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.

Diesel exhaust particles induce apoptosis via p53 and Mdm2 in J774A.1 macrophage cell line.

Diesel exhaust particles (DEP) are known to cause cardiopulmonary diseases due to their proinflammatory and cytotoxic effects. Continuous exposure to DEP potentiates chronic inflammatory processes and acute symptomatic responses in the respiratory tract. Recent studies have emphasized that alveolar cell apoptosis is a crucial step in chronic inflammation and lung injury. The phenomenon of apoptosis is a key event that successfully clears damaged cells, and its failure leads to the development of more serious diseases, such as lung cancer. The mechanism and molecular target of DEP-induced apoptosis in the respiratory tract remain unclear. In this study, J774A.1 macrophage cells were used to investigate the p53-mediated apoptotic pathway induced by DEP exposure. The results showed that murine double minute 2 (Mdm2), a negative regulator of p53, was downregulated at the protein level by DEP exposure. In contrast, the pro-apoptotic protein Bcl-2-associated X protein (Bax), an endogenous target of p53-dependent transcriptional activation, was continuously upregulated at the mRNA and protein levels by DEP exposure. Furthermore, pifithrin-alpha (p53 inhibitor) blocked DEP-induced apoptosis as well as p53 activation. Taken together, the findings of the present study suggest that DEP trigger apoptosis in J774A.1 macrophage cells via the activation of p53, followed by Bax.

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.

Array CGH reveals genomic aberrations in human emphysema.

Emphysema is the major component of chronic obstructive pulmonary disease (COPD), which is the fourth leading cause of death in the world. Several epidemiologic studies suggest that genetic factors may have an important role in the pathogenesis of emphysema. We analyzed the gene expression profiles of chromosomal aberrations using array comparative genomic hybridization (array CGH) in 32 patients with emphysema to identify the candidate genes that might be causally involved in the pathogenesis of emphysema. Copy number gains and losses were detected in chromosomal regions, and the corresponding genes were confirmed by real-time polymerase chain reaction. Several frequently altered loci were found, including a gain at 5p15.33 (60% of the study subjects), and a loss at 7q22.1 (31% of the study subjects). DNA gains were identified at a high frequency at 1p, 5p, 11p, 12p, 15q, 17p, 18q, 21q, and 22q, whereas DNA losses were frequently found at 7q and 22q. We found that the fold change levels were highest at the CYP4B1 (1p33), JUN (1p32.1), NOTCH2 (1p12-p11.2), SDHA (5p15.33), KCNQ1 (11p15.5-p15.4), NINJ2 (12p13.33), PCSK6 (15q26.3), ABR (17p13.3), CTDP1 (18q23), RUNX1 (21q22.12) and HDAC10 (22q13.33) gene loci. We also observed losses in the MUC17 (7q22.1), COMT (22q11.21) and GSTT1 (22q11.2) genes. These studies show that array CGH is a useful tool for the identification of gene alterations in cases of emphysema and that the aforementioned genes might represent potential candidate genes involved in the pathogenesis of emphysema.

Identification of DNA copy number aberrations by array comparative genomic hybridization in patients with ruptured intracranial aneurysms.

We aimed to use array comparative genomic hybridization (CGH) to identify chromosomal loci that contribute to the pathogenesis of ruptured intracranial aneurysms (IAs) in a Korean population and to confirm the results using real-time polymerase chain reaction (PCR). Twenty-three patients with ruptured IAs were enrolled in this study. Array CGH revealed copy number aberrations in 19 chromosomal regions. Chromosomal gains were identified at a high frequency in regions 1p12, 4q24, 5p15.31, 5p15.33, 6p12.2, 6q22.33, 7p21.1, 9q22.1, 10q24.32, 10q26.3, 12q13.13, 17p12, 18q12.3, 18q23, 19p13.3, 20q13.33, 21q11.2, and 21q22.3, whereas chromosomal losses were identified at 15q11.2 and 22q11.21. Real-time PCR confirmed the results of the array CGH studies of the COL6A2, GRIN3B, MUC17, and PRODH genes. This is the first study to identify candidate regions by array CGH in patients with IAs. The identification of genes that may predispose an individual to the development of IAs may lead to a better understanding of the mechanism of IA formation. Multicenter studies comparing cohorts of patients of different ethnicities are needed to better understand the mechanism of IA formation.

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.

Use of soybean protein hydrolysates for promoting proliferation of human keratinocytes in serum-free medium.

Human keratinocytes are generally cultured in media containing bovine pituitary extract (BPE), an animal product that can be a source of infectious contaminants. We investigated whether a safer plant product could replace BPE in the culture medium. Medium containing both BPE and soy protein hydrolysates (Bacto Soytone and Soy Hydrolysate) produced the largest number of viable cells, followed in descending order by medium supplemented only with BPE, only with the hydrolysates, and without supplementation (basal medium only). Soybean protein is thus an excellent source of nutrients for the growth of adherent keratinocytes, although they do not fully substitute for BPE.

Comparative genome hybridization array analysis for sporadic Parkinson's disease.

Parkinson disease (PD) is a common neurodegenerative disorder, characterized by the loss of midbrain dopamine neurons and Lewy body inclusions. We investigated array CGH to analyze gain or loss of genetic material from 30 patients with PD. We identified the frequent copy number variations in PD; gains in 1p21.1, 4p15.31, 5p15.33, 6q24.1, 7q35, 8q24.3, 10q26.3, 11p15.5-15.4, 12q21.2, 16p13.3, 18q12.3 and 22q13.31, and losses in 1p36.33, and 5q13.2. These findings enable a better description of genetic variations in PD, and could provide a foundation for understanding the critical regions of the genome that may be involved in the development of PD.

Altered DNA copy number in patients with different seizure disorder type: by array-CGH.

Epilepsy is one of the most common but genetically complex neurological disorders in children. Previous studies have showed that chromosomal abnormalities confer susceptibility to epilepsy. To identify new chromosomal abnormalities associated with epilepsy, DNA samples from patients with idiopathic generalized epilepsy (IGE), partial epilepsy (PE), and febrile seizures (FS) were analyzed using array comparative genome hybridization technique (array-CGH). Genomic aberrations were detected throughout whole chromosome. The most frequently altered loci were gains noted in: 1p (60%), 5p (55%), 8q (55%), 10q (55%), and losses in 7q (55%). The most frequent chromosomal aberrations for each seizure type were: IGE-1p (60%), 5p (55%), and 10q (55%), PE-11p (45%), 21q (45%) and FS-8q (55%), and losses in 7q (55%). To validate the array-CGH results, real time PCR was performed for several genes (EPM2AIP1, OSM, AFP, CYP19A1, SLC6A13, and COL6A2). The results from the real time PCR were consistent with those from the array-CGH. Therefore, we found that the three types of seizures disorder studied have different chromosomal aberrations. These results might be used for further investigation of the pathogenesis of epilepsy.

Cellular toxicity of various inhalable metal nanoparticles on human alveolar epithelial cells.

Nanoparticles (NPs) have a greater potential to travel through an organism via inhalation than any other larger particles, and could be more toxic due to their larger surface area and specific structural/chemical properties. The aim of this study was to evaluate in vitro biological effects of various inhalable metallic NPs (TiO2, Ag, Al, Zn, Ni). Human alveolar epithelial cells (A549) were exposed to various concentrations of NPs for 24 h. The extent of morphological damage was in the order of m-TiO2 > n-TiO2 > m-silica >> n-Ni approximately = n-Zn approximately = n-Ag approximately = n-Al and was affected in a dose-dependent manner. The extent of apoptotic damage measured with two-color flow cytometry was in the order of n-Zn > n- Ni > m-silica >> n- TiO2 > m- TiO2 > n-Al > n-Ag. The extent of apoptotic damage measured with DNA fragmentation was in the order of n-Zn approximately = m-silica > n- Ni >> m- TiO2 approximately = n- TiO2 approximately = n-Al > n-Ag, indicating no significant difference in the damages by both m-TiO2 and n-TiO2. The extents of apoptotic damages were also affected in a dose-dependent manner. Uptake of no other NPs but n-TiO2 and m-TiO2 into the cells was observed after 24 h exposure. The intracellular generation of ROS was significant with n-Zn but not with the other particles. These results demonstrated that various inhalable metallic NPs (TiO2, Ag, Al, Zn, Ni) could cause cell damages directly or indirectly. More detailed studies on the influence of size, structure, and composition of the NPs are needed to better understand their toxic mechanisms.

The merlin tumor suppressor interacts with Ral guanine nucleotide dissociation stimulator and inhibits its activity.

Neurofibromatosis type 2 (NF2) is the most commonly mutated gene in benign tumors of the human nervous system such as schwannomas and meningiomas. The NF2 gene encodes a protein called schwannomin or merlin, which is involved in regulating cell growth and proliferation through protein-protein interactions with various cellular proteins. In order to better understand the mechanism by which merlin exerts its function, yeast two-hybrid screening was performed and Ral guanine nucleotide dissociation stimulator (RalGDS), a downstream molecule of Ras, was identified as a merlin-binding protein. The direct interaction between merlin and RalGDS was confirmed both in vitro and in the NIH3T3 cells. The domain analyses revealed that the broad C-terminal region of merlin (aa 141-595) is necessary for the interaction with the C-terminal Ras-binding domain (RBD) of RalGDS. Functional studies showed that merlin inhibits the RalGDS-induced RalA activation, the colony formation and the cell migration in mammalian cells. These results suggest that merlin can function as a tumor suppressor by inhibiting the RalGDS-mediated oncogenic signals.

Natural Killer T cells in patients with major depressive disorder.

CD56 (Natural Killer T) cells showed a significant negative correlation with depressive symptom scale scores in acute and unmedicated patients with major depressive disorder. Decreased CD56 cells may reflect the severity of depressive symptoms but not the severity of anxiety symptoms in major depression.

The effect of adenosine 5'-triphosphate on calcium mobilization and cell proliferation in cervical cancer cells.

OBJECTIVE: To know the effect of adenosine 5'-triphosphate (ATP) on intracellular calcium level and cell proliferation in cervical cancer cells. STUDY DESIGN: Four different human cervical cancer cell lines (Caski, C33A, HeLaS3 and SiHa) were used in this study. The change of intracellular calcium level, cell proliferation and the activity of proliferation- and calcium-related transcription factors by extracellular ATP were examined in these cell lines. RESULTS: Extracellular ATP induced calcium mobilization, cell proliferation and the activation of NF-kappaB in all cell lines used. CONCLUSION: These results suggest that calcium mobilization and NF-kappaB dependent signaling pathway play an important role in the cell proliferation by ATP in cervical cancer.

Induction of nuclear factor-kappaB activation through TAK1 and NIK by diesel exhaust particles in L2 cell lines.

Diesel exhaust particles (DEPs) are known to induce allergic responses in airway epithelial cells, such as the production of various cytokines via nuclear factor-kappa B (NF-kappaB). However, the intracellular signal transduction pathways underlying this phenomenon have not been fully examined. This study showed that DEP induced NF-kappaB activity via transforming growth factor-beta activated kinase 1 (TAK1) and NF-kappaB-inducing kinase (NIK) in L2 rat lung epithelial cells. DEP induced the NF-kB dependent reporter activity approximately two- to three-fold in L2 cells. However, this effect was abolished by the expression of the dominant negative forms of TAK1 or NIK. Furthermore, it was shown that DEP induced TAK1 phosphorylation in the L2 cells. These results suggest that TAK1 and NIK are important mediators of DEP-induced NF-kappaB activation.

Silica induces human cyclooxygenase-2 gene expression through the NF-kappaB signaling pathway.

Silica is a causative factor of acute cell injury in pulmonary fibrosis. Inducible cyclooxygenase-2 (COX-2) was suggested to play a role in the process of inflammation and fibrosis. We report that silica induces COX-2 expression in WI-38 fibroblasts. Further analysis showed that silica activated the transcription of COX-2 gene primarily via a nuclear factor (NF)-kB binding site in the promoter. NF-kB-inducing kinase (NIK) and TGF-k activated kinase 1 (TAK1), the upstream signaling molecules of NF-kB, are involved in the silica-mediated COX-2 expression. The Electrophoretic Mobility Shift Assay (EMSA) showed that silica induced the direct binding of NF-kB on the putative binding site in COX-2 promoter. These results suggest that silica activates the human COX-2 gene transcription through the induction of NF-kB activity.

Anti-apoptotic role of phospholipase D isozymes in the glutamate-induced cell death.

Phospholipase D (PLD) plays an important role as an effector in a variety of physiological processes that reveal it to be a member of the signal transducing phospholipases. Recently, PLD2 was reported as a necessary intermediate in preventing apoptosis induced by hydrogen peroxide or hypoxia in rat pheochromocytoma (PC12) cells. The data presented here show that both PLD isozymes, PLD1 and PLD2 are also required in attenuating glutamate-induced cell death in PC12 cells. Treatment of PC12 cells with glutamate resulted in induction of apoptosis in these cells, which is accompanied by decreased PLD activity and increased ceramide concentration. Incubation of PC12 cells with exogenous C6-ceramide showed a time-dependent decrease of PLD activity. When cDNAs of PLD1 and PLD2 were transfected into PC12 cells respectively, overexpression of PLD1 or PLD2 resulted in inhibition of glutamate-induced apoptotic cell death. These data indicate that both PLD1 and PLD2 play a protective role against glutamate-induced cell death in PC12 cells.

TAK1 mediates lipopolysaccharide-induced RANTES promoter activation in BV-2 microglial cells.

Lipopolysaccharide (LPS), a part of the outer membrane of gram-negative bacteria activates the expression of the regulated upon activation, normal T cell expressed and secreted (RANTES), which plays an important role in the chemo-attraction of leukocytes during the inflammatory response. Recently, we found that LPS-induced RANTES production is mediated by the activation of NF-kappaB, but, the upstream regulatory mechanism involved in mediating this NF-kappaB activation was unclear. In this study, we investigated signal transducing molecules that mediate LPS-induced RANTES promoter activation and found the followings. First, LPS activates the RANTES gene promoter through NF-kappaB binding sites. Second, the expression of dominant negative mutants of TGF-beta-activated kinasel (TAK1) and NF-kappaB-inducing kinase (NIK), blocked the LPS-induced transcriptional activation of RANTES promoter. Moreover, the overexpression of TAK1 along with TAK1-binding protein 1 (TAB1), or NIK stimulated the transcriptional activation of RANTES in the absence of external stimuli. Third, we showed that endogenous TAK1 is phosphorylated by LPS stimulation, and that the association between TAK1 and tumour necrosis factor receptor-associated factor 6 (TRAF6) is constitutive and not induced by LPS treatment. These results indicate that NF-kappaB mediates LPS-triggered RANTES induction and that TAK1 as well as NIK, as NF-kappaB activators participates in LPS-triggered RANTES induction.

Inhibition of ras-mediated activator protein 1 activity and cell growth by merlin.

The neurofibromatosis type2 (NF2) gene encodes an intracellular-associated protein that is referred to as either merlin or schwannomin. Merlin/Schwannomin is a tumor suppressor protein that has been reported to block the Ras-mediated cell proliferation and -anchorage-independent cell growth. However, its inhibitory mechanism is uncertain. In this report, merlin was demonstrated to be effective in suppressing Ras-induced foci-formation and its associated AP-1 activity in NIH3T3cells. In addition, merlin blocked Ras-induced Rb phosphorylation, and inhibited the increase of cyclin D1 levels. It also blocked E2F-1-dependent transcription. These results suggest that merlin inhibits abnormal cell proliferation which is activated via Ras by repressing Rb phosphorylation, blocking the increase of the cyclin D1 protein level, and inhibiting the activation of AP-1- and E2F-1-dependent transcription in NIH3T3 cells.

Identification of the elements regulating the expression of the phospholipase C delta1.

Recently we cloned the phospholipase C deltal (PLC-delta1) promoter region and found that PLC-delta1 is selectively expressed in several tissues. In order to establish the common and cell-type specific transcriptional elements, 1.8 kilobase of the 5'-flanking region of PLC-delta1 was characterized in several cell lines. A transient transfection assay of the -1787-Luc construct in several cell lines demonstrated the potential transcriptional enhancement of reporter activities in the neuroblastoma cell [SK-N-BE(2)C] and kidney cell lines (Cos-7), but not in liver cell lines (Chang liver cell). Transient transfection assays of a series of 5' --> 3' deletion constructs of the PLC-delta1 promoter and electronic mobility shift assays suggested that the E-box and HFH3 binding sites are cell-type specific elements, and that Sp-1 is a major transcriptional activator of a majority of cell lines. Our findings, therefore, indicate that the combination of several elements within the 5'-flanking region of the PLC-delta1 gene dictates its restricted expression in several cell lines.