query: Depletion of nitric oxide is responsible for vasospasm.

title: Nitrite infusions to prevent delayed cerebral vasospasm in a primate model of subarachnoid hemorrhage.

text:
CONTEXT Delayed cerebral vasospasm causes permanent neurological deficits or death in at least 15% of patients following otherwise successful treatment for ruptured intracranial aneurysm. Decreased bioavailability of nitric oxide has been associated with the development of cerebral vasospasm.
 OBJECTIVE To determine whether infusions of nitrite will prevent delayed cerebral vasospasm.
 DESIGN, SETTING, AND SUBJECTS A total of 14 anesthetized cynomolgus monkeys had an autologous blood clot placed around the right middle cerebral artery. Cerebral arteriography was performed before clot placement and on days 7 and 14 to assess vasospasm. The study was conducted from August 2003 to February 2004.
 INTERVENTIONS A 90-mg sodium nitrite intravenous solution infused over 24 hours plus a 45-mg sodium nitrite bolus daily (n = 3); a 180-mg sodium nitrite intravenous solution infused over 24 hours (n = 3); or a control saline solution infusion (n = 8). Each was infused continuously for 14 days.
 MAIN OUTCOME MEASURES Nitrite, S-nitrosothiol, and methemoglobin levels in blood and cerebrospinal fluid and degree of arteriographic vasospasm.
 RESULTS In control monkeys, mean (SD) cerebrospinal fluid nitrite levels decreased from 3.1 (1.5) micromol/L to 0.4 (0.1) micromol/L at day 7 and to 0.4 (0.4) micromol/L at day 14 (P = .03). All 8 control monkeys developed significant vasospasm of the right middle cerebral artery, which was complicated by stroke and death in 1 animal. Sodium nitrite infusions increased the nitrite and methemoglobin levels (<2.1% of total hemoglobin) in the blood and cerebrospinal fluid without evoking systemic hypotension. Nitrite infusion prevented development of vasospasm (no animals developed significant vasospasm; mean [SD] reduction in right middle cerebral artery area on day 7 after subarachnoid hemorrhage of 8% [9%] in nitrite-treated monkeys vs 47% [5%] in saline-treated controls; P<.001). There was a negative correlation between the concentration of nitrite in cerebrospinal fluid and the degree of cerebral vasospasm (P<.001). Pharmacological effects of nitrite infusion were also associated with the formation of S-nitrosothiol in cerebrospinal fluid. There was no clinical or pathological evidence of nitrite toxicity.
 CONCLUSION Subacute sodium nitrite infusions prevented delayed cerebral vasospasm in a primate model of subarachnoid hemorrhage.

title: Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury.

text: Besides synthesizing nitric oxide (NO), purified neuronal NO synthase (nNOS) can produce superoxide (.O2-) at lower L-Arg concentrations. By using electron paramagnetic resonance spin-trapping techniques, we monitored NO and .O2- formation in nNOS-transfected human kidney 293 cells. In control transfected cells, the Ca2+ ionophore A23187 triggered NO generation but no .O2- was seen. With cells in L-Arg-free medium, we observed .O2- formation that increased as the cytosolic L-Arg levels decreased, while NO generation declined. .O2- formation was virtually abolished by the specific NOS blocker, N-nitro-L-arginine methyl ester (L-NAME). Nitrotyrosine, a specific nitration product of peroxynitrite, accumulated in L-Arg-depleted cells but not in control cells. Activation by A23187 was cytotoxic to L-Arg-depleted, but not to control cells, with marked lactate dehydrogenase release. The cytotoxicity was largely prevented by either superoxide dismutase or L-NAME. Thus, with reduced L-Arg availability NOS elicits cytotoxicity by generating .O2- and NO that interact to form the potent oxidant peroxynitrite. Regulating arginine levels may provide a therapeutic approach to disorders involving .O2-/NO-mediated cellular injury.

title: Lethal autoimmune myocarditis in interferon-gamma receptor-deficient mice: enhanced disease severity by impaired inducible nitric oxide synthase induction.

text:
BACKGROUND Interferon-gamma (IFN-gamma) is an essential cytokine in the regulation of inflammatory responses in autoimmune diseases. Little is known about its role in inflammatory heart disease.
 METHODS AND RESULTS We showed that IFN-gamma receptor-deficient mice (IFN-gammaR(-/-)) on a BALB/c background immunized with a peptide derived from cardiac alpha-myosin heavy chain develop severe myocarditis with high mortality. Although myocarditis subsided in wild-type mice after 3 weeks, IFN-gammaR(-/-) mice showed persistent disease. The persistent inflammation was accompanied by vigorous in vitro CD4 T-cell responses and impaired inducible nitric oxide synthase expression, together with evidence of impaired nitric oxide production in IFN-gammaR(-/-) hearts. Treatment of wild-type mice with the nitric oxide synthetase inhibitor N:-nitro-l-arginine-methyl-ester enhanced in vitro CD4 T-cell proliferation and prevented healing of myocarditis.
 CONCLUSIONS Our data provide evidence that IFN-gamma protects mice from lethal autoimmune myocarditis by inducing the expression of inducible nitric oxide synthase followed by the downregulation of T-cell responses.

title: Chronic Exercise Increases Both Inducible and Endothelial Nitric Oxide Synthase Gene Expression in Endothelial Cells of Rat Aorta

text: Chronic exercise upregulates endothelial nitric oxide synthase (eNOS) gene expression. Whether the expression of inducible nitric oxide synthase (iNOS) is affected by exercise is unknown. We therefore investigated the effects of chronic exercise on iNOS and eNOS expression in isolated rat aortic endothelial and smooth muscle cells separately. Five-week-old male Wistar rats were randomly divided into control and exercise groups. After 10 weeks of running training, animals were sacrificed under ether anesthesia. The standard curve quantitative competitive reverse transcriptase-polymerase chain reaction method was used to quantify NOS mRNA expression in isolated endothelial/smooth muscle cells. To evaluate the functional role of iNOS, we examined phenylephrine-induced vascular responses with or without pretreatment with aminoguanidine. We found that (1) expression of iNOS and eNOS mRNA in endothelial cells was increased by chronic exercise and (2) chronic exercise blunted phenylephrine-induced vascular responses, probably by increasing NO release via iNOS. Our results show that chronic exercise increases both iNOS and eNOS gene expression in endothelium. These alterations may be partially responsible for the change in vascular response after exercise.

title: Role of chicken melanoma differentiation-associated gene 5 in induction and activation of innate and adaptive immune responses to infectious bursal disease virus in cultured macrophages

text: The objective of the present study was to determine if chicken melanoma-differentiation-associated gene 5 (MDA5) senses infectious bursal disease virus infection to induce innate immunity that bridges to adaptive immunity. During IBDV infection in HD11 cells, IBDV titers and RNA loads increased up to 3.4 × 107 plaque-forming units (PFU)/mL and 1114 ng/µL, respectively, at 24 hours postinfection (hpi). IBDV infection in HD11 cells induced significantly upregulated (p < 0.05) expression levels of chicken MDA5 (59-fold), interferon-β (IFN-β) (693-fold), dsRNA-dependent protein kinase (PKR) (4-fold), 2’, 5’-oligoadenylate synthetase (OAS) (286-fold), myxovirus resistance gene (Mx) (22-fold), interleukin-1β (IL-1β) (5-fold), IL-6 (146-fold), IL-8 (4-fold), IL-10 (4-fold), inducible nitric oxide synthase (iNOS) (15-fold), and major histocompatibility complex class I (MHC class I) (4-fold). Nitric oxide production in the culture supernatants increased significantly (p < 0.05) up to 6.5 μM at 24 hpi. The expressed chMDA5 and IBDV-derived dsRNA were localized in the cytoplasm of HD11 cells during IBDV infection. ChMDA5-knockdown HD11 cells had significantly higher (p < 0.05) IBDV RNA loads at 24 hpi and significantly lower (p < 0.05) nitric oxide production and expression levels of chicken MDA5, IFN-β, PKR, OAS, Mx, IL-1β, IL-6, IL-8, IL-12(p40), IL-18, IL-10, iNOS, MHC class I and CD86 at 24 hpi. In addition, chMDA5 overexpression in HD11 cells resulted in significantly reduced (p < 0.05) IBDV titers and RNA loads and significantly increased (p < 0.05) nitric oxide production at 16 and 24 hpi. It also resulted in significantly higher (p < 0.05) expression levels of chicken MDA5, IFN-β, PKR, OAS, Mx, IL-1β, IL-6, IL-8, IL-12(p40), IL-10 and iNOS at 2 hpi. In conclusion, the results indicate that chMDA5 senses IBDV infection in chicken macrophages, and this is associated with IBDV-induced expression of IFN-β and initiation of an innate immune response that in turn activates the adaptive immune response and limits IBDV replication.

title: Crucial Role of Interferon Consensus Sequence Binding Protein, but neither of Interferon Regulatory Factor 1 nor of Nitric Oxide Synthesis for Protection Against Murine Listeriosis

text: Listeria monocytogenes is widely used as a model to study immune responses against intracellular bacteria. It has been shown that neutrophils and macrophages play an important role to restrict bacterial replication in the early phase of primary infection in mice, and that the cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) are essential for protection. However, the involved signaling pathways and effector mechanisms are still poorly understood. This study investigated mouse strains deficient for the IFN-dependent transcription factors interferon consensus sequence binding protein (ICSBP), interferon regulatory factor (IRF)1 or 2 for their capacity to eliminate Listeria in vivo and in vitro and for production of inducible reactive nitrogen intermediates (RNI) or reactive oxygen intermediates (ROI) in macrophages. ICSBP−/− and to a lesser degree also IRF2−/− mice were highly susceptible to Listeria infection. This correlated with impaired elimination of Listeria from infected peritoneal macrophage (PEM) cultures stimulated with IFN-γ in vitro; in addition these cultures showed reduced and delayed oxidative burst upon IFN-γ stimulation, whereas nitric oxide production was normal. In contrast, mice deficient for IRF1 were not able to produce nitric oxide, but they efficiently controlled Listeria in vivo and in vitro. These results indicate that (a) the ICSBP/IRF2 complex is essential for IFN-γ–mediated protection against Listeria and that (b) ROI together with additional still unknown effector mechanisms may be responsible for the anti-Listeria activity of macrophages, whereas IRF1-induced RNI are not limiting.

title: Lung Carcinogenesis by Diesel Exhaust Particles and the Carcinogenic Mechanism Via Active Oxygens.

text: In an experiment to clarify the involvement of oxygen radicals in lung carcinogenesis induced by diesel exhaust particles (DEP), we found that there is a strong relation between lung tumor response and formation of 8-hydroxydeoxyguanosine (8-OHdG) in lung DNA of mice administered DEP by repeated intratracheal instillation. Repeated intratracheal instillation of DEP also induced the activity of cytochrome P-450 reductase in the lungs as a representative enzyme of superoxide generation, and two types of nitric oxide (NO) synthase, cNOS and iNOS, in the lungs. On the other hand, activities of CuZn-superoxide dismutase (SOD) and Mn-SOD antioxidant enzymes were depressed by the instillation of DEP. These results suggest that generation of superoxide, hydroxyI radical, and nitric oxide are increased in epithelial cells in airways, and that the increased superoxide and nitric oxide react very easily to produce peroxynitrite (ONOO(-)). The peroxynitrite also produce hydroxyI radical. The hydroxyl radical may play an important role in carcinogenesis by DEP.

title: Vasodilator responses of coronary resistance arteries of exercise-trained pigs.

text:
BACKGROUND The purpose of this study was to test the hypothesis that vasodilator responses of porcine coronary resistance arteries are increased by exercise training.
 METHODS AND RESULTS Yucatan miniature swine were randomly divided into groups of exercise-trained (ET) and sedentary (SED) control pigs. ET pigs were placed on a progressive treadmill training program lasting 16 to 20 weeks, and SED pigs remained inactive during the same time period. Coronary resistance arteries 64 to 157 microns in diameter were isolated for in vitro evaluation of relaxation responses to the endothelium-independent dilators sodium nitroprusside (1 x 10(-10) to 1 x 10(-4) mol/L) and adenosine (1 x 10(-10) to 1 x 10(-5) mol/L) and to bradykinin (1 x 10(-13) to 3 x 10(-7) mol/L), an endothelium-dependent agent. Relaxation responses to adenosine and sodium nitroprusside were not altered by exercise training. Endothelium-dependent relaxation to bradykinin was enhanced in coronary resistance arteries from ET pigs (IC50: ET, 0.07 +/- 0.02 nmol/L; SED, 1.59 +/- 0.09 nmol/L). To determine whether prostanoids and/or the nitric oxide synthase pathway were involved in the ET-induced changes in bradykinin-induced vasodilation, responses to bradykinin were examined in coronary resistance arteries from both ET and SED pigs in the presence of indomethacin and in the presence of nitro-monomethyl L-arginine (L-NMMA). Both indomethacin and L-NMMA produced significant inhibition of the bradykinin-induced relaxation in vessels from both groups. Despite decreased bradykinin-induced relaxation after indomethacin, bradykinin-induced vasodilation was still enhanced in vessels from the ET group. L-NMMA caused greater inhibition of the bradykinin-induced relaxation in coronary resistance arteries from ET pigs relative to arteries from SED pigs and eliminated the training-induced enhancement of the bradykinin responses.
 CONCLUSIONS These results suggest that exercise training enhances bradykinin-induced vasodilation through increased endothelium-derived relaxing factor/nitric oxide production by the L-arginine/nitric oxide synthase pathway.

title: Detection and function of nitric oxide during the hypersensitive response in Arabidopsis thaliana: where there's a will there's a way.

text: Nitric oxide (NO) was identified as a key player in plant defence responses approximately 20 years ago and a large body of evidence has accumulated since then supporting its role as a signalling molecule. However, there are many discrepancies in current NO detection assays and the enzymatic pathways responsible for its synthesis have yet to be determined. This has provoked strong debates concerning the function of NO in plants, even questioning its existence in planta. Here we gather data obtained using the model pathosystem Arabidopsis/Pseudomonas, which confirms the production of NO during the hypersensitive response and supports is role as a trigger of hypersensitive cell death and a mediator of defence gene expression. Finally, we discuss potential sources of NO synthesis, focusing on the role of nitrite as major substrate for NO production during incompatible interactions.

title: Nerve-induced release of nitric oxide in the rabbit gastrointestinal tract as measured by in vivo microdialysis.

text: 1. Nitric oxide (NO) has been suggested as a gastrointestinal neurotransmitter, mediating the gastric receptive relaxation and the relaxation in the peristaltic reflex. The aim of the present study was to measure nerve-induced NO formation in vivo in the gastrointestinal tract. 2. Formation of the nitric oxide oxidation products nitrite and nitrate during vagal nerve stimulation were measured in the anaesthetized rabbit. Microdialysis probes were inserted into the wall of the stomach and proximal colon, and nitrite and nitrate in dialysate measured by capillary electrophoresis. 3. During bilateral vagal nerve stimulation there was an increase in nitrite and nitrate formation at the level of the stomach and in nitrite formation at the level of the colon. This increase was inhibited by intravenous administration of the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME 30 mg kg-1). Furthermore, L-NAME significantly increased nerve-induced gastric and colonic contractions, as well as spontaneous colonic contractions. 4. In summary, we present a new methodological procedure for quantification of small changes in nitric oxide formation in vivo. This study provides evidence that nitric oxide is released in the stomach and colonic wall during vagal nerve activity, at concentrations able to cause inhibition of smooth muscle contractions in vivo.

title: GAPDH Mediates Nitrosylation of Nuclear Proteins

text: S-nitrosylation of proteins by nitric oxide is a major mode of signalling in cells. S-nitrosylation can mediate the regulation of a range of proteins, including prominent nuclear proteins, such as HDAC2 (ref. 2) and PARP1 (ref. 3). The high reactivity of the nitric oxide group with protein thiols, but the selective nature of nitrosylation within the cell, implies the existence of targeting mechanisms. Specificity of nitric oxide signalling is often achieved by the binding of nitric oxide synthase (NOS) to target proteins, either directly or through scaffolding proteins such as PSD-95 (ref. 5) and CAPON. As the three principal isoforms of NOS--neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS)--are primarily non-nuclear, the mechanisms by which nuclear proteins are selectively nitrosylated have been elusive. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is physiologically nitrosylated at its Cys 150 residue. Nitrosylated GAPDH (SNO-GAPDH) binds to Siah1, which possesses a nuclear localization signal, and is transported to the nucleus. Here, we show that SNO-GAPDH physiologically transnitrosylates nuclear proteins, including the deacetylating enzyme sirtuin-1 (SIRT1), histone deacetylase-2 (HDAC2) and DNA-activated protein kinase (DNA-PK). Our findings reveal a novel mechanism for targeted nitrosylation of nuclear proteins and suggest that protein-protein transfer of nitric oxide groups may be a general mechanism in cellular signal transduction.

title: Mechanisms of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities.

text: Type 1 and type 2 diabetes are characterized by progressive beta-cell failure. Apoptosis is probably the main form of beta-cell death in both forms of the disease. It has been suggested that the mechanisms leading to nutrient- and cytokine-induced beta-cell death in type 2 and type 1 diabetes, respectively, share the activation of a final common pathway involving interleukin (IL)-1beta, nuclear factor (NF)-kappaB, and Fas. We review herein the similarities and differences between the mechanisms of beta-cell death in type 1 and type 2 diabetes. In the insulitis lesion in type 1 diabetes, invading immune cells produce cytokines, such as IL-1beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma. IL-1beta and/or TNF-alpha plus IFN-gamma induce beta-cell apoptosis via the activation of beta-cell gene networks under the control of the transcription factors NF-kappaB and STAT-1. NF-kappaB activation leads to production of nitric oxide (NO) and chemokines and depletion of endoplasmic reticulum (ER) calcium. The execution of beta-cell death occurs through activation of mitogen-activated protein kinases, via triggering of ER stress and by the release of mitochondrial death signals. Chronic exposure to elevated levels of glucose and free fatty acids (FFAs) causes beta-cell dysfunction and may induce beta-cell apoptosis in type 2 diabetes. Exposure to high glucose has dual effects, triggering initially "glucose hypersensitization" and later apoptosis, via different mechanisms. High glucose, however, does not induce or activate IL-1beta, NF-kappaB, or inducible nitric oxide synthase in rat or human beta-cells in vitro or in vivo in Psammomys obesus. FFAs may cause beta-cell apoptosis via ER stress, which is NF-kappaB and NO independent. Thus, cytokines and nutrients trigger beta-cell death by fundamentally different mechanisms, namely an NF-kappaB-dependent mechanism that culminates in caspase-3 activation for cytokines and an NF-kappaB-independent mechanism for nutrients. This argues against a unifying hypothesis for the mechanisms of beta-cell death in type 1 and type 2 diabetes and suggests that different approaches will be required to prevent beta-cell death in type 1 and type 2 diabetes.

title: Workshop: endothelial cell dysfunction leading to diabetic nephropathy : focus on nitric oxide.

text: Clinical manifestations of diabetic nephropathy are an expression of diabetic microangiopathy. This review revisits the previously proposed Steno hypothesis and advances our hypothesis that development of endothelial cell dysfunction represents a common pathophysiological pathway of diabetic complications. Specifically, the ability of glucose to scavenge nitric oxide is proposed as the initiation phase of endothelial dysfunction. Gradual accumulation of advanced glycated end products and induction of plasminogen activator inhibitor-1, resulting in the decreased expression of endothelial nitric oxide synthase and reduced generation of nitric oxide, are proposed to be pathophysiologically critical for the maintenance phase of endothelial dysfunction. The proposed conceptual shift toward the role of endothelial dysfunction in diabetic complications may provide new strategies for their prevention.

title: Endothelial KLF2 links local arterial shear stress levels to the expression of vascular tone-regulating genes.

text: Lung Krüppel-like factor (LKLF/KLF2) is an endothelial transcription factor that is crucially involved in murine vasculogenesis and is specifically regulated by flow in vitro. We now show a relation to local flow variations in the adult human vasculature: decreased LKLF expression was noted at the aorta bifurcations to the iliac and carotid arteries, coinciding with neointima formation. The direct involvement of shear stress in the in vivo expression of LKLF was determined independently by in situ hybridization and laser microbeam microdissection/reverse transcriptase-polymerase chain reaction in a murine carotid artery collar model, in which a 4- to 30-fold induction of LKLF occurred at the high-shear sites. Dissection of the biomechanics of LKLF regulation in vitro demonstrated that steady flow and pulsatile flow induced basal LKLF expression 15- and 36-fold at shear stresses greater than approximately 5 dyne/cm2, whereas cyclic stretch had no effect. Prolonged LKLF induction in the absence of flow changed the expression of angiotensin-converting enzyme, endothelin-1, adrenomedullin, and endothelial nitric oxide synthase to levels similar to those observed under prolonged flow. LKLF repression by siRNA suppressed the flow response of endothelin-1, adrenomedullin, and endothelial nitric oxide synthase (P < 0.05). Thus, we demonstrate that endothelial LKLF is regulated by flow in vivo and is a transcriptional regulator of several endothelial genes that control vascular tone in response to flow.

title: Reduced plasma concentrations of nitrogen oxide in individuals with essential hypertension.

text: Patients with essential hypertension exhibit blunted endothelium-dependent vasodilator responses, which may be largely attributable to reduced bioactivity of nitric oxide (NO). Therefore, we measured the end product of NO, nitrate plus nitrite (nitrogen oxide), and examined the relationship between the degree of hypertension and plasma nitrate plus nitrite levels in patients with essential hypertension. The combined plasma concentration of nitrate plus nitrite, end products of NO metabolism, was reduced in individuals with essential hypertension relative to that in control subjects (15.7+/-1.1 versus 22.8+/-1.4 mmol x L(-1), P<.001); individuals with borderline hypertension showed values that were intermediate between those of the other two groups (18.2+/-1.2 mmol x L(-1), P<.001). The plasma nitrogen oxide concentration showed significant inverse correlations with both systolic and diastolic blood pressures. The basal concentration of nitrogen oxide in the plasma was reduced, at least in the peripheral circulation, in individuals with essential hypertension.

title: Inducible nitric oxide synthase in pulmonary alveolar macrophages from patients with tuberculosis

text: The high-output pathway of nitric oxide production helps protect mice from infection by several pathogens, including Mycobacterium tuberculosis. However, based on studies of cells cultured from blood, it is controversial whether human mononuclear phagocytes can express the corresponding inducible nitric oxide synthase (iNOS;NOS2). The present study examined alveolar macrophages fixed directly after bronchopulmonary lavage. An average of 65% of the macrophages from 11 of 11 patients with untreated, culture-positive pulmonary tuberculosis reacted with an antibody documented herein to be monospecific for human NOS2. In contrast, a mean of 10% of bronchoalveolar lavage cells were positive from each of five clinically normal subjects. Tuberculosis patients' macrophages displayed diaphorase activity in the same proportion that they stained for NOS2, under assay conditions wherein the diaphorase reaction was strictly dependent on NOS2 expression. Bronchoalveolar lavage specimens also contained NOS2 mRNA. Thus, macrophages in the lungs of people with clinically active Mycobacterium tuberculosis infection often express catalytically competent NOS2.

title: Molecular mechanisms of hepatic ischemia-reperfusion injury and preconditioning.

text: Ischemia-reperfusion injury is, at least in part, responsible for the morbidity associated with liver surgery under total vascular exclusion or after liver transplantation. The pathophysiology of hepatic ischemia-reperfusion includes a number of mechanisms that contribute to various degrees in the overall injury. Some of the topics discussed in this review include cellular mechanisms of injury, formation of pro- and anti-inflammatory mediators, expression of adhesion molecules, and the role of oxidant stress during the inflammatory response. Furthermore, the roles of nitric oxide in preventing microcirculatory disturbances and as a substrate for peroxynitrite formation are reviewed. In addition, emerging mechanisms of protection by ischemic preconditioning are discussed. On the basis of current knowledge, preconditioning or pharmacological interventions that mimic these effects have the greatest potential to improve clinical outcome in liver surgery involving ischemic stress and reperfusion.

title: Preliminary assessment of inhaled nitric oxide for acute vaso-occlusive crisis in pediatric patients with sickle cell disease.

text:
CONTEXT Vaso-occlusion is central to the painful crises and acute and chronic organ damage in sickle cell disease. Abnormal nitric oxide-dependent regulation of vascular tone, adhesion, platelet activation, and inflammation contributes to the pathophysiology of vaso-occlusion. Nitric oxide may have promise as a mechanism-of-disease-based therapy for treatment of vaso-occlusion.
 OBJECTIVE To explore the efficacy and safety of inhaled nitric oxide (INO) for treatment of vaso-occlusive crisis in pediatric patients.
 DESIGN Prospective, double-blind, placebo-controlled, randomized clinical trial with enrollment between September 1999 and October 2001.
 SETTING Urban, tertiary care children's hospital in the United States.
 PARTICIPANTS Twenty patients aged 10 to 21 years with sickle cell disease and severe acute vaso-occlusive crisis.
 INTERVENTION Patients were randomly assigned to receive INO (80 ppm with 21% final concentration of inspired oxygen; n = 10), or placebo (21% inspired oxygen; n = 10) for 4 hours.
 MAIN OUTCOME MEASURES Change in pain at 4 hours of inhalation compared with preinhalation pain, measured on a 10-cm visual analog scale (VAS); secondary outcome measures were pain over 6 hours, parenteral narcotic use over 24 hours, duration of hospitalization, blood pressure, oxygen saturation, and methemoglobin concentration.
 RESULTS Preinhalation VAS pain scores were similar in the INO and placebo groups (P =.80). The decrease in VAS pain scores at 4 hours was 2.0 cm in the INO group and 1.2 cm in the placebo group (P =.37). Repeated-measures analysis of variance for hourly pain scores showed a 1-cm/h greater reduction in the INO group than the placebo group (P =.02). Morphine use over 6 hours was significantly less in the INO group (mean cumulative use, 0.29 vs 0.44 mg/kg; P =.03) but was not different over 4 hours (0.26 vs 0.32 mg/kg; P =.21) or 24 hours (0.63 vs 0.91 mg/kg; P =.15). Duration of hospitalization was 78 and 100 hours in the INO and placebo groups, respectively (P =.19). No INO toxicity was observed.
 CONCLUSIONS Results of this exploratory study suggest that INO may be beneficial for acute vaso-occlusive crisis. These preliminary results warrant further investigation.

title: Flow-Dependent Regulation of Kruppel-Like Factor 2 Is Mediated by MicroRNA-92a.

text:
BACKGROUND Upregulated by atheroprotective flow, the transcription factor Krüppel-like factor 2 (KLF2) is crucial for maintaining endothelial function. MicroRNAs (miRNAs) are noncoding small RNAs that regulate gene expression at the posttranscriptional level. We examined the role of miRNAs, particularly miR-92a, in the atheroprotective flow-regulated KLF2.
 METHODS AND RESULTS Dicer knockdown increased the level of KLF2 mRNA in human umbilical vein endothelial cells, suggesting that KLF2 is regulated by miRNA. In silico analysis predicted that miR-92a could bind to the 3' untranslated region of KLF2 mRNA. Overexpression of miR-92a decreased the expression of KLF2 and the KLF2-regulated endothelial nitric oxide synthase and thrombomodulin at mRNA and protein levels. A complementary finding is that miR-92a inhibitor increased the mRNA and protein expression of KLF2, endothelial nitric oxide synthase, and thrombomodulin. Subsequent studies revealed that atheroprotective laminar flow downregulated the level of miR-92a precursor to induce KLF2, and the level of this flow-induced KLF2 was reduced by miR-92a precursor. Furthermore, miR-92a level was lower in human umbilical vein endothelial cells exposed to the atheroprotective pulsatile shear flow than under atheroprone oscillatory shear flow. Anti-Ago1/2 immunoprecipitation coupled with real-time polymerase chain reaction revealed that pulsatile shear flow decreased the functional targeting of miR-92a precursor/KLF2 mRNA in human umbilical vein endothelial cells. Consistent with these findings, mouse carotid arteries receiving miR-92a precursor exhibited impaired vasodilatory response to flow.
 CONCLUSIONS Atheroprotective flow patterns decrease the level of miR-92a, which in turn increases KLF2 expression to maintain endothelial homeostasis.

title: expression by oxidized linoleic

text: Hypercholesterolemia is associated with impairments in endothelium-dependent vascular relaxations. Paradoxically, endothelial production of nitrogen oxides is increased in early stages of hypercholesterolemia. Prior work has shown that oxidized low density lipoprotein (LDL) has both stimulatory and inhibitory effects on endothelial nitric oxide synthase expression (eNOS) and has focused on lysophosphatidyl choline (LPC) as a component of oxidized LDL which may modulate this effect. Another biologically active component of oxidized LDL is 13-hydroperoxyoctadecadienoic acid (13-HPODE), an oxidized form of linoleic acid. The purpose of this study was to determine the effect of HPODE on the expression of eNOS in bovine aortic endothelial cells (BAECs). Twenty four hour treatment of endothelial cells with HPODE caused a dose-dependent increase in eNOS mRNA levels as assessed by Northern analysis. The time response studies show that HPODE treatment significantly increased eNOS mRNA levels at 12 and 24 h. Concomitant with the increase in eNOS mRNA levels, 20 microM HPODE treatment significantly increased eNOS protein content and enzyme activity. Nuclear run-on studies indicated that the rate of transcription of eNOS gene was significantly elevated 4 h after HPODE treatment when compared to control cultures. Also, actinomycin D studies demonstrated that the half-life of eNOS mRNA was increased from 6 h to 12 h by HPODE treatment. Thus, HPODE-induced up-regulation of eNOS expression is mediated by both transcriptional and posttranscriptional mechanisms. These observations suggest that endothelial cells may attempt to compensate for oxidative injury by increasing expression of eNOS in early stages of hypercholesterolemia.


query: Deregulation of HAND2 is a crucial step in endometrial carcinogenesis in mice.

title: Role of DNA Methylation and Epigenetic Silencing of HAND2 in Endometrial Cancer Development

text:
BACKGROUND Endometrial cancer incidence is continuing to rise in the wake of the current ageing and obesity epidemics. Much of the risk for endometrial cancer development is influenced by the environment and lifestyle. Accumulating evidence suggests that the epigenome serves as the interface between the genome and the environment and that hypermethylation of stem cell polycomb group target genes is an epigenetic hallmark of cancer. The objective of this study was to determine the functional role of epigenetic factors in endometrial cancer development.
 METHODS AND FINDINGS Epigenome-wide methylation analysis of >27,000 CpG sites in endometrial cancer tissue samples (n = 64) and control samples (n = 23) revealed that HAND2 (a gene encoding a transcription factor expressed in the endometrial stroma) is one of the most commonly hypermethylated and silenced genes in endometrial cancer. A novel integrative epigenome-transcriptome-interactome analysis further revealed that HAND2 is the hub of the most highly ranked differential methylation hotspot in endometrial cancer. These findings were validated using candidate gene methylation analysis in multiple clinical sample sets of tissue samples from a total of 272 additional women. Increased HAND2 methylation was a feature of premalignant endometrial lesions and was seen to parallel a decrease in RNA and protein levels. Furthermore, women with high endometrial HAND2 methylation in their premalignant lesions were less likely to respond to progesterone treatment. HAND2 methylation analysis of endometrial secretions collected using high vaginal swabs taken from women with postmenopausal bleeding specifically identified those patients with early stage endometrial cancer with both high sensitivity and high specificity (receiver operating characteristics area under the curve = 0.91 for stage 1A and 0.97 for higher than stage 1A). Finally, mice harbouring a Hand2 knock-out specifically in their endometrium were shown to develop precancerous endometrial lesions with increasing age, and these lesions also demonstrated a lack of PTEN expression.
 CONCLUSIONS HAND2 methylation is a common and crucial molecular alteration in endometrial cancer that could potentially be employed as a biomarker for early detection of endometrial cancer and as a predictor of treatment response. The true clinical utility of HAND2 DNA methylation, however, requires further validation in prospective studies. Please see later in the article for the Editors' Summary.

title: Exploring and exploiting the systemic effects of deregulated replication licensing.

text: Maintenance and accurate propagation of the genetic material are key features for physiological development and wellbeing. The replication licensing machinery is crucial for replication precision as it ensures that replication takes place once per cell cycle. Thus, the expression status of the components comprising the replication licensing apparatus is tightly regulated to avoid re-replication; a form of replication stress that leads to genomic instability, a hallmark of cancer. In the present review we discuss the mechanistic basis of replication licensing deregulation, which leads to systemic effects, exemplified by its role in carcinogenesis and a variety of genetic syndromes. In addition, new insights demonstrate that above a particular threshold, the replication licensing factor Cdc6 acts as global transcriptional regulator, outlining new lines of exploration. The role of the putative replication licensing factor ChlR1/DDX11, mutated in the Warsaw Breakage Syndrome, in cancer is also considered. Finally, future perspectives focused on the potential therapeutic advantage by targeting replication licensing factors, and particularly Cdc6, are discussed.

title: COX-2 and survivin are overexpressed and positively correlated in endometrial carcinoma.

text:
OBJECTIVES To investigate the expressions of survivin and Cyclooxygenase-2 (COX-2), and their possible correlations in the development of endometrial adenocarcinoma (EC). We also looked at their association with classical prognostic factors in EC. To our knowledge, this is the first time survivin expression is investigated in terms of its relation to COX-2 in the developmental pathway of EC.
 METHODS Archived tissue samples of 50 EC, 30 endometrial hyperplasia and 20 proliferative endometrium were selected and immunohistochemically analyzed for survivin and COX-2 expression.
 RESULTS Both survivin and COX-2 were overexpressed in hyperplasia and endometrial adenocarcinoma cases compared to proliferative endometrium, which was statistically significant (p=0.01, p=0.02, respectively). Among EC cases, survivin and COX-2 were strongly positive in 38 (76%) and 30 (60%) patients, respectively. Furthermore, we found survivin and COX-2 to be positively correlated, which was also statistically significant (p=0.0001, r=0.46). Neither survivin nor COX-2 expression was correlated with classical prognostic factors of endometrial carcinoma such as myometrial invasion, grade or lymph node metastasis (p>0.05). Neither COX-2 nor survivin had an impact on overall survival (p>0.05).
 CONCLUSIONS Both survivin and COX-2 are overexpressed, and they seem to be early events in the occurrence of EC. Moreover, protein products of these two genes are positively correlated. COX-2 and survivin might share a common molecular pathway or enhance each other's actions in the developmental pathway of EC. Molecular basis of such a relationship should be further investigated in endometrial carcinogenesis.

title: Single-Construct Polycistronic Doxycycline-Inducible Vectors Improve Direct Cardiac Reprogramming and Can Be Used to Identify the Critical Timing of Transgene Expression

text: Direct reprogramming is a promising approach in regenerative medicine. Overexpression of the cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2 (GHMT) directly reprogram fibroblasts into cardiomyocyte-like cells (iCMs). However, the critical timing of transgene expression and the molecular mechanisms for cardiac reprogramming remain unclear. The conventional doxycycline (Dox)-inducible temporal transgene expression systems require simultaneous transduction of two vectors (pLVX-rtTA/pLVX-cDNA) harboring the reverse tetracycline transactivator (rtTA) and the tetracycline response element (TRE)-controlled transgene, respectively, leading to inefficient cardiac reprogramming. Herein, we developed a single-construct-based polycistronic Dox-inducible vector (pDox-cDNA) expressing both the rtTA and TRE-controlled transgenes. Fluorescence activated cell sorting (FACS) analyses, quantitative RT-PCR, and immunostaining revealed that pDox-GMT increased cardiac reprogramming three-fold compared to the conventional pLVX-rtTA/pLVX-GMT. After four weeks, pDox-GMT-induced iCMs expressed multiple cardiac genes, produced sarcomeric structures, and beat spontaneously. Co-transduction of pDox-Hand2 with retroviral pMX-GMT increased cardiac reprogramming three-fold compared to pMX-GMT alone. Temporal Dox administration revealed that Hand2 transgene expression is critical during the first two weeks of cardiac reprogramming. Microarray analyses demonstrated that Hand2 represses cell cycle-promoting genes and enhances cardiac reprogramming. Thus, we have developed an efficient temporal transgene expression system, which could be invaluable in the study of cardiac reprogramming.

title: Roles of induced expression of MAPK phosphatase-2 in tumor development in RET-MEN2A transgenic mice

text: Germline mutations in the RET tyrosine kinase gene are responsible for the development of multiple endocrine neoplasia 2A and 2B (MEN2A and MEN2B). However, knowledge of the fundamental principles that determine the mutant RET-mediated signaling remains elusive. Here, we report increased expression of mitogen-activated protein kinase phosphatase-2 (MKP-2) in carcinomas developed in transgenic mice carrying RET with the MEN2A mutation (RET-MEN2A). The expression of MKP-2 was not only induced by RET-MEN2A or RET-MEN2B mutant proteins but also by the activation of endogenous RET by its ligand, glial cell line-derived neurotrophic factor (GDNF). MKP-2 expression was also evident in the MKK-f cell line, which was established from a mammary tumor developed in a RET-MEN2A transgenic mouse. Inhibition of MKP-2 attenuated the in vitro and in vivo proliferation of MKK-f cells, which was mediated by the suppression of cyclin B1 expression. Furthermore, we found that MKP-2 is highly expressed in medullary thyroid carcinomas derived from MEN2A patients. These findings suggest that the increased expression of MKP-2 may play a crucial role in oncogenic signaling downstream of mutant RET, leading to deregulation of cell cycle.

title: Multi-stage chemical carcinogenesis in mouse skin: Fundamentals and applications

text: For more than 60 years, the chemical induction of tumors in mouse skin has been used to study mechanisms of epithelial carcinogenesis and evaluate modifying factors. In the traditional two-stage skin carcinogenesis model, the initiation phase is accomplished by the application of a sub-carcinogenic dose of a carcinogen. Subsequently, tumor development is elicited by repeated treatment with a tumor-promoting agent. The initiation protocol can be completed within 1–3 h depending on the number of mice used; whereas the promotion phase requires twice weekly treatments (1–2 h) and once weekly tumor palpation (1–2 h) for the duration of the study. Using the protocol described here, a highly reproducible papilloma burden is expected within 10–20 weeks with progression of a portion of the tumors to squamous cell carcinomas within 20–50 weeks. In contrast to complete skin carcinogenesis, the two-stage model allows for greater yield of premalignant lesions, as well as separation of the initiation and promotion phases.

title: Epigenetic-induced repression of microRNA-205 is associated with MED1 activation and a poorer prognosis in localized prostate cancer

text: Deregulation of microRNA (miRNA) expression can have a critical role in carcinogenesis. Here we show in prostate cancer that miRNA-205 (miR-205) transcription is commonly repressed and the MIR-205 locus is hypermethylated. LOC642587, the MIR-205 host gene of unknown function, is also concordantly inactivated. We show that miR-205 targets mediator 1 (MED1, also called TRAP220 and PPARBP) for transcriptional silencing in normal prostate cells, leading to reduction in MED1 mRNA levels, and in total and active phospho-MED1 protein. Overexpression of miR-205 in prostate cancer cells negatively affects cell viability, consistent with a tumor suppressor function. We found that hypermethylation of the MIR-205 locus was strongly related with a decrease in miR-205 expression and an increase in MED1 expression in primary tumor samples (n=14), when compared with matched normal prostate (n=7). An expanded patient cohort (tumor n=149, matched normal n=30) also showed significant MIR-205 DNA methylation in tumors compared with normal, and MIR-205 hypermethylation is significantly associated with biochemical recurrence (hazard ratio=2.005, 95% confidence interval (1.109, 3.625), P=0.02), in patients with low preoperative prostate specific antigen. In summary, these results suggest that miR-205 is an epigenetically regulated tumor suppressor that targets MED1 and may provide a potential biomarker in prostate cancer management.

title: Stromal p16 expression differentiates endometrial polyp from endometrial hyperplasia

text: Endometrial polyps are very common benign endometrial lesions, but their pathogenesis is poorly understood, except for a few studies indicating the possibility of benign stromal neoplasm. Although the histopathological diagnosis of endometrial polyp on a surgical specimen is straightforward, it is often difficult to differentiate endometrial polyp from endometrial hyperplasia on a biopsy or curettage specimen. Presently, there is no immunohistochemical marker helpful in this differential diagnosis. In this study, we examined p16 expression in 35 endometrial polyps and 33 cases of endometrial hyperplasia that included 16 simple hyperplasias, 14 complex atypical hyperplasias, and 3 complex hyperplasias without atypia. Stromal p16 expression differed significantly between the two groups; it was seen in 31 (89 %) endometrial polyps, but in only 1 (3 %) endometrial hyperplasia. The percentage of p16-positive stromal cells ranged from 10 to 90 % (mean, 47 %) and the positive cells tended to be distributed around glands. Six cases of endometrial hyperplasia within an endometrial polyp were also examined and all cases showed stromal p16 expression. There was no difference in glandular p16 expression between endometrial polyps 33 (94 %) and hyperplasia 27 (82 %). The p16-immunoreactivity was mostly confined to metaplastic epithelial cells in both groups. Stromal p16 expression might be a peculiar characteristic of endometrial polyp and constitute a useful marker for the diagnosis, especially in fragmented specimens from biopsy or curettage. Stromal p16 expression might be a reflection of p16-induced cellular senescence, which has been documented in several benign mesenchymal neoplasms.

title: MicroRNAs: new tools for diagnosis, prognosis, and therapy in hepatocellular carcinoma?

text: MicroRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs involved in the regulation of gene expression and protein translation. Many studies have shown that they play a crucial role in driving organ and tissue differentiation during embryogenesis and in the fine-tuning of fundamental biological processes, such as proliferation and apoptosis. Growing evidence indicates that their deregulation plays an important role in cancer onset and progression as well, where they act as oncogenes or oncosuppressors. In this review, we highlight the most recent findings regarding the role of miRNAs in hepatocellular carcinoma (HCC) by analyzing the possible mechanisms by which they contribute to this neoplasm. Moreover, we discuss the possible role of circulating miRNAs as biomarkers, a field that needs urgent improvement in the clinical surveillance of HCC, and the fascinating possibility of using them as therapeutic targets or drugs themselves.

title: Liver receptor homolog-1 is essential for pregnancy

text: Successful pregnancy requires coordination of an array of signals and factors from multiple tissues. One such element, liver receptor homolog-1 (Lrh-1), is an orphan nuclear receptor that regulates metabolism and hormone synthesis. It is strongly expressed in granulosa cells of ovarian follicles and in the corpus luteum of rodents and humans. Germline ablation of Nr5a2 (also called Lrh-1), the gene coding for Lrh-1, in mice is embryonically lethal at gastrulation. Depletion of Lrh-1 in the ovarian follicle shows that it regulates genes required for both steroid synthesis and ovulation. To study the effects of Lrh-1 on mouse gestation, we genetically disrupted its expression in the corpus luteum, resulting in luteal insufficiency. Hormone replacement permitted embryo implantation but was followed by gestational failure with impaired endometrial decidualization, compromised placental formation, fetal growth retardation and fetal death. Lrh-1 is also expressed in the mouse and human endometrium, and in a primary culture of human endometrial stromal cells, reduction of NR5A2 transcript abundance by RNA interference abrogated decidualization. These findings show that Lrh-1 is necessary for maintenance of the corpus luteum, for promotion of decidualization and for formation of the placenta. It therefore has multiple, indispensible roles in establishing and sustaining pregnancy.

title: LncRNA-GAS5 induces PTEN expression through inhibiting miR-103 in endometrial cancer cells

text:
BACKGROUND Growth arrest-specific 5 (GAS5) was reported to be implicated and aberrantly express in multiple cancers. However, the expression and mechanism of action of GAS5 were largely poor understood in endometrial carcinoma.
 RESULTS According to the result of real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and flow cytometry analysis, we identified that GAS5 was down-regulated in endometrial cancer cells and stimulated the apoptosis of endometrial cancer cells. To investigate the expression of GAS5, PTEN and miR-103, RT-PCR was performed. And we found that the expression of PTEN was up-regulated when endometrial cancer cells overexpressed GAS5. The prediction of bioinformatics online revealed that GAS5 could bind to miR-103, which was further found to be regulated by GAS5. Finally, we found that miR-103 mimic could decrease the mRNA and protein levels of PTEN through luciferase reporter assay and western blotting, and GAS5 plasmid may reverse this regulation effect in endometrial cancer cells.
 CONCLUSION In summary, we demonstrate that GAS5 acts as an tumor suppressor lncRNA in endometrial cancer. Through inhibiting the expression of miR-103, GAS5 significantly enhanced the expression of PTEN to promote cancer cell apoptosis, and, thus, could be an important mediator in the pathogenesis of endometrial cancer.

title: Prediagnostic levels of C-peptide, IGF-I, IGFBP -1, -2 and -3 and risk of endometrial cancer.

text: Conditions related to chronic hyperinsulinemia, such as obesity, noninsulin dependent diabetes mellitus and polycystic ovary syndrome, are associated with an increased risk of endometrial cancer. Elevated plasma IGF-I and decreased levels of IGF-binding proteins have been shown to be associated with increased risk of several cancer types that are frequent in affluent societies. We investigated for the first time in a prospective study the association of pre-diagnostic blood concentrations of C-peptide (a marker of pancreatic insulin production), IGF-I, IGFBP-1, -2 and -3 with endometrial cancer risk. A case-control study was nested within 3 cohorts in New York (USA), Umeå (Sweden) and Milan (Italy). It included 166 women with primary invasive endometrial cancer and 315 matched controls, of which 44 case and 78 control subjects were premenopausal at recruitment. Endometrial cancer risk increased with increasing levels of C-peptide (ptrend = 0.0002), up to an odds ratio (OR) of 4.76 [95% confidence interval (CI) = 1.91-11.8] for the highest quintile. This association remained after adjustment for BMI and other confounders [OR for the top quintile = 4.40 (1.65-11.7)]. IGFBP-1 levels were inversely related to endometrial cancer [ptrend = 0.002; OR in the upper quintile = 0.30 (0.15-0.62)], but the association was weakened and lost statistical significance after adjustment for confounders [ptrend = 0.06; OR in the upper quintile = 0.49 (0.22-1.07)]. Risk was unrelated to levels of IGF-I, IGFBP-2 and IGFBP-3. Chronic hyperinsulinemia, as reflected by increased circulating C-peptide, is associated with increased endometrial cancer risk. Decrease in the prevalence of chronic hyperinsulinemia, through changes in lifestyle or medication, is expected to prevent endometrial cancer.

title: The nuclear receptors SF1 and LRH1 are expressed in endometrial cancer cells and regulate steroidogenic gene transcription by cooperating with AP-1 factors.

text: Excessive exposure to estradiol represents the main risk factor for endometrial cancer. The abnormally high estradiol levels in the endometrium of women with endometrial cancer are most likely due to overproduction by the tumour itself. Endometrial cancer cells express the genes encoding the steroidogenic enzymes involved in estradiol synthesis. Here we used RT-PCR and Western blot to show that the nuclear receptors SF1 and LRH1, two well-known regulators of steroidogenic gene expression in gonadal and adrenal cells, are also expressed in endometrial cancer cell lines. By transient transfections, we found that SF1 and LRH1, but not the related nuclear receptor NUR77, can activate the promoters of three human steroidogenic genes: STAR, HSD3B2, and CYP19A1 PII. Similarly, forskolin but not PMA, could activate all three promoters. In addition, we found that both SF1 and LRH1 can transcriptionally cooperate with the AP-1 family members c-JUN and c-FOS, known to be associated with enhanced proliferation of endometrial carcinoma cells, to further enhance activation of the STAR, HSD3B2, and CYP19A1 PII promoters. All together, our data provide novel insights into the mechanisms of steroidogenic gene expression in endometrial cancer cells and thus in the regulation of estradiol biosynthesis by tumour cells.

title: Deregulation of Scribble Promotes Mammary Tumorigenesis and Reveals a Role for Cell Polarity in Carcinoma

text: Loss of cell polarity proteins such as Scribble induces neoplasia in Drosophila by promoting uncontrolled proliferation. In mammals, the role that polarity proteins play during tumorigenesis is not well understood. Here, we demonstrate that depletion of Scribble in mammary epithelia disrupts cell polarity, blocks three-dimensional morphogenesis, inhibits apoptosis, and induces dysplasia in vivo that progress to tumors after long latency. Loss of Scribble cooperates with oncogenes such as c-myc to transform epithelial cells and induce tumors in vivo by blocking activation of an apoptosis pathway. Like depletion, mislocalization of Scribble from cell-cell junction was sufficient to promote cell transformation. Interestingly, spontaneous mammary tumors in mice and humans possess both downregulated and mislocalized Scribble. Thus, we demonstrate that scribble inhibits breast cancer formation and that deregulation of polarity pathways promotes dysplastic and neoplastic growth in mammals by disrupting morphogenesis and inhibiting cell death.

title: Large-scale hypomethylated blocks associated with Epstein-Barr virus-induced B-cell immortalization.

text: Altered DNA methylation occurs ubiquitously in human cancer from the earliest measurable stages. A cogent approach to understanding the mechanism and timing of altered DNA methylation is to analyze it in the context of carcinogenesis by a defined agent. Epstein-Barr virus (EBV) is a human oncogenic herpesvirus associated with lymphoma and nasopharyngeal carcinoma, but also used commonly in the laboratory to immortalize human B-cells in culture. Here we have performed whole-genome bisulfite sequencing of normal B-cells, activated B-cells, and EBV-immortalized B-cells from the same three individuals, in order to identify the impact of transformation on the methylome. Surprisingly, large-scale hypomethylated blocks comprising two-thirds of the genome were induced by EBV immortalization but not by B-cell activation per se. These regions largely corresponded to hypomethylated blocks that we have observed in human cancer, and they were associated with gene-expression hypervariability, similar to human cancer, and consistent with a model of epigenomic change promoting tumor cell heterogeneity. We also describe small-scale changes in DNA methylation near CpG islands. These results suggest that methylation disruption is an early and critical step in malignant transformation.

title: Stathmin overexpression identifies high-risk patients and lymph node metastasis in endometrial cancer.

text:
PURPOSE Overexpression of the oncogen Stathmin has been linked to aggressive endometrial carcinoma and a potential for PI3Kinase inhibitors in this disease. We wanted to validate the prognostic value of Stathmin expression in a large prospective multicenter setting. As lymph node sampling is part of current surgical staging, we also aimed to test if Stathmin expression in endometrial curettage specimens could predict lymph node metastasis.    EXPERIMENTAL DESIGN A total of 1,076 endometrial cancer patients have been recruited from 10 centers to investigate the biological tumor marker Stathmin in relation to clinicopathologic variables, including lymph node status and survival. Stathmin immunohistochemical staining was carried out in 477 hysterectomy and 818 curettage specimens.
 RESULTS Seventy-one percent of the patients (n = 763) were subjected to lymph node sampling, of which 12% had metastatic nodes (n = 94). Overexpression of Stathmin was detected in 37% (302 of 818) of the curettage and in 18% (84 of 477) of the hysterectomy specimens investigated. Stathmin overexpression in curettage and hysterectomy specimens were highly correlated and significantly associated with nonendometrioid histology, high grade, and aneuploidy. Stathmin analysis in preoperative curettage samples significantly correlated with, and was an independent predictor of, lymph node metastases. High Stathmin expression was associated with poor disease-specific survival (P ≤ 0.002) both in curettage and hysterectomy specimens.
 CONCLUSIONS Stathmin immunohistochemical staining identifies endometrial carcinomas with lymph node metastases and poor survival. The value, as a predictive marker for response to PI3Kinase inhibition and as a tool to stratify patients for lymph node sampling in endometrial carcinomas, remains to be determined.

title: Concurrent endometrial carcinoma in women with a biopsy diagnosis of atypical endometrial hyperplasia: a Gynecologic Oncology Group study.

text:
BACKGROUND Adenocarcinoma of the endometrium is the most common gynecologic malignancy in the United States, accounting for approximately 36,000 diagnoses of invasive carcinoma annually. The most common histologic type, endometrioid adenocarcinoma (EC), accounts for 75-80% of patients. The objective of this work was to estimate the prevalence of concurrent carcinoma in women with a biopsy diagnosis of the precursor lesion, atypical endometrial hyperplasia (AEH).
 METHODS This prospective cohort study included women who had a community diagnosis of AEH. Diagnostic biopsy specimens were reviewed independently by three gynecologic pathologists who used International Society of Gynecologic Pathologists/World Health Organization criteria. Study participants underwent hysterectomy within 12 weeks of entry onto protocol without interval treatment. The hysterectomy slides also were reviewed by the study pathologists, and their findings were used in the subsequent analyses.
 RESULTS Between November 1998 and June 2003, 306 women were enrolled on the study. Of these, 17 women were not included in the analysis: Two patients had unreadable slides because of poor processing or insufficient tissue, 2 patients had only slides that were not endometrial, the slides for 5 patients were not available for review, and 8 of the hysterectomy specimens were excluded because they showed evidence of interval intervention, either progestin effect or ablation. In total, 289 patients were included in the current analysis. The study panel review of the AEH biopsy specimens was interpreted as follows: 74 of 289 specimens (25.6%) were diagnosed as less than AEH, 115 of 289 specimens (39.8%) were diagnosed as AEH, and 84 of 289 specimens (29.1%) were diagnosed as endometrial carcinoma. In 5.5% (16 of 289 specimens), there was no consensus on the biopsy diagnosis. The rate of concurrent endometrial carcinoma for analyzed specimens was 42.6% (123 of 289 specimens). Of these, 30.9% (38 of 123 specimens) were myoinvasive, and 10.6% (13 of 123 specimens) involved the outer 50% of the myometrium. Among the women who had hysterectomy specimens with carcinoma, 14 of 74 women (18.9%) had a study panel biopsy consensus diagnosis of less than AEH, 45 of 115 women (39.1%) had a study panel biopsy consensus diagnosis of AEH, and 54 of 84 women (64.3%) had a study panel diagnosis of carcinoma. Among women who had no consensus in their biopsy diagnosis, 10 of 16 women (62.5%) had carcinoma in their hysterectomy specimens.
 CONCLUSIONS The prevalence of endometrial carcinoma in patients who had a community hospital biopsy diagnosis of AEH was high (42.6%). When considering management strategies for women who have a biopsy diagnosis of AEH, clinicians and patients should take into account the considerable rate of concurrent carcinoma.

title: Increased susceptibility to UV-induced skin carcinogenesis in polymerase eta-deficient mice.

text: Xeroderma pigmentosum variant (XPV) patients with mutations in the DNA polymerase eta (pol eta) gene are hypersensitive to sunlight and have greatly increased susceptibility to sunlight-induced skin cancer. Consistent with the ability of Pol eta to efficiently bypass UV light-induced cyclobutane pyrimidine dimers, XPV cells lacking Pol eta have diminished capacity to replicate UV-damaged DNA and are sensitive to UV light-induced killing and mutagenesis. To better understand these and other Pol eta functions, we generated Pol eta-deficient mice. Mice homozygous for a null mutation in pol eta are viable, fertile, and do not show any obvious spontaneous defects during the first year of life. However, fibroblasts derived from these mutant mice are sensitive to killing by exposure to UV light, and all Pol eta-deficient mice develop skin tumors after UV irradiation, in contrast to the wild-type littermate controls that did not develop such tumors. These results and biochemical studies of translesion synthesis by mouse Pol eta indicate that Pol eta-dependent bypass of cyclobutane pyrimidine dimers suppresses UV light-induced skin cancer in mice. Moreover, 37.5% of pol eta heterozygous mice also developed skin cancer during 5 months after a 5-month exposure to UV light, suggesting that humans who are heterozygous for mutations in pol eta may also have an increased risk of skin cancer.

title: KRAS gene amplification and overexpression but not mutation associates with aggressive and metastatic endometrial cancer

text:
BACKGROUND Three quarter of endometrial carcinomas are treated at early stage. Still, 15 to 20% of these patients experience recurrence, with little effect from systemic therapies. Homo sapiens v-Ki-ras2 Kirsten rat sarcoma viral oncogenes homologue (KRAS) mutations have been reported to have an important role in tumorigenesis for human cancers, but there is limited knowledge regarding clinical relevance of KRAS status in endometrial carcinomas.
 METHODS We have performed a comprehensive and integrated characterisation of genome-wide expression related to KRAS mutations and copy-number alterations in primary- and metastatic endometrial carcinoma lesions in relation to clinical and histopathological data. A primary investigation set and clinical validation set was applied, consisting of 414 primary tumours and 61 metastatic lesions totally.
 RESULTS Amplification and gain of KRAS present in 3% of the primary lesions and 18% of metastatic lesions correlated significantly with poor outcome, high International Federation of Gynaecology and Obstetrics stage, non-endometrioid subtype, high grade, aneuploidy, receptor loss and high KRAS mRNA levels, also found to be associated with aggressive phenotype. In contrast, KRAS mutations were present in 14.7% of primary lesions with no increase in metastatic lesions, and did not influence outcome, but was significantly associated with endometrioid subtype, low grade and obesity.
 CONCLUSION These results support that KRAS amplification and KRAS mRNA expression, both increasing from primary to metastatic lesions, are relevant for endometrial carcinoma disease progression.