Dimethyl sulfoxide-induced apoptosis in human leukemic U937 cells

The effects of two oral contraceptives, containing gestodene and either 20 micrograms or 30 micrograms ethinylestradiol, on hemostatic parameters was investigated in a six-month randomized study involving a total of 40 healthy women between the ages of 18 and 30 years. A large number of hemostatic parameters were measured, which were categorized as either pro-coagulatory, anti-coagulatory, profibrinolytic, anti-fibrinolytic or indicative of fibrin turnover. Additionally, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) were measured before and after venous occlusion and delta and ratio values calculated. Pro-coagulatory factors as well as reaction products reflecting in vivo coagulatory activity (thrombin-antithrombin III complex, prothrombin fragment 1 + 2) were found to increase. Among the anti-coagulatory parameters, only protein S concentration and protein S activity decreased, most notably in the 30 micrograms EE group. There was a corresponding increase in fibrinolytic activity reflected by reaction products of in vivo fibrinolysis (plasmin-antiplasmin 2-complex, fibrin-degradation products). Measurement of t-PA and PAI-1, before and after venous occlusion, revealed that the fibrinolytic response was more pronounced in the 20 micrograms EE group. There was also an increase in the threshold of fibrinolytic inhibition (ratio PAI-1) in both groups, which was less pronounced in the 20 micrograms EE group. Apart from isolated measurements, all parameters remained within their normal ranges and values returned to baseline in the follow-up cycle. It is concluded that both preparations had a balanced effect on the hemostatic system stimulating both pro-coagulant and fibrinolytic activity. No statistically significant differences were observed between the two groups; however, there was a trend towards greater fibrinolytic capacity in the 20 micrograms EE group.     

Characterization of a constitutive type III nitric oxide synthase in human U937 monocytic cells: stimulation by soluble CD23

1. Angiotensin II had a bimodal effect on human neutrophil migration. Low concentrations of angiotensin II stimulated random migration. At a concentration of 10(-10) M it caused a maximal increase of migration; migration increased from 47.2 +/- 2.1 microns in the absence of angiotensin II, to 73.1 +/- 2.2 microns with 10(-10) M angiotensin II present in the lower compartment of the Boyden chamber (n = 5, P < 0.001). Stimulation of migration by angiotensin II was partly chemotactic and partly chemokinetic. Angiotensin II concentrations of 10(-8) M and higher inhibited chemotactic peptide-stimulated chemotaxis. 2. The stimulant effect of angiotensin II on migration was completely dependent on extracellular Ca2+. In the presence of 1 mM Ca2+, angiotensin II stimulated migration to 76.1 +/- 1.7 microns, while migration in the absence of Ca2+ was 42.2 +/- 1.9 microns (n = 4, P < 0.001). Different types of calcium channel blockers either moderately or strongly inhibited angiotensin II-activated migration. Stimulation of migration by angiotensin II in intact cells required higher concentrations of Ca2+ than in electroporated cells. This supports the view that there is an influx of Ca2+ through the plasma membrane, and a requirement of calcium for an intracellular target. 3. Angiotensin II-stimulated migration was inhibited by pertussis toxin; from 71.6 +/- 2.0 microns in the absence, to 43.6 +/- 1.5 microns in the presence of pertussis toxin (n = 4, P < 0.001). Migration of electroporated neutrophils stimulated by angiotensin II was synergistically enhanced by GTP gamma S. This suggests that one or more G-proteins are involved in the activating effect of angiotensin II. 4. Inhibitors of soluble guanylate cyclase and antagonists of cyclic GMP-dependent kinase strongly inhibited the activating effect of angiotensin II. The results suggest that the activating effect of angiotensin II is mediated by cyclic GMP and by cyclic GMP-dependent kinase.     

Induction of apoptosis in leukemia U937 cells by 5'-deoxy-5'-methylthioadenosine, a potent inhibitor of protein carboxylmethyltransferase

We found dramatic changes in leukemia U937 cells treated with 5'-deoxy-5'-methylthioadenosine (MTA), a potent inhibitor of protein carboxylmethyltransferase (protein methylase II). Initiation of cell death was observed by 1 day after MTA treatment, and it was induced in a dose- and time-dependent manner. However, cell viability measured by trypan blue exclusion was not consistent with the actual percentage of cell death. These results indirectly indicated that the type of cell death is apoptosis rather than necrosis. Nuclear fragmentation and DNA condensation of MTA-treated U937 cells were analyzed by both fluorescent and electron microscopy. MTA-treated cells first began to arrest in the M phase of the cell cycle, and they then exhibited a mitotic-like nuclear fragmentation process with partially membraneless chromatin. Furthermore, agarose gel electrophoresis of DNA extracted from cells treated with MTA showed DNA laddering with production of fragments of approximately 200 bp multiples. These studies indicated that cell death induced by MTA has the characteristics of apoptosis, although nuclear fragmentation is atypical. It seems likely that the process of apoptosis in U937 cells induced by MTA correlates with incomplete assembly of the nuclear envelope, since MTA itself could inhibit the carboxylmethylation of nuclear lamin B and delayed incorporation of lamin B into the nuclear envelope.     

Gallium-67 radiotoxicity in human U937 lymphoma cells

Promising clinical results have been obtained with radiolabeled antibodies in lymphoma patients. The higher uptake by lymphomas of 67Gallium (67Ga) compared with monoclonal antibodies makes selective radiotherapy by the widely available 67Ga appealing. However, the gamma radiation of 67Ga used in scintigraphy is considered to be almost non-toxic to lymphoma cells. However, in addition to photon radiation 67Ga emits low energy Auger electrons and 80-90 keV conversion electrons which could be cytotoxic. The objective of the present study was the assessment of radiotoxicity of 67Ga on a lymphoid cell line: U937. Proliferation (MTT-assay) and clonogenic capacity (CFU-assay) were measured after 3 and 6 days incubation with 10, 20 and 40 microCi ml-1 67Ga. Growth inhibition was 36% after 3 days incubation and 63% after 6 days incubation with 40 microCi 67Ga ml-1. Clonogenic capacity was reduced by 51% after 3 days and 72% after 6 days incubation with 40 microCi ml-1 67Ga. A survival curve showed an initial shoulder and became steeper beyond 200-250 pCi cell-1 (low linear energy transfer type). Iso-effect doses of 67Ga and 90Yttrium (90Y) were determined. The iso-effect dose of 40 microCi 67Ga ml-1 (cumulative dose of conversion electrons 306 cGy) was 2.5 microCi 90Y ml-1 (cumulative dose 494 cGy) and the iso-effect dose of 80 microCi 67Ga ml-1 was 5.0 microCi 90Y/ml. The main cytotoxic effect of 67Ga seems to be induced by the 80 keV conversion electrons. We conclude that the conversion electrons of 67Ga have a cytotoxic effect on U937 cells and that in our experiments a 16-fold higher microCi-dose of 67Ga than of 90Y was needed for the same cytotoxic effect. We believe that 67Ga holds promise for therapeutic use.     

The PML/RARalpha fusion protein inhibits tumor necrosis factor-alpha-induced apoptosis in U937 cells and acute promyelocytic leukemia blasts

We investigated the effect of the acute promyelocytic leukemia (APL) specific PML/RARalpha fusion protein on the sensitivity to TNF-alpha-mediated apoptosis. The U937 leukemia cell line was transduced with PML/RARalpha cDNA. PML/RARalpha expression caused a markedly reduced sensitivity to TNF-alpha, even if apoptosis was triggered by agonistic antibodies to TNF-alpha receptors I and II (TNF-alphaRI, II). PML/RARalpha induced a 10-20-fold decrease of the TNF-alpha-binding capacity via downmodulation of both TNF-alphaRI and TNF-alphaRII: this may mediate at least in part the reduced sensitivity to TNF-alpha. Furthermore, the fusion protein did not modify Fas expression (CD95) or sensitivity to Fas-mediated apoptosis. The pathophysiological significance of these findings is supported by two series of observations. (a) Fresh APL blasts exhibit no TNF-alpha binding and are resistant to TNF-alpha-mediated apoptosis. Conversely, normal myeloblasts-promyelocytes show marked TNF-alphaR expression and are moderately sensitive to TNF-alpha-mediated cytotoxicity. Similarly, blasts from other types of acute myeloid leukemia (AML M1, M2, and M4 FAB types) show an elevated TNF-alpha binding. (b) The NB4 APL cell line, which is PML/RARalpha+, shows low TNF-alphaR expression capacity and is resistant to TNF-alpha-triggered apoptosis; conversely a PML/RARalpha- NB4 subclone (NB4.306) exhibits detectable TNF-alpha-binding capacity and is sensitive to TNF-alpha-mediated cytotoxicity. These studies indicate that the PML/RARalpha fusion protein protects against TNF-alpha-induced apoptosis, at least in part via downmodulation of TNF-alphaRI/II: this phenomenon may play a significant role in APL, which is characterized by prolonged survival of leukemic blasts.     

Down-modulation of CD4 antigen during programmed cell death in U937 cells

It has been hypothesized that programmed cell death (PCD), an active cell suicide process occurring in place of necrosis, can be associated with the pathogenesis of acquired immunodeficiency syndrome (AIDS). The entry of human immunodeficiency virus (HIV) into competent cells is mediated by the CD4 molecule present on the surface of certain lymphocyte subpopulations as well as on some cultured cell lines, e.g. U937 myelomonocytic cells. The present paper focuses on some specific aspects of PCD induced by the cytokine tumor necrosis factor (TNF). The results obtained indicate that the exposure of U937 cells to cycloheximide facilitates TNF-mediated PCD via a short term cell death program and modifies the expression of CD4 surface molecules. This change in surface antigen expression, manifested by internalization of the CD4 molecule, occurs in cells in which apoptosis has been triggered, but not in cells undergoing necrosis. These results indicate that the progression of cell death could be associated with specific alterations of certain surface molecules and could have a role in the entry of HIV into cells.     

Ethanol inhibits phosphatidylcholine and phosphatidylethanolamine biosynthesis in human leukemic monocyte-like U937 cells

The effect of ethanol (ETOH) on the incorporation of [14C]oleic acid (18:1) into lipid in human monocyte-like U937 cells was investigated. With increasing time of exposure to ETOH, the percentage of the label distributed into neutral lipid (NL) declined from 35 per cent (3 h) to 10 per cent (24 h) accompanied by increased incorporation into phospholipid (PL). [14C] 18:1 was preferentially incorporated into triglyceride (TG) and phosphatidylcholine (PC), comprising over 65 per cent and 50 per cent of the label associated with NL and PL, respectively. Low concentrations of ETOH (< or = 1.0 per cent; v/v) had no effect. At concentrations greater than 1.5 per cent, there was enhanced incorporation into TG and diacylglycerol (DAG) in a 24-h incubation period, while at 16 h the label in phosphatidylethanolamine (PE) was decreased. The effect of ETOH on the CDP-choline or ethanolamine pathway was examined by monitoring the incorporation of [3H]choline or [14C]ethanolamine into PC or PE, respectively. At low concentrations ETOH had no effect on either choline uptake or the incorporation into PC. Higher concentrations (> or = 1.5 per cent) for 3 and 6 h resulted in a slightly decreased choline uptake, and the reduction (40-50 per cent) of incorporation into PC suggests that the CDP-choline pathway was inhibited. There was a similar inhibition of the incorporation of [14C]ethanolamine into PE. When the cells were incubated for 3 h in the presence of 2 per cent ETOH and with labelled 18:1 and PL-base, the ratios of incorporation (base/18:1) into PC and PE fractions decreased, indicating that the major inhibition lay in blockage of the availability of the base moiety for PL formation. Analysis of the distribution of the label into metabolites revealed that ETOH inhibited the conversion of [14C]ethanolamine into [14C]phosphorylethanolamine. The reduction in incorporation was not due to the enhanced breakdown of base-labelled PL. Our results indicate that ETOH has an inhibitory effect on the CDP-choline or ethanolamine pathway.     

Induction of apoptosis and potentiation of TNF- and Fas-mediated apoptosis in U937 cells by the xanthogenate compound D609

The purpose of Study 1 was to examine the effect of dietary soy on the progression of MDA-MB-435 human breast cancer cell solid tumors in nude mice. When toasted soy chips were fed at levels of 5%, 10%, or 20% (wt/wt) in a high-fat, linoleic acid-rich diet for 12 weeks, there was a trend for larger mammary fat pad tumors to occur with increasing soy intake. However, compared with the controls the severity of macroscopic lung metastasis was reduced significantly in the groups fed 10% and 20% soy. Study 2 compared the effects of diets containing 23% corn oil (CO), 18% menhaden oil (MO) + 5% CO, 18% MO + 5% CO + 10% soy chips, and MO or soy-supplemented diets + indomethacin treatment in the same animal model. Feeding the 18% MO diet without soy or indomethacin reduced primary tumor growth; statistically significant effects were not observed in any of the other groups. All three of the groups with MO supplementation showed a reduction in the occurrence and severity of macroscopic lung metastases, together with the expected decreases in tumor prostaglandin E levels. These effects were most pronounced when MO was combined with indomethacin treatment. When indomethacin was given with dietary soy, the previously reported suppressive effect of the cyclooxygenase inhibitor on MDA-MB-435 cell tumor progression was lost, despite reductions in tumor prostaglandin E concentrations.     

mtDNA-depleted U937 cells are sensitive to TNF and Fas-mediated cytotoxicity

It has been proposed that TNF cytotoxicity is mediated by reactive oxygen intermediates generated by uncoupling of mitochondrial respiration. We have compared sensitive U937 cells and derived cell lines depleted of mtDNA for their ability to undergo TNF- and Fas-induced apoptosis. Cells lacking around 98% of mtDNA were still sensitive to TNF-induced apoptosis. U937 cells devoid of mtDNA (U937-rho degree) were resistant to TNF, but this was due to the loss of its 55 kDa receptor. U937-rho degree cells were also resistant to docosahexaenoic acid, which causes U937 cell death by lipid peroxidation. These cells were sensitive to anti-Fas toxicity. The results indicate that TNF and Fas-induced toxicity occurs by a mechanism mostly independent of mitochondrial free radical generation.     

Nitric oxide synthase plays a signaling role in TCR-triggered apoptotic death

A functional role for stimulated nitric oxide (NO) production was tested in the TCR-triggered death of mature T lymphocytes. In purified peripheral human T cell blasts or the 2B4 murine T cell hybridoma, apoptotic cell death induced by immobilized anti-CD3 was blocked by inhibitors of NO synthase (NOS) in a stereospecific and concentration-dependent manner. This effect appeared to be selective since apoptotic death induced by anti-Fas Ab or the steroid dexamethasone was not affected by NOS inhibitors. TCR-stimulated expression of functional Fas ligand was attenuated in a stereospecific manner by NOS inhibitors, but these compounds did not inhibit TCR-stimulated IL-2 secretion or CD69 surface expression. Nitrosylated tyrosines, a stable marker for NO generation, were immunochemically detected in T cells using flow cytometry. TCR signals induced NO production, as measured by an increase in nitrotyrosine-specific staining. NOS enzymatic activity was detected in lysates of 2B4 cells, and Western blot analysis suggests that the activity is due to expression of the neuronal isoform of NOS. Thus, T cells have the capacity to generate NO upon Ag signaling, which may affect signal transduction, Fas ligand surface expression, and apoptotic cell death of mature T lymphocytes.     

Enhanced expression of GTP-binding proteins in differentiated U937 monocytic cells: possible involvement of tyrosine kinase and protein kinase C

Seven Arxula adeninivorans strains originating from The Netherlands (CSIR 1136, CSIR 1138 and CBS 8244T), South Africa (CSIR 1147, CSIR 1148 and CSIR 1149) and Siberia (Ls3) were compared concerning their secretory glucoamylase. Within the spectrum of secretory polypeptides obtained by SDS-polyacrylamide gel electrophoresis, the glucoamylase corresponding polypeptide, could be identified by means of specific antibodies directed against the glucoamylase of strain Ls3. The molecular masses of the glucoamylases vary from 84 to 95 kDa. After endoglycosidase F treatment of the secretory proteins, the N-linked carbohydrate content for each glucoamylase could be quantified at about 25%. Glucoamylase activity staining of secretory proteins separated under nondenaturing conditions revealed one glucoamylase active protein for the Dutch strains and two active forms for the strains originating from South Africa and Siberia. Highest activities of glucoamylase can be measured at the end of the exponential growth phase for each strain. The Dutch strain CSIR 1138 secretes the highest activity. Optimum values for temperature and pH were determined and compared. By means of pulsed field gel electrophoresis and Southern analysis, the glucoamylase corresponding gene could be localized on the second of the four chromosomes of each yeast strain.     

Inhibition of human immunodeficiency type 1 virus replication in monocytic U-937 cells by superinfection with Chlamydia trachomatis

Status epilepticus is a neurological emergency associated with substantial morbidity and mortality. Experimental and clinical investigations suggest that prolonged seizure activity is associated with injury to vulnerable neurons. Compounds with neuroprotective properties may minimize such injury. Existing methods of inducing experimental status epilepticus result in seizure activity of variable duration and neuronal injury of variable degree. To minimize such variability, status epilepticus may be stopped with anticonvulsants, but this limits the ability to screen for independent neuroprotective properties. We have developed a simple and reliable non-pharmacological model of limbic status epilepticus in which the duration of status epilepticus is under direct experimental control. Status epilepticus is induced by continuous, unilateral hippocampal stimulation. Using this model, the degree of hippocampal pyramidal cell injury varies in direct proportion to status epilepticus duration across a range of 15-140 min. A progressive sequence of EEG changes unfolds with increasing status epilepticus duration, resembling that seen in other models. This model may serve as a reference against which the effects of potential neuroprotective compounds can be studied.     

The mitogen-activated protein kinase pathway inhibits ceramide-induced terminal differentiation of a human monoblastic leukemia cell line, U937

This communication describes an extracellular signal-regulated kinase kinase (MEK)-dependent signal transduction pathway that prevents the terminal differentiation of a hemopoietic cell line. Both PMA and the cell-permeable ceramide, C2-ceramide, caused differentiation of U937 cells, but with distinct cell morphology and CD11b/CD14 surface expression. While PMA activated extracellular signal-regulated kinase (ERK), a downstream kinase of Raf-MEK signaling, C2-ceramide activated c-Jun NH2-terminal kinase (JNK), an anchor kinase of stress-induced signaling. Furthermore, only C2-ceramide stimulated an induction of cell cycle arrest that was associated with stable expression of p21CIP1 and retinoblastoma nuclear phosphoprotein dephosphorylation. Expression of p21CIP1 and JNK activation were also observed in sphingosine-treated cells, whereas sphingosine did not induce detectable differentiation. Concomitant stimulation with C2-ceramide and PMA resulted in the PMA phenotype, and cell cycle arrest was absent. ERK activation was enhanced by C2-ceramide plus PMA stimulation, whereas the activation of JNK was aborted. Strikingly, the inhibition of MEK with PD98059 altered the phenotype of C2-ceramide- and PMA-stimulated U937 cells to that of cells treated with C2-ceramide alone. Thus, ERK and JNK pathways deliver distinct signals, and the ERK pathway is dominant to the JNK cascade. Furthermore, differentiation and cell cycle arrest caused by C2-ceramide rely on independent signaling pathways, and JNK is an unlikely signaling element for this differentiation. Importantly, during C2-ceramide and PMA costimulation, the JNK pathway is not simply blocked by ERK activation; rather, cross-talk between these MAP kinase pathways acts to simultaneously augment ERK activity and down-regulate JNK activity.     

The role of p53 and the CD95 (APO-1/Fas) death system in chemotherapy-induced apoptosis

To explore the pathway of p53 dependent cell death, we investigated if p53 dependent apoptosis following DNA damage is mediated by the CD95 (APO-1/Fas) receptor/ligand system. We investigated cell lines of solid human tumors upon treatment with clinically relevant chemotherapeutic drugs known to act via p53 accumulation. Treatment with these cytotoxic drugs led to an upregulation of both, the CD95 receptor (CD95) and the CD95L (CD95L). Induction of the CD95L occurred in p53 wild-type (wt), p53 mutant (mt) and in cell lines lacking p53 altogether (p53-/-). Thus, the regulation of the CD95L in response to chemotherapeutic drugs clearly involves p53 independent mechanisms. Most importantly, upregulation of CD95 occurred only in cell lines with wild-type p53, thereby strongly increasing the responsiveness towards CD95 mediated apoptosis. Thus, upregulation of the CD95 receptor seems to be dependent on intact wild-type p53. Apoptosis was mediated by cleavage of the receptor proximal caspase, caspase-8 (FLICE/MACH). Caspase-8 cleavage was observed, independent of the p53 status of the tumor cells and irrespective whether or not apoptosis was dependent on the CD95 system. Hence, additional effector pathways besides CD95/CD95L signaling are likely to contribute to drug-induced apoptosis.     

Stereospecific induction of apoptosis in U937 cells by N-octanoyl-sphingosine stereoisomers and N-octyl-sphingosine. The ceramide amide group is not required for apoptosis

We investigated the ability of N-octanoyl-sphingosine (C8-Cer) stereoisomers, N-octanoyl-DL-erythro-dihydrosphingosine (DL-e-DHC8-Cer), and a new ceramide derivative, N-octyl-D-erythro-sphingosine (D-e-C8-Ceramine), to induce apoptosis in U937 cells. We found the C8-Cer stereoisomers to be stereospecific with the D- and L-threo stereoisomers being severalfold more potent than the erythro in inducing nucleosomal fragmentation. The order of potency was: D-t-C8-Cer = L-t-C8-Cer > L-e-C8-Cer > D-e-C8-Cer > DL-e-DHC8-Cer. The importance of the carbonyl group in apoptosis was investigated by using a new ceramide derivative, D-e-C8-Ceramine, in which the carbonyl group was replaced by a methylene group. The carbonyl group was not necessary for triggering apoptosis. In fact, replacement of the carbonyl group decreased substantially the time required for cells to die, with maximum DNA fragmentation occurring at 6 h as opposed to the 18 h required by D-e-C8-Cer. To explore possible mechanisms by which these compounds trigger the apoptotic pathway, we tested their ability to increase the endogenous levels of cellular ceramide and to differentially activate a ceramide-activated protein kinase (CAPK). While the potent DNA fragmentation-inducing compounds D-e-C8-Ceramine and L-t-C8-Cer failed to increase the cellular ceramide levels, D-e-C8-Cer, D-t-C8-Cer and D-e-C8-Ceramine activated the CAPK equally. These studies suggest that the DNA fragmentation-inducing ability of the threo stereoisomers and D-e-C8-Ceramine cannot be attributed either to an increase in the activity of CAPK, or, as illustrated by D-e-C8-Ceramine and L-t-C8-Cer, to the differential elevation of endogenous ceramide. The phosphatase inhibitor okadaic acid failed to protect U937 cells from apoptosis induced by D-e-C8-Cer.     

Calphostin C triggers calcium-dependent apoptosis in human acute lymphoblastic leukemia cells

Recent studies have demonstrated that the naturally occurring perylenequinone antibiotic calphostin C is a potent inhibitor of protein kinase C and can induce apoptosis in some tumor cell lines by an as yet unknown mechanism. Here we demonstrate that calphostin C induces dose-dependent apoptosis in DT40 chicken lymphoma B-cells, and targeted disruption of lyn, syk, btk, PLCgamma2, or IP3R genes does not prevent or attenuate its cytotoxicity. In our study, calphostin C also induced rapid apoptosis in human acute lymphoblastic leukemia (ALL) cell lines ALL-1 (BCR-ABL+ pre-pre-B ALL), RS4;11 (MLL-AF4+ pro-B ALL), NALM-6 (pre-B ALL), DAUDI (Burkitt's/B-cell ALL), MOLT-3 (T-ALL), and JURKAT (T-ALL), whereas other potent PKC inhibitors did not. In biochemical studies, calphostin C was discovered to induce rapid calcium mobilization from intracellular stores of ALL cell lines, and its cytotoxicity against ALL cell lines was well correlated with the magnitude of this calcium signal. Calphostin C-induced apoptosis was markedly suppressed by BAPTA/AM, a cell-permeable Ca2+ chelator as well as NiCl2, an inhibitor of Ca2+/Mg2+-dependent endonucleases. Inhibition of the Ca2+/calmodulin-dependent phosphatase calcineurin with perfluoreperazine dimadeate (a calmodulin antagonist) or cyclosporin A (a specific inhibitor of calcineurin) also reduced the magnitude of calphostin C-induced apoptosis in ALL cell lines. Calphostin C was capable of inducing calcium mobilization and apoptosis in freshly obtained primary leukemic cells from children with ALL. Taken together, our results provide unprecedented evidence that calphostin C triggers a Ca2+-dependent apoptotic signal in human ALL cells.     

Early cytoplasmic acidification in retinamide-mediated apoptosis of human promyelocytic leukemia cells

The retinamide-mediated apoptosis of promyelocytic cells was investigated using pHi- and DNA-sensitive fluorescent probes (BCECF and Hoechst 33342). Acidification and apoptosis were observed during prolonged (0-12 h) exposure to retinamide (1 microM), but were absent in a retinamide-resistant clone. The analysis of experiments performed by simultaneous staining with the two dyes showed that acidification and apoptosis are correlated; the half-time for acidification is about 5 h while that for apoptosis is 80% longer. On the whole, these results suggest that apoptosis is preceded by an early mechanism of acidification in human leukemia cells treated by a retinoid of clinical interest in cancer chemo prevention and therapy.     

Induction of apoptosis in human leukemia K562 cells by alpha-anordrin

The apparent coexistence of primary biliary cirrhosis (PBC) and autoimmune hepatitis in the same patient raises unresolved problems for nosology and therapy. These are exemplified by a 45-year-old Japanese woman with overlapping clinical, serological and histological features of autoimmune cholangitis and autoimmune hepatitis. The classical serological test for PBC, antimitochondrial antibody (AMA) by immunofluorescence, was atypical. By immunoblotting there was reactivity with one of the enzymes of the 2-oxo-acid dehydrogenase complex (2-OADC) family, now recognized as autoantigens responsible for AMA reactivity. Also there was reactivity by immunofluorescence for antinuclear antibodies (ANA), one showing the typical speckled pattern of anti-Sp-100 and the other the peripheral pattern of antinuclear membrane antibody, both with titres > 10(6). There was also a positive result to the lupus erythematosus (LE) cell test. Treatment with ursodeoxycholic acid was beneficial. Thus while the clinical presentation suggested the overlapping syndrome of autoimmune hepatitis and PBC, PBC eventually proved to be the likely diagnosis. We suggest that apparent cases of overlapping PBC-autoimmune cholangitis-hepatitis syndromes, after detailed testing, will mostly align with PBC.     

Evidence against a direct role for the induction of c-jun expression in the mediation of drug-induced apoptosis in human acute leukemia cells

Previous reports have demonstrated that a variety of anticancer drugs, e.g., 1-beta-D-arabinofuranosylcytosine (ara-C), mitoxantrone, etoposide, camptothecin, and cisplatin, induce the expression of c-jun oncogene in leukemic cells prior to producing internucleosomal DNA fragmentation and the morphological features of apoptosis. This has led to the impression that the induction of c-jun expression may be directly involved in the molecular signaling of the final common pathway of programmed cell death or apoptosis. In the present study, we examined the role of c-jun expression in three different settings of anticancer drug-induced apoptosis in human leukemic cells. First, exposure of human myeloid leukemia HL-60 cells to high-dose ara-C for 4 h produced internucleosomal DNA fragmentation preceded by c-jun induction. However, pretreatment of HL-60 cells with staurosporine, a protein kinase C inhibitor, repressed c-jun yet enhanced DNA fragmentation and apoptosis due to ara-C. Second, in human pre-B leukemia 697/BCL-2 cells which are transfected with the cDNA of the bcl-2 oncogene and overexpress p26BCL-2, although ara-C or mitoxantrone treatment caused greater c-jun induction than in the 697/neo cells, significantly reduced endonucleolytic DNA fragmentation and apoptosis was observed in 697/BCL-2 cells. Finally, taxol-induced internucleosomal DNA fragmentation and morphological features of apoptosis in HL-60 cells were not associated with the induction of c-jun expression. These lines of evidence indicate that the induction of c-jun expression may not have a direct role in the molecular signaling of anticancer drug-induced apoptosis, and that the anticancer drug-induced apoptosis can occur by a mechanism that does not involve the induction of c-jun expression.