Françoise Bontemps

Substrate cycling between 5-amino-4-imidazolecarboxamide riboside and its monophosphate in isolated rat hepatocytes

AICA (5-amino-4-imidazolecarboxamide)-riboside is taken up by isolated rat hepatocytes and converted by adenosine kinase (ATP:adenosine 5-phosphotransferase, EC 2.7.1.20) into AICAR (ZMP), an intermediate of the de novo synthesis of purine nucleotides. We investigated if, in these cells, a cycle analogous to the adenosine-AMP substrate cycle operates between AICAriboside and ZMP. When 50 microM ITu, an inhibitor of adenosine kinase, was added to hepatocytes that had metabolized AICAriboside for 30 min, the concentration of ZMP decreased immediately. This was mirrored by a reincrease of AICAriboside. Rates of the ITu-induced decrease of ZMP and the increase of AICAriboside, calculated at different concentrations of ZMP, were first order, up to the highest concentration of ZMP (approx. 5 mumol/g of cells). Dephosphorylation of ZMP added to crude cytosolic extracts of rat liver displayed hyperbolic kinetics, with a Vmax of 0.65 mumol/min per g protein and an apparent Km of 5 mM, and was markedly inhibited by Pi, an inhibitor of IMP-GMP 5-nucleotidase (5-ribonucleotide phosphohydrolase, EC 3.1.3.5). We conclude that hepatocyte ZMP is continuously dephosphorylated, most likely by IMP-GMP 5-nucleotidase, into AICAriboside, which is rephosphorylated into ZMP by adenosine kinase. Substrate cycling was also shown to occur between other nucleoside analogs and their phosphorylated counterparts.

Stimulation by glycerate 2,3-bisphosphate: a common property of cytosolic IMP-GMP 5'-nucleotidase in rat and human tissues.

Glycerate 2,3-bisphosphate, a potent stimulator of the cytosolic 5-nucleotidase which preferentially hydrolyzes IMP and GMP in human erythrocytes (Bontemps et al., 1988, Biochem. J. 250, 687-696), also stimulates the dephosphorylation of IMP in cytosol fractions of rat heart, liver, brain, kidney, spleen and erythrocytes, and of human polymorphonuclear leucocytes, mixed peripheral blood lymphocytes, platelets and fibroblasts. Depending on the cell type, stimulation by 5 mM glycerate 2,3-bisphosphate varied from 1.5- to 12-fold. Where investigated, glycerate 2,3-bisphosphate had an approx. 5-fold higher affinity for the enzyme than its other stimulator, ATP. These observations provide a useful tool to distinguish IMP-GMP 5-nucleotidase from other 5-nucleotidases, and suggest a common origin of the cytosolic IMP-GMP 5-nucleotidase in various tissues.

2-Chloro-2'-deoxyadenosine inhibits DNA repair synthesis and potentiates UVC cytotoxicity in chronic lymphocytic leukemia B lymphocytes.

2-Chloro-2′-deoxyadenosine (CdA) is a deoxyadenosine analogue which targets enzymes involved in DNA synthesis, and hence might interfere with the resynthesis step of DNA repair. We tested this hypothesis in resting B cell chronic lymphocytic leukemia (B-CLL) lymphocytes, after firstly characterizing unscheduled DNA synthesis occurring in these cells. We observed that the spontaneous incorporation of [methyl-3H]thymidine (dThd) into DNA of B-CLL cells was not completely inhibitable by hydroxyurea (HU) which blocks DNA replication. In addition, in the presence of HU, dThd incorporation could be upregulated by UVC radiation or DNA alkylation, without re-entry of the cells into S phase. CdA was found to inhibit both spontaneous and upregulated DNA synthesis in B-CLL cells. Phosphorylation of CdA was essential to exert this effect. We finally observed a strong synergistic cytotoxicity between UV light and CdA, which was correlated with activation of caspase-3 and high molecular weight DNA fragmentation, two markers of apoptosis. Taken together, these observations indicate that in B-CLL cells CdA inhibits unscheduled DNA synthesis which represents the polymerizing step of a repair process responsive to DNA aggression. Inhibition of this process by CdA, together with a combined activation of the apoptotic proteolytic cascade by CdA and UV, may explain their synergistic cytotoxicity.

Identification of a Purine 5′-Nucleotidase in Human Erythrocytes

In 1975, Paglia and Valentine (1) demonstrated the existence in human erythrocytes of a specific pyrimidine 5′-nucleotidase, which they found inactive on purine nucleotides. A deficiency of this enzyme is known which provokes an hemolytic anemia (reviewed in 2). In accordance with the reported enzymic specificity, the erythrocytes of these patients have markedly elevated concentrations of cytidine and uridine nucleotides but not of purine nucleotides. Although it is evident that dephosphorylation of AMP and IMP should occur in erythrocytes (see Bontemps et al., this volume), the enzyme(s) catalysing this process have hitherto not been identified. This report describes the partial purification and the kinetic properties of a purine-specific 5′-nucleotidase present in human erythrocytes.

New Evidences for a Regulation of Deoxycytidine Kinase Activity by Reversible Phosphorylation

Recent studies indicate that deoxycytidine kinase (dCK), which activates various nucleoside analogues used in antileukemic therapy, can be regulated by post‐translational modification, most probably through reversible phosphorylation. To further unravel its regulation, dCK was overexpressed in HEK‐293 cells as a His‐tag fusion protein. Western blot analysis showed that purified overexpressed dCK appears as doublet protein bands. The slower band disappeared after treatment with protein phosphatase lambda (PP λ) in parallel with a decrease of dCK activity, providing additional arguments in favor of both phosphorylated and unphosphorylated forms of dCK.

Production of adenosine and nucleoside analogues by an exchange reaction catalyzed by adenosine kinase.

We have shown previously that rat liver adenosine kinase can catalyze an exchange reaction between adenosine (Ado) and AMP in the absence of ATP (1). This exchange reaction was potently stimulated by ADP. When measured in the absence of added ADP, the exchange could be due to a slight (0.001 %) contamination by ADP of analytical grade AMP (2). The ADP requirement of the Ado-AMP exchange intervenes in an ordered Bi Bi mechanism in which ATP is the first substrate to bind to the enzyme, and ADP the last product to dissociate. In the present work we have investigated (i) if Ado or AMP could be replaced by, respectively, nucleoside or nucleoside monophosphate analogs and (ii) if the Ado-AMP exchange is restricted to liver.

The AMP-adenosine cycle is active during normoxia and impaired in ATP depletion in isolated rabbit cardiomyocytes.

Adenosine (Ado), a breakdown product of adenine nucleotides, is a potent coronary vasodilator, which has been proposed to regulate coronary circulation according to myocardial oxygen demand (1). Recycling of Ado into AMP has been demonstrated to play a crucial role in the regulation of the concentration of Ado in isolated rat hepatocytes (2, 3). In the heart, however, the AMP — Ado substrate cycle has been considered either inactive (4), or operating at a very low rate (5), except for a recent study in normoxic guinea pig heart (6).

Production of adenosine and nucleoside analogs by the exchange reaction catalyzed by rat liver adenosine kinase

We have previously shown [8] that rat liver adenosine kinase can produce [14C]AMP from [14C]adenosine (Ado) and unlabelled adenosine monophosphate (AMP), in the absence of ATP, by an exchange reaction. In this study, we investigated whether Ado or AMP could be replaced in this exchange reaction by other nucleosides or nucleoside monophosphates (NMP), respectively. In the presence of 1 mM of the unlabelled NMP analogs 7-deazaadenosine (tubercidin) 5-monophosphate, 6-chloropurine riboside 5-monophosphate, or N6-methyl-AMP, [14C]AMP was formed from 20 microM [14C]Ado at up to 50% of the rate recorded with 1 mM unlabelled AMP. In the presence of 0.2 mM of the unlabelled analog nucleosides tubercidin, N6-methyladenosine, or 6-methylmercaptopurine riboside, [14C]Ado was generated from 1 mM [14C]AMP at up to 60% of the rate recorded with 0.2 mM unlabeled Ado. Small amounts of [14C]Ado were also formed from the natural nucleosides 5-amino-4-imidazolecarboxamide (AICA) riboside or 2-deoxyadenosine. Administration of therapeutic anticancer and antiviral nucleosides that can serve as substrates for the exchange reaction catalyzed by adenosine kinase might, thus, result in a net production of Ado, a potent autacoid with physiological effects in numerous tissues.