Regulated Phosphatidylserine Exposure on Platelets Mediates Fibrin Formation in Hemostasis and Thrombosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1645-1645
Author(s):  
Jialan Shi ◽  
Steven W. Pipe ◽  
Jan T. Rasmussen ◽  
Christian W. Heegaard ◽  
Gary E. Gilbert
Keyword(s):  
Flow Cytometry ◽  
Platelet Rich Plasma ◽  
Factor Xa ◽  
Annexin V ◽  
Vascular Wall ◽  
Fibrin Deposition ◽  
Exposure In Vivo

Abstract Thrombin-stimulated platelets support activity of phosphatidylserine (PS)-dependent blood coagulation reactions. However, only 2–6% of stimulated platelets expose sufficient PS to bind annexin V, leading to the supposition that procoagulant reactions are localized to the annexin V positive platelets and to microparticles shed by platelets. We hypothesized that thrombin-stimulated platelets expose sufficient PS to support the prothrombinase and factor Xase complexes but insufficient to meet the threshold for annexin V binding. We evaluated lactadherin, a PS-binding milk protein, as a reagent to detect platelet PS exposure. Thrombin or TRAP-treated platelets bound lactadherin with 3200 ± 700 sites/plt, but did not bind annexin V, as detected by flow cytometry. To confirm that lactadherin binding truly reported PS exposure, we performed activation experiments upon platelets loaded with 1-Palmitoyl-2- [6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-Glycero-3-Phospho-L-Serine (NBD-PS). Stimulation of platelets with 10 μM TRAP led to 8 ± 2 % PS exposure vs > 90% PS exposure for 10 μM A23187. PS exposure was maximal within 1 min of exposure to TRAP and was reversible with >70% of exposed PS re-internalized within 30 min. In situ platelet fibrin deposition was monitored utilizing a novel flow cytometry assay. Platelet rich plasma was recalcified and supplemented with 50 pM factor Xa, 100 μM GPRP. Platelet-bound fibrin, lactadherin and annexin V binding sites were measured at time intervals. Fibrin accumulated on lactadherin + platelets at a rate greater than or equal to the rate on platelet microparticles or annexin V + platelets. Lactadherin inhibited > 98% of prothrombinase and factor Xase activity on platelets while annexin V inhibition reached a plateau of ~ 80%. The localization and importance of regulated PS exposure in vivo was evaluated in mouse models. Anesthetized mice were injected intravenously with 1 μg of lactadherin and 1 μg annexin V. Three minutes after FeCl3 injury of exposed mesentery, mice were perfused with fixative. Immunohistochemistry showed veins edged with fibrin and decorated with platelets. Lactadherin co-localized with CD41+ platelets along the vascular wall but was less intense on platelets lodged within fibrin aggregates that extended into the vessel lumen and did not stain platelets in sequestered blood. Annexin V stained only scattered platelets and endothelial cells and did not correlate well to sites of fibrin deposition. To evaluate the hemostatic importance of platelet PS, 10 μg of lactadherin was injected intravenously prior to tail snip; bleeding volumes increased from 33 ± 29 μl to 128 ± 39 μl, n experimental = 8. Rose bengal/laser-induced carotid thrombosis delayed from 34 ± 9 min to 80 ± 20 min, n = 10 after 8–21 μg lactadherin. Four treated animals did not develop a thrombosis. In summary, use of lactadherin as a PS probe has revealed that the capacity for regulated PS exposure, at levels below the annexin V binding threshold, is a general platelet property and that low level PS exposure is sufficient to support thrombin and fibrin generation in vitro and in vivo. These results have implications about the mechanism through which PS exposure is regulated as well as for the potential value of PS exposure as a diagnostic or therapeutic target.

Microenvironment Regulation of IL23R/IL-23 Axis Drives Chronic Lymphocytic Leukemia (CLL) Progression

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 616-616
Author(s):  
Fortunato Morabito ◽  
Giovanna Cutrona ◽  
Anna Grazia Recchia ◽  
Marina Fabbi ◽  
Silvano Ferrini ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Biological Effects ◽  
Luciferase Reporter ◽  
Side Chain ◽  
Lymphoid Tissues ◽  
Stat3 Phosphorylation

Abstract Background : CLL displays a considerable degree of clinical heterogeneity, which is in part ascribable to clone-intrinsic biological features and that are also influenced by clone-extrinsic events related to the microenvironment. Among the dynamics-taking place within the CLL microenvironment, those finalized to the induction of an overly inflammatory milieu may significantly impact on the CLL natural history by hijacking the immunological microenvironment at the same time fostering clone fitness. IL-23 acts as a prototypical pro-inflammatory mediator representing a promising therapeutic target. We analyzed the ability of CLL cells to sense IL-23 through the IL-23R complex (consisting of IL12Rß1 and IL23R subunits) expression and correlated this feature with clinical outcome. Moreover, we investigated the synthesis of IL-23 within the CLL microenvironment, and tested the biological effects of the IL-23/IL-23R axis engagement and of its interference in vitro and in vivo. Methods : IL23R complex was detected by quadruple flow cytometry staining with CD19, CD5, IL23R, and IL12Rβ1 in prospectively enrolled CLL cases (O-CLL1 protocol, clinicaltrial.gov identifier NCT00917540). On human tissue specimens, lymph node and bone marrow samples from 16 CLL patients were selected for in situ immunolocalization analyses. NOD/Shi-scid/γcnull (NSG) mice were used for in vivo xenografts, in which activated autologous T cells (AAT), obtained by adding anti-CD3 and CD28 Dynabeads and rIL2 were co-injected with CLL cells. MiRNA analysis was performed by Agilent's Human V2 platform and by quantitative PCR. MirVANA microRNA mimics and inhibitors were purchased from Ambion, Inc. For 3'UTR luciferase reporter experiments, miRNA target reporter vectors were purchased from Origene. Results : By flow cytometry, circulating CLL cells of 281 cases variably expressed IL23R side chain while consistently lacking IL12Rß1 chain expression. The engagement of the uncoupled IL23R complex expression (i.e. IL23R but not IL12Rb1 expression) by IL23 did not activate downstream signaling pathways, such as the up-regulation of pSTAT3. The 3-year TTFT probability of patients with low IL23R expression (IL23R-low) was 91% as compared to 75% of IL23R-high cases [χ2 9.1, P=.003; HR=3.2, 95%CI (1.4-7.1)]; in a multivariate model, IL23R expression still remained independently associated with TTFT. We explored the potential control of IL23R expression in CLL cells by miRNA and found 15 miRNAs inversely associated with IL23R expression, five of which predicted as regulators (miRNA-146b-5p, miRNA-155, miRNA-324-5p, miRNA-532-3p and miRNA-630). Among these, miR-324-3p and miR-146b-5p were demonstrated to functionally regulate the expression of IL23R and IL12Rβ1 proteins in CLL cells, respectively. Within lymphoid tissues, in situ, CLL clones expressing IL23R side chain also showed expression of IL12Rß1, which varied according to the density of CD40L-expressing bystander elements suggesting a microenvironment-driven regulation of the IL-23R complex. To functionally test this hypothesis, CLL cells were co-cultured in the presence of NIH-3T3 transduced with CD40L or with AAT cells. A significant up-regulation was observed for both the IL12Rß1 and IL23R side chains, suggesting the environment co-stimulation as a mechanism of IL-23R complex regulation. Consistently, the IL-23R complex was upmodulated in CLL cells expressing IL-23R but not IL12Rß1, upon xenograft with autologous T cells into NOD-Scid mice. We then investigated the effect of IL-23R engagement by IL-23 in CLL cells and found that IL-23R activity correlated with CLL cell proliferation and survival in vitro via STAT3 phosphorylation. The trophic nature of IL-23-mediated stimuli over CLL cells was further demonstrated in vivo through the adoption of an anti-IL23p19 monoclonal antibody for clinical use, which proved to be effective in eradicating the xenografted CLL clone in the infiltrated tissues (spleen, liver and BM) by inhibiting proliferation and inducing apoptosis. Noteworthy, the therapeutic effect of IL-23 antagonism was demonstrated by histopathology, flow cytometry and BCR clonality. Conclusions : Overall, we demonstrated that IL-23/IL-23R axis is a novel microenvironment-regulated determinant in CLL pathobiology representing a strong prospect in disease prognostication and treatment. Disclosures No relevant conflicts of interest to declare.

Inhibition of Fas-mediated Apoptosis in Yac-1 Cell via Anti-Fas Ribozyme

2004 ◽  
Vol 36 (3) ◽  
pp. 199-205
Author(s):  
Min Zhang ◽  
Fang Liu ◽  
Wei He ◽  
Yong You ◽  
Ping Zou ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Proteolytic Activity ◽  
Caspase 3 ◽  
Hammerhead Ribozyme ◽  
Annexin V ◽  
In Vitro Transcription ◽  
Rt Pcr ◽  
Fas Mrna

Abstract To detect a new and more effective way against apoptosis mouse lymphomatic cell line-Yac-1 in which fas gene was expressed highly was used as a model for studying the effects of anti-Fas ribozyme on Fas-mediated apoptosis. A hammerhead ribozyme gene targeting the fas mRNA was synthesized and its in vitro transcription vector was constructed, which was transfected into Yac-1 cells using electroporation. Rz596 expression was detected using RT-PCR, and Fas expression in Yac-1 cells was detected using RT-PCR, Western blot and flow cytometry. After treated with anti-Fas antibody (JO2), Yac-1 cell viability was measured with MTT assay, caspase-3 proteolytic activity was detected, and cell apoptosis was measured according to annexin V apoptosis detecting kit. Anti-Fas ribozyme could cleave fas mRNA efficiently in vivo and in vitro. Fas expression in Yac-1 cells transfected with anti-Fas ribozyme was decreased remarkably and correlated with resistance to Fas-mediated apoptosis as determined by flow cytometry and caspase-3 proteolytic activity. Anti-Fas ribozyme was detected in cells transfected with pU6-RZ596 and pU6-dRZ596 and could remarkably decrease the Fas expression in Yac-1 cells, which made Yac-1 cells get rid of Fas-mediated apoptosis. Because of wide expression of fas in organs and tissues, our research was very useful for studying the inhibition of apoptosis of many organs and tissues in the future.

scholarly journals Platelet Storage Temperature Determines Recovery of GPVI-Function In Vivo

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 39-39
Author(s):  
Jeffrey Miles ◽  
Chomkan Usaneerungrueng ◽  
Ava M. Obenaus ◽  
Molly Y Mollica ◽  
Jake R Flynn ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Storage Temperature ◽  
Recovery Of Function ◽  
Platelet Rich Plasma ◽  
Integrin Activation ◽  
Platelet Storage ◽  
Platelet Aggregates ◽  
Contractile Forces

Background: Platelets (PLTs) are currently stored at 22°C (RT, room temperature) for clinical purposes. This approach ensures long circulation time but has numerous downsides, including limited storage time due to the risk of bacterial growth and increased costs due to bacterial testing or pathogen reduction processing. PLTs stored at 4°C were the standard of care in the 1960s and 1970s. In our previous study with healthy volunteers, we showed that humans who received cold-stored PLTs have a significantly weaker response to collagen (an agonist that acts predominantly via GPVI) compared to RT-stored PLTs. If and how cold-stored PLTs recover their function in vivo is poorly understood. Methods: We obtained human PLTs by an apheresis collection and sampled either at baseline (fresh) or after five days at RT or 4°C. To test the response to GPVI-dependent agonists, we stimulated platelet-rich plasma or washed PLTs with collagen and the GPVI-specific agonist convulxin (CVX) and tested for activated integrin and α-degranulation by flow cytometry. Platelet aggregation, in response to GPVI-dependent agonists, was tested by aggregometry. We checked for GPVI expression levels by flow cytometry and for signaling events downstream of GPVI by immunoblotting. To allow for recovery of function in vitro, we incubated either 4°C-stored, or RT-stored PLTs with fresh, platelet-depleted blood for 15min, and perfused the reconstituted whole blood through a microfluidic block and post device to quantify the contractile forces of platelet aggregates. Additionally, we performed platelet force measurements at the single cell level using a traction force microscopy approach. To validate a murine model of platelet storage and transfusion, we replicated functional studies in vitro by testing mouse PLTs for integrin activation and α-degranulation by flow cytometry. Platelet aggregation in response to collagen, CVX, and the GPVI-specific antibody JAQ-1 with crosslinking anti-IgG was also tested. To evaluate the platelet function after transfusion, we obtained whole blood from UbiC-GFP mice and isolated platelet-rich plasma followed by storage for 24 hours at either 4°C or RT. To allow tracking of stored PLTs in vivo, we transfused the UbiC-GFP PLTs into wild-type C57BL/6J mice and tested for integrin activation of endogenous and transfused PLTs. Results: In human PLTs, we found a significantly increased integrin response in 4°C-stored PLTs stimulated with collagen in flow cytometry studies in vitro. Similarly, the aggregation response of 4°C-stored PLTs to collagen was significantly increased compared to RT-stored PLTs in vitro. In line with these findings, we observed more PLCγ2 phosphorylation and Syk phosphorylation at baseline in 4°C-stored PLTs compared to RT-stored PLTs, suggesting more pre-activation downstream of GPVI. However, no differences in PLCγ2 phosphorylation or Syk-phosphorylation were found between RT and 4°C-stored PLTs after stimulation with CVX, and no significant differences in surface expression levels of GPVI were detected between RT and 4°C. Stored platelets in plasma showed superior function after 4°C-storage in aggregation and flow cytometry assays. In contrast, we found similar contractile forces of platelet aggregates when RT-stored or 4°C-stored PLTs were added to platelet-depleted fresh blood. Additionally, at the single cell level, we found a similar magnitude of platelet forces in RT-stored and 4°C-stored PLTs. Similar to human PLTs, mouse PLTs showed significantly more integrin activation, P-selectin exposure, and aggregation in 4°C-stored PLTs compared to RT. To test the recovery of function of stored mouse platelets in vivo, we transfused GFP-positive PLTs into GFP-negative wild-type mice. Contrary to our pre-transfusion results, we found a significantly lower integrin activation response to CVX in 4°C-stored platelets after transfusion, consistent with our previous results in healthy human volunteers. Summary: The in vivo recovery of function of stored PLTs is an underappreciated phenomenon in platelet storage biology, and most studies are solely based on functional in vitro data. Based on our post-transfusion results, storage temperature affects the ability to recover function in vivo significantly in human and mouse platelets. Whether these differences lead to differences in clinical outcomes needs to be investigated in clinical trials. Disclosures Sniadecki: Stasys Medical Corporation: Current equity holder in private company, Other: Co-founder; Curi Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

scholarly journals MicroRNA-29c Increases the Chemosensitivity of Pancreatic Cancer Cells by Inhibiting USP22 Mediated Autophagy

2018 ◽  
Vol 47 (2) ◽  
pp. 747-758 ◽  
Author(s):  
Limin Huang ◽  
Chaoquan Hu ◽  
Hui Cao ◽  
Xiaoliang Wu ◽  
Rongpin Wang ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Flow Cytometry ◽  
Annexin V ◽  
Luciferase Reporter ◽  
Pancreatic Cancer Cells ◽  
Reporter Assays ◽  
Direct Target ◽  
Induced Apoptosis

Background/Aims: Pancreatic cancer (PC) is an aggressive malignancy with a poor survival rate. Despite advances in the treatment of PC, the efficacy of therapy is limited by the development of chemoresistance. Here, we examined the role of microRNA-29c (miR-29c) and the involvement of autophagy and apoptosis in the chemoresistance of PC cells in vivo and in vitro. Methods: We employed qRT-PCR, western blot and immunofluorescence to examine the expression level of miR-29c, USP22 and autophagy relative protein. In addition, we used MTT assay to detect cell proliferation and transwell assay to measure migration and invasiveness. The apoptosis was determined using annexin V-FITC/PI apoptosis detection kit by flow cytometry. Luciferase reporter assays confirmed the relationship between USP22 and miR-29c. Results: miR-29c overexpression in the PC cell line PANC-1 enhanced the effect of gemcitabine on decreasing cell viability and inducing apoptosis and inhibited autophagy, as shown by western blotting, immunofluorescence staining, colony formation assays, and flow cytometry. Ubiquitin specific peptidase (USP)-22, a deubiquitinating enzyme known to induce autophagy and promote PC cell survival, was identified as a direct target of miR-29c. USP22 knockdown experiments indicated that USP22 suppresses gemcitabine-induced apoptosis by promoting autophagy, thereby increasing the chemoresistance of PC cells. Luciferase reporter assays confirmed that USP22 is a direct target of miR-29c. A xenograft mouse model demonstrated that miR-29c increases the chemosensitivity of PC in vivo by downregulating USP22, leading to the inhibition of autophagy and induction of apoptosis. Conclusions: Taken together, these findings reveal a potential mechanism underlying the chemoresistance of PC cells mediated by the regulation of USP22-mediated autophagy by miR-29c, suggesting potential targets and therapeutic strategies in PC.

The Activation of Fas Receptor by APO010, a Recombinant Form of Fas Ligand, Induces In Vitro and In Vivo Antimyeloma Activity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1515-1515 ◽  
Author(s):  
Enrique M. Ocio ◽  
Patricia Maiso ◽  
Mercedes Garayoa ◽  
Marc Dupuis ◽  
Atanasio Pandiella ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Fas Ligand ◽  
Annexin V ◽  
Recombinant Form ◽  
Caspase 8 ◽  
Fas Receptor ◽  
Healthy Donors

Abstract Background & Aims Fas receptor is expressed on the surface of many malignant cells and its activation represents a potentially relevant anticancer target. APO010 is a recombinant form of Fas Ligand with hexameric structure, which is currently being evaluated in Phase 1 clinical trials. In order to identify possible targeted indications, we tested the in vitro and in vivo anti-tumor efficacy of APO010 on multiple myeloma (MM) cells. Material & methods In vitro cytotoxicity was tested by MTT and Annexin V staining in 8 MM cell lines and PBMCs from 3 healthy donors. Other techniques used for mechanistic studies were propidium iodide uptake by flow cytometry, Western-blotting, BrdU uptake and gene expression profile analysis. The in vivo antimyeloma effect of APO010 was tested in a xenograft of human plasmocytoma in CB17-SCID mice. When tumors became palpable mice were randomized to receive APO010 15 μg/Kg ip × 5d/sem (n=7), APO010 5 μg/Kg ip × 5d/sem (n=8) or vehicle alone (n=8). Tumor volumes, clinical features and weight were monitored three times a week. Results Six of the 8 MM cell lines studied by MTT were highly sensitive to APO010 with IC-50 at 24h of 0.5–20 ng/ml (2.5–100 pM), whereas two were resistant (RPMI-8226 and OPM-1). This sensitivity was correlated with the expression of Fas receptor by flow cytometry. Activation of apoptosis was rapid (within two hours of incubation) with maximum effect at 10 hours, as determined by Annexin V staining. Interestingly, APO010 was not toxic against PBMCs (both resting and activated) from 3 healthy donors at doses effective against MM cell lines. The presence of the microenvironment, as simulated by the coculture of MM1S cells with IL-6, IGF-1 and BMSCs, was not able to abrogate the APO010 antimyeloma effect. The combination of APO010 with Doxorubicin and Bortezomib, and, to a less extent, with Melphalan and Lenalidomide, potentiated the efficacy of the drugs alone. Regarding the mechanism of action, APO010 antiproliferative activity is mediated through caspase dependent apoptosis (Annexin-V staining, and PARP, caspase-3, caspase-7, caspase-8 and caspase-9 cleavage) and is independent of variations on the cell cycle profile. In this sense, the presence of the pan-caspase or caspase-8 inhibitors (Z-VAD-FMK and Z-IETD-FMK respectively) were able to completely abrogate APO010-induced cell death. Treatment of MM1S cells with APO010 for just one hour induced changes in the expression of 52 genes, many of them implicated in regulation of transduction (n=16). Three of the 4 most upregulated genes were the 3 members of the nuclear receptor subfamily 4, group A (Nurr1, Nor1 and Nur77). Other upregulated transcription factors were members of the Fos/Jun family such as Jun, JunB or FosL2. Western-Blot studies revealed that APO010 also provoked cleavage of MCL-1 and BIM, a decrease of BID and an important downregulation of pAKT. In the in vivo studies, APO010 treatment inhibited tumor growth as compared with the control group (p=0.02) without differences among the two doses of APO010. No significant toxicity was observed regarding body weight loss or increase in liver enzymes. Conclusions These data show that Fas activation with APO010 induces in vitro and in vivo cytotoxicity in MM cell lines, mainly through transcriptional regulation. This study provides an initial rationale for the use of this compound for treatment of MM patients.

560 Alpha-tocopheryloxyacetic acid induces apoptosis of murine rhabdomyosarcoma in vitro while modulating innate and adaptive immune responses in vivo

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A594-A594
Author(s):  
Fernanda Szewc ◽  
Longzhen Song ◽  
Sean Rinella ◽  
Christopher Dubay ◽  
Emmanuel Akporiaye ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
T Cells ◽  
Tumor Growth ◽  
Live Cell Imaging ◽  
Control Diet ◽  
Annexin V ◽  
In Vivo Studies

BackgroundRelapsed pediatric sarcomas have a poor prognosis with no available curative options. Alpha-Tocopheryloxyacetic acid (a-TEA) is a redox-silent analog of alpha-tocopherol that induces apoptotic and immunogenic cell death of tumor cells at doses that are not harmful to healthy normal cells. In a first-in-human clinical trial, a-TEA was well tolerated in adults with advanced solid tumors (NCT02192346), but has not yet been studied in pediatric sarcoma. We used a murine model of rhabdomyosarcoma (M3-9-M RMS) to assess the in vitro and in vivo anti-tumor effects of a-TEA.MethodsIn vitro studies were performed on the M3-9-M RMS cell line to measure a-TEA-mediated apoptosis using flow cytometry (Annexin V+/7AAD+ cells) and live cell imaging (Annexin V+ cells). In vivo studies involved orthotopic implantation of luciferase+ M3-9-M tumor cells into syngeneic C57BL/6 recipients. Once tumors were palpable, mice were randomized to a control diet or a-TEA-supplemented chow for 21 days and evaluated for bioluminescence, tumor growth and overall survival. Gene expression of tumor-infiltrating and splenic T cells were analyzed by bulk RNA-Seq and flow cytometry respectively.ResultsM3-9-M RMS treatment with 2.5–100 uM a-TEA induced apoptosis in a dose-dependent manner within 24 hours (p < 0.05) as measured by flow cytometry and live cell imaging. In-vivo studies with the M3-9-M RMS mouse model showed that recipients of a-TEA chow had 30–40% reduced tumor growth (p<0.01) and bioluminescence (p<0.05), leading to prolonged survival (> 4 weeks) compared to recipients of matched control chow (p<0.05). Spleen cells isolated from a-TEA-fed tumor-bearing mice demonstrated increased levels of IFN??+ cells, CD4+ T-cells, Ki-67 proliferation, and decrease in splenic CD11b+ arginase-1+ (p<0.01) and PD-L1+ cells (p<0.05) compared to their counterparts on the control diet. Gene set enrichment analyses of excised RMS tumors after a-TEA treatment revealed increased gene expression of CD24, EP300, CXCR4, and c-Jun as compared to tumors from mice fed control chow.ConclusionsThese data indicate that a-TEA mediates apoptosis of RMS in vitro and suppresses in vivo tumor growth, leading to prolonged survival likely via enhanced activation of adaptive immunity through CD4+ T cells and suppression of innate immunity through regulation of myeloid cell subsets. Furthermore, a-TEA may have direct effects on tumor cell proliferation through EP300 and c-Jun as well as indirect effects on tumor growth by regulation of immune cell recruitment through CD24 and CXCR4 gene expression. Administration of a-TEA as a potential salvage treatment for RMS is warranted.AcknowledgementsThe study was supported by NIH TL1 TR002375 (FS), St. Baldrick’s Stand up to Cancer (SU2C) Pediatric Dream Team Translational Research Grant SU2C-AACR-DT-27-17, NIH/NCI R01 CA215461, American Cancer Society Research Scholar Grant RSG- 18-104-01-LIB, and the Midwest Athletes Against Childhood Cancer (MACC) Fund (CMC). SU2C is a division of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the scientific partner of SU2C. The contents of this article do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government.Ethics ApprovalThe University of Wisconsin-Madison Animal Care and Use Committee approved all protocols (M005915).

Mechanisms Underlying the Synergistic Interaction of Filanesib with Pomalidomide and Dexamethasone (FPD) in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1801-1801 ◽  
Author(s):  
Susana Hernández-García ◽  
Laura San-Segundo ◽  
Lorena González-Méndez ◽  
Montserrat Martín-Sánchez ◽  
Luis A Corchete ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Research Funding ◽  
Annexin V ◽  
Triple Combination ◽  
Advisory Committees ◽  
Cell Cycle Profile

Abstract Introduction: Filanesib (ARRY-520) is a novel inhibitor of the "kinesin spindle protein" (KSP), which has demonstrated efficacy in heavily pretreated patients with refractory MM, (Lonial et al, ASH 2013). Our preliminary studies demonstrated synergy with standard anti-MM agents, especially with pomalidomide and dexamethasone. This set the stage for a recently activated trial being run by the Spanish MM group investigating FPD in relapsed MM patients. In this abstract we investigate the mechanisms underlying the synergy of the combination. Methods: In vitro action of FPD was evaluated in MM cell lines by MTT assay, bioluminescence, Annexin V staining, cell cycle profile analysis and TMRE staining by flow cytometry. Synergy was quantified with the Calcusyn software. In vivo efficacy was assessed in a subcutaneous plasmacytoma model of MM1S in CB17-SCID mice (The Jackson Laboratory, Bar Harbor, ME, USA). The mechanism of action was analyzed by Western blot, flow cytometry, genomic techniques, immunohistochemistry and immunofluorescence techniques. Results: The triple combination of FPD resulted in clear synergy in multiple myeloma cell lines (MM1S, OPM2, and RPMI8226) with combination indices between 0.4-0.7, and abrogated the effect of the soluble cytokines IL-6 and IGF-I and the protective effect of the adhesion of plasma cells to BMSCs, HS-5 and TERT cells. FPD caused cell cycle arrest in G2/M and specific apoptosis of cells arrested in these proliferative phases (with apoptosis percentage of 5, 23, 58 and 88 for control, poma+dexa, filanesib and FPD, respectively) demonstrated by flow cytometry with DRAQ5 and Annexin-V. Thus, FPD and filanesib in monotherapy treatments induced a similar effect on the cell cycle profile (arrest in G2/M) with a concordant increase of cyclin B1 and phosphorylated Histone H3. Although a secondary increase of KSP protein levels would be expected, pomalidomide and dexamethasone induced a decrease of the levels of this protein, which was still present in the triple combination (FPD). This fact could be contributing to the potentiation observed with the combination. Attending to apoptosis mechanism, proapoptotic stimulus from the extrinsic and intrinsic apoptotic pathways were promoted by pomalidomide and dexamethasone and filanesib, and converged in the triple combination. In this regard, a decrease of MCL-1 (antiapoptotic protein) and a significant increase of the proapoptotic BCL2 family members of the intrinsic pathway like NOXA and BIMEL BIML, BIMS(this last one being the most potent proapoptotic isoform), tBID (extrinsic pathway) and Bax protein were observed. We confirmed that all these proteins were translocated into the mitochondria, resulting in a decrease of the mitochondrial membrane potential by TMRE, increase of permeability and a release of cytochrome C and AIF. These results were confirmed in vivo in a model of subcutaneous plasmacytoma in small (70 mm3) and large (2000 mm3) tumors. In this model we observed a significant reduction of tumor growth, which was correlated with a statistically significant improvement in survival. Changes induced by FPD in the gene expression profile were concordant with the in vitro results as several overexpressed genes belonging to the previous pathways were identified, such as spindle assembly checkpoint (CENP-E and CENP-F) and apoptosis (BCL2L11, gene that codifies BIM protein). Furthermore, IHC of tumors treated with FPD showed more apoptosis by TUNEL and a significant increase of monopolar spindles (2, 0, 53 and 140 per 10 high-power fields, for control, poma+dexa, filanesib and FPD, respectively). Conclusions: The synergy observed with filanesib in combination with pomalidomide and dexamethasone is the result of several coincidental mechanisms: a potentiation of the KSP inhibition with a subsequent increase in monopolar spindle formation and a simultaneous activation of the intrinsic and extrinsic pathways of apoptosis. In this regard, NOXA, BIM, BAX and tBID are probably the central players that, through different mechanisms, inhibit antiapoptotic proteins (MCL-1, BCL2 and BCL-XL) and promote mitochondrial outer membrane permeabilization and the release of apoptogenic factors such us cytochrome C and AIF. This work was funded in part by the company Array BioPharma. Disclosures Tunquist: Array BioPharma: Employment. Mateos:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy. Ocio:Jassen: Honoraria; Celgene: Honoraria, Research Funding; Pharmamar: Consultancy, Research Funding; MSD: Research Funding; Novartis: Consultancy, Research Funding; Mundipharma: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Array BioPharma: Consultancy, Research Funding.

scholarly journals V-ATPase Deficiency Aggravates Hypoxia-induced Spermatogenesis Reduction by Promoting Spermatocyte Apoptosis via the JNK/c-Jun Pathway in Mice

2021 ◽  
Author(s):  
yin jun ◽  
juan Wen He ◽  
Fei Han ◽  
Zhi qi Gao ◽  
Fang Deng ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Nuclear Translocation ◽  
Sperm Count ◽  
Tunel Staining ◽  
Hypoxia Exposure ◽  
Transcription Factor Eb ◽  
Exposure In Vivo

Abstract Spermatocyte apoptosis is the primary cause of poor outcome after hypoxia-triggered spermatogenesis reduction (HSR). The vacuolar H+-ATPase (V-ATPase) has been found to be involved in the regulation of hypoxia-induced GC-2 cells apoptosis. However, the mechanism of V-ATPase regulating spermatocyte apoptosis after HSR hasnot been well elucidated. In this study, HSRmodel was established by hypoxia exposure in vivo in V-ATPase-knockout (V-ATPase-/-) and wild-type (WT) mice to investigate theeffectof V-ATPase deficiency on spermatocyte apoptosis. GC-2, amouse pachytene spermatocyte-derived cell line, was introduced in vitro experiments. The sperm count and spermatogenic apoptosis were recorded after 60 d of hypoxia exposure in HSR model. The apoptosis of GC-2 cells was detected by flow cytometry and TUNEL staining. The expression of JNK/c-Jun was evaluated by RNA-seq or western blot. The expression of DR5 and caspase-8 was evaluated by RT-qPCR and western blot. The expression of V-ATPase was determined by western blot in the presence and absence ofLenti-transcription factor EB (TFEB).C-Jun interference was used for evaluating the role of JNK in regulating the apoptosis of GC-2 cells byTUNEL and flow cytometry. The in vivo results suggested that hypoxia induced spermatogenesis reduction and downregulation of V-ATPase. Moreover, V-ATPase deficiency resulted in moresevere spermatogenesis reduction after hypoxia exposure. The spermatogenesis reduction was associated with exacerbation of spermatocyte apoptosis. Hypoxia down-regulated the transcription of V-ATPase through inhibiting TFEB and its nuclear translocation. The mRNA and protein expressions of V-ATPaseincreased after TFEB overexpression in GC-2 cells. Moreover, V-ATPase deficiency enhanced JNK/c-Jun activation and related DR-apoptotic pathwayin GC-2 cells.However,inhibition of c-Jun attenuated V-ATPase deficiency-induced GC-2 cells apoptosis in vitro and HSR in vivo. In conclusion, JNK/c-Jun was involved in the enhancement of V-ATPase-mediated HSR in V-ATPase -/- mice. V-ATPase deficiency aggravates spermatocyte apoptosis, which may account forthe poor spermatogenesis outcomes of V-ATPase-/- mice. The discoveredfunction of V-ATPase modulating spermatocyte apoptosis indicates its potential therapeutic effect against HSR.

Microparticles Derived from Platelets (PMP), Endothelia (EMP), and Leukocytes (LMP) Exhibit Distinctive Hemostatic and Inflammatory Activities.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3699-3699
Author(s):  
Wenche Jy ◽  
Joaquin J. Jimenez ◽  
Lawrence L. Horstman ◽  
Lucia M. Mauro ◽  
Carlos Bidot ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Specific Activity ◽  
Platelet Rich Plasma ◽  
Leukocyte Activation ◽  
Clotting Time ◽  
Platelet Factor ◽  
Functional Activities

Abstract Introduction: PMP, EMP and LMP are known to be sensitive markers of thrombotic and inflammatory disorders, but their respective functional activities are obscure. Recent data indicate that these microparticles (MP) possess procoagulant, proaggregatory, and proinflammatory activities. In this study, we have compared these activities in MP derived from different cells. Methods: PMP were prepared by stimulating platelet-rich plasma with 10 μM ADP plus 5 μg/mL collagen. EMP were prepared by incubating renal endothelial cells (EC) with 10 ng/mL of TNF-α for 24 hrs. LMP were prepared by incubating U937 monocytic cells or neutrophils with 10 ng/mL LPS for 1 hr. Cells were removed by centrifugation (1000xg for 10 min), MP were sedimented (15,000xg for 30 min), pellets were washed twice, then resuspended to equal MP concentration (1 x 108 counts/μL, final concentration) based on counts by flow cytometry. The MP were then tested for (ia) tissue factor (TF) antigen expression (TF:Ag) by flow cytometry, (ib) TF activity by recalcified clotting time in presence of corn trypsin inhibitor, (ii) platelet factor 3 (PF3) procoagulant activity by RVVT [Thromb Res 80:471, 1995], (iii) von willebrand factor (vWF)-dependent platelet aggregating activity by a ristocetin flow cytometric method [J Thromb Haemost 3:1301, 2005], and (iv) binding of MP to leukocytes, induced expression of CD11b and enhanced transendothelial migration (TEM) [Front Biosci 9:3137, 2004]. Results: In vivo, we found that the relative abundance of PMP, EMP, and LMP numbers in normal plasma is 50–70%, 5–10%, and 5–15% of total MP, respectively. In vitro results were as follows: (i) LMP exhibited the highest TF:Ag per MP followed by EMP, >PMP. However, PMP produced the shortest recalcified clotting time, indicating PMP had the highest apparent TF activity. (ii) PF3 activity was highest in PMP, followed by EMP, > LMP. In view of the abundance of circulating PMP (50–70% of total MP), it appears that PMP are mainly responsible for hemostatic activity in cell-free coagulation. (iii) On the other hand, EMP had the highest specific activity in promoting vWF-dependent platelet aggregation, > PMP, > LMP. (iv) Both PMP and EMP showed high affinity in binding monocytes and neutrophils, and inducing expression of CD11b, as well as promoting TEM. LMP had little effect on leukocyte activation. Discussion: Our results show that MP of different cell origins have distinctive activities in promoting coagulation, platelet aggregation, leukocyte activation, and TEM. These differences may reflect their distinctive membrane compositions. Overall, PMP and EMP seem to play more active roles in hemostasis and inflammation, as judged by these measures. Since LMP expressed the highest TF:Ag, they may serve to initiate coagulation during leukocyte activation. But their net procoagulant and proinflammatory activities are relatively small. A better understanding of the functional activities of the growing number of recognized MP species is expected to provide new insights on their roles in hemostasis and inflammation.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Sign in / Sign up

Export Citation Format

Share Document