Immortalization of Rabbit Corneal Fibroblasts by Overexpression of Simian Virus 40 Large T antigen

Cho, Seung-Ju,Park, Yuk-Pheel,Lim, Heon-Man,Kim, Jae-Chan,Yang, Eun-Kyung,Park, Jung-Keug,Yoon, Do-Young,Lee, Hee-Gu 대한의생명과학회 2004 Biomedical Science Letters Vol.10 No.2

Immortalization of primary corneal cells has influence on pharmacy, medical and biological fields. Especially, investigation of immortalization mechanism using viral oncoproteins is useful for medical treatments, and these cell lines will be useful materials for toxic test of medical supplies and cell biological experiments. Rabbit corneal fibroblasts in culture undergo a finite number of divisions before they reach a terminally non-proliferating state known as replicative senescence. Therefore, we attempted to induce immortalization of rabbit corneal fibroblasts with SV 40 large T antigen. As a result of experiment, expression of SV 40 large T antigen was confirmed, and expression of proteins related to cell cycle repressor was decreased in the transfection group compared with non-transfection group. According to the results of cell cycle phase distribution test, SV 40 large T antigen-transfected cells had obtained higher proliferation rate than primary cells. It was confirmed that during induction of immortalization, SV 40 large T antigen was not able to increase telomerase activity. In conclusion, we made a rabbit corneal fibroblast cell line with SV40 large T antigen. This cell line will be useful for further studies of mammalian fibroblast biology, particularly with regard to angiogenesis and malignant transformation. In addition, this cell line offers opportunity for testing potential therapeutics and can be used for toxicity tests of materials or cosmetics. In the future, our cell line can potentially be utilized in a wide range of biology related fields.

Immortalization of Rabbit Corneal Fibroblasts by Overexpression of Simian Virus 40 Large T antigen

Cho, Seung Ju,Park, Yuk Pheel,Lim, Heon Man,Kim, Jae Chan,Yang, Eun Kyung,Park, Jung Keug,Yoon, Do Young,Lee, Hee Gu THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 2004 Journal of biomedical laboratory sciences Vol.10 No.2

Immortalization of primary corneal cells has influence on pharmacy, medical and biological fields. Especially, investigatior, of immortalization mechanism using viral oncoproteins is useful for medical treatments, and these cell lines will be useful materials for toxic test of medical supplies and cell biological experiments. Rabbit corneal fibroblasts in culture undergo a finite number of divisions before they reach a terminally non-proliferating state known as replicative senescence. Therefore, we attempted to induce immortalization of rabbit corneal fibroblasts with SV40 large T antigen. As a result of experiment, expression of SV40 large T antigen was confirmed, and expression of proteins related to cell cycle repressor was decreased in the transfection group compared with non-transfection group. According to the results of cell cycle phase distribution test, SV40 large T antigen-transfected cells had obtained higher proliferation rate than primary cells. It was confirmed that during induction of immortalization, SV40 large T antigen was not able to increase telomerase activity. In conclusion, we made a rabbit corneal fibroblast cell line with SV40 large T antigen. This cell line will be useful for further studies of mammalian fibroblast biology, particularly with regard to angiogenesis and malignant transformation. In addition, this cell line offers opportunity for testing potential therapeutics and can be used for toxicity tests of materials or cosmetics. In the future, our cell line can potentially be utilized in a wide range of biology related fields.

Immortalization of Rabbit Corneal Fibroblasts by Overexpression of Simian Virus 40 Large T antigen

Seung Ju Cho,Yuk Pheel Park,Heon Man Lim,Jae Chan Kim,Eun Kyung Yang,Jung Keug Park,Do Young Yoon,Hee Gu Lee 대한의생명과학회 2004 Biomedical Science Letters Vol.10 No.2

Immortalization of primary corneal cells has influence on pharmacy, medical and biological fields. Especially, investigation of immortalization mechanism using viral oncoproteins is useful for medical treatments, and these cell lines will be useful materials for toxic test of medical supplies and cell biological experiments. Rabbit corneal fibroblasts in culture undergo a finite number of divisions before they reach a terminally non-proliferating state known as replicative senescence. Therefore, we attempted to induce immortalization of rabbit corneal fibroblasts with SV40 large T antigen. As a result of experiment, expression of SV40 large T antigen was confirmed, and expression of proteins related to cell cycle repressor was decreased in the transfection group compared with non-transfection group. According to the results of cell cycle phase distribution test, SV40 large T antigen-transfected cells had obtained higher proliferation rate than primary cells. It was confirmed that during induction of immortalization, SV40 large T antigen was not able to increase telomerase activity. In conclusion, we made a rabbit corneal fibroblast cell line with SV40 large T antigen. This cell line will be useful for further studies of mammalian fibroblast biology, particularly with regard to angiogenesis and malignant transformation. In addition, this cell line offers opportunity for testing potential therapeutics and can be used for toxicity tests of materials or cosmetics. In the future, our cell line can potentially be utilized in a wide range of biology related fields.

Inhibition of BETs prevents heat shock-induced cell death via upregulating HSPs in SV40 large T antigen transfected cells

Zhou Nan,Zhang Ye,Lei Gongyun,Chen Yifan,Lin Ting,Liu Qin,Zhao Yinshuang,Mao Jiahui,Jiang Yongying,Mao Renfang 한국유전학회 2022 Genes & Genomics Vol.44 No.10

Background: Heat shock response is a protected mechanism against environmental changes for the organism, which must be tightly regulated. Bromodomain and extra terminal-containing protein family (BETs) regulate numerous gene expression in many physiological and pathological conditions, including viral infection. SV40 is considered as a highly human disease-associated virus. Objective: We aimed to explore whether BETs play a role in heat shock in SV40 large T antigen transfected cells. Methods: SV40LTA was transfected in HeLa cells using the Lipofectamine 8000. BETs inhibitor JQ1 and I-BET-762 was employed to treat transfected cells and HEK-293 T cells. Heat shock treatment was performed to determine the effect of JQ1 and I-BET-762 on these cells. Western blot and quantitative RT-PCR were carried out to assess the expression of HSP70 and other HSPs. Results: We found that inhibition of BETs by JQ1 and I-BET-762 protects cells from heat shock-induced death in HEK293T cells. Both JQ1 and I-BET-762 induce the expression of HSPs and HSF1 in HEK-293 T cells. However, neither JQ1 nor I-BET-762 fail to induce the expression of HSPs in either HeLa or HBL-1 cells. When SV40 large T antigen was transfected into HeLa cells, the induction of HSP70 expressing and the protection of heat shock-induced cell death are reproduced by JQ1 and IBET treatment in these transfected cells. Conclusions: Inhibition of BETs by JQ1 and I-BET-762 prevents heat shock-induced cell death via upregulating HSPs in SV40 large T antigen transfected cells. Our data indicate a novel function of BETs in SV40 large T antigen transformed cells, affecting HSPs and HSF1 as well as its function on heat shock response.

Establishment and evaluation of immortalized human epidermal keratinocytes for an alternative skin irritation test

Kim, Cho-Won,Kim, Chang Deok,Choi, Kyung-Chul Elsevier 2017 Journal of pharmacological and toxicological metho Vol.88 No.2

<P><B>Abstract</B></P> <P>Human skin is located at the outermost part of the body, and various cosmetics and chemicals that may come in contact with human skin need to be evaluated for their potential to cause irritation. Until recently, the Draize test was considered the standard method for skin irritation; however, this technique has disadvantages such as the need to sacrifice many rabbits and subjective scoring. Thus, to contribute to the development of an animal-free alternative skin irritation test, we investigated the cytotoxicity and inflammatory response to standard skin irritants in SV40 large T antigen-transformed human epidermal keratinocyte 2 cells (SV-HEK2 cells). In this study, we established an SV-HEK2 cell line immortalized by SV40 large T antigen (SV40 T) and characterized the inherent morphological and cytological properties. We next used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or neutral red uptake (NRU) assays of cell viability to investigate the optimal experimental conditions for determining SV-HEK2 cell viability after exposure to sodium dodecyl sulfate at 6.25×10<SUP>−3</SUP>% to 1×10<SUP>−1</SUP>% as a standard skin irritant. We then examined the viability of SV-HEK2 cells in response to five skin irritants (benzalkonium chloride, isopropanol, sodium dodecyl sulfate, Triton X-100 and Tween20) at 5×10<SUP>−3</SUP>% to 1×10<SUP>−1</SUP>% by MTT or NRU assay. Finally, we estimated the level of cytokines secretion in response to stimulation by skin irritants in SV-HEK2 cells. The results revealed that SV-HEK2 cells responded well to skin irritants in a concentration-dependent manner and that there was good correlation between irritant concentration and cytotoxicity (or cytokine secretion) when cells were exposed to skin irritants for 10min at room temperature (RT) using an MTT assay. Overall, these findings suggest that SV-HEK2 cells could be a good alternative <I>in vitro</I> model for skin irritation tests.</P>

SV40 T 항원의 온도조건부 변이형 유전자가 포함된 Amphotropic Retrovirus 에 의한 사람 태아 간세포의 불멸화

이정일(Joung Il Lee),동석호(Seok Ho Dong),김효종(Hyo Jong Kim),김병호(Byung Ho Kim),장영운(Young Woon Chang),장린(Rin Chang),성세라(Se Ra Seong),박재경(Jae Kyung Park),김승보(Seung Bo Kim),이상목(Sang Mok Lee) 대한내과학회 1999 대한내과학회지 Vol.57 No.1

N/A Human cells are almost never spontaneously immortalized in vitro. We tried to immortalize human fetal hepatocytes (h-FH) and evaluate the differentiational status and its change. Methods : Hepatocytes were isolated from a liver fragment of 20 week old fetus and infected with amphotropic recombinant retrovirus containing a temperature- sensitive mutant of SV40 large T antigen and neomycin phosphotransferase gene. G418 resistant colonies were cloned and expanded. The cells which were able to divide more than 30 times were used to analyze various functions. Results : The immortalization rate was 3.3 x 10-8 and two cell lines (C11, D21) were established. C11-60, C11-80, D21-30 and D21-60 (suffix number means the cell division counts) were evaluated. D21-30 was thougt to be imcompletely immortalized because a considerable portion of cells died during culture. The morphology was similar to that of epithelial cells except for D21-30 which looked like fibroblast. The cells grew rapidly at 33oC but stopped growing at 39oC. T antigen and p53 was expressed at 33oC but disappeared at 39oC, which suggest that T antigen binds to p53. Chromosomal changes were so marked that it was impossible to discriminate exact number. Albumin was secreted as about 1/10 as that of h-FH, but alpha-fetoprotein secretion stopped after immortalization. Telomerase was activated in both cell lines except for the incompletely immortalized cells D21-30. Telomere was elongated in competely immortalized cell lines, but it was rather shortened in D21-30 compared to that of h-FH. Macroscopic colonies did not develop in soft agar assay. Conclusions : We successfully immortalized human fetal hepatocytes. Although the cells are not likely to have oncogenicity, the functions are not so good, possibly due to marked chromosomal changes which are thought to occur before telomerase is activated during immortalization step.

Endometrial Cell Culture: Isolation, Characterization, and Immortalization

홍인선,김석현,궁미경,전진현,이영순,강경선,Hong, In-Sun,Kim, Seok-Hyun,Koong, Mi-Kyoung,Jun, Jin-Hyun,Lee, Yong-Soon,Kang, Kyung-Sun The Korean Society for Reproductive Medicine 2003 Clinical and Experimental Reproductive Medicine Vol.30 No.4

목 적: 본 실험의 목적은 자궁내막세포를 분리 및 배양법 확립과 함께 불멸화 시키는 것이다. 방 법: 자궁내막에서 상피세포(epithelial cells)와 기질세포(stromal cells)의 분리는 Satyawaroop 등(1979)의 방법에 기초를 두었다. 자궁내막에서 상피세포와 기질세포의 순수 분리도를 확인하고, 불멸화된 기질세포에서 SV40 large T antigen을 확인하기 위하여 면역형광 염색(immunocytochemistry)과 Western blot 기법을 이용하였다. 정상 기질세포의 경우 subconfluence (60%) 상태에서 transfection을 진행하였다. 순수 분리된 plasmid DNA와 Qiagen 사의 superfect를 이용하여 transfection을 실시하였다. 결 과: 본 연구에서 우리는 두 가지 형태의 자궁내막 세포의 분리 및 배양에 성공하였다. 상피세포는 다면체의 형태를 띠며, 선(grandular)조직의 조각으로부터 나선형으로 자란다.기질 세포는 길쭉한 형태를 띠며, 상피세포에 비해 오래 살고, 빠르게 증식하여 나란한 형태로 배열된 세포 다발(cell bundle)을 형성한다. 이렇게 분리된 세포들은 95%의 균질성을 보였으며, 면역형광염색과 western blot을 통해 확인 하였다. 한편 SV40(Simian Virus 40) large T 항원을 암호화 하고 있는 염기 서열을 포함한 플라스미드 벡터로 안정적인 트랜스펙션을 시킴으로써 불멸화 된 자궁내막의 기질 세포주를 확립하였다. 불멸화 된 세포는 그 세포가 유래한 정상의 세포와 동일한 표현형을 가지고 있었다. 결 론: 본 연구에서, 우리는 자궁내막에서 상피세포(epithelial cells)과 기질세포(stromal cells)를 분리하여 배양법을 확립하였다. 동시에 SV40 large T antigen을 이용하여 불멸화된 세포주를 확립하였다. 이렇게 확립된 세포주는 자궁의 생리작용 연구 및 자궁내막증(Endometriosis)과 자궁암(Endometrial cancer) 등과 같은 여러 자궁관련 질병 연구에 많은 도움이 될 것으로 사료된다.

온도조건부 SV40 T항원에 의한 불멸화 간세포주에서 T항원 비의존성 세포의 발생

동석호 ( Seok Ho Dong ),김병호 ( Byung Ho Kim ),남기덕 ( Gi Deog Nam ),장재영 ( Jae Young Jang ),김남훈 ( Nam Hoon Kim ),이상길 ( Sang Kil Lee ),주광로 ( Kwang Ro Joo ),김효종 ( Hyo Jong Kim ),장영운 ( Young Woon Chang ),이정일 ( 대한내과학회 2005 대한내과학회지 Vol.68 No.3

사람 간세포의 분리 및 SV40 Large T antigen에 의한 세포의 불멸화

허원희 ( Heo Won Hui ),윤승규 ( Yun Seung Gyu ),신주엽 ( Sin Ju Yeob ),류종순 ( Lyu Jong Sun ),왕진상 ( Wang Jin Sang ),김창욱 ( Kim Chang Ug ),최종영 ( Choe Jong Yeong ),정규원 ( Jeong Gyu Won ),선희식 ( Seon Hui Sig ) 대한소화기학회 2003 대한소화기학회 추계학술대회 Vol.2003 No.-

<목적> 간세포는 간의 해독 작용이나 운반체 단백질의 합성 저장과 같은 중요 기능을 수행한다. 간세포를 이용한 연구에는 간의 재생(regeneration)과 분화(differentiation) 기작과 간염 바이러스(hepatitis virus) 관련 연구 등이 있다. 특히 간염 바이러스 연구는 형질전환 동물로만 이용해야 한다는 한계를 안고있기에 새로운 신약 백신이 개발되었더라도 신약에 대한 간염 바이러스의 치료 효과를 입증하기가 어려운 실정에 있다. 이

Transfection of SV40 large T antigen induced an immortalization of human corneal epithelial cells with prolonged life span by regulating cell cycle-related genes

Cho Won Kim,Kyung Chul Choi 한국실험동물학회 2015 한국실험동물학회 학술발표대회 논문집 Vol.2015 No.8