A Comparison of Two 'In Vitro' Assays to Assess Radiosensitivity in Patients Receiving Radiotherapy

  • Sharon Mitchell

    Student thesis: Master's Thesis


    This thesis addresses the occurrence of normal tissue damage as a limiting factor in curative radiotherapy and the need for predictive radiosensitivity testing. Patients who display unusual sensitivity to radiation may also exhibit a poor DNA repair capacity. Consequently, in vitro assays which assess radiation-induced DNA damage and its subsequent repair may have potential in predicting a patient's response to radiotherapy. Nuclear Lysate Sedimentation (NLS) and Microgel Electrophoresis (MGE) were examined as rapid means to assess DNA damage and repair in human lymphocytes. NLS involves the centrifugation of histone-free DNA (HF-DNA) through a neutral 0-20% sucrose gradient - where DNA damage in a population of lymphocytes is detected as an upward shift in HF-DNA sedimentation distance. MGE involves the electrophoresis (in alkaline buffer) of cells embedded in agarose and lysed in situ - where DNA damage in single cells is detected as an increased migration of DNA material from the cell cavity site towards the anode, giving the appearance of a "comet" when stained and visualised using fluorescence microscopy.NLS and MGE were suitable for detecting DNA damage and repair after a clinically relevant dose and each exhibited similar dose response curves over 0.5 - 10 Gy (with MGE proving the more sensitive at doses > 2 Gy). However, the techniques showed: (i) no correlation between basal DNA damage, radiation-induced damage and repair proficiency and (ii) no significant differences in HFDNA sedimentation and electrophoretic behaviour between donor and patientgroups. Both assays detected individuals who displayed a poor DNA repair proficiency. The relationship of DNA damage and repair to radiosensitivity (determined from cell survival) was also investigated. The potential of MGE to study the molecular processes of DNA repair and predict in vivo tumour and normal tissue radiation response was discussed. The future of MGE as a simple, rapid screening test for unusual radiosensitivity was highlighted.
    Date of AwardApr 1994
    Original languageEnglish

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