Radiation-Induced Apoptosis of Lymphocytes in Peripheral Blood

말초혈액 내 림프구의 방사선에 의한 아포프토시스

  • Oh, Yoon-Kyeong (Department of Therapeutic Radiology Chosun University Medical School) ;
  • Lee, Tae-Bum (Medical Research Institute Chosun University Medical School) ;
  • Nam, Taek-Keun (Department of Therapeutic Radiology Chosun University Medical School) ;
  • Kee, Keun-Hong (Medical Research Institute Chosun University Medical School) ;
  • Choi, Cheol-Hee (Medical Research Institute Chosun University Medical School)
  • 오윤경 (조선대학교 의과대학 치료방사선과학교실) ;
  • 이태범 (조선대학교 의과대학 의학연구소) ;
  • 남택근 (조선대학교 의과대학 치료방사선과학교실) ;
  • 기근흥 (조선대학교 의과대학 의학연구소) ;
  • 최철희 (조선대학교 의과대학 의학연구소)
  • Published : 2003.03.01

Abstract

Purpose : This study quantitatively evaluated the apoptosis In human peripheral blood lymphocytes using flow cytometry, and investigated the possibility of using this method, with a small amount of blood, and the time and dose dependence of radiation-induced apoptosls. Materials and Methods : Peripheral blood lymphocyes were isolated from the heparinized venous blood of 11 healthy volunteers, 8 men and 3 women, with each 10 ml of blood being divided Into IS samples. The blood lymphocytes were Irradiated using a linear accelerator at a dose rate of 2.4 Gy/min, to deliver doses of 0.5, 1, 2 and S Gy. The control samples, and Irradiated cells, were maintained in culture medium for 24, 48 and 72 hours fellowing the Irradiation. The number of apoptotic cells after the in vitro X-irradiation was measured by flow cytometry after Incubation periods of 24, 48 and 72 hours. We also observed the apoptotic cells using a DNA fragmentation assay and electron microscopy. Results : The rate oi spontaneous apoptosis increased in relation to the time interval following irradiation (1.761 ${\pm}$0.161, 3.563${\pm}$0.554, 11.098${\pm}$2.849, at 24, 48, and 72 hours). The apoptotli cells also increased In the samples irradiated with 0.5, 1, 2 and 5 Gy, In a radiation dose and time interval after Irradiation manner, with the apoptosls being too great at 72 hours after Irradiation. The dose-response curves were characterized by an Initial steep Increase In the number of apoptotic cells for Irradiation doses below 2 Gy, with a flattening of the curves as the dose approached towards 5 Gy. Conclusion :The flow cytometric assay technique yielded adequate data, and required less than 1 mL of blood. The time and dose dependence of the radiation-induced apoptosis, was also shown. It is suggested that the adequate time Interval required for the evaluation of apoptosis would be 24 to 48 hours after blood sampling.

목적 : 방사선에 의해 유도되는 림프구의 아포프토시스를 정상 성인의 말초 혈액에서 유세포계측검사로 측정할 때 소량의 혈액으로도 검사가 가능한가를 알아보고 선량 증가와 방사선조사 후 시간 경과에 따른 반응 정도를 알아보고자 본 연구를 시행하였다. 대상 및 방법 : 건강한 성인 남녀 11명을 연구 대상으로 하여 말초혈액 10 mL에서 림프구를 분리하고 이를 각각 15개로 나누어서 실험하였다. 선형가속기를 이용하여 0.5, 1, 2, 5 GV의 방사선을 조사한 후 24, 48, 72시간 동안 배양하였다. 림프구의 아포프토시스를 정량적으로 측정하기 위해 유세포계측검사를 시행하였으며, 별도로 DNA fragmentation assay와 전자현미경검사를 이용하여 아포프토시스 소견을 추가로 관찰하였다. 결과 : 방사선을 조사하지 않았을 때의 자발성 아포프토시스율(%)은 배양 후 24, 48, 72시간이 경과함에 따라 증가하는 소견을 보였다(1.761${\pm}$0.16), 3.563${\pm}$0.564, 11.098${\pm}$2.8491. 또한 0.5, 1, 2, 5 eV의 방사선을 조사하여 24, 48, 72 시간 동안 배양한 후 측정한 아포프토시스율(%)은 선량 증가와 방사선조사 후 시간 경과에 따라 점차 증가하였다 방사선조사 후 24시간 후에 0.5~1, 1~2, 2~5 Gy구간의 아포프토시스율의 증가는 비교적 저 선량 영역인 0.5~1 1~2 Gy에서 2~5 Gy 구간보다 더 큰 기울기를 보였고, 48, 72시간 후에도 0.5~l Gy구간에서 가장 큰 기울기를 보였다.결론 : 유세포계측검사는 10 mL의 혈액으로 15개의 검사 결과를 낼 수 있었으므로 한 검사당 1 mL 미만의 혈액으로도 충분히 검사할 수 있겠으며, 방사선량 증가에 따라 반응의 정도도 증가하였으며, 아포프토시스 관찰 시기는 혈액 채취 후 24시간이나 48시간 후가 적절하다고 사료된다.

Keywords

References

  1. Hall EJ. Radiobiology for the radiologist. 5th ed. Philadelphia; Lippincott Williams & Wilkins, 2000:347-349
  2. Nakayama Y, Makino S, Fukuda Y, et al. Varied effects of thoracic irradiation on peripheral lymphocyte subsets in lung cancer patients. Intern Med 1995;34:959-965 https://doi.org/10.2169/internalmedicine.34.959
  3. Nollert J, Rudat V, Daniel V, Maier H, Dietz A. Effect of primary radiochemotherapy on cellular and subcellular immunologic parameters. HNO 1999;47:1058-1062 https://doi.org/10.1007/s001060050491
  4. Maier H, Daniel V, Heimlich F, Frank C, Opelz G. Cellular immune defect caused by postoperative irradiation in patients with squamous epithelial carcinomas of the upper aerodigestive tract. HNO 1995;43:364-370
  5. Louagie H, Eijkeren M, Philippe J, Thierens H, Ridder L. Changes in peripheral blood lymphocyte subsets in patients undergoing radiotherapy. Int J Radiat Biol 1999;75:767-771 https://doi.org/10.1080/095530099140113
  6. Choi YM, Kim JK, Lee HS, Hur WJ, Kim JM. Analysis of lymphocyte subsets in peripheral blood after radiotherapy. J Korean Soc Ther Radiol Oncol 1996;14:229-236
  7. Ahn SJ, Chung WK, Nah BS, Nam TK, Cho CK, Juhng SW. Study on the immunomodulatory effect of thymulin in the patients under radiotherapy. J Korean Cancer Assoc 1995;27:790-796
  8. Oh YK, Ha CS, Park HC, et al. Lymphopenia after mediastinal irradiation in lung cancer. J Korean Soc Ther Radiol Oncol 2002;20:34-40
  9. Abrams RA, Lichter AS, Bromer RH, Minna JD, Cohen MH, Deisseroth AB. The hematopoietic toxicity of regional radiotherapy. Cancer 1985;55:1429-1435 https://doi.org/10.1002/1097-0142(19850401)55:7<1429::AID-CNCR2820550702>3.0.CO;2-4
  10. Hallan E, Blomhoff HK, Smeland EB, Lomo J. Involvement of ICE (Caspase) family in gamma-radiation-induced apoptosis of normal B lymphocytes. SCand J Immunol 1997;46:601-608 https://doi.org/10.1046/j.1365-3083.1997.d01-173.x
  11. Kern P, Keilholz L, Forster C, Seegenschmiedt MH, Sauer R, Herrmann M. In vitro apoptosis in peripheral blood mononuclear cells induced by low-dose radiotherapy displays a discontinuous dose-dependence. Int J Radiat Biol 1999;75: 995-1003 https://doi.org/10.1080/095530099139755
  12. Vral A, Cornelissen M, Thierens H, et al. Apoptosis induced by fast neutrons versus $ ^{60}CO$ gamma-rays in human peripheral blood lymphocytes. Int J Radiat Biol 1998;73:289-295 https://doi.org/10.1080/095530098142383
  13. Bareham DR, Gale KL, Maves SR, Walker JA, Morrison DP. Radiation-induced apoptosis in human lymphocytes: potential as a biological dosimeter. Health Phys 1996;71:685-691 https://doi.org/10.1097/00004032-199611000-00007
  14. Seki H, Kanegane H, Iwai K, et al. Ionizing radiation induces apoptotic cell death in human ${TcR-gamma/delta}^+$ T and natural killer cells without detectable p53 protein. Eur J Immunol 1994;24:2914-2917 https://doi.org/10.1002/eji.1830241150
  15. Telford WG, King LE, Fraker PJ. Evaluation of glucocorticoid- induced DNA fragmentation in mouse thymocytes by flow cytometry. Cell Prolif 1991;24:447-459 https://doi.org/10.1111/j.1365-2184.1991.tb01173.x
  16. Mogil RJ, Shi Y, Bissonnette RP, Bromley P, Yamaguchi I, Green DR. Role of DNA fragmentation in T cell activation-induced apoptosis in vitro and in vivo. J Immunol 1994;152:1674-1683
  17. Luff JH. Improvement in epoxy resin embedding method. J Biophysic Biochem Cytol 1961;9:409-414 https://doi.org/10.1083/jcb.9.2.409
  18. Watson ML. Staining of tissue sections for electron microscopy with heavy metals. J Biophysic Biochem Cytol 1958;226:475-479
  19. Reynolds ES. The use of lead citrate at high pH as electron opaque stain in electron microscopy. J Cell Bioi 1963;17:208-212 https://doi.org/10.1083/jcb.17.1.208
  20. Lloyd DC. Advances in cytogenetic dosimetry. In: Ricks RC, Fry SA, eds. The medical basis for radiation accident preparedness. New York: Elsevier Science. 1990:479-488
  21. Muller WU, Streffer C. Biological indicators for radiation damage. Int J Radiat Bioi 1991;59:863-873 https://doi.org/10.1080/09553009114550771
  22. Sellins KS, Cohen JJ. Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes. J Immunol 1987;139:3199-3206
  23. Delic J, Morange M, Magdelenat H. Ubiquitin pathway involvement in human lymphocyte gamma-irradiation-induced apoptosis. Mol Cell Biol 1993;13:4875-4883
  24. Cregan SP, Boreham DR, Walker PR, Brown DL, Mitchel REJ. Modification of radiation-induced apoptosis in radiation- or hyperthermia-adapted human lymphocytes. Biochem Cell Biol 1994;72:475-482 https://doi.org/10.1139/o94-064
  25. Gorczyca W, Gong J, Darzynkiewicz Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res 1993b;53:1945-1951
  26. Birnboim HC, Jevcak JJ. Fluorometric method for rapid detection of DNA strand breaks in human White blood cells produced by low doses of radiation. Cancer Res 1981:41:1889-1892
  27. Kerr JFR, Winteford CM, Harmon BV. Apoptosis: Its significance in cancer and cancer therapy. Cancer 1994;73:2013-2026 https://doi.org/10.1002/1097-0142(19940415)73:8<2013::AID-CNCR2820730802>3.0.CO;2-J
  28. Eidus LK, Korystov YN, Dobrovinskaja OR, Shaposhnikova W. The mechanism of radiation-induced interphase death of lymphoid cells: a new hypothesis. Radiat Res 1990;123:17-21 https://doi.org/10.2307/3577652
  29. Hertveldt K, Philippe J, Thierens H, Cornelissen M, Vral A, Ridder LD. Flow cytometry as a quantitative and sensitive method to evaluate low dose radiation induced apoptosis in vitro in human peripheral blood lymphocytes. Int J Radiat Biol 1997;71:429-433 https://doi.org/10.1080/095530097144049
  30. oiner MC, Denekamp J, Maughan RL. The use of 'top-up' experiments to investigate the effect of very small doses per fraction in mouse skin. Int J Radiat Bioi Relat Stud Phys Chem Med 1986;49:565-580.
  31. Parkins CS, Fowler JF. The linear quadratic fit for lung function after irradiation with X-rays at smaller doses per fraction than 2 Gy. Br J Cancer Suppl 1986;7:320-323.
  32. Joiner MC, Johns H. Renal damage in the mouse: the response to very small doses per fraction. Radiat Res 1988;114:385-398 https://doi.org/10.2307/3577233