IQ domain GTPase-activating protein 1 mediates the process of injury and repair in bronchial epithelial cells.

The process of injury and repair in airway epithelium involves cell spreading and migration followed by cell proliferation. IQ domain GTPase-activating protein 1 (IQGAP1) acts in a series of cell processes, but has not been clarified in lung epithelial cells. In this study, a widely used model of injury and repair in vitro by scratching bronchial epithelial cells (BECs) was utilized to investigate the function of IQGAP1. The results showed that IQGAP1 was abundant in BECs of mouse, rat, pig and human. IQGAP1 was colocalized with tubulin cytoskeleton, but was destroyed by nocodazole, a microtubule disassembly reagent. IQGAP1 mRNA and protein expressions increased at 6-9 h after scratching. In addition, overexpression of IQGAP1 translocated ß-catenin from the cytoplasm into the nucleus and activated the Tcf/Lef signal. Scratching altered the associations of IQGAP1 with ß-catenin, adenomatous polyposis coli (APC) and cytoplasmic linker protein-170 (CLIP-170). Silencing IQGAP1 expression by small interference RNA (siRNA) blocked the wound closure. It is concluded that IQGAP1 signal is involved in the wound closure of BECs induced by scratching.

[Mutations of p53 gene in 41 cases of human brain gliomas].

BACKGROUND & OBJECTIVE: p53 gene plays an important role in regulating cell cycle, maintaining completeness of cellular genomes, inducing cell differentiation and apoptosis. p53 gene mutation occurs usually in gliomas, especially astrocytomas. This study was to investigate p53 gene mutation and its correlation to the development of glioma. METHODS: p53 gene mutation in 41 specimens of human gliomas was analyzed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing. RESULTS: p53 gene mutations were found in 17 (41.5%) of the 41 specimens of gliomas. The p53 mutations located on exons 5-8: 7 cases (41.2%) on exon 5; 1 (5.9%) on exon 6; 4 (23.5%) on exon 7; 5 (29.4%) on exon 8. The mutation rate of p53 gene was significantly higher in grade III-IV gliomas than in grade I-II gliomas (P<0.01). DNA sequencing indicated that the p53 gene mutations were point mutations and deletions of exons in the 17 positive cases, including 13 cases (76.5%) of missense mutation, 2 cases (11.8%) of samesense mutation, and 2 cases (11.8%) of frame shift mutation. G_A and A-->G were major base mutations (55.6%). CONCLUSIONS: p53 mutations always locate on exons 5 and 8. Missense mutation is major mutant type of p53 gene. p53 mutation plays an important role in the development and malignant transformation of human gliomas.

[Activation and prognostic significance of AKT, NF-kappaB and STAT3 in breast cancer with lymph node metastasis and estrogen receptor expression].

BACKGROUND & OBJECTIVE: Being downstream targets of the phosphatase and tensin homolog deleted on chromosome ten (PTEN) and epidermal growth factor receptor (EGFR) pathways, serine/threonine kinase AKT, nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription-3 (STAT3), play important roles in cell proliferation, apoptosis and oncogenesis. This study was to investigate the activation and prognostic values of AKT, NF-kappaB and STAT3 in breast cancer with lymph node metastasis and estrogen receptor (ER) expression. METHODS: The expression of phosphatized AKT (p-AKT), NF-kappaB (p-NF-kappaB) and STAT3 (p-STAT3), and the expression of EGFR, PTEN, HER-2, and Ki67 in tissue samples from 130 breast cancer patients with lymph node metastasis and ER expression were detected by immunohistochemistry. RESULTS: The activity of AKT (p-AKT) and NF-kappaB (p-NF-kappaB) were correlated to HER-2 overexpression (P=0.023 and P=0.017) and histological grade of breast cancer (P=0.035 and P=0.004). The expression of p-AKT, p-NF-kappaB and p-STAT3 had no correlation to tumor size, EGFR overexpression, and proliferation index assessed by Ki67. The expression of p-AKT was positively correlated to that of p-NF-kappaB (r=0.43, P<0.001) and negatively correlated to that of p-PTEN (r=-0.20, P=0.002). The overexpression of p-AKT and p-NF-kappaB were significantly related to shorter survival (P=0.002 and P=0.003). The up-regulation of p-NF-kappaB expression was also related to enhanced risk of recurrence and metastasis (P=0.006). Cox multivariate analysis showed that the expression of p-AKT and p-NF-kappaB were correlated to shorter overall survival (OS) (P=0.017 and P=0.008) and disease-free survival (DFS) (P=0.005 and P=0.012), while the expression of p-STAT3 had no correlation to OS (P=0.332) and DFS (P=0.237). CONCLUSIONS: The activation of AKT and NF-kappaB, but not STAT3, significantly contributes to the progression of breast cancer. Activated AKT and NF-kappaB may indicate poor prognosis of breast cancer with lymph node metastasis and ER expression.

Synergistic anti-tumor effect of recombinant chicken fibroblast growth factor receptor-1-mediated anti-angiogenesis and low-dose gemcitabine in a mouse colon adenocarcinoma model.

AIM: To evaluate whether the combination of recombinant chicken fibroblast growth factor receptor-1 (FGFR-1) protein vaccine (cFR-1) combined with low-dose gemcitabine would improve anti-tumor efficacy in a mouse CT26 colon adenocarcinoma (CT26) model. METHODS: The CT26 model was established in BABL/c mice. Seven days after tumor cell injection, mice were randomly divided into four groups: combination therapy, cFR-1 alone, gemcitabine alone, and normal saline groups. Tumor growth, survival rate of tumor-bearing mice, and systemic toxicity were observed. The presence of anti-tumor auto-antibodies was detected by Western blot analysis and enzyme-linked immunospot assay, microvessel density (MVD) of the tumors and tumor cell proliferation were detected by Immunohistochemistry staining, and tumor cell apoptosis was detected by TdT-mediated biotinylated-dUTP nick end label staining. RESULTS: The combination therapy results in apparent decreases in tumor volume, microvessel density and tumor cell proliferation, and an increase in apoptosis without obvious side-effects as compared with either therapy alone or normal control groups. Also, both auto-antibodies and the antibody-producing B cells against mouse FGFR-1 were detected in mice immunized with cFR-1 vaccine alone or with combination therapy, but not in non-immunized mice. In addition, the deposition of auto-antibodies on endothelial cells from mice immunized with cFR-1 was observed by immunofluorescent stain-ing, but not on endothelial cells from control groups. Synergistic indexes of tumor volume, MVD, cell apoptosis and proliferation in the combination therapy group were 1.71 vs 1.15 vs 1.11 and 1.04, respectively, 31 d after tumor cell injection. CONCLUSION: The combination of cFR-1-mediated anti-angiogenesis and low-dose gemcitabine synergistically enhances the anti-tumor activity without overt toxicity in mice.

[Spatial-temporal distribution of glycogen synthase kinase 3beta and adenomatous polyposis coli protein are involved in the injury and repair of airway epithelial cells induced by scratching].

To investigate the roles of glycogen synthase kinase 3beta (GSK3beta) and adenomatous polyposis coli (APC) protein in wound repair of airway epithelial cells (AECs), we established a wound model of airway epithelium in vitro. Then the following tests were undertaken: (1) Western blot was used to detect the levels of total GSK3beta and phosphorylated GSK3beta in human bronchial epithelial (16HBE) cells; (2) The localizations of APC protein was observed by using immunofluorescence technique; (3) Immunoprecipitation was used to investigate the relationship between APC protein and GSK3beta during the repair of 16HBE cells. The results were as follows: (1) The level of phosphorylated GSK3beta increased 0.5 h after scratching (P<0.05), reached a maximum at 6 h (P<0.05), and maintained until 12 h, while the total level of GSK3beta remained constant; (2) Results of immunofluorescence study showed that APC protein clustered with tubulin in the region of the migrating leading cells 6 h after scratching, which was dissimilar with that in the cells 0 h after scratching; (3) GSK3beta and APC protein were immunoprecipitated and analysed on SDS-PAGE. We found that GSK3beta and APC protein were precipitated, indicating that the two proteins existed in a complex. After scratching, dissociation of the two proteins occurred. Taken together, we conclude that scratching caused a decrease in phosphorylation of GSK3beta, and that reduced phosphorylation of GSK3beta promoted APC protein to bind to the plus ends of microtubules and stabilize the growing ends. These observations suggest that APC protein and GSK3beta may synergistically play an important role in the repair of airway epithelium.

Glycogen synthase kinase 3beta induces cell cycle arrest in a cyclin D1-dependent manner in human lung adenocarcinoma cell line A549.

The effect of glycogen synthase kinase 3beta (GSK3beta) has been repeatedly implicated in cell proliferation, but studies on the effect of GSK3beta in different cell lines with different stimuli have drawn different conclusions. To investigate the direct effect of GSK3beta on cell growth in human lung adenocarcinoma cell line A549, we changed its activity by transient transfection with two kinds of GSK3beta mutant plasmids, constitutively active form S9A-GSK3beta and dominant negative form KM-GSK3beta. Twenty-four hours later, cell counting, flow cytometry and Western blot detection were made respectively. The results showed that enhancing GSK3beta activity caused a decrease in cell number, as well as a higher percentage of cells at G(1) phase. Further, the expression of cyclin D1 was down-regulated by GSK3beta. Taken together, our observations suggest that GSK3beta may induce G(1) cell cycle arrest in a cyclin D1-dependent fashion and therefore possibly plays a growth-inhibitory role in A549 cells.

Role of glycogen synthase kinase 3 in squamous differentiation induced by cigarette smoke in porcine tracheobronchial epithelial cells.

Epidemiological evidence suggests that cigarette smoke induces squamous metaplasia in human tracheobronchial epithelium that can progress to lung squamous carcinoma. But it is not well understood how tracheobronchial epithelial cells transduce the signals that mediate cigarette smoke-induced squamous differentiation or squamous metaplasia. In the present study, we found that in vitro cigarette smoke components notably inhibited glycogen synthase kinase 3 (GSK3) and induced the expression of involucrin, a marker of squamous differentiation. The inactivation of GSK3 by two highly selective inhibitors, lithium and SB216763, also significantly enhanced involucrin expression in cultured porcine tracheobronchial epithelial cells (PTBECs). Moreover, we demonstrated that cigarette smoke components significantly promoted activator protein-1 (AP-1) binding activities to the upstream regulatory region of involucrin gene, and similar results were observed by further studies through using GSK3 inhibitors to imitate the effects of cigarette smoke components. Taken together, we conclude that GSK3 is involved in involucrin expression induced by cigarette smoke in PTBEC probably via negatively regulating AP-1 activity, implying a possible mechanism responsible for squamous differentiation induced by cigarette smoke.

[Dynamic changes of adenomatous polyposis coli protein and glycogen synthase kinase 3beta in the repair of the injured airway epithelial cells in smoking mice].

To investigate the roles of adenomatous polyposis coli (APC) protein and glycogen synthase kinase 3beta (GSK3beta) of smoking murine model in the repair of the injured airway epithelial cells (AECs) in different stages, 30 male Kun-Ming mice were randomly divided into two groups, the control group and the smoking group. There were 24 mice in smoking group, and 6 animals were separately killed at the end of the 1st, 4th, 8th and 12th week after smoking. Then the following tests were undertaken: (1) HE staining of lung section to observe the morphological changes of the bronchi in the smoking mice. (2) Immunohistochemical staining of APC protein and GSK3beta in the AECs. (3) Western blot was used to detect the levels of APC protein, GSK3beta and phosphorated GSK3beta (p-GSK3beta) in pulmonary tissue. (4) Observing the localizations of APC protein and GSK3beta in the AECs by immunofluorescence technique. The results showed: (1) AECs showed changes of predominant injury (1-, 4-week), repair (8-week) and reinjury (12-week) along with smoking time prolonged. The experimental results indicated that the model of smoking mice was duplicated successfully. (2) Immunohistochemical results showed that the expression of APC protein in the AECs increased after 1-week smoking (0.458 +/- 0.062 vs 0.399 +/- 0.060, P< 0.05 vs control), but was significantly decreased at the end of the 4th week (0.339+/- 0.056, P<0.01 vs control) and increased at the end of the 8th and 12th week (0.387 +/- 0.041, 0.378 +/- 0.037, P<0.05 vs 4-week). The expression of GSK3beta in the AECs of smoking mice obviously decreased (P<0.01 or P<0.05 vs control). (3) Western blot showed that the expressions of APC protein and GSK3beta in lung tissue were consistent with the results of immunohistochemistry; and the levels of p-GSK3beta in all smoking models were higher than that in control. (4) The results of immunofluorescence showed that APC protein was localized mainly near the regions of epithelial cell membrane at the end of the 1st and 8th week after smoking, which were dissimilar with the localization in control, and this change was not seen in the location of GSK3beta. Taken together, these results demonstrate that the expressions and localizations of APC protein, GSK3beta and the activity of GSK3beta are dynamically changed in the AECs with experimental smoking injury at different phases, suggesting that APC protein and GSK3beta may be involved in the regulation of migration and proliferation of AECs, and play an important role in the process of repair of airway epithelium injury.

Significance of Survivin and PTEN expression in full lymph node-examined gastric cancer.

AIM: To study the relationship between Survivin and PTEN expression and lymph node metastasis, depth of invasion and prognosis of gastric cancer patients in China. METHODS: Specimens of gastric cancer tissue were collected from the Affiliated Hospital of Jianghan University. All the 140 patients had complete examination data. All lymph nodes were found by the fat-clearing method. The interrupted serial 4-micron sections, routine hematoxylin and eosin staining and immunohistochemical methods were used to detect the lymph node metastases. Gastric cancer tissue microarray was performed to test the expression of Survivin and PTEN (17A) in gastric cancer by immunohistochemical method. All data were processed using chi2 test, Fisher's exact test, Kaplan-Meyer Log-rank method and Cox multivariate analysis (SPSS 12.0 software). RESULTS: One hundred and eighteen specimens were used in our tissue microarray (utilization rate was 82.4%). A total of 7580 lymph nodes were found. Metastases were found in 90 specimens and 1618 lymph nodes were detected. The positive rate of Survivin and PTEN expression was 52.5% (62/118) and 76.2% (90/118), respectively. A highly positive correlation was found between Survivin and PTEN expression (chi2 = 4.17, P = 0.04). Survivin expression was positively correlated with UICC N stage (chi 2 = 8.69, P = 0.03) and histological classification (chi2 = 4.41, P = 0.04) by chi2 tests. PTEN expression was positively correlated with depth of invasion (P = 0.02) and histological classification (chi2 = 5.47, P = 0.02). But Survivin and PTEN expressions were not related with prognosis of gastric cancer patients. A significant correlation between lymph node metastasis and prognosis was demonstrated by Cox multivariate analysis (chi2 = 4.85, P = 0.028). CONCLUSION: Survivin is positively correlated with PTEN expression in gastric cancer and is a molecular marker of lymph node metastasis while PTEN expression is a molecular marker of advanced gastric cancer. UICC N stage is the most important prognostic factor of gastric cancer in China.

Role of glycogen synthase kinase 3 in squamous differentiation of pig airway epithelial cells: A primary study.

To investigate if glycogen synthase kinase 3 (GSK3) is involved in squamous differentiation of airway (tracheobronchial) epithelial cells, primary pig airway epithelial cells were treated with lithium chloride, a highly selective inhibitor of GSK3. Change in morphology of cells was monitored under microscopy, and expression of beta-catenin, phosphorylated GSK3 and involucrin, a squamous differentiation marker, were dectected by Western blotting, while expression of mRNA of another squamous differentiation marker, small proline-rich protein, was detected by RT-PCR. Further, luciferase reporter assay was used to assess the activation of beta-catenin/Tcf signaling. The results demonstrated that lithium was able to induce a squamous morphology of the cells, and to enhance the expression of involucrin and small proline-rich protein mRNA. Moreover, lithium increased inhibitory phosphorylation of GSK3, augmented nuclear translocation of beta-catenin in a dose- and time-dependent manner. Activation of beta-catenin/Tcf signaling was observed after the elevation of squamous differentiation markers. Taken together, these data suggest that GSK3 is possibly involved in squamous differentiation of pig airway epithelial cells.

[Expressions of CD147 and matrix metalloproteinase-2 in breast cancer and their correlations to prognosis].

BACKGROUND & OBJECTIVE: Matrix metalloproteinases (MMPs) play an important role in invasion and metastasis of cancers. CD147, an inducer of MMPs, can activate the expression of multiple MMPs in cancer cells and interstitial cells. This study was to investigate the correlations of CD147 and MMP-2 expressions in tumor and adjacent tissue to metastasis, invasion, and prognosis of breast cancer. METHODS: Expressions of CD147 and MMP-2 in 112 specimens of breast cancer and adjacent tissue were detected by EnVision immunohistochemistry. RESULTS: Positive rate of CD147 was 49.1% in breast cancer tissues, and 0 in adjacent tissues and interstitial cells. Positive rate of MMP-2 was 33.1% in breast cancer tissues, and 53.6% in normal adjacent tissues. The intensity of MMP-2 staining in normal tissue was positively related with tumor size, lymph node metastasis, and clinical stage (P < 0.01). CD147 staining in cancer tissue was positively related with lymph node metastasis, estrogen receptor(ER) expression, and pathologic grade (P < 0.01). Univariate analysis indicated that the overall survival rate was significantly lower in patients with positive expression of CD147 or MMP-2 than in patients with negative expression (P < 0.05). Multivariate analysis showed that CD147 was a risk predictor; the higher expression of CD147, the shorter survival time of the patients. CONCLUSIONS: MMP-2 and its inducer CD147 maybe participate in invasion and metastasis of breast cancer. CD147 may be an independent prognostic factor and, when used in combination with pathologic grading and clinical staging, may increase accuracy of predicting prognosis of patients with breast cancer.

Alterations of MLH1 and microsatellite instability in esophageal squamous cell carcinomas.

Human MLH1 is one of the DNA mismatch repair genes and located in chromosome 3p21.3. Alterations of the MLH1 were associated with various kinds of human tumors. The present study was to investigate allelic loss of MLH1 and microsatellite instability (MSI) in esophageal squamous cell carcinomas (ESCC), and to estimate the correlation between MSI status and allelic loss of the MLH1 gene. The MSI of 14 microsatellite markers and mRNA expression of MLH1 were assessed in a large cohort of patients with ESCC by using denaturing polyacrylamide gel electrophoresis (PAGE) and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. Thirty-five percent of tumors displayed MSI in at least one tested marker. MSI in D3S1611, an intragenic marker of the MLH1, was observed in 66.7% of tumors. No down-expression of MLH1 was found at the transcriptional level. The presence of MSI did not correlate with tumor stage, degree of differentiation, lymphonode-metastasis, age and sex of the patients, neither with allelic loss of the MLH1 gene. The data suggested that LOH of MLH1 is common in ESCC, but not lead to the alteration of MLH1 at the level of RNA expression. MSI in tested microsatellite markers occurs frequently in ESCC but is not associated with allelic loss of the MLH1 gene.

[Regulatory effects on apoptotic activities of gastric cancer cell line by over-expression of Smac gene].

OBJECTIVE: To investigate the effects of overexpression of second mitochondria-derived activator of caspases (Smac) gene on apoptosis of gastric cancer cells. METHODS: Under the induction of liposome, MKN-45 cells were transfected by Smac gene and incubated with G418 for subclone selection. Reverse transcriptase-polymerase chain reaction and Western blot were used to determine cellular Smac gene expression. After induction of apoptosis by mitomycin (MMC), cell viabilities were analyzed using trypan blue stain. Apoptosis was measured by electronic microscopy, acridine orange-ethidium bromide fluorescent staining and in situ terminally labelled transferase technique (TUNEL). Western blot and colorimetry were used to assess cellular caspase-3 expression and its activity. RESULTS: The Smac mRNA and protein levels in MKN-45/Smac subclone cells (subclone consistently expressing Smac gene) were significantly higher than those in MKN-45 (P < 0.01). When compared with those in MKN-45, cell viabilities of MKN-45/Smac were reduced by 10.0% to 30.8% (P < 0.01), after treatment with 10 microg/ml MMC for 6 to 24 hours. Some of the MKN-45/Smac cells showed characteristic morphologic changes of apoptosis, their apoptotic rate being increased by 21.2% (P < 0.01). After treatment with MMC, caspase-3 expression and its activity in MKN-45/Smac cells were significantly higher than those in MKN-45 (P < 0.01). CONCLUSIONS: Overexpression of Smac in gastric cancer cell line significantly improves expression and activity levels of caspase-3 after induction by MMC. Such apoptosis-inducing effect establishes a novel strategy for regulating the apoptosis activity of gastric cancer.

[Allelic loss and down-regulation of FHIT gene expression in esophageal squamous cell carcinoma].

BACKGROUND & OBJECTIVE: Deletions and translocations involving the short arm of chromosome 3 (3p14) have been observed frequently in esophageal cancer. Fragile histidine triad (FHIT) gene is located in 3p14.2, and its deletion or abnormal expression was found in many kinds of cancers. The study was to investigate the alterations of FHIT gene, and its significance in esophageal squamous cell carcinoma (ESCC). METHODS: The deletion of FHIT gene in 80 cases of ESCC was evaluated by microsatellite analysis, and the mRNA expression of FHIT gene in 20 cases of ESCC was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Intragenic markers of FHIT gene, D3S3356, D3S3378, and D3S3361, showed homozygous in all samples. D3S1234 and D3S1540, located near FHIT, presented high heterozygosity. In the tested informative cases, loss of heterozygosity (LOH) of D3S1234 was detected in 30 out of 52 tumors (57.69%), and that of D3S1540 was observed in 38 out of 56 carcinomas (67.86%). Reduced expression of FHIT mRNA occurred in 15 of 20 (75.00%) cases, and was often accompanied with LOH. However, the FHIT down-regulation was not always coincident with LOH. CONCLUSIONS: The abnormal expression of FHIT gene occurred frequently in ESCC. LOH was the main factor leading to down-regulation of FHIT expression. Epigenetic mechanism might be associated with reduced expression of FHIT in a part of ESCC cases.