Accumulating evidence suggests that sustained oxidative stress affects many biochemical and molecular processes and is responsible for altered metabolic functions in various pathophysiological conditions including cancer, cardiovascular disease and aging. ? ? In Phase I, we hypothesized that an immunoassay for the DNA binding activity of NF-?B, a redox-sensitive transcription factor, could serve as a useful biomarker for cellular oxidative stress provided a rapid, sensitive and reliable assay for its measurement was available. To test this hypothesis, we (1) developed a rapid and sensitive chemiluminescent immunoassay (CI) for quantitative measurement of NF-?B (2) validated the CI by comparison with conventional electrophoretic mobility shift assay EMSA) (3) validated the utility of NF-?B as a marker for cellular oxidative stress in nuclear and cytosolic extracts from prostrate cancer cells and from human lymphocytes and (4) performed a pilot study (n=20) in which values for the DNA-binding activity of NF-?B in peripheral lymphocytes were compared with urinary isoprostanes (known biomarkers for systemic oxidative stress). ? ? In Phase II, we plan to achieve the following goals: (1) further optimization of parameters for CI of the DNA-binding activity of transcription factors, using NF-?B as the model. (2) develop a quantitative transcription factor (TF) mini-immunoarray using multiple redox-sensitive transcription factors to obtain a 'signature"""""""" of cellular oxidative stress status (3) establish selected redox -sensitive transcription factors as markers for cellular oxidative stress by quantification of the DNA binding activity of TFs in human subjects with extensive smoking history, in conjunction with an existing NIH funded clinical trial. (4) compare DNA binding activity of TFs with other putative markers of cellular oxidative stress including oxidized protein in tissue samples derived from an existing NIH SBIR study. ? ?
