Methionine dependence, the inability of cultured cells to grow when methionine is replaced by its immediate precursor homocysteine (Met-Hcy+ medium), is a frequent component of the oncogenically-transformed phenotype. Normal cells grow under this condition. However, methionine-dependent cells synthesize normal or higher amounts of methionine and maintain a normal pool size of endogenously-synthesized methionine but are deficient in utilizing this pool for S-adenosyemethionine (SAM) synthesis. In contrast exogenously-supplied methionine is utilized normally for SAM biosynthesis. We propose here to further understand the reduced ability of methionine-dependent cells to utilize endogenously-synthesize methionine for SAM biosynthesis by 1) further measurements of key methionine metabolities under various conditions using high pressure liquid chromatography of extracts of cells labeled with isotopically-pure (35 S) - homocysteine that we have synthesized and 2) understanding with the relationship between methionine synthetase and SAM synthetase by determining if the two enzymes are on a complex in normal cells and if the complex is disrupted in methionine-dependent cells and thereby disrupting use of endogenously-synthesized methionine. To further understand the relationship between methionine dependence and oncogenic transformation we will 1) investigate the relationship of methionine dependence to cellular methylation, in particular DNA methylation and the effect of altering DNA methylation by 5-azacytidine, a specific inhibitor of DNA methylation on oncogenic transformation itself, 2) Methylate, in vitro, oncogene-containing DNA, including DNA from azacytidine-treated cells to determine the effect of methylation on the activity of the oncogenes as measured by DNA-mediated transfection of normal cells and 3) determine if reduced SAM biosynthesis from endogenously synthesized methionine is diagnostic for oncogenic transformation in culture by labeling many cell lines derived from human tumors with isotopically-pure (35 S) - homocysteine and determining by high pressure liquid chromatography the amount of SAM formed.
| Vescio, R A; Redfern, C H; Nelson, T J et al. (1987) In vivo-like drug responses of human tumors growing in three-dimensional gel-supported primary culture. Proc Natl Acad Sci U S A 84:5029-33 |
| Stern, P H; Hoffman, R M (1986) The chemical synthesis of high specific-activity [35S]adenosylhomocysteine. Anal Biochem 158:408-12 |
| Freeman, A E; Hoffman, R M (1986) In vivo-like growth of human tumors in vitro. Proc Natl Acad Sci U S A 83:2694-8 |
| Stern, P H; Hoffman, R M (1986) Enhanced in vitro selective toxicity of chemotherapeutic agents for human cancer cells based on a metabolic defect. J Natl Cancer Inst 76:629-39 |
| Hoffman, R M (1985) Altered methionine metabolism and transmethylation in cancer. Anticancer Res 5:1-30 |
