Incorporation of non-natural (fraudulent) nucleoside analogs into DNA is an effective chemotherapeutic strategy used in many cancer protocols, but a significant proportion of patients do not benefit from these agents. The initial steps of cellular response to incorporation of fraudulent nucleosides into DNA, especially incorporation that does not directly destroy the integrity of DNA, remain poorly characterized. The long-term objective of this project is to characterize cellular components involved in recognition of chemotherapy-induced DNA damage and to define how these components differ between patients. The efficacy of chemotherapy can then be improved by finding a rational way to modulate and redirect the behavior of these components. The Pl's preliminary studies have shown that non-natural nucleosides (e.g., cytarabine, 5-fluorouridine, deoxythioguanosine) incorporated into the DNA of leukemic cells are bound by a nuclear complex containing high mobility group proteins 1 and 2 (HMGBI/2), heat shock cognate protein 70 (HSC70), disulfide isomerase ERp60, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The goal of the proposed study is to characterize the role of this putative DNA damage sensor in the dynamic cellular response to genotoxic insult caused by incorporation of fraudulent nucleosides into DNA. The roles of the individual components of the sensor protein complex in the integral cellular response (changes in gene expression, protein modification, and protein localization) will be explored by using global and real-time techniques.
In Aim 1, cells proficient and deficient in components of the complex will be compared for their sensitivity to drugs that exert their cytotoxicity via incorporation into DNA.
In Aim 2, the functional role of the complex and its individual components will be tested by using genomics and proteomics approaches.
In Aim 3, the intranuclear localization and molecular proximity of the components will be assessed in the living cells.
In Aim 4, the DNA of pediatric patients will be analyzed for functionally significant mutations and polymorphsims in the genes that encode the components of the complex. Characterization of a mechanism by which cells detect incorporation of fraudulent nucleosides into DNA will provide further insights into the therapeutic effect of this class of anticancer drugs and establish a basis for rational drug design and for the search for molecules that redirect the behavior of these targets.
