This project aims to (1) determine the mutation rate (f) in cells from human blood samples; and (2) determine whether f can be reduced by anti-oxidants. f is critical in the risk of developing cancer, but 60 years after f was first measured in bacteria, there still has been no test to measure this parameter in human cells. In preliminary data f was measured in human B-lymphoblastoid cell lines (BLCLs), using the PIG-A gene (Xp22.1) as a sentinel. A broad spectrum of mutations inactivate PIG-A, and rare cells with the mutant phenotype--lack of surface expression of glycosylphosphatidylinositol (GPI)-Iinked proteins (e.g. CD48, CD55, and CD59)--are readily identified by flow cytometry. Pre-existing PIG-A mutants are first eliminated from the population by sorting, followed by in vitro expansion, and then determination of the frequency (f) of cells lacking GPl-linked proteins, f is calculated as f = f/d, where d = # of cell divisions in vitro. In normal BLCLs f ranged from 2.5 to 29.6 x 10-7 mutations/cell division, and was increased in genetic cancer predisposition syndromes and by exposure to radiation. While BLCLs can be produced from any individual, it is critical to measure f in primary cells. Here it is hypothesized that: (a) f can be reproducibly measured in T lymphocytes; and (b) that f is amenable to pharmacologic modulation. After optimization of the reproducibility of the assay, f will be measured in T cells derived from blood samples from normal individuals and stimulated by lectin, interleukin-2, and anti-CD3/anti-CD28. The effects of antioxidants will be determined by incubating cells with dehydroascorbic acid, which greatly increases intracellular ascorbic acid (AA) and avoids undesired in vitro artefacts. Preliminary data suggests that this assay will be as reliable as with BLCLs and that AA will reduce f. These studies will begin to elucidate the relationship between f and cancer risk and provide a highly relevant in vitro parameter with which to evaluate chemoprevention.
