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1 J. Biomed. Lab. Sci. 10 (2004) Immortalization of Rabbit Corneal Fibroblasts by Overexpression of Simian Virus 40 Large T antigen Seung Ju Cho 1, Yuk Pheel Park 1, Heon Man Lim 2, Jae Chan Kim 3, Eun Kyung Yang 4, Jung Keug Park 5, Do Young Yoon 1 and Hee Gu Lee 1 1 Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology (KRIBB), P.O. Box 115, Daejeon , Korea, 2 Department of Biology, Chungnam National University, College of National Sciences, #220, Gung-dong, Yuseong-gu, Daejeon , Korea, 3 Department of Ophthalmology, Yongsan Hospital, Chung-Ang University, College of Medicine, # Hangangro-3-ga, Yongsan-gu, Seoul , Korea, 4 Bioland Ltd. #39-4, Songjeong, Byongchon, Chonan, Chungnam , Korea, 5 Department of Chemical and Biochemical Engineering, Dongguk University, College of Engineering, #26, 3-ga, Pil-dong, Chung-gu, Seoul , Korea 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. Key Words: Immortalization, SV40 large T antigen, Telomerase 서 각막은눈의가장바깥쪽에있는투명한무혈관조직으로, 안구를보호하는방어막의역할과광선을굴절시켜망막으로도달시키는창의역할, 즉눈으로들어오는빛의양을조절한다. 하지만각막세포는제한적인세포분열을하므로, 각막의손상은결국복제노화 (replicative senescence) 단계에 * 논문접수 : 2004 년 4 월 26 일수정재접수 : 2004 년 5 월 17 일 별책요청저자 : Hee Gu Lee, Ph.D., Cell Biology Laboratory, Korea Research Institute of Bioscience and Biotechnology (KRIBB), P.O. Box 115, Daejeon , Korea Tel: , Fax: hglee@kribb.re.kr 론 이르게된다 11). 특히토끼의일차각막섬유아세포 (primary rabbit corneal fibroblast cells) 는복제노화로가기전 20~25 계대의세포분열을하게되고, 이러한세포분열의계대수는기원이되는동물의수명과비례적인관계가있다 10,14). 일반적인동물일차세포에서는계대가많이증가할수록세포의복제노화나성장억류 (cell growth-arrest) 를일으킨다 2,17). 따라서일차세포의불멸화 (immortalization) 는세포복제노화나성장억류에관한유전자의발현감소를야기하는데, 이러한세포의불멸화수단으로써 Simian virus 40 (SV40) 과같은 DNA 종양바이러스 (tumor virus) 가많이이용된다. 특히바이러스유전자중 SV40 large T antigen (SV- 40T) 은다양한세포들의형질전환을야기하는강력한종양형성단백질이다. SV40T는 708개의아미노산으로구성되어있고, 크게두

2 부분으로나누어 N-말단의 Rb 결합부위와 C-말단의 p53 결합부위로이루어져있다. Rb와 p53은세포내에서종양억제단백질 (tumor suppressor protein) 로알려져있다. SV40T 는이러한 Rb와 p53과의결합을통해 Rb와 p53을매개로하는세포성장의억류를막는다. 또한 SV40T는세포내에서 DNA helicase로서의기능이나여러유전자의전사활성또는억제자로서의기능을하게된다 12). 세포의성장활성, 억류및세포사 (apoptosis) 등은세포내의 telomere 길이와밀접한관계가있는것으로알려져있다. 일반적으로동물일차세포들은 telomere의길이를유지시키는 telomerase의 catalytic subunit (telomerase reverse transcriptase, TERT) 발현이극히드물게일어난다. 세포가노화로갈수록 telomere의길이가짧아지는현상에관한 telomere 단축이론 (telomere shortening theory) 은세포의복제노화에대한설명에서많이적용된다 3,6). 세포의 telomere 단축은 DNA 손상을야기하고결과적으로세포성장억류나노화를일으킨다 20). 하지만 telomere 단축이론은세포종류에따라제한적으로적용된다. 즉 telomerase의활성이낮음에도불구하고세포의성장이활발한경우도있다 4,9). 세포의불멸화는의학적, 약학적그리고생물학적분야에많은영향을미친다. 특히바이러스유전자를이용한불멸화의기작에관한연구는그바이러스에관련된질병치료연구에많은영향을미칠뿐만아니라, 이러한방법으로제작된세포는새로운의약품들이개발되고있는현시대에많은용도로쓰일수있다. 새로운의약품후보물질들을위해서는인간안구자극테스트가필수적이며, 특히각막에손상을주는정도를파악하기위해각막세포주의 in vitro 독성실험이기본적으로수행되어야한다. 따라서본연구에서는토끼각막섬유아세포를대상으로 SV40T 유전자를이용하여불멸화를유도하였고, 그것을통한형질분석과정으로써세포의성장곡선및형태의변화, 종양억제단백질인 Rb 와세포주기관련단백질들의발현변화, telomerase의활성도변화를알아보았다. 그결과유도한세포가일차세포와비교하여 SV40T에의해세포주기의활성을야기한세포주임을확인하였다. 재료및방법 1. 세포배양및플라스미드토끼의일차각막섬유아세포 (primary rabbit corneal fibroblast cell, 중앙대학교의과대학 ) 및 SV40T를이용하여형질전환시킨토끼각막섬유아세포를 DMEM (Sigma, USA) 배지에 10% FBS (Hyclone, USA) 을넣어서 5% CO 2 배양기에서배양하였다. 또한 SV40T 유전자를포함하고있는 psv3-neo 플라스미드를형질전환에사용하였다. 2. 형질전환및세포주확립 100 mm 배양용기에 세포를깔아두고, 다음날배양용기에부착된세포가 70% 차지하였을때 PBS로 3번세척하였다. DNA (psv3-neo) 10 µg에무혈청배지 (Serum free media) 를첨가하여 500 µl가되도록하고, Lipofectamine (GIBCO BRL, USA) 30 µl에 475 µl의무혈청배지를첨가하여상온에서 15분간반응시켰다. 15분후 DNA를첨가한배지와 Lipofectamine을첨가한배지를혼합하여상온에서 30 분간반응시켰다. 이때세척한세포를다시무혈청배지로세척하고, 30분후 DNA와 Lipofectamine이섞인배지에 5 ml의무혈청배지를첨가하여총 6 ml을잘혼합한후세척한세포에혼합하였다. 37, 5% CO 2 배양기에서 6시간동안배양후일반적인배양배지로교체하였고, 48시간후부터 0.8 mg/ml의 G418 (Sigma, USA) 로형질전환된세포들을선별하였다. 3. 중합효소연쇄반응 (PCR) 토끼의일차각막섬유아세포와형질전환된세포에서 genomic DNA를추출하여주형 (template) 으로사용하였고, 각각 1 pmol의 SV40T 특이적 primer, 1 µl의 dntp 혼합물및 Taq polymerase (Solgent, Korea) 를첨가하여전체볼륨을 20 µl로맞춘후반응시켰다. SV40T의특이적 primer로써, first fragment primer pairs: 5'-AAA GGG AAT AAG GGC GAC AC-3' (sense) and 5'-GCA AAA CAG GAG GCA CAT TT-3' (antisense); second fragment primer pairs: 5'-GGG AGG TGT GGG AGG TTT T-3' (sense) and 5'-CTT TGG TCC CGG ATC TTT GT-3' (antisense); third fragment primer pairs: 5'-GCT CCC ATT CAT CAG TTC CA-3' (sense) and 5'-GGA TGA TCT GGA CGA AGA GC-3' (antisense) 로제작하였다. PCR을위해혼합물을 95 에서 5분간반응시킨후 94 1분, 58 1분, 72 1분반응을 30회반복하였으며, 72 에서 7분간반응시켰다. 반응후 agarose gel에서전기영동한후 EtBr 염색을통해확인하였다. 4. Southern Hybridization SV40T에대한 probe를제조하기위해 SV40T의두번째단편 primer를이용하여 RCR을수행하였고동위원소를이용하여표지하였다. 토끼의일차각막섬유아세포및형질전환된세포에서 genomic DNA를추출하였다. 이 genomic DNA는제한효소인 Xho I의단일처리및 Cla I과 Sal I의혼합처리를병행하여 37 에서 2시간동안반응시켰고, 0.8% agarose gel에서전기영동하여 EtBr 염색을통해확인하였다. 확인후 gel은물로탈색하였고, depurination 용액 (0.2 N HCl) 을 15분간처리하였다. 다시 denaturation 용액

3 (0.5 N NaOH, 1.5 M NaCl) 을 15분간처리하고, 물로세척후 15분간 neutralization 용액 (0.5 M Tris-Cl, ph 8.0, 1.5 M NaCl) 을처리하였다. Capillary transfer 방법으로 DNA를 nitrocellulose filter 에이동시킨후, 그 filter와 SV40T probe를 hybridization 시켰으며, autoradiography를통해감광하였다. 5. Western blot analysis 배양하던토끼의일차각막섬유아세포와형질전환세포를수집하여 PBS로세척하고, protease inhibitors (5 mg/ml의 aprotinin, 5 mg/ml의 leupeptin) 를첨가한 lysis buffer (20 mm HEPES, ph 7.9, 100 mm KCl, 300 mm NaCl, 10 mm EDTA, 1% Triton-100) 로세포를용해시켰다. 세포의 lysate를획득하여단백질농도를정량하고, SDS-PAGE (polyacrylamide gel electrophoresis) gel에서전기영동하였다. 전기영동을통해분리된단백질들을 gel로부터 PVDF (polyvinylidene difluoride, Amersham Biosciences, UK) membrane 으로이동시켰으며, 이 membrane에 5% blocking 용액 [nonfat powdered milk (DIFCO, USA) in TBS (Tris-buffered saline)] 을 1시간동안처리하였다. 그후일차항체를실온에서 1시간 30분간처리후 TBST [0.05% Tween-20 (usb, USA) in TBS] 를이용하여 membrane을세척하고, peroxidase가결합된이차항체를실온에서 30분간처리한뒤 ECL (Amersham Biosciences, UK) 로발색시켰다. 6. 세포주기분석배양하던토끼의일차각막섬유아세포와형질전환세포의 confluency가 70~80% 되었을때수집하여 PBS로세척하고, 70% EtOH로 -20 에서 16~18시간동안고정시켰다. PBS 로세척한후 RNase (100 µg/ml in PBS) 가첨가된 propidium iodide (20 µg/ml in DMSO) 를이용하여실온에서 30분간반응시킨후유세포분석기 (Flow cytometry) 로세포주기를분석하였다. 7. TRAP assay Telomerase 활성은 TRAPeze Detection Kit (Intergen, USA) 를사용하여 PCR-based telomeric repeat amplification protocol (TRAP) 에의해분석되었다. 토끼의일차각막섬유아세포와형질전환세포를수집하여 PBS로세척하였다 ~ 10 6 세포에 1 CHAPS lysis Buffer (10 mm Tris-HCl, ph 7.5, 1 mm MgCl 2, 1 mm EGTA, 0.1 mm Benzamidine, 5 mm β- mercaptoethanol, 0.5% CHAPS, 10% Glycerol) 200 µl씩첨가하여혼합한후 4 에서 30분동안반응시켜서세포의 lysate를획득하였다. 동위원소를이용한 TS primer (Substrate oligonucleotide, in TRAPeze Detection Kit) 의 end-labeling은 10 mci/ ml γ- 32 P-ATP, TS Primer, 10 Kinase Buffer, T4 Polynucleotide Kinase, dh 2 O를첨가하여 37 에서 20분동안반응하고 85 Fig. 1. Growth curves of primary and SV40 large T antigentransfected rabbit corneal fibroblasts. Primary normal rabbit corneal fibroblasts were stably transfected with SV40 large T antigen (SV40T). The transfected cells were selected in medium containing G418. Population doublings (PD) were measured as a serial cultivation with split ratio 1:4. The growth of the rabbit corneal fibroblasts (RCF) slowed down around 14~15 PD and ceased at 22~23 PD. SV40 large T antigen-transfected rabbit corneal fibroblasts [RCF(SV40T)] were still growing, in contrast with primary rabbit corneal fibroblasts (RCF). 에서 5분동안반응시켜수행하였다. 10 TRAP Reaction Buffer, 50 dntp Mix, 32 P-TS Primer, TRAP primer Mix, Taq polymerase, dh 2 O를첨가하여 Master Mix를준비하고 RNasefree PCR tube에분주하였으며세포의 lysate 및 heat-inactivated extracts를첨가하였다. PCR tube를 30 에서 30분간둔후 94 30초, 59 30초반응을 27~30회반복하였다. 반응된각혼합물은 10~12.5% non-denaturing PAGE gel에서전기영동하고, autoradiography를통해감광하였다. 결 1. 세포의형질전환및확인 토끼의일차각막섬유아세포를형질전환시키고세포의선별과정을거친후형질분석을수행하였다. 형질분석과정에서일차세포와형질전환세포의세포성장곡선을비교한결과, 일차세포는 20~22 계대정도로분열되는반면형질전환세포는계속적인분열이일어남을확인하였다 (Fig. 1). 세포의형태에서는섬유아세포의형태가형질전환후짧아진것을확인할수있었다 (Fig. 2). 또한 PCR 및 southern hybridization을통해 SV40T를이용한형질전환세포에서의 SV40T 유전자를확인하였다 (Fig. 3). 이를통해제작한세포가 SV40T를함유하고있음을알수있었다. 과 2. 세포의성장관련단백질발현의비교 형질분석은세포내에임의로도입한 SV40T 유전자의

4 A Fig. 2. Morphology of primary and SV40 large T antigentransfected rabbit corneal fibroblasts. Phase-contrast photomicrographs of primary normal cultures of rabbit corneal fibroblasts (A) and SV40 large T antigen-transfected rabbit corneal fibroblasts (B). An extended life span and the development of a more in vivolike morphology compared to primary rabbit corneal fibroblasts were also observed in SV40 large T antigen-transfected rabbit corneal fibroblasts. Morphological change at senescent fibroblast cells through microscopic observation was similar to the result observed in other known fibroblast cell lines. Primary rabbit corneal fibroblsts (A): passage 19, SV40 large T antigen-transfected rabbit corneal fibroblasts (B): passage 61. B 확인및그유전자에대한발현을알아보고, 세포내에있는 SV40T의목표물질이자, 동시에세포주기에관여하는단백질인 Rb의인산화및 cyclin-dependent kinase inhibitors (CDKIs) 의발현을확인하는것으로진행되었다. 세포에도입된 SV40T DNA를확인하였으며, 그 DNA에대한단백질의발현을 western blotting으로확인하였다. SV40T 발현에대한양성대조군 (positive control) 으로서 SV40T가세포내부에서발현되는 COS-7 세포의 lysate를이용하였고, 실험결과일차세포와는달리형질전환된세포에서만 SV40T가발현됨을확인하였다 (Fig. 4A). 이전의보고에의하면 Rb의인산화 (phosphorylation) 는 SV40T에의한세포의불멸화에서중요한역할을한다. 그러므로 SV40T의세포내목표물질인 Rb의발현과 SV40T를통한 Rb의인산화정도및 Rb 의 upstream에서세포주기관련단백질인 p16(ink4a), p21 (WAF1) 및 p27(kip1) 의발현을확인하였다 (Fig. 4). 실험결과, 일차세포와는달리형질전환된세포에서 SV40T에의한 Rb의인산화를확인하였다. p16(ink4a) 의경우 SV40T가세포내로삽입되었을때발현이증가하는것으로알려져 A B Fig. 3. Confirmation of SV40 large T antigen gene in primary and SV40 large T antigen- transfected rabbit corneal fibroblasts by PCR and Southern Hybridization. It was confirmed that one candidate expresses SV40 large T antigen by using genomic DNA PCR (A) and southern hybridization (B). PCR was performed with genomic DNA isolated from the primary rabbit corneal fibroblasts and SV40 large T antigen-transfected rabbit corneal fibroblasts using specific primers for SV40 large T antigen. Southern probe for SV40 large T antigen was used DNA fragment (687 bp) synthesized by PCR. SV40 large T antigen DNA fragment was labeled with random priming. The genomic DNA were treated with restriction enzyme (Xho I, or Cla I and Sal I). A, SV40 large T antigen partial product by PCR; lane 1, primary rabbit corneal fibroblasts (RCF); lane 2, SV40 large T antigen-transfected rabbit corneal fibroblasts [RCF (SV40T)]. B, Southern hybridization; lane 1, primary rabbit corneal fibroblasts (RCF); lane 2, SV40 large T antigen-transfected rabbit corneal fibroblasts [RCF (SV40T)]/ Xho I; lane 3, SV40 large T antigen-transfected rabbit corneal fibroblasts [RCF(SV40T)]/Cla I and Sal I

5 A B Fig. 4. Expressions of SV40 large T antigen and cell-cycle related proteins. Each expression was measured by western blot analysis. SV40 large T antigen was expressed highly in the SV40 large T antigen-transfected rabbit corneal fibroblasts as expected (A). To address the molecular mechanism of immortalization, expressions of markers like p21(waf1), p27(kip1), and p16(ink4a) were examined. In the SV40 large T antigen-transfected rabbit corneal fibroblasts, expressions of the p21(waf1) and p27(kip1) proteins were undetectable (A). Interestingly, expression of the p16(ink4a) was elevated highly whenever SV40 large T antigen is expressed (A). Unlike primary cells, increase in phosphorylated Rb was observed because of the inhibition effect on Rb by SV40 large T antigen (B). α-tubulin was used for standardization of protein concentration. lane 1, primary rabbit corneal fibroblasts (RCF); lane 2, SV40 large T antigen-transfected rabbit corneal fibroblasts [RCF (SV40T)]; lane 3, COS-7 cell lysate (SV40 large T antigen positive control), MCF-7 cell lysate [p21(waf1) positive control], HeLa cell lysate [p27(kip1) and p16(ink4a) positive control]. A B Fig. 5. Cell-cycle phase distribution. Analysis of cell cycle was obtained using Flow cytometry. According to the results of cell cycle phase distribution test, primary rabbit corneal fibroblasts had high degree of G1/G0 phase. In contrast, SV40 large T antigen-transfected rabbit corneal fibroblasts showed low degree of G1/G0 phase and high degree of S phase than primary rabbit corneal fibroblasts. From the results, SV40 large T antigen-transfected cells had obtained higher proliferation rate than primary rabbit corneal fibroblasts. Histograms of primary rabbit corneal fibroblasts (RCF, left) and SV40 large T antigen-transfected rabbit corneal fibroblasts [RCF (SV40T), right] (A). Data of the computer analysis of % cell-cycle phase distribution are shown (B)

1 2 3 4 5 6 7 3. 세포주기및 telomerase 활성비교 형질분석과정에서유세포분석기를이용한일차세포와형질전환된세포들의세포주기를비교한결과, 형질전환세포에서 G0/G1 기 (phase) 상태가상대적으로낮게나오고 S 기상태가높게나오는것을확인하였다 (Fig. 5). 이결과, 형질전환된세포들의세포주기활성이일차세포보다더크다는것을알수있었다.

The activity of telomerase was analyzed with primary rabbit corneal fibroblasts and SV- 40 large T antigen-transfected rabbit corneal fibroblasts by using the TRAPeze telomerase detection kit.

6 세포주기및 telomerase 활성비교 형질분석과정에서유세포분석기를이용한일차세포와형질전환된세포들의세포주기를비교한결과, 형질전환세포에서 G0/G1 기 (phase) 상태가상대적으로낮게나오고 S 기상태가높게나오는것을확인하였다 (Fig. 5). 이결과, 형질전환된세포들의세포주기활성이일차세포보다더크다는것을알수있었다. 또한 TRAP assay를통해 telomerase 활성을측정한결과일차세포와형질전환된세포에서의차이가거의없음을확인하였다 (Fig. 6). 이것은 SV40T 를이용한토끼각막섬유아세포의불멸화에있어서, 불멸화가 telomerase 의활성에는영향을미치지못하는것으로예상하였다. 고 찰 Fig. 6. Analysis of telomerase activity. The activity of telomerase was analyzed with primary rabbit corneal fibroblasts and SV- 40 large T antigen-transfected rabbit corneal fibroblasts by using the TRAPeze telomerase detection kit. In telomerase activity assay, the SV40 large T antigen-transfected rabbit corneal fibroblasts, unexpectedly, shown no significant differences with primary cells. So we assumed that SV40 large T antigen was not able to increase telomerase activity in rabbit corneal fibroblasts. lane 1, primary rabbit corneal fibroblasts (control); lane 2, primary rabbit corneal fibroblasts (heat-inactivated negative control); lane 3, SV40 large T antigen-transfected rabbit corneal fibroblasts; lane 4, SV40 large T antigen-transfected rabbit corneal fibroblasts (heat-inactivated negative control); lane 5, lysis buffer only (negative control); lane 6~7, quantitation controls (lane 6, 1 µg; lane 7, 2 µg). 있고, 결국 SV40T를이용한형질전환세포에서의발현증가를확인하였다. p21(waf1) 과 p27(kip1) 의경우일차세포와달리형질전환된세포에서발현의감소를확인하였다. p21(waf1) 의발현에대한양성대조군으로 MCF-7 세포의 lysate를이용하였고, p16(ink4a) 및 p27(kip1) 에대한양성대조군으로는 HeLa 세포의 lysate를이용하였다. 이결과, 형질전환된세포의세포주기가활성화되었음을의미하였다. 따라서세포의성장관련단백질발현에관련된실험의결과, 일차세포와는달리 SV40T에의해형질전환된세포에서의세포주기활성이더큰것으로나타났으며, 이것은세포의불멸화를의미하는것이다. 일차세포의불멸화는많은다양한방법으로수행되어왔다. 세포성장관련신호를자극하는방법이나, SV40T 혹은 E6/E7과같은 DNA 종양바이러스의유전자를도입하는방법등이그것이다 21,22). 세포치료학분야에서세포증식의활성을변화시키는세포의불멸화기작에대하여연구하는것이필수적이기때문에, 본연구에서는토끼의일차각막섬유아세포에 SV40T를도입하여불멸화를유도하였고, 그불멸화된세포의형질분석과정에서토끼의일차각막섬유아세포와형질전환된세포사이에서의세포주기조절기전의변화를비교분석하였다. SV40T의세포내목표물질로써 Rb의발현변화및 upstream에서의관련물질인 CDKIs 의발현변화를비교하였으며, 일차세포의형질전환에따른 telomerase의활성도를측정하였다. Rb는세포내에서전사인자인 E2F와결합하여 E2F의역할을통제한다. 이러한 Rb는세포주기의 G1에서 S 기로진행할때 cyclin/cdk 복합체 [ 주로 cyclin D1/cdk4 (or cdk6) 와 cycline/cdk2] 에의해인산화되고, 그로인해, 세포주기의 S 기에필요한유전자의전사를유도하는 E2F의기능이활성화된다 6). SV40T는세포내에서 Rb family들과함께결합하여세포성장의억류를막는다. 즉, SV40T에의해 Rb로부터 E2F가격리되면세포주기에서 S 기로진행하기위한특정유전자가발현된다 13,18). 세포내에서 SV40T는 Rb와직접적인결합이이루어지므로 Rb에관련된 CDKIs를분석한결과일차세포와달리형질전환된세포에서 p21(waf1) 과 p27(kip1) 의발현이감소된것을확인하였다. 따라서토끼각막섬유아세포의 SV- 40T에의한세포의불멸화에서, p21(waf1) 과 p27(kip1) 의발현이억제되고그것에따른 Cyclin/cdk 복합체가활성화되어 Rb family들의인산화가유도될것으로예상하였고, 그것

7 을 western blotting 결과로써확인할수있었다. 일반적으로인간의암세포에서는 p16(ink4a) 에변이가생기거나혹은메틸화되면 p16(ink4a) 의활성이억제될뿐만아니라발현도억제되는데, 그럼에도불구하고 SV40T에의해형질전환된세포에서는 p16(ink4a) 의발현이높게측정될수있다 7,15,16). 이현상은토끼각막섬유아세포에서도나타났는데그정확한기작은아직밝혀진바없다. 결과적으로 SV40T 는토끼각막섬유아세포에서 Rb의인산화를통해세포주기의 S 기로의전환을유도하였고, 그로인해 SV40T에의한세포의불멸화에서 Rb의인산화는그기작에중요한역할을하는것을알수있었다. 전형적인세포배양조건에서 SV40T를통한세포의불멸화는한가지중요한특성을내포한다. 그것은세포의 colony 형성을촉진한다는것이다. 즉세포의부착관련분자 (adhesion molecules) 들의발현이조절됨으로써세포의모양이 colony 형태로전환되고, 세포의부착과는독립적으로세포가성장하게된다 5). 토끼각막섬유아세포에서도형질전환후확인한세포의모양에서일차세포와는달리세포의크기가작아지고형태가짧아지는현상을확인하였다. 이는일차세포에유전자도입후항생제를이용한세포선별과정에서, 항생제가세포에미치는영향을배제할순없지만, SV40T에의한 colony 형태의유도가더큰원인일것으로예상한다. 불멸화가유도된세포의 telomerase 활성은높게측정되는것이일반적이나, SV40T를통해유도된토끼각막섬유아세포는낮게측정되었다. 이러한현상은세포의종류마다차이가나타날수있고, 현재까지알려진원인으로는 telomere 의확장을유도하는데중요한역할을하는 telomerase에있어서, 특정종류의세포에서는그주요요소가 TERT가아닌다른인자가 telomerase의능력을조절할수있기때문이다. 그인자에는 TRF1, TIN2, 그리고 POT1 혹은그외 telomerase 전체효소를구성하는다른구성인자가포함될수있다 1,8,19). 본연구에서는 SV40T를이용하여토끼각막섬유아세포의불멸화를유도하였고, 이것은세포성장과관련하여 Rb 를매개로하는세포성장억류를차단하는기작을통해이루어짐을확인하였다. 이러한각막섬유아세포의불멸화를유도하여제작된세포는새롭게개발되는의약품독성검사에이용될수있을것이고, 현재발생하는바이러스에의한질병에관한연구및다른세포생물학적실험에많은도움이될것으로기대된다. 감사의말본연구는보건복지부제품화연구지원 (01-PJ1-PG4-01PT ) 사업의지원에의하여이루어졌음. 참고문헌 1) Baumann P and Cech TR (2001): Pot1, the putative telomere end-binding protein in fission yeast and humans. Science, 292(5519): ) Brown JP, Wei W and Sedivy JM (1997): Bypass of senescence after disruption of p21(cip1/waf1) gene in normal diploid human fibroblasts. Science, 277(5327): ) Campisi J (1997): The biology of replicative senescence. Eur J Cancer, 33(5): ) Dickson MA, Hahn WC, Ino Y, Ronfard V, Wu JY, Weinberg RA, Louis DN, Li FP and Rheinwald JG (2000): Human keratinocytes that express htert and also bypass a p16 (INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics. Mol Cell Biol, 20(4): ) Elenbaas B, Spirio L, Koerner F, Fleming MD, Zimoniic DB, Donaher JL, Popescu NC, Hahn WC and Weinberg RA (2001): Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells. Genes Dev, 15(1): ) Harley CB, Futcher AB and Greider CW (1990): Telomeres shorten during ageing of human fibroblasts. Nature, 345 (6274): ) Huschtscha LI and Reddel RR (1999): p16(ink4a) and the control of cellular proliferative life span. Carcinogenesis, 20(6): ) Kim S, Kaminker P and Campisi J (1999): TIN2, a new regulator of telomere length in human cells. Nat Genet, 23(4): ) Kiyono T, Foster SA, Koop JI, McDougall JK, Galloway DA and Klingelhutz AJ (1998): Both Rb/p16(INK4a) inactivation and telomerase activity are required to immortalize human epithelial cells. Nature, 396(6706): ) Martin GM, Sprague CA and Epstein CJ (1970): Replicative life-span of cultivated human cells. Effects of donor's age, tissue, and genotype. Lab Invest, 23(1): ) Medrano EE, Yang F, Boissy R, Farooqui J, Shah V, Matsumoto K, Nordlund JJ and Park HY (1994): Terminal differentiation and senescence in the human melanocyte: repression of tyrosine-phosphorylation of the extracellular signalregulated kinase 2 selectively defines the two phenotypes. Mol Biol Cell, 5(4): ) Pipas JM and Levine AJ (2001): Role of T antigen interact

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