IUBMB

Life, 55(8): 467472, August 2003

Review Article
Apoptosis in Yeasts
Martin Weinberger, Lakshmi Ramachandran and William C. Burhans
Dept. of Cancer Genetics and Biophysical Therapies Program, Roswell Park Cancer Institute, Bualo, NY 14263, USA

Summary Although yeasts lack some elements of the complex apoptotic machinery of metazoan cells, recent studies show that many features of apoptosis, including a caspase-like activity, can be induced in these organisms by DNA damage and other apoptotic triggers. These remarkable ndings provide a compelling argument for increased eorts to bring the powerful genetic approaches available to yeast researchers more directly to bear on questions related to apoptosis and its induction or inhibition by drugs. Yeasts may provide a particularly useful model for understanding connections between DNA damage, cell cycle regulation and apoptosis. Here we summarize these recent ndings and explore their implications, particularly for the development of more eective therapeutic strategies for treating cancer. IUBMB Life, 55: 467472, 2003 Keywords Apoptosis; yeast; S. pombe; S. cerevisiae; metacaspase;
DNA damage.

APOPTOSIS PATHWAYS CONSERVED IN YEASTS Apoptosis plays important roles in the development and homeostasis of all metazoans. These roles include tissue remodeling during development, the elimination of viralinfected cells or autoreactive immune cells, and the elimination of neoplastic cells or cells that have suered irreparable DNA damage. The enhanced genome tness underlying the evolution of an active cell death program with these roles in multicellular organisms is clear. Whether a similar cell death program exists in yeasts has been controversial, due in part to the apparent absence in yeasts of genes encoding the metazoan apoptotic machinery, as well as less obvious explanations for how cell suicide might contribute to the evolutionary tness of unicellular organisms.
Received 6 February 2003; accepted 7 August 2003 Address correspondence to William C. Burhans, Dept. of Cancer Genetics and Biophysical Therapies Program, Roswell Park Cancer Institute Bualo, NY 14263, USA. Tel: 716-845-7691. Fax: 716-8451579. E-mail: wburhans@acsu.bualo.edu
ISSN 1521-6543 print/ISSN 1521-6551 online # 2003 IUBMB DOI: 10.1080/15216540310001612336

Within the past year, however, it was convincingly shown that budding yeast harbor a gene encoding a caspase-like protein (1). Caspases are, of course, important components of the apoptotic machinery in metazoans that were previously thought to be absent from yeasts. Existence of both budding and ssion yeast metacaspase genes was rst suggested by enhanced algorithms that predict homology of distantly related proteins based on protein secondary structure, in contrast to previous attempts to identify yeast caspase genes that relied on primary sequence information (2). The predicted budding yeast metacaspase genewhich has been designated MCA1/YCA1 (2, 3)was subsequently shown to encode a protein functionally similar to metazoan caspases (1). These similarities include an enzymatic activity that cleaves uorescent caspase substrates typically used to detect caspase activity in mammals, activation of this activity by a selfcleavage event similar to that which occurs during the activation of mammalian caspases, inhibition of caspase activity by the broad-range caspase inhibitor zVAD-fmk, induction of caspase activity in conjunction with other markers of apoptosis by hydrogen peroxide, which also induces apoptosis in mammals, and eects on viability associated with overexpression or deletion of the metacaspase gene that clearly indicate activation of the metacaspase contributes to cell death. Prior to the discovery of this caspase-like protein, a number of studies showed that expression of pro-apoptotic members of the bcl-2 family of proteins that regulate apoptosis in mammals can be lethal in both budding and ssion yeast, and that this lethality can be blocked by co-expression of antiapoptotic proteins of the bcl-2 family (reviewed in (4)). The failure of earlier attempts to detect unmistakable homologues of elements of the apoptotic machinery in either budding or ssion yeast suggested to some investigators that, although yeasts might serve as useful experimental tools for studying apoptotic pathways reconstituted in these organisms, the reconstitution of these pathways did not reect the existence of an apoptotic program in these organisms (5). However, in budding (6) and ssion (7) yeasts, the loss of viability caused by expressing pro-apoptotic members of the

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Bcl-2 family is accompanied by a number of morphological and ultrastructural changes similar, if not identical, to those observed in mammalian cells undergoing apoptosis. These include chromatin condensation and margination, DNA fragmentation detected by the typical TUNEL assay employed in apoptotic mammalian cells, loss of asymmetric distribution of plasma membrane phosphatidylserine, and plasma membrane blebbing. Furthermore, in addition to blocking the lethality induced by expression of these proteins, the coexpression of anti-apoptotic Bcl-2 family proteins blocks the appearance of these apoptotic-like features. The apoptotic-like phenotypes of yeasts described above, as well as other apoptotic markers, have also been detected in budding yeast cells exposed to hydrogen peroxide (8) or in association with the lethal eects of a temperature-sensitive mutation in the CDC48 gene, whose product is involved in vesicle tracking (9), or with the truncation or deletion of several genes encoding proteins involved in mRNA stability (10). Interestingly, overexpression of a mutant form of the mammalian orthologue of Cdc48p induces apoptosis in mammals (11), clearly suggesting conservation of a cell death pathway regulated by Cdc48p or that responds to disruption of Cdc48p function. The lethal eects of hydrogen peroxide or the cdc48 mutation that causes the apoptotic phenotype in budding yeast can be attenuated by blocking protein synthesis (8), thus establishing that these eects correspond to an active cell death process. Shifting strains with this cdc48 mutation or other temperature-sensitive mutations to the nonpermissive temperature also induces another commonly observed feature of apoptosis in mammals, the production of reactive oxygen species (ROS) (8); (Fig. 1). Growth of the cdc48 strain at nonpermissive temperatures in anaerobic conditions or in the presence of oxygen radical scavengers partly suppresses both ROS production and the temperature sensitivity of this mutation, as well as DNA fragmentation, which is consistent with a causal role for ROS in the apoptotic phenotype of this strain. In fact, similar to the induction of ROS by the pro-apoptotic protein Bax during apoptosis in mammals (12), Bax expression in budding yeast also stimulates ROS production, and the lethal eects of Bax expression are attenuated by treatment with free radical scavengers (8). As in mammals, ROS induction by Bax in budding yeast occurs in mitochondria. Other well-documented (albeit sometimes contradictory) mitochondrial features of the apoptotic phenotype induced by Bax in budding yeast include membrane permeability changes and release of cytochrome c (reviewed in (13) and references therein). Interestingly, a recent study indicates that some of these features are regulated by a protein implicated in autophagy in budding yeast, which suggests another parallel between the apoptosis phenotype in yeast and mammalian cells (13). Cytochrome c is also released in budding yeast cells exposed to acetic acid, which exhibit many of the other apoptotic markers described above (14), and

Figure 1. Budding yeast cells producing ROS during apoptosis. Cells containing the orc2-1 mutation in the Origin Recognition Complex required for initiation of DNA replication were shifted to the nonpermissive temperature of 378C for 6 h and then stained with propidium iodide, which detects loss of membrane integrity associated with cell death, and the ROS-sensitive probe 2,7-dichlorodihydrouorescein diacetate, which penetrates live cells but does not uoresce unless oxidized by ROS. ROS-producing cells are indicated by green uorescence, and propidium iodide-stained cells uoresce red. M. Weinberger and W. Burhans, manuscript in preparation.

during induction by mating pheromone of an apoptotic-like phenotype in budding yeast (15). In both these latter cases, mutational inactivation of pathways leading to synthesis of cytochrome c inhibits the appearance of other apoptotic markers (15, 16). Cytochrome c release also occurs during apoptosis in budding yeast cells deleted of the ASF1/CIA1 gene encoding a histone chaperone (17). The human homologue of this protein (CIA1) interacts with the largest subunit of the transcription complex TFIID, which, in addition to its role in transcription, has been implicated in apoptosis in mammalian cells (18). Yet another similarity between apoptosis in metazoans and the apoptotic phenotype of yeasts is the capacity for induction of both by DNA damage. For example, UV radiation can induce at least some aspects of the apoptotic phenotype in budding yeast, including DNA fragmentation indicated by a population of cells with a sub-G1 content of DNA (19). In addition, exposure of budding yeast cells to a DNA damaging antitumor agent causes the specic destruction by the proteasome of the DNA replication protein Cdc6, the mammalian homologue of which is also destroyed by proteasome and caspase-dependent pathways in mammalian cells undergoing apoptosis (20). Thus, at least one of the substrates destroyed as part of the apoptotic program induced

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by DNA damage and other apoptotic triggers in mammals is similarly attacked in yeast. In mammals, the ubiquitin/ proteasome pathway has both pro- and anti-apoptotic activities (21), and the destruction of Cdc6 in budding yeast and mammals suggests that the pro-apoptotic role of the proteasome is conserved in yeasts. Finally, inactivation of the budding yeast telomere-binding protein Cdc13p causes lethal eects associated with induction of various markers of apoptosisincluding activation of the budding yeast metacaspasein conjunction with DNA damage caused by the loss of Cdc13p function (22).

WHY CELL SUICIDE IN YEASTS? All these ndings (many of which were reviewed in more detail recently by Madeo et al. (23) and Jin and Reed (4)) clearly point to the conservation of at least some elements of apoptotic pathways in yeasts, including those induced by DNA damage. It is important to emphasize that in several cases, the apoptotic phenotype in yeasts has been shown to be an active process that requires protein synthesis. Its also the case that the appearance of apoptotic markers is not a generalized, nonspecic signature of cell death. For example, UV-induced apoptosis in budding yeast is conned to a narrow ux of UV light, higher uxes producing a more necrotic-like cell death (19). In particular, the discovery in budding yeast of a molecule with caspase-like activity that can, at least in some circumstances, promote cell death, provides a compelling argument against the notion that similarities between apoptosis in mammals and the apoptotic phenotype of yeasts are merely coincidental. In the absence of solid evidence for elements of the apoptotic machinery in yeast, this notion was mostly sustained by a theoretical argument suggesting the absence of a clear selective advantage to the evolution of a cellular suicide program in a unicellular organism. What was missing from this argument is the fact that, in contrast to laboratory conditions, yeasts mostly exist as colonies in the wild, where the evolution of apoptosis occurred in an environment of nutrient resources far more scarce than exists in most laboratory asks. In this context, it is reasonable to expect that (as has been proposed by others (23)), evolutionary tness could be enhanced by a cell suicide program that eliminates sick or damaged cells consuming scarce nutrients, thus making these nutrients more available to healthier members of the colony whose viability and reproductive capacity depend on these nutrients.

YEAST AS MODEL ORGANISMS FOR UNDERSTANDING APOPTOSIS Whatever the explanation for apoptosis in yeastsand there are other models in addition to the one described abovethe now compelling evidence that it does, in fact, occur in these organisms has important implications. For yeast

reseachers, these include the need to consider the potential role of ROS and other aspects of apoptosis in the interpretation of phenotypes associated with various mutations, particularly when these phenotypes are related to DNA damage responses. Perhaps the larger implications, however, are for eorts to understand and eectively treat cancer. The induction of apoptosis is an important component of the mechanisms of most chemotherapeutic drugs, and defects in apoptotic pathways in tumor cells contribute to their tumor phenotype and to anticancer drug resistance. Yeasts have been employed to great advantage as model organisms for the dissection of pathways and mechanisms of other conserved cell biological processes relevant to the problem of cancer, most notably, cell cycle regulation and checkpoint responses to DNA damage. Now these same approaches can more vigorously be applied to understanding apoptosis and the eects of antitumor drugs. In fact, there are fundamental connections between DNA damage, cell cycle regulation and apoptosis that may be particularly amenable to investigation in yeasts. In both metazoans and yeasts, sub-lethal levels of DNA damage invoke checkpoints that inhibit the cyclin-dependent kinases (CDKs) required to drive the cell cycle forward, thus blocking cell cycle progression while this damage is repaired. In metazoan cells with irreparable DNA damage, however, checkpoint proteins instead contribute to apoptosis. Much of the focus of research in this area has been on p53, which is one element of the metazoan apoptotic machinery that is not found in yeasts. However, the DNA damage checkpoint kinase ATM (2428), the downstream checkpoint kinase Chk2 ((24, 2932) and references therein), and the checkpoint protein Rad9p ((33) and references therein) also play important roles in apoptosis, and all these proteins are conserved in yeasts. Furthermore, all these proteins function in p53-independent apoptotic pathways induced by DNA damage, whichbecause of their lack of a requirement for p53are perhaps more likely related to apoptotic pathways in yeasts. For example, although ATM clearly plays a role in apoptosis involving phosphorylation of p53, it can also contribute to apoptosis in the absence of functional p53 (25). Human Rad9p also plays a role in DNA damage-induced apoptosis in cells lacking p53 (34), downstream of ATM, which phosphorylates Rad9p (35) and its upstream regulator, the tyrosine kinase c-abl (36, 37), in response to DNA damage. In budding yeast, the functions of ATM and the related checkpoint kinase ATR are shared by the MEC1 and TEL1 genes. Interestingly, Mec1p is required for the apoptotic response recently observed in budding yeast upon inactivation of the CDC13 gene (22). The ssion yeast version of Rad9p also has been implicated in pathways regulating apoptosis (38). In fact, both the ssion yeast and human versions of Rad9p contain a region homologous to the Bcl-2 homology 3 (BH3) death domain shared by Bax and other pro-apoptotic members of the Bcl-2 family of apoptosis regulators, and this region is required for the p53-independent apoptotic response

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that occurs when Rad9p derived from either ssion yeast or humans is overexpressed in mammalian cells (34, 38). Rad9pinduced apoptosis is likely due to interactions of the BH3 death domain region with anti-apoptotic Bcl-2 proteins that inactivate their anti-apoptotic function. Furthermore, expression of human anti-apoptotic Bcl-2 proteins in ssion yeast can enhance resistance to the lethal eects of DNA damage (34). These ndings clearly suggest that pathways regulated by Bcl-2 family members in response to DNA damage are, in fact, functionally conserved in yeasts. Another potential connection between checkpoints and apoptosis in yeast involves the budding yeast RAD9 gene, which is also required for DNA damage checkpoints and is conserved in mammals, but is not equivalent to the human or ssion yeast rad9 gene. Inactivation of this gene attenuates the lethal eects of the orc2-1 temperature-sensitive mutation in the second subunit of the Origin Recognition Complex (39), which interacts with Cdc6, the initiation protein destroyed by cell death pathways in budding yeast and mammalian cells (20). Not surprisingly, the lethal eects of the orc2-1 mutation are accompanied by some of the markers of apoptosis described above, such as chromatin condensation and fragmentation (39), as well as production of ROS (M. Weinberger and W. Burhans, unpublished observations; see Fig. 1.). Clearly, the highly conserved and extensively characterized DNA damage response and checkpoint regulatory pathways in yeasts provide fertile ground for continued exploration of the roles of checkpoint proteins in apoptosis. Although the specic details are complex and sometimes contradictory, there is signicant overlap between other aspects of cell cycle regulation and apoptosis that are poorly understood. Relevant here, for example, are the numerous reports that elevated expression of oncogenic proteins sensitizes mammalian cells to apoptotic triggers, including DNA damage, at the same time that it promotes aberrant cell proliferation. Although the mechanism underlying this increased sensitivity involves elements of proliferative and/or apoptotic response pathways that appear to be absent from yeast, such as E2F and p53 (reviewed in (40)), ultimately, this increased sensitivity may require the activation of CDKs, whose functions are, of course, highly conserved in yeast. For example, in mammalian cells suering normally sublethal levels of DNA damage, overexpression of the proto-oncogene myc appears to switch p53 from its checkpoint regulatory mode, where CDKs are inhibited by p53-dependent expression of the CDK inhibitor p21, to an apoptotic mode through a mechanism involving myc-dependent repression of the p21 promoter (41). This is consistent with an extensive literature implicating p21 in the regulation of apoptosis that mostly (but not always) argues for an anti- rather than pro-apoptotic function of this protein (reviewed in (42)). Although numerous possibilities exist for downstream components of this switch, at least in some cases, they include the activation of CDKs due to the loss of p21,

because apoptosis associated with reduction in levels of p21 can be blocked by dominant-negative CDK mutants or chemical inhibitors of CDK activity (43). In fact, there is a rapidly growing body of evidence which argues that activation of CDKs is a requisite feature of many (but not all) apoptotic responses in mammals ((44, 45)). Whether CDK activation accompanies apoptosis in yeasts is not yet known. However, the destruction of the DNA replication protein Cdc6 induced by lethal levels of DNA damage in budding yeast (20), similar to its destruction during apoptosis in mammals (20, 46), is consistent with this possibility. Budding yeast Cdc6 and its ssion yeast and Xenopus homologues share with p21 the ability to inhibit CDKs, either through direct interactions, or indirectly through their role in establishing DNA replication forks required for S phase checkpoints that restrain mitosis until DNA replication is complete ((47, 48) and references therein). Furthermore, a recent report describes a direct role for human Cdc6 in mitotic restraint through a Chk1-dependent checkpoint that may also inhibit CDK activity (49). In this context, one might expect that destruction of Cdc6 by the apoptotic machinery in mammals or yeasts would facilitate the activation of CDKs during apoptosis. Loss of restraint of CDK activity by Cdc6 could, for instance, explain how Cdc6 destruction during apoptosis in human cells contributes to the apoptotic program, as suggested by the attenuation of this program that occurs when Cdc6 destruction is blocked (46).

YEAST AND GENOMICS-BASED TOOLS FOR APOPTOTIC PATHWAY ANALYSIS AND ANTICANCER DRUG DISCOVERY Delineation of complex apoptotic pathways regulated by checkpoint and other proteins (perhaps including CDKs) in yeasts could facilitate the development of more eective therapies for treating cancer, and in particular therapeutic strategies that might eliminate the large fraction of tumors which harbor defects in p53 pathways. The genetic tractability and relatively simple culture requirements of yeasts make them particularly suitable for genomics-based gene and drug discovery and pathway analyses based on high-throughput genetic and chemical screens. In fact, yeast-based screens of mammalian cDNA libraries cloned into yeast expression vectors have identied several interesting inhibitors of Bax (50, 51), including Ku70 (52), a protein conserved in yeasts which plays a role in the repair of double-strand breaks in DNA. Similarly, a recent screen in ssion yeast for inhibitors of the pro-apoptotic Bcl-2 family member Bak identied the mammalian HMGB1 protein as a potent inhibitor of Bakinduced cell death in ssion yeast, and of apoptosis induced by a variety of triggers, including UV radiation, in mammalian cells (53). This study also showed that HMBG1 expression is elevated in breast tumors, and thus may contribute to tumor formation by blocking apoptosis. These ndings underscore

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the utility of this general approach for identifying genes that regulate apoptosis, which are often disregulated in cancer cells. Yeasts can also be employed to screen chemical libraries for compounds that modify the toxic eects of mammalian proor anti-apoptotic proteins expressed in yeasts, as well as compounds that alter phenotypes associated with mutations in various yeast genes implicated in apoptosis. So far, the focus of yeast genetic screens for regulators of apoptosis has been on elements of mammalian apoptotic pathways reconstituted in yeasts. A broader approach that detects interactions with, or between, yeast genes required for apoptosis in these organisms (for example, genes encoding checkpoint proteins or metacaspases) now is clearly warranted. Similarly, chemical screens for compounds that modify elements of endogenous apoptotic pathways in yeast may help to identify novel drugs and drug targets. This approach may be particularly promising when the endpoint of these screens is an endogenous marker of apoptosis rather than growth arrest or an undened cell death. Although many elements of the mammalian apoptotic machinery have been identied and characterized in the past several years, our understanding of this machinery remains incomplete, often confused, and sometimes distressingly contradictory. Contributing factors are the complexity of mammalian genomes and frequent reliance on experiments performed with cultured tumor cells that harbor numerous and complex changes in genetic information, many of which obscure the mechanisms underlying apoptosis. Apoptotic pathways conserved in yeasts likely include the features of apoptosis that are fundamentally important to this process, regardless of the type of cell or alterations that occur during neoplastic transformation. Thus, investigation of these pathways in the simpler and easier to manipulate experimental systems provided by yeasts will likely provide a framework for better understanding the extraordinarily complex and often contradictory elements of apoptosis in mammalian cells.

ACKNOWLEDGEMENTS We are grateful to Frank Madeo and Joel Huberman for critically evaluating our manuscript. Research in our laboratory is supported by NIH grants CA-84086 and CA-81326 and by shared resources funded by the Roswell Park Cancer Center support grant P30CA-16065.

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