Abstract
The present study investigated the effect of cladribine (CLA) and six of its derivatives containing a formamidine group at position 6 (CLA-FDM, CLA-FPAZ, CLA-FPIR, CLA-FPIP, CLA-FHEX, and CLA-FMOR) on acute promyelocytic, lymphoblastic, and acute monocytic leukemia cells. The role of ATR kinase in deoxycytidine kinase (dCK) activation in response to DNA damage was assessed. The presence of DNA lesions was assessed by measurement phosphorylation of H2AX and by using the alkaline comet assay with proteinase K post-treatment following assessment of the cell cycle. Apoptotic events such as alterations in intracellular calcium concentration, caspase-3/7 activity and increased sub-G1 cell population were measured. CLA derivatives were highly effective against leukemic cells, showing high cytotoxicity, causing DNA fragmentation, and inducing DNA-protein cross-links in leukemic cells. CLA-FMOR showed the highest efficacy. CLA derivatives increased the levels of intracellular calcium ions, caspase-3/7 and the percentage of sub-G1 apoptotic cells and blocked cells in the S phase of the cell cycle to a greater extent than free CLA. The selective ATR inhibitor VE-821 significantly suppressed the increase in dCK activity and decreased basal dCK activity. The present results suggested that ATR kinase controls dCK activity in response to synthetic CLA derivatives.
Highlights
The present study investigated the effect of cladribine (CLA) and six of its derivatives containing a formamidine group at position 6 (CLA-FDM, CLA-FPAZ, CLA-FPIR, CLA-FPIP, CLA-FHEX, and CLAFMOR) on acute promyelocytic, lymphoblastic, and acute monocytic leukemia cells
CLA is resistant to deamination by adenosine deaminase (ADA) due to the presence of a chlorine atom in the structure of the purine ring, but is efficiently phosphorylated by deoxycytidine kinase (dCK) and 5′-nucleotidases (5′-NT) to 2-chlorodeoxyadenosine monophosphate (2-CdAMP) and to triphosphate 2- chlorodeoxyadenosine (2-CdATP)
According to protective effect of VE-821 observed in cytotoxicity assays, we found that activation of caspase-3/7 in response to cladribine and new derivatives was significantly reduced in the presence of the ATR inhibitor, indicating a decrease in apoptosis
Summary
The present study investigated the effect of cladribine (CLA) and six of its derivatives containing a formamidine group at position 6 (CLA-FDM, CLA-FPAZ, CLA-FPIR, CLA-FPIP, CLA-FHEX, and CLAFMOR) on acute promyelocytic, lymphoblastic, and acute monocytic leukemia cells. The presence of DNA lesions was assessed by measurement phosphorylation of H2AX and by using the alkaline comet assay with proteinase K post-treatment following assessment of the cell cycle Apoptotic events such as alterations in intracellular calcium concentration, caspase-3/7 activity and increased sub-G1 cell population were measured. Cladribine modifies level of histones, inhibits the action of anti-apoptotic proteins, reduces the mitochondrial membrane potential, increases the production of reactive oxygen species in cells and causes intracellular calcium growth, resulting in the release of apoptosis-inducing factors such as cytochrome C, second mitochondria-derived activator of caspases/direct IAP-binding protein with low PI, and apoptosis-inducing factor[4,7,8]. ATR promotes cell cycle arrest and repair of DNA or induces apoptosis if the repair systems are overwhelmed (activating CHK-1 kinase and phosphorylating many proteins that are part of the DDR pathway: H2AX, BRCA1/2 (breast cancer type 1/2 susceptibility protein), RAD51 and p53)[14]. The role of ATR in dCK activation in response to cladribine derivatives was investigated
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