Loss of clusterin both in serum and tissue correlates with the tumorigenesis of esophageal squamous cell carcinoma via proteomics approaches

AIM: To identify the differentially secreted proteins or polypeptides associated with tumorigenesis of esophageal squamous cell carcinoma (ESCC) from serum and to find potential tumor secreted biomarkers.METHODS: Proteins from human ESCC tissue and its matched adjacent normal tissue; pre-surgery and postsurgery serum; and pre-surgery and normal control serum were separated by two-dimensional electrophoresis (2-DE)to identify differentially expressed proteins. The silverstained 2-DE were scanned with digital ImageScanner and analyzed with ImageMaster 2D Elite 3.10 software. A cluster of protein spots differentially expressed were selected and identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). One of the differentially expressed proteins, clusterin, was downregulated in cancer tissue and pre-surgery serum, but it was reversed in post-surgery serum. The results were confirmed by semi-quantitative reverse-transcription (RT)-PCR and western blot.RESULTS: Comparisons of the protein spots identified on the 2-DE maps from human matched sera showed that some proteins were differentially expressed, with most of them showing no differences in composition, shape or density.Being analyzed by MALDI-TOF-MS and database searching,clusterin was differentially expressed and down-regulated in both cancer tissue and pre-surgery serum compared with their counterparts. The results were also validated by RTPCR and western blot.CONCLUSION: The differentially expressed clusterin may play a key role during tumorigenesis of ESCC. The 2DE-MS based proteomic approach is one of the powerful tools for discovery of secreted markers from peripheral.

Expression of MRP14 gene is frequently down-regulated in Chinese human esophageal cancer

Migration inhibitory factor-related protein 14 (MRP 14) is one of calcium-binding proteins,referred as S 100A9. The heterodimeric molecule formed by MRP 14 with its partner MRP8 (S 100A8) is the major fatty acid carrier in neutrophils.The MRP8/14 complex has been also implicated in the intracellular transport of arachidonic acid and its precursors in keratinocytes. We show here the involvement of MRP14 in human esophageal cancer. In an initial study,mRNA differential display - reverse transcription polymerase chain reaction (DD-PCR) was performed with two esophageal carcinomas,one esophageal adenocarcinoma and matched normal adjacent mucosa. DD-PCR with the arbitrary primer OPA3 showed that one cDNA band was highly expressed in normal tissues,but disappeared or substantially decreased in tumor counterparts. It was later identified to be the 3'-end of migration inhibitory factor-related protein 14 (MRP14).Northern blotting,RT-PCR and Western blotting corroborated the down-regulation of MRP14 in 58/64 squamous cell carcinomas and 2/2 adenocarcinomas as compared with adjacent normal epithelia of the esophagus. MRP14 was undetectable in 3/3 esophageal-carcinoma cell lines. Immunochemistry demonstrated that expression of MRP14 was restricted to normal esophageal epithelia. No mutation was found in the genomic DNA of the MRP14 gene by PCR and directed DNA sequencing. Our finding suggested that the reduction of MRP14 expression is a frequent event in Chinese human esophageal cancer.

Translocation of annexin I from cellular membrane to the nuclear membrane in human esophageal squamous cell carcinoma

AIM: To investigate the alteration of the annexin I subcellular localization in esophageal squarnous cell carcinoma (ESCC)and the correlation between the translocation and the tumorigenesis of ESCC.METHODS: The protein localization of annexin I was detected in both human ESCC tissues and cell line via the indirect immunofluorescence strategy.RESULTS: In the normal esophageal epithelia the annex in I was mainly located on the plasma membrane and formed a consecutive typical trammels net. Annexin I protein also expressed dispersively in cytoplasm and the nuclei without specific localization on the nuclear membrane. In esophageal cancer annexin I decreased very sharply with scattered disappearance on the cellular membrane, however it translocated and highly expressed on the nuclear membrane,which was never found in normal esophageal epithelia. In cultured esophageal cancer cell line annexin I protein was also focused on the nuclear membrane, which was consistent with the result from esophageal cancer tissues.CONCLUSION: This observation suggests that the translocation of annexin I protein in ESCC may correlate with the tumorigenesis of the esophageal cancer.

Enhancement of germ cell apoptosis induced by ethanol in transgenic mice overexpressing Fas Ligand

It was suggested that chronic ethanol exposure could result in testicular germ cell apoptosis, but the mechanism is still unclear. In the present study, we use a model of transgenic mice ubiquitously overexpressing human FasL to investigate whether Fas ligand plays a role in ethanol-induced testicular germ cell apoptosis. Both wild-type (WT)mice and transgenic (TG) mice were treated with acute ethanol (20% v/v) by introperitoneal injection for five times.After ethanol injection, WT mice displayed up-regulation of Fas ligand in the testes, which was shown by FITCconjugated flow cytometry and western blotting. Moreover, TG mice exhibited significantly more apoptotic germ cells than WT mice did after ethanol injection, which was demonstrated by DNA fragmentation, PI staining flow cytometry and TUNEL staining. In addition, histopathological examination revealed that degenerative changes of epithelial component of the tubules occurred in FasL overexpressing transgenic mice while testicular morphology was normal in wild-type mice after acute ethanol exposure, suggesting FasL expression determines the sensitivity of testes to ethanol in mice. In summary, we provide the direct evidences that Fas ligand mediates the apoptosis of testicular germ cells induced by acute ethanol using FasL transgenic mice.

Downregulation of retinoic acid receptor-β_z expression is linked to aberrant methylation in esophageal squamous cell carcinoma cell lines

AIM:To study the role of hypermethylation in the loss of retinoic acid receptor β2(RARβ2) in esophageal squamous cell carcinoma (ESCC).METHODS:The role of hypermethylation in RARβ2 gene silencing in 6 ESCC cell lines was determined by methylation-specific PCR (MSP),and its methylation status was compared with RARβ2 mRNA expression by RT-PCR.The MSP results were confirmed by bisulfite sequencing of RARβ2promoter regions.RESULTS:Methylation was detected in 4 of the 6 cell lines,and the expression of RARβ2 was markedly downregulated in 3 of the 4 methylated cell lines. The expression of RARβ2 was restored in one RARβ2-downregulated cell line with the partial demethylation of promoter region of RARβ2 after 5-aza-2′-deoxycytidine (5-aza-dc) treatment.CONCLUSION:The methylation of the 5′ region may play an important role in the downregulation of RARβ2 in some ESCC cell lines, suggesting that multiple mechanisms contribute to the loss of RARβ2expression in ESCC cell lines.This study may have clinical applications for treatment and prevention of ESCC.

Gene expression profiles at different stages of human esophageal squamous cell carcinoma

AIM: To characterize the gene expression profiles in differentstages of carcinogenesis of esophageal epithelium.METHODS: A microarray containing 588 cancer relatedgenes was employed to study the gene expression profileat different stages of esophageal squamous cell carcinomaincluding basal cell hyperplasia, high-grade dysplasia,carcinoma in situ, early and late cancer. Principle componentanalysis was performed to search the genes which wereimportant in carcinogenesis.RESULTS: More than 100 genes were up or down regulatedin esophageal epithelial cells during the stages of basal cellhyperplasia, high-grade dysplasia, carcinoma in situ, earlyand late cancer. Principle component analysis identified aset of genes which may play important roles in the tumordevelopment. Comparison of expression profiles betweenthese stages showed that some genes, such as P160ROCK,JNK2, were activated and may play an important role inearly stages of carcinogenesis. CONCLUSION: These findings provided an esophagealcancer-specific and stage-specific expression profiles,showing that complex alterations of gene expression underliethe development of malignant phenotype of esophagealcancer cells.

Loss of myeloid-related proteins 8 and myeloid-related proteins 14 expression in human esophageal squamous cell carcinoma correlates with poor differentiation

To study the expression of myeloid-related proteins(MRP)8 and myeloid-related proteins(MRP)14 in humanesophageal squamous cell carcinoma and to investigate ifthere was any correlation between MRP8 and MRPI4expression level and histopathological grade in thesetumors.

Gene expression profiles of hepatoma cell line HLE

AIM: To investigate the global gene expression of cancer related genes in hepatoma cell line HLE using Atlas Human Cancer Array membranes with 588 well-characterized human genes related with cancer and tumor biology.METHODS: Hybridization of cDNA blotting membrane was performed with 32P-labeled cDNA probes synthesized from RNA isolated from Human hepatoma cell line HLE and noncirrhotic normal liver which was liver transplantation donor.AtlasImage, a software specific to array, was used to analyze the result. The expression pattern of some genes identified by Atlas arrays hybridization was confirmed by reverse transcription polymerase chain reaction (RT-PCR)in 24 pairs of specimens and Northern blot of 4 pairs of specimens.RESULTS: The differential expression of cell cycle/growth regulator in hepatocellular carcinoma (HCC) showed a stronger tendency toward cell proliferation with more than 1.5-fold up-regulation of Cyclin C, ERK5, ERK6, E2F-3, TFDP2 and CK4. The anti-apoptotic factors such as Akt-1 were up-regulated, whereas the promotive genes of apoptosis such as ABL2 were down-regulated. Among oncogene/tumors suppressors, SKY was down-regulated. Some genes such as Integrin beta 8, Integrin beta 7, DNA-PK, CSPCP,byglycan, Tenacin and DNA Topo were up-regulated. A number of genes, including LAR, MEK1, eps15, TDGF1,ARHGDIA were down-regulated. In general, expression of the cancer progression genes was up-regulated, while expression of anti-cancer progression genes was downregulated. These differentially expressed genes tested with RT-PCR were in consistent with cDNA array findings.CONCLUSION: Investigation of these genes in HCC is helpful in disclosing molecular mechanism of pathogenesis and progression of HCC. For the first time few genes were discovered in HCC. Further study is required for the precise relationship between the altered genes and their correlation with the pathogenesis of HCC.

Alterations in expression,proteolysis and intracellular localizations of clusterin in esophageal squamous cell carcinoma

AIM: To investigate biogenesis and intracellular Iocalizations of clusterin to elucidate the potential molecular mechanisms implicated in tumorigenesis of esophageal mucosa.METHODS: Semi-quantitative RT-PCR for multi-region alteration analysis, Western blot for different transcriptional forms and immunohistochemical staining for intracellular Iocalizations of clusterin were carried out in both tissues and cell lines of ESCC.RESULTS: The N-terminal deletions of the clusterin gene and the appearance of a 50-53 ku nuclear clusterin, an uncleaved, nonglycosylated, and disulfide-linked isoform,were the major alterations in cancer cells of esophagus.Naturally the 40 ku clusterin was located in the connective tissue of the lamina propria of epithelial mucosa and right under the basal membrane of epithelia, but it was disappeared in stromal mucosa of esophagus and the pre-matured clusterin was found positive in cancerous epithelia.CONCLUSION: The N-terminal deletion of clusterin may be essential for its alterations of biogenesis in ESCC.

Overexpression of ETS2 in human esophageal squamous cell carcinoma

AIM: To study the expression pattern of ETS2 (erythroblastosisvirus oncogene homolog 2) in human esophageal squamouscell carcinoma (ESCC).METHODS: Reverse transcription polymerase chain reaction(RT-PCR) and Northern blot were performed to examinethe expression level of ETS2 mRNA in 37 pairs of ESCCtissue samples. Western blot and immunohistochemistrywere carried out to check the expression level of ETS2 proteinin 30 pairs of ESCC tissue specimens.RESULTS: RT-PCR and Northern blot analysis showed thatETS2 mRNA upregulated in 75.7 % (28/37) examined ESCCtissues relative to matched normal tissues. From those 37cases, 14 cases were randomly selected to perform Westernblot and the results revealed that ETS2 protein overexpressedin 71.4 % (10/14) checked ESCC tissues compared with thecorresponding normal tissues. Moreover, the expressionpatterns of ETS2 protein in those 14 cases were identical tothose of ETS2 mRNA displayed by RT-PCR or Northern Blot.Immunohistochemistry analysis showed that the expressionlevel of ETS2 protein rose in 75 % (12/16) tumor epithelialcells contrasted to the normal cells. Altogether the expressionlevel of ETS2 protein increased in 73.3 % (22/30) checkedESCC tissue samples contrary to their normal counterparts.CONCLUSION: The results suggested that ETS2overexpressed in paired human ESCC tissue samples at bothmRNA and protein levels and may be associated with thetumorigenesis of esophagus.

Expression patterns of esophageal cancer deregulated genes in C57BL/6J mouse embryogenesis

To investigate the expression patterns of esophagealsquamous cell cancer deregulated genes in mid to late stagesof C57BL/6J mouse embryogenesis, and the correlationbetween these genes in embryonic development andtumorigenesis of esophageal squamous cell cancer.

Delay-dependent criteria for the robust stability of systems with time-varying delay

The problem of delay-dependent robust stability for systems with time-varying delay has been considered. By using the S-procedure and the Park's inequality in the recent issue, a delay-dependent robust stability criterion which is less conservative than the previous results has been derived for time-delay systems with time-varying structured uncertainties. The same idea has also been easily extended to the systems with nonlinear perturbations. Numerical examples illustrated the effectiveness and the improvement of the proposed approach. Keywords Delay-dependent criteria - Robust stability - Time-varying structured uncertainties - Nonlinear perturbations - Linear matrix inequality This work was supported by the Doctor Subject Foundation of China (No. 2000053303).

Parameter-dependent Lyapunov functional for systems with multiple time delays

The separation of the Lyapunov matrices and system matrices plays an important role when one uses parameter-dependent Lyapunov functional handling systems with polytopic type uncertainties. The delay-dependent robust stability problem for systems with polytopic type uncertainties is discussed by using parameter-dependent Lyapunov functional. The derivative term in the derivative of Lyapunov functional is reserved and the free weighting matrices are employed to express the relationship between die terms in the system equation such that the Lyapunov matrices are not involved in any product terms with the system matrices. In addition, the relationships between the terms in the Leibniz Newton formula are also described by some free weighting matrices and some delay-dependent stability conditions are derived. Numerical examples demonstrate that the proposed criteria are more effective than the previous results.

Delay-dependent robust stability for neutral systems with mixed discrete-and-neutral delays

This paper focuses on the problem of delay-dependent robust stability of neutral systems with different discrete-and-neutral delays and time-varying structured uncertainties. Some new criteria are presented, in which some free weighting matrices are used to express the relationships between the terms in the Leibniz-Newton formula. The criteria include the information on the size of both neutral-and-discrete delays. It is shown that the present results also include the results for identical discrete-and-neutral delays as special cases. A numerical example illustrates the improvement of the proposed methods over the previous methods and the influences between the discrete and neutral delays.

CARDIAC RISK STRATIFICATION IN PATIENTS WITH CONGESTIVE HEART FAILURE:A CATECHOLAMINES-β-ADRENOCEPTOR-cAMP PATHWAY

Objective To investigate the stratification risk of catecholamines-β-adrenoceptor (β-AR)-cAMP pathway for cardiogenic death events in patients with congestive heart failure (CHF). Methods A total of 83 identified CHF patients with a baseline and follow-up plasma levels of norepinephrine (NE) and epinephrine (E), lymphocytes β-AR density (Bmax), and intralymphocyte cAMP content in peripheral blood were followed up. Major cardiogenic death events were registered. Results The period between the initial entry and the last follow-up measurement were 51±16 months, the total duration of clinical follow-up after the last measurement were 14±8 months. During follow-up, 39 patients died of cardiogenic (sudden death 17 patients, worsening heart failure 22 patients). Persistence of high NE, E, and cAMP from baseline to follow-up were confirmed as risk predicting factors of cardiovascular events. Persistence NE above 4.0 nmol/L, E above 3.5 nmol/L, and the intralymphocyte cAMP content above 3.5 pmd·mg-1·pro-1 from baseline to follow-up were significant adverse prognostic predictors. The major cardiogenic death events rates per 100 patients-years were 1.33 and 4.82 in patients with NE below and above 4.0 nmol/L (HR: 2.91; 95% CI: 1.08-7.33; P = 0.015); were 1.42 and 4.36 in the patients with E levels below and above 3.5 nmol/L (HR: 2.64; 95% CI: 1.02-6.41; P = 0.019); were 1.81 and 4.67 in the patients with the intralymphocyte cAMP content below and above 3.5 pmd·mg-1·pro-1 (HR: 2.79; 95% CI: 1.04-6.83; P = 0.017), but difference was not significant between the β-AR density below and above median. Conclusions Persistent increase in circulating catecholamines and intralymphocyte cAMP content may increase the long-term mortality in CHF patients.

On absolute stability of Lur'e control systems with multiple non-linearities using linear matrix inequalities

Necessary and suffcient conditions for the existence of a Lyapunov function in the Lur ’ e form to guarantee the absolute stability of Lur’ e control systems with multiple non-linearities are discussed in this paper. It simplifies the existence problem to one of solving a set of linear matrix inequalities (LMIs). If those LMIs are feasible, free parameters in the Lyapunov function, such as the positive definite matrix and the coefficients of the integral terms, are given by the solution of the LMIs. Otherwise, this Lyapunov function does not exist. Some sufficient conditions are also obtained for the robust absolute stability of uncertain systems. A numerical example is provided to demonstrate the effectiveness of the proposed method.

Robust impulsive synchronization of linear discrete dynamical networks

This paper aims to study robust impulsive synchronization problem foruncertain linear discrete dynamical network. For the discrete dynamical networks with unknown butbounded linear coupling, by introducing the concept of uniformly positive definite matrix functions,some robust impulsive controllers are designed, which ensure that the state of a discrete dynamicalnetwork globally asymptotically synchronizes with an arbitrarily assigned state of an isolate nodeof the network. This paper also investigates the synchronization problem where the network couplingfunctions are uncertain but bounded nonlinear functions. Finally, two examples are simulated toillustrate our results.

EXTRAOCULAR CELLULAR PHOTOTRANSDUCTION

Photobiomodulation(PBM)is a modulation of monochromatic light or laser irradiation(LI)on biosystems.It is reviewed from the viewpoint of extraocular phototransduction in this paper.It was found that LI can induce extraocular phototransduction,and there may be an exact correspondence relationship of LI at different wavelengths and in different dose zones,and cellular signal transduction pathways.The signal transduction pathways can be classified into two types so that the Gs protein-mediated pathways belong to pathway 1,and the other pathways such as protein kinase Cs-mediated pathways and mitogen-activated protein kinase-mediated pathways belong to pathway 2.Almost all the present pathways found to mediate PBM belong to pathway 2,but there should be a pathway 1-mediated PBM.The previous studies were rather preliminary,and therefore further work should be done.

Higher Level Orderings on Modules

The aim of this paper is to investigate higher level orderings on modulesover commutative rings. On the basis of the theory of higher level orderings on fields andcommutative rings, some results involving existence of higher level orderings are generalized to thecategory of modules over commutative rings. Moreover, a strict intersection theorem for higherlevel orderings on modules is established.