OXIDIZED DNA BASES AS BIOMARKERS OF TUMOR PROGRESSION

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. An early theory of carcinogenesis proposes that DNA is damaged by environmental factors, which lead to changes in the DNA code. These changes are called mutations. Once enough mutations accumulate, cells can no longer function properly, and cancer arises. The facts that smoking causes lung cancer, and that sunlight exposure causes skin cancer support this theory. However, such a direct environmental link is not evident for breast cancer in humans. A more recent theory suggests that the metabolism of oxygen produces free radicals that cause carcinogenic DNA damage. In order to validate the latter theory, it is necessary to measure the products of free radical damage in cells. Damage to cells caused by reactive oxygen is called oxidation. In some breast cancers, increased levels of oxidative DNA damage have been associated with tumor progression. However, the most commonly used marker of DNA oxidation, called 8-oxoG, is known to be chemically unstable, and is difficult to measure accurately. Importantly, 8-oxoG is itself easily oxidized to form several secondary oxidation products. We propose to develop new technology that will allow detection of the products in rat and human breast cancer cells. Such an assay can be used as a foundation for making diagnostics for use in future human clinical and epidemiological studies. An additional benefit of these biomarkers will be their eventual application to chemoprevention in animals and humans. AMS is a desirable technique due to its sensitivity and robustness as a method for high-throughput measurement. In comparison, less quantitative MS methods such as FT-MS lack the precision of AMS for routine operations, especially at the expected level of a few DNA adducts per cell. The combination of these two techniques will allow for the fractionation and detection of protein polypeptides at or below attomole levels, which is necessary for small forensic samples. This project is complementary aims to those of Project 3 of the AMS RR grant, particularly with regard to the use of AMS to further understand cancer etiology using DNA-based markers.