Microsoft Word - 14-김영권.doc

Size: px

Start display at page:

Download "Microsoft Word - 14-김영권.doc"

은진 팽
6 years ago
Views:

1 J. Exp. Biomed. Sci. 12 (2006) Analysis of Telomerase Activity by HPV E6/E7 Expression in SW13 Young-Kwon Kim 1, and Yuk-Pheel Park 2 1 Department of Biomedical Laboratory Science, Konyang University, Daejeon , Korea. 2 Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology, Daejeon , Korea Cervical cancer is one of the most prevalent cancers developed in women worldwide, and human papillomavirus (HPV) type 16 is the most common agent linked to human cerivical carcinoma. Viral oncogenes E6 and E7 are selectively ratined and expressed in carcinoma cells infected with human papillomavirus type 16 and cooperated with each other in immortalization and transformation of primary keratinocytes. Because of HPV oncogenesis mechanism was not completely solved, the more studies be required thoroughly. In the present study, to investigate 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, SW13. Expressions of Inserted genes were measured by RT-PCR. E6, E7 and htert genes were well expressed in each cell lines comparing with the control groups. By analyzing the cell morphology under the microscope, htert clone size was a more smaller than the mock control but oncogene expressed clones were slightly lengthened the marginal region. In addition, htert cells has also, a tendency of brief dividing time compared to the mock control. To determine whether telomerase activity associated with a HPV oncogenesis by oncoprotein expression, we performed the PCR based TRAP assay and Northern blot analysis. In TRAP assay data, telomerase activities in htert and oncogene clones were more increased than the mock control. In addition, SW13/ E6/E7 cells appeared a extremely increased activity than any other clones. Induced TERT mrna by E6/E7 wasn't, however, detected in Northern blotting. In conclusion, these findings suggest that telomerase activity closely associated the HPV oncogenesis and E6/E7 co-expression is a most important factor of telomerase activity. Key Words: Human papillomavirus (HPV), Oncogenes, Telomerase activity 서 자궁경부암은전세계적으로여성에게서나타나는가장흔한질병의하나로써인유두종바이러스 (human papilloma virus) 16형의감염이가장중요한원인으로알려져있다. 이바이러스의감염에는 E6와 E7의발암단백질이감염된자궁경부암세포에서선택적으로유지되고발현되어, primary 각질세포의형질전환및불멸화에관여하는것으로알려져있다 (Hudson et al., 1990; Hallbert et al., 1992; Woodworth et al., 1992). HPV 바이러스의감염시, E6와 E7 발암단백질이종양억제단백질인 (tumor suppressor) p53과 Rb의기능을불활성화시켜세포주기를활성화시킴으로써, 발암과정에관여하는 * 논문접수 : 2006년 11월 2일수정재접수 : 2006년 12월 8일 교신저자 : 김영권, ( 우 ) 대전광역시서구가수원동 685, 건양대학교 Tel: , Fax: ykkim3245@konyang.ac.kr 론 것으로알려져있으며 (Rapp and Chen, 1998; Thomas et al., 1999; Zwerschke et al., 2000; Kubbutat et al., 2000), E7 단백질의경우, prb와결합하여 Rb 단백질의종양억제기능을억제하고 (Dyson et al., 1989; Munger et al., 1989), E6 단백질은 ubiquitin E3 ligase인 E6AP와결합하여 p53을 ubiquitin 의존적인단백질파괴기작으로 p53을파괴시키는것으로보고되고있다 (Scheffiner et al., 1990; Werness et al., 1990; Scheffiner et al., 1993). 최근 10년간의연구결과들에서는 HPV 바이러스의 E6 발암단백질이 htert의전사를증가시키고 telomerase의활성을증가시킨다는결과들이많이도출되어왔다. Telomerase 는정상세포에서는불활성화상태이나다양한암세포, 불멸화된세포들에게서 90% 이상의활성을보여암화과정에밀접한관련이있는단백질로많이주목을받아왔다. HPV 발암단백질이 primary 세포들을불멸화시키는과정에있어서, in vitro 상의 HCK (human cervical keratinocyte), HFK (human foreskin keratinocyte), HMEC (human mammary epithelial cells) 등의다양한세포들에발암단백질이과발현되면, 세포가노화과정

2 을극복하여죽지않는불멸화세포가형성됨을보여주었다 (Klingelhutz et al., 1996; Kiyono et al., 1998; Sprague et al., 2002; Baege et al., 2002). 또한이 primary 세포들이불멸화되는과정에서세포의노화에직접관련된 telomerase의활성이증가되어 telomere를지속적으로합성하게되고, 따라서죽지않는세포가형성되는것이다. 또한 htert promoter 연구에서도, HPV E6 단백질이 c-myc 비-의존적인상태로도 htert의전사를증가시킬수있음이보고되었다 (Gewin et al., 2001). 결론적으로기존연구들의결과에서, telomerase가 HPV 발암기전에관련이있다는것을시사하고있으나기존연구들에서나타난문제점은, telomerase의효과가각연구팀에서이루어진시스템과세포에따라다른경향을보이고있다. 따라서본연구에서는 telomere 비의존적인 TERT subunit 자체의기능을분석하기위한것으로, HPV 발암기전에서의 E6, E7 발암단백질들과 catalytic subunit (TERT) 의상호작용및 telomerase 의기능을분석하고자하였다. 재료및방법 1. 세포배양및발현벡터제작 Telomerage negative 세포주, SW13은 ATCC에서구입하였으며, L-15 Leiboviz (Sigma, St Louis, MO) 배지에 10% FBS (Hyclone, Logan, Utah) 을넣어서 5% CO 2 배양기에서배양하였다. htert, HPV E6m, HPV E7, HPV E6/E7 발현벡터를형질전환시켜만들어진 SW13 세포주들은 DMEM (Sigma, St Louis, MO) 에 10% FBS를첨가한배양액을이용하였다. 각유전자의발현벡터는성균관대의이한웅박사팀에서얻은 htert full length/pcdna3과 ptarget/e6m, ptarget/e7, ptarget/e6e7을사용하였다. 2. 형질전환및세포주확립 SW13 세포를 cells/100 mm dish의분주한후, 다음날형질전환시켰다. 각각의플라스미드 5 mg에무혈청배지 (serum free media) 를첨가하여 500 ml가되도록하고, Lipofectamine (GIBCO, Grand Island, NY) 30 ml에 475 ml의무혈청배지를첨가하여상온에서 15분간반응시켰다. 15분후 DNA를첨가한배지와 Lipofectamine을첨가한배지를혼합하여상온에서 30분간반응시키고, 이때준비된세포는무혈청배지로 2회세척후, 반응이끝난 DNA-Lipofectamine 혼합물에 5 ml의배지를첨가하여총 6 ml을잘혼합한후세척한세포에도말하였다. 37 5% CO 2 배양기에서 6시간반응시킨후일반배지로교체하였으며, 24시간이후부터는 0.8 mg/ml의 G418 (Sigma, St Louis, MO) 로약 2주동안형질전환된세포들을선별한뒤, 확보된세포들을 96 well plate 의각 well당 0.5~1개의세포를분주하여단일클론으로선 별하였다. 3. 역전사효소-중합효소연쇄반응 (RT-PCR) 다양한세포들에게서 RNAzolB를이용하여 total RNA를추출하였다. 추출한 RNA로부터 cdna를합성하기위하여 MMLV reverse transcriptase (Promega, Madision, WI) 를사용하였다. Total RNA로부터 Poly(A+) mrna를분리하기위하여 oligo dt mm을사용하였으며, 얻어진 5 µg의 mrna, 5 µl RT buffer, 1 mm dntp, 200 unit 역전사효소를혼합하여 37 에서 1시간 30분동안반응시켰다. 마지막으로 95 에서 5분간가열하여역전사효소들을불활성화시킴으로서 cdna를얻어내었다. 확보한 cdna 2 ml과각각 1 pmol 의특이적 primer, 1 ml의 dntp 혼합물및 Taq polymerase (intron Biotech, Sungnam, Kyungki-Do, Korea) 를첨가하여연쇄반응을하였으며, 각유전자의특이적 primer는다음과같다. 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 ATT ATG GTT TCT GAG AAC A-3'). PCR이끝난산물을 agarose gel에서전기영동한후 EtBr 염색을통해확인하였다. 4. Northern hybridization 다양한세포 ( cell) 를 Trypsin-EDTA 를사용하여모두수확한후, guanidinium 방법을사용하여 total RNA를추출하였다. 추출한 RNA는 260 nm에서정량한후, 8 mg의 RNA를 1% formaldehyde gel에 running하였다. RNA gel을 nylon membrane에 transfer한후, UV에서 cross-linking하였다. 이 blot을이용하여 htert mrna 발현을확인하였다. 이때사용한 probe는 htert 1950/3210 bp이며 sequencing을통하여 specificity를검증하였다. 5. TRAP (Telomerase Repeats Amplification Protocol) assay Telomerase 의활성을측정하기위하여 Intergen사의 TRAPeze kit를사용하였다. SW-13/Mock 세포를비롯한확보된세포주들을수확하여 1X PBS로 1회수세한후, 새 microfuge tube에옮겼다. 1X CHAPS lysis buffer (10 mm Tris-HCl, ph 7.5, 1 mm MgCl 2, 1 mm EGTA, 0.1 mm benzamidine, 5 mm b-mercaptoethanol, 0.5% CHAPS, 10% glycerol) 200 µl를첨가하여 ice에서 30분동안반응시킨후, 12,000 rpm에서 10분동안원심분리하였다. 용출한 cell extracts 를정량한후, TRAP reaction에사용하였다. TS primer (Substrate oligonucleotide, in

TRAPeze Detection Kit) 의동위원소 end-labeling은 10 mci/ml g- 32 P-ATP, TS primer, 10X kinase buffer, T4 polynucleotide kinase 를 37 에서 20분동안반응시킨후 85 에서 5분간효소를불활성화시켜사용하였다.

PCR tube를 30 에서 30분간반응시킨후, 94 에서 30초, 59 30초반응을 27~40회반복하였다. 반응된각혼합물은 15% neutral-polyacrylamide gel에서전기영동하고 autoradiography 를통해감광하였다. 결과및고찰 1.

먼저 telomerase subunit인 pcdna3-htert 유전자와 ptarget-e6mutants, ptarget-e6/e7 유전자의벡터를이용하여 telomerase negative 세포, SW-13에 liposome을이용한 transfection을하였다.

또한이때 HeLa, Caski, SiHa, SW13 모세포주, K562 세포들은각각의음성및양성대조군으로사용되었다. 본연구에사용된 HPV 유전자는 16형타입이므로 HPV oncogene E6, E7이양성대조군인 Caski, SiHa에서도나타났으며 HPV 18형타입인 HeLa 에서는나타나지않았다.

3 TRAPeze Detection Kit) 의동위원소 end-labeling은 10 mci/ml g- 32 P-ATP, TS primer, 10X kinase buffer, T4 polynucleotide kinase 를 37 에서 20분동안반응시킨후 85 에서 5분간효소를불활성화시켜사용하였다. 10X TRAP reaction buffer, 50X dntp mix, 32 P-TS primer, TRAP primer mix, Taq polymerase를혼합한 Master mix를준비하고 RNase-free PCR tube에분주하였으며 0.5~1 mg 농도의 cell extracts를첨가하였다. PCR tube를 30 에서 30분간반응시킨후, 94 에서 30초, 59 30초반응을 27~40회반복하였다. 반응된각혼합물은 15% neutral-polyacrylamide gel에서전기영동하고 autoradiography 를통해감광하였다. 결과및고찰 1. HPV16 E6/E7 유전자및 htert 유전자를과발현하는 SW13 세포주확립본연구에서는 HPV 바이러스 oncogenesis 기작에서의 telomerase 의기능을분석하고자다음의세포주들을확립하 였다. 먼저 telomerase subunit인 pcdna3-htert 유전자와 ptarget-e6mutants, ptarget-e6/e7 유전자의벡터를이용하여 telomerase negative 세포, SW-13에 liposome을이용한 transfection을하였다. 약 2주동안 G418에내성을지니는세포들을확보한후클로닝하여 SW/hTERT #31, SW/E6m #29, SW/E6E7 #30의단일클론들을확보하였다. 확보된세포들의 total RNA를이용하여 RT-PCR 을수행한결과, 각각의세포가 insert gene인 HPV oncogene, E6m, E7, E6/E7과 htert 유전자를발현하고있음을확인하였다. 또한이때 HeLa, Caski, SiHa, SW13 모세포주, K562 세포들은각각의음성및양성대조군으로사용되었다. 본연구에사용된 HPV 유전자는 16형타입이므로 HPV oncogene E6, E7이양성대조군인 Caski, SiHa에서도나타났으며 HPV 18형타입인 HeLa 에서는나타나지않았다. 또한 K562 세포는 htert의발현이높은양성대조군으로사용되어역시 htert의발현을나 A A B 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 SW/ transfectants. 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 were extracted with a chaps lysis buffer. And 0.5 mg of cell extracts were used TRAP-PCR based assay. (B) Total RNAs were extracted by guanidinium method in SW13 transfectants. And the Northern blot analysis was performed using the htert specific probe

4 타내었다 (Fig. 1A). 또한 SW-13/Mock 세포주와비교하여확보된클론들의세포형태를현미경을통해분석하였다. SW/hTERT 세포의경우, SW13/Mock 세포와비교하였을때, 세포형태의변화는거의없었으나세포의크기가작아졌고, 세포의분열시간이조금빨라졌다. 반면에 HPV oncogene들이발현되는 SW13 의경우세포의형태가 Mock 세포에비해많이변하였으며특히 SW/E6E7의경우는세포가바닥에부착하는정도가심해졌고세포의모양이길쭉하게변했다. SW13/Mock의경우도원래의 SW13 세포에비해모양이낫모양처럼길쭉하게변한세포들이많이형성된특징을보임을알수있었다 (Fig. 1B). 2. Telomerase 의활성과 HPV oncogenesis 의관련성확립된세포주들의특성분석및 telomerase 의관련성을분석하기위하여 Fig. 2에서나타난것처럼 telomerase 활성및 htert의발현을 TRAP assay와 Northern blot analysis를이용하여분석하였다. Fig. 2A와같이바이러스발암단백질 E6/ E7을도입하였을때, telomerase의활성이뚜렷이증가하였으며, 이는 htert 유전자를발현시킨클론에서보다도더높은활성을보였다. Telomerase의활성에있어서 HPV oncogene E6/E7 모두의발현이크게영향을미침을알수있었고, 이결과는 telomerase가 HPV 바이러스의 oncogenesis 과정에밀접하게관련됨을시사하고있다. 또한 HPV E6/E7에의해 htert의발현의증가도확인할수있는지알아보기위하여 Northern blot analysis를수행하였다 (Fig. 2B). 그러나 HPV E6/E7에의해유도된 htertmrna의발현은확인할수없었다. 이결과는본연구에서사용된세포가 telomerase의활성및 TERT 발현이전혀없는 negative 세포이므로그에따른현상일가능성을지니고있거나 HPV 발암단백질이 TERT mrna의전사물발현과관련없이단백질수준에서의조절작용에관여하여 telomerase의활성을증가시켰을가능성을내포하고있다 (Gewin et al., 2001). 따라서본연구에서는 mrna 수준에서의발현의증가는확인할수없었으나, 분명한사실은 E6/E7 발암유전자가 telomerase 활성을뚜렷이증가시킨다는것이며, 이는 HPV oncogenesis에 telomerase 가밀접하게관여할것임을시사하고있음을확인하였다. 본연구에서는 HPV 발암기전에서의 E6, E7 발암단백질들과 catalytic subunit (TERT) 의상호작용및 telomerase의기능을분석하기위하여 telomerase negative 세포주인 SW13에 HPV E6, E7 발암유전자및 htert를과발현시킨세포주들을확립하였다. 기존의 htert promoter 연구에서, HPV E6 단백질이 c-myc 비-의존적인상태로도 htert의전사를증가시킬수있음이보고되어 E6, E7 발암단백질과 TERT 의상호작용이 HPV oncogenesis에중요할것으로사료된다 (Gewin and Galloway, 2001). 본연구에서는 E6, E6/E7의두발암단백질이결합된유전자가각각 telomerase의활성을증가시킬수있었으며 (Fig. 2A), 특히 E6/E7 유전자는 htert 유전자를과발현시킨세포주보다도높은 telomerase 활성을지니고있었다. 따라서 E6/E7 발암단백질이 TERT의발현에도영향을미칠것 (Klingelhutz et al., 1996; Kiyono et al., 1998; Sprague et al., 2002; Baege et al., 2002) 으로사료되어 TERT mrna를 Northern blot analysis로확인하였다 (Fig. 2B). 그러나 SW13/E6/E7 세포에서 htert mrna를확인할수없었으며, 이는 telomerase는일차적으로전사수준에서조절되는단백질이지만, 단백질수준에서의조절도많이이루어지므로, E6/E7 발암단백질에의한 TERT 전사발현이외에도 telomerase의발현에영향을미칠가능성은충분한것으로사료된다. 결론적으로 E6 와 E7 발암단백질을 telomerase negative 세포주에과발현시켰을때, telomerase 활성이증가하는것으로보아 telomerase 의발현에 E6, E7 발암단백질이관여할것임을시사하며, 이는또한 telomerase가 HPV oncogenesis에도관여할수있을것으로사료된다. REFERENCES Baege AC, Berger A, Schlegel R, Veldman T, Schelegel R. Cervical epithelial cells transduced with the papillomavirus E6/E7 oncogenes maintain stable levels of oncoprotein expression but exhibit progressive, major increases in htert gene expression and telomerase activity. Am J Pathol : Dyson N, Howley PM, Munger, K, Harlow E. The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science : Gewin L, Galloway DA. E box-dependent activation of telomerase by human papillomavirus type 16 E6 dose not require induction of c-myc. J Virol : 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 : 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 Virol : Huibregtse JM, Scheffner M, Howley PM. A cellular protein mediates association of p53 with the E6 oncoprotein of human papillomavirus types 16 or 18. EMBO J :

5 Kiyono T, Foster SA, Koop JI, McDougall JK, Galloway DA, Klingelhutz AJ. Both Rb/p16 INK4a inactivation and telomerase activity are required to immortalize human epithelial cells. Nature : Klingelhutz AJ, Foster SA, McDougall JK. Telomerase activation by the E6 gene product of human papillomavirus type 16. Nature : Kubbutat HG, Vousden KH. New HPV E6 binding proteins: Dangerous liaisons? Trends Microbiol : Munger K, Werness 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 : Rapp L, Chen JJ. The papillomavirus E6 proteins. Biochem Biophysics Acta : Scheffner M, Huibregtse JM, Vierstra RD, Howley PM. The HPV-16 E6 and E6-AP complex functions as a ubiquitinprotein liase in the ubiquitination of p53. Cell : Scheffner M, Werness BA, Huibregtse JM, Levine AJ, Howley PM. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell : Sprague DL, Phillips SL, Mitchell CJ, Berger KL, Lace M, Turek LP, Klingelhutz AJ. Telomerase activation in cervical keratinocytes containing stably replicating human papillomavirus type 16 episomes. Virology : Thomas M, Pim D, Banks L. The role of the E6 p53 interaction in the molecular pathogenesis of HPV. Oncogene : Werness BA, Levine AJ, Howley PM. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science : Woodworth CD, Cheng S, Simpson S, Hamacher L, Chow LT, Dipaolo JA. Recombinant retroviruses encoding human papillomavirus type 18 E6 and E7 genes stimulate proliferation and delay differentiation of human keratinocytes early after infection. Oncogene : Zwerschke W, Jansen-Durr P. Cell transformation by the E7 oncoprotein of human papillomavirus type 16: Interactions with nuclear and cytoplasmic target proteins. Adv Cancer Res :

대한임상검사과학회지 39(1):1-6, 2007 으로알려져있다. 이바이러스의감염에는 E6와 E7 의발암단백질이감염된자궁경부암세포에서선택적으로유지되고발현되어, pnmaη 각질세포의형질전환및불멸화에관여하는것으로알려져있다 (Hudson 등, 1990; Halbert 등, 1

대한임상검사과학회지 39(1):1-6, 2007 으로알려져있다. 이바이러스의감염에는 E6와 E7 의발암단백질이감염된자궁경부암세포에서선택적으로유지되고발현되어, pnmaη 각질세포의형질전환및불멸화에관여하는것으로알려져있다 (Hudson 등, 1990; Halbert 등, 1 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