Mass spectrometry analysis of intact protein N-glycosylation signatures of cells and sera in pancreatic adenocarcinomas. / èƒ°è ºç™Œç»†èƒžå’Œè¡€æ¸ å®Œæ•´è›‹ç™½è´¨N-糖基化特征的质谱分析.

Pancreatic cancer is among the most malignant cancers, and thus early intervention is the key to better survival outcomes. However, no methods have been derived that can reliably identify early precursors of development into malignancy. Therefore, it is urgent to discover early molecular changes during pancreatic tumorigenesis. As aberrant glycosylation is closely associated with cancer progression, numerous efforts have been made to mine glycosylation changes as biomarkers for diagnosis; however, detailed glycoproteomic information, especially site-specific N-glycosylation changes in pancreatic cancer with and without drug treatment, needs to be further explored. Herein, we used comprehensive solid-phase chemoenzymatic glycoproteomics to analyze glycans, glycosites, and intact glycopeptides in pancreatic cancer cells and patient sera. The profiling of N-glycans in cancer cells revealed an increase in the secreted glycoproteins from the primary tumor of MIA PaCa-2 cells, whereas human sera, which contain many secreted glycoproteins, had significant changes of glycans at their specific glycosites. These results indicated the potential role for tumor-specific glycosylation as disease biomarkers. We also found that AMG-510, a small molecule inhibitor against Kirsten rat sarcoma viral oncogene homolog (KRAS) G12C mutation, profoundly reduced the glycosylation level in MIA PaCa-2 cells, suggesting that KRAS plays a role in the cellular glycosylation process, and thus glycosylation inhibition contributes to the anti-tumor effect of AMG-510.

Diagnostic Potential of Serum Glycome Analysis in Lung Cancer: A Glycopattern Study.

Lung cancer is the leading cause of cancer-related death, with high morbidity and mortality rates due to the lack of reliable methods for diagnosing lung cancer at an early stage. Low-dose computed tomography can help detect abnormal areas in the lungs, but only 16% of cases are diagnosed early. Tests for lung cancer markers are often employed to determine genetic expression or mutations in lung carcinogenesis. Serum glycome analysis is a promising new method for early lung cancer diagnosis as glycopatterns exhibit significant differences in lung cancer patients. In this study, we employed a solid-phase chemoenzymatic method to systematically compare glycopatterns in benign cases, adenocarcinoma before and after surgery, and advanced stages of adenocarcinoma. Our findings indicate that serum high-mannose levels are elevated in both benign cases and adenocarcinoma, while complex N-glycans, including fucose and 2,6-linked sialic acid, are downregulated in the serum. Subsequently, we developed an algorithm that utilizes 16 altered N-glycans, 7 upregulated and 9 downregulated, to generate a score based on their intensity. This score can predict the stages of cancer progression in patients through glycan characterization. This methodology offers a potential means of diagnosing lung cancer through serum glycome analysis.

Novel Approach to Enriching Glycosylated RNAs: Specific Capture of GlycoRNAs via Solid-Phase Chemistry.

Ribonuclease (RNA) modifications can alter cellular function and lead to differential immune responses by acting as discriminators between RNAs from different phyla. RNA glycosylation has recently been observed at the cell surface, and its dysregulation in disease may change RNA functions. However, determining which RNA substrates can be glycosylated remains to be explored. Here, we develop a solid-phase chemoenzymatic method (SPCgRNA) for targeting glycosylated RNAs, by which glycosylated RNA substrates can be specifically recognized. We found the differential N-glycosylation of small RNAs in hTERT-HPNE and MIA PaCa-2 cancer cells using SPCgRNA. RNA-Seq showed that the changes in glyco-miRNAs prepared from SPCgRNA were consistent with those of traditional methods. The KEGG signaling pathway analysis revealed that differential miRNA glycosylation can affect tumor cell proliferation and survival. Further studies found that NGI-1 significantly inhibited the proliferation, migration, and circulation of MIA PaCa-2 and promoted cell apoptosis. In addition, ß-1,4-galactosyltransferase 1 (B4GALT1) not only affected the expression level of glycosylated miRNAs hsa-miR-21-5p but also promoted cell apoptosis and inhibited the cell cycle possibly through the p53 signaling pathway, while B4GALT1 and p53 were also affected following the hsa-miR-21-5p increase. These results suggest that B4GALT1 may catalyze miRNAs glycosylation, which further promotes cancer cell progression.

Recent development of analytical methods for disease-specific protein O-GlcNAcylation.

The enzymatic modification of protein serine or threonine residues by N-acetylglucosamine, namely O-GlcNAcylation, is a ubiquitous post-translational modification that frequently occurs in the nucleus and cytoplasm. O-GlcNAcylation is dynamically regulated by two enzymes, O-GlcNAc transferase and O-GlcNAcase, and regulates nearly all cellular processes in epigenetics, transcription, translation, cell division, metabolism, signal transduction and stress. Aberrant O-GlcNAcylation has been shown in a variety of diseases, including diabetes, neurodegenerative diseases and cancers. Deciphering O-GlcNAcylation remains a challenge due to its low abundance, low stoichiometry and extreme lability in most tandem mass spectrometry. Separation or enrichment of O-GlcNAc proteins or peptides from complex mixtures has been of great interest because quantitative analysis of protein O-GlcNAcylation can elucidate their functions and regulatory mechanisms in disease. However, valid and specific analytical methods are still lacking, and efforts are needed to further advance this direction. Here, we provide an overview of recent advances in various analytical methods, focusing on chemical oxidation, affinity of antibodies and lectins, hydrophilic interaction, and enzymatic addition of monosaccharides in conjugation with these methods. O-GlcNAcylation quantification has been described in detail using mass-spectrometric or non-mass-spectrometric techniques. We briefly summarized dysregulated changes in O-GlcNAcylation in disease.

Identification of key differentially expressed genes between ER-positive/HER2-negative breast cancer and ER-negative/HER2-negative breast cancer using integrated bioinformatics analysis.

BACKGROUND: Treatment strategies for various subtypes of breast cancer (BC) are different based on their distinct molecular characteristics. Therefore, it is very important to identify key differentially expressed genes (DEGs) between ER-positive/HER2-negative BC and ER-negative/HER2-negative BC. METHODS: Gene expression profiles of GSE22093 and GSE23988 were obtained from the Gene Expression Omnibus database. There were 74 ER-positive/HER2-negative BC tissues and 85 ER-negative/HER2-negative BC tissues in the two profile datasets. DEGs between ER-positive/HER2-negative tissues and ER-negative/HER2-negative BC tissues were identified by the GEO2R tool. The common DEGs among the two datasets were detected with Venn software online. Next, we made use of the Database for Annotation, Visualization and Integrated Discovery to analyze enriched Kyoto Encyclopedia of Gene and Genome (KEGG) pathways and gene ontology terms. Then, the protein-protein interactions (PPIs) of these DEGs were visualized by Cytoscape with the Search Tool for the Retrieval of Interacting Genes. Of the proteins in the PPI network, Molecular Complex Detection plug-in analysis identified nine core upregulated genes and one core downregulated gene. UALCAN and Gene Expression Profiling Interactive Analysis were applied to determine the expression of these 10 genes in BC. Furthermore, for the analysis of overall survival among those genes, the Kaplan-Meier method was implemented. RESULTS: Ninety-three common DEGs (63 upregulated and 30 downregulated) were identified. KEGG pathway enrichment analysis showed that upregulated DEGs were particularly enriched in the progesterone-mediated oocyte maturation pathway. In addition, PGR might be a prognostic biomarker for ER-positive/HER2-negative BC. CCND1 is a poor prognostic biomarker for ER-positive/HER2-negative BC and ER-negative/HER2-negative BC. Moreover, TFF1, AGR2 and EGFR might be predictive biomarkers of node metastasis in ER-positive/HER2-negative BC and ER-negative/HER2-negative BC. CONCLUSIONS: CCND1, AGR2, PGR, TFF1 and EGFR are the key DEGs between ER-positive/HER2-negative BC and ER-negative/HER2-negative BC. Further studies are required to confirm the functions of the identified genes.

First-Line Nivolumab Plus Ipilimumab in Advanced Non-Small-Cell Lung Cancer (CheckMate 568): Outcomes by Programmed Death Ligand 1 and Tumor Mutational Burden as Biomarkers.

PURPOSE: CheckMate 568 is an open-label phase II trial that evaluated the efficacy and safety of nivolumab plus low-dose ipilimumab as first-line treatment of advanced/metastatic non-small-cell lung cancer (NSCLC). We assessed the association of efficacy with programmed death ligand 1 (PD-L1) expression and tumor mutational burden (TMB). PATIENTS AND METHODS: Two hundred eighty-eight patients with previously untreated, recurrent stage IIIB/IV NSCLC received nivolumab 3 mg/kg every 2 weeks plus ipilimumab 1 mg/kg every 6 weeks. The primary end point was objective response rate (ORR) in patients with 1% or more and less than 1% tumor PD-L1 expression. Efficacy on the basis of TMB (FoundationOne CDx assay) was a secondary end point. RESULTS: Of treated patients with tumor available for testing, 252 patients (88%) of 288 were evaluable for PD-L1 expression and 98 patients (82%) of 120 for TMB. ORR was 30% overall and 41% and 15% in patients with 1% or greater and less than 1% tumor PD-L1 expression, respectively. ORR increased with higher TMB, plateauing at 10 or more mutations/megabase (mut/Mb). Regardless of PD-L1 expression, ORRs were higher in patients with TMB of 10 or more mut/Mb (n = 48: PD-L1, ≥ 1%, 48%; PD-L1, < 1%, 47%) versus TMB of fewer than 10 mut/Mb (n = 50: PD-L1, ≥ 1%, 18%; PD-L1, < 1%, 5%), and progression-free survival was longer in patients with TMB of 10 or more mut/Mb versus TMB of fewer than 10 mut/Mb (median, 7.1 v 2.6 months). Grade 3 to 4 treatment-related adverse events occurred in 29% of patients. CONCLUSION: Nivolumab plus low-dose ipilimumab was effective and tolerable as a first-line treatment of advanced/metastatic NSCLC. TMB of 10 or more mut/Mb was associated with improved response and prolonged progression-free survival in both tumor PD-L1 expression 1% or greater and less than 1% subgroups and was thus identified as a potentially relevant cutoff in the assessment of TMB as a biomarker for first-line nivolumab plus ipilimumab.

Notch3 functions as a regulator of cell self-renewal by interacting with the ß-catenin pathway in hepatocellular carcinoma.

The Notch signaling pathway plays a role in cell proliferation, differentiation. Emerging data have revealed aberrant Notch3 expression in hepatocellular carcinoma (HCC). However, whether Notch3 plays a role in tumorigenesis or tumor progression is unclear. In this study, we found that over 71.8% of the cases studied had high Notch3 expression levels (n = 32); Notch3 expression positively correlated with alpha-fetoprotein (AFP) levels (p = 0.0311) and negatively correlated with the differentiation grade (p = 0.042). We demonstrated that the patients with higher levels of Notch3 expression commonly had a poor prognosis. We discovered that Notch3 expression is inversely correlated with ß-catenin content but positively associated with the protein level of Nanog. In parallel, we found that Notch3 attenuation resulted in the upregulation of ß-catenin and the downregulation of Nanog in the hepatoma cell lines QGY7701 and HepG2. The downregulation of Notch3 enhanced the sensitivity to cisplatin in the QGY7701 and HepG2 cells and inhibited the ability of QGY7701 cells to form tumors. The Notch3-positive cells had higher levels of aldehyde dehydrogenase (ALDH) activity, and a tendency to differentiate into Notch3-negative cells. In conclusion, our study demonstrated that Notch3 plays a role in modulating the stemness of tumor cells via the inactivation of the Wnt/ß-catenin pathway.

Diagnostic evaluation of ductal carcinoma in situ of the breast: ultrasonographic, mammographic and histopathologic correlations.

The aims of the study described here were to illustrate the spectrum of ultrasonographic features of ductal carcinoma in situ (DCIS) and to evaluate the ability of ultrasonography (US) to predict the grade and recurrence of DCIS on the basis of mammographic and histopathologic findings. We retrospectively evaluated the ultrasonographic features of 129 DCIS lesions from 127 consecutive women and compared these with their mammographic and histopathologic features. The mean size of DCISs on ultrasonography and mammography (MMG) was 3.67 ± 1.40 and 4.00 ± 1.74 cm, respectively, which do not differ statistically (p = 0.09). Despite the statistical difference in Breast Imaging Reporting and Data System (BI-RADS) classification on US and MMG (p = 0.000), the median BI-RADS classification is category 4c on both US and MMG (p = 0.01). There was no statistically significant difference in the distribution of microcalcification on MMG and US. Clusters <5 mm in greatest diameter are easily seen on MMG. At US, a scattered/linear distribution on MMG had a higher level of visibility than clustered distribution on MMG. The correlation between tumor size and DCIS with micro-invasion evaluated using US is higher than that obtained using MMG (p = 0.001 and 0.024, respectively). When US was used for the detection of DCIS, diagnostic accuracy was significantly associated with higher Van Nuys groups, the presence of micro-invasion and comedo carcinoma (p = 0.000, 0.022 and 0.011, respectively). However, mammographic diagnostic accuracy was found not to associate with higher Van Nuys groups, the presence of micro-invasion and comedo carcinoma (p = 0.054, 0.093 and 0.256, respectively). Ultrasonography may play an important role both in detecting DCIS and in evaluating its histopathologic features. Detection of DCIS using MMG alone may be suboptimal for patients with dense breasts, especially among Chinese women.

[Post-transplant cell immune reconstitution in humanized NOD/SCID mice].

OBJECTIVE: To investigate the cell immune reconstitution in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice by the transplantation of human umbilical cord blood (HUCB) CD34⁺; cells. Methods CD34⁺; cells were isolated from HUCB by magnetic activated cell sorting (MACS), and then were transplanted into NOD/SCID mice following the irradiation of sublethal doses via the lateral tail vein. Human CD45⁺; CD3⁺; CD56⁺; cell populations in the peripheral blood of mice were dynamically analyzed by flow cytometry (FCM) 4, 6, 8 and 10 weeks after transplantation. After 10 weeks, the expression of human ALU gene was detected by PCR in the bone marrow of mice, and the expressions of human CD3⁺; CD56⁺; cells were examined by immunohistochemical staining in the spleen tissues. RESULTS: After irradiation, the nucleated cells and giant cells in the marrow cavity of NOD/SCID mice were reduced significantly or completely demolished. The effect of myeloablative pretreatment was ideal. Human CD45⁺; CD3⁺; CD56⁺; cells were found by FCM in the peripheral blood of all surviving mice in transplantation group 4, 6, 8, and 10 weeks after the transplantation. The population of the human lymphocytes varied over time, peaked at the 8th week, and remained at a high level later. At the 10th week, the human ALU sequence could be detected in the bone marrow of all surviving mice in transplantation group, and human CD3⁺; CD56⁺; cells could be observed in the spleen tissues. All mice which received no transplantation died within 2 weeks after irradiation. CONCLUSION: The hu-SRC-NOD/SCID model was successfully established in irradiation-induced NOD/SCID mice by the transplantation of HUCB CD34⁺; cells, and its cell immune system was effectively rebuilt.

Gray-scale ultrasonography combined with elastography imaging for the evaluation of papillary thyroid microcarcinoma: as a prognostic clinicopathology factor.

Ultrasonography (US) is the preferred imaging modality for papillary thyroid microcarcinoma (PTMC). The aim of this study was to evaluate the importance of gray-scale ultrasound combined with elastography to predict extrathyroidal extension and cervical lymph node (LN) metastasis in patients with PTMC. We retrospectively evaluated gray-scale ultrasonic and elastographic results from 119 consecutive cases of PTMC with 138 nodules and correlated the histopathological findings. The results indicated that pathological extrathyroidal extension was significantly associated with T staging on US, extrathyroidal extension on US, bilaterality on US, boundary, strain ratio and hard malignancy as measured with the Rago score. Central LN metastasis on pathology was significantly associated with central LN metastasis on US, lateral LN metastasis on US, multifocality on US and bilaterality on US. Lateral LN metastasis on US was significantly associated with lateral LN metastasis on pathology. On multivariate analysis, T staging on US, extrathyroidal extension on US and hard malignancy as measured with the Rago score were significantly associated with pathological extrathyroidal extension. Lateral LN metastasis on US and bilaterality on US were independent factors in predicting central LN metastasis on pathology. Lateral LN metastasis on US was the predictive factor for lateral LN metastasis on pathology. US should be helpful in the diagnosis of PTMC and in the evaluation of possible PTMC recurrence on US in routine clinical practice.

[Reconstitution of humoral immunity by the transplantation of human umbilical cord blood CD34+ cells into NOD/ SCID mice].

OBJECTIVE: To investigate the immune reconstitution by the transplantation of human umbilical cord blood CD34+ cells in the NOD/SCID mouse. METHODS: Mononuclear cells (MNC) were isolated from human fresh cord blood and CD34+ hematopoietic stem cells were selected by magnetic activated cell sorting method. The selected cells were transplanted via tail vein injection into 16 NOD/SCID mice after sublethal whole-body irradiation. Four mice were sacrificed respectively at 4th, 6th, 8th and 10th week after the transplantation, the harvested spleen and peripheral blood cells were used to cell phenotype analysis and humoral immune analysis, respectively. There were 14 mice in another two groups, 7 mice did not receive the transplantation after irradiation, 7 were used as blank control (no irradiation, no transplantation). RESULTS: The mice without transplantation all died within 2 weeks after irradiation. The survival rate of the mice with transplantation was 37.5% at 6th week after the irradiation, while the survival rate of blank control was 100%. At 4th, 6th, 8th and 10th week, the percentage of human CD45+ cells in transplantation group were 4.7 +/- 1.23, 9.22 +/- 2.07, 12.34 +/- 2.38, 8.14 +/- 2.36, respectively, and the percentage of CD19+ B lymphocytes were 1.07 +/- 0.50, 2.17 +/- 0.95, 3.34 +/- 0.90, 1.67 +/- 0.90, respectively. 10 weeks after the transplantation, human CD19+ B lymphocytes distribution were found in the transplanted mice spleen. CONCLUSION: The human-mouse chimeric immune model can be built in irradiated NOD/ SCID mice by the transplantation of human cord blood CD34+ cells. CD34+ cell differentiation declined with time, which might be due to the lack of appropriate cytokines.

Inflammatory biomarkers and fatigue during radiation therapy for breast and prostate cancer.

PURPOSE: Biomarkers of radiation-induced behavioral symptoms, such as fatigue, have not been identified. Studies linking inflammatory processes to fatigue in cancer survivors led us to test the hypothesis that activation of the proinflammatory cytokine network is associated with fatigue symptoms during radiation therapy for breast and prostate cancer. EXPERIMENTAL DESIGN: Individuals with early-stage breast (n = 28) and prostate cancer (n = 20) completed questionnaires and provided blood samples for determination of serum levels of interleukin 1beta (IL-1beta) and IL-6 at assessments conducted before, during, and after a course of radiation therapy. Serum markers of proinflammatory cytokine activity, including IL-1 receptor antagonist and C-reactive protein, were examined in a subset of participants. Random coefficient models were used to evaluate the association between changes in cytokine levels and fatigue. RESULTS: As expected, there was a significant increase in fatigue during radiation treatment. Changes in serum levels of inflammatory markers C-reactive protein and IL-1 receptor antagonist were positively associated with increases in fatigue symptoms (Ps < 0.05), although serum levels of IL-1beta and IL-6 were not associated with fatigue. These effects remained significant (Ps < 0.05) in analyses controlling for potential biobehavioral confounding factors, including age, body mass index, hormone therapy, depression, and sleep disturbance. CONCLUSIONS: Results suggest that activation of the proinflammatory cytokine network and associated increases in downstream biomarkers of proinflammatory cytokine activity are associated with fatigue during radiation therapy for breast and prostate cancer.

[Pathological study of testicular injury induced by high power microwave radiation in rats].

OBJECTIVE: To explore the pathological characteristics and the dynamic change regularity of the testis induced by high power microwave (HPM) radiation. METHODS: One hundred and sixty-five male Wistar rats were exposed to 0, 3, 10, 30 and 100 mW/cm2 HPM radiation for five minutes, and changes of testicular morphology and teratogenic ratio of epididymal spermatozoa were observed through light microscope and electron microscope at 6 h, 1, 3, 7, 14, 28 and 90 d after radiation. RESULTS: Injury of testicular spermatogenic cells in rats might be induced by 3 to approximately 100 mW/cm2 HPM radiation, and the main pathological changes were degeneration, necrosis, shedding of spermatogenic cells, formation of multinuclear giant cells, decrease or loss of sperm and interstitial edema. Injury of spermatogenic cells underwent such phases as death and shedding, cavitation, regeneration and repair, characterized by being focalized, inhomogenous and phased. And the severity of pathological changes of the testis increased with power density. There was only scattered degeneration, necrosis, shedding of spermatogenic cells in the seminiferous tubule one day after 3 mW/cm2 radiation, and the pathological changes six hours after 10 mW/cm2 radiation was similar to those one day after 3 mW/cm2 radiation, but with the formation of multinuclear giant cells, and the above-mentioned pathological changes aggravated from one day to seven days after radiation. There was a significant increase in degeneration, necrosis, shedding of spermatogenic cells, as well as a significant decrease in spermatozoa and focal necrosis in simple seminiferous tubules six hours after 30 and 100 mW/cm2 radiation, and the subsequent changes were similar to those of 10 mW/cm2 radiation. There was a significant increase in teratogenic ratio of epididymal spermatozoa at 3 d, 1 to approximately 7 d, 6 h to approximately 7 d after 3, 10, 30 and 100 mW/cm2 microwave radiation respectively (P < 0.01 or P < 0.05). CONCLUSION: HPM radiation may cause injury of testicular spermatogenic cells in rats, which has a positive correlation to radiation dosage and time.

Effect of recombinant human interleukin-11 on expressions of interleukin-11 receptor alpha-chain and glycoprotein 130 in intestinal epithelium cell line-6 after neutron irradiation.

AIM: To explore the effect of recombinant human interleukin-11 (rhIL-11) on the expressions of interleukin-11 receptor alpha-chain (IL-11Ralpha) and an additional signal transducer glycoprotein 130 (gp130) in intestinal epithelium cell line-6 (IEC-6) after neutron irradiation. METHODS: Cultured IEC-6 cells were exposed to 4.0Gy neutron and treated with 100 ng/mL rhIL-11 12 h prior to or immediately after irradiation. The apoptosis and necrosis rates and expressions of IL-11Ralpha and gp130 were observed by flow cytometry, immunohistochemistry, Western blot and image analysis. RESULTS: The apoptosis rate of IEC-6 cells was increased by irradiation at 6 h (P < 0.01), IL-11 stimulation resulted in a decreased apoptosis rate in irradiated IEC-6 cells (P < 0.05). In normal control IEC-6 cells, intense immunoreactivity of IL-11Ralpha was located within the cell membrane and cytoplasm. The level of IL-11Ralpha expression significantly decreased at 6 h after irradiation (P < 0.01) and restored at 24 h after irradiation. In IEC-6 cells treated with both radiation and rhIL-11, the level of IL-11Ralpha expression was higher than that of irradiated cells (P < 0.05). When it came to gp130 protein, it was located in the cytoplasm of IEC-6 cells. After irradiation, we found a progressive decrease in the expression of gp130 protein (P < 0.05) in 48 h post-radiation, while in rhIL-11-stimulated cells, it came back to normal level at 24 h after irradiation and decreased at 48 h, but was still higher than that of only irradiated cells (P < 0.05). CONCLUSION: rhIL-11 can protect IEC-6 cells from neutron irradiation. The protective effect of rhIL-11 might be connected with its ability to up-regulate the expressions of specific ligand-binding subunit IL-11Ralpha and signal-transducing subunit gp130.

Mad2beta, an alternative variant of Mad2 reducing mitotic arrest and apoptosis induced by adriamycin in gastric cancer cells.

Mad2beta is an alternative splicing variant of spindle checkpoint gene mad2, which was previously found by us and was related to the drug resistance in gastric cancer cells. In this paper, we explored the molecular mechanisms that Mad2beta variant promoted the formation of multidrug resistance in gastric cancer cells. We found that Mad2beta variant was detected only in the two human drug resistant gastric cancer cell sublines SGC7901/VCR and SGC7901/ADR, and it did not appear in its parental cell line SGC7901 and other detected gastric cancer cell lines. Expressions of Mad2 mRNA and protein in SGC7901 cells transfected with Mad2beta, SGC7901/VCR and SGC7901/ADR were significantly lower than that in SGC7901 cells. Moreover, SGC7901 cells overexpressing Mad2beta variant became more resistant to adriamycin, vincristine and mitomycin by abrogating mitotic arrest and apoptosis. This suggests that expression of Mad2beta variant decreases the relative expression of efficient MAD2, which may help gastric cancer cells to develop the phenotype of multidrug resistance.

[The expression of TNF-alpha in the intestine of mice irradiated by neutron and gamma rays].

AIM: To study the expression of tumor necrosis factor alpha (TNF-alpha) in the intestine of mice irradiated by neutron and gamma rays. METHODS: 350 male BALB/c mice were irradiated with neutron and gamma rays of different doses, and sacrificed at 6 and 12 hours, 1, 2, 3, 4, 5, 7, 10, 14, 21 and 28 days after irradiation. The TNF-alpha in the mice intestinal tissue was detected by means of immunohistochemical staining and image analysis. RESULTS: In normal control mice, TNF-alpha was expressed in the cytoplasm of macrophages in intestinal villus interstitium, submucosa and lymph tissue. After 2.5Gy neutron radiation, TNF-alpha was decreased progressively within 2 days, increased obviously in macrophages and crypt cells during the 3rd-7th day, reached the peak at the 5th day and recovered to normal level at the 14th day. TNF-alpha was decreased progressively within 4 days after 4.0 and 5.5Gy neutron and 12Gy gamma ray irradiation. TNF-gamma was increased obviously in 6-12 hours, decreased on the first day, increased at the 2nd-5th day, peaked at the third day and recovered at the 10th day after 5.5Gy gamma ray irradiation. CONCLUSION: Neutron and gamma ray radiation induce different expression profile of endogenous TNF-gamma in small intestine, which may be related with the pathologic courses of irradiation-induced damage and repair of intestine.

[Changes of the expression of beta1-adrenergic receptor and M2-muscarinic acetylcholine receptor in rat hearts after high power microwave radiation].

OBJECTIVE: To investigate the effect of high power microwave (HPM) radiation on the expression of beta(1)-adrenergic receptor (beta(1)-AR) and M(2)-muscarinic acetylcholine receptor (M(2)-AchR) in cardiomyocytes. METHODS: S-band HPM device of mean power density 2 approximately 90 mW/cm(2) was used to irradiate 150 healthy Wistar male rats. Immunohistochemistry and image analysis were used to study the pathological characteristics of heart tissue and the expression of beta(1)-AR and M(2)-AchR. RESULTS: Radiation of over 10 mW/cm(2) made myocardial fibers disordered in arrangement, degeneration even sarcoplasm condensation, Pace cells necrosis, and Purkinje cells lysis in a dose-dependent manner (r = 0.968, P < 0.05). beta(1)-AR expression in endocardium, membrane and cytoplasm of myocardium of left ventricle was increased on d1 after radiation, peaked on d3 (P < 0.05) and recovered on d14. M(2)-AchR expression was peaked on d1 (P < 0.01) and recovered on d14. CONCLUSION: Certain degree intensity of HPM radiation may cause heart injury, and increased expressions of beta(1)-AR and M(2)-AchR, which may play an important role in the pathophysiology of heart injury induced by HPM.

[Effect of a hypothetical gene Af116609 on multi-drug resistance of gastric cancer cells].

OBJECTIVE: To investigate the effect of gene Af116609 on gastric cancer multi-drug resistance (MDR) by introducing it into gastric cancer multi-drug resistant (MDR) cell line SGC7901/VCR. METHODS: Gene Af116609 was cloned from SGC7901/VCR by RT-PCR and its differential expression between gastric cancer MDR cells and its parental cells was displayed by Northern blot. The gene was introduced to gastric cancer cells by transfection of recombinant eukaryotic expression vector by electroporation. MTT assay in vitro was applied to investigate its effect on multi-drug resistance phenotype of gastric cancer cells. RESULTS: The full length CDS of gene Af116609, as long as 327 bp, was cloned from gastric cancer MDR cell line SGC7901/VCR and its sequence was coincident with the hypothetical gene Af116609 in GenBank. It was overexpressed in MDR cells than its parental cells at mRNA level. In the MTT assay in vitro, the drug sensitive cells transfected with sense eukaryotic expression vector showed upregulated targeted gene, with increased resistance to vincristine, 5-fliorouracil and arabinoside, and decreased resistance to adriamycin, but no influence on resistance to methotrexate. However, the drug resistant cells transfected with anti-sense eukaryotic expression vector, showed down regulated targeted gene, with less resistance to all the five anticancer drugs to different degrees. CONCLUSION: Gene Af116609 is related to MDR phenotype of gastric cancer cells and may become a candidate molecular target to reverse the MDR of gastric cancer.

Regulation of drug sensitivity of gastric cancer cells by human calcyclin-binding protein (CacyBP).

BACKGROUND: Calcyclin-binding protein (CacyBP) was previously identified as an upregulated gene in a multidrug-resistant gastric cancer cell line, SGC7901/ADR, compared to its parental cells, SGC7901, by subtractive hybridization. The aim of this study was to explore the role of CacyBP in multidrug resistance (MDR) in gastric cancer cells. METHODS: The cDNA encoding CacyBP was generated by reverse-transcription-polymerase chain reaction (RT-PCR), and mouse antisera against CacyBP was raised using recombinant CacyBP as the immunogen. The expression of CacyBP in gastric cancer cells was determined by Northern and Western blots. Sense and antisense vectors for CacyBP were introduced into SGC7901 and SGC7901/ADR cells, respectively. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was performed to evaluate the drug sensitivity of gastric cancer cells. Flow cytometry was employed to determine adriamycin accumulation and retention in gastric cancer cells. RESULTS: Northern and Western blots demonstrated upregulation of CacyBP in SGC7901/ADR cells compared to SGC7901 cells. SGC7901-CacyBP and SGC7901/ADR-anCacyBP cells were prepared, in which CacyBP was genetically increased and decreased, respectively. As compared with SGC7901, SGC7901-CacyBP cells exhibited significantly increased ( P < 0.01) IC(50) values for vincristine, adriamycin, and 5-fluorouracil. Meanwhile, as compared with SGC7901/ADR, SGC7901/ADR-anCacyBP cells exhibited significantly decreased ( P < 0.01) IC(50) values for these three drugs. SGC7901-CacyBP and SGC7901/ADR-anCacyBP cells displayed no obvious difference ( P > 0.05) in intracellular adriamycin content compared to their corresponding parental cells. CONCLUSIONS: Upregulation of CacyBP is associated with MDR in gastric cancer cells. CacyBP could regulate the responses of gastric cancer cells to chemotherapy. But the underlying mechanisms of CacyBP-related MDR need further identification.