Cancer is a genetic disease where the progression of a tumor from benign to highly aggressive is determined by complex genetic changes associated with sequential clonal expansion of tumor cells with different and new somatic mutations. These mutations frequently lead to activation or overexpression of oncogene products. In particular, ionizing radiation has been associated with mutations due to chromosomal rearrangements such as translocations, gene deletions and genomic sequence amplifications. The molecular mechanisms underlying these rearrangements are unknown. The long term goal of this project is to elucidate the molecular mechanisms of genomic sequence amplification in general and double minute chromosome formation in particular, since double minutes are particularly associated with tumor cells. This project proposes to exploit a model system where resistance to increasing levels of the drug methotrexate is due to increased copies of a double minute chromosome with a single copy of the DHFR gene. Many independent methotrexate-resistant isolates have been developed containing different double minutes. Half of these isolates were subjected to ionizing radiation prior to selection. The other half are unirradiated controls.
The specific aim of this proposal is to demonstrate that double minute chromosomes result from interstitial or terminal deletion of chromosomes, where the chromosome fragment improperly condenses at mitosis which leads to pulverization and smaller, more easily amplified double minutes. We will employ molecular biological approaches to identify the factors contributing to double minute formation.