KAI1/CD82 decreases Rac1 expression and cell proliferation through PI3K/Akt/mTOR pathway in H1299 lung carcinoma cells.

Although the KAI1/CD82 protein has been reported to inhibit cell metastasis in many studies, its mechanism of action has not yet been fully elucidated. In the present study, we investigated the possible effects of KAI1/CD82 on the metastatic phenotype in H1299 lung carcinoma cells. These studies were based on the pivotal role that the acquisition of motile phenotype plays on the initial steps of metastasis. KAI1/CD82-mediated morphological changes were observed using phase contrast microscopy. We report here, that a KAI1/CD82-induced phenotypic change was involved in the decrease of Rac1 expression and GTPase activity. However, we found that KAI1/CD82 did not regulate Rac1 mRNA levels. This suggests the existence of another regulatory mechanism of Rac1 protein maturation or activation. To identify the signaling pathway of Rac1 regulation, we investigated the PI3K/Akt/mTOR pathway, since the PI3K/Akt pathway regulates Rac1 activation and mTOR is known to play a regulatory role in protein translation. H1299/CD82-transfectants showed lower mTOR expression and cell growth than the control group. The data obtained from this study suggested that KAI1/CD82 decreased the metastatic phenotype of H1299 lung carcinoma cells by down-regulating Rac1 expression through the PI3K/Akt/mTOR pathway.

HOX gene analysis of endothelial cell differentiation in human bone marrow-derived mesenchymal stem cells.

Human bone marrow-derived mesenchymal stem cells (hMSCs) have been shown to possess multilineage differentiation potential. HOX genes function in transcriptional regulators, and are involved in stem cell differentiation. The aim of the present study was to demonstrate HOX genes that are related to angiogenesis. To identify the expression patterns of 37 HOX genes in the endothelial cell differentiation of hMSCs, we analyzed HOX genes through profiling with multiplex RT-PCR. The results showed that the expression patterns of four HOX genes, HOXA7, HOXB3, HOXA3, and HOXB13, significantly changed during angiogenesis. The expression levels of HOXA7 and HOXB3 were dramatically increased, whereas those of HOXA3 and HOXB13 were decreased during endothelial cell differentiation. When further analysis of the expressions of these HOX genes was performed with real-time PCR and an immunoblot assay, the expression patterns were also found to be well-matched with the results of multiplex RT-PCR. Here, we report that HOXA7, HOXB3, HOXA3, and HOXB13 might be involved in the angiogenesis of hMSCs.

DNA profiling by array comparative genomic hybridization (CGH) of peripheral blood mononuclear cells (PBMC) and tumor tissue cell in non-small cell lung cancer (NSCLC).

Lung tumor cell DNA copy number alteration (CNA) was expected to display specific patterns such as a large-scale amplification or deletion of chromosomal arms, as previously published data have reported. Peripheral blood mononuclear cell (PBMC) CNA however, was expected to show normal variations in cancer patients as well as healthy individuals, and has thus been used as normal control DNA samples in various published studies. We performed array CGH to measure and compare genetic changes in terms of the CNA of PBMC samples as well as DNA isolated from tumor tissue samples, obtained from 24 non-small cell lung cancer patients. Contradictory to expectations, our studies showed that the PBMC CNA also showed chromosomal variant regions. The list included well-known tumor-associated NTRK1, FGF8, TP53, and TGFbeta1 genes and potentially novel oncogenes such as THPO (3q27.1), JMJD1B, and EGR1 (5q31.2), which was investigated in this study. The results of this study highlighted the connection between PBMC and tumor cell genomic DNA in lung cancer patients. However, the application of these studies to cancer prognosis may pose a challenge due to the large amount of information contained in genetic predisposition and family history that has to be processed for useful downstream clinical applications.

HOX gene analysis in the osteogenic differentiation of human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the capacity to differentiate into osteoblasts during osteogenesis. Several studies attempted to identify osteogenesis-related genes in hMSCs. Although HOX genes are known to play a pivotal role in skeletogenesis, their function in the osteogenesis of hMSCs has not yet been investigated in detail. Our aim was to characterize the expression of 37 HOX genes by multiplex RT-PCR to identify the ones most probably involved in osteogenic differentiation. The results showed that the expression patterns of four HOX genes were altered during this process. In particular, the expression levels of HOXC13 and HOXD13 were dramatically changed. Real-time PCR and Western blot analysis were performed in order to further analyze the expression of HOXC13 and HOXD13. The qRT-PCR results showed that transcription of HOXC13 was up-regulated by up to forty times, whereas that of HOXD13 was down-regulated by approximately five times after osteogenic differentiation. The Western blot results for the HOXC13 and HOXD13 proteins also corresponded well with the real-time PCR result. These findings suggest that HOXC13 and HOXD13 might be involved in the osteogenic differentiation of hMSCs.

COX-2 expression and inflammatory effects by diesel exhaust particles in vitro and in vivo.

Recent studies have shown that diesel exhaust particles (DEP) have adverse effects on the respiratory tract in vitro and in vivo, related to various pro-inflammatory cytokines and inflammatory mediators. The inflammation induced by the production of cyclooxygenase (COX)-2, an important mediator of inflammation and tumor promotion, and excess eicosanoids may be central to the pathogenesis of DEP-induced airway inflammation. However, the role of COX-2 in the pathogenesis of DEP-induced lung inflammation remains unclear, especially in vivo. In this study, we demonstrated that treatment with 50 microg/ml of DEP for 24h induced the expression of the COX-2 gene at both the transcriptional and protein levels, which led to an increase in the release of prostaglandin E(2) (PGE(2)) in A549 cells. In addition, the increased levels of COX-2 and PGE(2) by DEP exposure were significantly suppressed by treatment with 50 pg/ml of dexamethasone (Dex). We also showed that exposure to 25 mg/kg of DEP induced the expression of the COX-2 protein in mouse lung tissues, and this increased COX-2 expression was attenuated by pretreatment with 5 mg/kg of Dex. These findings suggest that COX-2 may play an important role in the pathogenesis of DEP-induced pulmonary inflammation, which is effectively inhibited by glucocorticoid treatment.

Effect of KAI1/CD82 on the beta1 integrin maturation in highly migratory carcinoma cells.

The KAI1/CD82 protein has been documented as the tumor metastasis suppressor in many types of human cancers. KAI1/CD82 regulates cell motility and invasiveness; however, the mechanism by which this occurs remains to be fully established. Several studies have shown that KAI1/CD82 modulates integrin-dependent signaling. It was suggested that KAI1/CD82 might function to attenuate the beta1 integrin function of inducing cellular migration. A wound-healing and modified Boyden chamber assays were performed to investigate the mechanism of the KAI1/CD82-mediated inhibition of cell migration. It was found that the migratory ability of H1299/CD82 was inhibited. The immunoblotting and biotinylation assays revealed that H1299/CD82 showed significantly decreased expression of the mature form of beta1, which was functional at the cell surface. It was confirmed that KAI1/CD82 regulates the maturation of the beta1 integrin using CD82-specific si-RNA. These results support a model in which KAI1/CD82 attenuates the maturation of the beta1 integrin precursor and thereby suppresses cell migration.

Role of tumor necrosis factor-alpha, interleukin-8, and dexamethasone in the focal adhesion kinase expression by human nucleus pulposus cells.

STUDY DESIGN: Human nucleus pulposus cells from intervertebral disc specimens were cultured to study the effects of tumor necrosis factor (TNF)-alpha and interleukin (IL)-8 on the focal adhesion kinase (FAK) expression by these cells. The effect of co-stimulation with dexamethasone on the FAK expression by nucleus pulposus cells was also studied. OBJECTIVES: To evaluate the possible role of activated FAK expressed by the human nucleus pulposus cells and its correlation with inflammatory cytokines (TNF-alpha, IL-8) and dexamethasone. SUMMARY OF BACKGROUND DATA: There have been no reported studies showing the correlation between the activated FAK expression by human nucleus pulposus cells with inflammatory cytokines and dexamethasone. METHODS: The FAK expression in cultured human nucleus pulposus cells was studied, and Western blot and immunofluorescence analysis were performed to assess its relation to TNF-alpha, IL-8, and dexamethasone. RESULTS: Treatments of TNF-alpha and IL-8 up-regulated the activated FAK expression. Dexamethasone attenuated the cytokine-induced FAK expression. The effects of inflammatory cytokines on the FAK expression were found to be concentration dependent, with greater correlation shown by IL-8 than TNF-alpha. CONCLUSION: TNF-alpha and IL-8 stimulation up-regulated the FAK expression of human nucleus pulposus cells, and the coadministration of dexamethasone attenuated it.

Analysis of neuron-like differentiation of human bone marrow mesenchymal stem cells.

The objective of the study was to evaluate differentiation of human bone marrow mesenchymal stem cells into true or pseudo neurons after treating with chemical induction medium in vitro. The morphological changes were assessed using interference contrast microscopy. Immunocytochemistry and Western blotting were performed using neuronal markers. Further evaluation was conducted with proteomic profiling, DNA microarray analysis and the whole-cell patch clamp test. After three hours of treatment with chemical induction medium, nearly three-fourths of the hMSCs changed to cells with a neuronal phenotype. The results of immunocytochemistry and Western blotting showed a high expression of neuronal markers in these cells at 3 h which decreased at 24 h. The proteomics analysis showed no change of proteins related to neuronal differentiation. DNA microarray showed downregulation of neuron related genes. The patch clamp test was unable to demonstrate any similarity to true neurons. Our findings suggest that neuron-like cells derived from chemical induction of hMSCs are not the genuine neurons as they resemble true neurons phenotypically but are different in genotypic and electrophysiological characteristics.

KAI1/CD82 suppresses tumor invasion by MMP9 inactivation via TIMP1 up-regulation in the H1299 human lung carcinoma cell line.

We conducted a study on the mechanism of KAI1/CD82-mediated suppression of tumor invasiveness and metastasis, and examined its effect on MMP-9 activity and the TIMP1 levels in H1299 human non-small cell lung carcinoma cells. The H1299 human lung carcinoma cells were transfected with pcDNA3.1-CD82 and stable transfectant clones that had a high KAI1/CD82 expression were obtained. We performed Western blot analysis, cell invasion assay, gelatin zymography, and RT-PCR to assess the KAI1/CD82 expression and tumor invasiveness, the MMP-9 activity, the MMP-9 mRNA and protein levels, and the TIMP1 levels in the H1299/CD82 transfectant cells and compared the results with those of the control groups. The H1299/CD82 transfectants exhibited significant suppression of cell invasion, reduced MMP9 enzyme activity, elevated MMP9 mRNA and MMP-9 protein levels, and elevated TIMP1 levels. It may be postulated that KAI1/CD82 over-expression in the H1299 non-small cell lung carcinoma cells suppresses the tumor invasiveness and metastatic potential by inducing MMP9 inactivation via the up-regulation of TIMP1.