Grant: Operating Grant

Radiation therapy and many of the commonly used cancer chemotherapeutic drugs target DNA for cytotoxicity. The subsequent cellular response to these cancer treatments in both malignant and normal cells/tissues determines the efficiency of the treatment. This response is a complex cellular process involving multiple DNA repair pathways that are specifically activated depending on the type of DNA damage. These pathways have been shown to have great potential as targets for cancer therapy and our understanding of how the cells respond to DNA damage will play a crucial role in the development of new anticancer strategies. We have developed a unique set of techniques that combines cellular fractionation protocols allowing purification of different cellular compartments, as well as mass spectrometry techniques that allows identification and quantification of thousands of proteins at the same time. We used these techniques to characterize the changes in cellular localization of proteins following treatment of human cancer cells with etoposide, a chemotherapeutic drug that causes DNA damage. Initial preliminary experiments provided unexpected new evidence showing that DNA damage alters the properties of a protein complex normally involved in DNA replication during cell division, the MCM complex. In this application, we propose to apply this novel approach combining quantitative proteomics and protein biochemistry to identify and characterize other members of the complex and elucidate the mechanism in which they play a role in the cellular response to DNA damage. It is hoped that this information will lead to a better understanding of how different classes of chemotherapy drugs can cause specific DNA damage in normal and malignant cells and identify more direct therapeutic intervention by identifying specific molecules/interactions that can serve as targets for drugs designed to selectively inhibit proliferation of cancer cells.