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

This paper is aimed to investigate the effect of fluid shear stress on the tight junction of laryngeal squamous carcinoma (Hep2) cells and to explore the potential molecular mechanism. Hep2 cells were selected and subjected to the fluid shear stress of 1.4 dyn/cm2 for different time, respectively. The morphological changes of Hep2 cells under shear stress were observed using inverted microscope. The cell-cell junctions were examined by transmission electron microscope (TEM). The expressions of tight junction proteins (including Occludin, Claudin-5 and ZO-1) and the distribution of Claudin-5 were examined by Western blot assay and laser scanning confocal microscope, respectively. The results indicated that Hep2 cells turned to spindle-like shapes after exposed to shear stress, and showed the trend of the recovering to original shapes when the shear stress was cancelled. The cell-cell junctions were tight under the shear flow condition, and the permeability was reduced under the condition of 1.4 dyn/cm shear flow. The expressions of tight junction proteins were enhanced with increased duration of shear flow, but reduced after removing shear flow. The result of Claudin-5 expression by immufluorescence assay was consistent with that by Western blot. The Claudin-5 mainly distributed in the cytoplasm under static condition, while it located at the intercellular after shear flow stimulation, and it appeared intercellular and cytoplasm after stopping shear flow stimulation. Therefore, it can be concluded that shear stress changes the morphology of laryngeal squamous carcinoma Hep2 cells, and upregulates the tight junction.
Blotting, Western ; Carcinoma, Squamous Cell ; pathology ; Claudin-5 ; metabolism ; Hep G2 Cells ; Humans ; Laryngeal Neoplasms ; pathology ; Occludin ; metabolism ; Stress, Mechanical ; Tight Junctions ; Zonula Occludens-1 Protein ; metabolism

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

This study was purposed to investigate the difference of zo-1 gene promoter methylation between healthy individuals and acute leukemia patients. BS-PCR method was used to detect the status of zo-1 gene methylation in healthy individuals, acute leukemia patients and leukemic cell line NB4 cells. The results showed that zo-1 gene was hypomethylated in bone marrow samples from healthy individuals (1.9%). In newly diagnosed AL and relapsed patients, the rate of zo-1 gene methylation was 93.2% and 66.9% respectively, while it was 16.4% in AL patients in complete remission, which was much higher than that in healthy individuals. There was significant difference between them. It is concluded that as compared with healthy individuals, zo-1 gene in acute leukemia patients is hypermethylated and with different degrees in various phases of leukemia. Analysis of zo-1 gene methylation status may be useful to monitor the development of acute leukemia.
Acute Disease ; Cell Line, Tumor ; DNA Methylation ; Humans ; Leukemia ; genetics ; Membrane Proteins ; genetics ; Phosphoproteins ; genetics ; Promoter Regions, Genetic ; 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

After a series of studies, we found that the intestinal permeability was increased, tight junction protein (zonula occluden-1) obviously decreased and redistributed, accompanied by an increase in expression of myosin light chain (MLC) phosphorylation in severely burned rats. After using inhibitor of MLC kinase (ML-9 2 mg/kg) or of Rho-associated kinase (Y-27632 2 mg/kg), above-mentioned changes could be alleviated. Therefore, to regulate the MLC phosphorylation of tight junction protein and perijunctional actin-myosin ring may be one of the key links to lessen the intestinal epithelium permeability after burn injury.
Animals ; Burns ; metabolism ; Intestinal Mucosa ; metabolism ; Intestines ; metabolism ; Membrane Proteins ; metabolism ; Myosin Light Chains ; metabolism ; Permeability ; Phosphoproteins ; metabolism ; Phosphorylation ; Rats ; Zonula Occludens-1 Protein ; rho-Associated Kinases ; metabolism

OBJECTIVETo explore the effects of acrylamide on the permeability of blood cerebrospinal fluid barrier (BCB) and tight junction protein ZO-1 of choroid plexus in rats and to provide a theoretical basis for explaining the mechanism of nerve injury induced by acrylamide.

METHODSThirty two male Sprague-Dawley rats were randomly divided into ACR and control groups. ACR group was exposed to 20 mg/kg ACR daily for 5 days a week by intraperitoneal injection (i.p.) for 4 weeks. Control group was exposed to normal saline. The neurobehavioral tests (including sensatory and motor functions) were performed every week. At the end of exposure, Evan blue (EB) and Sodium fluorescein (NaFI) content in rat CSF were detected for determining the BCB permeability, Real-time PCR was used to measure the expression levels of ZO-1 mRNA in the epithelium cells of choroid plexus, and laser scanning confocal microscope (LSCM) was utilized to observe the distribution of ZO-1 protein.

RESULTSNeurobehavioral tests showed that the tail-flick latencies of ACR group were 27.77% and 53.71% as long as control group in the 3rd week and 4th week, respectively (P < 0.05). The hind lamb splay distances of ACR group were 131.76% and 153.77% as long as control group in the 3rd week and 4th week, respectively (P < 0.05). Evan blue (EB) and Sodium fluorescein (NaFI) content of ACR group were significantly higher than those of control group (P < 0.05). In the 4th week, the expression level of ZO-1 mRNA in ACR group was 0.21 +/- 0.07, which was significantly lower than that (0.31 +/- 0.11) in control group (P < 0.05). In the 4th week, the ZO-1 protein expression level of choroid plexus in ACR group was significantly lower than that in control group (P < 0.05).

CONCLUSIONAcrylamide could increased the BCB permeability of rats, which may be involved in the central nervous injury induced by ACR.

Acrylamide ; toxicity ; Animals ; Blood-Brain Barrier ; drug effects ; Choroid Plexus ; metabolism ; Male ; Permeability ; drug effects ; Rats ; Rats, Sprague-Dawley ; 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