Activation du cycle cellulaire par la 2-chloro-2'-désoxyadénosine dans la lignée leucémique EHEB : un nouveau mécanisme impliqué dans l'induction de l'apoptose
(2005)
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- Authors
- Supervisors
- Bontemps, Françoise
- Abstract
- 2-Cholor-2’-deoxyadenosine (CdA, cladribine) is a chemotherapeutic agent belonging to the family of purine analogues, which is particularly active in B-cell chronic lymphocytic leukemia (C-CLL). CdA has to be converted into CdATP to expert its cytotoxic effects. CdATP, the active metabolite of CdA, inhibits several enzymes involved in DNA synthesis, such as ribonucleotide reductase and DNA polymerases, and also activates apoptotic pathways in both resting and dividing lymphocytes. Inhibition of DNA synthesis, leading to DNA lesions, is considered the mechanism by which CdA exert its cytotoxicity in dividing cells. The aim of the present work was to further investigate the mechanism of action of CdA. We performed this study in the B-CLL cell lime EHEB. <BR> EHEB cells were founds less sensitive to CdA than B-CLL lymphocytes and other human lymphoblastic cell lines. Therefore, the first part of this work was devoted to unravel the mechanism(s) of resistance of EHEB cells to CdA. Results showed that inhibition of ribonucleotide reductase by CdA, which plays a major role in inhibition of DNA synthesis, is low in EHEB cells, probably because of the low capacity of EHEB cells to accumulate CdATP. Moreover, whereas DNA synthesis is slightly inhibited after a 4 h-incubation with low CdA, it is surprisingly 2- to 3-fold augmented after a 24 h-incubation. This effect, which is maximal at concentration of CdA close to its IC50, is explained by an increase of the proportion of cells in the S phase of the cell cycle. <BR> If reduced accumulation of CdATP could account, at least partially, for the resistance of EHEB cells to CdA, it could not explain why CdA produces in this cell line an increase of the number of cells in the S phase of the cell cycle. Flow cytometry experiments revealed that the increase in the proportion of cells in S phase is due to a stimulation by CdA of the passage of cells from G1 to S phase. In accordance with this result, we observed an increased of cdk2 activity, a protein kinase that plays a major role in the control of G1/S transition, and of the phosphorylation of its target, the Rb protein. We also determined that the increase of cdk2 activity is correlated with an increase of its expression and with a decrease in the level of its inhibitors p21 and p27. In order to know whether this cdk2 activation, was involved in CdA-induced apoptosis, we tested the effect of roscovitine, a cdk2-inhibitor, on the activation of caspase-3. At a concentration which completely blocks the CdA-induced activation of the cell cycle, roscovitine also significantly reduces the CdA-induced activation of caspase-3, indicating that cdk2 and/or cell cycle activation play a role in apoptosis provoked by CdA in this cell line. In conclusion, our studies have led to the identification of a new mode of cellular response to CdA, involving activation of cdk2 and the cell cycle, which appears to contribute to apoptotic cell death
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Citations
Cardoen, S. (2005). Activation du cycle cellulaire par la 2-chloro-2′-désoxyadénosine dans la lignée leucémique EHEB : un nouveau mécanisme impliqué dans l’induction de l’apoptose. https://hdl.handle.net/2078.5/110712