenuated c-Raf activation, reintroduction of wild-type DJ-1, but not C106S DJ-1, into DJ-1 knock-out cells restored c-Raf activation in a DJ-1 binding activity in a c-Raf-dependent manner. DJ-1 was not responsible for activation of c-Raf in phorbol myristate acetate-treated cells. Furthermore, DJ-1 stimulated self-phosphorylation activity of c-Raf in vitro, but DJ-1 was not a target for Raf kinase. Oxidation of Cys-106 in DJ-1 was not affected by EGF treatment. These findings showed that DJ-1 is a positive regulator of the EGF/Ras/ERK pathway through targeting c-Raf.     

Interferon-dependent activation of the serine kinase PI 3'-kinase requires engagement of the IRS pathway but not the Stat pathway

Several signaling pathways are activated by interferon alpha (IFNalpha) in hematopoietic cells, including the Jak-Stat and the insulin receptor substrate (IRS) pathways. It has been previously shown that IFNalpha activates the phosphatidylinositol (PI) 3'-kinase via an interaction of the p85 subunit of PI 3'-kinase with IRS proteins. Other studies have proposed that Stat-3 also functions as an adapter for p85. We sought to identify the major pathway that regulates IFNalpha activation of the PI3'-kinase in hematopoietic cells. Our data demonstrate that IFNalpha induces the interaction of p85 with IRS-1 or IRS-2, but not Stat-3, in various hematopoietic cell lines in which IRS-1 and/or IRS-2 and Stat-3 are activated by IFNalpha. In addition, inhibition of PI 3'-kinase activity by preincubation of cells with the PI 3'-kinase inhibitor LY294002 does not affect IFN-dependent formation of SIF complexes that contain Stat-3. To determine whether phosphorylation of tyrosine residues in the IFN receptor is required for activation of the PI 3'-kinase, we performed studies using mouse L929 fibroblasts transfected with mutated human IFNAR1 and/or IFNAR2 subunits of the Type I IFN receptor, lacking tyrosine phosphorylation sites. The serine kinase activity of the PI-3K was activated by human IFNalpha in these cells, suggesting that phosphorylation of the Type I IFN receptor is not essential for PI3K activation. We then determined whether IFNalpha activates the Akt kinase, a known downstream target for PI 3'-kinase that mediates anti-apoptotic signals. Akt was activated by insulin or IGF-1, but not IFNalpha, in the IFNalpha-sensitive U-266 myeloma cell line. Altogether, our data establish that the IRS pathway and not the Stat pathway, is the major pathway regulating engagement of PI 3'-kinase in hematopoietic cells. Furthermore, the selective activation of Akt by insulin/IGF-1 suggests the existence of distinct regulatory activities of PI3'-kinase in growth factor versus interferon signaling.     

Herpes simplex virus 1 blocks caspase-3-independent and caspase-dependent pathways to cell death

Earlier reports have shown that herpes simplex virus 1 (HSV-1) mutants induce programmed cell death and that wild-type HSV blocks the execution of the cell death program triggered by viral gene products, by the effectors of the immune system such as the Fas and tumor necrosis factor pathways, or by nonspecific stress agents such as either osmotic shock induced by sorbitol or thermal shock. A report from this laboratory showed that caspase inhibitors do not block DNA fragmentation induced by infection with the HSV-1 d120 mutant. To identify the events in programmed cell death induced and blocked by HSV-1, we examined cells infected with wild-type virus or the d120 mutant or cells infected and exposed to sorbitol. We report that: (i) the HSV-1 d120 mutant induced apoptosis by a caspase-3-independent pathway inasmuch as caspase 3 was not activated and DNA fragmentation was not blocked by caspase inhibitors even though the virus caused cytochrome c release and depolarization of the inner mitochondrial membrane. (ii) Cells infected with wild-type HSV-1 exhibited none of the manifestations associated with programmed cell death assayed in these studies. (iii) Uninfected cells exposed to osmotic shock succumbed to caspase-dependent apoptosis inasmuch as cytochrome c was released, the inner mitochondrial potential was lost, caspase-3 was activated, and chromosomal DNA was fragmented. (iv) Although caspase-3 was activated in cells infected with wild-type HSV-1 and exposed to sorbitol, cytochrome c outflow, depolarization of the inner mitochondrial membrane, and DNA fragmentation were blocked. We conclude that although d120 induces apoptosis by a caspase-3-independent pathway, the wild-type virus blocks apoptosis induced by this pathway and also blocks the caspase-dependent pathway induced by osmotic shock. The block in the caspase-dependent pathway may occur downstream of caspase-3 activation.     

Activation of B-Raf and regulation of the mitogen-activated protein kinase pathway by the G(o) alpha chain

Many receptors coupled to the pertussis toxin-sensitive G(i/o) proteins stimulate the mitogen-activated protein kinase (MAPK) pathway. The role of the alpha chains of these G proteins in MAPK activation is poorly understood. We investigated the ability of Galpha(o) to regulate MAPK activity by transient expression of the activated mutant Galpha(o)-Q205L in Chinese hamster ovary cells. Galpha(o)-Q205L was not sufficient to activate MAPK but greatly enhanced the response to the epidermal growth factor (EGF) receptor. This effect was not associated with changes in the state of tyrosine phosphorylation of the EGF receptor. Galpha(o)-Q205L also potentiated MAPK stimulation by activated Ras. In Chinese hamster ovary cells, EGF receptors activate B-Raf but not Raf-1 or A-Raf. We found that expression of activated Galpha(o) stimulated B-Raf activity independently of the activation of the EGF receptor or Ras. Inactivation of protein kinase C and inhibition of phosphatidylinositol-3 kinase abolished both B-Raf activation and EGF receptor-dependent MAPK stimulation by Galpha(o). Moreover, Galpha(o)-Q205L failed to affect MAPK activation by fibroblast growth factor receptors, which stimulate Raf-1 and A-Raf but not B-Raf activity. These results suggest that Galpha(o) can regulate the MAPK pathway by activating B-Raf through a mechanism that requires a concomitant signal from tyrosine kinase receptors or Ras to efficiently stimulate MAPK activity. Further experiments showed that receptor-mediated activation of Galpha(o) caused a B-Raf response similar to that observed after expression of the mutant subunit. The finding that Galpha(o) induces Ras-independent and protein kinase C- and phosphatidylinositol-3 kinase-dependent activation of B-Raf and conditionally stimulates MAPK activity provides direct evidence for intracellular signals connecting this G protein subunit to the MAPK pathway.     

Induction of apoptosis in luteinized granulosa cells by the MAP kinase kinase (MEK) inhibitor PD98059.

Our objective is to test the hypothesis that inhibition of mitogen-activated protein (MAP) kinase kinase (MEK) with PD98059 in human luteinized granulosa cells will block epidermal growth (EGF)-stimulated MAP kinase activity and induce apoptosis. Luteinized granulosa cells from human in vitro fertilization aspirates were cultured and treated with the following: (1) vehicle; (2) PD98059; (3) EGF; (4) PD98059 + EGF. Treatment with PD98059 suppressed MAP kinase activity, inhibited MAP kinase phosphorylation by Western blot analysis, blocked nuclear translocation of phosphorylated MAP kinase by confocal microscopy, and increased percentages of subdiploid apoptotic nuclei by flow cytometry. Our data are the first evidence that a relationship may exist between the MAP kinase pathway and control of apoptosis in human luteinized granulosa cells. These results support the hypothesis that suppression of the MAP kinase pathway may lead to apoptosis in these cells.     

Sphingosine 1-phosphate activates Erk-1/-2 by transactivating epidermal growth factor receptor in rat-2 cells

We investigated a signaling pathway leading to activation of extracellular signal-regulated protein kinase (Erk) 1 and 2 in Rat-2 cells stimulated with sphingosine 1-phosphate (S1P). S1P treatment transiently activated Erk-1/-2 in a dose-dependent manner, and its activation was blocked by pertussis toxin, expression of RasN17, or inhibition of Raf or MEK-1/-2. S1P-induced activation of Erk-1/-2 was also suppressed by the inhibition of epidermal growth factor (EGF) receptor tyrosine kinase with the specific inhibitor AG1478, suggesting that activation of EGF receptor tyrosine kinase was involved in the signaling pathway. S1P-induced Erk-1/-2 activation was enhanced up to 2-fold by inhibiting protein kinase C (PKC) with GF109203X, and PKC inhibition in the absence of S1P treatment also activated Erk-1/-2. The stimulatory effects of Erk-1/-2 activation by PKC inhibition was blocked by treating cells with AG1478, suggesting the involvement of PKC in the regulation of EGF receptor tyrosine kinase activation that leads to Erk-1/-2 activation. Together, these results suggest that S1P activates the EGF receptor through a PKC-dependent pathway that links Ras signaling to the activation of Erk-1/-2 in Rat-2 cells.