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Deoxycytidine kinase (dCK) is an enzyme involved in the deoxynucleoside salvage pathway; is phosphorylates deoxycytidine, but also deoxyaadenosine and deoxyguanosine. In addition, dCK phosphorylates and activates several nucleoside analogues used as chemotherapeutic agents. Previous studies have shown that dCK is a phosphoprotein and that its activity is modulated by phosphorylation of its Ser-74 residue. However, the signaling pathways regulating this phosphorylation are still not very well documented. In order to define them better, we studied the effects of hyperosmotic stress, the only condition known to decrease dCK activity. The first results showed that hyperosmotic stress, triggered by addition of 500 mM sorbitol, induced a bimodal effect on dCK activity in the leukemic cells CCRF-CEM : a decrease in dCK activity was observed after 30 min, followed by a return to the initial activity and finally by a second, steeper, decrease after 240 min. Given that the effect of hyperosmotic stress was higher at this time, we started by investigating its action after 240 min. We observed that the decrease in dCK activity at this point was not only due to its dephosphorylation on Ser-74, but also to its partial degradation. Use of protease inhibitors revealed that this degradation was partly due to its cleavage by caspases. Furthermore, sorbitol induced the catabolism of adenylic nucleotides and the degradation of Mcl-1, an anti-apoptotic protein, indicating its cytotoxicity in leukemic cells. Since dCK degradation complicated the analysis of the signaling pathways involved in the regulation of its activity, we continued our work by studying dCK regulation by sorbitol after 30 min in CCRF-CEM cells, when dCK is not yet degraded. In order to determine whether dCK dephosphorylation on Ser-74 induced by osmotic stress was due to the inhibition of a protein kinase or the stimulation of a protein phosphatase (PP), we used the different protein kinase and phosphatase effectors. The results indicated that neither the p70S6 kinase nor the p38 kinase, whose activities are modified by osmotic stress, would be involved in dCK inactivation by sorbitol. Finally, we showed that neither PP2A nor PP1 would play a major role in dCK regulation by osmotic stress induced by sorbitol.

Deoxycytidine Kinase --- Neoplasms --- Osmotic Pressure --- Apoptosis

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Deoxycytidine kinase (dCK) catalyzes the phosphorylation of deoxycytidine, deoxyadenosine and deoxyguanosine, using ATP or UTP as phosphate donor. This is the rate-limiting reaction in the salvage of deoxyribonucleosides, which supplies cells with dNTPs for DNA synthesis. In addition, dCK phosphorylates and activates a number of nucleoside analogues used in anticancer and antiviral chemotherapy. Studies of the mechanisms that control the activity of the enzyme are thus of particular interest. Recently, it has been demonstrated in our lab that dCK is a phosphoenzyme containing at least four phosphorylation sites. Moreover, phosphorylation of Ser-74 was found to be essential for the control of dCK activity. However, the signalling pathway leading to the phosphorylation and the activation of dCK is not known yet.
The first objective of my study was to analyze in EHEB cells whether GSK-3 was implicated in the regulation of dCK. Indeed, TDZD-8, a specific inhibitor of GSK-3, was able to suppress the activation of dCK induced by the nucleoside analogue 2-chloro-2’-deoxyadenosine (CdA). First, we have shown that TDZD-8 also reduced the activation of dCK induced by several other genotoxic agents, such as genistein and aphidicolin. However, other inhibitors of GSK-3, such as Li+ and alsterpaullone, were unable to reduce dCK activation by CdA or others agents, despite the fact that they were found to inhibit GSK-3 in vitro as well as in intact cells. These results disagreed with a role of GSK-3 in the regulation of dCK activity. This conclusion was strengthened by our observation that CdA, which activates dCK, did not modify GSK-3 activity. Moreover, GSK-3 was not able to phosphorylate recombinant dCK in vitro. Concerning the TDZD-8, we have shown that its effect in intact cells could neither be explained by a direct effect on dCK activity nor by inhibition of CdA metabolism. Thus, we suggest that TDZD-8 reduces dCK activation not via inhibition of GSK-3, but via inhibition of another protein kinase that remains to identify.
The second step of our study was to examine whether the transcription factor p53 was involved in the regulation of dCK activity. Indeed, it had been reported that pifithrin-, a pharmacological inhibitor of p53, abolished the activation of dCK by CdA. The hypothesis of a role of p53 in the control of dCK activity was attractive because the majority of the agents that activate dCK are genotoxic and induce p53 activation. However, the effect of pifithrin- reported in the literature was not reproduced in our lab. Moreover, we showed that elevation of p53 was not always followed by an activation of dCK. Finally, we noted that inhibition of protein kinases responsible for the phosphorylation and the activation of p53 did not affect dCK activation by CdA. Taken together, these results indicate that the p53 pathway is not involved in the control of dCK activity. La désoxycytidine kinase (dCK) catalyse la phosphorylation de la désoxycytidine, de la désoxyadénosine et de la désoxyguanosine, l'étape limitante de la voie de récupération des désoxynucléosides qui mène à la formation des dNTPs, précurseurs de l'ADN. En plus de ce rôle physiologique, la dCK phosphoryle et active un grand nombre d'analogues de nucléosides utilisés en chimiothérapie anticancéreuse et antivirale, d'où l'intérêt porté à cette enzyme. Il a été récemment démontré au laboratoire que l'activité de la dCK était régulée par phosphorylation, en particulier de la Ser-74. La voie de signalisation qui mène à la phosphorylation et l'activation de la dCK n'est cependant pas encore élucidée.
Le premier objectif de mon travail était d'analyser si la GSK-3 était impliquée dans la régulation de l'activité de la dCK. En effet, un inhibiteur spécifique de cette protéine kinase, le TDZD-8, était capable de supprimer l'activation de cette enzyme provoquée par l'analogue de nucléoside 2-chloro-2'-désoxyadénosine (CdA). Nous avons commencé par montrer que le TDZD-8 supprimait aussi l'activation de la dCK induite par d'autres agents génotoxiques, comme la génistéine et l'aphidicoline. Malheureusement, ces effets ne purent être reproduits par d'autres inhibiteurs de la GSK-3 (Li+, alsterpaullone, SB-216763), qui pourtant semblaient efficaces aussi bien in vitro que dans la cellule intacte. Ces résultats mettaient en doute un rôle de la GSK-3 dans la régulation de la dCK. D'autre part, la GSK-3 n'est pas capable de phosphoryler directement la dCK recombinante in vitro. De plus, la CdA qui active la dCK ne modifie pas l'activité de la GSK-3, ainsi que nous l'avons constaté après avoir mis au point le dosage de cette protéine kinase. Nous en concluons qu’il est peu probable que la GSK-3 joue un rôle dans la régulation de l'activité de la dCK. Quant à l'effet du TDZD-8, nous avons montré qu'il ne peut s'expliquer ni par un effet direct sur l'activité de la dCK, ni par une inhibition du métabolisme de la CdA. Nous suggérons qu'il inhibe une autre protéine kinase que la GSK-3, qui serait elle réellement impliquée dans le contrôle de l'activité de la dCK.
Le second objectif de mon travail était d'examiner si le facteur de transcription p53 jouait un rôle dans la régulation de l'activité de la dCK. La littérature rapportait en effet que l'activation de la dCK par la CdA était supprimée par la pifithrin-, un inhibiteur de l'activité transcriptionnelle de p53. Cette hypothèse était plausible puisque la plupart des agents qui activent la dCK sont génotoxiques et activent p53. D'une part, l'effet de la pifithrin- décrit dans la littérature n'a pu être reproduit au laboratoire. D'autre part, nous avons montré que l'activation de p53 n'était pas toujours suivie d'une activation de la dCK. Finalement, l'inhibition des protéines kinases responsables de la phosphorylation et de l'activation de p53 n'affecte pas l'activation de la dCK. Ces résultats suggèrent que la voie p53 n'est pas impliquée dans le contrôle de l'activité de la dCK.

glycogen synthase kinase 3 beta --- Tumor Suppressor Protein p53 --- Deoxycytidine Kinase --- Dideoxyribonucleases --- Humans --- Tumor Cells, Cultured --- Leukemia, Lymphoid