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Endocrinology |
Departments of Pathology [A. G., O. W. R., M. B. C.] and Urology [M. B. C.], University of Iowa, Iowa City, Iowa 52242, and Veterans Affairs Medical Center, Iowa City, Iowa 52240 [M. B. C.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Fas (CD95) receptor is a type I transmembrane protein and belongs to the tumor necrosis factor receptor family (4 , 14) . Fas was identified as a cell surface receptor that mediates cell death after ligation with agonistic anti-Fas antibodies (15 , 16) . Because functional Fas is expressed on the surface of diverse cancer cells, it potentially provides an approach for the rapid and irreversible killing of tumor cells, although methods of proper targeting of the therapeutic Fas ligand specifically to the Fas receptors on the tumor cells still have to be devised to prevent deleterious side effects (17) . Some of the pathways leading to Fas-mediated apoptosis have been characterized in detail. Engagement of Fas results in the formation of the DISC, a complex of Fas, Fas-associating protein with death domain, and pro-caspase-8 (4) . Activated caspase-8 is released from the DISC (18) and has been shown to directly activate the executioner caspases (19) . At the same time, caspase-8 has been reported to cleave Bid, a proapoptotic member of the Bcl-2 family, which then induces cyto c release, thus forming a link between Fas-mediated apoptosis and the mitochondrial pathway (20) . In certain cell types, the direct activation of downstream caspases by the DISC appears to be sufficient for the execution of Fas-mediated apoptosis because Bcl-2 does not protect against Fas killing in these cell types (21, 22, 23) . However, in other cell systems, Bcl-2 or Bcl-XL was reported to protect against Fas-mediated apoptosis (12 , 24, 25, 26, 27) . Thus, depending on the cell type studied, Fas-mediated apoptosis can be dependent or independent on the mitochondrial pathway, and sometimes the mitochondrial pathway at least appears to contribute to Fas-mediated apoptosis by amplifying the effects of caspase-8 on activation of downstream caspases (6 , 28 , 29) .
We have shown previously that in the human prostatic carcinoma cell lines PC3, ALVA31, DU145, and JCA1, the pathway(s) leading to Fas-mediated apoptosis is intact (30) . PC3 and ALVA31 are sensitive to treatment with anti-Fas mAb, whereas DU145 and JCA1 are only sensitive under combined treatment with anti-Fas mAb and CHX. CHX is necessary to convert DU145 and JCA1 from Fas-resistant to Fas-sensitive because of a labile-dominant inhibitory protein(s) presumably acting at the apex of the apoptotic cascade (31 , 32) .
In this study, we performed experiments to determine whether the mitochondrial pathway is involved in Fas-mediated apoptosis of prostatic carcinoma cell lines. Our results indicate that activation of the Fas pathway in prostatic carcinoma cells induces a cascade that includes activation of caspase-8, Bid cleavage, cyto c release, and activation of caspase-9. Experiments using a cell-free system indicate that the apoptotic executioner events downstream from cyto c are intact in PC3, ALVA31, and JCA1.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of Cytosols for cyto c Release.
Mitochondria-free cytosol for the detection of cyto c release
was prepared as described (11)
. Briefly, cells were
grown for different times in the presence of 0.5 µg/ml anti-Fas mAb
(IPO-4), harvested, washed, and lysed in ice-cold buffer M [20
mM HEPES (pH 7.5), 10 mM KCl, 1.5
mM MgCl2, 1 mM EGTA, 1
mM EDTA, 1 mM DTT, 250 mM sucrose,
0.1 mM PMSF, 2 µg/ml pepstatin, 2 µg/ml leupeptin, and
2 µg/ml aprotinin] at about 2 x 106 cells per 100 µl by homogenization in a
small glass homogenizer with a Teflon pestle (50 strokes on ice). The
ho-mogenates were spun at 16,000 x g for
20 min at 4°C, and the supernatants were used for anti-cyto c Western
blot analysis. As a proof for the loading of equal amounts of protein,
we also performed blots using anti-actin mAb.
Western Blotting.
Western blot detection of proteins was performed as described
previously (30)
. Briefly, 10–20 µg of proteins were
separated on 4–20% gradient SDS-PAGE and blotted to nitrocellulose
membrane (Novex, San Diego, CA). Membranes were incubated with the
corresponding monoclonal antibodies: anti-cyto c, anti-caspase-3
(Transduction Laboratories, San Diego, CA), anti-actin mAb (Sigma
Chemical Co., St. Louis, MO), anti-caspase-8, anti-caspase-10 (Upstate,
Lake Placid, NY), goat polyclonal anti-Bid (R&D Systems, Minneapolis,
MN), and rabbit antibody to caspase-7 (kindly provided by Dr. Yuri
Lazebnik, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
Anti-caspase-9 antibodies were monoclonal or rabbit polyclonal (as
indicated in the figure legends; PharMingen, San Diego, CA, or
Oncogene, La Jolla, CA). The blots were incubated with a goat antimouse
or goat antirabbit IgG conjugated with horseradish peroxidase, and
immunoreactive bands were visualized by incubation of the membrane with
SuperSignal chemiluminescence reagent (Pierce, Rockford, IL).
Preparation of Cytoplasmic Extracts.
Cytoplasmic extracts for use in the cell-free system were
prepared essentially as described (33)
. Briefly, cells
were harvested by trypsinization; trypsinization was stopped by adding
fetal bovine serum to a final concentration of 50%. Cells were washed
once in complete RPMI 1640, twice in ice-cold PBS, once in KPM buffer
[50 mM PIPES (pH 7.0), 50 mM KCl, 2
mM MgCl2, 1 mM EGTA, 1
mM DTT, 10 µg/ml cytochalasin B, 0.1 mM PMSF,
2 µg/ml pepstatin, 2 µg/ml leupeptin, and 2 µg/ml aprotinin].
The cell pellet was resuspended in about 1 volume of KPM buffer and
lysed by three cycles of freezing and thawing in liquid nitrogen. The
lysate was centrifuged at 16,000 x g for 20
min at 4°C. The supernatant (15–25 mg/ml protein) was stored at
-70°C.
Isolation of Nuclei.
Nuclei were prepared essentially as described previously
(34)
. Briefly, 10 µM cytochalasin B was
added to adherent growing prostate carcinoma cells, and incubation was
continued for 30 min (for preparation of Jurkat nuclei this step was
omitted). Cells were harvested, washed twice with PBS and once with
Nuclei Buffer [10 mM PIPES (pH 7.4), 10 mM
KCl, 2 mM MgCl2, 1 mM
DTT, 10 µg/ml cytochalasin B, 0.1 mM PMSF, 2 µg/ml
pepstatin, 2 µg/ml leupeptin, and 2 µg/ml aprotinin], and gently
lysed with a Dounce homogenizer, and the homogenate was layered over
30% sucrose in Nuclei Buffer and pelleted by centrifugation at
800 x g for 10 min. For the preparation of
radioactive-labeled nuclei, ALVA31 cells were harvested, and
5 x 106 cells were reseeded in
162-cm2 tissue culture flasks in complete RPMI
1640 + 2 µCi/ml [3H]thymidine.
After 24 h of incubation, labeled cells were harvested, and nuclei
were prepared as described above. The degree of labeling was about 1
cpm/nucleus.
Caspase Enzymatic Assays.
To measure caspase activity, cytoplasmic extracts were diluted
to a protein concentration of 2.5 mg/ml with Dilution Buffer containing
an ATP-regeneration system [10 mM HEPES (pH 7.0), 5
mM EGTA, 50 mM NaCl, 2 mM MgCl, 1
mM DTT, 2 mM ATP, 10 mM
phosphocreatine, and 50 µg/ml creatine kinase] and activated with 5
µM cyto c (Sigma) and 1 mM dATP (Promega
Corp., Madison, WI) in a total volume of 15 µl. After incubation at
37°C for 45 min, the extracts were incubated for 30 min at room
temperature in 200 µl Assay Buffer [50 mM PIPES-KOH (pH
7.2), 0.1 mM EDTA, and 10% glycerol] with 20
µM fluorescent substrates: Ac-DEVD-AMC (CPP32 subfamily
substrate), Ac-YVAD-AMC (interleukin-1ß-converting enzyme subfamily
substrate), and Ac-VEID-AMC (caspase-6 substrate; all from Calbiochem,
San Diego, CA). Fluorescence was measured with a FL600 fluorimeter
(Bio-Tek Instruments, Inc., Burlington, VT).
Radioactive DNA Fragmentation Assay.
Radioactive nuclei were prepared as described above. Prior to
use in the cell-free system, 5 x 104 nuclei were distributed in 0.5 ml
microcentrifuge tubes and were washed once in Dilution Buffer. The
nuclei were incubated in cytoplasmic extracts (7.5 mg/ml) in the
presence or absence of 10 µM cyto c and 1 mM
dATP in a total volume of 10 µl for 4 h at 37°C (650
nuclei/µg protein). After incubation, the DNA of the nuclei was
harvested on a glass fiber membrane, and the retained
radioactivity was measured by scintillation counting. Experiments were
run in triplicate or pentuplicate for each condition. The percentage of
DNA fragmentation was calculated as follows: [(cpm of nuclei in pure
extracts) - (cpm of nuclei in extracts + cyto
c/dATP)/(cpm of nuclei in pure extracts)] x 100.
4'6-Diamidino-2-phenylindole Staining and DNA Laddering.
A total of 3 x 105
nuclei from frozen stocks (1.5 µl of 2 x 108 nuclei/ml) was incubated for 4 h in
cytoplasmic extracts at a protein concentration of 7.5 mg/ml in a total
volume of 50 µl (800 nuclei/µg protein) in the presence or absence
of 10 µM cyto c and 1 mM dATP. If zVAD-fmk
was used, it was added to the extracts first, and then cyto c and dATP
were added. After incubation, a sample of 5 x 104 nuclei was fixed in 4% PBS-buffered
paraformaldehyde solution in the presence of 2 µg/ml
4'6-diamidino-2-phenylindole (Sigma) and observed using a fluorescence
microscope (BH Series; Olympus, New Hyde Park, NY) at excitation
wavelength 350 nm. DNA was prepared by incubation of the remaining
nuclei in Lysis Buffer [50 mM Tris-HCl (pH 8.0), 10
mM EDTA, 0.2% SDS, and 0.5 mg/ml proteinase K] overnight
at 37°C, and precipitation of the DNA by adding 0.1 volume of 3
M NaOAc and 2 volumes of ice-cold ethanol. The dried pellet
was dissolved in 20 µl of TE [50 mM Tris-HCl (pH 8.0), 1
mM EDTA], and RNase A was added at 0.1 mg/ml. After
incubation for 1 h at 37°C, the DNA was analyzed on a 1.5%
agarose gel (at 4 V/cm) containing 0.5 µg/ml ethidium bromide.
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RESULTS |
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Involvement of Bid, cyto c, and Caspase-9 in Fas-mediated Apoptosis
of Prostatic Carcinoma Cell Lines.
To determine whether the cyto c pathway is involved in
Fas-mediated apoptosis in prostatic carcinoma cell lines, PC3, DU145,
and ALVA31 were treated with anti-Fas mAb for different times, and
cytosol was prepared by gentle lysis. Western blots revealed increasing
amounts of cyto c in the cytosols of PC3 and ALVA31 during anti-Fas
treatment, whereas in DU145 an increase of cyto c was observed only
under combined treatment with anti-Fas mAb and CHX (Fig. 1A)
. The increase of cyto c in the cytosols of PC3 and ALVA31
was already observed after 2 h of anti-Fas treatment when
morphologically no cell death could be detected. For PC3 we also
demonstrate that the pan-caspase-inhibitor zVAD-fmk prevents cyto c
release into the cytosol (Fig. 1A)
. Additionally, we
detected activation of caspase-9 in anti-Fas-treated PC3 and ALVA31, as
judged by the decrease of the proenzyme band (Fig. 1B)
. In
DU145, the level of pro-caspase-9 decreased only after combined
treatment with anti-Fas and CHX.
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. Note that Bid processing was prevented in the presence of the caspase
inhibitor zVAD-fmk, as shown for PC3 (Fig. 2B)
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cyto c Induces Caspase Activity in Cytoplasmic Extracts.
Because anti-Fas treatment triggers cyto c release into the
cytosol, we analyzed the effect of cyto c on cytoplasmic extracts from
PC3, DU145, ALVA31, and JCA1. After incubation of cytoplasmic extracts
with 5 µM cyto c and 1 mM dATP at 37°C for
45 min, we detected caspase-3-like activity in activated extracts from
all four cell lines when using Ac-DEVD-AMC as substrate (Fig. 3)
. Extracts from JCA1 displayed levels of DEVDase activity comparable
with those from Jurkat, whereas extracts from PC3, ALVA31, and DU145
displayed lower activity. cyto c-activated extracts also displayed
activity with Ac-VEID-AMC, a selective substrate for caspase-6
(37)
, which was about three times lower than DEVDase
activity, and we did not detect any activity with the caspase-1
substrate Ac-YVAD-AMC (data not shown). Thus, we found caspase activity
characteristic for members of the CPP32 subfamily, but activity
specific for caspases of the interleukin-1ß-converting enzyme
subfamily was not detected.
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. Activated ALVA31 extracts induced strong DNA fragmentation (60%) in
nuclei, whereas PC3 and JCA1 extracts induced only 20–30% DNA
fragmentation. cyto c-activated DU145 extracts did not induce any
significant DNA fragmentation. To determine whether the inability
of DU145 extracts to induce apoptosis in isolated nuclei was
specific for the stimulation of apoptotic activity by cyto c, we also
stimulated cytoplasmic extracts by the addition of active recombinant
caspase-8. Extracts from PC3, JCA1, and ALVA31, but not extract from
DU145, induced morphological changes and DNA fragmentation in nuclei
when active recombinant caspase-8 was added to the cell-free system
(data not shown). In summary, stimulation of cytoplasmic extracts from
ALVA31, PC3, and JCA1 with cyto c or active caspase-8 induced nuclear
apoptotic activity, whereas DU145 extracts were inactive.
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Activation Status of Caspases in cyto c-stimulated Extracts.
cyto c treatment induced caspase activity in the cell extracts
from all four cell lines, although there were differences in the level
of activity (Fig. 3)
. Furthermore, remarkable differences in the
nuclear apoptotic activity were also detected in the cytoplasmic
extracts from the four different cell lines (Fig. 4)
which, however,
did not correlate with the differences in caspase activity. Therefore,
the activation status of individual caspases was determined. Western
blotting revealed that caspase-9 was activated in all extracts when
cyto c and dATP were added (Fig. 6)
. We detected comparable expression and activation of caspase-7 in all
activated extracts (Fig. 7A)
. In contrast, levels of pro-caspase-3 and its activated
p17 subunit varied considerably in extracts from the different cell
lines (Fig. 7B)
. High levels of the p17 subunit were
detected in JCA1 extracts, moderate levels in PC3 and in ALVA31
extracts, and DU145 extracts displayed the lowest level of active
caspase-3.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(45)
. However, it also has been reported that
certain drugs induce apoptosis in PC3 but do not trigger cyto c
release, suggesting that cyto c release is an inducer-dependent
phenomenon (45)
.
In this study, we examined whether cyto c is involved in
Fas-mediated apoptosis of human prostatic carcinoma cell lines and
investigated the cyto c signaling pathway in these cell lines. We found
that after Fas ligation, cyto c is released into the cytosol in PC3 and
ALVA31 (Fig. 1A)
. cyto c could be detected in the cytosol as
early as 2 h after Fas ligation. We have shown previously that in
the Fas-sensitive prostatic carcinoma cell lines PC3 and ALVA31,
apoptotic features such as phosphatidylserine exposure and DNA
fragmentation occur after at least 6 h of treatment with agonistic
anti-Fas mAb (30)
. Additionally, we have demonstrated that
in PC3 cells, activation of caspase-8 occurs between 1 and 2 h of
anti-Fas treatment, and activation of caspase-7 occurs after 4 h
of treatment (31)
. Detection of increased cyto c levels in
the cytosol after 2 h of Fas ligation indicates that cyto c
release is one of the early events in Fas-mediated apoptosis in
prostatic carcinoma cell lines. We did not detect cytosolic cyto c
increase in the Fas-resistant cell line DU145 after treatment with
anti-Fas mAb, but cyto c release was observed after 4–8 h of combined
treatment of DU145 with anti-Fas mAb and CHX (Fig. 1A)
.
Because cyto c release in Fas-mediated apoptosis is considered to be
mediated by activated caspase-8 (20
, 36)
, this time course
is consistent with our previous finding that caspase-8 is activated
after only 
4 h of combined treatment with anti-Fas mAb and CHX
(31)
. These results also support our hypothesis that the
putative inhibitory factor(s), which is responsible for Fas resistance
in DU145, acts at the apex of the cascade, presumably at the level of
caspase-8 activation (31)
.
Recent studies of Fas-mediated apoptosis have implicated the
cleavage of Bid by caspase-8, resulting in the translocation of the
truncated Bid to the mitochondria, where it induces the release of cyto
c (20
, 36)
. In this report, we demonstrate that Bid is
processed after Fas-mediated activation of caspase-8 in PC3, DU145, and
ALVA31. In PC3, Bid cleavage and cyto c release can be inhibited by the
caspase inhibitor zVAD-fmk (Fig. 1A
and Fig. 2B
).
The primary target of zVAD-fmk presumably is the inhibition of
caspase-8 activation prior to any perturbation of mitochondria,
resulting in the inhibition of all downstream biochemical effects
including cyto c release (38)
. This result suggests that
after Fas ligation and activation of caspase-8, Bid is cleaved by
caspase-8, and the resulting truncated Bid triggers cyto c release from
the mitochondria into the cytosol of prostatic carcinoma cell lines.
However, it cannot be entirely ruled out that caspase-8 first activates
downstream caspases such as caspase-7, which then induces cyto c
release by activating cytosolic factors other than Bid, as was reported
recently (29)
.
After cyto c release into the cytosol, caspase-9 is activated
by the formation of the apoptosome (7)
, and by this the
executioner phase of apoptosis is initiated, e.g., by the
activation of caspase-7, as has been reported previously in
Fas-mediated apoptosis of prostatic carcinoma cell lines
(31)
. In fact, we were able to detect the processing of
caspase-9 after Fas ligation (Fig. 1B)
.
Cell-free systems have been successfully applied previously in the dissection of biochemical mechanisms during the apoptotic process, such as the identification and characterization of the "apoptosome" (35) , apoptosis-inducing factor (13) , and the DNA fragmentation factor ICAD (inhibitor of caspase-activated DNase; Ref. 46 ). It has become clear that the apoptotic pathways acting in the cytoplasm function independently from the nucleus, and thus cell-free systems appear to be appropriate model systems that represent at least part of the apoptotic machinery and signaling mechanisms (47, 48, 49) .
We used a cell-free system to determine whether cyto c can
trigger apoptotic activity in the cytosol of the prostatic cancer cell
lines. Incubation of isolated nuclei together with cyto c-activated
extracts from PC3, JCA1, and ALVA31 resulted in nuclear apoptotic
features such as characteristic morphological changes and DNA
fragmentation (Fig. 4)
. Thus, in these cell lines, the pathways leading
from cyto c release to the execution of nuclear apoptosis appear to be
intact. The degree of DNA fragmentation induced by activated extracts
from the different cell lines varied considerably. Caspase-3-like
activity was essential for the induction of DNA fragmentation in
isolated nuclei because we found its complete inhibition by 1
µM zDEVD-fmk (Fig. 5)
. However, the level of caspase
activity in a certain cell extract was not correlated with the degree
of DNA fragmentation induced by this extract. For example, cyto
c-activated JCA1 extracts exhibited a high level of active caspase-3
(Fig. 7B)
and displayed high DEVDase activity (Fig. 3)
but
induced only moderate DNA fragmentation in nuclei (Fig. 4)
, whereas
ALVA31 extracts induced strong DNA fragmentation activity but had much
lower levels of active caspase-3. Thus, additional factors other than
just the level of activated caspase-3 appear to influence the capacity
of cytoplasmic extracts to induce DNA fragmentation in the cell-free
system.
It should be mentioned that thus far we have been unable to
detect activated caspase-3 in extracts from PC3 and DU145 treated with
anti-Fas or anti-Fas/CHX in culture (31)
. However, we
report here that cell-free activation of cytoplasmic extracts with cyto
c (Fig. 7B)
and also with active recombinant caspase-8 (not
shown) does result in activated caspase-3. Because caspase activation
in cell-free systems apparently recapitulates the selectivity observed
in treated intact cells (49)
, our cell-free experiments
might indicate that caspase-3 is activated during Fas-mediated
apoptosis in PC3 and DU145 on a low level that can be detected under
cell-free conditions but not in extracts from anti-Fas-treated cells
because of a lack of detection sensitivity.
Consistent with previous studies (31
, 43)
, the
cell-free experiments show that caspase-7 is expressed at similar
levels and is activated at a high level in all prostate carcinoma cells
examined (Fig. 7A)
and thus appears to be the dominant
executioner caspase in these cell lines. However, high levels of active
caspase-7 are obviously not sufficient to confer nuclear apoptotic
activity because, for instance, stimulated DU145 extracts possess high
levels of activated caspase-7 but do not induce oligonucleosomal DNA
fragmentation in isolated nuclei.
The inability of cytoplasmic DU145 extracts to trigger nuclear
apoptotic events in the cell-free system was shown to not be specific
for a certain signaling pathway because neither cyto c nor caspase-8
was found to induce nuclear apoptotic events in cytoplasmic extracts
from DU145. In contrast, caspase-8 induced strong nuclear apoptotic
activity in extracts from PC3, JCA1, and ALVA31 (data not shown).
Mixing experiments did not show any evidence for the presence of an
inhibitory factor in DU145 extracts (Fig. 5)
. Thus, the inactivity of
DU145 extracts in the cell-free system has to be attributed to a
deficiency of an activity. Because DU145 cells do undergo DNA
fragmentation when cultured in the presence of anti-Fas mAb and CHX,
the inactivity observed in the cell-free system might be an artifact.
Alternatively, the nuclear apoptotic activity in DU145 depends on a
factor(s) that is in place after induction of apoptosis in living cells
but not after induction of apoptosis in cytoplasmic extracts. A recent
study suggests that indeed there might be differences between the
activation processes occurring in treated intact cells and in cell-free
systems (49)
.
In conclusion, our data suggest that in prostatic carcinoma cell lines, Fas ligation triggers a cascade that leads from activated caspase-8 over the processing of Bid to the release of cyto c into the cytosol and activation of caspase-9. The release of cyto c is an early event in Fas-mediated apoptosis in prostatic carcinoma cell lines because it can be observed as early as 2 h after Fas ligation. The results from the cell-free system suggest that the apoptotic executioner signaling pathway(s) induced by cyto c is usually in place and comprises the activation of caspase-9, caspase-7, and caspase-3. In the cell-free system, caspase-3-like activity is essential for the nuclear apoptotic activity of extracts exerted on isolated nuclei, but additional factors other than just the level of active executioner caspases appear to influence the capability to induce DNA fragmentation.
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FOOTNOTES |
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1 Supported in part by NIH Grant CA76673. 
2 To whom requests for reprints should be
addressed, at Department of Pathology, 1117 ML, University of Iowa,
Iowa City, IA 52242-1087. Phone: (319) 335-8232; Fax: (319) 335-8916;
E-mail: michael-cohen{at}uiowa.edu
3 The abbreviations used are: cyto c, cytochrome
c; DISC, death-inducing signaling complex; mAb,
monoclonal antibody; CHX, cycloheximide; PMSF, phenylmethylsulfonyl
fluoride; AMC, aminomethylcoumarin; Ac, acetyl; fmk,
fluoromethylketone; z, benzyloxycarbonyl; VAD, Val-Ala-Asp; YVAD,
Tyr-Val-Ala-Asp; DEVD, Asp-Glu-Val-Asp; VEID, Val-Glu-Ile-Asp; CPP32,
32-kDa cystein protease.
Received 9/14/99. Accepted 2/17/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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