Expression of TIMP1, TIMP2 Genes by Ionizing Radiation

이온화 방사선에 의한 TIMP1, TIMP2 유전자 발현 측정

  • Park Kun-Koo (Department of Molecular Genetics, Asan Institute for Life Science, College of Medicine) ;
  • Jin Jung Sun (Department of Molecular Genetics, Asan Institute for Life Science, College of Medicine) ;
  • Park Ki Yong (Department of Molecular Genetics, Asan Institute for Life Science, College of Medicine) ;
  • Lee Yun Hee (Department of Molecular Genetics, Asan Institute for Life Science, College of Medicine) ;
  • Kim Sang Yoon (Department of Otolaryngology, Asan Medical Center, College of Medicine, University of Ulsan) ;
  • Noh Young Ju (Department of Radiation Oncology, College of Medicine, University of Ulsan) ;
  • Ahn Seung Do (Department of Radiation Oncology, College of Medicine, University of Ulsan) ;
  • Kim Jong Hoon (Department of Radiation Oncology, College of Medicine, University of Ulsan) ;
  • Choi Eun Kyung (Department of Radiation Oncology, College of Medicine, University of Ulsan) ;
  • Chang Hyesook (Department of Radiation Oncology, College of Medicine, University of Ulsan)
  • 박건구 (아산생명과학연구소 분자유전연구실) ;
  • 진정선 (아산생명과학연구소 분자유전연구실) ;
  • 박기영 (아산생명과학연구소 분자유전연구실) ;
  • 이연희 (아산생명과학연구소 분자유전연구실) ;
  • 김상윤 (울산대학교 의과대학 서울중앙병원 이비인후과) ;
  • 노영주 (울산대학교 의과대학 서울중앙병원 방사선종양학과) ;
  • 안승도 (울산대학교 의과대학 서울중앙병원 방사선종양학과) ;
  • 김종훈 (울산대학교 의과대학 서울중앙병원 방사선종양학과) ;
  • 최은경 (울산대학교 의과대학 서울중앙병원 방사선종양학과) ;
  • 장혜숙 (울산대학교 의과대학 서울중앙병원 방사선종양학과)
  • Published : 2001.06.01

Abstract

Purpose : Expression of TIMP, intrinsic inhibitor of MMP, is regulated by signal transduction in response to genotoxins and is likely to be an important step in metastasis, angiogenesis and wound healing after ionizing radiation. Therefore, we studied radiation mediated TIMP expression and its mechanism in head and neck cancer cell lines. Materials and Methods : Human head and neck cancer cell lines established at Asan Medical Center were used and radiosensitivity $(D_0)$, radiation cytotoxicity and metastatic potential were measured by clonogenic assay, n assay and invasion assay, respectively. The conditioned medium was prepared at 24 hours and 48 hours after 2 Gy and 10 Gy irradiation and expression of TIMP protein was measured by Elisa assay with specific antibodies against human TIMP. hTIMP1 promoter region was cloned and TIMP1 luciferase reporter vector was constructed. The reporter vector was transfected to AMC-HN-1 and -HN-9 cells with or without expression vector Ras, then the cells were exposed to radiation or PMA, PKC activator. EMSA was peformed with oligonucleotide (-59/-53 element and SP1) of TIMP1 promoter. Results : $D_0$ of HN-1, -2, -3, -5 and -9 cell lines were 1.55 Gy, 1.8 Gy, 1.5 Gt, 1.55 Gy and 2.45 Gy respectively. n assay confirmed cell viability, over $94\%$ at 24hrs, 48hrs after 2 Gy irradiation and over 73% after 10 Gy irradiation. Elisa assay confirmed that cells secreted TIMP1, 2 proteins continuously. After 2 Gy irradiation, TIMP2 secretion was decreased at 24hrs in HN-1 and HN-9 cell lines but after 10 Gy irradiation, it was increased in all cell lines. At 48hrs after irradiation, it was increased in HN-1 but decreased in HN-9 cells. But the change in TIMP secretion by RT was mild. The transcription of TIMP1 gene in HN-1 was induced by PMA but in HN-9 cell lines, it was suppressed. Wild type Ras induced the TIMP-1 transcription by 20 fold and 4 fold in HN-1 and HN-9 respectively. The binding activity to -59/-53, AP1 motif was increased by RT, but not to SP1 motif in both cell lines. Conclusions : We observed the difference of expression and activity of TIMPs between radiosensitive and radioresistant cell line and the different signal transduction pathway between in these cell lines may contribute the different radiosensitivity. Further research to investigate the radiation response and its signal pathway of TIMPs is needed.