Cell survival and DNA damage in fibroblasts following irradiation in vivo

The in vitro radiosensitivity of fibroblasts derived from patients undergoing radiotherapy has been investigated by a number of groups for possible prediction of normal tissue effects. Some studies have suggested a weak correlation between radiosensitivity and late normal tissue effects, such as fibrosis, but others have not. One possible reason may be that radiosensitivity in vivo is not always reflected by radiosensitivity in vitro. We are investigating whether heterogeneity in the normal tissue response of individual soft tissue sarcoma patients receiving pre-operative radiotherapy can be assessed by determining the number of micronuclei (DNA damage) in fibroblasts obtained and assayed directly from their skin after irradiation. The micronuclei are counted in binucleate cells in primary cultures of the fibroblasts at 72 hrs after treatment with cytochalasin B. This endpoint is dose responsive and in rats we have demonstrated that the presence of micronuclei can be detected months after irradiation. The assay can thus provide data for fibroblasts irradiated with fractionated doses in situ in tissue or for fibroblasts irradiated in vitro following outgrowth from the tissue. We have demonstrated that fibroblasts obtained directly from irradiated skin at surgery (approx 5-6 weeks after the end of radiotherapy) from a small number of soft tissue sarcoma patients given nominally similar preoperative irradiation (50Gy in 20 fractions to the tumor) show significant variability in response. Estimates of radiation dose to the skin have suggested that much of this variability may be dose related but further studies are underway with skin samples from regions given carefully measured doses. Comparisons with micronucleus formation for fibroblasts from the same patient irradiated in vitro are also underway. Our results demonstrate that DNA damage in fibroblasts in irradiated tissue can be assessed directly ex vivo and that DNA damage can be detected at 1-2 months after irradiation