Application of glycan microarrays in cancer research / 生物工程学报

Glycosylation is one of the common post-translational modifications of proteins to regulate the ability of tumor invasion, metastasis and tumor heterogeneity by interacting with glycan-binding proteins such as lectins and antibodies. Glycan microarray can be constructed by chemical synthesis, chemical-enzyme synthesis or natural glycan releasing. Glycan microarray is an essential analytical tool to discover the interaction between glycan and its binding proteins. Here we summarize the standard techniques to construct glycan microarray for the application in cancer vaccine, monoclonal antibody and diagnostic markers.

Screening of proteins interacting with RSK4 in breast cancer by affinity purification and liquid chromatography/mass spectrometry / 中国癌症杂志

Background and purpose:As a tumor suppressor gene, ribosomal S6 kinase 4 (RSK4) plays important roles in inhibiting cell proliferation, migration and inducing cell apoptosis. However, the proteins interacting with RSK4 are still unknown. This study aimed to screen proteins interacting with RSK4 in breast cancer cell line MDA-MB-231 by lfag-tag affnity puriifcation and LC-MS/MS (liquid chromatography/mass spectrometry).Methods:The pcDNA3.1/EGFP-RSK4-Flag eukaryotic expression vector was constructed by inserting full lengthRSK4 gene into vector pcDNA3.1/EGFP-Flag. And then the recombinant plasmids were transferred into MDA-MB-231 cells. Real-timelfuorescent quantitative polymerase chain reaction (RTFQ-PCR) and Western blot were used to detect the expression of RSK4 in MDA-MB-231 cells. Affnity puriifcation and LC-MS/MS were applied to screen proteins interacting with RSK4, and the related action mechanism of RSK4 with its interacted proteins was detected based on bioinformatics gene ontology (GO) and ingenuity pathway analysis (IPA).Results:Twenty-four proteins, such as serine/threonine-pro-tein kinase 38 (STK38)/serine/threonine-protein kinase 38-like (STK38L), MOB kinase activator 2 (MOB2) and protein arginineN-methyltransferase 5 (PRMT5), were successfully identiifed by Flag-tag affnity puriifcation followed by LC-MS/MS analysis, which probably interacted with RSK4. Bioinformatics analysis of the identiifed proteins suggested the proteins interacting with RSK4 were involved in diverse biological pathways, such as apoptosis and cell migration. Conclusion:According to bioinformatics results of proteins interacting with RSK4 identiifed by affnity puriifcation and LC-MS/MS, biological networks of RSK4 are involved in apoptosis and migration in breast cancer cells.

Stathmin for promoting cell proliferation and invasion ability in human SMMC-7721 hepatocellular carcinoma cells / 重庆医学

Objective To construct the stable stathmin-overexpression SMMC-7721 hepatocellular carcinoma cells and to explore the effect of stathmin-overexpression on the cell proliferation and metastasis in SMMC-7721 cells .Methods By using liposome , Flag-pcDNA3 .1 and Flag-pcDNA3 .1-stathmin plasmid were transfected into SMMC-7721 cells respectively ,the stable Flag-pcD-NA3 .1 expression cells(control group) and the stable stathmin-overexpression cells(experimental group) were established after an-tibiotic resistant gene screening ,and the cell lines were identified by Western Blot .Subsequently ,the cell proliferation was detected by cell count kit(CCK-8) and the soft agar assay ,the apoptosis and cell cycle were determined by the flow cytometry (FCM ) ,and the cell motility and invasion were analyzed by the Transwell assay in vitro .Results The stathmin protein expression of the experi-mental group was significantly increased compared with the control group (0 .76 ± 0 .12 vs .0 .16 ± 0 .05 ,P<0 .05) ,which indicated that the stathmin-overexpression human SMMC-7721 hepatocellular carcinoma cell line was successfully constructed .CCK-8 and the soft agar assay showed that the cell proliferation of the experimental group was higher than that of the control group (0 .29 ± 0 .03 vs .0 .60 ± 0 .05 ,P< 0 .01);additionally ,the apoptotic ratio of the experimental group was decreased compared with the control group[(11 .57 ± 1 .09)% vs .(5 .80 ± 0 .33)% ,P<0 .05] ,the cell cycle was arrested in the stage G2/M ;the Transwell experiment results verified that the cell motility and the invasive ability of the experimental group were obviously reinforced compared with the control group[transmenbrane cell numbers in migrant assay :(54 .03 ± 7 .21) vs .(130 .45 ± 14 .13);transmenbrane cell numbers in invasive assay :(17 .75 ± 2 .52) vs .(57 .76 ± 8 .50) respectively] ,the differences had statistical significance(P<0 .01) .Conclusion The overexpression of stathmin promotes the cell proliferation and the invasive ability in SMMC-7721 hepatocellular carcinoma cells .

Serum glycoprotein profiling by lectin affinity microarray to distinguish the various stages of primary liver carcinogenesis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To identify specific serum glycoprotein profiles that correspond to the carcinogenic process of primary liver cancer (PLC) by analyzing a population with high-incidence of PLC using lectin affinity microarray.</p><p><b>METHODS</b>Serum samples were collected from individuals classified as high risk for PLC (including patients with liver cirrhosis and hepatitis B) and development of PLC was recorded. Healthy individuals served as normal controls. The serum samples were subjected to glycoprotein profling by using lectin microarrays and the results were confirmed by lectin blot. Between-group differences were statistically analyzed.</p><p><b>RESULTS</b>PLC carcinogenesis was found to be correlated with enhanced affinity for AAL, ACL, ConA, LCA, MPL, NML, PHA-E, PHA-L, PSA, RCA-I, STL, VAL,WGA, and SNA (P less than 0.05). These data implied that changes in specific glycan structures, such as aFuc, GlcNAc, GalNAc, mannose, bisecting GlcNAc and terminal beta1-4 Gal, may be involved in PLC carcinogenesis . The PLC group showed significantly different results for all detected lectins, except SNA (P less than 0.05). However, among the PLC group, the SNA affinity was not significantly different for the hepatitis B group (P =0.443, P more than 0.05).</p><p><b>CONCLUSION</b>Glycans may be associated with the carcinogenic process of PLC and may be developed as diagnostic and prognostic biomarkers of PLC in the future.</p>

Alteration of the glycan profile of serum glycoproteins during the seroconversion process in hepatitis B virus-infected patients treated with antiviral therapy and its clinical significance / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To use a lectin microarray to study the alteration of glycan affinity profiles of serum glycoproteins during the hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) seroconversion in patients with chronic hepatitis B (CHB) following treatment with antiviral therapy,and to explore its biological significance.</p><p><b>METHODS</b>CHB patients were divided into the following four groups:untreated HBeAg-positive,HBeAg seroconversion after anti-HBV therapy,HBsAg loss after anti-HBV therapy,and healthy individuals (controls).Serum samples were collected from each participant,depleted of high abundance proteins and analyzed by a lectin microarray containing 50 lectins.The lectin-affinity glycan profiles of serum proteins were partially verified by lectin blotting.Between-group differences were analyzed by one-way analysis of variance,and pairwise comparisons were carried out with the Student-Newman-Keuls (SNK) method.</p><p><b>RESULTS</b>The results from the lectin microarray and lectin blotting assay showed significantly reduced affinity for 16 lectins in the untreated HBeAg-positive group compared to the control group (P less than 0.05);in addition,the specific glycan profiles of the untreated HBeAg-positive group included decreased terminal and core fructose,GalNAc alpha-Thr/Ser (T,Tn-antigen),GalNac alpha,terminal beta1-4,and beta-D galactose,bisecting and/or GlcNAc,mannose and Sia.However,the HBeAg seroconversion after anti-HBV therapy group showed enhanced binding of PSA,MPL and the above-mentioned 16 lectins (P less than 0.05),suggesting that the reduced serum glycoprotein glycan structures returned to normal or slightly higher than healthy levels after the therapy-induced seroconversion.Comparison of the group with HBsAg loss after anti-HBV therapy to the group with HBeAg seroconversion after anti-HBV therapy showed the binding ability of ten lectins (AAL,ACL,HAL,HPL,RCA-I,LEL,STL,PHA-E,NML and PCL) were weakened to near control levels and six lectins (VAL,LCA,GNL,PSA,MPL and JAC) were significantly strengthened (all P less than 0.05). These findings implied that the glycan containing terminal fructose, GalNacalpha, terminal beta1-4 galactose,and bisecting GlcNAc glycan structures dropped to near control levels, while the terminal beta-D-galactose residues and core fructose structure increased significantly.</p><p><b>CONCLUSION</b>The glycan structures of serum glycoproteins are closely related to HBeAg and HBsAg seroconversion in CHB patients.It is possible that a special lectin binding glycan involving the terminal beta-D-galactose residues and core fructose may act as sugar markers associated with the disappearance of serum HBsAg during anti-HBV therapy for CHB.</p>

Evaluation of isotopic labeling of lysine residues of peptides for quantitative proteomics / 生物工程学报

To evaluate the reagent 2-methoxy-4,5-dihydro-1H-imidazole used for isotopic labeling in quantitative proteomics, we synthesized 2-methoxy-4,5-dihydro-1H-imidazole and its tetradeuterated analog in three steps. Prior to tryptic cleavage, bovine serum albumin (BSA) was reduced and alkylated. Tryptic peptides were derivatized with an equal volume of either DO or D4 and D4-derivatized peptides were mixed with at variable ratio (from 10:1 to 1:5) prior to MS and MS/MS analysis. We used matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and Electro spray ionization-mass spectrometry (ESI-MS) to evaluate the quantitative capability of labeling. The specificity of the reagent is excellent: only lysine side chains were modified among tryptic peptides. MALDI and ESI ionization modes not only could achieve the quantification of differentially expressed proteins but also facilitate the de novo sequencing. This side-chain modification can be used for quantitative analysis with proteomic strategies involving liquid chromatography. Reverse phase liquid chromatography (RPLC) kept a good resolution, and the introduction of D atoms did not introduce a variation of retention time between heavy and light peptides in RPLC.

Value of macrophage migration inhibitory factor in the diagnosis of hepatocdlular carcinoma / 中华消化外科杂志

Objective To investigate the diagnostic value of macrophage migration inhibitory factor (MIF) for hepatocellular carcinoma (HCC).MethodsThe research was divided into 2 parts,including testing research and confirmatory research.The clinical data of 269 patients with HCC ( group A) and 390 individuals (including 135 patients with hepatic cirrhosis,106 with benign hepatic diseases and 149 healthy individuals,control group A) who were admitted to the Zhongshan Hospital of Fudan University from January to May,2004,and 173 patients with hepatic cancer (group B) and 257 individuals (including 86 patients with hepatic cirrhosis,79 with benign hepatic diseases and 92 healthy individuals,control group B ) who were admitted from August to December,2004,and 80 patients with HCC who received radical hepatic resection in January 2005 were retrospectively analyzed.Samples of plasma of patients in the group A and individuals in the control group A were collected before operation.Samples of plasma of patients received radical hepatic resection were collected preoperatively and at postoperative day 3,7 and 30.HCC and adjacent issues of patients in the group A were collected.The levels of MIF in the plasma and tissues were detected by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry,respectively.Non-normal distribution data were described as M( QR).Differences between the groups were analyzed by using the Mann-Whitney U test,and the relationship between the levels of MIF in the plasma and tissues was detected by the Spearman correlation coefficient.The diagnostic value of MIF was analyzed by the ROC curve.ResultsThe levels of MIF in the plasma of patients in the group A and individuals in the control group A were 85.7 μg/L (58.8 μg/L) and 15.5 μg/L(31.6 μg/L),respectively.The levels of MIF in the plasma of the patients with hepatic cirrhosis,benign hepatic diseases and healthy individuals were 24.9 μg/L (12.6 μg/L),12.5 μg/L(7.3 μg/L) and 13.2 μg/L (7.7 μg/L),respectively.There was a significant difference in the level of MIF between the group A and the control group A (F =54.235,P ＜ 0.05 ).The area under the ROC curve reached peak when the level of MIF in the plasma was 35.3μg/L.Compared with the control group B,the vdues of AUC,sensitivity and specificity were 92.1％,90.7％ and 93.4％ in the group B.The levels of MIF of the patients with HCC before operation and at 3,7,and 30 days after operation were 81.0 μg/L(54.0 μg/L),76.1 μg/L(47.5 μg/L),50.9 μg/L (40.7 μg/L) and 18.7 μg/L ( 15.1 μg/L),respectively.The levels of MIF decreased with time passed by,and were back to normal at 30 days after the operation.The median expressions of MIF in the HCC and adjacent issues were 0.083 and 0.007,respectively,with a significant difference ( U =3.975,P ＜ 0.05).The expression of MIF in the plasma was positively correlated with its expression in the HCC tissue ( r =0.759,P ＜ 0.05 ).ConclusionMIF plays an important role in the genesis and development of HCC and has potential to be one of the molecular markers for the diagnosis of HCC.

HCC serum associated proteins screened by SELDI-TOF-MS analysis / 中华检验医学杂志

Objective To screen potential serum HCC associated proteins with low molecular weight and low abundance for better understanding the pathological mechanism of HCC and discovering new biomarkers.Methods All serum samples were collected from 81 HBV-related HCC patients,43 chronic hepatitis B patients and 36 cirrhosis patients.Serum protein fingerprint profiles were first generated by selected WCX2 protein chip integrating with SELDI-TOF-MS,and then normalized and aligned by Ciphergen SELDI Software 3.1.1 with Biomarker Wizard.Comparative analysis of the intensity of corresponding protein fingerprint peaks in normalized protein spectra was performed.Some protein peaks with significant difference among HCC,cirrhosis and chronic hepatitis B groups were found.The reproducibility of the SELDI system was assessed before serum protein fingerprint profiles analysis.Results The intra-and inter-assay CV for intensity and m/z in this SELDI system were 17.46% and 0.024%,and 17.74% and 0.024% respectively.Total 128 protein fingerprint peaks between 2 000 to 30 000 Da were identified under the condition of signal to noise＞5 and minimum threshold for cluster＞20%.Eighty-seven proteins were found to significantly expressed between HCC and cirrhosis groups(P＜0.05).Of the above differential proteins,forty-five proteins had changes greater than two fold,including 15 up-regulated proteins and 30 downregulated proteins in HCC sera.Between HCC and chronic hepatitis B groups,nine of fifty-two differential proteins(P＜0.05) had intensities of more than two folds,including 2 up-regulated proteins and 7 downregulated proteins in HCC sera.Between cirrhosis and chronic hepatitis B groups,twenty-eight of seventynine significantly differential proteins(P＜0.05) changed greater than two folds in intensity,including 17 up-regulated proteins in cirrhosis seru and 11 down-regulated proteins in chronic hepatitis B sera.Analysis of above leading differential proteins among three diseases using subtraction difference mode,the 5 common down-regulated proteins 2 870,3 941,2 688,3 165 and 5 483 m/z in HCC sera and 2 common up-regulated proteins 3 588 and 2 017 m/z in cirrhosis and HCC sera were screened.But no statistic difference in the level of protein 2 017m/z was found between HCC group and normal group inour previous study.Conclusion Because the interference of unspecific proteins from hepatitis B and cirrhosis could be eliminated partly in HCC sera through subtraction difference analysis,these 6 common differential proteins (2 870,3 941,2 688,3 165,5 483,3 588 m/z)have obvious advantages of increased specificity for evaluating the pathological state of HCC and might become promising candidate biomarkers in the diagnosis of HCC.

Screening of serum biomarkers related to the tumor recurrence following liver transplantation for hepatocellular carcinoma / 中华器官移植杂志

Objective To appraise and compare protein expression profiles in sera of patients without or with recurrence following liver transplantation for hepatocellular carcinoma (HCC) using SELDI-TOF-MS technique,and establish the diagnostic and predictive model. Methods A total of 76 sera (41 from disease free survival patients and 35 from recurrence individuals) were collected pretransplantation and differentially expressed proteins were identified by SELDI-TOF-MS. The intensity values for each peak were analyzed by Biomarker Wizard Software to screen serum proteome biomarkers related to the recurrence post-transplantation. By using Biomarker Patterns Software, the classification trees were generate. from randomly selected samples (30 fingerprints obtained from each group). The sensitivity and specificity of best decision tree were then chosen for blind test with 16 samples (5 from recurrence individuals and 11 from recurrence-free survival patients). Results There were significant differences only in tumor size and the presence of vascular invasion between recurrence group and recurrence-free survival group (P＜0.05). According to serum protein fingerprints, a total of 368 protein peaks were identified at the mass-to-charge ratio (M/Z) value ranging from 2000 to 300 00. There were 22 significant differential proteins between two groups. Among them, 9 proteins were up-regulated and 13 proteins were down-regulated -espectively in recurrence group. The intensity values of differential proteins were input into BPS for classification tree analysis and the best performing tree could distinguish two groups successfully. As a result of blind assessment for this model,a sensitivity of 80.0 % (4/5) and specificity of 72.7 % (8/11) were obtained. Conclusion Some of differential proteins screened by SELDI-TOF-MS technique in the serum may be correlated with the prognoses of liver transplantation patients with HCC. The decision tree may be useful for the clinical application of formulating the indication for liver transplantation, detecting extrahepatic micrometastasis and setting up the diagnostic and treatment strategies.

Selective blockage of abnormal Wnt pathway inhibits the growth and invasion of hepatocarcinoma cells / 复旦学报（医学版）

Objective To construct the recombinant expression vector encoding antisense Tcf fragment for the blockage of abnormal Wnt pathway, and to investigate its effect on the biological behaviors of human hepatocarcinoma cells. Methods Antisense expression vector was transfected into hepatocarcinoma cells SMMC-7721 with GeneJammer. RT-PCR and Western blot were used to detect Tcf expression. Cell proliferation and motility were compared by growth curves and Transwell plate assay. Cell apoptosis was determined by Annexin V and cell cycle was examined by fluorescent staining. Results The stable transfection of antisense Tcf in SMMC-7721 cells significantly reduced Tcf expression at both mRNA and protein levels. Compared with parental and mock-transfected 7721 (7721-vector) cells, antisense Tcf RNA transfected cells 7721-pTas showed much decreased activities of proliferation, migration and invasion in vitro. Furthermore, the apoptosis rate of 7721-pTas cells [(26.34±2.07)%] was significantly higher than that of 7721-vector cells [(6.53±1.02)%] and parental SMMC-7721 cells [(4.33±0.68)%] (P<0.001). The percentages of G0-G1 phase antisense transfected cells were 20.24% and 20.95%, higher than parental SMMC-7721 and 7721-vector cells, and percentages of S phase antisense transfected cells were 11.8% and 11.38%, lower than parental SMMC-7721 and 7721-vector cells, respectively. Conclusions Antisense RNA suppress the growth ability of liver cancer cells by inducing cell apoptosis and impeding the progress of cell cycle, which suggests that selective blockage of abnormal Wnt signal pathway by antisense Tcf RNA may be a potential new gene therapy for liver cancer.

Stathmin: a potential tumor biomarker / 肿瘤

Stathmin, a ubiquitously expressed cytosolic protein(Mr=19×10~3), is also called oncogene protein 18 (Op18). Stathmin is involved in the assembly of microtubule (MT) and spindle by binding the tubulin protein. It plays a key role in cell proliferation, differentiation, regeneration, and migration and has regulatory effects on the signal transduction. Recently, it is reported that stathmin is overexpressed in a variety of human malignancies. It induces tumor cell migration and invasion by regulating MT depolymeri-zation. Its post-translational modification influences the interaction with p53 protein and is involved in the initiation and progression of malignant tumor. Stathmin alone or in combination with chemotherapeutics has been used for tumor therapy. The internal association of stathmin with cancer etiology is still unknown. So, further studies are needed to determine the role of stathmin as potential tumor biomarker and a drug target in tumor therapy.

Progress in differentiation of hepatocytes from embryonic stem cells / 国际生物医学工程杂志

Acute liver failure and end-stage liver diseases are llfe-threatening.So far,orthotopic liver transplantation is the unique treatment available for these late-stage liver diseases,which,however,is limited by the lack of donor liver.While hepatocyte transplantation,bioartificial liver and tissue engineered liver are potential alternative treatments,the limited availability of functional hepatoeytes is the major hurdle.Due to their extensive capacity for self-renewal and pluripotency to differentiate into cells,embryonic stem cells represent a potential unlimited cell source for therapy.The clinical application of embryonic stem cells in liver diseases requires well-defined and efficient protocols for differentiation,characterization and purification in vitro.Hepatic differentiation of embryonic stem cells in different culttire systelns and the problems remained are reviewed in this article.

Detection of pigment epithelium derived factor gene mutation in a human malignant melanoma cell line A375 / 中华皮肤科杂志

Objective To detect the mutations of pigment epithelium derived factor (PEDF) gene in a human malignant melanoma cell line A375. Methods A375 cells and control melanocytes obtained from circumcised prepuce were cultured, genomic DNA was extracted from these cells. All eight exons of PEDF gene were scanned by single strand conformation polymorphism analysis ofpolymerase chain reaction products (PCR-SSCP) in both A375 cells and control melanocytes. DNA sequencing was performed for the PCR products separated into electrophoretic bands with altered mobility. Results Altered mobility was observed with SSCP analysis in amplicons of exon 3, 4, 5, 6 and 7, with the most obvious alteration occurred in exon 5 and 6. DNA sequencing revealed mutations in both exon 5 and 6. The common type of mutations was single base-deletion in exon 5 and single base-substitution in exon 6. Conclusion Mutations of PEDF gene may contribute to the development of human malignant melanoma.

Overexpression and Effect on Apoptosis of the 150-ku Oxygen-regulated Protein(ORP150) in Human Hepatocellular Carcinoma / 生物化学与生物物理进展

In previous study, the 150-ku oxygen-regulated protein(ORP150) was identified as a candidate glycoprotein related to hepatocellular carcinoma.In order to further validate the expression level of ORP150 in hepatocellular carcinoma, protein expression was determined by Western blot and cell immunochemistry, and messenger RNA(mRNA) expression was detected by quantitative real-time polymerase chain reaction.The effect of ORP150 on apoptosis and invasive potential of hepatocellular carcinoma cells was evaluated using the small interference RNA(siRNA) technique.Both the protein and mRNA expression levels of ORP150 were significantly upregulated in hepatocellular carcinoma cell lines compared with a non-tumor human liver cell line.After transfection with the specific siRNA of ORP150, significantly greater apoptosis of hepatocellular carcinoma cells was induced compared with untransfected cells.However, no significant effect on invasive potential was found.Overexpression of ORP150 was associated with hepatocellular carcinoma, and ORP150 might promote the proliferation of hepatocellular carcinoma cells by inhibiting apoptosis.ORP150 could be a potential therapeutic target for hepatocellular carcinoma.

Alteration of CK8 Both in Its Expression Level and Its Glycan Parts Related to HCC Metastatic Ability / 生物化学与生物物理进展

A glycomic method was used to screen the aberrantly ?1-6 fucosylated glycoproteins related to HCC metastasis and analyze the alteration of CK8 both in its expression level and its glycan parts associated with metastatic ability. Based on the approach, 2-DE coupled with lectin affinity blot, lectin affinity precipitation followed by MALDI-TOF-MS/MS, the lens culinaris agglutinin (LCA) affinity glycoprotein profiles from MHCC97-L and MHCC97-H cells, two higher metastatic HCC cell lines, were obtained, in which a differentially displayed protein spot was indicated when compared with Hep3B, in the region within 55～60 ku in molecular mass and 4～6 in isoelectric point. The identification result was CK8 by MALDI-TOF-MS/MS. To confirm the relation between increased core-fucosylation of CK8 and HCC metastasis, LCA affinity precipitation was used to extract the ?1-6 fucosylated glycoproteins, followed by Western blot. And it was found that CK8 was highly fucosylated in both MHCC97-L and MHCC97-H cells compared to Hep3B. Immunofluorescence analysis and Western blot were used to detect its intracellular localization and its protein expression levels, indicating that CK8 distributed in cytoplasm and increased protein expressions in MHCC97-L and MHCC97-H cell lines. And further lectin binding studies found that CK8 has a high affinity for Con A in both MHCC97-H and Hep3B cells, indicating that CK8 was a glycoprotein with high-mannose type N-glycans. But the amount of the lectin RCA-1 binding to CK8 was greater in MHCC97-H than Hep3B, suggesting that CK8 contained the increased terminal galactose residues ?-1, 4-linked to GlcNAc in MHCC97-H. All the results suggested that the increase of CK8 in its protein expression level, core-fucosylation and terminal gal ?1,4 GlcNAc disaccharides might be related to HCC metastatic ability.

Alterations of beta-catenin and Tcf-4 instead of GSK-3beta contribute to activation of Wnt pathway in hepatocellular carcinoma / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>The goal of this study is to investigate the inappropriate activation of Wnt pathway in the hepatocarcinogenesis.</p><p><b>METHODS</b>We analyzed the alterations of three key components of Wnt pathway, beta-catenin, glycogen synthase kinase 3beta (GSK-3beta) and T cell factor 4 (Tcf-4), in 34 samples of hepatocellular carcinoma (HCC) and paracancerous normal liver by immunohistochemistry, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), direct sequencing, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization.</p><p><b>RESULTS</b>We found 61.8% (21/34) of all the HCCs examined showed an abnormal beta-catenin protein accumulation in the cytoplasm or nuclei. RT-PCR-SSCP and direct sequencing showed that beta-catenin exon 3 mutations existed in 44.1% (15/34) of the HCCs. No mutations of GSK-3beta or Tcf-4 were detected in HCCs. Moreover, mRNA of beta-catenin and Tcf-4 but not GSK-3beta was found to be over expressed in HCCs. On analyzing the relationship between alterations of beta-catenin or Tcf-4 and C-myc or Cyclin D1 expression, we found that the mutations of beta-catenin as well as over expression of beta-catenin or Tcf-4 gene were independently correlated with C-myc gene over expression in HCCs.</p><p><b>CONCLUSIONS</b>Our present findings strongly suggest mutations of beta-catenin as well as over expression of beta-catenin and Tcf-4 gene activate the Wnt pathway in HCC independently with the target gene most likely to be C-myc.</p>

Expression of telomerase hTERT in human non-small cell lung cancer and its correlation with c-myc gene / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the expression of human telomerase catalytic subunit, hTERT, in human non-small cell lung cancer (NSCLC) and its correlations to c-myc gene.</p><p><b>METHODS</b>hTERT and c-myc mRNA expressions were detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Statistical correlation analysis was made to estimate whether there was interrelation between them.</p><p><b>RESULTS</b>Positive rate of hTERT expression in 51 surgically resected lung cancer specimens was 86.3%, significantly higher than that in adjacent non-neoplastic lung tissues and benign lesions, which were 14.3% and 27.3% respectively. No statistical significance was observed between the frequency of hTERT expression and histologic types, degree of differentiation, TNM stages, tumor size or lymph nodes metastases. Correlation analysis revealed that the expression of c-myc gene was significantly related to that of hTERT (correlation coefficient, r = 0.633, P < 0.001).</p><p><b>CONCLUSIONS</b>hTERT may be a useful tumor marker in diagnosing lung cancer. Significant correlation between the expression of hTERT and c-myc mRNA indicates that the activation and up-regulation of hTERT might be conferred by over-expression of c-myc gene.</p>

Recombinant human heparin-binding neurite-promoting factor expressed with yeast stimulates neurites outgrowth / 中华医学杂志(英文版)

<p><b>OBJECTIVES</b>Heparin-binding neurite-promoting factor (HBNF) is a heparin-binding protein primarily found in the brain, which can stimulate neurite outgrowth in vitro. We expressed recombinant human heparin-binding neurite-promoting factor (hrHBNF) using a yeast system, and observed its activity in stimulating neurite outgrowth in vitro.</p><p><b>METHODS</b>cDNA encoding mature human HBNF was amplified from total RNA isolated from an 18-week aborted human fetal brain by RT-PCR method. After amplification, the HBNF cDNA gene was cloned into pPIC9K, a shuttle expression vector for yeast system. The positive clone of expression vector bearing HBNF cDNA gene was obtained by screening. Verified recombinant vector was then used to transform Pichia strain GS115 by electroporation. His(+) transformants were selected on minimal dextrose medium (MD) plates which were histidine free. His(+) yeast recombinants with multi-copy inserts were screened in vivo by their resistance to G418. PCR analysis was used to confirm the integration of the HBNF cDNA gene into the Pichia genome. Secreted expression of hrHBNF protein in culture medium was obtained when the positive clone containing the HBNF cDNA gene was induced by methanol. The hrHBNF product purified by gel chromatography was added to cultured rat pheochromocytoma (PC12) cells to observe its ability to stimulate neurite outgrowth.</p><p><b>RESULTS</b>In the recombinant expression vector, the insert was sequenced to show exactly the sequence encoding human HBNF according to Genbank data. The HBNF cDNA gene was cloned downstream to the alpha-factor, and its open reading frame was in frame with the alpha-factor signal sequence in pPIC9K. SDS-PAGE showed that the molecular weight of the induced expression product was about 18 kDa, consistent with that of human HBNF reported in the literature. The protein product did promote neurite outgrowth in cultured rat pheochromocytoma (PC12) cells.</p><p><b>CONCLUSION</b>Recombinant human heparin-binding neurite-promoting factor can be expressed with a yeast system, and its product possesses the biological activity to promote neurite outgrowth.</p>

Gene expression profile of human hepatocellular carcinoma cell lines with different metastatic potentials / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To compare gene expression profile of human hepatocellular carcinoma (HCC) cell lines with different metastatic potentials, so as to screen for metastasis-related genes.</p><p><b>METHODS</b>Gene expression profile of MHCC97-L and HCCLM3, two HCC cell lines with similar genetic background but different in spontaneous metastatic potentials, were studied by cDNA microarray.</p><p><b>RESULTS</b>From 1,626 screened genes, 25 differentially expressed genes were found, 18 showed decreased expression including the decreased expression of cell cycle control genes Rb2, mismatch repair gene hMSH2, and signal transduction gene protein kinase C beta 2 and 7 increased expression including signal transduction gene MAP kinase kinase 6, cell proliferation gene E25, immunity related gene SP40, 40, etc in HCCLM3.</p><p><b>CONCLUSION</b>The genes, being closely associated with cancer metastasis, could be considered as potential markers to predict metastasis and targets for anti-metastasis intervention.</p>

Urinary type IV collagen: a specific indicator of incipient diabetic nephropathy / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To determine whether urinary type IV collagen can serve as an indicator specific for diabetic nephropathy.</p><p><b>METHODS</b>Using a novel sandwich ABC-ELISA to measure type IV collagen directly, the 24-hour urinary type IV collagen excretion rate was determined in 120 diabetic patients and some groups of controls. Urinary albumin determinations were made with a RIA kit at the same time. A total of 13 diabetic patients with microalbuminuria underwent percutaneous renal biopsy for definitive diagnosis of diabetic nephropathy. Type IV collagen and TGF-beta 1 immunoreactivities were detected with ABC methods in renal biopsies.</p><p><b>RESULTS</b>Urinary type IV collagen excretion was significantly increased in diabetic patients with microalbuminuria, especially those with albumin excretion above 200 mg/24 h. By comparison, collagen excretion was equivalent to that in healthy controls when measured in diabetics with normalbuminuria and in patients with primary glomerular disease, primary hypertension, or coronary heart disease. Urinary type IV collagen excretion in diabetics was negatively correlated with creatinine clearance. In renal biopsies from subjects with elevated collagen excretion, the glomeruli showed pathological changes typical of diabetic nephropathy. Also, excessive type IV collagen and TGF-beta 1 immunoreactivity were detected in the glomeruli, Bowman's capsule and interstitium.</p><p><b>CONCLUSIONS</b>Excretion of type IV collagen, possibly reflecting increased production or decreased degradation of this protein, may be a clinically useful indicator of incipient diabetic nephropathy.</p>