benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and acadesine

5-Aminoimidazole-4-carboxamide (AICA) riboside, a precursor of purine nucleotide biosynthesis, induces apoptosis in Jurkat cells. Incorporation of AICAriboside into the cells is necessary for this effect since addition of nitrobenzylthioinosine, a nucleoside-transport inhibitor, completely protects Jurkat cells from apoptosis. Adenosine, but not other nucleosides, also protects Jurkat cells from AICAriboside-induced apoptosis. The apoptotic effect is caspase-dependent since caspases 9 and 3 are activated and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) blocks apoptosis. Furthermore, AICAriboside induces mitochondrial cytochrome c release. AICAriboside, when phosphorylated to AICAribotide (ZMP), is a specific activator of the AMP-activated protein kinase (AMPK) in certain cell types. However, AICAriboside does not activate AMPK in Jurkat cells. Moreover, 5-iodotubercidin, an inhibitor of AICAriboside phosphorylation, does not inhibit apoptosis in Jurkat cells. These results indicate that AICAriboside induces apoptosis independently of ZMP synthesis and AMPK activation in Jurkat cells.

Topics: Adenosine; Affinity Labels; Amino Acid Chloromethyl Ketones; Aminoimidazole Carboxamide; Apoptosis; Caspases; Cyclic AMP-Dependent Protein Kinases; Cytochrome c Group; Enzyme Inhibitors; Humans; Jurkat Cells; Phosphorylation; Ribonucleosides; Thioinosine

Acadesine, 5-aminoimidazole-4-carboxamide (AICA) riboside, induced apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells in all samples tested (n = 70). The half-maximal effective concentration (EC(50)) for B-CLL cells was 380 +/- 60 microM (n = 5). The caspase inhibitor Z-VAD.fmk completely blocked acadesine-induced apoptosis, which involved the activation of caspase-3, -8, and -9 and cytochrome c release. Incubation of B-CLL cells with acadesine induced the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), indicating that it is activated by acadesine. Nitrobenzylthioinosine (NBTI), a nucleoside transport inhibitor, 5-iodotubercidin, an inhibitor of adenosine kinase, and adenosine completely inhibited acadesine-induced apoptosis and AMPK phosphorylation, demonstrating that incorporation of acadesine into the cell and its subsequent phosphorylation to AICA ribotide (ZMP) are necessary to induce apoptosis. Inhibitors of protein kinase A and mitogen-activated protein kinases did not protect from acadesine-induced apoptosis in B-CLL cells. Moreover, acadesine had no effect on p53 levels or phosphorylation, suggesting a p53-independent mechanism in apoptosis triggering. Normal B lymphocytes were as sensitive as B-CLL cells to acadesine-induced apoptosis. However, T cells from patients with B-CLL were only slightly affected by acadesine at doses up to 4 mM. AMPK phosphorylation did not occur in T cells treated with acadesine. Intracellular levels of ZMP were higher in B-CLL cells than in T cells when both were treated with 0.5 mM acadesine, suggesting that ZMP accumulation is necessary to activate AMPK and induce apoptosis. These results suggest a new pathway involving AMPK in the control of apoptosis in B-CLL cells and raise the possibility of using acadesine in B-CLL treatment.

Topics: Adenosine; Amino Acid Chloromethyl Ketones; Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Antimetabolites, Antineoplastic; Apoptosis; B-Lymphocytes; Caspases; Cyclic AMP-Dependent Protein Kinases; Cysteine Proteinase Inhibitors; Enzyme Activation; Enzyme Inhibitors; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; MAP Kinase Signaling System; Mitochondria; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Neoplasm Proteins; Neoplastic Stem Cells; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Ribonucleosides; Ribonucleotides; T-Lymphocytes; Tubercidin; Tumor Cells, Cultured

We have recently shown that conditions known to activate AMP-activated protein kinase (AMPK) in primary beta-cells can trigger their apoptosis. The present study demonstrates that this is also the case in the MIN6 beta-cell line, which was used to investigate the underlying mechanism. Sustained activation of AMPK was induced by culture with the adenosine analogue AICA-riboside or at low glucose concentrations. Both conditions induced a sequential activation of AMPK, c-Jun-N-terminal kinase (JNK) and caspase-3. The effects of AMPK on JNK activation and apoptosis were demonstrated by adenoviral expression of constitutively active AMPK, a condition which reproduced the earlier-described AMPK-dependent effects on pyruvate kinase and acetyl-coA-carboxylase. The effects of JNK activation on apoptosis were demonstrated by the observations that (i). its inhibition by dicumarol prevented caspase-3 activation and apoptosis, (ii). adenoviral expression of the JNK-interacting scaffold protein JIP-1/IB-1 increased AICA-riboside-induced JNK activation and apoptosis. In primary beta-cells, AMPK activation was also found to activate JNK, involving primarily the JNK 2 (p54) isoform. It is concluded that prolonged stimulation of AMPK can induce apoptosis of insulin-producing cells through an activation pathway that involves JNK, and subsequently, caspase-3.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Chloromethyl Ketones; Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Apoptosis; Carrier Proteins; Caspase 3; Caspases; Cell Line; Dicumarol; Enzyme Activation; Enzyme Inhibitors; Glucose; Insulin; Islets of Langerhans; JNK Mitogen-Activated Protein Kinases; Mice; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Protein Serine-Threonine Kinases; Rats; Recombinant Fusion Proteins; Ribonucleosides