원발성 폐암에서 LKB1 단백질 발현 소실에 따른 임상 양상 및 조직병리학적 특성

황기은 ( Ki Eun Hwang ),조향정 ( Hyang Jeong Jo ),이강규 ( Kang Kyoo Lee ),심혁 ( Hyeok Shim ),송정섭 ( Jung Sub Song ),신정현 ( Jeong Hyun Shin ),신성남 ( Seong Nam Shin ),박성훈 ( Seong Hoon Park ),홍경만 ( Kyeong Man Hong ) 대한결핵 및 호흡기학회 2008 Tuberculosis and Respiratory Diseases Vol.64 No.5

연구배경: LKB1 (STK11)유전자는 Peutz-Jeghers syndrome에서 생식세포 돌연변이가 있으면 소화기와 폐를 포함한 타 장기의 암 발생 위험도가 증가한다고 알려져 있으며, 또한 종양 억제 기능이 있다고도 알려지고 있다. 하지만 현재까지 폐암에서 LKB1 유전자의 생물학적 기능이 명확하게 밝혀져 있지 않아, 저자들은 폐암조직에서 LKB1 단백질 발현소실과 임상양상 및 조직병리와의 연관관계를 알아보고자 하였다. 방법: 1998년 3월부터 2006년 3월까지 본원에 내원하여 원발성 폐암으로 진단받고 근치적 절제술을 시행 받은 77명의 환자를 대상으로 하였다. 파라핀에 포매된 조직을 택하여 면역조직화학염색법으로 LKB1 단백질 발현을 확인하였고, 정상 기관지 상피세포 세포질에서의 단백질 발현과 비슷한 정도의 발색을 갖는 종양세포가 전체 종양에서 30% 이상인 경우를 양성으로 판정하였다. 결과: LKB1 발현 양성은 40% (31/77)였고, 남성, 흡연, 편평상피암인 경우에 LKB1 발현 음성률이 통계적으로 유의하게 높았다. 종양위치가 중앙부위일수록 LKB1 발현 음성률이 증가하는 경향이 있으며, 종양 위치가 말초 부위인 경우 흡연력이 있는 군에서 LKB1 발현 음성률이 통계적으로 유의하게 높았다. TNM 병기가 진행할수록 LKB1 발현 음성률이 증가하는 경향이 있었으며, T2 병기 이상, N 병기가 진행할수록 LKB1 발현 음성률이 높아지는 경향이 있었으나, 통계적 유의성은 보이지 않았다. 결론: 원발성 폐암환자에서 LKB1 발현소실은 성별, 흡연력, 조직병리 형태와 의미있는 상관관계를 보였으나, 예후인자로서의 의의는 찾지 못했다. 하지만 환자의 숫자가 적어 추후의 연구가 이루어져야 할 것으로 사료된다. Background: LKB1(STK11) is a serine/threonine kinase that functions as a tumor growth suppressor. The functions of LKB1 in lung cancer are not completely understood. This study evaluated the relationship between LKB1 protein expression and the clinicopathological features in lung cancer tissues. Methods: The expression of LKB1 was studied in paraffin-embedded tumor blocks, which were obtained from 77 patients who had undergone surgery at Wonkwang University Hospital. The expression of the LKB1 protein was considered positive if the staining intensity in the tumor tissue adjacent to the normal airway epithelium was>30%. Results: The LKB1 expression was positive in 31 (40%) of samples. Loss of LKB1 expression was significantly associated with being male, smoking history, and squamous cell carcinoma. In the peripheral sites, the loss of LKB1 expression was strongly associated with a smoking history. A loss of LKB1 expression was more frequently associated with progression according to TNM staging, particularly more than T2, N progression. Conclusion: There was a significant relationship between the loss of the LKB1 protein and gender, smoking history, and histological type in primary lung cancer. Although LKB1 expression was not found to be a significant prognostic factor, further studies with a larger cohort of patient`s lung cancer tissue samples will be needed to confirm this. (Tuberc Respir Dis 2008;64:362-368)

B형 간염바이러스 S 표면항원에 대한 사람 단일클론 항체의 생산 및 특성연구

홍경만,진병래,홍효정,정홍근 大韓免疫學會 1996 大韓免疫學會誌 Vol.18 No.3

For the prevention of hepatitis B virus infection, we have constructed a human hybridoma (23HN) secreting a monoclonal antibody (mAb) with specificity for the common 'a' determinant of hepatitis B surface antigen (HBsAg). HBsAg-primed human B lymphocytes were prepared by panning and subsequently activated by Epstein-Barr virus transformation. The transformed B cells were fused with human-mouse heterohybridoma cells as a fusion partner, and the resulting hybridoma cell clones were isolated and examined for the antibody production. One hybridoma clone 23HN produced the human mAb for 4 months. The heavy and light chains of the mAb were shown to have yl and ic isotypes, respectively. For the characterization of 23HN, the antibody was purified, and its size and purity was confirmed by 10% SDS-PAGE and Western analysis. The epitope analysis showed that 23HN binds to both adr and ay subtypes of hepatitis B virus surface antigen linked by disulfide bridges, suggesting that it re-cognizes a common 'a' antigenic determinant. Competition binding assay using three murine mAbs H67, F39.20 and F5.28.6, which bind to the common 'a' antigenic determinant but their fine epitopes are different from each other, showed that 23HN competes effectively with F39.20, suggesting that the fine epitope of 23HN is same as that of F39.20. The antigen binding affinity of 23HN was 6 10' M-1. From these results, we expect that the 23HN human mAb may be useful in the prevention or therapy of HBV infection.

Expression of a Protein-Arginine N-Methyltransferase, HRMT1L1

Hong, Kyeong-Man,Park, Hyun,Paik, Moon-Kee 圓光大學校 醫科學硏究所 1998 圓光醫科學 Vol.14 No.2

Protein methylase Ⅰ (or protein-arginine N-methyltransferase) catalyzes the methylation of arginine residues in glycine-arginine-rich (GAR) domains of eucaryotic proteins. Members of this family of methyl accepting substrates include hnRNP A1, nucleolin and fibrillarin Mammalian protein methylase Ⅰ genes, PRMT1, HRMT1L1 and PRMT3, were cloned recently. But the biological function of these enzymes is not clear yet, so it would be necessary to produce monoclonal antibodies against protein methylase Ⅰ enzymes for the study of their function. In this study, HRMT1L1, a human protein methylase Ⅰ gene, was cloned and was expressed in bacteria and the recombinant protein was purified for the production of monoclonal antibody. Total human HRMT1L1 cDNA was amplified by PCR from human placental cDNA library. The BamHI site was included in 5' sequence of the 5' primer. The amplified PCR product was cloned to T-vector and sequenced to confirm the nucleotide sequence. The HRMT1L1-T-vector was digested with BamHI and EcoRI and the HRMT1L1 cDNA was cloned to Trx fusion expression vector and was sequenced again. Transformed bacteria with Trx-HRMT1L1 vector was induced to Trx-HRMT1L1 fusion protein, The induced fusion protein was purified with His-bind chromatography and was digested with recombinant enterokinase. The digested proteins were passed through His-bind column to remove TrxA protein. The purified HRMT1L1 recombinant protein will be used to produce monoclonal antibody.

Cloning and Expression of Protein-arginine Methyltransferase Ⅰ

Hong, Kyeong-Man,Choi, Yong-Bock,Chang, Yoo-Jung,Kim, Han-Soo,Lee, Sang-Do,Pak, Gun-Suk,Kim, Jeong-Kyu,Rhee, Kang-Ill,Park, Hyun,Kim, Kyung-Suk,Park, Moon-Kee 圓光大學校 醫科學硏究所 1999 圓光醫科學 Vol.15 No.2

Purpose: PRMT I gene was cloned recently, but the biological function of these enzymes has not been clear yet. This study was designed to produce recombinant PRMT1 in Escherichia coli for the future production of monoclonal antibody. Methods: Partial human PRMT1 cDNA was amplified by PCR from human placental cDNA library. The hPRMT1 was cloned to T-vector, again to Trx fusion vector for expression. The transformed bacteria with Trx-hPRMT1 vector were induced. The induced fusion protein was purified with His-bind chromatography and was digested with recombinant enterokinase. Digested proteins were passed through His-bind column to remove TrxA protein. Results: The recombinant fusion protein, Trx-hPRMT1, was expressed in E.coli. And the pure recombinant hPRMT1 was purified from His-bind chromatography and the digestion with enterokinase. Conclusions: The purified recombinant hPRMT1 protein was adequate to use for the ^production of monoclonal antibody.

열안정형 카테콜-O-메틸전이효소 유전자의 클로닝

홍경만,최용복,정갑용,지은정,장현신,박현,백문기 圓光大學校 醫科學硏究所 1998 圓光醫科學 Vol.14 No.2

Catechol-O-methyltransferase (COMT; EC 2.1.1.6) is the enzyme which catalyzes the transfer of methyl group to the catecholamine neurotransmitters from its methyl donor S-adenosyl-L-methionine. Partially purified COMT from rat liver has been used to measure the concentration of catecholamines in the blood through solvent extraction and thin layer chromatography after converting ^3H-methyl derivatives of catecholamines. To improve this inconvenient and fluctuating method, an attempt was made to use COMT gene for the measurement of catecholamines. Specific primers, COMT5P (5'-TGC TCA GAG GTG CTT TGA AG-3') and COMT3P (5'-GGA GCC GCA GAA GGT CA G-3'), were used to amplify COMT gene from human placenta cDNA library. The amplified COMT gene through 35 cycles of polymerase chain reaction was cloned into T-vector and the nucleotide sequences are determined by automatic sequencer. Human COMT has two common variants, a thermostable high activity form which has valine at amino acid 158 and a thermolabile low activity form which Has methionine at the same position. The cloned COMT gene in this study has both variants. Clone 4 and 5 have valine at amino acid 158 (guanine at nucleotide 472) and clone 1-3 have methionine at this site (adenine at nucleotide 472). In addition to this, there are other DNA polymorphisms in COMT gene at nucleotide 101, 102 (amino acid 34, a structural mutation cysteine/serine) and nucleotide 186 (a silent mutation). All of the clones have cysteine at amino acid 34 (guanine and adenine at nucleotides 101 and 102) and cytosine at nucleotide 186 except clone 3 which has thymine at this nucleotide. Clone 4 and 5 are thermostable high activity variants, suggesting being more useful for the measurement of catecholamines after expressing the gene.

간흡충 cystein proteinase 유전자의 클로닝

박현,홍경만 圓光大學校 醫科學硏究所 1998 圓光醫科學 Vol.14 No.2

Clonorchis sinensis is one of the most important parasitic trematode in China, Korea, Japan and Southeast Asia. When intermediate host eat raw fish which is infected with it metacercariae, the worm excyst in gut. migrate along bile duct and grow up to adult worm. Recently, cysteine proteinases in trematoda parasites were reported to be specific antigens which reacted with the sera of the infected patients. In our preliminary data, the cysteine proteinase in Clonorchis sinensis also reacted with the sera of Clonorchiasis patients. So we think that the cysteine protinase of C. sinensis could be possible candidate as an antigen for the serodiagnosis of Clonorchiasis patients. Therefore, we cloned cysteine proteinase cDNA to find out the cysteine proteinase which could react with the sera of Clonorchiasis patients. In this study, we cloned a cysteine proteinase gene from adult C. sinensis. And also we compared the expression of the cysteine proteinase between C. sinensis and P. westermani, and then were inspected to a specificity of that gene. Cysteine proteinase cDNA fragment from adult Clonorchis sinensis was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate oligonucleotide primers derived from conserved cysteine proteinase sequences. The amplified yielded a cDNA fragment of approximately 500 bp in length, the amplified product was directly cloned into a T- vector and was sequenced. Sequence analysis and aligment showed significant similarity to other eukaryotic cysteine proteinases and conservation of the cysteine, histidine, and asparagine residues that formed the catalytic triad. To study the cross reactivity with the sera of the patients of Paragonimiasis, we did PCR with C. sinensis and P. westermani cDNA Library. It was only amplified in cysteine of C. sinensis. These results indicated that the cysteine proteinase gene of C. sinensis might be used as an antigen forbe for diagnosis.