Mitochondrial pathways for ROS formation and myocardial injury: the relevance of p66Shc and monoamine oxidase

Although mitochondria are considered the most relevant site for the formation of reactive oxygen species (ROS) in cardiac myocytes, a major and unsolved issue is where ROS are generated in mitochondria. Respiratory chain is generally indicated as a main site for ROS formation. However, other mitochondrial components are likely to contribute to ROS generation. Recent reports highlight the relevance of monoamine oxidases (MAO) and p66^Shc. The importance of these systems in the irreversibility of ischemic heart injury will be discussed along with the cardioprotective effects elicited by both MAO inhibition and p66^Shc knockout. Finally, recent evidence will be reviewed that highlight the relevance of mitochondrial ROS formation also in myocardial failure and atherosclerosis.     

Deletion of the lifespan determinant p66(Shc) improves performance in a spatial memory task, decreases levels of oxidative stress markers in the hippocampus and increases levels of the neurotrophin BDNF in adult mice

Deletion of the p66(Shc) gene in mice results in reduced levels of oxidative stress and longer lifespan. Reactive oxygen species (ROS) can lead to tissue damage, particularly in the brain. In this study we extended previous findings on the behavioral phenotype of the p66(Shc-/-) mice. Cognitive performance of adult and old p66(Shc-/-) and p66(Shc+/+) mice was tested in a Morris water maze (MWM) task while general reactivity and pain sensitivity were assayed at adulthood, respectively, in an open field and by means of a tail flick test. Levels of brain-derived neurotrophic factor (BDNF), a neurotrophin involved in several aspects of synaptic plasticity, emotionality and pain sensitivity, were assessed in selected brain areas. P66(Shc-/-) adult subjects, compared to WT, overall showed a better performance in the MWM, lower emotionality and a higher pain threshold, in addition to increased basal levels of BDNF in the hippocampus, as well as decreased levels of oxidative stress markers in the same brain area. Although all aged subjects failed to learn the cognitive task, aged p66(Shc-/-) mice were characterized by a better physical performance. These results suggest an interaction between the p66(Shc) gene and specific signaling pathways involved in behavioral adaptation to stress and aging.     

Ablation of the gene encoding p66Shc protects mice against AGE-induced glomerulopathy by preventing oxidant-dependent tissue injury and further AGE accumulation

Aims/hypothesis AGEs have been implicated in renal disease associated with ageing, diabetes and other age-related disorders. Reactive oxygen species (ROS) promote formation of AGEs, which cause AGE-receptor-mediated ROS generation with activation of signalling pathways leading to tissue injury and further AGE accumulation. ROS generation is regulated by the Src homology 2 domain-containing transforming protein C1 (Shc1) isoform p66^Shc, whose deletion has been shown to protect from tissue injury induced by ageing, diabetes, hyperlipidaemia and ischaemia-reperfusion by preventing oxidative stress. This study was aimed at assessing the role of p66^Shc in the modulation of oxidative stress and oxidant-dependent renal injury induced by AGEs. Methods For 10 weeks, male p66 ^shc knockout (KO) and wild-type (WT) mice were injected with 60 μg/day albumin modified or unmodified by (CML). Mice were then killed for the assessment of renal function and structure, as well as systemic and renal tissue oxidative stress. Results Upon CML injection, KO mice, in contrast to WT mice, showed no or only mild forms of proteinuria, glomerular hypertrophy, mesangial expansion, glomerular sclerosis, renal/glomerular cell apoptosis and extracellular matrix upregulation. Moreover, KO mice had lower circulating and tissue AGEs than WT mice and unchanged plasma isoprostane 8-epi-prostaglandin- levels, renal/glomerular CML, 4-hydroxy-2-nonenal, AGE receptor and NAD(P)H oxidase 4 (NOX4) content (and expression of the corresponding genes), and nuclear factor κB activation (NFκB). Mesangial cells from KO mice exposed to CML showed no or slight increase in ROS levels and NFκB activation, again at variance with WT cells. Conclusions/interpretation These data indicate that p66^Shc participates in the pathogenesis of AGE-dependent glomerulopathy by mediating AGE-induced tissue injury and further AGE formation through ROS-dependent mechanisms involving NFκB activation and upregulation of Nox4 expression and NOX4 production. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Structure and function of the p130Cas-related protein family

Abstract

Adhesive interactions play a critical role in inflammation and the immune response by regulating dynamic cellular processes including adhesion, migration, recognition, activation, proliferation, and even apoptosis. Aberration in such interactions can lead to chronic inflammatory disorders such as rheumatoid arthritis or other autoimmune diseases. These adhesive interactions act through the regulation of intracellular signal transduction pathways, which have been intensively studied in the last decade. Understanding such signaling pathways is essential not only to elucidate how adhesive interactions regulate cell behavior, but also to gain important insight into the pathophysiology of various diseases and to identify novel therapeutic targets. p130^Cas and its related proteins play a significant role in integrin-mediated cell adhesion. Recent success in generating mutant mice lacking p130^Cas has provided insight into the central function of this molecule. This article will focus on recent advances in our understanding of structure and function of this unique protein family.     

Epinephrine-induced Ca2+ influx in vascular endothelial cells is mediated by CNGA2 channels

Epinephrine, through its action on β-adrenoceptors, may induce endothelium-dependent vascular dilation, and this action is partly mediated by a cytosolic Ca²⁺ ([Ca²⁺]_i) change in endothelial cells. In the present study, we explored the molecular identity of the channels that mediate epinephrine-induced endothelial Ca²⁺ influx and subsequent vascular relaxation. Patch clamp recorded an epinephrine- and cAMP-activated cation current in the primary cultured bovine aortic endothelial cells (BAECs) and H5V endothelial cells. L-cis-diltiazem and LY-83583, two selective inhibitors for cyclic nucleotide-gated channels, diminished this cation current. Furthermore, this cation current was greatly reduced by a CNGA2-specific siRNA in H5V cells. With the use of fluorescent Ca²⁺ dye, it was found that epinephrine and isoprenaline, a β-adrenoceptor agonist, induced endothelial Ca²⁺ influx in the presence of bradykinin. This Ca²⁺ influx was inhibited by L-cis-diltiazem and LY-83583, and by a β₂-adrenoceptor antagonist ICI-118551. CNGA2-specific siRNA also diminished this Ca²⁺ influx in H5V cells. Furthermore, L-cis-diltiazem and LY-83583 inhibited the endothelial Ca²⁺ influx in isolated mouse aortic strips. L-cis-diltiazem also markedly reduced the endothelium-dependent vascular dilation to isoprenaline in isolated mouse aortic segments. In summary, CNG channels, CNGA2 in particular, mediate β-adrenoceptor agonist-induced endothelial Ca²⁺ influx and subsequent vascular dilation.     

Shear stress inhibition of H<Subscript>2</Subscript>O<Subscript>2</Subscript> induced p66<Superscript>Shc</Superscript> phosphorylation by ASK1–JNK inactivation in endothelium

Shear stress protects endothelium from a variety of risk factors for vascular disease. Here, we demonstrate a novel mechanism whereby shear stress inhibited reactive oxygen species (ROS)-triggered signaling cascades in endothelial cells. Stimulation of bovine aortic endothelial cells (BAECs) with H₂O₂ induced a 3.07-fold increase in p66^Shc phosphorylation. This response was fully blocked by pretreatment of cells with specific JNK but not p38 or ERK MAP kinase inhibitor. Further study showed that knocking down of apoptosis signal-regulating kinase 1 (ASK1) by siRNA transfection in cells dramatically inhibited phosphorylation of JNK and p66^Shc elicited by H₂O₂. Pre-perfusion of BAECs cultured in silastic tubes with laminar flow generated by a servo-pump system for 30 min also significantly suppressed H₂O₂-induced phosphorylation of p66^Shc. This was accompanied by quantitatively similar inhibition of ASK1 and JNK phosphorylation and activation. These results suggested that shear stress protects endothelium against oxidant stress by suppression of ASK1–JNK-mediated p66^Shc phosphorylation.     

The hypermethylation and protein expression of p16 INK4A and DNA repair gene O6-methylguanine-DNA methyltransferase in various uterine cervical lesions

Purpose This study is aimed at investigating the significance of gene promoter methylation status and protein expression of p16 ^INK4A and O⁶-methylguanine-DNA methyltransferase (MGMT) in the various uterine cervical lesions.Materials and methods Methylation status by using methylation-specific polymerase chain reaction (MS-PCR) and protein expression by using immunohistochemistry for p16 ^INK4A and MGMT genes were performed in cervical squamous intraepithelial neoplasms (CIN), invasive squamous cell carcinomas (SCC), adenocarcinomas and non-neoplastic cervices.Results None of 20 non-neoplastic cervices showed p16 ^INK4A and MGMT gene hypermethylation, whereas at least one of these genes was hypermethylated with 50.0% (5/10) of CIN I, 65.0% (13/20) of CIN II–III, 70.2% (33/47) of SCC and 85.0% (17/20) of adenocarcinoma. p16 ^INK4A protein was totally negative in non-neoplastic cervices, but positive with 90.0% of CIN I, 100% of CIN II–III and adenocarcinoma, and 78.7% of SCC. MGMT protein was expressed in 10% of non-neoplastic cervices, but significantly increased in SCC (42.5%) and adenocarcinoma (70.0%). The protein expression of p16 ^INK4A and MGMT was not related to their gene promoter methylation status.Conclusions The hypermethylation of p16 ^INK4A and MGMT genes in the uterine cervix may indicate the presence of malignant cells, and p16 ^INK4A immunostaining is useful in grading CIN and diagnosing invasive SCC and adenocarcinoma.     

p21<Superscript>WAF1/CIP1</Superscript> is more effective than p53 in growth suppression of mouse renal carcinoma cell line Renca in vitro and in vivo

Purpose Although there are many controversial reports about the effect of p53 and p21^WAF1/CIP1 overexpression in different human tumor cells, the p53 gene is shown to be a more effective candidate for cancer gene therapy because of its more pronounced ability to induce apoptosis. In the present study, we present the effect of p53 and p21^WAF1/CIP1 overexpression on mouse renal carcinoma cells in vitro and in vivo.Methods p53 and p21^WAF1/CIP1 genes were introduced into Renca cells using adenoviral vectors (Ad5CMV-p53 and Ad5CMV-p21). The induction of apoptosis was measured using Annexin V assay and DNA fragmentation analysis. The expression of proteins was examined using immunocytochemistry and Western blot methods. The ability of adenoviral vectors to inhibit tumorigenicity of Renca cells, as well as the growth of pre-established tumors was measured.Results In vitro growth assays revealed higher growth suppression after Ad5CMV-p21 infection. Although both vectors induced apoptosis, Ad5CMV-p53 was slightly more efficient. In vivo studies in Balb/c mice, demonstrated that tumorigenicity was completely suppressed by Ad5CMV-p21. Besides this, Ad5CMV-p21 significantly inhibited the growth of established tumors, while Ad5CMV-p53 did not.Conclusions These data suggest that p21^WAF1/CIP1 is a more potent growth suppressor than p53 of mouse tumor cells Renca. The divergent responses of tumor cells to p21^WAF1/CIP1 overexpression could be due to various networks that differ between species.     

p34<Superscript>cdc2</Superscript> expression is an independent indicator for lymph node metastasis in colorectal carcinoma

Purpose The significance of p34^cdc2 expression in human tumors has not been fully explained. The aim of the current study was to elucidate the clinicopathologic significance of immunohistochemical p34^cdc2 expression in carcinoma of the colon and rectum.Methods The immunohistochemical expression of p34^cdc2 was examined in 90 consecutive colorectal tumor cases, and p34^cdc2 expression and the clinicopathologic features of the patients and their tumors were compared.Results Lymph node metastasis was significantly more frequent in tumors expressing p34^cdc2 (47.8%, 11 of 23 tumors) than in tumors not expressing p34^cdc2 (22.4%, 15 of 67 tumors; P=0.020). Multivariate analysis demonstrated that tumor depth (P=0.008) and p34^cdc2 expression (P=0.022) were independently associated with lymph node metastases of colorectal carcinomas.Conclusions The immunohistochemical expression of p34^cdc2 is independently associated with lymph node metastasis in colorectal carcinoma.     

Structure and function of the p130Cas-related protein family

Adhesive interactions play a critical role in inflammation and the immune response by regulating dynamic cellular processes including adhesion, migration, recognition, activation, proliferation, and even apoptosis. Aberration in such interactions can lead to chronic inflammatory disorders such as rheumatoid arthritis or other autoimmune diseases. These adhesive interactions act through the regulation of intracellular signal transduction pathways, which have been intensively studied in the last decade. Understanding such signaling pathways is essential not only to elucidate how adhesive interactions regulate cell behavior, but also to gain important insight into the pathophysiology of various diseases and to identify novel therapeutic targets. p130^Cas and its related proteins play a significant role in integrin-mediated cell adhesion. Recent success in generating mutant mice lacking p130^Cas has provided insight into the central function of this molecule. This article will focus on recent advances in our understanding of structure and function of this unique protein family.     

Astrocyte-induced cortical vasodilation is mediated by D-serine and endothelial nitric oxide synthase

Astrocytes play a critical role in neurovascular coupling by providing a physical linkage from synapses to arterioles and releasing vaso-active gliotransmitters. We identified a gliotransmitter pathway by which astrocytes influence arteriole lumen diameter. Astrocytes synthesize and release NMDA receptor coagonist, D-serine, in response to neurotransmitter input. Mouse cortical slice astrocyte activation by metabotropic glutamate receptors or photolysis of caged Ca²⁺ produced dilation of penetrating arterioles in a manner attenuated by scavenging D-serine with D-amino acid oxidase, deleting the enzyme responsible for D-serine synthesis (serine racemase) or blocking NMDA receptor glycine coagonist sites with 5,7-dichlorokynurenic acid. We also found that dilatory responses were dramatically reduced by inhibition or elimination of endothelial nitric oxide synthase and that the vasodilatory effect of endothelial nitric oxide synthase is likely mediated by suppressing levels of the vasoconstrictor arachidonic acid metabolite, 20-hydroxy arachidonic acid. Our results provide evidence that D-serine coactivation of NMDA receptors and endothelial nitric oxide synthase is involved in astrocyte-mediated neurovascular coupling.     

The role of XPB in cell apoptosis and viability and its relationship with p53, p21 and c-myc in hepatoma cells

BACKGROUND: Accumulation of DNA damage has been implicated in hepatocarcinogenesis. XPB plays a pivotal part in repairing damaged DNA. However, up to now, the biological effect of XPB on hepatoma cells remains elusive. MATERIALS AND METHODS: Here, we investigated the role of XPB in the apoptosis and the viability of hepatoma cells by using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labelling and cell viability assay; we also investigated their relationship with p53, p21(waf1/cip1) and c-myc by using the RT-PCR and Western blot. RESULTS: Compared with the control cells HepG2/pcDNA3.1 or HepG2, XPB-transfected HepG2 cells (HepG2/pcDNA3.1-XPB) displayed lower viability, weaker activity and higher apoptosis index. At the same time, an increased expression of p21(waf1/cip1) mRNA, protein and p53 protein in addition to a decreased expression of c-myc mRNA and protein were detected in HepG2/pcDNA3.1-XPB cells. CONCLUSIONS: Our results indicated that XPB could inhibit the proliferation of hepatoma cells and had a positive effect on the expression of p53 and p21(waf1/cip1) but a negative effect on c-myc.     

Status of p53 phosphorylation and function in sensitive and resistant human cancer models exposed to platinum-based DNA damaging agents

Purpose Resistance to chemotherapeutic drugs is a hallmark of many human cancers, which can occur independent of p53 gene status; however, the presence of wild-type p53 in chemorefractory tumors confers greater resistance to cisplatin, but such tumors do not display complete cross-resistance to the platinum analog (1R,2R-diaminocyclohexane)(trans-diacetato)(dichloro)platinum^IV (DACH-Ac-Pt). In this article we examine DNA damage-induced phosphorylation of p53 and downstream p53-dependent transactivation events in cisplatin-sensitive and cisplatin-resistant human cancer cell lines possessing wild-type p53.Methods Western-blot analysis was utilized to study the effect of cisplatin and the analog on p53 phosphorylation and p53-dependent target genes.Results In response to CDDP and DACH-Ac-Pt, both CDDP-sensitive and CDDP-resistant models demonstrated time- and dose-dependent inductions of total p53 protein and an increase in Ser-15 phosphorylation, which was more pronounced with CDDP. Although phosphorylation of p53 at Ser-392 was also observed in CDDP-treated sensitive and resistant cells, it was weak or absent in response to DACH-Ac-Pt. Lack of Ser-392 phosphorylation by DACH-Ac-Pt, however, did not affect the induction of p21^WAF1/CIP1 or Mdm2. Similarly, inductions of p21^WAF1/CIP1 and Mdm2 were observed in sensitive cells exposed to cisplatin. In marked contrast, cisplatin-mediated induction of p21^WAF1/CIP1 was minimal or absent in resistant cells, but that of Mdm2 was unaffected. Wortmannin, a PI3-kinase (PI3-K) inhibitor, caused a dose-dependent inhibition of total p53 accumulation, Ser-15 phosphorylation and p21^WAF1/CIP1 transactivation in response to both CDDP and DACH-Ac-Pt, indicating that members of the PI3-K family are involved in phosphorylation of p53 and that transactivation of p21^WAF1/CIP1 is p53 dependent.Conclusion These studies demonstrate that cisplatin and DACH-Ac-Pt differentially phosphorylate p53 through independent DNA damage-induced pathways, and that the kinase-mediated phosphorylation of p53 at Ser-15 or Ser-392 is unaltered in resistance. Moreover, the phosphorylation status of Ser-392 on its own does not appear to correlate with p21^WAF1/CIP1 or Mdm2 induction in these studies; however, a lack of increase in p21^WAF1/CIP1 by cisplatin, but not DACH-Ac-Pt, provides a correlation with resistance and its circumvention, and implicates the role for cyclin-dependent kinase inhibitor in the differential cytotoxic effects of the two platinum agents against resistant cells.     

Relationship between intracellular localization of p34<Superscript>cdc2</Superscript> protein and differentiation of esophageal squamous cell carcinoma

Purpose G2/M cyclins including cyclins A and B can exert their biologic functions of mitosis and proliferation of the tumor cells by being combined by protein kinase p34cdc2. The aim of the current study was to elucidate the clinicopathologic significance of immunohistochemical expression of p34^cdc2 in esophageal squamous cell carcinoma (ESCC), which has not been resolved.Methods Immunohistochemical expression of p34^cdc2 was examined for 91 cases of ESCC, and the relationship between the type of p34^cdc2 expression and the clinicopathologic features of the patients and tumors was analyzed.Results Forty-one ESCCs demonstrated cytoplasm dominant expression of p34^cdc2 and the other 50 ESCCs showed nuclei dominant p34^cdc2 expression. This differential expression pattern of p34^cdc2 did not reflect a prognostic aspect; however, the proportion of keratinizing tumors ESCCs with cytoplasm dominant expression of p34^cdc2 was significantly higher than that among ESCCs presenting nuclei-dominant p34^cdc2 expression (P=0.006).Conclusion Cellular differentiation in squamous cell carcinoma of the esophagus may be mediated by an intracellular localization of p34^cdc2.     

Hydrogen peroxide-induced vascular relaxation in porcine coronary arteries is mediated by Ca2+-activated K+ channels

Summary Hydrogen peroxide (H₂O₂) elicited concentration-dependent relaxation of endothelium-denuded rings of porcine coronary arteries. The relaxation induced by the H₂O₂ was markedly attenuated by 10μM 1H-[1,2,4]oxadiazolo [4,3,a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase, or by 100nM charybdotoxin, an inhibitor of large-conductance Ca²⁺-activated K⁺ (K_Ca) channels. A combination of the ODQ and charybdotoxin abolished the H₂O₂-induced relaxation. Pretreatment with 25 μM of an Rp stereoisomer of adenosine-3′,5′-cyclic monophosphothioate (Rp-cAMPS), 20μM glibenclamide, or 1mM 4-aminopyridine did not affect the vascular response to H₂O₂. The presence of catalase at 1000U/ml significantly attenuated the H₂O₂-induced relaxation. Exposure of cultured smooth muscle cells to H₂O₂ activated K_Ca channels in a concentration-dependent manner in cell-attached patches. Pretreatment with catalase significantly inhibited the activation of K_Ca channels. Rp-cAMPS did not inhibit the H₂O₂-induced activation of K_Ca channels. The activation of K_Ca channels by H₂O₂ was markedly decreased in the presence of ODQ. However, even in the presence of ODQ, H₂O₂ activated K_Ca channels in a concentration-dependent manner. In inside-out patches, H₂O₂ significantly activated K_Ca channels through a process independent of cyclic guanosine 3′,5′-monophosphate (cGMP). In conclusion, H₂O₂ elicits vascular relaxation due to activation of K_Ca channels, which is mediated partly by a direct action on the channel and partly by activation of soluble guanylate cyclase, resulting in the generation of cGMP.     

p53 and p14 increase sensitivity of gastric cells to H. pylori-induced apoptosis

H. Pylori infection of the gastric mucosa is associated with increased epithelial cell apoptosis. In vitro, interferon- and TNF- have been shown to increase the sensitivity of cells to apoptosis induced by H. Pylori. The p53 tumor suppressor gene is frequently mutated in many cancers, including gastric cancer. Since p53 protein can induce apoptosis, we sought to determine whether or not p53 increases the ability of gastric epithelial cells to undergo apoptosis in response to H. Pylori-induced cell injury. Human gastric epithelial cell lines, AGS (p53 wild-type) cells and AGS cells infected with HPV E6 gene (AGS-E6) to inactivate p53 were exposed to H. Pylori. The p53, p21, and p14^ARF proteins were measured in gastric epithelial cells by immunoelectrophoresis. Gastric epithelial cell apoptosis was measured by DNA end-labeling assay (TUNEL) and subG₀ cell fractions using flow cytometry, and by agarose gel electrophoresis of DNA. Exposure to H. Pylori increased the levels of p53, p21, and p14^ARF proteins two fold in AGS cells. Gastric AGS cells with fragmented DNA increased from 1.1% to 68% in after exposure to H. Pylori for 24 hr. However, AGS-E6 cells were relatively resistant to apoptosis induced by H. Pylori (only 15% of cells underwent apoptosis). In additional experiments, mouse embryonic fibroblasts (MEFs) were used to further investigate the role of ARF in stabilizing p53 after exposure to H. Pylori. Wild-type and p19^ARF–/– MEFs were exposed to H. Pylori and evaluated for activation of p53, p19^ARF, and apoptosis. As with AGS cells, H. Pylori stimulated a 2-fold increase in p53 and p19^ARF in wild-type MEFs; however, there was no increase in p53 in ARF-null MEFs. H. Pylori easily stimulated apoptosis in wild-type MEFs, although, the absence of p19^ARF significantly reduced the ability of H. Pylori to induce apoptosis in these cells. Activation of ARF by H. Pylori is important in stabilizing p53 resulting in increased apoptosis. Thus, inactivation of either ARF or p53 in gastric cells may reduce their ability to undergo apoptosis in response to injury induced by H. Pylori.     

Clinical significance of apoptotic index in non-small cell lung cancer: correlation with p53, mdm2, pRb and p21WAF1/CIP1 protein expression

Purpose: The aim of this study was to assess the prognostic relevance of apoptotic index (AI), considered alone or together with expression of several proteins controlling G1 check point (p53, mdm2, pRb and p21^WAF1/CIP1) in non-small cell lung cancer (NSCLC) patients. Methods: Study group included 50 NSCLC patients who underwent curative pulmonary resection. Apoptosis was detected with the use of TUNEL technique and AI was defined as the number of apoptotic cells per 1,000 tumor cells. The expression of p53, mdm2, pRb and p21^WAF1/CIP1 was assessed immunohistochemically. Results: The mean and median AI calculated for all 50 patients was 14 and 9, respectively. Patients with lower (<14) and higher (≥14) AI constituted 35 (70%) and 15 (30%) of cases, respectively. AI was not correlated with patient clinical characteristics, and expression of p53, pRb and p21^WAF1/CIP1 . However, lower AI was correlated with over-expression of mdm2 protein (P=0.04). Median survival for patients with lower and higher AI was 43 months and 22 months, respectively, and 5-year survival probability—60 and 25%, respectively (P=0.03). In multivariate analysis, the only variable associated with shortened survival was AI (P=0.03, HR=2.9, 95% CI 1.95–3.86). Conclusions: These results suggest that AI correlates with mdm2 protein expression and influences survival in NSCLC.