장액성난소암세포주의확립및특성분석 연세대학교대학원 의학과 채두병
장액성난소암세포주의확립및특성분석 지도교수김재훈 이논문을석사학위논문으로제출함 2008 년 6 월 연세대학교대학원 의학과 채두병
채두병의석사학위논문을인준함 심사위원 인 심사위원 인 심사위원 인 연세대학교대학원 2008 년 6 월
감사의글 본논문을완성함에있어서많은조언과지도를해주신김재훈교수님께진심으로감사를드립니다. 또한김동욱교수님과백용한교수님의지도와자문에깊은감사를드립니다. 아울러본인이산부인과전문의의길을갈수있도록어려운일이있을때마다격려와가르침을주신연세의대산부인과학교실교수님들께또한감사의말씀을올립니다. 마지막으로지금까지의사로성장할수있도록키워주신부모님과사랑하는아내신연정과아들채희건에게이논문을바칩니다. 저자씀
< 차례 > 국문요약 1 I. 서론 3 II. 재료및방법 5 1. 환자병력 5 2. 세포주의확립및배양조건 6 3. 형태학적관찰 6 4. 세포주의 Mycoplasma 감염측정 7 5. 성장속도측정 7 6. 종양표지자분비능검사 8 7. 인간백혈구항원 (HLA) 분석 8 8. 종양억제유전자의변이분석 9 9. 항암화학약물감수성검사 10 10. 종양형성능력측정 10 11. cdna microarray를이용한유전자발현연구 11 12. 2-DE 및 MALDI-TOF MS를이용한단백질발현연구 12 III. 결과 14 IV. 고찰 26 V. 결론 32 참고문헌 32 영문요약 37
그림차례 Figure 1. Phase-contrast microscopy of YDOV-139 cell line 15 Figure 2. Transmission electron microscopy of YDOV-139 cell line 15 Figure 3. The growth curve of YDOV-139 cell line 16 Figure 4. Chemosensitivity test of YDOV-139 cell line 19 Figure 5. Xenograft of YDOV-139 cells to nude mice 20 Figure 6. Hierarchical clustering of YDOV-139 cell line gene expression 21 Figure 7. Proteomic comparison between YDOV-139 cell line and HOSE using 2-DE 25
표차례 Table 1. BRCA2 mutation 18 Table 2. Selected groups of upregulated genes expressed at least 10 folds higher in YDOV-139 compared with HOSE 22 Table 3. Selected groups of downregulated genes expressed at least 10 folds higher in HOSE compared with YDOV-139 23 Table 4. Identification of proteins that were differentially expressed in YDOV-139 and HOSE using 2-DE and MALDI-TOF MS 25
< 국문요약 > 장액성난소암세포주의확립및특성분석 목적 : 암세포주는생체외 (in vitro) 실험및생체내 (in vivo) 실험을가능하게하는중요한암연구수단이다. 특히불균질성과복잡성을특징으로하는난소암을연구하기위해서는다양한난소암세포주의확보가중요하다. 최근 YDOV-139로명명된세포주를확립하고특성을분석하였다. 연구방법 : 67세장액성난소암환자의복수에서세포를분리하여 DMEM 배양액으로세포주 (YDOV-139) 를확립하였으며현재지속적계대배양중이다. 위상차현미경및전자현미경에의한세포주의형태학적관찰, 성장속도측정, 종양표지자분비능검사, 인간백혈구항원분석, p53과 BRCA1,2들의유전자변이분석, 이종이식을통한종양형성능력관찰, 항암제감수성검사, 유전자및단백질발현연구등을시행하였다. 결과 : 세포들은세포간의접촉에의한억제현상없이부착성단층으로성장하였으며위상차현미경소견상세포들은방추형, 타원형혹은다각형의형태를보였다. 세포주의배가시간은 120시간이었다. 난소암종양표지자분비능 1
검사에서 CA 125, CA 19-9는정상범위보다증가하였고 CEA, CA 15-3 는증가하지않았거나큰차이를보이지않았다. 유전자변이분석에서 BRCA1의변이는없었으나 BRCA2는다양한 polymorphism 과 missense mutation 이발견되었고, p53 유전자의변이는발견되지않았다. 또한이종이식에의한종양형성이관찰되었고, 항암제감수성검사상 gemcitabine이가장높은 chemosensitivity index를보였다. cdna microarray를이용한유전자발현연구에의하면정상난소상피세포에비해 2520개의유전자가통계학적으로유의한발현차이를보였으며, 이차원젤전기영동을이용한단백질발현연구에서도정상난소상피세포와비교하여발현차이를보인 23개의단백질을확인하였다. 결론 : 본연구는성공적으로확립된장액성난소암세포주를소개하고그분자생물학적특성을분석하였다. 확립된세포주의특성은난소암의병리기전연구, 세포기능연구, 항암제감수성연구그리고암에대한새로운치료모델개발에유용한가치를제공할수있을것이다. ------------------------------------------------------- 핵심되는말 : 난소암, 세포주특성화, 유전자발현, 단백질학 2
장액성난소암세포주의확립및특성분석 < 지도교수김재훈 > 연세대학교대학원의학과 채두병 I. 서론 근래의우리나라여성암발병빈도의변화는유방암과난소암의증가로요약할수있다. 서구형암으로알려진난소암의증가는주요부인암이자궁경부암에서난소암으로자리이동함을뜻한다. 1 난소암은부인암중사망률이가장높은치명적인암이다. 질병초기에특징적인증상이없고효과적인집단검진법 (screening) 이없어조기진단이쉽지않기때문이다. 따라서대부분의환자들은 III기이상의진행된상태에서발견된다. 광범위한수술과새로운약물치료로완치된환자가늘어날것으로기대했지만수십년동안의통계는난소암의생존률변화가거의없음을보고하고있다. 2 2007년도미국통계청 (American Cancer Society; www.cancer.org) 3
자료에의하면난소암은미국여성암 (female cancer) 중발생률 8위이고사망률 4위이다. 매년 2만3천여명의신환과 1만6천여명의사망환자가발생하고있다. 우리나라의경우 2005년도한국중앙암등록본부자료에의하면여성암중발생률 9위로연평균 1,300 여명이새롭게진단받고있고사망률 9위이며매년 800 여명이사망하고있다. 3 난소암연구가쉽지않은이유는난소암특유의불균질성 (heterogeneity) 과복잡성 (complexity) 때문이다. 같은아형의난소암환자에서도개체마다다른유전적변이와암관련단백질의다양한발현이관찰된다. 이러한이유로난소암을대표하는고유의질환모델을만들기는매우어렵다. 따라서다양한특징을지닌난소암세포주를많이확보하여야만난소암의다양성과복잡성을극복하여효과적인연구를진행할수있을것이다. 세포주는생체외 (in vitro) 실험및동물을이용한생체내 (in vivo) 실험을가능하게하여, 질병발현에영향을주는생물학및유전학적요소를이해하고질병의합리적인연구계획을개발하는근거로써유용하다. 종양학분야에서는암발생기전에대한연구뿐만아니라암의진단과감시에사용하는단세포군항체의생산및항암제감수성검사에이용하여왔으며, 최근에는암관련유전자윤곽연구 (profiling study) 및암표지자선별연구에주로사용하고있다. 4-7 이에최근재발을반복하지만항암화학요법에민감하게반응하는 4
67세장액성난소암환자의복수에서세포주 (YDOV-139) 를확립하고세포주의특성을연구하기위해위상차현미경및전자현미경에의한형태학적관찰, 성장속도측정, 종양표지자분비능검사, 인간백혈구항원 (HLA) 분석, p53과 BRCA1,2들의유전자변이분석, 종양형성능력관찰, 항암제감수성검사, 유전자및단백질발현연구를분석하였다. II. 재료및방법 1. 환자병력 세포주는 2005년 5월재발성난소암으로진단받은 67세여자환자의복수에서유래되었다. 환자는 2006년 4월난소암의심하에병기결정개복술로양측난소난관절제술, 대망절제술및다중골반내생검을시행하였으며조직검사상난소의장액성난소암으로진단받았고 FIGO 병기상 IIIc였다. 수술후 paclitaxel과 carboplatin으로총 6차례보조항암화학요법시행받으며 2005년 5월재발성난소암진단하에 belotecan에의한총 9회항암화학요법, 2006년 1월부터 paclitaxel과 cisplatin에의한 6회항암화학요법, 2006년 10월부터는 docetaxel과 cisplatin에의한 6회항암화학요법 2007년 9월부터 gemcitabine과 cisplatin에의한 6회항암화학요법을시행받았으며관해후현재까지외래추적관찰중이다. 5
2. 세포주의확립및배양조건 채취한복수 (Ascites) 를 ficoll(sigma, St. Louis, MO, USA) 을이용하여 18 에서 1500 rpm, 35분원심분리한후암세포를분리하였으며, Phosphate buffered saline(pbs) 로 2회세척한후다시원심분리하였다. 10% Fetal bovine serum(fbs), antimyotics가함유된 DMEM 배양액 (Gibco, Grand island, NY, USA) 에세포를부유시킨후, 조직배양접시에옮겨 37, 5% CO 2 세포배양기에서배양하였다. 배양된세포들이배양접시바닥에 75% 정도덮였을때 PBS로 2회세척을한뒤, 1 mm EDTA가함유된 0.25% trypsin으로처리하여계대배양하였으며현재까지 80회이상진행중이다. 3. 형태학적관찰 배양된세포를 PBS로세척한후위상차현미경 (Olympus, Toko, Japan) 으로 40배, 100배에서관찰하였다. 또한전자현미경을이용한미세구조관찰을위해배양된세포를 0.1 M PBS(pH7.4) 로완충된 2% glutaraldehyde 와 paraformaldehyde로전고정 (prefix) 및 phosphate buffered 1.33% osminum tetroxide(oso 4 ) 로후고정 (postfix) 한후 alcohol로탈수과정을거처 propylene oxide로치환하였다. Epon 혼합액 (EPON812, MNA, DDSA, DMP30) 에포매 (embedding) 한후초박절편을만들어 Uranyl acetate와 lead citrate로이중염색하여 Philips CM 10 transmission electron microscopy (TEM; Philips Scientifics, 6
Eindhoven, The Netherlands) 을이용하여미세구조를관찰하였다. 4. 세포주의 Mycoplasma 감염측정 계대배양한세포주의 Mycoplasma 감염여부를측정하기위해 Mycoplasma detection kit(e-myco TM mycoplasma PCR detection kit; intron Biotechnology Inc., Seongnam, Korea) 를이용하였다. 약 100개의세포로부터얻은 DNA templet과 2.5 U i-taq DNA polymerase, 250 mm dntps, 10 mm Tris-Hcl(pH 8.3), 50 mm KCl, 1.5 mm MgCl 2, 10 pmol primers, loading buffer, chemical stabilizer I 를포함하는 e-myco TM PCR 통합 용액을혼합하여 20 μl로맞춘후가열방법으로 templet을추출하였으며 initial denaturation을위해 94 에서 1분, cycle denaturation을위해 94 에서 30초, cycle annealing을위해 60 에서 20초, cycle extension을위해 72 에서 1분그리고 final extension을위해 72 에서 5분간의조건으로 35 주기의중합효소연쇄반응 (PCR) 을실시하였다. 1.2% agarose gel에서 30분간전기영동한후 ethinum bromide로염색하여 Mycoplasma DNA의증폭여부를확인하여감염여부를검사하였고 3회이상반복검사하였다. 5. 성장속도측정 50 회계대배양후 Cell Titer 96 Aqueous one Solution cell proliferation Assay kit(promega, Madison, WI, USA) 을이용하여성장 7
속도및성장곡선을측정하였다. 96 well plate에 3 x 10 3 의세포를 100 μl의배지에잘섞어서분주한후첫 4일간은매일, 그후 10일까지이틀간격으로측정하였다. 측정시 20 μl의 MTS solution을 96 well plate 의각 well 에넣고, 37 배양기에서 4시간동안배양한후 490 nm에서 ELISA reader(el311, Bio-tek Instruments, NY, USA) 기를이용하여 OD값을측정하였다. 이후성장곡선으로부터배가시간 (doubling time) 을구하였으며모든검사는 3회반복측정하였다. 6. 종양표지자분비능검사 종양세포 1.5 10 6 개를 4일간배양시킨후불순물을제거한배양상청액에서난소종양표지자인 CA 125, CA 19-9, CA 15-3 그리고 CEA의분비여부를면역효소분석법으로 Architect system (Abbott Diagnostics, Abbott park, IL, USA) 에서측정하였다. 정상대조군은세포가없는배양상청액을이용하였으며모든검사를 2회반복시행하였다. 7. 인간백혈구항원 (human leukocyte antigen, HLA) 분석 HLA Class I ABC 는 PCR 후 sequencing 반응에의해염기서열을분석하는 allele specific PCR-SBT(sequencing based typing) 방법으로검사하였으며, Class II DR은 PCR 후 sequence specific oligonucleotide probe를이용한 southern blot하는 allele specific PCR-SSOP(sequence 8
specific oligonucleotide probe) (INNO-Lipa HLA DRB1 plus, Innogenetics, Belgium) 방법을사용하였다. 8. 종양억제유전자의변이분석 배양세포주에서종양억제유전자인 BRCA1, BRCA2 그리고 p53의 exon 4의변이여부를검사하였다. BRCA1, BRCA2는 PCR- DHPLC(PCR based denaturing high performance liquid chromatography) 방법으로분석하였으며이는세포주에서 QIAamp DNA Blood Mini kit (QIAGEN, Valencia, CA, USA) 를이용하여 DNA를적출후 2 μl DNA templete ( 약 50.100 ng/l), 10 pmol 의각 primer, 200 µm dntps, 표준 PCR 용액 (1.5 mm MgCl 2 ), 그리고 5U Taq DNA polymerase의혼합액 25 μl로 81개의 primer pair에대한중합효소연쇄반응 (PCR) 을실시하였다 (Takara Bio Inc., Kyoto, Japan). Thermal cycling은 GeneAmp 2700 thermocycler(applied Biosystems Inc., Foster City, CA, USA) 를 이용하였고, WAVE Maker System (Transgenomic Inc., San Jose, CA, USA) 을이용하여 DHPLC 분석을시행하였다. 중합효소연쇄반응 (PCR) 에의한 BRCA1, BRCA2의증폭체 (amplicons) 의적출및쌍방향 sequencing은 BigDyeDeoxy terminator cycle sequencing kit(applied Biosystems Inc., Foster City, CA, USA) 을이용하고, Sequencing 반응을 ABI 3100 Genetic Analyzer(Applied Biosystems Inc., Foster City, CA, USA) 으로분석하였다. p53 exon4 변이는 DNA 추출물의 PCR-RFPL(Restriction Fragment 9
Length Polymorphism) 을확인하는방법으로조사하였다. 9. 항암화학약물감수성검사 세포주를 96 well plate에 3일간배양한후예비실험을통해각항암제별로실험군과대조군에서세포의성장속도차이가명확한농도를 PPC(Peak Plasma Concentration) 로결정하였다. 실험에는상용항암화학약물인 bleomycin, carboplatin, cisplatin, docetaxel, doxorubicin, etoposide, 5-FU(fluorouracil), gemcitabine, irinotecan, oxaliplatin, paclitaxel, topotecan의 12종을사용하였으며, 예비실험을통해얻은 PPC를기본으로 0.2 x, 1 x, 5 x에서항암화학약물감수성검사를시행하였고대조군과비교한실험군의 ATP(adenosine triphosphate) 양을측정하여세포의사멸정도를측정하였다. Chemosensitivity Index(CI) 는 300 SUM (% cell suppression 0.2 x - 5 x) 로계산하였다. 10. 종양형성능력측정 3 마리의 BALB/c nu/nu 누드마우스 (Orient Bio Inc., Seongnam, Korea) 의왼쪽등에 5 X 10 6 개의종양세포를함유한 PBS 200 μl를 피하접종한후종양형성능력및성장을관찰하였다. 10
11. cdna microarray를이용한유전자발현연구 유전자발현은 cdna microarray 기술을이용하여관찰하였다. Trizol Reagent(Invitrogen Life Technologies, Carlsbad, CA, USA) 을이용하여세포에서 RNA를추출하였고 Illumina Amplification Kit(Ambion, Inc.,San Diego, CA, USA) 을이용하여 Biotynylated crna를만든후 RNeasy kit(qiagen, Valencia, CA, USA) 를이용해정제한 crna를 ND-1000 Spectrophotometer(NanoDrop, Wilmington, DE, USA) 로정량화하였다. 750 ng의 crna 표본을 Sentrix Human Ref-6-V2 Expression Bead Chips(Illumina, Inc., San Diego, CA, USA) 에 hybridization 시킨후배열신호 (array signal) 를 Amersham fluorolink streptavidin- Cy3(GE Healthcare Bio-Sciences, Little Chalfont, UK) 로감지하였다. Illumina BeadArray Reader를이용하여스캔한후 Illumina BeadStudio 프로그램으로분석하였다. 26827개의 probes가분석되었으며선별된 probes에대해실험에서발생하는시스템적변동 (systematic variation) 을줄이기위해 Quantile Normalization 방법으로보정했다. 두그룹즉 HOSE(human ovarian surface epithelial cell) 와 YDOV-139간의발현차이를살펴보기위해 one-way analysis of variance(anova) 및 LPE-test를이용하여분석하였다. 또한다수의유전자들을한꺼번에검정하면서생길수있는 multiple test을피하기위해 Benjamini-Hochberg FDR(False Discovery Rate) 으로 p-value값을보정했다. 11
본연구에서는 ANOVA p-value < 0.05 이며, Fold (YDOV-139/HOSE) > 2 값을만족하는 2520개의 probes를 Hierarchical cluster(euclidean method, complete linkage) 를통해관찰하였으며모든통계적분석은 Avadis Prophetic version.3.3(strand Genomics, Bangalore, India) 으로하였다. 12. 2-DE 및 MALDI-TOF MS를이용한단백질발현연구 단백질발현및동정은 2-DE(2-Dimentional gel electrophoresis) 과 MALDI-TOF(Matrix assisted laser desorption ionization Time of flight) MS (Mass spectrometry) 을이용하였다. 배양된세포에서단백질을추출하여 2D Quant Kit(Amersham Biosciences Co., Piscataway, NJ, USA) 를이용하여정량화하였으며 20 에 24시간동안 IPG gel strips (Amersham Biosciences Co., Piscataway, NJ, USA) 를재수화하고, 13 cm immobilized nonlinear ph 3-10 NL strip에단백질 100 μg을 Isoelectric focusing(ief) 하였다. IEF는 Ettan IPGphor II(Amersham Biosciences Co., Piscataway, NJ, USA) 를이용하여 20 에서 50 V, 500 V, 1000 V 그리고 8000 V에총 60 kvhrs로시행되었다. IEF 후각 strip들은 6 M Urea, 75 mm Tris-HCl, 29.3% Glycerol, 2% SDS이함유된완충액및 1% DTT에 15분간, 그후 15분간은 2.5% iodaacetamide를넣어평형화시켰다. 10% SDS gel에평형화된 IPG gel strip을넣고 0.5% agarose 용매로덮은후전기영동한후 silver staining하였으며, ImageScanner, ImageMaster 4.01 software(amersham Biosciences Co., 12
Piscataway, NJ, USA) 로스캔및분석하였다. 정상대조군으로 HOSE를사용하였다. 질량분석을위해단백질들은 modified porcine trypsin을이용하여작은단편으로효소적으로분해하였다. 젤조각으로부터 SDS, 유기용매, 염색시약등의불순물을제거하기위하여 50% acetonitrile로세척한후, trypsin(8-10 ng/ μl ) 으로 reswelling하고 8-10시간동안 37 에서 incubation하였으며단백질분해반응은 5 μl 0.5% trifluoroacetic acid의첨가에의해종결되었다. Trypsin에의해잘려진단백질단편들은수용액상태로회수되었고, C18ZipTips(Millipore) 을이용하여 1-5 μl부피로탈염및농축되었으며, 동량의 50% aqueous acetonitrile에포화된 α-cyano-4-hydroxycinnamic acid와혼합되어, 질량분석을위하여 target plate 위에적하되었다. 단백질동정 (MALDI-TOF analysis and database search) 은질량분석기 Ettan MALDI-TOF(Amersham Biosciences Co., Piscataway, NJ, USA) 를사용하였다. Target plate 상에적하되어있는단백질단편들은 337 nm 의 N 2 laser 조사에의해기화된다음, 20 Kv injection pulse에의해가속되었고 300 laser shots의누적 peaks에의해각단백질 spot에대한 mass spectrum을구하였다. Mass spectrum의분석을위해서 trypsin의자가분해에의해생성된펩타이드의 ion peak m/z(842.510, 2211.1046) 를표준 peaks로이용하였으며분석이완료된 mass spectrum으로부터단백질동정을위하여 Rockefeller 대학에서개발한 ProFound 검색엔진 (http://129.85.19.192/profound_bin/webprofound.exe) 13
을이용하였다. III. 결과 1. 세포주의확립 세포배양 5일후부터세포성장이관찰되었으며, 1 X 10 5 / cm 2 농도에서는세포간의접촉에의한억제현상없이세포간에쌓이는부착성단층으로성장하는모습을관찰할수있었다. 세포파종 2주후첫계대가이루어졌으며, 이후계대부터는 1주에 1회로세포성장이빨라지고연속적계대가유지되었다. 확립된세포주를 YDOV-139로지정하였으며, 현재 80회이상계대배양중이다. 2. 형태학적특성 위상차현미경소견상세포들은비교적크고둥근핵을가진방추형, 타원형혹은다각형의형태를보였다. 또한핵 (nucleus) 안에작은인 (nucleolus) 이다수관찰되었다 (Fig. 1). 전자현미경소견상장액성난소암의특징인세포질에비해큰핵, 세포질내풍부한당원, 미토콘드리아및 secretory vesicles, 그리고세포표면의풍부한미세융모가관찰되었다. 15000 배율에서는세포질내에서풍부한당원및미토콘드리아와함께 desmosomal-tonofilament 복합체도관찰되었다 (Fig. 2). 14
A Figure 1. B Phase-contrast microscopy of YDOV-139 cell line (A, x 40; B, x 100) Polygonal appearance with large and round nucleus. A Figure 2. B Transmission electron microscopy of YDOV-139 cell line. A : Magnification x 6,000 The tumor cell reveal high N/C ratio. The tumor cell shows abundant, short and blunt mcirovilli in the cell surface(red arrow) and abundant mitochondria, glycogen and amorphous annulated lamellae, possible secretory vesicles (yellow arrowhead) in the cytoplasm. B : Magnification x 15,000 Higher magnification shows desmosomal-tonofilament complexs(red arrow), abundant mitochondria and glycogen (yellow arrowhead). 15
3. 세포주의 Mycoplasma 감염측정 배양중 Mycoplasma 감염은발견되지않았다. 4. 성장속도 세포주의성장은 10% 우태아혈청 (FBS) 에서지수성장곡선을보였고, 세포증식의평균배가시간은 120 시간이었으며 95% 이상의생존력을 보였다 (Fig. 3). 350 300 FBS 0% FBS 10% 250 % of control 200 150 100 50 0 0 1 2 3 4 6 8 10 day Figure 3. The growth curve of YDOV-139 cell line. Cells were cultured under the conditions of 5% CO 2 incubator at 37 C. 16
5. 종양표지자분비능검사 세포주의배양상청액에서난소암종양표지자를측정한결과 CA 125와 CA 19-9은각각 2487.8 U/ml, 2476.6 U/ml으로정상보다높았으며 CA 15-3은 13.1 U/ml, CEA는 5.3 ng/ml로측정되었다. 표지자들의혈중농도기준치 CA 125: 35 U/ml, CA 19-9: 31.3 U/ml, CA 15-5: 36 U/ml 그리고 CEA: 5.0 ng/ml 이상과비교하였을때 CA 125, CA 19-9의분비는증가하였으며 CA 15-3, CEA는증가하지않았거나큰차이를보이지않았다. 6. 인간백혈구항원 (human leukocyte antigen, HLA) 분석 Allele specific PCR-SSOP와 allele specific PCR-SBT 방법으로 HLA Class I과 II를검사하였으며 HLA Class I의유전형 (ABC allele1/allele2) 은 A*24/A*31 B*07/B*35 Cw03*(09)/w*07 이었고 HLA Class II의유전형은 allele1에서 DRB1*01, allele2에서 DRB1*15로나타났다. 7. 종양억제유전자의변이분석 p53 유전자 exon 4 는 wild type 을보였다. BRCA1 에서는유전자 변이가발견되지않았으나 BRCA2 에서는다양한 polymorphism 과 missense mutation 이발견되었다 (Tab. 1). 17
Table 1. BRCA2 mutation Mutation: location change (amino acid change) Mutation type Mutation effect Exon 10: 1093 A>C (N289H) Missense Mutation Polymorphism Exon 10: 1342 C>A (H372N) Missense Mutation Polymorphism Exon 10: 1593 A>G (S455S) Polymorphism Polymorphism Exon 11: 2457 T>C (H743H) Polymorphism Polymorphism Exon 11: 3199 A>G (N991D) Missense Mutation Unclassified variant Exon 14 (int14): 7663+53 C>T Polymorphism Polymorphism Exon 17 (int16): 8034-14 T>C Polymorphism Polymorphism Exon 22 (int21): 8983-66 T>C Polymorphism Polymorphism A: adenine T: thymine G: guanine C: cytosine N: Asparagine H: Histidine S: Serine D: Aspartic acid 8. 항암화학약물감수성검사 PPC에의한 12종의상용항암제의억제율 (inhibition rate) 은 gemcitabine(49.1%), doxorubicin(42.4%), irinotecan(37.9%), 5-FU(30.0%), topotecan(26.1%), etoposide(25.5%), bleomycin(21.3%), oxaliplatin(19.4%), docetaxel(18.7%), cisplatin(17.1%), paclitaxel(9.0%), carboplatin(6.9%) 순이었다. 약물농도에대하여약물감수성을나타내는 CI값은 gemcitabine(ci=157.3), irinotecan(ci=161.1), doxorubicin(ci=184.4), 5-FU (CI=201.9), oxaliplatin(ci=210.4), etoposide(ci=221.2), topotecan(ci=221.7), docetaxel(ci=226.7), cisplatin(ci=228.1), bleomycin(ci=238.1), carboplatin (CI=248.8) 그리고 paclitaxel (CI=282.6) 순으로나타났다 (Fig. 4). 18
Inhibition Rate (%) 60 49.1 50 42.4 37.9 40 30 20 10 0 Gemcitabine Irinotecan Doxorubicin 5-FU 30 19.4 Oxaliplatin Etoposide 25.5 26.1 21.3 18.7 17.1 Topotecan Docetaxel Cisplatin Bleomycin Carboplatin 6.9 9 Paclitaxel Gemcitabine Irinotecan Doxorubicin 5-FU Oxaliplatin Etoposide Topotecan Docetaxel Cisplatin Bleomycin Carboplatin Paclitaxel 300 250 200 150 100 153.7 161.1 184.4 Chemosensitivity Index (Rank) 210.4 201.9 221.7 221.2 228.1 226.7 248.8 238.1 282.6 Gemcitabine(1) Irinotecan (2) Doxorubicin (3) 5-FU(4) Oxaliplatin (5) Etoposide (6) Topotecan (7) 50 Docetaxel(8) 0 Gemcitabine(1) Irinotecan (2) Doxorubicin (3) 5-FU(4) Oxaliplatin (5) Etoposide (6) Topotecan (7) Docetaxel(8) Cisplatin (9) Bleomycin (10) Carboplatin (11) Paclitaxel (12) Cisplatin (9) Bleomycin (10) Carboplatin (11) Paclitaxel (12) Figure 4. Chemosensitivity test of YDOV-139 cell line. Inhibition rate(%) and chemosensitivty index(rank) were analyzed 9. 종양형성능력 누드마우스에 YDOV-139를접종한 7일후접종부위에서피하종양이형성되었으며 7주동안종양의크기는서서히증가하였다. 접종후 8주에최대직경은 6 mm였고, 3마리의누드마우스모두에서형성되었다 (Fig. 5). 19
Figure 5. A Xenograft of YDOV-139 cells to nude mice. B Formation of tumor mass was noted in 8 weeks (A) with the largest diameter of 6mm (B). 10. cdna microarray을이용한유전자발현연구 cdna microarray을이용하여 YDOV-139 와 HOSE의유전자발현차이를비교분석하였다. Hierarchical clustering( 계층학적묶음 ) 분석에서약 2520개의유전자가통계적으로유의한발현차이를보였다 (> 2-fold, P < 0.05). 이들유전자중 1108개는발현증가 (up regulation) 되었으며, 1412개는발현감소 (down regulation) 되었다 (Fig. 6). 20
Figure 6. Hierarchical clustering of YDOV-139 cell line gene expression. N: HOSE C1: YDOV-139 Upregulated signals are expressed in red while downregulated signals are expressed in green. 또한이들유전자와난소암의기능적경로의연관성을보고자 10 배이상통계적으로의미있게증가및감소를보인유전자들을 기능학적으로분류하였다 (Tab.2 & Tab.3). 21
Table 2. Selected groups of upregulated genes expressed at least 10 folds higher in YDOV-139 compared with HOSE TargetID Definition Symbol Fold* Accession Cell adhesion ILMN_23335 epithelial V-like antigen 1 EVA1 49.33 NM_005797.2 Cell cycle control ILMN_24793 cell division cycle 2, G1 to S and G2 to M CDC2 15.50 NM_001786.2 Mitosis ILMN_15944 SMC4 structural maintenance of chromosomes 4-like 1 (yeast) SMC4L1 17.22 NM_001002799.1 Cell proliferation & differentiation ILMN_38732 PREDICTED: paternally expressed 10 PEG10 13.14 XM_499343.2 ILMN_4070 tumor-associated calcium signal transducer 1 TACSTD1 102.39 NM_002354.1 ILMN_3786 midkine (neurite growth-promoting factor 2) MDK 43.95 NM_001012334.1 Cell structure ILMN_7397 spondin 1, extracellular matrix protein SPON1 12.25 NM_006108.1 ILMN_13755 claudin 7 CLDN7 67.35 NM_001307.3 T-cell mediated immunity V-set domain containing T cell activation ILMN_29440 inhibitor 1 VTCN1 42.62 NM_024626.1 Nucleotide and nucleic acid metabolism ILMN_5449 centromere protein A, 17kDa CENPA 13.37 NM_001809.2 ILMN_30154 Thymidine kinase 1, soluble TK1 32.13 NM_003258.1 Oncogenesis ILMN_16236 runt-related transcription factor 3, transcript variant 2 RUNX3 32.26 NM_004350.1 ILMN_20794 lipocalin 2 (oncogene 24p3) LCN2 111.19 NM_005564.2 Protein biosynthesis and modification ILMN_26083 chromosome 12 open reading frame 46 C12orf46 34.62 NM_152321.1 ILMN_1007 protein phosphatase 2 (formerly 2A), regulatory subunit B (PR 52), beta isoform PPP2R2B 42.00 NM_181676.1 Proteolysis ILMN_30268 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 SERPINA1 10.67 NM_001002236.1 ILMN_14210 protease, serine, 8 (prostasin) PRSS8 55.04 NM_002773.2 ILMN_11560 kallikrein 6 (neurosin, zyme) KLK6 58.23 NM_002774.3 22
Signal transduction ILMN_2234 gap junction protein, beta 3, 31kDa (connexin 31) GJB3 10.93 NM_001005752.1 ILMN_3066 insulin-like growth factor binding protein 5 IGFBP5 27.97 NM_000599.2 ILMN_17250 solute carrier family 1 (glial high affinity glutamate transporter), member 3 SLC1A3 32.87 NM_004172.3 Ion transport ILMN_14229 * YDOV-139 / HOSE solute carrier organic anion transporter family, member 2A1 SLCO2A1 12.19 NM_005630.1 Table 3. Selected groups of downregulated genes expressed at least 10 folds higher in HOSE compared with YDOV-139 TargetID Definition Symbol Fold* Accession Amino acid biosynthesis chromosome 10 open reading frame ILMN_5150 C10orf65-13.15 NM_138413.2 65 Apoptosis ILMN_28684 tumor necrosis factor receptor superfamily, member 19 TNFRSF19-13.84 NM_148957.2 Carbohydrate metabolism ILMN_15812 solute carrier family 2 (facilitated glucose transporter), member 3 SLC2A3-63.30 NM_006931.1 ILMN_9544 aconitase 1, soluble ACO1-11.62 NM_002197.1 Cell proliferation and differentiation ILMN_3875 vascular cell adhesion molecule 1 VCAM1-25.88 NM_001078.2 ILMN_25295 junctional adhesion molecule 3 JAM3-29.95 NM_032801.3 Cell structure ILMN_676 vimentin VIM -111.60 NM_003380.2 ILMN_1052 tropomodulin 1 TMOD1-22.59 NM_003275.1 ILMN_17132 talin 2 TLN2-10.30 NM_015059.1 ILMN_4516 Fibronectin 1 FN1-23.01 NM_054034.2 ILMN_1137 collagen, type IV, alpha 6 COL4A6-15.62 NM_001847.1 ILMN_137533 collagen, type V, alpha 2 COL5A2-51.67 NM_000393.2 ILMN_17073 claudin 11 (oligodendrocyte transmembrane protein) CLDN11-10.04 NM_005602.4 Homeostasis ILMN_24349 aquaporin 1 (Colton blood group) AQP1-34.12 NM_198098.1 Immunity and defense ILMN_16098 Thrombospondin 2 THBS2-104.73 NM_003247.2 ILMN_12111 complement factor H CFH -14.79 NM_001014975.1 ILMN_14902 C1q and tumor necrosis factor related protein 1 C1QTNF1-11.03 NM_030968.2 Nucleotide and nucleic acid metabolism 23
ILMN_6405 REC8-like 1 (yeast) REC8L1-39.25 NM_005132.1 Tumor suppressor ILMN_1781 Wilms tumor 1 WT1-13.94 NM_024425.2 Protein biosynthesis and modification ILMN_28424 galactose-3-o-sulfotransferase 1 GAL3ST1-11.40 NM_004861.1 ILMN_5690 myosin, light polypeptide kinase MYLK -12.60 NM_005965.3 ILMN_29283 eef1a2 binding protein DKFZp434B1231-24.70 NM_178275.3 ILMN_6510 glutaminyl-peptide cyclotransferase (glutaminyl cyclase) QPCT -27.34 NM_012413.3 Proteolysis ILMN_3680 kallikrein 11 KLK11-51.34 NM_006853.2 ILMN_28025 macrophage stimulating 1 (hepatocyte growth factor-like) MST1-18.39 NM_020998.2 Signal transduction ILMN_4516 Fibronectin 1 FN1-23.01 NM_054034.2 ILMN_15406 Phosphoinositide-3-kinase, catalytic, delta polypeptide PIK3CD -17.29 NM_005026.2 ILMN_18672 disabled homolog 2, mitogenresponsive phosphoprotein DAB2-22.09 NM_001343.1 (Drosophila) ILMN_27192 RAB7, member RAS oncogene family-like 1 RAB7L1-10.14 NM_003929.1 ILMN_139202 doublecortin domain containing 2 DCDC2-20.77 NM_016356.1 Transport ILMN_7795 SEC24 related gene family, member D (S. cerevisiae) SEC24D -11.24 NM_014822.1 ILMN_9543 hemoglobin, delta HBD -145.18 NM_000519.3 ILMN_2381 folate receptor 3 (gamma) FOLR3-49.94 NM_000804.2 * YDOV-139 / HOSE 11. 2-DE 및 MALDI-TOF MS를이용한단백질발현연구 2-D electrophoresis로 YDOV-139 와 HOSE의단백질발현연구에서여러단백질이출현및발현증가또는발현감소및소실되는발현차이를보였다 (Fig. 7). 이중발현증가된 17개또한발현감소된 6개총23개의단백질을 MALDI-TOF MS를이용하여확인하였다 (Tab. 4). 24
A B Figure 7. Proteomic comparison between YDOV-139 cell line and HOSE using 2-DE (A)YDOV-139, (B) HOSE Table 4. Identification of proteins that were differentially expressed in YDOV-139 and HOSE using 2-DE and MALDI-TOF MS Spot No. Identification % Coverage pi Mw (kda) <Up-regulated proteins in YDOV-139> 1 FUBP1 (Far upstream element binding protein1) 20 6.9 68.81 2 Far upstream element binding protein 27 7.2 67.71 3 Far upstream element binding protein 14 7.2 67.71 4 Chaperonin 49 5.7 61.21 5 ER-60 protein (ethylene responsive catalase) 41 5.9 57.1 6 Chaperonin containing T-complex protein 1, subunit 2 34 6.0 57.81 7 ENO1 protein (enolase 1) 46 5.9 29.19 8 Fumarate hydratase precursor 25 9.0 54.79 9 S-adenosylhomocysteine hydrolase 27 6.0 48.27 10 Actin related protein2 isoform b 20 6.3 45.03 11 Albumin-like protein 19 5.7 53.43 12 Thioredoxin-like protein 22 5.2 37.76 13 B23 nucleophosmin (280 AA) 28 4.7 31.09 14 Pyrophosphatase 1 31 5.5 33.10 15 NP (nucleoside phosphorylase) 31 6.5 32.33 16 5 -methythioadenosine phosphorylase 32 6.8 31.73 25
17 Albumin-like protein 16 5.7 53.43 <Down-regulated proteins in YDOV-139> 18 VLA-3 alpha subunit 8 6.1 114.5 19 Keratin 7 39 5.4 51.46 20 VAT ( Vesicle amine transport) protein 1 28 6.2 4167 21 Annexin 5 chain A 64 4.9 35.84 22 Chloride intracellular channel 4 60 5.5 28.9 23 Nicotinamide N-methyltransferase 23 5.6 30.1 pi: isoelectric point IV. 고찰 난소암을임상적으로진단하고치료하기위해난소암의분자학적, 세포학적경로를특징화하고규명하는많은새로운연구및기술의발전들이요구되며다양한세포주의확립은매우유용하다. 8, 9 또한최근에는배양기술의발전에힘입어조직으로부터확립된 세포주이외에환자의체액에서도세포주들이확립되고있다. 10 본 연구에서도환자의복수에서장액성난소암세포주를확립하고그분자생물학적특성을규명하였다. 난소암세포주확립과정은검체의종류, 검체의처리과정, 비종양세포의오염비율, 성장인자의존도그리고배양액함유성분등여러가지요건에영향을받으며, 장기간배양시과도한 subcloning때문에확립된많은세포주에서표현형적불안정을보이고점차적으로기원한세포의특징이소실되기도한다. 11,12 본연구에서확립된세포주는성장인자의추가없이표준 media에서잘배양되었으며확립된세포주는형태학적으로장액성상피성난소암의소견을보였고또한이종이식에서종양형성능력을확인할수있듯이기원세포의특징을유지하고있었다. 26
본연구의예측배가시간은 120 시간으로기존보고된다른여러 난소암세포주의 18-66 시간과차이가있었다. 13,14 보고된여러 난소암세포주는난소조직에서대부분확립되었으며, 일부는항암제치료후진행된환자에서확립된세포주도있었으나배가시간이차이가나는조건은설명할수없었다. 난소암은진단당시에이미상당히진행된경우가대부분이며조기진단을위한선별검사의중요성이특히강조되는질환이다. CA125는난소암세포주를쥐에주입하여형성된단세포군항체 (OC125) 에의하여발견된종양표지자로상피성난소암의약 80% 에서증가되어있으며초기난소암 (I기또는 II기 ) 인경우는약 60% 에서진행성난소암 (III기또는 IV기 ) 인경우는 90% 이상에서증가되어되어있다. Coelemic epithelium에서기원된조직에서발현되므로암이외에임신초기그리고내막증, 골반염증을포함한복막이자극되는양성질환에서도증가한다. CEA는진행된상피성암의경우 30-65%, CA 19-9는 17-25% 까지증가가보고되었다. 이러한종양표지자단독의특이성저하와높은위양성때문에암조기진단을위하여 CA125와함께 CA19-9, CA15-5, CEA등같은 다른종양표지자들을병합검사한다. 15-17 본세포주에서상기 표지자중 CA 125 및 CA 19-9 가증가되었으며이는상기두가지표지자가장빈번히증가하는진행된장액성상피세포난소암의임상학적특징에부합된다. HLA는인간의조직적합항원을반영하며유전자는 6번염색체의 27
단완에위치한다. 약 3500 Kb로이루어져있고 Class II(DR) Class III ClassI(B,C,A) 의순서로배열되어있다. HLA 유전자는광대한다형현상 (polymorphism) 이존재하여각 locus에대하여각기다른 HLA-allele를특징지우며또한특정 HLA 분자에결합하는 peptide들을결정하고각개인들은 Haptotype이라일컫는특정 HLA allele들의조합이유전된다. HLA class I 분자는종양특이세포독성 T 세포의항원체표시자로암세포에대한면역감시에대한중요한역할을하는데암의경우면역체계서암세포를비자가 (non-self) 로인식하여면역반응을유발하며조직적합항원의변형은면역체계서암세포에대한인지를변화시켜면역감시를피하고암발생을진행하게한다. HLA-A2 frequency 와 Haplotype A33-B14-DR1처럼일부 HLA cass I, II haplotype들은난소암발생위험성과연관된다는 보고가있다. 18-20 본세포주의 HLA 유전적표현형은현재까지 보고된이러한 haplotype과무관하였으나추후더많은세포주를대상으로연관성에대한연구가필요하다. 같은종류의암이라도발암유전자 (HER-2/nu, K-ras, c-myc), 종양억제유전자 (p53, BRCA1, BRCA2) 또는부조화교정유전자 (mismatch repair gene- hmlh1, hmlh2, hpms1, hpms2) 등의변이정도가다르며이로인해생물학적특성이달라지고암화과정에서또한발암유전자및종양억제유전자등에의하여성장인자의분비및수용체증가발현 (overexpression) 등다양한세포주기조절의변화가일어난다. 이러한변화는임상학적으로진단및치료전략을 28
세우는데있어서중요한표적이될수있어종양유전자와종양억제유전자가난소암발생에미치는영향에관해서많은연구들이있어왔다. 21-23 17q21 염색체에존재하는 BRCA1와 13q12-q13 염색체에존재하는 BRCA2는 Rad51 등과함께핵산의이중구조파손을교정및유전자안정을유지하는역할을하며유전성유방암 / 난소암과관련이있는것으로알려져있다. BRCA1과 BRCA2의유전적이상은난소암의 10% 정도에서발견되며, BRCA1 및 BRACA2 이상이있는여성에서 70세까지 20-40% 와 10-20% 에서난소암이발생하는것으로보고되고 있다. 24, 25 본세포주에서 BRCA1 의유전적이상은관찰되지 않았으며, BRCA 2의경우 exon10, 11, 14, 17, 22 에서 polymorphism이나타났으며, exon10(1093번째, 1342번째염기서열 ) exon11(3199번째염기서열 ) 에서 missense mutation을보였다. 종양억제유전자인 p53 유전자는염색체 17p13.3에위치하며 11개의 exon으로이루어져있으며세포성장억제하고 DNA 손상복귀및 apoptosis등에관여하며난소암에있어서가장흔한유전적이상으로알려져있다. 진행된난소암의경우약 50~80% 까지보고되는반면조기난소암의경우엔약 10~20% 정도만보고되고 있다. P53 변이가발현된 cell line 은 wild type 의 p53 cell line 보다더 종양형성능력이높다고보고되었으며, 상피성난소암에서 p53 은 생존에대한독립적예측인자로서유용하다는보고도있듯이 p53 안정화에대한노력은난소암치료에도움이될것이다. 26, 27 본 29
세포주에서 p53은 wild type을보였다. 진행된난소암에있어서항암제치료에대하여많은난항이존재한다. 현재표준치료로진행된 III기또는 IV기난소암에서수술후 paclitaxel과백금제 (platinum based) 항암제가적용되며. 반응률은 70-80% 로써 20-30% 의환자에서는암이계속진행되거나, 치료 6개월이내에재발하는저항성이나타난다. 또한진행된난소암의 5년생존률은 30-40% 로보고되고있다. 이러한항암제저항성및 5년생존률에대하여난소암의임상적병태생리학적분류및치료이외에분자유전학적분류및치료의필요성이제기되고있으며, 이를근거로한맞춤형치료에대한논의가있다. 28, 29 본연구에서세포주의항암제검사상수술후표준치료 항암제인 paclitaxel과백금제 (platinum based) 항암제가 gemcitabine, irinotecan, doxorubicin, 5-FU와같은항암제보다낮은억제율 (inhibition rate) 및 CI값을보였으며이는 Chemosensitive 하지만재발을반복하는환자의임상양상과연관성이제기될수도있으며앞서논한맞춤형치료모델이유용할수도있다. 분자생물학적발현연구는암진단및치료에있어서잠재력이높은강력한연구방법이다. 현재까지의여러연구에서난소암의복합성때문에전통적인조직학적방법으로는명확하게구별이안되었던부분을분자적표현형으로구별확인할수있으며, 또한새로운분자표시및치료제표적의확인을가능하게해줄것으로 기대된다. 30, 31 30
현재임상학적으로난소암에서유전자발현연구는정상난소조직과난소암조직을구별하게하는유전자집합을밝히는데유용하며환자의임상학적예후에대한연관성이밝혀지지는않았으나항암제감수성과연관하여유용하였으며 78-86% 의예측성적을보였다는보고가있다. 32, 33 그러나유전자수준의발현분석이세포의실질적단백질수준발현을대표하지는못하고있으며유전자발현분석과단백질발현분석을함께한보완적복합분석이난소암의생물학적연구에필요로한다. 34 본연구에서는 cdna microarray을이용하여세포주특유의유전자발현을확인할수있었으며, 또한 2-DE 및 MALDI-TOF MS를이용하여단백질발현을분석하였다. Hierarchical clustering( 계층학적묶음 ) 분석에서약 2520개의유전자가다른발현을나타냈으며 YDOV-139에서유전자발현은 HOSE와현저히구별되었다. 또한단백질발현연구에서도 YDOV-139와 HOSE사이에발현차이를보이는 26개의단백질을확인하였다. 이런연구들이모여정상세포와여러많은난소암세포표본에서단백질발현을관찰하는것이기능적역할을확인해주고난소암진단및치료에있어서암표지자및치료표적자로이용될수있는방법을제시해줄것으로기대된다. 추후과정에서본연구의분자생물학적으로발현된양상이추가적인연구를통하여선택과정또여러많은난소암표본을통한유용성확인 (validation) 과정을필요로할것이다. 31
V. 결론 본연구에서성공적으로장액성난소암세포주 (YDOV-139) 를확립하고특성을분석하였다. 확립된세포주의특성은난소암의병리기전연구, 세포기능연구, 항암제감수성연구그리고현재치료가어려운암에대한새로운치료모델개발에유용한가치를제공할수있을것이다. 참고문헌 1. 이효표, 송용상, 김재욱, 남궁성은, 목정은, 문형등. 한국부인암등록사업조사보고서 (2003. 1. 1-2003.12.31) 대한산부회지, 2006; 49(4): 762-812. 2. Reynolds EA, Moller KA. A review and an update on the screening of epithelial ovarian cancer. Curr Probl Cancer. 2006 Sep-Oct;30(5):203-32. 3. Central Cancer Registry Center in Korea. Annual Report of the Central Cancer Registry in Korea (2005.1-2005.12). Ministry of Health and Welfare, Republic of Korea. 2007. 4. Shih IeM, Kurman RJ. Ovarian tumorigenesis: a proposed model based on morphological and molecular genetic analysis. Am J Pathol. 2004;164(5): 1511-8. 5. Garson K, Shaw TJ, Clark KV, Yao DS, Vanderhyden BC. Models of ovarian cancer--are we there yet? Mol Cell Endocrinol 2005;239: 15-26. 6. Biade S, Marinucci M, Schick J, Roberts D, Workman G, Sage EH, et 32
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Abstract Establishment and characterization of a serous ovarian cancer cell line Doo Byung Chay Department of Medicine The Graduate School, Yonsei University (Directed by Professor Jae Hoon Kim) Objective: Cancer cell line is a valuable tool in cancer studies because it can substitute an in vitro experiment for a human body. As for ovarian cancer because of the extreme heterogeneity and complexity, many cell lines with their own biologic properties are needed. A new cell line, designated YDOV-139, was established and the characteristics were determined. Methods: The tumor cells were obtained from ascites of a 67-year-old Korean woman with recurrent ovarian cancer. Primary cell cultures were performed with DMEM media and the cells are maintained by serial passages. Cell line characterization was performed by cell line morphologies, growth kinetics, tumor marker secretion, HLA typing, mutational analysis of BRCA1, BRCA2 and p53 genes. Tumorigenicity and chemosensitivity test of the cultured cells were also performed. Gene expression profile and proteomics were studied using cdna 37
microarray and 2D electrophoresis. Results: The cells showed a polygonal appearance with large, round nucleus by phase-contrast microscopy. The average population doubling times were 120 hours and the tumor marker secretion assay revealed highly elevated CA125 and CA19-9. P53 gene was wild type and while no mutations were detected with BRCA1, polymorphism and missense mutation were detected with BRCA2. When transplanted into nude mice, cell successfully induced tumor and gemcitabine had the highest chemosensitivity index. Compared to HOSE(Human Ovarian Surface Epithelial cell), 2520 differentially expressed genes were noted by gene expression profile using cdna microarray and 23 proteins were identified by 2D electrophoresis. Conclusions: Biological characteristics of this cell line may be an important research resource for studying ovarian cancer pathophysiology, cell biology and chemosensitivity as well as for developing a new strategy model against ovarian cancer treatment. -------------------------------------------------------------------------------------- Key Words: Ovarian cancer, Cell line characterization, Gene expression, Proteomics. 38