Radiation Oncology Journal

The Korean Society for Radiation Oncology (대한방사선종양학회)
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Identification of Differentially Expressed Radiation-induced Genes in Cervix Carcinoma Cells Using Suppression Subtractive Hybridization

자궁경부암세포에서 방사선조사시 차등 발현되는 유전자 동정

  • Kim Jun-Sang (Cancer Research Institute, College of Medicine, Chungnam National University) ;
  • Lee Young-Sook (Cancer Research Institute, College of Medicine, Chungnam National University) ;
  • Lee Jeung Hoon (Department of Dermatology, College of Medicine, Chungnam National University) ;
  • Lee Woong-Hee (Department of Biology, College of Natural Sciences, Chungnam National University) ;
  • Seo Eun Young (Department of Dermatology, College of Medicine, Chungnam National University) ;
  • Cho Moon-June (Cancer Research Institute, College of Medicine, Chungnam National University)
  • 김준상 (충남대학교 의과대학 암공동연구소) ;
  • 이영숙 (충남대학교 의과대학 암공동연구소) ;
  • 이증훈 (충남대학교 의과대학 피부과교실) ;
  • 이웅희 (충남대학교 자연과학대학 생물학과) ;
  • 성은영 (충남대학교 의과대학 피부과교실) ;
  • 조문준 (충남대학교 의과대학 암공동연구소)
  • Published : 2005.03.01
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Abstract

Purpose : A number of genes and their products are Induced early or late following exposure of cells to ionizing radiation. These radiation-Induced genes have various effects on irradiated cells and tissues. Suppression subtractive hybridization (SSH) based on PCR was used to Identify the differentially expressed genes by radiation in cervix carcinoma cells. Materials and Methods : Total RNA and poly $(A)^+$ mRNA were Isolated from Irradiated and non-irradiated HeLa cells. Forward- and reverse-subtracted cDNA libraries were constructed using SSH. Eighty-eight clones of each were used to randomly select differentially expressed genes using reverse Northern blotting (dot blot analysis). Northern blotting was used to verify the screened genes. Results : Of the 17t clones, 10 genes in the forward-subtracted library and 9 genes In the reverse-subtracted library were identified as differentially expressed radiation-induced genes by PCR-select differential screening. Three clones from the forward-subtracted library were confirmed by Northern blotting, and showed increased expression in a dose-dependent manner, including a telomerase catalytic subunit and sodium channel-like protein gene, and an ESTs (expressed sequence tags) gene. Conclusion : We Identified differentially expressed radiation-induced genes with low-abundance genes with SSH, but further characterization of theses genes are necessary to clarify the biological functions of them.

목적 : 자경경부암세포에서 polymeric chain reaction (PCR)원리를 이용한 suppression subtractive hybridization (SSH) 방법으로 방사선조사 시 차등 발현되는 유전자를 동정하고자 하였다. 대상 및 방법 : 자궁경부암세포주인 HeLa세포주에 방사선조사 전과 후 총 RNA와 poly $(A)^+$ mRNA를 분리였다. SSH방법으로 forward 및 reverse-subtracted cDNA libraries를 만들었다. 차등 발현된 유전자를 screening하기 위해 reverse Northern blotting (dot blot analysis)을 이용하여 각각의 library에서 88개의 클론을 선택하였고 Nothern blotting으로 확인 후 sequencing하였다. 결과 : screening상 176개 클론 중 forward-subtracted library에서 10개의 유전자가 reverse-subtracted library에서 9개의 유전자가 동정되었다. forward-subtracted library로부터 3개의 유전자가 Northern blotting에 의하여 확인되었고 이중 telomerase catalytic subunit and sodium channel-like protein 유전자와 1개의 ESTs (expressed sequence tags) 유전자가 방사선선량에 따라 증가하쳐다. 결론 : 본 연구를 통해 자궁경부암세포주에서 방사선에 의해 유도되는 유전자를 SSH 방법을 통해 동정할 수 있었다. 그러나 이러한 유전자가 어떤 생물학적인 기능을 갖고 있는지에 대한 계속적인 연구가 필요하다

Keywords

References

  1. Tkeshelashvili VT, Bokhman JV, Kuznetzov VV, Maximov SJ, Chkuaseli GT. Geographic peculiarities of endometrial and cervical cancer incidence in five continents (review). Eur J Gynaecol Oncol 1993;14:89-94
  2. Ponten J, Adami HO, Bergstrom R, et al. Strategies for global control of cervical cancer. Int J Cancer 1995;60:1-26 https://doi.org/10.1002/ijc.2910600102
  3. Holbrook N, Fornace A. Response to adversity: molecular control of gene activation following genotoxic stress. New Biol 1991;3:825-833
  4. Weichselbaum RR, Hallahan D, Fuks Z, Kufe D. Radiation induction of immediate early genes: effectors of the radiation-stress response. Int J Radiat Oncol Biol Phys 1994;30:229-234 https://doi.org/10.1016/0360-3016(94)90539-8
  5. Fornace AJ Jr, Nebert DW, Hollander MC, et al. Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents. Mol Cell Biol 1989;9:4196-4203 https://doi.org/10.1128/MCB.9.10.4196
  6. Papathanasiou MA, Kerr NC, Robbins JH, et al. Induction by ionizing radiation of the gadd45 gene in cultured human cells:lack of mediation by protein kinase C. Mol Cell Biol 1991;11:1009-1016 https://doi.org/10.1128/MCB.11.2.1009
  7. Schmidt-Ullrich RK, Valerie KC, Chan W, McWilliams D. Altered expression of epidermal growth factor receptor and estrogen receptor in MCF-7 cells after single and repeated radiation exposures. Int J Radiat Oncol Biol Phys 1994;29:813-819 https://doi.org/10.1016/0360-3016(94)90570-3
  8. Hong JH, Chiang CS, Campbell IL, Sun JR, Withers HR, McBride WH. Induction of acute phase gene expression by brain irradiation. Int J Radiat Oncol Biol Phys 1995;33:619-626 https://doi.org/10.1016/0360-3016(95)00279-8
  9. Lowe SW, Schmitt EM, Smith SW, Osborne BA, Jacks T. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 1993;362:847-849 https://doi.org/10.1038/362847a0
  10. Diatchenko L, Lukyanov S, Lau YF, Siebert PD. Suppression subtractive hybridization: a versatile method for identifying differentially expressed genes. Methods Enzymol 1999;303:349-380 https://doi.org/10.1016/S0076-6879(99)03022-0
  11. Diatchenko L, Lau YC, Campbell AP, et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA 1996;93:6025-6030 https://doi.org/10.1073/pnas.93.12.6025
  12. Coleman CN. Beneficial liaisons: radiobiology meets cellular and molecular biology. Radiother Oncol 1993;28:1-15 https://doi.org/10.1016/0167-8140(93)90179-C
  13. Hallahan DE, Sukhatme VP, Sherman ML, Virudachalam S, Kufe D, Weichselbaum RR. Protein kinase C mediates x-ray inducibility of nuclear signal transducers EGR1 and JUN. Proc Natl Acad Sci USA 1991;88:2156-2160 https://doi.org/10.1073/pnas.88.6.2156
  14. Rubin P, Johnston CJ, Williams JP, McDonald S, Finkelstein JN. A perpetual cascade of cytokines postirradiation leads to pulmonary fibrosis. Int J Radiat Oncol Biol Phys 1995;33:99-109 https://doi.org/10.1016/0360-3016(95)00095-G
  15. Alwine, JC, Kemp DJ, Stark GR. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci USA 1977;74:5350-5354 https://doi.org/10.1073/pnas.74.12.5350
  16. Liang P, Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 1992;257:967-971 https://doi.org/10.1126/science.1354393
  17. Appel M, Bellstedt DU, Gresshoff PM. Differential display of eukaryotic mRNA: meeting the demands of the new millennium. J Plant Physiol 1999;154:561-570 https://doi.org/10.1016/S0176-1617(99)80227-2
  18. Kurian KM, Watson CJ, Wyllie AH. DNA chip technology. J Pathol 1999;187:267-271 https://doi.org/10.1002/(SICI)1096-9896(199902)187:3<267::AID-PATH275>3.0.CO;2-
  19. Fordor, SP, Rava RP, Huang XC, Pease AC, Holmes CP, Adams CL. Multiplexed biochemical assays with biological chips. Nature 1993;364:555-556 https://doi.org/10.1038/364555a0
  20. Velculescu VE, Zhang L, Vogelstein B, Kinzler JW. Serial analysis of gene expression. Science 1995;270:484-487 https://doi.org/10.1126/science.270.5235.484
  21. Meyerson M, Counter CM, Eaton EN, et al. Weinberg RA. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 1997;90:785-795 https://doi.org/10.1016/S0092-8674(00)80538-3
  22. Nakamura TM, Morin GB, Chapman KB, et al. Telomerase catalytic subunit homologs from fission yeast and human. Science 1997;277:955-959 https://doi.org/10.1126/science.277.5328.955
  23. Lin Z, Lim S, Viani MA, Sapp M, Lim MS. Down-regulation of telomerase activity in malignant lymphomas by radiation and chemotherapeutic agents. Am J Pathol 2001;159:711-719 https://doi.org/10.1016/S0002-9440(10)61742-7
  24. Kim HR, Kim YJ, Kim HJ, Kim SK, Lee JH. Change of telomerase activity in rectal cancer with chemoradiation therapy. J Korean Med Sci 2000;15:167-172 https://doi.org/10.3346/jkms.2000.15.2.167
  25. Harada K, Kurisu K, Arita K, et al. Telomerase activity in central nervous system malignant lymphoma. Cancer 1999;86:1050-1055 https://doi.org/10.1002/(SICI)1097-0142(19990915)86:6<1050::AID-CNCR22>3.0.CO;2-K
  26. Sawant SG, Gregoire V, Dhar S, et al. Telomerase activity as a measure for monitoring radiocurability of tumor cells. FASEB J 1999;13:1047-1054 https://doi.org/10.1096/fasebj.13.9.1047
  27. Yamamoto K, Okamoto A, Isonishi S, Ochiai K, Ohatake Y. A novel gene, CRR9, which was up-regulated in CDDP-resistant ovarian tumor cell line, was associated with apoptosis. Biochem Biophys Res Commun 2001;280:1148- 1154 https://doi.org/10.1006/bbrc.2001.4250
  28. Eichholtz-Wirth H, Marx K. Clonal variability of radiation-i nduced cisplatin resistant HeLa cells. Anticancer Res 1998;18:2989-2991
  29. Altshculer DL, Ribeiro-Neto F. Mitogenic and oncogenic properties of the small G protein Rap1b. Proc Natl Acad Sci USA 1998;95:7475-7479 https://doi.org/10.1073/pnas.95.13.7475
  30. Shoshani T, Faerman A, Mett I, et al. Identification of a novel hypoxia-inducible factor 1-responsive gene, RTP801, involved in apoptosis. Mol Cell Biol 2002;22:2283-2293 https://doi.org/10.1128/MCB.22.7.2283-2293.2002