Seung-Chul Choi

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.

Expression of NDRG2 is related to tumor progression and survival of gastric cancer patients through Fas-mediated cell death

Although N-myc downstream regulated gene 2 (NDRG2) has been known to be a tumor suppressor gene, the function of this gene has not been elucidated. In the present study, we investigated the expression and function of NDRG2 in human gastric cancer. Among seven gastric cancer and two non-cancer cell lines, only two gastric cancer cell lines, SNU-16 and SNU-620, expressed NDRG2, which was detected in the cytoplasm. Interestingly, NDRG2 was highly expressed in normal gastric tissues, but gastric cancer patients were divided into NDRG2-positive and -negative groups. The survival rate of NDRG2-negative patients was lower than that of NDRG2-positive patients. We confirmed that the loss of NDRG2 expression was a significant and independent prognostic indicator in gastric carcinomas by multivariate analysis. To investigate the role of NDRG2 in gastric cancer cells, we generated a NDRG2-silenced gastric cancer cell line, which stably expresses NDRG2 siRNA. NDRG2-silenced SNU-620 cells exhibited slightly increased proliferation and cisplatin resistance. In addition, inhibition of NDRG2 decreased Fas expression and Fas-mediated cell death. Taken together, these data suggest that inactivation of NDRG2 may elicit resistance against anticancer drug and Fas-mediated cell death. Furthermore, case studies of gastric cancer patients indicate that NDRG2 expression may be involved in tumor progression and overall survival of the patients.

A proinflammatory cytokine interleukin-32β promotes the production of an anti-inflammatory cytokine interleukin-10

A new proinflammatory cytokine interleukin‐32 (IL‐32) has six isoforms. Although IL‐32 can be detected in sera from patients suffering from Crohn’s disease and rheumatoid arthritis, it is unclear which isoforms are involved. To this end, we investigated the functions of the most abundant IL‐32β by generating K562‐IL‐32β stable cell lines. This report confirms, using IL‐32 small interfering RNA, that IL‐32β induces an anti‐inflammatory cytokine IL‐10 in K562‐IL‐32β cells and U937 promonocytic cells, which express endogenous IL‐32β upon phorbol 12‐myristate 13‐acetate (PMA) treatment, and monocyte‐derived dendritic cells (DC) upon lipopolysaccharide (LPS) treatment. Interleukin‐32β was induced in monocyte‐derived macrophages by LPS and in monocyte‐derived DC by LPS, poly(I:C), or anti‐CD40 antibody, but was not induced by PMA. We showed that IL‐32β expression was increased in a time‐dependent manner in monocyte‐derived DC upon LPS treatment and peaked at 24 hr. Production of IL‐10 was exactly coincident with IL‐32β expression, but IL‐1β and tumour necrosis factor‐α production peaked at 6 hr after LPS treatment, then steeply declined. Interleukin‐12 p40 was induced at 9 hr and gradually increased until 48 hr, at which time IL‐32β and IL‐10 were no longer increased. Knock‐down of IL‐32β by IL‐32 small interfering RNA led to the decrease of IL‐10, but the increase of IL‐12 in monocyte‐derived DC, which means that IL‐32β promotes IL‐10 production, but limits IL‐12 production. We also showed that IL‐10 neutralization increases IL‐12, IL‐1β and tumour necrosis factor‐α production, which implies that IL‐10 suppresses such proinflammatory cytokines. Taken together, our results suggest that IL‐32β upregulates the production of an anti‐inflammatory cytokine IL‐10, and then IL‐10 suppresses proinflammatory cytokines.

Up-regulation of Mac-2 binding protein by hTERT in gastric cancer

Mac‐2 binding protein (Mac‐2BP) is a secreted tumor antigen that is elevated in many cancers and implicated in tumor metastasis, as well as cell adhesion and immune functions. We focused on the human telomerase reverse transcriptase (hTERT) induced Mac‐2BP expression and the relationship between Mac‐2BP expression and the progression of gastric cancer. A cDNA expression array analysis was performed on the telomerase‐negative cell line, SW13, which was engineered to overexpress hTERT when compared with the parental SW13 cell. hTERT‐induced Mac‐2BP expression was confirmed via RT‐PCR and Northern blotting. ELISA and flow cytometric analyses revealed that Mac‐2BP protein was increased by 2‐ to 4‐fold in hTERT‐overexpressing cells compared with the mock control. Mac‐2BP expression was significantly reduced when the overexpressed hTERT was neutralized by the introduction of hTERT‐specific siRNA. These results suggest that Mac‐2BP expression is modulated by hTERT. Mac‐2BP levels in both gastric cancer cells and tumor tissues were determined via Northern blot analysis and immunohistochemistry. Mac‐2BP protein was highly expressed in most gastric cancer cell lines, and gastric tumor tissues were stained more densely than normal tissues. The intracellular and secreted Mac‐2BP levels were also evaluated via ELISA, indicating that Mac‐2BP was expressed and secreted more abundantly in gastric cancer patients than in healthy donors. The elevated serum Mac‐2BP level in gastric tumor patients was also significantly associated with distant metastasis (p = 0.05) and higher tumor stage (p = 0.04). Our findings suggest that Mac‐2BP is induced by hTERT, and that it may prove to be a useful prognostic marker for the detection of malignant progression of metastatic stomach cancers.

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.

Impaired responses of leukemic dendritic cells derived from a human myeloid cell line to LPS stimulation

Several myeloid leukemia-derived cells have been reported to possess the ability to differentiate into dendritic cells (DC). MUTZ-3, a myeloid leukemia cell line, responds to GM-CSF, IL-4 and TNF-α, and acquires a phenotype similar to immature monocyte-derived DC (MoDC). In the present study, MUTZ-3-derived DC (MuDC) showed high level expression of HLA class II molecules, CD80 and CD86, and were able to function as potent antigen presenting cells as previously reported. Interestingly, MuDC maturation was induced by CD40-mediated stimulation, but not by LPS stimulation. We analyzed CCR1, CCR7 and Toll-like receptor (TLR) expressions in MuDC, and measured IL-10 and IL-12 production after maturation stimuli. Although MuDC expressed the mRNA for TLR4, a major component of the LPS receptor system, they did not show an enhanced level of CCR7 or cytokine production after LPS stimulation. In contrast, they responded to CD40 stimulation, which resulted in increased levels of CD83, CD86 and CCR7. Moreover, while LPSstimulated MoDC could potently stimulate NK cells in a DC-NK cell co-culture, LPS-stimulated MuDC failed to stimulate primary NK cells. Taken together, our findings suggest that, although MuDC express TLR4, unlike TNF-α and IL-1β, LPS does not stimulate MuDC to acquire mature phenotypes, and they may have impaired activity to initiate innate immune response.

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.