Amplification of oncogenes is a very frequent phenomenon in human tumors, and the amplification of drug efflux transporters is a major cause of multi-drug resistance in cancer. We have shown by fluorescence in situ hybridization (FISH) that amplification of the DHFR gene in CHO cells is usually initiated by chromosome breaks. Two additional important observations have recently been reported: 1) the tumor suppressor, p53, is apparently involved in a damage-sensing signal transduction pathway that prevents replication until damage can be repaired, and 2) cells with wild-type p53 cannot undergo amplification. These data invoke a unifying model in which tumor cells that have no active p53 replicate through single-strand lesions, leading to chromosome breaks which can then initiate amplification. However, our data does not address amplification mediated by double minute chromosomes, as occurs in human and murine cells. In addition, information is still lacking relative to subsequent amplification events that lead to higher amplicon copy numbers. Furthermore, the simple model outlined above is not consistent with the observation that CHO cells, which can amplify their DNA (implying a lack of wild-type p53) nevertheless exhibit the damage-arrest phenotype (implying the presence of p53 activity).
Specific aims of the proposed project are: 1) to use two-color FISH analysis to obtain a detailed picture of subsequent rearrangements that occur after initial chromosome breaks; 2) to determine whether amplification of the DHFR gene is also initiated by chromosome breaks during spontaneous amplification and after DNA damaging treatments that increase the frequency of amplification; 3) to test whether amplification in other cell lines in which the amplicons are carried on double minutes is also initiated by chromosome breaks; and 4) to determine the status of p53 in appropriate Chinese hamster cells and to determine whether an acute change in p53 activity can regulate a cell's ability to amplify the DHFR gene.
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