Erythrocyte survival is promoted by plasma and suppressed by a Bak-derived BH3 peptide that interacts with membrane-associated Bcl-XL

Blood ◽  
2002 ◽  
Vol 99 (9) ◽  
pp. 3439-3448 ◽  
Author(s):  
Melanie Walsh ◽  
Robert J. Lutz ◽  
Thomas G. Cotter ◽  
Rosemary O'Connor
Keyword(s):  
Cell Death ◽  
Intracellular Calcium ◽  
Culture System ◽  
Caspase Inhibitor ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Erythrocyte Survival

Abstract Erythrocytes have a defined lifespan in vivo, and the signals that maintain their survival in circulation or trigger their death are unknown. Here, we investigated the control of erythrocyte survival and death in an in vitro culture system where erythrocytes survived for 10 days in serum-free medium in the presence or absence of bovine serum. Death of the cells in culture was correlated with increased exposure of phosphatidylserine and increased levels of intracellular calcium. Cell death could be suppressed by supplementing the medium with human plasma or serum, resulting in a doubling of the lifespan to 20 days. Freshly isolated erythrocytes and cultured erythrocytes were both found to express Bcl-XL and, to a lesser extent, Bak in membrane protein extracts. Treatment of the cells with a Bak-derived BH3 peptide fused to the internalization sequence of the antennapedia protein, which has previously been shown to enter cells by diffusion and antagonize Bcl-XL, resulted in substantial cell death in erythrocyte cultures. BH3-induced death was accompanied by an immediate increase in accumulation of intracellular calcium and could be suppressed by plasma, but not by the caspase inhibitor zVAD. A BH3 peptide mutated at amino acid 78 of full-length Bak required for heterodimerization with Bcl-XL had no effect on cell viability or calcium levels. We conclude that the BH3 peptide accelerates erythrocyte death through antagonization of Bcl-XL. The data suggest that erythrocyte survival is promoted by survival factors in plasma and by membrane-associated Bcl-XL.

scholarly journals The role of neutrophil membrane glycoprotein GP-150 in neutrophil adherence to endothelium in vitro

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 167-178 ◽  
Author(s):  
JM Harlan ◽  
PD Killen ◽  
FM Senecal ◽  
BR Schwartz ◽  
EK Yee ◽  
...  
Keyword(s):  
Calcium Ionophore ◽  
Membrane Glycoprotein ◽  
Ionophore A23187 ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Endothelial Monolayers ◽  
Neutrophil Adherence

Abstract We have previously described two patients with a congenital defect in neutrophil function characterized by an inability to form pus. The patients' neutrophils lack a membrane glycoprotein of mol wt 150,000 daltons (GP-150) on analysis by SDS-PAGE. This glycoprotein is part of a membrane antigen complex recognized by the murine monoclonal antibody (MoAb) 60.3. Addition of MoAb 60.3 to normal neutrophils produces defects in chemotaxis and phagocytosis in vitro similar to those observed in the patients. Since neutrophil adherence to vascular endothelium is prerequisite to neutrophil emigration in vivo, we examined the interaction of the patients' neutrophils and normal neutrophils treated with MoAb 60.3 with cultured endothelium. Adherence was determined as the percentage of 51Cr-labeled purified peripheral blood neutrophils which remained adherent to plastic wells or endothelial monolayers after a 45-minute incubation at 37 degrees C. The percentage of neutrophils from patient 1 remaining adherent to uncoated, fibronectin-coated, or laminin-coated plastic was similar to that observed in normal neutrophils (55% to 84% adherence with normal neutrophils v 73% to 78% adherence with the patient's neutrophils and 63% to 82% adherence with MoAb 60.3-treated normal neutrophils). The adherence of the neutrophils from patient 1 and MoAb 60.3-treated normal neutrophils to human or bovine endothelium in serum-free medium was also not significantly different from that observed in normal neutrophils (less than 10% adherence with normal, MoAb 60.3-treated, and patient neutrophils). In medium containing 10% autologous or heterologous human plasma, however, the adherence of neutrophils from patient 1 or MoAb 60.3-treated normal neutrophils to endothelial monolayers was significantly reduced (35% +/- 7% of normal neutrophils in seven experiments). Although phorbol myristate acetate (PMA) (10 ng/mL) and calcium ionophore A23187 (10(-5) mol/L) markedly increased the adherence of normal neutrophils to endothelial monolayers in serum- free medium (40% to 85% adherence), neither agent increased the adherence of the neutrophils from patient 1 or normal neutrophils treated with MoAb 60.3 (less than 5% adherence). The adherence of PMA- activated neutrophils from patient 2 to endothelial monolayers was also markedly decreased when compared with that of normal neutrophils. Postsecretory cell-free supernatants from PMA-activated normal neutrophils failed to augment adherence of neutrophils from patient 1 (less than 5% adherence).(ABSTRACT TRUNCATED AT 400 WORDS)

Oncolytic HSV and PI3K inhibitor BKM120 synergize to promote killing of prostate cancer stem-like cells.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e16503-e16503
Author(s):  
Lei Wang
Keyword(s):  
Prostate Cancer ◽  
Tumor Model ◽  
Complete Regression ◽  
Novel Therapies ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Anti Tumor Activity

e16503 Background: Novel therapies to override chemo-radiation resistance in prostate cancer (PCa) are needed. Prostate cancer sphere-forming cells (PCSCs) (also termed prostate cancer stem-like cells) likely participate in tumor progression and recurrence and are important therapeutic targets. Methods: We established PCSC-enriched spheres by culturing human (DU145) and murine (TRAMP-C2) PCa cells in growth factor-defined serum-free medium, and characterized stem-like properties of clonogenicity and tumorigenicity in vivo. The efficacy of two different oHSVs (G47∆ and MG18L) in PCSCs was tested alone and in combination with radiation, chemotherapy, and inhibitors of PI3K, Wnt, and NOTCH in vitro, and G47∆ with BKM120 in a PCSC-derived tumor model in vivo. Results: PCSCs were more tumorigenic than serum-cultured parental cells. Human and murine PCSCs were sensitive to oHSV and BKM120 killing in vitro, while the combination was synergistic. In contrast, oHSV combined with radiation, docetaxel, Wnt, or NOTCH inhibitors was not. In athymic mice bearing DU145 PCSC-derived tumors, combination intra-tumoral G47∆ and systemic BKM120 induced complete regression of tumors in 2 of 7 animals, and exhibited superior anti-tumor activity compared to either monotherapy alone, with no detectable toxicity. Conclusions: oHSV synergizes with BKM120 in killing PCSCs in vitro and the combination markedly inhibits tumor growth even inducing regression in vivo.

Cytotoxicity of the Type 4 Phosphodiesterase Inhibitor CD160130 for Freshly Isolated Human CLL Cells In Vitro.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3129-3129 ◽  
Author(s):  
J. Brice Weinberg ◽  
Ning Jiang ◽  
Alicia D. Volkheimer ◽  
Youwei Chen ◽  
Karen M. Bond ◽  
...  
Keyword(s):  
Cell Death ◽  
The United States ◽  
Pde4 Inhibitor ◽  
Community Control ◽  
Serum Free Medium ◽  
Free Medium ◽  
Normal Peripheral Blood ◽  
Serum Free ◽  
Normal Controls

Abstract B cell chronic lymphocytic leukemia (CLL), the most common subtype of leukemia in the United States of America and in Europe, is treatable but incurable. New drugs are needed for its management. Phosphodiesterase inhibitors (PDEi) block catabolism of cyclic nucleotides resulting in accumulation of cellular cAMP and cGMP. These PDEi also decrease production of inflammatory cytokines such as tumor necrosis factor. Furthermore, they inhibit expression of inducible nitric oxide synthase (NOS2) mRNA and NO production. Researchers have previously noted that nonspecific PDE inhibitors such as theophylline as well as a relatively specific PDE4 inhibitor (rolipram) cause CLL cell death in vitro, while relatively sparing normal peripheral blood mononuclear cells (PBMC). The purpose of this study was to evaluate the effectiveness of CD160130 in the killing of freshly isolated CLL cells in vitro. CD160130 is a novel, orally available heterocyclic pyrimido-indole derivative that is relatively PDE4-specific. Patients were from the V.A. and Duke University Medical Centers, and normal controls were from the community. Control normal PBMC were isolated by ficoll-Hypaque centrifugation, and CLL cells were purified using negative selection with antibodies. We determined cytotoxicity using the MTS colorimetric assay. Samples from 10 CLL patients (6 male and 4 female) and 10 normal controls were examined. Nine of 10 patients were stage 0 at presentation, and 1 was stage 2. They had been followed 6.1 yr (median; range 0.7–19.1 yr). One of 10 was CD38 positive, and 6 of 10 were Zap-70 positive. Of nine analyzed, one had unmutated IgVH gene, and 9 were mutated. Six patients had not been treated, and 4 had been treated with chlorambucil, fludarabine, and/or rituximab. Four had normal cytogenetics by FISH analysis; one had trisomy 12; three had 13q14 del; and one had 17p del. Of seven determined, two had elevated CLL cell lipoprotein lipase mRNA elevated. CD160130 induced cell death in a dose-dependent fashion in all patients’ samples, with a mean cytotoxicity of 96% at 12.5 uM. The mean ED50 for killing was 233 nM in media with FBS and 314 nM in serum-free medium, while that for PBMC was higher at 7500 nM with FBS and 5210 nM with serum-free medium. The agent was 32.2 fold more potent for killing of CLL cells compared to PBMC in medium with FBS and 16.6 fold more potent for CLL cells in serum-free medium. In summary, the PDE4 inhibitor CD160130 potently kills freshly isolated CLL cells in vitro in the presence or absence of serum. The killing is relatively selective for CLL cells compared to normal PBMC. In vivo trials of CD160130 in patients with CLL should help determine the toxicity and efficacy in patients.

A Serum-Free in vitro Culture System for Crayfish Organs

1986 ◽  
Vol 41 (4) ◽  
pp. 472-476 ◽  
Author(s):  
Gerd Gellissen ◽  
Marco Traub ◽  
Klaus-Dieter Spindler
Keyword(s):  
Culture System ◽  
Amino Acid Mixture ◽  
Acid Mixture ◽  
Serum Free Medium ◽  
Free Medium ◽  
Astacus Leptodactylus ◽  
Serum Free ◽  
In Vitro Culture System

Midgut gland and hypodermis of the crayfish Astacus leptodactylus have been cultured in a serum-free medium for several days. The medium consists of 1 part of van Harreveld solution and 1 part of an amino acid mixture supplemented with 1.2 mᴍ Na2HP04, 12 mᴍ Hepes and 80 mᴍ glucose. The antibiotics penicillin (15 mg/l) and streptomycin (25 mg/1) were added for long term culturing. This medium, called TG medium, allows the maintainance of the tissues for more than 100 h without any loss of their viability with respect to protein synthesis and secretion.

scholarly journals Ascorbic acid can promote the generation and expansion of neuroepithelial-like stem cells derived from hiPS/ES cells under chemically defined conditions through promoting collagen synthesis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rui Bai ◽  
Yun Chang ◽  
Amina Saleem ◽  
Fujian Wu ◽  
Lei Tian ◽  
...  
Keyword(s):  
Stem Cells ◽  
Culture System ◽  
Collagen Synthesis ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Induction System ◽  
Defined Culture

Abstract Introduction Spinal cord injury (SCI) is a neurological, medically incurable disorder. Human pluripotent stem cells (hPSCs) have the potential to generate neural stem/progenitor cells (NS/PCs), which hold promise in the treatment of SCI by transplantation. In our study, we aimed to establish a chemically defined culture system using serum-free medium and ascorbic acid (AA) to generate and expand long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) differentiated from hPSCs effectively and stably. Methods We induced human embryonic stem cells (hESCs)/induced PSCs (iPSCs) to neurospheres using a newly established in vitro induction system. Moreover, lt-NES cells were derived from hESC/iPSC-neurospheres using two induction systems, i.e., conventional N2 medium with gelatin-coated plates (coated) and N2+AA medium without pre-coated plates (AA), and were characterized by reverse transcription polymerase chain reaction (RT-PCR) analysis and immunocytochemistry staining. Subsequently, lt-NES cells were induced to neurons. A microelectrode array (MEA) recording system was used to evaluate the functionality of the neurons differentiated from lt-NES cells. Finally, the mechanism underlying the induction of lt-NES cells by AA was explored through RNA-seq and the use of inhibitors. Results HESCs/iPSCs were efficiently induced to neurospheres using a newly established induction system in vitro. lt-NES cells derived from hESC/iPSC-neurospheres using the two induction systems (coated vs. AA) both expressed the neural pluripotency-associated genes PAX6, NESTIN, SOX1, and SOX2. After long-term cultivation, we found that they both exhibited long-term expansion for more than a dozen generations while maintaining neuropluripotency. Moreover, the lt-NES cells retained the ability to differentiate into general functional neurons that express β-tubulin at high levels. We also demonstrated that AA promotes the generation and long-term expansion of lt-NES cells by promoting collagen synthesis via the MEK-ERK1/2 pathway. Conclusions This new chemically defined culture system was stable and effective regarding the generation and culture of lt-NES cells induced from hESCs/iPSCs using serum-free medium combined with AA. The lt-NES cells induced under this culture system maintained their long-term expansion and neural pluripotency, with the potential to differentiate into functional neurons. Graphical abstract

scholarly journals The role of neutrophil membrane glycoprotein GP-150 in neutrophil adherence to endothelium in vitro

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 167-178
Author(s):  
JM Harlan ◽  
PD Killen ◽  
FM Senecal ◽  
BR Schwartz ◽  
EK Yee ◽  
...  
Keyword(s):  
Calcium Ionophore ◽  
Membrane Glycoprotein ◽  
Ionophore A23187 ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Endothelial Monolayers ◽  
Neutrophil Adherence

We have previously described two patients with a congenital defect in neutrophil function characterized by an inability to form pus. The patients' neutrophils lack a membrane glycoprotein of mol wt 150,000 daltons (GP-150) on analysis by SDS-PAGE. This glycoprotein is part of a membrane antigen complex recognized by the murine monoclonal antibody (MoAb) 60.3. Addition of MoAb 60.3 to normal neutrophils produces defects in chemotaxis and phagocytosis in vitro similar to those observed in the patients. Since neutrophil adherence to vascular endothelium is prerequisite to neutrophil emigration in vivo, we examined the interaction of the patients' neutrophils and normal neutrophils treated with MoAb 60.3 with cultured endothelium. Adherence was determined as the percentage of 51Cr-labeled purified peripheral blood neutrophils which remained adherent to plastic wells or endothelial monolayers after a 45-minute incubation at 37 degrees C. The percentage of neutrophils from patient 1 remaining adherent to uncoated, fibronectin-coated, or laminin-coated plastic was similar to that observed in normal neutrophils (55% to 84% adherence with normal neutrophils v 73% to 78% adherence with the patient's neutrophils and 63% to 82% adherence with MoAb 60.3-treated normal neutrophils). The adherence of the neutrophils from patient 1 and MoAb 60.3-treated normal neutrophils to human or bovine endothelium in serum-free medium was also not significantly different from that observed in normal neutrophils (less than 10% adherence with normal, MoAb 60.3-treated, and patient neutrophils). In medium containing 10% autologous or heterologous human plasma, however, the adherence of neutrophils from patient 1 or MoAb 60.3-treated normal neutrophils to endothelial monolayers was significantly reduced (35% +/- 7% of normal neutrophils in seven experiments). Although phorbol myristate acetate (PMA) (10 ng/mL) and calcium ionophore A23187 (10(-5) mol/L) markedly increased the adherence of normal neutrophils to endothelial monolayers in serum- free medium (40% to 85% adherence), neither agent increased the adherence of the neutrophils from patient 1 or normal neutrophils treated with MoAb 60.3 (less than 5% adherence). The adherence of PMA- activated neutrophils from patient 2 to endothelial monolayers was also markedly decreased when compared with that of normal neutrophils. Postsecretory cell-free supernatants from PMA-activated normal neutrophils failed to augment adherence of neutrophils from patient 1 (less than 5% adherence).(ABSTRACT TRUNCATED AT 400 WORDS)

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