SELDI-TOF-MS Analysis of Transcriptional Activation Protein Binding to Response Elements Regulating Carcinogenesis Enzymes

The risk from chemical carcinogens and environmental toxins is dependent on the metabolic balance between bioactivation and detoxification enzymes. Therefore, agents that alter enzyme expression are critical factors in toxicity. Enhancement or suppression of enzyme activities through gene expression is in part regulated by interactions between specific DNA promoter response elements and specific transcription proteins. DNA-protein interactions are dependent upon translocation of proteins from the cytoplasm to the nucleus and the affinity of proteins for binding to transcription promoter sequences. A key factor in both processes is the intracellular redox state, which influences protein-protein interactions and protein-DNA binding and can be altered by exposure to electrophiles, antioxidants and oxidative stress. Oxidative stress levels can be readily detected by measurable effects on the intracellular glutathione (GSH): glutathione disulfide redox potential, the major intracellular redox buffer. Alterations in the GSH redox pool can directly affect enzyme activity by altering disulfide bonds in the transcription factors regulating enzyme expression. These may affect: 1) specific DNA-protein and protein-protein interactions, 2) cyst(e)ine redox state within transcriptional proteins and 3) translocation of transcription proteins from cytoplasmic to nuclear compartments within the cell. The studies reported here are designed to investigate the relative changes in enzyme expression in response to cellular redox potential changes using the new proteomics technology of surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI). Treatment of HeLa and HT29 human cell lines to increase the expression of enzymes that are upregulated by oxidative stress was used as a model system to determine the efficacy of the SELDI technology in measuring changes in transcriptional protein binding to transcriptional response elements. An important goal is to determine whether the SELDI will allow simultaneous studies of multiple transcriptional protein-DNA interactions in response to controlled oxidative stress. This will provide a better understanding of the effect of electrophilic carcinogens and oxidants on the balance between activation and detoxification mechanisms in chemical carcinogenesis.