The tumor suppressor gene, p53, appears to be a critical regulator of normal cell proliferation and apoptosis. This gene is also the most frequently mutated gene in human tumors. We have obtained from Dr. Tyler Jacks (Center for Cancer Research, MIT) wild-type mouse embryonic fibroblasts (p53 +/+) and corresponding transfected cells which are homozygous p53-deficient (p53-/-). Both cell lines have been immortalized and are readily grown in culture. Since the only difference between these two lines is their p53 status, these cell lines represent a novel and excellent model for studying cellular responses unambiguously attributed to p53 function. We have initiated studies to examine differences in biochemistry and energetics between these cell lines in the early stages of apoptosis using high resolution nuclear magnetic resonance (NMR). For the first phase of this research, we are examining possible compositional differences between cultured p53-/-and p53+/+ cells. We are in the process of measuring a statistically relevent number of one-dimensional (1D) proton NMR and two-dimensional proton NMR total correlated spectroscopy (TOCSY) spectra of packed, intact p53-/- and p53+/+ cells and of their chloroform/methanol extracts. We have begun to measure 1D phosphorous NMR spectra of perchloric acid and chloroform / methanol extracts to determine the phospholipid profile for these cells. Results obtained to date have not yet revealed any statistically significant differences between these cell lines. Future studies will include examination of the effects of Adriamycin and/or ionizing radiation on these cell lines while perfused in the NMR tube.
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