Papers by Françoise Bontemps
Toxicology and Applied Pharmacology, 2016
Gentamicin, an aminoglycoside used to treat severe bacterial infections, may cause acute renal fa... more Gentamicin, an aminoglycoside used to treat severe bacterial infections, may cause acute renal failure. In the renal cell line LLC-PK1, gentamicin accumulates in lysosomes, induces alterations of their permeability, and triggers the mitochondrial pathway of apoptosis via activation of caspase-9 and -3 and changes in Bcl-2 family proteins. Early ROS production in lysosomes has been associated with gentamicin induced lysosomal membrane permeabilization. In order to better understand the multiple interconnected pathways of gentamicin-induced apoptosis and ensuing renal cell toxicity, we investigated the effect of gentamicin on p53 and p21 levels. We also studied the potential effect of gentamicin on proteasome by measuring the chymotrypsin-, trypsin-and caspase-like activities, and on endoplasmic reticulum by determining phopho-eIF2α, caspase-12 activation and GRP78 and 94. We observed an increase in p53 levels, which was dependent on ROS production. Accumulation of p53 resulted in accumulation of p21 and of phospho-eIF2α. These effects could be related to an impairment of proteasome as we demonstrated an inhibition of trypsin-and caspase-like activities. Moderate endoplasmic reticulum stress could also participate to cellular toxicity induced by gentamicin, with activation of caspase-12 without change in GRP74 and GRP98. All together, these data provide new mechanistic insights into the apoptosis induced by aminoglycoside antibiotics on renal cell lines.
Recent Investigations on the Control of Glycogen Metabolism in the Liver
Springer eBooks, 1976
The major factor that controls glycogen metabolism in the liver is the concentration of phorphory... more The major factor that controls glycogen metabolism in the liver is the concentration of phorphorylase alpha. Indeed, this enzyme catalyzes the limiting step of glycogen breakdown and, by controlling the activity of synthetase phosphatase, also regulates glycogen synthesis. The formation of phosphorylase alpha is stimulated by cAMP, by glycogen, and presumably also by some still ill-defined ionic changes. The ininactivation of phosphorylase is greatly stimulated by glucose and inhibited by AMP and glycogen. Glycogen synthesis is proportional to the concentration of synthetase alpha, which in normally fed animals is formed only when most of the phosphorylase is in the beta form. The inactivation of glycogen synthetase is stimulated by cAMP, an elevated concentration of which puts a double lock on glycogen synthetase by activating phosphorylase alpha (and thereby preventing synthetase activation) and by inactivating glycogen synthetase. The effect of cAMP, 5'-AMP, glucose, and glycogen can presently be explained in molecular terms. The main missing link is in the ionic effect whose elucidation might lead to the understanding of the mode of action of insulin.
Springer eBooks, 1989
Glycerate 2,3.bisphosphate, a potent stimulator of the cytosolic 5'-nucleotidase which preferenti... more 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 eytosolic IMP-GMP 5'-nudeotidase in various tissues.
Influence of Ethanol on the Production of Allantoin by the Perfused Rat Liver
Springer eBooks, 1984
Ingestion of alcohol is traditionally considered a predisposing or precipitating factor in the go... more Ingestion of alcohol is traditionally considered a predisposing or precipitating factor in the gouty attack. Studies performed in man by Lieber et al. (1962) have shown that the administration of ethanol provokes an increase in the serum concentration of uric acid, which was attributed to a decrease in its renal excretion. More recently, ethanol infusion has been shown to enhance the production of uric acid by the liver, as measured from arteriovenous concentration differences during hepatic catheterisation in human volunteers (Grunst et al., 1973).
Production of Adenosine and Nucleoside Analogues by an Exchange Reaction Catalyzed by Adenosine Kinase
Springer eBooks, 1995
We have previously shown [8] that rat liver adenosine kinase can produce [14C]AMP from [14C]adeno... more 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.
Journal of Biological Chemistry, Feb 1, 2006
Deoxycytidine kinase (dCK) catalyzes the rate-limiting step of the deoxyribonucleoside salvage pa... more Deoxycytidine kinase (dCK) catalyzes the rate-limiting step of the deoxyribonucleoside salvage pathway in mammalian cells and plays a key role in the activation of numerous nucleoside analogues used in anti-cancer and antiviral chemotherapy. Although compelling evidence indicated that dCK activity might be regulated by phosphorylation/dephosphorylation, direct demonstration was lacking. Here we showed that dCK overexpressed in HEK 293T cells was labeled after incubating the cells with [ 32 P]orthophosphate. Sorbitol, which was reported to decrease dCK activity, also decreased the labeling of dCK. These results indicated that dCK may exist as a phosphoprotein in vivo and that its activity can be correlated with its phosphorylation level. After purification of 32 P-labeled dCK, digestion by trypsin, and analysis of the radioactive peptides by tandem mass spectrometry, the following four in vivo phosphorylation sites were identified: Thr-3, Ser-11, Ser-15, and Ser-74, the latter being the major phosphorylation site. Site-directed mutagenesis and use of an anti-phospho-Ser-74 antibody demonstrated that Ser-74 phosphorylation was crucial for dCK activity in HEK 293T cells, whereas phosphorylation of other identified sites did not seem essential. Phosphorylation of Ser-74 was also detected on endogenous dCK in leukemic cells, in which the Ser-74 phosphorylation state was increased by agents that enhanced dCK activity. Our study provided direct evidence that dCK activity can be controlled by phosphorylation in intact cells and highlights the importance of Ser-74 for dCK activity. Deoxycytidine kinase (dCK 4 ; EC 2.7.1.74) catalyzes the phosphorylation of deoxycytidine, deoxyguanosine, and deoxyadenosine, with ATP or UTP as phosphoryl donor. This reaction is the rate-limiting step of the deoxyribonucleoside salvage pathway that supplies cells with pre
Exogenous S-Adenosyl-Methionine Methylates Phospholipids Located on the Outer Cell Surface of Isolated Rat Hepatocytes
Springer eBooks, 1998
S-adenosyl-methionine (AdoMet) is a naturally occurring molecule, produced endogenously from ATP ... more S-adenosyl-methionine (AdoMet) is a naturally occurring molecule, produced endogenously from ATP and methionine (Met) by AdoMet synthetase. It acts as a methyl donor for a large variety of substrates, including hormones, proteins, nucleic acids, phospholipids and several small molecules, such as glycine or guanidinoacetate.1 Transmethylation from AdoMet yields S-adenosylhomocysteine (AdoHcy), of which the homocysteine moiety serves as precursor of glutathione via the transsulfuration pathway.
Pediatric Research, 1985
I n human e r y t h r o c y t e s , a pyrimidine 5'-nucleotidase has keen c h a r a c t e r i z e... more I n human e r y t h r o c y t e s , a pyrimidine 5'-nucleotidase has keen c h a r a c t e r i z e d (P a g l i a & Valentine, Curr. Top. Hematol 3:75, 1980) but the enzyme(s) responsible f o r the d e p h o s p h o r y l a t i~n of AMP and IMP had h i t h e r t o not been i d e n t i f i e d. Haemolysates i n 10 mM Tris-maleate b u f f e r pH 7, incubated w i t h 1 mM [ I~c ] IMP,
Purine and Pyrimidine Metabolism in Man V, 1986
Cancer research, Jan 15, 1989
The mechanism of the depletion of ATP, recorded in the erythrocytes of adenosine deaminase-defici... more The mechanism of the depletion of ATP, recorded in the erythrocytes of adenosine deaminase-deficient children and of leukemia patients treated with deoxycoformycin, was investigated in normal human erythrocytes treated with this inhibitor of adenosine deaminase. Deoxyadenosine, which accumulates in both clinical conditions, provoked a dose-dependent accumulation of dATP, depletion of ATP, and increases in the production of inosine plus hypoxanthine. Concomitantly, there was an increase of AMP and IMP, but not of adenosine, indicating that catabolism proceeded by way of AMP deaminase. A series of nucleoside analogues (9-beta-D-arabinofuranosyladenine, N6-methyladenosine, 6-methylmercaptopurine ribonucleoside, tubercidin, ribavirin, and N-1-ribosyl-5-aminoimidazole-4-carboxamide riboside) also stimulated adenine nucleotide catabolism and increased AMP and IMP to various extents. The effects of deoxyadenosine and of the nucleoside analogues were prevented by 5'-iodotubercidin, an i...
Proceedings of the National Academy of Sciences, 1983
Pediatric Research, 1985
I n human e r y t h r o c y t e s , a pyrimidine 5'-nucleotidase has keen c h a r a c t e r i z e... more I n human e r y t h r o c y t e s , a pyrimidine 5'-nucleotidase has keen c h a r a c t e r i z e d (P a g l i a & Valentine, Curr. Top. Hematol 3:75, 1980) but the enzyme(s) responsible f o r the d e p h o s p h o r y l a t i~n of AMP and IMP had h i t h e r t o not been i d e n t i f i e d. Haemolysates i n 10 mM Tris-maleate b u f f e r pH 7, incubated w i t h 1 mM [ I~c ] IMP,
Inhibition of the ERK Pathway Promotes Apoptosis Induced by 2-Chloro-2′-Deoxyadenosine in the B-Cell Leukemia Cell Line Eheb
Nucleosides, Nucleotides and Nucleic Acids, 2006
2-Chloro-2'-deoxyadenosine (CdA) is a nucleoside analogue active in B-cell chronic lympho... more 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.
Influence of Phosphorylation of THR-3, SER-11, and SER-15 on Deoxycytidine Kinase Activity and Stability
Nucleosides, Nucleotides and Nucleic Acids, 2010
Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxyribonucleosides and in the acti... more 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.
Leukemia & Lymphoma, 2012
Structural alterations of TP53 and ATM in chronic lymphocytic leukemia p53 is a transcription fac... more Structural alterations of TP53 and ATM in chronic lymphocytic leukemia p53 is a transcription factor which, in the absence of stress signals, is expressed at very low levels due to its ubiquitination by Mdm2, leading to nuclear export and proteasomemediated degradation. After genotoxic stress and activation of sensor kinases, notably ATM, DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia-related kinase (ATR), p53 and Mdm2 become phosphorylated, which prevents Mdm2 from binding to p53 and allows p53 stabilization and accumulation in the nucleus [19-22]. Once activated, p53 binds to DNA and stimulates the transcription of genes, i.e. P21 , PCNA , GADD45 , BAX , NOXA , MDM2 or miR-34a , which are implied in cell-cycle arrest and apoptosis [23-26]. p53 plays a central role in mediating the action of DNA-damaging anti-leukemic agents [6,27-29]. In CLL, TP53 can be deleted and/or mutated [30-33]. Th e incidence of TP53 aberrations varies with stage of the disease:
Leukemia, 1999
Because 2-chloro-2′-deoxyadenosine (CdA) is active in Bchronic lymphocytic leukemia (B-CLL), and ... more Because 2-chloro-2′-deoxyadenosine (CdA) is active in Bchronic 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.
Journal of Clinical Investigation, 1986
The exact pathway whereby the initial catabolism of the adenine nucleotides proceeds from AMP and... more The exact pathway whereby the initial catabolism of the adenine nucleotides proceeds from AMP and the possibility of a recycling of adenosine were investigated in human erythrocytes. Adenine nucleotide catabolism, reflected by the production of hypoxanthine, is very slow under physiologic conditions and can be greatly increased by suppression of glucose or alkalinization of the medium. Experiments with inhibitors of adenosine deaminase and adenosine kinase demonstrated that under physiologic conditions the initial catabolism of AMP proceeds by way of a deamination of AMP, followed by dephosphorylation of inosine monophosphate, and that no recycling occurs between AMP and adenosine. Under glucose deprivation,-75% of the 20-fold increase of the catabolism of the adenine nucleotides proceeded by way of a dephosphorylation of AMP followed by deamination of adenosine, and a small recycling of this nucleoside could be evidenced. Inhibition of adenosine transport showed that the dephosphorylation of AMP occurred intracellularly. When the incubation medium was alkalinized in the presence of glucose, the 15-fold increase in the conversion of AMP to hypoxanthine proceeded exclusively by way of AMP deaminase but a small recycling of adenosine could also be evidenced. The threefold elevation of intraerythrocytic inorganic phosphate (PI) during glucose deprivation and its 50% decrease during alkalinization as well as experiments in which extracellular P1 was modified, indicate that the dephosphorylation of red blood cell AMP is mainly responsive to variations of AMP, whereas its deamination is more sensitive to Pi.
Protein phosphatase 2A regulates deoxycytidine kinase activity via Ser-74 dephosphorylation
FEBS Letters, 2014
Deoxycytidine kinase (dCK) is a critical enzyme for activation of anticancer nucleoside analogs. ... more Deoxycytidine kinase (dCK) is a critical enzyme for activation of anticancer nucleoside analogs. Its activity is controlled via Ser-74 phosphorylation. Here, we investigated which Ser/Thr phosphatase dephosphorylates Ser-74. In cells, the PP1/PP2A inhibitor okadaic acid increased both dCK activity and Ser-74 phosphorylation at concentrations reported to specifically target PP2A. In line with this, purified PP2A, but not PP1, dephosphorylated recombinant pSer-74-dCK. In cell lysates, the Ser-74-dCK phosphatase activity was found to be latent, Mn(2+)-activated, responsive to PP2A inhibitors, and diminished after PP2A-immunodepletion. Use of siRNAs allowed concluding definitively that PP2A constitutively dephosphorylates dCK in cells and negatively regulates its activity.
AICA-riboside (acadesine), an activator of AMP-activated protein kinase with potential for application in hematologic malignancies
Expert Opinion on Investigational Drugs, 2010
Despite considerable advances, B-cell chronic lymphocytic leukemia (CLL) is incurable with standa... more Despite considerable advances, B-cell chronic lymphocytic leukemia (CLL) is incurable with standard approaches. Thus, there remains a need for new therapies, particularly for patients who develop chemoresistance to DNA-targeting treatments. AICA-riboside (acadesine) is a nucleoside with a wide range of metabolic effects, including release of adenosine and activation of AMP-activated protein kinase (AMPK), which was initially developed as a cardioprotective agent. More recently, it has been shown that AICA-riboside induces apoptosis in various models of leukemia, including CLL. The literature data show that apoptosis induced by AICA-riboside in CLL is not dependent on a functionally normal p53 pathway. Moreover, AICA-riboside is active towards resting and proliferative models of leukemia cells, including resistant phenotypes. Finally, studies in healthy subjects and during coronary artery bypass graft surgery show that AICA-riboside is devoid of serious toxicity. This paper reviews the mechanisms of action of AICA-riboside in normal and malignant cells and discusses how AICA-riboside could impact CLL treatment. We propose that AICA-riboside, which displays a relative selectivity and a favorable toxicity profile, may offer a new treatment option for CLL.
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Papers by Françoise Bontemps