Nuclear Medicine and Molecular Imaging

  • 제42권5호
  • /
  • Pages.383-393
  • /
  • 2008
  • /
  • 1869-3474(pISSN)
  • /
  • 1869-3482(eISSN)
대한핵의학회 (The Korean Society of Nuclear Medicine)
권호 표지

조직 특이 발현 Sodium Iodide Symporter 유전자 이입에 의한 방사성옥소 간암세포 치료와 광학영상을 이용한 치료효과 평가

Radioiodine Therapy of Liver Cancer Cell Following Tissue Specific Sodium Iodide Symporter Gene Transfer and Assessment of Therapeutic Efficacy with Optical Imaging

  • 장병국 (경북대학교 의과대학 핵의학교실) ;
  • 이유라 (경북대학교 의과대학 핵의학교실) ;
  • 이용진 (경북대학교 의과대학 핵의학교실) ;
  • 안손주 (경북대학교 의과대학 핵의학교실) ;
  • 류민정 (경북대학교 의과대학 핵의학교실) ;
  • 윤선미 (경북대학교 의과대학 핵의학교실) ;
  • 이상우 (경북대학교 의과대학 핵의학교실) ;
  • 유정수 (경북대학교 의과대학 핵의학교실) ;
  • 조제열 (경북대학교 치과대학 구강생화학교실) ;
  • 이재태 (경북대학교 의과대학 핵의학교실) ;
  • 안병철 (경북대학교 의과대학 핵의학교실)
  • Jang, Byoung-Kuk (Department of Nuclear Medicine, School of Medicine, Kyungpook National University) ;
  • Lee, You-La (Department of Nuclear Medicine, School of Medicine, Kyungpook National University) ;
  • Lee, Yong-Jin (Department of Nuclear Medicine, School of Medicine, Kyungpook National University) ;
  • Ahn, Sohn-Joo (Department of Nuclear Medicine, School of Medicine, Kyungpook National University) ;
  • Ryu, Min-Jung (Department of Nuclear Medicine, School of Medicine, Kyungpook National University) ;
  • Yoon, Sun-Mi (Department of Nuclear Medicine, School of Medicine, Kyungpook National University) ;
  • Lee, Sang-Woo (Department of Nuclear Medicine, School of Medicine, Kyungpook National University) ;
  • Yoo, Jeong-Soo (Department of Nuclear Medicine, School of Medicine, Kyungpook National University) ;
  • Cho, Je-Yeol (Department of Biochemistry, School of Dentistry, Kyungpook National University) ;
  • Lee, Jae-Tae (Department of Nuclear Medicine, School of Medicine, Kyungpook National University) ;
  • Ahn, Byeong-Cheol (Department of Nuclear Medicine, School of Medicine, Kyungpook National University)
  • 발행 : 2008.10.31
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

목적: 조직 특이 프로모터를 이용하면 특정 암조직내에서만 원하는 치료유전자를 발현시킬 수 있다. 나트륨 옥소 공동 수송체(sodium iodide symporter: NIS) 유전자는 옥소를 섭취하는 특성을 가져 방사성옥소를 이용한 치료용 유전자로 사용될 수 있다. 광학 영상용 유전자인 luciferase (Luc) 유전자를 세포에 이입하면 비침습적으로 유전자가 이입된 세포의 상태를 평가할 수 있다. 본 연구는 간암 특이성을 나타내는 AFP 프로모터에 의해 발현이 조절되는 NIS유전자와 CMV프로모터에 의해 발현되는 Luc유전자를 간암세포에 이입하여 NIS유전자 이입에 의한 방사성옥소 유전자치료의 효과를 알아보고, 종양사멸 정도를 광학 리포터 유전자 발현으로 알아보고자 하였다. 대상 및 방법: AFP enhancer와 GSTP 프로모터를 연결하여 AFP프로모터를 제작하였으며 이를 NIS유전자와 연결하였다. 또한 CMV 프로모터에 조절 받는 Luc 유전자를 동시에 삽입하여 AFP-NIS-CMV-Luc 유전자 발현 벡터를 생산하였다. 실험 대상 세포주로는 간암세포주인 HepG2와 Huh-7 세포와 사람 대장암세포주인 HCT-15 세포를 이용하였다. AFP-NIS-CMV-Luc 발현벡터를 Liposome을 이용해 실험대상 세포주 내로 이입하였으며, 방사성옥소 섭취율과 방사성옥소의 유출량을 측정하였다. 또한 Luciferase 발현 정도를 luminometer로 측정하였으며, clonogenic assay를 통하여 I-131에 대한 세포주에 따른 사멸효과 차이를 알아보았다. AFP-NIS-CMV-Luc 유전자 이입 세포주를 누드마우스에 대퇴부 피하에 주입하여 I-131 축적여부를 감마카메라 영상을 획득하였다. 결과: AFP-NIS-CMV-Luc 유전자 발현 벡터를 제작하였다. AFP-NIS-CMV-Luc 유전자가 이입된 HepG2와 Huh-7 세포의 방사성옥소 섭취율은 유전자 이입이 되지 않은 대조군 HepG2와 Huh-7 세포에 비하여 높았으며, $KClO_4$를 처리시 옥소 섭취가 저해되었다. 대장암 세포주인 HCT-15세포에 AFP-NIS-CMV-Luc유전자를 이입 시 방사성옥소의 섭취률은 증가되지 않았다. 30분간 방사성옥소를 섭취시킨 AFP-NIS-Luc 유전자가 이입된 HepG2와 Huh-7 세포에서의 방사성옥소의 유출반감기는 약 4분과 6분으로 각각 나타났다. AFP-NIS-CMV-Luc 유전자가 이입된 HepG2, Huh-7세포의 Luc 유전자의 발현은 241, 441 $RLU/2\;{\times}\;10^5$ cells로 나타났으며, 대조군 HepG2와 Huh구세포에서의 Luc 유전자의 발현은 74, $RLU/2\;{\times}\;10^5$ cells로 나타났다. HCT-15 세포는 AFP-NIS-CMV-Luc 유전자 이입에 따라 I-131에 의한 세포 사멸능이 증가되지 않았으나, HepG2 및 Huh-7 세포는 FP-NIS-CMV-Luc 유전자 이 입에 따라 I-131에 의한 세포 사멸능이 증가되었으며, Huh-7세포의 경우 0.5mCi의 I-131을 투여한 경우 모든 세포가 사멸하였다. AFP-NIS-CMV-Luc 유전자가 이입된 Huh-7 세포수가 많을수록 방사성옥소 섭취율이 증가하며 luciferase활성도도 높게 나타났다. AFP-NIS-CMV-Luc 유전자가 이입된 Huh-7 세포를 이식한 누드마우스에 I-131 감마카메라 영상에서 종양이식부위에 방사능 축적을 관찰 할 수 있었다. 결론: AFP프로모터의 의하여 NIS유전자가 발현되며, CMV프로모터에 의한 Luc 유전자가 발현되는 벡터를 제작하였으며, 이 벡터를 이입한 경우 간암세포에서만 I-131의 세포 독성이 증가하는 효과를 나타내었다. 또한 Luc유전자를 이용하여 비침습적인 광학 영상으로 세포사멸 효과를 확인할 수 있었다. 간암특이 프로모터에 조절되는 치료 유전자와 광학리포터 유전자를 한 벡터에 동시에 이입하면 간암 특이 유전자 치료와 그 치료효과를 비침습적으로 평가할 수 있을 것으로 생각된다.

Purpose: Cancer specific killing can be achieved by therapeutic gene activated by cancer specific promotor. Expression of sodium iodide symporter (NIS) gene causes transportation and concentration of iodide into the cell, therefore radioiodine treatment after NIS gene transfer to cancer cell could be a form of radionuclide gene therapy. luciferase (Luc) gene transfected cancer cell can be monitored by in vivo optical imaging after D-luciferin injection. Aims of the study are to make vector with both therapeutic NIS gene driven by AFP promoter and reporter Luc gene driven by CMV promoter, to perform hepatocellular carcinoma specific radiodiodine gene therapy by the vector, and assessment of the therapy effect by optical imaging using luciferase expression. Materials and Methods: A Vector with AFP promoter driven NIS gene and CMV promoter driven Luc gene (AFP-NIS-CMV-Luc) was constructed. Liver cancer cell (HepG2, Huh-7) and non liver cancer cell (HCT-15) were transfected with the vector using liposome. Expression of the NIS gene at mRNA level was elucidated by RT-PCR. Radioiodide uptake, perchlorate blockade, and washout tests were performed and bioluminescence also measured by luminometer in these cells. In vitro clonogenic assay with 1-131 was performed. In vivo nuclear imaging was obtained with gamma camera after 1-131 intraperitoneal injection. Results: A Vector with AFP-NIS-CMV-Luc was constructed and successfully transfected into HepG2, Huh-7 and HCT-15 cells. HepG2 and Huh-7 cells with AFP-NIS-CMV-Luc gene showed higher iodide uptake than non transfected cells and the higher iodide uptake was totally blocked by addition of perchlorate. HCT-15 cell did not showed any change of iodide uptake by the gene transfection. Transfected cells had higher light output than control cells. In vitro clonogenic assay, transfected HepG2 and Huh-7 cells showed lower colony count than non transfected HepG2 and Huh-7 cells, but transfected HCT-15 cell did not showed any difference than non transfected HCT-15 cell. Number of Huh-7 cells with AFP-NIS-CMV-Luc gene transfection was positively correlated with radioidine accumulation and luciferase activity. In vivo nuclear imaging with 1-131 was successful in AFP-NIS-CMV-Luc gene transfected Huh-7 cell xenograft on nude mouse. Conclusion: A Vector with AFP promoter driven NIS and CMV promoter driven Luc gene was constructed. Transfection of the vector showed liver cancer cell specific enhancement of 1-131 cytotoxicity by AFP promoter, and the effect of the radioiodine therapy can be successfully assessed by non-invasive luminescence measurement.

키워드

참고문헌

  1. Carrasco N. Iodide transport in the thyroid gland. Biochem Biophys Acta 1993;1154:65-82 https://doi.org/10.1016/0304-4157(93)90017-I
  2. Dai G, Levy O, Carrasco N. Cloning and characterization of the thyroid iodide transporter. Nature 1996;379:458-60 https://doi.org/10.1038/379458a0
  3. Smanik PA, Liu Q, Furminger TL, Ryu K, Xing S, Mazzaferri EL, Jhiang SM. Cloning of the human sodium iodide symporter. Biochem Biophys Res Commun 1996; 226:339-45 https://doi.org/10.1006/bbrc.1996.1358
  4. Smanik PA, Ryu KY, Theil KS, Mazzaferri EL, Jhiang SM. Expression, exon-intron organization, and chromosome mapping of the human sodium iodide symporter. Endocrinology 1997;138: 3555-8 https://doi.org/10.1210/en.138.8.3555
  5. Spitzweg C, Morris JC. Approaches to gene therapy with sodium/iodide symporter. Exp Clin Endocrinol Diabetes 2001; 109:56-9 https://doi.org/10.1055/s-2001-11020
  6. Mazzaferri EL. An overview of the management of papillary and follicular thyroid carcinoma. Thyroid 1999;9:421-7 https://doi.org/10.1089/thy.1999.9.421
  7. Spitzweg C, Harrington KJ, Pinke LA, Vile RG, Morris JC. Clinical review 132: The sodium iodide symporter and its potential role in cancer therapy. J Clin Endocrinol Metab 2001; 86:3327-35 https://doi.org/10.1210/jc.86.7.3327
  8. Dingli D, Russell SJ, Morris JC 3rd. In vivo imaging and tumor therapy with the sodium iodide symporter. J Cell Biochem 2003;90:1079-86 https://doi.org/10.1002/jcb.10714
  9. Hart IR. Tissue specific promoters in targeting systemically delivered gene therapy. Semin Oncol 1996;23:154-8
  10. Adams JY, Johnson M, Sato M, Berger F, Gambhir SS, Carey M, Iruela-Arispe ML, Wu L. Visualization of advanced human prostate cancer lesions in living mice by a targeted gene transfer vector and optical imaging. Nat Med 2002;8:891-7 https://doi.org/10.1038/nm743
  11. Cengic N, Baker CH, Schutz M, Goke B, Morris JC, Spitzweg C. A novel therapeutic strategy for medullary thyroid cancer based on radioiodine therapy following tissue-specific sodium iodide symporter gene expression. J Clin Endocrinol Metab 2005;90:4457-64 https://doi.org/10.1210/jc.2004-2140
  12. Hadjantonakis AK, Dickinson ME, Fraser SE, Papaioannou VE. Technicolour transgenics: imaging tools for functional genomics in the mouse. Nat Rev Genet 2003;4:613-25
  13. Kang JH, Lee DS, Paeng JC, Lee JS, Kim YH, Lee YJ, Hwang DW, Jeong JM, Lim SM, Chung JK, Lee MC. Development of a sodium/iodide symporter-transgenic mouse for imaging of cardiomyocyte-specific reporter gene expression. J Nucl Med 2005;46:479-83
  14. Serganova I, Blasberg R. Reporter gene imaging: potential impact on therapy. Nucl Med Biol 2005;32:763-80 https://doi.org/10.1016/j.nucmedbio.2005.05.008
  15. Cassidy PJ, Radda GK. Molecular imaging perspectives. J R Soc Interface 2005;2:133-44 https://doi.org/10.1098/rsif.2005.0040
  16. Nakabayashi H, Koyama Y, Suzuki H, Li HM, Sakai M, Miura Y, Wong NC, Nishi S. Functional mapping of tissue-specific elements of the human alpha-fetoprotein gene enhancer. Biochem Biophys Res Commun 2004;318:773-85 https://doi.org/10.1016/j.bbrc.2004.04.096
  17. 통계청. 2002년 사망원인 통계 결과. 통계청 홈페이지 (www. nso.go.kr)
  18. 대한민국 보건복지부, 국립암센터. 한국중앙암등록사업 23차 2002년도 보고서. 2003
  19. Lai EC, Fan ST, Lo CM, Chu KM, Liu CL, Wong J. Hepatic resection for hepatocellular carcinoma. An audit of 343 patients. Ann Surg 1995;221:291-8 https://doi.org/10.1097/00000658-199503000-00012
  20. Colleoni M, Audisio RA, De Braud F, Fazio N, Martinelli G, Goldhirsch A. Practical consideration in the treatment of hepatocellular carcinoma. Drugs 1998;55:367-82 https://doi.org/10.2165/00003495-199855030-00004
  21. Lo HW, Day CP, Hung MC. Cancer-specific gene therapy. Adv Genet 2005;54:235-55
  22. Dilber MS, Gahrton G. Suicide gene therapy: possible applications in haematopoietic disorders. J Intern Med 2001;249:359-67 https://doi.org/10.1046/j.1365-2796.2001.00808.x
  23. JY Cho. Molecular imaging using sodium iodide symporter. Korean J Nucl Med 2004;38:152-160
  24. Scholz IV, Cengic N, Baker CH, Harrington KJ, Maletz K, Bergert ER, Vile R, Göke B, Morris JC, Spitzweg C. Radioiodine therapy of colon cancer following tissue-specific sodium iodide symporter gene transfer. Gene Ther 2005;12:272-80 https://doi.org/10.1038/sj.gt.3302410
  25. Dwyer RM, Bergert ER, O'Connor MK, Gendler SJ, Morris JC. Adenovirus-mediated and targeted expression of the sodiumiodide symporter permits in vivo radioiodide imaging and therapy of pancreatic tumors. Hum Gene Ther 2006;17:661-8 https://doi.org/10.1089/hum.2006.17.661
  26. Haberkorn U, Kinscherf R, Kissel M, Kubler W, Mahmut M, Sieger S, Eisenhut M, Peschke P, Altmann A. Enhanced iodide transport after transfer of the human sodium iodide symporter gene is associated with lack of retention and low absorbed dose. Gene Ther 2003;10:774-80 https://doi.org/10.1038/sj.gt.3301943
  27. Willhauck MJ, Sharif Samani BR, Klutz K, Cengic N, Wolf I, Mohr L, Geissler M, Senekowitsch-Schmidtke R, Goke B, Morris JC, Spitzweg C. Alpha-fetoprotein promoter-targeted sodium iodide symporter gene therapy of hepatocellular carcinoma. Gene Ther 2008;15:214-23 https://doi.org/10.1038/sj.gt.3303057
  28. Elisei R, Vivaldi A, Ciampi R, Faviana P, Basolo F, Santini F, Traino C, Pacini F, Pinchera A. Treatment with drugs able to reduce iodine efflux significantly increases the intracellular retention time in thyroid cancer cells stably transfected with sodium iodide symporter complementary deoxyribonucleic acid. J Clin Endocrinol Metab 2006;91:2389-95 https://doi.org/10.1210/jc.2005-2480
  29. Chung JK. General Perspectives for Molecular Nuclear Imaging. Korean J Nucl Med 2004;38:111-114
  30. Weissleder R. A clearer vision for in vivo imaging. Nat Biotechnol 2001;19:316-7 https://doi.org/10.1038/86684
  31. Wu JC, Sundaresan G, Iyer M, Gambhir SS. Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice. Mol Ther 2001;4:297-306 https://doi.org/10.1006/mthe.2001.0460