Jong-Tae Kim

Expression and regulation of NDRG2 (N-myc downstream regulated gene 2) during the differentiation of dendritic cells

We searched for genes with expressions specific to human monocyte‐derived dendritic cells (DCs) using differential display reverse transcription‐polymerase chain reaction, and found that N‐myc downstream regulated gene 2 (NDRG2), a member of a new family of differentiation‐related genes, was expressed in DCs. While DCs derived from CD34+ progenitor cells also showed strong NDRG2 expression, the corresponding mRNA expression was absent in other cell lines including monocytes, B cells, and NK cells. The inhibition of DC differentiation by dexamethasone or vitamin D3 treatment down‐regulated the expression of the NDRG2 gene in DCs. In addition, gene expression was induced in a myelomonocytic leukemia cell line, which is capable of differentiating into DCs in cytokine‐conditioned culture. The level of NDRG2 gene expression in DCs was significantly higher than that of other members of the NDRG gene family. Finally, in contrast to the stable NDRG2 expression in CD40‐stimulated DCs, the induction of DC maturation by lipopolysaccharide (LPS) resulted in the down‐regulation of NDRG2 gene expression. This down‐regulation is likely to be due to a modification and subsequent destabilization of NDRG2 mRNA, because co‐treating with actinomycin D and LPS significantly blocked this LPS effect. Taken together, our results indicate that NDRG2 is expressed during the differentiation of DCs, and that NDRG2 gene expression is differentially regulated by maturation‐inducing stimuli.

NDRG2 suppresses cell proliferation through down‐regulation of AP‐1 activity in human colon carcinoma cells

Recently, the anti‐tumor activity of N‐myc downstream‐regulated gene 2 (NDRG2) was elucidated, but the molecular mechanism of how NDRG2 works as a tumor suppressor is not well known. To determine the function of NDRG2 as a tumor suppressor, we established stable cell lines expressing NDRG2 protein or its mutant forms, and studied their effects on tumor cell growth. Interestingly, constitutive expression of wild‐type NDRG2 induced the growth retardation of SW620 colon carcinoma cells. Introduction of NDRG2 into SW620 cells induced the decrease of c‐Jun phosphorylation at Ser63, followed by the attenuation of activator protein‐1 (AP‐1) function as a transcriptional activator. Subsequently, the down‐regulation of cyclin D1, which is known as a major target for AP‐1 transcription activator, resulted in cell cycle arrest at G1/S phase. Additionally, treatment of NDRG2‐siRNA on NDRG2‐expressing cells has induced the recovery of c‐Jun phosphorylation and cyclin D1 expression. Cell proliferation of those cells was also increased compared with untreated cells. NDRG2 mutants of which the phosphorylation sites at C‐terminal region were removed by deletion or site‐directed mutagenesis have shown no effect on cyclin D1 expression and could not induce cell growth retardation. In conclusion, NDRG2 modulates intracellular signals to control cell cycle through the regulation of cyclin D1 expression via phosphorylation pathway.

NDRG2 expression decreases with tumor stages and regulates TCF/β-catenin signaling in human colon carcinoma

NDRG (N-Myc downstream-regulated gene)-2 is a member of the NDRG family. Although it has been suggested that NDRG2 is involved in cellular differentiation and tumor suppression, its intracellular signal and regulatory mechanism are not well known. Here, we show the differential expression of NDRG2 in human colon carcinoma cell lines and tissues by reverse transcription–polymerase chain reaction and immunohistochemical analyses with monoclonal antibody against NDRG2. NDRG2 was strongly expressed in normal colonic mucosa and colonic adenomatous tissues (25 of 25) but not in all invasive cancer tissues [44 of 99 (44%)]. Most distinctive results indicated that the high expression level of NDRG2 has a positive correlation with tumor differentiation and inverse correlation with tumor invasion depth and Dukes’ stage of colon adenocarcinoma. To investigate the roles of NDRG2 in tumorigenesis, we used in vitro cell culture system. SW620 colon cancer cell line with a low level of intrinsic NDRG2 protein was transfected with NDRG2-expressing plasmid. TOPflash luciferase reporter assay showed that the transcriptional activity of T-cell factor (TCF)/lymphoid enhancer factor (LEF) was reduced by NDRG2 introduction, but not by the introduction of mutant NDRG2 generated by deletion or site-directed mutagenesis. Intracellular β-catenin levels were slightly reduced in the NDRG2-transfected SW620 cells and this regulation of β-catenin stability and TCF/LEF activity were mediated through the modulation of glycogen synthase kinase-3beta activity by NDRG2 function. Our results suggest that NDRG2 might play a pivotal role as a potent tumor suppressor by the attenuation of TCF/β-catenin signaling for the maintenance of healthy colon tissues.

Up‐regulation and clinical significance of serine protease kallikrein 6 in colon cancer

Kallikrein‐related peptidase 6 (KLK6) encodes a trypsin‐like serine protease that is up‐regulated in several cancers, although the putative functions of KLK6 in cancer have not been elucidated. In the current study, overexpression of KLK6 was identified in colon cancer, and the possibility that KLK6 may be a suitable candidate as a tumor marker was examined.

Upregulation and secretion of macrophage inhibitory cytokine-1 (MIC-1) in gastric cancers

BACKGROUND Macrophage inhibitory cytokine-1 (MIC-1), a distant member of the transforming growth factor (TGF)-beta superfamily, has been reported to be upregulated and secreted from several cancers. We examined MIC-1 expression and secretion in gastric cancers. METHODS MIC-1 mRNA and protein levels in cancer tissues and cell lines were analyzed by RT-PCR and Western blot. MIC-1 expression in cancer tissues and its secretion in serum were analyzed using immunohistochemistry and ELISA. RESULTS MIC-1 was significantly upregulated in gastric cancer tissues and cell lines. MIC-1 was secreted from gastric SNU620 cells and its levels in the serum of cancer patients were 10-fold higher than those of healthy controls. In addition, the staining of MIC-1 expression was strongly increased in metastatic gastric cancers. CONCLUSIONS MIC-1 was obviously overexpressed in gastric cancers and MIC-1 secretion into blood may be useful for the prediction of gastric cancer progression.

Expression of human NDRG2 by myeloid dendritic cells inhibits down-regulation of activated leukocyte cell adhesion molecule (ALCAM) and contributes to maintenance of T cell stimulatory activity

We reported previously that N‐myc downstream‐regulated gene 2 (NDRG2), a member of a new family of differentiation‐related genes, is expressed specifically in dendritic cells (DC) differentiated from monocytes, CD34+ progenitor cells, and the myelomonocytic leukemic cell line. In this study, we demonstrate that NDRG2 protein expression is detected, not only in in vitro‐differentiated DC but also in primary DC from lymph nodes, thymus, and skin when anti‐NDRG2 antibodies are used. As predicted from previous studies investigating the mRNA expression pattern of several types of cell lines, progenitor cells, and DC, NDRG2 protein was expressed strongly in DC. Its expression was detected at significant levels after differentiation from progenitor cells. RNA interference of NDRG2 demonstrated that activated leukocyte cell adhesion molecule (ALCAM) expression is down‐regulated specifically in DC differentiated from NDRG2 small interfering RNA (siRNA)‐transfected monocytes. This was consistent with our observation that U937 cells transfected with NDRG2 became resistant to the GM‐CSF/IL‐4‐induced ALCAM reduction. Furthermore, DC, which had differentiated from NDRG2 siRNA‐transfected monocytes, showed a reduced ability to induce T cell proliferation. Taken together, our results indicate that NDRG2 is able to preserve ALCAM expression during DC differentiation from monocytes under cytokine culture conditions and that its expression helps DC maintain costimulatory signals necessary for T cell stimulation.

Upregulation of the cycline kinase subunit CKS2 increases cell proliferation rate in gastric cancer.

PurposeCKS2 was identified as an upregulated gene in gastric cancer via our DNA microarray. This study was to verify the upregulation of CKS2 in many gastric cancer patients and to examine the CKS2-mediated cellular response.MethodsCKS2 upregulation was analyzed using reverse transcriptase PCR, real-time PCR, and immunohistochemical and clinicopathological analyses. GFP-CKS2 or CKS2-siRNA was used to analyze the cellular localization and proliferation.ResultsThe strong upregulation of mRNA and protein levels of CKS2 was identified. In CKS2-overexpressing cells, tumor suppressor p53 and p21cip1 were downregulated and cell growth was increased. In contrast, CKS2-siRNA-transfected cells showed an increased tumor suppressor expression and decreased cell growth.ConclusionsWe showed that CKS2 was significantly upregulated in gastric cancers and a high level of CKS2 was highly correlated with histologic tumor differentiation and pathological grade of the tumor size, lymph node, and metastasis stage. We suggest that the cell cycle regulator CKS2 might be deeply involved in gastric cancer progression.

NDRG2 and PRA1 interact and synergistically inhibit T-cell factor/β-catenin signaling

NDRG2a and PRA1 colocalize by fluorescence microscopy (View Interaction: 1, 2, 3)

NDRG2 is one of novel intrinsic factors for regulation of IL-10 production in human myeloid cell

N-myc downstream-regulated gene 2 (NDRG2) implicated in cellular growth and differentiation was previously reported as it is specifically expressed in primary and in vitro-differentiated dendritic cells (DCs) from monocytes and CD34(+) progenitor cells. However, its function has yet to be investigated in DCs. Here, the novel NDRG2 function about modulation of cytokines in DC was observed in this study. The secretion of IL-10 was not found in the monocyte-derived DC cells with high level of NDRG2 expression, but IL-10 was abundantly secreted up to 1ng/ml in the monocyte-derived macrophages with low level of NDRG2 expression, and further confirmed that the expression of IL-10 was dramatically increased in NDRG2-silenced DCs under presence of LPS, and significantly reduced in the NDRG2-overexpressed U937 cells under stimulation of PMA. The secretion of IL-12p70 was significantly reduced in the siNDRG2 introduced DC cells. The intracellular signaling of IL-10 secretion was markedly inhibited by SB203580, inhibitor of p38 MAPK, in the LPS-activated DCs and phosphorylation of p38 MAPK was decreased in the NDRG2 introduced U937 cells under PMA-stimulation. Taken together, NDRG2 might have a pivotal role as one of intrinsic factors for the modulation of p38 MAPK phosphorylation, and subsequently involve in controlling of IL-10 production.

Cystatin SN inhibits auranofin-induced cell death by autophagic induction and ROS regulation via glutathione reductase activity in colorectal cancer

Cystatin SN (CST1) is a specific inhibitor belonging to the cystatin superfamily that controls the proteolytic activities of cysteine proteases such as cathepsins. Our previous study showed that high CST1 expression enhances tumor metastasis and invasiveness in colorectal cancer. Recently, auranofin (AF), a gold(I)-containing thioredoxin reductase 1 (TrxR1) inhibitor, has been used clinically to treat rheumatoid arthritis. AF is a proteasome-associated deubiquitinase inhibitor and can act as an anti-tumor agent. In this study, we investigated whether CST1 expression induces autophagy and tumor cell survival. We also investigated the therapeutic effects of AF as an anti-tumor agent in colorectal cancer (CRC) cells. We found that CRC cells expressing high levels of CST1 undergo increased autophagy and exhibit chemotherapeutic resistance to AF-induced cell death, while those expressing low levels of CST1 are sensitive to AF. We also observed that knockdown of CST1 in high-CST1 CRC cells using CST1-specific small interfering RNAs attenuated autophagic activation and restored AF-induced cell mortality. Conversely, the overexpression of CST1 increased autophagy and viability in cells expressing low levels of CST1. Interestingly, high expression of CST1 attenuates AF-induced cell death by inhibiting intracellular reactive oxygen species (ROS) generation, as demonstrated by the fact that the blockage of ROS production reversed AF-induced cell death in CRC cells. In addition, upregulation of CST1 expression increased cellular glutathione reductase (GR) activity, reducing the cellular redox state and inducing autophagy in AF-treated CRC cells. These results suggest that high CST1 expression may be involved in autophagic induction and protects from AF-induced cell death by inhibition of ROS generation through the regulation of GR activity.