Ribonucleoside diphosphate reductase catalyzes the reduction of all four ribonucleoside diphosphates to their deoxynucleotide counterparts. It functions in the only de novo pathway for the in vivo synthesis of all four deoxyribonucleoside triphosphates for DNA synthesis and its activity is essential for cell cycle regulation. Recently, I have selected a series of S49 T lymphoma cell lines resistant to hydroxyurea--an inhibitor of ribonucleotide reductase that binds to the tyrosyl radical on the M2 subunit which is necessary for catalytic activity. These cell lines have increased deoxynucleotide pools, increased CDP reductase activity due to increased M2 activity, increased M2 protein and appear to be amplified at the genomic and messenger level; however, they retain normal cell cycle dependent variation in ribonucleotide reductase activity. Surprisingly, these cell lines cannot be arrested in the G1 phase of the cell cycle when exposed to cyclic AMP or agents that increase endogenous cyclic AMP, as wild type S49 cells can. The experiments outlined in this proposal will investigate the mechanism of cyclic AMP resistance in hydroxyurea resistant cells by: 1) determining if cyclic AMP resistance is a uniform characteristic of both """"""""amplification"""""""" and """"""""single step"""""""" mutation to resistance to hydroxyurea; 2) pursuing our observation that the M2 subunit of ribonucleotide reductase is a target for cyclic AMP dependent protein kinase by determining whether it is phosphorylated in vivo and whether phosphorylation inactivates the enzyme; and, 3) determining whether cyclic AMP inhibits transcription of M2 specific messenger RNA. The results of these experiments should determine how cyclic AMP generates cell cycle arrest in S49 T lymphoma cell lines and provide insight into the mechanisms of action of cyclic AMP and cell cycle regulation. Recent studies link cyclic AMP and ribonucleotide reductase to viral transformation, to the protein products of oncogenes and to the effect of growth factors. Thus these studies have significant implication for lymphoid proliferation, immune responsiveness and carcinogenesis.
| Albert, D A; Rozengurt, E (1993) Opposite effects of cyclic AMP on the expression of the genes encoding ribonucleotide reductase in Swiss 3T3 and RAT 1 cells. Biochem Biophys Res Commun 196:642-9 |
| Albert, D A; Rozengurt, E (1992) Synergistic and coordinate expression of the genes encoding ribonucleotide reductase subunits in Swiss 3T3 cells: effect of multiple signal-transduction pathways. Proc Natl Acad Sci U S A 89:1597-601 |
| Albert, D A; Nodzenski, E (1992) Ribonucleotide reductase gene expression during cyclic AMP-induced cell cycle arrest in T lymphocytes. Exp Cell Res 203:476-82 |
| Albert, D A; Nodzenski, E; Cruz, G H et al. (1991) Deoxyadenosine- and cyclic AMP-induced cell cycle arrest and cytotoxicity. Exp Cell Res 197:75-81 |
| Albert, D A; Nodzenski, E; Yim, G et al. (1990) Effect of cyclic AMP on the cell cycle regulation of ribonucleotide reductase M2 subunit messenger RNA concentrations in wild-type and mutant S49 T lymphoma cells. J Cell Physiol 143:251-6 |
