Radiation-induced DNA double-strand breaks and rejoining in malignant glioma cells

Purpose: This study was performed to standardize experimental conditions fo r the quantification by pulsed-field gel electrophoresis (PFGE) of radiatio n-induced DNA double-strand breaks (dsb) and rejoining in a human malignant brain tumour xenograft model. Materials and methods: Because no correlation was found between DNA dsb ind uction or rejoining and clinical radiation response for six fresh glioblast oma (GBM) specimens, assay conditions were examined in detail. SF-767 human GBM cells were implanted into the flanks of athymic mice. Resulting tumour s were irradiated in nice, dissociated mechanically or using an enzyme cock tail, and assayed for DNA dsb induction and repair. In other experiments, e xcised tumour portions were irradiated and allowed to repair either as chun ks (> 50 mm(3) pieces), as minced tumour (similar to mm(3) pieces), or as s ingle-cell suspensions. Finally, the effect of holding excised tumours in v itro for times of up to 72 h before irradiation and assay For DNA dsb and c ell survival was studied. Results and conclusions: The method of tumour dissociation had no effect on results; however, both the configuration of specimens during irradiation a nd the in vitro maintenance time markedly affected the experimental outcome . Chunks irradiated in vitro had DNA dsb results that were very similar to those obtained when tumours were irradiated in situ, while minced pieces or single-cell suspensions resulted in steeper dose-response curves. When tum our chunks were maintained at 4 degrees C in medium, DNA dsb induction was not affected for 24h and DNA dsb rejoining remained constant for 48 h but t hen decreased. Cell survival, however, decreased continually during the 72 h in vitro maintenance time.