Apoptosis and Cell Proliferation, страница 3

The second messenger ceramide,a product of membrane-linked acid sphingomyelinase activation, may act as a signal for plasma membrane damage8.And a powerful caspase-activating system is mediated by cytokine receptors of the tumor necrosis factor family, notablyfas/apo-1/CD95, TNF receptor I, and others. These receptors, on receiving a death stimulus from binding their ligand,initiate a series of protein-protein interactions, building acomplex (the death initiating signalling complex or DISC)which eventually recruits and activates caspase 89.These mechanisms for coupling cell injury to apoptosis havemostly depended on activation of pre-formed proteins.Apoptosis can also be initiated (and forestalled) by transcriptional mechanisms, although rather little is known aboutmost of them. An outstanding example is the Drosophilagene reaper, transcriptionally activated around two hoursprior to developmental and injury-induced deaths in thisorganism.

Drosophila apoptosis can occur without reapertransactivation, but requires very substantially enhancedstimuli, suggesting that reaper adjusts a threshold for apoptosis initiation10. Another gene whose transcription can initiate death is the familiar immediate early gene c-myc11.Transcriptional activation of c-myc initiates entry into DNAsynthesis and is required for sustained re-entry in repeatedcell cycles, but c-myc activation in the absence of concurrentcytokine support triggers apoptosis. This can also be interpreted as a “threshold regulatory” effect: – c-myc expressionincreases the cellular requirement for survival factors such asIGF-1.Impressive confirmation of the significance of these pathways to apoptosis is available from study of transformingviruses. These are hardened survivors in the labyrinth of cellregulation, and have found keys to allow escape from celldeath in a variety of ways.

Thus the transforming papovavirus SV40, adenovirus type 12, Human Papilloma Virus type16 and Epstein-Barr Virus all have proteins that inactivateapoptosis through inactivation of p53 or binding of BAX12.Even lytic viruses possess mechanisms to postpone death,such as the cowpox crmA serpin protein and the baculovirusp35 protein, which are caspase inhibitors.So far so good: there are transcriptional and non-transcriptional pathways for activation of apoptosis, and they playthrough common effector events mediated by caspases andregulated by members of the BCL2 family.

Underlying thissimple scheme, however, is an extraordinary complexity.Thus, inactivation of fas signalling appears to neuter the ability of both c-myc and p53 to initiate apoptosis13,14. Maybefas signalling is yet another example of “threshold regulation”. New proteins have been discovered that are recruitedto the DISC but appear to inhibit rather than activatedeath15, some of them of viral origin. Many of the proteinsmentioned above have alternative splice variants that haveopposite effects.

And we still have little idea of the relevanceof intracellular location or of cell lineage to the activity ofmost of the apoptosis proteins. Susceptibility to apoptosiscan be influenced by many other gene products, includingoncoproteins such as RAS and ABL16, but in some cases asingle oncoprotein may either increase or decrease susceptibility depending on the context.

Perhaps it is not surprisingthat a cellular function as important and irreversible as deathshould be subject to a huge range of coarse and fine controls.The reagents and protocols in this book should help unravelthese.Andrew H. Wyllie FRS,Professor of Experimental Pathology,Sir Alastair Currie CRC Laboratories, University Medical School,Edinburgh, ScotlandReferences1. Kerr, J.

R. F., Wyllie, A. H., Currie, A. R. (1972) Apoptosis: a basic biologicalphenomenon with wide-ranging implications in tissue kinetics. Br. J.Cancer 26, 239–257.2. Hengartner, M. O., Horvitz, H. R. (1994) The ins and outs of programmedcell death during C. elegans development. Phil. Trans. R. Soc. Lond. B 345,243–248.3. Thornberry, N.A. (1997) The caspase family of cysteine proteases. Brit.Med.

Bull. 53, 478–490.4. Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., Nagata, S.(1998) A caspase-activated DNAse that degrades DNA during apoptosis,and its inhibitor ICAD. Nature 391, 43–50.5. Zou, H., Henzel, W. J., Liu, X., Lutschg, A., Wang, X. (1997) Apaf-1, a humanprotein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase 3. Cell 90, 405–413.6. Oltvai, Z.

N., Milliman, C. L., Korsmeyer, S. J. (1993) Bcl-2 heterodimerisesin vivo with a conserved homologue BAX, that accelerates programmed celldeath. Cell 74, 609–619.7. Miyashita, T., Reed, J. C. (1995) Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80, 293–299.8. Jarvis, D. W., Kolesnick, R. N., Fornari, F. A., Traylor, R. S., Gewirtz, D. A.,Grant, S.

(1994) Induction of apoptotic DNA degradation and cell death byactivation of the sphingomyelin pathway. Proc. Natl. Acad. Sci. USA 91,73–77.9. Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O’Rourke, K., Shevchenko, A.,Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer,P. H., Peter, M. E., Dixit, V. M. (1996) FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD 95 (FAS/APO-1) death-inducingsignalling complex. Cell 85, 817–827.10. White, K., Tahaoglu, E., Steller, H.

(1996) Cell killing by the Drosophila genereaper. Science 271, 805–807.11. Evan, G. I., Wyllie, A. H., Gilbert, C. S., Land, H., Brooks, M., Littlewood, T.,Waters, C., Hancock, D. (1992) Induction of apoptosis in fibroblasts byc-myc protein. Cell 69, 119–128.12. Young, L. S., Dawson, C. W., Eliopoulos, A. G.

(1997) Viruses and apoptosis.Brit. Med. Bull. 53, 509–521.13. Hueber, A. O., Zornig, M., Lyon, D., Suda, T., Nagata, S., Evan, G. I. (1997)Requirement for the CD95 receptor-ligand pathway in c-myc-inducedapoptosis. Science 278, 1305–1309.14. Krammer, P. H. (1997) The tumor strikes back: new data on expression ofthe CD-95 (APO-1/Fas) receptor/ligand system may cause paradigmchanges in our view on drug treatment and tumor immunology. Cell Deathand Differentiation 4, 362–364.15. Irmler, M., Thorne, M., Hanne, M., Schneider, P., Hofmann, B., Steiner, V.,Bodmer, J. L., Schroter, M., Burns, K., Mattmann, C., Rimoldi, D.,French, L. E., Tschopp, J.

(1997) Inhibition of death receptor signals bycellular FLIP. Nature 388, 190–195.16. Evan, G. (1997) A question of DNA repair. Nature 387, 450.VIIt!ewN:sucdorpwnetseLatNew antibody for the reliable detection of apoptosis.– A unique tool for the easy and reliable determination of early apoptotic events in epithelial cells. –M30 CytoDEATH*Cat.

No. 2 140 322Cat. No. 2 140 34950 tests250 testsTypeMonoclonal antibody, clone M30, IgG2b, mouseUseful forDetection of apoptosis in epithelial cells and tissues (formalin grade)SamplesAdherent cells, tissue samples (routinely fixed and paraffin - embedded tissue sections, cryostat sections)MethodDetect apoptosis by applying the M30-antibody to fixed samples, then using secondary detection systems. Suitable for immunohistochemistry, immunocytochemistry, and flow cytometryTime2 h for immunofluorescence on cells, 3.5h for staining of tissues (excluding dewaxing)Background: During Apoptosis, vital intracellular proteinsare cleaved.

The proteases that mediate this process are called caspases (Cysteinyl-aspartic acid proteases). Caspasesare expressed as zymogenes, which are activated by differentapoptosis inducers. Once activated, a single caspase activatesa cascade of caspases.for immunofluorescence on cells:1. Fix cells.2. Add M30 antibody.3. Add Anti-Mouse-Ig-Fluorescein.4. Analyze under a fluorescence microscope.Until recently cytokeratins, in particular cytokeratin 18,were not known to be affected by early events of apoptosis.Recently , it has been shown that the M30 antibody recognizes a specific caspase cleavage site within cytokeratin 18 thatis not detectable in native CK18 of normal cells (Leers et al.,in preparation).

Consequently, the M30 CytoDEATH antibody is a unique tool for the easy and reliable determinationof very early apoptotic events in single cells and tissue sections.Staining procedure for immunohistochemistry and flow cytometry (FCM)Wash cells with PBSFix cells in ice-cold pure methanol (–20°C) for 30 minWash cells twice with PBS/BSAIncubate with M30 working solution (for 30 min, at RT)Significance of reagent: Use the M30 CytoDEATH antibody for the determination of early apoptotic events in cellsand tissue sections by detection of a specific epitope of cytokeratin 18 that is presented after cleavage by caspases.Wash cells twice with PBSIncubate with Anti-Mouse-Ig-Fluorescein3 (for 30 min, at RT)Test principlefor formalin-embedded tissue:1. Dewax formalin-fixed, paraffin-embedded tissue sections.2. Retrieve antigen by heating in citric acid buffer.3.

Add M30 antibody.4. Add Anti-Mouse-Biotin.5. Add Streptavidin-POD.6. Add substrate solution (DAB or AEC).7. Counterstain with Harries hematoxilin.8. Analyze under a light microscope.VIIIWash cells twice with PBSFor flow cytometry, dilute cells in PBS, and store the samples in the darkuntil analysis.1. Anti-Mouse-Biotin, Cat. No.

1089 2852. Streptavidin-POD, Cat. No. 1089 1533. Anti-Mouse-Ig-Fluorescein, Cat. No. 1214 616* The M30 CytoDEATH antibody is made under a license agreement form BEKIAB/BEKI Diagnostics AB, Sweden.Staining procedure for immunohistochemistryIncubate paraffin-embedded sections (over night) at 37°CDewax formalin-fixed, paraffin-embedded tissue sections:2x xylol, 2x ethanol (96%), 1x ethanol (70%), 1x methanol/H2O2 (3%)(10 min, RT). Rinse for 10 min in demineralized waterSpecificity: The M30 CytoDEATH antibody binds to a caspase-cleaved, formalin-resistant epitope of the cytokeratin18 (CK 18) cytoskeletal protein.The immunoreactivity of the M30 CytoDEATH antibodyconfined to the cytoplasma of apoptotic cells.Antibody supplied as: Mouse monoclonal antibody (cloneM30), lyophilized, stabilized.