Indeed, in intact cells J774 macrophages, HeLa cells and AG1518 fibroblasts the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride MSDH causes lysosomal rupture, enhanced
Trang 1Lysosomal enzymes promote mitochondrial oxidant production,
Ming Zhao1, Fernando Antunes1,2, John W Eaton1,3and Ulf T Brunk1
1
Division of Pathology II, Faculty of Health Sciences, Linko¨ping University, Sweden;2Grupo de Bioquı´mica e Biologia Teo´ricas – Instituto Bento da Rocha Cabral and Department of Chemistry and Biochemistry, Faculty of Sciences, University of Lisbon, Portugal;
3
James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA
Exposure of mammalian cells to oxidant stress causes early
(iron catalysed) lysosomal rupture followed by apoptosis
or necrosis Enhanced intracellular production of reactive
oxygen species (ROS), presumably of mitochondrial origin,
is also observed when cells are exposed to nonoxidant
pro-apoptotic agonists of cell death We hypothesized that ROS
generation in this latter case might promote the apoptotic
cascade and could arise from effects of released lysosomal
materials on mitochondria Indeed, in intact cells (J774
macrophages, HeLa cells and AG1518 fibroblasts) the
lysosomotropic detergent O-methyl-serine dodecylamide
hydrochloride (MSDH) causes lysosomal rupture, enhanced
intracellular ROSproduction, and apoptosis Furthermore,
in mixtures of rat liver lysosomes and mitochondria, selective
rupture of lysosomes by MSDH promotes mitochondrial
ROSproduction and cytochrome c release, whereas MSDH
has no direct effect on ROSgeneration by purifed
mito-chondria Intracellular lysosomal rupture is associated with the release of (among other constituents) cathepsins and activation of phospholipase A2 (PLA2) We find that addi-tion of purified cathepsins B or D, or of PLA2, causes substantial increases in ROSgeneration by purified mito-chondria Furthermore, PLA2) but not cathepsins B or
D) causes rupture of semipurified lysosomes, suggesting an amplification mechanism Thus, initiation of the apoptotic cascade by nonoxidant agonists may involve early release of lysosomal constituents (such as cathepsins B and D) and activation of PLA2, leading to enhanced mitochondrial oxidant production, further lysosomal rupture and, finally, mitochondrial cytochrome c release Nonoxidant agonists
of apoptosis may, thus, act through oxidant mechanisms Keywords: apoptosis; cathepsins; lysosomes; lysosomotropic detergents; oxidative stress
In the last two decades, the phenomenon of apoptosis has
attracted great interest and many intricate molecular events
underlying the process have been elucidated [1–8] Several
crucial steps are thought to involve mitochondrial release
of pro-apoptotic factors, although the exact mechanisms
involved in this release are less well understood
In this regard, there is substantial evidence that, at least
in some circumstances, the discharge into the cytosol of
lysosomal constituents may be an early and, perhaps,
initiating event in apoptosis, and that mitochondrial release
of pro-apoptotic factors might be a consequence of earlier
lysosomal destabilization [9–18] In further, albeit indirect,
support of this, it was recently found that activation of the
pro-apoptotic tumour supressor protein, p53, also results
in early lysosomal rupture, although through still unknown mechanisms [14]
In the case of simple oxidant-induced apoptosis, lyso-somal rupture occurs in two sequential phases [19,20], where the second one includes activation of phospholipase A2 (PLA2) with production of free arachidonic acid (AA) [21,22] Theoretically, released lysosomal enzymes, PLA2, and AA all might be capable of destabilizing mitochondrial membranes Interestingly, over-expression of the anti-apoptotic protein, Bcl-2, abrogates the secondary phase of lysosomal rupture, the activation of PLA2, and the mitochondrial release of cytochrome c [19,21,22] However, the precise mechanisms through which Bcl-2 mediates these effects are presently unknown
Remarkably, in apoptosis caused by a number of nonoxidative agents, there appears to be increased intracel-lular generation of reactive oxygen species (ROS), probably
of mitochondrial origin [23–30] Although the mechanisms responsible for enhanced mitochondrial ROSproduction during the process of apoptosis remain unknown, this phenomenon raises the possibility that internally generated ROS, like exogenously added oxidants, may act through a common pathway–lysosomal destabilization
The present investigations were aimed at identifying intracellular events that might connect exposure of cells to nonoxidative agonists of apoptosis and intracellular ROS production As mentioned above, there is abundant evi-dence that) at least in some circumstances ) lysosomal rupture might be an early, perhaps even initiating, event in
Correspondence to M Zhao, Division of Pathology II,
Faculty of Health Sciences, Linko¨ping University,
SE-581 85 Linko¨ping, Sweden.
Fax: +46 13 22 15 29, Tel.: +46 13 22 15 15,
E-mail: ming.zhao@inr.liu.se
Abbreviations: AA, arachidonic acid; DHE, dihydroethidium; HRP,
horseradish peroxidase; LE, lysosomal enzymes; LEF,
lysosome-mitochondria enriched fraction; MSDH, O-methyl-serine
dodecylamide hydrochloride; PLA2, phospholipase A2;
ROS, reactive oxygen species.
(Received 28 April 2003, revised 11 July 2003,
accepted 24 July 2003)
Trang 2the apoptotic cascade Therefore, in the present
investiga-tions we have used a synthetic lysosomotropic detergent,
O-methyl-serine dodecylamide hydrochloride (MSDH) to
specifically induce lysosomal rupture and ensuing apoptosis
[12,31,32] This was done in order to determine whether
internal oxidative stress of mitochondrial origin might arise
as a consequence of lysosomal rupture and act as an
amplifying loop causing further lysosomal breach Here, we
present evidence that released lysosomal enzymes) both
directly and through activation of PLA2) may trigger
enhanced mitochondrial production of superoxide and
hydrogen peroxide, and cause the release of cytochrome c
Materials and methods
Materials
Chemicals were from Sigma unless stated otherwise RPMI
1640 medium, Hepes, foetal bovine serum, glutamine,
penicillin, and streptomycin were from Gibco BODIPY
FL phallacidin and dihydroethidium (DHE) were from
Molecular Probes Monoclonal anti-cytochrome c Igs were
from Pharmingen, and horseradish peroxidase
(HRP)-conjugated goat anti-mouse Igs were from DAKO Percoll
was from Amersham Pharmacia Biotech
Cell cultures
Human foreskin fibroblasts (AG-1518, passages 14–20;
Coriell Institute, Camden, NJ, USA), J774 cells (a murine
histiocytic lymphoma cell line), and human epithelial cells
(HeLa) were cultured at 37C in humidified air with 5%
CO2 in RPMI 1640 medium supplemented with 2 mM
glutamine, 50 IUÆmL)1 penicillin-G, 50 lgÆmL)1
strepto-mycin, and 10% foetal bovine serum Cells were
subcul-tured once a week Twenty-four hours before experiments,
cells were trypsinized and seeded into 35-mm Petri dishes or
96-well plates (Costar, Cambridge, MA, USA) at a density
of 10 000 cells per cm2
Apoptosis assays
DNA fragmentation was assessed using propidium iodide
staining of nuclear DNA, essentially as described by
Nicoletti et al [33] Briefly, cell pellets from individual
wells were gently resuspended in 1.5 mL of a hypotonic
and membrane-disrupting solution of propidium iodide
(50 lgÆmL)1 in 0.1% sodium citrate with 0.1% Triton
X-100) in 12· 75 mm polypropylene tubes The tubes were
kept overnight in the dark at 4C before flow-cytometric
analyses The propidium iodide-induced red fluorescence of
suspended individual nuclei was measured by flow
cyto-fluorometry, using the FL3 channel Nuclei with partly
degraded DNA were counted, and their frequency was
expressed as a percentage of the total number of nuclei
analysed in at least 10 000 cells
Actin staining
AG1518 fibroblasts were seeded in 35-mm Petri dishes and
cultured for 24 h before being exposed to 30 lMMSDH in
ordinary medium for 3 h Cellular actin was stained with
BODIPY FL phallacidin Cells were fixed for 10 min in 4% formaldehyde in NaCl/Pi, permeabilized for 10 min in 0.3% Triton X-100 in phosphate-buffered saline (NaCl/Pi), and stained for 30 min with BODIPY FL phallacidin (final concentration 0.6 lgÆmL)1) at 37C After staining, cells were washed twice in NaCl/Pi, and visualized and documented (kEX 495 nm; kEM 520 nm) using a Nikon microphot-SA fluorescence microscope with a Hamamatsu ORCA-100 color digital camera and Adobe PHOTOSHOP software
Evaluation of oxidative stress AG1518 fibroblasts, J774 and HeLa cells were seeded in 96-well plates and cultured for 24 h under standard conditions before being exposed to 30 lM MSDH and
10 lMDHE (in complete medium) Fluorescence intensity, indicating oxidation of DHE was assayed at various periods
of time after addition of MSDH and DHE on a VICTOR
1420 (Wallac Sverige AB, Upplands Va¨sby, Sweden) fluorescent plate-reader (kEX 485 nm; kEM 620 nm) In some experiments, cells were observed and documented under green light excitation (kEX 546 nm; kEM 590 nm) using fluorescence microscopy as described above
Preparation of rat liver lysosome-mitochondria enriched fraction
Livers were removed from 160–200-g female Sprague– Dawley rats (starved overnight), homogenized in 0.3M sucrose (1 : 9, w/v) and centrifuged at 500 g for 10 min The supernatants were again centrifuged at 3500 g for
10 min, the pellets discarded, and the lysosome/mitochon-dria-containing supernatants centrifuged at 10 000 g for
10 min The pellets were washed, suspended and re-centri-fuged at 10 000 g for 10 min and finally resuspended in the sucrose solution to a protein concentration of
1.5 mgÆmL)1 The resultant lysosome/mitochondria enriched fraction (LEF) was found to be stable (no release
of lysosomal enzymes) for up to 4 h in the homogenization solution at 4C, while some release of lysosomal enzymes occurred within 2 h at 37C
Preparation of a purified mitochondria fraction Mitochondria were purified from rat liver using a combi-nation of differential and Percoll gradient centrifugation [34,35] All procedures were carried out at 4C Briefly, fresh liver was minced and homogenized in M-SHE buffer (0.21M mannitol, 0.07M sucrose, 10 mM Hepes pH 7.4,
1 mM EDTA, 1 mM EGTA, 0.15 mMspermine, 0.75 mM spermidine) Unbroken cells and nuclei were pelleted at
500 g for 10 min The supernatant was centrifuged at
10 000 g to pellet mitochondria and lysosomes which were resuspended and washed twice with M-SHE buffer A 2-mL suspension was then layered onto 37.5 mL of Percoll solution (50% Percoll, 50% 2· M-SHE) and centrifuged for 1 h at 50 000 g in a Ti-60 rotor The brown mitochondrial band was collected, either by fractionating the gradient or by direct syringe aspiration The purified mitochondria were pooled, diluted 10-fold with M-SHE buffer, again pelleted by centrifugation and, finally,
Trang 3resuspended in M-SHE buffer to a protein concentration
of 1.5 mgÆmL)1 The degree of lysosomal contamination
of the purified mitochondria fraction was estimated by
assaying b-galactosidase/protein and compared to that
of LEF
Enzymatic detection of lysosomal integrity
and estimation of fraction purity
The integrity of lysosomes in the LEF preparation was
assessed by assaying released b-galactosidase LEF
(200 lL) was incubated for 3 h at 37C with either
PLA2 (0.2 UÆmL)1), 30 lM MSDH, 2.5 lgÆmL)1
cathep-sin B, or 2.5 lgÆmL)1cathepsin D and then centrifuged at
14 000 g for 10 min Stock solutions of the cathepsins were
made up in NaCl/PipH 6.0, whereas MSDH and PLA2
were in NaCl/PipH 7.4 The supernatants were removed,
and 1 mL distilled water with Triton X-100 (final
concen-tration 0.1%) was added to the pellets to cause complete
lysis of remaining intact lysosomes Activities of
b-galac-tosidase were measured as described previously [22] on the
ruptured lysosomal pellet and on the supernatant The
results were expressed as percentage released over total
b-galactosidase
Mitochondrial generation of H2O2 Mitochondrial production of H2O2was assayed essentially
as described elsewhere [36] Briefly, 1.33 UÆmL)1 HRP, 0.066 mgÆmL)1 q-hydroxyphenylacetate, 0.013 mgÆmL)1 superoxide dismutase, and 1 mg mitochondrial protein were added to 2.4 mL respiratory buffer (0.07M sucrose, 0.23M mannitol, 30 mM Tris/HCl, 4 mM MgCl2, 5 mM
KH2PO4, 1 mMEDTA, 0.5% BSA, pH 7.4) in a spectro-fluorophotometer cuvette at 37C Succinate (final concen-tration 6.67 mM) and antimycin A (final concentration 0.83 lgÆmL)1) were added, and H2O2-induced fluorescence recorded (kEX320 nm; kEM400 nm) during the first 10 min after mixing
Western blotting for cytochromec Two-hundred microlitres LEF, or purified mitochondria, were incubated for 3 h at 37C with either 30 lM MSDH, PLA2 (0.2 UÆmL)1), 2.5 lgÆmL)1cathepsin B, or 2.5 lgÆmL)1cathepsin D Stock solutions of the cathepsins were made up in NaCl/PipH 6.0, while MSDH and PLA2 were in NaCl/Pi pH 7.4 Following centrifugation at
14 000 g for 10 min, the supernatants were separated by
Fig 1 MSDH induces apoptosis and stress fibre formation in fibroblasts (A) Cells were seeded into 35-mm Petri dishes at a density of
10 000 cellsÆcm)2 After 24 h, 30 l M MSDH was added to complete culture medium (2 mL), and cells were incubated for another
10 h under standard culture conditions The Nicoletti technique for apoptotic nuclei was applied One representative experiment out of three is shown (B) Cells were seeded in 35-mm Petri dishes and kept for 24 h before being exposed to 30 l M MSDH for 3 h Actin staining was then performed as described in Materials and methods.
Trang 4SDS/PAGE (12% acrylamide) and transferred onto
Immo-bilon membranes (2 h; 200 mA) Membranes then were
incubated at room temperature for 1 h in blocking buffer
[5% low-fat milk powder in Tris-buffered saline (TBS)] and
for another 2 h in dilution buffer (0.5% low-fat milk
powder in TBS) containing a 1 : 400 dilution of a
mono-clonal anti-cytochrome c Ig After washing in TBSwith
0.06% Tween 20, Immobilon membranes were incubated
for 1 h at room temperature in a buffer containing a
1 : 1500 dilution of peroxidase-conjugated secondary
antibodies After washing, peroxidase-dependent chemilu-minescence was detected by using enhanced chemilumines-cence Western blotting reagents and hyperfilm according to the manufacturer’s instructions (Amersham Pharmacia Biotech)
Statistical analysis All experiments were repeated at least three times Values are given as arithmetic mean ± SD Significance
Fig 2 MSDH induces intracellular ROS production Cells were seeded into 96-well plates at a density of 10 000 cellsÆcm)2 After 24 h, cells were exposed simultaneously to 30 l M MSDH and 10 l M DHE under otherwise standard culture conditions while control cells were exposed to DHE only (A) Fluorescence intensity arising from oxidized dihydroethidium in J774, HeLa and AG1518 cells was measured at indicated time points (B) J774 cells were visualized and photographed after 3 h exposure to MSDH (n ¼ 3) Very similar results were obtained with HeLa and AG1518 cells under the same conditions although detectable oxidant generation occurred earlier.
Trang 5of differences between groups was determined using
Student’s two-tailed t-test *P£ 0.05; **P£ 0.01;
***P£ 0.001
Results
Cultured cells exposed to the synthetic lysosomotropic
detergent, MSDH, undergo lysosomal rupture and ensuing
apoptosis or necrosis depending upon the extent of
lysosomal destabilization [12] In the present experiments,
we induced apoptosis in fibroblasts, J774 cells, and HeLa cells by exposing them to 30 lM MSDH After 8 h of MSDH exposure, nuclear propidium iodide staining and flow cytometry (to detect DNA fragmentation) revealed apoptotic nuclei appearing as a broad, hypodiploid DNA smear in front of a narrow peak of diploid DNA (Fig 1A shows results in fibroblasts) At an early stage in this process, well before the appearance of frank apoptosis, fibroblasts showed significantly increased numbers of stress fibres (Fig 1B)
Fig 3 MSDH induces mitochondrial ROS production by rupturing lysosomes Purified mitochondria (1.0 mg proteinÆmL)1) or a lysosome/mito-chondria-enriched fraction (1.0 mg proteinÆmL)1) were incubated with either of MSDH (30 l M ), PLA2 (0.2 UÆmL)1), or cathepsin B or D (12.5 lgÆmL)1; pH 6.0) for 3 h (A) H 2 O 2 production, (B) cytochrome c release, and (C) lysosomal stability were assayed as described in Materials and methods (n ¼ 3).
Trang 6Because oxidative stress has been reported to induce
stress fibre formation [37], we suspected that the MSDH
exposure might be causing increased intracellular generation
of ROS This latter was monitored by following changes in
DHE-induced fluorescence When oxidized, this compound
intercalates into DNA and RNA, resulting in an increase in
quantum yield Fluorescence intensity was measured
kineti-cally at indicated time points Increased ROSproduction
occurred after 1 h of MSDH-exposure in fibroblasts
(AG1518) and epithelial cells (HeLa), but was significant
only after 3 h in macrophages (J774) (Fig 2A) Note that in
fibroblasts and HeLa cells, and also in J774 cells (results not
shown), the oxidation of DHE eventually reached a steady
state consistent with only a transient production of ROS
Figure 2B shows DHE-exposed J774 cells after 3 h
expo-sure to MSDH, when there were still no morphological
signs of apoptosis
Theoretically, the increased oxidant generation might
arise from effects of released lysosomal enzymes (directly or
by activation of PLA2) on mitochondrial ROSproduction
or, alternatively, from direct effects of MSDH on the
mitochondria To discriminate between these possibilities,
we added MSDH to purified rat liver mitochondria
(4.5-fold purified from lysosomal contamination as compared to
the LEF preparation, results not shown) Under these
conditions, no changes in mitochondrial production of
H2O2(Fig 3A) or release of cytochrome c (Fig 3B) took
place Because we previously observed that lysosomal
contents cause activation of PLA2 in J774 cells [22], we
also exposed mitochondria to that enzyme and found it to
enhance mitochondrial production of ROS(Fig 3A) and to
release cytochrome c as well (Fig 3B) These findings strongly suggest that MSDH affects mitochondria by first destabilizing lysosomes and causing the release of hydrolytic enzymes which, in turn, attack mitochondria or activate PLA2 Activated PLA2 may further promote this cascade
of events, attacking both mitochondrial and lysosomal membranes and causing further lysosomal rupture This supposed sequence of events was confirmed by adding MSDH to a lysosome/mitochondria-enriched rat liver fraction, where it was found to induce enhanced mito-chondrial production of H2O2 (Fig 3A), release of cyto-chrome c (Fig 3B), and lysosomal rupture (Fig 3C)
To test further the idea that released lysosomal hydrolases might enhance mitochondrial ROSproduction, release of cytochrome c, and activation of PLA2 (all of which may promote the apoptotic cascade), we tested the effects of two lysosomal cathepsins (cathepsin B, a cysteine protease, and cathepsin D, an aspartic protease)
on purified mitochondria Both proteases caused substan-tial increases in mitochondrial production of H2O2 (Fig 3A) and release of cytochrome c (Fig 3B) However, neither cathepsin B nor D caused detectable lysosomal rupture in LEF preparations (Fig 3C), although, as expected, both MSDH and PLA2 did induce lysosomal rupture (Fig 3C)
Thus, cathepsins B and D do not directly cause rupture of lysosomes in an LEF preparation However, the possibility remains that the intracellular release of other lysosomal hydrolases may do so, or that lysosomal proteases might secondarily destabilize lysosomes through, for example, enhanced oxidative stress or activation of PLA2 following
Fig 4 The lysosomal/mitochondrial axis theory of apoptosis Both the internal and external pathways may involve lysosomal rupture Released lysosomal enzymes (LE) may: (a) attack mitochondria directly, inducing oxidative stress and release of cytochrome c (this study and [12,20–22,49– 52]); (b) activate lytic pro-enzymes, such as PLA2, which may attack both mitochondria or lysosomes (this study and [22]); (c) activate Bid [53]; (d) directly activate caspases [15,16,54,55] It is also possible that released lysosomal enzymes backfire on still intact lysosomes, causing further rupture Caspase 8 may somehow induce lysosomal rupture [56,57] or the activation of death receptors may cause production of sphingosine [58], which is a lysosomotropic detergent [59]; while p53 causes lysosomal labilization by unknown mechanisms [14] Other mechanisms may also be involved in lysosomal labilization in relation to apoptosis.
Trang 7mitochondrial attack by cathepsins and PLA2 Indeed, low,
steady-state oxidative stress has been shown to destabilize
lysosomes [20] and relocation of lysosomal enzymes to the
cytosol was earlier shown to activate PLA2 [22]
Discussion
We previously suggested that oxidative stress-induced
apoptosis might be initiated by iron-catalysed lysosomal
rupture [9,10] It has since been found that early release to
the cytosol of lytic lysosomal enzymes may be characteristic
of apoptosis caused by a variety of stimuli [10,12–14,
19,21,22,38–40] In these latter circumstances, it appears
that relocation of lysosomal enzymes to the cytosol may, as
in the case of oxidant-induced apoptosis, precede changes
of mitochondrial membrane potential, release of
cyto-chrome c, and all the morphological signs of apoptosis
These considerations raised the question of whether there
might be some ROS-dependent mechanisms common to
apoptosis caused by oxidants and that caused by
nonoxi-dant agents
In most cells, the predominant source of intracellular
ROSgeneration is the mitochondrial electron transport
chain which, even under normal conditions, may leak
1–2% of all electrons as ROS[41–43] (although there is
controversy regarding this estimate and the absolute
percentage may well be lower [44]) Not only will exogenous
oxidants, such as H2O2, directly induce apoptosis, but
enhanced intracellular production of ROSoccurs when cells
are exposed to a number of pro-apoptotic agents including
tumour necrosis factor-a [23], ceramide [24], growth factor
withdrawal, HIV infection, and lipopolysaccharide [25–30]
In these cases it is unclear whether such oxidative stress is
the cause or an effect of apoptosis
We hypothesized that released lysosomal enzymes or
PLA2 directly or indirectly activated by such enzymes [22]
might attack mitochondria and induce not only release
of cytochrome c, but also enhanced formation of ROS
Released arachidonic acid may further exaggerate this
process [45] These ROSof mitochondrial origin could
promote further lysosomal rupture but could also have the
secondary effect of maintaining any cytochrome c released
by the mitochondria in the oxidized form (although we
should note that the cellular cytoplasm contains an
abun-dance of reducing agents which could counteract this)
Cytochrome c is involved in the activation of caspase-9
[7,46] and is considered a key component of the apoptotic
cascade Ordinarily, any cytochrome c released from
mitochondria in oxidized form would rapidly be reduced
by the reductive cytosolic milieu However, it has been
proposed that cytochrome c needs to remain oxidized
in order to promote apoptosis [46], and the oxidizing
equivalents generated by mitochondria may have precisely
this effect
MSDH is a lysosomotropic detergent that rapidly induces
specific lysosomal rupture and therefore is a very useful tool
for detailed kinetic studies of the consequences of lysosomal
rupture The pKa of MSDH is 5.8–5.9 [31,32], allowing it to
accumulate in charged form intralysosomally (pH 4.5)
due to proton trapping [47], while its accumulation in the
cytosol (pH 7.2) is negligible In protonated, charged
form MSDH acts as a much stronger detergent than when
uncharged, further targeting the action of this agent to the lysosomal compartment [31]
We previously reported that released lysosomal enzymes activate PLA2 causing further lysosomal fragmentation [22] The new data presented here confirm and extend those findings and show that relocated lysosomal enzymes work
in concert with activated PLA2, causing the release of cytochrome c, enhanced mitochondrial formation of ROS, and promoting further lysosomal degradation With regard
to the mechanisms involved in enhanced mitochondrial ROSproduction, one particularly likely possibility is that of generation of free fatty acids At least in pancreatic beta cell mitochondria, free fatty acids have been shown to increase ROSgeneration, perhaps through electron leak involving complex I of the respiratory chain [48] Whether the progressive lysosomal destabilization is dependent exclu-sively on upstream actions of cathepsins B and D, or whether other lysosomal constituents might similarly desta-bilize mitochondria and lysosomes is not yet clear Our present understanding concerning the involvement
of lysosomes in apoptosis is summarized in Fig 4 As shown, the initiation of apoptosis by exogenous oxidants, and by at least some other agonists, may involve early lysosomal rupture The release of lysosomal enzymes (LE) into the cell cytoplasm may set off a cascade of intracellular degradative events These LE may: (a) attack mitochondria directly, inducing release of cytochrome c; (b) directly and/
or indirectly cause enhanced formation of mitochondrial ROS(and further oxidant-induced lysosomal destabiliza-tion); (c) activate lytic pro-enzymes, such as PLA2, which in turn would attack both mitochondria and lysosomes; (d) activate Bid and/or other pro-apoptotic proteins; and (e) directly activate pro-caspases Notably, this sequence of early events (except for cytochrome c release) may be relatively independent of the classical apoptotic cascade involving caspase activation In many circumstances, this
lysosomal-mitochondrial axis apoptotic pathway, invol-ving combined effects of caspases, lysosomal hydrolases and mitochondrial ROSgeneration, may be of central import-ance in the final execution of the apoptotic cascade wherein
a lysosomal/mitochondrial cross-talk may constitute an amplifying loop
Acknowledgements
We thank G Dubowchik (Bristol-Myers Squibb; Pharmaceutical Research Institute) for the kind gift of MSDH This study was supported by a grant from the Swedish Cancer Foundation (grant no 4296) JWE was the recipient of a visiting professorship from the Linko¨ping University Hospital and is supported by The Common-wealth of Kentucky Research Challenge Trust Fund.
References
1 Nicholson, D.W (2000) From bench to clinic with apoptosis-based therapeutic agents Nature 407, 810–816.
2 Nicholson, D.W (2001) Apoptosis Baiting death inhibitors Nature 410, 33–34.
3 Savill, J & Fadok, V (2000) Corpse clearance defines the meaning
of cell death Nature 407, 784–788.
4 Baumann, S., Krueger, A., Kirchhoff, S & Krammer, P.H (2002) Regulation of T cell apoptosis during the immune response Curr Mol Med 2, 257–272.
Trang 85 Yuan, J & Yankner, B.A (2000) Apoptosis in the nervous system.
Nature 407, 802–809.
6 Kaufmann, S.H & Hengartner, M.O (2001) Programmed cell
death: alive and well in the new millennium Trends Cell Biol 11,
526–534.
7 Hengartner, M.O (2000) The biochemistry of apoptosis Nature
407, 770–776.
8 Zakeri, Z & Lockshin, R.A (2002) Cell death during
develop-ment J Immunol Methods 265, 3–20.
9 Zdolsek, J., Zhang, H., Roberg, K & Brunk, U (1993) H 2 O 2
-mediated damage to lysosomal membranes of J-774 cells Free
Radic Res Commun 18, 71–85.
10 O¨llinger, K & Brunk, U.T (1995) Cellular injury induced by
oxidative stress is mediated through lysosomal damage Free
Radic Biol Med 19, 565–574.
11 Brunk, U.T., Dalen, H., Roberg, K & Hellquist, H.B (1997)
Photo-oxidative disruption of lysosomal membranes causes
apoptosis of cultured human fibroblasts Free Radic Biol Med.
23, 616–626.
12 Li, W., Yuan, X., Nordgren, G., Dalen, H., Dubowchik, G.M.,
Firestone, R.A & Brunk, U.T (2000) Induction of cell death by
the lysosomotropic detergent MSDH FEBS Lett 470, 35–39.
13 Brunk, U.T., Neuzil, J & Eaton, J.W (2001) Lysosomal
involvement in apoptosis Redox Report 6, 91–97.
14 Yuan, X.M., Li, W., Dalen, H., Lotem, J., Kama, R., Sachs, L &
Brunk, U.T (2002) Lysosomal destabilization in p53-induced
apoptosis Proc Natl Acad Sci USA 99, 6286–6291.
15 Ishisaka, R., Utsumi, T., Kanno, T., Arita, K., Katunuma, N.,
Akiyama, J & Utsumi, K (1999) Participation of a cathepsin
1-type protease in the activation of caspase-3 Cell Struct Funct.
24, 465–470.
16 Katunuma, N., Matsui, A., Le, Q.T., Utsumi, K., Salvesen, G &
Ohashi, A (2001) Novel procaspase-3 activating cascade mediated
by lysoapoptases and its biological significances in apoptosis Adv.
Enz Reg 41, 237–250.
17 Bursch, W (2001) The autophagosomal-lysosomal compartment
in programmed cell death Cell Death Differ 8, 569–581.
18 Salvesen, G.S (2001) A lysosomal protease enters the death scene.
J Clin Invest 107, 21–22.
19 Zhao, M., Eaton, J.W & Brunk, U.T (2000) Protection against
oxidant-mediated lysosomal rupture: a new anti-apoptotic activity
of Bcl-2? FEBS Lett 485, 104–108.
20 Antunes, F., Cadenas, E & Brunk, U.T (2001) Apoptosis
induced by exposure to a low steady-state concentration of
H 2 O 2 is a consequence of lysosomal rupture Biochem J 365,
549–555.
21 Zhao, M., Eaton, J.W & Brunk, U.T (2001) Bcl-2
phosphory-lation is required for inhibition of oxidative stress-induced
lyso-somal leak and ensuing apoptosis FEBS Lett 509, 405–412.
22 Zhao, M., Brunk, U.T & Eaton, J.W (2001) Delayed
oxidant-induced cell death involves activation of phospholipase A2 FEBS
Lett 509, 399–404.
23 Obrador, E., Navarro, J., Mompo, J., Asensi, M., Pellicer, J.A &
Estrela, J.M (1998) Regulation of tumour cell sensitivity to
TNF-induced oxidative stress and cytotoxicity: role of glutathione.
Biofactors 8, 23–26.
24 Andrieu-Abadie, N., Gouaze, V., Salvayre, R & Levade, T (2001)
Ceramide in apoptosis signaling: relationship with oxidative stress.
Free Radic Biol Med 31, 717–728.
25 Muller, J.M., Ziegler-Heitbrock, H.W.L & Baeuerle, P.A (1993)
Nuclear factor kappa B, a mediator of lipopolysaccharide effects.
Immunobiology 187, 233–256.
26 Albrecht, H., Tschopp, J & Jongeneel, C.V (1994) Bcl-2 protects
from oxidative damage and apoptotic cell death without
inter-fering with activation of NF-kappa B by TNF FEBS Lett 351,
45–48.
27 Atabay, C., Cagnoli, C.M., Kharlamov, E., Ikonomovic, M.D & Manev, H (1996) Removal of serum from primary cultures of cerebellar granule neurons induces oxidative stress and DNA fragmentation: protection with antioxidants and glutamate receptor antagonists J Neurosci Res 43, 465–475.
28 Degli Esposti, M & McLennan, H (1998) Mitochondria and cells produce reactive oxygen species in virtual anaerobiosis: relevance to ceramide-induced apoptosis FEBS Lett 430, 338–342.
29 Garcia-Ruiz, C., Colell, A., Mari, M., Morales, A & Fernandez-Checa, J.C (1997) Direct effect of ceramide on the mitochondrial electron transport chain leads to generation of reactive oxygen species Role of mitochondrial glutathione J Biol Chem 272, 11369–11377.
30 Dobmeyer, T.S., Findhammer, S., Dobmeyer, J.M., Klein, S.A., Raffel, B., Hoelzer, D., Helm, E.B., Kabelitz, D & Rossol, R (1997) Ex vivo induction of apoptosis in lymphocytes is mediated
by oxidative stress: role for lymphocyte loss in HIV infection Free Radic Biol Med 22, 775–785.
31 Firestone, R.A., Pisano, J.M & Bonney, R.J (1979) Lysosomo-tropic agents 1 Synthesis and cytotoxic action of lysosomoLysosomo-tropic detergents J Med Chem 22, 1130–1133.
32 Wilson, P.D., Firestone, R.A & Lenard, J (1987) The role of lysosomal enzymes in killing of mammalian cells by the lyso-somotropic detergent N-dodecylimidazole J Cell Biol 104, 1223–1229.
33 Nicoletti, I., Migliorati, G., Pagliacci, M.C., Grignani, F & Riccardi, C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry J Immunol Methods 139, 271–279.
34 Hempel, S.L., Buettner, G.R., O’Malley, Y.Q., Wessels, D.A & Flaherty, D.M (1999) Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2¢,7¢-dichloro-dihydrofluorescein diacetate, 5 (and 6)-carboxy-2¢,7¢-dichlorodi-hydrofluorescein diacetate, and dihydrorhodamine 123 Free Radic Biol Med 27, 146–159.
35 Gasnier, F., Rousson, R., Lerme, F., Vaganay, E., Louisot, P & Gateau-Roesch, O (1993) Use of Percoll gradients for isolation of human placenta mitochondria suitable for investigating outer membrane proteins Anal Biochem 212, 173–178.
36 Hyslop, P.A & Sklar, L.A (1984) A quantitative fluorimetric assay for the determination of oxidant production by poly-morphonuclear leukocytes: its use in the simultaneous fluorimetric assay of cellular activation processes Anal Biochem 141, 280– 286.
37 Huot, J., Houle, F., Marceau, F & Landry, J (1997) Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vas-cular endothelial cells Circ Res 80, 383–392.
38 Brunk, U.T & Svensson, I (1999) Oxidative stress, growth factor starvation and Fas activation may all cause apoptosis through lysosomal leak Redox Report 4, 3–11.
39 Yuan, X.M., Li, W., Brunk, U.T., Dalen, H., Chang, Y.H & Sevanian, A (2000) Lysosomal destabilization during macrophage damage induced by cholesterol oxidation products Free Radic Biol Med 28, 208–218.
40 Neuzil, J., Zhao, M., Ostermann, G., Sticha, M., Gellert, N., Weber, C., Eaton, J.W & Brunk, U.T (2002) Alpha-tocopheryl succinate, an agent with in vivo anti-tumour activity, induces apoptosis by causing lysosomal instability Biochem J 362, 709–715.
41 Boveris, A & Chance, B (1973) The mitochondrial generation of hydrogen peroxide General properties and effect of hyperbaric oxygen Biochem J 134, 707–716.
42 Chance, B., Sies, H & Boveris, A (1979) Hydroperoxide meta-bolism in mammalian organs Physiol Rev 59, 527–605.
Trang 943 Liu, Y., Fiskum, G & Schubert, D (2002) Generation of reactive
oxygen species by the mitochondrial electron transport chain.
J Neurochem 80, 780–787.
44 Hansford, R.G., Hogue, B.A & Mildaziene, V (1997)
Depen-dence of H 2 O 2 formation by rat heart mitochondria on substrate
availability and donor age J Bioenerg Biomembr 29, 89–95.
45 Scorrano, L., Penzo, D., Petronilli, V., Pagano, F & Bernardi, P.
(2001) Arachidonic acid causes cell death through the
mitochon-drial permeability transition Implications for tumor necrosis
factor-alpha aopototic signaling J Biol Chem 276, 12035–12040.
46 Hancock, J.T., Desikan, R & Neill, S.J (2001) Does the redox
status of cytochrome C act as a fail-safe mechanism in the
regu-lation of programmed cell death? Free Radic Biol Med 31, 697–
703.
47 de Duve, C., de Barsy, T., Poole, B., Trouet, A., Tulkens, P & Van
Hoof, F (1974) Commentary Lysosomotropic agents Biochem.
Pharmacol 23, 2495–2531.
48 Koshkin, V., Wang, X., S cherer, P.E., Chan, C.B & Wheeler,
M.B (2003) Mitochondrial functional state in clonal pancreatic
beta-cells exposed to free fatty acids J Biol Chem 278, 19709–
19715.
49 Roberg, K & O¨llinger, K (1998) Oxidative stress causes
reloca-tion of the lysosomal enzyme cathepsin D with ensuing apoptosis
in neonatal rat cardiomyocytes Am J Pathol 152, 1151–1156.
50 Roberg, K., Johansson, U & O¨llinger, K (1999) Lysosomal
release of cathepsin D precedes relocation of cytochrome c and
loss of mitochondrial transmembrane potential during apoptosis
induced by oxidative stress Free Radic Biol Med 27, 1228–1237.
51 Roberg, K (2001) Relocalization of cathepsin D and cytochrome
c early in apoptosis revealed by immunoelectron microscopy Lab.
Invest 81, 149–158.
52 Roberg, K., Kagedal, K & O¨llinger, K (2002) Microinjection of cathepsin D induces caspase-dependent apoptosis in fibroblasts.
Am J Pathol 161, 89–96.
53 Stoka, V., Turk, B., Schendel, S.L., Kim, T.H., Cirman, T., Snipas, S.J., Ellerby, L.M., Bredesen, D., Freeze, H., Abraham-son, M., Bromme, D., Krajewski, S , Reed, J.C., Yin, X.M., Turk,
V & Salvesen, G.S (2001) Lysosomal protease pathways to apoptosis Cleavage of bid, not pro-caspases, is the most likely route J Biol Chem 276, 3149–3157.
54 Zhou, Q & Salvesen, G.S (1997) Activation of pro-caspase-7 by serine proteases includes a non-canonical specificity Biochem.
J 324, 361–364.
55 Katunuma, N., Matsui, A., Kakegawa, T., Murata, E., Asao, T & Ohba, Y (1999) Study of the functional share of lysosomal cathepsins by the development of specific inhibitors Adv Enzyme Regul 39, 247–260.
56 Guicciardi, M.E., Deussing, J., Miyoshi, H., Bronk, S.F., Svingen, P.A., Peters, C., Kaufmann, S.H & Gores, G.J (2000) Cathepsin
B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c J Clin Invest.
106, 1127–1137.
57 Cuvillier, O., Edsall, L & Spiegel, S (2000) Involvement of sphingosine in mitochondria-dependent Fas-induced apoptosis of type II Jurkat T cells J Biol Chem 275, 15691–15700.
58 Werneburg, N.W., Guicciardi, M.E., Bronk, S.F & Gores, G.J (2002) Tumor necrosis factor-a-associated lysosomal permeabili-zation is cathepsin B dependent Am J Physiol Gastrointest Liver Physiol 283, 947–956.
59 Ka˚gedal, K., Zhao, M., Svensson, I & Brunk, U.T (2001) Sphingosine-induced apoptosis is dependent on lysosomal pro-teases Biochem J 359, 335–343.