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Zbigniew Darzynkiewicz

Publications and source records attributed to Zbigniew Darzynkiewicz.

At least 55 records · Page 3Linked to original sources
PubMed2004

Simple, semiautomatic assay of cytostatic and cytotoxic effects of antitumor drugs by laser scanning cytometry: effects of the bis-intercalator WP631 on growth and cell cycle of T-24 cells.

BACKGROUND: Common assays of drug-induced cytotoxicity on adherent cells rely on cell trypsinization followed by count of live and dead cells. To estimate the cell cycle effects, cellular DNA content is analyzed by flow cytometry. This procedure is laborious and time consuming. The alternative viability assays, e.g., based on reduction of 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide, although rapid and convenient, do not provide information about individual cells or cell cycle effects and may be biased by growth imbalance. METHODS: The bladder carcinoma T-24 cells were seeded onto eight-chamber microscope slide-based tissue culture vessels. The novel antitumor drug, the bis-intercalating anthracycline WP631, was administered at various concentrations to different chamber cultures on the same slide; the control cultures were left untreated. After 24, 48, and 72 h, the cultures were fixed, and cellular DNA was stained with 4,6-diamidino-2-phenyl indole (DAPI). The slides were scanned by laser scanning cytometry (LSC) to obtain the number of attached cells per culture chamber and reveal their cell cycle distribution. RESULTS: The cell growth and viability plots in the absence and presence of WP621 were constructed from the frequency of the attached cells per chamber. A 50% reduction in cell number was observed at the 75 nM concentration of WP321. Mitotic and postmitotic cells were identified based on high intensity of maximal pixel of DAPI fluorescence. An increase in proportion of cells in G2 was seen at 75-300 nM of WP631. Relatively few (<12%) apoptotic cells, identified by the presence of DNA strand breaks, remained attached in the WP631-treated cultures. CONCLUSIONS: Because late apoptotic cells detach during culturing, the cells that remain attached in the multi-chamber cultures represent predominantly live cells; the deficit in their number compared with the untreated cultures, recorded by LSC during scanning, provides information about the degree of cytostatic and cytotoxic effects of the studied drug. The possibility to demonstrate the cell cycle distribution, including a distinction between G2 and M cells, provides an additional advantage of this assay. Other parameters that may be associated with the cell cycle perturbation or with induction of apoptosis also can be measured in the same cultures by using the multiparameter capabilities of LSC. Each measured cell can be relocated for imaging or measurement after subsequent staining with other probes.

Antineoplastic Agents↗
PubMed2004

Assessment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors topotecan and mitoxantrone and by the DNA cross-linking agent cisplatin.

BACKGROUND: DNA double-strand breaks (DSBs) in chromatin, whether induced by radiation, antitumor drugs, or by apoptosis-associated (AA) DNA fragmentation, provide a signal for histone H2AX phosphorylation on Ser-139; the phosphorylated H2AX is denoted gammaH2AX. The intensity of immunofluorescence (IF) of gammaH2AX was reported to reveal the frequency of DSBs in chromatin induced by radiation or by DNA topoisomerase I (topo 1) and II (topo 2) inhibitors. The purpose of this study was to further characterize the drug-induced (DI) IF of gammaH2AX, and in particular to distinguish it from AA gammaH2AX IF triggered by DNA breaks that occur in the course of AA DNA fragmentation. METHODS: HL-60 cells in cultures were treated with topotecan (TPT), mitoxantrone (MTX), or with DNA cross-linking drug cisplatin (CP); using multiparameter flow and laser-scanning cytometry, induction of gammaH2AX was correlated with: 1) caspase-3 activation; 2) chromatin condensation, 3) cell cycle phase, and 4) AA DNA fragmentation. The intensity of gammaH2AX IF was compensated for by an increase in histone/DNA content, which doubles during the cell cycle, and for the "programmed" H2AX phosphorylation, which occurs in untreated cells. RESULTS: In cells treated with TPT or MTX, the increase in DI-gammaH2AX IF peaked at 1.5 or 2 h, and was maximal in S- or G(1)-phase cells, respectively, for each drug. In cells treated with CP, compared with TPT, the gammaH2AX IF was less intense, peaked later (3 h) and showed no cell cycle-phase specificity. In the presence of phosphatase inhibitor calyculin A, a continuous increase in the TPT-induced gammaH2AX IF was still seen past 1.5 h, and after 3 h gammaH2AX IF was 2.7- to 3.4-fold higher than in the absence of the inhibitor. The AA gammaH2AX IF was distinguished from the DI-gammaH2AX IF by: 1) its greater intensity; 2) its prevention by caspase inhibitor zVAD-FMK; and 3) the concurrent activation of caspase-3 in the same cells. A decrease in AA gammaH2AX IF coinciding with AA chromatin condensation was seen in the late stages of apoptosis. CONCLUSIONS: Multiparameter analysis of gammaH2AX IF, caspase-3 activation, cellular DNA content, and chromatin condensation allowed us to distinguish the DI from AA H2AX phosphorylation and relate them to the cell cycle phase and stage of apoptosis. With a comparable degree of ds DNA breaks, the cells arrested at the G1 or G2/M checkpoint were less prone to undergo apoptosis than the cells replicating DNA. H2AX phosphorylation seen in CP-treated cells may be associated with DNA repair that involves nucleotide excision repair (NER) and nonhomologous end joining (NHEJ). When the primary drug-induced lesions do not involve ds DNA breaks, but ds DNA breaks are formed during DNA repair, as in the case of CP, analysis of H2AX phosphorylation may reflect extent of the repair process.

Antineoplastic Agents↗
PubMed2004

Histone H2AX phosphorylation induced by selective photolysis of BrdU-labeled DNA with UV light: relation to cell cycle phase.

BACKGROUND: The induction of DNA double-strand breaks (DSBs) in chromatin triggers histone H2AX phosphorylation (on Ser-139) by ATM-, ATR-, or DNA-dependent protein kinases (DNA-PK). Phosphorylated H2AX, denoted as gammaH2AX, can be detected immunocytochemically using an antibody that is specific to the Ser-139-phosphorylated epitope. We previously reported that the induction DSBs by DNA topoisomerase I or II inhibitors can be monitored in individual cells by measuring gammaH2AX immunofluorescence (IF) by cytometry. The present study explored whether the detection of gammaH2AX IF can serve as a marker of the presence of the DNA precursor bromodeoxyuridine (BrdU) that is incorporated into DNA. METHODS: HeLa cells growing on microscope slides were incubated with BrdU for 1 h, rinsed free of the precursor, and incubated for different periods for up to 12 h. The cells were then briefly incubated with Hoechst 33342 (to sensitize BrdU-labeled DNA to ultraviolet [UV] light), irradiated with 300 nm UV light to photolyze BrdU-labeled DNA, transferred back into culture for an additional hour, and fixed. Cells were concurrently immunostained for gammaH2AX (Alexa Fluor 633) and cyclin A (fluorescein isothiocyanate); their DNA was counterstained with 4,6-diamidino-2-phenylindole. The intensities of cellular far red (gammaH2AX), green (cyclin A), and blue (DNA) fluorescences were measured by laser scanning cytometry. RESULTS: After a 1-h pulse of BrdU followed by exposure to UV, nearly all cells with S-phase DNA content had many-fold higher gammaH2AX IF than G(1) or G(2)/M cells. The nonirradiated cells had minimal ("programmed") expression of gammaH2AX, whereas the irradiated cells incubated without BrdU had uniformly elevated levels of gammaH2AX IF independent of the cell cycle phase. Pulse-chase experiments showed that the cohort of BrdU-labeled (gammaH2AX-positive) cells progressed through G(2)/M and into G(1) phase after 8 and 12 h of growth in BrdU-free medium, respectively. Bivariate analysis of gammaH2AX versus cyclin A expression for the gated S-phase cells showed a correlation between these variables, suggesting that the rate of BrdU incorporation (DNA replication) correlates with expression of cyclin A. CONCLUSIONS: Photolysis of BrdU-labeled DNA induces DSBs and leads to H2AX phosphorylation. Detection of gammaH2AX IF indicates the presence of the incorporated BrdU, is compatible with concurrent detection of other intracellular antigens, and can be used to demonstrate cell cycle kinetics.

Benzimidazoles↗
PubMed2004

Analysis of cell cycle by flow cytometry.

Described are four widely used procedures to analyze the cell cycle by flow cytometry. The first two are based on univariate analysis of cellular DNA content following cell staining with either propidium iodide (PI) or 4',6'-diamidino-2-phenylindole (DAPI) and deconvolution of the cellular DNA content frequency histograms. This approach reveals distribution of cells in three major phases of the cycle (G1 vs S vs G2/M) and makes it possible to detect apoptotic cells with fractional DNA content. The third approach is based on the bivariate analysis of DNA content and proliferation-associated proteins. The expression of cyclin D, cyclin E, cyclin A, or cyclin B1 vs DNA content is presented as an example. This approach allows one to distinguish, for example, G0 from G1 cells, identify mitotic cells, or relate expression of other intracellular proteins to the cell cycle position. The fourth procedure relies on the detection of 5'-bromo-2'-deoxyuridine (BrdU) incorporation to label the DNA-replicating cells.

Apoptosis↗
PubMed2004

Treatment of Jurkat acute T-lymphocytic leukemia cells by onconase (Ranpirnase) is accompanied by an altered nucleocytoplasmic distribution and reduced expression of transcription factor NF-kappaB.

Onconase (Ranpirnase), a novel ribonuclease isolated from Rana pipiens oocytes, was reported to suppress cancer cell growth in vitro, reduce tumor size in animals, and augment cytotoxicity of several chemotherapeutic agents. Since onconase is currently in phase III clinical trials tested in treatment of mesothelioma, much emphasis has been placed on the mechanism of its anti-tumor activity. Previous studies have shown that onconase-responsive cells become arrested at the G1/S checkpoint of the cell cycle and also undergo apoptosis. A proposed mechanism for these effects is that the enzymatic activity of onconase targets cellular RNAs, in particular tRNA, with an accompanying inhibition of protein synthesis. In the present study, we have investigated the time- and dose-dependent effects of onconase on growth of Jurkat SN acute T-lymphocytic leukemia cells. Significant suppression of cell proliferation became evident after 72 and 96 h of treatment, and was most pronounced at the highest concentration (10 microg/ml; 8.3x10(-7) M) of onconase. This reduction of cell proliferation, however, was not accompanied by measurable changes in distribution of cells at different phases of the cell cycle, but was paralleled by the induction of apoptosis, as assayed by flow cytometry, and with a modest decrease in the expression of a cell cycle regulatory retinoblastoma protein (Rb). Further biochemical analysis revealed that growth suppression was closely coordinated with a down-regulation in the steady state and subcellular distribution of NF-kappaB, a transcription factor known to be functionally associated with cell survival. The reduction in expression of NF-kappaB by onconase appeared to coincide or even precede growth suppression, suggesting a causal relationship. To further test the hypothesis that cellular localization and expression of NF-kappaB may be critical to cellular response to onconase, we also studied the growth effects of onconase in Jurkat-BalphaM cells, which, unlike the parent SN T cells, contain a stably transfected dominant-negative IkappaB gene. Growth suppression by onconase in BalphaM cells was more pronounced and occurred earlier compared to SN cells, although still did not affect changes in cell cycle phase distribution. Contrary to expectation, however, diminution in NF-kappaB expression by onconase was even more pronounced in BalphaM cells, suggesting that this transcription factor, while presumably prevented from dissociation from its inhibitory protein IkappaB in these cells, is even more efficiently targeted for degradation by onconase. These results implicate NF-kappaB and its turnover as important determinants in the anti-proliferative/apoptotic effects of onconase in acute T-lymphocytic leukemia cells.

Antineoplastic Agents↗
PubMed2003

BUBR1 deficiency results in abnormal megakaryopoiesis.

The physiologic function of BUBR1, a key component of the spindle checkpoint, was examined by generating BUBR1-mutant mice. BUBR1(-/-) embryos failed to survive beyond day 8.5 in utero as a result of extensive apoptosis. Whereas BUBR1(+/-) blastocysts grew relatively normally in vitro, BUBR1(-/-) blastocysts exhibited impaired proliferation and atrophied. Adult BUBR1(+/-) mice manifested splenomegaly and abnormal megakaryopoiesis. BUBR1 haploinsufficiency resulted in an increase in the number of splenic megakaryocytes, which was correlated with an increase in megakaryocytic, but a decrease in erythroid, progenitors in bone marrow cells. RNA interference-mediated down-regulation of BUBR1 also caused an increase in polyploidy formation in murine embryonic fibroblast cells and enhanced megakaryopoiesis in bone marrow progenitor cells. However, enhanced megakaryopoiesis in BUBR1(+/-) mice was not correlated with a significant increase in platelets in peripheral blood, which was at least partly due to a defect in the formation of proplatelet-producing megakaryocytes. Together, these results indicate that BUBR1 is essential for early embryonic development and normal hematopoiesis.

Animals↗
PubMed2003

Heme oxygenase-1 attenuates glucose-mediated cell growth arrest and apoptosis in human microvessel endothelial cells.

Heme oxygenase-1 (HO-1) is a stress protein that has been suggested to participate in defense mechanisms against agents that may induce oxidative injury, such as heme and inflammatory molecules. Incubation of endothelial cells in a high-glucose (33 mmol/L) medium for 7 days resulted in a decrease of HO activity by 34% and a decrease in HO-1 and HO-2 proteins compared with cells exposed to low glucose (5 mmol/L) (P<0.05) or cells exposed to mannitol (33 mmol/L). Overexpression of HO-1 was coupled with an increase in HO activity and carbon monoxide synthesis, decreased cellular heme, and acceleration in all phases of the cell cycle (P<0.001). The rate of cell cycle or cell birth rate was increased by 29% (P<0.05) in cells overexpressing HO-1 but decreased by 23% (P<0.05) in cells underexpressing HO-1 compared with control cells. Exposure to high glucose significantly decreased cell-cycle progression in control cells and in cells underexpressing HO-1 but did not decrease cell-cycle progression in cells overexpressing HO-1. High glucose induced p21 and p27 in control cells but not in cells overexpressing HO-1. The addition of tin-mesoporphyrin (SnMP), an inhibitor of HO activity, reversed the HO-1-mediated decrease of p21 and p27 in cells overexpressing HO-1. These findings identify a novel effect of HO-1 on endothelial cell growth and indicate that heme metabolism and HO-1 expression regulate signaling systems in cells exposed to high glucose, which controls cell-cycle progression.

Apoptosis↗
PubMed2003

Dual functionality of cyclooxygenase-2 as a regulator of tumor necrosis factor-mediated G1 shortening and nitric oxide-mediated inhibition of vascular smooth muscle cell proliferation.

BACKGROUND: Cyclooxygenase (COX)-2 contributes to vascular smooth muscle cell (VSMC) proliferation induced by tumor necrosis factor (TNF) and angiotensin II. The present study demonstrates, however, that depending on prevailing conditions, COX-2-derived prostanoids may also inhibit VSMC proliferation. METHODS AND RESULTS: TNF-alpha stimulated proliferation of VSMCs by shortening the G1 phase of the cell cycle. This effect was abolished by NS-398, a selective COX-2 inhibitor. Addition of TNF did not affect the protein-to-DNA ratio, measured by flow cytometry, suggesting that TNF does not induce VSMC hypertrophy. Inhibition of nitric oxide synthase (NOS) activity attenuated TNF-mediated increases in prostaglandin (PG) I2 synthesis, whereas thromboxane (TX) A2 production and COX-2 protein expression were unaffected. Moreover, inhibition of NOS activity increased TNF-mediated proliferation by approximately 23%. Thus, NO preferentially stimulates PGI2 production, suggesting that production of NO by VSMCs challenged with TNF limits the ability of the cytokine to increase proliferation. NO donors increased COX-2 protein expression and PGI2 synthesis, had no effect on TXA2 production, and decreased cell numbers by 50%, indicating that expression of COX-2 per se might not be sufficient to support proliferation. The effects of NO donors were prevented when COX-2 activity was inhibited with NS-398. CONCLUSIONS: The COX-2-dependent proliferative response of VSMCs to TNF was modulated in an NO-dependent manner, and PGI2 derived from COX-2 might contribute to the antiproliferative effect of NO donors.

Animals↗
PubMed2003

Interactions of chlorophyllin with acridine orange, quinacrine mustard and doxorubicin analyzed by light absorption and fluorescence spectroscopy.

The present study was designed to estimate the ability of chlorophyllin (CHL) to interact with two acridine mutagens, quinacrine mustard (QM) and acridine orange (AO), and with the antitumor anthracycline doxorubicin (Dox). To this end, aqueous solutions of QM, AO or Dox during titration with CHL were subjected to spectrophotometry and spectrofluorimetry to detect possible interactions between these reagents. The data indicate that CHL forms complexes with AO, QM or Dox in these solutions. The presence of the complexes was manifested by a bathochromic shift of the absorption spectra, as well as by strong quenching of the fluorescence of each of these mutagens in the presence of CHL. CHL, thus, may serve as an interceptor of these mutagenic acridines in different in vivo or in vitro applications. Its ability to interact with Dox may potentially be utilized to detoxify patients overdosed with this or similar drugs.

Acridine Orange↗
PubMed2003

In vitro model of "wound healing" analyzed by laser scanning cytometry: accelerated healing of epithelial cell monolayers in the presence of hyaluronate.

BACKGROUND: In vitro models of "wound healing" rely on analysis of confluent cell cultures that are mechanically wounded, e.g., by scratching the cell monolayer. Damage and removal of cells during wounding provides mitogenic signals to the adjacent cells and induces their migration to close the wound. The progress of healing is generally estimated by microscopy or time-lapse cinematography by assessing cell proliferation and/or migration that leads to the wound closure. METHODS: The aim of the present study was to adapt laser scanning cytometry (LSC) to measure cellular changes related to damage and recovery of a monolayer of primary epithelial cells from rat kidneys growing with and without hyaluronate ( approximately 6 x 10(6) average molecular weight). Because x-y coordinates of the cell position on the slide were recorded by LSC, the apoptotic and proliferative changes in individual cells induced by wounding and wound closure could be correlated, by multiparameter analysis, with the cell location with respect to the wound. RESULTS: The initial change, observed as soon as 4 h after scratching and seen among the cells at the wound edge, was the appearance of apoptotic cells, characterized by cell shrinkage, typically condensed chromatin, and activation of caspases, the latter detected by binding of fluorochrome-labeled inhibitor of caspases. Their frequency was reduced to up to sixfold in the presence of hyaluronate. Cell proliferation, measured by frequency of cells incorporating bromodeoxyuridine, also reflected by percentage of cells in S, G(2), and mitosis, was higher in proximity of the wound but was not significantly affected by hyaluronate. However, the monolayer gap closure was accelerated in the presence of hyaluronate. CONCLUSIONS: By offering the means to measure apoptosis and proliferation in relation to the cell position (distance) with respect to the wound in cell monolayer and to relocate them for visual inspection, LSC is uniquely suited to quantitatively analyze in vitro the process of wound healing. Hyaluronate, the ubiquitous component of intercellular matrix, preparations of which are being used in the clinic to suppress inflammatory reactions in tissues and promote healing, accelerated the healing process by protecting cells from apoptosis and stimulating cell migration to close the gap in the cell monolayer.

Animals↗
PubMed2003

NF-kappaB inhibitor sesquiterpene parthenolide induces concurrently atypical apoptosis and cell necrosis: difficulties in identification of dead cells in such cultures.

BACKGROUND: Apoptosis and necrosis ("accidental cell death") are distinct modes of cell death. The feature that often distinguishes apoptotic from necrotic cells is preservation of the plasma membrane integrity, reflected by ability of the former cells to exclude cationic dyes such as propidium iodide (PI) for a certain length of time. During necrosis, the plasma membrane is rapidly ruptured and necrotic cells stain intensely with PI. While studying cytostatic effects of the anti-inflammatory sesquiterpene parthenolide (PRT), we have noticed that, concurrent with apoptosis, the cells were dying by necrosis in the same cultures. Furthermore, because apoptosis was atypical, reflected by rapid loss of plasma membrane integrity, it was difficult to distinguish apoptotic from necrotic cells based on this feature. METHODS: Three methods were used to distinguish apoptosis from necrosis: (a) HL-60 cells treated with PRT were subjected to analysis of caspases activation using antibody that detects activated (cleaved) caspase-3, (b) apoptotic cells were identified by binding of fluorochrome-labeled inhibitor of caspases FAM-VAD-FMK combined with the PI exclusion assay, and (c) cellular DNA and RNA were differentially stained with acridine orange (AO). RESULTS: Apoptotic cells were characterized by (a) caspase-3 activation detected immunocytochemically and (b) binding of FAM-VAD-FMK followed by or concurrent with (c) loss of ability to exclude PI, (d) deficit in DNA content, and (e) relatively little changed RNA content. Necrotic cells showed (a) no evidence of caspase-3 activation, (b) no binding of FAM-VAD-FMK, (c) inability to exclude PI, (d) rapid loss of RNA, and (e) unchanged DNA content. CONCLUSIONS: Identification of apoptotic cells versus necrotic cells was possible either based on the evidence of caspase-3 activation, by labeling with FAM-VAD-FMK combined with PI or by differential staining of cellular DNA and RNA with AO. The data indicate that plasma membrane appears to be one of the targets of PRT, because its integrity is lost very early during cell death, which is reflected by atypical apoptosis and by primary necrosis (lysis of the membrane).

Acridine Orange↗
PubMed2003

Caspase-mediated cell death in hematological malignancies: theoretical considerations, methods of assessment, and clinical implications.

Apoptosis, the caspase-mediated cell death, plays an important role in the etiology, pathogenesis and therapy of a variety of diseases. Abnormalities of apoptosis regulation, resulting in either its inhibition or enhancement, play a key role in the development of various malignant hematological disorders. Several routine and new therapeutic strategies in Oncohematology are based on apoptosis modulation. Cytotoxic effects of most antineoplastic drugs are based on induction of apoptosis. The accurate estimate of incidence of apoptosis, therefore, is of importance in Oncohematology. In this review we provide an overview of the methods designed to measure the incidence of apoptosis, including the recently developed assays that are based on detection of caspases activation. We also review recent findings on the role of caspase-mediated cell death in hematological malignancies and discuss their clinical implications, including new therapeutical strategies that evolve from these findings.

Animals↗
PubMed2003

DEVDase detection in intact apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)2-cresyl violet.

Using a bisubstituted caspase-3 target sequence: aspartate-glutamate-valine-aspartate, (z-DEVD)2 peptide derivative of the fluorophore, cresyl violet, we have obtained a cell permeant, fluorogenic, caspase substrate capable of detecting the site-specific presence of functionally active, caspase-3 and caspase-7 up-regulation within intact apoptotic cells. Addition of this substrate to induced and noninduced cell culture populations allows for the rapid site-specific detection of caspase up-regulation without the requirement for a wash step. We demonstrate here the use of (z-DEVD)2-cresyl violet substrate for the detection of apoptosis induction in Jurkat, THP-1, and MCF-7 cells using fluorescence microscopy and 96-well fluorescence plate reader analysis. Intracellular up-regulated DEVDase activity, which was clearly visible by fluorescence microscopy and 96-well fluorescence plate reader measurements, showed greater than 6-fold increases in fluorescence output in induced versus noninduced Jurkat cell samples. A simple fluorogenic substrate conversion method is demonstrated here for detecting apoptosis induction within intact living cells.

Apoptosis↗
PubMed2003

Direct evidence that Bcr-Abl tyrosine kinase activity disrupts normal synergistic interactions between Kit ligand and cytokines in primary primitive progenitor cells.

We previously reported that chronic myelogenous leukemia (CML) primitive granulocyte-monocyte (GM) progenitors have a greatly reduced requirement for kit ligand (KL) to achieve optimal growth with granulocyte colony-stimulating factor (G-CSF) + granulocyte-monocyte colony-stimulating factor (GM-CSF). Conversely, others have demonstrated that unlike normal, CML CD34+ progenitors can proliferate in response to KL as a sole stimulus. To address these seemingly paradoxical findings, we examined the growth responses of CML CD34+ GM progenitors to various cytokines with and without a potent inhibitor of Bcr-Abl tyrosine kinase activity, PD173955. The heightened growth responses of CML GM progenitors to KL alone and to G-CSF + GM-CSF were abrogated by 10 nM PD173955 while having no effect on normal GM progenitors. While normal GM progenitors exhibited the expected synergistic response when KL was added to G-CSF + GM-CSF, CML GM progenitors had a minimal response; however, some synergism was restored by 10 nM PD173955. Normal erythroid progenitors require the synergistic interaction between KL and a saturating amount of erythropoietin (EPO, 1 unit) for optimal growth. In contrast, CML erythroid progenitors had up to 50% of optimal growth in KL alone, and, only a subthreshold amount of EPO (0.1 unit) was needed with KL to achieve 85% of the optimal response; these heightened growth responses were largely abrogated by 10 nM PD173955. Thus, direct evidence is provided that constitutively activated Bcr-Abl kinase pathways in primitive CML progenitors cooperate with single growth factors producing a heightened growth response, and, in so doing, disrupt the normally required synergistic interactions between KL and other cytokines to achieve activation and optimal growth of primitive progenitors. Coupled with our previous findings that a larger than normal proportion of CML primitive progenitors are at a later stage of maturation, we propose that this disruption of normal synergistic responses leads to increased progenitor recruitment into a committed pool by a process of accelerated maturation.

Antigens, CD34↗
PubMed2003

Relative abundance and patterns of correlation among six cytokines in pleural fluid measured by cytometric bead array.

Several cytokines play significant roles in the development and pathogenesis of pleural effusion. Little is known, however, about possible interactions between individual cytokines in terms of regulation of their relative abundance in the effusion. We studied 93 patients presenting with pleural effusion to the National Sanyo Hospital (68 men and 25 women; mean age, 64 years). Twenty-two patients had tuberculous pleurisy, 40 had malignant pleuritis, and 31 had effusions due to an etiology other than tuberculosis or cancer (miscellaneous). Pleural fluid concentrations of IL-2, IL-4, IL-5, IL-10, TNF-alpha, and INF-gamma were simultaneously measured by cytometric bead array (CBA). The ratios of IL-4/IL-5, IL-4/TNF-alpha, IL-2/TNF-alpha, and IL-10/TNF-alpha were lower in patients with tuberculosis pleurisy compared with other patients. In all three groups of patients significant correlation was seen between abundance of IL-2 vs. IL-4, IL-5, IL-10, or TNF-alpha, between IL-4 vs. IL-10, and between TNF-alpha vs. INF-gamma. In malignant pleural fluid patients, the significant correlation was between IL-2 vs. IL-4, TNF-alpha, or INF-gamma, between IL-4 vs. INF-gamma, and between TNF-alpha vs. INF-gamma. In tuberculosis pleural fluid patients, the significant correlation was between IL-2 vs. TNF-alpha, between IL-4 vs. IL-10, and between TNF-alpha vs. INF-gamma. In miscellaneous pleural fluid patients, the significant correlation was between IL-2 vs. IL-4, IL-10, or TNF-alpha, between IL-4 vs. IL-10, TNF-alpha, and between IL-10 vs. TNF-alpha. No significant correlation was observed between other pairs of cytokines. Strong correlation in abundance between particular cytokines in pleural fluids suggests cross-talk between them, in terms that an altered level of one of them provides a feedback mechanism regulating synthesis and/or secretion of another one. Such interactions may play important roles in pathogenesis and severity of the effusion. The CBA methodology provides a convenient tool to investigate these interactions.

Adenosine Deaminase↗