대한임상검사학회지 : 39 권제 1 호, 1-6, 2007 ISSN 1738-3544 인간파펼로마바이러스 E6Æ7 에의한 Telomerase 활성 건양대학교임상병리학과 1 한국생명공학연구원세포생물학설 2 긴여쿼 1. 서축워 1. 키산하 1. 바육핑 2 = C> L: "1 C>':"'::: C 그 :> ~I --, -, """ Analysis of Telomerase Activity by HPV E6/E7 Expression in SW13 Young-Kwon Kim 1 Choong-Won Scol Sang-Ha Kim1 and Yuk-Pheel Park2 Department 01 Biomedical Laboratory Science, Konyang University, Da밍eon 302-718, Korea 1 Laboratory 01 Cell Biology, Korea Research Institute 01 Bioscience and Biotechnology, Da밍eon 305-333, Korei Cervical cancer is one of the most prevalent cancers dεveloped in women worldwidε, and human papi1lomavirus(hpv) type 16 is the most common agent li따ced to human cerivical carcinoma. Viral oncogenes E6 and E7 are selectively retained and expressed in carcinoma cells infected with human papillomavirus type 16 and cooperatε with εach other in thε immortalization and transformation of primary keratinocytes. Because the HPV oncogenesis mechanism was not completely solved, more thorough studies are required. ln the present study, we investigated the telomere independent role of telomerase in HPV oncogenesis, we constructed the E6 mutant, E7, E6/E7 and htert over-expressed stable cells with a telomerase negative cell line, SWI3. Expressions of inserted genes were measured by RT-PCR. E6, E7 and htert genes were well expressed in each cell lines whεn compared with the control groups. By analyzing the cell morphology under the microscope, htert clone sizε was a smaller than thε mock control but oncogene expressed clones had a slightly lengthened marginal region. ln addition, htert cells also has a tεndency of brief dividing time compared to the mock control. To determine whether telomerase activity was associated with a HPV oncogenesis by oncoprotεin expression, wε performed the PCR based TRAP assay and a Northεm blot analysis. In TRAP assay data, tε10mεrasε activitiεs in hter T and oncogenε clones mcrεased compared to thε mock control. In addition, SWI3/E6/E7 cells showed an extremely increased activity compared to the othεr clones. lnduced htert mrna by E6/E7 wasn t, however, detected in Northem blotting. In conclusion, these findings suggest that telomerase activity is closεly associated with the HPV oncogenesis and E6/E7 co-expression is a most important factor of telomerase activity. Key Won:ls : Human papillomavirus, HPV, Oncogεne, Telomerase activity 1. 서론 교신저자 : 김영권, ( 우 )302-718 대전시서구가수원동 685 건양대학교임상병리학과 Tel: 042-600-6371, 017-435-3245 E-mail y셈이 m3245@konyang.ac.kr 자궁경부암은전세계적으로여성에게서나타나는가 장흔한질병의하나로써인유두종바이러스 (human papilloma virus, HPV) 16 형의감염이가장중요한원인
대한임상검사과학회지 39(1):1-6, 2007 으로알려져있다. 이바이러스의감염에는 E6와 E7 의발암단백질이감염된자궁경부암세포에서선택적으로유지되고발현되어, pnmaη 각질세포의형질전환및불멸화에관여하는것으로알려져있다 (Hudson 등, 1990; Halbert 등, 1992; Woodworth 등, 1992). HPV 바이러스의감염시, E6와 E7 발암단백질이종양억제단백질인 (tumor suppressor) p53과 Rb의기능을불활성화시켜세포주기를활성화시킴으로써, 발암과정에관여하는것으로알려져있으며 (Rapp와 Chen, 1998; Thomas 등, 1999; Zwerschke 등, 2000; Kubbutat 등, 2000), E7 단백질의경우, prb와결합하여 Rb 단백질의종양억제기능을억제하고 (Dyson 등, 1989; Munger 등, 1989), E6 단백질은 ubiquitin E3 ligase 인 E6AP와결합하여 p53을 ubiquitin 의존적인단백질파괴기작으로 p53을파괴시킨다고보고되고있다 (Scheffiner 등, 1990; Wemess 등, 1990; Scheffiner 등, 1993). 최근 10년간의연구결과중에서는 HPV 바이러스의 E6 발암단백질이 htert의전사를증가시키고 telomerase의활성을증가시킨다는결과들이많이도출되어왔다. Telomerase는정상세포에서는불활성화상태이나다양한암세포, 불멸화된세포들에게서 90% 이상의활성을보여암화과정에밀접한관련이있는단백질로많이주목을받아왔다. HPV 발암단백질이 primary 세포들을불멸화시키는과정에있어서, in vitro 상의 HCK (human cervical keratinocyte ), HFK(human foreskin keratinocytε), HMEC(human mammary εpithεlial cε l1 s) 등의다양한세포들에발암단백질이과발현되면, 세포가노화과정을극복하여죽지않는불멸화세포가형성됨을보여주었다 (Kl ingelhutz, 등, 1996; Kiyono 등, 1998; Sprague 등, 2002; Baege 등, 2002). 또한이 primary 세포들이불멸화되는과정에서세포의노화에직접관련된 telomerase의활성이증가되어 telomere를지속적으로합성하게되고, 따라서죽지않는세포가형성되는것이다. 또한 htert promoter 연구에서도, HPV E6 단백질이 c-myc 비의존적인상태로도 htert의전사를증가시킬수있음이보고되었다 (Gewin 등, 2001). 결론적으로기존의연구결과중에서, telomerase가 HPV 발암기전에관련이있다는것을시사하고있으나기존의연구결과중에서나타난문제점은, telomerase 의효과가각연구팀에서 이루어진시스템과세포에따라다른경향을보이고있다. 따라서본연구에서는 telomere 비의존적인 TERT subunit 자체의기능을분석하기위한것으로, HPV 발암기전에서의 E6, E7 발암단백질들과 catalytic subunit (TERT) 의상호작용및 telomerase의기능을분석하고자하였다. 11. 재료및방법 1. 서 포배양및발현벡터제작 Telomerase negative 세포주, SW13은 ATCC에서구입하였으며, L-15 Leiboviz(Sigma, St Louis, MO) 배지에 10% FBS(Hyclone, Logan, Utah) 를넣어서 5% C02 배양기에서배양하였다. htert, HPV E6m,- HPV E7, HPV E6/E7 발현벡터를형질전환시켜만들어진 SW13 세포주들은 DMEM(Sigma, St Louis, MO) 에 10% FBS를첨가한배양액을이용하였다. 각유전자의발현벡터는성균관대의이한웅박사팀에서분양받은 htert fu11 1εngth/ pcdna3과 ptarget/e6m, ptarget/eτ ptarget/ E6E7을사용하였다. 2. 형질전환및세포주확립 SW13 세포를 5 x 10 6 cells/l 00 mm dish에분주한후, 다음날형질전환시켰다. 각각의플라스미드 5 mg에무혈청배지 (serum free media) 를첨가하여 500 ml가되도록하고, Lipofectamine(GIBCO, Grand Island, NY) 30 ml에 475 ml의무혈청배지를첨가하여상온에서 15분간반응시켰다. 15분후 DNA를첨가한배지와 lipofectamine 을첨가한배지를혼합하여상온에서 30분간반응시키고, 이때준비된세포는무혈청배지로 2회세척후, 반응이끝난 DNA-lipofectamine 혼합물에 5 ml의배지를첨가하여총 6 ml을잘혼합한후세척한세포에도말하였다. 37 0 C 5%C02 배양기에서 6시간반응시킨후일반배지로교체하였으며, 24시간이후부터는 0.8 mg/ml의 G418 (Sigma, St Louis, MO) 로약 2주동안형질전환된세포들 2
Korean Soc. Clin. Lab. Sci. 39(1):1-6, 2007 을선별한뒤, 확보된세포들을 96 well plate의각 well 당 0.5~1 개의세포를분주하여단일클론으로선별하였다. 3. 역전사효소-중합효소연쇄반응 (RT-PCR) 하였다. 5. TRAPπelomerase Repeats Amplification Proto 1) assay 다양한세포들에게서 RNAzol B를이용하여 total RNA를추출하였다. 추출한 RNA로부터 cdna를합성하기위하여 hα1l V reverse transcriptase(promega, Madision, WI) 를사용하였다. Total RNA로부터 Poly(A+) mrna를분리하기위하여 oligo dt 0.125 mm을사용하여얻어진 mrna 5 μg, R T buffer 5 μl, 1 mm dntp, 200 unit와역전사효소를혼합하여 37 0 C 에서 1 시간 30분동안반응시켰다. 마지막으로 95 0 C 에서 5분간가열하여역전사효소들을불활성화시킴으로써 cdna를얻어내었다. 확보한 cdna 2 μl와특이적 pnmer 각각 1 pmol, dntp 혼합물 I11L및 Taq polymerase(intron Biotech, Sungnam, Kyungki-Do, Korea) 1 μl를첨가하여연쇄반응을하였으며, 각유전자의특이적 pnmer는다음과같다. htert sense primer(5 -ATG AAG TTC CTG CAC TGG CTG AT-3 ), antisense primer( 5 -AGT TGA GCA CGC TGA ACA GT-3 ), E6 sense primer( 5 -GAA GAT CTC TAT GTT TCA GGA CCA CAG-3), E6 antisense primer(5 ' -TTA CAG CTG GGT TTT CTC T-3); E7 sense primer( 5 -GGA GAT CTC ATG CAT GGA GAT ACA CCT-3 ), antisense primer( 5 -GGG TCG ACG A TT ATG GTT TCT GAG AAC A-3'). PCR이끝난산물을 agarose gel 에서전기영동한후 ethidium bromider 염색을통해확인하였다. Telomerase의활성을측정하기위하여 Intergen 사의 TRAPeze 키트를사용하였다. SW-13/Mock 세포를비롯한확보된세포주들을수확하여 lx PBS로 1 회수세한후, 새 microfuge tube에옮겼다. lx CHAPS lysis buffer(10 mm Tris-HCl, ph 7.5, 1 mm MgCb, 1 mm EGT A, 0.1 mm benzamidine, 5 mm b-mercaptoethanol, 0.5% CHAPS, 10% glycerol) 200 μl를첨가하여얼음에서 30분동안반응시킨후, 12,000 rpm에서 10분동안원섬분리하였다. 용출한 cell extracts를정량한후, TRAP reaction에사용하였다. TS primer(substrate oligonucleotide, in TRAPeze Detection Kit) 의동위원소 end-labeling은 10 mci/ml g-32p-atp, TS primer, 10X kinase buffer, T4 polynucleotide kinasε 를 3rc 에서 20 분동안반응시킨후 85 에서 5분간효소를불활성화시켜사용하였다. 10X TRAP reaction buffer, 50X dntp mix, 32P-TS primer, TRAP primer mix, Taq polymerase를혼합한 master mix를준비하고 RNase-감ee PCR tube 에분주하였으며 0.5~ 1 mg 농도의 cell extracts를첨가하였다. PCR tube를 30 0 C 에서 30분간반응시킨후, 94 0 C 에서 30초, 59 OC 30초반응을 27~40회반복하였다. 반응된각혼합물은 15% neutral-polyacrylamide gel 에서전기영동하고 autoradiography를통해감광하였다. 4. Northern hybridization 다양한세포 (5 x 10 6 cell) 를 Trypsin-EDTA를사용하여모두수확한후, guanidinium 방법을사용하여 total RNA 를추출하였다. 추출한 RNA는 260 nm에서정량한후, 8 mg의 RNA를 1 % formaldehyde gel 에 running 하였다. F A gel을 nylon mεmbrane에 transfer한후, UV에서 cross-linking 하였다. 이 blot을이용하여 htert mrna 발현을확인하였다. 이때사용한 probe는 htert 1950/3210 bp 이며 sequencing을통하여 specificity를검증 111. 결과및고찰 1. HPV16 E6/E7 유전자및 htert 유전자를과발현 하는 SW13 세포주확립본연구에서는 HPV 바이러스 oncogenesis 기작에서의 telomerase의기능을분석하고자다음의세포주틀을확립하였다. 먼저 telomerase subunit 인 pcdna3-htert유전자와 ptarget-e6mutants, ptarget-e6/e7 유전자의 3
대한임상검사과학회지 39(1 ):1-6, 2007 벡터를이용하여 telomerase negative 세포, SW-13 에 liposome을이용한 trans fecti on을하였다. 약 2주동안 G418 에내성을지니는세포들을확보한후클로닝하여 SW/hTERT #31, SW/E6m #29, SW/E6E7 #30의단일클론들을확보하였다. 확보된세포들의 total RNA를이용하여 RT-PCR을수행한결과, 각각의세포가 insert gene '?J HPV oncogene, E6m, E7, E6/E7과 htert 유전자를발현 E6"1I E6"1 ht똥홉x E6 t생 uíant E7 g 짧합융 H 하고있음을확인하였다. 또한이때 HeLa, Caski, SiHa, SW13 모세포주, K562 세포들은각각의음성및양성대조군으로사용되었다. 본연구에사용된 HPV 유전자는 16 형타입이므로 HPV oncogene E6, E7 이양성대조군인 Caski, SiHa에서도나타났으며 HPV 18 형타입인 HeLa에서는나타나지않았다. 또한 K562 세포는 htert의발현이높은양성대조군으로사용되어역시 htert의발현을 A Co 擬 f 빼앵용흉 u 양 나타내였다 (Fig. la). 또한 SW-13/Mock 세포주와비교하여확보된클론들 의세포형태를현미경을통해분석하였다. SW/hTERT 세 포의경우, SW13/Mock 세포와비교하였을때, 세포형태 의변화는거의없었으나세포의크기가작아졌고, 세포의분열시간이조금빨라졌다. 반면에 HPV oncogene들이발현되는 SW13 의경우세포의형태가 Mock 세포에비해많이변하였으며특히 SW/E6E7 의경우는세포가바닥에부착하는정도가심해졌고세포의모양이길쭉하게변했다. SW13/Mock의경우도원래의 SW13 세포에비해모양이낫모양처럼길쭉하게변한세포들이많이형성된특정을알수있었다 (Fig. 18). B Fig. 1. Construction of HPV oncogenes and htert overexpressed SW13 clones. (A) The identification of E6, E7 and htert expression in stable clones of SW13. E6 mutants, E7 and E6/E7 oncogenes and htert expression were detected by RT-PCR. (B) Representative photographs show the cell phenotype in S W /transfectants. 을시사하고있다. 또한 HPV E6/E7 에의해 htert 의발 2. Telomerase의활성과 HPV oncogenesis의관련성확립된세포주들의특성분석및 te10merase의관련성을분석하기위하여 Fig.2 에서나타난것처럼 telomerase 활성및 htert의발현을 TRAP assay와 Northem b10t analysis를이용하여분석하였다. Fig. 2A와같이바이라스발암단백질 E6/E7을도입하였을때, telomerase의활성이뚜렷이증가하였으며, 이는 htert 유전자를발현시킨클론에서보다도더높은활성을보였다. Telomεrase 의활성에있어서 HPV oncogene E6/E7 모두의발현이크게영향을미침을알수있었고, 이결과는 telomεrasε 가 HPV 바이러스의 oncogenesis 과정에밀접하게관련됨 현의증가도확인할수있는지알아보기위하여 Northem b10t analysis를수행하였다 (Fig. 28). 그러나 HPV E6/E7 에의해유도된 htert mrna의발현은확인할수없었다. 이결과는본연구에서사용된세포가 telomerase의활성및 TERT 발현이전혀없는 negative세포이므로그에따른현상일가능성을지니고있거나 HPV 발암단백질이 TERT mrna의전사물발현과관련없이단백질수준에서의조절작용에관여하여 te10merase 의활성을증가시킬가능성도내포하고있다 (Gewin 등, 2001). 따라서본연구에서는 mrna 수준에서의발현의증가는확인할수없었으나, 분명한사실은 E6/E7 발암유전자가 tεlomerase 활성을뚜렷이증가시킨다는것이며, 4
Korean Soc. Clin. Lab. Sci. 39(1):1-6, 2007 A B TRAP products Internal Standard""" '" 폼쳤 * 생쌓홍 4 짧 HE 훌 t + +- +- + + l8 판 괴쏠 * 밸칸 } 활환혔철환繼鐵 물맴릎 18 릎 Fig. 2. Telomerase activity is closely related with human papilloma virus oncogenesis. (A) Telomerase activity was measured by TRAPeze detection kit (Intergen). Total cell lysates wεrε εxtracted with a chaps lysis buffìε r. And O.5mg of cell extracts were used T~ 아 P-PCR based assay. (8) Total RNAs wεre extractεd by guanidinium mεthod in SW13 transfectants. And the Northem blot analysis was performεd using thε htert specific probe. 이는 HPV oncogenesis 에 telomerase 가밀접하게관여할 것임을시사하고있음을확인하였다. 본연구에서는 HPV 발암기전에서의 E6, E7 발암단백 질들과 catalytic subunit(htert) 의상호작용및 telomerase 의기능을분석하기위하여 telomerase negative 세포주인 SW13 에 HPV E6, E7 발암유전자빛 htert 를과발현시 킨세포주들을확립하였다. 기존의 htert promoter 연구에서, HPV E6 단백질이 c-myc 비의존적인상태로도 htert의전사를증가시킬수있음이보고되어 E6, E7 발암단백질과 htert의상호작용이 HPV oncogenesis 에중요할것이라사료된다 (Gewin와 Galloway, 2001). 본연구에서는 E6, E6/E7의두발암단백질이결합된유전자가각각 telomerase의활성을증가시킬수있었으며 ((Fig. 2A), 특히 E6/E7 유전자는 htert 유전자를과발현시킨세포주보다도높은 telomerase 활성을지니고있었다. 따라서 E6/E7 발암단백질이 TERT의발현에도영향을미칠것 (Klingelhutz, 등 1996; Kiyono 등, 1998; Sprague 등 02; Baege 등 2002) 으로사료되어 TERT mrna를 northem blot analysis로확인하였다 (Fig. 2B). 그러나 SWI3/E6/E7 세포에서 htert mrna를확인할수없었으며, 이 telomerase는일차적으로전사수준에서조절되는단백질이지만, 단백질수준에서의조절도많이이루어지므로, E6/E7 발암단백질에의한 TERT 전사발현이외에도 telomerase 의발현에영향을미칠가능성은충분한것으로사료된다. 결론적으로 E6와 E7 발암단백질음 telomerase negative 세포주에과발현시켰을때, telomerase 활성이증가하는것으로보아 telomerasε 의발현에 E6, E7 발암단백질이관여할것임을시사하며, 이는또한 telomerase가 HPV oncogenesls에도관여할수있을것으로사료된다. 참고문헌 1. Baege AC, Berger A, Schlegel R, Veldman T, Schelegel R. Cεrvical epithelial cells transduced with the papillomavirus E6/E7 oncogenes maintain stable levels of oncoprotεin expression but exhibit progressive, m매 or incrεases in htert gεne expression and telomerase activity. Am J Pathol 160: 1251-1257, 2002. 2. Dyson N, Howley PM, Munger K, Harlow E. The human papi110ma virus-16 E7 oncoprotein is able to bind to thε retinoblastoma genε product. Science 243: 934-937, 1989. 3. Gewin L, Galloway DA. E box-dependent activation 5
대한임상검사과학회지 39(1 ):1-6, 2007 of telomerase by human papillomavirus type 16 E6 dose not require induction of c-myc. J γirol 75: 7198-7201, 2001. 4. Halbert CL, Demers GW, Galloway DA. The E6 and E7 genes of human papillomavirus type 6 have weak immortalizing activity in human epithelial cells. J Virol 66: 2125-2134, 1992. 5. Hudson JB, Bedell MA, McCance DJ, Laimins LA. Immortalization and altered differentiation of human keratinocytes in vitro by the E6 and E7 open reading frames of human papillomavirus type 18. J ηrol 64: 519-526, 1990. 6. Huibregtse JM, Scheffner M, Howley PM. A cellular protein rr빼iates association of p53 with the E6 oncoprotein of human papillomavirus types 16 or 18. EMBO J 10:4129-4135, 1991. 7. Kiyono T, Foster SA, Koop 11, McDougall JK, Galloway DA, Klingelhutz A1. Both Rb/p 16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells. Nature 396:84-88, 1998. 8. Klingelhutz AJ, Foster SA, McDougall JK. Telomerase activation by the E6 gene product of human papillomavirus type 16. Nature 380:79-82, 1996. 9. Kubbutat HG, Vousden KH. New HPV E6 binding proteins: Dangerous liaison Trends Microbiol 6: 173-175, 2000. 10. Munger K, Wemess BA, Dyson N, Phelps WC, Harlow E, Howley PM. Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J 8:4099-4105, 1989. 11. Rapp L Chen J1. The papillomavirus E6 proteins. Biochem Biophysics Acta 1378:1-19, 1998. 12. Scheffner M, Huibregtse JM, Vierstra RD, Howley PM. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein liase in the ubiquitination of p53. Cell 75:495-505, 1993. 13. Scheffner M, Wemess BA, Huibregtse JM, Levine AJ, Howley PM. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63:1129-1136, 1990. 14. Sprague DL, Phillips SL, Mitchell CJ, Berger KL, Lace M, Turek LP, Klingelhutz A1. Telomerase activation in cervical keratinocytes containing stably replicating human papillomavirus type 16 episomes. Virology 301:247-254, 2002. 15. Thomas M, Pim D, Banks L. The role of the E6 p53 interaction in the molecular pathogenesis of HPV. Oncogene 18:7690-7700, 1999. 16. Wemess BA, Levine AJ, Howley PM. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 248:76-79, 1990. 17. Woodworth CD, Cheng S, Simpson S, Hamacher L, Chow L T, Dipaolo JA. Recombinant retroviruses encoding human papillomavirus type 18 E6 and E7 genes stimulate proliferation and delay differentiation of human keratinocytes early after infection. 0.ηcogene 7 :619-626, 1992. 18. Zwerschke W, Jansen-Durr P. Cell transformation by the E7 oncoprotein of human papillomavirus type 16: Interactions with nuclear and cytoplasmic targεt proteins. A찌J Cancer Res 78:1-29, 2000. 6