Chronic myelogenous leukaemia CD34+ cells exit G0/G1 phases of cell cycle more rapidly than normal marrow CD34+ cells

To investigate the mechanisms behind the leukaemic expansion of chronic myelogenous leukaemia (CML), we examined the cell cycle status and activation kinetics of purified subpopulations of CD34⁺ cells from normal and CML bone marrow (BM). Propidium iodide staining was used to assess cell cycle status of fresh cells or those stimulated with cytokines. Although the cell cycle status of fresh low-density cells from CML and normal BM was similar, a larger percentage of CML CD34⁺ cells were cycling than those from normal BM. The HLA-DR⁻ compartment of CML CD34⁺ cells, a fraction enriched for normal, non-leukaemic progenitors, contained a higher percentage of quiescent cells than the CD34⁺ HLA-DR⁺ fraction. When the activation of CD34⁺ cells was examined in response to SCF or IL-3 alone, or SCF+IL-3+IL-6, CML CD34⁺ cells exited G₀/G₁ more rapidly than normal CD34⁺ cells. Interestingly, although normal BM CD34⁺ cells failed to cycle in response to IL-6 alone, or in the absence of exogenous cytokines, 30% of CML cells cycled under these conditions. No differences in the degree of apoptosis were documented among CML and normal CD34⁺ cells in these cultures. These data suggest that enhanced cell cycle activation of CML CD34⁺ cells, by either autocrine stimuli or via enhanced sensitivity to exogenous stimuli, may be partially responsible for the pronounced cellular expansion characteristic of CML.     

Liposomal 1,25 (OH)2 vitamin D3 compounds block proliferation and induce differentiation in myelomonocytic leukaemia cells

The vitamin D₃ derived hormone 1,25 (OH)₂ vitamin D₃ (1,25 D₃) is able to induce growth arrest and differentiation in myelomonocytic leukaemia cells. In order to allow for specific delivery to leukaemic cells the lipophilic compound was incorporated into the lipid membranes of liposomes. Liposomal 1,25 D₃ reduced proliferation as measured by ³H-thymidine incorporation in HL60 leukaemia cells by up to 60%. When liposomes were prepared at different concentrations of 1,25 D₃ 65% inhibition was achieved at 48 n M . The MC 1288 stereoisomer of 1,25 D₃ was more potent and had the same activity at 48 n M .
The effect of the liposomal compounds was specific to myeloid cells as they reduced proliferation in myelomonocytic HL60, monoblastic U937 and monocytic Mono Mac 6 cells but not in the T-cell lines Jurkat and Molt 4.
The antiproliferative effect of liposomal 1,25 D₃ was associated with an induction of differentiation since treated HL60 cells showed a monocytic morphology, increased expression of CD14 and decreased expression of CD33.
When peripheral blood leukaemic cells from M4 and M5 acute myeloid leukaemia (AML) patients were admixed with liposomal compounds an antiproliferative effect was seen in all five cases, including the two cases where free compounds led to enhanced growth. Liposomal delivery of 1,25 (OH)₂ vitamin D₃ may offer a novel approach to treatment of myelomonocytic leukaemia.     

In leukaemic CD5+ B cells the expression of BCL-2 gene family is shifted toward protection from apoptosis

This study further investigated the mechanisms that control apoptosis in leukaemic CD5⁺ B cells, and focused on the Bcl-2 gene family. The pattern of expression of Bcl-2, Bcl-xL, Bcl-xS and Bax genes, selected because of their interrelated role in the control of apoptosis, was analysed in a series of CD5⁺ B-cell chronic lymphoid leukaemias.
Cells from 34 patients with chronic lymphoid leukaemia of B-cell type (23 B-chronic lymphocytic leukaemia (B-CLL) and 11 mantle cell lymphoma (MCL) in leukaemic phase) were investigated. High levels of Bcl-2 mRNA were observed by Northern blot and high levels of Bcl-2 protein were detected by cytofluorograph analysis with a specific monoclonal antibody (MAb) in all cases. Strong Bax expression was detected by RT-PCR in 20/23 B-CLL cases; Bax was also observed in 8/11 MCL in leukaemic phase with variable degree of intensity. In both B-CLL and MCL samples the presence of Bax protein was confirmed by cytofluorograph analysis. RT-PCR detected high levels of Bcl-xL in 16/23 B-CLL and in 8/11 MCL in leukaemic phase, whereas Bcl-xS was detectable in low to trace amounts respectively in 13/23 B-CLL and in 6/11 MCL in leukaemic phase.
According to the functional role of Bcl-2, Bcl-xL, Bcl-xS and Bax, these data indicate that the pattern of Bcl-2 family genes expression in leukaemic CD5⁺ B cells is skewed toward prevention of apoptosis and may thus favour the relentless accumulation of CD5⁺ leukaemic B cells.     

Changes in the proteome of human bronchial epithelial cells following stimulation with leucotriene E4 and transforming growth factor-β1

Background and objective: Activated bronchial epithelial cells exert considerable potential to maintain a microenvironment in the airway wall that promotes airway inflammation and remodelling. Cysteinyl leucotrienes (CysLT) and transforming growth factor‐β₁ (TGF‐β₁) are both increased in asthmatic airways and may influence the pathophysiology of disease. However, the consequences of activation of bronchial epithelial cells by these mediators are not fully understood. A proteomic‐based approach was used to characterize the inflammatory pathways in bronchial epithelial cells after stimulation with CysLT and TGF‐β₁. Methods: Human bronchial epithelial cells (BEAS‐2B) were stimulated with 1 ng/mL TGF‐β₁ and 50 nmol/L leucotriene E₄ (LTE₄) for 48 h and whole‐cell lysates were subjected to two‐dimensional gel electrophoresis. Proteins showing statistically significant differential expression were identified by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry and database searching. Results: Stimulation with LTE₄ increased the expression of three proteins and five proteins showed decreased expression. Of the latter group, two were definitively identified as heat shock protein (Hsp90α) and stress‐70 protein. Hsp90α forms a heterocomplex with the glucocorticoid receptor (GR) and a significant decrease in GR following LTE₄ stimulation was confirmed. TGF‐β₁ downregulated 18 intracellular proteins, including lamin A/C, glyceraldehyde‐3‐phosphate dehydrogenase, protein DJ‐1, voltage‐dependent calcium channel gamma‐7 subunit, heterogeneous nuclear ribonucleoprotein A2/B1 and stress‐70 protein. Conclusions: The current findings suggest that by downregulating GR and Hsp90α, CysLT may interfere with the action of glucocorticoids. Overall, the results confirm the complex role of bronchial epithelium in aspects of airway inflammation and remodelling.     

The novel histamine H2 receptor antagonist FRG-8813 prevents delay of wound repair induced by hydrogen peroxide in a rabbit gastric epithelial cell system

Abstract The effects of a novel histamine H₂ receptor antagonist (FRG-8813) on the restoration process of gastric epithelial wounds were assessed using an in vitro wound healing model. FRG-8813 (1, 10 mol/L) was added to a complete confluent monolayer cell sheet after artificial wounding. The restoration process was analysed by a time-lapse video system and cell migration, proliferation and apoptosis were assessed. Hydrogen peroxide (1, 3 mmol/L) inhibited restoration after wounding by suppressing cell migration and proliferation and induced epithelial cell apoptosis around the wound. The addition of FRG-8813 abolished the hydrogen peroxide-induced retardation and prevented apoptosis, although FRG-8813 itself did not enhance wound healing. FRG-8813 may act as a radical scavenger as well as having an anti-secretory action and may have favourable effects on peptic ulcer healing.     

Alpha(2) adrenoceptors regulate proliferation of human intestinal epithelial cells

BACKGROUND AND AIMS: Previous studies on rodents have suggested that catecholamines stimulate proliferation of the intestinal epithelium through activation of alpha(2) adrenoceptors located on crypt cells. The occurrence of this effect awaits demonstration in humans and the molecular mechanisms involved have not yet been elucidated. Here, we examined the effect of alpha(2) agonists on a clone of Caco2 cells expressing the human alpha(2A) adrenoceptor. METHODS: Cells were transfected with a bicistronic plasmid containing the alpha2C10 and neomycin phosphotransferase genes. G418 resistant clones were assayed for receptor expression using radioligand binding. Receptor functionality was assessed by testing its ability to couple Gi proteins and to inhibit cAMP production. Mitogen activated protein kinase (MAPK) phosphorylation was followed by western blot, and cell proliferation was estimated by measuring protein and DNA content. RESULTS: Permanent transfection of Caco2 cells allowed us to obtain a clone (Caco2-3B) expressing alpha(2A) adrenoceptors at a density similar to that found in normal human intestinal epithelium. Caco2-3B retained morphological features and brush border enzyme expression characteristic of enterocytic differentiation. The receptor was coupled to Gi2/Gi3 proteins and its stimulation caused marked diminution of forskolin induced cAMP production. Treatment of Caco2-3B with UK14304 (alpha(2) agonist) induced a rapid increase in the phosphorylation state of MAPK, extracellular regulated protein kinase 1 (Erk1), and 2 (Erk2). This event was totally abolished in pertussis toxin treated cells and in the presence of kinase inhibitors (genistein or PD98059). It was unaffected by protein kinase C downregulation but correlated with a transient increase in Shc tyrosine phosphorylation. Finally, sustained exposure of Caco2-3B to UK14304 resulted in modest but significant acceleration of cell proliferation. None of these effects was observed in the parental cell line Caco2. CONCLUSION: The results obtained in the present study support a regulatory role for alpha(2) adrenoceptors in intestinal cell proliferation.     

Netrin-1 prevents ischemia/reperfusion-induced myocardial infarction via a DCC/ERK1/2/eNOSs1177/NO/DCC feed-forward mechanism

We have recently shown that a novel endothelial mitogen netrin-1 potently stimulates nitric oxide (NO) production via a DCC-ERK1/2 dependent mechanism. In view of the well-established cardioprotective role of NO, the present study investigated whether netrin-1 is cardioprotective via NO signaling in the heart. Netrin-1 receptor DCC was abundantly expressed in the C57BL/6J mouse hearts. Perfusion of heart with netrin-1 (100 ng/mL) using a Langendorff system significantly increased NO production. Under ischemia/reperfusion (I/R), netrin-1 induced a substantial reduction in infarct size (21.8 ± 4.9% from 42.5 ± 3.6% in the controls), which was accompanied by an augmented production of NO. Pre-perfusion with DCC-antibody, U0126 (MEK1/2 inhibitor), L-NAME or PTIO (NO scavenger) attenuated protective effects of netrin-1 on infarct size and NO production, indicating upstream roles of DCC and ERK1/2 in NO production, as well as an essential role of NO in cardioprotection. Netrin-1 induced reduction in infarct size was significantly attenuated in DCC+/− mice, confirming an intermediate role of DCC. In additional experiments we found netrin-1 increased ERK1/2 and eNOS_s1177 phosphorylation, and DCC protein expression, which was diminished by I/R. Furthermore, netrin-1-induced DCC upregulation was NO and ERK1/2-dependent, implicating a feed-forward mechanism. DAF-AM staining revealed enhanced NO production in both cardiac endothelial cells (ECs) and myocytes. In primarily isolated cardiomyocytes, netrin-1 also increased NO production, DCC abundance and ERK1/2 phosphorylation. Of note, cardiac apoptosis was significantly attenuated by netrin-1, which was reversed by DCC-antibody, U0126, L-NAME or PTIO. In summary, our data clearly demonstrate that netrin-1 potently protects the heart from I/R injury by stimulating NO production from cardiac ECs and myocytes. This potent effect is mediated by a DCC/ERK1/2/eNOS_s1177/NO/DCC feed-forward mechanism in both cell types.     

Alcohol Induction of Ferritin Expression in a Human Hepatoblastoma Cell Line (HEP G2)

Hyperferritinemia, an unclear mechanism, is frequently observed in chronic alcoholics. The aim of this work was to study the effect of alcohol on ferritin expression in a human hepatoblastoma cell line, HepG2. This cell line proved to be sensitive to alcohol, since alcohol increased gamma-GT activity both in cells and media. The most striking result was the increase of ferritin in cells and media by alcohol. Moreover, this effect was specific, since it contrasted with a decrease in total protein synthesis and secretion, a decrease in transferrin excretion and a lack of effect on orosomucoid. In our model, alcohol was able to induce, in a specific manner, ferritin expression.     

Identification of biclonal (duplex) leukaemic cells expressing either CD4+/CD8- or CD4-/CD8+ from a patient with adult T-cell leukaemia/lymphoma

A 24-year-old Japanese woman was admitted to our hospital in 1987 with a chief complaint of skin eruptions, and was diagnosed as having chronic ATLL. In 1993 the leucocyte count increased gradually to 126.0x10⁹/l with 91.5% abnormal lymphocytes expressing two different types of antigenicity, either CD4⁺/CD8^- or CD4^-/CD8⁺. Monoclonal integration of human T-cell lymphotropic virus type-I proviral DNA was detected at different sites of the genomic DNA in each cell type. These studies clearly indicate that CD4⁺/CD8^- and CD4^-/CD8^- leukaemic cells originated from two independent clones.     

Effect of 1,25-(OH)2D3 (a vitamin D analogue) on passively sensitized human airway smooth muscle cells

BACKGROUND AND OBJECTIVES: In asthma, airway smooth muscle cell (ASMC) hyperplasia plays an important role in airway remodelling. Increased expression of matrix metalloproteinases-9 (MMP-9), a disintegrin and metalloprotease 33 (ADAM33) in ASMCs are also relevant to asthmatic airway remodelling. 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) has potent antiproliferative properties in vitro in various cell types; however, its role in ASMCs is not well understood. This study investigated the effect of 1,25-(OH)(2)D(3) on passively sensitized human bronchial (airway) smooth muscle cell (HASMC) proliferation and MMP-9 and ADAM33 expressions. METHODS: The effect of 1,25-(OH)(2)D(3) on cell proliferation was examined by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium-bromide colorimetry assay; cell cycle analysis by flow cytometry; and immunocytochemical staining for proliferating cell nuclear antigen (PCNA). The expression of MMP-9 and ADAM33 in HASMCs was investigated by real-time quantitative PCR and Western Blot analysis. RESULTS: 1,25-(OH)(2)D(3) effectively suppressed passively sensitized HASMC proliferation, proliferating cell nuclear antigen expression and G(1)/S transition in HASMCs passively sensitized with asthmatic serum. Further analysis showed that 1,25-(OH)(2)D(3) significantly down-regulated the expressions of protein for MMP-9 and ADAM33, as well as their mRNA levels in passively sensitized HASMCs. CONCLUSIONS: 1,25-(OH)(2)D(3) has direct inhibitory effects on passively sensitized HASMCs in vitro, including inhibition of cell proliferation and expression of MMP-9 and ADAM33, suggesting a possible beneficial role for 1,25-(OH)(2)D(3) in preventing and treating asthmatic airway remodelling.     

Established IL-2-dependent double-negative (CD4- CD8-) TCRαβ/CD3+ ATL cells: induction of CD4 expression

Summary. We established IL-2-dependent T cells from an adult T-cell leukaemia (ATL) patient whose leukaemic cells changed from CD4 single-p-positive in the initial phase to double-negative (CD4^- CD8^-) at the time of exacerbation. The cells termed SO-4 were of ATL cell origin and showed the double-negative TCRαβ/CD3⁺ T-cell phenotype. SO-4 cells acquired CD4 antigen expression following stimulation with concanavlin A (ConA) or immobilized anti-CD3 antibody. The induction was inhibited by herbimycin A, an inhibitor of protein tyrosine kinase (PTK) activity. No CD4 mRNA was detectable in unstimulated SO-4 cells but a 3.0 kb signal specific for CD4 mRNA was detected after stimulation. These findings indicate that SO-4 cells return to their original phenotype (CD4 single-positive) by stimulation involving PTK. The results indicate that there is a pathway of phenotypic cycling between CD4 single-positive and double-negative T cells.     

Functional characteristics of apheresis-derived platelets treated with ultraviolet light combined with either amotosalen-HCl (S-59) or riboflavin (vitamin B2) for pathogen-reduction

Background  To examine if different pathogen-reduction technologies (PRTs) induce different degrees of platelet (PLT) storage lesion.
Design  Twenty-seven split triple-dose apheresis PLTs were PRT treated using ultraviolet light with either riboflavin (M) or psoralen (I) or remained untreated (C). Samples taken on days (d) 0 to 8 were analysed for PLT count, blood gas (pH, pO₂ and pCO₂), metabolism (lactate, glucose, ATP content), in vitro function [swirling, hypotonic shock response (HSR) and aggregation], activation (p-selectin expression) and cellular integrity (JC-1 signal, annexin A5 release).
Results  Platelet counts of all study groups remained unchanged during storage indicating that PRT treatment did not induce relevant cell lysis. Although M units demonstrated the highest values for HSR until d5, PRT treatment lowered all parameters examined with significant differences to untreated controls by d7 of storage. During final storage, M was significantly superior over I for HSR, aggregation with TRAP-6 as agonist (collagen was similar), annexin A5 release and JC-1 signal. Regarding blood gas and metabolic analysis, the most evident effect of PRT was an elevated glycolytic flux combined with higher acidity due to increased lactate accumulation. Most likely due to impaired O₂ consumption, pH and ATP decreased more rapidly in I relative to C and M.
Conclusion  Pathogen reduction technology-treated PLTs remained comparable to untreated units throughout 7 days of storage. Mitochondria-based oxidative respiration appeared up-regulated after the riboflavin-based PRT. Compared to the psoralen-based PRT, this resulted in significantly better ATP maintenance and in vitro function during the last storage period (d7, d8).     

Ultrastructural analysis of the distribution of the vitronectin receptor (αvβ3) in human platelets and megakaryocytes reveals an intracellular pool and labelling of the α-granule membrane

The vitronectin receptor (VnR or α_vβ₃) belongs to the cytoadhesin subclass of the integrin family. This subclass consists of two receptors which have the β₃ subunit in common: GP IIb–IIIa complexes (or α_IIbβ₃) and VnR. We report the subcellular distribution of VnR within human platelets as determined by immunogold staining of ultrathin frozen sections and transmission electron microscopy. Monoclonal antibodies directed against: (i) the α_v subunit (LM142, AMF7, CLB-706), or (ii) an epitope specific to the complex (LM609) were used. Although VnR is present on platelets, it is a minor component. We therefore first compared several different staining procedures to detect this integrin. Optimal localization of VnR was obtained using a multistep procedure in which biotinylated anti-mouse IgG and a monoclonal anti-biotin antibody provided staining enhancement. Results showed that although present on the surface, α_vβ₃ was mostly detected in internal membrane systems including those of α-granules. Occasionally, platelet sections showed special vesicular structures covered by gold particles. These were often localized at the edge or immediately under the plasma membrane and their origin remains unclear. An internal pool of α_vβ₃ was confirmed by flow cytometry and by using platelets from a patient with type I Glanzmann's thrombasthenia arising from a GP IIb gene defect. We also investigated the presence of VnR in megakaryocytes (MK) obtained from normal human bone marrow. A fluorescence study showed VnR in small MK with unilobulated nuclei, suggesting that synthesis of this integrin occurs early during megakaryocytopoiesis. In mature cells, VnR expression had decreased relative to GP IIb–IIIa, although intracellular staining was present in EM and α-granules were again labelled.     

Redox regulation of apoptosis in interleukin-3-dependent haemopoietic cells: absence of alteration in both mitochondrial membrane potential (Δψm) and free radical production during apoptosis induced by IL3 withdrawal

Apoptosis in haemopoietic cells has been linked to the production of free radicals and reduction in mitochondrial transmembrane potential, Δψ_m. We have found that apoptosis in murine IL3-dependent haemopoietic cells induced by IL3 withdrawal lowers a principal source of anti-oxidant (reduced glutathione) but does not increase free radicals nor affect Δψ_m. Increased free radicals are, however, produced during IL3 signalling. Artificially increasing intracellular free radicals by depleting internal glutathione mimics IL3 signalling in that cells maintain shape and are transiently stimulated into S-phase in the absence of IL3. Conversely, depletion of free radicals by anti-oxidants accelerates apoptosis both in the presence and absence of IL3. Our studies suggest that redox conditions rather than free radicals or mitochondrial membrane potential per se may play a significant role in maintaining cell and replicative integrity, and in determining apoptosis in haemopoietic cells.     

Susceptibility of the different developmental stages of the asexual (schizogonic) erythrocyte cycle of Plasmodium chabaudi chabaudi to hyperimmune serum, immunoglobulin (Ig)G1, IgG2a and F(ab'')2 fragments

The mechanisms by which antibodies interfere with Plasmodium growth are still under debate. Characterizing the asexual erythrocyte stages susceptible to antibodies from hyperimmune individuals is therefore a relevant contribution to vaccine research. In this study, using a virulent and synchronous murine malaria parasite, Plasmodium chabaudi chabaudi AJ, we have shown that trophozoites and circulating schizonts are not the main targets for antibodies from hyperimmune serum. In drug-cured mice challenged with a high inoculum of ring-infected erythrocytes, parasitemias do not decline until the moment of erythrocyte rupture, suggesting that effector mechanisms operate immediately prior to reinvasion. Confirming these findings, treatment of primary-infected mice with hyperimmune serum inhibited the generation of new ring forms, but did not alter the numbers of schizont-infected erythrocytes, despite the fact that these cells were recognized by immunoglobulin (Ig)G antibodies. When these mice were treated with IgG1 or IgG2a purified from hyperimmune serum, both subclasses limited reinvasion, but IgG2a showed a stronger protective activity. The fact that Fc digestion decreases but does not abrogate protection suggests that both Fc-dependent and independent mechanisms participate in this process. Treatment with cobra venom factor did not interfere with the antibody-mediated protection, ruling out the participation of the complement system in both lysis and phagocytosis of merozoites or infected erythrocytes. Therefore, in mice suffering from P. c. chabaudi AJ malaria, merozoite neutralization seems to be a major mechanism of protection conferred by hyperimmune serum antibodies. However, FcgammaR-mediated interactions, or other mechanisms not yet defined, may also contribute to inhibit erythrocyte reinvasion.     

Characterization of the thromboxane synthase pathway product 12-oxoheptadeca-5(Z)-8(E)-10(E)-trienoic acid as a thromboxane A2 receptor antagonist with minimal intrinsic activity

Thromboxane synthase forms thromboxane (TX) A₂ and 12(S)-hydroxyheptadeca-5(Z)-8(E)-10(E)-trienoic acid (HHT) at equimolar amounts. Twelve-oxoheptadeca-5(Z)-8(E)-10(E)-trienoic acid (Oxo-HT) is the primary metabolite of HHT and has been described to be an inhibitor of platelet aggregation. Functional studies, Schild analysis and competitive binding studies were performed to clarify its mode of action. Oxo-HT was prepared biosynthetically as well as chemosynthetically, purified and characterized by gas chromatography and mass spectrometry. Platelet activation was assessed by determination of shape change, aggregation, fibrinogen binding and P-selectin expression using optical aggregometry and flow cytometry. Oxo-HT 0.1 n M to 50 μM did not induce platelet activation. Furthermore, it had no effect on platelet activation induced by thrombin, ADP or PAF. In contrast, Oxo-HT inhibited platelet aggregation, fibrinogen binding and P-selectin expression induced by U46619 in a competitive manner. Schild analysis for U46619-induced fibrinogen binding and P-selectin expression revealed pA₂ values of 6.1 and 6.6, respectively, which correspond to K_d values of approximately 0.8 μM and 0.3 μM , respectively. Oxo-HT also inhibited U46619 induced shape change (IC₅₀ ? 10 μM ). However, Oxo-HT over a concentration range of 0.1–1 μM enhanced the partial shape change induced by low concentrations of U46619. Thus Oxo-HT seems to possess a minimal agonistic potential, which alone is not sufficient to trigger a platelet activation but can enhance low levels of platelet activation. Oxo-HT blocked the binding of [³H]SQ 29548 in a concentration-dependent manner, whereas HHT did not displace [³H]SQ 29548. The K_d of Oxo-HT determined from competition binding studies was 7.7 μM , about 10–25-fold higher than the apparent K_d determined by Schild analysis. This discrepancy might be due to a desensitization of the TXA₂ receptor triggered by the minimal intrinsic activity of Oxo-HT. We conclude that Oxo-HT is a naturally occurring specific TXA₂ receptor antagonist with minimal intrinsic activity. Oxo-HT may contribute to the regulation of TXA₂-induced platelet activation in vivo.