[Show abstract][Hide abstract] ABSTRACT: T lymphoma invasion and metastasis 1 (Tiam1) is a potential modifier of tumor development and progression. Our previous study in vitro and in nude mice suggested a promotion role of Tiam1 on invasion and metastasis of colorectal cancer (CRC). In the present study, we generated Tiam1/C1199-CopGFP transgenic mice to investigate the tumorigenetic, invasive and metastatic alterations in the colon and rectum of wild-type and Tiam1 transgenic mice under 1,2-dimethylhydrazine (DMH) treatment.
Transgenic mice were produced by the method of pronuclear microinlectlon. Whole-body fluorescence imaging (Lighttools, Edmonton, Alberta, Canada), PCR, and immunohistochemical techniques (IHC) were applied sequentially to identify the transgenic mice. The carcinogen DMH (20 mg/kg) was used to induce colorectal tumors though intraperitoneal (i.p.) injections once a week for 24 weeks from the age of 4 weeks on Tiam1 transgenic or non-transgenic mice.
We successfully generated Tiam1/C1199-CopGFP transgenic mice and induced primary tumors in the intestine of both wild type and Tiam1 transgenic mice by DMH treatment. In addition, Tiam1 transgenic mice developed larger and more aggressive neoplasm than wild-type mice. Moreover, immunohistochemical staining revealed that upregulation of Tiam1 was correlated with increased expression of β-Catenin and Vimentin, and downregulation of E-Cadherin in these mice.
Our study has provided in vivo evidence supporting that Tiam1 promotes invasion and metastasis of CRC, most probably through activation of Wnt/β-catenin signaling pathway, in a Tiam1 transgenic mouse model.
PLoS ONE 09/2013; 8(9):e73077. DOI:10.1371/journal.pone.0073077 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the changes in methylation levels of the promoters of the tumor suppressor gene Wilms' tumor gene on the X-chromosome (WTX) and its possible role in gastric cancer.
WTX promoter methylation levels were detected in 20 pairs of specimens of gastric cancer and matched normal tissues and in 3 gastric cancer cell lines (MGC803, SCG7901, and BGC823) using the Sequenom MassARRAY quantitative analysis system. The gastric cancer cell line BGC823 was treated with 5-aza-2'-deoxycytidine (5-aza-dC) for demethylation and the changes in the level of WTX promoter methylation were investigated.
WTX promoter methylation levels were very low and showed no significant differences among normal gastric tissues, gastric cancer tissues and the 3 gastric cancer cell lines. In BGC823 cells, treatment with 5-aza-dC did not obviously affect the promoter methylation levels of WTX.
High methylation levels of WTX promoters are rare in gastric cancer.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 03/2013; 33(3):318-21.
[Show abstract][Hide abstract] ABSTRACT: Cyclooxygenase-2 (Cox-2) is an inducible enzyme that converts arachidonic acid to prostaglandins, and it is hypothesized to induce carcinogenesis and metastasis in colorectal cancer. Our previous data also indicated that a higher expression level of Cox-2 was correlated with colorectal cancer metastasis. The Cox-2 protein was detected in the glandular cavity of colorectal cancer and the surrounding interstitial tissues. The usefulness of the Cox-2 gene as a gene therapy target and diagnostic marker remains unknown. In this study, a method using immuno-PCR and real-time PCR followed by supramolecular immunobead real-time PCR was established and used to detect the expression of Cox-2 in serum samples of nude mice with colorectal carcinoma. In addition, we established a Cox-2 gene stable knockdown colorectal cell line (SW480-EGFP-Cox-2 shRNA) using lentiviral vector-mediated RNA interference (RNAi) technology and established an imageable colorectal cancer metastasis mouse model. We found that the proliferation, invasion and tumorigenesis of SW480-EGFP-Cox-2 shRNA cells were attenuated compared with SW480 cells. In vivo experiments also demonstrated that angiogenesis in the Cox-2 knockdown colorectal cancer cells was decreased. The whole body optical imaging revealed that the SW480-EGFP-Cox-2 shRNA cells had an abrogated ability to develop metastases in the lymph nodes, lungs or liver in vivo. The improved immunobead PCR assay detected significantly lower Cox-2 protein levels in the serum samples of the SW480-EGFP-Cox-2 shRNA group compared with those of the SW480-EGFP-Cox-2-Ctrl shRNA group. In conclusion, our results indicated that the knockdown of Cox-2 expression suppressed the proliferation and invasion of colorectal cancer cells both in vitro and in vivo. This study also demonstrated that silencing Cox-2 in vivo reduced the metastastic potential of colorectal cancer. Thus, Cox-2 is a promising marker for the diagnosis of colorectal metastasis and a potential therapeutic target for colorectal cancer.
[Show abstract][Hide abstract] ABSTRACT: To study the pathogenic and tumorigenic effect of 1,2-dimethylhydrazine (DMH) on the colon and ovaries of mice.
Sixty ICR female mice were randomly divided into groups A and B for intraperitoneal injection of DMH (20 mg/kg) and saline (control) once a week for 24 weeks, respectively. The mice were sacrificed at 12, 16, 20, 24, 28 and 32 weeks after the first DMH injection for pathological examination of the colon and ovaries.
In group A, colorectal adenomas were found in 7, colorectal adenocarcinomas in 5, and hemorrhagic lesions of the ovaries with chronic inflammatory in 21 mice. Choriocarcinoma in the ovaries were detected in one mouse at 28 weeks and in another at 32 weeks. No obvious pathological changes were found in group B following the injections.
Intraperitoneal injection of DMH may induce colon tumors and ovarian diseases in mice.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 06/2011; 31(6):999-1002.
[Show abstract][Hide abstract] ABSTRACT: To establish the BGC-823/WTX-EGFP gastric cancer cell line with stable expression of Wilms tumor gene on the X chromosome (MTX) for functional analysis of WTX gene.
The full-length WTX cDNA was amplified from human embryonic kidney 293FT cells and cloned into the pEGFP-N1 vector containing the reporter gene of green fluorescence protein. The recombinant pEGFP-WTX expression vector, after digestion by restriction enzyme to identify the size of target gene fragment, was transfected into 293FT cells and the expression of fluorescent reporter gene was observed under fluorescence microscope. pEGFP-WTX vector was transfected into human gastric cancer BGC-823 cell line to establish BGC-823/WTX-EGFP cell line stably expressing WTX. Quantitative RT-PCR and immunocytochemical staining were used to detect the expression of WTX in both BGC-823/WTX-EGFP and control BGC-823 cells.
The recombinant pEGFP-WTX plasmid was successfully constructed and verified by PCR and sequencing. The mRNA and protein expressions of MTX were significantly increased in BGC-823/WTX-EGFP cells as compared with those in the control cells.
The full-length WTX expression vector (pEGFP-WTX) and BGC-823/WTX-EGFP gastric cancer cell line have been successfully established to facilitate further functional study of WTX gene.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 03/2011; 31(3):392-6.
[Show abstract][Hide abstract] ABSTRACT: To establish an animal model visualizing orthotopic growth and metastasis of colorectal cancer.
pEGFP-N1 plasmid was transfected into human colorectal carcinoma cell line SW480 so that the resultant SW480/EGFP cells emitted fluorescence that could be detected externally by fluorescence stereo microscope. SW480/EGFP cells were inoculated subcutaneously in nude mice, and the orthotopic tumor growth was evaluated in real time. Whole-body visualization models of orthotopically implanted colorectal carcinoma was established surgically, and the tumor growth and metastasis are evaluated by conventional pathological methods.
SW480/EGFP cells stably expressed high-levels of enhanced green fluorescent protein. Subcutaneous injection of SW480/EGFP cells resulted in tumor growth in nude mice, and the emitted fluorescence could be quantitatively detected and imaged with fluorescence stereo microscope to visualize real-time tumor growth. Visualization animal model was established successfully with surgical orthotopic implantation (SOI) of the tumor, and all mice survived. After two weeks, all the mice developed colorectal carcinoma without metastasis, but 4 weeks later, 75%percnt; of the mice developed peritoneal tumor metastasis and 50% had liver metastasis. The whole-body visualization animal model was successfully validated by pathological detection.
Whole-body visualization model of orthotopic and metastatic tumor growths provides a reliable means for observing the behavior of human colorectal carcinoma and can be helpful to study the growth and metastasis patterns of the cancer.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 09/2007; 27(8):1161-3, 1166.
[Show abstract][Hide abstract] ABSTRACT: To confirm the role of Tiam1 (T lymphoma invasion and metastasis 1) gene in the proliferation and metastasis of colorectal cancer.
Proliferative and metastatic abilities of Tiam1 transfectant were investigated by subcutaneous injection of cells and surgical orthotopic transplantation (SOI) in mice.
The expression of Tiam1 led to a pronounced increase in HT29/Tiam1 cell growth starting from day 7, up to 2.5 fold increase of tumor volume at day 20 post injection. Tumors in the HT29/Tiam1 group receiving surgical orthotopic implantation were significantly heavier than those in HT29/mock group (t = -14.916, P < 0.01). In vivo metastasis assay by SOI showed that in HT29/Tiam1 group, 7/7 of mice developed peritoneal metastases and 4/7 had hepatic lesions. In addition, one of the seven HT29/Tiam1 group mice had tumors in lung, spleen and lymph nodes. In the HT29/mock group, only 2/7 of animals had peritoneal metastases and none produced detectable tumor in the liver.
Tiam1 gene plays an important role in the proliferation, invasion and metastasis of colorectal cancer. It may serve as a useful clinical marker for tumor progression and metastasis of colorectal cancer.
Zhonghua bing li xue za zhi Chinese journal of pathology 06/2007; 36(6):390-3.
[Show abstract][Hide abstract] ABSTRACT: To evaluate the effects of amino acids (AA) on the development of in vitro cultured preimplantation embryos of Kunming mice, and define the optimal AA concentration for embryo culture.
Totally 630 zygotes were collected from the oviducts of superovulated female Kunming mice, which were cultured in protein-free potassium simplex optimized medium (mKSOM) supplemented with Eagle's essential amino acids and Eagle's non-essential amino acids of different concentrations (mKSOM, mKSOM+1/16AA, mKSOM+1/8AA, mKSOM+1/4AA, mKSOM+1/2AA, mKSOM+AA, and mKSOM+2AA).
The embryos cultured with the amino acids showed higher development rate to both 8-cell embryo stage and blastocyst stage than those cultured without amino acids. The correlation of amino acid concentration with 8-cell and blastocyst development rates conformed to the cubic model, with the highest development rate to both of the two stages observed at half of the amino acid concentration.
Amino acids can promote the development of preimplantation Kunming mouse embryos, but excessively high concentration of amino acids impair embryo development possibly because of metabolic and osmotic pressure changes of the embryos as well as toxicity of ammonium resulting from the metabolism of amino acids.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 04/2005; 25(3):241-5.
[Show abstract][Hide abstract] ABSTRACT: To explore the distribution of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) in SARS autopsy tissues at the molecular level.
In situ hybridization was used to detect the expression and location of SARS-CoV RNA polymerase gene in autopsy tissues from SARS-Cov-infected subjects, including the lung, spleen, lymph nodes, pituitary, pancreas, parathyroid, adrenal glands, gastrointestinal tract, skin, brain, liver, kidney, blood vessels, striated muscles of the limbs, bone marrow, heart, ovary, uterus and testicles.
SARS-CoV RNA was detected in the cytoplasm of the alveolar epithelia, infiltrating mononuclear phagocytes in the lungs, serous gland epithelium of the trachea/bronchus, monocytes in the spleen and lymph nodes, acinar cells in the pancreas, acidophilic cells in the parathyroid and pituitary, adrenal cortical cells, epithelia of the alimentary tracts, gastric parietal cells, sweat gland cells, brain neurons, hepatocytes near the central vein, epithelia of the distal renal tubules, bone marrow promyelocytes, and endothelia of the small veins.
SARS-CoV invades various organs of the body and distributes in a similar fashion to CD13, the receptor of human coronavirus 229E. The detection of SARS-CoV in the sweat glands, alimentary tracts and epithelia of the distal convoluted tubules of the kidney may help identify the transmission routes of SARS-CoV.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 12/2003; 23(11):1125-7.
[Show abstract][Hide abstract] ABSTRACT: To investigate the presence and distribution of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) in autopsy tissues obtained from patients died of SARS.
Immunohistochemical technique was applied in 4 fatal SARS cases to examine the autopsy tissues including the lungs, spleen, lymph nodes, brain, pituitary, heart, liver, kidney, pancreas, trachea, esophagus, gastrointestinal tract, adrenal glands, parathyroids, skin and bone marrow.
Immunohistochemistry identified positive monoclonal antibody against SARS-CoV nuceeocapsid (N) protein in the alveolar epithelium and the infiltrating monocytes or macrophages in the lung, spleen and lymph nodes; the presence of the antibody was also detected in the serous gland epithelium of the trachea/bronchus, squamous epithelium of the esophagus, the gastric parietal cells, the epithelium of the intestinal tract, acidophilic cells in the parathyroids and pituitary, acinus cells in the pancreas, adrenal cortical cells, sweat gland cells, small vessel endothelium, bone marrow promyelocytes, epithelial cells of the distal convoluted tubule of the kidney, brain neurons, and the hepatocytes near the central vein.
A variety of organs and tissues can be infected by SARS-CoV, and the positive expression of SARS-CoV N protein in the epithelial cells of the gastrointestinal tract, the distal convoluted tubule of the kidney and the sweat gland cells is significant for studying the transmission routes of SARS.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 12/2003; 23(11):1128-30.
[Show abstract][Hide abstract] ABSTRACT: To explore the role and mechanism of apoptosis in severe acute respiratory syndrome (SARS).
Klenow-FragELTM DNA Fragmentation Detection Kit and immunohistochemical alkaline phosphatase detection reagent kit were used to detect cell apoptosis and expressions of CD68, CD20, CD4, CD8 and CD45RA in the pathological tissues of SARS patients.
Apoptotic cells increased significantly in the spleen, lung and lymph nodes of SARS patients as compared with normal tissues. The apoptotic cells included pneumocytes, lymphocytes and monocytes, and CD68+ monocytes were observed in abundance in the lung, spleen and lymph nodes of SARS patients. In the lung tissue of the patients, few CD20+/CD45RA+ B cells and CD4+/CD8+ T cells were spotted, and CD20+/CD45RA+ B cells along with CD4+/CD8+ T cells were also significantly decreased in the spleen and lymph nodes, where few conserved B and T cells underwent apoptosis.
Apoptosis is a general phenomenon in SARS, and the invasive cells in the pathological tissues are primarily monocytes, suggesting that apoptosis and invasion of monocyte play important roles in the progression of SARS. The cell apoptosis and decreased number of T cell and B cells in the lungs and CD4+/CD8+ T cells and CD20+/CD45RA+ B cells in the spleen and lymph nodes indicate that the SARS virus may exercise immune cell-killing effect to some extent during its pathogenesis.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 09/2003; 23(8):770-3.
[Show abstract][Hide abstract] ABSTRACT: To study the expression of the immune cell markers and their active antigens in the involved tissues of patients with severe acute respiratory syndrome (SARS).
Specimens of the lungs, spleens and lymph nodes were obtained from autopsy of 3 SARS cases to investigate the expression of the immune cells and their activities with double immunohistochemical staining (DAB and AP) by using 14 immune cell markers and their active antigens.
A large quantity of proliferated macrophages could be observed in the lungs, spleens and lymph nodes of the SARS patients, some of which were positive for CD25 marker (active macrophages). In the lungs of the 3 patients, localized necrosis occurred where infiltration of CD45RO (+) T lymphocytes was observed, with only scarce Ki67 (+) T lymphocytes (active T lymphocytes) and B lymphocytes. In the lymph nodes, scattered T lymphocytes positive for Ki67 and CD45RO markers (active T lymphocytes) were seen, but the T lymphocytes subpopulations were obviously decreased, which was especially so with CD4+ and CD8+ T lymphocytes and NK cells.
Significantly enhanced activity of the macrophages occurs in SARS as the major reactive cells, but T lymphocyte subsets are obviously decreased, indicating the important roles of the macrophages and T lymphocytes in the pathogenesis of SARS.
Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA 09/2003; 23(8):774-6, 780.