ABSTRACT
To clarify the role of lipid rafts in 2-chloro-2'-deoxyadenosine (2CdA; Cladribine)-induced apoptosis, the effects of disruption of lipid rafts by methyl-beta-cyclodextrin (MbetaCD) and filipin on 2CdA-induced apoptosis were investigated in four human acute lymphoblastic leukemia (ALL) cell lines comprised of T cells (MOLT-4, Jurkat) and B cells (NALM, BALL-1). The disruption of lipid rafts significantly inhibited 2CdA-induced apoptosis, indicating the crucial role of lipid rafts in the induction of apoptosis in leukemia cells. These reagents significantly inhibited 2CdA-induced elevation of the intracellular calcium concentration ([Ca(2+)](i)) in MOLT-4 cells, and 2CdA-induced apoptosis was partly inhibited by the Ca(2+) chelators BAPTA-AM and EGTA, and the L-type Ca(2+) channel blocker nifedipine. On the other hand, they had no effects on the cellular uptake of 2CdA. These results indicated that lipid rafts partly contributed to 2CdA-induced apoptosis by regulating Ca(2+) influx via the plasma membrane.
Subject(s)
Apoptosis/drug effects , Cladribine/pharmacology , Membrane Microdomains/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Calcium/metabolism , Cell Line, Tumor , Chelating Agents/pharmacology , Cladribine/antagonists & inhibitors , Drug Screening Assays, Antitumor , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Filipin/pharmacology , Humans , Membrane Microdomains/drug effects , Nifedipine/pharmacology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Time Factors , beta-Cyclodextrins/pharmacologyABSTRACT
The mechanism of apoptosis induced by 2-chloro-2'-deoxyadenosine (2CdA) in human leukemia cell line MOLT-4 was investigated. 2CdA induced increases of 3'-OH ends of genomic DNA, ladder-like DNA fragmentation and phosphatidylserine translocation to the outer membrane, which are apoptotic characteristics. These apoptotic phenomena induced by 2CdA were inhibited by cycloheximide (CHX; a protein synthesis inhibitor), deoxycytidine (dC; a substrate of deoxycytidine kinase), acetyl Ile-Glu-Thr-Asp aldehyde (Ac-IETD-CHO; a caspase-8 inhibitor) and acetyl Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO; a caspase-3 inhibitor). The protein synthesis-dependent expression of Fas and Fas ligand (Fas-L) was detected by treatment with 2CdA. The proteolytic processing of procaspases-8 and -3 to produce active fragments, caspases-8 (p18) and -3 (p17), respectively, was observed after treatment with 2CdA, and suppressed by cycloheximide. Increases in the activities of caspases-8 and -3 were observed after 2CdA treatment. Their activation was also dependent on protein synthesis. These results indicated that 2CdA-induced apoptosis was triggered by phosphorylation of 2CdA followed by the protein synthesis-dependent expression of Fas and Fas-L and activation of caspases-8 and -3.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Caspases/metabolism , Cladribine/pharmacology , Leukemia/drug therapy , Leukemia/metabolism , fas Receptor/drug effects , Antineoplastic Agents/antagonists & inhibitors , Blotting, Western , Caspase 3 , Caspase 8 , Caspase 9 , Caspase Inhibitors , Cladribine/antagonists & inhibitors , Cycloheximide/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , DNA Fragmentation , DNA, Neoplasm , Deoxycytidine/pharmacology , Enzyme Activation/drug effects , Flow Cytometry , Fluorometry , Humans , In Situ Nick-End Labeling , Leukemia/enzymology , Oligopeptides/pharmacology , Phosphorylation/drug effects , Tumor Cells, CulturedABSTRACT
Interleukin-10 failed to modify either the percentage of bcl-2+ cells or the number of bcl-2 molecules, or to reduce 2-chlorodeoxyadenosine- and fludarabine-induced apoptosis. The cytokine at 0.1 ng/mL induced an increase of cell survival both in the absence or in the presence of 2-chlorodeoxyadenosine, while no difference was appreciated with fludarabine.
Subject(s)
Apoptosis/drug effects , Gene Expression Regulation, Leukemic/drug effects , Interleukin-10/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Neoplasm Proteins/biosynthesis , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Antimetabolites, Antineoplastic/pharmacology , Cladribine/antagonists & inhibitors , Cladribine/pharmacology , Drug Interactions , Drug Screening Assays, Antitumor , Humans , Neoplasm Proteins/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Tumor Cells, Cultured/drug effects , Vidarabine/analogs & derivatives , Vidarabine/pharmacologyABSTRACT
2-chloroadenosine induced DNA fragmentation and cell death in human thymocytes primarily by Ca(2+)-dependent mechanisms. Incubation of human thymocytes with 2-chlorodeoxyadenosine (5-1000 nM) also induced cell death (apoptosis) which was dependent on macromolecule synthesis and involved activation of an endonuclease which was inhibited by Zn2+. The effect of 2-chlorodeoxyadenosine was prevented by addition of dipyridamole, a strong nucleoside transport inhibitor, or of deoxycytidine, previously shown to compete for uptake by deoxycytidine kinase. 2-Chlorodeoxyadenosine-induced apoptosis did not involve increases in the cytosolic Ca2+ concentration, but required the presence of intracellular Ca2+. It was not inhibited by activators of protein kinase C previously shown to inhibit Ca(2+)-dependent cell death. Addition of 2-chlorodeoxyadenosine induced an increase in the amount of p53 in human thymocytes, while 2-chloroadenosine had no effect. These data suggest that 2-chloroadenosine and 2-chlorodeoxyadenosine induce cell death in human thymocytes via different signalling pathways.
