Defective Molecular Timer in the Absence of Nucleotides
Leads to Inefficient Caspase Activation
Honghao Zhang1, Raghu Gogada1, Neelu Yadav1, Ravi K. Lella1, Mark Badeaux2, Mary Ayres3, Varsha
Gandhi3, Dean G. Tang2, Dhyan Chandra1*
1Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York, United States of America, 2Department of Molecular Carcinogenesis,
The University of Texas MD Anderson Cancer Center, Smithville, Texas, United States of America, 3Department of Experimental Therapeutics, The University of Texas MD
Anderson Cancer Center, Houston, Texas, United States of America
In the intrinsic death pathway, cytochrome C (CC) released from mitochondria to the cytosol triggers Apaf-1 apoptosome
formation and subsequent caspase activation. This process can be recapitulated using recombinant Apaf-1 and CC in the
presence of nucleotides ATP or dATP [(d)ATP] or using fresh cytosol and CC without the need of exogenous nucleotides.
Surprisingly, we found that stored cytosols failed to support CC-initiated caspase activation. Storage of cytosols at different
temperatures led to the loss of all (deoxy)nucleotides including (d)ATP. Addition of (d)ATP to such stored cytosols partially
restored CC-initiated caspase activation. Nevertheless, CC could not induce complete caspase-9/3 activation in stored
cytosols, even with the addition of (d)ATP, despite robust Apaf-1 oligomerization. The Apaf-1 apoptosome, which functions
as a proteolytic-based molecular timer appeared to be defective as auto-processing of recruited procaspase-9 was inhibited.
Far Western analysis revealed that procaspase-9 directly interacted with Apaf-1 and this interaction was reduced in the
presence of physiological levels of ATP. Co-incubation of recombinant Apaf-1 and procaspase-9 prior to CC and ATP
addition inhibited CC-induced caspase activity. These findings suggest that in the absence of nucleotide such as ATP, direct
association of procaspase-9 with Apaf-1 leads to defective molecular timer, and thus, inhibits apoptosome-mediated
caspase activation. Altogether, our results provide novel insight on nucleotide regulation of apoptosome.
Citation: Zhang H, Gogada R, Yadav N, Lella RK, Badeaux M, et al. (2011) Defective Molecular Timer in the Absence of Nucleotides Leads to Inefficient Caspase
Activation. PLoS ONE 6(1): e16379. doi:10.1371/journal.pone.0016379
Editor: Amit Singh, University of Dayton, United States of America
Received October 16, 2010; Accepted December 13, 2010; Published January 27, 2011
Copyright: ? 2011 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by a National Institutes of Health K01 award CA123142 to DC, National Cancer Institute Center grant CA16056 to
Roswell Park Cancer Institute, and institutional start-up support to DC. The authors also acknowledge support by grants from the National Institutes of Health
(ES015888 and ES015893-01A1 to DGT, and CA57629 and CA85915 to VG). The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Caspases, the core enzymes responsible for executing apoptotic
cell death, are synthesized as inactive zymogens and divided into
initiator (caspase-2, -8, -9, and –10) and effector or executioner
(caspase-3, -6, and –7) caspases. Active initiator caspases generated
in response to apoptosis signals induce intrachain cleavage of
effector caspases, which undergo reorganization of active site loops
to become active . Activation of initiator caspases such as
caspase-9 requires the adaptor protein Apaf-1 , which contains
caspase-recruitment domain (CARD), nucleotide binding and
oligomerization domain (NOD), and WD40 repeats for CC
interaction. The released CC from mitochondria binds to and
induces oligomerization of Apaf-1 to form the ‘apoptosome’, a
heptameric complex with molecular masses of ,700–1400 kDa
[3,4,5]. Procaspase-9 subsequently becomes activated within the
apoptosome either involving proximity-induced dimerization or
‘‘induced conformational changes’’ .
Among various factors that regulate apoptosome formation and
caspase activation, (d)ATP plays a critical role. Cell-free and
recombinant protein reconstitution experiments have demonstrat-
ed that (d)ATP initiates Apaf-1 oligomerization following Apaf-1
binding to CC [5,6,7]. Truncated Apaf-1, i.e., Apaf-591, which
lacks the WD-40 repeats but retains the NOD, binds to ADP
molecule, which locks Apaf-1 in a conformationally inactive state
. Full-length Apaf-1 is capable of binding one molecule of
dATP. CC binding to Apaf-1 induces hydrolysis of dATP to dADP
coupled with exchange for dATP to initiate apoptosome assembly
[9,10]. Thus, nucleotide binding and exchange are critical for the
regulation of apoptosome formation and caspase activation. It is of
interest that once functional apoptosome is assembled, recruited
procaspase-9 is processed within the apoptosome. The processed
caspase-9 fragment possesses lower affinity for apoptosome and is
continuously replaced by procaspase-9. Therefore, the Apaf-1
apoptosome functions as proteolytic-based ‘‘molecular timer’’,
wherein the autoprocessing of procaspase-9 starts the timer and
intracellular levels of procaspase-9 sets the overall duration of the
Most mammalian cells have an intracellular ATP and
nucleotide pool in millimolar range [13,14,15,16,17]. For
example, the cytoplasmic levels of ATP alone can be as high as
3–8 mM [13,14,15,16], which explains our recent observations
that freshly purified cytosol does not require exogenous (d)ATP to
initiate apoptosome assembly and caspase activation by CC .
Here we report that cytosols stored at low temperatures fail to fully
support the CC-mediated caspase activation. Loss of (d)ATP
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causes stable association of procaspase-9 with the apoptosome.
Altogether, degradation of nucleotides leads to dysfunctional
molecular timer, and thus, blocking sustained activation of
caspase-9 on the apoptosome.
dATP is required for CC-initiated caspase activation by
recombinant Apaf-1 but not by freshly purified cytosol
Exogenous (d)ATP is required for caspase activation in
reconstitution experiments using recombinant proteins or purified
cytosol [3,4,5,7]. On the other hand, we recently found that in
reconstitution experiments using freshly purified cytosol, exoge-
nous (d)ATP was not required for CC-initiated caspase activation
[16,18]. To clarify this seeming ‘discrepancy’, we first purified
recombinant Apaf-1, procaspase-9, and procaspase-3 from insect
cells (Sf9) (Fig. 1A) and used theses purified proteins in CC-
initiated caspase assays (Fig. 1B). The results showed that (d)ATP
was absolutely required for Apaf-1/CC-initiated caspase activa-
tion (Fig. 1B, and data not shown). For example, in the absence of
dATP, there was no cleavage of procaspase-9 or procaspase-3
(Fig. 1B). However, in the presence of dATP, the p35 caspase-9
was generated through Apaf-1-dependent autocatalytic cleavage of
procaspase-9 at Asp315(Fig. 1B). In the presence of both dATP
and procaspase-3, the p37 caspase-9 was also generated (Fig. 1B)
as a result of caspase-3-mediated cleavage of procaspase-9 at
Asp330[16,19]. Procaspase-3 was cleaved by activated caspase-9 to
generate active p20/p17 fragments (Fig. 1B).
In contrast to the above reconstitution experiments using
purified proteins, CC, in a concentration-dependent manner,
initiated caspase-9 and -3 cleavage in fresh cytosol purified from
GM701 cells without requiring exogenous dATP (Fig. 1C).
Likewise, similar levels of procaspase-9 and -3 cleavage were
observed in fresh HeLa cell cytosol in the absence or presence of
dATP (Fig. 1D). Measurements of caspase-3 and caspase-9
activities (data not shown) also revealed that CC initiates caspase
activation without the need of exogenous (d)ATP in fresh cytosols
from GM701, NHDF (normal human dermal fibroblasts) and
HMEC (human mammary epithelial cells) cells. Similar to cell-free
reconstitution experiments using fresh cytosols from multiple cells
including MDA-MB231, LNCaP, PPC-1, PC3, Du145, NHP,
K562, HCT116, and C2F3 (see Materials and Methods for
descriptions of cells) also revealed CC-initiated caspase activation
without requiring exogenous nucleotides (data not shown) .
The above observations suggest that freshly purified cytosols
contain sufficient amounts of nucleotides to assist in the CC-
initiated caspase activation. In support, direct measurements of
(d)NTP levels in 6 tumor cell lines and 3 primary cell strains
revealed that these cells contain ATP levels of 1.5–5 mM and
possessed an NTP pool of ,3–7 mM and a dNTP pool of ,
10–80 mM .
Stored cytosols fail to support CC-initiated caspase
To determine whether cytosol could be stored for later use to
investigate the mechanisms of functional apoptosome assembly, we
tested the capability of cytosol stored at different temperatures.
Much to our surprise, when we performed similar cell-free
reconstitution experiments using purified GM701 cytosols stored
for 5 days at 4uC, we found that addition of CC alone could not
support caspase activation, whereas complete caspase-9/3 process-
ing was observed in fresh cytosol (Fig. 2A; Fig. 3A). Substrate
cleavage assays also demonstrated no increase in caspase-9 (Fig. 2B)
or caspase-3 (Fig. 2C) activity in stored cytosols. We then performed
cell-free reconstitution experiments using either freshly purified
cytosol from HCT116 cells or HCT116 cytosol stored at 4uC for 5
days. As shown in Fig. 2D and 2E, stored cytosol failed to support
the CC-initiated caspase activation. We then examined GM701
cytosolsstored at220uC or280uC fordifferent periods oftime.We
observed that cytosols stored at 220uC for 15 days could not
support CC-initiated caspase activation (Fig. 3B). Cytosols stored at
280uC for 1 month supported CC-induced caspase activation,
whereas storage of cytosols at 280uC for longer periods of time
failed to activate caspases (data not shown).
To examine if storage of cytosol leads to degradation of protein
required for apoptosome assembly, we performed Western
blotting to detect their levels and demonstrated that stored
cytosols possess similar levels of Apaf-1, procaspase-9, and
procaspase-3 (Fig. 3C), suggesting that failure of stored cytosols
to support the CC-initiated caspase activation is not caused by
quantitative differences in these three main constituents of the
apoptosome. To determine whether during storage of cytosol, the
main components of apoptosome might have undergone confor-
mational changes, and therefore, become functionally inactive, we
performed reconstitution experiments using stored cytosols by
adding CC along with recombinant Apaf-1 or procaspase-9 or
procaspase-3. We demonstrated that addition of CC in the
presence/absence of each individual recombinant protein did not
significantly alter caspase-9 or caspase-3 activation (Fig. 2B–C).
These experiments indicate that failure of stored cytosols to
support the CC-initiated caspase activation does not seem to be
associated with degradation/inactivation of the core protein
components of the Apaf-1-apoptosome.
(Deoxy)nucleotides become degraded and exogenous
dATP partially restores CC-mediated caspase activation in
caspase activation (Fig. 1B), we reasoned that failure of stored cytosols
to support CC-initiated caspase activation might be associated with
degradation of nucleotides including (d)ATP. To test this possibility,
purified cytosols from GM701 cells were kept at 4uC, 220uC, or
280uC for various periods of time followed by measurement of
(deoxy)nucleotides concentrations. As shown in Table 1 and 2, storage
at 4uC for 2 days led to .90% loss and storage for $4 days resulted in
nearly complete degradation of all (deoxy)nucleotides. Similarly,
storage at 220uC for 4 days led to $40% loss and for 15 days to
,85% loss of most (deoxy)nucleotides including ATP (Table 1 and 2).
dATP appeared to be slightly more stable as 15-day storage resulted in
,60% of its loss (Table 2). Even storage at 280uC for 2 months led to
nearly 50% loss of all (deoxy)nucleotides (Table 1 and 2). These results
practiced in laboratory research, may lead to rapid degradation of
To determine whether loss of (d)ATP might be responsible for
the failure of stored cytosols to support the CC-mediated caspase
activation, we added dATP back to the cytosols stored at 4uC for 5
days (Fig. 3A) or at 220uC for 15 days (Fig. 3B), and in every case,
we observed a CC concentration-dependent generation of p35
caspase-9 and p24/p20 caspase-3 bands. Substrate cleavage assays
for caspase-9 (Fig. 2B) and caspase-3 (Fig. 2C) also showed partial
restoration of their activity by addition of exogenous dATP.
Similar results were obtained using ATP in the reconstitution
experiments (data not shown).
Surprisingly, dATP addition did not result in full caspase
activation to the same level as observed in fresh cytosols (Fig. 2B–
C). Furthermore, increasing CC to $10 mg/ml did not enhance
caspase cleavage (Fig. 3A, lanes 10 and 11). Most strikingly, we
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observed that dATP addition to stored cytosols resulted in
prominent generation of the p35 caspase-9 band without the p37
band (Fig. 3A and B). Since the p35 caspase-9 is generated through
Apaf-1-dependent cleavage of procaspase-9 at Asp315and the p37
caspase-9 results from caspase-3-mediated cleavage of procaspase-9
at Asp330[16,19], these observations suggest that exogenous dATP
appeared to have initiated relatively normal apoptosome activation
of procaspase-9 in stored cytosols but caspase-3 mediated caspase-9
cleavage/activation was inhibited. Itisknownthat thep24fragment
of caspase-3 has a high affinity for XIAP and further processing to
mature p20/p17 fragments is required for full caspase activity
[4,20,21,22]. Interestingly, majority of dATP/CC-induced process-
ing of caspase-3 in cytosols stored at 4uC for 5 days (Fig. 3A) or at
220uC for 15 days (Fig. 3B) was p24, suggesting that caspase-3
activation by p35 caspase-9 was incomplete, which could be
responsible for a lack of generation of the p37 caspase-9.
Stored cytosols support CC-initiated Apaf-1
oligomerization and procaspase-9 recruitment but
display defective caspase-9 activation in the apoptosome
The preceding experiments have established that, 1) cytosols
stored at low temperatures rapidly lose (deoxy)nucleotides
including (d)ATP; 2) loss of (deoxy)nucleotides is responsible for
the failure of stored cytosols to support CC-initiated caspase
activation as exogenous dATP could partially restore the CC-
initiated caspase activation in stored cytosols; and 3) dATP does
not support complete processing and activation of caspases. To
further elucidate why dATP fails to fully support the CC-initiated
caspase cleavage/activation in stored cytosols, we carried out gel
filtration analysis to evaluate whether stored cytosols are capable
of supporting Apaf-1 oligomerization, which is considered to be
the rate-limiting step in apoptosome assembly and apoptosome-
mediated caspase activation. As shown in Fig. 4, CC alone
initiated robust Apaf-1 oligomerization in fresh cytosol. Interest-
ingly, CC alone in cytosolic samples stored at 220uC for 15 days,
which retained ,15% ATP and 36% dATP (Table 1 and 2),
induced significant Apaf-1 oligomerization similar to that observed
in fresh cytosols and addition of exogenous dATP did not
dramatically increase Apaf-1 oligomerization (Fig. 4). Similar
results were obtained in gel-filtration experiments using cytosols
stored at 280uC for 3 months (not shown). These latter
observations suggest that the residual amounts of (d)ATP in the
cytosolic samples stored at 220uC for 15 days or at 280uC for 3
months are sufficient for CC-triggered Apaf-1 oligomerization.
Figure 1. Fresh cytosol does not require exogenous nucleotides for CC-initiated caspase activation. A, Recombinant Apaf-1,
procaspase-9 (procasp-9), or procaspase-3 (procasp-3) was expressed in insect cells and subsequently purified. These proteins were separated on
SDS-PAGE and stained with Coomassie blue. B, Reconstitution experiments were performed to test the activity of purified proteins by using
recombinant Apaf-1 (165 ng), procaspase-9 (130 ng), and procaspase-3 (100 ng) with or without CC (15 mg/ml) and dATP (200 mM) in a total reaction
mixture of 30 ml. At the end of incubation at 30uC for 120 min, aliquots were subjected to Western blotting for caspase-9 and caspase-3. C, Fresh
GM701 cytosol (250 mg) incubated for 150 min at 37uC with increasing amount of CC was used in Western blotting for caspase-9 and caspase-3. D,
Fresh HeLa cytosol incubated with CC (15 mg/ml) in the absence or presence of dATP (200 mM) was subjected to Western blotting for caspase-9 and
caspase-3. Procasp-9, procaspase-9; procasp-3, procaspase-3; CC, cytochrome c.
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Altogether, the gel-filtration studies suggest that failure of stored
cytosols to fully support the CC-initiated caspase activation is not
due to the lack of Apaf-1 oligomerization.
During apoptosome assembly, Apaf-1 oligomerization is accom-
panied by recruitment and activation of procaspase-9 to generate
p35/p37 caspase-9 fragments, which remain in the apoptosome
complex and exhibit caspase processing activity (CPA) in the
presence of exogenous procaspase-9 and procaspase-3 [23,24].
Recent studies from the Bratton’s group  further suggest that
auto-processing of caspase-9 starts the molecular timer and is
constantly replaced by procaspase-9 until all intracellular procas-
pase-9 are processed. Processed caspase-9 when present on the
apoptosome, recruits and activates procaspase-3 . We found
that CC-induced Apaf-1 oligomerization in fresh cytosols was
accompanied by the presence of both p35 and p37 caspase-9
fragments in high M.W. fractions with little procaspase-9 (Fig. 4).
We also observed prominent p20 caspase-3 but not procaspase-3 in
the oligomeric fractions in the CC-activated fresh cytosol (Fig. 4).
Cytosols that had been stored at 220uC (Fig. 4) or 4uC (not shown)
initiated Apaf-1 oligomerizationuponadditionof CC or CC/dATP
leading to the recruitment of procaspase-9 with the high M.W.
fractions, but no processed p35/p37 caspase-9 was observed.
Similarly, prominent procaspase-3 was detected in the oligomeric
fractions with no cleaved caspase-3 bands in the 220uC cytosols
(Fig. 4). These results suggest that in stored cytosols, recruitment of
procaspase-9 to the apoptosome is not significantly diminished but
the molecular timer  is dysfunctional such that recruited
procaspase-9 does not undergo auto-processing, and thus, does not
dissociate from the apoptosome.
Consistent with gel filtration analysis, measurement of caspase-
processing activity (CPA) revealed (Fig. 5A–B) that the CC-triggered
apoptosome in fresh cytosol (Fig. 4, fractions 10-14) possessed readily
detectable CPA whereas apoptosome formed in the 220uC cytosol
with the addition of exogenous dATP (Fig. 4, fractions 10–14) did not
show significant CPA, indicating non-functional apoptosome.
Procaspase-9 interacts with Apaf-1 in absence of
nucleotides and co-incubation of Apaf-1 with
procaspase-9 prior to CC addition inhibits caspase
To understand how procaspase-9 recruitment to the apopto-
some leads to defective molecular timer in absence of
Figure 2. Stored cytosols require exogenous nucleotides to support CC-initiated caspase activation. A, GM701 cytosols (250 mg) either
freshly purified or stored at 4uC for 5 days were incubated with CC (15 mg/ml) for 150 min at 37uC. At the end of incubation, reaction mixtures were
subjected to Western blotting for caspase-9 and caspase-3. B and C, GM701 cytosols (250 mg) either freshly purified or stored at 4uC for 5 days were
incubated with CC in the absence or presence of dATP (200 mM). Some reaction mixtures were supplemented with recombinant Apaf-1 (A1) or
procaspase-9 (C9) or procaspase-3 (C3) to assess if these molecules were inactivated during storage. D and E, HCT116 cytosols either freshly purified
or stored at 4uC for 5 days were incubated with CC. At the end of incubation, aliquots were used in activity assays for caspase-9 and caspase-3.
Procasp-9, procaspase-9; procasp-3, procaspase-3; CC, cytochrome c.
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nucleotides, we performed Far-Western analysis in the presence
or absence of physiological levels of ATP (2 mM) using
immobilized recombinant Apaf-1. Since intracellular concen-
tration of ATP is in millimolar range and ATP also regulates
apoptosome function, we utilized ATP instead of dATP, which
generally exists in micromolar range. We observed that
procaspase-9 directly interacted with Apaf-1 in the absence of
ATP (Fig. 6A). However, the interaction between procaspase-9
and Apaf-1 was drastically reduced in the presence of
physiological levels of ATP (Fig. 6A). These results suggest that
degradation of nucleotides during storage of cytosols might have
promoted the association of procaspase-9 with Apaf-1, which
leads to defective apoptosome assembly and function. To further
confirm this notion, we incubated recombinant Apaf-1 with
procaspase-9 at 4uC for 30 min followed by addition of
procaspase-3, CC and ATP to induce apoptosome formation.
We observed that CC-induced caspase-9 and caspase-3 activa-
tion was inhibited upon pre-incubation of Apaf-1 with procas-
pase-9 (Fig. 6B).
Our present work first reveals distinct component requirements
when performing cell-free reconstitution of the apoptosome
activity under different conditions (Figure 7A). Along the way,
we uncover interesting biochemical mechanisms regulating
apoptosome activation by nucleotides. In the absence of
nucleotides such as in stored cytosols, auto-processing of caspase-
9 seems to be inhibited thereby abrogating the molecular timer
 leading to incomplete caspase-9 processing and inhibition of
caspase-3 activation by the apoptosome (Fig. 7B).
Nucleotides such as (d)ATP are absolutely required for
(Fig. 7A, left). In such systems, if procaspase-9 and procaspase-3
using recombinant proteins
Figure 3. Exogenous dATP restores CC-initiated caspase activation. A, GM701 cytosols stored at 4uC for 5 days were incubated with
increasing amounts of CC in the absence (lanes 1–6) or presence (lanes 7–11) of dATP (1 mM). B, Cytosol stored at 220uC for 15 days were incubated
with CC in the absence or presence of ATP (1 mM). At the end of incubation, samples were subjected to Western blotting for caspase-9 or caspase-3.
C, Cytosols stored at 4uC for 5 days were subjected to Western blotting to detect the levels of indicated proteins. Procasp-9, procaspase-9; Procasp-3,
procaspase-3; CC, cytochrome c.
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are present, both p35 and p37 cleaved caspase-9 bands as well as
active p20/p17 caspase-3 fragments can be observed. In contrast,
when using freshly purified cytosols to perform cell-free reconsti-
tution experiments, only CC is required to initiate the apoptosome
activation due to the presence of sufficient amounts of endogenous
nucleotides (Fig. 7A, middle). In such systems, both p35 and p37
caspase-9 and p24/p20/p17 caspase-3 bands are frequently
observed (Fig. 1C–D). However, the use of cytosols stored at low
temperatures in cell-free reconstitution assays shows defective CC-
initiated caspase activation (Fig. 7A, right). Direct measurement of
(deoxy)nucleotides demonstrates that storage of cytosols at low
temperatures leads to their rapid loss. Strikingly, the degree of
nucleotide loss correlates well with the failure of stored cytosols to
support the CC-initiated caspase activation. Thus, cytosols that
have been stored at 4uC for 5 days, which have completely lost
(deoxy)nucleotides, fail to induce caspase activation. However,
addition of exogenous (d)ATP partially restores the CC-triggered
In stored cytosols, addition of exogenous dATP did not support
full caspase activation evidenced by activity measurement (Fig. 2B–
C) and only p35 caspase-9 and mainly p24 caspase-3 are
generated in such systems (Fig. 7A, right). These observations
suggest that storage of cytosols at low temperatures may lead to
not only degradation of (deoxy)nucleotides but also degradation or
alteration of some other components important for apoptosome
activation. Mechanistic studies demonstrate that cytosols stored at
4uC for 5 days can support robust CC-initiated Apaf-1
oligomerization and procaspase-9 recruitment upon addition of
exogenous dATP. These observations suggest that the initial step
of apoptosome assembly is normal in stored cytosols, as also
supported by the generation of p35 caspase-9 (Fig. 7A, right).
Then why can’t exogenous dATP support full apoptosome-
mediated activation of caspase-9?
We find that although apoptosome formation and caspase-9
recruitment to the apoptosome are not affected, caspase-9
processing is severely compromised. Procaspase-9 was found to
Table 1. Changes in nucleotide levels during storage at different temperatures*.
Storage Storage Nucleotide levels (%)
temperaturetime ATPUTP GTP CTP
Fresh- 35.8 (100)11.5 (100)10.0 (100) 2.4 (100)
280uC 1 month36.1 (101) 12.1 (105)10.4 (104)2.4 (104)
2 months18.8 (53) 6.2 (54)5.3 (53) 1.1 (46)
220uC 4 days20.2 (56) 7.2 (62)6.2 (62) 1.1 (46)
8 days14.1 (39)5.3 (46) 4.6 (46)0.7 (29)
15 days 5.5 (15)1.9 (17) 1.6 (16)0.3 (12)
4uC 2 days 2.4 (7) 0.9 (8)0.8 (8) 0.0 (0)
4 days 1.1 (3) 0.1 (1)0.2 (2)0.0 (0)
8 days 0.7 (2)0.1 (1) 0.2 (2) 0.0 (0)
*Cytosols obtained from GM701 cells were stored at different temperatures for the time intervals indicated and then nucleotides levels were measured using HPLC.
Values represent the mean (nmoles/mg protein) derived from two independent measurements (values in the parentheses represent percentage changes compared to
the fresh cytosol).
Table 2. Changes in deoxynucleotide levels during storage at different temperatures*.
Storage Storage Deoxynucleotide levels (%)
temperature timedATP dTTPdCTP dGTP
Fresh- 0.103 (100) 0.547 (100)0.097 (100) 0.027 (100)
280uC 1 month 0.090 (87)0.483 (88) 0.090 (93)0.023 (85)
2 months 0.043 (42)0.282 (52) 0.045 (46)0.012 (44)
220uC 4 days 0.057 (55)0.330 (60)0.020 (21) 0.010 (37)
8 days0.057 (55) 0.253 (46)0.010 (10)0.007 (26)
15 days 0.037 (36) 0.090 (16)0.000 (0) 0.000 (0)
4uC 2 days 0.003 (3) 0.040 (7)0.003 (3) 0.000 (0)
4 days 0.000 (0)0.007 (1) 0.000 (0)0.000 (0)
8 days0.000 (0)0.007 (1)0.000 (0) 0.003 (11)
*Cytosols obtained from GM701 cells were stored at different temperatures for the time intervals indicated and then deoxynucleotides levels were measured using
HPLC. Values represent the mean (nmoles/mg protein) derived from two independent measurements (values in the parentheses represent percentage changes
compared to the fresh cytosol).
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be associated with the apoptosome, but a very low level of caspase
activity was observed. These findings suggest the requirement of
procaspase-9 processing upon recruitment to the apoptosome for
full caspase activity. Indeed, Bratton and colleagues  have
clearly demonstrated that for sustained apoptosome-mediated
caspase activation and apoptosis, recruited procaspase-9 must
undergo auto-processing (i.e., like a timer) followed by replace-
ment with new procaspase-9. Our findings suggest that in stored
cytosols, auto-processing of recruited procaspase-9 does not seem
to occur, thus abrogating the molecular timer and leading to
sequestration and maintenance of procaspase-9 on the apopto-
some (Fig. 7B). Although the precise molecular mechanisms of the
stable association of procaspase-9 with the apoptosome in stored
cytosols remain to be elucidated, we have observed that
procaspase-9 interaction with Apaf-1 is significantly stronger in
the absence of nucleotides, suggesting that loss of nucleotides in
stored cytosols promotes stable association of procaspase-9 with
the Apaf-1 apoptosome leading to dysfunctional molecular timer
and inefficient procaspase-9 processing and caspase-3 activation.
Indeed, prior incubation of Apaf-1 with procaspase-9 leads to
defective caspase activation (Fig. 6). Although the timer mecha-
nism suggests that cells can escape apoptosis in the event of low
amounts of apoptosome activation [11,12], the present findings
further support the physiological importance of molecular timer,
which may be regulated by (d)ATP.
Cancer cells frequently avert apoptosis by upregulating various
prosurvival molecules such as XIAP and Hsp70, which promote
cell survival by inhibiting caspase activation [24,25,26,27,28,
29,30]. For example, XIAP binds with active caspase-9 and
caspase-3 to inhibit the caspase cascade, and thus, apoptosis.
Similarly, Hsp70 has been shown to abrogate apoptosome
formation and function. It is also important to note that Hsp70
Figure 4. Defective apoptosome formation and caspase activation in stored cytosols. GM701 cytosols either freshly prepared or stored at
220uC for 15 days were incubated with CC in the absence or presence of dATP. At the end of incubation, samples were fractionated on superose-6
gel filtration column on AKTA FPLC machine. Fractions were collected and 20 ml of different fractions (fraction numbers and M.W. standards
are indicated on top) were analyzed by Western blotting for the molecules indicated. Procasp-9, procaspase-9; procasp-3, procaspase-3; CC,
Nucleotide Regulation of Apoptosome
PLoS ONE | www.plosone.org7 January 2011 | Volume 6 | Issue 1 | e16379
plays an important role in nucleotide exchange and is essential for
functional apoptosome assembly . It is possible that in the
absence of (d)ATP, Hsp70 may associate with the apoptosome,
and is not available for performing nucleotide exchange during the
complex formation. Therefore, in the absence of nucleotide
exchange, the recruited procaspase-9 is not active and continues to
associate with the apoptosome. During stress and apoptotic
stimulation, when mitochondria-mediated ATP generation is
compromised and the levels of nucleotides are reduced ,
procaspase-9 may stably associate with Apaf-1 apoptosome
inhibiting caspase activation and apoptosis. Therefore, these
findings further provide evidence that nucleotide pools, especially
the ATP levels, play an important role in regulating apoptotic cell
death by targeting apoptosome.
Materials and Methods
Cells and reagents
GM701 (immortalized human fibroblasts) cells kindly provided
by Dr. M. King (Thomas Jefferson University) were cultured in
Dulbecco’s Minimum Essential Medium (DMEM; Gibco Grand
Island, NY) supplemented with 10% heat-inactivated fetal bovine
serum (FBS) and antibiotics. Human prostate cancer cells, PC3,
LNCaP, Du145, and PPC1 were purchased from ATCC (Rock-
ville, MD) and cultured in RPMI 1640 supplemented with 10%
FBS. MDA-MB231 (breast cancer) and HeLa (cervical cancer)
cells were obtained from ATCC and cultured in MEM with 10%
FBS. SKOV-3 (ovarian cancer) obtained from ATCC and
cultured in McCoy’s 5a medium with 10% FBS. K562 (leukemia),
C2F3 (myofibroblasts) were procured from ATCC and cultured in
RPMI with 10% FBS. HCT116 (colon cancer) cells were provided
by Dr. B. Vogelstein and cultured in DMEM supplemented with
10% FBS. NHDF-neo (normal human dermal fibroblasts-
neonatal), HMEC (human mammary epithelial cells), and NHP
(normal human prostate) cells were purchased from Lonza
(Walkersville, MD USA) and cultured in company recommended
medium [16,18,32,33,34,35]. Primary antibodies (Ab) used in this
study were: rabbit (Rb) pAb to caspase-3 (Biomol, Cat # SA320);
mAbs to CC (BD PharMingen, Cat # 556433), Rb pAbs to Apaf-
1 (BD PharMingen, Cat # 559683); and Rb pAb to caspase-9
(Chemicon, Cat # AB16970), mAb to actin (MP Biomedicals, Cat
# 69100). All secondary antibodies, and enhanced chemilumi-
nescence (ECL) reagents were from GE Healthcare. DEVD-AFC
(Cat # P-409), and LEHD-AFC (Cat # P-445) were bought from
Biomol. All other chemicals were purchased from Sigma (St.
Louis, MO) unless specified otherwise.
Subcellular fractionation and Western blotting
The cytosol and mitochondria were purified as described
previously [16,18,32,33,34,35]. Various types of cells were harvest-
ed, washed twice in ice-cold PBS, and resuspended in homogenizing
buffer (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM
MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA and 1 mM
dithiothreitol) containing 250 mMsucrose and a mixture of protease
inhibitors (1 mM PMSF, 1% aprotinin, 1 mM leupeptin, 1 mg/ml
pepstatin A and 1 mg/ml chymostatin). After 30 min incubation on
ice, cells were homogenized using a glass Pyrex homogenizer (type A
pestle, 140 strokes) and centrifuged twice at 1000 g for 5 min at 4uC.
The resulting supernatant was centrifuged at 10,000 g for 20 min to
obtain mitochondria as pellet. Supernatant was further subjected to
centrifugation at 100,000 g for 1 h to obtain cytosolic fraction (i.e.,
S100 fraction). Protein concentration was determined by Micro-
BCA kit (Pierce, Rockford, IL).
For Western blotting, samples were loaded on SDS polyacryl-
amide gel. After gel electrophoresis and protein transfer, the
membrane was probed or reprobed, after stripping, with various
primary and corresponding secondary antibodies. Western
blotting was performed using ECL as previously described
Far Western analysis was performed as described previously
. Apaf-1 or BSA was immobilized on PVDF membrane
through gel electrophoresis. The membrane was blocked with 5%
nonfat dry milk overnight and then incubated with either
procaspase-9 alone or procaspase-9 in the presence of ATP and
1% BSA for 1 hour in Buffer A (20 mM HEPES-KOH, pH 7.5,
Figure 5. Oligomerized Apaf-1 in stored cytosol fails to activate caspase-3. A–B, GM701 cytosols either freshly prepared or stored at 220uC for 15
superose-6 gel filtration column on AKTA FPLC machine. 20 ml of fractions 10–14 were used for caspase processing activity (CPA) measurement (A) by
incubating with 100 nM recombinant procaspase-3 (C3) and 100 nM of procaspase-9 (C9) for 90 min at 30uC followed by DEVDase activity measurement. (B)
CPA measurement in fractions 11–13. Shown are the mean 6 S.D (n=2). CPA activities are presented as arbitrary unit.
Nucleotide Regulation of Apoptosome
PLoS ONE | www.plosone.org8 January 2011 | Volume 6 | Issue 1 | e16379
10 mM KCl, 1.5 mM MgCl2, and 1 mM each of EDTA, EGTA,
and DTT) at room temperature. After thorough washing with
buffer A, the membrane was probed for caspase-9.
Caspase activity measurements
Caspase-9 (i.e., LEHDase) or caspase-3 (i.e., DEVDase) activities
were determined as described previously [16,18,32,33,34,35]. Briefly,
reconstituted cytosolic proteins were added to the caspase reaction
mixture containing 30 mM fluorogenic peptide substrates, DEVD-
AFC (for caspase-3) or LEHD-AFC (for caspase-9), 50 mM of
HEPES, pH 7.4, 10% glycerol, 0.1% CHAPS, 100 mM NaC1,
1 mM EDTA, and 10 mM DTT, in a total volume of 100 ml and
incubated at 37uC for 90 min. Production of 7-amino-4-trifluor-
omethyl-coumarin (AFC) was monitored on spectrofluorimeter using
excitation wavelength 400 nm and emission wavelength 505 nm.
The results were presented as fold activation over the control.
All cell-free reactions were performed in homogenizing buffer
(20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2,
1 mM sodium EDTA, 1 mM sodium EGTA and 1 mM
dithiothreitol) containing 250 mM sucrose and a mixture of
protease inhibitors in a total volume of 100 ml [16,18]. Briefly,
cytosols were activated by adding bovine CC (15 mg/ml) with or
without dATP and incubated for 150 min at 37uC. In some
experiments, cytosols were first stored at different temperatures
and then activated by bovine CC with or without dATP. In some
reconstitution experiments, cytosols were incubated with recom-
binant Apaf-1, procaspase-9, and procaspase-3 alone or in
different combinations as indicated in the Figure legends. After
incubation, samples were either used for substrate cleavage assays
for caspase-9 (LEHDase) and caspase-3 (DEVDase) or for caspase
processing by Western blotting.
Analysis of apoptosome complexes
Control, CC-activated, or CC/dATP-activated cytosols were
fractionated by size-exclusion chromatography using Superose 6
column (GE Healthcare) calibrated with thyroglobulin (669 kDa),
ferritin (440 kDa), aldolase (158 kDa), bovine serum albumin
(67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and
Figure 6. Procaspase-9 interacts with Apaf-1 in the absence of ATP, whereas physiological levels of ATP disrupt Apaf-1-procaspase-
9 interaction, and prior incubation of Apaf-1 with procaspase-9 leads to defective apoptosome. A, 500 ng of recombinant Apaf-1 or
bovine serum albumin (BSA) were immobilized on PVDF membrane. Membrane was then incubated with procaspase-9 (500 ng) in the absence or
presence of physiological levels of ATP (2 mM) followed by Western blotting for caspase-9. Left panel represents coomassie stained gel image. B, To
induce defective apoptosome formation, recombinant Apaf-1 was pre-incubated with procaspase-9 for 30 min at 4uC followed by the addition of
procaspase-3, CC, and ATP (200 mM). Caspase-3 activity presented as DEVDase activity. Control, Apaf-1+procaspase-9+procaspase-3; apoptosome,
Apaf-1+procaspase-9+procaspase-3+CC+ATP. Apopto, apoptosome.
Nucleotide Regulation of Apoptosome
PLoS ONE | www.plosone.org9 January 2011 | Volume 6 | Issue 1 | e16379
ribonuclease A (13.7 kDa) (all obtained from GE Healthcare).
Apoptosome complexes (,700 kDa) were eluted using column
buffer (20 mM HEPES, 0.1% (w/v) CHAPS, 5 mM DTT, 5%
(w/v) sucrose pH 7.0) supplemented with 50 mM NaCl. Different
fractions were then analyzed by Western blotting for changes
in the distribution of Apaf-1, processing of caspases [3,5,16]. For
caspase processing activity, 20 ml of fractions 11–14 were
incubated with 100 nM of recombinant procaspase-9 and
procaspase-3for 90 minfollowed
by DEVDase activity
Purification of recombinant Apaf-1, procaspase-9, and
Apaf-1, procaspase-9, and procaspase-3 were cloned into
pFastbacH, a modified version of pFastbac with a C-terminal 6
histidine tag, expressed in insect cells (Sf9) and purified on Nickel
agarose as previously described [12,16,36,37].
Determination of intracellular dNTP pools
The levels of dNTP from various cytosols were determined as
described previously [16,38]. Briefly, nucleotides were extracted
from cytosolic samples using 60% methanol. The DNA polymer-
ase assay was utilized to quantify dNTPs in the cell extracts. The
Klenow fragment of DNA polymerase I lacking exonuclease
activity (USB, Cleveland, OH) was used to start a reaction in a
mixture that contained 100 mM HEPES buffer (pH 7.3), 10 mM
MgCl2, 7.5 mg BSA, and synthetic oligonucleotides of defined
sequences as templates annealed to a primer, [3H]dTTP or
[3H]dATP and either standard dNTP or the extract from 1 or
26106cells. Reactants were incubated for 1 h and applied to filter
discs. After washing, the radioactivity on the disks was determined
by liquid scintillation counting and compared with that in the
standard dNTP samples [16,38].
Measurement of nucleotide triphosphates by HPLC
To measure the NTPs levels, nucleotides were extracted
using perchloric acid. The nucleotide extracts were neutral-
ized with KOH . The neutralized extracts were applied to
an anion-exchange Partisil-10 SAX column and eluted at a
flow rate of 1.5 ml/min with a 50-min concave gradient (curve
7; Waters 600E System Controller; Waters Corp.) from 60%
0.005 M NH4H2PO4(pH 2.8) and 40% 0.75 M NH4H2PO4
(pH 3.6) to 100% 0.75 M NH4H2PO4(pH 3.6). The column
elute was monitored by UV absorption at 256 nm, and the
nucleoside triphosphates were quantified by electronic inte-
gration with reference to external standards. The lower limit
of sensitivity of this assay was 10 pmol in an extract of 56106
cells corresponding to a cellular concentration of 1 mM
We thank Drs. S. Bratton and G. Salvesen for providing reagents and Drs.
S. Bratton and S. Malladi for critical reading of and helpful comments on
this manuscript. We thank Drs. D. Schultz, S. Malladi, and A. Martin for
the help with protein purification.
Conceived and designed the experiments: HZ VG DGT DC. Performed
the experiments: HZ RG NY MA RKL. Analyzed the data: DC VG DGT
HZ. Contributed reagents/materials/analysis tools: DC DT MB VG RKL.
Wrote the paper: DC NY DGT.
Figure 7. Nucleotide regulates apoptosome-mediated caspase activation. A, Summary of cell-free apoptosome reconstitution assays using
recombinant proteins, or fresh or stored cytosols. Dashed arrows in ‘Stored cytosol’ indicate attenuated caspase activation. B, Defective molecular
timer in the absence of nucleotides leads to inefficient caspase activation. See Discussion for details. Casp-9, caspase-9.
Nucleotide Regulation of Apoptosome
PLoS ONE | www.plosone.org10 January 2011 | Volume 6 | Issue 1 | e16379
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