A model system will be developed to study the effects of X- irradiation-induced chromosomal rearrangements on tumor cell clonal variability specifically with respect to the development of resistance to chemotherapeutic agents by gene amplification, and other mutations. During cancer treatment, the emergence of drug resistant clonal variants in the population of tumor cells is a major cause of failure of chemotherapy. The general hypothesis to be tested is that chromosome breakage and rearrangement facilitate induction of resistance to chemotherapeutic agents. To study this process, novel cell fusion techniques will be employed to: 1) Test whether ionizing radiation-induced translocations of the chromosomal region encompassing the dihydrofolate reductase (DHFR) gene (associated with resistance to the chemotherapeutic agent methotrexate (MTX) can lead to alterations in the frequency of DHFR gene amplification. Preliminary evidence indicates that CHO cell lines carrying these chromosomal rearrangements become resistant to MTX by gene amplification much more frequently (several orders of magnitude) than do normal CHO cells. 2) Test whether these differences are due to the new chromosomal location of the translocated DHFR gene, or to mutations either to the translocated chromosomal fragment or to other regions. 3) Test whether specific treatment combinations can be employed to decrease the fraction of clonal variants in tumor populations. The procedures will employ novel techniques based on cell fusion that allow the generation of unlimited numbers of CHO cell strains differing only in the chromosomal location of a CHO chromosome fragment carrying the DHFR gene. Gene amplification will be selected by resistance to methotrexate, quantified by hybridization analysis (Southern), and characterized by orthogonal field gel electrophoresis. In-situ hybridization will locate the introduce X- ray fragment containing the DHFR gene. Both tissue culture monolayer and multicellular spheroids will be used to monitor the effects of different treatment combinations on percentage of clonal variants in tumor cell populations.
|Ahn, S Y; Nevaldine, B; Hahn, P J (1991) Direct measurement by pulsed-field gel electrophoresis of induction and rejoining of X-ray-induced double-strand breaks in cultured mouse cells. Int J Radiat Biol 59:661-75|