Ribonucleotide reductase catalyzes the rate-limiting step in the de novo synthesis of the dNTPs that are required for DNA replication. This enzyme consists of two non-identical protein subunits [a non-heme iron (NHI) and effector-binding (EB) subunit. The synthesis/degradation of the two protein subunits are not coordinately regulated as the cells traverse the cell cycle. The separate genes which encode for these subunits are likewise not under coordinate control. The individual subunits can be specifically and independently inhibited/inactivated by defined classes of drugs. Since the development of drug resistance is a common phenomenon in tumor cells and the mechanisms of resistance can be varied and multiple, drug resistance at the ribonucleotide reductase site can be developed at either subunit or both subunits simultaneously. The mechanisms of resistance will be based on changes quantitatively or qualitatively in the non-heme iron or effector-binding subunits. In the proposed studies, we will in a systematic manner investigate the mechanisms of resistance which are developed at the ribonucleotide reductase site in mouse leukemia L1210 cells. We will determine the levels of resistance to specific ribonucleotide reductase inhibitors, cross-resistance patterns, the levels of enzyme activity and subunit concentrations, the properties of the subunits and the enzyme in the resistance cells, the levels of the mRNAs for the NHI and EB subunits and the gene copy number and DNA sequence if warranted for each subunit compared to the wild-type cells. The overall goal of this project is to define the biochemical and molecular mechanisms by which resistance to inhibitors of ribonucleotide reductase that are specifically and independently directed at either the non-heme iron or effector-binding subunits are developed. Further, these resistant cell lines will be useful in discerning subtle differences, at the cellular level, in the mechanisms of inhibition at the ribonucleotide reductase site between apparently similar drugs.
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