Renilla luciferase-labeled Annexin V: a new probe for detection of apoptotic cells.

The Ca(2+)-dependent binding of Annexin V to phosphatidylserine on cell surfaces is a reliable marker for apoptosis that is widely used in flow cytometry based apoptosis assays. In this paper, we report a new class of Annexin V-based probes for apoptosis. Luciferase from Renilla reniformis (RLuc) was linked to Annexin V and expressed successfully in a soluble form in Escherichia coli BL21 (DE3). The new probe, Rluc/Annexin V, was purified and functionally assayed for detection of apoptosis in actinomycin D-induced apoptotic Jurkat cells. Moreover, the spontaneous apoptosis in neutrophils was shown using the new probe. The results indicate that Rluc/Annexin V can bind to the apoptotic cells, and the signal of Renilla luciferase can be detected by luminometric measurements. The availability of Rluc/Annexin V may be of potential commercial interest for improving current apoptosis assays.

[1]  F. Grespi,et al.  Molecular analysis of neutrophil spontaneous apoptosis reveals a strong role for the pro-apoptotic BH3-only protein Noxa , 2011, Cell Death and Differentiation.

[2]  G. Lenoir,et al.  In Cystic Fibrosis Homozygotes and Heterozygotes, Neutrophil Apoptosis Is Delayed and Modulated by Diamide or Roscovitine: Evidence for an Innate Neutrophil Disturbance , 2010, Journal of Innate Immunity.

[3]  F. Loison,et al.  Neutrophil spontaneous death is mediated by down-regulation of autocrine signaling through GPCR, PI3Kγ, ROS, and actin , 2010, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Rainer Fischer,et al.  Eukaryotic expression and secretion of EGFP-labeled annexin A5. , 2008, Protein expression and purification.

[5]  D. Zheng,et al.  Actinomycin D enhances TRAIL-induced caspase-dependent and -independent apoptosis in SH-SY5Y neuroblastoma cells , 2007, Neuroscience Research.

[6]  M. Farinacci Improved apoptosis detection in ovine neutrophils by annexin V and carboxyfluorescein diacetate staining , 2007, Cytotechnology.

[7]  M. Linial,et al.  Apoptotic cell thrombospondin-1 and heparin-binding domain lead to dendritic-cell phagocytic and tolerizing states. , 2006, Blood.

[8]  S. Orrenius,et al.  Apoptosis: a basic biological phenomenon with wide‐ranging implications in human disease , 2005, Journal of internal medicine.

[9]  Adeline R. Whitney,et al.  Spontaneous neutrophil apoptosis and regulation of cell survival by granulocyte macrophage‐colony stimulating factor , 2005, Journal of leukocyte biology.

[10]  K. Schulze-Osthoff,et al.  New Approaches and Therapeutics Targeting Apoptosis in Disease , 2005, Pharmacological Reviews.

[11]  Zbigniew Darzynkiewicz,et al.  Cytometry of the cell cycle: Cycling through history , 2004, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[12]  S. Edwards,et al.  Anaplasma phagocytophilum Reduces Neutrophil Apoptosis In Vivo , 2003, Infection and Immunity.

[13]  E. Gulbins,et al.  Actinomycin D-induced apoptosis involves the potassium channel Kv1.3. , 2002, Biochemical and biophysical research communications.

[14]  R. William,et al.  Neutrophil apoptosis is delayed in patients with inflammatory bowel disease. , 2000, Shock.

[15]  J. Marshall,et al.  Maternal neutrophil apoptosis in normal pregnancy, preeclampsia, and normotensive intrauterine growth restriction. , 1999, American journal of obstetrics and gynecology.

[16]  Ruedi Aebersold,et al.  Molecular characterization of mitochondrial apoptosis-inducing factor , 1999, Nature.

[17]  J. Ernst,et al.  Preparation and characterization of an endogenously fluorescent annexin for detection of apoptotic cells. , 1998, Analytical biochemistry.

[18]  O. Shimomura,et al.  The use of Renilla luciferase, Oplophorus luciferase, and apoaequorin as bioluminescent reporter protein in the presence of coelenterazine analogues as substrate. , 1997, Biochemical and biophysical research communications.

[19]  Marty W. Mayo,et al.  TNF- and Cancer Therapy-Induced Apoptosis: Potentiation by Inhibition of NF-κB , 1996, Science.

[20]  D. Nicholson ICE/CED3-like Proteases as Therapeutic Targets for the Control of Inappropriate Apoptosis , 1996, Nature Biotechnology.

[21]  D. Green,et al.  Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl , 1995, The Journal of experimental medicine.

[22]  C Haanen,et al.  A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. , 1995, Journal of immunological methods.

[23]  J. Ernst,et al.  Annexins possess functionally distinguishable Ca2+ and phospholipid binding domains. , 1994, Biochemical and biophysical research communications.

[24]  C. Winterford,et al.  Apoptosis. Its significance in cancer and cancer Therapy , 1994, Cancer.

[25]  J. H. Dierendonck,et al.  A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA. , 1993, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[26]  C. Haslett,et al.  Different populations of macrophages use either the vitronectin receptor or the phosphatidylserine receptor to recognize and remove apoptotic cells. , 1992, Journal of immunology.

[27]  V. Fadok,et al.  Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. , 1992, Journal of immunology.

[28]  I Nicoletti,et al.  A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. , 1991, Journal of immunological methods.

[29]  L. Tessarollo,et al.  An improved method for the detection of DNA fragmentation. , 1991, Journal of immunological methods.

[30]  C. Haslett,et al.  Phagocytosis of aged human neutrophils by macrophages is mediated by a novel "charge-sensitive" recognition mechanism. , 1989, The Journal of clinical investigation.

[31]  C. Sarraf,et al.  Proportions of Mitotic and Apoptotic Cells In A Range of Untreated Experimental Tumours , 1988, Cell and tissue kinetics.

[32]  A. Wyllie,et al.  Macrophage recognition of cells undergoing programmed cell death (apoptosis). , 1985, Immunology.

[33]  A. Wyllie,et al.  Hormone-induced cell death. 2. Surface changes in thymocytes undergoing apoptosis. , 1984, The American journal of pathology.

[34]  P. Henson,et al.  Phagocytosis of senescent neutrophils by human monocyte-derived macrophages and rabbit inflammatory macrophages , 1982, The Journal of experimental medicine.

[35]  F. Sarkar,et al.  Molecular mechanism(s) of actinomycin-D induced sensitization of pancreatic cancer cells to CD95 mediated apoptosis. , 2002, International journal of oncology.

[36]  J. Giorgi,et al.  Sensitive method for measuring apoptosis and cell surface phenotype in human thymocytes by flow cytometry. , 1994, Cytometry.

[37]  X. M. Sun,et al.  Quantification of apoptosis and necrosis by flow cytometry. , 1993, Acta oncologica.

[38]  C. Haslett,et al.  Phagocyte recognition of cells undergoing apoptosis. , 1993, Immunology today.