Elevated neutrophil membrane expression of proteinase 3 is dependent upon CD177 expression.

Proteinase 3 (PR3) is a major autoantigen in anti-neutrophil cytoplasmic antibodies (ANCA)-associated systemic vasculitis (AASV), and the proportion of neutrophils expressing PR3 on their membrane (mPR3+) is increased in AASV. We have shown recently that mPR3 and CD177 are expressed on the same cells in healthy individuals. In this study we try to elucidate mechanisms behind the increased mPR3 expression in AASV and its relationship to CD177. All neutrophils in all individuals were either double-positive or double-negative for mPR3 and CD177. The proportion of double-positive neutrophils was increased significantly in AASV and systemic lupus erythematosus patients. The proportion of mPR3+/CD177+ cells was not correlated to general inflammation, renal function, age, sex, drug treatment and levels of circulating PR3. AASV patients had normal levels of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. Pro-PR3 was found to constitute 10% of circulating PR3 but none of the mPR3. We found increased mRNA levels of both PR3 and CD177 in AASV, but they did not correlate with the proportion of double-positive cells. In cells sorted based on membrane expression, CD177-mRNA was several-fold higher in mPR3+ cells. When exogenous PR3 was added to CD177-transfected U937 cells, only CD177+ cells bound PR3 to their membrane. In conclusion, the increased membrane expression of PR3 found in AASV is not linked directly to circulating PR3 or PR3 gene transcription, but is dependent upon CD177 expression and correlated with the transcription of the CD177 gene.

Specific inhibition of basal mitogen-activated protein kinases and phosphatidylinositol 3 kinase activities in leukemia cells: a possible therapeutic role for the kinase inhibitors.

OBJECTIVE: The roles of phosphatidylinositol 3 (PI3K) and mitogen-activated protein kinases (MAPK) have been widely studied in terms of the differentiation process induced by several drugs (phorbol ester, vitamin D-3, retinoic acid, etc.), but their exact functions in leukemic cells' phenotype and their potential therapeutic role remain incompletely clarified. MATERIALS AND METHODS: In order to investigate this query, leukemia cells were cultured in presence of kinase inhibitors (KIs). Proliferation, apoptosis, and differentiation were analyzed at the cellular and molecular levels, using flow cytometry and reverse transcriptase quantitative polymerase chain reaction. RESULTS: SB203580, a P38 MAPK inhibitor, had no effect on cell proliferation, whereas LY294002, a PI3K inhibitor, and PD098059, a selective inhibitor of mitogen-activated extracellular regulated kinase (MEK) phosphorylation, arrested cells in G(0)/G(1). However, LY294002 and PD098059 acted using different mechanisms: LY294002 decreased the expression of phosphorylated S6RP, whereas PD098059 increased P21/waf1 antigen expression. SP600125, an inhibitor of N-terminal c-jun kinases, arrested cells in G(2) and induced an endoreplicative process. SP600125 increased p21 at both the mRNA and protein levels. G(2) blockage is dependent on the PI3K pathway and the endoreplicative process is dependent on the PI3K and extracellular regulated kinase (ERK) pathways and mRNA synthesis. On the other hand, PD098059 potentiated the apoptotic process induced by either SP600125 or LY294002. Modulation of the expression of CD11, CD15, CD18, and CD54 was cell-dependent. CONCLUSION: Our results suggest that KIs modulate proliferation of leukemia cells and that the MEK/ERK inhibitor, PD098059, in combination with either SP600125 or LY294002, could have clinical value.

The broad-spectrum caspase inhibitor Boc-Asp-CMK induces cell death in human leukaemia cells.

Synthetic caspase inhibitors and particularly broad-spectrum caspase inhibitors can prevent cells from death or at least slow down cell death process and abrogate some apoptotic hallmarks [Kitanaka, C., Kuchino, Y., 1999. Caspase-independent programmed cell death with necrotic morphology. Cell Death and Differentiation 6, 508-515]. However, not all synthetic caspase inhibitors diminish cell death. We have found that the broad-spectrum caspase inhibitor Boc-Asp-CMK induced cell death at micromolar concentrations in human leukaemia cells. Interestingly, low concentrations of Boc-Asp-CMK induced cell death with apoptotic hallmarks. Increasing concentrations of Boc-Asp-CMK led to necrotic cell death. The switch between apoptosis and necrosis seemed to depend upon the degree of inhibition of executioner caspases, including caspase-3/7 with Boc-Asp-CMK. Interestingly, caspase-3 processing was not inhibited even for the highest concentration of Boc-Asp-CMK used. We assume, that toxic properties of Boc-Asp-CMK can be attributed to the chloromethylketone residuum in its molecule, as its analogue Boc-Asp-FMK with fluoromethylketone residuum was more than 13 times less toxic. Our results further indicated that toxicity of Boc-Asp-CMK might arise from its interference with mitochondrial metabolism.

Topoisomerase II beta levels are a determinant of melphalan-induced DNA crosslinks and sensitivity to cell death.

The role of topoisomerase (topo) II in DNA repair has yet to be fully elucidated. Current evidence suggesting a role for topo II in the repair of DNA damage has been obtained by using in vitro model systems or inferred from correlative data in drug resistant cell lines. In this study we directly examined the role of topo IIalpha and beta in mediating the repair of melphalan-induced crosslinks in cellular DNA. To accomplish this, we used siRNA technology to knock down either topo IIalpha or beta in human chronic myelogenous leukemia K562 and histiocytic lymphoma U937 cell line. Our data demonstrate that topo IIbeta levels, (but not alpha), are a determinant of melphalan-induced crosslinks and sensitivity to melphalan. Furthermore, we show that knocking down topo IIbeta inhibits the repair of melphalan-induced crosslinks in K562 cells. These studies represent the first direct evidence that topo IIbeta participates in the repair of DNA damage induced by an alkylating agent in cellular DNA. Finally, these results suggest non-redundant roles for these two isoforms in mediating repair of DNA crosslinks.

Modulation of the p38 MAPK (mitogen-activated protein kinase) pathway through Bcr/Abl: implications in the cellular response to Ara-C.

The chimaeric protein Bcr/Abl, the hallmark of chronic myeloid leukaemia, has been connected with several signalling pathways, such as those involving protein kinase B/Akt, JNK (c-Jun N-terminal kinase) or ERKs (extracellular-signal-regulated kinases) 1 and 2. However, no data about the p38 MAPK (mitogen-activated protein kinase) have been reported. Here, we present evidence showing that Bcr/Abl is able to modulate this signalling pathway. Transient transfection experiments indicated that overexpression of Bcr/Abl in 293T cells is able to activate p38 MAPK or induce p73 stabilization, suggesting that c-Abl and Bcr/Abl share some biological substrates. Interestingly, the control exerted by Bcr/Abl on the p38 MAPK pathway was not only mediated by the tyrosine kinase activity of Bcr/Abl, as the use of STI571 demonstrated. In fact, Bcr alone was able to induce p38 MAPK activation specifically through MKK3 (MAP kinase kinase 3). Supporting these observations, chronic myeloid leukaemia-derived K562 cells or BaF 3 cells stably transfected with Bcr/Abl showed higher levels of phosphorylated p38 MAPK compared with Bcr/Abl-negative cells. While Bcr/Abl-negative cells activated p38 MAPK in response to Ara-C (1-beta-D-arabinofuranosylcytosine), Bcr/Abl-positive cells were unable to activate p38 MAPK, suggesting that the p38 MAPK pathway is not sensitive to Abl-dependent stimuli in Bcr/Abl-positive cells. Our results demonstrate that the involvement of Bcr/Abl in the p38 MAPK pathway is a key mechanism for explaining resistance to Ara-C, and could provide a clue for new therapeutic approaches based on the use of specific Abl inhibitors.

Atypical protein kinase C zeta as a target for chemosensitization of tumor cells.

Exposure of tumor cells to cytotoxic agents simultaneously activates a variety of intracellular signaling pathways. Some of these pathways involve enzymes from the protein kinase C (PKC) family of serine/threonine kinases. This family includes isoenzymes that negatively influence cell death, whereas other demonstrate an opposite effect. The present study analyzes the role of the zeta atypical PKC isoform in tumor cell response to cytotoxic agents. Using a histone H1 phosphorylation assay, we showed that both tumor necrosis factor alpha and etoposide activate PKCzeta in U937 human leukemic cells. Stable transfection of a kinase-dead, dominant-negative PKCzeta mutant in U937 cells decreases Bcl-2 expression while increasing the expression of Bax and several procaspases. This transfection also prevents etoposide-induced nuclear factor-kappaB nuclear translocation and accumulation of X-linked inhibitor of apoptosis protein. PKCzeta inhibition accelerates the occurrence of apoptosis in leukemic cells exposed to etoposide and tumor necrosis factor alpha. This sensitization was confirmed in vitro by use of a clonogenic assay. In addition, PKCzeta inhibition sensitized tumor cells grown in nude mice to etoposide. These results indicate that PKCzeta isoform is a protective signals that is activated in tumor cells exposed to a cytotoxic agent. This inducible resistance factor thus appears an attractive target for chemosensitization of tumor cells.

Pharmacological inhibitors of the mitogen-activated protein kinase (MAPK) kinase/MAPK cascade interact synergistically with UCN-01 to induce mitochondrial dysfunction and apoptosis in human leukemia cells.

Interactions between the checkpoint abrogator UCN-01 and several pharmacological inhibitors of the mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK pathway have been examined in a variety of human leukemia cell lines. Exposure of U937 monocytic leukemia cells to a marginally toxic concentration of UCN-01 (e.g., 150 nM) for 18 h resulted in phosphorylation/activation of p42/44 MAPK. Coadministration of the MEK inhibitor PD184352 (10 microM) blocked UCN-01-induced MAPK activation and was accompanied by marked mitochondrial damage (e.g., cytochrome c release and loss of DeltaPsi(m)), caspase activation, DNA fragmentation, and apoptosis. Similar interactions were noted in the case of other MEK inhibitors (e.g., PD98059; U0126) as well as in multiple other leukemia cell types (e.g., HL-60, Jurkat, CCRF-CEM, and Raji). Coadministration of PD184352 and UCN-01 resulted in reduced binding of the cdc25C phosphatase to 14-3-3 proteins, enhanced dephosphorylation/activation of p34(cdc2), and diminished phosphorylation of cyclic AMP-responsive element binding protein. The ability of UCN-01, when combined with PD184352, to antagonize cdc25C/14-3-3 protein binding, promote dephosphorylation of p34(cdc2), and potentiate apoptosis was mimicked by the ataxia telangectasia mutation inhibitor caffeine. In contrast, cotreatment of cells with UCN-01 and PD184352 did not substantially increase c-Jun-NH(2)-terminal kinase activation nor did it alter expression of Bcl-2, Bcl-x(L), Bax, or X-inhibitor of apoptosis. However, coexposure of U937 cells to UCN-01 and PD184352 induced a marked increase in p38 MAPK activation. Moreover, SB203580, which inhibits multiple kinases including p38 MAPK, partially antagonized cell death. Lastly, although UCN-01 +/- PD184352 did not induce p21(CIP1), stable expression of a p21(CIP1) antisense construct significantly increased susceptibility to this drug combination. Together, these findings indicate that exposure of leukemic cells to UCN-01 leads to activation of the MAPK cascade and that interruption of this process by MEK inhibition triggers perturbations in several signaling and cell cycle regulatory pathways that culminate in mitochondrial injury, caspase activation, and apoptosis. They also raise the possibility that disrupting multiple signaling pathways, e.g., by combining UCN-01 with MEK inhibitors, may represent a novel antileukemic strategy.

Pharmacologic mitogen-activated protein/extracellular signal-regulated kinase kinase/mitogen-activated protein kinase inhibitors interact synergistically with STI571 to induce apoptosis in Bcr/Abl-expressing human leukemia cells.

Interactions between the kinase inhibitor STI571 and pharmacological antagonists of the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) cascade have been examined in human myeloid leukemia cells (K562 and LAMA 84) that express the Bcr-Abl kinase. Exposure of K562 cells to concentrations of STI571 that minimally induced apoptosis (e.g., approximately 200 nM) resulted in early suppression (i.e., at 6 h) of p42/44 MAPK phosphorylation followed at later intervals (i.e., > or =24 h) by a marked increase in p42/44 MAPK phosphorylation/activation. Coadministration of a nontoxic concentration of the MEK1/2 inhibitor PD184352 (5 microM) prevented STI571-mediated activation of p42/44 MAPK. Cells exposed to STI571 in combination with PD184352 for 48 h demonstrated a very dramatic increase in mitochondrial dysfunction (e.g., loss of DeltaPsim and cytosolic cytochrome c release) associated with procaspase-3 activation, poly(ADP-ribose) polymerase cleavage, and the appearance of the characteristic morphological features of apoptosis. Similar results were obtained using other pharmacological MEK1/2 inhibitors (e.g., PD 98059 and U0126) as well as another leukemic cell line that expresses Bcr-Abl (e.g., LAMA 84). However, synergistic induction of apoptosis by STI571 and PD184352 was not observed in human myeloid leukemia cells that do not express the Bcr-Abl kinase (e.g., HL-60 and U937) nor in normal human peripheral blood mononuclear cells. Synergistic potentiation of STI571-mediated lethality by PD184352 was associated with multiple perturbations in signaling and apoptotic regulatory pathways, including caspase-dependent down-regulation of Bcr-Abl and Bcl-2; caspase-independent down-regulation of Bcl-x(L) and Mcl-1; activation of JNK, p38 MAPK, and p34(cdc2); and diminished phosphorylation of Stat5 and CREB. Significantly, coexposure to PD184352 strikingly increased the lethality of a pharmacologically achievable concentration of STI571 (i.e., 1-2 microM) in resistant K562 cells expressing marked increases in Bcr-Abl protein levels. Together, these findings raise the possibility that treatment of Bcr-Abl-expressing cells with STI571 elicits a cytoprotective MAPK activation response and that interruption of the latter pathway (e.g., by pharmacological MEK1/2 inhibitors) is associated with a highly synergistic induction of mitochondrial damage and apoptosis. They also indicate that in the case of Bcr-Abl-positive cells, simultaneous interruption of two signal transduction pathways may represent an effective antileukemic strategy.

SIRT6 Is a Positive Regulator of Aldose Reductase Expression in U937 and HeLa cells under Osmotic Stress: In Vitro and In Silico Insights.

SIRT6 is a protein deacetylase, involved in various intracellular processes including suppression of glycolysis and DNA repair. Aldose Reductase (AR), first enzyme of polyol pathway, was proposed to be indirectly associated to these SIRT6 linked processes. Despite these associations, presence of SIRT6 based regulation of AR still remains ambiguous. Thus, regulation of AR expression by SIRT6 was investigated under hyperosmotic stress. A unique model of osmotic stress in U937 cells was used to demonstrate the presence of a potential link between SIRT6 and AR expression. By overexpressing SIRT6 in HeLa cells under hyperosmotic stress, its role on upregulation of AR was revealed. In parallel, increased SIRT6 activity was shown to upregulate AR in U937 cells under hyperosmotic milieu by using pharmacological modulators. Since these modulators also target SIRT1, binding of the inhibitor, Ex-527, specifically to SIRT6 was analyzed in silico. Computational observations indicated that Ex-527 may also target SIRT6 active site residues under high salt concentration, thus, validating in vitro findings. Based on these evidences, a novel regulatory step by SIRT6, modifying AR expression under hyperosmotic stress was presented and its possible interactions with intracellular machinery was discussed.

Requirement of protein kinase (Krs/MST) activation for MT-21-induced apoptosis.

Fas is a well characterized apoptosis-inducing factor. One of our synthetic compounds, MT-21, induced apoptosis in human leukemia HL-60 cells similar to Fas. MT-21 activated caspase-3, an important cysteine aspartic protease for apoptosis induction. MT-21 also activated c-Jun-NH2-terminal kinase (JNK), a member of mitogen activated protein kinase (MAPK) superfamily that is involved in the regulation of cell growth, differentiation and cell death. Moreover, MT-21 treatment resulted in the activation of a 36 kDa kinase which uses myelin basic protein (MBP) as a substrate. However, MAPK and p38 were not activated by treatment with MT-21. The 36 kDa MBP kinase was shown to be a proteolytic product derived from the Krs protein with a molecular weight of 60 kDa. The Krs protein is a Ser/Thr protein kinase whose activity is enhanced by digestion of its C-terminal regulatory domain by caspase-3. When a kinase-inactive mutant form of Krs protein was overexpressed in HL-60 cells, JNK activation and apoptosis induction by MT-21 were suppressed. Furthermore, overexpression of dominant negative c-Jun also suppressed apoptosis induction by MT-21. These findings indicate that MT-21 induces apoptosis by the activation of JNK via the Krs protein, which is activated by caspase cleavage.

Beta-hydroxyisovalerylshikonin induces apoptosis in human leukemia cells by inhibiting the activity of a polo-like kinase 1 (PLK1).

beta-Hydroxyisovalerylshikonin (beta-HIVS), which was isolated from the plant, Lithospermum radix, induces apoptosis in various lines of human tumor cells. To identify genes involved in beta-HIVS-induced apoptotic process, we performed cDNA array analysis and found that beta-HIVS suppresses the expression of the gene for a polo-like kinase 1 (PLK1) that is involved in control of the cell cycle. When U937 and HL60 cells were treated with 10(-6) M beta-HIVS for 0.5 h, both the amount of PLK1 itself and the kinase activity of this enzyme were decreased. By contrast, Bcr-Abl-positive K562 cells were resistant to the induction of apoptosis by beta-HIVS and this compound did not suppress the kinase activity of PLK1 in these cells. However, simultaneous treatment of K562 cells with both beta-HIVS and STI571, which selectively inhibits the protein tyrosine kinase (PTK) activity of Bcr-Abl, strongly induced apoptosis. Moreover, beta-HIVS increased the inhibitory effect of STI571 on PTK activity. Treatment of K562 cells with antisense oligodeoxynucleotides (ODNs) specific for PLK1 sensitized these cells to the beta-HIVS-induced fragmentation of DNA. These results suggest that suppression of the activity of PLK1 via inhibition of tyrosine kinase activity by beta-HIVS might play a critical role in the induction of apoptosis.

Catecholamines potentiate LPS-induced expression of MMP-1 and MMP-9 in human monocytes and in the human monocytic cell line U937: possible implications for peri-operative plaque instability.

Plaque destabilization leading to myocardial infarction is observed after surgery even if the intervention is of noncardiovascular nature. Mediators of peri- or postoperative stress responsible for such events could include catecholamines and lipopolysaccharide (LPS). Monocytes may be involved in destabilization of atherosclerotic plaques by production of matrix metalloproteinases (MMP). We examined whether catecholamines could affect the expression of MMPs in human monocytes/macrophages and whether catecholamines could modulate LPS-stimulated expression of particular MMPs in these cells. Epinephrine and norepinephrine up-regulated MMP-1 and potentiated LPS-induced expression of MMP-1 in peripheral blood monocytes and monocyte-derived macrophages. We further characterized this effect employing the monocytic cell line U937 and showed that catecholamines potentiate LPS-induced effects on MMP-1 and MMP-9 antigen and activity. mRNA levels of the respective MMPs also increased. These effects did not result from higher mRNA stability but rather from increased transcription possibly induced by enhanced DNA binding of AP-1 and were mediated by either beta1- or beta 2-receptors. If this mechanism is also effective in vivo, our findings might, at least in part, help to explain the observation that cardiac events are important causes of morbidity and mortality after noncardiac surgery and support the findings that peri-operative beta-blockade has been shown to reduce postoperative mortality from cardiac events.

C-reactive protein stimulates MMP-1 expression in U937 histiocytes through Fc[gamma]RII and extracellular signal-regulated kinase pathway:: an implication of CRP involvement in plaque destabilization.

OBJECTIVE: It has been shown that plasma level of C-reactive protein (CRP) is an independent predictor for acute coronary syndromes and is associated with plaque weakening. However, the underlying mechanisms are not well understood. In this study, we investigated the effect of CRP on the expression of matrix metalloproteinase-1 (MMP-1) that has been implicated in plaque vulnerability by human U937 histiocytes and monocyte-derived macrophages. METHODS AND RESULTS: Enzyme-linked immunosorbent assay of MMP-1 in conditioned medium showed that treatment of U937 cells with 100 microg/mL of CRP for 24 hour led to a 3- to 5-fold increase in MMP-1 secretion. CRP also markedly stimulated MMP-1 release from human monocyte-derived macrophages. In contrast, CRP had no effect on tissue inhibitor of metalloproteinase-1 (TIMP-1) secretion. Northern blot showed that CRP upregulated MMP-1 mRNA expression. Collagenase activity assay showed that CRP increased collagen-degrading activity in cell-conditioned medium. Furthermore, results showed that the stimulation of MMP-1 secretion by CRP was inhibited by anti-CD32, but not by anti-CD64 antibody, and by mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitor PD98059. Finally, Western blot showed that CRP stimulated phosphorylation of extracellular signal-regulated kinase. CONCLUSIONS: This study demonstrates that CRP stimulates MMP-1 expression by U937 cells through FcgammaRII and extracellular signal-regulated kinase pathway. These findings suggest that CRP may promote matrix degradation and thus contribute to plaque vulnerability.

Induction of apoptosis in human hematopoietic U937 cells by granulocyte-macrophage colony-stimulating factor: possible existence of caspase 3-like pathway.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced apoptosis in human hematopoietic U937 cells by itself and in a synergistic manner with tumor necrosis factor (TNF). GM-CSF-induced apoptosis was not inhibited by caspase inhibitors YVAD-CMK, DEVD-CHO and z-VAD-FMK, under the condition that these inhibitors potently suppressed TNF-induced apoptosis. Both GM-CSF and TNF induced caspase 3-like activity in this cell line though the time course was distinct between two cytokines, and combined stimulation of cells with GM-CSF plus TNF induced additive or synergistic activation of caspase 3-like activity. Amount of immunoreactive cleaved forms of caspase 3 recognized by specific antibody was completely dissociated with its enzymatic activity when the cells were stimulated with GM-CSF, but not with TNF. These results indicate that GM-CSF induces apoptosis of U937 cells via unknown pathway, which seems to be mediated by caspase 3-like activity, yet not caspase 3 itself, resistant to the caspase inhibitors, and synergistically interacts with conventional caspase 3 pathway of TNF. Possible involvement of caspases 1 and 8 (-like activity) but not caspase 7 in this pathway was also suggested.

The expression of extracellular-superoxide dismutase is increased by lysophosphatidylcholine in human monocytic U937 cells.

Extracellular-superoxide dismutase (EC-SOD) [EC 1.15.1.1] is a secretory glycoprotein with an affinity for heparin-like proteoglycans. This enzyme locates in blood vessel walls at high levels and may be important for the antioxidant capability of vascular walls. Oxidative process plays an important role in atherogenesis. Lysophosphatidylcholine (lysoPC) is generated during oxidation of low-density lipoprotein (LDL) and is located within atherosclerotic plaques. Recently, lysoPC has been reported to induce transcription of a variety of cellular genes. In this study, we observed that lysoPC significantly increased the expression of EC-SOD mRNA and protein in human monocytic U937 cells, but not those of CuZn-SOD or Mn-SOD. Induced EC-SOD by lysoPC had a high affinity for heparin, and may bind to the endothelial cell surface. Very recently, it has been reported that exogenous addition of EC-SOD or overexpression of EC-SOD prevented endothelial cell-mediated oxidative modification of LDL. Therefore, it is speculated that EC-SOD is induced by lysoPC-stimulated monocytes as a feedback mechanism in vascular homeostasis.

Parvovirus H-1-induced cell death: influence of intracellular NAD consumption on the regulation of necrosis and apoptosis.

The autonomous parvovirus H-1 exerts tumor-suppressive effects in living organisms and has been shown to specifically interfere with the survival of transformed cells in culture. The mechanism(s) by which H-1 virus induces death of transformed cells is not yet well understood. It has recently been reported that H-1 virus induces apoptotic cell death in the human monocytic U937 cell line, as assessed by biochemical and morphological changes of infected cells (Rayet, B., Lopez-Guerrero, J.-A., Rommelaere, J., Dinsart, C., 1998. Induction of programmed cell death by parvovirus H-1 in U937 cells: connection with the TNFalpha signalling pathway. J. Virol. 72, 8893-8903). Here we show that parvovirus H-1 infection induced early biochemical changes pointing to apoptotic events also in the transformed human keratinocyte cell line, HeLa, and the transformed rat fibroblast cell line, P1. Morphologic changes, however, and in particular the early breakdown of plasma membrane integrity, suggested that apoptosis did not go to completion, leading to necrotic cell death as the major result of parvovirus infection of HeLa and P1 cells. Parvovirus infection of these, and to a significantly lesser extent of U937 cells, was accompanied by rapid depletion of intracellular NAD stores. Inhibition of NAD-consuming enzymes interfered with parvovirus-induced NAD depletion and increased the proportion of H-1 virus-infected cells displaying apoptotic features of cell death. In contrast, a similar prevention of NAD depletion through stimulation of NAD production had little influence on the cell death pathway, suggesting that NAD-consuming enzymes may promote necrosis in a direct way rather than through inducing the overall drop of intracellular NAD.

Differential influence of etoposide on two caspase-2 mRNA isoforms in leukemic cells.

Etoposide (VP-16) is an anticancer agent that induces apoptosis in human leukemic cell lines such as U937 and HL60. We performed RNase protection assays, with two distinct cRNA panels covering most of caspase and BCL-2-related genes, using total RNA from cell lines exposed to various concentrations of the drug. Our results show that VP-16 down-regulates expression of most surveyed genes with the noticeable exception of casp-2S mRNA that is up regulated whereas casp-2L mRNA is decreased. Since these mRNAs are produced by the alternative splicing of exon 9, we devised a reverse transcriptase-polymerase chain reaction method using primers from exons 8 and 10 to demonstrate that VP-16 stimulates the production of exon 9-containing sequences, irrespective of active transcription. However, this effect is specific of the 3'-end of the CASP-2 gene since no difference in the relative amounts of the 5'-end of the mRNA species is detected. Nevertheless, the level of full-length casp-2L mRNA together with that of procaspase-2L protein, which is pro-apoptotic, are decreased under VP-16 treatment, suggesting that an early cell response to treatment by cytotoxic agents is to down-regulate expression of selected pro-apoptotic proteins.

Degradation of glyceraldehyde-3-phosphate dehydrogenase induced by acetylleucine chloromethyl ketone in U937 cells.

We examined whether any changes were induced in cellular proteins by an inhibitor of acylpeptide hydrolase (ACPH) (EC 3.4.19.1), acetylleucine chloromethyl ketone (ALCK), which was shown in our previous report to induce apoptosis of human U937 cells. Extract prepared from U937 cells in 0.05% Triton X-100-PBS was incubated with ALCK at 37 degrees, and then analyzed using SDS-PAGE. A 36kDa protein in the cell extract was decreased markedly during the incubation period. This protein was purified and identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) by its specific enzyme activity, N-terminal amino acid sequence, and Western blotting. Incubation of purified GAPDH with ALCK resulted in a decrease of GAPDH activity, but not in a decrease in the amount of GAPDH. The ALCK-induced GAPDH decrease in the cell extract was abrogated by co-incubation with a serine protease inhibitor, diisopropyl fluorophosphate, suggesting that GAPDH was first inactivated by ALCK, and subsequently degraded by a serine protease(s). GAPDH degradation was also observed in U937 cell cultures in the presence of ALCK. The significance of GAPDH inhibition in the apoptotic process is discussed.

Urinary trypsin inhibitor, a Kunitz-type protease inhibitor, modulates tumor necrosis factor-stimulated activation and translocation of protein kinase C in U937 cells.

Urinary trypsin inhibitor (UTI) is a Kunitz-type protease inhibitor. We have reported that UTI inhibited TNF-induced urokinase (uPA) production via a protein kinase C (PKC)-dependent mechanism. It is likely that UTI suppresses tumor cell invasion and metastasis by a mechanism, possibly by inhibiting uPA production. In the present study, we attempted to determine how UTI is associated with PKC, and how UTI is involved in uPA-dependent tumor cell invasion and metastasis. The increments of membrane-associated PKC activity by TNF were subsequently accompanied by a rapid loss of cytosol-associated PKC activity in U937 leukemia cells. Semi-quantitative immunoblotting of membrane and cytosol fractions showed that the translocation of PKC-alpha, -beta, and -epsilon were blocked by the addition of UTI in cells stimulated with TNF but not in cells stimulated with PMA, demonstrating that PKC itself is not sensitive to UTI. This effect was dependent on the carboxyl-terminus of UTI. In addition, UTI neither inhibited TNF binding to cellular receptors nor inactivate PKC and uPA activities directly. Taken together, the experiments suggest that the carboxyl-terminus of UTI may inhibit the PKC-signalling pathways upstream of diacylglycerol by a mechanism, possibly by interrupting the coupling of receptor and effector systems. UTI was shown to have an interesting new function besides being a protease inhibitor. This is the first report that UTI has a selective inhibition of TNF-activated PKC. We conclude that UTI suppresses tumor cell invasion and metastasis by a mechanism that UTI inhibits TNF-stimulated uPA production via a PKC-dependent mechanism.

The leukocyte integrins are regulated by transcriptional and post-transcriptional mechanisms in a leukemic cell that overexpresses protein kinase C-zeta.

Overexpression of protein kinase C-zeta (PKC-zeta) in the leukemic myeloid cell line U937 (U937-PKC-zeta cells), previously shown to induce leukemic cell differentiation, resulted in nearly complete downregulation of leukocyte integrins CD11a, CD11b, CD11d, and CD18, but not CD11c from the cell surface. The steady-state level of mRNAs for the downregulated leukocyte integrins was not detectable by Northern analysis. Nuclear run-on analysis revealed that transcription of all the leukocyte integrin genes except CD11c was reduced 70-90% as compared to control U937-Vector cells [U937 cells transfected with the empty vector pSV2M(2)6]. Transfection analysis of CD11-promoter-luciferase constructs confirmed that transcription of the leukocyte integrin genes was drastically downregulated in U937-PKC-zeta cells. The two c-jun binding sites in the CD11c promoter were essential for continued expression of CD11c in U937-PKC-zeta cells. Additionally, the 3' untranslated region (3' UTR) from CD11b, when fused to the luciferase gene, lead to the destabilization of this chimeric mRNA in U937-PKC-zeta cells. This indicates that downregulation of CD11b expression in U937-PKC-zeta cells is also the result of reduced stability of CD11b mRNA. Thus, overexpression of PKC-zeta in U937 cells leads not only to leukemic cell differentiation, but also to differential regulation of the leukocyte integrins.