Subcellular proteome analysis of camptothecin analogue NSC606985-treated acute myeloid leukemic cells

We reported previously that NSC606985, a camptothecin analogue, induces apoptosis of acute myeloid leukemia (AML) cells through proteolytic activation of protein kinase Cdelta. Here, we analyzed protein expression profiles of fractionated nuclei, mitochondria, raw endoplasmic reticula, and cytosols of NSC606985-induced apoptotic AML cell line NB4 cells by two-dimensional electrophoresis combined with MALDI-TOF/TOF tandem mass spectrometry. In total, 90 unique deregulated proteins, including 16 compartment-compartment translocated ones, were identified. They contributed to multiple functional activities such as DNA damage repairing, chromosome assembly, mRNA processing, biosynthesis, modification, and degradation of proteins. More interestingly, several increased oxidative stress-related proteins mainly presented in mitochondria, while upregulated glycolysis proteins mainly occurred in the nuclei. With their functional analyses, the possible roles of these deregulated proteins in NSC606985-induced apoptosis were discussed. Collectively, these discoveries would shed new insights for systematically understanding the mechanisms of the camptothecin-induced apoptosis.     

Regulation of cadmium-induced apoptosis by PKCδ in U937 human promonocytic cells

Pulse treatment with cadmium chloride followed by recovery caused apoptosis in U937 human promonocytic cells. In addition, the treatment-induced PKCδ translocation from cytosol to membrane fraction, which was already detected at 30 min of treatment; and also caused PKCδ cleavage to give a 41-kDa fragment, which was detected at 3–6 h of recovery, concomitantly with the execution of apoptosis. All these effects were reduced by the PKCδ-specific inhibitor rottlerin. By contrast, rottlerin did not prevent the cadmium-provoked stimulation of the stress response (as measured by HSP70 expression), nor inhibited the generation of apoptosis by heat-shock, which failed to cause PKCδ translocation. Cadmium chloride rapidly induced p38^MAPK activation, which was not affected by rottlerin. By contrast, the p38^MAPK inhibitor SB203580 reduced PKCδ translocation and cleavage, indicating that p38^MAPK activation precedes and regulates PKCδ activation. It is concluded that PKCδ mediates apoptosis induction by cadmium ions via early membrane translocation, and also possibly through late kinase proteolytic cleavage and phosphorylation on tyrosine residues.     

SIRT3, PP2A and TTP protein stability in the presence of TNF‐α on vincristine‐induced apoptosis of leukaemia cells

The contribution of vincristine (VCR)‐induced microtubule destabilization to evoke apoptosis in cancer cells remains to be resolved. Thus, we investigated the cytotoxic mechanism of VCR on U937 and HL‐60 human leukaemia cell lines. We discovered that VCR treatment resulted in the up‐regulation of TNF‐α expression and activation of the death receptor pathway, which evoked apoptosis of U937 cells. Moreover, VCR induced microtubule destabilization and mitotic arrest. VCR treatment down‐regulated SIRT3, and such down‐regulation caused mitochondrial ROS to initiate phosphorylation of p38 MAPK. p38 MAPK suppressed MID1‐modulated degradation of the protein phosphatase 2A (PP2A) catalytic subunit. The SIRT3‐ROS‐p38 MAPK‐PP2A axis inhibited tristetraprolin (TTP)‐controlled TNF‐α mRNA degradation, consequently, up‐regulating TNF‐α expression. Restoration of SIRT3 and TTP expression, or inhibition of the ROS‐p38 MAPK axis increased the survival of VCR‐treated cells and repressed TNF‐α up‐regulation. In contrast to suppression of the ROS‐p38 MAPK axis, overexpression of SIRT3 modestly inhibited the effect of VCR on microtubule destabilization and mitotic arrest in U937 cells. Apoptosis of HL‐60 cells, similarly, went through the same pathway. Collectively, our data indicate that the SIRT3‐ROS‐p38 MAPK‐PP2A‐TTP axis modulates TNF‐α expression, which triggers apoptosis of VCR‐treated U937 and HL‐60 cells. We also demonstrate that the apoptotic signalling is not affected by VCR‐elicited microtubule destabilization.     

Protein kinase Cδ and caspase‐3 modulate TRAIL‐induced apoptosis in breast tumor cells

This report describes that protein kinase C delta (PKCδ) overexpression prevents TRAIL‐induced apoptosis in breast tumor cells; however, the regulatory mechanism(s) involved in this phenomenon is(are) incompletely understood. In this study, we have shown that TRAIL‐induced apoptosis was significantly inhibited in PKCδ overexpressing MCF‐7 (MCF7/PKCδ) cells. Our data reveal that PKCδ inhibits caspase‐8 activation, a first step in TRAIL‐induced apoptosis, thus preventing TRAIL‐induced apoptosis. Inhibition of PKCδ using rottlerin or PKCδ siRNA reverses the inhibitory effect of PKCδ on caspase‐8 activation leading to TRAIL‐induced apoptosis. To determine if caspase‐3‐induced PKCδ cleavage reverses its inhibition on caspase‐8, we developed stable cell lines that either expresses wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) or caspase‐3 cleavage‐resistant PKCδ mutant (MCF‐7/cas‐3/PKCδ mut) utilizing MCF‐7 cells expressing caspase‐3. Cells that overexpress caspase‐3 cleavage‐resistant PKCδ mutant (MCF‐7/cas‐3/PKCδmut) significantly inhibited TRAIL‐induced apoptosis when compared to wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) expressing cells. In MCF‐7/cas‐3/PKCδmut cells, TRAIL‐induced caspase‐8 activation was blocked leading to inhibition of apoptosis when compared to wild‐type PKCδ (MCF‐7/cas‐3/PKCδ) expressing cells. Together, these results strongly suggest that overexpression of PKCδ inhibits caspase‐8 activation leading to inhibition of TRAIL‐induced apoptosis and its inhibition by rottlerin, siRNA, or cleavage by caspase‐3 sensitizes cells to TRAIL‐induced apoptosis. Clinically, PKCδ overexpressing tumors can be treated with a combination of PKCδ inhibitor(s) and TRAIL as a new treatment strategy. J. Cell. Biochem. 111: 979–987, 2010. © 2010 Wiley‐Liss, Inc.     

Comparative proteomic analysis of hypoxia-treated and untreated human leukemic U937 cells

We reported recently that moderate hypoxia and hypoxia-mimetic agents could induce growth arrest and differentiation of leukemic cells via the mediation of hypoxia-inducible factor 1 alpha (HIF-1alpha), but the exact molecular mechanisms remain largely unknown. In this study, human acute promonocytic leukemic U937 cells were incubated under 2% O2 or in 50 microM of the hypoxia mimetic agent cobalt chloride (CoCl2) and normal oxygen for 24 h, and their protein expression profiles were compared by 2-DE coupled with MALDI-TOF/TOF MS/MS. We identified 62 and 16 proteins that were significantly deregulated by hypoxia and CoCl2 treatment, respectively. These proteins were mainly involved in metabolism, gene expression regulation, signal transduction, cell proliferation, differentiation and apoptosis. As an example, N-myc downstream regulated gene 1 (NDRG1), a putative differentiation-related gene, was up-regulated in both 2% O2- and CoCl2-treated U937 cells. Moreover, enforced HIF-1alpha expression also elevated NDRG1 mRNA and protein in U937 cells. These data will provide some clues for understanding mechanisms by which leukemic cells response to hypoxia.     

Signaling defect in the activation of caspase-3 and PKCdelta in human TUR leukemia cells is associated with resistance to apoptosis

Exposure of the two related human leukemic cell lines U937 and TUR to chemotherapeutic compounds resulted in opposite effects on induction and resistance to apoptosis. Incubation of U937 cells with 1-beta-d-arabinofuranosylcytosine or the etoposide VP-16 was accompanied by growth arrest in G0/G1 of the cell cycle and an accumulation of a population in the sub-G1 phase which exhibited characteristics typical for the apoptotic pathway. In contrast, human TUR leukemia cells demonstrated no significant effects after a similar treatment with Ara-C and VP-16. Thus, TUR cells continued to proliferate in the presence of these anti-cancer drugs and the number of apoptotic cells as evaluated by propidium iodide staining and the detection of internucleosomal DNA fragmentation was significantly reduced when compared to the parental U937 cells. Similar effects were observed upon serum-starvation demonstrating resistance to apoptosis in TUR cells. Whereas induction of apoptosis is regulated by a network of distinct factors including the activation of proteolytically active caspases, we investigated these pathways in both cell lines. U937 cells demonstrated activation of the 32-kDa caspase-3 upon drug treatment by cleavage into the 20-kDa activated form. However, there was no 20-kDa caspase-3 fragment detectable in TUR cells. Simultaneously, the enzymatic activity of caspase-3 was significantly increased in drug-treated U937 cells as measured in vitro by enhanced metabolization of a fluorescence substrate and in vivo by cleavage of an appropriate substrate for caspase-3, namely, protein kinase Cdelta. In contrast, there was little if any caspase-3 activation detectable in drug-treated TUR cells. Taken together, these data suggest a signaling defect in the activation of the caspase-3 proteolytic system in TUR cells upon treatment with chemotherapeutic compounds which is associated with resistance to apoptosis in these human leukemia cells.     

Protein kinase C activation modulates pro- and anti-apoptotic signaling pathways

Activation of protein kinase C (PKC) by TPA in human U937 myeloid leukemia cells is associated with induction of adherence, differentiation, and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these differentiating cells about 25% of U937 cells accumulated in the subG1 phase after TPA treatment. This effect proved to be phorbol ester-specific, since other compounds such as retinoic acid or vitamin D3 failed to induce apoptosis in conjunction with differentiation. Only a specific inhibitor of PKC, GF109203X, but not the broad-spectrum kinase inhibitor staurosporine or a tyrosine kinase inhibitor genistein could reverse the induction of apoptosis. Bryostatin-1, another specific PKC activator with distinct biochemical activity failed to induce apoptosis. Moreover, bryostatin-1 completely abolished the induction of apoptosis in U937 cells even if added 8 hours after TPA treatment. Apart from apoptosis induced by various chemotherapeutic drugs, TPA-related cell death is not mediated by an autocrine Fas-FasL loop and could not be prevented by a blocking antibody to the Fas receptor. However, a 75% reduction in the number of apoptotic cells after TPA stimulation was achieved by preincubation with a blocking antibody to the TNFalpha receptor. Tetrapeptide cleavage assays revealed a four-fold increase in the DEVD-cleavage activity in U937 cells compared to a three-fold increase in TUR cells. Immunoblotting demonstrated that TUR cells did not activate significant levels of caspase-3 or -7, whereas in U937 cells a 20-kDa cleavage product corresponding to activated caspase-3 was detectable after 3 d TPA exposure. Moreover, immunoblots revealed a strongly reduced expression of the adaptor molecule APAF-1, which is required for cytochrome c-dependent activation of caspase-9 and subsequently caspase-3. APAF-1 proved to be inducible after PKC activation with phorbol ester in U937, but not in TUR cells. Thus, APAF-1 expression may, at least in part, be regulated by PKC activity and reduced APAF-1 levels are associated with resistance to various inducers of apoptosis. Furthermore, TPA exposure of U937 cells is associated with increased levels of the pro-apoptotic proteins Bak and Bcl-xs, whereas simultaneously a decline in the Bcl-2 expression was noticable.     

Signaling Defect in the Activation of Caspase-3 and PKCδ in Human TUR Leukemia Cells Is Associated with Resistance to Apoptosis

Exposure of the two related human leukemic cell lines U937 and TUR to chemotherapeutic compounds resulted in opposite effects on induction and resistance to apoptosis. Incubation of U937 cells with 1-β- -arabinofuranosylcytosine or the etoposide VP-16 was accompanied by growth arrest in G₀/G₁of the cell cycle and an accumulation of a population in the sub-G₁phase which exhibited characteristics typical for the apoptotic pathway. In contrast, human TUR leukemia cells demonstrated no significant effects after a similar treatment with Ara-C and VP-16. Thus, TUR cells continued to proliferate in the presence of these anti-cancer drugs and the number of apoptotic cells as evaluated by propidium iodide staining and the detection of internucleosomal DNA fragmentation was significantly reduced when compared to the parental U937 cells. Similar effects were observed upon serum-starvation demonstrating resistance to apoptosis in TUR cells. Whereas induction of apoptosis is regulated by a network of distinct factors including the activation of proteolytically active caspases, we investigated these pathways in both cell lines. U937 cells demonstrated activation of the 32-kDa caspase-3 upon drug treatment by cleavage into the 20-kDa activated form. However, there was no 20-kDa caspase-3 fragment detectable in TUR cells. Simultaneously, the enzymatic activity of caspase-3 was significantly increased in drug-treated U937 cells as measuredin vitroby enhanced metabolization of a fluorescence substrate andin vivoby cleavage of an appropriate substrate for caspase-3, namely, protein kinase Cδ. In contrast, there was little if any caspase-3 activation detectable in drug-treated TUR cells. Taken together, these data suggest a signaling defect in the activation of the caspase-3 proteolytic system in TUR cells upon treatment with chemotherapeutic compounds which is associated with resistance to apoptosis in these human leukemia cells.     

PKCδ knockdown inhibits free fatty acid induction of endothelial cell apoptosis

The mechanisms whereby free fatty acids induce endothelial cell apoptosis are not yet understood. The present study aimed to investigate the role of PKCδ in free fatty acid–induced endothelial cell apoptosis. In addition, we looked for evidence of apoptosis‐related interactions between PKCδ and Fas signal pathway. Human umbilical vein endothelial cells were treated with various concentrations of free fatty acids and transiently transfected with PKCδ siRNA or Fas siRNA to inhibit PKCδ or Fas expression. Cell proliferation was determined through colorimetric assays, and apoptosis was quantified using flow cytometry. Protein expression was determined from cell lysates using Western blots with antibodies against p‐PKCδTyr512, PKCδ, and Fas. Statistical analyses were performed. Free fatty acids had multiple effects on human umbilical vein endothelial cells, including concentration‐dependent inhibition of cell proliferation, induction of apoptosis, increased Fas expression, and increased PKCδ expression and phosphorylation. Inhibition of PKCδ mRNA expression by PKCδ siRNA led to a reduction in both free fatty acid–induced apoptosis and Fas expression. However, Fas siRNA treatment inhibited Fas, but not PKCδ, expression in human umbilical vein endothelial cells. The free fatty acid–induced apoptosis in endothelial cells are possibly mediated by PKCδ and may involve upregulation of its downstream Fas. Copyright © 2012 John Wiley & Sons, Ltd.     

PU.1, a novel capase-3 substrate, partially contributes to chemotherapeutic agents-induced apoptosis in leukemic cells

PU.1 is one of key regulators of hematopoietic cell development, a tightly-regulated lineage-specific process. Here we provide the first evidence that PU.1 protein is cleaved into two fragments of 24 kDa and 16 kDa during apoptosis progression in leukemic cell lines and primary leukemic cells. Further experiments with specific capase-3 inhibitor Z-DEVD-fmk and the in vitro proteolytic system confirmed that PU.1 is a direct target of caspase-3. Using site-directed mutagenesis analyses, the aspartic acid residues at positions 97 and 151 of PU.1 protein were identified as capsase-3 target sites. More intriguingly, the suppression of PU.1 expression by small interfering RNAs (siRNAs) significantly inhibits DNA-damaging agents NSC606985 and etoposide-induced apoptosis in leukemic cells, together with the up-regulated expression of anti-apoptotic bcl-2 gene. These results would provide new insights for understanding the mechanism of PU.1 protein in hematopoiesis and leukemogenesis.     

Caffeine induces matrix metalloproteinase‐2 (MMP‐2) and MMP‐9 down‐regulation in human leukemia U937 cells via Ca2+/ROS‐mediated suppression of ERK/c‐fos pathway and activation of p38 MAPK/c‐jun pathway

Caffeine attenuated invasion of human leukemia U937 cells with characteristic of decreased protein expression and mRNA levels of matrix metalloproteinase‐2 (MMP‐2) and MMP‐9. Down‐regulation of MMP‐2 and MMP‐9 in U937 cells was abrogated by abolishment of caffeine‐elicited increase in intracellular Ca²⁺ concentration and ROS generation. Pretreatment with BAPTA‐AM (Ca²⁺ chelator) and N‐acetylcysteine (ROS scavenger) abolished caffeine‐induced ERK inactivation and p38 MPAK activation. Moreover, caffeine treatment led to MAPK phosphatase‐1 (MKP‐1) down‐regulation and protein phosphatase 2A catalytic subunit (PP2Ac) up‐regulation, which were involved in cross‐talk between p38 MAPK and ERK. Transfection of constitutively active MEK1 or pretreatment with SB202190 (p38 MAPK inhibitor) restored MMP‐2 and MMP‐9 protein expression in caffeine‐treated cells. Caffeine treatment repressed ERK‐mediated c‐Fos phosphorylation but evoked p38 MAPK‐mediated c‐Jun phosphorylation. Knock‐down of c‐Fos and c‐Jun by siRNA reflected that c‐Fos counteracted the effect of c‐Jun on MMP‐2/MMP‐9 down‐regulation. Taken together, our data indicate that MMP‐2/MMP‐9 down‐regulation in caffeine‐treated U937 cells is elicited by Ca²⁺/ROS‐mediated suppression of ERK/c‐Fos pathway and activation of p38 MAPK/c‐Jun pathway. J. Cell. Physiol. 224: 775–785, 2010. © 2010 Wiley‐Liss, Inc.     

Subtilase cytotoxin produced by locus of enterocyte effacement‐negative Shiga‐toxigenic Escherichia coli induces stress granule formation

Subtilase cytotoxin (SubAB) is mainly produced by locus of enterocyte effacement (LEE)‐negative strains of Shiga‐toxigenic Escherichia coli (STEC). SubAB cleaves an endoplasmic reticulum (ER) chaperone, BiP/Grp78, leading to induction of ER stress. This stress causes activation of ER stress sensor proteins and induction of caspase‐dependent apoptosis. We found that SubAB induces stress granules (SG) in various cells. Aim of this study was to explore the mechanism by which SubAB induced SG formation. Here, we show that SubAB‐induced SG formation is regulated by activation of double‐stranded RNA‐activated protein kinase (PKR)‐like endoplasmic reticulum kinase (PERK). The culture supernatant of STEC O113:H21 dramatically induced SG in Caco2 cells, although subAB knockout STEC O113:H21 culture supernatant did not. Treatment with phorbol 12‐myristate 13‐acetate (PMA), a protein kinase C (PKC) activator, and lysosomal inhibitors, NH₄Cl and chloroquine, suppressed SubAB‐induced SG formation, which was enhanced by PKC and PKD inhibitors. SubAB attenuated the level of PKD1 phosphorylation. Depletion of PKCδ and PKD1 by siRNA promoted SG formation in response to SubAB. Furthermore, death‐associated protein 1 (DAP1) knockdown increased basal phospho‐PKD1(S916) and suppressed SG formation by SubAB. However, SG formation by an ER stress inducer, Thapsigargin, was not inhibited in PMA‐treated cells. Our findings show that SubAB‐induced SG formation is regulated by the PERK/DAP1 signalling pathway, which may be modulated by PKCδ/PKD1, and different from the signal transduction pathway that results in Thapsigargin‐induced SG formation.     

Extracellular-superoxide dismutase expression during monocytic differentiation of U937 cells

Leukemic cell lines, such as U937, THP-1, and HL60 cells, can differentiate into macrophages following exposure to various agents including 12-O-tetradecanoylphorbol-13-acetate (TPA) in vitro. It is well known that TPA enhances reactive oxygen species (ROS) generation through the activation of NADPH oxidase (NOX), and ROS act as mediators in TPA signaling. Extracellular-superoxide dismutase (EC-SOD) is a major anti-oxidative enzyme that protects the cells from damaging effects of superoxide. Recently, the reduction of Cu/Zn-SOD and the induction of Mn-SOD by TPA in leukemic cells have been reported; however, the regulation of EC-SOD by TPA remains poorly understood. Here, we explored the regulation of EC-SOD during the monocytic differentiation of U937 cells by TPA. We observed the reduction of EC-SOD and Cu/Zn-SOD, whereas the induction of Mn-SOD during the differentiation of U937 cells. The reduction of EC-SOD and Cu/Zn-SOD was attenuated by pretreatments with GF109203X (an inhibitor of protein kinase C, PKC), diphenyleneiodonium (an inhibitor of NOX), and U0126 (an inhibitor of mitogen-activated protein kinase kinase, MEK/extracellular-signal regulated kinase, ERK). Interestingly, pretreatment with BAY11-7082 (an inhibitor of nuclear factor-κB, NF-κB) suppressed the reduction of Cu/Zn-SOD, but not of EC-SOD. Furthermore, we also determined the involvement of newly synthesized protein and the instability of mRNA in the reduction of EC-SOD. Overall, our results suggest that the expression of EC-SOD is decreased by TPA through intracellular signaling consisting of PKC, NOX-derived ROS and MEK/ERK, but not of NF-κB signaling.     

Superoxide dismutase 1 encoding mutations linked to ALS adopts a spectrum of misfolded states

Background

Oxidative stress is a key pathophysiological mechanism contributing to degenerative processes in many neurodegenerative diseases and therefore, unraveling molecular mechanisms underlying various stages of oxidative neuronal damage is critical to better understanding the diseases and developing new treatment modalities. We previously showed that protein kinase C delta (PKCδ) proteolytic activation during the late stages of oxidative stress is a key proapoptotic signaling mechanism that contributes to oxidative damage in Parkinson's disease (PD) models. The time course studies revealed that PKCδ activation precedes apoptotic cell death and that cells resisted early insults of oxidative damage, suggesting that some intrinsic compensatory response protects neurons from early oxidative insult. Therefore, the purpose of the present study was to characterize protective signaling pathways in dopaminergic neurons during early stages of oxidative stress.

Results

Herein, we identify that protein kinase D1 (PKD1) functions as a key anti-apoptotic kinase to protect neuronal cells against early stages of oxidative stress. Exposure of dopaminergic neuronal cells to H₂O₂ or 6-OHDA induced PKD1 activation loop (PKD1S744/748) phosphorylation long before induction of neuronal cell death. Blockade of PKCδ cleavage, PKCδ knockdown or overexpression of a cleavage-resistant PKCδ mutant effectively attenuated PKD1 activation, indicating that PKCδ proteolytic activation regulates PKD1 phosphorylation. Furthermore, the PKCδ catalytic fragment, but not the regulatory fragment, increased PKD1 activation, confirming PKCδ activity modulates PKD1 activation. We also identified that phosphorylation of S916 at the C-terminal is a preceding event required for PKD1 activation loop phosphorylation. Importantly, negative modulation of PKD1 by the RNAi knockdown or overexpression of PKD1^S916A phospho-defective mutants augmented oxidative stress-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 or constitutively active PKD1 plasmids attenuated oxidative stress-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury.

Conclusion

Collectively, our results demonstrate that PKCδ-dependent activation of PKD1 represents a novel intrinsic protective response in counteracting early stage oxidative damage in neuronal cells. Our results suggest that positive modulation of the PKD1-mediated compensatory protective mechanism against oxidative damage in dopaminergic neurons may provide novel neuroprotective strategies for treatment of PD.     

Decrease in CD93 (C1qRp) expression in a human monocyte-like cell line (U937) treated with various apoptosis-inducing chemical substances

Human CD93, a receptor for complement component 1, subcomponent q phagocytosis (C1qRp), has been shown to be selectively expressed by cells of a myeloid lineage and was originally reported to be involved in the C1q-mediated enhancement of phagocytosis in innate and adaptive immune responses. The modulation of CD93 expression has been investigated in various cells, particularly in granulocytes and monocytes . We previously reported that a protein kinase C activator (PKC), phorbol myristate acetate (PMA), effectively up-regulated CD93 expression on several cultured cell lines and that its regulation was mainly controlled by a PKC delta-isoenzyme. However, the expression pattern of CD93 in myeloid cells with apoptotic properties remains poorly understood. In this study, we examined the modulation of CD93 expression on a human monocyte-like cell line (U937) treated with various apoptosis-inducing chemical substances : an RNA-synthesis inhibitor, actinomycin D (ActD); a DNA topoisomerase I inhibitor, camptothecin (CPT); a protein-synthesis inhibitor, cycloheximide (CHX); a DNA topoisomerase II inhibitor, etoposide (EPS); and a DNA-synthesis inhibitor, mitomycin C (MMC). Apoptosis was monitored using two-color flow cytometry with Annexin V and 7-amino actinomycin D (7AAD). The above-mentioned substances sufficiently induced the early and late stages of apoptosis, identified as Annexin V positive (+)/7AAD negative (-) cells and Annexin V positive (+)/7AAD positive (+) cells, respectively, in U937 cells after 6 hr of treatment. The modulation of CD93 expression on U937 cells during the early stage of apoptosis, gated as Annexin V positive (+)/7AAD negative (-) cells, was then investigated using a CD93 mAb (mNI-11), originally established in our laboratories, and flow cytometry using a fluorescence-activated cell sorter (FACS). The mean fluorescence intensity (MFI) of the cells that stained positive for CD93 mAb (mNI-11) among the treated U937 cells showed a dramatic decrease in expression. In addition, the expressions of HLA-class I (HLA-A, B, C), HLA-class II (HLA-DR), CD18 (lymphocyte function-associated antigen-1 beta; LFA-1beta) and CD54 (intercellular adhesion molecule-1; ICAM-1) were also markedly decreased on the treated U937 cells identified as Annexin V positive (+)/7AAD negative (-) cells (early stage of apoptosis). Interestingly, the expression patterns of CD93 on the U937 cells treated with the above-mentioned chemical substances closely resembled those of HLA-class I (HLA-A, B, C). An immunoblotting analysis showed that the expression of a surface antigen (molecular size, about 97 kDa) targeted by the CD93 mAb (mNI-11) on the U937 cells treated with various apoptosis-inducing chemical substances had clearly decreased. On the other hand, an enzyme-linked immunoassay (EIA) showed that although PMA-treated U937 cells had strongly secreted soluble CD93 (sCD93) into the culture supernatant, the secretion of sCD93 in the culture supernatant of the U937 cells treated with the above-mentioned chemical substances was not enhanced, compared with that of untreated U937 cells. Importantly, however , the U937 cells with apoptotic properties induced by various apoptosis-inducing chemical substances also rapidly (in 30 min) and strongly secreted sCD93 into the culture supernatant in the presence of PMA. Taken together, these findings indicate that the expression of the CD93 molecule identified by CD93 mAb (mNI-11) is dramatically decreased on U937 cells with apoptotic properties, and that the decrease in CD93 expression on U937 cells treated with apoptosis-inducing chemical substances may be a good model for analyzing the regulation of CD93 expression on apoptotic myeloid cells.     

Phorbol ester inhibits DNA damage-induced apoptosis in U937 cells through activation of protein kinase C

The effects of phorbol 12-myristate 13-acetate (PMA) on DNA damage-induced apoptosis were examined in promyelocytic leukemia cells, U937, in comparison with other differentiation-inducing agents to clarify the role of protein kinase C (PKC) vis-a-vis cellular differentiation in apoptosis. The apoptosis of U937 cells was observed at as early as 1-1.5 h following UV irradiation, with most cells being in apoptotic state at 3 h. Pretreatment with PMA for as short as 5 min was sufficient to inhibit apoptosis induced by UV irradiation, whereas apparent changes in cell cycle distributions and expression of differentiation markers by PMA were not observed until 12 h and 48 h, respectively. The inhibition of apoptosis by PMA was completely abolished by the pretreatment with calphostin C, a PKC inhibitor, and 4 alpha-phorbor 12,13-didecanoate, which is unable to activate PKC, did not protect U937 cells against apoptosis induced by UV irradiation. Other differentiation inducers, such as cyclic AMP and active vitamin D3, did not affect the UV-induced apoptosis of U937 cells. Taken together, it was suggested that PMA inhibits DNA damage-induced apoptosis through the activation of PKC rather than as a result of differentiation of U937 cells.