The goal of this research continues to be to understand the relationships between the induction and subsequent repair of the lesions in DNA of mammalian cells exposed to ionizing radiation, and radiobiological factors observed at the cell or tissue level which are thought to be important to human cancer radiotherapy. These factors would include cellular recovery mechanisms and heterogeneity in intrinsic cell sensitivity. This proposal seeks to continue an approach to these questions which has involved testing these relationships in model systems: initially in cultured mammalian cells exposed in vitro and then by extension to cells in normal and tumor tissues of experimental animals irradiated in vivo. Specifically, the following aims are proposed. The first is to develop pulsed-field gel electrophoresis (PFGE) for the analysis of radiation-induced DNA lesions. This will involve isolating the DNA probes necessary for detecting large, restriction enzyme generated, 5Mbp, DNA fragments in cultured hamster, human, and mouse cells so that PFGE can be used to investigate DNA damage and repair mechanisms in such cells. The second specific aim concerns the characterization of DNA damage using PFGE in cells from normal and tumor tissues of mice irradiated in vivo.
This aim will take advantage of the DNA probes developed under aim 1 to allow extension of PFGE technology to analysis of DNA lesions in cells irradiated in vivo. Two studies will be conducted that extend our previous observations on the differences in strand break induction and repair in various mouse tissues: (a), PFGE will be used to address whether the in vivo environment can modulate the induction of double-strand breaks (DSBs); and (b), different tissues will be tested for their relative ability to rejoin DSBS. Hopefully, the results of this research will not only enhance our understanding of the basic cellular and molecular mechanisms that influence the mammalian cell response to radiation but will also provide new tools that could be developed for predicting a human tumor's clinical response to radiotherapy.
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