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An L1210 cell line (Y8) selected for resistance to deoxyadenosine contains ribonucleotide reductase that is not subject to inhibition by dATP. In addition, the Y8 cells have other phenotypic expressions that include increased sensitivity to apoptosis induced by various agents such as radiation, doxorubicin, anisomycin and roscovitine. The Y8 cells were found to be more sensitive to apoptosis induced by methotrexate (MTX), tiazofurin (TZ), deoxyguanosine (dGuo) and N-(phosphonoacetyl)-L-aspartate (PALA). Deoxyguanosine, at concentrations that did not cause apoptosis in the Y8 cells, prevented the apoptotic response of the Y8 cells to MTX and TZ. Deoxycytidine had no effect. Since caspase-3 activation is involved in apoptotic pathways, the effects of the caspase-3 inhibitor, Ac-DEVD-CHO, were studied on the dGuo-, MTX- or TZ-induced apoptosis in the Y8 cells. Ac-DEVD-CHO caused a marked decrease in the fraction of cells in the early phase of apoptosis. However, there was a corresponding increase in the fraction of cells in the late apoptotic/necrotic stages of cell death. This is in marked contrast to the dGuo-induced decrease in apoptosis seen in the MTX- and TZ-treated Y8 cells in which there were no increases in the late apoptotic/necrotic fraction of cells. These data show that alterations of nucleotide pools in the Y8 cells cause marked increases in the apoptotic response which may indicate that the Y8 cells are much more susceptible to the effects of misincorporation of nucleotides into DNA than are the parental WT L1210 cells.

Recent studies have implicated nucleotides in diverse and unexpected functions related to p53 levels, p53-dependent G0/G1 cell cycle arrest, and the role of dATP in the activation of the caspase-induced apoptosis. Using deoxyadenosine-resistant L1210 cells (ED2 and Y8) that had ribonucleotide reductase that was not sensitive to inhibition by dATP and also exhibited other metabolic alterations, the properties of these cells with respect to the role(s) of nucleotides in these functions were explored. In the ED2 and Y8 cells that did not express p53 protein, the pools of UTP, CTP, ATP, and GTP were markedly decreased. The decreased cellular levels of UTP and CTP did not result in these cells being more sensitive to either PALA or acivicin. The ED2 and Y8 cells did not block in G0/G1 in response to PALA treatment even though the basal cellular concentrations of UTP and CTP were reduced 50 to 80%. While it has been shown that dATP in combination with cytochrome c is involved in the apoptotic pathway, the concentration of exogenous deoxyadenosine required to induce apoptosis in the parental L1210 cells was far in excess of the concentration required to inhibit cell growth. Deoxyadenosine did not cause an increase in apoptosis in the deoxyadenosine-resistant Y8 cells. These data suggest that the new roles ascribed to nucleotides may be specific for the particular cell type under very specific conditions.

The 2'-deoxy-2'-methylene derivatives of adenosine (MdAdo), guanosine (MdGuo), tubercidin (MdTu), cytidine (MdCyd) and uridine (MdUrd) were synthesized as mechanism-based inhibitors directed at ribonucleotide reductase. It was shown that MdCyd 5'-diphosphate irreversibly inactivated ribonucleotide reductase from Escherichia coli (Baker et al., J Med Chem 34: 1879-1884, 1991). In studies reported here, MdAdo/EHNA, MdGuo and MdCyd inhibited L1210 cell growth with IC50 values of 3.4, 10.6 and 1.4 microM, respectively. Since MdAdo is a substrate for adenosine deaminase, the presence of EHNA was required to give maximal growth inhibition. 8-Aminoguanosine was not required to maximize the cytotoxic effects of MdGuo. The 2'-deoxy-2'-methylene derivatives of tubercidin and uridine did not inhibit L1210 cell growth at concentrations as high as 50 microM (MdTu) or 100 microM (MdUrd). L1210 cell lines resistant to hydroxyurea (directed at the non-heme iron subunit of ribonucleotide reductase) or deoxyadenosine (directed at the effector binding subunit of ribonucleotide reductase) were not resistant to MdCyd. An L1210 cell line that was highly resistant to dGuo due to the loss of a relatively specific deoxyribonucleoside kinase (Cory et al., J Biol Chem 268: 405-409, 1993) had a 6.6-fold increase in the IC50 value toward MdCyd, but showed only a 2-fold increase in resistance to MdGuo. Another L1210 cell line that was markedly deficient in adenosine kinase activity was highly resistant to MdAdo. Analysis by flow cytometry showed that MdCyd showed the transit of the cells through the G2/M phase of the cell cycle resulting in the buildup of the G2/M population. MdAdo, MdGuo and MdCyd inhibited the incorporation of [14C]cytidine into DNA without an effect on RNA synthesis or total cellular uptake of [14C]cytidine. The conversion of [14C]cytidine to deoxycytidine nucleotides was partially inhibited by MdGuo, but not by MdAdo or MdCyd. These data show that the 2'-deoxy-2'-methylene derivatives of adenosine, guanosine and cytidine are activated via specific nucleoside kinases and that the modes of action of these compounds are not identical.

L1210 cell lines, selected for resistance to deoxyadenosine due to the loss of allosteric inhibition of ribonucleotide reductase by dATP, had altered steady-state levels of the mRNAs for c-myc, fos, and p53. Wild-type L1210 cells had constitutive steady-state levels of c-myc and p53 with little or no fos mRNA. Two different deoxyadenosine-resistant cell lines (Y8 and ED2) had elevated steady-state levels of c-myc and fos but essentially no p53 mRNA. Hydroxyurea-resistant L1210 cells had the same levels of c-myc, fos, and p53 as the wild-type cells. There was no amplification of the gene for c-myc in the Y8 or ED2 cell lines. The half-life for c-myc mRNA was essentially the same in the wild-type and the Y8 and ED2 cells. Nuclear runoff experiments showed that the rates of transcription for c-myc in the Y8 and ED2 cells were elevated and could account for the increased steady-state levels of c-myc in these two cell lines. The transcription rate for p53 mRNA was not decreased in the Y8 and ED2 cells and therefore did not account for the loss of the steady-state levels of p53 in the cells. Cycloheximide treatment of the Y8 and ED2 cells resulted in a marked increase in the steady-state p53 mRNA level, indicating that a protein which was rapidly turned over was responsible for the extremely short half-life of p53 mRNA in these two cell lines.