Death caspase-1
Steroid hormones coordinate multiple cellular changes, yet the mechanisms by which these systemic signals are refined into stage- and tissue-specific responses remain poorly understood. The Drosophila gene Eip93F, more familiarly termed E93 determines the nature of a steroid-induced biological response. E93 mutants possess larval salivary glands that fail to undergo steroid-triggered programmed cell death, and E93 is expressed in cells immediately before the onset of death. E93 protein is bound to the sites of steroid-regulated and cell death genes on polytene chromosomes, and the expression of these genes is defective in E93 mutants. Furthermore, expression of E93 is sufficient to induce programmed cell death. It is proposed that the steroid induction of E93 determines a programmed cell death response during development (Lee, 2000).
The nuclear localization of E93
in larval salivary glands provided an opportunity to determine if E93
binds to the salivary gland polytene chromosomes and, if so, to
identify the sites bound by the protein. Salivary glands were
dissected 12-14 hr after puparium formation, fixed, squashed,
and photographed to acquire accurate cytology of the banding and
puffing patterns for mapping. The chromosomes were then stained with
affinity-purified E93 antibodies, and these patterns were compared
with the original set of photographs to allow accurate mapping of the
bound sites. E93 clearly binds to the polytene chromosomes in a
reproducible and site-specific manner and is consistently detected
at 65 chromosome sites, many of which contain ecdysone-regulated
genes or programmed cell death genes. Among these sites are the 74EF
and 75B early puffs, which contain the E74 and E75
ecdysone-inducible genes, as well as the 93F puff, which contains
E93. In addition, 1B, 21C, 59F, and 99B are bound by E93 and
contain the programmed cell death genes dredd, crq,
dcp-1, and drICE, respectively. The 2B5 early puff,
containing the BR-C ecdysone-inducible gene, and 75CD,
containing βFTZ-F1 and the programmed cell death genes
rpr, hid, and grim, were not bound by E93.
These data indicate that E93 may directly regulate the genes in bound
chromosome loci and may either encode a site-specific DNA binding
protein or a chromatin-associated protein that functions as a
transcriptional regulator (Lee, 2000).
A specific inhibitor of mammalian CPP-32 caspase, Ac-DEVD-CHO, completely inhibits Poly(adenosine diphosphate-ribose) (PARP) cleavage by DCP-1, whereas the ICE-specific inhibitor Ac-YVAD-CHO is ineffective. Therefore, DCP-1 is biochemically more closely related to caspases CED-2 and CPP-32 than to ICE. DCP-1 also cleaves p35 in an identical manner to that of CED-3 (Song, 1997).
Expression of the cell death regulatory protein Reaper (RPR) in the developing Drosophila eye
results in a smaller than normal eye, owing to excessive cell death. Mutations in thread (th) are
dominant enhancers of RPR-induced cell death. thread encodes a protein homologous to
baculovirus inhibitors of apoptosis (IAPs), called Drosophila IAP1 (DIAP1/Thread).
Overexpression in the eye of DIAP1 or a related protein, DIAP2, suppresses normally occurring
cell death as well as death due to overexpression of rpr or head involution defective. IAP
death-preventing activity localizes to the N-terminal baculovirus IAP repeat region, a repeat motif found in both
viral and cellular proteins associated with death prevention (Hay, 1995).
The baculovirus inhibitor of apoptosis gene, iap, can impede cell death in insect cells. iap
can also prevent cell death in mammalian cells. The ability of iap to regulate programmed cell death in widely
divergent species raised the possibility that cellular homologs of iap might exist. Consistent with this
hypothesis, Drosophila and human genes that encode IAP-like proteins (dILP and hILP) have been isolated.
Like baculovirus IAP, both dILP and hILP contain amino-terminal baculovirus IAP repeats (BIRs) and carboxy-terminal
RING finger domains. Human ilp encodes a widely expressed cytoplasmic protein that can suppress apoptosis
in transfected cells. An analysis of the expressed sequence tag database suggests that hilp is one of several
human genes related to iap. Together these data suggest that iap and related cellular genes play an
evolutionarily conserved role in the regulation of apoptosis (Ducket, 1996).
ced-9 (Drosophila homolog: death executioner Bcl-2 homologue), a member of the bcl-2 gene family in Caenorhabditis elegans plays a central role in preventing cell death in worms.
Overexpression of human bcl-2 can partially prevent cell death in C. elegans. However, it remains to be elucidated whether
ced-9 can regulate cell death when expressed in other organisms. The CED-9 protein is co-localized
with BCL-2 in COS cells and Drosophila Schneider's L2 (SL2) cells, suggesting that the site of CED-9 action is located to
specific cytoplasmic compartments. Overexpression of ced-9 only poorly protects cells from the death induced by ced-3 in HeLa cells, but ced-9 significantly reduces the cell death induced by ced-3 in Drosophila SL2 cells. Apoptosis of SL2 cells induced by reaper, a Drosophila cell-death gene, is partially prevented by
ced-9, bcl-2 and bcl-xL. These results suggest that the signaling pathway that is required for the anti-apoptotic function of
bcl-2 family members, including ced-9, is conserved in Drosophila cells. In addition, SL2 cells provide a unique systems
for dissecting the main machinery of cell death (Hisahara, 1998).
Site-specific proteases play critical roles in regulating many cellular processes. To identify novel site-specific proteases, their regulators, and substrates, a general reporter system in Saccharomyces cerevisiae has been designed in which a transcription factor is linked to the intracellular domain of a transmembrane
protein by protease cleavage sites. Here, the efficacy of this approach has been explored by using as a model, the caspases, a family of aspartate-specific cysteine proteases.
Introduction of an active caspase into cells that express a caspase-cleavable reporter results in the release of the transcription factor from the
membrane and subsequent activation of a nuclear reporter. Known caspases activate the reporter; an activator of caspase activity stimulates
reporter activation in the presence of an otherwise inactive caspase, and caspase inhibitors suppress caspase-dependent reporter activity. Although low or moderate levels of active caspase expression do not compromise yeast cell growth, higher level expression leads to lethality. This observation has been exploited to isolate clones from a Drosophila embryo cDNA library that block DCP-1 caspase-dependent yeast cell death. Among these
clones, the known cell death inhibitor DIAP1 has been identified. Using bacterially synthesized proteins, it has been shown that glutathione S-transferase-DIAP1
directly inhibits DCP-1 caspase activity but that it has minimal effect on the activity of a predomainless version of a second Drosophila caspase, drICE (Hawkins, 1999).
In Drosophila, the induction of apoptosis requires three closely linked genes, reaper, head involution defective, and grim. The products of these genes induce apoptosis by activating a caspase pathway. Two very similar Drosophila caspases, DCP-1 and drICE, have been previously identified. DCP-1 has a substrate specificity that is remarkably similar to that of human caspase 3 and Caenorhabditis elegans CED-3, suggesting that DCP-1 is a death effector caspase. drICE and DCP-1 have similar yet different enzymatic specificities. Although expression of either in cultured cells induces apoptosis, neither protein is able to induce DNA fragmentation in Drosophila SL2 cells. Ectopic expression of a truncated form of dcp-1 (DeltaN-dcp-1) in the developing Drosophila retina under an eye-specific promoter results in a small and rough eye phenotype, whereas expression of the full-length dcp-1 (fl-dcp-1) has little effect. However, expression of either full-length drICE (fl-drICE) or truncated drICE (DeltaN-drICE) in the retina shows no obvious eye phenotype. Although active DCP-1 protein cleaves full-length DCP-1 and full-length drICE in vitro, GMR-DeltaN-dcp-1 does not enhance the eye phenotype of GMR-fl-dcp-1 or GMR-fl-drICE flies. Significantly, GMR-rpr and GMR-grim, but not GMR-hid, dramatically enhance the eye phenotype of GMR-fl-dcp-1 flies. These results indicate that Reaper and Grim, but not HID, can activate DCP-1 in vivo (Song, 2000).
The
proapoptotic proteins encoded by rpr, hid, and
grim all require caspase activity to kill cells. Whether coexpression of caspases and these
proapoptotic genes could lead to significantly enhanced cell killing was investigated.
For this purpose, flies carrying GMR-rpr,
GMR-hid, and GMR-grim were crossed to
GMR-fl-dcp-1 and GMR-fl-drICE flies. Two
different GMR-fl-dcp-1 transgenic fly lines were crossed to
GMR-rpr46, GMR-hid1M, and GMR-grim
flies, with identical results. Likewise, two GMR-fl-drICE
transgenic fly lines were crossed to GMR-rpr46,
GMR-hid1M, and GMR-grim flies, again with
identical results. Flies carrying one copy of GMR-fl-dcp-1 or
GMR-fl-drICE have almost normal eye morphology. Flies transgenic for GMR-hid1M, GMR-grim, or
GMR-rpr46 have a mild but easily detectable eye phenotype. The coexpression of hid and full-length
drICE produces no obvious enhancement of the eye phenotype,
but rather an additive effect of the two transgenes. Also,
the expression of hid together with full-length
dcp-1 enhanced the eye phenotype only weakly,
comparable to what is seen for coexpression of many other proapoptotic
gene combinations. In stark contrast, expression of
either rpr or grim together with GMR-fl-dcp-1 yields a dramatically enhanced eye phenotype
that cannot be simply explained by additive effects. rpr produces a stronger effect than grim. The expression of rpr also enhances the eye
phenotype of GMR-fl-drICE flies, whereas
grim is not very effective. This finding is
consistent with the observation that drICE is activated in rpr-transfected S2 cells. Among the different
cell types of the Drosophila retina, the pigment cells
appear to be particularly sensitive to DCP-1. Both the truncated and
the full-length DCP-1 cause pigment cell death.
Judging by the complete loss of eye color, all pigment cells are
eliminated in flies that coexpress DCP-1 with either rpr
and grim. In order to further investigate
the specificity of this interaction, GMR-fl-dcp-1 flies were also crossed
to a transgenic line with strong
hid expression: GMR-hid 10 flies. Again, the eye
phenotype observed for this combination is not significantly enhanced.
Overall, rpr and grim were found to interact with
dcp-1 much more strongly than hid and interact
more effectively with dcp-1 than with drICE.
Taken together, these observations suggest that dcp-1 is
rate limiting for cell killing by rpr and grim, but not hid. Therefore, it is proposed that rpr and
grim function upstream of dcp-1 in vivo (Song, 2000).
These results indicate that Reaper and Grim, but not Hid, can lead
to DCP-1 activation. Several other observations also indicate that
rpr and grim have cell killing properties that
are distinct from those of hid. For example, the Ras/MAPK
pathway inhibits hid-induced cell death but has no effect on
rpr- or grim-induced death (Bergmann, 1998). In
addition, mutations in the diap1 gene of
Drosophila have been isolated that enhance rpr- and grim-induced cell killing but suppress hid-induced cell killing (J. Agapite, K. McCall, and H. Steller, unpublished data cited in Song, 2000). The easiest interpretation of all these
observations is that rpr and grim kill cells by
activating the same (set of) caspases and that hid activates
a distinct caspase. Since it has
been recently shown that rpr, hid, and grim induce cell death by inhibiting the antiapoptotic
activity of diap1, diap1 must
control at least two distinct caspase pathways. According to this
model, Reaper and Grim and HID would interact selectively with specific DIAP1-(pro)caspase complexes. The binding of Reaper, Grim, or HID to
the relevant DIAP1-(pro)caspase complex is thought to result in caspase
activation. This model is consistent with a variety of findings from
both invertebrate and vertebrate systems. However, the possibility that
rpr and grim may also activate DCP-1 through a
DIAP1-independent pathway cannot be ruled out. Although several Drosophila caspases have been described, these results
indicate that additional caspases, in particular ones activated by HID, remain to be identified (Song, 2000).
See the embryonic expression pattern of Dcp-1 at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
Preblastoderm embryos, prior to zygotic gene transcription, contain large and uniform amounts of DCP-1 mRNA. Therefore dcp-1 is maternally expressed. At later stages, transcripts continue to be present throughout the embryo. Toward the end of embryogenesis, dcp-1 expression becomes more restricted. Some regions of the embryo, including the head, some cells within the central nervous system, the developing gonads, and portions of the gut, contain higher levels of DCP-1 mRNA. Strong expression can be seen in cells along the midline of the central nervous system (Song, 1997).
Drosophila metamorphosis is characterized by diverse
developmental phenomena, including cellular proliferation, tissue
remodeling, cell migration, and programmed cell death. Cells undergo
one or more of these processes in response to the hormone
20-hydroxyecdysone (ecdysone), which initiates metamorphosis at the end
of the third larval instar and before puparium formation (PF) via a
transcriptional hierarchy.
Additional pulses of ecdysone further coordinate these processes during
the prepupal and pupal phases of metamorphosis. Larval tissues such as
the gut, salivary glands, and larval-specific muscles undergo
programmed cell death and subsequent histolysis. The imaginal discs
undergo physical restructuring and differentiation to form rudimentary
adult appendages such as wings, legs, eyes, and antennae. Ecdysone also
triggers neuronal remodeling in the central nervous system (White, 1999).
Wild-type patterns of gene expression in
D. melanogaster during early metamorphosis were examined by assaying whole
animals at stages that span two pulses of ecdysone. Microarrays were constructed containing
6240 elements that included more than 4500 unique cDNA expressed
sequence tag (EST) clones along with a number of
ecdysone-regulated control genes having predictable expression
patterns. These ESTs represent
approximately 30% to 40% of the total estimated number of genes in the
Drosophila genome. In order to gauge
expression levels, microarrays were hybridized with fluorescent probes
derived from polyA+ RNA isolated from developmentally
staged animals. The time points examined are relative to PF, which
last approximately 15 to 30 min, during which time the larvae cease to move
and evert their anterior spiracles. Nineteen arrays were examined representing
six time points relative to PF: one time point before the late larval
ecdysone pulse; one time
point just after the initiation of this pulse (4 hours BPF), and time
points at 3, 6, 9, and 12 hours after PF (APF). The prepupal pulse of
ecdysone occurs 9 to 12 hours APF (White, 1999).
In order to manage, analyze, and disseminate the large amount of data,
a searchable database was constructed that includes
the average expression differential at each time point. The analysis
set consists of all elements that reproducibly fluctuate in
expression threefold or more at any time point relative to PF, leaving
534 elements containing sequences represented by 465 ESTs and control
genes. More than 10% of the genes represented by the
ESTs display threefold or more differential expression during early
metamorphosis. This may be a conservative estimate of the percentage of
Drosophila genes that change in expression level during
early metamorphosis, because of the stringent criteria used for their
selection (White, 1999).
To interpret these data, genes were grouped according to similarity of
expression patterns by two methods. The first relied on pairwise
correlation statistics, and the second relied on the use
of self-organizing maps (SOMs). Differentially expressed genes fall into two main categories. The first
category contains genes that are expressed at >18 hours BFP (before
the late larval ecdysone pulse) but then fall to low or undetectable
levels during this pulse. These genes are potentially repressed by ecdysone and
make up 44% of the 465 ESTs identified in this set. The second
category consists of genes expressed at low or undetectable levels
before the late larval ecdysone pulse but then are induced during this pulse. These genes are
potentially induced by ecdysone and make up 31% of the 465 ESTs.
Consequently, 75% of genes that changed in expression by threefold or
more do so during the late larval ecdysone pulse that marks the
initial transition from larva to prepupa. This result is consistent
with the extreme morphological changes that are about to occur in these
animals. There are clearly discrete subdivisions of gene expression within these
categories (White, 1999).
Larval-specific tissues such as larval muscles, the
midgut, and the salivary glands undergo programmed cell death during
metamorphosis. Genes involved in programmed cell death were identified
in these experiments. The apoptosis-activating reaper gene
has previously been shown to be ecdysone-inducible, and
this is reflected in the data. Expression of the
Drosophila caspase-1 gene is also observed during the prepupal ecdysone pulse
but not during the late larval pulse. This gene is also an
activator of apoptosis, and mutants display melanotic tumors and larval
lethality. Induction of a cell death inhibitor
gene, thread (also known as Diap1), is
observed during the late larval pulse but not the prepupal phase. The DIAP1 protein includes inhibitor-of-apoptosis (IAP) domains
and has been identified as a factor that can block reaper activity. Because different tissues begin apoptosis at
different stages of development, changes in the expression of
inhibitors and activators of apoptosis are expected to be tissue-specific. For
example, the expression profiles observed for the
caspase-1 activator and the Diap1 inhibitor are
those expected in tissues such as the larval salivary glands.
Tissue-specific information on the induction of these genes will be
important to an understanding of the coordination of apoptosis during
metamorphosis (White, 1999).
In Drosophila oogenesis, the programmed cell death of germline cells occurs predominantly at three distinct stages: stage 2a/2b (germarium), stage 8 (midoogenesis), and stages 10-13 (late oogenesis). These cell deaths are subject to distinct regulatory controls, since cell death during early and midoogenesis is stress-induced, whereas the cell death of nurse cells in late oogenesis is developmentally regulated. This report shows that the effector caspase Drice is activated during cell death in both mid- and late-oogenesis, but that the level and localization of activity differ depending on the stage. Active Drice forms localized aggregates during nurse cell death in late oogenesis; however, active Drice is found more ubiquitously and at a higher level during germline cell death in midoogenesis. Because Drice activity is limited in late oogenesis, an examination was performed to see whether another effector caspase, Dcp-1, could drive the unique morphological events that occur normally in late oogenesis. Premature activation of the effector caspase, Dcp-1, results in a disappearance of filamentous actin, rather than the formation of actin bundles, suggesting that Dcp-1 activity must also be restrained in late oogenesis. Overexpression of the caspase inhibitor DIAP1 suppresses cell death induced by Dcp-1 but has no effect on cell death during late oogenesis. This limited caspase activation in dying nurse cells may prevent destruction of the nurse cell cytoskeleton and the connected oocyte (Peterson, 2003).
The cytoskeletal events induced by expression of activated Dcp-1 (tdcp-1) are significantly different from those normally seen in nurse cells during late oogenesis. Normally during stage 10B of oogenesis, actin bundles form in the cytoplasm of nurse cells, connecting the plasma membrane with the nucleus. After actin bundle formation, actin-myosin-based contraction occurs and drives nurse cell cytoplasm dumping. Expression of tdcp-1 does not lead to actin bundles or nurse cell dumping. Instead, actin forms clumps and disappears as the egg chambers degenerate. Similar cytoskeletal events have been reported for egg chambers that degenerate during stage 8 in response to other stimuli. It is possible that factors required for actin bundle formation are not expressed during midoogenesis. However, egg chambers from midway mutants that degenerate prematurely during stages 8-9 do show actin bundles. Thus, midway, which encodes an acyl coenzyme (A:diacylglycerol acyltransferase) may normally act to control the timing of actin bundle formation and nurse cell death. It has been reported that dcp-1 germline clone mutants are defective in actin bundle formation and nurse cell dumping; however, recent findings suggest that these effects are due to a neighboring gene affected by the P-element alleles and that dcp-1 activity is not required for these cytoskeletal events. Nonetheless, because nurse cell nuclear breakdown has already begun prior to dumping, it has been suggested that the cytoskeletal events are part of the nurse cell death process (Peterson, 2003).
The formation of actin bundles in the cytoplasm of dying late-stage nurse cells differs from the actin rearrangements that have been reported in apoptotic cells and shows similarity to the actin structures in autophagic MCF-7 cells. In cultured mammalian cells undergoing apoptosis, filamentous actin relocalizes to form a dense network in the cytoplasm at the base of the membrane blebs, and later disappears. However, in MCF-7 cells undergoing autophagy, actin forms fibers stretching from the nucleus to the plasma membrane, resembling the actin bundles seen in nurse cells. Autophagic cell death has been observed in groups of cells that die, such as cells of the Drosophila salivary gland during metamorphosis. Caspase activity is required for Drosophila salivary gland autophagy, but it is not known if caspase activity is regulated differently in autophagy and apoptosis. There are several modes of cell death, including deaths that show properties of both autophagy and apoptosis; nurse cell death may fall into this class. Indeed, autophagic vacuoles have been reported in nurse cells, although follicle cells appear to engulf nurse cell material as well (Peterson, 2003).
Egg chambers degenerating during midoogenesis in response to nutrient deprivation or tdcp-1 expression do not show expression of the altered form of cytochrome c, suggesting that this may be a stage-specific event. One explanation is that the factors required to alter cytochrome c may not be expressed in midoogenesis. Alternatively, a mitochondria-independent cell death pathway may be utilized in midoogenesis. The lack of cytochrome c alteration suggests that cytochrome c involvement is not necessary for Drosophila apoptosis when a high level of caspase activity is present. However, it is possible that cytochrome c plays no direct role in apoptotic signaling in Drosophila, and this antibody simply recognizes cytochrome c in dividing or otherwise altered mitochondria (Peterson, 2003).
Interestingly, egg chambers prior to stage 8 were largely resistant to any apoptotic effects of activated Dcp-1. Stage 8 has been shown to be a checkpoint stage for a number of signals, including reduced food availability, ecdysone signaling, treatment with chemicals, ectopic death of follicle cells, or abnormal egg chamber development. Because vitellogenesis begins during stage 8, it has been suggested that the state of the egg chambers is monitored before making the investment of vitellogenesis. However, it is interesting that the egg chambers prior to stage 8 are well-protected from strong death-inducing stimuli, including expression of a truncated caspase. This stage-specific protection may be the result of a high level of caspase inhibitors like IAPs early in oogenesis. Indeed, transcriptional downregulation of Drosophila IAPs during stage 8 has been reported. Overexpression of DIAP1 can indeed block cell death induced by expression of truncated Dcp-1 in midoogenesis (Peterson, 2003).
The limited activation of Drice and the unusual cytoskeletal events that normally occur in late oogenesis suggest that caspase activity is carefully controlled during nurse cell death in late oogenesis. One model to explain the observed cytoskeletal differences is that only a subset of the usual cytoskeletal targets of caspases are cleaved in late oogenesis. This limited cleavage would allow for the formation and persistence of cytoplasmic actin bundles, which are necessary for proper nurse cell cytoplasm transfer. Alternatively, the cytoskeletal events may be controlled by Damm or Decay or may be caspase-independent. However, these models would still require that the activity of Drice and Dcp-1 be curtailed to prevent disassembly of actin cytoskeleton (Peterson, 2003).
Overexpression of the caspase inhibitor DIAP1 does not affect normal nurse cell death. This suggests that nurse cell death may be caspase-independent, or utilize caspases that are not readily inhibitable by DIAP1. Alternatively, mechanisms may exist to compartmentalize caspase activation or to degrade DIAP1, even when it is overexpressed. Support for this idea comes from the observations that full-length DIAP1 does not inhibit naturally occurring cell death in the eye as well as a version of DIAP1 lacking the RING finger. The RING finger has been shown to be critical for rapid turnover of DIAP1 protein (Peterson, 2003).
The controlled caspase activation that occurs in nurse cells during late oogenesis may explain why this form of cell death is not regulated by the cell death activators: Reaper, Hid, and Grim. Reaper, Hid, and Grim induce apoptosis in many cell types by triggering the degradation of DIAP1. Embryos homozygous for the H99 chromosomal deletion, which removes reaper, hid, and grim, are completely lacking in normal programmed cell death (White, 1994). However, flies carrying H99 germline clones undergo normal nurse cell death, indicating that nurse cell death is regulated differently from the vast majority of cell deaths in Drosophila. Perhaps the Reaper, Hid, Grim/DIAP1 mechanism of apoptosis induction would not permit such localized and limited caspase activation. This regulation of caspase activity may be necessary for the systematic destruction of nurse cells while the oocyte is protected from active caspases and other dangerously cleaved proteins (Peterson, 2003).
There are no significant abnormalities related to cell death in dcp-1 mutants. Thus zygotic DCP-1 function is not required for most embryonic cell deaths in Drosophila, perhaps because of the existence of additional capspases. However, because DCP-1 has significant maternal expression, it is also possible that sufficient DCP-1 protein is present during embryogenesis for cell death to occur (Song, 1997).
dcp-1 mutation causes lethality during larval stages. Although most of the dcp-1 mutants die before the third instar larval stage, some reach that stage and display several abnormalities. Mutant larvae lack imaginal discs and gonads. In addition, they have fragile trachea. However, the most prominent phenotype of these larvae is the presence of melanotic tumors located in various parts of the body (Song, 1997).
During Drosophila oogenesis, nurse cells transfer their cytoplasmic contents to developing oocytes and
then die. Loss of function for the dcp-1 gene, which encodes a caspase, causes female sterility by
inhibiting this transfer. dcp-1- nurse cells are defective in the cytoskeletal reorganization and nuclear
breakdown that normally accompany this process. Breakdown of the nuclear envelope is a central event during apoptosis and is accompanied by caspase-mediated cleavage of nuclear lamins. To address whether nuclear lamins are degraded as nurse cells become permeable, egg chambers were examined for the distribution of Lamin Dm0. Loss of Lamin Dm0 signal in control nurse cells takes place by stage 11, at which time lamin staining appears to be a diffuse cytoplasmic cloud around the nuclei. Mutant nurse cells continue to show distinct nuclear envelope staining as late as stage 14. Thus dcp-1 mutants are defective in the cleavage or dissociation, or both, of nuclear lamins. This failure in lamin breakdown is a likely cause of the defect in nuclear permeability revealed by a beta-Gal marker. Lamin breakdown is likely to be directly due to DCP-1 protease activity. Actin is localized to the plasma membrane during early stages in control and mutant egg chambers. During stage 10B in control egg chambers, actin bundles form throughout the cytoplasm, connecting the nuclei and plasma membrane. In contrast, actin in many dcp-1 mutant egg chambers remains associated with the plasma membrane, even in stage 14 egg chambers. Therefore, dcp-1 activity is required for the proper formation of cytoplasmic actin bundles in nurse cells. The dcp-1- phenotype suggests that the cytoskeletal and nuclear events in the nurse cells make use of the machinery normally associated with apoptosis and that apoptosis of the nurse cells is a necessary event for oocyte development (McCall, 1998).
In Drosophila, the APAF-1 homolog ARK is required for the activation of the initiator caspase DRONC, which in turn cleaves the effector caspases DRICE and DCP-1. While the function of ARK is important in stress-induced apoptosis in Drosophila S2 cells, since its removal completely suppresses cell death, the decision to undergo apoptosis appears to be regulated at the level of caspase activation, which is controlled by the IAP proteins, particularly DIAP1. This study further dissects the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. The induction of apoptosis is dependent in each case on expression of ARK and DRONC and surviving cells continue to proliferate. A difference was noted in the effects of silencing the executioner caspases DCP-1 and DRICE; knock-down of either or both of these have dramatic effects to sustain cell survival following depletion of DIAP1, but have only minor effects following cellular stress. These results suggest that the executioner caspases are essential for death following DIAP1 knock-down, indicating that the initiator caspase DRONC may lack executioner functions. The apparent absence of mitochondrial outer membrane permeabilization (MOMP) in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted (Kiessling, 2006).
While a requirement for ARK and DRONC was observed under all of the pro-apoptotic conditions in S2 cells, a difference was noted in the effects of knock-down of the executioner caspases DCP-1 and DRICE. Knock-down of either or both of these has dramatic effects to sustain cell survival following knock-down of DIAP1, but has only minor effects following cellular stress (Kiessling, 2006).
Several possible explanations were envisioned for this difference. One possibility is that knock-down of DIAP1 leads to caspase activation uniquely through permitting DRONC function to activate DCP-1 and DRICE, while stressors somehow engage other caspase activation pathways (and other caspases). This, however, is inconsistent with the observation that stress-induced apoptosis is clearly dependent on ARK and DRONC. Alternatively, it may be that stress-induced death also involves inhibition of other IAPs, such as DIAP-2 and dBRUCE, which may have a wider spectrum of effects to engage additional caspases not affected by DIAP1 alone. Previously, however, it was noted that knock-down of DIAP-2 does not trigger apoptosis, but greatly enhances susceptibility to death induced by stressors such as were used in this study. This argues that DIAP-2 function, at least, continues following such stress (such that its knock-down has an effect) and thus it is less likely to be an important explanation for the current effects (Kiessling, 2006).
The final possibility is perhaps the most interesting. The knock-down of DIAP1 leads to death, presumably through permitting low levels of ongoing (and otherwise repressed) caspase activation to function and any subsequent effect may depend on amplification, as active executioner caspases cleave and activate others. Therefore, knock-down of even one caspase in the cell may dampen this amplification so that cells survive. In contrast, the induction of apoptosis by stress may involve not only blockade of DIAP1 function (through the N-termini of Reaper, Hid, Grim and Sickle) but also another signal that amplifies caspase activation upstream of ARK. Such an upstream effect has been suggested by studies of the so-called 'GH3' region in these proteins, that appears to be required for death in Drosophila cells and can function to promote the mitochondrial pathway in vertebrate systems. Reaper, Hid, Grim, and probably Sickle are necessary for stress-induced apoptosis in Drosophila, and therefore their effects are likely to depend on ARK and ARK-DRONC interactions. Nevertheless, this line of reasoning suggests that they function not only to de-repress caspases (though blocking DIAP1), but also to do something else to bypass full dependence on DCP-1 and DRICE, perhaps by amplifying caspase activation at the level of the ARK-DRONC interaction. While speculative, this possibility is intriguing, and suggests that the induction of apoptosis in Drosophila may prove to be more complex than the simple models indicate (Kiessling, 2006).
A genome-wide RNA interference screen was performed to systematically identify regulators of apoptosis induced by DNA damage in Drosophila cells. Forty-seven double- stranded RNAs were identified that target a functionally diverse set of genes, including several with a known function in promoting cell death. Further characterization uncovers 10 genes that influence caspase activation upon the removal of Drosophila inhibitor of apoptosis 1. This set includes the Drosophila initiator caspase Dronc and, surprisingly, several metabolic regulators, a candidate tumor suppressor, Charlatan, and an N-acetyltransferase, ARD1. Importantly, several of these genes show functional conservation in regulating apoptosis in mammalian cells. These data suggest a previously unappreciated fundamental connection between various cellular processes and caspase-dependent cell death (Yi, 2007).
The genes that are specifically involved in caspase-dependent cell death were classified. Substantial induction of caspase activity was observed 8 h after treatment with a topoisomerase II inhibitor, doxorubicin (dox), to induce dose-dependent cell death. Any RNAi suppressing this activity implicates the target gene in early regulation of caspase activation. In addition to dcp-1 RNAi, knockdown of dronc and jra (the Drosophila homolog of c-Jun) significantly suppressed caspase-3/7-like activity in the presence of dox, whereas the negative control, RNAi against calpain A, a calcium-dependent cysteine protease, did not affect this pathway (Yi, 2007).
This analysis was expanded to all of the genes identified in the initial RNAi screen and 20 dsRNAs were discovered that suppressed caspase activation induced by DNA damage. Interestingly, 12 of these genes were found to be epistatic to diap1 (Yi, 2007).
diap1 epistatic analysis was performed to further categorize the genes. DIAP1, the fly orthologue of the mammalian inhibitors of apoptosis proteins, is a direct inhibitor of caspases, and deficiency in DIAP1 leads to rapid caspase activation and apoptosis in vivo. Thus, apoptosis induced by the loss of DIAP1 presents an alternative apoptotic assay independent of DNA damage. Silencing of genes that regulate activation of the core apoptotic machinery may provide protection against apoptosis induced by both DNA damage and the loss of DIAP1. RNAi against dcp-1 partially suppressed cell death induced by the depletion of DIAP1 in Kc cells. Also, dronc RNAi potently protected cells against apoptosis induced by deficiency in DIAP1. Altogether, 32 of the genes confirmed from the primary screen provided significant protection against cell death induced by the silencing of DIAP1 (Yi, 2007).
Interestingly, 12 dsRNAs suppressed caspase-3/7-like activity after dox treatment and protected against cell death induced by diap1 RNAi, suggesting that these genes are required for apoptosis induced by multiple stimuli. To confirm that these genes are necessary for the full activation of caspases, it was determined whether these dsRNAs could suppress spontaneous caspase activity induced by diap1 RNAi. Maximal induction of caspase activity by diap1 RNAi was observed after 24 h, and this effect was completely suppressed by dsRNA against dcp-1. Importantly, ablating 10/12 dsRNAs resulted in the significant suppression of caspase activity compared with diap1 RNAi only (Yi, 2007).
In addition to dronc RNAi, dsRNAs targeting chn and dARD1 provided the strongest suppression of spontaneous caspase activity. Consistent with the observation that RNAi against chn protects against DNA damage-induced cell death, the mammalian orthologue neuron-restrictive silencer factor (NRSF)/RE1-silencing transcription factor (REST) was recently identified as a candidate tumor suppressor in epithelial cells (Westbrook, 2005). Previous work indicates that Chn and NRSF/REST function as a transcriptional repressor of neuronal-specific genes (Chong, 1995; Schoenherr, 1995; Tsuda, 2006), suggesting that cellular differentiation may render cells refractory to caspase activation and apoptosis. Also, several metabolic genes, CG31674, CG14740, and CG12170, were identified that may be involved in the general regulation of caspase activation. It has been demonstrated that NADPH produced by the pentose phosphate pathway regulates the activation of caspase-2 in nutrient-deprived Xenopus laevis oocytes. Together with these results, these observations provide further evidence for an intimate link between the regulation of metabolism and induction of apoptosis (Yi, 2007).
To further explore the significance of these findings, whether silencing the mammalian orthologues of the fly genes identified from the RNAi screen confers protection against dox-induced cell death was investigated in mammalian cells. A set of mammalian orthologues was selected that are believed to be nonredundant. The list includes the orthologues of dMiro, which functions as a Rho-like GTPase; dARD1, which functions as an N-acetyltransferase; CG12170, which functions as a fatty acid synthase; and Chn, which functions as a transcriptional repressor (RHOT1, hARD1, OXSM, and REST, respectively; FlyBase). In addition, Plk3, a mammalian orthologue of Polo, was tested since dsRNA targeting polo potently protected against dox treatment (Yi, 2007).
The ability of siRNAs targeting a gene of interest to protect against DNA damage was tested in HeLa cells. As a positive control, cells were transfected with siRNAs targeting Bax or Bak, two central regulators of mammalian cell death. Indeed, silencing of Bax or Bak resulted in significant protection against dox- induced cell death. It was observed that plk3 RNAi provided partial protection against dox treatment, which is consistent with previous studies implicating Plk3 in stress-induced apoptosis. Interestingly, the knockdown of hARD1 dramatically enhanced cell survival in the presence of dox to levels similar to that of Bak. This protective effect was also evident at the morphological level. In cells transfected with a nontargeting control siRNA, dox treatment resulted in typical apoptotic morphology, including cell rounding and membrane blebbing. In direct contrast, cells transfected with siRNAs against hARD1 maintained a normal and healthy morphology and continued to proliferate in the presence of dox (Yi, 2007).
To examine whether the protection provided by siRNAs targeting hARD1 and plk3 is associated with the suppression of caspase activation, caspase activity was measured in these cells treated with dox. RNAi against plk3 provided partial suppression of caspase activity, again supporting the observed protection phenotype. Interestingly, the depletion of REST resulted in some suppression of caspase activity in the presence of dox even though the protection against cell death was not statistically significant. Consistent with the viability assay, complete suppression of caspase-3/7 activity was observed in cells transfected with hARD1 siRNA. These results indicate that hARD1 is required for caspase-dependent cell death induced by DNA damage. Furthermore, all four siRNAs targeting hARD1 were individually capable of providing robust protection against cell death, strongly suggesting that these siRNAs target hARD1 specifically (Yi, 2007).
Because the silencing of hARD1 dramatically suppressed activation of the downstream caspases, whether activation of the upstream caspases in response to dox treatment is also perturbed was also examined. Remarkably, hARD1 RNAi inhibited the cleavage of caspase-2 and -9 in cells treated with dox, whereas caspase cleavage was readily detected in control cells. Thus, it is proposed that hARD1 regulates the signal transduction pathway apical to the apoptotic machinery in the DNA damage response itself or the activation of upstream caspases (Yi, 2007).
Consistent with the results of the caspase-3/7 assay, silencing of hARD1 completely inhibited the appearance of activated caspase-3 induced by dox. This assay was used for a hARD1 complementation experiment to demonstrate the proapoptotic role of hARD1 in response to DNA damage. A new siRNA pool was used, targeting the 5' untranslated region of hARD1 (5'si); this treatment inhibited caspase-3 cleavage induced by dox treatment. Furthermore, caspase-3 cleavage was observed in reconstituted hARD1 knockdown cells. Because six out of six siRNAs against hARD1 provided strong protection against DNA damage–induced apoptosis and complementation of hARD1-sensitized cells to caspase activation, it is concluded that the functional role of ARD1 for dox-induced apoptosis is evolutionally conserved from Drosophila to mammals (Yi, 2007).
In summary, this study used an unbiased RNAi screening platform in Drosophila cells to identify genes involved in promoting DNA damage-induced apoptosis. Forty-seven dsRNAs were isolated that suppress cell death induced by dox. These genes encode for known apoptotic regulators such as Dronc, the Drosophila orthologue of the known proapoptotic transcriptional factor c-Jun, and an ecdysone-regulated protein, Eip63F-1, thereby validating the primary screen. Furthermore, this study implicates a large class of metabolic genes that were previously not suspected to have a role in modulating caspase activation and apoptosis, such as genes involved in fatty acid biosynthesis (CG11798), amino acid/carbohydrate metabolism (CG31674), citrate metabolism (CG14740), complex carbohydrate metabolism (CG10725), and ribosome biosynthesis (CG6712). These results support the proposal that the cellular metabolic status regulates the threshold for activation of apoptosis and thus plays a critical role in the decision of a cell to live or die (Yi, 2007).
Of particular interest is the identification of ARD1. Evidence is presented that RNAi against ARD1 provides protection against cell death and leads to the suppression of caspase activation induced by DNA damage in fly cells and HeLa cells. Furthermore, deficiency in dARD1 renders fly cells resistant to the spontaneous caspase activity and cell death associated with loss of Diap1. Importantly, substantial evidence is provided that hARD1 is required for caspase activation in the presence of DNA damage in mammalian cells. Cleavage of initiator and executioner caspases are suppressed in hARD1 RNAi cells treated with dox, suggesting that hARD1 functions further upstream of caspase activation, and the complementation of hARD1 knockdown cells restores caspase-3 cleavage. These data indicate that ARD1 is necessary for DNA damage-induced apoptosis in flies and mammals (Yi, 2007).
ARD1 functions in a complex with N-acetyltransferase to catalyze the acetylation of the Nα-terminal residue of newly synthesized polypeptides and has been implicated in the regulation of heterochromatin, DNA repair, and the maintenance of genomic stability in yeast. These studies suggest that ARD1 may be involved in regulating an early step in response to DNA damage. It is anticipated that future studies will focus on determining whether ARD1 functions in similar processes in mammals. The diversity of genes identified in our screen illustrates the complex cellular integration of survival and death signals through multiple pathways (Yi, 2007).
Death caspase-1:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| References
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