1. Statement of work: Objective: 1 To explore common changes in genetic transcription in response to specific bioactive food components. Studies will utilize two human cancer cell lines, LNCaP cells (prostate cancer) and MCF7 cells (breast cancer). The impact of soy related compounds (genestein, equol, etc), polyphenols (resveratrol, tea poly phenols, etc) or other bioactives indole-3-carbinol, DIM, etc) at various micro molar doses for 48 hours on potentially each cell line will be studied in enough detail to enable a reliable indication of the genes affected. RT-PCR will also be carried out in Dr. Wang's lab to verify the gene identification resulting from the microarray analysis, a necessary quality control step. The target estimate for the final evaluation would include up to 5 dose levels for each compound and each exposure repeated up to 5 times in replicate to give reasonable statistics making the total samples as high as 100.Objective: 2 To demonstrate the feasibility of applying proteomics to the investigation of response to bioactive food components in human cancer cell lines LNCaP and MCF7. These studies will evaluate the concordance in response with regard to specific gene messages and their corresponding proteins. Samples from the genomics studies will be divided and the cells lysed for protein fractionation and proteomic analysis. The fingerprint response will then be compared with the genomic data. This will be one of the first studies of this kind and will help determine the rigorousness of the proteomic approach to the long range study of human nutrition.Approach and Goal: Proteomics refers to the newly-developed technologies that enable the very rapid and reproducible generation of protein expression profiles known as phenomic fingerprints that are used to monitor multiple protein markers associated with different biological states. The technique involves protein chip technology coupled with matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry (TOF-MS). This technique may revolutionize the early stages of biomarker discovery and characterization for diseases such as cancer. However, as yet, few studies have compared the effects of diet on gene expression and corresponding protein patterns in humans. The issue being investigated here is the overall effect of nutrients on cell growth and whether the alterations in gene expression are mirrored in protein patterns. We will likely be able to demonstrate differences in the expression of specific cellular proteins and define other gene and protein biomarkers that may reflect dietary differences.The DCP/NCI agrees to provide an additional $12,000 to support ongoing investigations concerning the impact of dietary constituents on cancer cell line genomic expression patterns and collection of additional samples for proteomic characterization. Analysis will be performed in the laboratory of Dr. Tom Wang at the USDA, The NCI agrees to provide $12,000 for part of the laboratory costs associated with these analysis.The DCP/NCI agrees to provide an additional $10,000 to support ongoing investigations concerning the impact of dietary constituents on cancer cell line genomic and proteomic expression patterns. Analysis will be performed in the laboratories of Drs. Earl Harrison and Tom Wang at the USDA, The NCI agrees to provide $10,000 for part of the laboratory costs associated with these analysis
