calpain and Leukemia

Growth arrest specific 2 (GAS2) modulates cell cycle, apoptosis, and Calpain activity. GAS2-Calpain2 axis is required for the growth of BCR-ABL(+) hematopoietic cells and chronic myeloid leukemia cells. However, the expression of GAS2 in acute leukemia patients remains unclear and what role GAS2-Calpain2 axis plays in these leukemic cells is not known yet. In this study, GAS2 was found to have significantly higher expression in 16 various leukemic cell lines than in control cells. Using THP-1 cells (from acute myeloid leukemia patient, AML) and Jurkat cells (from acute lymphoid leukemia patient, ALL) as models, we found that GAS2 silence led to elevated Calpain activity, decreased cellular growth, and inhibition of colony-forming cell (CFC) production; and these effects could be rescued by GAS2 re-expression. Moreover, GAS2 silence prevented tumor formation of THP-1 cells in nude mice. In both THP-1 and Jurkat cells, GAS2 interacted with Calpain2 rather than Calpain1. The dominant negative form of GAS2 (GAS2DN, GAS2Δ171-313) had similar effects on leukemic cells through the activation of Calpain. Importantly, Calpain2 silence abolished the proliferation inhibition induced by GAS2 targeting. We also found that GAS2 was aberrantly expressed and Calpain activity was decreased in clinical isolates from acute leukemia patients. Taken together, our results demonstrated the deregulation of GAS2 in both AML and ALL and the requirement of GAS2-Calpain2 axis for the growth of leukemic cells, which will help to understand the molecular pathogenesis of hematological malignancies and possibly to develop novel approaches to treat these deadly diseases.

Topics: Biomarkers, Tumor; Calpain; Cell Line, Tumor; Cell Proliferation; Humans; Jurkat Cells; Leukemia; Microfilament Proteins; Signal Transduction

Topics: Animals; Calpain; Cell Transformation, Neoplastic; GATA1 Transcription Factor; Humans; Leukemia; Megakaryocytes; Mutation; Positive Transcriptional Elongation Factor B; Ribonucleoproteins, Small Nuclear

Megakaryocyte morphogenesis employs a "hypertrophy-like" developmental program that is dependent on P-TEFb kinase activation and cytoskeletal remodeling. P-TEFb activation classically occurs by a feedback-regulated process of signal-induced, reversible release of active Cdk9-cyclin T modules from large, inactive 7SK small nuclear ribonucleoprotein particle (snRNP) complexes. Here, we have identified an alternative pathway of irreversible P-TEFb activation in megakaryopoiesis that is mediated by dissolution of the 7SK snRNP complex. In this pathway, calpain 2 cleavage of the core 7SK snRNP component MePCE promoted P-TEFb release and consequent upregulation of a cohort of cytoskeleton remodeling factors, including α-actinin-1. In a subset of human megakaryocytic leukemias, the transcription factor GATA1 undergoes truncating mutation (GATA1s). Here, we linked the GATA1s mutation to defects in megakaryocytic upregulation of calpain 2 and of P-TEFb-dependent cytoskeletal remodeling factors. Restoring calpain 2 expression in GATA1s mutant megakaryocytes rescued normal development, implicating this morphogenetic pathway as a target in human leukemogenesis.

Topics: Actinin; Animals; Blotting, Western; Calpain; Cell Differentiation; Cell Nucleus; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Cytoskeleton; Flow Cytometry; GATA1 Transcription Factor; Humans; Immunoprecipitation; Leukemia; Megakaryocytes; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Morphogenesis; Mutation; Positive Transcriptional Elongation Factor B; Protein Binding; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleoproteins, Small Nuclear; RNA, Messenger; Transcription, Genetic

3β,16β,17α-Trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-β-D-xylopyranosyl)-(1→3)-2-O-acetyl-α-L-arabinopyranoside (OSW-1) is a natural product with potent antitumor activity against various types of cancer cells, but the exact mechanisms of action remain to be defined. In this study, we showed that OSW-1 effectively killed leukemia cells at subnanomolar concentrations through a unique mechanism by causing a time-dependent elevation of cytosolic Ca(2+) prior to induction of apoptosis. A mechanistic study revealed that this compound inhibited the sodium-calcium exchanger 1 on the plasma membrane, leading to an increase in cytosolic Ca(2+) and a decrease in cytosolic Na(+). The elevated cytosolic Ca(2+) caused mitochondrial calcium overload and resulted in a loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase-3. Furthermore, OSW-1 also caused a Ca(2+)-dependent cleavage of the survival factor GRP78. Inhibition of Ca(2+) entry into the mitochondria by the uniporter inhibitor RU360 or by cyclosporin A significantly prevented the OSW-1-induced cell death, indicating the important role of mitochondria in mediating the cytotoxic activity. The extremely potent activity of OSW-1 against leukemia cells and its unique mechanism of action suggest that this compound may be potentially useful in the treatment of leukemia.

Topics: Biological Products; Calcium; Calcium Channels; Calpain; Caspase 3; Cell Death; Cell Line, Tumor; Cholestenones; Cyclosporine; Cytochromes c; Cytosol; Drug Screening Assays, Antitumor; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Activation; Extracellular Space; Heat-Shock Proteins; Homeostasis; Humans; Leukemia; Lymphoma; Membrane Potential, Mitochondrial; Mitochondria; Saponins; Sodium-Calcium Exchanger; Thapsigargin; Time Factors

It is now generally recognised that different modes of programmed cell death (PCD) are intimately linked to the cancerous process. However, the mechanism of PCD involved in cancer chemoprevention is much less clear and may be different between types of chemopreventive agents and tumour cell types involved. Therefore, from a pharmacological view, it is crucial during the earlier steps of drug development to define the cellular specificity of the candidate as well as its capacity to bypass dysfunctional tumoral signalling pathways providing insensitivity to death stimuli. Studying the cytotoxic effects of violacein, an antibiotic dihydro-indolone synthesised by an Amazon river Chromobacterium, we observed that death induced in CD34(+)/c-Kit(+)/P-glycoprotein(+)/MRP1(+) TF1 leukaemia progenitor cells is not mediated by apoptosis and/or autophagy, since biomarkers of both types of cell death were not significantly affected by this compound. To clarify the working mechanism of violacein, we performed kinome profiling using peptide arrays to yield comprehensive descriptions of cellular kinase activities. Pro-death activity of violacein is actually carried out by inhibition of calpain and DAPK1 and activation of PKA, AKT and PDK, followed by structural changes caused by endoplasmic reticulum stress and Golgi apparatus collapse, leading to cellular demise. Our results demonstrate that violacein induces kinome reprogramming, overcoming death signaling dysfunctions of intrinsically resistant human leukaemia cells.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Calcium-Calmodulin-Dependent Protein Kinases; Calpain; Cell Death; Cell Line, Tumor; Cell Survival; Cyclic AMP-Dependent Protein Kinases; Death-Associated Protein Kinases; Endoplasmic Reticulum Stress; Golgi Apparatus; Humans; Indoles; Leukemia; Proto-Oncogene Proteins c-akt

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. In this video, calpain activity in fixed and living murine 32 Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32 Dkit cells are shown to have elevated activity compared to normal splenocytes.

Topics: Acrylates; Animals; Calpain; Cell Line, Tumor; Coumarins; Dipeptides; Flow Cytometry; Leukemia; Mice; Spleen

The caspase-mediated cleavage of a limited number of cellular proteins is a common feature of apoptotic cell death. This cleavage usually inhibits the function of the target protein or generates peptides that actively contribute to the death process. In the present study, we demonstrate that the cyclin-dependent kinase inhibitor p27Kip1 is cleaved by caspases in human leukemic cells exposed to apoptotic stimuli. We have shown recently that p27Kip1 overexpression delayed leukemic cell death in response to cytotoxic drugs. In transient transfection experiments, the p23 and the p15 N-terminal peptides generated by p27Kip1 proteolysis demonstrate an anti-apoptotic effect similar to that induced by the wild-type protein, whereas cleavage-resistant mutants have lost their protective effect. Moreover, stable transfection of a cleavage-resistant mutant of p27Kip1 sensitizes leukemic cells to drug-induced cell death. Altogether, these results indicate that proteolysis of p27Kip1 triggered by caspases mediates the anti-apoptotic activity of the protein.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Base Sequence; Calpain; Caspase 3; Caspase 6; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cysteine Proteinase Inhibitors; Etoposide; Humans; Leukemia; Leupeptins; Microtubule-Associated Proteins; Molecular Sequence Data; Mutation; Nucleic Acid Synthesis Inhibitors; Oligopeptides; Protein Serine-Threonine Kinases; Thimerosal; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Side-chain modified vitamin D analogs including 20-Epi-22-oxa-24a,26a,27a-trihomo-1alpha,2 5-dihydroxyvitamin D3 (KH1060), and 1,24-dihydroxy-22-ene-24-cyclopropyl-vitamin D3 (MC903) were originally designed to aid in the treatment of hyperproliferative disorders including psoriasis and cancer. Here we demonstrate that these analogs, as well as the 6-cis-locked conformer, 1alpha,25-dihydroxy-lumisterol3 (JN) prime NB4 cells for monocytic differentiation. Previously, the action of MC903 and KH1060 was presumed to be mediated by the nuclear vitamin D receptor (VDRnuc). Differentiation in response to all analogs was shown to be inhibited by 1beta,25-dihydroxyvitamin D3 (HL), the antagonist to the nongenomic activities of 1,25D3. These data suggest that although MC903 and KH1060 may bind the VDRnuc, that the differentiative activities of these agents requires nongenomic signaling pathways. Here we show that 1alpha,25(OH)2-d5-previtamin D3 (HF), JN, KH1060, and MC903 induce expression of PKC alpha and PKC delta and translocation of both isoforms to the particulate fraction, and PKC alpha to the nuclear fraction. The full differentiation response with combinations of analogs and TPA was inhibited 50% by the membrane permeable Ca2+ chelator, 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) or calpain inhibitor I. These data demonstrate that intracellular free calcium and the calcium-dependent protease, calpain play critical roles in monocytic differentiation. Intracellular calcium appears to be most critical in the 1,25D3-priming stage of differentiation, while calpain is essential in the TPA maturation response.

Topics: Biological Transport; Calcitriol; Calcium; Calpain; Cell Differentiation; Cell Nucleus; Cellular Senescence; Humans; Intracellular Membranes; Isoenzymes; Leukemia; Monocytes; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-delta; Receptors, Calcitriol; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vitamin D

N-terminal peptides from the calpain small subunit were shown to have dose-dependent chemotactic activity toward several types of leukemia cells: T cell, B cell, monocyte and granulocyte/monocyte line leukemia cells. In order to prove that chemotaxis is mediated via receptors, a fluorescein-labeled probe was prepared from one of the N-terminal peptides and its interaction with peripheral leukocytes was estimated by means of flow cytometry, resulting in staining not only of neutrophils but also of most of the monocytes and more than half of the T and B lymphocytes. The results indicate that calpain-derived N-terminal peptides may be involved in defense mechanisms, inducing chemotaxis of immunocytes as well as that of neutrophils.

Topics: Amino Acid Sequence; B-Lymphocytes; Calpain; Chemotactic Factors; Chemotaxis, Leukocyte; Granulocytes; Humans; In Vitro Techniques; Leukemia; Macromolecular Substances; Molecular Sequence Data; Monocytes; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Receptors, Formyl Peptide; Receptors, Immunologic; Receptors, Peptide; Structure-Activity Relationship; T-Lymphocytes; Tumor Cells, Cultured