TNF-α 유전자형과 방광암과의 관계

정필두,김은정,엄민식,서정원,윤석중,김종석,이상철,김원재 충북대학교 의학연구소 2001 忠北醫大學術誌 Vol.11 No.2

연구목적: TNF-α는 일부 종양의 종양화 과정과 관련이 있는 것으로 알려져 있다. 본 연구는 TNF-α 발현에 영향을 미치는 TNF-α 촉진자 -308 부위의 유전적 다형성이 방광암과 관련이 있는지 유무를 알고자 시행하였다. 대상 및 방법: 유전자 분석을 위하여 환자 113명 및 대조군 109명으로부터 혈액을 채취하여 genomic DNA를 분리한 후 PCR-RFLP 및 direct DNA sequencing을 통하여 TNF-α유전자의 다형성을 조사하여 방광암의 발생, 병기 및 분화도와 비교 검토하였다. 결과: TNF-α 촉진자 -308 부위의 유전형은 대조군에서는 GG형이 83.5%(90례 및 GA형이 16.5%(19례)로 관찰되었으며 AA형은 없었다. 환자군에서는 GG 형이 85.4%(97례), GA형 및 AA형은 각각 13.1%(15례)및 0.8%(1례)에서 관찰되었다. 두 군 모두에서 GG형이 가장 많이 나타났으며 다음으로 GA형을 보이고 AA형은 1례의 방광암 환자에서만 관찰되었다. -308부위의 경우도 두 군 사이에 유전자형의 차이는 없었다(p=0.259) 분화도별 분포를 보면 grade I이 20례, grade II가 49례, grade Ⅲ은 34례였고 병기별로 표재성인 경우가 90례였으며 침윤성은 14례였다. 분화도가 나빠질수록 GA형이 증가하였다(p=0.04). 그러나 병기와 TNF-α promoter -308부위의 유전자형 사이에는 유의한 상관 관계가 없었다(p=0.123). 결론: 방광암 환자의 혈액에서 GA genotype이 관찰되는 경우, 분화도가 나쁠 가능성이 매우 높기 때문에 좀 더 적극적인 치료와 세밀한 추적관찰을 함으로써 방광암으로 인한 사망과 암의 진행을 예방할 수 있을 것으로 기대한다. Purpose : Tumor necrosis factor-alpha (TNF-α) is involved in tumorigenesis of several cancers as an endogenous tumor promoter. The purpose of this study was to investigate whether genetic polymorphism of TNF-α promoter region (-308) was associated with human bladder tumor. Materials and Methods: The DNA from 113 and 109 respective blood samples of bladder tumor Patients and control group was analyzed by PCR-based restriction fragment length polymorphism (RFLP) and direct DNA sequencing methods to characterize the genetic polymorphism of -308 promoter region of the TNF-α gene in bladder tumor patients. We compared the association of bladder tumor with genetic Polymorphism of TNF-α promoter region(-308) in relation to the stage, grade, recurrence and progressio. Results : Eighty-six percents(97/113) of bladder tumor patients and 83.5% (90/109) of control group showed genotype GG at -308 region of TNF-α. Difference in genetic variations of TNF-α promoter (-308) did not exist between bladder tumor patients and control group(p=0.259). Tumor grade was significantly related to the GA genotype (p=0.04). The higher is the grade in bladder tumor, the more frequent was the GA genotype. Tumor stage, recurrence and progression were not significantly associated with genetic polymorphism of TNF-α promoter region (-308). Conclusion: The GA genotype of TNF-a promoter region (-308) had a significant impact on TNF-α production and was related to higher grade tumor compared to GG genotype. TNF-α serum levels in bladder tumor patients were significantly higher than controls. These data suggested that TNF-α might involve the tumorigenesis of the bladder rather than treatment or prevention of bladder tumor.

비소 세포형 폐암에서의 p53과 bcl-2 유전자의 발현에 관한 연구

명나혜,서필원,이민철 단국대학교 1998 論文集 Vol.32 No.-

We investigated immunohistochemical expression of bcl-2 and p53 genes in 30 resected human non-small cell lung cancers. We attempted to know how frequently these gene alterations occur and the correlation between their immunoreactivity and clinicopathologic parameters such as sex, histologic type, differentiation, and stage. Also, the p53 immunoreactivity was compared with that of bcl-2 to detect their inverse or positive relationship. By ABC(avidin biotin complex) immunostainings using p53 and bcl-2 primary monoclonal antibodies on the paraffin blocks, p53 gene mutation and bcl-2 overexpression were found in 29 (96.6%) and 22(73.3%) 30 cases, respectively. Both immunopositivities were detected in 21 cases(70%), whereas neither immunopositivity in only one (3.3%) case. Almost all clinicopathologic parameters revealed no significant correlation with both immunoreactivities of p53 and bcl-2 genes except for the significantly higher overepression of bcl-2 gene in the squamous cell type than that of the non-squamous cell type ant there was neither inverse nor positive relationship between p53 and bcl-2 immuno-expressions. In conclusion, most cases of the non-small cell lung cancers are found to undergo both p53 mutation and bcl-2 overexpression, but their expressivity does not have any relationship with most of clinicopathologic variables. The two genetic alterations seem to be involved coexistently in the molecular pathogenesis of the non-small cell lung cancers.

OGGI 유전자의 다형성이 방광암에 미치는 영향

김은정,정필두,정춘구,서정원,윤석중,김원재 충북대학교 의학연구소 2001 忠北醫大學術誌 Vol.11 No.2

연구목적 : 손상된 유전자의 회복은 암의 발생 및 예방에 중요한 역할을 한다. 잘못된 염기의 제거 및 회복에 중요한 역할을 하는 OGGI 유전자의 다형성이 방광암에 어떠한 작용을 하는지를 환자-대조군 연구를 통해서 조사하였다. 대상 및 방법: 방광암 환자 168명과 건강인 672명을 대조군으로 하여 genomic DNA를 이용하여 SSCP (single-stranded conformational polymorphism), direct DNA sequencing 및 restriction fragment length polymorphism (RFLP) 방법을 통해 환자 및 대조군사이의 OGGI 유전자의 다형성을 조사하였다. 결과: 건강 한국인에서 OGGI 유전자의 유전형을 조사한 결과 econ 6의 Pro324Pro 부위(lb type)와 exon 7의 Ser326Cys 부위 (la type)에 유전자의 다형성이 있음을 발견하였다. Codon 324는 silent polymorphic site였으나 codon 326은 C→G로 nucleotide가 바뀌어 amino acid가 serine→cysteine으로 바뀌는 polymorphic site였다. 대조군에서 codon 326은 Ser326Ser 형이 127례 (18.9%), Ser326Cys 형이 363례 (54.0%) 및 Cys326Cys 형이 182례 (27.1%)이었으며, 168명의 방광암 환자군에서는 Ser326Ser 형이 44례 (26.2%), Ser326Cys 형이 87례 (51.8%)이고 0ys3260ys 형이 37례 (22.0%) 이었다(p=0.034) 즉 Cys326Cys 형 및 Ser326cys 형에 비하여 Ser326Ser 형에서 방광암이 생길 확율이 1.52배 (95% CI=0.439-0.969) 높았다 특히 40세 미만의 남자의 경우 Ser326Ser 형에서 방광암 발생확률이 다른 유전형에 비하여 6.1배 높았다. 결론: OGGI 유전자의 유전형은 서구인과 판이하게 달랐으며 방광암에서는 이 유전자의 변이가 빈번할 뿐아니라 codon 326의 Ser326Ser 형에서는 다른 유전자형에 비하여 방광암이 발생할 확률이 1.5배 이상 높았다. 특히 한국인 40세 미만의 남자에서는 방광암 발생확률이 6배이상 높았다. 본 연구결과 OGGI codon 326의 유전적 다형성은 방광암의 종양화 과정과 관계가 있을 것으로 여겨지며 특히 40세 미만의 한국 남자의 경우는 더욱 밀접한 관계가 있을 것으로 사료된다. Purpose: A repair of damaged DNA has been shown to be involved in the susceptibility to cancer development and prevention. Therefore, it is worth investigating genetic polymorphisms of the OGGl gene associated with the gene repair mechanism. In this study, we examined a possible association of genetic ploymorphisms in OGGl with the risk of bladder tumor. Materials and Methods: Hospital based, case-control investigation was carried out in 168 primary bladder tumor patients and 672 control subjects. We performed the SSCP, PCR-based restriction fragment length polymorphism (RFLP) and direct DNA sequencing to characterize the genetic polymorphism of OGGI in both cases and oontrols. Results: We found two polymorphic sites in OGGl. A Ser/Cys polymorphism at codon 326 (la type) in exon 7 was associated with an exchange of amino acid. Another polymorphic site at codon 324 (1b type) in econ 6 was silent. The association between codon 326 Polymorphism and the risk of the bladder tumor was examined by a age-sex adjusted analysis. We found that the distribution of OGGl Ser326Cys genotypes of controls(Ser/Ser, 18.9% ; Ser/Cys, 54.0% ; Cys/Cys, 27.1%) was significantly different from that of bladder tumor patients (36.2%, 51.8% and 22.0%, respectively) (p = 0.034, adjusted OR = 0.652, 95% Cl = 0.44 - 0.97). Especially, bladder tumor risk in Korean male under 40 years old was approximately 6 times higher than over 40 years old males. Conclusion : Our data suggested that Ser326cys polymorphism at codon 326 of OGGl male below 40 years old in Korea significantly increased the risk of tumorigenesis in the urinary bladder (p = 0.015, adjusted OR = 0.165, 95% CI = 0.04 - 0.75) Our results suggest that the OGGl Ser326Cys Polymorphism might play a role in the tumorigenesis of the bladder.

Background for the stereospecific condensation of two acetaldehydes using 2-oxoglutarate dehydrogenase E1 subunit from Vibrio vulnificus

Pil-Won SEO,Hye-Jin JO,Jin-Byung PARK,Jeong-Sun KIM 한국생물공학회 2019 한국생물공학회 학술대회 Vol.2019 No.4

Structures of Zymomonas 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase with and without a Substrate Analog at the Phosphate-Binding Loop

( Pil-won Seo ),( Ho-chang Ryu ),( Do-heon Gu ),( Hee-sae Park ),( Suk-youl Park ),( Jeong-sun Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.8

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, which catalyzes aldol cleavage and condensation reactions, has two distinct substrate-binding sites. The substrate-binding mode at the catalytic site and Schiff-base formation have been well studied. However, structural information on the phosphate-binding loop (P-loop) is limited. Zymomonas mobilis KDPG aldolase is one of the aldolases with a wide substrate spectrum. Its structure in complex with the substrate-mimicking 3-phosphoglycerate (3PG) shows that the phosphate moiety of 3PG interacts with the P-loop and a nearby conserved serine residue. 3PG-binding to the P-loop replaces water molecules aligned from the P-loop to the catalytic site, as observed in the apostructure. The extra electron density near the P-loop and comparison with other aldolases suggest the diversity and flexibility of the serine-containing loop among KDPG aldolases. These structural data may help to understand the substrate-binding mode and the broad substrate specificity of the Zymomonas KDPG aldolase.

Phase determination of the UDP-N-acetylmuramic acid :L-alanine ligase (MurC) crystal from Mycobacterium bovis

Pil-Won Seo,Jeong-Sun Kim 한국구조생물학회 2018 Biodesign Vol.6 No.3

Bacterial peptidoglycan is necessary for bacterial survival against environmental osmotic pressure and is a relevant target for development of anti-bacterial drugs. Formation of a covalent bond between a carbohydrate and an amino acid is a key chemical process for peptidoglycan bio-synthesis. UDP-N-acetylmuramic acid (UDPMurNAc):L-alanine ligase (MurC) is an ATP-dependent amide bond ligase to form an UDP-MurNAc-L-alanine required in bacterial peptidoglycan. To provide a structural background for development of tuberculosis-specific antibiotics, Mycobacterium bovis MurC (MbMurC), which has sequence identity of 100% to M. tuberculosis, was cloned and expressed. The purified protein was crystallized from the precipitant of 0.1 M HEPES (pH 7.0), 0.2 M NaCl, 24% (w/v) polyethylene glycol 1.5K, and 10% (v/v) 2-methyl-2,4-pentanediol. Diffraction data were collected to 2.3 Å resolution. The crystal belonged to the primitive monoclinic space P2 1 with unit-cell parameters a = 65.30 Å, b = 76.70 Å, c =103.96 Å, α = γ = 90°, and β = 106.0°. The spatial positions of the two protein molecules in the asymmetric unit were determined by molecular replacement using the sequentially related Yersinia pestis MurC structure.

Phase determination of the Fructose-6-phosphate aldolase 1 crystal from Aeromonas media

Pil-Won Seo,김정선 한국구조생물학회 2022 Biodesign Vol.10 No.4

Fructose-6-phosphate aldolase 1 (FSA) from Aeromonas media (AmFSA) is a class I aldolase characterized by the formation of a Schiff-base intermediate between a conserved lysine residue and a carbonyl substrate. FSA has shown high stereoselectivity and broad substrate spectrum. To provide a structural background for engineering as a catalyst for desired reaction product, AmFSA was cloned and expressed. The purified protein was crystallized from the precipitant of 0.1 M sodium acetate (pH 5.0), 1.4 M ammonium sulfate, and 10 mM β-mercaptoethanol. Diffraction data were collected to 2.1 Å resolution. The crystal belonged to the orthorhombic space group C2221 with unit-cell parameters a = 93.80 Å, b = 175.27 Å, c = 150.99 Å, and α = β = γ = 90°. The spatial positions of the five protein molecules in the asymmetric unit were determined by molecular replacement using the sequentially related Escherichia coli FSA structure.

Engineering of Vibrio SucA for Better Enantioselectivity and Productivity

Pil-Won SEO,Hye-Jin CHO,Jin-Byung PARK,Jeong-Sun KIM 한국생물공학회 2020 한국생물공학회 학술대회 Vol.2020 No.10

Structure-Guided Generation of a Redox-Independent Blue Fluorescent Protein from mBFP

Seo, Pil-Won,Jo, Eun-Seo,You, Sung-Hwan,Cheong, Dae-Eun,Kim, Geun-Joong,Kim, Jeong-Sun Elsevier 2019 Journal of molecular biology Vol.431 No.17

<P><B>Abstract</B></P> <P>Fluorescent proteins, such as the green fluorescent protein, are used for detection of cellular components and events. However, green fluorescent protein and its derivatives have limited usage under anaerobic conditions and require a long maturation time. On the other hand, the NADPH-dependent blue fluorescent protein (BFP) without oxidative modification of residues is instantly functional in both aerobic and anaerobic systems. BFP proteins belong to a short-chain dehydrogenase/reductase (SDR) protein family, and their fluorescent property changes with reaction time in the presence of a substrate. With the aim of developing a better fluorescent reporter independent of redox state, we elucidated the crystal structure of a tetrameric mBFP from soil metagenomes with and without NADPH. Apart from the previously known regions, structure-guided mutational studies have identified several residues that contribute to the fluorescence of mBFP, including two aromatic residues (F97 and Y157) near the nicotinamide moiety of the bound NADPH. A single histidine mutation at Y157 (Y157H) has conferred more stabilized, time-independent fluorescence even in the presence of substrates. Furthermore, we discovered another SDR protein that can also emit blue fluorescence. These results open a new possibility for the development of BFP as a stable cellular reporter for widespread use, independent of subcellular environments.</P> <P><B>Highlights</B></P> <P> <UL> <LI> mBFP is an NADPH-dependent blue fluorescent protein. </LI> <LI> Its fluorescence changes by oxidizing or reducing putative substrates. </LI> <LI> Structure-guided mBFP derivative emits a constant fluorescence with substrates. </LI> <LI> mBFP can be used as a stable cellular reporter regardless of redox states. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

폐암세포주에서 NF-κB 활성 억제를 통한 Proteasome 억제제 MG132의 TRAIL-유도성 Apoptosis 감작 효과

서필원 ( Pil Won Seo ),이계영 ( Kye Young Lee ) 대한결핵 및 호흡기학회 2008 Tuberculosis and Respiratory Diseases Vol.65 No.6

연구배경: 정상세포는 보호되고 종양세포에 독성을 보인다고 알려진 TNF유전자족으로 새로이 확인된 TRAIL이 폐암세포에서 보이는 아포프토시스 효과를 확인하고, 아포프토시스로부터 세포를 보호하는 전사인자 NF-κB가 TRAIL에 의하여 활성화 되는 정도를 평가하여 MG132의 NF-κB활성억제가 TRAIL 유도성 아포프토시스를 감작시키는지를 확인하기 위하여 본 연구를 시행하였다. 방법: A549(wt p53) 및 NCI-H1299(null p53) 폐암세포주를 사용하였다. 세포독성 검사는 MTT assay를 이용하였고 아포프토시스는 Annexin V assay와 FACS 분석을 이용하였다. NF-κB 전사활성은 luciferase reporter gene assay를 이용하였고 IκBα 분해는 western blot을 이용하였으며, TRAIL에 의해 활성화된 NF-κB와 DNA 결합은 electromobility shift assay와 anti-p65 antibody를 이용한 supershift assay로 확인하였다. 결과: 1) TRAIL 100 ng/ml 농도에서 wild-type p53인 A549 폐암세포는 34.4%, p53 null인 NCI-H1299 폐암세포는 26.4%의 세포사를 관찰하였다. 2) Luciferase reporter gene assay로서 TRAIL에 의한 NF-κB의 활성이 A549 IgGκB-luc세포에서 2.45배 증가하고 NCI-H1299 IgGκB-luc세포에서는 1.47배 증가함을 관찰하여 TRAIL에 의하여 NF-κB가 활성화됨을 확인하였다. 3) MG132의 전처치로 TRAIL에 의한 NF-κB의 활성이 A549 세포와 NCI-H1299 세포에서 각각 기저수준의 0.24, 0.21배로 강력히 억제되었다. 4) TRAIL단독으로 30% 전후의 세포독성이 MG132 전처치 후 TRAIL을 투여하면 두 세포주 모두에서 80% 이상의 세포독성이 관찰되어 MG132가 TRAIL유도성 아포프토시스에 감작효과가 있음을 확인하였다. 결론: 이상의 결과로 TRAIL에 상대적인 내성을 보이는 폐암세포주에서 MG132가 NF-κB 활성억제로서 TRAIL유도성 아포프토시스를 강화시키는 효과가 있음을 확인할 수 있었다. 따라서 본 연구는 향후 폐암치료에 있어서 TRAIL유도성 아포프토시스가 이용될 수 있는 가능성을 확인한 기초자료가 된다고 생각된다. Background: TRAIL (TNF-related apoptosis inducing ligand) is a newly identified member of the TNF gene family which appears to have tumor-selective cytotoxicity due to the distinct decoy receptor system. TRAIL has direct access to caspase machinery and induces apoptosis regardless of p53 phenotype. Therefore, TRAIL has a therapeutic potential in lung cancer which frequently harbors p53 mutation in more than 50% of cases. However, it was shown that TRAIL also could activates NF-κB in some cell lines which might inhibit TRAIL-induced apoptosis. This study was designed to investigate whether TRAIL can activate NF-κB in lung cancer cell lines relatively resistant to TRAIL-induced apoptosis and inhibition of NF-κB activation using proteasome inhibitor MG132 which blocks IκBα degradation can sensitize lung cancer cells to TRAIL-induced apoptosis. Methods: A549 (wt p53) and NCI-H1299 (null p53) lung cancer cells were used and cell viability test was done by MTT assay. Apoptosis was confirmed with Annexin V assay followed by FACS analysis. To study NF-κB-dependent transcriptional activation, a luciferase reporter gene assay was used after making A549 and NCI-H1299 cells stably transfected with IgGκ-NF-κB luciferase construct. To investigate DNA binding of NF-κB activated by TRAIL, electromobility shift assay was used and supershift assay was done using anti-p65 antibody. Western blot was done for the study of IκBα degradation. Results: A549 and NCI-H1299 cells were relatively resistant to TRAIL-induced apoptosis showing only 20~30% cell death even at the concentration 100 ng/ml, but MG132 (3μM) pre-treatment 1 hour prior to TRAIL addition greatly increased cell death more than 80%. Luciferase assay showed TRAIL-induced NF-κB transcriptional activity in both cell lines. Electromobility shift assay demonstrated DNA binding complex of NF-κB activated by TRAIL and supershift with p65 antibody. IκBα degradation was proven by western blot. MG132 completely blocked both TRAIL-induced NF-κB dependent luciferase activity and DNA binding of NF-κB. Conclusion: This results suggest that inhibition of NF-κB can be a potentially useful strategy to enhance TRAIL-induced tumor cell killing in lung cancer. (Tuberc Respir Dis 2008;65:476-486)