Mimosine, a non-protein amino acid, acts as a reversible inhibitor of DNA replication, and is widely used to synchronize cells at G1 phase of the cell cycle. We tested the possibility that mimosine might have an apoptotic effect on two types of AML cells: the monoblastic U-937 and the promyelocytic HL-60 cell lines. We show that mimosine induces apoptosis in both cell lines, with U-937 cells being more sensitive. The apoptotic effect of mimosine was antagonized by the addition of exogenous iron, indicating that it may act through iron chelation. Its mode of action was thus compared to that of desferrioxamine (DFO), a therapeutic iron chelating agent. Mimosine and DFO differed in their sensitivity to the suppressive effect of exogenous sources of iron in the form of hemin and ferrous sulfate suggesting different targets of action. Addition of another metal ion cupric sulfate was also able to antagonize the apoptotic effect of mimosine, undermining the notion that apoptosis is mediated through inhibition of ribonucleotide reductase, since this enzyme is solely dependent on iron for its activity. Moreover, when higher concentrations of iron were added to mimosine, cell death shifted from apoptosis to necrosis. Induction of apoptosis by both mimosine and DFO caused an early reduction in mitochondrial transmembrane potential and increase in caspase-3 activity, while only mimosine induced oxidative stress. In summary, our results imply that besides its known effect on DNA synthesis and G1 arrest, mimosine also activates apoptosis through an intrinsic pathway as well as reactive oxygen species production and thus elicits its anticancer effect by multiple pathways.

The antileukemic activity of nonsteroidal antiestrogens was investigated. Tamoxifen, clomiphene and nafoxidine caused a decrease in viability of the estrogen receptor-negative T-lymphoblastic leukemia cell line CCRF/CEM, nafoxidine being the most active. A combination of clomiphene and genistein resulted in a synergistic cytotoxic effect when applied to Molt-3, another T-lymphblastic leukemic cell line. The antiestrogens arrested the cells at G(0)/G(1) phase and induced apoptosis. Using the CCRF/VCR(1000) cell line, which is resistant to vincristine, it was observed that the effect of nafoxidine on modulating drug resistance was manifested at a lower concentration than that causing a direct cytotoxic effect. Nafoxidine inhibited the Pgp pump activity as measured by rhodamine 123 efflux. Combination with verapamil was found to be more effective in abrogating the pump activity. This study points to the multifactorial activities of nonsteroidal antiestrogens against lymphoblastic leukemia and implies their potential use in clinical treatment as antileukemic drugs.

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 I-125-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. (C) 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc.

This study investigates the possible involvement of serine proteases in interferon-gamma (IFN-gamma) activity on WISH cells. It was observed that inhibition of (3)H-thymidine incorporation induced by IFN-gamma was abrogated by the serine protease inhibitors Nalpha-tosyl-L-lysyl-chloromethane and soybean trypsin inhibitor, both of which act mainly on trypsin. Phenylmethyl sulfonyl fluoride also had a partial inhibitory effect. Other protease inhibitors specific to the cysteine, the aspartic, and the metalloprotease families were not effective. Kinetic analysis revealed that a trypsin-like protease is involved in IFN-gamma activity for up to 7 h. Trypsin-like activity induced by IFN-gamma was detected in the particulate fraction but not in the cytosolic fraction, whereas chymotrypsin activity was not enhanced in either the cytosolic or particulate fractions under similar conditions. Following separation on a gelatin substrate gel, two trypsin-like protease activities located in the particulate fraction were found to increase in response to IFN-gamma treatment. Hence, it seems that a specific membrane-associated trypsin-like protease activity induced by IFN-gamma may play a role in the action of the cytokine on thymidine incorporation in WISH cells.

Conference Abstract| October 01 2000 Involvement of a TPCK-Sensitive Enzyme in Cell Death Induced in a Monocytic Cell line A. Lichstenstein; A. Lichstenstein 1Department of Clinical Biochemistry, Faculty of Health Sciences and Soroka Medical Center, Ben-Gurion University, Beer Sheva, Israel Search for other works by this author on: This Site PubMed Google Scholar J. Mazar; J. Mazar 1Department of Clinical Biochemistry, Faculty of Health Sciences and Soroka Medical Center, Ben-Gurion University, Beer Sheva, Israel Search for other works by this author on: This Site PubMed Google Scholar A. Dvilansky; A. Dvilansky 1Department of Clinical Biochemistry, Faculty of Health Sciences and Soroka Medical Center, Ben-Gurion University, Beer Sheva, Israel Search for other works by this author on: This Site PubMed Google Scholar I. Nathan I. Nathan 1Department of Clinical Biochemistry, Faculty of Health Sciences and Soroka Medical Center, Ben-Gurion University, Beer Sheva, Israel Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (2000) 28 (5): A378. https://doi.org/10.1042/bst028a378a Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation A. Lichstenstein, J. Mazar, A. Dvilansky, I. Nathan; Involvement of a TPCK-Sensitive Enzyme in Cell Death Induced in a Monocytic Cell line. Biochem Soc Trans 1 October 2000; 28 (5): A378. doi: https://doi.org/10.1042/bst028a378a Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu nav search search input Search input auto suggest search filter All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 2000 Biochemical Society2000 Article PDF first page preview Close Modal You do not currently have access to this content.

The antitumoral activity of non-steroidal antiestrogens on promyelocytic leukemia HL60 and T lymphoblastic MOLT3 cell lines was studied. Tamoxifen and its derivatives, clomiphene and nafoxidine, caused reduction of cell viability in a dose-dependent manner. These drugs showed differences in their potency following four days incubation, with nafoxidine being the most efficient inhibitor and tamoxifen the least active. Apoptosis was induced as assessed by the DNA ladder pattern and formation of pre G0/G1 population as detected by flow cytometry analysis of DNA. The effect of these drugs was abrogated by antioxidants: alpha-tocopherol was most effective in antagonizing the drugs' effect. N-acetyl L-cysteine reversed mainly the decrease in cell viability caused by the drugs, but was less active on induction of apoptosis. GF109203X, a protein kinase inhibitor, attenuated apoptosis induced by clomiphene in MOLT3 cells. The results suggest that the antileukemic activity of the antiestrogens is mediated by oxidative stress and protein kinase C (PKC) activation. Triphenylethylene antiestrogens and their derivatives may be used as antileukemic drugs which kill cells by apoptosis mediated by oxidative stress and activation of PKC.

The effect of various differentiation inducers on membrane cell dynamics was studied using HL-60 and K562 leukemic cell lines. Membrane lipid dynamics was measured by the steady-state fluorescence polarization (P) method utilizing either 1,6-diphenyl-1,3,5-hexatriene (DPH) or the trimethyl ammonium derivative of DPH (TMA-DPH), which ascertains anchorage of the label to the membrane-water-lipid interface. Decrease in membrane microfluidity was observed in HL-60 cells undergoing differentiation into macrophages by 1,25-dihydroxyvitamin D3 and by K562 cells induced to differentiate by DMSO. Sodium butyrate caused an increase in membrane fluidity in K562 cells undergoing differentiation into erythroid-like cells while in HL-60 cells a dual effect was observed. At 0.4 mM concentration, in which the cells were induced to differentiate along the monocyte pathway, a decrease in membrane fluidity was observed, while at 1 mM concentration an increase in membrane fluidity occurred. Interferon-gamma (IFN-gamma) induced an increase in membrane fluidity in both cell lines. Using HL-60 cells fluorescently labeled by TMA-DPH, similar results indicating fluidization of the membrane following IFN-gamma treatment were obtained. Advanced fluorescence lifetime measurements, evaluated either by phase modulation spectrofluorometry or by single photon correlation fluorometry confirmed that the decrease in fluorescence polarization by IFN-gamma resulted from membrane fluidization and not from elongation of the probe's excited state lifetime. It is suggested that the inducer mode of action, and not the differentiation route, determine the outcome of changes in membrane microviscosity.

Aging and Alzheimer's disease (AD) have been the subject of many studies. It has been suggested that chromosomal alterations may be involved in the etiology and/or pathogenesis of ageing and AD. The purpose of the present study was to examine the effect of diepoxybutane (DEB) on lymphocyte chromosomal instability in the elderly. We examined lymphocytes cytogenetically with, as well as, without DEB treatment, in a group of 12 elderly (range of age 72-96 years), nine of them suffering from AD type. Without DEB treatment six of the donors expressed chromosomal instability in at least 6% of the analyzed cells. After treatment with DEB, lymphocytes showed an increase in the chromosomal instability in up to 20% of the analyzed in eight donors. The sex chromosomes were the main chromosomes involved in the acquired chromosomal abnormalities. It is not clear from this study whether this chromosomal instability is related to the AD. The significance of the involvement of sex chromosomes either in ageing or in AD, as well as, the question whether the chromosomal instability is the cause of or part of ageing processes, has to be addressed.

A fluorescent analog of human recombinant interferon-gamma (IFN-gamma) was prepared for the first time. The recovered pyrene-labeled IFN-gamma (py-IFN-gamma), with an estimated seven pyrene molecules per IFN-gamma, retained over half of its original biological activity. Binding of py-IFN-gamma to human amnion WISH cells showed appreciable enhancement in fluorescence polarization from 0.055 to 0.215 and in fluorescence lifetime from 56 to 80 ns. The ratio of the vibronic peaks did not change, indicating that the pyrene molecules remained in water environment even after binding. Py-IFN-gamma provides a novel tool for unraveling the mechanism of the initial interaction between this antiproliferative lymphokine and its target, cancer cell membrane receptors. Its fluorescence could provide the means to follow receptor recycling when it occurs.

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. © 1997 Wiley‐Liss, Inc.

Ionizing radiation is currently used for prevention of transfusion associated graft versus host disease (TAGVHD). As radiation damage is associated with the production of activated oxygen species, the aim of this study was to observe the immediate effect of ionizing radiation on red cell membrane and intracellular oxidative defense systems. Neonatal and iron deficiency (IDA) cells, known for their increased sensitivity to oxidative stress, were chosen and compared with normal cells. Irradiation was performed in doses of 1500 cGy, 3000 cGy and 5000 cGy. GSH and methemoglobin levels and the activity of different antioxidant enzymes, measured under optimal in vitro conditions, were preserved in all cells after irradiation. Only radiation at the highest does of 5000 cGy, caused significant potassium leakage in neonatal cells and insignificant increase in IDA cells. Thus, cells with increased sensitivity to oxidative stress are more susceptible to damage by ionizing radiation than normal cells.

American Journal of HematologyVolume 52, Issue 2 p. 121-121 Letters and CorrespondenceFree Access Factor XI deficiency in a Bedouin family Lev Hatskelzon, Corresponding Author Lev Hatskelzon Department of Hematology and Department of Gynecology and Obstetrics, Soroka Medical Center, Ben-Gurion University of the Negev, Beer-Sheva, IsraelDepartment of Hematology and Department of Gynecology and Obstetrics, Soroka Medical Center, Ben-Gurion University of the Negev, Beer-Sheva, IsraelSearch for more papers by this authorA. Dvilansky, A. Dvilansky Department of Hematology and Department of Gynecology and Obstetrics, Soroka Medical Center, Ben-Gurion University of the Negev, Beer-Sheva, IsraelSearch for more papers by this authorGershon Holcberg, Gershon Holcberg Department of Hematology and Department of Gynecology and Obstetrics, Soroka Medical Center, Ben-Gurion University of the Negev, Beer-Sheva, IsraelSearch for more papers by this author Lev Hatskelzon, Corresponding Author Lev Hatskelzon Department of Hematology and Department of Gynecology and Obstetrics, Soroka Medical Center, Ben-Gurion University of the Negev, Beer-Sheva, IsraelDepartment of Hematology and Department of Gynecology and Obstetrics, Soroka Medical Center, Ben-Gurion University of the Negev, Beer-Sheva, IsraelSearch for more papers by this authorA. Dvilansky, A. Dvilansky Department of Hematology and Department of Gynecology and Obstetrics, Soroka Medical Center, Ben-Gurion University of the Negev, Beer-Sheva, IsraelSearch for more papers by this authorGershon Holcberg, Gershon Holcberg Department of Hematology and Department of Gynecology and Obstetrics, Soroka Medical Center, Ben-Gurion University of the Negev, Beer-Sheva, IsraelSearch for more papers by this author First published: June 1996 https://doi.org/10.1002/(SICI)1096-8652(199606)52:2<121::AID-AJH11>3.0.CO;2-KCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume52, Issue2June 1996Pages 121-121 ReferencesRelatedInformation

American Journal of HematologyVolume 52, Issue 4 p. 328-329 Letters and CorrespondenceFree Access Translocation T(4;21) associated with the Pelger-Hüet anomaly in a patient with Ph chromosome-negative CML Tikva Yermiahu, Corresponding Author Tikva Yermiahu Hematology Laboratory, Faculty of Health Sciences, Ben-Gurion University of the Negev-Soroka Medical Center, Beer Sheva, IsraelHematology Laboratory, Faculty of Health Sciences, Ben-Gurion University of the Negev-Soroka Medical Center, Beer Sheva, IsraelSearch for more papers by this authorAlexander Dvilansky, Alexander Dvilansky Hematology Laboratory, Faculty of Health Sciences, Ben-Gurion University of the Negev-Soroka Medical Center, Beer Sheva, IsraelSearch for more papers by this authorEsther Manor, Esther Manor Hematology Laboratory, Faculty of Health Sciences, Ben-Gurion University of the Negev-Soroka Medical Center, Beer Sheva, IsraelSearch for more papers by this author Tikva Yermiahu, Corresponding Author Tikva Yermiahu Hematology Laboratory, Faculty of Health Sciences, Ben-Gurion University of the Negev-Soroka Medical Center, Beer Sheva, IsraelHematology Laboratory, Faculty of Health Sciences, Ben-Gurion University of the Negev-Soroka Medical Center, Beer Sheva, IsraelSearch for more papers by this authorAlexander Dvilansky, Alexander Dvilansky Hematology Laboratory, Faculty of Health Sciences, Ben-Gurion University of the Negev-Soroka Medical Center, Beer Sheva, IsraelSearch for more papers by this authorEsther Manor, Esther Manor Hematology Laboratory, Faculty of Health Sciences, Ben-Gurion University of the Negev-Soroka Medical Center, Beer Sheva, IsraelSearch for more papers by this author First published: August 1996 https://doi.org/10.1002/(SICI)1096-8652(199608)52:4<328::AID-AJH18>3.0.CO;2-9Citations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume52, Issue4August 1996Pages 328-329 ReferencesRelatedInformation

The roles of protein phosphorylation and dephosphorylation in the tumor necrosis factor (TNF) cytotoxic and antiproliferative effects on L-929-transformed fibroblasts were explored, Genistein and erbstatin, specific inhibitors of tyrosine kinase, had antiproliferative but not cytotoxic effects on the cells by themselves and synergistically enhanced the cytotoxic and antiproliferative effects of TNF-alpha. Immunoblot analysis with a monoclonal antiphosphotyrosine antibody revealed that TNF, administered for 5-180 min, induced tyrosine dephosphorylation of two pairs of membranal proteins, 34-36 kDa and 50-52 kDa, and potentiated tyrosine phosphorylation of a 115-kDa protein in both the cytosolic and membranal fractions of the cells. A very brief exposure (30 sec) to TNF induced rapid phosphorylation of several proteins, whereas genistein, but not inhibitors of other protein kinases, enhanced this effect of TNF, The results suggest that TNF activity could be potentiated by the inhibition of tyrosine phosphorylation and point to specific proteins that are dephosphorylated on tyrosine in response to TNF.

The effect of various tyrosine protein kinase inhibitors on processes involved in the antiproliferative effect of interferon-gamma on WISH cells was studied. Following 24 hr treatment interferon-gamma inhibited thymidine incorporation into DNA and thymidine kinase activity, but no significant effect on cell number was observed. The isoflavonoid, genistein, which is a specific inhibitor of tyrosine protein kinase, reversed the inhibition in thymidine incorporation caused by the cytokine in a dose dependent manner. Prunetin, a member of the same group, did not significantly antagonize this effect. N alpha-tosyl-L-lysyl-chloromethane, a serine protease inhibitor which also serves as a tyrosine protein kinase inhibitor, partially reversed the effect of interferon-gamma at a concentration of 100 microM. The bioflavonoid, quercetin, a non-specific tyrosine protein kinase inhibitor, at a concentration of 30 microM completely abolished the action of interferon-gamma on thymidine incorporation. Genistein completely reversed the inhibition of thymidine kinase exerted by interferon, while quercetin had only a slight effect. However, the drugs could not antagonize the antiproliferative effect of interferon following 48 hr incubation, as measured by reduction of cell number. The results indicate that tyrosine protein kinase may play a role in the effects of interferon on thymidine metabolism and thymidine kinase activity. The differential effects of the inhibitors on thymidine metabolism and cell proliferation could support dissociation between the effect of interferon-gamma on these processes. Alternatively, this dissociation of effects could point to the limited use of inhibitors in clarifying modes of action as described.