Radiation Oncology Journal

The Korean Society for Radiation Oncology (대한방사선종양학회)
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Gene Expression Profiles in Cervical Cancer with Radiation Therapy Alone and Chemo-radiation Therapy

자궁경부암의 방사선치료 및 방사선항암화학 병용치료에 따른 유전자발현 조절양상

  • Lee Kyu Chan (Department of Radiation Oncology Kachon Medical School) ;
  • Kim Meyoung-kon (Departments of Biochemistry College of Medicine, Korea University) ;
  • Kim Jooyoung (Department of Radiation Oncology Kachon Medical School) ;
  • Hwang You Jin (Laboratory of Molecular Biology, Kachon Medical School,) ;
  • Choi Myung Sun (Radiation Oncology, College of Medicine, Korea University) ;
  • Kim Chul Yong (Radiation Oncology, College of Medicine, Korea University)
  • 이규찬 (가천의과대학교 방사선종양학과) ;
  • 김명곤 (고려대학교 의과대학 생화학교실) ;
  • 김주영 (가천의과대학교 방사선종양학과) ;
  • 황유진 (가천의과대학교 분자생물학연구실) ;
  • 최명선 (고려대학교 방사선종양학교실) ;
  • 김철용 (고려대학교 방사선종양학교실)
  • Published : 2003.03.01
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Abstract

Purpose : To analyze the gene expression Profiles of uterine ceulcal cancer, and its variation after radiation therapy, with or without concurrent chemotherapy, using a CDNA microarray. Materials and Methods :Sixteen patients, 8 with squamous ceil carcinomas of the uterine cervix, who were treated with radiation alone, and the other 8 treated w14h concurrent chemo-radiation, were Included in the study. Before the starling of the treatment, tumor biopsies were carried out, and the second time biopsies were peformed after a radiation dose of 16.2$\~$27 Gy. Three normal cervix tissues were used as a control group. The microarray experiments were peformed with 5 groups of the total RNAs extracted individually and then admixed as control, pre-radiation therapy alone, during-radiation therapy alone, pre-chemoradiation therapy, and during-chemoradlation therapy. The 33P-iabeled CDNAS were synthesized from the total RNAs of each group, by reverse transcription, and then they were hybridized to the CDNA microarray membrane. The gene expression of each microarrays was captured by the intensity of each spot produced by the radioactive isotopes. The pixels per spot were counted with an Arrayguage, and were exported to Microsoft Excel The data were normalized by the Z transformation, and the comparisons were peformed on the Z-ratio values calculated. Results : The expressions of 15 genes, including integrin linked kinase (ILK), CDC28 protein kinase 2, Spry 2, and ERK 3, were increased with the Z-ratio values of over 2.0 for the cervix cancer tissues compared to those for the normal controls. Those genes were involved In cell growth and proliferation, cell cycle control, or signal transduction. The expressions of the other 6 genes, Including G protein coupled receptor kinase 5, were decreased with the Z-ratio values of below -2.0. After the radiation thorapy, most of the genes, with a previously Increase expressions, represented the decreased expression profiles, and the genes, with the Z-ratio values of over 2.0, were cyclic nucleotlde gated channel and 3 Expressed sequence tags (EST). In the concurrent chemo-radiation group, the genes involved in cell growth and proliferation, cell cycle control, and signal transduction were shown to have increased expressions compared to the radiation therapy alone group. The expressions of genes involved in anglogenesis (angiopoietln-2), immune reactions (formyl peptide receptor-iike 1), and DNA repair (CAMP phosphodiesterase) were increased, however, the expression of gene involved In apoptosls (death associated protein kinase) was decreased. Conclusion : The different kinds of genes involved in the development and progression of cervical cancer were identified with the CDNA microarray, and the proposed theory is that the proliferation signal stalls with ILK, and is amplified with Spry 2 and MAPK signaling, and the cellular mitoses are Increased with the increased expression oi Cdc 2 and cell division kinases. After the radiation therapy, the expression profiles demonstrated 4he evidence of the decreased cancer cell proliferation. There was no sigificant difference in the morphological findings of cell death between the radiation therapy aione and the chemo-radiation groups In the second time biopsy specimen, however, the gene expression profiles were markedly different, and the mechanism at the molecular level needs further study.

목적 : 동시에 대량으로 유전자발현 양상을 검사할 수 있는 cDNA microarray 기법을 이용하여 자궁경부암에서 특징적으로 나타나는 유전자발현 양상을 알아보고, 방사선치료 및 방사선 항암화학요법 병용치료시의 유전자발현 변화양상을 파악하고자 하였다. 대상 및 방법 :자궁경부 편평상피암으로 확진된 후 근치목적 방사선치료를 단독으로 시행한 8명과 항암화학요법을 병행한 8명에서 채취한 종양조직을 대상으로 하고, 정상 자궁경부 3례를 대조군으로 하였다 조직 생검은 치료 전과 외부 방사선치료 16.2$\~$27 Gy에 두 번하였다. 항암화학요법을 병용한 경우, 5-FU 1,000 mg/m$^{2}$을 제 1일부터 5일까지 정주하고, clsplatin 60 mg/m$^{2}$을 제 1일에 정주하였다. cDNA microarray는 종양조직에서 추출한 total RNA를 역전사(reverse transcription)방법을 이용하여 (P-33)을 표지한 cDNAS를 제작, nylon membrane에 hybridization하였다. 이후 membrane을 phosphor-imager screens에 옮겨 1$\~$5일 동안 노출시킨 후 이미지를 스캔하였다. 유전자의 발현정도는 각 스팟(spot)들의 방사능 강도로 나타나는데, 각 스팟의 픽셀(pixel)을 Arrayguage를 사용하여 산출한 후 엑셀파일로 저장하였다. 유전자의 발현정도 비교는 원 자료(original data)를 Z-변환을 통해 보정(normalized)한 후 Z-ratio값을 산출하여 시행하였다. 결과 : 대조군에 비해 자궁경부암에서 Z-ratio 2.0 이상으로 유의한 발현증가를 보인 유전자들은 integrin-linked kinase, CDC28 protein kinase 2, Spry 2, ERK 3 등 15개로 주로 세포성장과 증식, 세포주기, 신호전달 등에 관련된 유전자들이었으며, Z-ratio -2.0 이하의 유의한 발현감소는 G protein-coupled receptor kinase 6외 6개였다. 방사선 단독치료를 시행한 후 Z-ratio 2.0 이상 발현이 증가한 것은 cyclic nucleotlde gated channel외 3개의 Expressed sequence tags (EST)들이었고, Z-ratio -2.0 이하의 발현감소를 보인 것들에는 치료전 종양세포에서 발현이 증가되었던 세포성장과 증식, 세포주기, 신호전달 등에 관련된 유전자들이 포함되었다 방사선치료와 항암화학요법을 병용했을 때는 방사선 단독치료에 비하여 세포성장과 증식 및 신호전달 관련 유전자들이 상대적으로 높게 발현되었으며, 이외에도 혈관형성(angiopoietin-2), 면역반응(formyl peptide receptor-like 1), DNA 손상회복에 관련된 유전자(CAMP phosphodiesterase)의 발현은 증가되고 세포고사(death associated protein kinase)에 관련된 유전자는 발현 감소를 보였다. 결론 : 자궁경부암에서분열과 증식 및 신호전달에 관여하는 여러 종류의 유전자들 발현이 동시다발적으로 증가되어 있다는 것과 방사선치료를 시행하면 이들 유전자의 발현이 감소하여 종양세포의 분열과 증식이 저해된다는 것을 확인하였다. 방사선 단독치료와 항암화학요법 병용치료를 비교하면 그 유전자 발현양상이 다르므로 향후 이번연구에서 나타난 유전자들에 대한 추가 연구가 필요하며, 이는 개별화된 맞춤형 치료법을 개발하는데 기초자료로 사용될 수 있을 것으로 기대된다.

Keywords

References

  1. Parkin DM, Pisani P, Ferlay J. Estimates of the worldwide incidence of 25 major cancers in 1990. Int J Cancer 1999;80:827-841 https://doi.org/10.1002/(SICI)1097-0215(19990315)80:6<827::AID-IJC6>3.0.CO;2-P
  2. Central Cancer Registry Center in Korea. Ministry of Health and Welfare, Republic of Korea. Annual report of the central cancer registry in Korea (1999. 1. -1999. 12.) April, 2001
  3. Morris M, Eifel PJ, Lu J, et al. Pelvic radiation with concurrent chemotherapy compared with pelvic and para-aortic radiation for high-risk cervical cancer. N Engl J Med. 1999;340:1137-1143 https://doi.org/10.1056/NEJM199904153401501
  4. Kang OC, Choi EK, Chung WK, et al. Concurrent chemoraiotherapy in locally advanced carcinoma of the uterine cervix. J Korean Soc Ther Radiol 1998;16:311-323
  5. SChena M, Shalon D, Davis RW, Brown PO. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 1995;270:467-470 https://doi.org/10.1126/science.270.5235.467
  6. Wang K, Gan L, Jeffery E, et al. Monitoring gene expression profile changes in ovarian carcinomas using cDNA microarray. Gene 1999;229:101-108 https://doi.org/10.1016/S0378-1119(99)00035-9
  7. Bertucci F, Houlgatte R, Benziane A, et al. Gene expression profiling of primary breast carcinomas using arrays of candidate genes. Hum Mol Genet 2000;9:2981-2991 https://doi.org/10.1093/hmg/9.20.2981
  8. Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 2000;403:503-511 https://doi.org/10.1038/35000501
  9. Park GH, Choe J, Choo HJ, Park YG, Sohn J, Kim MK. Genome-wide expression profiling of 8-chloroadenosine- and 8-chloro-cAMP-treated human neuroblastoma cells using radioactive human cDNA microarray. Exp Mol Med 2002;34(3):184-193 https://doi.org/10.1038/emm.2002.27
  10. Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 1998;95(25):14863-14868 https://doi.org/10.1073/pnas.95.25.14863
  11. Villaret DB, Wang T, Dillon D, et al. Identification of genes overexpressed in head and neck squamous cell carcinoma using a combination of complementary DNA subtraction and microarray analysis. Laryngoscope 2000;110:374-381 https://doi.org/10.1097/00005537-200003000-00008
  12. Shirota Y, Kaneko S, Honda M, Kawai HF, Kobayashi K. Identification of differentially expressed genes in hepatocellular carcinoma with cDNA microarrays. Hepatology 2001;33:832-840 https://doi.org/10.1053/jhep.2001.23003
  13. Bertucci F, Bernard K, Loriod B, et al. Sensitivity issues in DNA array-based expression measurements and perfor- mance of nylon microarrays for small samples. Hum Mol Genet 1999;8:1715-1722 https://doi.org/10.1093/hmg/8.9.1715
  14. Yoo HS. 21C Frontier Cancer Genome Project of Korea. J Korean Cancer Asso 2001;33(S):4
  15. Achary MP, Jaggernauth W, Gross E, Alfieri A, Klinger HP, Vikram B. Cell lines from the same cervical carcinoma but with different radiosensitivities exhibit different eDNA microarray patterns of gene expression. Cytogenet Cell Genet 2000;91:39-43 https://doi.org/10.1159/000056815
  16. Kitahara O, Katagiri T, Tsunoda T, Harima V, Nakamura V. Classification of sensitivity or resistance of cervical cancers to ionizing radiation according to expression profiles of 62 genes selected by eDNA microarray analysis. Neoplasia 2002:4(4):295-303
  17. Voganathan TN, Costello P, Chen X, et al. Integrin-linked kinase (ILK): a 'hot' therapeutic target. Biochem Pharmacol 2000;60(8):1115-1119 https://doi.org/10.1016/S0006-2952(00)00444-5
  18. White DE, Cardiff RD, Dedhar S, Muller WJ. Mammary epithelial-specific expression of the integrin-linked kinase (ILK) results in the induction of mammary gland hyperplasias and tumors in transgenic mice. Oncogene 2001;20(48):7064-7072 https://doi.org/10.1038/sj.onc.1204910
  19. SCandurro AB, Weldon CW, Figueroa VG, Alam J, Beckman BS. Gene microarray analysis reveals a novel hypoxia signal transduction pathway in human hepatocellular carcinoma cells. Int J Oncol 2001;19(1):129-135
  20. D'Amico M, Hulit J, Amanatullah DF, et al. The integrinlinked kinase regulates the cyclin D1 gene through glycogen synthase kinase 3beta and cAMP-responsive element-binding protein-dependent pathways. J Bioi Chem 2000;275(42): 32649-32657 https://doi.org/10.1074/jbc.M000643200
  21. Dedhar S, Williams B, Hannigan G. Integrin-linked kinase (ILK) : a regulator of integrin and growth-factor signaling. Trends Cell Bioi 1999;9(8):319-323 https://doi.org/10.1016/S0962-8924(99)01612-8
  22. Voganathan N, Vee A, Zhang Z, et al. Integrin-linked kinase, a promising cancer therapeutic target: biochemical and biological properties. Pharmacol Ther 2002;93(2-3):233
  23. Persad S, Attwell S, Gray V, et al. Inhibition of integrinlinked kinase (ILK) suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTENmutant prostate cancer cells. Proc Natl Acad Sci USA 2000; 97(7):3207-3212 https://doi.org/10.1073/pnas.060579697
  24. Graff JR, Deddens JA, Konicek BW, et al. Integrin-linked kinase expression increases with prostate tumor grade. Clin Cancer Res 2001;7(7):1987-1991
  25. Marotta A, Tan C, Gray V, et al. Dysregulation of integrin- linked kinase (ILK) signaling in colonic polyposis. Oncogene 2001;20(43):6250-6257 https://doi.org/10.1038/sj.onc.1204791
  26. Zwick E, Bange J, Ullrich A. Receptor tyrosine kinase signaling as a target for cancer intervention strategies. Endocr Relat Cancer 2001;8(3):161-173 https://doi.org/10.1677/erc.0.0080161
  27. Ostman A, Bohmer FD. Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatases. Trends Cell Bioi 2001;11(6):258-266 https://doi.org/10.1016/S0962-8924(01)01990-0
  28. Majercakova P. Role of protein phosphorylation in the development of tumors. Sb Lek 2000;101(3):215-228
  29. Chernoff J. Protein tyrosine phosphatases as negative regulators of mitogenic signaling. J Cell Physiol 1999;180(2):173-181 https://doi.org/10.1002/(SICI)1097-4652(199908)180:2<173::AID-JCP5>3.0.CO;2-Y
  30. Fischer EH. Cell signaling by protein tyrosine phosphorylation. Adv Enzyme Regul 1999;39:359-369 https://doi.org/10.1016/S0065-2571(98)00014-4
  31. Bixby JL. Ligands and signaling through receptor-type tyrosine phosphatases. UBMB Life 2001;51(3):157-163
  32. Smits VA, Medema RH. Checking out the G(2)/M transition. Biochem Biophys Acta 2001;1519(1-2):1-12 https://doi.org/10.1016/S0167-4781(01)00204-4
  33. Arellano M, Moreno S. Regulation of CDK!cyciin complexes during the cell cycle. Int J Biochem Cell Bioi 1997;29(4):559573
  34. Gross I, Bassit B, Benezra M, Licht JD. Mammalian sprouty proteins inhibit cell growth and differentiation by preventing\ ras activation. J Bioi Chem 2001 ;276(49):46460-46468 https://doi.org/10.1074/jbc.M108234200
  35. Egan JE, Hall AB, Vatsula BA, Bar-Sagi D. The bimodal regulation of epidermal growth factor signaling by human Sprouty proteins. Proc Natl Acad Sci USA 2002;99(9):6041-6046 https://doi.org/10.1073/pnas.052090899
  36. Wong ES, Fong CW, Lim J, et al. Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and endocytosis, and consequently enhances Ras/ERK signaling. EMBO J 2002;21(18):4796-4808 https://doi.org/10.1093/emboj/cdf493
  37. Holash J, Maisonpierre PC, Compton D, et al. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF. Science 1999;284(5422):1994-1998 https://doi.org/10.1126/science.284.5422.1994
  38. Vajkoczy P, Farhadi M, Gaumann A, et al. Microtumor growth initiates angiogenic sprouting with simUltaneous expression of VEGF, VEGF receptor-2, and angiopoietin-2. J Clin Invest 2002;109(6):777-785 https://doi.org/10.1172/JCI0214105