Annexin-V Apoptosis Assay Research Articles

Differentiation-Defective Class IV GCSF Receptor Promotes Resistance to Apoptosis Through Induction of Anti-Apoptotic Bcl-2 Family Proteins

Abstract 3819 INTRODUCTION.Granulocyte colony stimulating factor (GCSF) drives the production, proliferation and differentiation of myeloid progenitor and precursor cells via the GCSF Receptor (GCSFR). The Class I GCSFR constitutes the full-length, wild type receptor. Alternative splicing events create a truncated isoform, the Class IV GCSFR, which lacks 3 of the 4 distal tyrosine residues in the C-terminal domain, and results in impaired differentiation. A role for the Class IV receptor in human myeloid leukemia has been suggested from two clinical observations: 1) Bone marrow cells from patients with monosomy 7 overexpress the Class IV receptor and demonstrate increased proliferative response to GCSF; 2) Increased relapse rates were found in children and adolescents with AML whose blasts overexpress the Class IV receptor. Additionally, multiple splicing factors have recently been discovered to be mutated in myelodysplastic syndrome (MDS). We have discovered that the S34F mutation in the splicing factor U2AF1 is associated with exon skipping of the GCSFR. Yet, little is known about the biological properties of this Class IV isoform. METHODS.The IL-3 dependent murine Ba/F3 cell line was transfected with cDNA to express either human Class I (Ba/F3 GRI) or Class IV (Ba/F3 GRIV) GCSFR. Proliferation of Ba/F3 GRI and GRIV cells was measured using MTT assay after 48 hours of exposure to varying doses of GCSF. Cells were serum- and cytokine-starved for 0, 4, 8 and 16 hours. Cell count and viability were determined by trypan blue exclusion. Members of the mitochondrial apoptosis pathway were also evaluated by western blot analysis. Time-dependent changes in GCSF-mediated phosphoprotein signaling events were studied by western blotting. Samples were also stained with propidium iodide and used for cell cycle analysis by flow cytometry or used to perform AnnexinV apoptosis assay. RESULTS.Flow cytometry results demonstrated a G0/G1 cell cycle arrest at 8 hrs for both Ba/F3 GRI and GRIV cells with a similar magnitude of effect. However, longer starvation of 16 hrs resulted in increased cell death, more pronounced in the GRI than GRIV cells (46% v. 9% in the sub-G1 phase of the cell cycle, respectively). This pattern of apoptotic resistance in the GRIV cells was also seen in cell number and viability (viability of GRI and GRIV cells at 16 hrs was 67% ± 4.9% v. 90% ± 4%, respectively). These results were validated by flow cytometry using the AnnexinV apoptosis assay: proportion of GRI and GRIV cells staining positive for both AnnexinV and 7-AAD was 71% and 32%, respectively. To determine the molecular basis for this resistance to apoptosis, we performed western blotting to analyze Bcl-2, Mcl-1, Puma, GSK3ß, Bcl-xLand other Bcl-2 family members. Of these, the most prominent differences were seen in Bcl-2 and Mcl-1. The Class IV cells demonstrated increased levels of Bcl-2 throughout 16 hrs of starvation. The Class IV cells also displayed persistence of Mcl-1 for a longer duration than the GRI cells, correlating with the observed pattern of viability and cell cycle data. When these starved cells were stimulated with GCSF, there was a greater time dependent increase of phopho-p38 MAPK in the GRI cells. CONCLUSIONS.Here, we show that the Class IV GCSFR isoform confers both a proliferative and survival or anti-apoptotic advantage. Increased levels of both Mcl-1 and Bcl-2 may contribute to the apoptotic resistance seen in the Ba/F3 GRIV cells. Because there were no differences in activation state of GSK3ß, an alternate pathway for regulating Mcl-1 levels must be operative. The p38 MAPK stress response has been implicated in affecting downstream Mcl-1 and Bcl-2 regulation. p38 activation is important for chemotherapy induced cancer cell death, and chemotherapeutic resistance has been linked to overexpression of Bcl-2 or Mcl-1 in various cancer types. It is intriguing that Class IV expression has been linked to increased relapse rates in AML patients. Thus, we speculate that there may be a synergistic, protective effect of decreased p38 activation coincident with enhancement of the anti-apoptotic Bcl-2 and Mcl-1 proteins in the Class IV cells. Further elucidation of the mechanism for apoptotic resistance conferred by the Class IV GCSFR will help define its contribution to MDS transformation and provide candidate targets for novel therapeutics or prevention of disease progression in patients with primary or secondary MDS/AML. Disclosures:No relevant conflicts of interest to declare.

Sorafenib in combination with ionizing radiation has a greater anti-tumour activity in a breast cancer model

High expression of vascular endothelial growth factor (VEGF) in patients with breast cancer has been associated with a poor prognosis, indicating that VEGF could be linked to the efficacy of chemotherapy and radiotherapy. It has also been suggested that radiation resistance is partly due to tumour cell production of angiogenic cytokines, particularly VEGF receptor (VEGFR). This evidence indicates that inhibition of VEGFR might enhance the radiation response. Sorafenib tosylate (Bay 54-9085) is an oral, small-molecule multikinase inhibitor of several targets including RAF/MEK/ERK MAP kinase signalling, VEGFR-2, VEGFR-3 and platelet-derived growth factor receptor-beta. Sorafenib has shown clinical efficacy in treating solid tumours such as renal cell and hepatocellular carcinomas. However, strategies are yet to be identified to prolong and maximize the anticancer effect of this multikinase inhibitor. The objective of this study was to determine whether a combination of Sorafenib and radiation will enhance the treatment response in vitro and in vivo. Radio-modulating effect of Sorafenib was assessed by performing clonogenic assays. In addition, cell cycle analyses as well as annexin-V apoptosis assays were performed 24 and 48 h after treatment, respectively. To confirm our in-vitro results, tumour growth delay assays were performed. Our results showed a strong and supra-additive antitumour effect of radiation combined with Sorafenib in vitro (dose enhancement factor of 1.76). The combined therapy demonstrated a strong and significant G2/M cell cycle arrest (combined treatment vs. irradiated alone: P<0.0008). Moreover, annexin-V staining showed a significant increase in the level of apoptosis (combined treatment vs. irradiated alone: P<0.0004). Study of the syngeneic model demonstrated the superior potency of the Sorafenib combined with radiotherapy. Our results demonstrate that higher antitumour activity can be achieved when radiation and Sorafenib are combined.

Direct Induction of Apoptosis Using an Optimal Mitochondrially Targeted p53

Targeting the tumor suppressor p53 to the mitochondria triggers a rapid apoptotic response as efficiently as transcription-dependent p53. (1, 2) p53 forms a complex with the antiapoptotic Bcl-XL, which leads to Bak and Bax oligomerization resulting in apoptosis via mitochondrial outer membrane permeabilization. (3, 4) Although p53 performs its main role in the mitochondrial outer membrane, it also interacts with different proteins in the mitochondrial inner membrane and matrix. (5, 6) To further investigate mitochondrial activity of p53, EGFP-p53 was fused to different mitochondrial targeting signals (MTSs) directing it to the mitochondrial outer membrane ("XL-MTS" from Bcl-XL; "TOM-MTS" from TOM20), the inner membrane ("CCO-MTS" from cytochrome c oxidase), or matrix ("OTC-MTS" from ornithine transcarbamylase). Fluorescence microscopy and a p53 reporter dual luciferase assay demonstrated that fusing MTSs to p53 increased mitochondrial localization and nuclear exclusion depending on which MTS was used. To examine if the MTSs initiate mitochondrial damage, we fused each individual MTS to EGFP (a nontoxic protein) as negative controls. We performed caspase-9, TUNEL, annexin-V, and 7-AAD apoptosis assays on T47D breast cancer cells transfected with mitochondrial constructs. Except for EGFP-XL, apoptotic potential was observed in all MTS-EGFP-p53 and MTS-EGFP constructs. In addition, EGFP-p53-XL showed the greatest significant increase in programmed cell death compared to its nontoxic MTS control (EGFP-XL). The apoptotic mechanism for each construct was further investigated using pifithrin-α (an inhibitor of p53 transcriptional activity), pifithrin-μ (a small molecule that reduces binding of p53 to Bcl-2 and Bcl-XL), and overexpressing the antiapoptotic Bcl-XL. Unlike the MTSs from TOM, CCO, and OTC, which showed different apoptotic mechanisms, we conclude that p53 fused to the MTS from Bcl-XL performs its apoptotic potential exclusively through the p53/Bcl-XL specific pathway.

TRPV2 Expression and Its Role in Proliferation of Human Multiple Myeloma Cell Lines

Abstract 5003Multiple myeloma (MM) is a disease typically characterized by repeated relapse that needs several new-drugs throughout its clinical course. Therefore, increasing knowledge of the pathways involved in the pathophysiology of MM will contribute to identified new therapeutic strategies. Specific signaling pathways as: nuclear factor kappa B (NFkB), farnelsyl-transferase (FTI), mitogenen-activated protein kinase (MAPK), proteosoma and others have been found to be disregulated in MM patients and cell lines. These findings have allowed the design of specific inhibitors that have been assessed in preclinical and clinical studies. Recent data have highlighted the contribute of ion channels (K+, Cl−, Na+ and Ca2+) in the regulation of cell proliferation, chemo-resistance, migration and invasion. Transient Receptor Potential Vanilloid type-2 (TRPV2) is a cation channel, member of the TRPV family, and its expression in human cancer cells and tissues has been reported for gliomas, prostate, bladder and pancreas, while no data were available in MM or in other hematological malignancies. TRPV2 expression has been found to influence cancer cell responses to chemo-therapeutic drugs as well as proliferation, migration and apoptosis, in part by increasing intracellular Ca2+ influx and/or by regulating specific signal pathways, like MAPK associated pathways. TRPV2 responds to noxious heat with an activation threshold of >52°C as well as to changes in osmolarity and membrane stretch; in addition, TRPV2 is activated by agonists such as 2-aminoethoxydiphenyl borate, as well as cannabinoids. The aim of this study was, firstly, to evaluate if TRPV2 was expressed in three human MM cell lines established models (RPMI, SKO-007, U266). Secondly we investigated on the role of TRPV2 activation in regulating the proteosoma inhibitor Bortezomib activity, in MM cell lines. By Real-Time PCR analysis we demonstrated that TRPV2 gene was expressed, with comparable relative levels, in the MM cell lines studied, and similar results were obtained at protein levels by Western blot analysis. Moreover, by immunofluorescence and FACS analysis we found that the percentage of TRPV2+ cells was 11% in RPMI, 3% in U266 and 2.6% in SKO-007. These data indicate that TRPV2 was expressed at low levels in MM cell lines. Since lost of TRPV2 expression was found to be associated with higher cell proliferation rate, in other tumor cell lines, and increasing of TRPV2 transcription and/or activity by specific agonists induced cell death and anti-proliferative effects, we evaluated the biological effects of TRPV2 activation in MM cell lines by MTT assay. The results showed that, after three incubation days, TRPV2 activation induced a decrease of cell viability of 70% in RPMI, 60% in U266 and 55% in SKO-007, compared to control. To evaluate if the reduced cell viability was dependent by an anti-proliferative and/or apoptotic process, the three cell lines treated with the TRPV2 agonist were analyzed by 5-bromo-2-deoxyuridine (BrdU) incorporation assay for proliferation and by Annexin-V apoptosis assays. Data shown a decrease of BrdU+ cells in TRPV2 agonist treated cells (40% in RPMI, 30% in U266 and 28% in SKO-007) compared to control, while no significant differences were observed by Annexin-V analysis. Moreover, to evaluate a putative role of TRPV2 in influencing Bortezomib cytotoxicity, RPMI, SKO-007 and U266, were co-treated with the TRPV2 agonist in combination or not with Bortezomib, for three days. By a dose-response MTT analysis we determined that TRPV2 activation reduced MM cell lines viability more than 40% in RPMI, 20 % in U266 and 15% in SKO-007, compared to Bortezomib (5 ng/ml) alone. Summarizing, these preliminary data demonstrated that the TRPV2 cation channel was expressed in human MM cell lines and that activation of TRPV2 could play a role in increasing Bortezomib cytotoxicity, by inhibiting cell proliferation rather than increasing apoptosis of MM cells. These data may open new perspectives in combination therapy, albeit the molecular mechanisms undergone TRPV2 activation needs to be clarified. Disclosures:No relevant conflicts of interest to declare.

Overexpression of VEGF189 in breast cancer cells induces apoptosis via NRP1 under stress conditions

The existence of multiple VEGF-A isoforms raised the possibility that they may have distinct functions in tumor growth. We have previously published that VEGF189 and VEGF165 contribute to breast cancer progression and angiogenesis, but VEGF165 induced the most rapid tumor uptake. Since VEGF165 has been described as a survival factor for breast tumor cells, we questioned here the effects of VEGF189 on the survival/apoptosis of MDA-MB-231 cells. We used clones which overexpress VEGF189 (V189) or VEGF165 (V165) isoforms and compared them to a control one (cV). Overexpression of VEGF189 resulted in increased cell apoptosis, as determined by Annexin-V apoptosis assay, under serum starvation and doxorubicin treatment, while VEGF 165 was confirmed to be a survival factor. Since MDA-MB-231 highly express NRP1 (a co-receptor for VEGF-A), we used short hairpin RNA (shRNA) to knockdown NRP1 expression. V189shNRP1 clones were characterized by reduced apoptosis and higher necrosis, as compared to V189shCtl, under stress conditions. Unexpectedly, NRP1 knock-down had no effect on the survival or apoptosis of V165 cells. VEGF189 showed greater affinity towards NRP1 than VEGF165 using a BIAcore binding assay. Finally, since endogenously produced urokinase-type plasminogen (uPA) has been found to prevent apoptosis in breast cancers, we analyzed the level of uPA activity in our clones. An inhibition of uPA activity was observed in V189shNRP1 clones. Altogether, these results suggest a major role of NRP1 in apoptosis induced by VEGF189 in stress conditions and confirm VEGF165 as a survival factor.

Abstract 1077: Galectin-1 is implicated in G1/S-checkpoint control following irradiation - possible explanation of Galectin-1 mediated radioresistance

Abstract Purpose: The purpose of the conducted study was to examine a possible therapeutic relevance of Galectin 1 (Gal-1), a beta-galactoside binding protein. Gal-1 is overexpressed in high grade gliomas and leads to increased radioresistance in glioma cell culture models. Inhibition of Gal-1 increases radiosensitivity and thus is a potential therapeutic target for radiation sensitization. P53 has been shown to negatively regulate Gal-1, hence radiosensitivity could possibly be cell cycle mediated. Experimental Design: We used flow cytometry based cell cycle analysis to investigate cell cycle distribution of LN 229 glioma cells following irradiation. LN229 control cell lines and shRNA mediated Gal-1 knockdown LN 229 cell line were exposed to 6 Gy of irradiation and cell cycle analysis was performed. Furthermore Western blot analysis was carried out, probing for multiple proteins implicated in cell proliferation. The phosphorylation status of a variety of phosphorylation sites of p53, Chk1/2 and AKT were examined. Additionally p53-Gal-1 double-knockdown cell lines were created and clonogenic survival assays as well as AnnexinV apoptosis assays were conducted to analyze the influence on radiation sensitivity. Results: The results of the flow cytometry based cell cycle analysis show significant differences between the LN 229 control cell line and the Gal-1 knockdown cell line after irradiation. 24 h after irradiation 77 % of the control line cells are in G1 whereas only 60 % of the Gal-1 knockdown cells are in G1. Previous data has identified differences in the phosphorylation status of Akt in control vs. Gal-1 knockdown glioma cell lines. Phosphorylation of Akt is induced after irradiation. Western blot analysis indicates a reduction of phosphorylated Chk1/2 in Gal1 knockdown cell lines after irradiation. The phosphorylation state of the p53 phosphorylation site, correlating with Chk1/2 activity, exhibit anticipated results. P53-Gal-1 double-knockdown cell lines demonstrate significantly reduced clonogenic survival compared to control cell lines. Conclusions: The finding that less Gal-1 knockdown cells are in G1 phase after irradiation and that there is a reduction in phosphorylated Chk1/2 leads to the conclusion that Gal-1 knockdown abrogates G1 arrest through a Chk1/2 mediated pathway. That means in reverse that Gal-1 can mediate cell cycle arrest, thus potentially can explain Gal-1 mediated radioresistance. Further analysis of Chk1/2 independent phosphorylation sites of p53 is going to be conducted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1077.

Engineering Human Tumor-specific Cytotoxic T Cells to Function in a Hypoxic Environment

Hypoxia occurs in many tumors and reduces the effectiveness of radio- and chemotherapy. Hypoxia also impedes immune responses to tumors, reducing T lymphocyte production of cytokines such as interleukin-2 (IL-2) and interferon gamma, as well as the survival and proliferation of these cells. We constructed a lentiviral vector encoding a bidirectional hypoxia-inducible responsive element (HRE) derived from human vascular endothelial growth factor, which drives the hIL-2 gene and a marker gene. We used a model of human B cell lymphoma to show that tumor-specific T cells modified with this vector upregulate hIL-2 expression when oxygen tension is low in vitro and in vivo. The consequence of this effect is to increase T-cell survival and proliferation whilst sustaining effector function, even in O(2) concentrations as low as 1%. The phenotype of the transduced cells is unchanged, as is their ability to migrate to tumor. HRE-IL-2-modified cytotoxic T lymphocytes (CTLs) produce faster and more complete tumor regression than parental CTLs and increase overall survival. Hypoxia-resistant T cells may thus be of value in the treatment of human tumors in which areas of hypoxia may otherwise account for resistance to this therapeutic strategy.

BSc2118, a Novel Proteasome Inhibitor, Shows Anti-Tumor Activity in Multiple Myeloma and Mantle Cell Lymphoma.

The ubiquitin-proteasome pathway was recently identified as a promising new therapeutic target in cancer treatment. An increased proteasome activity was described in certain cancer types, especially in multiple myeloma (MM) and also in mantle cell lymphoma (MCL). The proteasome inhibitor bortezomib has been approved for the treatment of refractory MM and has also shown reproducible activity in MCL. Recently we described a novel tripeptide compound, BSc2118, with inhibitory activity against all three proteolytic activities (post-glutamyl peptide hydrolase-like, trypsin-like and chymotrypsin-like activities) of the 20S proteasome (Cancer Res 2006; 66:7598–605). We investigated the in vitro effects of BSc2118 in MM and MCL cell lines by MTT cell viability and AnnexinV apoptosis-assays. Furthermore, the intracellular chymotrypsin-like proteasome activity and NF-κB activity were detected in MM and MCL cells by detection of proteasome-degraded peptides or NF-κB p65 subunit. In MM cell lines OPM-2 and U266 we could show a significant time and dose-dependent reduction of cell viability by BSc2118 with an IC50 at 48hrs of 52nM and 65nM, respectively, whereas the MM cell line RPMI-S was less sensitive with an IC50 of 287nM. Using AnnexinV assay, a dose-dependent induction of apoptosis by BSc2118 was shown after 48hrs incubation. Comparably, in MCL we also found a time and dose-dependent reduction of cell viability in the cell lines HBL-2, JeKo-1 and Granta-519 with an IC50 of 82nM, 130nM and 262nM, respectively. Furthermore, BSc2118 induced apoptosis in all three MCL cell lines. Additionally, we detected a significant dose-dependent inhibition of intracellular chymotrypsin-like proteasome activity in MM and MCL cells, and a dose-dependent inhibition of TNFα-induced NF-κB activation in the MM cell lines OPM-2 and RPMI-S. This is the first report of anti-tumor effects of the novel proteasome inhibitor BSc2118 in MM and MCL cells. The compound effectively reduces the cell survival and shows a high pro-apoptotic activity in the MM cell lines OPM-2 and U266 and a significant activity in MCL cell lines HBL-2 and JeKo-1. Mechanisms of action are the inhibition of proteasome and NF-κB activity. Since previous clinical trials have shown an activity of the proteasome inhibitor bortezomib in MM and MCL, and adverse effects of other proteasome inhibitors may differ, our preclinical data support the idea to consider BSc2118 as a promising new agent in anti-tumor drug development.

Evidence that apoptosis of activated T cells occurs in spontaneous tolerance of liver allografts and is blocked by manipulations which break tolerance.

Fully allogeneic liver grafts from piebald virol glaxo to dark agouti rats are spontaneously tolerated, whereas kidney transplants between these strains are rejected. Liver tolerance is broken by donor irradiation or peritransplant corticosteroid treatment of recipient rats, both of which interfere with the activation of recipient cells. In this study we used a combination of immunohistochemical staining, reverse transcription-polymerase chain reaction, and terminal deoxynucleotide transferase-mediated dUTP nick end labeling and Annexin-V apoptosis assays to compare donor cell migration, cytokine profiles, and leukocyte apoptosis in grafts and lymphoid organs from tolerant liver and rejecting kidney recipients. We then examined the effect on apoptosis of treatments which abrogate liver tolerance. Liver transplantation in this tolerant strain combination is accompanied by rapid migration of many passenger leukocytes to the recipient spleen and lymph node, concurrent with a marked but transient increase in the amount of mRNA for the cytokines interleukin-2 and interferon-gamma. Apoptotic cells appear promptly in the spleen, their numbers reaching a peak 2 days earlier than has been previously shown for the graft infiltrate. Both CD4+ and CD8+ T cells undergo apoptosis and apoptotic cells are most concentrated among CD25+ T cells. In contrast, renal transplant rejection is associated with limited donor cell migration to lymphoid tissues and significantly less up-regulation of interleukin-2 and interferon-gamma in the spleen. Few apoptotic cells are detected in spleen or graft infiltrate during rejection, whereas apoptotic renal tubular and glomerular cells are found from day 5. Either recipient steroid treatment or donor irradiation significantly reduced the number of apoptotic cells in liver graft infiltrates and recipient spleen. Taken together, these findings suggest that a mechanism akin to activation-induced cell death, with apoptosis of alloreactive recipient cells may be responsible for the induction of spontaneous liver transplant tolerance.

Flt3 ligand can promote survival and macrophage development without proliferation in myeloid progenitor cells

Flt3 ligand elicits a variety of effects on early hemopoietic progenitors by occupying its cognate receptor, Flt3, a member of the type III tyrosine kinase receptor family. The cytokines macrophage colony-stimulating factor (M-CSF) and stem cell factor (SCF) bind to related members of this tyrosine kinase receptors family, c-fms and c-kit, respectively. The relative effects of the cytokines M-CSF, SCF, and Flt3L on the proliferation and development of the late myeloid progenitors granulocyte-macrophage colony-forming cells (GM-CFC) were investigated. Distinct biologic responses were stimulated by ligand binding to these different tyrosine kinase receptors in enriched GM-CFC. M-CSF stimulated GM-CFC to proliferate and develop into macrophages. SCF, on the other hand, stimulated GM-CFC to develop into neutrophils. Flt3 ligand had a relatively small proliferative effect on enriched GM-CFC compared to SCF and M-CSF and had no ability to either stimulate colony formation or synergize with these two cytokines in promoting DNA synthesis, colony formation, or expansion in liquid culture. Flt3 ligand, however, was capable of maintaining the clonogenic potential of GM-CFC and acted as an anti-apoptotic agent as assessed using the Annexin-V apoptosis assay. GM-CFC cultured in Flt3 ligand eventually formed macrophages and neutrophils in liquid culture. Labeling with the membrane-associated cell tracker dye PKH26 indicated that the majority of the enriched GM-CFC responded to Flt3 ligand by undergoing limited proliferation and macrophage development, whereas other cells survived but did not proliferate and differentiate into macrophages. Thus, Flt3 ligand promoted survival and stimulated development without proliferation in primary-enriched myeloid progenitor cells.