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Review Article

Apoptosis in YeastsMartin Weinberger, Lakshmi Ramachandran and William C. BurhansDept. of Cancer Geneticsand Biophysical Therapies Program, Roswell Park Cancer Institute, Buffalo, NY 14263, USA

Summary

Although yeasts lack some elements of the complex apoptoticmachinery of metazoan cells, recent studies show that many featuresof apoptosis, including a caspase-like activity, can be induced in theseorganisms by DNA damage and other apoptotic triggers. Theseremarkable findings provide a compelling argument for increasedefforts to bring the powerful genetic approaches available to yeastresearchers more directly to bear on questions related to apoptosisand its induction or inhibition by drugs. Yeasts may provide aparticularly useful model for understanding connections betweenDNA damage, cell cycle regulation and apoptosis. Here wesummarize these recent findings and explore their implications,particularly for the development of more effective therapeuticstrategies 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 viral-

infected cells or autoreactive immune cells, and the elimination

of neoplastic cells or cells that have suffered irreparable DNA

damage. The enhanced genome fitness underlying the evolu-

tion 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 forhow cell suicide might contribute to the evolutionary fitness of

unicellular organisms.

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 fission yeast metacaspase genes was first 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 fluor-

escent caspase substrates typically used to detect caspase

activity in mammals, activation of this activity by a self-

cleavage 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 effects 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 fission yeast, and

that this lethality can be blocked by co-expression of anti-

apoptotic proteins of the bcl-2 family (reviewed in (4)). The

failure of earlier attempts to detect unmistakable homologuesof elements of the apoptotic machinery in either budding or

fission 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 reflect the existence of

an apoptotic program in these organisms (5).

However, in budding (6) and fission (7) yeasts, the loss of

viability caused by expressing pro-apoptotic members of the

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 Buffalo, NY 14263, USA. Tel: 716-845-7691. Fax: 716-845-

1579. E-mail: wburhans@acsu.buffalo.edu

IUBMB Life, 55(8): 467472, August 2003

ISSN 1521-6543 print/ISSN 1521-6551 online # 2003 IUBMB

DOI: 10.1080/15216540310001612336

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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 mem-

brane blebbing. Furthermore, in addition to blocking the

lethality induced by expression of these proteins, the co-

expression 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 effects of a temperature-sensitive

mutation in the CDC48 gene, whose product is involved in

vesicle trafficking (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 effects 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 effects correspond to an active

cell death process.

Shifting strains with this cdc48 mutation or other tempera-

ture-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 ofoxygen 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 effects 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 some-

times contradictory) mitochondrial features of the apoptotic

phenotype induced by Bax in budding yeast include membranepermeability 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

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 ofcytochrome 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 homo-

logue 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 apopulation 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 specific 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

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-dichlorodihydrofluorescein

diacetate, which penetrates live cells but does not fluoresce

unless oxidized by ROS. ROS-producing cells are indicated by

green fluorescence, and propidium iodide-stained cells fluor-

esce red. M. Weinberger and W. Burhans, manuscript in

preparation.

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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

effects associated with induction of various markers of

apoptosisincluding activation of the budding yeast meta-

caspasein conjunction with DNA damage caused by the loss

of Cdc13p function (22).

WHY CELL SUICIDE IN YEASTS?

All these findings (many of which were reviewed in more

detail recently by Madeo etal. (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, nonspecific signature

of cell death. For example, UV-induced apoptosis in budding

yeast is confined to a narrow flux of UV light, higher fluxes

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 circum-

stances, 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 ofa 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 flasks. In this context, it is reasonable to expect

that (as has been proposed by others ( 23)), evolutionary fitness

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 UNDERSTANDINGAPOPTOSIS

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 efforts to

understand and effectively treat cancer. The induction of

apoptosis is an important component of the mechanisms of

most chemotherapeutic drugs, and defects in apoptotic path-

ways 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 effects 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 functionin 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 apoptoticresponse recently observed in budding yeast upon inactivation

of the CDC13 gene (22). The fission yeast version of Rad9p

also has been implicated in pathways regulating apoptosis

(38). In fact, both the fission 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 fission yeast or

humans is overexpressed in mammalian cells (34, 38). Rad9p-

induced 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, expres-

sion of human anti-apoptotic Bcl-2 proteins in fission yeast

can enhance resistance to the lethal effects of DNA damage

(34). These findings 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

fission yeast rad9 gene. Inactivation of this gene attenuates the

lethal effects 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 effects 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

specific details are complex and sometimes contradictory,

there is significant overlap between other aspects of cell cycle

regulation and apoptosis that are poorly understood. Relevant

here, for example, are the numerous reports that elevatedexpression 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 suffering 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 inhibitorp21, 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 fission 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 check-

point 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 activa-

tion 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 FORAPOPTOTIC PATHWAY ANALYSIS AND ANTICANCERDRUG DISCOVERY

Delineation of complex apoptotic pathways regulated bycheckpoint and other proteins (perhaps including CDKs) in

yeasts could facilitate the development of more effective

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 identified 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 inDNA. Similarly, a recent screen in fission yeast for inhibitors

of the pro-apoptotic Bcl-2 family member Bak identified the

mammalian HMGB1 protein as a potent inhibitor of Bak-

induced cell death in fission 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 findings 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 effects of mammalian pro-

or 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 metacas-

pases) 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 undefined cell death.

Although many elements of the mammalian apoptotic

machinery have been identified 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 path-

ways in the simpler and easier to manipulate experimental

systems provided by yeasts will likely provide a framework forbetter 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 labora-

tory 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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