Previous studies indicate unrestrained cell cycle progression in liver lesions from hepatocarcinogenesis-susceptible Fisher 344 (F344) rats and a block of G(1)-S transition in corresponding lesions ...from resistant Brown Norway (BN) rats. Here, the role of the Forkhead box M1B (FOXM1) gene during hepatocarcinogenesis in both rat models and human hepatocellular carcinoma (HCC) was assessed.
Levels of FOXM1 and its targets were determined by immunoprecipitation and real-time PCR analyses in rat and human samples. FOXM1 function was investigated by either FOXM1 silencing or overexpression in human HCC cell lines. Activation of FOXM1 and its targets (Aurora Kinose A, Cdc2, cyclin B1, Nek2) occurred earlier and was most pronounced in liver lesions from F344 than BN rats, leading to the highest number of Cdc2-cyclin B1 complexes (implying the highest G(2)-M transition) in F344 rats. In human HCC, the level of FOXM1 progressively increased from surrounding non-tumorous livers to HCC, reaching the highest levels in tumours with poorer prognosis (as defined by patients' length of survival). Furthermore, expression levels of FOXM1 directly correlated with the proliferation index, genomic instability rate and microvessel density, and inversely with apoptosis. FOXM1 upregulation was due to extracellular signal-regulated kinase (ERK) and glioblastoma-associated oncogene 1 (GLI1) combined activity, and its overexpression resulted in increased proliferation and angiogenesis and reduced apoptosis in human HCC cell lines. Conversely, FOXM1 suppression led to decreased ERK activity, reduced proliferation and angiogenesis, and massive apoptosis of human HCC cell lines.
FOXM1 upregulation is associated with the acquisition of a susceptible phenotype in rats and influences human HCC development and prognosis.
The DNA-repair gene DNA-dependent kinase catalytic subunit (DNA-PKcs) favours or inhibits carcinogenesis, depending on the cancer type. Its role in human hepatocellular carcinoma (HCC) is unknown.
...DNA-dependent protein kinase catalytic subunit, H2A histone family member X (H2AFX) and heat shock transcription factor-1 (HSF1) levels were assessed by immunohistochemistry and/or immunoblotting and qRT-PCR in a collection of human HCC. Rates of proliferation, apoptosis, microvessel density and genomic instability were also determined. Heat shock factor-1 cDNA or DNA-PKcs-specific siRNA were used to explore the role of both genes in HCC. Activator protein 1 (AP-1) binding to DNA-PKcs promoter was evaluated by chromatin immunoprecipitation. Kaplan-Meier curves and multivariate Cox model were used to study the impact on clinical outcome.
Total and phosphorylated DNA-PKcs and H2AFX were upregulated in HCC. Activated DNA-PKcs positively correlated with HCC proliferation, genomic instability and microvessel density, and negatively with apoptosis and patient's survival. Proliferation decline and massive apoptosis followed DNA-PKcs silencing in HCC cell lines. Total and phosphorylated HSF1 protein, mRNA and activity were upregulated in HCC. Mechanistically, we demonstrated that HSF1 induces DNA-PKcs upregulation through the activation of the MAPK/JNK/AP-1 axis.
DNA-dependent protein kinase catalytic subunit transduces HSF1 effects in HCC cells, and might represent a novel target and prognostic factor in human HCC.
Abstract Background: Recent lines of research have boosted awareness of the immunological facets of schizophrenia. However, associations with protein tyrosine phosphatase regulators have never been ...reported. The aim of our study was to investigate the expression and promoter status methylation of phosphatase SHP-1, a key negative regulator of the inflammatory process, in Peripheral blood mononuclear cells (PBMCs) of Schizophrenic patients. Methods: We enrolled fifty-four (28 men and 26 women) unmedicated first episode subjects (SC) who met DSM-IV and thirty-eight (22 men and 16 women) healthy controls (HC). The SC psychopathological status was assessed using the Positive and Negative Syndrome Scale. We evaluated SHP-1 expression by Quantitative Real-time PCR (qPCR) and Western blotting (WB) methods and promoter status methylation through PCR bisulfate. IKK/NFkB signaling was detected by WB, and medium and plasma levels of pro-inflammatory cytokines (IL-1β, IL-2, and TNF-α) by the ELISA method. SHP-1 was silenced by treating cells with specific siRNA. Results: We found a significantly lower level of SHP-1 gene expression in PBMCs from SC vs. HC, consistently with which the promoter region analyzed presented significant hypermethylation. Silencing of SHP-1 expression induced higher activation of IKK/NF-kB signaling and pro-inflammatory cytokine production in ex vivo PBMCs from both SC and HC. Linear regression among patients generated a model in which SHP-1 expression explained 30% of the clinical negative symptom variance (adjusted R (2) = 0.30, ANOVA p < 0.001). Conclusions: Our findings are the first to suggest that impairment of SHP-1 expression is involved in the physiopathology of schizophrenia, opening fruitful new avenues for ameliorating treatment at least of negative symptoms.
Background & Aims: Although the natural history and pathologic characteristics of human hepatocellular carcinoma (HCC) are well documented, the molecular pathogenesis of HCC remains poorly ...understood. Here, we define the role for Ras and Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathways in human HCC. Methods: Promoter and genomic status of Ras and Jak/Stat inhibitors were assessed in 80 HCCs by methylation-specific polymerase chain reaction and microsatellite analysis. Activation of Ras and Jak/Stat signaling pathways was determined by DNA sequencing, Western blot, and immunoprecipitation analysis. Suppression of Ras and Jak/Stat pathways in HCC cell lines was evaluated by viability and apoptosis assays. Results: Activation of Ras and Jak/Stat pathways was enhanced in all HCCs when compared with nonneoplastic surrounding and normal livers coincidently with the suppression of at least 1 Ras (RASSF1A and/or NORE1A) and 2 Jak/Stat inhibitors (cytokine-inducible SH2-protein CIS; suppressor of cytokine signaling SOCS1, 2, 3; and SH2-containing phosphatases SHP1). HCC associated with cirrhosis showed significantly higher frequency of RASSF1A, CIS, and SOCS1 promoter methylation than HCC without cirrhosis (P < .002, P < .02, and P < .02, respectively). Furthermore, aberrant methylation of NORE1A and SOCS3 promoters was observed only in a subclass of HCC with poor survival, suggesting that inactivation of these 2 genes might be involved in HCC progression. Combined treatment of HCC cell lines with Ras and Jak/Stat inhibitors as well as with the demethylating agent zebularine induced a strong apoptotic response. Conclusions: These data demonstrate the ubiquitous activation of Ras and Jak/Stat pathways in HCC and suggest the potential use of Ras and Jak/Stat inhibitors and demethylating agents as therapeutic modality for human liver cancer.
Certain antidepressant drugs exert an anxiolytic action in both humans and rodents. The effects of long-term treatment with imipramine or mirtazapine, two antidepressant drugs with different ...mechanisms of action, on the response of cortical cholinergic neurons to foot-shock stress or to the anxiogenic drug FG 7142 were investigated in freely moving rats. Chronic treatment with imipramine or mirtazapine reduced the increase in cortical acetylcholine output induced by foot-shock stress by ∼50%. The same treatment also reduced the sensitivity of cortical cholinergic neurons to the stimulatory effect of acute administration of FG 7142. In contrast, the administration of a single dose of either antidepressant 40
min before foot shock or FG 7142 injection failed to increase the threshold of excitability of cortical cholinergic neurons. These results demonstrate that long-term treatment with either imipramine or mirtazapine reduces the sensitivity of cortical cholinergic neurons to stress or to an anxiogenic drug with an efficacy similar to that of acute administration of benzodiazepines. The neurochemical mechanism responsible for regulation of cholinergic neuron sensitivity might contribute to the modulation of cognitive function associated with emotional and affective disorders.
Previously, we showed that activation of the β-catenin/Wnt pathway is a dominant event during c-Myc/E2F1 hepatocarcinogenesis. Majority of c-Myc/E2F1 HCCs displayed nuclear accumulation of β-catenin ...in the absence of β-catenin mutations, suggesting that alterations in other members of the Wnt pathway might be responsible for nuclear localization of β-catenin. Here, we investigated the mechanisms responsible for nuclear translocation of wild-type β-catenin and addressed the potential contribution of the Wnt pathway in c-Myc/E2F1 hepatocarcinogenesis.
Status of the members of the Wnt pathway was determined through microsatellite and Western blot analysis.
Majority of c-Myc/E2F1 HCCs exhibited multiple abnormalities in the Wnt pathway regardless of the presence of β-catenin mutations. The observed abnormalities included overexpression of Wnt-1, Frizzled 1 and 2 receptors, Dishevelled-1, downregulation of Secreted frizzled-related protein-1, GSK-3β inactivation, microsatellite instability at the Axin locus as well as induction of β-catenin target genes, such as glutamine synthetase, glutamate transporter-1, and Wisp-1. HCCs with β-catenin activation displayed significantly higher proliferation rate and larger tumor size when compared with β-catenin negative tumors.
The data demonstrate that multiple abnormalities in the members of the Wnt pathway lead to nuclear accumulation of β-catenin and suggest that activation of Wnt pathway provides proliferative advantages in c-Myc/E2F1-driven hepatocarcinogenesis.
Co-expression of c-Myc and TGF-α in the mouse liver accelerates hepatocarcinogenesis and enhances DNA damage due to chronic oxidative stress. Dietary supplementation with vitamin E (VE) inhibits ...hepatocarcinogenesis and reduces chromosomal alterations in the same mice. Here we investigated the sources of reactive oxygen species (ROS) production in c-Myc/TGF-α transgenic mice.
Inducible nitric oxide synthase (iNOS) and NADPH oxidase levels were determined in c-Myc, TGF-α and c-Myc/TGF-α mice by RT-PCR, western blot analysis and immunohistochemistry.
iNOS and nitrotyrosines levels were higher in the three transgenic lines when compared with wild-type mice. Preneoplastic and neoplastic lesions from c-Myc, TGF-α and c-Myc/TGF-α transgenic mice displayed upregulation of NADPH oxidase subunits p47-, 67-phox, Rac1, HSP 70, and HO-1. Importantly, dietary supplementation with vitamin E abolished iNOS expression, lowered nitrotyrosines, p47-, p67-phox, and Rac1 levels, and suppressed HSP 70 and HO-1 proteins in c-Myc/TGF-α livers.
The results suggest that iNOS and NADPH oxidase are involved in ROS generation during c-Myc/TGF-α hepatocarcinogenesis and are inhibited by VE treatment. The data provide additional evidence for the potential use of VE in treatment of chronic liver diseases and HCC prevention.
Background & Aims
: Human liver cancer can be divided into 2 categories that are characterized by activation of β-catenin and genomic instability. Here we investigate whether similar categories exist ...among 5 transgenic models of liver cancer, including c-myc, transforming growth factor-α, E2F-1, c-myc/transforming growth factor-α, and c-myc/E2F-1 mice.
Methods
: The random amplified polymorphic DNA method was used to assess the overall genomic instability, and chromosomal loci affected by genomic alterations were determined by microsatellite analysis. β-Catenin mutations and deletions were analyzed by polymerase chain reaction and sequencing screening. Cellular localization of β-catenin and expression of α-fetoprotein, a prognostic marker of hepatocellular carcinoma, were investigated by immunohistochemistry.
Results
: Liver tumors from the transgenic mice could be divided into 2 broad categories characterized by extensive genomic instability (exemplified by the c-myc/transforming growth factor-α mouse) and activation of β-catenin (exemplified by the c-myc/E2F-1 mouse). The c-myc/transforming growth factor-α tumors displayed extensive genomic instability with recurrent loss of heterozygosity at chromosomes 1, 2, 4, 6, 7, 9, 12, 14, and X and a low rate of β-catenin activation. The genomic instability was evident from the early dysplastic stage and occurred concomitantly with increased expression of α-fetoprotein. The c-myc/E2F-1 tumors were characterized by a high frequency of β-catenin activation in the presence of a relatively stable genome and low α-fetoprotein levels.
Conclusions
: We have identified 2 prototype experimental models, i.e., c-myc/transforming growth factor-α and c-myc/E2F-1 mice, for the 2 categories of human hepatocellular carcinoma characterized by genomic instability and β-catenin activation, respectively. These mouse models will assist in the elucidation of the molecular basis of human hepatocellular carcinoma.
E-cadherin is a cell–cell adhesion molecule that plays a pivotal role in the development and maintenance of cell polarity. Disruption of E-cadherin-mediated adhesion represents a key step toward the ...invasive phenotype in a variety of solid tumors, including hepatocellular carcinoma (HCC). Here, we investigate whether deregulation of E-cadherin occurs along the multistep process of hepatocarcinogenesis in transgenic mouse models, including c-Myc, E2F1, c-Myc/TGF-α and c-Myc/E2F1 mice. Liver tumors from the transgenic mouse lines could be divided into two categories based on E-cadherin levels. Of 28, 20 (71.4%) c-Myc HCCs showed marked reduction of E-cadherin expression when compared with wild-type livers. In contrast, all of c-Myc/TGF-α and the majority of E2F1 and c-myc/E2F1 preneoplastic and neoplastic lesions exhibited overexpression of E-cadherin. Downregulation of E-cadherin was associated with promoter hypermethylation in seven of 20 c-Myc HCCs (35%), while no loss of heterozygosity at the E-cadherin locus was detected. Nuclear accumulation of β-catenin did not correlate with E-cadherin downregulation. Furthermore, c-Myc HCCs with reduced E-cadherin displayed upregulation of hypoxia-inducible factor-1α and vascular endothelial growth factor proteins. Importantly, loss of E-cadherin was associated with increased cell proliferation and higher microvessel density in c-Myc tumors. Taken together, these data suggest that loss of E-cadherin might favor tumor progression in relatively more benign HCC from c-Myc transgenic mice by stimulating neoplastic proliferation and angiogenesis under hypoxic conditions.
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![[351] ROLE OF PERTURBATION ...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/0168-8278_1600-0641/small "[351] ROLE OF PERTURBATION OF METHIONINE METABOLISM AND GLOBAL DNA METHYLATION IN THE GENERATION OF GENOMIC INSTABILITY IN TRANSGENIC MOUSE MODELS OF LIVER CANCER")
