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Signal transduction pathways to apoptosis
!
Signal transduction pathways to apoptosis
these regulate cellular metabolism and gene expression. Thus, it is perhaps not surprising that several familiar signal transduction pathways (Fig. 1) have recently been implicated in the positive and negative regulation of apoptosis in cells of diverse tissue origins. Regulation by Ca z÷
Recent work has demonstrated that a number of si~lalling events, including cytosolic Caz÷rises, cAMP accumulation, activation of protein kinase C, activation of protein tyrosine kinases, and production of ceramide, regulate apoptosis in diverse model systems. However, in some cells these signals promote apoptosis, whereas in others thc~,block the response. This review discusses these observations and proposes explanations for how a given set of signal transduction systems might be involved in multiple cellular responses.
Apoptosis Is a highly regulated process of cell death. Like cell proliferation and differentiation, It is controlled by hormonal and other receptor-mediated cues (Box 1). Receptor.mediated cellular control mechanisms In general act through a limited set of signal transductlon systems that regulate the release of 'second messenger' molec,,les and the activation of protein kinase/phosphatase 'c~sf'adcs'; by altering the phosphorylatlon status of key target proteins,
BOX I - CHARACTERISTICS OF APOPTOSlS
David McConkey is at the Dept of Cell Biology, Box 173, The Universityof Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA;and Sten Orrenius is at the Institute of Env,onmental Medicine, Karolinska Institutet, Box 2i0, 5-171 77 Stockholm, Sweden. 370
The elimination of cells through apoptosls is important in development, normal cell turnover, hormone-induced tissue atrophy, and pathological processes such as T-cell depletion in AIDS and neural degeneration in Huntington's chorea and Alzheimer's disease. Cells undergoing apop. tosis show characteristic morphological changes, in. cluding plasma and nuclear membrane blebbing, cell shrinkage, and chromatin condensation and fragmen. ration ~6. These changes distinguish apoptosis from cell death by necrosis. In most cells, the biochemical chardcteristics of the apoptosis response include endogenous endonuclease and pro. tease activation - leading to th~ production at first of large (50-300 kb) DNA ;ragments and later of oligonucleosomal DNA fragments 67,68('ladders') - aswell as transglutaminase activation. All mammalian cells (with the possible exception of blastomeres) appear to constitutively express the basic enzymatic machinery that mediates .~poptotic cell death, and Ca2÷is implicated in the activation of these processesin many experimental models, suggesting that it may play a major regulatory role in the response.
© 1994 ElsevierScienceLtd 0962.8924/94/$07.00
Activation of various isozymes of phospholipase C (PLC) is a common consequence of the binding of surface receptors to their ligands, and leads to the generation of the second messengers diacylglycerol and inositol (1,4,S)-trisphosphate [Ins(1,4,S)P3]. Diacylglycerol promotes the activation of a family of serine/threonine protein kinases known collectively as protein kinase C (PKC, discussed below), whereas Ins(1,4,5)P3 promotes Ca z+ release from intracellul,~r stores via Ins(1,4,S)P3 receptors located in the endoplasmic reticulum (ER) and nuclear membrane. The effects of lns(1,4,5)P3 can be mimicked by pharmacological agents: thapsigargin increases cytosolic Ca 2+ by inhibiting the ATP-dependent ER Ca z+ pump, thereby promoting Ca 2+ release from intracellular stores and influx of extracellular Ca 2+ via a capacitative mechanism; and Ca 2+ionophores such as ionomycin or A23187 increase intracellular Ca 2+by facilitating Ca z+ influx across the plasma membrane. In lymphocytes, combined treatment of cells with Ca 2+ ionophores and PKC activators (i.e. phorbol esters) can reproduce all the downstream biological re. sponses normally promoted by PLC stimulation. Several lines of evidence indicate that the cytosollc Ca 2+ concentration can regulate apoptosis. In many cells, apoptosls Is induced by treatment with thapslgargin or Ca 2* lonophores 14. The process can also be triggered in thymocytes (immature T cells) or primed mature T cells by stimulation of the T-cell receptor s,6 and In neurons by stimulation of glutamate (NMDA) receptors ~, and both these responses involve sustained Ca 2~ increases. Certain chemical toxins 8,9 may also promote apoptosis by stimulating PLC and disrupting lntraceilular Ca 2~' homeostasis, leading to nonphysiologlcal Ca 2. increases that promote endonuclease activation and apoptotlc cell death. Intracellular or extraceUular Ca 2. chelators, CaZ÷-channei blockers and calmodulln antagonists can all delay or abolish apoptosis in several model systems2"~,~0, Consistent with these effects, overexpression of the Ca2*-binding protein calbindln D-28K can block apoptotic cell death ~1. Finally, recent work suggests that the protective effects of the anti-apoptosis oncoprotein Bcl.2 involve alterations In Ca 2÷ compartmentalization 12, Together, these observations indicate that Ca 2. is a frequent trigger of apoptosis In diverse experimental systems. In other cellular systems, however, increases in cytosolic Ca 2÷ block apoptotic cell death. For example, treatment of haematopoietic cells dependent on interleukin (IL) 3 with Ca 2÷ ionophores blocks endogenous endonuclease activation and cell death following withdrawal of IL-3I'~. In addition, Ca 2÷ ionophores block apoptosis in aged neutrophils~4; and membrane depolarization in neurons, which leads to Ca 2÷ increases, can prevent apoptosis in TRENDS IN CELL BIOLOGY VOL. 4 OCTOBER 1994
- m response to withdrawal of nerve growth factor (NGF) in dependent cells is. The cellular targets for Ca 2+in initiating apoptosis are the subject of intense investigation (Fig. 2), One important insight has come from the observation that the immunosuppressants cyclosporin A and FKS06 can block apoptosis in some model systemsl6.]L Because these agents act by inhibiting the CaZ+--calmodulin-dependent protein phosphatase calcineurin TM,the finding strongly suggests that activation of the phosphatase may be involved in Caz+triggered apoptosis. Other targets may include CaZ+--calmodulin-dependent protease(s) and the endonuclease that is responsible for degrading chromatin during apoptosis: activation of the CaZ*-dependent neutral protease calpain has recently been shown to be involved in glucocorticoid- and irradiationinduced thymocyte apoptosis ~9, and the Ca2+dependency of endonuclease activation in a variety of model systems is well established. In addition, C # + increases may promote apoptosis by altering the activity of particular transcription factors such as Fos, Jun, Nur7720 or the cAMP-response-element-binding protein CREB, leading to changes in gene expression that may be required for the response. Protein kinase C
Most of the evidence supporting a role for PKC in apoptosis comes from studies with phorbol esters, a class of tumour promoters that act by binding to the dlacylglycerol-bindtng site on the enzyme and promoting its activation. Phorbol ester treatment stimulates apoptosls in thymocytes 2t and some (but not all) T-cell lines (D. J. McConkey, unpublished). J. Lord and colleagues (pers. commun.) have shown that phorbol esters that selectively promote activation of the PKC-I~ isoform can also stimulate apoptosis in myeloid leukaemia cells. Furthermore, the glucocortl. cold-induced apoptosls in thymocytes may involve selective activation and transiocation of PKC-~=~2, Conversely, phorbol esters and other activators of PKC can block apoptotlc cell death in thymocytes s,~t, leukaemic B cells2s,24, a human mammary adenocarcinema cell line (BT-20)2s, human synovlal cells 2°, IL.3-dependent haematopoietic cells 27, and kidney epithelial cells2s. Phorbol esters can also block apop. tosis mediated by tumour necrosis factor (TNF)2s,29. Moreover, PKC antagonists can stimulate :~poptosisa°; indeed, the protein kinase inhibitor staurosporine is being used widely as a 'universal' trigger of apoptosis (see for example Ref. 31). Whether or not the actions of these inhibitors, which are notoriously nonspecific, require inhibition of PKC has not yet been determined. Again, identification of the targets of phorbol esters and PKC that are important to the regulation of apoptosis is a current goal. Several groups are analysing PKC movement/relocation in apoptotic cells, with the focus on translocation to and enzymatic activation within the nucleus. Likely targets in the nucleus include AP-1, a transcription factor composed of Fos-Jun dimers that serves as a common target for signal transduction through Ras. The effects of phorbol esters and other PKC activators in TP,~NDSIN CELLBIOLOGYVOL. 4 OCTOBER1994
A .,.lCa2~
/" /
Calmodulin Calcineurin
Phorbol esters Diacylglycerol
xx ~
J. _~_
A TcAMP
/ /
PKA /
Change in chromatin structure Protease activation Endonuclease activation DNA fragmentation Cell death FIGURE 1
Signal transduction pathways in thymocyteapoptosis. Physiologicalor pathological agents that elevate the cytosolicCa2+concentration stimulate apoptosis in thymocytesvia a mechanismthat appears to involve calmodulindependent processes,including in some casesthe Ca2+-calmodulin-dependent protein phosphatasecalcineurin.Alternatively,prostaglandin Eor pharmacological stimulators of cAMP production can promote endonucleaseactivation and cell death via activation of cAMP-dependentprotein kinase (PKA). Phorbol estersand glucocorticoids may promote apoptosis in thymocytesvia activation of various isoforms of protein kinaseC (PKC),or they can block apoptosistriggered by the other pathways, presumablyalso via PKC and possibly Ras,which is activated by phorbol ester treatment in lymphocytes.
lymphocytes require Ras activation. Phorbol esters and diacylglyceroi may activate Ras through PKC in some systems. Additionally, recent work has shown that they can directly bind to and activate the Ras GTP-GDP exchange protein Vav, leading to Ras activation independently of PKC'~2. On the one hand, an inhibitory role for Ras in apoptosis is suggested by the finding that active Ras blocks apoptosis in a rat fibroblast cell line, an effect that may be mediated by reducing the availability of Ca2*-dependent nuclease a:~. On the other hand, active Ras promotes apoptosis and nuclease avail. ability in a murine fibroblast line (lOTl/2), an effect that can be countered by overexpression of bcl-2 (D. J. McConkey, unpublished). Similarly, Ras activation mediates Fas-induced apoptosis in Jurkat cells (D. R. Green and colleagues, pers. commun.). Thus, like PKC activators, Ras is implicated in both pro meting and inhibiting apoptosis. cAMP
Studies of programmed cell death within the secondary palatal epithelium during palatal fusion provided the first direct evidence for a role for cAMP in promoting apoptosis "~4.Pharmacological cAMP agonists are also known to be cytotoxic to certain lymphoid lines in vitro 3s, and the effects of cAMP involve changes in protein phosphorylation that lead to apoptosis :~'. Furthermore, agents that elevate cAMP stimulate DNA fragmentation and apoptosis in thymocytes via activation of cAMP-dependent protein kinase (PKA)w. However, evidence that cAMP can block apoptosis in other model systems is also 371
cytoplasmic protein tyrosine kinases (PTKs). It is perhaps not surprising, therefore, that PTKs are now being implicated in the regldation of apoptosis. The strongest evidence for their involvement comes from the observation that transfected cells overproducing a constitutively active form of the PTK Abl are resistant to induction of apoptosis by growth factor withdrawal or treatment with chemotherapeutic chemica:~4z, Moreover, treatment of the apoptosis-resistant chronic myc!ogenous le-!:~-~,,a (CML) cell line K562 with antisense oligonucleotides to abl restores its ability to undergo apoptosis 4~. These observations appear clinically relevant, as they may explain why CML cells develop resistance to chemotherapy. Like the other signalling molecules, PTKs have been implicated in promoting apoptosis as well as inhibiting it. Ionizing radiation promotes PTK activation that appears to be required for apoptosis in B cells44; and engagement of the Thy-1 antigen on CD4*CD8 ÷ thymocytes leads to potentiation of T-cell-receptor-mediated apoptosis, via activation of Protein tyrosine kinases the FI"K p56 t'* and increases in substrate tyrosine All physiological survival factors that suppress phosphorylation 4s. More recently, we have found apoptosis act through receptors that regulate that engagement of the T-cell surface antigens CD4 or CD8 also promotes T-cell-receptor-mediated apoptosis via a Extracellular similar mechanism 4~'.In this system one stimulus of the important PTK substrates is PLC. Given their ubiquitous roles, it is likely that the components of the PLC and Ras ~Ins(4,5)p 2 DAG signalling pathways will represent genPt.C.coupled ~ / / eral targets of FrKs in apoptosis. surface receptor o ~ GTP-blndlng / / ~. proteins....... (= / emerging. Analogues of cAMP inhibit apoptosis in neurons induced by withdrawal of NGP s, in aged neutrophils (C. Haslett, pets. conmmn.), and in T-cell hybridomas following triggering of the T-cell receptor as. Thus, elevations in cAMP can have opposite effects on apoptosis depending on the cellular context. The substrates targeted by cAMP-activated protein kinases and phosphatases remain largely unknown. One extremely good candidate is the transcription factor CREB, which mediates the effects of cAMP in many physiological responses. Additionally, cAMP can interfere with the PLC pathway, leading to inhibition of Ca z÷ responses and diacylglycerol production 39. cAMP inhibits the Ras pathway in fibroblasts via phosphorylation and inhibition of the protein serine/threonine kinase Raf-14°,4~,whereas in neurons cAMP can stimulate Ras. Both these observations are consistent with the divergent effects of cAMP on apoptosis in these models.
Ceramlde Some receptors stimulate sphingo. myeUnase and thus cause the hydrolysis o~:' spldngomyelhl when they bind ~",,\2-'~ ~ ; i::i:,=::~_-~ receptor "J their ligands, leading to the release of ionoohc dlacylglycerol and ceramide. Recent work suggests that ceramkte is a second messenger that may activate a protein klnase cascade 47. The Fas antigen and the TNF receptor trigger apoptosis in sensitive target cells, and share a region Proteases J of similarity in their cytoplasmic tails Phospholipases(?) ~ '~, Transcriptionalregulation that has been imphcated in this proProtein kinases (?) . . . . . . . . ~----I ~" Chromatinunfolding cess. Recent attempts to identify the Protein phosphatases / Endonuclease activation signalling pathways underlying the Cytoskeleton / / actions of TNF and Fas have suggested NUCLEUS that ceramide is a potent stimulator of apoptosls. A rapid increase in ceramide FIGURE2 is observed in TNF-treated flbroblasts, Targets for Caz+action in apoptosis. Ligand binding to surface receptors or treatment with and addition of synthetic ceramide Caz* ionophoresor thapsigargin (an ERC#* ATPaseinhibitor) can result in sustainedcytosolic analogues is sufficient to reproduce all Ca2~increasesthat oromote apoptosis in diverse cell types. Surfacereceptors are coupled to the the events observed following TNF Caz* signalling pathway by heterotrimeric G proteins and/or protein tyrosine kinase(s)that treatment 2'~,4", Increases in ceramide activatevarious phospholipaseC isozymes,thereby leading to the production of Ins(1,4,S)P3and have also been observed during Fasdiacylglycerol (DAG) via hydrolysisof phosphatidylinositolbisphosphate [Ptdlns(4,S)Pz]. mediated apoptosis in Jurkat T cells Ins(1,4,S)P~ promotes releaseof Ca2÷from the endoplasmicreticulum (ER)and, indirectly, also (D. R. Green, pets. commun.), in this Caz* influx across the plasma membranethrough a capacitative mechanism. Both the emptying system, Fas engagement can apparently of the ERCaa* store and the subsequent stimulation of Ca2÷influx can be mimicked by be mimicked by exogenous sphingothapsigargin. The influx of extracellularCaz* ,:an also be achieved by treatment with Caz• myelinase, and it appears that Ras is the ionophores. Ca2÷can mediateall the cytosolic and nuclearevents of apoptosis in thymocytes downstream target in the response. and in some other cell types. Howevar, in other models Ca2. appears to block apoptosis. However, the observation that protein 372
TRENDSIN CELLBIOLOGYVOL. 4 OCTOBER1994
m kinase inhibitors fail to block Fas-mediated killing leaves a role for a ceramide-activated ldnase cascade in question at present.
Role of pH changes Recent evidence suggests that chemotherapeutic agents for cancer stimulate endonuclease activation in target cells. Analysis of the biochemical mechanisms underlying this response in Chinese hamster ovary (CHO) cells indicated that changes in the cytosolic Ca 2+concentration were not involved, but implicated intracellular acidification as the trigger49. A pH-sensitive endonuclease (DNase II) may mediate DNA fragmentation in systems where Caz+ does not appear to be involveds°. However, the biochemical mechanism(s) underlying intracellular acidification have not been established, and the pH at which the enzyme is maximally active (pH 5.5) is usually seen only in the lysosomal compartment of cells. Oxygen radicals The production of overwhelming levels of reactive oxygen species ('oxidative stress') is thought to be an important mechanism of cytotoxicity in a number of systems. Oxidative 'bursts' may also serve roles in cellular activation, particularly In macrophages, and nitric oxide is an important and common second messenger. Oxygen radical production is now also being implicated in the control of apoptosls. Early work suggested that oxidative stress could lead to a response similar to apoptosls in isolated hepatocytesSL In addition, low concentrations of the oxidant hydrogen peroxide induce apoptosis in HL.60 cells, while necrosis occurs when mtillmolar amounts o f the oxidant are present s2. However, antloxidants may protect cells from apoptosls (see Ref. 53 for a recent review). For example, apoptotle cell death induced by TNF can be prevented by antloxidants such as N.acetylcystelne (NAC) and thloredoxin s4,ss or overexpresslon of mltochondrlal superoxlde dismutase s6. Moreover, we have recently found that antloxidants such as the nltroxlde oxygen radical spin trap 5,5'.dlmethyl-l-pyrrollne-N-oxlde (DMPO), and Its tetramethyl form TMPO, can inhibit thymocyte apoptosls Induced by glucocortlcolds, thapslgargln, and the topoisomerase II inhibitor etoposide (A. F. G. Slater, S. Nobel a r d S. Orrenlus, unpublished). The recent discovery that the Bci-2 protein has antioxidant properties further emphasizes the probable importance of oxidative events in apoptosis sT,ss. These results suggest that oxidation Is required at some point(s) if a cell is to undergo apoptosis successfully. There are several possible roles for oxidation in apoptosis. First, protein oxidation may be essential to change gene transcription such that the apoptotic pathway is initiated. Several transcription factors, e.g. Fos and Jun sg, have crucial cysteine residues involved in DNA binding and thiol oxidation could cause a large decrease in the affinity of DNA binding. By contrast, the DNA-binding activity of the transcription factor NF-KB is activated by oxidative events (via enhanced degradation of its inhibitory factor lgB), thereby initiating transcription TRENDS IN CELLBIOLOGYVOL. 4 OCTOBER1994
of NF-~B-responsive genes ~. However, it is also possible that oxidative events are required at later stages in apoptosis, for example to initiate either cell shrinkage or changes in higher-order chromatin structure.
Crosstalk and genetics influence diverse responses to apoptosis signals One recurrent theme of this review is that signal transduction pathways have contradictory effects on apoptosis depending upon the experimental model in question. Indeed, disparate cell responses to receptot engagement have been noted for several models of apoptosis. For example, triggering of the T-cell receptor can lead to either apoptosis or differentiation of thymocytes, and treatment with TNF or antibodies to the Fas antigen can lead to either apoptosis or proliferation depending on the cellular systern. It is increasingly clear tha' !. ~~liferation, differentiation and apoptosis are closely related alternative cellular responses that share many of the same mob ecular mechanisms. An important challenge for ongoing research is to determine precisely how they differ. Experiments with cytokines or pharmacological mimics of second messengers indicate that cell proliferation usually requires at least two independent signals. This is perhaps best demonstrated by T cells, for which treatment with both a Ca2+ionophore and a phorbol ester, but not either alone, efficiently stimulates IL-2 production and proliferation. The molecular basis for this requirement for two signals is well documented and involves elements within the IL-2 promoter that selectively respond to Ca z÷ o: PKC61. With this in mind, we propose that apoptosis may result from imbalanced signal transduction, when an lntracellular Ca 2÷ increase or other signal transduction pathway is initiated in the absence of an appropriate second (or multiple) signal(s) (Fig. 1). Phorbol esters often block apoptosls, suggesting that PKC and Ras are targets for one such second signal. The presence or absence of crosstalk among sig. nailing pathways may therefore be one general mechanism for promoting divergent cellular effects in response to a shared set of signal transduction pathways. Recent work on the developmental regulation of bcl-2 expression suggests that cells can be induced to be more or les predisposed to undergo apoptosis in response to a particular signal. In the thymus, the CD4+CD8+ subpopulation of cells is known to be sensitive to diverse apoptotic stimuli, whereas CD4-CD8- precursors and CD4÷CD8"/CD4"CD8 ÷ mature thymocytes are not. Interestingly, bcl-2 expression is relatively high in the CD4-CD8- precursors, it drops off in the CD4+CD8' cells, and then its expression is elevated again in the m a t t e cells. This pattern precisely parallels the sensitivity of the cells to apoptosis 6z. It appears that Bcl-2 is regulated similarly in B cells during development. It is possible that alterations in other apoptosis-active gene products occur during development or tumour progression, where changes in sensitivity to apeptosis are observed. Thus, genetic programming may alter the manner in which a particular signal is ;eceived 373
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by the cell, and perhaps an apoptotic signal is translated into a differentiation signal within the appropriate genetic context. Determination of the relative contributions of crosstalk and programming to the regulation of apoptosis clearly requires further investigation. Signal transduction pathways ultimately influence gene regulators. For example, Fos63, Myc64 and Nur772° are transcription factors that have been implicated in some forms of apoptosis and in growth factor signal transduction pathways. Additionally, the tumour suppressor protein p53 can promote apoptosis 6s, and it is known that p53 is a phosphoprotein. Precisely how signal transduction pathways determine whether these factors mediate cell division or death is an important question for the future.
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No more slipped discs? C u r r e n t Protocols o n CD-ROM
Greene Publishing Associates, and John Wiley and Sons, 1994. Current Protocols in Molecular Biology/Current Protocols in Immunology on CD-ROM (CD containing manual and licensed software, a program manual containing hard copy of ull figures, and quarterly updates on CD) .~4 90. 00. One.year licence renewal $245.00. Most people have become familiar with the hard-back Current Protocols (CP) manuals over recent years. These huge volumes (with the ingenious me~'.al ratchet to allow easy photocopying) adorn the shelves of many labs and are regularly updated with the very latest techniques. As the fields of molecular biology and immunology seemingly grow exponentially, the size of the CP manuals increase accordingly. With volumes weighing in at around 3.4 kg each, a trip to the photocopier with both molecular biology volumes could be dangerously bad for the back. An additional problem occurs as manuals grow ever larger: user friendliness is reduced and people are often driven to smaller, more specialist manuals, and so lose the benefits of a broad-based manual. Many publishers have promised the panacea of the ultimate manual: 'hands.on" protocols from people who use them all the time, with extensive references and background information. However, few have been able to deliver. With the CD-ROM versions of CP, there is light at the end of the tunnel, not to mention
64 EVAN,G. I. et al. (1992) Cell69, 119-128 65 YONISH-ROUACH,E. et al. (1991) Nature352, 345-347 66 WYLLIE,A. H., KERR,J. F. R. and CURRIE,A. R.(1980)/nt. Rev. Cytol. 68, 251-305 67 BROWN,D. G., SUN,X. M. and COHEN,G. M. (1993)/. Biol. Chem.268, 3037-3039 68 WYLUE,A. H. (1980) Nature284, 555-556
a much reduced chance of that slipped disc! We installed CP in Molecular Biology and CP in Immunology on CD-ROM easily in just a few minutes, all the software being included on the disk*. A user directory is established on the hard disk, which allows rapid use of CP thereafter. The manual opens as a series of contents pages, and you can progress through levels of these by clicking with the mouse onto the desired chapters, subject headings or specific sections. The layout of the control panels is clear, with push bars for printing, returning to the contents pages, page flicking and so on. The excellent, tiered structure of the hardcopy manual has been retained, so introductory sections lead into detailed protocols, with supporting lists of reagent and literature. Using the manual like a book, it is possible to browse through tiers of pages, but several aids are F~resentto make life far easier. The particular strength of CPon CD. RaM is the ability to search the complete volume with various levels of stringency. This process will search for the keyword or phrases you desire, and will even suggest a list of related words (this is particularly useful when the spelling is uncertain). All the matches are rapidly brought up on screen, listed from best to worst match, and you can browse your way through. Several additional features for getting around the manual allow you to exploit its contents. A cross.referencing system comes into its own for moving around quickly. Text that is cross.referenced is underlined in the manual and a 'double-click' with the mouse on this text takes you immediately to the desired section. You can read, save or print this and then simply return Lo your original position by closing the page. At any stage, you can leave yourself and others a note of modifications or comments that you have on a particular page. This 'sticky notes' ~eature, which appears on the screen as a familiar yellow r~ote pad,
TRENDS IN CELLBIOLOGYVOL. 4 OCTOBER1994
allows you to annotate your manual. Similarly, you can leave 'bookmarks', the electronic equivalent of earmarked pages, so that upon your return you can flick from section to section extremely easily. Bookmarks and sticky page notes can be given customized titles, and are retained in the manual unlessdeleted by the user. In such a way, you can create your own index of sections that are used repeatedly. The manual is particularly useful for manipulating text. The fonts and sizes of text can all be altered to suit you. Ever felt like making your own labbook with all those frequently used techniques? Now is your chance. Any page, section or line can be copied, either by printing directly, or by saving and dumping into a word processor. What this means is that an almost unlimited source of information can be easily manipulated. There will no doubt be many graduate students facing a blank thesis page who will gain 'inspiration' from such a facilityl Extolling the virtues of this manual is easy, and pages can be written about the features it contains, but we could find very little to fault it. As is often the case with diagrams on CD.ROM, the figures are of low resolution and a hard copy of each is provided in the accompanying manual. This dG~; not often interfere with its use, bt~t will hopefully be improved upon in later versions of the manual. For most people, a major limitation of CP on CD-ROM is the price. CP on CD.ROM will certainly be most useful on a computer network, but it is not clear what the total yeady subscription will be for a site licence. In summary, CP in Molecular Biology and CPin It.~munologyon CD-ROM are excellent manuals, and while they contain the same text as the hardcopy version, the benefit of the CDRaM version is the ease of use. Many publishers entering the electronic age could learn a lesson from the degree of user.friendliness within this software.
*The softwarecan be run on either Apple Macintosh or IBM-PCcompatible computers. The CD wastested on a MacintoshII ci with an external CD600drive.
M~rk HIrst and Chades Bangham Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, UIC 375
Signal transduction pathways to apoptosis
these regulate cellular metabolism and gene expression. Thus, it is perhaps not surprising that several familiar signal transduction pathways (Fig. 1) have recently been implicated in the positive and negative regulation of apoptosis in cells of diverse tissue origins. Regulation by Ca z÷
Recent work has demonstrated that a number of si~lalling events, including cytosolic Caz÷rises, cAMP accumulation, activation of protein kinase C, activation of protein tyrosine kinases, and production of ceramide, regulate apoptosis in diverse model systems. However, in some cells these signals promote apoptosis, whereas in others thc~,block the response. This review discusses these observations and proposes explanations for how a given set of signal transduction systems might be involved in multiple cellular responses.
Apoptosis Is a highly regulated process of cell death. Like cell proliferation and differentiation, It is controlled by hormonal and other receptor-mediated cues (Box 1). Receptor.mediated cellular control mechanisms In general act through a limited set of signal transductlon systems that regulate the release of 'second messenger' molec,,les and the activation of protein kinase/phosphatase 'c~sf'adcs'; by altering the phosphorylatlon status of key target proteins,
BOX I - CHARACTERISTICS OF APOPTOSlS
David McConkey is at the Dept of Cell Biology, Box 173, The Universityof Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA;and Sten Orrenius is at the Institute of Env,onmental Medicine, Karolinska Institutet, Box 2i0, 5-171 77 Stockholm, Sweden. 370
The elimination of cells through apoptosls is important in development, normal cell turnover, hormone-induced tissue atrophy, and pathological processes such as T-cell depletion in AIDS and neural degeneration in Huntington's chorea and Alzheimer's disease. Cells undergoing apop. tosis show characteristic morphological changes, in. cluding plasma and nuclear membrane blebbing, cell shrinkage, and chromatin condensation and fragmen. ration ~6. These changes distinguish apoptosis from cell death by necrosis. In most cells, the biochemical chardcteristics of the apoptosis response include endogenous endonuclease and pro. tease activation - leading to th~ production at first of large (50-300 kb) DNA ;ragments and later of oligonucleosomal DNA fragments 67,68('ladders') - aswell as transglutaminase activation. All mammalian cells (with the possible exception of blastomeres) appear to constitutively express the basic enzymatic machinery that mediates .~poptotic cell death, and Ca2÷is implicated in the activation of these processesin many experimental models, suggesting that it may play a major regulatory role in the response.
© 1994 ElsevierScienceLtd 0962.8924/94/$07.00
Activation of various isozymes of phospholipase C (PLC) is a common consequence of the binding of surface receptors to their ligands, and leads to the generation of the second messengers diacylglycerol and inositol (1,4,S)-trisphosphate [Ins(1,4,S)P3]. Diacylglycerol promotes the activation of a family of serine/threonine protein kinases known collectively as protein kinase C (PKC, discussed below), whereas Ins(1,4,5)P3 promotes Ca z+ release from intracellul,~r stores via Ins(1,4,S)P3 receptors located in the endoplasmic reticulum (ER) and nuclear membrane. The effects of lns(1,4,5)P3 can be mimicked by pharmacological agents: thapsigargin increases cytosolic Ca 2+ by inhibiting the ATP-dependent ER Ca z+ pump, thereby promoting Ca 2+ release from intracellular stores and influx of extracellular Ca 2+ via a capacitative mechanism; and Ca 2+ionophores such as ionomycin or A23187 increase intracellular Ca 2+by facilitating Ca z+ influx across the plasma membrane. In lymphocytes, combined treatment of cells with Ca 2+ ionophores and PKC activators (i.e. phorbol esters) can reproduce all the downstream biological re. sponses normally promoted by PLC stimulation. Several lines of evidence indicate that the cytosollc Ca 2+ concentration can regulate apoptosis. In many cells, apoptosls Is induced by treatment with thapslgargin or Ca 2* lonophores 14. The process can also be triggered in thymocytes (immature T cells) or primed mature T cells by stimulation of the T-cell receptor s,6 and In neurons by stimulation of glutamate (NMDA) receptors ~, and both these responses involve sustained Ca 2~ increases. Certain chemical toxins 8,9 may also promote apoptosis by stimulating PLC and disrupting lntraceilular Ca 2~' homeostasis, leading to nonphysiologlcal Ca 2. increases that promote endonuclease activation and apoptotlc cell death. Intracellular or extraceUular Ca 2. chelators, CaZ÷-channei blockers and calmodulln antagonists can all delay or abolish apoptosis in several model systems2"~,~0, Consistent with these effects, overexpression of the Ca2*-binding protein calbindln D-28K can block apoptotic cell death ~1. Finally, recent work suggests that the protective effects of the anti-apoptosis oncoprotein Bcl.2 involve alterations In Ca 2÷ compartmentalization 12, Together, these observations indicate that Ca 2. is a frequent trigger of apoptosis In diverse experimental systems. In other cellular systems, however, increases in cytosolic Ca 2÷ block apoptotic cell death. For example, treatment of haematopoietic cells dependent on interleukin (IL) 3 with Ca 2÷ ionophores blocks endogenous endonuclease activation and cell death following withdrawal of IL-3I'~. In addition, Ca 2÷ ionophores block apoptosis in aged neutrophils~4; and membrane depolarization in neurons, which leads to Ca 2÷ increases, can prevent apoptosis in TRENDS IN CELL BIOLOGY VOL. 4 OCTOBER 1994
- m response to withdrawal of nerve growth factor (NGF) in dependent cells is. The cellular targets for Ca 2+in initiating apoptosis are the subject of intense investigation (Fig. 2), One important insight has come from the observation that the immunosuppressants cyclosporin A and FKS06 can block apoptosis in some model systemsl6.]L Because these agents act by inhibiting the CaZ+--calmodulin-dependent protein phosphatase calcineurin TM,the finding strongly suggests that activation of the phosphatase may be involved in Caz+triggered apoptosis. Other targets may include CaZ+--calmodulin-dependent protease(s) and the endonuclease that is responsible for degrading chromatin during apoptosis: activation of the CaZ*-dependent neutral protease calpain has recently been shown to be involved in glucocorticoid- and irradiationinduced thymocyte apoptosis ~9, and the Ca2+dependency of endonuclease activation in a variety of model systems is well established. In addition, C # + increases may promote apoptosis by altering the activity of particular transcription factors such as Fos, Jun, Nur7720 or the cAMP-response-element-binding protein CREB, leading to changes in gene expression that may be required for the response. Protein kinase C
Most of the evidence supporting a role for PKC in apoptosis comes from studies with phorbol esters, a class of tumour promoters that act by binding to the dlacylglycerol-bindtng site on the enzyme and promoting its activation. Phorbol ester treatment stimulates apoptosls in thymocytes 2t and some (but not all) T-cell lines (D. J. McConkey, unpublished). J. Lord and colleagues (pers. commun.) have shown that phorbol esters that selectively promote activation of the PKC-I~ isoform can also stimulate apoptosis in myeloid leukaemia cells. Furthermore, the glucocortl. cold-induced apoptosls in thymocytes may involve selective activation and transiocation of PKC-~=~2, Conversely, phorbol esters and other activators of PKC can block apoptotlc cell death in thymocytes s,~t, leukaemic B cells2s,24, a human mammary adenocarcinema cell line (BT-20)2s, human synovlal cells 2°, IL.3-dependent haematopoietic cells 27, and kidney epithelial cells2s. Phorbol esters can also block apop. tosis mediated by tumour necrosis factor (TNF)2s,29. Moreover, PKC antagonists can stimulate :~poptosisa°; indeed, the protein kinase inhibitor staurosporine is being used widely as a 'universal' trigger of apoptosis (see for example Ref. 31). Whether or not the actions of these inhibitors, which are notoriously nonspecific, require inhibition of PKC has not yet been determined. Again, identification of the targets of phorbol esters and PKC that are important to the regulation of apoptosis is a current goal. Several groups are analysing PKC movement/relocation in apoptotic cells, with the focus on translocation to and enzymatic activation within the nucleus. Likely targets in the nucleus include AP-1, a transcription factor composed of Fos-Jun dimers that serves as a common target for signal transduction through Ras. The effects of phorbol esters and other PKC activators in TP,~NDSIN CELLBIOLOGYVOL. 4 OCTOBER1994
A .,.lCa2~
/" /
Calmodulin Calcineurin
Phorbol esters Diacylglycerol
xx ~
J. _~_
A TcAMP
/ /
PKA /
Change in chromatin structure Protease activation Endonuclease activation DNA fragmentation Cell death FIGURE 1
Signal transduction pathways in thymocyteapoptosis. Physiologicalor pathological agents that elevate the cytosolicCa2+concentration stimulate apoptosis in thymocytesvia a mechanismthat appears to involve calmodulindependent processes,including in some casesthe Ca2+-calmodulin-dependent protein phosphatasecalcineurin.Alternatively,prostaglandin Eor pharmacological stimulators of cAMP production can promote endonucleaseactivation and cell death via activation of cAMP-dependentprotein kinase (PKA). Phorbol estersand glucocorticoids may promote apoptosis in thymocytesvia activation of various isoforms of protein kinaseC (PKC),or they can block apoptosistriggered by the other pathways, presumablyalso via PKC and possibly Ras,which is activated by phorbol ester treatment in lymphocytes.
lymphocytes require Ras activation. Phorbol esters and diacylglyceroi may activate Ras through PKC in some systems. Additionally, recent work has shown that they can directly bind to and activate the Ras GTP-GDP exchange protein Vav, leading to Ras activation independently of PKC'~2. On the one hand, an inhibitory role for Ras in apoptosis is suggested by the finding that active Ras blocks apoptosis in a rat fibroblast cell line, an effect that may be mediated by reducing the availability of Ca2*-dependent nuclease a:~. On the other hand, active Ras promotes apoptosis and nuclease avail. ability in a murine fibroblast line (lOTl/2), an effect that can be countered by overexpression of bcl-2 (D. J. McConkey, unpublished). Similarly, Ras activation mediates Fas-induced apoptosis in Jurkat cells (D. R. Green and colleagues, pers. commun.). Thus, like PKC activators, Ras is implicated in both pro meting and inhibiting apoptosis. cAMP
Studies of programmed cell death within the secondary palatal epithelium during palatal fusion provided the first direct evidence for a role for cAMP in promoting apoptosis "~4.Pharmacological cAMP agonists are also known to be cytotoxic to certain lymphoid lines in vitro 3s, and the effects of cAMP involve changes in protein phosphorylation that lead to apoptosis :~'. Furthermore, agents that elevate cAMP stimulate DNA fragmentation and apoptosis in thymocytes via activation of cAMP-dependent protein kinase (PKA)w. However, evidence that cAMP can block apoptosis in other model systems is also 371
cytoplasmic protein tyrosine kinases (PTKs). It is perhaps not surprising, therefore, that PTKs are now being implicated in the regldation of apoptosis. The strongest evidence for their involvement comes from the observation that transfected cells overproducing a constitutively active form of the PTK Abl are resistant to induction of apoptosis by growth factor withdrawal or treatment with chemotherapeutic chemica:~4z, Moreover, treatment of the apoptosis-resistant chronic myc!ogenous le-!:~-~,,a (CML) cell line K562 with antisense oligonucleotides to abl restores its ability to undergo apoptosis 4~. These observations appear clinically relevant, as they may explain why CML cells develop resistance to chemotherapy. Like the other signalling molecules, PTKs have been implicated in promoting apoptosis as well as inhibiting it. Ionizing radiation promotes PTK activation that appears to be required for apoptosis in B cells44; and engagement of the Thy-1 antigen on CD4*CD8 ÷ thymocytes leads to potentiation of T-cell-receptor-mediated apoptosis, via activation of Protein tyrosine kinases the FI"K p56 t'* and increases in substrate tyrosine All physiological survival factors that suppress phosphorylation 4s. More recently, we have found apoptosis act through receptors that regulate that engagement of the T-cell surface antigens CD4 or CD8 also promotes T-cell-receptor-mediated apoptosis via a Extracellular similar mechanism 4~'.In this system one stimulus of the important PTK substrates is PLC. Given their ubiquitous roles, it is likely that the components of the PLC and Ras ~Ins(4,5)p 2 DAG signalling pathways will represent genPt.C.coupled ~ / / eral targets of FrKs in apoptosis. surface receptor o ~ GTP-blndlng / / ~. proteins....... (= / emerging. Analogues of cAMP inhibit apoptosis in neurons induced by withdrawal of NGP s, in aged neutrophils (C. Haslett, pets. conmmn.), and in T-cell hybridomas following triggering of the T-cell receptor as. Thus, elevations in cAMP can have opposite effects on apoptosis depending on the cellular context. The substrates targeted by cAMP-activated protein kinases and phosphatases remain largely unknown. One extremely good candidate is the transcription factor CREB, which mediates the effects of cAMP in many physiological responses. Additionally, cAMP can interfere with the PLC pathway, leading to inhibition of Ca z÷ responses and diacylglycerol production 39. cAMP inhibits the Ras pathway in fibroblasts via phosphorylation and inhibition of the protein serine/threonine kinase Raf-14°,4~,whereas in neurons cAMP can stimulate Ras. Both these observations are consistent with the divergent effects of cAMP on apoptosis in these models.
Ceramlde Some receptors stimulate sphingo. myeUnase and thus cause the hydrolysis o~:' spldngomyelhl when they bind ~",,\2-'~ ~ ; i::i:,=::~_-~ receptor "J their ligands, leading to the release of ionoohc dlacylglycerol and ceramide. Recent work suggests that ceramkte is a second messenger that may activate a protein klnase cascade 47. The Fas antigen and the TNF receptor trigger apoptosis in sensitive target cells, and share a region Proteases J of similarity in their cytoplasmic tails Phospholipases(?) ~ '~, Transcriptionalregulation that has been imphcated in this proProtein kinases (?) . . . . . . . . ~----I ~" Chromatinunfolding cess. Recent attempts to identify the Protein phosphatases / Endonuclease activation signalling pathways underlying the Cytoskeleton / / actions of TNF and Fas have suggested NUCLEUS that ceramide is a potent stimulator of apoptosls. A rapid increase in ceramide FIGURE2 is observed in TNF-treated flbroblasts, Targets for Caz+action in apoptosis. Ligand binding to surface receptors or treatment with and addition of synthetic ceramide Caz* ionophoresor thapsigargin (an ERC#* ATPaseinhibitor) can result in sustainedcytosolic analogues is sufficient to reproduce all Ca2~increasesthat oromote apoptosis in diverse cell types. Surfacereceptors are coupled to the the events observed following TNF Caz* signalling pathway by heterotrimeric G proteins and/or protein tyrosine kinase(s)that treatment 2'~,4", Increases in ceramide activatevarious phospholipaseC isozymes,thereby leading to the production of Ins(1,4,S)P3and have also been observed during Fasdiacylglycerol (DAG) via hydrolysisof phosphatidylinositolbisphosphate [Ptdlns(4,S)Pz]. mediated apoptosis in Jurkat T cells Ins(1,4,S)P~ promotes releaseof Ca2÷from the endoplasmicreticulum (ER)and, indirectly, also (D. R. Green, pets. commun.), in this Caz* influx across the plasma membranethrough a capacitative mechanism. Both the emptying system, Fas engagement can apparently of the ERCaa* store and the subsequent stimulation of Ca2÷influx can be mimicked by be mimicked by exogenous sphingothapsigargin. The influx of extracellularCaz* ,:an also be achieved by treatment with Caz• myelinase, and it appears that Ras is the ionophores. Ca2÷can mediateall the cytosolic and nuclearevents of apoptosis in thymocytes downstream target in the response. and in some other cell types. Howevar, in other models Ca2. appears to block apoptosis. However, the observation that protein 372
TRENDSIN CELLBIOLOGYVOL. 4 OCTOBER1994
m kinase inhibitors fail to block Fas-mediated killing leaves a role for a ceramide-activated ldnase cascade in question at present.
Role of pH changes Recent evidence suggests that chemotherapeutic agents for cancer stimulate endonuclease activation in target cells. Analysis of the biochemical mechanisms underlying this response in Chinese hamster ovary (CHO) cells indicated that changes in the cytosolic Ca 2+concentration were not involved, but implicated intracellular acidification as the trigger49. A pH-sensitive endonuclease (DNase II) may mediate DNA fragmentation in systems where Caz+ does not appear to be involveds°. However, the biochemical mechanism(s) underlying intracellular acidification have not been established, and the pH at which the enzyme is maximally active (pH 5.5) is usually seen only in the lysosomal compartment of cells. Oxygen radicals The production of overwhelming levels of reactive oxygen species ('oxidative stress') is thought to be an important mechanism of cytotoxicity in a number of systems. Oxidative 'bursts' may also serve roles in cellular activation, particularly In macrophages, and nitric oxide is an important and common second messenger. Oxygen radical production is now also being implicated in the control of apoptosls. Early work suggested that oxidative stress could lead to a response similar to apoptosls in isolated hepatocytesSL In addition, low concentrations of the oxidant hydrogen peroxide induce apoptosis in HL.60 cells, while necrosis occurs when mtillmolar amounts o f the oxidant are present s2. However, antloxidants may protect cells from apoptosls (see Ref. 53 for a recent review). For example, apoptotle cell death induced by TNF can be prevented by antloxidants such as N.acetylcystelne (NAC) and thloredoxin s4,ss or overexpresslon of mltochondrlal superoxlde dismutase s6. Moreover, we have recently found that antloxidants such as the nltroxlde oxygen radical spin trap 5,5'.dlmethyl-l-pyrrollne-N-oxlde (DMPO), and Its tetramethyl form TMPO, can inhibit thymocyte apoptosls Induced by glucocortlcolds, thapslgargln, and the topoisomerase II inhibitor etoposide (A. F. G. Slater, S. Nobel a r d S. Orrenlus, unpublished). The recent discovery that the Bci-2 protein has antioxidant properties further emphasizes the probable importance of oxidative events in apoptosis sT,ss. These results suggest that oxidation Is required at some point(s) if a cell is to undergo apoptosis successfully. There are several possible roles for oxidation in apoptosis. First, protein oxidation may be essential to change gene transcription such that the apoptotic pathway is initiated. Several transcription factors, e.g. Fos and Jun sg, have crucial cysteine residues involved in DNA binding and thiol oxidation could cause a large decrease in the affinity of DNA binding. By contrast, the DNA-binding activity of the transcription factor NF-KB is activated by oxidative events (via enhanced degradation of its inhibitory factor lgB), thereby initiating transcription TRENDS IN CELLBIOLOGYVOL. 4 OCTOBER1994
of NF-~B-responsive genes ~. However, it is also possible that oxidative events are required at later stages in apoptosis, for example to initiate either cell shrinkage or changes in higher-order chromatin structure.
Crosstalk and genetics influence diverse responses to apoptosis signals One recurrent theme of this review is that signal transduction pathways have contradictory effects on apoptosis depending upon the experimental model in question. Indeed, disparate cell responses to receptot engagement have been noted for several models of apoptosis. For example, triggering of the T-cell receptor can lead to either apoptosis or differentiation of thymocytes, and treatment with TNF or antibodies to the Fas antigen can lead to either apoptosis or proliferation depending on the cellular systern. It is increasingly clear tha' !. ~~liferation, differentiation and apoptosis are closely related alternative cellular responses that share many of the same mob ecular mechanisms. An important challenge for ongoing research is to determine precisely how they differ. Experiments with cytokines or pharmacological mimics of second messengers indicate that cell proliferation usually requires at least two independent signals. This is perhaps best demonstrated by T cells, for which treatment with both a Ca2+ionophore and a phorbol ester, but not either alone, efficiently stimulates IL-2 production and proliferation. The molecular basis for this requirement for two signals is well documented and involves elements within the IL-2 promoter that selectively respond to Ca z÷ o: PKC61. With this in mind, we propose that apoptosis may result from imbalanced signal transduction, when an lntracellular Ca 2÷ increase or other signal transduction pathway is initiated in the absence of an appropriate second (or multiple) signal(s) (Fig. 1). Phorbol esters often block apoptosls, suggesting that PKC and Ras are targets for one such second signal. The presence or absence of crosstalk among sig. nailing pathways may therefore be one general mechanism for promoting divergent cellular effects in response to a shared set of signal transduction pathways. Recent work on the developmental regulation of bcl-2 expression suggests that cells can be induced to be more or les predisposed to undergo apoptosis in response to a particular signal. In the thymus, the CD4+CD8+ subpopulation of cells is known to be sensitive to diverse apoptotic stimuli, whereas CD4-CD8- precursors and CD4÷CD8"/CD4"CD8 ÷ mature thymocytes are not. Interestingly, bcl-2 expression is relatively high in the CD4-CD8- precursors, it drops off in the CD4+CD8' cells, and then its expression is elevated again in the m a t t e cells. This pattern precisely parallels the sensitivity of the cells to apoptosis 6z. It appears that Bcl-2 is regulated similarly in B cells during development. It is possible that alterations in other apoptosis-active gene products occur during development or tumour progression, where changes in sensitivity to apeptosis are observed. Thus, genetic programming may alter the manner in which a particular signal is ;eceived 373
24 FORBES,I. J., ZALEWSKI,P. D., GIANNAKIS,C. and COWLED, P. A. (1992) Exp. Cell Res. 198, 367-372 2.5 BELLOMO,G. et aL (1992) Cancer Res.52, 1342-1346 26 PEROTI'I,M. et al. (1990) FEBSLett. 259, 331-334 27 RA!OTTE,D., HADDAD, P., HAMAN,A., CRAGOE,E. J., Ir and HOANG,T. (1992) I. Biol. Chem. 267, 9980-9987 28 KOSEKI,C., HERZUNGER,D. and AL-AWQATI,Z. (1992)I. Cell Biol. 119, 1327-1333 29 OBEID,L. M., LINARDIC,C. M., KAROLAK,L. A. and HANNUN, Y. A. (1993) Science259, 1769-1771 30 IARVIS,W. D., TURNER,A. I., POVIRK,L. F., TRAYLOR,R. S. and GRANT,S. (1994) Cancer Res. 54, 1707-1714 31 JACOBSEN,M. D., BURNE,J. F., KING, M. P., MIYASHITA,T., REED,J. C. and RAFF,M. C. (1993) Nature 361,365-369 32 GULBINS,E. et aL (1994) Mol. Cell. Biol. 14, 4749--4758 33 ARENDS,M. I., McGREGOR,N. I., TAFT, E. I. H. and WYLLIE,A. H. (1993) Br. J. Cancer68, 1127-1133 34 PP,AI"r, R. M. and MARTIN,G. R. (1975) Prec. NatlAcad. Sci. References USA 72, 874-877 1 WYLLIE,A. H., MORRIS,R. H., SMITH,A. L. and DUNLOP, D. 35 DANIEL,V., LII"WACK,G. and TOMKINS.G. M. (1973) Prec. (1984) ]. Pathol. 142, 67-77 Natl Acad. Sci. USA 70, 76-79 2 McCONKEY,D. J., HARTZELL,P., NICOTERA,P. and 36 DUPREZ,E., GJERTSEN,B. T., BERNARD,O., LANOTI"E,M. and ORRENIUS,S. (1989) FASEBJ. 3, 1843-1849 DOSKELAND,S. O. (1993)I. Biol. Chem. 268, 8332-8340 3 MARTIKAINEN,P. and ISAACS,J. (1990) Prostate 17, 37 McCONKEY,D. I., ORRENIUS,S. and IONDAL, M. (1990) 175-187 I. Immunol. 145, 1227-1230 4 IIANG,S., CHOW, S. C., NICOTERA,P. and ORRENIUS,S. 38 LEE,M. R., LIEU, M. L., YANG,Y. F., and LAI, M. Z. (1993) (1994) Exp. Cell Res.212, 84-92 I. Immunol. 151, 5208-5217 S SMITH,C. A., WILLIAMS,G. T., KINGSTON,R., JENKINSON,E. J. 39 KAMMER,G. M. (1988) Immunol. Today9, 222-229 and OWEN, J. J. T. (1989) Nature 337, 181-183 40 WU, I. etal. (1993) Science262, 1065-1068 6 McCONKEY,D. J., HARTZELL'P., PEREZ,I. F., ORRENIUS,S. 41 GRAVES,L. M. et al. (1993) Proc. Natl Acad. Sci. USA 90, and JONDAL'M. (1989)I. Immunol. 143, 1801-1806 10300-10304 7 CHOI,D W (1992) Science258, 241-243 42 EVANS,C. A., OWEN.LYNCH,J., WHETTON,A. D. and DIVE,C. 8 WARING,P. (1990)I. Biol. Chem. 265, 14476-14480 (1993) Cancer Res, 53, 1735-1738 9 AGARWAL,M. L., LARKIN,H. E., ZAIDI, 5. I. A., MUKHTAR,H. 43 McGAHON,A., BISSONNETTE,R., SCHMITr,M., and OLEINICK,N. L. (1993) Cancer Res.53, 5897-5902 COTTER,K. M., GREEN,D. R. and COTTER,T. G. (1994) Blood 10 DOWD,D. R., MacDONALD, P. N., KOMM, B. S., 83, 1179=1187 HAUSSLER,M. R. and MIESFELD,R. (1991) J. Blol. Chem. 266, 44 UCKUN,F. M. et al. (1992) Proc. NatlAcod. ';el. USA 89, 18423-18426 9005-9009 11 DOWD,D, R., MacDONALD, P. N,, KOMM, B. S., 45 NAKASHIMA,I. et al. (1991) I. Immunol. 147, 1153-1162 HAUSSLER,M. R. and MIESFELD,R. (1992) Mol. Endocrlnol. 6, 46 McCONKEY,D. I. et al. I. Immunol. (in press) 1843-1848 47 HANNUN,Y. A, and BELL,R. M, (1989) Science243, 12 BAFFY,G., MIYASHITA,T., WILLIAMSON,J. R. and REED,J. C. 500-507 (1993) I. Biol. Chem. 268, 6511-6519 48 IARVIS,W. D, etal. (1994)Prec. NatlAcod. Scl. USA91, 13 RODRIGUEZ.TARDUCHY,G., COLLINS,M. and 73-77 LOPEZ.RIVAS,A. (.1990) EMBOl. 9, 2997-3002 49 BARRY,M. A. and EASTMAN,A. (1992) Biochem. Bioph~. Res, 14 WHYTE,M. K. B., HARDWlCK,S. I., MEAGHER,L. C., SAVlLL'J. S. Commun, 166, 782-789 and HASLETT,C. (1993)J. Gin. Invest. 92, 446-455 50 BARRY,M, A, and EASTMAN,A, (1993) Arch. Biochem. Biophvs. 15 EDWARDS,S. N., BUCKMASTER,A. E. and TOLKOVSKY, A. M, 300, 44(~-450 (1991) j. Iveurochem. 57, 2140-2143 51 McCONKEY,D, I,, HARTZELL,P., NICGrERA,P., WYLLIE,A, H. 16 SIll,Y., SAHAI,B. M. and GREEN,D. R. (1989) Nature 339, and ORRENIUS,S. (1988) ToxicoL Left. 42,123-130 615-616 52 LENNON, S, V,, MARTIN, S, l, and COI"I'ER,T. G. (1991) Cell 17 DAWSON,T. M., STINER,J. P., DAWSON,V. L., UHL, G. R. TissueKinet. 24, 203-214 and SNYDER,S. H. (19q3) Proc.Nail Acad. $ci. USA 88, 53 BUTTKE,T, M, and 5ANDSTROM,P, A, (1994) ImmunoL Today 6368-6371 15, 7-10 18 LIU,J., FARMER,J. D., Jr, LANE,W. S., FRIEDMAN,J., 54 CHANG,D. J,, RINGOLD,G, M. and HELLER,R. A. (1992) WEISSMAN, I, and SCHREIBER,S. L, (1991) Cell 66, 807-815 Biochem. Bioph),s. Res. Commun.188, 538-546 19 SQUIER,M. K. T., MILLER,A. C, K., MALKINSON,A, M. and 55 MATSUDA,M,, MASUTANI,H. and NAKAMURA,H. (1991) COHEN,J. J. (1994)J. Cell. PhysioL 159, 2z9-237 I. Immunol. 147, 3837-3841 20 WORONICZ,J. D., CALNAN, B., NGO, V. and WINOTO, A. 56 HIROSE,K,, LONGO, D, L,, OPPENHEIM,I. I. and ~1994) Nature 367, 277-281 MATSUSHIMA,K. (1993)FASEBI, 7, 361-368 21 KIZAKI,H., TADAKUMA,T., ODAKA,C,, MURAMATSU,l. and 57 KANE,D, l, et aL (1993) Science262, 1274-1277 ISHIMURA,Y. (1989) I. Immunol. 143, 1790--1794 58 HOCKENBERY,D. M., OLTVAI,Z. N., YIN, X. M., 22 IWATA,M., ISEKI,R., SATe, K., TOZAWA,Y. and OHOKA,Y. MILLIMAN,C. L, and KORSMEYER,S. I. (1993) Cell 75, (1994) Int. Immunol. 6, 431-438 241-251 23 McCONKEY,D. !. et al. (19Ol) I. Immunol. 146, 1072-1076
by the cell, and perhaps an apoptotic signal is translated into a differentiation signal within the appropriate genetic context. Determination of the relative contributions of crosstalk and programming to the regulation of apoptosis clearly requires further investigation. Signal transduction pathways ultimately influence gene regulators. For example, Fos63, Myc64 and Nur772° are transcription factors that have been implicated in some forms of apoptosis and in growth factor signal transduction pathways. Additionally, the tumour suppressor protein p53 can promote apoptosis 6s, and it is known that p53 is a phosphoprotein. Precisely how signal transduction pathways determine whether these factors mediate cell division or death is an important question for the future.
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No more slipped discs? C u r r e n t Protocols o n CD-ROM
Greene Publishing Associates, and John Wiley and Sons, 1994. Current Protocols in Molecular Biology/Current Protocols in Immunology on CD-ROM (CD containing manual and licensed software, a program manual containing hard copy of ull figures, and quarterly updates on CD) .~4 90. 00. One.year licence renewal $245.00. Most people have become familiar with the hard-back Current Protocols (CP) manuals over recent years. These huge volumes (with the ingenious me~'.al ratchet to allow easy photocopying) adorn the shelves of many labs and are regularly updated with the very latest techniques. As the fields of molecular biology and immunology seemingly grow exponentially, the size of the CP manuals increase accordingly. With volumes weighing in at around 3.4 kg each, a trip to the photocopier with both molecular biology volumes could be dangerously bad for the back. An additional problem occurs as manuals grow ever larger: user friendliness is reduced and people are often driven to smaller, more specialist manuals, and so lose the benefits of a broad-based manual. Many publishers have promised the panacea of the ultimate manual: 'hands.on" protocols from people who use them all the time, with extensive references and background information. However, few have been able to deliver. With the CD-ROM versions of CP, there is light at the end of the tunnel, not to mention
64 EVAN,G. I. et al. (1992) Cell69, 119-128 65 YONISH-ROUACH,E. et al. (1991) Nature352, 345-347 66 WYLLIE,A. H., KERR,J. F. R. and CURRIE,A. R.(1980)/nt. Rev. Cytol. 68, 251-305 67 BROWN,D. G., SUN,X. M. and COHEN,G. M. (1993)/. Biol. Chem.268, 3037-3039 68 WYLUE,A. H. (1980) Nature284, 555-556
a much reduced chance of that slipped disc! We installed CP in Molecular Biology and CP in Immunology on CD-ROM easily in just a few minutes, all the software being included on the disk*. A user directory is established on the hard disk, which allows rapid use of CP thereafter. The manual opens as a series of contents pages, and you can progress through levels of these by clicking with the mouse onto the desired chapters, subject headings or specific sections. The layout of the control panels is clear, with push bars for printing, returning to the contents pages, page flicking and so on. The excellent, tiered structure of the hardcopy manual has been retained, so introductory sections lead into detailed protocols, with supporting lists of reagent and literature. Using the manual like a book, it is possible to browse through tiers of pages, but several aids are F~resentto make life far easier. The particular strength of CPon CD. RaM is the ability to search the complete volume with various levels of stringency. This process will search for the keyword or phrases you desire, and will even suggest a list of related words (this is particularly useful when the spelling is uncertain). All the matches are rapidly brought up on screen, listed from best to worst match, and you can browse your way through. Several additional features for getting around the manual allow you to exploit its contents. A cross.referencing system comes into its own for moving around quickly. Text that is cross.referenced is underlined in the manual and a 'double-click' with the mouse on this text takes you immediately to the desired section. You can read, save or print this and then simply return Lo your original position by closing the page. At any stage, you can leave yourself and others a note of modifications or comments that you have on a particular page. This 'sticky notes' ~eature, which appears on the screen as a familiar yellow r~ote pad,
TRENDS IN CELLBIOLOGYVOL. 4 OCTOBER1994
allows you to annotate your manual. Similarly, you can leave 'bookmarks', the electronic equivalent of earmarked pages, so that upon your return you can flick from section to section extremely easily. Bookmarks and sticky page notes can be given customized titles, and are retained in the manual unlessdeleted by the user. In such a way, you can create your own index of sections that are used repeatedly. The manual is particularly useful for manipulating text. The fonts and sizes of text can all be altered to suit you. Ever felt like making your own labbook with all those frequently used techniques? Now is your chance. Any page, section or line can be copied, either by printing directly, or by saving and dumping into a word processor. What this means is that an almost unlimited source of information can be easily manipulated. There will no doubt be many graduate students facing a blank thesis page who will gain 'inspiration' from such a facilityl Extolling the virtues of this manual is easy, and pages can be written about the features it contains, but we could find very little to fault it. As is often the case with diagrams on CD.ROM, the figures are of low resolution and a hard copy of each is provided in the accompanying manual. This dG~; not often interfere with its use, bt~t will hopefully be improved upon in later versions of the manual. For most people, a major limitation of CP on CD-ROM is the price. CP on CD.ROM will certainly be most useful on a computer network, but it is not clear what the total yeady subscription will be for a site licence. In summary, CP in Molecular Biology and CPin It.~munologyon CD-ROM are excellent manuals, and while they contain the same text as the hardcopy version, the benefit of the CDRaM version is the ease of use. Many publishers entering the electronic age could learn a lesson from the degree of user.friendliness within this software.
*The softwarecan be run on either Apple Macintosh or IBM-PCcompatible computers. The CD wastested on a MacintoshII ci with an external CD600drive.
M~rk HIrst and Chades Bangham Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, UIC 375