Nucleoside analogs induce proteasomal down-regulation of p21 in chronic lymphocytic leukemia cell lines.

Nucleoside analogs (NAs) represent an important class of anticancer agents that induce cell death after conversion to triphosphate derivatives. One of their most important mechanisms of action is the activation of p53, leading to apoptosis through the intrinsic pathway. Classically, the activation of p53 also induces p21 accumulation, which leads to cell cycle arrest at the G1/S transition. In previous work, we observed that 2-chloro-2'-deoxyadenosine (CdA), a NA with high activity in lymphoid disorders, including chronic lymphocytic leukemia (CLL), promotes the G1/S transition in the CLL cell line EHEB at cytotoxic concentrations. This finding led us to investigate the p21 response to NAs in these cells. We show here that CdA, but also fludarabine, gemcitabine, and cytarabine, strongly reduced the p21 protein level in EHEB cells as well as in JVM-2 cells, another CLL cell line. This p21 depletion occurred despite induction of p53 and increase of p21 mRNA and was prevented by proteasome inhibitors. Increase of proteasomal degradation caused by NAs appeared to be ubiquitin-independent. Also, NAs induced in these cells an increase of cyclin-dependent kinase (Cdk2) activity and a monoubiquitination of cell proliferating nuclear antigen (PCNA), two processes that are negatively regulated by p21. These changes were not observed with other p53 activators, like etoposide and nutlin-3a that increased the p21 protein level. In conclusion, our study reveals that NAs can induce an alternative pattern of cellular response in some cell models.

Influence of phosphorylation of THR-3, SER-11, and SER-15 on deoxycytidine kinase activity and stability.

Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxyribonucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We have recently shown that dCK is a phosphoprotein. Four in vivo phosphorylation sites were identified: Thr-3, Ser-11, Ser-15, and Ser-74. Site-directed mutagenesis demonstrated that phosphorylation of Ser-74, the major phosphorylated residue, strongly influences dCK activity in eucaryotic cells. Here, we show that phosphorylation of the three other sites, located in the N-terminal extremity of the protein, does not significantly modify dCK activity, but phosphorylation of Thr-3 could promote dCK stability.

Study of the efficacy of a pronucleotide of 2-chloro-2'-deoxyadenosine in deoxycytidine kinase-deficient lymphoma cells.

2-Chloro-2 '-deoxyadenosine (CdA, cladribine) is a nucleoside analogue (NA) used for the treatment of lymphoproliferative disorders. Phosphorylation of the drug to CdAMP by deoxycytidine kinase (dCK) and its subsequent conversion to CdATP is essential for its efficacy. DCK deficiency is a common mechanism of resistance to NA, which could be overcome by the pronucleotide approach. The latter consists of using the nucleoside monophosphate conjugated to a lipophilic group enabling CdAMP to enter the cells by passive diffusion. In this study, we show that cycloSaligenyl-2-chloro-2 '-deoxyadenosine monophosphate (cycloSal-CdAMP) is 10-fold more potent that CdA in a dCK-deficient lymphoma cell line. These results suggest that the use of cycloSal-nucleotides could be a strategy to counteract resistance caused by dCK deficiency.

Inhibition of the ERK pathway promotes apoptosis induced by 2-chloro-2'-deoxyadenosine in the B-cell leukemia cell line EHEB.

2-Chloro-2'-deoxyadenosine (CdA) is a nucleoside analogue active in B-cell chronic lymphocytic leukemia (B-CLL). Although the mechanism of action of CdA has been extensively investigated in leukemic cells, the possibility that this nucleoside analogue interacts with the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway has never been explored. In this study, we show that CdA, at concentrations close to the IC50, activated the ERK pathway in the B-cell line EHEB. Because activation of this pathway is assumed to exert anti-apoptotic effect, we combined CdA with inhibitors of the ERK pathway. The latter were found to enhance CdA-induced apoptosis. These results suggest that the efficacy of CdA could be strengthened by combination with inhibitors of the ERK pathway.

Identification of phosphorylation sites on human deoxycytidine kinase after overexpression in eucaryotic cells.

Compelling evidence suggests that deoxycytidine kinase (dCK), a key enzyme in the salvage of deoxyribonucleosides and in the activation of clinically relevant nucleoside analogues, can be regulated by reversible phosphorylation. In this study, we show that dCK overexpressed in HEK-293T cells was labelled after incubation of the cells with [32P]orthophosphate. Tandem mass spectrometry allowed the identification of 4 in vivo phosphorylation sites, Thr3, Ser11, Ser15, and Ser74. These results provide the first evidence that dCK is constitutively multiphosphorylated in intact cells. In addition, site-directed mutagenesis demonstrated that phosphorylation of Ser74, the major in vivo phosphorylation site, is crucial for dCK activity.

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 lambda) in parallel with a decrease of dCK activity, providing additional arguments in favor of both phosphorylated and unphosphorylated forms of dCK.

Effects of 2-chloro-2'-deoxyadenosine on the cell cycle in the human leukemia EHEB cell line.

To explain why 2-chloro-2'-deoxyadenosine (CdA) is unable to block DNA synthesis and cell cycle progression, and paradoxically enhances progression from G1 into S phase in the CdA-resistant leukemia EHEB cell line, we studied its metabolism and effects on proteins regulating the transition from G1 to S phase. A low deoxycytidine kinase activity and CdATP accumulation, and a lack of p21 induction despite p53 phosphorylation and accumulation may account for the inability of CdA to block the cell cycle. An alternative pathway involving pRb phosphorylation seems implicated in the CdA-induced increase in G1 to S phase progression.

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.

Resistance to 2-chloro-2'-deoxyadenosine of the human B-cell leukemia cell line EHEB.

The effects of 2-chloro-2'-deoxyadenosine (CdA, cladribine), an adenosine deaminase-resistant analogue toxic for both proliferating and resting lymphoid cells, were investigated in the human leukemia cell line EHEB, which was derived from a patient with B-cell chronic lymphocytic leukemia. These cells were found to be less sensitive to CdA than B-cell chronic lymphocytic leukemia lymphocytes (approximately 25-fold) and other human lymphoblastic cell lines (10-1000-fold). Phosphorylation of CdA by deoxycytidine kinase and intracellular accumulation of 2-chloro-2'-deoxyadenosine triphosphate (CdATP) were similar in EHEB cells and in other CdA-sensitive cell lines. In contrast, the inhibitory effect of CdA on ribonucleotide reductase activity, which was investigated in situ by the conversion of cytidine into deoxyribonucleotides and its incorporation into DNA, was much less pronounced in EHEB cells than in other human lymphoblastic cells. Accordingly, concentrations of deoxynucleoside triphosphates did not decrease and even tended to rise. Unexpectedly, incorporation of thymidine and deoxycytidine into DNA was increased severalfold after a 24-h incubation with CdA. CdA also increased the activities of deoxycytidine kinase and thymidine kinase approximately 4-fold. Analysis of the cell cycle by flow cytometry showed that after 24 h, CdA provoked an increase in the proportion of cells in S phase, synthesizing DNA. We conclude that the EHEB cell line is resistant to the cytotoxic action of CdA not only because of a lack of inhibition of ribonucleotide reduction but also because CdA, in contrast with its known effects, provokes in this cell line an increase in the proportion of cells replicating their DNA. Unraveling of the mechanism of this effect may shed light on clinical resistance to CdA.

Metabolism and cytotoxic effects of 2-chloroadenine, the major catabolite of 2-chloro-2'-deoxyadenosine.

EHEB cells, a continuous cell line derived from a patient with B cell chronic lymphocytic leukemia (B-CLL), synthesized, when incubated with tritiated 2-chloro-2'-deoxyadenosine (CdA), labeled mono-, di-, and triphosphate ribonucleosides at a much higher rate than CdA deoxyribonucleotides. Further analysis revealed that these ribonucleotides were formed from labeled 2-chloroadenine (CAde), which contaminated commercial tritiated CdA at a proportion of 2-3%. Since CAde is the major catabolite of CdA measured in plasma after oral or intravenous administration of CdA to patients, its metabolism and in particular its potential cytotoxicity were investigated both in EHEB cells and in B-CLL lymphocytes. Phosphorylation of CAde was inhibited by adenine, indicating that its initial metabolism most probably proceeds via adenine phosphoribosyltransferase (EC 2.4.2.7). In both cell types, chloro-ATP was the major metabolite formed from CAde and its concentration increased proportionally at least up to 50 microM CAde. At high concentration, CAde metabolism was accompanied by a decrease in intracellular ATP. Cytotoxicity of CAde, evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, showed an IC(50) of 16 microM in EHEB cells and 5 microM in B-CLL lymphocytes. At cytotoxic concentrations, apopain/caspase-3 activation and high molecular weight DNA fragmentation were observed, indicating that CAde cytotoxicity results from induction of apoptosis. However, since CAde cytotoxicity requires higher concentrations than CdA, it probably does not play a role in the therapeutic effect of CdA in the treatment of hematologic malignancies.

Potentiation of antitumor effects of cyclophosphamide derivatives in B-chronic lymphocytic leukemia cells by 2-chloro-2'-deoxyadenosine.

Because 2-chloro-2'-deoxyadenosine (CdA) is active in B-chronic lymphocytic leukemia (B-CLL), and may interfere with DNA repair, we investigated the potentiating effect of CdA on the cytotoxicity induced in vitro in B-CLL lymphocytes by cyclophosphamide (CP) derivatives, which induce DNA damage by DNA cross-linking. Exposure to CdA at clinically achievable concentrations for 2 h, followed by mafosfamide (MAF) or 4-hydroxycyclophosphamide (4HC) for 22 h, resulted in synergistic cytotoxicity in the majority of B-CLL samples tested. Synergy between CdA and MAF was observed in cell samples of sensitive/untreated patients, as well as in cells of resistant/pretreated patients, particularly at the highest concentrations of MAF. In the cells treated with CdA and MAF, we observed loss in ATP and hallmarks of apoptosis, as evidenced by cellular morphology and high molecular weight DNA fragmentation. The synergy could be explained neither by an influence of MAF on the phosphorylation of CdA, nor by an increase in the incorporation of CdA into DNA in the presence of MAF. The in vitro synergy between CdA and CP derivatives provides a rationale for the use of this association in B-CLL patients.

Novel evidence for an ecto-phospholipid methyltransferase in isolated rat hepatocytes.

Phospholipids of isolated rat hepatocytes were labelled by preincubation with either 2 microM -methyl-14C-S-adenosylmethionine (AdoMet) or 2 microM [methyl-14C]methionine. Subsequent addition of phospholipase C to the suspension removed 95% of the radioactivity from phospholipids methylated by [methyl-14C]AdoMet within a few minutes, but was without effect on phospholipids methylated by [methyl-14C]methionine radioactivity from the latter could, nevertheless, be removed by phospholipase C after permeabilization of the cells with digitonin. The results clearly show that the methyl group of exogenous AdoMet, contrary to that of methionine, is transferred on to phospholipids located on the external face of the plasma membrane. Accordingly, pretreatment of isolated hepatocytes with trypsin prevented the methylation of phospholipids from exogenous AdoMet by 60-80%, whereas it was almost without effect when exogenous methionine was the methyl donor. Our data corroborate previous work [Bontemps and Van den Berghe (1997) Biochem. J. 327, 383-389], which indicated that AdoMet methylates hepatocyte phospholipids without penetrating the cells.

Metabolism of exogenous S-adenosylmethionine in isolated rat hepatocyte suspensions: methylation of plasma-membrane phospholipids without intracellular uptake.

Administration of S-adenosylmethionine (AdoMet), the main biological methyl donor, has been shown to exert favourable effects on liver disorders in man and animal models. The mechanism of action of AdoMet has, however, remained elusive, mainly owing to controversies with respect to its capacity to enter intact liver cells. Incubation of isolated rat hepatocytes with 2 or 50 microM -methyl-14C-AdoMet showed that it was utilized predominantly to methylate cellular phospholipids, forming mainly phosphatidylcholine, although less than 0.2% of labelled AdoMet was found inside the cells. The concentration of neither AdoMet nor S-adenosylhomocysteine (AdoHcy), its demethylation product, was significantly elevated inside the cells. A slight elevation of intracellular AdoMet was only recorded on incubation with concentrations of AdoMet above 200 microM. AdoHcy, which does not penetrate cells, inhibited phospholipid methylation from [methyl-14C]AdoMet but not from [methyl-14C]Met. Elevation of intracellular AdoHcy by adenosine dialdehyde, an inhibitor of AdoHcy hydrolase, inhibited phospholipid methylation from [methyl-14C]Met, but virtually not at all from [methyl-14C]AdoMet. Taken together, these data indicate that exogenous AdoMet does not penetrate hepatocytes significantly but is utilized for phospholipid methylation on the outer surface of the plasma membrane.

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.

Labelling of uric acid and allantoin in different purine organs and urine of the rat.

We studied the incorporation of 14C-formate into uric acid and allantoin in different organs (liver, lung, kidney, spleen), isolated hepatocytes, perfused liver and urine of the rat. Allantoin had a higher specific radioactivity than uric acid after 14C-formate load in the liver in vivo. This was found to be a strictly hepatic phenomenon and not due to the influence of other tissues.

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.

Pleural amyloidosis: thoracoscopic aspects.

We describe the case of a 60 year old man with primary amyloidosis, who suffered from peripheral neuropathy and cardiomyopathy and who presented with recurrent right pleural effusion. For this reason, he was admitted to hospital for thoracoscopic examination with pleurodesis. Macroscopic examination of the parietal pleura revealed a diffuse inflammation and light brown deposits that where covered with nodules. Biopsy specimens confirmed the amyloid deposition. This macroscopic appearance is described for the first time, and suggests that a local pleural synthesis of amyloid substance may occur during the course of systemic amyloidosis.

Existence and role of substrate cycling between AMP and adenosine in isolated rabbit cardiomyocytes under control conditions and in ATP depletion.

BACKGROUND: Adenosine, a physiological coronary vasodilator, has been proposed to regulate coronary circulation according to myocardial oxygen demand. In the present study, we investigated the mechanisms of adenosine formation and utilization in isolated rabbit cardiomyocytes and, in particular, the existence and the role of substrate cycling between AMP and adenosine in the regulation of its concentration. METHODS AND RESULTS: Rabbit cardiomyocytes were isolated by collagenase perfusion and incubated in HEPES-buffered Krebs-Henseleit solution at 37 degrees C, pH 7.4, in control conditions and in ATP depletion achieved by inhibiting glycolysis with 5 mmol/L iodoacetate. Under control conditions, adenosine accumulated at a rate of 4 pmol.min-1.10(-6) cells. The 13-fold elevation of adenosine accumulation induced by iodotubercidin (ITu), an inhibitor of adenosine kinase, proves that adenosine is normally recycled into AMP. This recycling involves 95% of the adenosine formed. In ATP depletion, adenosine accumulated at the rate of 335 pmol.min-1.10(-6) cells and was no longer rephosphorylated after 20 minutes, as shown by the absence of effect of ITu after this time interval. Moreover, adenosine was deaminated, as indicated by the twofold increase of its accumulation induced by deoxycoformycin (dCF), an inhibitor of adenosine deaminase. Both in control conditions and in ATP depletion, adenosine-dialdehyde, an inhibitor of S-adenosylhomocysteine (SAH) hydrolase, had no significant effect on adenosine formation, indicating that the transmethylation pathway is not an important source of adenosine in rabbit cardiomyocytes. CONCLUSIONS: The results indicate that recycling of adenosine into AMP is essential for the maintenance of low, nonvasodilatory concentrations of the nucleoside under control conditions and that interruption of recycling plays an important role in elevating adenosine during ATP depletion.

Kinetic studies of rat liver adenosine kinase. Explanation of exchange reaction between adenosine and AMP.

Rat liver adenosine kinase can catalyze an exchange reaction between adenosine and AMP in the absence of ATP (Bontemps, F., Mimouni, M., and Van den Berghe, G. (1993) Biochem. J. 290, 679-684), suggesting a classical ping-pong mechanism. Contrary to expectations, formation of a phosphorylenzyme intermediate could not be demonstrated by incubating the enzyme with [gamma-32P] ATP. Although initial velocity measurements in function of the concentration of adenosine or Mg.ATP, at various fixed concentrations of Mg.ATP or adenosine, generated parallel line patterns, inhibition studies revealed that competitive inhibition was only observed between ADP and ATP. This indicates an Ordered Bi Bi mechanism in which ATP binds first to the enzyme, and ADP is released last. The adenosine-AMP exchange reaction was found to be potently stimulated by ADP, and the basal exchange reaction, i.e. measured in the absence of added ADP, could be accounted for by a slight (0.001%) contamination by ADP of analytical grade AMP. The ADP requirement of the adenosine-AMP exchange reaction explains its occurrence in an Ordered Bi Bi mechanism. Stimulation of the exchange reaction between AMP and adenosine by increasing concentrations of ADP/ATP, and stimulation followed by inhibition of the exchange reaction between ADP and ATP by increasing concentrations of AMP/adenosine, corroborated the proposed mechanism.