We have demonstrated a previously unsuspected lesion in cells treated with methotrexate, the incorporation of deoxyuridylate into DNA. This is a result of gross explansion of intracellular deoxyuridine nucleotide pools, including deoxyuridine triphosphate, and apparently is common to all cells limited in thymidylate. We have also shown that the incorporation and consequent efforts of the cell to remove deoxyuridylate from DNA is accompanied by fragmentation of DNA and cell death. We propose to continue these studies as follows: 1. Complete the studies which indicate that incorporation of deoxyuridylate into DNA can result in fragmentation of DNA and cell death, both of which are characteristic effects of methotrexate. 2. Determine the consequences of deficient intracellular deoxythymidine triphosphate without the great increase in deoxyuridylate that usually accompanies low deoxythymidine triphosphate. 3. Characterize in greater detail the DNA defect(s) caused by MTX and by incorporation of deoxyuridylate. 4. Determine the relationship of results with methotrexate to the mechanism of action of analogs of deoxyuridine and other inhibitors of DNA synthesis. 5. Explore the possibility that the single strand breaks or gaps in DNA caused by methotrexate or deoxyuridylate may play a part in other processes involving DNA; in particular, recombination and amplification. It is hoped that the results of these studies will improve our understanding of how this important chemotherapeutic agent acts, and facilitate rational use of the drug, clinically and in the laboratory.
| Goulian, M; Bleile, B M; Dickey, L M et al. (1986) Mechanism of thymineless death. Adv Exp Med Biol 195 Pt B:89-95 |
| Ingraham, H A; Dickey, L; Goulian, M (1986) DNA fragmentation and cytotoxicity from increased cellular deoxyuridylate. Biochemistry 25:3225-30 |
