Growth, Differentiation and Sexuality

170 B.C.K. Lu
enterthePCDpathway,withallthehallmarksof
apoptosis. Moreover, the PCD induced in cdc13-1
involves the mitochondrial event, because the
mitochondria-deficient mutant Š ◦ can rescue
cdc13-1 cells and suppresses cdc13-1p-induced
caspase activation (Qi et al. 2003). However, cell
death of cdc13-1 during DNA damage arrest has
recently been demonstrated to be independent of
caspase activation, and death by apoptosis has been
called into question (Wysocki and Kron 2004).
Other death pathways need to be investigated.
Cell-cycle progression is highly regulated by
checkpoint controls in all organisms (see Harris,
Chap. 3, this volume). For initiation of DNA replication
in the budding yeast, ORC1 and ORC2 (origin
recognition complex) are required to assemble the
pre-replicative complex (preRC) on the chromatin
during G1 phase (Watanabe et al. 2002; Burhans
et al. 2003). Inactivation of ORC genes in yeast (as
in orc1-4/orc1-4 diploid cells) will trigger RAD9dependent
DNA-damage checkpoint control and
cell-cycle arrest at G2/M boundary, and the celldeath
pathway ensues when cell-cycle arrest is abrogatedafterthe10-hcatastrophictime(Watanabe
et al. 2002). Ts-mutant orc2-1/orc2-1 diploid cells
also die at non-permissive temperatures (Watanabe
et al. 2002). Several checkpoint proteins (e.g.,
Mec1 and ScRad9 of S. cerevisiae, and Cdc2, Rad3
and SpRad9 of S. pombe) and their requirement
for apoptotic pathways have been established (reviewed
in Burhans et al. 2003). Interestingly, the
G1/S checkpoint works by cell-cycle arrest at G1,
during which pre-RC and licensing proteins are
assembled on the chromatin. Defects in the checkpoint
or ORC geneswillleadtoabrogationofthe
arrest and start of the S phase, and only then is
the apoptotic pathway triggered (Burnhans et al.
2003).
Human cell-cycle gene 1 (hCCG1) appears to
be a regulator of apoptosis and its product has
been identified to be a histone acetyltransferase.
The factor hCIA1 (CCG1 interacting factor A) is
a histone chaperone. The yeast homolog of hCIA is
ASF1 (anti-silencing factor-1). Thus, ASF1/CIA1 is
a yeast histone chaperone that is essential for cellcycle
progression. The defective asf1/cia1 mutant
cells arrest and die predominantly at G2/M. The
dead asf1/cia1 cells not only exhibit characteristics
of apoptosis, but also include autophagic bodies in
the vacuole with some hints of necrosis, as found
by Yamaki et al. (2001). These authors suggest that
yeast “may have evolved a prototypal active cell
death system”.
D. Cytological Phenotypes of Apoptosis
ThecytologicalphenotypesofPCDthathavebeen
found, be it induced by expression of heterologous
proapoptotic genes, by external stimuli, or by genetic
defects, exhibit almost all of the hallmarks of
apoptosis found in metazoan organisms. These are
chromatin condensation, DNA fragmentation (as
demonstrated by TdT-mediated dUTP nick end labeling,
or TUNEL assay), nuclear and cytoplasmic
fragmentation, phosphatidylserine externalization
and vacuolization (Sato et al. 1994; Ink et al. 1997;
James et al. 1997; Madeo et al. 1997; Ligr et al. 1998;
Fröhlich and Madeo 2000), activation of caspase
activity (Qi et al. 2003) and increased production
of ROS (Fröhlich and Madeo 2000; Gross et al. 2000;
Poliaková et al. 2002; Ludovico et al. 2002; Qi et al.
2003). It should be noted that nucleosomal ladders
have not been found in all cases examined,
with the exception of Mucor racemosus (Roze and
Linz 1998). It is also interesting to note that in rare
cases autophagic vesicles have been associated with
apoptotic cell death (Yamaki et al. 2001), suggesting
a crosstalk between apoptotic and autophagic
pathways (see below).
Apoptotic and necrotic cells can be distinguished
by cytological criteria. Phosphatidylserine
(PS) externalization and its localization can be detected
by a fluorescent FITC-conjugate annexin V.
However, annexin V can also enter the necrotic
cells and bind to PS on the inner leaflet of the
plasma membrane. Thus, this technique would
not be discriminating. The DNA stain propidium
iodite (PI) is not permeable to apoptotic cells but
it is to necrotic cells. Thus, the apoptotic cells will
be annexin V positive and PI negative, while the
necroticcellswillbepositiveforboth.Themost
discriminating stain for apoptotic cells will be the
TUNEL assay, where apoptotic cells will be positive
and the necrotic cells negative (Chen et al. 2003).
The distinction between apoptotic and autophagic
celldeathislessclearcut.Iattempttodefinethese
in a following section in this chapter.
E. Caspase-Like Proteins Found
Caspases (cysteine aspartases) may have an ancient
origin (Boyce et al. 2004). A caspase-like or metacaspase
gene has been discovered in S. cerevisiae,
named YCA1 (for yeast caspase-1). Its protein
product behaves like a bona fide caspase. Like
mammalian caspases, the yeast YCA1p proenzyme
(about 52 kDa) is activated by proteolysis. A 12-