Distinct roles of c-Abl and Atm in oxidative stress response are mediated by protein kinase C delta

c-Abl and Atm have been implicated in cell responses to DNA damage and oxidative stress. However, the molecular mechanisms by which they regulate oxidative stress response remain unclear. In this report, we show that deficiency of c-Abl and deficiency of ATM differentially altered cell responses to oxidative stress by induction of antioxidant protein peroxiredoxin I (Prx I) via Nrf2 and cell death, both of which required protein kinase C (PKC) delta activation and were mediated by reactive oxygen species. c-abl-/- osteoblasts displayed enhanced Prx I induction, elevated Nrf2 levels, and hypersusceptibility to arsenate, which were reinstated by reconstitution of c-Abl; Atm-/- osteoblasts showed the opposite. These phenotypes correlated with increased PKC delta expression in c-abl-/- osteoblasts and decreased PKC delta expression in Atm-/- cells, respectively. The enhanced responses of c-abl-/- osteoblasts could be mimicked by overexpression of PKC delta in normal cells and impeded by inhibition of PKC delta, and diminished responses of Atm-/- cells could be rescued by PKC delta overexpression, indicating that PKC delta mediated the effects of c-Abl and ATM in oxidative stress response. Hence, our results unveiled a previously unrecognized mechanism by which c-Abl and Atm participate in oxidative stress response.     

Intravitreal delivery of AAV-NDI1 provides functional benefit in a murine model of Leber hereditary optic neuropathy

Leber hereditary optic neuropathy (LHON) is a mitochondrially inherited form of visual dysfunction caused by mutations in several genes encoding subunits of the mitochondrial respiratory NADH-ubiquinone oxidoreductase complex (complex I). Development of gene therapies for LHON has been impeded by genetic heterogeneity and the need to deliver therapies to the mitochondria of retinal ganglion cells (RGCs), the cells primarily affected in LHON. The therapy under development entails intraocular injection of a nuclear yeast gene NADH-quinone oxidoreductase (NDI1) that encodes a single subunit complex I equivalent and as such is mutation independent. NDI1 is imported into mitochondria due to an endogenous mitochondrial localisation signal. Intravitreal injection represents a clinically relevant route of delivery to RGCs not previously used for NDI1. In this study, recombinant adenoassociated virus (AAV) serotype 2 expressing NDI1 (AAV-NDI1) was shown to protect RGCs in a rotenone-induced murine model of LHON. AAV-NDI1 significantly reduced RGC death by 1.5-fold and optic nerve atrophy by 1.4-fold. This led to a significant preservation of retinal function as assessed by manganese enhanced magnetic resonance imaging and optokinetic responses. Intraocular injection of AAV-NDI1 overcomes many barriers previously associated with developing therapies for LHON and holds great therapeutic promise for a mitochondrial disorder for which there are no effective therapies.     

Age-dependent increase of heme oxygenase-1 gene expression in the liver mediated by NFkappaB

Heme, the iron-porphyrin coordination complex, released from the degradation of hemoproteins, is a strong prooxidant. It is enzymatically degraded by heme oxygenase to free iron, carbon monoxide and biliverdin. Biliverdin and its reduced metabolite bilirubin are two potent physiological antioxidants. Here we show a progressive increase of steady-state levels of the mRNA encoding the inducible isoform of this enzyme (heme oxygenase-1) in the rat liver during aging. We had previously reported that aging is associated with increased activation of the nuclear factor kappaB (NFkappaB). We now provide evidence to establish that overexpression of NFkappaB in transfected liver-derived HepG2 cells can cause a marked induction of the endogenous heme oxygenase-1 (HO-1) mRNA and activation of the cotransfected HO-1 gene promoter. Taken together, these results support the conclusion that enhanced oxidative stress during aging is accompanied by compensatory induction of the antioxidant enzyme HO-1 through activation of the NFkappaB pathway.     

Fas-mediated apoptosis in cholangiocarcinoma cells is enhanced by 3,3'-diindolylmethane through inhibition of AKT signaling and FLICE-like inhibitory protein

Stimulation of Fas-mediated apoptosis has been promoted as a potential therapy for many cancers, including cholangiocarcinoma. We have previously reported that Fas-resistant, but not Fas-sensitive, cholangiocarcinoma cells are tumorigenic in nude mice. The present studies sought to identify molecular targets that promote Fas-mediated apoptosis in cholangiocarcinoma. We found that Fas-resistant cholangiocarcinoma cells exhibited increased constitutive phosphorylation of AKT compared with Fas-sensitive cells. Increased phosphorylation of AKT was also demonstrated in human cholangiocarcinoma tumors and was evident in a mouse xenograft cholangiocarcinoma model. Furthermore, we found that 3,3'-diindolylmethane (DIM), a vegetable autolysis product, promoted Fas-mediated apoptosis of cholangiocarcinoma cells. DIM inhibited phosphorylation of AKT and activation of FLICE-like-inhibitory-protein (FLIP). Inhibition of phosphatidylinositol 3-kinase/AKT decreased FLIP activation and promoted Fas-mediated apoptosis. By contrast, adenovirus-mediated constitutively activated AKT protected cholangiocarcinoma cells from Fas-mediated apoptosis. Decreased activation of extracellular signal-regulated kinase and nuclear factor-kappaB and increased activation of caspase-3, -8, and -9 were associated with inhibition of AKT and FLIP. These results support AKT and FLIP as potential molecular targets and DIM as a potent compound for cholangiocarcinoma intervention.     

Characterization of the modes of action of deoxynivalenol (DON) in the human Jurkat T-cell line

Abstract

Deoxynivalenol (DON) is one of the most abundant mycotoxins worldwide and mostly detected in cereals and grains. As such, DON poses a risk for many adverse health effects to human and animals. In particular, immune cells are very sensitive to DON, with the initiating step leading to toxicity being a binding to the eukaryotic 60S ribosomal subunit and induction of ribotoxic stress. The present study aimed to: (1) extend insight into the mechanism of action (MOA) of DON in immune cells; and (2) understand why immune cells are more sensitive to DON than most other cell types. Previously published microarray studies have described the effects of DON on immune cells. To build upon these findings, here, immunocytological and biochemical studies were performed using human T-lymphocyte Jurkat cells that were exposed for 3?h to 0.5?µM DON. Induction of ER stress by DON was confirmed by immunocytology demonstrating increased protein expression of two major ER stress markers ATF3 and DDIT3. T-cell activation was confirmed by induction of phosphorylation of protein kinases JNK and AKT, activation of NF-κB (p65), and increased expression of NFAT target gene NUR77; each of these are known inducers of the T-cell activation response. Induction of an oxidative stress response was also confirmed by monitoring the nuclear translocation of major oxidative stress markers NRF2 and KEAP1, as well as by changes (i.e. decreases) in cell levels of reduced glutathione. Lastly, this study showed that DON induced cleavage of caspase-3, an event known to mediate apoptosis. Taken together, these results allowed us to formulate a potential mechanism of action of DON in immune cells, i.e. binding to eukaryotic 60S ribosomal subunit?→?ribotoxic stress?→?ER stress?→?calcium release from the ER into cytoplasm?→?T-cell activation and oxidative stress?→?apoptosis. It is proposed that immune cells are more sensitive to DON than other cell types due to the induction of a T-cell activation response by increased intracellular calcium levels.     

A novel 90-kDa tyrosine-phosphorylated protein associated with TCR complex in thymocytes

Ligation of the TCR-CD3 complex initiates a cascade of tyrosine phosphorylation that results in T cell activation. Initial activation of tyrosine kinases depends on the phosphorylation of activation motifs on CD3 chains. We previously found that a 90-kDa protein was tyrosine phosphorylated upon TCR cross-linking and the induction of the phosphorylation was dependent on the structure of the CD3 complex. In this study, we further characterized p90 phosphorylation. Phosphorylation of p90 was induced only by stimulation through the TCR-CD3 complex but not by other kinds of stimulation including CD28- or hydrogen peroxide-mediated activation and was dynamically regulated. Phosphorylated p90 was associated with the TCR-CD3 complex upon T cell activation. In a normal T cell population, thymocytes but not splenic T cells induced the tyrosine phosphorylation of p90 upon TCR cross-linking. These results suggest that p90 is a novel phosphoprotein associated with the TCR-CD3 complex and may play a role in TCR signaling during thymocyte differentiation.     

Thymosin-β4 (Tβ4) blunts PDGF-dependent phosphorylation and binding of AKT to actin in hepatic stellate cells

Hepatic stellate cell transdifferentiation is a key event in the fibrogenic cascade. Therefore, attempts to prevent and/or revert the myofibroblastic phenotype could result in novel therapeutic approaches to treat liver cirrhosis. The expression of platelet-derived growth factor (PDGF)-β receptor and the proliferative response to platelet-derived growth factor-ββ (PDGF-ββ) are hallmarks of the transdifferentiation of hepatic stellate cells (HSC). In this communication, we investigated whether thymosin-β4 (Tβ4), a chemokine expressed by HSC could prevent PDGF-BB-mediated proliferation and migration of cultured HSC. Using early passages of human HSC, we showed that Tβ4 inhibited cell proliferation and migration and prevented the expression of PDGF-β receptor (PDGF-βr), α-smooth muscle actin and α1(I) collagen mRNAs. Tβ4 also inhibited the reappearance of PDGF-βr after its PDGF-BB-dependent degradation. These PDGF-dependent events were associated with the inhibition of AKT phosphorylation at both T308 and S473 amino acid residues. The lack of AKT phosphorylation was not due to the inhibition of PDGF-βr phosphorylation, the activation of phosphoinositide 3-kinase (PI3K), pyruvate dehydrogenase kinase isozyme 1 (PDK1), and mammalian target of rapamycin (mTOR). We found that PDGF-BB induced AKT binding to actin, and that Tβ4 prevented this effect. Tβ4 also prevented the activation of freshly isolated HSC cultured in the presence of Dulbecco's modified Eagle's medium or Dulbecco's minimal essential medium containing 10% fetal bovine serum. In conclusion, overall, our findings suggest that Tβ4 by sequestering actin prevents binding of AKT, thus inhibiting its phosphorylation. Therefore, Tβ4 has the potential to be an antifibrogenic agent.     

Uncoupling of oxidative phosphorylation prevents the urinary alcohol level cycling caused by feeding ethanol continuously at a constant rate

The mechanism of the UAL cycle in the intragastric feeding model of alcoholic liver disease in the rat was investigated by administering dinitrophenol (DNP) with ethanol in the diet. The question was: is the rate of oxidative phosphorylation fluxuation essential for the cycle to occur? The question has been partially answered by showing that rotenone, which inhibits complex I, blocks the cycle by preventing the generation of NAD from NADH. This would inhibit ATP generation from complex I but would not affect oxidative phosphorylation by complex 2 and 3. Since the rate of O2 consumption is normal at the troughs of the cycle and decreases at the peaks of the cycle and the levels of ATP are reduced at the peaks of the cycle, it is likely that the rate of oxidative phosphorylation also cycles. Since 2-4 dinitrophenol (DNP) uncouples oxidative phosphorylation, it was anticipated that feeding it with ethanol would prevent the cycle from occurring. This proved to be the case. In addition, DNP caused energy wasting and prevented the increase in serum alanine aminotranspeptidase caused by ethanol feeding, probably by preventing the hypoxia which occurs at the peaks of the cycle.     

Hydrogen peroxide activates NFkappaB and the interleukin-6 promoter through NFkappaB-inducing kinase

Aging is associated not only with oxidant stress, but also with increased interleukin-6 (IL-6) levels. To determine if oxidative stress could contribute to the age-associated increase IL-6 expression, we exposed LNCaP prostate carcinoma cells and HeLa cervical carcinoma cells to H2O2 as an oxidant challenge. We found that H2O2 induced IL-6 expression through activation of the IL-6 promoter. Furthermore, H2O2-induced activation of the promoter was mediated through nuclear factor-kappaB (NFkappaB) secondary to H2O2-induced phosphorylation and degradation of IkappaBalpha. NFkappaB-inducing kinase (NIK) is upstream of the IkappaB kinase complex that induces IkappaBalpha degradation. Accordingly, we explored if H2O2 induces IL-6 expression through NIK. In addition to H2O2 inducing NIK autophosphorylation, transfection of LNCaP cells with a dominant negative NIK diminished H2O2-mediated NFkappaB and IL-6 promoter activity. Taken together, these results demonstrate that H2O2 induces the IL-6 promoter by activating NFkappaB through NIK. These data provide a candidate mechanism through which oxidant challenge induces IL-6 gene expression with age.     

Imp2 controls oxidative phosphorylation and is crucial for preserving glioblastoma cancer stem cells

Growth of numerous cancer types is believed to be driven by a subpopulation of poorly differentiated cells, often referred to as cancer stem cells (CSCs), that have the capacity for self-renewal, tumor initiation, and generation of nontumorigenic progeny. Despite their potentially key role in tumor establishment and maintenance, the energy requirements of these cells and the mechanisms that regulate their energy production are unknown. Here, we show that the oncofetal insulin-like growth factor 2 mRNA-binding protein 2 (IMP2, IGF2BP2) regulates oxidative phosphorylation (OXPHOS) in primary glioblastoma (GBM) sphere cultures (gliomaspheres), an established in vitro model for CSC expansion. We demonstrate that IMP2 binds several mRNAs that encode mitochondrial respiratory chain complex subunits and that it interacts with complex I (NADH:ubiquinone oxidoreductase) proteins. Depletion of IMP2 in gliomaspheres decreases their oxygen consumption rate and both complex I and complex IV activity that results in impaired clonogenicity in vitro and tumorigenicity in vivo. Importantly, inhibition of OXPHOS but not of glycolysis abolishes GBM cell clonogenicity. Our observations suggest that gliomaspheres depend on OXPHOS for their energy production and survival and that IMP2 expression provides a key mechanism to ensure OXPHOS maintenance by delivering respiratory chain subunit-encoding mRNAs to mitochondria and contributing to complex I and complex IV assembly.     

Phosphoinositol 3-kinase, a novel target molecule for the inhibitory effects of juglone on TPA-induced cell transformation

Juglone (5-hydroxy-1,4-naphthalenedione) from black walnut trees induces apoptosis and inhibits proliferation of various malignant cells. Here, we investigated whether juglone affects 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation through the phosphoinositol 3-kinase (PI3K) pathway. The results showed that TPA- and endothelial growth factor (EGF)-induced anchorage-independent colony formation were suppressed in a dose-dependent manner by treatment of JB6 CI41 mouse skin epidermal cells with juglone (2.5 and 5 μM). We demonstrated that juglone suppressed PI3K activity via direct binding to PI3K by sepharose 4B pull-down assay and western blot analysis. Juglone significantly suppressed TPA-induced protein kinase B (AKT) and c-Jun phosphorylation and c-fos activation, but not mitogen-activated protein-kinase kinase (MEK), extracellular signaling-regulated kinase (ERK) or 90 kDa ribosomal protein S6 kinase (RSK) phosphorylation. Juglone significantly blocked activator protein-1 (AP-1) and cyclooxygenase-2 (COX-2) activation more than the PI3K inhibitors LY294002 and wortmannin. Overall, these results showed the anticancer efficacy of juglone targeting PI3K to prevent TPA-induced tumorigenesis.     

Mutations in the zinc finger domain of IKK gamma block the activation of NF-kappa B and the induction of IL-2 in stimulated T lymphocytes

Mutations in the zinc finger of I kappa B kinase gamma (IKK gamma) are associated with hypohidrotic ectodermal dysplasia-immune deficiency (HED-ID) in which the major immune deficit is the inability to switch Ab heavy chain class. However, the pathophysiologic role of the mutations has not been fully delineated. Since help from activated Th cells is essential in Ab class switching, we sought to examine how these mutations affect T cell activation. Using a human T cell line that was null for IKK gamma, we generated cells stably expressing two of the reported mutations, namely, D406V and C417R. Cells expressing either mutation failed to induce IL-2 following stimulation with PMA/ionomycin while the induction of IL-2 was restored in cells reconstituted with the wild type IKK gamma. The lack of IL-2 upregulation correlated with the lack of NF-kappaB activation as evidenced by the inability to induce I kappa B alpha degradation, NF-kappaB binding to DNA and the expression of a reporter gene. However, both mutations did not prevent the incorporation of IKK gamma into the IKK complex and, interestingly, the induced phosphorylation of I kappa B alpha at S32 and S36 and its subsequent ubiquitination were not affected. The suppression of IL-2 induction was solely due to the inhibition of NF-kappaB activation as the mutations did not impair the activation of AP-1 and NFAT. Our data indicated that the failure of T cells to undergo activation in response to TCR stimuli may play a role in the pathophysiology of HED-ID and also showed that IKK gamma has a role in the post-ubiquitination processing of I kappa B alpha.     

Glucose transporter protein 1 expression in mucoepidermoid carcinoma of salivary gland: correlation with grade of malignancy

Mucoepidermoid carcinoma (MEC), the most common primary salivary malignancy, shows great variability in clinical behaviour, thus demanding investigation to identify of prognostic markers. Since Warburg’s studies, unrestricted cell growth during tumorigenesis has been linked to altered metabolism, implying hypoxic stimulation of glycolysis and diminished contribution of mitochondrial oxidative phosphorylation to cellular ATP supply. Hypothesizing that the study of MEC metabolic status could lead to the discovery of prognostic markers, we investigated by immunohistochemistry the expression of glucose transporter 1 (Glut‐1), mitochondrial antigen and peroxiredoxin I (Prx I) in samples of MEC from different histological grades. Our results showed that mitochondrial antigen and Prx I were expressed in the majority of the MEC cases independent of the histological grade. In contrast Glut‐1 expression increased significantly as the tumours became more aggressive. These results suggested that oxidative phosphorylation may contribute to ATP supply in all stages of MEC progression, and that the relative contribution of glycolysis over mitochondria for cellular ATP supply increases during MEC progression, favouring growth under low oxygen concentration. In addition, the observed high Prx I protein levels could provide protection to tumour cells against reactive oxygen species generated as a consequence of mitochondrial function and hypoxia‐reoxygenation cycling. Altogether our findings suggest that upregulation of Glut‐1 and Prx I constitute successful adaptive strategies of MEC cells conferring a growth advantage over normal salivary gland cells in the unstable oxygenation tumour environment.     

Ack1 tyrosine kinase activation correlates with pancreatic cancer progression

Pancreatic cancer is a significant cause of cancer mortality worldwide as the disease has advanced significantly in patients before symptoms are evident. The signal transduction pathways that promote this rapid progression are not well understood. Ack1 or TNK2, an ubiquitously expressed oncogenic non-receptor tyrosine kinase, integrates signals from ligand-activated receptor tyrosine kinases to modulate intracellular signaling cascades. In the present study, we investigated the Ack1 activation profile in a pancreatic cancer tumor microarray, and observed that expression levels of activated Ack1 and pTyr284-Ack1 positively correlated with the severity of disease progression and inversely correlated with the survival of patients with pancreatic cancer. To explore the mechanisms by which Ack1 promotes tumor progression, we investigated the role of AKT/PKB, an oncogene and Ack1-interacting protein. Ack1 activates AKT directly in pancreatic and other cancer cell lines by phosphorylating AKT at Tyr176 to promote cell survival. In addition, the Ack1 inhibitor AIM-100 not only inhibited Ack1 activation but also suppressed AKT tyrosine phosphorylation, leading to cell cycle arrest in the G1 phase. This effect resulted in a significant decrease in the proliferation of pancreatic cancer cells and induction of apoptosis. Collectively, our data indicate that activated Ack1 could be a prognostic marker for ascertaining early or advanced pancreatic cancer. Thus, Ack1 inhibitors hold promise for therapeutic intervention to inhibit pancreatic tumor growth.     

Complex regulation of the cyclin-dependent kinase inhibitor p27kip1 in thyroid cancer cells by the PI3K/AKT pathway: regulation of p27kip1 expression and localization

Functional inactivation of the tumor suppressor p27(kip1) in human cancer occurs either through loss of expression or through phosphorylation-dependent cytoplasmic sequestration. Here we demonstrate that dysregulation of the PI3K/AKT pathway is important in thyroid carcinogenesis and that p27(kip1) is a key target of the growth-regulatory activity exerted by this pathway in thyroid cancer cells. Using specific PI3K inhibitors (LY294002, wortmannin, and PTEN) and a dominant active AKT construct (myrAKT), we demonstrated that the PI3K/AKT pathway controlled thyroid cell proliferation by regulating the expression and subcellular localization of p27. Results obtained with phospho-specific antibodies and with transfection of nonphosphorylable p27(kip1) mutant constructs demonstrated that PI3K/AKT-dependent regulation of p27(kip1) mislocalization in thyroid cancer cells occurred via phosphorylation of p27(kip1) at T157 and T198 (but not at S10 or T187). Finally, we evaluated whether these results were applicable to human tumors. Analysis of 100 thyroid carcinomas indicated that p27(kip1) phosphorylation at T157/T198 and cytoplasmic mislocalization were preferentially associated with activation of the PI3K/AKT pathway. Thus the PI3/AKT pathway and its effector p27(kip1) play major roles in thyroid carcinogenesis.