Regulation of the salvage pathway of deoxynucleotides synthesis in apoptosis induced by growth factor deprivation

Abstract

Here we describe changes in dNTP metabolism that precede DNA fragmentation in a model of apoptosis driven by deprivation of the cytokine interleukin 3 (IL-3). In haemopoietic BAF3 cells, IL-3 withdrawal leads to a rapid decrease in the size of dATP, dTTP and dGTP pools without affecting dCTP levels. This imbalance in dNTP pools precedes DNA fragmentation and is accompanied by down-regulation of enzymes controlling the de novo and salvage pathways of dNTP synthesis, ribonucleotide reductase and thymidine kinase (TK) respectively. Readdition of IL-3 results in a rapid, protein synthesis-independent restoration of normal dNTP pools, enhanced TK activity and increased precursor incorporation through the salvage pathway. Up-regulation of TK activity after IL-3 readdition is prevented by the protein kinase C (PKC) inhibitor staurosporin, but not by tyrosine kinase inhibitors. Furthermore activation of PKC by phorbol esters mimics the stimulatory effect of IL-3 on TK activity, suggesting that PKC might be involved in regulating this effect. These results indicate that regulation by IL-3 of the salvage pathway of dNTP synthesis plays a role in the maintenance of cellular dNTP pool balance and suggests that alterations in dNTP metabolism after IL-3 deprivation could be a relevant event in the commitment of haemopoietic cells to apoptosis.

Overexpression of a Heterologous Thymidine Kinase Delays Apoptosis Induced by Factor Deprivation and Inhibitors of Deoxynucleotide Metabolism

“…Control cells treated for 3 h with 5 M MTX exhibited a 66% decrease in dTTP and dGTP levels, whereas dATP and dCTP remained unchanged. In IL-3 deprivation experiments, the levels of dATP, dGTP, and dTTP after 8 h in the absence of IL-3 decreased to about 40 -50% of the initial levels, whereas the dCTP level was only slightly changed, as reported previously (11). Interestingly, in cells overexpressing HSV-1 TK, treatment with MTX had no effect on the intracellular dATP, dGTP, and dTTP levels and had little effect on the dCTP pool (67% of the level found in untreated cells).…”

“…2B). Furthermore, the clone overexpressing HSV-1 TK was able to maintain TK activity after deprivation of IL-3, in contrast to what was observed in puro3 cells (data not shown) and parental BAF3 cells (11). We have also determined the effect of HSV-1 TK overexpression on the viability of cells treated with inhibitors of dNTP metabolism such as MTX and FdUrd, which rapidly deplete the cells of dTTP.…”

“…Although mammalian TK expression is cell cycle-regulated, with peak gene expression occurring immediately before entry into S-phase (38), we have demonstrated that IL-3 also regulates TK activity posttranslationally (11). Serum has also been shown to regulate the phosphorylation state and activity of TK in HL-60 cells (24).…”

“…Previous results from our laboratory have shown that inhibition of thymidylate synthase (an enzyme of the de novo synthesis of dNTP) with 5-fluoro-2Ј-deoxyuridine, which perturbs deoxyribonucleotide metabolism, drives the IL-3-dependent cell line BAF3 to enter apoptosis even in the presence of IL-3 (10). Furthermore, removal of IL-3 from BAF3 cell cultures leads to an early imbalance in dNTP pools during apoptosis (11).…”

Thymidylate synthase inhibition triggers glucose‐dependent apoptosis in p53‐negative leukemic cells

“…Second, dATP may be essential for activation of the apoptosome [5], and the mitochondrial dATP/ATP transporter adenine nucleotide translocator (ANT) is a component of the mitochondrial Permeability Transition Pore Complex, that may be involved in the onset of apoptosis in several systems [6]. Third, changes in dNTP levels have been found to precede DNA fragmentation in apoptosis driven by starvation of the survival factor interleukin-3 [7].…”