leupeptins and Leukemia--Myeloid

Ethacrynic acid, a diuretic, inhibits glutathione S-transferase P1-1 (GSTP1-1) activity and induces cell death in malignant cells at high concentrations. To improve ethacrynic acid activity, ethacrynic acid oxadiazole analogs 6s and 6u were synthesized. Although both compounds have greater antiproliferative effects than ethacrynic acid in human HL-60 cells, 6u has a reduced ability to inhibit GSTP1-1 activity. The mechanisms of both 6s- and 6u-induced cell death as well as the role of GSTP1-1 in their actions were studied. Both 6s and 6u equally induced apoptosis in HL-60 cells due to the activation of caspase-3, -9, and -8, which was correlated with the downregulation of antiapoptotic proteins c-FLIP, Mcl-1, and XIAP. The caspase inhibitor Z-VAD-FMK blocked the reduction of XIAP, but not of c-FLIP and Mcl-1, in 6s-treated cells. The reduction of c-FLIP and Mcl-1 by 6s was not blocked by the proteasomal inhibitor MG132, but was correlated with inhibition of the phosphorylation of extracellular signal-regulated kinase (ERK) and eIF4E. Both 6s and 6u decreased the intracellular glutathione (GSH) levels. N-acetylcysteine blocked reduction in the levels of Mcl-1, c-FLIP, and intracellular GSH as well as apoptosis in HL-60 cells treated by either compound. Silencing of GSTP1-1 in K562 cells sensitized, but overexpression of GSTP1-1 in Raji cells blocked, apoptosis induction by either compound. GSH conjugation at the methylene group abrogated the ability of inducing apoptosis. These data suggest that the methylene group plays an important role in the downregulation of c-FLIP and Mcl-1 proteins and apoptosis induction, which is inactivated by GSTP1-1 by forming GSH conjugates.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspases; Cell Line, Tumor; Enzyme Inhibitors; Ethacrynic Acid; Gene Expression Regulation, Leukemic; Glutathione; Glutathione S-Transferase pi; HL-60 Cells; Humans; Jurkat Cells; K562 Cells; Leukemia, Myeloid; Leupeptins; Myeloid Cell Leukemia Sequence 1 Protein; Oxadiazoles; Signal Transduction; X-Linked Inhibitor of Apoptosis Protein

ST1926 is an atypical retinoid and a promising anti-tumour agent with selective apoptotic activity on the leukaemic blast. The anti-tumour activity of the compound has been associated with its capacity to induce DNA double stranded breaks. Target profiling by affinity chromatography coupled to mass spectrometry led to the identification of histone H2A.Z as a protein capable of binding ST1926 specifically. The result was confirmed by studies involving Surface Plasmon Resonance (SPR). This indicates that H2A.Z is a primary target of ST1926 and links the perturbations of the histone pathway observed by microarray analysis to the DNA damage and apoptotic responses caused by the atypical retinoid. Comparison of the whole-genome gene-expression profiles of the ST1926-sensitive NB4 and the ST1926-resistant NB4.437r cell lines demonstrated differential expression of numerous genes. Network analysis of the data indicated enrichment of the cellular pathways controlling cAMP (cyclic adenosine-monophosphate)-dependent signal transduction, proteasome-dependent protein degradation and nuclear histones in NB4.437r cells. Pharmacological inhibition of cAMP-dependent protein kinase A with H89 partially reverted resistance of NB4.437r cells to ST1926. Conversely, inhibition of the proteasome with MG132 or bortezomib blocked the apoptotic response afforded by ST1926 in the NB4 cell line. This last effect was associated with a dramatic reduction in the DNA damage caused by the atypical retinoid. The results corroborate the idea that DNA damage is an important determinant of ST1926 apoptotic activity. More importantly, they demonstrate a proactive role of the proteasome in the DNA damaging and ensuing apoptotic response observed upon the challenge of acute myeloid leukaemia cells with ST1926.

Topics: Acute Disease; Adamantane; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Survival; Cinnamates; Cyclic AMP-Dependent Protein Kinases; Drug Resistance, Neoplasm; Enzyme Inhibitors; Gene Expression Profiling; Gene Expression Regulation, Leukemic; Histones; Humans; Isoquinolines; Leukemia, Myeloid; Leupeptins; Oligonucleotide Array Sequence Analysis; Proteasome Endopeptidase Complex; Protein Binding; Signal Transduction; Sulfonamides; Surface Plasmon Resonance

Long-term culture of phorbol ester (TPA)-differentiated and growth-arrested human U937 leukemia cells was associated with expression of c-jun transcription factors and vimentin intermediate filaments until the cells entered a retrodifferentiation program. This retrodifferentiation process revealed a reversion of the senecent differentiated cells back to undifferentiated and proliferative active young cells. A significant protein ubiquitination was detectable before retrodifferentiation and rejuvenation indicating a proteolytic down-modulation of differentiation markers. Thus, proteolytic activity significantly increased during retrodifferentiation, however, proteasomal protein expression remained unaltered. In order to investigate proteasomal associates, (ADP-ribose)polymerase-1 (PARP-1) expression progressively increased to maximal levels at the time of retrodifferentiation suggesting a possible regulatory association. Indeed, PARP-1 immunoprecipitations demonstrated a co-immunoprecipitation of proteolytically active 20S proteasome with maximal levels during retrodifferentiation. Inhibition of PARP and the proteasome by 3-aminobenzamide and MG-132, respectively, revealed about 90% of apoptotic cells by cell cycle analysis at the time of retrodifferentiation whereas control cells doubled. In contrast, a similar PARP and proteasome inhibition within 5d after TPA-induced differentiation demonstrated little if any apoptotic effects. More specifically, down-modulation of PARP-1 by an antisense PARP-1 vector construct underwent a rapid differentiation and aging and revealed no detectable retrodifferentiation in contrast to control vector-transfected U937 cells. In conclusion, retrodifferentiation of growth-arrested U937 monocytic cells requires proteasomal protein degradation and activity of PARP-1.

Topics: Apoptosis; Benzamides; Cell Differentiation; Cell Proliferation; Cellular Senescence; DNA, Antisense; Humans; Leukemia, Myeloid; Leupeptins; Monocytes; Phenotype; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Proteasome Endopeptidase Complex; Tetradecanoylphorbol Acetate; U937 Cells

The human herpes virus 8 (HHV8)-encoded viral FLICE (Fas-associating protein with death domain-like interleukin-1-converting enzyme) inhibitory protein (vFLIP) is believed to protect cells against death receptor-mediated apoptosis. In the present study we demonstrate that expression of HHV8 vFLIP in a growth factor-dependent TF-1 leukemia cell line protects against growth factor withdrawal-induced apoptosis. Unlike vector-expressing cells, those expressing HHV8 vFLIP maintain their mitochondrial membrane potential upon withdrawal from growth factor and also exhibit a block in the activation of caspases. The protective effect of HHV8 vFLIP is associated with its ability to activate the nuclear factor-kappa B (NF-kappaB) pathway and is missing in the vFLIP encoded by equine herpes virus 2 that lacks this activity. Inhibition of the NF-kappaB pathway by IkappaB superrepressor, lactacystin, MG132, arsenic trioxide, and phenylarsine oxide reverse the protection against growth factor withdrawal-induced apoptosis conferred by HHV8 vFLIP. HHV8 vFLIP up-regulates the expression of Bcl-x(L), an antiapoptotic member of the Bcl2 family, which is a known target of the NF-kappaB pathway. Collectively, the above results suggest that HHV8 vFLIP-induced NF-kappaB activation may contribute to cellular transformation seen in association with HHV8 infection by preventing the apoptosis of cells destined to die because of growth factor deprivation.

Topics: Acetylcysteine; Acute Disease; Apoptosis; Arsenic Trioxide; Arsenicals; bcl-X Protein; Cell Transformation, Viral; Enzyme Activation; Gene Expression Regulation, Viral; Granulocyte-Macrophage Colony-Stimulating Factor; Herpesvirus 8, Human; Humans; I-kappa B Proteins; Leukemia, Myeloid; Leupeptins; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; NF-kappa B; Oxides; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins; Recombinant Proteins; Rhadinovirus; Transcription, Genetic; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Viral Proteins

Acute myelogenous leukemia (AML) is typically a disease of stem progenitor cell origin. Interestingly, the leukemic stem cell (LSC) shares many characteristics with normal hematopoietic stem cells (HSCs) including the ability to self-renew and a predominantly G(0) cell-cycle status. Thus, although conventional chemotherapy regimens often ablate actively cycling leukemic blast cells, the primitive LSC population is likely to be drug-resistant. Moreover, given the quiescent nature of LSCs, current drugs may not effectively distinguish between malignant stem cells and normal HSCs. Nonetheless, based on recent studies of LSC molecular biology, we hypothesized that certain unique properties of leukemic cells could be exploited to induce apoptosis in the LSC population while sparing normal stem cells. In this report we describe a strategy using treatment of primary AML cells with the proteasome inhibitor carbobenzoxyl-l-leucyl-l-leucyl-l-leucinal (MG-132) and the anthracycline idarubicin. Comparison of normal and leukemic specimens using in vitro culture and in vivo xenotransplantation assays shows that the combination of these two agents induces rapid and extensive apoptosis of the LSC population while leaving normal HSCs viable. Molecular genetic studies using a dominant-negative allele of inhibitor of nuclear factor kappaB (IkappaBalpha) demonstrate that inhibition of nuclear factor kappaB (NF-kappaB) contributes to apoptosis induction. In addition, gene-expression analyses suggest that activation of p53-regulated genes are also involved in LSC apoptosis. Collectively, these findings demonstrate that malignant stem cells can be preferentially targeted for ablation. Further, the data begin to elucidate the molecular mechanisms that underlie LSC-specific apoptosis and suggest new directions for AML therapy.

Topics: Acute Disease; Alleles; Animals; Antibiotics, Antineoplastic; Apoptosis; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Gene Expression Regulation, Leukemic; Graft Survival; Hematopoietic Stem Cells; Humans; I-kappa B Proteins; Idarubicin; Leukemia, Myeloid; Leukocytes; Leupeptins; Mice; Mice, Inbred NOD; Mice, SCID; Multienzyme Complexes; Neoplasm Proteins; Neoplasm Transplantation; Neoplastic Stem Cells; NF-kappa B; Proteasome Endopeptidase Complex; Recombinant Fusion Proteins; Tumor Suppressor Protein p53