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NEOPLASIA
From the Department of Immunology and Cell Biology,
Department of Hematology/Oncology, University of Münster, D-48149
Münster, Germany, and Department of Radiation Oncology,
University of Tübingen, D-72076, Tübingen, Germany.
Diverse death stimuli including anticancer drugs trigger apoptosis
by inducing the translocation of cytochrome c from the outer
mitochondrial compartment into the cytosol. Once released, cytochrome c
cooperates with apoptotic protease-activating factor-1 and
deoxyadenosine triphosphate in caspase-9 activation and initiation of
the apoptotic protease cascade. The results of this study show that on
death induction by chemotherapeutic drugs, staurosporine and triggering
of the death receptor CD95, cytochrome c not only translocates into the
cytosol, but furthermore can be abundantly detected in the
extracellular medium. The cytochrome c release from the cell is a rapid
and apoptosis-specific process that occurred within 1 hour after
induction of apoptosis, but not during necrosis. Interestingly,
elevated cytochrome c levels were observed in sera from patients with
hematologic malignancies. In the course of cancer chemotherapy, the
serum levels of cytochrome c in the majority of the patients grew
rapidly as a result of increased cell death. These data suggest that
monitoring of cytochrome c in the serum of patients with tumors might
serve as a useful clinical marker for the detection of the onset of
apoptosis and cell turnover in vivo.
(Blood. 2001;98:1542-1548) Damaged cells can die through different mechanisms.
Two major forms of cell death are necrosis and apoptosis.1
Apoptosis, also known as programmed cell death, is the form of cell
elimination commonly occurring during development as well as in many
physiologic and pathologic processes.2,3 In contrast,
necrosis occurs mostly when noxious stimuli disintegrate the function
of various cellular compartments leading to plasma membrane damage,
mitochondrial dysfunction, and cell lysis.
The induction of the endogenous death machinery can be initiated via 2 principal signaling pathways.4 One involves the ligation
of death receptors, such as CD95 and tumor necrosis factor-receptor (TNF-R1), which on binding of the adapter protein FADD, recruit procaspase-8 into the death-inducing signaling complex. Another pathway
that is triggered by a number of apoptotic stimuli such as anticancer
drugs or irradiation is essentially controlled at the mitochondrion. An
initial event is the release of cytochrome c from mitochondria into the
cytosol.5,6 Once released, cytochrome c together with
deoxyadenosine triphosphate binds to apoptotic protease-activating
factor-1 (Apaf-1), leading to an unmasking of its caspase recruitment
domain and the subsequent binding and autoproteolytic activation of
procaspase-9. The complex of procaspase-9, cytochrome c, and Apaf-1 is
known as the apoptosome. Similarly to caspase-8, active caspase-9 then
proteolytically activates downstream effector caspases, which by
degrading various cellular proteins propagate the apoptotic signal.
Both the death receptor and the mitochondrial pathway can synergize and
amplify their own signals by positive feedback loops. First, the
BH3-only Bax-interacting protein Bid, which is a substrate of
caspase-8, becomes activated in the death receptor-mediated pathway and
induces the release of cytochrome c from mitochondria.7,8
Activated Bid triggers a conformational change of another proapoptotic
molecule, Bax, which leads to its oligomerization and subsequent
insertion into the outer mitochondrial membrane and finally to
cytochrome c release.9,10 Second, caspase-9 can activate
procaspase-8 via the caspase-3 and caspase-6 cascade, thus amplifying
the receptor-derived signal and via Bid cleavage also the mitochondrial
pathway.11,12
A crucial step controlling the apoptosome pathway is the release of
cytochrome c. It is a rapid and presumably irreversible process that
appears to be both energy- and caspase-independent.13 The
mechanism by which cytochrome c translocates to the cytosol during
apoptosis has not been elucidated in detail and is still a matter of
debate. Much of the controversy has focused on the mode of action of
the proapoptotic Bcl-2 family members such as Bad, Bak, Bax, and Bid,
which cause the release of cytochrome c, whereas the antiapoptotic
proteins Bcl-2 and Bcl-xL suppress the translocation of
cytochrome c.5-10,14 The functions of the proapoptotic Bcl-2 members have been proposed to involve the
formation of pores in the outer mitochondrial membrane through which
cytochrome c diffuses. Other models suggest that these proteins affect
channels in the outer or the inner mitochondrial membrane, such as the permeability transition pore, thereby inducing hyperpolarization and
permeability transition.15 It has been proposed that these events cause the entry of water and solutes, matrix swelling, and
rupture of the outer membrane, which allows the passive release of
cytochrome c. However, it has been observed that in many cell types the
release of cytochrome c occurs before or in the absence of a change in
mitochondrial permeability,13 suggesting that this process
involves additional or other mechanisms than opening of the
permeability transition pore.
In the present study, our results show that cytochrome c not only
enters the cytoplasm on apoptosis induction, but furthermore is also
released from the "committed to die cell" into the extracellular medium. The externalization of cytochrome c is a rapid and
apoptosis-specific process because it was not observed on necrosis
induced by diverse triggers. A release of cytochrome c into the
extracellular medium was detected during apoptosis induced by various
stimuli such as staurosporine, anticancer drugs, and CD95 ligation in
different cell types. Moreover, even in the serum of patients with
hematologic malignancies, elevated cytochrome c levels were found,
which increased on induction of antitumor chemotherapy. These results
suggest that monitoring of extracellular cytochrome c levels may be
used as a marker to monitor cell death in vivo.
Materials and cell culture
Serum sample processing
Cell extracts, immunoprecipitation, and immunoblotting To determine the cellular cytochrome c content, cells were collected by centrifugation, washed in cold phosphate-buffered saline (PBS), and extracted in cold lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100 containing 3 µg/mL aprotinin, 3 µg/mL leupeptin, 3 µg/mL pepstatin, and 2 mM phenylmethylsulfonyl fluoride). Extracellular fractions and cell lysates were precleared by centrifugation at 10 000g, 4°C for 15 minutes prior to immunoprecipitation. Supernatants were kept at 70°C until use.
Immunoprecipitations were performed in a volume of 4 mL in a rotator at
4°C for 4 hours using anticytochrome c mAb 6H2.B4 (Pharmingen Europe,
Hamburg, Germany) at a final concentration of 0.5 µg/mL. The
cytochrome c-mAb complexes were precipitated for 1 hour with 40 µL
of a 50% slurry of protein G-Sepharose in PBS. Precipitates were
harvested by short centrifugation (2000 rpm, 10 seconds, 4°C) and
washed 4 times with cold washing buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, 1 µg/mL aprotinin, 1 µg/mL leupeptin). Proteins were eluted by boiling the precipitates in sodium
dodecyl sulfate (SDS)-loading buffer containing  -mercaptoethanol, separated under reducing conditions on a 12% SDS-polyacrylamide gel
electrophoresis (SDS-PAGE), and subsequently transferred to a
polyvinylidene difluoride membrane (Amersham Buchler, Braunschweig, Germany). Equal loading was confirmed by staining of the proteins with
Ponceau S. Subsequently, membranes were blocked for 1 hour with 5%
nonfat dry milk powder in Tris-buffered saline (50 mM Tris, pH 7.4, 150 mM NaCl) and 0.05% Tween-20 (TBST) and then immunoblotted with
anticytochrome c mouse mAb 7H8.2C12 (Pharmingen Europe) for 2 hours.
After washing in TBST the blots were incubated with antimouse
horseradish peroxidase-conjugated secondary antibody for 1 hour.
Finally, the membranes were washed extensively in TBST and developed
using enhanced chemiluminescence reagents (Amersham Buchler). The
quantity of immunoprecipitated cytochrome c was determined by
densitometric analysis using the National Institutes of Health
(Bethesda, MD) image software. The values of the patients' sera were
always normalized with the amount of cytochrome c from healthy controls
that were incorporated in each Western blot analysis. The relative
cytochrome c levels indicate the ratio of the densitometric values from
the patients and the mean values of control. Sera from control persons
revealed only low levels of detectable cytochrome c, and mean values
did not reveal large interindividual differences (< 9%).
Measurement of cell death For determination of apoptosis, Jurkat and L929 cells were seeded in microtiter plates and treated with the cytotoxic agents for the indicated time points. Apoptotic, hypodiploid nuclei were measured as described previously.16 Briefly, apoptotic nuclei were prepared by lysing cells in a hypotonic lysis buffer (1% sodium citrate, 0.1% Triton X-100, 50 µg/mL propidium iodide) and subsequently analyzed by flow cytometry. In parallel, cell death as assessed by membrane damage was determined by the uptake of propidium iodide (2 µg/mL in PBS; Sigma) into nonfixed cells. After 10 minutes, red fluorescence (FL-3) was measured by flow cytometry using a FACScalibur (Becton Dickinson, Heidelberg, Germany) and CellQuest analysis software (Becton Dickinson, Franklin Lakes, NJ).Cell viability was also determined by monitoring the release of lactate dehydrogenase (LDH), which was measured by the central laboratory of the medical clinic. To obtain total LDH activity, cells were lysed with 1% Triton X-100. The percentage of LDH release represents the fraction of LDH activity found in the supernatants with respect to the overall enzyme activity.
One of the earliest events in apoptosis induced by death receptor-independent mechanisms is the translocation of cytochrome c from the mitochondrial intermembrane space into the cytosol.5,6 Released cytochrome c then activates the apoptosome-dependent death machinery by binding to Apaf-1, resulting in the subsequent recruitment and activation of procaspase-9.17 Our initial experiments in cells overexpressing a green fluorescent protein-tagged version of cytochrome c revealed that following translocation of cytochrome c the fluorescent signal was rapidly lost during apoptosis (data not shown). Therefore, we investigated whether cytochrome c was degraded or could be detected in the extracellular medium on induction of cell death. To our surprise, large amounts of cytochrome c were found in the
culture medium of Jurkat T cells on apoptosis induction by several
stimuli including the protein kinase inhibitor staurosporine, agonistic
anti-CD95 antibody, and the anticancer drugs etoposide and doxorubicin
(Figure 1A). This event was accompanied
by a decrease of cytochrome c in the corresponding cellular fractions.
The weaker cytochrome c release observed in anticancer drug-treated
cells could be attributed to a slower progression of cell death. These results are consistent with previous data showing that cytochrome c
release into the cytosol, caspase activation, and final cell death
occur more slowly in drug-induced than in CD95 receptor-mediated cell
death.18 In response to staurosporine and anti-CD95, 2 potent apoptosis inducers, almost all of the cytochrome c was detected
in the culture medium. The extracellular release of cytochrome c in
response to the proapoptotic agents was dose dependent (Figure 1A). The
concentration dependency correlated with the induction of apoptosis in
Jurkat cells as described previously.11,19 Similar to
Jurkat T-cells, in murine L929 cells cytochrome c was readily released
in cultures following induction of apoptosis by several stimuli (data
not shown). To analyze whether the released cytochrome c was present in
a soluble form or associated with apoptotic bodies or other membrane
fractions, we cleared the supernatants by ultracentrifugation.
Cytochrome c was not recovered in the pellets but was still present in
the supernatants (Figure 1B), indicating that it was indeed released as
a soluble protein.
The release of cytochrome c from the cell was a rapid process. Already
1 hour after apoptosis induction with staurosporine, cytochrome c could
be detected in the medium (Figure 2A). A
parallel measurement of the activity of LDH, a cytoplasmic enzyme and
clinical marker of cell damage, indicated that the release of LDH into supernatants from apoptotic cells occurred much later and was less
pronounced than cytochrome c release. Only approximately 20% of total
LDH was detected in the supernatants of apoptotic cells after 15 hours
of staurosporine treatment (Figure 2A). To compare the kinetics of the
release of cytochrome c and LDH, cytochrome c levels were further
quantified by densitometric analysis (Figure 2A). A comparison of both
events showed that the extracellular release of cytochrome c was
stronger and occurred much earlier than the release of LDH (Figure 2B).
The fast kinetics of cytochrome c release corresponded to a recent
study demonstrating that the translocation of this molecule into
cytosol is an all-or-nothing event that, once induced, is completed
within a few minutes.13 Parallel to cytochrome c detection
in the extracellular medium, the progression of apoptosis was monitored
by flow cytometry. As shown in Figure 2C, formation of hypodiploid
nuclei was not observed until 5 hours after apoptosis triggering. This
agrees with observations that cytochrome c release into the cytosol
precedes caspase activation and other morphologic changes of apoptosis including phosphatidylserine exposure, cell shrinkage, and membrane blebbing.13,17,20 Therefore, detection of cytochrome c in the extracellular medium can be regarded as an early and sensitive death indicator.
Because during necrotic cell death, contrary to apoptosis, marked
morphologic changes of mitochondria are observed, we wanted to compare
the degree of cytochrome c release in both types of cell death. To this
end, 2 different death models were used: (1) The stimulation of L929
cells with TNF-
Because the release of cytochrome c could be induced by a variety of
apoptotic stimuli, we postulated that it should be also detected in
vivo, for instance during anticancer drug therapy. To examine this
hypothesis we screened human sera from oncologic patients receiving
combined chemotherapy. The serum cytochrome c levels were monitored
from 17 patients hospitalized mostly due to hematologic malignancies
and treated with various chemotherapy protocols (Table 1). For
standardization, sera from 8 healthy control persons were included in
the analysis, but did not show significant differences in cytochrome c
levels (< 9%). The patients' samples were loosely categorized into
those with strongly elevated (
Thus, the majority of the monitored patients responded to chemotherapy with an increase of serum cytochrome c levels (Table 1). The increase of cytochrome c in the serum could be observed within a few hours after the onset of chemotherapy (data not shown). In most patients cytochrome c levels started to decrease later in the course of chemotherapy, reaching levels similar to or even lower than those in the controls (Figure 4). In a large number of patients the cytochrome c levels were already high before the start of the chemotherapy, which may indicate an increased cell turnover and augmented spontaneous apoptosis. Similar to cytochrome c, in most patients high serum levels of LDH were observed prior to therapy (Table 1). However, although LDH levels increased during therapy in 6 patients, its kinetics was different from that of cytochrome c (Table 1). This finding indicates that the release of cytochrome c and LDH activity reflects different (patho)physiologic processes. LDH, a cytosolic protein, is most likely released on cell lysis, eg, during necrosis. In contrast, cytochrome c needs to pass from mitochondria to the cytoplasm, before reaching extracellular compartment. Thus, based on our experimental data, we propose that serum LDH activity is more indicative of necrotic processes in vivo, whereas the release of cytochrome c characterizes apoptotic events. So far, it has only been described that cytochrome c is released from
the mitochondria to the cytosol. The exact mechanism of this key event
is not understood. Several models including rupture of the outer
mitochondrial membrane and permeability transition15 and
the escape of cytochrome c through megachannels or pores formed by
proapoptotic Bcl-2 family members have been
proposed.7,9,10 It is possible that related mechanisms of
externalization may be responsible for the release of cytochrome c into
the extracellular medium. However, it is unlikely that cytochrome c is
released by a nonspecific mechanism such as cell lysis, because a
release of LDH occurred at later time points (Figure 2). It has been
reported that the tripeptide glutathione is specifically extruded
during apoptosis,23 although it is unlikely that
cytochrome c, a 14.5-kd protein, is externalized by a related
mechanism. In addition, there are several examples of proteins, such as
HIV-Tat, thioredoxin, interleukin 1 In summary, we show that cytochrome c, the key regulator of apoptosome pathway, is released not only into cytoplasm, but moreover leaves the cell. This externalization of cytochrome c is an early and apoptosis-specific event, which occurs not only in experimental settings but also in vivo during chemotherapy of tumor patients. The kinetics of cytochrome c release differs from that of LDH indicating that both parameters are indicative for distinct physiologic processes. The noninvasive assessment of cytochrome c release may therefore provide a useful assay to detect apoptosis and to determine cell turnover and treatment efficacy in several clinical disorders.
The authors thank Dr W. Fiers (University of Ghent, Belgium) for
the gift of TNF-
Submitted June 15, 2000; accepted April 25, 2001.
Supported in part by grants from the Deutsche Forschungsgemeinschaft (SFB 293), the IZKF of the University of Münster, and the Deutsche Krebshilfe.
K.S.-O. and M.L. share equal senior authorship.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Marek Los, Department of Immunology and Cell Biology, University of Münster, Röntgenstrasse 21, D-48149 Münster, Germany; e-mail: los{at}uni-muenster.de.
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© 2001 by The American Society of Hematology.
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  C. W. Bell, W. Jiang, C. F. Reich III, and D. S. Pisetsky The extracellular release of HMGB1 during apoptotic cell death Am J Physiol Cell Physiol, December 1, 2006; 291(6): C1318 - C1325. [Abstract] [Full Text] [PDF]
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 S. Maddika, E. P. Booy, D. Johar, S. B. Gibson, S. Ghavami, and M. Los Cancer-specific toxicity of apoptin is independent of death receptors but involves the loss of mitochondrial membrane potential and the release of mitochondrial cell-death mediators by a Nur77-dependent pathway J. Cell Sci., October 1, 2005; 118(19): 4485 - 4493. [Abstract] [Full Text] [PDF]
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 H. Dussmann, D. Kogel, M. Rehm, and J. H. M. Prehn Mitochondrial Membrane Permeabilization and Superoxide Production during Apoptosis. A SINGLE-CELL ANALYSIS J. Biol. Chem., April 4, 2003; 278(15): 12645 - 12649. [Abstract] [Full Text] [PDF]
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H. Dussmann, M. Rehm, D. Kogel, and J. H. M. Prehn Outer mitochondrial membrane permeabilization during apoptosis triggers caspase-independent mitochondrial and caspase-dependent plasma membrane potential depolarization: a single-cell analysis J. Cell Sci., February 1, 2003; 116(3): 525 - 536. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
(TNF-
2-fold) cytochrome c levels compared
to control donors (Figure 