Ilana Nathan

Serum Amyloid A, an Acute Phase Protein, Inhibits Platelet Activation

The effect of serum amyloid A, an acute phase protein, on platelet function was studied. Serum amyloid A was isolated and purified from sera of patients with recent trauma. Serum amyloid A inhibited thrombin-induced gel-filtered platelet aggregation. However, it did not inhibit aggregation induced by collagen or adenosine diphosphate, nor did it influence the aggregation of platelet-rich plasma activated with thrombin. Further studies of its effect on thrombin-induced activities showed that serum amyloid A, at concentrations of 25 to 100 micrograms/ml (which are found in mild acute events), suppressed the increase of cytosolic [Ca2+], thromboxane generation, and carbon 14-labeled serotonin release in a dose-dependent fashion. Serum amyloid A did not interfere with the clotting or amidolytic activities of thrombin. Therefore, the data suggest a protective role for serum amyloid A in thromboembolic disease by specific interaction with thrombin-induced platelet activation. Amyloid A protein also markedly inhibited thrombin-induced platelet aggregation. Because amyloid A is homologous to the N-terminal portion of serum amyloid A, the observed activity probably resides in that part of the molecule. This finding may be of importance in localization of the active site on serum amyloid A.

Appraisal of the MTT-based Assay as a Useful Tool for Predicting Drug Chemosensitivity in Leukemia

The MTT-based assay relies upon the cellular reduction of tetrazolium salts to their intensely colored formazans. The test is easy to perform in hematological malignancies and is adaptable for high throughput of samples, although there are some minor limitations in its application resulting from metabolic interference. This class of assays are highly accurate for predicting drug resistance, whereas their predictive value for drug sensitivity depends on the type of disease and drug or drug combination used. They have been found to predict clinical response to fludarabine FLD in B-CLL and were useful for predetermining clinical potential of a single drug or drug combination in AML patients. Extensive studies with ALL patients have supported their advantage for selecting effective drug treatment of the disease. To conclude, pretreatment chemosensitivity assays may help in the selection of chemotherapeutic drugs with the greatest likelihood for clinical effectiveness, and in the exclusion of uneffective therapy. This can lead to improved disease management, response, survival and use of financial resources.

Novel role of 1,25(OH)2D3 in induction of erythroid progenitor cell proliferation

OBJECTIVE Burst-forming unit erythroid and colony-forming unit erythroid growth in vitro is lower in studies of continuous ambulatory peritoneal dialysis patients than healthy controls. Burst-forming unit erythroid growth was potentiated by addition of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and normalized by erythropoietin (Epo) therapy, suggesting an interaction between Epo and 1,25(OH)(2)D(3) at the stem cell level. The objective of this study was to determine the mechanism by which 1,25(OH)(2)D(3) enhances the stimulatory effect of Epo on the growth of erythroid precursor cells. MATERIALS AND METHODS We examined the effect of 1,25(OH)(2)D(3) and Epo on stem cell proliferation. Proliferation of TF1 cells of erythroid origin was measured by the XTT method, 3[H] thymidine incorporation, and cell counting by trypan blue exclusion; cord blood (CB) stem cells were counted. Epo receptor (EpoR) quantitation was evaluated by 125I-Epo binding and Scatchard analysis, immunoprecipitation, and Western blotting. Expression of EpoR mRNA was measured by reverse transcriptase polymerase chain reaction. RESULTS The stem cell factor-dependent CB stem cells and the TF1 cells responded to Epo and 1,25(OH)(2)D(3) by increased proliferation, while their simultaneous addition potentiated cell proliferation in a synergistic manner (25.67% +/- 4.8% of Epo proliferation at day 10 for CB cells; p < 0.005). 1,25(OH)(2)D(3) produced an up-regulation of EpoR number in TF1 cells and increased the expression of EpoR mRNA (p < 0.01). CONCLUSIONS The increase in EpoR expression induced by 1,25(OH)(2)D(3) might explain the synergistic interaction between Epo and 1,25(OH)(2)D(3) in stem cells.

Diagnosis of Cell Death by Means of Infrared Spectroscopy

Fourier transform infrared (FTIR) spectroscopy has been established as a fast spectroscopic method for biochemical analysis of cells and tissues. In this research we aimed to investigate FTIRs utility for identifying and characterizing different modes of cell death, using leukemic cell lines as a model system. CCRF-CEM and U937 leukemia cells were treated with arabinoside and doxorubicin apoptosis inducers, as well as with potassium cyanide, saponin, freezing-thawing, and H(2)O(2) necrosis inducers. Cell death mode was determined by various gold standard biochemical methods in parallel with FTIR-microscope measurements. Both cell death modes exhibit large spectral changes in lipid absorbance during apoptosis and necrosis; however, these changes are similar and thus cannot be used to distinguish apoptosis from necrosis. In contrast to the above confounding factor, our results reveal that apoptosis and necrosis can still be distinguished by the degree of DNA opaqueness to infrared light. Moreover, these two cell death modes also can be differentiated by their infrared absorbance, which relates to the secondary structure of total cellular protein. In light of these findings, we conclude that, because of its capacity to monitor multiple biomolecular parameters, FTIR spectroscopy enables unambiguous and easy analysis of cell death modes and may be useful for biochemical and medical applications.

Release of platelet 5-hydroxytryptamine by plasma taken from patients during and between migraine attacks

&NA; Experiments were carried out in order to further delineate the pathophysiology of the fall of plasma 5‐hydroxytryptamine (5‐HT) during a migraine attack. Platelets from normal subjects were incubated with 14 C‐labelled 5‐HT, and the release of 5‐HT was measured following exposure of these platelets to plasma taken from migraine patients during an attack or at headache‐free intervals. Plasma taken during attacks released significantly more 5‐HT. It is concluded that factor (s) exist in the serum during migraine attacks, which can cause 5‐HT release from normal platelets. The identification of this factor may be important.

A molecular mechanism for mimosine-induced apoptosis involving oxidative stress and mitochondrial activation.

Mimosine, a non-protein amino acid, is mainly known for its action as a reversible inhibitor of DNA replication and, therefore, has been widely used as a cell cycle synchronizing agent. Recently, it has been shown that mimosine also induces apoptosis, as mainly reflected in its ability to elicit characteristic nuclear changes. The present study elucidates the mechanism underlying mimosine’s apoptotic effects, using the U-937 leukemia cell line. We now demonstrate that in isolated rat liver mitochondria, mimosine induces mitochondrial swelling that can be inhibited by cyclosporine A, indicative of permeability transition (PT) mega-channel opening. Mimosine-induced apoptosis was accompanied by formation of hydrogen peroxide and a decrease in reduced glutathione levels. The apoptotic process was partially inhibited by cyclosporine A and substantially blocked by the antioxidant N-acetylcysteine, suggesting an essential role for reactive oxygen species formation during the apoptotic processes. The apoptosis induced by mimosine was also accompanied by a decrease in mitochondrial membrane potential, cytochrome c release and caspase 3 and 9 activation. Our results thus imply that mimosine activates apoptosis through mitochondrial activation and formation of H2O2, both of which play functional roles in the induction of cell death.

Membrane dynamic alterations associated with activation of human platelets by thrombin.

Two fluorescent probes, N-carboxymethylisatoic anhydride, which binds to membrane proteins, and 1,6-diphenyl-1,3,5-hexatriene, a lipophilic label, have been used to follow membrane microenvironmental changes. Activation of human platelets by thrombin resulted in a simultaneous increase in values of fluorescence polarization (P) of both probes during the stages of shape change and secretion, which further increased during platelet aggregation. The similar pattern of changes in P for both probes indicates the interdependence of lipids and proteins in the activated platelet membrane.

1,25-Dihydroxyvitamin D-3 alters membrane phospholipid composition and enhances calcium efflux in HL-60 cells

1,25-Dihydroxyvitamin D-3 (1,25(OH)2D3) had direct effects on the HL-60 cell membrane. Treatment of HL-60 cells with 1,25(OH)2D3 for short time periods (2-4 hours) caused an increase in calcium efflux. This phenomenon was found to be unrelated to new protein synthesis since it was not inhibited in the presence of RNA and protein synthesis inhibitors. The treatment of the HL-60 cells with 1,25(OH)2D3 for four hours caused changes in their membrane phospholipid composition. The phosphatidylcholine:phosphatidylethanolamine ratio increased from 1.2 to 1.5. Thus the alteration in the phospholipid composition in the membrane induced by 1,25(OH)2D3 may be responsible for the changes in the permeability of the membrane to calcium ions.

Mechanism of the antitumoral activity of deferasirox, an iron chelation agent, on mantle cell lymphoma.

Abstract Mantle cell lymphoma (MCL) characterized by the t(11;14)(q13;q32) translocation, resulting in cyclin D1 overexpression, is one of the most challenging lymphomas to treat. Iron chelators, such as deferasirox, have previously been shown to exhibit anti-proliferative properties; however, their effect on MCL cells has never been investigated. We showed that deferasirox exhibited antitumoral activity against the MCL cell lines HBL-2, Granta-519 and Jeko-1, with 50% inhibitory concentration (IC50) values of 7.99 ± 2.46 μM, 8.93 ± 2.25 μM and 31.86 ± 7.26 μM, respectively. Deferasirox induced apoptosis mediated through caspase-3 activation and decreased cyclin D1 protein levels resulting from increased proteasomal degradation. We also demonstrated down-regulation of phosphor-RB (Ser780) expression, which resulted in increasing levels of the E2F/RB complex and G1/S arrest. Finally, we showed that deferasirox activity was dependent on its iron chelating ability. The present data indicate that deferasirox, by down-regulating cyclin D1 and inhibiting its related signals, may constitute a promising adjuvant therapeutic molecule in the strategy for MCL treatment.

DISTAMYCIN-A DERIVATIVES POTENTIATE TUMOR-NECROSIS-FACTOR ACTIVITY VIA THE MODULATION OF TYROSINE PHOSPHORYLATION

The cytotoxic activities of 2 novel distamycin‐A derivatives, FCE 24517 and FCE 25450A, alone and in combination with tumor‐necrosis factor‐α (TNF), were studied. Both drugs, especially FCE 25450A, analyzed extensively here, inhibited the growth of HL60 promyelocytic cells, and human SV80 and murine L929 transformed fibroblasts in a dose‐dependent manner. The growth‐inhibitory potential of sequential exposure to the distamycin‐A analogs and TNF was determined. A 4‐hr treatment of L929 fibroblasts with 100–1,000 ng/ml FCE 25450A, followed by 2 ng/ml TNF, resulted in a synergistic anti‐proliferative effect. The synergism of FCE 24517 with TNF was less profound. Experiments to elucidate the mechanism underlying the cooperation revealed that FCE 25450A pre‐treatment almost completely abolished the elevated tyrosine phosphorylation of a 137‐kDa and other membranal proteins and prevented the de‐phosphorylation of another protein band observed in L929 cells in the presence of TNF. FCE 25450A alone induced no changes in the phosphotyrosine profile of the cells. The effect of FCE 25450A was counteracted by the tyrosine‐phosphatase inhibitor orthovanadate. In parallel, the inhibitor also diminished the anti‐proliferative action of the FCE 25450A/TNF combination. These findings suggest that, beyond their cytotoxic effects as single agents, the distamycin derivatives increase the sensitivity of cells to TNF. This effect is governed via the inhibition of TNF‐induced tyrosine phosphorylation of specific proteins which are probably involved in the development of TNF resistance. Thus, protein de‐phosphorylation might provide an additional mechanism of action of these novel distamycin‐A‐derived drugs. Int. J. Cancer 72:810–814, 1997.