Background : The expressions of osteopontin (OPN), zonula occludens-1 (ZO-1) and E-cadherin, known as cell adhesion-associated substances, were examined in adenoma and adenocarcinoma of the colon. The relationship of their expressions with clinicopathologic factors was examined to investigate the roles of these proteins in the development, invasion or metas- tasis of colon adenocarcinoma. Methods : The expressions of OPN, ZO-1, and E-cadherin were examined in 54 cases of adenoma and 67 cases of adenocarcinoma of the colon by immunohistochemical staining. Results : The expression of OPN in colon adenocarcinoma correlated with staging (p=0.012) and distant metastasis (p=0.021). The expression of ZO-1 was closely related with tumor cell differentiation (p<0.001), and the reduced expression of E-cadherin was associated with tumor cell differentiation (p=0.05) and lymph node metastasis (p<0.001). Co-expression of ZO-1 and E-cadherin was significantly associated with tumor cell differentiation, and the expressions of ZO-1 and E-cadherin were reduced or lost in all cases (5 cases) of poorly differentiated adenocarcinoma. Conclusions : Our data suggest that OPN is involved in the process of invasion and metastasis of colon adenocarcinoma, and ZO-1- and E-cadherin-mediated cell adhesion may play an important role in the differentiation of colon adenocarcinoma.
Adenocarcinoma* ; Adenoma* ; Cadherins* ; Cell Adhesion ; Cell Differentiation ; Colon* ; Lymph Nodes ; Neoplasm Metastasis ; Osteopontin* ; Zonula Occludens-1 Protein

OBJECTIVETo investigate the clinical significance of ZO-1 gene methylation level in MDS progression in order to provide a theoretical basis for evaluating progrosis of MDS patients.

METHODSThe methylation specific PCR (MS-PCR) was performed to evaluate the ZO-1 gene methylation status in bone marrow samples of normal persons as control (NC). MDS and AML patients, the bisulfite sequencing PCR (BSP) was applied to detect the ZO-1 gene methylation status in serial bone marrow samples of MDS-RA, MDS-RAEB and AML stages of a MDS patients.

RESULTSThe possitive rate of ZO-1 gene methylation in samples of NC, MDS and AML patients displayed significant difference; in sample of NC group the positive of ZO-1 gene methylation was not observed, but the positive rate of ZO-1 gene methylation in samples of AML patients was highest (65.0%), the proportion of ZO-1 gene methylation in myeloid blast count of MDS/AML patients was higher (P=0.000). The serial samples in one MDS patient showed that along with progress of disease, the positive rate of ZO-1 gene methylation in MDS-RA, MDS-RAEB and AML stages was found to be obvious different (P=0.000), the positive rate of ZO-1 gene methylation in AML stage was highest (64.65%).

CONCLUSIONThe high methylation in promoter region of ZO-1 gene has been found in MDS/AML patients, and along with clonal proliferation, the positive rate of ZO-1 methylation and positive froguency of methylation sites increase graduatly which suggests that the MDS progresses in a certain degree, and the ZO-1 gene methylation level may be used as an new indicator for monitoring desease progression from MDS to AML.

Bone Marrow ; DNA Methylation ; Disease Progression ; Humans ; Leukemia, Myeloid, Acute ; Methylation ; Myelodysplastic Syndromes ; Polymerase Chain Reaction ; Zonula Occludens-1 Protein

OBJECTIVETo observe the effect of SIRT1 on intestinal barrier function of epithelial Caco-2 cells under hypoxia and investigate its mechanism.

METHODSCaco-2 cells were randomly divided into three groups: normoxia group (Nx), hypoxia group (Hx,1%O2 for 6 h) and hypoxia plus 40 μmol/L Resveratrol (agonist of SIRT1) group (Hx+Res). Transepithelial electrical resistance (TER) was determined. mRNA and protein expressions of SIRT1 and tight junctions (ZO-1, Occludin, Claudin-1) were examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.

RESULTSBoth mRNA and protein expressions of SIRT1 were significantly reduced in Hx group as compared with Nx group (0.40±0.02 vs. 0.70±0.07, P=0.001; 0.37±0.03 vs. 0.76±0.03, P=0.001). The mRNA and protein expressions of SIRT1 were significantly increased in Hx+Res group as compared with Hx group(0.50±0.02 vs. 0.40±0.02, P=0.026; 0.54±0.02 vs. 0.37±0.03, P=0.011). The expression levels of ZO-1, Occludin and Claudin-1 in Hx group were lower than those in Nx group (P<0.05), however, pretreatment with Resveratrol could attenuate the decreased expression of above 3 molecules under hypoxia(P<0.05). TERs of Nx group, Hx group and Hx+Res group were (142±7) Ohm/cm(2), (94±3) Ohm/cm(2) and (119±7) Ohm/cm(2) respectively. Compare with the Nx group, the TER of Hx group was significantly decreased(P<0.05). TER of Hx+Res group was significantly increased compare with Hx group, but it was still significantly lower than that in Nx group(P<0.05).

CONCLUSIONSExpression of SIRT1 is significantly reduced under hypoxia. Activation of SIRT1 can maintain the epithelial barrier function through regulating the expression of tight junctions under hypoxia.

Caco-2 Cells ; Cell Hypoxia ; Claudin-1 ; metabolism ; Epithelial Cells ; metabolism ; Humans ; Intestinal Mucosa ; cytology ; Occludin ; metabolism ; Sirtuin 1 ; metabolism ; Zonula Occludens-1 Protein ; metabolism

OBJECTIVEThe purpose of this study was to assess weather the immortalized mouse brain endothelial cell line Bend.3 displays the comparative barrier characteristics as the primary brain microvascular endothelial cells (BEMC).

METHODSImmortalized mouse brain endothelial cell line, Bend.3 cells were cultured in transwell inserts and their restrictive characteristics were assessed by transendothelial electrical resistance (TEER) and horseradish peroxidase (HRP) permeability assays. Western blot and direct fluorescent staining methods were used to detect the tight junction protein expression and F-actin distribution.

RESULTSThe TEER in Bend.3 cells increased with the prolonged culture time and increased to 82.3+/-6.0 Omega cm2 10 days after culture, which was significantly higher than that 3 days after culture (37.3+/-3.1 Omega cm2; P<0.05). There were significant differences in the permeability rates for HRP 3 and 10 days after culture (4.3+/-0.20)% vs (2.2+/-0.05)% (P<0.05). Western blot indicated high level expression of tight junction proteins occludin and ZO-1 in Bend.3 cells 10 days after culture. F-actin was visualized around the cell membrane and presented scrobiculate linear fluorescence 10 days after culture.

CONCLUSIONSBend.3 cells have similar barrier characteristics to BEMC, and their barrier function may reach to the best effect 10 days after culture.

Actins ; analysis ; Animals ; Blood-Brain Barrier ; Cell Line ; Electric Impedance ; Endothelial Cells ; metabolism ; Horseradish Peroxidase ; metabolism ; Membrane Proteins ; analysis ; Mice ; Phosphoproteins ; analysis ; Zonula Occludens-1 Protein

OBJECTIVE: To observe the time-course expression of zonula occludens-1 (ZO-1) in cerebral cortex after traumatic brain injury (TBI). METHODS: The TBI model of mouse was established. The mice were divided in 1 h, 3 h, 6 h, 12 h, 24 h, 3 d, 7 d after TBI, sham and control groups. The permeability of the blood brain barrier was evaluated by measuring the extravasation of Evans blue (EB) dye. The expression of ZO-1 in cerebral cortex in the injured area was detected by Western blotting and immunohistochemistry. RESULTS: The extravasation of EB dye of injured cortex gradually increased from 1 h, peaked at 1-3 d and approximately decreased to normal at 7 d after TBI. Western blotting revealed that the expression of ZO-1 gradually decreased after 1 h, was at the lowest at 1-3 d, and then significantly increased after 7 d but was still lower than that of normal and sham groups. The result of immunohistochemistry showed that ZO-1 had strong expression in vessel of normal cortex, gradually decreased after TBI, and almost disappeared at 3 d after TBI and gradually recovered to normal level later. CONCLUSION The expression of ZO-1 in the injured cortex after TBI initially decreases and then increases. The negative correlation between ZO-1 expression and EB extravasation after TBI could be used as a new indicator for wound age estimation.
Animals ; Blood-Brain Barrier ; Blotting, Western ; Brain Injuries/physiopathology* ; Cerebral Cortex/metabolism* ; Immunohistochemistry ; Mice ; Permeability ; Tight Junctions/metabolism* ; Zonula Occludens-1 Protein/metabolism*

The study was aimed to identify a new leukemia related gene zo-1 from leukemia and to explore its mechanism in leukemia. Methylation specific PCR (MSP) was used for testing gene zo-1 methylation in leukemia cells. The gene zo-1 specific siRNA was designed according to its sequence, and transfected into THP-1 cell, and the cells were cultured for 48 hours before harvesting. The effect of zo-1 siRNA was monitored by RT-PCR. The cellular proliferation activity was assayed by CCK-8, the apoptosis was detected by Annexin-V-fluorescence in isothiocyanate (FITC) assay, and cell cycle was observed by propidium iodide (PI). The results indicated that the gene zo-1 in patients with acute leukemia was hypermethylated, while the gene zo-1 in healthy persons was unmethylated. The THP-1 cells with unmethylation of zo-1 gene promoter overexpressed the gene zo-1, while the Molt4 and HL-60 cells with hypermethylation of gene zo-1 promoter did not express the gene zo-1. The silenced zo-1 gene in Molt4 and HL-60 leukemia cell lines could be reactivated by demethylation treatment with 5-AZA-dC. The oligofectamine-transfected siRNA for zo-1 gene successfully inhibited the expression of gene zo-1 in THP-1 cells, but did not interfere with cell proliferation, cell cycle and apoptosis. It is concluded that gene zo-1 is a leukemia-related gene. Gene zo-1 in acute leukemia was hypermethylated, the methylation status of gene zo-1 regulates the expression of gene zo-1. Lack of gene zo-1 expression in THP-1 cells does not influence the cell proliferation, apoptosis and cell cycle, which suggests that the methylation of gene zo-1 may be involved in the genesis of acute leukemia, its mechanism is worthy to be studied.
CpG Islands ; DNA Methylation ; Gene Silencing ; HL-60 Cells ; Humans ; Leukemia ; genetics ; Membrane Proteins ; metabolism ; Phosphoproteins ; metabolism ; RNA, Small Interfering ; genetics ; Zonula Occludens-1 Protein

OBJECTIVETo explore ZO-1 gene expression and methylation in leukemia cells and the involvement of ZO-1 gene in leukemogenesis.

METHODSRestriction landmark genomic scanning (RLGS) was used to identify new leukemia related gene, and methylation specific PCR (MSP) for ZO-1 methylation status. ZO-1 specific siRNA was designed and prepared by in vitro transcription and transfected into K562 cells, the transfected cells were cultured for 48 hours before harvesting. The effect of ZO-1 siRNA was monitored by Northern blot. Cellular proliferation capacity was assayed by CCK-8, cell apoptosis by Annexin V-fluorescence in isothiocyanate (FITC) assay, and cell cycle by phosphatidylinositol (PI).

RESULTSThe intensified spots in RLGS gel were subjected to bioinformatics analysis and one of the candidate spots was proved to be ZO-1 gene. In fresh leukemia cells, Molt4 cells and HL-60 cells, ZO-1 was hypermethylated, causing it reduced or silenced. ZO-1 gene was highly expressed with no methylation in normal peripheral blood MNC and K562 cells. There was a good correlation between promoter methylation and the gene silence. The silenced gene can be re-activated by demethylation treatment with 5-AZA-dC in most leukemia cell lines. RNA interference for ZO-1 gene in K562 cells did not interfere with cell proliferation, cell cycle and apoptosis.

CONCLUSIONZO-1 gene methylation might be involved in the tumorigenesis of acute leukemia.

Animals ; DNA Methylation ; HL-60 Cells ; Humans ; K562 Cells ; Leukemia ; genetics ; pathology ; Membrane Proteins ; genetics ; metabolism ; Mice ; Phosphoproteins ; genetics ; metabolism ; Zonula Occludens-1 Protein

OBJECTIVETo observe the effect of 25-hydroxyvitamin D3 on the permeability and ZO-1 expression in normal human airway epithelial cells.

METHODSMTT assay was used to assess the viability of human airway epithelial cell line 16HBE following a 24-hour exposure to different concentrations of 25-hydroxy vitamin D3, and the transepithelial electrical resistance (TER) of the cell monolayer was measured using a Millicell-ERS voltohmmeter. Real-time quantitative RT-PCR was employed to determine the changes of ZO-1 mRNA expression in the cells following the exposures.

RESULTSExposure to 25-hydroxyvitamin D3 resulted in significantly increased permeability of 16HBE cells, but the exspression of ZO-1 showed no obvious changes. 25-hydroxyvitamin D3 at 4×10(-9) mol/L showed the strongest effect in increasing the permeability of cell monolayer.

CONCLUSION25-hydroxyvitamin D3 increases the permeability of normal bronchial airway epithelial cell monolayer in vitro, but this effect is not mediated by upregulation of ZO-1 expression.

Bronchi ; cytology ; metabolism ; Calcifediol ; pharmacokinetics ; pharmacology ; Cell Line ; Cell Membrane Permeability ; drug effects ; Epithelial Cells ; cytology ; metabolism ; Humans ; RNA, Messenger ; genetics ; metabolism ; Zonula Occludens-1 Protein ; genetics ; metabolism

Recently, several studies showed that gastrointestinal tract may be associated with pathophysiology of Parkinson's disease (PD). Intestine tight junction protein zonula occluden-1 (ZO-1) is an important component of intestinal barrier which can be degraded by matrix metallopeptidase 9 (MMP-9). In our previous study, a significant decline in ZO-1 was observed along with enhanced MMP-9 activity in the duodenum and distal colon of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. In this study, the protective effect of simvastatin on ZO-1 was investigated using an MPTP mouse model of PD. Seven days after the end of MPTP application, the expression level of ZO-1 was evaluated by immunohistochemistry. The protein expression levels of ZO-1 and MMP9 were detected by Western blotting. Meanwhile, MMP-9 activity was analyzed by gelatin zymography. MPTP treatment led to a decrease in the expression of ZO-1, which was accompanied by elevated MMP-9 activity. Treatment with simvastatin could partly reverse the MPTP-induced changes in ZO-1 expression and reduce MMP-9 protein and activity. Taken together, these findings suggest that simvastatin administration may partially reverse the impairment of ZO-1 induced by MPTP via inhibiting the activity of MMP9, fortify the impaired intestinal barrier and limit gut-derived toxins that pass across the intestinal barrier.
Animals ; Disease Models, Animal ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; pharmacology ; Mice ; Parkinson Disease ; metabolism ; Simvastatin ; pharmacology ; Zonula Occludens-1 Protein ; drug effects

OBJECTIVETo investigate the methylation status of zonula occludens-1 (ZO-1) gene promoter and its clinical significance in children with stage IV non-Hodgkin lymphoma (NHL) and to provide a basis for further etiological study and early diagnosis of this disease.

METHODSFifty-five children with a confirmed diagnosis of stage IV NHL (40 cases of T-NHL and 15 cases of B-NHL) were selected as the case group, and 20 children with diseases other than hematologic malignancies were selected as the control group. Bone marrow samples were collected from these subjects. Methylation-specific PCR (MS-PCR) was applied to evaluate the methylation status of ZO-1 gene promoter, and the integrated optical density (IOD) was determined. RT-PCR was used to measure the mRNA expression of ZO-1.

RESULTSMS-PCR showed that the methylated bands of ZO-1 gene promoter were found in 39 (70.9%) of 55 patients in the case group before treatment, while no ZO-1 gene promoter methylation was detected in the control group. With close tracking of 47 cases in the study group, consisting of 32 cases of T-NHL and 15 cases of B-NHL, the rates of ZO-1 gene promoter methylation prior to treatment were 72% and 67%, respectively, (P>0.572). The cases of T-NHL and B-NHL showed no significant changes in methylation rate in the early and middle phases of chemotherapy (P>0.05), but they showed significant changes in methylation rate in the late phase of chemotherapy (P<0.05). RT-PCR showed that the NHL cases carrying methylated ZO-1 gene had no mRNA expression of ZO-1, while all children in the control group had mRNA expression of ZO-1. There was no linear relationship between the total number of peripheral blood leukocytes and ZO-1 gene IOD (r=0.093, P=0.575); a positive correlation was found between the number of malignant cells in bone marrow and ZO-1 gene IOD (r=0.669, P<0.001).

CONCLUSIONSZO-1 gene shows a hypermethylation status in children with NHL, and the methylation level is positively correlated with the number of malignant cells in bone marrow. ZO-1 may be used as a novel molecular marker in early diagnosis, outcome assessment, prognostic evaluation, and detection of minimal residual disease.

Adolescent ; Child ; Child, Preschool ; DNA Methylation ; Female ; Humans ; Infant ; Lymphoma, Non-Hodgkin ; genetics ; Male ; Promoter Regions, Genetic ; Zonula Occludens-1 Protein ; genetics