In most organisms, the ribonucleoside diphosphates (purine and pyrimidine) serve as precursors for biosynthesis of deoxyribonucleotides (see Ribonucleotide Reductases and Deoxyribonucleotide Biosynthesis And Degradation). In some organisms, however, those precursors are the respective triphosphates.
Vertebrate cells contain a number of the various enzyme activities of IMP biosynthesis in the form of multifunctional enzymes. This was first suspected when cloned vertebrate cDNAs encoding purine synthetic enzymes were found to complement multiple genetic defects in purine synthesis after transformation into Escherichia coli (1). By this means, it was found that a single enzyme catalyzes the second, third, and fifth reactions shown in Figure 2. Similar evidence indicated that the sixth and seventh reactions are catalyzed by a bifunctional enzyme. Moreover, the two transformylase enzymes (reactions 3 and 9) in some animals constitute part of a tightly associated multienzyme complex that also contains several activities of tetrahydrofolate metabolism and single-carbon mobilization. The metabolic rationale for all these enzyme associations has not been established, but it may well involve the cell’s attempt to utilize scarce or unstable intermediates more efficiently by facilitating their transfer from active site to active site within the same reaction sequence.
Disruption of the purine nucleotide metabolismgenerally results in an accumulation and/or a lack ofribonucleotides or deoxyribonucleotides or metabolic intermediateswith potentially cytotoxic consequences. The observed decreasedexpression of the 3 purine metabolism enzymes affects both synthesis and the salvage pathway of purine metabolism andmay also affect purine nucleotide homeostasis in TRAIL-resistantHBL-2/R cells. Such an imbalance may represent a selectivedisadvantage for the affected cells. Such a ‘weakness’ may not beapparent under normal circumstances but may become critical understress or unfavorable conditions. As the proliferation rates ofHBL-2/R and HBL-2 cells are comparable, the proposed imbalance inpurine nucleotide metabolism in TRAIL-resistant cells is possiblymild and/or well compensated . However, this‘weakness’ may become apparent due to lack of building blocks forDNA and RNA synthesis in the environment or upon further disruptionof purine metabolism. Since both pathways of purine metabolism arecompromised in TRAIL-resistant MCL cells, these cells should bevulnerable to further inactivation of purine nucleotide metabolismenzymes. Therefore, drugs that target (already disbalanced) purinemetabolism should be highly cytotoxic to TRAIL-resistant cells(compared to non-malignant cells) and may therefore be selectivelyeffective in the elimination of TRAIL-resistant MCL cells inexperimental therapy. There are several approved inhibitors ofpurine metabolism, such as methotrexate (inhibits purine synthesis via dihydrofolate reductase) (), ribavirin and mycophenolic acid(inhibitors of IMPDH2) (,) or forodesine (a novel inhibitor ofPNP) (,), available for clinical use.
The delicate balance of enzyme activities andconcentrations of products and intermediates are critical forpurine (nucleotide) homeostasis. The inhibition of PNP results inthe accumulation of its substrate, 2′-deoxyguanosine which isfurther phosphorylated to deoxyguanosine triphosphate (dGTP). Ahigh intracellular concentration of dGTP inhibits cellproliferation and induces apoptosis (–). If APRT is inhibited, accumulatedadenine is oxidized to insoluble 2,8-dihydroxyadenine. Accumulationof this precipitate results in cell death (). Similarly, the inhibition of IMPDH2leads to depletion of guanosine nucleotides, which blocks DNAsynthesis and cell division (,).
The adaptation of cancer cells to cytostatic andcytotoxic drugs is associated to a certain degree with extensivechanges in the cell phenotype. Some of the molecular changes,although seemingly unrelated to the mechanism of resistance, canprovide a selective disadvantage to the cells and such a ‘weakness’may be used as a potential therapeutic target. By the presentedproteomic analysis of the changes associated with resistance toTRAIL in MCL HBL-2 cells, we demonstrated the downregulation of alltypes of TRAIL receptors and identified the altered expression ofseveral proteins including 3 enzymes of the purine metabolismpathway. This downregulated pathway potentially represents a‘weakness’ of the TRAIL-resistant MCL cells and has potential as atherapeutic target for the selective elimination of such cells inthe future.
The catabolism of purine nucleotides leads to theliberation of free purine bases by PNP (downregulated in HBL-2/Rcells). In the salvage pathway the free bases are reconverted backto nucleoside-5′-monophosphates in a reaction with activated sugar(PRPP) catalyzed by APRT (downregulated in HBL-2/R cells) orhypoxanthine-guanine phosphoribosyltransferase () (). Ribonucleotides are converted by ribonucleotide reductaseinto the corresponding deoxyribonucleotides.
The synthesis of purine nucleotidesrequires 5-phosphoribosyl-1-pyrophosphate (PRPP), ATP, glutamine,glycine, CO, aspartate and formate to create the firstfully formed nucleotide, inosine-5′-monophosphate (IMP). IMPrepresents a branch point for purine biosynthesis, since it can beconverted either to guanosine-5′-monophosphate (GMP) by IMPDH2(downregulated in HBL-2/R cells) or to adenosine-5′-monophosphate().
The downregulation of the 3 key enzymes of purinemetabolism can have a profound effect on nucleotide homeostasis inTRAIL-resistant lymphoma cells. Purine nucleotides, the buildingblocks for synthesis of DNA, RNA and enzyme co-factors, arerecruited either from purine synthesis from lowmolecular weight precursors or by recycling of free nucleobases inthe so-called salvage pathway. Both pathways lead to the productionof nucleoside-5′-phosphates (). Both pathways can supply cellular demand independently;however, their importance in different tissues is variable. Inleukemic and lymphoma cells the salvage pathway is considered themajor source of purine nucleotides (,).
To confirm the results of proteomic analysis by anindependent method we verified the decreased expression of the 2proteins involved in purine metabolism, namely PNP and APRT, bywestern blot analysis in HBL-2 and HBL-2/R cell lysates ().
Scheme of the purine metabolismpathways, showing the position of IMPDH2, APRT and PNP in purinenucleotide biosynthesis, adopted from a previous study (35). The de novo synthesis ofpurine nucleotides begins with the phosphorylation ofribose-5-phosphate to form PRPP. In a number of reactions, PRPPcreates the first fully formed nucleotide, IMP. IMP is converted byIMPDH2 to GMP. PNP catalyzes the reversible cleavage of purinenucleosides, releasing purine nucleobases (adenine, hypoxanthine,xanthine and guanine). In the salvage pathway the free nucleobasescan be reconverted back to nucleoside-5′-monophosphates in areaction with activated sugar (PRPP) catalyzed by APRT. IMPDH2,inosine-5′-monophosphate dehydrogenase 2; APRT, adeninephosphoribosyltransferase; PNP, purine nucleoside phosphorylase;PRPP, 5-phosphoribosyl-1-pyrophosphate; IMP,inosine-5′-monophosphate; GMP, guanosine-5′-monophosphate; dADP,deoxyadenosine diphosphate; ADP, adenosine diphosphate; GDP,guanosine diphosphate; dGDP, deoxyguanosine diphosphate; AMP,adenosine monophosphate; XMP, xanthosine monophosphate.
What is the relationship between deficiencies in two obscure purine catabolic enzymes and defective immune function? A clue came when it was found that erythrocytes of ADA-deficient patients contained high levels of deoxyadenosine and dATP. Since human erythrocytes are without nuclei, the presence of a DNA precursor seemed gratuitous. Subsequently, it was found that dATP accumulates in many tissues. It arises because ADA acts on deoxyadenosine as well as adenosine. Lymphoid tissues have very high activities of purine salvage enzymes, which reutilize products released in nucleic acid breakdown of cells undergoing apoptosis. Accumulation of deoxyadenosine, when its catabolism is blocked, leads to its conversion to dATP in these tissues, and it accumulates in both red and white blood cells. dATP is a potent allosteric inhibitor of ribonucleotide reductase, and its accumulation can block the white cell proliferative response that results from immunochallenge by inhibiting an essential step in replication of DNA—the synthesis of its precursors. There are also indications that excessive accumulation of dATP leads to an ATP deficiency in some cells. By contrast, in PNP-defective cells, the deoxyribonucleotide that accumulates is primarily dGTP, which is a less potent inhibitor of ribonucleotide reductase. This may explain the somewhat milder immune dysfunction associated with PNP deficiency. However, a completely different mechanism for the toxic effect has recently come to light from in vitro studies showing that dATP, in combination with cytochrome c, triggers a chain of protease activation steps leading ultimately to apoptosis (10).
It is not yet clear why this deficiency accelerates purine synthesis. One model states that in the absence of these salvage pathways, GMP levels decline which, in turn, modulates the feedback inhibition of PRPP amidotransferase by this nucleotide. Alternatively, it has been proposed that decreased flux through this salvage pathway causes PRPP to accumulate and this, in turn, accelerates flux through PRPP amidotransferase by substrate level control.