SpringerProtocols: Abstract: In Vitro and In Vivo Apoptosis Detection Using Membrane Permeant Fluorescent-Labeled Inhibitors of Caspases

In Vitro and In Vivo Apoptosis Detection Using Membrane Permeant Fluorescent-Labeled Inhibitors of Caspases

By: Brian W. Lee², Michael R. Olin³, Gary L. Johnson², Robert J. Griffin⁴

Abstract

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Apoptosis detection methodology is an ever evolving science. The caspase family of cysteine proteases plays a central role in this environmentally conserved mechanism of regulated cell death. New methods that allow for the improved detection and monitoring of the apoptosis-associated proteases are key for further advancement of our understanding of apoptosis-mediated disease states such as cancer and Alzheimer’s disease. From the use of membrane permeant fluorescent-labeled inhibitors of caspases (FLICA) probe technology, we have demonstrated their successful use as tools in the detection of apoptosis activity within the in vitro and in vivo research setting. In this chapter, we provide detailed methods for performing in vitro apoptosis detection assays in whole living cells, using flow cytometry, and 96-well fluorescence plate reader analysis methods. Furthermore, novel flow cytometry-based cytotoxicity assay methods, which incorporate the FLICA probe for early apoptosis detection, are described. Inclusion of this sensitive apoptosis detection probe component into the flow-based cytotoxicity assay format results in an extremely sensitive cytotoxicity detection mechanism. Lastly, in this chapter, we describe the use of the FLICA probe for the in vivo detection of tumor cell apoptosis in mice and rats. These early stage in vivo-type assays show great potential for whole animal apoptosis detection research.

Affiliation(s): (2) Immunochemistry Technologies, LLC, Bloomington, MN
(3) University of Minnesota College of Veterinary Medicine, St. Paul, MN
(4) University of Minnesota Medical School, Minneapolis, MN

Book Title: Apoptosis and Cancer: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 414 | Pub. Date: Oct-11-2007 | Page Range: 109-135 | DOI: 10.1007/978-1-59745-339-4_10

Subject: Cancer Research

Key Words: FLICA - in vivo - in vitro - apoptosis - caspase - tumor imaging - detection - cytotoxicity assay

References

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1.	Kerr, J.F.R., Wyllie, A.H., and Currie, A.R. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257.

2.	Wyllie, A.H., Kerr, J.F.R., and Currie, A.R. (1981) Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251–306.

3.	Earnshaw, W.C., Martins, L.M., and Kaufmann, S.H. (1999) Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem. 68, 383–424.

4.	Nicholson, D.W. (1999) Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ. 6, 1028–1042.

5.	Heemels, M.T. (ed.) (2000) Nature insight apoptosis. Nature 407, 770–816.

6.	Degterev, A., Boyce, M., and Yuan, J. (2003) A decade of caspases. Oncogene 22, 8543–8567.

7.	Fink, S.L. and Cookson, B.T. (2005) Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. Infect. Immun. 73, 1907–1916.

8.	Lavrik, I.N., Golks, A., and Krammer, P.H. (2005) Caspases: pharmacological manipulation of cell death. J. Clin. Invest. 115, 2665–2672.

9.	Thornberry, N.A. and Lazebnik, Y. (1998) Caspases: enemies within. Science 281, 1312–1316.

10.	Thornberry, N.A. et al. (1992) A novel heterodimeric cysteine protease is required for interleukin-1UPbeta processing in monocytes. Nature 356, 768–774.

11.	Thornberry, N.A. et al. (1997) A combinatorial approach defines specificities of members of the caspase family and granzyme B. J. Biol. Chem. 272, 17907–17911.

12.	Fuentes-Prior, P. and Salvesen, G.S. (2004) The protein structures that shape caspase activity, specificity, activation and inhibition. Biochem. J. 384, 201–232.

13.	Sarin, A., Wu, M.L., and Henkart, P.A. (1996) Different interleukin-1UPbeta converting enzyme (ICE) family protease requirements for the apoptotic death of T lymphocytes triggered by diverse stimuli. J. Exp. Med. 184, 2445–2450.

14.	Armstrong, R.C. et al. (1996) Fas-induced activation of the cell death-related protease CPP32 is inhibited by Bcl-2 and by ICE family protease inhibitors. J. Biol. Chem. 271, 16850–16855.

15.	Garcia-Calvo, M., Peterson, E.P., Leiting, B., Ruel, R., Nicholson, D.W., and Thornberry, N.A. (1998) Inhibition of human caspases by peptide-based macromolecular inhibitors. J. Biol. Chem. 273, 32608–32613.

16.	Sun, X.M., MacFarlane, M., Zhuang, J., Wolf, B.B., Green, D.R., and Cohen, G.M. (1999) Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis. J. Biol. Chem. 274, 5053–5060.

17.	Ekert, P.G., Silke, J., and Vaux, D.L. (1999) Caspase inhibitors. Cell Death Differ. 6, 1081–1086.

18.	Clark, P., Dziarmaga, A., Eccles, M., and Goodyer, P. (2004) Rescue of defective branching nephrogenesis in renal coloboma syndrome by the caspase inhibitor, z-VAD-FMK. J. Am. Soc. Nephrol. 15, 299–305.

19.	Nakano, M. et al. (2004) Caspase-3 inhibitor prevents apoptosis of human islets immediately after isolation and improves islet graft function. Pancreas 29, 104–109.

20.	Nedev, H.N., Klaiman, G., LeBlanc, A., and Saragovi, H.U. (2005) Synthesis and evaluation of novel dipeptidyl benzoyloxymethyl ketones as caspase inhibitors. Biochem. Biophys. Res. Commun. 336, 397–400.

21.	Rauber, P., Angliker, H., Walker, B., and Shaw, E. (1986) The synthesis of peptidylfluoromethanes and their properties as inhibitors of serine proteases and cysteine proteases. Biochem. J. 239, 633–640.

22.	Powers, J.C., Asgian, J.L., Ekici, O.D., and James, K.E. (2002) Irreversible inhibitors of serine, cysteine, and threonine proteases. Chem. Rev. 102, 4639–4750.

23.	Wu, J.C. and Fritz, L.C. (1999) Irreversible caspase inhibitors: tools for studying apoptosis. Methods 17, 320–328.

24.	Bedner, E., Smolewski, P., Amstad, P., and Darzynkiewicz, Z. (2000) Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp. Cell Res. 259, 308–313.

25.	Amstad, P.A., Yu, G.L., Johnson, G.L., Lee, B.L., Dhawan, S., and Phelps, D.J. (2001) Detection of caspase activation in situ by fluorochrome-labeled caspase inhibitors. Biotechniques 31, 608–616.

26.	Smolewski, P., Bedner, E., Du, L., Hsieh, T.C., Wu, J.M., Phelps, D.J., and Darzynkiewicz, Z. (2001) Detection of caspase activation by fluorochrome-labeled inhibitors: multiparameter analysis by laser scanning cytometry. Cytometry 44, 73–82.

27.	Smolewski, P., Grabarek, J., Halicka, H.D., and Darzynkiewicz, Z. (2002) Assay of caspase activation in situ combined with probing plasma membrane integrity to detect three distinct stages of apoptosis. J. Immunol. Methods 265, 111–121.

28.	Smolewski, P., Grabarek, J., Lee, B.W., Johnson, G.L., and Darzynkiewicz, Z. (2002) Kinetics of HL-60 cell entry to apoptosis during treatment with TNF-a or camptothecin assayed by the stathmo-apoptosis method. Cytometry 47, 143–149.

29.	Wolbers, F., Buijtenhuijs, P., Haanen, C., and Vermes, I. (2004) Apoptotic cell death kinetics in vitro depend on the cell types and the inducers used. Apoptosis 9, 385–392.

30.	Grunewald, S., Paasch, U., Said, T.M., Sharma, R.K., Glander, H.J., and Agarwal, A. (2004) Caspase activation in human spermatozoa in response to physiological and pathological stimuli. Fertil. Steril. 83, 1106–1112.

31.	Fiala, M., Lin, J., Ringman, J., Arab, V.K., Tsao, A., Patel, A., Lossinsky, A.S., Graves, M.C., Gustavson, A., Sayre, J., Sofroni, E., Suarez, T., Chiappelli, F., and Bernard, G. (2005) Ineffective phagocytosis of amyloid-B by macrophages of Alzheimer’s disease patients. J. Alzheimer Dis. 7, 221–232.

32.	Mizukami, S., Kikuchi, K., Higuchi, T., Urano, Y., Mashima, T., Tsuruo, T., and Nagano, T. (1999) Imaging of caspase-3 activation in HeLa cells stimulated with etoposide using a novel fluorescent probe. FEBS Lett. 453, 356–360.

33.	Hug, H., Los, M., Hirt, W., and Debatin, K.M. (1999) Rhodamine 110-linked amino acids and peptides as substrates to measure caspase activity upon apoptosis induction in intact cells. Biochemistry 38, 13906–13911.

34.	Mack, A., Furmann, C., and Hacker, G. (2000) Detection of caspase-activation in intact lymphoid cells using standard caspase substrates and inhibitors. J. Immunol. Methods 241, 19–31.

35.	Komoriya, A., Packard, B.Z., Brown, M.J., Wu, M.L., and Henkart, P.A. (2000) Assessment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic substrates. J. Exp. Med. 191, 1819–1828.

36.	Telford, W.G., Komoriya, A., and Packard, B.Z. (2002) Detection of localized caspase activity in early apoptotic cells by laser scanning cytometry. Cytometry 47, 81–88.

37.	Lee, B.W., Johnson, G.L., Hed, S.A., Darzynkiewicz, Z., Talhouk, J.W., and Mehrotra, S. (2003) DEVDase detection in intact apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)2-cresyl violet. Biotechniques 35, 1080–1085.

38.	Li, X., Du, L., and Darzynkiewicz, Z. (2000) During apoptosis of HL-60 and U-937 cells caspases are activated independently of dissipation of mitochondrial electrochemical potential. Exp. Cell Res. 257, 290–297.

39.	Duan, R.W., Garner, D.S., Williams, S.D., Funckes-Shippy, C.L., Spath, I.S., and Blomme, E.A. (2003) Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts. J. Pathol. 199, 221–228.

40.	Salvioli, S., Ardizzoni, A., Franceschi, C., and Cossarizza, A. (1997) JC-1 but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett. 411, 77–82.

41.	Poot, M., Zhang, Y.Z., Kramer, J.A., Wells, K.S., Jones, L.J., Hanzel, D.K., Lugade, A.G., Singer, V.L., and Haugland, R.P. (1996) Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. J. Histochem. Cytochem. 44, 1363–1372.

42.	Brunner, K.T., Mauel, J., Cerottini, J.C., and Chapuis, B. (1968) Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labeled allogenic target cells in vitro; inhibition by isoantibody and by drugs. Immunology 14, 181–196.

43.	Ward, P.W., Bonaparte, M.I., and Barker, E. (2004) HLA-C and HLA-E reduce antibody-dependent natural killer cell-mediated cytotoxicity of HIV-infected primary T cell blasts. AIDS 18, 1769–1779.

44.	Barber, D.L., Wherry, E.J., and Ahmed, R. (2003) Cutting edge: rapid in vivo killing by memory CD8 T cells . J. Immunol. 171, 27–31.

45.	Yang, S. and Haluska, F.G. (2004) Treatment of melanoma with 5-fluorouracil or dacarbazine in vitro sensitizes cells to antigen-specific CTL lysis through perforin/granzyme- and Fas-mediated pathways. J. Immunol. 172, 4599–4608.

46.	Schwarer, A.P., Jiang, Y.Z., Deacock, S., Brookes, P.A., Barret, A.J., Goldman, J.M., Batchlor, J.R., and Lechler, R.I. (1994) Comparison of helper and cytotoxic antirecipient T cell frequencies in unrelated bone marrow transplantation. Transplantation 58, 1198–1203.

47.	Russell, J.H. and Ley, T.J. (2002) Lymphocyte-mediated cytotoxicity. Annu. Rev. Immunol. 20, 323–370.

48.	Pross, H., Callewaert, D., and Rubin, P. (1986) Assays for NK cell cytotoxicity-their values and pitfalls, in Immunobiology of Natural Killer Cells (E. Lotzova and R.B. Herberman eds.) Vol. I. CRC Press, Boca Raton, FL, pp. 1–16.

49.	Jerome, K.R., Sloan, D.D., and Aubert, M. (2003) Measurement of CTL-induced cytotoxicity: the caspase 3 assay. Apoptosis 8, 563–571.

50.	Slezak, S.E. and Horan, P.K. (1989) Cell-mediated cytotoxicity: a highly sensitive and informative flow cytometric assay. J. Immunol. Methods 117, 205–214.

51.	Radosevic, K., Garritsen, H.S.P., Van Graft, M., De Grooth, B.G., and Greve, J. (1990) A simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells. J. Immunol. Methods 135, 81–89.

52.	Hatam, L., Schuval, S., and Bonagura, V.R. (1994) Flow cytometric analysis of natural killer cell function as a clinical assay. Cytometry 16, 59–68.

53.	Lee-MacAry, A.E., Ross, E.L., Davies, D., Laylor, R., Honeychurch, J., Glennie, M.J., Snary, D., and Wilkinson, R.W. (2001) Development of a novel flow cytometric cell-mediated cytotoxicity assay using the fluorophores PKH-26 and TO-PRO-3 iodide. J. Immunol. Methods 252, 83–92.

54.	Hoppner, M., Luhm, J., Schlenke, P., Koritke, P., and Frohn, C. (2002) A flow-cytometry based cytotoxicity assay using stained effector cells in combination with native target cells. J. Immunol. Methods 267, 157–163.

55.	Olin, M.R., Choi, K.H., Lee, J., and Molitor, T.W. (2005) γδ T-lymphocyte cytotoxic activity against Mycobacterium bovis analyzed by flow cytometry. J. Immunol. Methods 297, 1–11.

56.	Kienzle, N., Oliver, S., Buttigieg, K., and Kelso, A. (2002) The fluorolysis assay, a highly sensitive method for measuring the cytolytic activity of T cells at very low numbers. J. Immunol. Methods 267, 98–108.

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