Seong-Yong Kim

Exploration of replicative senescence-associated genes in human dermal fibroblasts by cDNA microarray technology

The aging process is known to be regulated by specific genes in various organisms, including yeast, the nematode C. elegans, fruitflies and mice. To explore the novel genes involved in aging process, we applied cDNA microarray technology to a replicative senescence model of human dermal fibroblasts (HDF). Eighty-four genes, including inflammatory genes, cell cycle regulatory genes, cytoskeletal genes, and metabolic genes were found to show more than two fold expressional differences in young and old fibroblasts. Furthermore, 31 genes were confirmed to be up- or down-regulated during replicative senescence by semi-quantitative RT-PCR. The overexpressions of several genes including CD36, putative lymphocyte G0/G1 switch gene (G0S2), tumor protein D52-like 1 (TPD52L1), chemokine (C-X-C motif) ligand 6, myxovirus resistant gene 1 (MX1), and the down-regulation of the immunoglobulin superfamily containing leucine-rich repeat (ISLR), neurotrimin, insulin-like growth factor 2 associated protein (IGF2A), and apoptosis-related RNA binding protein (NAPOR3) were newly identified. These results suggest that fibroblasts show the deregulation of various cellular processes, such as inflammatory response, mitosis, cell adhesion, transport, signal transduction, and metabolism during replicative senescence.

Identification of amyloid

Amyloid β-peptide (Aβ), a causative molecule in the pathogenesis of Alzheimers disease and the main component of senile plaques, is known to be neurotoxic in vitro and in vivo. The mechanisms involved in this Aβ-mediated neurotoxicity are not fully understood, although there is evidence to suggest the involvement of oxidative stress, alterations in calcium homeostasis, and/or of CDK activators. Many studies have suggested that Aβ may exert its toxic effect via the activation of transcription factors. Therefore, we investigated Aβ- responsive genes in human neuroblastoma CHP134 cells using 3.1K human DNA microarrays. Among the several genes overexpressed or repressed by Aβ, RTP801, Hi95/sestrin 2, and stanniocalcin 2 were confirmed to be Aβ-mediated overexpression in the cells by semiquantitative RT-PCR. Transient expression of the sense RTP801 gene in CHP134 cells increased sensitivity to Aβ cytotoxicity and the expression of the antisense RTP801 gene protected the cells from the Aβ toxicity. These results suggest that RTP801 might play important roles in Aβ toxicity and the pathogenesis of Alzheimers disease.

{\beta}-peptide

The induction of inducible NO synthase (iNOS) by group IIA phospholipase A2 (PLA2) involves the stimulation of a novel signaling cascade. In this study, we demonstrate that group IIA PLA2 up-regulates the expression of iNOS through a novel pathway that includes M-type secretory PLA2 receptor (sPLA2R), phosphatidylinositol 3-kinase (PI3K), and Akt. Group IIA PLA2 stimulated iNOS expression and promoted nitrite production in a dose- and time-dependent manner in Raw264.7 cells. Upon treating with group IIA PLA2, Akt is phosphorylated in a PI3K-dependent manner. Pretreatment with LY294002, a PI3K inhibitor, strongly suppressed group IIA PLA2-induced iNOS expression and PI3K/Akt activation. The promoter activity of iNOS was stimulated by group IIA PLA2, and this was suppressed by LY294002. Transfection with Akt cDNA resulted in Akt protein overexpression in Raw264.7 cells and effectively enhanced the group IIA PLA2-induced reporter activity of the iNOS promoter. M-type sPLA2R was highly expressed in Raw264.7 cells. Overexpression of M-type sPLA2R enhanced group IIA PLA2-induced promoter activity and iNOS protein expression, and these effects were abolished by LY294002. However, site-directed mutation in residue responsible for PLA2 catalytic activity markedly reduced their ability to production of nitrites and expression of iNOS. These results suggest that group IIA PLA2 induces nitrite production by involving of M-type sPLA2R, which then mediates signal transduction events that lead to PI3K/Akt activation.

responsive genes by cDNA microarray technology: Involvement of RTP801 in amyloid

Mammalian cells contain two forms of thioredoxin reductase (TrxR), cytosolic TrxR1 and mitochondrial TrxR2. To investigate the biological roles of TrxR2, we generated stable HeLa cell lines expressing a dominant negative form of TrxR2 (TrxR2DN) under the control of the tetracycline-off system. We observed that TrxR2DN-induced cells, following stimulation with EGF, produced more hydrogen peroxide than uninduced cells. The extent of protein tyrosine phosphorylation of many proteins including ERK was higher in TrxR2DN-induced cells than in uninduced cells when stimulated with fetal bovine serum or EGF. Induction of TrxR2DN also resulted in the increased rate of progression of G1 to S phase in cell cycle and cell proliferation and affected the expression of many proteins involved in cell cycle. These results suggest that TrxR2 participates in the regulation of protein tyrosine phosphorylation and cell growth as a component of the mitochondria specific H2O2-eliminating system that includes peroxiredoxin III and thioredoxin 2.

{\beta}-peptide

Abstract The purpose of this study was to investigate the effect of green tea catechin on enzyme activities and gene expression of antioxidative system in rat liver exposed to microwaves. Sprague-Dawley male rats 100±10 g body weight were randomly divided into control group and microwave exposed group: Microwave exposed group was further divided into three groups: catechin free diet (MW) group, 0.25% catechin (MW-0.25C) group and 0.5% catechin (MW-0.5C) group. The rats were irradiated with microwave at frequency of 2.45 GHz for 15 min and then the changes in the pattern of antioxidative defense system and gene expression were investigated for 16 days (the 2nd, 4th, 6th, 8th and 16th days), and compared with the control group. The activity of superoxide dismutase (SOD) in MW group was lower on the 4th day after irradiation and increased in the catechin supplementation group were on the 8th day, compared with control group. The activity of glutathone peroxidase (GSHpx) in MW group was lower than that in the control group on the 8th day after irradiation, but increased to the level of the control group on the 16th day and those of MW-0.25C and MW-0.5C groups showed the same level as the control group but that was higher than the control group from 6 days after irradiation. The content of thiobarbituric acid reactive substances (TBARS) in liver of MW group was increased to 1.3, 1.5, and 1.7 fold of the control group at 2, 4, and 6 days after irradiation respectively but that in MW-0.25C and MW-0.5C groups was increased to 1.1, 1.3 and 1,3 fold of the control group at 2, 4, and 6 days but recovered to the level of the control group at 16 days after irradiation. The level of SOD gene expression in MW group was lower than that in the control group but that of MW-0.25C and MW-0.5C group were higher than the MW group. The GSHpx gene expression in MW group was expressed lower than in the control group, but expressed at a higher level in the MW-0.25C and MW-0.5C groups. It is suggested that the damage of liver tissues was alleviated and function rapidly recovered to the normal level due to probable to the correction of imbalances in the antioxidative system with the administration of green tea catechin

toxicity

The mammalian group IIA secretory phospholipase A2 (sPLA2) is believed to play an important role in inflammation and cell injury. The present study underlines the importance of group IIA sPLA2 in the regulation of iNOS. Treatment of cells with sPLA2 induced protein expression and mRNA accumulation of iNOS in a dose‐dependent manner. The pretreatment of cells with ρ‐BPB or SCA, selective sPLA2 inhibitors, inhibited sPLA2‐induced iNOS expression. sPLA2 stimulated the simultaneous activation of two classes of mitogen‐activated protein kinases ERK and JNK, but did not stimulate p38 MAPK. PD98059, a selective MEK inhibitor, inhibited sPLA2‐induced nitrite production and iNOS expression as well as ERK phosphorylation. In addition, pretreatment of ρ‐BPB or SCA also resulted in inhibition of sPLA2‐induced ERK phosphorylation. The sPLA2 signaling mechanisms involving the activation of transcription factor NF‐κB were studied in the same cells. That stimulation of cells with sPLA2 caused NF‐κB activation in a time‐dependent manner was shown by the detection of NF‐κB‐specific DNA‐protein binding and by IκBα degradtion. sPLA2‐induced NF‐κB activation was prevented in the presence of ρ‐BPB. Furthermore, the NF‐κB inhibitor PDTC suppressed sPLA2‐induced nitrite production and iNOS expression as well as IκBα degradation. The results strongly suggest that group IIA sPLA2 induces iNOS in macrophages and that this induction occurs through ERK and NF‐κB.

Akt as a Mediator of Secretory Phospholipase A2 Receptor-Involved Inducible Nitric Oxide Synthase Expression

The CDKN2 (MTS1/p16INK4A) gene, encoding cyclin dependent kinase inhibitor, was found to be homozygously deleted at a high frequency in cell lines from many different types of cancer and some primary cancers. To determine the frequency of CDKN2 mutations in most common human cancers in Korea, PCR and PCR-SSCP analyses for the exon 2 of CDKN2 were performed on each set of 20 formalin-fixed and paraffin-embedded tumor tissues of stomach adenocarcinomas, lung cancers, cervix cancers and hepatocellular carcinomas. No mutations in exon 2 of CDKN2 were found in 20 stomach adenocarcinomas. In contrast to rare mutations in stomach adenocarcinomas, a high frequency of CDKN2 mutations was identified in other 3 cancers, 11 of 20 (55%) lung cancers (7 of 10 NSCLCs and 4 of 10 SCLCs), 14 of 20 (70%) cervix cancers and 11 of 20 (55%) hepatocellular carcinomas. These results suggest that mutations of the CDKN2 gene might be an important genetic change in NSCLCs, cervix cancers and hepatocellular carcinomas.

Involvements of mitochondrial thioredoxin reductase (TrxR2) in cell proliferation

Oxidative stress has been implicated in a wide range of cellular damage which includes DNA oxidation, membrane lipid peroxidation, and apoptosis. In our study, we found that overexpression of PLC-beta1 in NIH3T3 fibroblasts protected them from cell death occuring in response to oxidative stress. Cell death caused by treatment with prooxidant tert-butylhydroperoxide (TBH), H2O2, or CdCl2 was considerably suppressed in PLC-beta1 overexpressed NIH/beta1-14 cells in comparison to control NIH/neo cells. However, overexpression of PLC-beta1 failed to protect the cells from toxicity by diamide or KCN. In addition, while accumulation of c-fos mRNA was observed within 30 min of TBH treatment in vector transfected NIH/neo cells, TBH-induced c-fos mRNA generation was completely suppressed in NIH/beta1-14 cells, while that of c-jun and GAPDH was not affected. These findings suggest that PLC-beta1 may play a role in process that can protect cells from oxidative stress-induced cell death.

Effects of green tea catechin on enzyme activities and gene expression of antioxidative system in rat liver exposed to microwaves

PURPOSE Treatment with arsenic trioxide (As(2)O(3)) results in a wide range of cellular effects that includes induction of apoptosis, inhibition of cell growth, promotion or inhibition of cellular differentiation, and inhibition of angiogenesis through a variety of mechanisms. The mechanisms of As(2)O(3)-induced cell death have been mainly studied in hematological cancers, and those mechanisms in solid cancers have yet to be clearly defined. In this study, the mechanisms by which As(2)O(3) induces apoptosis in human colorectal adenocarcinoma HT-29 cells were investigated. MATERIALS AND METHODS To examine the levels of apoptosis, HT-29 cells were treated with As(2)O(3) and then we measured the percentage of Annexin V binding cells, the amount of ROS production and the mitochondrial membrane potential. Western blot analysis was performed to identify the activated caspases after As(2)O(3) exposure, and we compared the possible target molecules of apoptosis. As(2)O(3) treatment induced the loss of the mitochondrial membrane potential and an increase of ROS, as well as activation of caspase-3, -7, -9 and -10. RESULTS As(2)O(3) induced apoptosis via the production of reactive oxygen species and the loss of the mitochondrial membrane potential. As(2)O(3) induced the activation of caspase-3, -7, -9 and -10. Furthermore, As(2)O(3) treatment downregulates the Mcl-1 and Bcl-2 expressions, and the release of cytochrome c and an apoptosis-inducing factor (AIF). Pretreating the HT-29 cells with N-acetyl-L-cysteine, which is a thiol-containing antioxidant, inhibited the As(2)O(3)-induced apoptosis and caspase activation. CONCLUSION Taken together, these results suggest that the generation of reactive oxygen species (ROS) by As(2)O(3) might play an important role in the regulation of As(2)O(3)-induced apoptosis. This cytotoxicity is mediated through a mitochondria-dependent apoptotic signal pathway in HT-29 cells.

Group IIA secretory phospholipase A2 stimulates inducible nitric oxide synthase expression via ERK and NF-κB in macrophages

The CDKN2 (MTS1/p16(INK4A)) and MTS2/p15(INK4B) genes, encoding cyclin dependent kinase inhibitors, were found to be homozygously deleted at high frequency in cell lines from many different types of cancer and some primary cancers. To determine the frequency of CDKN2 and MTS2 mutations in human stomach, liver, and cholangio-cancers, molecular analyses of CDKN2 and MTS2 were performed on 4 stomach cancer cell lines, 14 primary stomach adenocarcinomas, 11 hepatocellular carcinomas, and 5 cholangiocarcinomas. Two (50%) of the four stomach cancer cell lines (SNU1, SNU5, SNU16 and Kato III) had mutations of the CDKN2 and MTS2 gene: SNU16, a homozygous deletion; SNU5, a nonsense mutation, CGA to TGA (Arg to stop) at codon 72 of the CDKN2 gene. No mutations were observed in the 14 primary stomach cancer tissues. In contrast to the mutations of CDKN2 and MTS2, Northern blot analysis showed that expression of CDKN2 was absent or decreased in all the remaining four stomach cancer cell lines and 11: of the 14 (79%) primary stomach adenocarcinomas. Five of the 11 (45%) hepatocellular carcinomas and one of the 5 (20%) cholangiocarcinomas have possible mutations in CDKN2 exon 2 and MTS2. One of hepatocellular carcinoma was expressed mobility shift on PCR-SSCP analysis and a missense mutation, GAC to GAA (Asp to Glu) at codon 105 of CDKN2 gene. These results suggest that mutations or inactivation of the CDKN2 gene may be a critical genetic change in the formation of stomach, hepatocellular, and cholangiocarcinomas.