STP-A11, an oncoprotein of Herpesvirus saimiri augments bothNF-κB and AP-1 transcription activity through TRAF6

정수남,조일래,안원근,전병학,이복수,박기랑,정영화 생화학분자생물학회 2007 Experimental and molecular medicine Vol.39 No.1

Herpesvirus saimiri (HVS), a member of the γ- herpesvirus family, encodes an oncoprotein called Saimiri Transforming Protein (STP) which is required for lymphoma induction in non-human primates. However, a detailed mechanism of STP-A11-induced oncogenesis has not been revealed yet. We first report that STP-A11 oncoprotein interacts with TNF-receptor-associated factor (TRAF) 6 in vivo and in vitro. Mutagenesis analysis of the TRAF6-binding motif 10PQENDE15 in STP-A11 reveals that Glu (E)12 residue is critical for binding to TRAF6 and NF-κB activation. Interestingly, co-expression of E12A mutant, lack of TRAF6 binding, with cellular Src (Src) results in decreased transcriptional activity of Stat3 and AP-1, a novel target of STP-A11 compared to that enhances the association of TRAF6 with Src and induces the translocation of both TRAF6 and Src to a nonionic detergent-insoluble fraction. Taken together, these studies suggest that STP-A11 oncoprotein up-regulates both NF-κB and AP-1 transcription activity through TRAF6, which would ultimately contribute cellular transformation.

Antitumor activity of recombinant adeno-associated virus(rAAV) against human lung large cell carcinoma(NCI-460) transplanted into nude mice

김동규,황석연,이남진,홍성희,조영화,안미영,박기랑,강종구 한국실험동물학회 2005 한국실험동물학회 학술발표대회 논문집 Vol.2005 No.-

후박 추출물의 유전독성평가

이승호,류재면,서임권,이태희,김윤배,문성권,정경환,박기랑,황석연 한국독성학회 2007 Toxicological Research Vol.23 No.1

생쥐 턱밑샘의 발생, 분화 및 노화에 따른 C-type natriuretic peptide와 natriuretic peptide 수용기의 발현양상

복세미(Se-Mi Bok),김택헌(Tak-Heun Kim),박기랑(Kee-Rang Park),조의식(Eui-Sic Cho) 대한해부학회 2005 Anatomy & Cell Biology Vol.38 No.4

C-type natriuretic peptide (CNP)는 natriuretic peptide family의 하나로서 주로 중추신경계통과 혈관내피세포에서 국소적으로 합성되는 것으로 알려져 있으나 최근 다른 말초기관에서도 합성되어 세포의 성장과 분화 조절에 관여하는 것으로 알려지고 있다. 본 연구자들은 CNP가 정상 성체 턱밑샘의 사이관과 곱슬과립세관세포에서 국소적으로 합성되어 분포하며 생물학적 활성을 보임을 보고한 바 있으나, 발생 및 분화, 그리고 노화에 따르는 발현 및 활성에 대해서는 알려진 바 없다. 따라서 본 연구는 턱밑샘의 발생 및 분화과정에서 CNP와 그 수용기의 발현양상과 노화과정에서 그 수용기의 생물학적 활성변화를 알아보고자, 임신14, 16, 18일된 C57BL/6N 생쥐 배자와 출생 후 1일, 2주, 그리고 1, 2, 12, 24개월된 성체의 턱밑샘을 대상으로 RT-PCR, in situ hybridization, 면역조직화학법 그리고 cGMP assay를 시행하였다. CNP는 임신14일에 턱밑샘의 원기인 외배엽성상피싹 종말부위에서 강하게 발현되었으며 출생 전까지 높게 유지되었으나 출생 후 현저히 감소되었다. CNP는 성체 턱밑샘의 사이관과 곱슬과립세관에 분포하고 있었으며 이들 관계통에서 NPRC는 특이적으로 발현되었으나 NPRB는 발현되지 않았다. RT-PCR결과 턱밑샘에서 CNP전사체는 임신16일부터 출생 후 2개월까지 점차 감소하였으나 ANP전사체는 증가하였다. CNP에 특이적인 수용기인 NPRB와 NPRC는 CNP와 동일한 발현양상이었으나 NPRA는 약하게 발현되었다. 또한 CNP는 치아싹, 혀, 앞위턱뼈, 뼈형성 부위와 같은 두개안면 조직에서도 발현되었으며, 이들 부위에서도 NPRC는 발현되었으나 NPRB는 발현되지 않았다. 출생 후 2개월된 턱밑샘의 관세포는 CNP에 의해 현저히 많은 cGMP를 생성하였으나, 샘꽈리는 CNP보다는 ANP와 BNP에 의해 많은 cGMP를 생성하였다. 노화에 따라 턱밑샘의 cGMP 활성능력은 전반적으로 감소되었으며 출생 후 1개월된 턱밑샘에서는 CNP, 출생 후 2개월 이후로는 ANP에 의한 cGMP 활성이 우세하였다. 이상의 결과로 보아 CNP는 턱밑샘에서 NPRC를 매개로 하는 발생 및 분화유도 기능을 주로 수행하며, 성체이후에는 NPRB를 매개로 하는 관계통 유지에 관여하고 노화에 따라서 활성이 점차 감소하는 것으로 추정된다. C-type natriuretic peptide (CNP), a member of natriuretic peptide family, is mainly synthesized in the endothelium and central nervous system. But CNP is also involved in the growth and differentiation of other peripheral organs. Although we have reported the local synthesis and localization of CNP in the adult submandibular glands (SMG), it is not known that the expression and biological activity of CNP following the morphogenesis, differentiation and aging. This study aimed to examine the expression of CNP and its receptors in the developing and differentiating stages of mouse SMG, and the changes of biological activity of its receptors with aging. The SMG, obtained from 14, 16, 18 days-old embryos (E) and 1 day, 2 weeks, 1, 2, 12, and 24 month-old C57BL/6N mouse, were processed for RT-PCR, in situ hybridization, immunohistochemistry and cGMP assay. CNP was strongly expressed in the epithelial clusters of primitive SMG, which was maintained before birth but was markedly decreased after birth. CNP was localized in the intercalated duct and granular convoluted tubules of adult SMG, where NPRC was specifically expressed but NPRB was not. CNP mRNA was gradually decreased from E16 to 2 M but ANP mRNA was opposed. NPRB and NPRC were the same pattern of the expression of CNP but NPRA was weakly expressed. In addition, CNP mRNA was also expressed in the craniofacial tissues such as tooth germs, tongue, premaxilla and bone forming area in which NPRC was specifically expressed but NPRB was not. In the SMG of 2 M, the membrane of duct cells markedly produced cGMP by CNP whereas acini produced cGMP by ANP and BNP rather than CNP. The biological activity of cGMP production of SMG gradually decreased with age. cGMP production was dominant by CNP in SMG of 1M but was by ANP after 2M. These results shows that CNP may play roles both in the morphogenesis and differentiation via NPRC and in the maintenance of duct system via NPRB in the mouse SMG and that the biological activity of its receptors may decreased with aging.

선박 Night Vision 시스템용 Pedestal의 제어부 개발

김정근(Jung-Keun Kim),김종민(Jong-Min Kim),박기랑(Ki-Rang Park),백승훈(Seung-Hun Baek),송세훈(Se-Hun Song),진상훈(Sang-Hun Jin),정인(In Jung),황승욱(Seung-Wook Hwang),진강규(Gang-Gyoo Jin) 한국마린엔지니어링학회 2006 한국마린엔지니어링학회 학술대회 논문집 Vol.2006 No.-

This paper presents the design of a night vision system for vessels. Both a hardware system and software modules for stabilization control are developed. In order to stabilize each control axis, the two-degree of freedom(TDF) PID controller is designed and its parameters are tuned using a real-coded genetic algorithm(RCGA). Simulation demonstrates the effectiveness of the proposed system.

Selection and Characterization of Tenascin C Targeting Peptide

김미영,정선주,Ok Ran Kim,Yong Seok Choi,이희란,박기랑,이춘택,강건욱 한국분자세포생물학회 2012 Molecules and cells Vol.33 No.1

Since tenascin C is a factor expressed highly in the tu-mor-associated matrix, it would be a desirable first step for targeting the tumor-specific microenvironment. In fact, a high level of tenascin C expression has been re-ported in most solid tumors, including lung cancer, colon cancer and glioblastoma. Therefore, the targeted binding of tenascin C in tumor stroma would inhibit tumor metastasis by modulating cancer cell growth and migration. We isolated a peptide that bound to tenascin C by phage display peptide library selection, and the selected peptide specifically recognized tenascin C protein in xenograft mouse tissue. We also observed exclusive staining of tenascin C by the selected peptide in tumor patient tissues. Moreover, the peptide reduced tenascin C-induced cell rounding and migration. We propose that the tenascin C targeting peptide may be useful as a specific anti-cancer diagnostic and therapeutic tool for most human solid tumors.

Transcriptional targeting of gene expression in breast cancerby the promoters of protein regulator of cytokinesis 1 andribonuclease reductase 2

Hye Jin Yun,조영화,Youngsun Moon,박영우,윤혜경,Yeun-Ju Kim,Sung-Ha Cho,이영일,Bong-Su Kang,김원재,박기랑,설원기 생화학분자생물학회 2008 Experimental and molecular medicine Vol.40 No.3

For cancer gene therapy, cancer-specific overexpression of a therapeutic gene is required to reduce side effects derived from expression of the gene in normal cells. To develop such an expression vector, we searched for genes over-expressed and/or specifically expressed in cancer cells using bioinformatics and have selected genes coding for protein regulator of cytokinesis 1 (PRC1) and ribonuclease reductase 2 (RRM2) as candidates. Their cancer-specific expressions were confirmed in both breast cancer cell lines and patient tissues. We compared each promoter’s cancer-specific activity in the breast normal and cancer cell lines using the luciferase gene as a reporter and confirmed cancer-specific expression of both PRC1 and RRM2 promoters. To test activities of these promoters in viral vectors, the promoters were also cloned into an adeno-associated viral (AAV) vector containing green fluorescence protein (GFP) as the reporter. The GFP expression levels by these promoters were various depending on cell lines tested and, in MDA-MB-231 cells, GFP activities derived from the PRC1 and RRM2 promoters were as strong as that from the cytomegalovirus (CMV) promoter. Our result showed that a vector containing the PRC1 or RRM2 promoter could be used for breast cancer specific overexpression in gene therapy.

A novel way of therapeutic angiogenesis using anadeno-associated virus-mediated angiogenin gene transfer

조영화,박현,조의식,김원재,Bong-Su Kang,박병용,Yeun-Ju Kim,Young-Ill Lee,장수익,박기랑 생화학분자생물학회 2007 Experimental and molecular medicine Vol.39 No.3

To develop a novel therapeutic angiogenesis for the treatment of cardiovascular diseases, angiogenin (ANG1) was examined as a potential therapeutic gene. An adeno-associated virus (AAV)-mediated gene delivery system was used to measure the therapeutic efficacy of ANG1. Using a triple cotransfection technique, rAAV-ANG1-GFP, rAAVVEGF- GFP and rAAV-GFP vectors were produced, which were then used to infect human umbilical vein endothelial cells (HUVECs) in order to evaluate in vitro angiogenic activities. Their protein expres - sions, tagged with green fluorescent protein (GFP),were monitored by confocal microscopy. The functional activities were measured using woundhealing HUVEC migration assays. The number of migrated cells stimulated by both the expressed ANG1 and the VEGF in rAAV-infected HUVECs increased almost twice the number observed in the expressed GFP control. In vivo angiogenic activities of the expressed ANG1 or VEGF were determined using mouse angiogenesis assays. The angiogenic activities of ANG1 or VEGF expressed in the injected mice were increased by 1.36 and 2.16 times, respectively, compared to those of the expressed GFP control. These results demonstrate that the ex - pressed ANG1 derived from rAAV infection has in vitro and in vivo angiogenic activities and suggest that the rAAV-ANG1 vector is a potential strategy for therapeutic angiogenesis.

폐암의 유전자 치료법을 위한 암특이적인 PRC1 프로모터

조영화(Young-Hwa Cho),윤혜진(Hye Jin Yun),권희충(Heechung Kwon),김희종(Heejong Kim),조성하(Sung-Ha Cho),강봉수(Bong-Su Kang),김연주(Yeun-Ju Kim),설원기(Wongi Seol),박기랑(Keerang Park) 한국생명과학회 2008 생명과학회지 Vol.18 No.10

우리는 최근에 PRC1 프로모터가 유방암 유전자치료를 위하여 전사 표적이 된 유전자의 발현을 조절할 수 있는 후보 프로모터임을 보고하였다. 우리는 본 실험에서 PRC1이 폐암 유전자 치료에서도 적용이 가능한지 조사하였다. 특정 프로모터가 루시퍼라제 유전자와 연결된 플라스미드를 이용한 형질전환 assay에서 PRC1 프로모터는 정상폐세포주에서는 활성을 보이지 않으나 폐암세포주에선 약 30 배의 활성을 보였다. 이는 이미 암특이적인 발현으로 알려진 BIRC5 (survivin) 프로모터와 유사한 결과였다. 또한, 바이러스 벡터를 이용한 실험에서 PRC1은 CMV 프로모터에 비해 아데노 부속바이러스에서 약 75%, 아데노바이러스에서 약 66%의 활성을 보였다. 이와 대조적으로, PRC1 프로모터를 함유한 이 들 두 종류의 바이러스는 정상 폐세포에서는 20%정도의 낮은 활성을 보였다. 흥미롭게도, 인간 폐종양세포를 이식한 생쥐모델을 사용한 결과에서는 PRC1 프로모터가 CMV 프로모터와 비슷한 생체 활성을 보였다. 종합하면, 이상의 결과는 PRC1이 폐암 유전자치료를 위한 전사표적 유전자의 발현을 위한 프로모터로 사용 가능함을 암시한다. We have recently reported the PRC1 promoter as a promoter candidate to control expression of transcriptionally targeted genes for breast cancer gene therapy. We tested whether the PRC1 promoter could be also applied for the lung cancer gene therapy. In the transient transfection assay with naked plasmids containing the luciferase fused to the PRC1 promoter, the promoter showed little activity in the normal lung cell line, MRC5. However, in the lung cancer A549 cells, PRC1 showed approximately 30-fold activation which was similar to the survivin promoter, the gene whose promoter has been already reported as a candidate for the gene therapy of lung cancer. In viral systems, the PRC1 promoter showed approximately 75% and 66% of transcriptional activity compared to the CMV promoter in the adeno-associated virus (AAV) and the adenovirus (AV) systems, respectively. However, the PRC1 promoter in either AAV or AV showed approximately 20% activity compared to the CMV promoter in the normal lung cells. In addition, human lung tumor xenograft mice showed that the PRC1 promoter activity was as strong as the CMV activity in vivo. Taken together, these results suggested that PRC1 might be a potential promoter candidate for transcriptionally targeted lung cancer gene therapy.

Improved Production Efficiencies of Various Adeno-Associated Virus (AAV) Serotypes and a Novel Universal AAV Titration Method

Young-Hwa Cho(조영화),Yejin Choi(최예진),Jung-Hee Yun(윤정희),Nam Hee Kim(김남희),Mira Choi(최미라),Young-Kook Choi(최영국),Kyung-Hee Kim(김경희),Young-Ill Lee(이영일),Beom Jun Lee(이범준),Keerang Park(박기랑) 한국생명과학회 2012 생명과학회지 Vol.22 No.6

AAV는 매우 안전하고 효율적인 유전자전달방법으로 인정되어 왔다. 그러나, AAV가 가진 생물의약품으로서 단점은 효율적이고 재현성 높은 생산법이 취약하고, 또한 다양한 혈청형을 간단한 한 가지 공통적 방법으로 신뢰성 있게 정량하는 방법이 개발되어야 하는 것이다. 따라서, 본 연구에서는 AAV2와 아데노바이러스를 동시에 감염하는 종래의 생산법에 의한 효율성과 새로운 생산법, 즉 AAV2 감염 후 pHelper 플라스미드를 transfection 하는 방법을 통한 생산효율성을 비교하였고, HEK293과 293T를 생산세포주로 하여 시간에 따른 생산효율성도 분석하였다. 그 결과 AAV2와 pHelper DNA를 포함한 새로운 생산법은 기존의 방법에 비해 10배 이상 높은 생산효율성을 보였고, 293T에서 AAV2를 10 MOI로 감염한 후 5일째에 가장 높은 생산효율성을 보였는데, 생산세포 한개 당 1.61×10? virus genomes (v.g.)을 생산하는 결과였다. 따라서 이 생산조건을 다른 혈청형 생산에 적용한 결과, 모든 혈청형에서 생산세포주 한 개 당 10? v.g. 이상을 생산하는 효율성을 보였다. 한편, 다양한 AAV 혈청형을 한 가지 공통적인 방법으로 정량하기 위해 the universal PCR 프라이머를 제작하였고, 그것을 이용하여 신뢰성 높고 10개 분자까지도 증폭이 가능한 결과를 모든 혈청형에서 얻었다. 그러므로 이 한 쌍의 정량용 the universal 프라이머는 임상시험용 아데노바이러스벡터에 존재하는 AAV오염을 검출하는 것에도 사용 가능하다. Adeno-associated virus (AAV) has been considered to be a very safe and efficient gene delivery system. However, the major obstacles to therapeutic usage of AAV have been to achieve highly efficient and reproducible production processes, and also to develop a reliable quantifying method of various serotypes with a simple protocol. We compared the efficiency of the conventional production protocol of AAV2 and adenovirus (Ad) co-infection to that of a new method containing AAV2 infection followed by pHelper transfection. We tested HEK293 and 293T, and further examined the time-dependent changes of AAV2 production. The new method of AAV2 and pHelper DNA gave about ten times higher production efficiency than that of the conventional protocol. The highest production efficiency in 293T was achieved as 1.61 × 10? virus genomes (v.g.)/cell by the new method of 10 MOI of AAV2 infection and 5 days post-infection. This protocol of the highest efficiency was then applied to produce various AAV serotypes and showed the efficiencies higher than 10? v.g./cell. Next, we designed the universal PCR primers of highly conserved regions for various AAV serotypes to develop a simple and reliable titration method. The universal primers could amplify all the tested AAV serotypes with similar sensitivities by ten molecular copies. Therefore, this pair of universal primers can be further utilized to detect AAV contaminants in therapeutic adenoviral vectors.