Comparison of Paclitaxel-, 5-Fluoro-2'-deoxyuridine-, and
Epidermal Growth Factor (EGF)-induced Apoptosis
EVIDENCE FOR EGF-INDUCED ANOIKIS*
Timothy J.
Kottke,
April L.
Blajeski
,
L. Miguel
Martins§¶,
Peter W.
Mesner Jr.,
Nancy E.
Davidson
,
William C.
Earnshaw§**,
Deborah K.
Armstrong

, and
Scott H.
Kaufmann§§
From the Division of Oncology Research, Mayo Clinic and the
Department of Pharmacology, Mayo Graduate School,
Rochester, Minnesota 55905, the § Institute of Cell & Molecular Biology, University of Edinburgh,
Edinburgh EH9 3JR, United Kingdom, and the
Johns Hopkins
Oncology Center, Baltimore, Maryland 21287
 |
ABSTRACT |
Epidermal growth factor (EGF), a hormone that
stimulates proliferation of many cell types, induces apoptosis in some
cell lines that overexpress the EGF receptor. To evaluate the mechanism of EGF-induced apoptosis, MDA-MB-468 breast cancer cells were examined
by microscopy, flow cytometry, immunoblotting, enzyme assays, and
affinity labeling after treatment with EGF, paclitaxel, or
5-fluoro-2'-deoxyuridine (5FUdR). Apoptosis induced by all three agents
was accompanied by activation of caspases-3, -6, and -7, as indicated
by disappearance of the corresponding zymogens from immunoblots,
cleavage of substrate polypeptides in situ, and detection
of active forms of these caspases in cytosol and nuclei using
fluorogenic assays and affinity labeling. Further analysis indicated
involvement of the cytochrome c/Apaf-1/caspase-9 pathway of
caspase activation, but not the Fas/Fas ligand pathway. Interestingly,
caspase activation was consistently lower after EGF treatment than
after paclitaxel or 5FUdR treatment. Additional experiments revealed
that the majority of cells detaching from the substratum after EGF (but
not paclitaxel or 5FUdR) were morphologically normal and retained the
capacity to readhere, suggesting that EGF-induced apoptosis involves
cell detachment followed by anoikis. These observations not only
indicate that EGF- and chemotherapy-induced apoptosis in this cell line
involve the same downstream pathways but also suggest that
detachment-induced apoptosis is responsible for the paradoxical
antiproliferative effects of EGF.
 |
INTRODUCTION |
EGF1 is a 6-kDa
polypeptide that binds to a 170-kDa cell surface receptor (the EGFR)
expressed on a wide variety of normal and neoplastic cells (reviewed in
Refs. 1-4). The interaction of these two molecules results in
activation of the EGFR tyrosine kinase, which in turn activates the
mitogen-activated protein kinase and Janus kinase signaling pathways
(reviewed in Refs. 1-4; see also Refs. 5 and 6). In most target cells,
these events result in proliferation (1-4). In a number of cell lines, however, EGF paradoxically inhibits proliferation (7-12). Studies of
A431 epidermoid carcinoma cells and MDA-MB-468 breast cancer cells
suggest that this inhibition of proliferation results from EGF-induced
apoptosis (5, 13, 14). Because the mechanism by which EGF induces
apoptosis is not known, the physiological significance of these
findings is unclear.
Apoptosis is a morphologically and biochemically distinct form of PCD
that occurs in many cell types after withdrawal of trophic stimuli
(15-18) or treatment with a wide variety of cytotoxic agents (19, 20).
Current models separate the apoptotic process into at least two
distinct phases, initiation and execution. The initiation phase
involves biochemical changes that might be unique to each apoptotic
stimulus (21). In contrast, the execution phase involves a series of
stereotypic morphological and biochemical changes (19) that appear to
result from the action of cysteine-dependent aspartate-directed proteases called caspases (22).
In most cell types, caspases are constitutively expressed as zymogens
that require proteolytic cleavage for activation (23, 24). Three
canonical pathways of caspase activation have been identified. First,
caspases can be activated by granzyme B, a major serine protease in
cytotoxic lymphocyte granules (reviewed in Refs. 25 and 26). Second,
ligation of cell surface death receptors, such as Fas (the cell surface
polypeptide also known as CD95 or Apo-1) or the type 1 tumor necrosis
factor
receptor, results in binding of adaptor molecules, which in
turn recruit procaspases-2, -8, and -10 to membrane-associated
signaling complexes, leading to proximity-induced activation of at
least some of these caspases (reviewed in Refs. 27-32). After
activation, these upstream caspases appear capable of directly cleaving
precursors of effector caspases (reviewed in Refs. 27-32). Finally, in
some model systems, mitochondria are induced to release cytochrome
c, which interacts with the cytosolic docking protein
Apaf-1, thereby facilitating binding and activation of procaspase-9
(reviewed in Refs. 31-35). After activation, caspase-9 is thought to
proteolytically activate caspase-3 and possibly caspase-7 (34, 36).
The mechanism by which caspases are activated after treatment with
chemotherapeutic agents has been the subject of considerable investigation. The Fas/FasL pathway (reviewed in Refs. 27-30, 32, and
37) has been implicated in the initiation of apoptosis in methotrexate-
or doxorubicin-treated CEM T cell leukemia cells (38, 39), etoposide-
or teniposide-treated Jurkat T cells (40), bleomycin-treated HepG2
hepatoma cells (41), and 5-fluorouracil-treated Gc3/cl colon cancer
cells (42), but other studies have found no evidence for Fas/FasL
interactions in drug-induced apoptosis (43-45). Likewise, release of
cytochrome c to the cytosol has been observed early in the
process of drug-induced apoptosis in some model systems (46-49) but
not others (50, 51).
Once caspase activation is initiated, the work of disassembling the
cell then falls to caspases-3, -6, and -7 and the downstream activities
that they activate (24, 31, 32, 52). These enzymes cleave a number of
cellular polypeptides, inactivating some and producing enzymatically
active forms of others (29, 32). The net result of these cleavages is
the disassembly of key structural components of the nucleus and
cytoskeleton; inhibition of the processes of DNA repair, replication,
and transcription; and activation of one or more endonucleases that
irreversibly damage the genome (reviewed in Refs. 31 and 32).
Collectively, these events contribute to the biochemical and
morphological changes that make up the apoptotic phenotype.
Previous studies from our laboratories have demonstrated that
MDA-MB-468 breast cancer cells undergo apoptosis after treatment with
5FUdR, paclitaxel, or EGF (13, 53, 54). The caspase substrates PARP and
lamin B1 were cleaved in all cases, although the cleavage
was less extensive in EGF-treated cultures. The identity of the
activated caspases and the mechanism of their activation were not
addressed in these earlier studies. More recently, Chin et
al. (5) reported that EGF treatment of MDA-MB-468 cells resulted
in enhanced expression and activation of caspase-1 without any
activation of caspase-3. In view of previous results indicating that
caspase-1 plays a limited role in apoptosis under many circumstances (55, 56), it was unclear whether the EGF-induced expression of
caspase-1 was sufficient to explain the apoptosis observed in these
cells. Accordingly, the present studies were undertaken to 1)
characterize the complement of caspases activated during apoptosis
induced by paclitaxel, 5FUdR, and EGF; and 2) compare the mechanisms
involved in caspase activation after these treatments.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Reagents were obtained from the following
suppliers: paclitaxel, 5FUdR, Hoechst 33258, and poly(HEMA) from Sigma;
EGF from Life Technologies, Inc.; ECL and SuperSignalTM ULTRA enhanced
chemiluminescent reagents from Amersham Pharmacia Biotech and Pierce,
respectively; DEVD-AFC from BioMol (Plymouth, MA); YVAD-AFC and
VEID-AFC from Enzyme Systems Products (Dublin, CA); and Z-EK(bio)D-aomk
from the Peptide Institute (Osaka, Japan). PD153035 was a kind gift from Dr. W. Karnes, Jr. (Mayo Clinic).
Monoclonal antibodies to caspase-2 and FAK (Transduction Laboratories,
Lexington, KY), cytochrome c (Pharmingen, La Jolla, CA) and
Fas (Kamiya, Tukwila, WA) as well as polyclonal anti-glutathione S-transferase
(Biotrin International, Dublin, Ireland)
were purchased from the indicated suppliers. C-2-10 murine monoclonal anti-PARP and rabbit anti-caspase-6 were kindly provided by Drs. G. Poirier (Laval University School of Medicine, Ste-Foy, Quebec, Canada)
and J. Reed (Burnham Institute, La Jolla, CA). Polyclonal sera that
recognize lamin A, lamin B1, the nucleolar protein B23, and
the large subunits of caspase-3 and caspase-7 were generated as
described previously (49, 57).
Cell Culture--
MDA-MB-468 cells (American Type Culture
Collection, Manassas, VA) were cultured in improved minimal essential
medium (Biofluids, Rockville, MD) supplemented with 5%
heat-inactivated fetal bovine serum, 100 units/ml penicillin G, 100 µg/ml streptomycin, and 2 mM glutamine (medium A) in a
humidified atmosphere containing 5% (v/v) CO2. When cells
were 40-60% confluent, medium A was replaced by medium A containing
100 µM 5FUdR, 100 ng/ml EGF, or diluent; and incubation
was continued for 48 or 72 h, respectively. Alternatively, after
medium A containing 100 nM paclitaxel was added, cells were incubated for 24 h, washed, and incubated in drug-free medium A
for 24 h. These treatments were previously shown to induce
apoptosis in this cell line (13, 53, 54). At the completion of the treatment, nonadherent cells were removed with the tissue culture medium, and adherent cells were released by trypsinization. Cells were
sedimented at 200 × g for 10 min and processed for the
various biochemical assays described below. Alternatively, in some
experiments, nonadherent cells were sedimented at 100 × g, resuspended in fresh medium A, and seeded on new tissue
culture plates. After an additional 72 h, the nonadherent and
adherent fractions were collected, counted on a hemacytometer in the
presence of 0.2% trypan blue, and fixed for morphological examination
as described below.
To assess the effect of growth under conditions in which cells could
not adhere, semiconfluent MDA-MB-468 cells were released by
trypinization and suspended in drug-free medium A at a concentration of
3-5 × 105 cells/ml. Aliquots (1 ml) in medium A were
incubated for up to 72 h in 17 × 100-mm test tubes. Under
these conditions, >95% of HL-60 human leukemia cells survive (58). In
other experiments, tissue culture plates were treated with poly(HEMA)
exactly as described by Folkman and Moscona (59). After the diluent was evaporated, cells were plated, allowed to adhere for 14-16 h, and
treated with 100 ng/ml EGF for 72 h. Adherent and nonadherent cells were then separately harvested.
Jurkat cells (kindly provided by Drs. C. M. Eischen and P. Leibson, Mayo Clinic) were grown in RPMI 1640 medium containing 5%
heat-inactivated fetal bovine serum, 100 units/ml penicillin G, 100 µg/ml streptomycin, and 2 mM glutamine at concentrations below 1 × 106/ml. To examine the effects of the ZB4
blocking antibody, Jurkat cells were treated for 24 h with 20 ng/ml CH-11 in the absence or presence of 1 µg/ml ZB4.
Immunoblotting--
Sedimented cells were washed once with
ice-cold RPMI medium containing 10 mM HEPES (pH 7.4 at
4 °C) and lysed by vigorous vortexing in 6 M guanidine
hydrochloride containing 250 mM Tris-HCl (pH 8.5 at
4 °C), 10 mM EDTA, 150 mM
-mercaptoethanol, and 1 mM phenylmethylsulfonyl
fluoride. After sonication, samples were treated with iodoacetamide to
block free sulfhydryl groups and then dialyzed sequentially into 4 M urea and 0.1% (w/v) SDS as described previously (57).
After an aliquot was removed for determination of protein (60), the
sample was lyophilized to dryness; resuspended at a concentration of 5 mg protein/ml in SDS sample buffer consisting of 4 M
deionized urea, 2% (w/v) SDS, 62.5 mM Tris-HCl (pH 6.8 at
21 °C), and 1 mM EDTA; and heated to 65 °C for 20 min. Aliquots containing 50 µg of total cellular protein were
subjected to SDS-PAGE on gels with 5-15% (w/v) acrylamide gradients,
transferred to nitrocellulose or polyvinylidene difluoride, and probed
with antibodies as described (24).
Cell Fractionation, Caspase Assays, and Affinity
Labeling--
Cytosol and nuclei were prepared at 4 °C as recently
reported (24) except that the previously described sedimentation of nuclei through 2.1 M sucrose was omitted because
tenaciously adherent cytoskeletal components in MDA-MB-468 cells
precluded purification by this technique. Instead, cells homogenized in
Buffer A (25 mM HEPES (pH 7.5 at 4 °C), 5 mM
MgCl2, 1 mM EGTA supplemented immediately
before use with 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A, and 10 µg/ml leupeptin) were sedimented at
800 × g. The supernatant was removed and sedimented at
280,000 × gmax to prepare cytosol (24).
After the crude nuclei in the 800 × g pellet were
resuspended in Buffer A and resedimented at 800 × g,
they were stored in small aliquots at
70 °C in Buffer A containing
5 mM EDTA and 2 mM dithiothreitol.
Cleavage of DEVD-AFC, VEID-AFC, and YVAD-AFC by caspases present in
cytosol and nuclei was assayed as described previously (24). All
reactions were run as end point assays using 100 µM substrate concentrations, 2-h reaction times at 37 °C, and 50-µg aliquots of cytosolic or nuclear protein. Standards containing 0-1500
pmol of AFC were utilized to determine the amount of fluorochrome released. Control experiments indicated that the assays were linear with respect to incubation time and enzyme content under these conditions. Cytosol from etoposide-treated HL-60 leukemia cells served
as a positive control for DEVD-AFC and VEID-AFC cleavage, and cytosol
from THP.1 monocytic leukemia cells was a positive control for YVAD-AFC
cleavage (24).
For affinity labeling of active caspases, aliquots containing the
indicated amounts of nuclear or cytosolic protein were incubated for
1 h at room temperature with 1 µM Z-EK(bio)D-aomk
(24), diluted with 1/2 volume of 3× concentrated SDS sample
buffer, heated to 95 °C for 3 min, subjected to one-dimensional
SDS-PAGE on 16% (w/v) acrylamide gels, transferred to nitrocellulose,
probed with peroxidase-labeled streptavidin, and visualized using ECL
reagents. Two-dimensional analysis was performed using isoelectric
focusing for the first dimension and SDS-PAGE for the second dimension as described (24), except that precast pH 4-7 Immobiline gels (Pharmacia, Uppsala, Sweden) were utilized for isoelectric focusing. Labeled polypeptides were visualized using peroxidase-coupled streptavidin followed by SuperSignalTM ULTRA chemiluminescent
substrate. As a control, recombinant caspases expressed in Sf9
cells (24) were subjected to the same analysis.
Fluorescence Microscopy and Quantitation of Apoptotic
Cells--
Adherent and nonadherent cells were collected separately,
sedimented at 200 × g for 10 min, washed with ice-cold
calcium- and magnesium-free phosphate-buffered saline, fixed in 3:1
(v/v) methanol:acetic acid, stained with 1 µg/ml Hoechst 33258, and examined by fluorescence microscopy as described previously (13, 58). A
minimum of 300 cells/sample were scored for apoptotic changes
(fragmentation of the nucleus into multiple discrete fragments). Samples were photographed using Eastman Kodak Co. Elite II ASA 400 film
and a 1/4 s exposure time.
Flow Cytometry--
Adherent and nonadherent cells were
collected separately, sedimented at 200 × g for 10 min, washed with ice-cold phosphate-buffered saline, fixed in 50%
ethanol, treated with 1 mg/ml RNase A, stained with 100 µg/ml
propidium iodide, and subjected to flow cytometry as described
previously (61).
Statistical Analysis--
Experiments were replicated as
indicated in the figure legends. The statistical significance of
differences between treated and untreated cells was assessed using
two-sided t tests and a post hoc Bonferroni correction to
take into account the effect of multiple comparisons (62).
 |
RESULTS |
EGF-induced Apoptosis Is Mediated by the EGFR Tyrosine
Kinase--
Although EGF stimulates the proliferation of most cells,
it paradoxically inhibits the growth of a number of cell lines (5, 7-14). To begin to elucidate the mechanism of this latter effect, MDA-MB-468 cells were treated with 100 ng/ml EGF, a concentration previously shown to result in apoptosis in this cell line (13). To
provide a basis for comparison, MDA-MB-468 cells were also treated with
the chemotherapeutic agents paclitaxel (100 nM) or 5FUdR
(100 µM) under conditions previously shown to induce PCD in these cells (53, 54). Initial examination revealed that treatment
with paclitaxel for 24 h resulted in accumulation of cells in
mitosis. The paclitaxel-treated cells had a tetraploid DNA content
(Fig. 1B) and contained
condensed chromosomes when examined after staining with Hoechst 33258 (Fig. 1B, inset). In contrast, 5FUdR caused a marked
decrease of cells in G2, with a concomitant accumulation of
cells in G1 and early S phase (Fig. 1C).
Finally, treatment with EGF for 24 h caused a decrease in the S
phase population (cf. Fig. 1, A and D)
with accumulation of cells in G1 and G2 phases
of the cell cycle. Further EGF treatment, however, was associated with
recovery of the S phase population (Fig. 1D, inset) followed
by detachment of a portion of the cells from the tissue culture plate
(Fig. 1E) (13).

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Fig. 1.
Effect of paclitaxel (Taxol), 5FUdR, and EGF
on cell cycle distribution and adhesion. A-D,
MDA-MB-468 cells were treated with diluent (A), 100 nM paclitaxel for 24 h (B), 100 µM 5FUdR for 48 h (C), or 100 ng/ml EGF
for 24 h (D). At the end of the incubation, adherent
cells were harvested and subjected to flow cytometry after staining
with propidium iodide. B, inset, cells treated with 100 nM paclitaxel for 24 h were fixed, stained with
Hoechst 33258, and photographed as described under "Experimental
Procedures." Arrowhead, nonmitotic cell. D,
inset, percentage of cells in S phase at various times after
addition of 100 ng/ml EGF to log phase MDA-MB-468 cells. E,
effect of PD153035 on EGF-induced detachment of MDA-MB-468 cells. Cells
were treated with 5 µM PD153035 or diluent beginning 30 min prior to addition of EGF for 72 h. Results are representative
of three (A-D) or four (E) experiments.
Error bars, ±1 S.D. *, p < 0.002 when
compared with control. All other points were not significantly
different from control.
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To confirm that the detachment process initiated by EGF was mediated by
the EGFR, cells were treated with EGF in the presence or absence of
PD153035 at a concentration (5 µM) that has been observed
to inhibit the EGFR tyrosine kinase (63, 64) without itself inducing
apoptosis (64). PD153035 inhibited the EGF-induced detachment of
MDA-MB-468 cells (Fig. 1E), suggesting that signaling through the EGFR is required for EGF-induced apoptosis. In contrast, PD153035 did not have any effect on paclitaxel- or 5FUdR-induced detachment (data not shown).
Comparison of Polypeptide Cleavages after Treatment with EGF,
Paclitaxel, or 5FUdR--
In further experiments, nonadherent and
adherent cells were separated and examined for evidence of caspase
activation. Nonadherent cells harvested after treatment with paclitaxel
or 5FUdR (Fig. 2, lanes 4 and
6, respectively) contained diminished levels of full-length
PARP and the lamins. Fragments of PARP, lamin A, and lamin
B1 were detected (Fig. 2, arrowheads). The same
polypeptides were also cleaved in nonadherent cells resulting from EGF
treatment (Fig. 2, lane 8), although cleavage was less
complete than in paclitaxel- or 5FUdR-treated cells. In all cases, the
corresponding polypeptides remained intact in cells that remained
adherent (Fig. 2, lanes 3, 5, and 7), suggesting
that proteolysis was occurring concomitant with or subsequent to
detachment of cells from the substratum. FAK, another putative caspase
substrate (32), was also diminished in the nonadherent cells, although
discrete fragments of FAK were not detectable using this antibody (Fig.
2).

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Fig. 2.
Effect of paclitaxel, 5FUdR, and EGF on
selected caspase substrates. MDA-MB-468 cells that became
nonadherent (NA) or remained adherent (A) after
treatment with 100 nM paclitaxel (48 h), 100 µM 5FUdR (48 h), or 100 ng/ml EGF (72 h) were harvested
and subjected to immunoblotting with reagents that recognized the
caspase substrates PARP, lamins A and C, lamin B1, and FAK
(32). The nucleolar protein B23, which is not cleaved during apoptosis
(65), served as a loading control. Lanes 1 and 2 contained adherent cells treated with diluent for 48 and 72 h,
respectively. All lanes were loaded with 50 µg of protein. All blots
in this figure and Fig. 3 are from the same experiment.
Arrowheads, previously described proteolytic fragments of
PARP, lamin A, and lamin B1 (66-68). The lower intensity
of the signal for fragments of lamins A and B1 as compared
with the intact polypeptides (cf. lanes 5 and 6)
suggests that multiple epitopes recognized by the polyclonal sera have
been lost from these carboxyl-terminal fragments. Results are
representative of three to six experiments with each treatment.
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The cleavage of PARP, lamin A, and lamin B1 to their
signature fragments (65-68) suggested that caspases had been activated by the three treatments. Based on the observation that PARP is cleaved
efficiently by caspases-3 and -7 (69), whereas lamin A is cleaved
preferentially by caspase-6 (67), we initially focused on these three
caspases. Immunoblotting of the same cell lysates revealed that the
precursors for caspases-3, -6, and -7 were extensively or
quantitatively decreased in nonadherent cells after treatment with
paclitaxel or 5FUdR (Fig. 3, A-C,
lanes 4 and 6). In the case of caspases-6 and -7, fragments corresponding to the large subunits of the active enzymes
were detectable in some apoptotic cell fractions (Fig. 3, B
and C, arrow). Cleavage of the procaspases was less complete
in the nonadherent cells resulting from EGF treatment but was
nonetheless evident (Fig. 3, A-C, lane 8). Two closely
spaced isoforms of procaspase-1 were also detectable in MDA-MB-468
cells (Fig. 3, D, lanes 1 and 2). In cells that
remained adherent after treatment with paclitaxel, 5FUdR, or EGF,
levels of these isoforms remained constant (Fig. 3D, lanes 5 and 7) or decreased slightly (Fig. 3D, lane 3).
Although these procaspase-1 species decreased markedly in nonadherent
cells (Fig. 3D, lanes 4, 6, and 8), bands
corresponding to the large subunit of active caspase-1 were not
detectable even upon prolonged exposure of the blot. Procaspase-2,
which did not appear to be cleaved after any of the treatments (Fig.
3E, lanes 4, 6, and 8), served as a loading
control.

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Fig. 3.
Effect of paclitaxel, 5FUdR, and EGF on
caspases. A-E, blots shown in Fig. 2 were reprobed
with antibodies to procaspases. MDA-MB-468 cells that became
nonadherent (NA) or remained adherent (A) after
treatment with paclitaxel, 5FUdR, or EGF were subjected to
immunoblotting with reagents that recognized procaspase-3 (panel
A), procaspase-7 (panel B), procaspase-6 (panel
C), or procaspase-1 (panel D). All lanes were loaded
with 50 µg of protein. To confirm equal loading, blots were reprobed
with antibodies to procaspase-2 (panel E), a polypeptide
that does not appear to be activated in chemotherapy-induced PCD in
other model systems (24, 44, 96). Arrowheads, active large
subunits resulting from proteolytic cleavages of procaspase-7 and
procaspase-6, respectively. Results are representative of three
separate experiments.
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Comparison of Active Caspases after Treatment with EGF, Paclitaxel,
or 5FUdR--
To determine whether the disappearance of procaspases
reflected the generation of enzymatically active proteases, cleavage of
fluorogenic substrates that correspond to preferred cleavage sites of
caspase-3 (DEVD-AFC) and caspase-6 (VEID-AFC) was examined (Fig.
4, A and B).
Activities that cleaved both of these substrates were markedly
increased in cytosol of cells that became nonadherent after treatment
with paclitaxel or 5FUdR (Fig. 4, A and B, solid bars). Smaller, but statistically significant, increases in
activity were also observed in cells that remained adherent after these two treatments, suggesting that caspase activation precedes detachment after exposure to these drugs. Increased DEVD-AFC and VEID-AFC cleavage
activities were also detected after EGF treatment. Two differences,
however, were noted. First, the activities observed in cytosol of
nonadherent cells after EGF treatment were consistently ~2-fold lower
than activities observed after drug treatment (Fig. 4, A and
B, solid bars). Second, the activities were not
significantly increased in cells that remained adherent after EGF
treatment.

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Fig. 4.
Paclitaxel-, 5FUdR-, and EGF-induced changes
in activities that cleave tetrapeptide derivatives. Aliquots of
cytosol (solid bars) and nuclei (hatched bars)
from adherent (A) and nonadherent (NA) MDA-MB-468
cells were incubated with DEVD-AFC, a substrate preferred by caspases-3
and -7 (panel A); VEID-AFC, a substrate preferred by
caspase-6 (panel B); or YVAD-AFC, a substrate preferred by
caspase-1 (panel C). The amount of fluorescent product
released was quantitated as described under "Experimental
Procedures." Error bars, ±1 S.D. in five (A),
four (B), or three (C) independent experiments.
*, p 0.005 (A), p 0.02 (B), or p = 0.05 (C) compared
with control cytosol or nuclei, respectively. All other points were not
significantly different from their controls.
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Because of the suggestion that caspase-1 might play a role in
triggering EGF-induced apoptosis, activity that cleaved YVAD-AFC, a
fluorogenic substrate of caspase-1, was also measured after paclitaxel,
5FUdR, or EGF treatment. This activity increased less than 2-fold after
treatment (Fig. 4C) and was very low compared with the other
activities (compare y axes in Figs. 4, A-C),
suggesting that caspase-1 activation is not a major feature of
apoptosis in this cell line.
Recent results (24, 49) indicate that active caspases can also be
detected in nuclei of apoptotic leukemia cells. Consistent with these
results, activities that cleave DEVD-AFC and VEID-AFC (but not
YVAD-AFC) were easily detected in nuclei from nonadherent MDA-MB-468
cells harvested after treatment with paclitaxel, 5FUdR or EGF (Fig. 4,
A and B, hatched bars). These activities were also elevated in nuclei from adherent cells after paclitaxel or 5FUdR.
To provide more complete identification of the caspases that are
activated in MDA-MB-468 cells, cytosol and nuclei were reacted with the
affinity label Z-EK(bio)D-aomk (24, 49), subjected to one-
and two-dimensional gel electrophoresis, and reacted with peroxidase-coupled streptavidin. Results of the one-dimensional analysis (Fig. 5A) revealed
that multiple active caspase species were detectable in cytosol and
nuclei after all three treatments. Interestingly, none of these species
migrated with the large subunit of active caspase-1 (Fig. 5A,
cf. lane 0 with lanes 4-6 and
9-11). Although samples from EGF-treated cells usually
contained less signal than samples from paclitaxel- or 5FUdR-treated
cells, qualitative differences among the treatments were not
discernible at this level of resolution.

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Fig. 5.
Affinity labeling of active caspases after
treatment of MDA-MB-468 cells with paclitaxel, 5FUdR, or EGF.
A, nonadherent and adherent cells were collected after
treatment with 100 nM paclitaxel for 24 h followed by
a 24-h incubation in drug-free medium, 100 µM 5FUdR for
48 h, or 100 ng/ml EGF for 72 h. In each case, cytosol and
crude nuclei prepared as described under "Experimental Procedures"
were incubated with 1 µM zEK(bio)D-aomk, subjected to
one-dimensional SDS-PAGE followed by blotting with peroxidase-coupled
streptavidin. Mobilities of the large subunits of recombinant caspases
(determined as described previously in Ref. 24) are indicated in
lane 0 for comparison. B, after affinity labeling
as described for A, cytosol and nuclei were subjected to
isoelectric focusing followed by SDS-PAGE as indicated. Numbered
arrowheads indicate species present in cytosol from 5FUdR-treated
cells but not other cells. The circled species in
paclitaxel- and EGF-treated nuclei appears to be diminished in
5FUdR-treated nuclei but enhanced in 5FUdR-treated cytosol.
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In further experiments, the active caspase species present in nuclei
and cytosol were compared by two-dimensional gel electrophoresis (Fig.
5B). Previous results in leukemia cells have demonstrated that the major caspase species detected by this technique are active
forms of caspases-3 and -6 (24, 52). Two major and several minor
caspase species were detected in cytosol of paclitaxel- and EGF-treated
cells by this method. Interestingly, several additional species (Fig.
5B, numbered arrowheads) were detected in the cytosol from
5FUdR-treated cells. With one exception (Fig. 5B, arrowhead 1, 5FUdR), all of these species were detectable in nuclei as well. In general, nuclei contained higher amounts of many of the more weakly
labeled species, raising the possibility that certain species have been
specifically targeted or activated in this organelle. In addition, it
appeared that one caspase species (Fig. 5B, indicated by
arrowhead 2 in cytosol and circles in nuclei) was
preferentially present in cytosol of 5FUdR-treated cells and nuclei of
paclitaxel and EGF-treated cells, raising the possibility that nuclear
accumulation of some caspase species might vary with the apoptotic
stimulus. Despite these differences, the overall impression was that
caspase species activated by the three treatments were generally similar.
Evaluation of the Potential Role of Fas/FasL Interactions in
MDA-MB-468 Cell Death--
Further experiments were performed to
evaluate possible pathways that might participate in the activation of
these caspases. Recent studies have implicated the Fas/FasL pathway in
some models of chemotherapy-induced PCD (see the Introduction). To
evaluate the potential role of this pathway in MDA-MB-468 cells,
cultures were treated with CH-11, a cross-linking anti-Fas antibody
that induces apoptosis in other cells (Ref. 70 and Fig.
6D). This treatment failed to
induce PCD in MDA-MB-468 cells (Fig. 6, B and E),
suggesting that these cells are resistant to cytotoxic effects of Fas
ligation. To further evaluate the potential role of the Fas/FasL system
in MDA-MB-468 PCD, the effects of the blocking anti-Fas antibody ZB4
(44, 71) were evaluated. Although ZB4 blocked induction of apoptosis in
thymidine-deprived human colon cancer cells (42) and in Jurkat cells
subjected to T cell receptor cross-linking (44) or Fas ligation (Fig.
6F), it did not alter the number of MDA-MB-468 cells that
detached from the plates (Fig. 6E) or developed apoptotic
morphological changes (Fig. 6F) after treatment with 5FUdR,
paclitaxel, or EGF.

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Fig. 6.
Evaluation of the potential role of Fas/FasL
interactions in MDA-MB-468 cell PCD. A-D, morphology
of MDA-MB-468 cells (A and B) or Jurkat cells
(C and D) before (A and C)
or after (B and D) treatment for 16 h with
1000 ng/ml (A and B) or 20 ng/ml (C
and D) CH-11 agonistic anti-Fas antibody. Results identical
to those in B were also observed after treatment of
MDA-MB-468 cells with 1000 ng/ml CH-11 in the presence of 100 µM cycloheximide for 24 h. E, bar graph
showing percentage of MDA-MB-468 cells that detach from the substratum
after treatment with 100 nM paclitaxel, 100 µM 5FUdR, or 100 ng/ml EGF in the absence or presence of
1 µg/ml ZB4 blocking antibody. F, bar graph showing effect
of the blocking anti-Fas antibody ZB4 (1 µg/ml) on the percentage of
nonadherent MDA-MB-468 cells that display apoptotic morphology after
treatment with 100 nM paclitaxel, 100 µM
5FUdR, or 100 ng/ml EGF. Shown for comparison is the effect of 1 µg/ml ZB4 antibody on Jurkat cells treated with 20 ng/ml CH-11
anti-Fas antibody. Error bars, ±1 S.D. in four separate
experiments. *, p 0.02 relative to untreated cells
(E) or to cells subjected to equivalent treatment except for
omission of cytotoxic agent (F).
|
|
Release of Cytochrome c to Cytosol in Drug-treated MDA-MB-468
Cells--
Because the preceding experiments failed to provide
evidence that the Fas/FasL pathway was involved in initiation of
EGF-induced apoptosis, we examined the possibility that activation of
downstream caspases might involve the release of cytochrome
c from mitochondria to cytosol (see the Introduction).
Cytochrome c was readily detectable in cytosol from
nonadherent MDA-MB-468 cells after treatment with paclitaxel, 5FUdR, or
EGF (Fig. 7). Thus, paclitaxel, 5FUdR,
and EGF all belong on the growing list of agents that are capable of
inducing cytochrome c release from mitochondria.

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Fig. 7.
Detection of cytochrome c in
cytosol of nonadherent MDA-MB-468 cells after treatment with
paclitaxel, 5FUdR, or EGF. After treatment, cytosol was prepared
from adherent (A) and nonadherent (NA) cells as
described under "Experimental Procedures." Samples containing 50 µg of protein were subjected to SDS-PAGE followed by blotting with
anti-cytochrome c antibody. Results are representative of
four independent experiments. To confirm equal loading, blots were
reprobed antiserum that recognizes the cytosolic enzyme glutathione
transferase .
|
|
Effects of Paclitaxel 5FUdR and EGF on Bcl-2 Family
Members--
Recent studies have suggested that the release of
cytochrome c from mitochondria can be induced by Bax, a
pro-apoptotic member of the Bcl-2 family (72, 73). Previous studies
also demonstrated that paclitaxel treatment is associated with the
appearance of a slow-migrating hyperphosphorylated species of Bcl-2
(74) that has a lower affinity for Bax (75). One model suggests that
the resulting increase in free Bax mediates the induction of apoptosis by paclitaxel, although this concept remains controversial (76, 77). An
alternative mechanism of triggering apoptosis involves induction of bax
expression, e.g. after antibody ligation of the EGFR or
inhibition of EGFR kinase activity in colon cancer cells (78). To
assess the potential role of these types of alterations, MDA-MB-468
cells treated with paclitaxel, 5FUdR, or EGF were harvested at various
times and reacted with anti-Bcl-2 antiserum. Results of these studies
(Fig. 8A) readily confirmed
the existence of a paclitaxel-induced alteration in Bcl-2 migration but
failed to provide any evidence that 5FUdR or EGF induced a similar
change. Additional experiments failed to provide any evidence that EGF caused alterations in levels of the antiapoptotic Bcl-2 family members
Bcl-xL and Mcl-1 or the proapoptotic homologs Bad, Bax, or
Bak (Fig. 8B). Accordingly, it does not appear that
EGF-induced alterations in the expression of these polypeptides
contribute to activation of the apoptotic process.

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Fig. 8.
Effect of EGF treatment on Bcl-2 family
members. A, MDA-MB-468 cells were incubated with 100 nM paclitaxel (top), 100 µM 5FUdR
(middle), or 100 ng/ml EGF (bottom) for the
indicated period of time. Adherent cells were then harvested and
subjected to SDS-PAGE followed by immunoblotting with anti-Bcl-2
antiserum. In addition to the usual species (a), a slower
migrating species (b) appeared in paclitaxel-treated cells
as described previously (74, 75). In contrast, this species was not
observed after 5FUdR or EGF treatment. B, after MDA-MB-468
cells were incubated with 100 ng/ml EGF for the indicated period of
time, adherent cells were harvested, subjected to SDS-PAGE, and blotted
with antisera that recognize the indicated Bcl-2 family member. To
confirm equal loading, blots were reprobed with antiserum that
recognizes the nucleolar protein B23 (not shown). Blots were derived
from two experiments and were representative of three to five
experiments examining each polypeptide.
|
|
EGF Treatment Induces Anoikis--
In a final series of
experiments, we attempted to further evaluate the observation that
EGF-treated cells displayed lower levels of active caspases (Fig. 4),
less cleavage of caspase precursors (Fig. 3) and less degradation of
caspase substrates (Fig. 2). Morphological examination revealed that
80-90% of the cells detaching from the tissue culture plates after
treatment with paclitaxel or 5FUdR had fragmented nuclei (Fig.
9, A and B). Even
at paclitaxel concentrations as low as 5 nM, which caused
<10% of the cells to detach from the plate, ~80% of the
nonadherent cells were frankly apoptotic (data not shown). In contrast,
at all time points up to 96 h after addition of EGF, 50-70% of
the nonadherent MDA-MB-468 cells appeared morphologically normal (Figs.
6F and 9C), although the number of floating cells
progressively increased. When cell viability was analyzed by trypan
blue exclusion, a similar distinction between drug- and EGF-induced
apoptosis was observed. Forty-eight hours after initiation of
paclitaxel or 5FUdR treatment, 47 ± 8% (mean ± S.D.;
n = 3) and 41 ± 9% of the nonadherent cells were nonviable, respectively. In contrast, only 23 ± 4% of the
nonadherent cells stained with trypan blue 48 h after addition of
EGF. Upon further incubation, however, the EGF-treated cells also died, as evidenced by the fact that 46 ± 10% and 62 ± 1% of the
nonadherent cells took up trypan blue at 96 and 144 h,
respectively. Coupled with the results of the caspase assays in Fig. 4,
these observations raised the possibility that EGF might initially
cause detachment of viable cells, which then become apoptotic due to
the lack of interactions with the substratum. This model suggested
three testable predictions: 1) MDA-MB-468 cells would become apoptotic
if adhesion to a substratum were prevented by other means; 2) EGF might
have a larger effect if the strength of the adhesion between MDA-MB-468 cells and the substratum were diminished; and 3) EGF-treated cells might survive if they could reattach. Experiments were performed to
test each of these predictions.

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Fig. 9.
Many of the cells that become nonadherent
during EGF treatment are morphologically normal and retain the ability
to reattach to tissue culture plates. A-C, morphology
of nonadherent MDA-MB-468 cells after treatment with 100 nM
paclitaxel, 100 µM 5FUdR, or 100 ng/ml EGF. D,
MDA-MB-468 cells require interactions with a substratum to survive.
Cells that were 80% confluent were trypsinized and seeded at low
density or transferred to polystyrene test tubes and cultured a density
of 5 × 105/ml. At the indicated times, cells in the
tissue culture plates (adherent + nonadherent) and tubes were harvested
and stained with Hoechst 33258. E, MDA-MB-468 cells cultured
on poly(HEMA)-coated tissue culture plates are more sensitive to EGF.
Tissue culture plates were treated with the indicated concentration of
poly(HEMA). After evaporation of the diluent, cells were plated on
poly(HEMA) and allowed to adhere overnight. After a subsequent 72 h in the absence (open bars) or presence (closed
bars) of 100 ng/ml EGF, the adherent and nonadherent cell
populations were manually counted. F, cells that detach in
the presence of EGF can readhere. Nonadherent cells obtained after
scraping untreated cells from a plate (control) or harvested after
treatment with paclitaxel, 5FUdR, or EGF (see panels A-C)
were sedimented at 100 × g, resuspended in drug-free
medium, and incubated on fresh tissue culture plates for 72 h.
Trypan blue-excluding attached cells were then counted and compared
with the total number of cells present. Error bars, ±1 S.D.
from seven (D) or three (E and F)
independent experiments. * in D, p 0.005 compared with the same time point in plate. ** in F,
p 0.04 compared with paclitaxel-treated or
5FUdR-treated cells. Statistical analyses of data in E are
described in Footnote 2.
|
|
First, semiconfluent MDA-MB-468 cells were trypsinized and seeded in
fresh tissue culture plates or in test tubes to which they could not
adhere. Over the course of 3 days, a substantial fraction of the cells
cultured in test tubes became apoptotic (Fig. 9D),
establishing that this cell line requires contact with its substratum
for survival.
Next, MDA-MB-468 cells were cultured on plates coated with various
concentrations of poly(HEMA), a hydrophobic polymer that diminishes
cell-substrate adhesion (59). As the amount of poly(HEMA) coating was
increased, EGF treatment caused more cell detachment (Fig.
9E),2 establishing
that cell-substratum interactions play an important role in the effect
of EGF on these cells. Morphological examination of cells that detached
from the poly(HEMA)-treated plates revealed that the percentage of
apoptotic cells was the same in the absence and presence of EGF,
suggesting that it was detachment rather than EGF treatment per
se that was toxic.
Finally, MDA-MB-468 cells that detached after treatment with
paclitaxel, 5FUdR, or EGF were sedimented, resuspended in drug-free medium, seeded on fresh tissue culture plates, and subsequently examined for viability (Fig. 9F). Fewer than 5% of the
paclitaxel- or 5FUdR-treated cells attached and remained viable. In
contrast, ~40% of the EGF-treated cells reattached and excluded
trypan blue 72 h after EGF removal.
 |
DISCUSSION |
In the present study, we have compared the apoptotic pathways
activated by paclitaxel, 5FUdR, or EGF in MDA-MB-468 breast cancer
cells. Our results indicate that the vast majority of cells detaching
from the tissue culture plates after treatment with paclitaxel or 5FUdR
are frankly apoptotic, whereas 50-70% of the cells becoming
nonadherent after EGF treatment are morphologically normal (Fig. 9,
A-D). Further experiments have demonstrated that the cells
becoming nonadherent after EGF treatment can reattach and survive (Fig.
9F). In contrast, cells that detach after treatment with
paclitaxel or 5FUdR cannot. Despite these differences, all of the
treatments appear to activate the cytochrome c/Apaf-1
caspase-3 pathway, as indicated by the release of cytochrome
c to the cytosol (Fig. 7) and the appearance of active
caspases-3, -6, and -7 (Figs. 2-5). These observations have
potentially important implications for the understanding of drug- and
hormone-induced PCD in human breast cancer cells.
Recent studies (reviewed in Refs. 79-82) have demonstrated that
cell-substratum interactions can provide survival signals for many cell
types. Conversely, removal of certain cell lines from their substrata
can result in apoptosis. These recent descriptions of the process
termed "anoikis" lead us to propose the following sequence of
events following EGF treatment: 1) EGF-treated cells adhere less
tightly to the tissue culture plates; 2) a fraction of cells detach
from the substratum (Figs. 1E, 6E, and
9C) and lose integrin-mediated signaling; 3) those cells
that do not reattach in a timely fashion initiate the process of
anoikis, which includes release of cytochrome c to the
cytosol (Fig. 7) and subsequent activation of downstream caspases
(Figs. 2-5).
A number of observations support this model. First, MDA-MB-468 cells
clearly undergo apoptosis when deliberately deprived of the opportunity
to attach to a substratum (Fig. 9D). Second, treatment of
the tissue culture plates with poly(HEMA), which decreases the adhesion
of the MDA-MB-468 cells,3
enhances the effect of EGF (Fig. 9E). Third, MDA-MB-468
cells that detach during EGF treatment can be rescued by replating in EGF-free medium (Fig. 9F). Fourth, EGF-treated cells show
little increase in caspase activity until they have detached, whereas paclitaxel- or 5FUdR-treated cells contain elevated levels of DEVD-AFC
and VEID-AFC cleavage activity before detachment (Fig. 4). Finally,
when MDA-MB-468 cells are cultured in suspension as illustrated in Fig.
9D, EGF fails to exert any pro- or anti-apoptotic effect
that can be distinguished from the effect of detachment alone.3 Collectively, all of these observations support the
view that EGF-induced apoptosis results from the loss of
cell-substratum attachment. Although this process of anoikis has been
implicated in a variety of physiological processes (79, 80, 82), the present report appears to be the first implicating anoikis in the
antiproliferative effects of any hormone. Further studies are required
to determine whether detachment-induced apoptosis plays a similar role
in the antiproliferative effects of other hormones.
The present studies do not address the question of how EGF treatment
causes detachment. EGF is known to enhance motility of various
epithelial cells (83-86), providing one potential explanation for the
decreased adherence. A change in the affinity of integrins for their
ligands (87), either in association with altered motility or
independent of this process, might also contribute to the decreased adhesion. Further studies are required to examine these possibilities.
While the present studies were in progress, Chin et al. (5)
reported that EGF treatment of serum-deprived MDA-MB-468 cells resulted
in enhanced procaspase-1 expression. Based in part on these results,
these investigators proposed that EGF-induced apoptosis was triggered
by procaspase-1 autoactivation. Our results are not compatible with
this model. First, when MDA-MB-468 cells were cultured in
serum-containing medium, immunoblotting failed to provide evidence that
EGF treatment enhanced procaspase-1 polypeptide levels (Fig. 3D,
lanes 1, 2, and 7) even though this treatment induced
apoptosis (Figs. 6F and 9C). Second, assays for
cleavage of the caspase-1 substrate YVAD-AFC failed to demonstrate an
EGF-induced increase in the low level basal activity of this enzyme
(Fig. 4C). Finally, an affinity labeling technique with
nanogram sensitivity (24) failed to provide evidence for active
caspase-1 in cytosol or nuclei of EGF-treated cells (Fig.
5A). Although these observations argue against a role for
caspase-1 in EGF-induced apoptosis of the MDA-MB-468 cells, we
nonetheless observed decreased levels of procaspase-1 concomitant with
PCD in this cell line (Fig. 3D). Several potential
explanations could account for these observations, including the
possibility that caspase-1 is exported out of the MDA-MB-468 cells upon
activation (88) or the possibility that procaspase-1 has been
proteolytically cleaved at one or more sites that do not result in its
catalytic activation.
In contrast to procaspase-1, cleavage of procaspases-3, -6, and -7 (Fig. 3, A-C) is clearly accompanied by their activation, as evidenced by the generation of protease species of appropriate molecular weight that react with the affinity label zEK(bio)D-aomk (Fig. 5), appearance of activities that cleave DEVD-AFC and VEID-AFC (Fig. 4, A and B), and degradation of caspase
substrates to fragments that are known to reflect activity of
caspases-3, -6, and/or -7 in situ (Fig. 2). In short, four
separate pieces of evidence (Figs. 2-5) support the view that EGF
treatment is accompanied by activation of these downstream caspases.
The cohort of effector caspases activated during EGF treatment is very
similar to the cohort activated by paclitaxel or 5FUdR (Fig. 5),
although the specific activities of these caspases in vitro
(Fig. 4, A and B) and in situ (Fig. 2)
are lower, presumably reflecting the lower percentage of cells that are
undergoing apoptosis after EGF treatment (Figs. 6F and 9C). These observations not only provide additional
support for previous claims that different stimuli activate the same
downstream effectors of PCD (44, 52, 89, 90) but also provide the first
characterization of the caspases that are activated as cells undergo anoikis.
Additional experiments were designed to examine, in a preliminary
fashion, the potential role of various pathways in the activation of
these downstream caspases. Although a number of studies have provided
evidence that the Fas/FasL pathway can participate in the induction of
apoptosis by chemotherapeutic agents in some Fas-expressing cells (see
the Introduction), this pathway does not appear to contribute to PCD in
MDA-MB-468 cells. Treatment with a blocking anti-Fas antibody failed to
attenuate the effects of 5FUdR, paclitaxel, and EGF in this cell line
(Fig. 6, E and F). Furthermore, ligation of Fas
by exogenous antibodies did not induce PCD in MDA-MB-468 cells (Fig. 6,
B and E). Although these observations are
difficult to reconcile with a model in which drug treatment induces PCD
by activating the Fas/FasL pathway in this cell line, they do not rule
out the possibility that another death receptor (e.g. death
receptor 5 (91)) and its ligand might be involved in MDA-MB-468 cell
death, nor do they rule out the possibility that the Fas/FasL pathway
might play a role in chemotherapy-induced apoptosis in other cell
lines. These issues require further investigation.
Recent studies have provided evidence that a pathway involving
cytochrome c, Apaf-1, and procaspase-9 can also lead to
activation of downstream caspases (32-36). To evaluate the possible
activation of this pathway, the distribution of cytochrome c
was examined by immunoblotting. Results of these experiments (Fig. 7)
revealed that cytochrome c was readily detectable in cytosol
of cells that became apoptotic after treatment with EGF as well as
paclitaxel or 5FUdR (Fig. 7). Because of the lack of sensitivity of
currently available anti-procaspase-9 antibodies, we were unable to
directly confirm that procaspase-9 was cleaved in these cells.
Nonetheless, the observations in Fig. 7 are highly suggestive that the
cytochrome c/Apaf-1/procaspase-9 pathway contributes to
activation of the downstream caspases after treatment with any of these agents.
Current evidence suggests that this pathway can be triggered by
alterations in the expression or phosphorylation of certain Bcl-2
family members. In particular, increased levels of Bax (78, 92, 93),
decreased levels of Bcl-2 (94), and altered phosphorylation of Bcl-2
(74, 75, 95) have been observed in various model systems prior to the
onset of apoptosis. To search for similar changes in MDA-MB-468 cells,
whole cell lysates were probed with antibodies that recognize six Bcl-2
family members. Consistent with previous reports (74, 75, 77, 95),
paclitaxel treatment caused a transient reduction in the mobility of
part of the Bcl-2 molecules present in MDA-MB-468 cells (Fig.
8A). In contrast, changes in Bcl-2 phosphorylation were not
evident after treatment with 5FUdR or EGF (Fig. 8A).
Moreover, changes in phosphorylation state or levels of
Bcl-xL, Mcl-1, Bak, Bax, or Bad were not discernible during
treatment with EGF (Fig. 8B), suggesting that other
alterations must trigger the apoptotic cascade.
In summary, the present studies have demonstrated that EGF treatment
induces the process of anoikis in MDA-MB-468 cells. As is the case with
drug-induced PCD, anoikis in these cells involves release of cytochrome
c from mitochondria and activation of downstream caspases,
including caspases-3, -6, and -7. The changes occurring between
treatment with EGF and activation of downstream caspases do not appear
to involve activation of the Fas/FasL pathway or alterations in
levels of commonly studied Bcl-2 family members. Although the
intervening steps remain unidentified at present, EGF-treated
MDA-MB-468 cells might provide a useful model for studying the process
by which loss of integrin-mediated signaling results in caspase
activation and cell death.
 |
ACKNOWLEDGEMENTS |
We thank Drs. John Reed and Guy Salvesen for
antibodies to caspase-6, Bcl-xL, and Mcl-1; Dr. Guy Poirier
for C-2-10 anti-PARP; Wendy Deveraux and Phyllis Svingen for technical
assistance with some of the experiments; and Deb Strauss for
secretarial assistance.
 |
FOOTNOTES |
*
This work was supported in parts by United States Public
Health Service Grants U01 CA66084 (to N. E. D., D. K. A., and
S. H. K.) and R01 CA69008 (to S. H. K. and W. C. E.) and by a
grant from the Welcome Trust (to W. C. E.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Supported in part by a predoctoral fellowship from "Programa
Gulbenkian de Doutoramento em Biologia e Medicina." Current address: Signal Transduction Laboratory, Imperial Cancer Research Fund, London
WC2A 3PX, United Kingdom.
**
Principal Fellow of the Welcome Trust.

Recipient of an American Cancer Society Clinical Oncology
Career Development Award.
§§
Scholar of the Leukemia Society of America. To whom
correspondence should be addressed: Division of Oncology Research, 1301 Guggenheim, Mayo Clinic, 200 First St., S.W., Rochester, MN 55905. Tel.: 507-284-8950; Fax: 507-284-3906; E-mail:
Kaufmann.Scott{at}Mayo.edu.
2
One-way analysis of variance indicated that
increasing poly(HEMA) thickness was associated with increasing cell
detachment when samples were grown either in the absence
(p = 0.001) or presence (p < 0.0001)
of EGF. At each poly(HEMA) concentration, more cells were detached in
the presence of EGF than its absence (p < 0.01).
3
T. J. Kottke and S. H. Kaufmann,
unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
EGF, epidermal
growth factor;
EGFR, EGF receptor;
5FUdR, 5-fluoro-2'-deoxyuridine;
caspase, cysteine-dependent aspartate-directed protease;
AFC, 7-amino-4-trifluoromethylcoumarin;
DEVD-AFC, aspartylglutamylvalinylaspartyl-AFC;
VEID-AFC, valinylglutamylisoleucylaspartyl-AFC;
YVAD-AFC, tyrosinylvalinylalanylaspartyl-AFC;
FAK, focal adhesion kinase;
FasL, Fas ligand;
PARP, poly(ADP-ribose) polymerase;
PCD, programmed cell
death;
poly(HEMA), poly(2-hydroxyethyl methacrylate);
Z-EK(bio)D-aomk, N-(N
-benzyloxycarbonylglutamyl-N
-biotinyllysyl)
aspartic acid [(2,6-dimethylbenzoyl)oxy] methyl ketone.
 |
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