In Seong Choe

Dendritic cell-tumor coculturing vaccine can induce antitumor immunity through both NK and CTL interaction.

Immunization of dendritic cells (DC) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL) that are responsible for protection and regression. We show here that immunization with bone marrow-derived DC cocultured with tumor cells can induce a protective immunity against challenges to viable tumor cells. In this study, we further investigated the mechanism by which the antitumor activity was induced. Immunization of mice with DC cocultured with murine colon carcinoma. CT-26 cells, augmented CTL activity against the tumor cells. Concomitantly, an increase in natural killer (NK) cell activity was also detected in the same mice. When DC were fixed with paraformaldehyde prior to coculturing with tumor cells, most of the CTL and NK cell activity diminished, indicating that DC are involved in the process of presenting the tumor antigen(s) to CTL. NK cell depletion in vivo produced markedly low tumor-specific CTL activity responsible for tumor prevention. In addition, RT-PCR analysis confirmed the high expression of INF-gamma mRNA in splenocytes after vaccination with DC cocultured with tumors, but low expression in splenocytes from NK-depleted mice. Most importantly, the tumor protective effect rendered to DC by the coculturing with CT-26 cells was not observed in NK-depleted mice, which suggests that DC can induce an antitumor immune response by enhancing NK cell-dependent CTL activation. Collectively, our results indicate that NK cells are required during the priming of cytotoxic T-cell response by DC-based tumor vaccine and seem to delineate a mechanism by which DC vaccine can provide the desired immunity.

Inhibition of glucocorticoid-mediated, caspase-independent dendritic cell death by CD40 activation

Glucocorticoids (GC) are potent anti‐inflammatory and immunosuppressive agents that act on a variety of immune cells, including T cells, monocytes/macrophages, osteoclasts, and dendritic cells (DC). However, the mechanism(s) by which GC exert anti‐inflammatory effects is still largely unknown. It is already well known that GC treatment inhibits DC maturation and interleukin (IL)‐12 production by DC. In this study, we investigated the apoptosis induction of DC by a synthetic GC, dexamethasone (Dex). The stimulation with Dex resulted in DC apoptosis in a dose‐ and time‐dependent manner as it was measured by determining annexin V‐positive cells and mitochondrial potential. In contrast, monocytes that are precursor cells of DC are resistant to Dex‐mediated apoptosis. The Dex‐induced apoptosis of DC was independent of caspase activation because it was not inhibited by the broad caspase inhibitor, Z‐VAD‐fmk. It is interesting that agonistic CD40 antibody completely inhibited Dex‐induced cell death, whereas other inflammatory stimuli did not show the same effect, suggesting that CD40 signaling may selectively modulate GC‐mediated DC apoptosis. Taken together, our findings revealed an important role of GC and CD40 signaling in the regulation of immune responses in which DC play a key role in the inflammatory process of various immunomediated diseases.

S100A6 (calcyclin) enhances the sensitivity to apoptosis via the upregulation of caspase-3 activity in Hep3B cells.

S100A6 (calcyclin) is a small calcium‐binding protein which has been implicated in several cellular processes such as cell cycle progression, cytoskeleton rearrangement, and exocytosis. Also the upregulation of S100A6 has been reported in a variety of tumors and linked to metastasis. However, exact intracellular roles of S100A6 related with apoptosis have not been clarified yet. Here we demonstrated that the upregulation of S100A6 enhances the cell death rate compared to the control under the apoptotic conditions. In exogenously S100A6 induced Hep3B cells, cell viability was significantly decreased compared with mock and S100A6‐knockdown cells under calcium ionophore A23187 treatment. The exogenously introduced S100A6 significantly affected the caspase‐3‐like activity in programmed cell death through the enhanced caspase‐3 expression, which was verified by promoter assay in wild or mutant S100A6‐transfected Hep3B cells. Next, the promoter activity of caspase‐3 was increased by 2.5‐folds in wild‐type S100A6‐transfected cells compared to mutant 2 (E67K, mutant of EF‐hand motif) or control. Our results suggest that S100A6 might be involved in the processing of apoptosis by modulating the transcriptional regulation of caspase‐3. J. Cell. Biochem. 103: 1183–1197, 2008.

S100A6 protein as a marker for differential diagnosis of cholangiocarcinoma from hepatocellular carcinoma

A new marker useful for differential diagnosis of cholangiocarcinoma (CC) from hepatocellular carcinoma (HCC) has been identified and investigated. cDNA clone for S100A6 (calcyclin) was identified as a upregulated gene in intrahepatic tumors by differential dot hybridization and Northern blot analysis using normal and tumor intrahepatic tissues. Significant increase in the expression level of S100A6 mRNA was recognized in intrahepatic tissues in three of three CC cases but none of six primary HCC. Immunohistochemical examination of the tumor tissues using monoclonal antibody specific to S100A6 showed that 14 of 18 (77.8%) CC tissue samples were positively stained but only two of 20 (10%) specimens from the patients with primary HCC were weakly stained. These results suggest that the levels of S100A6 mRNA expression and protein in the tissue specimens examined by Northern blot and immunohistochemical staining may be additional useful marker for the differentiation of CC from HCC.

Involvement of NF-κB in the regulation of S100A6 gene expression in human hepatoblastoma cell line HepG2

Abstract S100A6 (calcyclin) is an acidic calcium binding protein with two EF-hand motifs and overexpressed in several tumors including intrahepatic carcinoma. TNFα, a strong NF-κB activator required for hepatocyte proliferation during liver regeneration, triggered the expression of S100A6 mRNA in human hepatoblastoma cell line HepG2. Transient expression of NF-κB (p65) increased S100A6 promoter activity and expression of inhibitor of NF-κB (IκBα) decreased TNFα-induced S100A6 promoter activity. To confirm the involvement of NF-κB in S100A6 promoter activation, we analyzed serially deleted promoter constructs of the S100A6 gene by luciferase reporter assay and found a NF-κB-responsive DNA fragment at the position between −584 and −361. Electrophoretic mobility shift assays showed that TNFα induced p65 binding to a potential NF-κB binding site at −460/−451. Furthermore, treatment of cells with CAPE (caffeic acid phenethyl ester), a specific NF-κB (p65) inhibitor, decreased NF-κB binding and promoter activity. These results suggest that NF-κB transcription factor contributes to the activation of S100A6 gene expression in response to TNFα in HepG2 cells.

Cloning and expression of human cDNA encoding human homologue of pituitary tumor transforming gene.

Recently, a potent transforming gene which was exclusively expressed in rat pituitary tumor but not in normal pituitary had been isolated and named as pituitary tumor transforming gene (PTTG). A cDNA clone encoding human homologue of rat PTTG was isolated from human fetal liver cDNA library. It contained an open reading frame of 603 base pairs predicting a protein composed of 201 amino acids with a calculated molecular weight of 26 kDa. The deduced protein showed about 85% homology (78% identity, 7% favored substitution) with the rat PTTG. Northern blot analysis showed that the cDNA hybridized to 1.0 kb mRNA species which was expressed in fetal liver and several cancer cell lines. These results suggest that the presence of the human homologue of rat PTTG gene may not be restricted to pituitary tumor.

Overexpression and clinical significance of carcinoembryonic antigen-related cell adhesion molecule 6 in colorectal cancer.

BACKGROUNDnCarcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) inhibits anoikis and affects the malignant phenotype of cancer cells. In this study, we analyzed CEACAM6 as a gene that is highly upregulated in colon cancer tissues, and examined the assertion that CEACAM6 might be a suitable candidate tumor marker for the diagnosis of colon cancer.nnnMETHODSnCEACAM6 gene expression in human colon tissues was performed by tissue microarray and analyzed using RT-PCR (each of normal and tumor tissue, n=40) and immunohistochemical and clinicopathological (colon cancer patients, n=143) analyses.nnnRESULTSnCEACAM6 transcriptional and translational levels were significantly upregulated in human tumor tissues compared to non-tumor regions, and clinicopathological analysis revealed a significant correlation between CEACAM6 protein expression and Dukes stage (p<0.001). High expression levels of CEACAM6 were significantly associated with lower overall survival (p<0.001) and shorter recurrence-free survival (p<0.001). We demonstrated that knockdown of CEACAM6 with CEACAM6-specific small interfering RNA in colorectal cancer cells attenuated invasivity (35%); conversely, the overexpression of CEACAM6 increased invasiveness.nnnCONCLUSIONSnCEACAM6 is significantly upregulated in colon cancer tissues and is closely associated with poor prognosis, indicating that CEACAM6 might be used as a tumor biomarker and a potential therapeutic target for colon cancer.

ISOLATION AND CHARACTERIZATION OF CDNA CLONE FOR HUMAN LIVER 10-FORMYLTETRAHYDROFOLATE DEHYDROGENASE

A cDNA clone encoding 10‐formyltetrahydrofolate dehydrogenase (10‐FTHFDH) was isolated from a human fetal liver cDNA library. It contained the open reading frame of 2,709 base pairs and predicted a protein comprising 902 amino acids with a calculated molecular weight of 98,700 Da. The deduced protein showed about 93.6% homology (90.5% identity, 3.1% favored substitutions) when compared with rat 10‐FTHFDH. The distribution of 10‐FTHFDH transcript in various human tissues was studied by Northern blot analysis using poly(A+) RNAs from different tissues. The 10‐FTHFDH transcript with an approximate size of 2.7 kb was mainly expressed in human kidney, skeletal muscle, and liver and rarely expressed in other tissues.

Protective antitumor activity through dendritic cell immunization is mediated by NK cell as well as CTL activation

Dendritic cells (DCs) are potent professional antigen-presenting cells (APC) capable of inducing the primary T cell response to antigen. Although tumor cells express target antigens, they are incapable of stimulating a tumor-specific immune response due to a defect in the costimulatory signal that is required for optimal activation of T cells. In this work, we describe a new approach using tumor-DC coculture to improve the antigen presenting capacity of tumor cells, which does not require a source of tumor-associated antigen. Immunization of a weakly immunogenic and progressive tumor cocultured with bone marrow-derived DCs generated an effective tumor vaccine. Immunization with the cocultured DCs was able to induce complete protective immunity against tumor challenges and was effective for the induction of tumor-specific CTL (cytotoxic T lymphocyte) activity. Furthermore, high NK cell activity was observed in mice in which tumors were rejected. In addition, immunization with tumor-pulsed DCs induced delayed tumor growth, but not tumor eradication in tumor-bearing mice. Our results demonstrate that coculture of DCs with tumors generated antitumor immunity due to the NK cell activation as well as tumor-specific T cell. This approach would be useful for designing tumor vaccines using DCs when the information about tumor antigens is limited.

Effect of High Blood Flow on the Expression of Endothelial Constitutive Nitric Oxide Synthase in Rats with Femoral Arteriovenous Shunts

The effect of high blood flow on the expression of endothelial nitric oxide synthase has been investigated in the femoral arteriovenous shunt (AVS) rats created by inserting U-shaped polyurethane tubes in the left femoral arteries and veins. Three days after inserting the femoral AVS, the mean aortic blood flow rate in the abdominal aorta of the AVS rats was about 2.0 times higher than that in the control rats (110.0 +/- 8.4 ml/min vs 52.7 +/- 2.7 ml/min, p < 0.001). The competitive reverse transcriptase-polymerase chain reaction (RT-PCR) data revealed that the mRNA expression level of the endothelial constitutive nitric oxide synthase (ecNOS) was increased in the aortas of the femoral AVS rats compared to that in the control rats. Western blot analysis using a monoclonal antibody against ecNOS revealed that the ecNOS protein levels were markedly increased in the aortas of femoral AVS rats, but ecNOS protein levels in aortas without endothelium were not significantly increased. Inducible nitric oxide synthase (iNOS) protein was not expressed in the aortic tissues with and without endothelium in the control rats. This iNOS expression was not increased by the high blood flow in the femoral AVS rats. These findings suggest that high blood flow could up-regulate the expression levels of ecNOS mRNA and proteins in femoral arteriovenous shunt rats.