benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Leukemia--Erythroblastic--Acute

To examine the p53-mediated biological activities and signalling pathways, we generated stable transfectants of the p53-null IW32 murine erythroleukemia cells expressing the temperature-sensitive p53 mutant DNA, tsp53(val135). Two clones with different levels of p53 protein expression were selected for further characterization. At permissive temperature, clone 1-5 cells differentiated along the erythroid pathway, and clone 3-2 cells that produced greater levels (3.5-fold) of p53 underwent apoptosis. Apoptosis of 3-2 cells was accompanied by mitochondrial cytochrome c release and caspase activation as well as by cleavage of caspase substrates. Bax protein was induced to a similar extent in these clones by wild-type p53; expression of p21(Cip1/Waf1) and p27(Kip1) proteins was also increased. However, significantly lesser extent of induction for both CDK inhibitors was detected in the apoptotic 3-2 clone. The general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD.fmk) blocked the p53-induced apoptosis in 3-2 cells, with a concomitant elevation of p27(Kip1), suggesting that p27(Kip1) protein underwent caspase-dependent proteolysis in the apoptotic 3-2 cells. Together these results linked a pathway involving cytochrome c release, caspase activation and p27(Kip1) degradation to the p53-induced apoptosis in IW32 erythroleukemia cells.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Cycle Proteins; Clone Cells; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Cytochrome c Group; Enzyme Inhibitors; Eukaryotic Cells; Leukemia, Erythroblastic, Acute; Mice; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

K562 erythroleukemia cells undergo apoptosis when induced to differentiate along the erythroid lineage with hemin. This event, characterized by DNA fragmentation, correlated with downregulation of the survival protein, BCL-xL, and decrease in mitochondrial transmembrane potential (deltapsi[m]) that ultimately resulted in cell death. Reorientation of phosphatidylserine (PS) from the cells inner-to-outer plasma membrane leaflet and inhibition of the aminophospholipid translocase was observed upon hemin-treatment. Constitutive expression of BCL-2 did not inhibit hemin-induced alterations in lipid asymmetry or decrease in deltapsi[m], and only moderately prevented DNA fragmentation. BCL-2, on the other hand, effectively inhibited actinomycin D-induced DNA fragmentation, the appearance of PS at the cells outer leaflet and the decrease in deltapsi[m]. The caspase inhibitor, z.VAD.fmk, blocked DNA fragmentation by both hemin and actinomycin D, but inhibited PS externalization only in the actinomycin D-treated cells. These results suggest that, unlike pharmacologically-induced apoptosis, PS externalization triggered by differentiation-induced apoptosis occurs by a mechanism that is associated with a decrease in deltapsi[m], but independent of BCL-2 and caspases.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-X Protein; Caspase Inhibitors; Cell Differentiation; Cell Membrane; Cysteine Proteinase Inhibitors; Dactinomycin; DNA Fragmentation; Erythropoiesis; Gene Expression; Genes, bcl-2; Hemin; Humans; K562 Cells; Leukemia, Erythroblastic, Acute; Phosphatidylserines; Proto-Oncogene Proteins c-bcl-2

Since the CTL secreted granule protease granzyme B can activate multiple target caspases, it has been proposed that this pathway is responsible for CTL-induced cytolysis of Fas-negative targets. However, target lysis via the granule exocytosis pathway is completely resistant to caspase inhibitors. To test the possibility that granzymes trigger a postcaspase cytoplasmic apoptotic pathway leading to lysis, we have examined the caspase dependence of several cytoplasmic changes associated with apoptotic death. Rapid prelytic phosphatidylserine externalization was induced in Jurkat target cells by both the Fas ligand (FasL)/Fas and the granule exocytosis effector pathways. This was specifically blocked by peptide ketone caspase inhibitors when induced by the former, but not by the latter, pathway. A rapid prelytic loss of target mitochondrial psi was also induced by both CTL effector pathways, and this was also specifically blocked by caspase inhibitors when induced by the FasL/Fas, but not by the granule exocytosis, pathway. Similarly, target membrane blebbing induced by CTL via the FasL/Fas, but not via the granule exocytosis, effector pathway was specifically blocked by caspase inhibitors. In contrast to the above nonnuclear damage, CTL-induced target staining by the lipid probe FM1-43 reflecting plasma membrane endocytosis was blocked by caspase inhibitors. Thus, when caspase activation is blocked, the granule exocytosis pathway triggers several parameters of target apoptotic damage in addition to lysis, suggesting that granzymes directly trigger a postcaspase cytoplasmic apoptotic death pathway.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Cell Membrane; Cell Nucleus; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytoplasmic Granules; Cytotoxicity Tests, Immunologic; Cytotoxicity, Immunologic; Exocytosis; Fas Ligand Protein; fas Receptor; Humans; Jurkat Cells; Killer Cells, Natural; Leukemia, Erythroblastic, Acute; Ligands; Membrane Glycoproteins; Mitochondria; Oligopeptides; Phosphatidylserines; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured