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).
In multicellular organisms, apoptotic cells induce compensatory proliferation of neighboring cells to maintain tissue homeostasis. In the Drosophila wing imaginal disc, dying cells trigger compensatory proliferation through secretion of the mitogens Decapentaplegic (Dpp) and Wingless (Wg). This process is under control of the initiator caspase Dronc, but not effector caspases. This study shows that a second mechanism of apoptosis-induced compensatory proliferation exists. This mechanism is dependent on effector caspases which trigger the activation of Hedgehog (Hh) signaling for compensatory proliferation. Furthermore, whereas Dpp and Wg signaling is preferentially employed in apoptotic proliferating tissues, Hh signaling is activated in differentiating eye tissues. Interestingly, effector caspases in photoreceptor neurons stimulate Hh signaling which triggers cell-cycle reentry of cells that had previously exited the cell cycle. In summary, dependent on the developmental potential of the affected tissue, different caspases trigger distinct forms of compensatory proliferation in an apparent nonapoptotic function (Fan, 2008).
In developing wing discs in which apoptosis was induced by expression of the pro-apoptotic gene hid, loss of the caspase inhibitor DIAP1, or by X-ray treatment, the accumulation of two major mitogens, Dpp and Wg, has been observed in dying cells. Key for this finding is the simultaneous expression of the caspase inhibitor P35. Under these conditions, the dying cells were kept alive ('undead'), allowing accumulation of Dpp and Wg. This accumulation appears to be dependent on the initiator caspase Dronc, because it cannot be blocked by expression of P35 which inhibits effector caspases but not Dronc. In addition, the Drosophila homolog of the tumor suppressor p53, Dp53, has been implicated downstream of Dronc for compensatory proliferation. Notably, these studies on mechanisms of compensatory proliferation were carried out in developing larval wing imaginal discs in Drosophila. Cells in wing discs proliferate extensively during larval stages, and the majority of these cells does not differentiate before they reach pupal development. Hence, the mechanisms of compensatory proliferation have so far only been investigated in situations where most cells are proliferating. Interestingly, apoptosis-induced compensatory proliferation in differentiating eye tissue of third-instar larvae. However, it is unclear whether this form of compensatory proliferation is controlled by a similar mechanism as reported for larval proliferating wing discs (Fan, 2008).
This study revealed that there are at least two distinct mechanisms that promote compensatory proliferation in response to apoptotic activity. The general difference between these two mechanisms lies in the developmental context of the tissue in which compensatory proliferation occurs. In proliferating wing and eye tissues, compensatory proliferation induced by extensive apoptosis is dependent on Dronc and Dp53, which induce Dpp and Wg expression. In contrast, in differentiating eye tissue, apoptosis induces compensatory proliferation through a novel mechanism requiring the effector caspases DrICE and Dcp-1, which induce Hh signaling in a nonapoptotic function (Fan, 2008).
When cells stop proliferating and become committed to adopt cell fate, dramatic changes in gene expression are occurring. Given these changes in developmental plasticity, it is not surprising that distinct mechanisms of apoptosis-induced compensatory proliferation are employed in proliferating versus differentiating tissues. However, it should be noted that the proliferating capacity of differentiating tissues is rather restricted. In GMR-hid eye discs, although hid is expressed in all cells posterior to the MF, compensatory proliferation occurs only in cells that are still undifferentiated. Yet, even though they are undifferentiated they have withdrawn from the cell cycle and, under normal developmental conditions (i.e., without GMR-hid), they would soon be recruited to adopt cell fate. However, the apoptotic environment causing increased Hh signaling appears to be able to trigger reentry of these cells into the cell cycle (Fradkin, 2008).
Interestingly, the Hh signal is specifically increased in photoreceptor neurons requiring a nonapoptotic activity of effector caspases. Hh signaling can then nonautonomously induce proliferation of undifferentiated cells at the basal side of the eye disc. However, overexpression of Hh posterior to the MF in wild-type eye discs alone is not sufficient to induce a comparable wave of compensatory proliferation as in GMR-hid eye discs. This suggests that cell-cycle reentry requires activation of additional factors/pathways stimulated in apoptotic cells (Fradkin, 2008).
Although hid can stimulate increased Hh expression in photoreceptor neurons throughout the posterior half of the eye disc, compensatory proliferation is restricted to a certain distance (six to ten ommatidial columns) from the MF. This corresponds to approximately 6-15 hr of developmental time, and might be the time required for cell-cycle reentry. Similarly, when mammalian cells that have exited the cell cycle are stimulated to reenter the cell cycle, they need about 8 hr to do this. The reason for this delay is unknown. Studying compensatory proliferation in GMR-hid eye discs might provide a genetic model to address this interesting problem (Fradkin, 2008).
It is not clear whether this novel effector caspase-, Hh-dependent pathway of compensatory proliferation also applies to other, or even all, differentiating tissues. However, what this study shows is that there are at least two distinct mechanisms of apoptosis-induced compensatory proliferation. It is also possible that other mechanisms of compensatory proliferation in different developmental contexts are going to be uncovered in the future. Interestingly, in developing larval wing discs, P35-dependent compensatory proliferation has been implicated in cell competition. This suggests that, even in tissue with the same developmental potential, compensatory proliferation can occur with distinct mechanisms (Fradkin, 2008).
How cells sense different developmental contexts and operate distinct proliferating mechanisms in response to apoptotic stress is unknown. Specifically, where is the specificity and selectivity for distinct caspases coming from in tissues of different developmental potential? What are the mechanisms engaged by these caspases to trigger secretion of either Dpp and Wg or Hh? These are questions which need to be addressed in the future (Fan, 2008).
This study has several implications for tumorigenesis. First, many tumors develop when quiescent cells reenter the cell cycle. The mechanisms for cell-cycle reentry are largely unknown. Second, evasion from apoptosis is a hallmark of cancer. Many tumor cells are induced to undergo apoptosis. However, they do not die, because they downregulate essential components of the apoptotic pathway such as Apaf-1 and caspases. Thus, these undead tumor cells might secrete mitogens which might induce compensatory proliferation similar to the Drosophila case. In this way, undead cells might contribute to the growth of the tumor. A similar argument can be made for chemotherapy, which in many cases attempts to activate the apoptotic program in a tumor cell. If the death of the tumor cell is blocked, or slow, mitogens might be produced and the tumor growth could be even more severe. This is very obvious in the apoptotic wing or anterior eye discs in Drosophila when apoptosis is blocked by P35. Under these conditions, overgrown wing and eye tissues are observed. Thus, evasion of apoptosis might directly contribute to tumor growth. Finally, although increased Hh signaling can lead to various cancers, how Hh induces cellular proliferation and tissue overgrowth is not well understood. Mutations in Patched1, a negative regulator of sonic Hh, frequently give rise to human tumors. The exact cause is unknown. These data imply that Hh signaling might be involved in cell-cycle reentry allowing cells to resume proliferation (Fan, 2008).
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).
Members of the caspase family of cysteine proteases coordinate cell death through restricted proteolysis of diverse protein substrates and play a conserved role in apoptosis from nematodes to man. However, while numerous substrates for the mammalian cell death-associated caspases have now been described, few caspase substrates have been identified in other organisms. This study used a proteomics-based approach to identify proteins that are cleaved by caspases during apoptosis in Drosophila D-Mel2 cells, a subline of the Schneider S2 cell line. This approach identified multiple novel substrates for the fly caspases and revealed that bicaudal/betaNAC is a conserved substrate for Drosophila and mammalian caspases. RNAi-mediated silencing of bicaudal expression in Drosophila D-Mel2 cells resulted in a block to proliferation, followed by spontaneous apoptosis. Similarly, silencing of expression of the mammalian bicaudal homologue, betaNAC, in HeLa, HEK293T, MCF-7 and MRC5 cells also resulted in spontaneous apoptosis. These data suggest that bicaudal/betaNAC is essential for cell survival and is a conserved target of caspases from flies to man (Creagh, 2009).
At present, three substrates for Drosophila caspases, DIAP1, Lamin DmO, and the Drosophila Apaf-1 homologue, ARK, have been identified. A proteomics-based screen resulted in the identification of 14 proteins that underwent caspase-dependent alterations to their relative mobilities on two-dimensional (2D) SDS-PAGE gels. Two of these substrates were cloned and confirmed that they are efficiently cleaved by the Drosophila caspases DrICE and DCP-1. Bicaudal and its human homologue, βNAC, appear to be essential for cell proliferation and cell survival as RNAi-mediated silencing of the expression of these proteins resulted in a block to cell division, followed by spontaneous apoptosis. This suggests that, as well as targeting substrate proteins that contribute to the ordered destruction of the cell, caspases also inactivate key proteins such as βNAC that are essential for cell survival (Creagh, 2009).
Interestingly, βNAC has also been implicated as a negative regulator of programmed cell death in the nematode C. elegans and ablation of this gene product results in massive and unscheduled apoptosis in developing worm embryos. NAC functions to bind short nascent polypeptides as they emerge from the ribosome. The latter event prevents inappropriate interactions with cellular proteins and non-specific binding by the signal recognition particle and consequent targeting to the ER. NAC also prevents the targeting of non-translating ribosomes to the ER. This fundamental role of NAC is reflected in the catastrophic phenotype of null mutations affecting the βNAC-coding sequence gene in a range of species. Loss of βNAC in developing mice leads to post-implantation lethality and mutation of Drosophila bicaudal promotes developmental arrest, which is associated with duplication of the posterior embryonic regions in the place of the anterior embryonic segments (Markesich, 2000). RNAi-mediated silencing of the C. elegans βNAC homologue, ICD-1, also results in developmental arrest associated with massive cell death (Bloss, 2000). Thus, the disablement of βNAC function through caspase-dependent proteolysis may contribute substantially to cellular demise (Creagh, 2009).
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 both vertebrates and invertebrates, developing organs and tissues must be precisely patterned. One patterning mechanism is Notch/Delta-mediated lateral inhibition. Through the process of lateral inhibition, Drosophila sensory organ precursors (SOPs) are selected and sensory bristles form into a regular pattern. SOP cell fate is determined by high Delta expression and following expression of neurogenic genes like neuralized. SOP selection is spatially and temporally regulated; however, the dynamic process of precise pattern formation is not clearly understood. In this study, using live-imaging analysis, it was shown that the appearance of neuralized-positive cells is random in both timing and position. Excess neuralized-positive cells are produced by developmental errors at several steps preceding and accompanying lateral inhibition. About 20% of the neuralized-positive cells show aberrant cell characteristics and high Notch activation, which not only suppress neural differentiation but also induce caspase-dependent cell death. These cells never develop into sensory organs, nor do they disturb bristle patterning. This study reveals the incidence of developmental errors that produce excess neuralized-positive cells during sensory organ development. Notch activation in neuralized-positive cells determines aberrant cell fate and typically induces caspase-dependent cell death, as detected using SCAT3, a fluorescence resonance energy transfer (FRET) indicator for effector caspase activity. Apoptosis is utilized as a mechanism to remove cells that start neural differentiation at aberrant positions and timing and to ensure robust spacing pattern formation (Koto, 2011).
The Drosophila sensory organ is a typical model for the study of Notch/Delta-mediated lateral inhibition. Tracking the process of cell fate determination in each cell lineage is presumed to be effective in revealing the mechanisms behind precise pattern formation (Koto, 2011).
The first finding in this study is that bristle patterning starts in a random fashion: about 20% of neuralized-positive cells are fated to become aberrant SOP-like cells, and Notch signaling is involved in determining the fate of SOP-like cells. However, the mechanisms proposed for the production of SOP-like cells in previous reports do not coincide perfectly with the current observations. Previous studies showed that the conventional model of Notch/Delta-mediated lateral inhibition is not sufficient to produce the precise bristle pattern but that cell-autonomous interaction or filopodia-mediated intermittent Notch/Delta signaling makes lateral inhibition robust enough to suppress the neural differentiation of surrounding cells. In contrast, the current results suggest that lateral inhibition from adjacent SOPs is not the sole source of Notch activation in SOP-like cells, because a portion of SOP-like cells preceded the nearby SOPs. Also, SOP-like cells showed ongoing Notch activity even in the absence of adjacent SOPs. One possible explanation for these SOP-like cells failing to develop into sensory organs may be that they appear too early in the developmental time course and cannot complete the developmental program to become sensory organs in a cell-autonomous manner. In any case, the decrease in SOP-like cells in the N55e11 heterozygous mutant reliably suggests that Notch activation in cells that start neural differentiation contributes to the determination of their cell fate as aberrant SOP-like cells (Koto, 2011).
Dynamic oscillation of the Notch effector gene Hes1 has been observed in neural progenitors of the developing mouse brain with the aid of a short-half-life indicator using ubiquitinated firefly luciferase. In Drosophila sensory organ development, the technical limitations of the GFP reporter make it difficult to confirm this type of oscillation pattern in Notch signaling. However, given that Notch oscillation occurs in cells in the proneural stripe regions at the beginning of SOP selection, it is conceivable that SOP-like cells might be the product of fluctuating Notch signaling at inappropriate times, developmentally speaking (Koto, 2011).
The second finding in this study is that a program of caspase-dependent cell death specifically eliminates SOP-like cells. Ablating an incipient SOP removes Notch/Delta-mediated lateral inhibition and allows a nearby epithelial cell to become the SOP, as has also been observed in the embryonic central nervous system of grasshoppers. However, the adjacent SOP-like cells never develop into sensory organs, suggesting that the fate of SOP-like cells is irreversible. By observing the nuclear morphology along with an indicator for caspase activation, it was noted that in the process of sensory organ development, only SOP-like cells showed the typical features of programmed cell death. These results indicate that programmed cell death ensures robust pattern formation by eliminating aberrantly differentiated cells (Koto, 2011).
The significance of programmed cell death in pattern formation has been well studied, especially in the development of the fly eye. Each ommatidium is composed of eight photoreceptor neurons and six support cells, consisting of four cone cells and two primary pigment cells. Between each ommatidium, remaining cells form the interommatidial lattice. Excess pigment cells are eliminated through programmed cell death. Notch functions within the interommatidial lattice to induce cell death, and the primary pigment cells send a survival signal to adjacent cells. The life-and-death fate of interommatidial cells is decided by their position and the cells to which they are attached. In the case of sensory organ formation, Notch signaling is crucial in determining the aberrant cell fate of SOP-like cells. However, Notch activation alone seems insufficient to induce programmed cell death, because the surrounding epithelial cells do not disappear, even though they exhibit high levels of Notch activation during sensory organ development. Therefore, some factor that marks neural differentiation in SOP-like cells may be required to induce cell suicide. This study found that ectopic neuralized expression did not induce the aberrant cell fate or cell death in epithelial cells, suggesting that neuralized itself is not essential in determining the aberrant cell fate of SOP-like cells. Therefore, to determine how apoptosis is induced in SOP-like cells, the effect of Notch activation in neuralized-positive cells was examined at the one-cell stage using the temporal and regional gene expression targeting (TARGET) system with tub-GAL80ts. As reported previously, activated Notch induced the multiple-sockets phenotype. At the same time, about 50% of neuralized-positive cell lineages died, accompanied by nuclear fragmentation, causing a dramatic bald phenotype that was observed in the adult flies. These findings suggest that the combination of neural differentiation in the SOP lineage and Notch activation switches on cell death signaling. One possible future approach to searching for the killing factor expressed in SOP-like cells would be gene profiling using laser microdissection (Koto, 2011).
When the apoptotic pathway is blocked, the inhibition of cell death results in cell fate transformation. In C. elegans, cell death survivors in ced-3 mutants exhibit an ambiguous cell fate. The most disruptive alternative cell fate occurs when the remaining cells differentiate into tumor-like proliferating cells, as shown in the development of the Drosophila serotonin lineage. Under apoptosis-deficient conditions, other types of cell death occur, such as necrosis or autophagic cell death. These alternate reactions could mask the incidence of programmed cell death; therefore, it is possible that the role of the apoptotic pathway has been missed in the case of sensory organ development. This study has shown that SOP-like cells differentiate into epithelial cells when the cell death pathway is blocked. Time-lapse imaging made it possible to trace the transient fate of dying SOP-like cells, revealing the contribution of programmed cell death in the SOP selection process. Although the function of apoptosis has been emphasized in various developmental processes, the principle message is that several pathways exist to overcome the appearance of excess or aberrant cells and to make the developmental process more robust. This study reveals that programmed cell death plays an important role in overcoming innately induced developmental errors and contributing to robust neural cell selection (Koto, 2011).
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 this screen illustrates the complex cellular integration of survival and death signals through multiple pathways (Yi, 2007).
A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. A systematic study was conducted of Drosophila cell death-related genes to determine their requirement in the regulation of starvation-induced autophagy. It was discovered that six cell death genes--death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53--as well as Ras-Raf-mitogen activated protein kinase signaling pathway components had a role in autophagy regulation in Drosophila cultured cells. During Drosophila oogenesis, it was found that autophagy is induced at two nutrient status checkpoints: germarium and mid-oogenesis. At these two stages, the effector caspase Dcp-1 and the inhibitor of apoptosis protein Bruce function to regulate both autophagy and starvation-induced cell death. Mutations in autophagy-related genes Atg1 and Atg7 (Atg signifies an autophagy-related gene) resulted in reduced DNA fragmentation in degenerating midstage egg chambers but did not appear to affect nuclear condensation, which indicates that autophagy contributes in part to cell death in the ovary. This study provides new insights into the molecular mechanisms that coordinately regulate autophagic and apoptotic events in vivo (Hou, 2008).
Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved mechanism for the degradation of long-lived proteins and organelles. During autophagy, cytoplasmic components are sequestered into double membrane structures called autophagosomes, which then fuse with lysosomes to form autolysosomes, where degradation occurs. Currently, there are 31 autophagy-related (Atg) genes in yeast, and 18 Atg proteins are essential for autophagosome formation. Most yeast Atg genes have orthologues in higher eukaryotes and encode proteins required for autophagy induction, autophagosome nucleation, expansion, and completion, and final retrieval of Atg protein complexes from mature autophagosomes (Hou, 2008).
Depending on the physiological and pathological conditions, autophagy has been shown to act as a pro-survival or pro-death mechanism in vertebrates. In the case of growth factor withdrawal, starvation, and neurodegeneration, autophagy has been shown to function in cell survival. In contrast, autophagy has been found to act as a cell death mechanism in derived cell lines where caspases or apoptotic regulators are impaired. The nature and perhaps level of the stress stimulus may also be important in determining whether autophagy promotes cell survival or cell death (Hou, 2008).
Overlaps between components in apoptosis and autophagic pathways have been described. Upstream signal transducers in apoptotic pathways, including TNF-related apoptosis-inducing ligand (TRAIL), TNF, Fas-associated protein with death domain (FADD), and death-associated protein kinase (DAPK), have been shown to play a role in autophagy regulation. In addition, two recent studies demonstrate physical and functional interactions between components of apoptosis and autophagy. First, the antiapoptosis protein, Bcl-2, suppresses autophagy through a direct interaction with Beclin 1, a protein required for autophagy. Second, Atg5, which is cleaved by calpain, associates with Bcl-XL, leading to cytochrome c release and caspase activation. Further examples and discussion of the connections between apoptosis and autophagy can be found in several recent reviews on this topic. The current findings indicate that there is a complex relationship between apoptosis and autophagy, but the regulatory mechanisms underlying the crosstalk between the two processes are still largely unknown (Hou, 2008 and references therein).
Autophagy is observed in several Drosophila tissues during development, and thus Drosophila is useful as a model to study autophagy in the context of a living organism. 14 Drosophila annotated genes share significant sequence identity with the yeast Atg genes, and, overall, eight Drosophila Atg homologues have already been shown to be required for autophagy function. In addition, recent studies demonstrated the role of autophagy in Drosophila physiological cell death. Loss of Atg genes, including Atg1, Atg2, Atg3, Atg6, Atg7, Atg8, Atg12, and Atg18, inhibit proper degradation of salivary glands during development. Overexpression of Atg1 induces premature salivary gland cell death in a caspase-independent manner). In contrast, caspase activity was required for Atg1-mediated apoptotic death in the fat body. Mutation of Atg7 results in an inhibition of DNA fragmentation in the midgut but leads to an increase of DNA fragmentation in the adult Drosophila brain. Together, these results further suggest that the mechanistic role of autophagy in cell death and the interrelations between autophagy and apoptosis may be tissue and/or context dependent (Hou, 2008).
The adult Drosophila ovary contains 15-20 ovarioles comprised of developing egg chambers, which consist of 16 germ line cells (15 nurse cells and 1 oocyte) surrounded by a layer of somatic follicle cells. The germ line cells originate from stem cells that undergo mitosis to form 16-cell cysts in a specialized region called the germarium. In the late stage of oogenesis, the nurse cells support the development of the oocyte by transferring to it their cytoplasmic contents. After this 'dumping' event, the nurse cells undergo cell death, and their remnants are engulfed by the surrounding follicle cells. In addition to this late-stage developmental cell death, egg chambers can be induced to die at two earlier stages, during germarium formation (in region 2) and mid-oogenesis, by factors such as nutrient deprivation, chemical insults, and altered hormonal signaling. In some respects, cell death during Drosophila oogenesis is similar to the death of Drosophila larval salivary glands. Both nurse cells and salivary gland cells are large and polyploid, and the entire tissues undergo cell death simultaneously. Notably, morphological features of autophagy have been described during mid-oogenesis cell death in a related species, Drosophila virilis, which suggests that the cell death process in ovaries and salivary glands share additional similarities (Hou, 2008).
Previous studies have focused on characterizing the role of autophagy genes in cell death and determining the paradoxical functions of autophagy (pro-survival and pro-death) in various cell lines and organisms. However, a systematic approach that investigates the involvement of cell death genes in starvation-induced autophagy has not been conducted. This study presents RNAi analyses to determine whether known cell death-related genes in Drosophila play a role in autophagy regulation in the lethal (2) malignant blood neoplasm (l(2)mbn) cell line. Drosophila genetics was used to investigate a role for the effector caspase death caspase-1 (Dcp-1) and the inhibitor of apoptosis (IAP) family member Bruce in autophagy regulation in vivo during Drosophila oogenesis. Further, the function was studied of autophagy genes Atg7 and Atg1 in starvation-induced germ line cell death in the Drosophila ovary (Hou, 2008).
All Reaper, Hid, Grim (RHG) family members, Rpr, Hid, Grim, and Skl, bind to Drosophila IAP-1 (DIAP1) and inhibit its antiapoptotic activities. To test whether DIAP1 (encoded by th) is a putative downstream mediator of Hid-dependent autophagy in l(2)mbn cells, dsRNA was designed specifically to target th. th-dsRNA-treated cells showed no difference in LysoTracker green (LTG) fluorescence levels compared with Hs-dsRNA (negative control)-treated cells. Interestingly, the data showed that reduced expression of Bruce, another IAP family member protein, further increased the LTG fluorescence levels after starvation treatment (confirmed using nonoverlapping dsRNAs). RNAi of Bruce expression also resulted in an increase in GFP-LC3 puncta (refering to GFP tagged microtubule associated protein 1 light chain 3B) after starvation treatment. These results suggest that Bruce, instead of DIAP1, could be the downstream target of Hid during starvation induced autophagy in l(2)mbn cells (Hou, 2008).
To investigate the requirement of caspases, the final effectors of apoptosis, in starvation-induced autophagy, gene-specific dsRNAs were designed corresponding to seven different Drosophila caspases. RNAi of just one caspase, Dcp-1, but not others resulted in a decrease in the percentage of LTGhigh cells after starvation treatment. A second dsRNA against Dcp-1, nonoverlapping with the first dsRNA, yielded a similar result. Reduction of Dcp-1 expression by RNAi was determined using QRT-PCR. Consistent with the LTG derived data, RNAi-mediated knockdown of Dcp-1 resulted in a decrease in GFP-LC3-positive cells after starvation treatment. These results indicate that Dcp-1 functions as a positive regulator of autophagy in D. melanogaster l(2)mbn cells (Hou, 2008).
Key outstanding questions that need to be addressed are how autophagy and apoptosis pathways interact with each other, and whether common regulatory mechanisms exist between these two processes. This study showed that six known cell death genes and the Ras-Raf-MAPK signaling pathway not only function in apoptosis but also act to regulate autophagy in D. melanogaster l(2)mbn cells. The possibility cannot be ruled out that additional cell death genes that were screened may also function in autophagy but were not detected in the assay that was used because of insufficient knockdown by RNAi, a long half-life of the corresponding proteins, and/or functional redundancy (Hou, 2008).
Consistent with in vitro data, the involvement of Hid in autophagy regulation has been demonstrated in Drosophila. Overexpression of Hid induced autophagy in the fat body, larval epidermis, midgut, salivary gland, Malpighian tubules, and trachea epithelium. Further, expression of the constitutively active Ras form (RasV12), which has been shown to inhibit Hid activity in apoptosis, can also block Hid-induced autophagy. In Drosophila salivary glands, the Ras signaling pathway has also been shown to inhibit the autophagy process. Based on loss-of-function findings and these previous gain-of-function studies, it was speculated that the Ras-Raf-MAPK pathway acts upstream to inhibit Hid activity in autophagy (Hou, 2008).
Poor nutrition has a dramatic effect on egg production in Drosophila. Flies fed on a protein-deprived diet showed an increase in cell death in germaria and midstage egg chambers. These two stages have been proposed to serve as nutrient status checkpoints where defective egg chambers are removed before the investment of energy into them. The molecular mechanisms of germarium cell death are still largely unknown, and Daughterless, a helix-loop-helix transcription factor, was the only known regulator involved in cell death of germaria. Nurse cell death during mid-oogenesis is also different from most developmental cell death in other Drosophila tissues because apoptotic regulators such as rpr, hid, or grim are not required for cell death in these cells. However, the activity of caspases, particularly Dcp-1, was shown to be required for mid-oogenesis cell death. The current findings implicate several additional genes, Dcp-1, Bruce, Atg7, and Atg1, in nutrient deprivation-induced cell death in the germarium, as well as during mid-oogenesis (Hou, 2008).
Other forms of cell death, such as autophagic cell death, have been proposed previously to be involved in the elimination of defective egg chambers during mid-oogenesis. Known signaling pathways, including the insulin and ecdysone pathways, have been shown to be required not only for the survival of nurse cells in mid-oogenesis; they are also known to regulate the autophagy process, supporting the notion that autophagy plays a role in mid-oogenesis cell death. Features of autophagy were observed during D. virilis mid-oogenesis cell death as shown by monodansylcadaverine staining and transmission electron microscopy. The results using GFP-LC3 and LTG demonstrate that autophagy occurs in degenerating midstage egg chambers and also in germaria of nutrient deprived Drosophila. It was found that mutation of Atg7 results in a significant decrease of autophagy in dying mid-stage egg chambers and in germaria of starved flies, further supporting the presence of autophagy during these stages (Hou, 2008).
The role of autophagy in cell survival or cell death is still not well resolved and is likely to be context dependent. The results show that autophagy contributes to the cell death process in the ovary. Loss of Atg7 or Atg1 activity in both dying midstage egg chambers and germaria leads to decreased TUNEL staining, which indicates a reduction in DNA fragmentation. Consistent results were observed previously in the larval midguts of Atg7 mutants, which also showed an inhibition of DNA fragmentation. Interestingly, lack of autophagy function does not appear to affect nuclear DNA condensation in nurse cells. Nurse cells in degenerating stage 8 egg chambers of starved Atg7 mutants or Atg1 GLCs appeared to still have condensed nuclei, as shown by DAPI staining. Thus, based on Atg7 and Atg1 mutant analyses, autophagy contributes to DNA fragmentation but not all aspects of nurse cell death. Future studies are required to determine how autophagy is connected to known pathways leading to DNA fragmentation and chromatin condensation during cell death (Hou, 2008).
The IAP family member Bruce was shown previously to repress cell death in the Drosophila eye (Vernooy, 2002). Bruce was also shown to protect against excessive nuclear condensation and degeneration, perhaps by limiting excessive caspase activity, during sperm differentiation (Arama, 2003). Other IAP family members have been shown to bind caspases via a BIR domain and inhibit apoptosis. The presence of a BIR domain in Bruce suggests that it may also have caspase-binding activity. This study found that lack of Bruce function resulted in an increase in both LTR and TUNEL staining in germaria and degenerating midstage egg chambers. Thus, the Bruce mutant-degenerating phenotype in ovaries suggests that Bruce might function normally to restrain or limit caspase activity in this tissue. Because it was found that Dcp-1 and Bruce are both required for the regulation of autophagy and DNA fragmentation in germaria and dying midstage egg chambers, it is possible that Bruce acts to bind and degrade Dcp-1 in nurse cells under nutrient-rich conditions. Future studies using epistasis and protein interaction analyses will be required to test this prediction. The possibility cannot be ruled out that other IAP proteins, such as DIAP1, and other caspases also play a role during these stages. However, at least in response to starvation signals, Bruce and Dcp-1 play a nonredundant dual role in the regulation of autophagy and cell death in the ovary (Hou, 2008).
Numerous studies have linked caspase function to apoptosis, but recent findings indicate that caspases are also required for nonapoptotic processes including immunity and cell fate determination (for reviews see Kumar, 2004; Kuranaga, 2007). Tnis study has shown that Dcp-1 is also required for starvation-induced autophagy. In the ovary, it appears that both apoptotic and autophagic events occur in the germaria and midstage egg chambers after nutrient deprivation. It is possible that Dcp-1 coordinates autophagy and apoptosis at these two nutrient status checkpoints to ensure elimination of defective egg chambers in the most efficient manner possible. Dcp-1 mutants exhibit intact nuclei in stage 8 defective egg chambers, which indicates a block in both DNA fragmentation and nuclear condensation, and further supports a dual regulatory role for Dcp-1 in mid-oogenesis cell death. Dcp-1 might function to induce autophagosome formation while coordinately acting upon alternate proteolytic targets to complete execution of apoptosis. Future studies to elucidate upstream regulators and downstream substrates of Dcp-1 in cells undergoing autophagy or apoptosis will help to establish the regulatory mechanisms governing the crosstalk between these two cellular processes. Given the multiple cellular effects associated with autophagy, these results also have important therapeutic implications for the use of modulators of caspase or IAP activity in the treatment of cancer and other diseases (Hou, 2008).
Death caspase-1:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| References
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