1 Department of Physiology, National Cheng-Kung University Medical College, and 2 Department of Nursing, Chang-Jung Christian University, Tainan, Taiwan 70101, Republic of China
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ABSTRACT |
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We previously demonstrated that collagen gel overlay induced cell remodeling to form lumen and apoptosis in Madin-Darby canine kidney cells. In the present study, we established that collagen gel overlay-induced apoptosis was initiated at areas exclusive of cell remodeling within 24 h (first phase) and extended into areas of cell remodeling within 48 h (second phase). Collagen gel overlay-induced apoptosis was accompanied by selective proteolysis of focal adhesion kinase (FAK), talin, p130cas, and c-src. Upon collagen gel overlay, FAK was initially degraded into a 90-kDa product during the first phase and subsequently into a 80-kDa product during the second phase. Collagen gel overlay-induced apoptosis of focal adhesion complex proteins and apoptosis of the first phase could be blocked only by a protease inhibitor cocktail. In addition, we found that both DEVD-fmk and ZVAD-fmk inhibited secondary proteolysis of FAK, but only ZVAD-fmk blocked collagen gel overlay-induced apoptosis of the second phase. Finally, collagen gel overlay-induced apoptosis and proteolysis of focal adhesion complex proteins were completely inhibited by the combination of protease inhibitor cocktail and ZVAD-fmk. Taken together, collagen gel overlay induces two phases of apoptosis; the first phase is dependent on proteolysis of focal adhesion complex proteins, and the second phase on activation of caspases.
Madin-Darby canine kidney cells; proteolysis; focal adhesion kinase
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INTRODUCTION |
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THE BASOLATERAL MEMBRANE of epithelial cells normally adheres immediately to a sheet of basement membrane composed of laminin, type IV collagen, and proteoglycan (34). Proper interactions with the extracellular matrix may result in survival and stabilization and may facilitate differentiation of the polarized cells. In contrast, application of collagen gel overlay to epithelial cells results in rapid membrane remodeling of cells and elicited lumen formation within 24 h (13, 32, 33). In addition, collagen gel overlay also triggers apoptosis of epithelial cells (33). However, the cellular mechanisms involved in collagen gel overlay-induced apoptosis have not been examined.
The focal adhesion complex has been recognized as an important site of
signal transduction affecting cellular behavior such as migration,
proliferation, differentiation, and survival. A central member of the
focal adhesion complex is the tyrosine kinase pp125FAK
(focal adhesion kinase, FAK) that plays a key role in the assembly of
various signaling proteins recruited to focal adhesions. FAK interacts
with a number of cellular proteins, including Src, Grb2, phosphatidylinositol-3 kinase, paxillin, talin, and p130cas
(5, 7, 29, 30, 38, 39). It has been established that
FAK plays important roles in cell cycle progression (40), cell migration (2), cell adhesion (27), and
prevention of apoptosis (9, 14). On the other
hand, c-src, grb2, and p130cas have been
implicated in FAK signaling in regulating these cellular functions.
Talin is involved in FAK-mediated cytoskeletal organization (4). Other proteins, such as -actinin, vinculin, and
Crk, are also present in focal adhesion complex (24).
Several pieces of evidence suggest that FAK could be a target for
proteases, either cysteine proteases or caspases. Crouch et al.
(7) showed that FAK serves as a specific, early and novel
target for proteolysis during c-myc-induced cell death, the
suppression of which was clearly linked to cell survival. Wen
et al. (35) also demonstrated that FAK was sequentially cleaved into two different fragments early in Apo-2L-induced
apoptosis possibly mediated by caspases-3/caspase-8. In
addition to FAK, other focal adhesion complex proteins also play
important roles in maintenance of cellular functions or in
apoptosis. During platelet aggregation, activation of the
cysteine protease calpain serves to degrade the focal adhesion complex
proteins, such as c-src and talin, thereby mediating the
postaggregation response (31). Chan et al.
(3) showed that during ultraviolet irradiation-induced apoptosis, the focal adhesion complex proteins FAK,
p130cas, and c-src were proteolyzed and that
overexpression of FAK can partially rescue the ultraviolet-induced
apoptosis. Recently, it was shown that overexpression of
constitutive activated FAK rescued cells from apoptosis induced
by disruption of cell-matrix interaction (9). On the other
hand, fibroblasts underwent apoptosis when the interaction of
FAK with the cytoplasmic domain of the 1-integrin was
inhibited by microinjection of peptide identical to the FAK binding
site on the integrin or by microinjection of an antibody interfering
with this binding (14). These data indicate that FAK plays
an important role in maintaining cell survival.
In the present study, we established that collagen gel overlay-induced apoptosis could be classified into two phases. The first phase of apoptosis was observed at the areas exclusive of cell remodeling within 24 h. The second phase of apoptosis could be seen within areas of cell remodeling by 48 h. In addition, we also found that collagen gel overlay induced two phases of degradation of FAK. FAK was initially degraded into a 90-kDa product during the first 24 h and subsequently into a 80-kDa product during the second phase. These observations prompted the examination of whether proteolysis of FAK is the cause or result of collagen gel overlay-induced apoptosis. In this study, the association of FAK proteolysis with collagen gel overlay-induced apoptosis was explored.
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MATERIALS AND METHODS |
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Cell line. Madin-Darby canine kidney (MDCK) cells (purchased from ATCC) were regularly maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) at 37°C in humidified 5% CO2 atmosphere.
Preparation of hydrated collagen gel. Type I collagen was prepared from tail tendons of 1-mo-old rats according to the established procedure described previously (17, 22, 33). The final concentration of type I collagen stock was 1% dissolved in 0.025 N acetic acid. For the preparation of agarose gel, a stock of 1% agarose solution in 0.025 N acetic acid was made. In preparing collagen gel, we mixed three volumes of collagen stock with 5.7× DMEM (1 vol), 2.5% NaHCO3 (0.5 vol), 0.1 M HEPES (1 vol), 0.17 M CaCl2 (0.1 vol), 1 N NaOH (0.1 vol), and 4.3 vol of 1× culture medium (DMEM + 10% FCS) under chilled conditions. The mixtures were dispensed on the cultured cells at 2 ml/60-mm dish and placed in an incubator (5% CO2 in air, 37°C) to allow for the gelation. The agarose gel was prepared by the same protocol as described (33). After gelation, each culture was supplemented with 1.5 ml of culture medium containing DMEM and 10% FCS. The culture medium was replaced every other day.
Preparation of cell lysates. MDCK cells cultured to 70-80% confluence were overlaid with type I collagen gel. At the appropriate time points, cells were harvested from both gel and dish. To release cells that adhered to the gel, we removed the collagen gel and incubated the gel with 3 mg/ml of type II collagenase (Worthington Biochemical) at 37°C for 10 min. After digestion, the sample was then centrifuged at 800 g, and cells in the pellet were collected (on gel). On the other hand, cells that remained on the dish were washed twice by chilled PBS and then lysed by lysis buffer containing 150 mM NaCl, 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5% Nonidet P-40 (on dish). Cells on gel and on dish were either pooled together or kept separated, depending on the purpose of the experiment. The lysate was further homogenized by the method of sonication.
Western blot analysis.
We used the following antibodies to assess the protein levels in focal
adhesion complex: anti-FAK kinase domain and anti-paxillin (Transduction Laboratories); anti-FAK NH2-terminal
domain; anti-vinculin and anti--actinin (Sigma); anti-talin
(Biogenesis); and anti-c-src and anti-p130cas
(provided by Dr. H.-C. Chen, National Chung-Hsing University, Taichung,
Taiwan). Protein concentration was measured by the method of Lowry et
al. (21) using bovine serum albumin as the standard. For
Western blot, 20 or 50 µg of cell homogenate from specific samples
was resolved by 5% SDS-PAGE (for talin and vinculin) or 10% SDS-PAGE
(for FAK, paxillin,
-actinin, c-src, and
p130cas) and electrophoretically blotted onto
nitrocellulose paper. The nitrocellulose paper was incubated with the
specific antibody listed above, and then immunocomplexes were detected
with horseradish peroxidase-conjugated IgG, and finally the
immunocomplexes were made visible by fluorograghy with enhanced
chemiluminescence detection kit (Amersham-Pharmacia International PLC).
Determination of apoptosis. The apoptosis was assessed by flow cytometry using propidium iodide as described previously (19, 33). MDCK cells were first treated with trypsin-EDTA (0.25%-2mM in PBS) to obtain a better cell suspension. The trypsin-digested cells were then washed with PBS-1 mM EDTA and fixed in 70% alcohol. After fixation, cells were treated with RNase (100 µg/ml in PBS) and stained with propidium iodide (40 µg/ml in PBS). The mixed cells were incubated in the dark at room temperature for 30 min and analyzed by flow cytometry using a FACScan (Becton Dickinson, Mountain View, CA) with excitation set at 488 nm. Data were analyzed by Cell FIT software and represented as either histograms or numbers. The hypodiploid DNA peak of apoptotic cells can be distinguished from the normal diploid DNA peak on the fluorescence profiles of propidium iodide-stained cells.
Treatment with protease inhibitors. To elucidate what type of protease(s) might be involved in collagen gel overlay-induced proteolysis of focal adhesion complex proteins, we employed protease inhibitor cocktail (Complete, Boehringer Mannheim), calpain inhibitor I (ALLN, Calbiochem-Novabiochem), PMSF, and pepstatin A (both purchased from Sigma). Cells were pretreated for 1 h and then overlaid with collagen gel in the presence of the inhibitors for 24-48 h. At appropriate time points, cells were harvested for analysis of cell cycle or Western blotting, depending on the purpose of experiments.
Treatment with caspase inhibitors. To elucidate whether caspases were involved in collagen gel overlay-induced apoptosis, we employed tetrapeptide caspase inhibitors such as YVAD-CHO, cell-permeable DEVD-fmk, and ZVAD-fmk (all purchased from Calbiochem-Novabiochem). Cells were preincubated with either inhibitor alone or inhibitors in combination for 1 h and then overlaid with collagen gel in the presence of the same inhibitor(s) for 48 h. At appropriate time points, cells were harvested for analysis of cell cycle or Western blotting, depending on the purpose of experiments.
Statistics. All data are expressed as means ± SE of at least three independent experiments. One-way ANOVA was used to test for statistical differences. P < 0.05 was taken to be statistically significant.
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RESULTS |
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Collagen gel overlay-induced apoptosis and cell remodeling
could be separated.
MDCK cells cultured to 70-80% confluence were overlaid with
collagen gel, and the morphology of cells was observed under light microscope. Within 24 h of collagen gel overlay, MDCK cells
underwent morphological changes and remodeled into lumen-containing
structures (13, 32, 33). Meanwhile, a small proportion of
MDCK cells disintegrated into tiny vesicles that resembled
apoptotic bodies in the area where cell remodeling was not present
(Fig. 1A). Between 24 and
48 h of collagen gel overlay, apoptotic bodies increased in
the area of cell remodeling (Fig. 1B). The remodeled and
apoptotic cells could be separated by the removal of collagen gel.
As shown in Fig. 1, C and D, apoptotic bodies
remaining on dish were markedly reduced after the removal of collagen
gel, indicating that apoptosis is associated with collagen gel.
To quantitate apoptosis in the population of cells adhering to
gel or remaining on dish, we used FACScan analysis. Our results showed
that almost all cells adhering to gel were apoptotic (93.5 ± 2.7% at 24 h and 94.7 ± 1.5% at 48 h), whereas cells
remaining on dish were mostly viable, with apoptosis ratio of
4.9 ± 1.5% at 24 h and 28.5 ± 2.7% at 48 h (Fig. 1E).
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Collagen gel overlay induced selective proteolysis of focal
adhesion complex proteins.
We examined whether there were changes in the focal adhesion complex
proteins in MDCK cells overlaid by collagen gel. After collagen gel
overlay, cells adhering to both dish and gel were harvested at
different time points, and the lysates were analyzed by Western
blotting using a monoclonal antibody against kinase domain of FAK. We
observed that FAK underwent distinct proteolytic changes with time in
response to collagen gel overlay (Fig.
2A, top). A
significant decrease in 125-kDa FAK was observed within 8 h of
collagen gel overlay, along with the appearance of a major degraded
product of 90 kDa. The intensity of FAK was gradually decreased
8-48 h after collagen gel overlay. The degraded fragment of FAK
appeared to increase in intensity from 8 to 24 h (first phase),
and another degraded product appeared between 24 and 48 h (second
phase) with a molecular weight of 80 kDa. The pattern of FAK
degradation was also analyzed by polyclonal antibody against NH2-terminal 2-17 amino acids of FAK. This antibody
detected two degraded products of FAK that were similar to those
recognized by anti-FAK kinase domain antibody (Fig. 2A,
middle). The first degradation product of FAK (90 kDa) was
increased from 8 to 24 h and decreased in 48 h, whereas the
second degradation product (80 kDa) was increased 16-48 h after
collagen gel overlay. In contrast, FAK remained intact even
after agarose gel overlay for 72 h (Fig. 2B).
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Cells adhering to collagen gel exhibited proteolysis of focal
adhesion complex proteins.
We collected cells either adhering to gel or remaining on dish
separately and analyzed the integrity of their focal adhesion complex
proteins in these two different populations. Although the quantity of
cells adhering to gel increased with time, the amount adhering to gel
in a 60-mm dish was too low for the assessment of total protein content
within 24 h of collagen gel overlay. We therefore had to load all
the lysate of each sample into each well for Western blot analysis. In
contrast, for the analysis of focal adhesion complex proteins in cells
left on the dish, an equal amount of total protein was loaded for each
sample. As shown in Fig. 4A,
cells adhering to the gel exhibited only a degraded form of FAK, the
intensity of which increased with time. In contrast, cells
left on the dish showed intact FAK, talin, and p130cas with
little sign of proteolysis (Fig. 4B). Taking these data together, we found that cells adhering to the gel were almost apoptotic and exhibited completely degraded FAK,
p130cas, and talin throughout the time course, whereas
cells left on the dish displayed little apoptosis or intact
focal adhesion complex proteins.
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First-phase apoptosis and FAK proteolysis
could be blocked by protease inhibitor cocktail.
We previously showed that cycloheximide prevented collagen gel-induced
cell remodeling and apoptosis within 36 h
(33). We examined whether cycloheximide might prevent
collagen gel overlay-induced proteolysis of focal adhesion complex
proteins. MDCK cells cultured to 70-80% confluence were
pretreated with cycloheximide (1 µg/ml) for 1 h and overlaid
with collagen gel in the presence of cycloheximide for another 36 h, and the biochemical changes of focal adhesion complex proteins were
assessed. The results showed that collagen gel overlay-induced cleavage
of FAK, talin, p130cas, and c-src was completely
blocked by cycloheximide within 36 h of collagen gel overlay (Fig.
5).
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Second-phase apoptosis and FAK proteolysis
could be blocked by ZVAD-fmk.
To elucidate whether caspases were involved in collagen gel
overlay-induced apoptosis, we employed the following
tetrapeptide caspase inhibitors: YVAD-CHO, cell-permeable DEVD-fmk, and
ZVAD-fmk. Cells were preincubated with either inhibitor alone or
inhibitors in combination for 1 h and then overlaid with collagen
gel in the presence of the same inhibitor for 48 h. None of the
caspase inhibitors affected collagen gel overlay-induced cell
remodeling. Among these caspase inhibitors, only ZVAD-fmk attenuated
collagen gel overlay-induced apoptosis in a dose-dependent
manner (Fig. 7A).
Nevertheless, it did not completely block collagen gel overlay-induced apoptosis. Interestingly, we found that DEVD-fmk and ZVAD-fmk but not YVAD-CHO partially blocked proteolysis of FAK induced by
collagen gel overlay (Fig. 7B). These inhibitors
selectively blocked the formation of degraded product 80 kDa during the
second phase. The pattern of FAK degradation resulting from the
presence of these caspase inhibitors within 48 h of collagen gel
overlay is reminiscent of that observed at 24 h in the absence of
any inhibitor. Taken together, protease inhibitor cocktail and
ZVAD-fmk might inhibit the first and second phase of apoptosis,
respectively. To test this possibility, we employed a combination of
protease inhibitor cocktail and ZVAD-fmk. As shown in Fig.
8A, both protease inhibitor
cocktail and ZVAD-fmk only partially inhibited collagen gel-induced
apoptosis within 48 h. However, a combination of them completely block collagen gel overlay-induced apoptosis from
39.1 ± 1.7% to 5.1 ± 0.2%. Interestingly, the proteolysis
of focal adhesion complex proteins, including FAK, talin, and
p130cas, was also completely blocked by a combination of
protease inhibitor and ZVAD-fmk (Fig. 8B).
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DISCUSSION |
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In this study, we first defined the model in which collagen gel overlay could induce two different phases of apoptosis. Collagen gel overlay-induced apoptosis of the first phase was observed in cells that lost their intact focal adhesion complex proteins. These cells were excluded from those that formed lumen but remained adhering to collagen gel even when they developed apoptosis during 24-h overlay. The second phase of apoptosis occurred to the cells that had remodeled to form lumen and contacted to collagen gel directly during 24-48 h overlay. The induction of two-phase apoptosis by collagen gel overlay has not been reported before. Interestingly, the cellular mechanism involved in apoptosis of these two phases is also different. In the first phase of apoptosis, activation of protease(s) other than caspases that cleaves focal adhesion complex proteins plays important roles. On the other hand, caspases are required for execution of apoptosis in the second phase. Because MDCK cells are heterogeneous in their phenotypic property, it is possible that the two collagen gel overlay-induced phases of apoptosis in MDCK cells may reflect the heterogeneity of the cell line. To rule out this possibility, we employed a single clone of MDCK, clone II 3B5 (17). 3B5 cells exhibited apoptosis and cell remodeling as well as FAK proteolysis in response to collagen gel overlay (data not shown). Therefore, we tend to believe that cell heterogeneity is not the cause.
In the present study, we demonstrated that FAK, the central component of focal adhesion, was cleaved into 90-kDa fragments as early as 8 h during the phase of proteolysis; a further degradation product of 80 kDa appeared from 24- to 48-h overlay. Compared with the previous reports that demonstrated proteolysis of FAK induced by activation of caspases, the degradation pattern of FAK in our study may not be similar. Levkau et al. (20) demonstrated that during growth factor deprivation-induced apoptosis in vascular endothelial cells, FAK was cleaved by caspase activation within 2 h. Wen et al. (35) also demonstrated in Apo-2- and Fas-induced Jurkat or H460 cell apoptosis, respectively, that FAK was degraded at a similar time course to Levkau's finding. In these studies, they found several putative caspase cutting sites in the COOH terminus of FAK, which generated 33- or 41-kDa COOH-terminal degraded fragments, and the predicted size was similar to their experiments (20, 35). These studies showed that the different degraded products of FAK were caused by the activation of different caspases such as caspase-3 or -6. In our study, using anti-NH2-terminal antibody of FAK, we detected a 90-kDa (first phase) and an 80-kDa (second phase) degraded product after collagen gel overlay of 24 and 48 h, respectively. Most importantly, we found that caspase inhibitors did not prevent the collagen gel overlay-induced first phase of apoptosis. These results rule out the involvement of caspases in collagen gel overlay-induced first phase of apoptosis or FAK proteolysis.
Recent studies have demonstrated that proteases other than caspases, such as calpain and cathepsin D, may contribute to the induction of apoptosis (16, 25, 26, 28, 36, 37). In these studies, only calpain has been reported to cleave FAK and thereby contribute to the induction of apoptosis (37). On the other hand, it has also been shown that activation of calpain may induce FAK proteolysis, the result of which is associated with platelet activation and adhesion, but not cell death (15). We have tried to delineate whether a specific protease is involved in collagen gel overlay-induced apoptosis by testing the effects of different protease inhibitors. However, none of the inhibitors of serine protease, aspartate protease, or calpain can prevent collagen gel overlay-induced proteolysis of FAK or apoptosis. Nevertheless, a protease inhibitor cocktail can completely block collagen gel overlay-induced apoptosis and degradation of FAK in the first phase. Proteolysis of FAK is therefore responsible for collagen gel-induced apoptosis during the first phase.
It is of interest to know whether focal adhesion complex proteins other
than FAK were degraded after collagen gel overlay. We found that only
proteins present upstream of the focal adhesion complex such as FAK,
talin, c-src, and p130cas were affected.
Conversely, proteins participating in the downstream of the
integrin signal or associated with cytoskeletal proteins, such as
paxillin, vinculin, and -actinin, remained intact. The selective
proteolysis of focal adhesion complex proteins induced by collagen gel
overlay may result in a blockade of FAK downstream signaling that
regulates cell viability (3, 11, 12, 14). Chen and Guan
(5) demonstrated that FAK could form a stable complex with
its potential substrate phosphatidylinositol 3-kinase. The
latter was known to be involved in antiapoptotic signal (18, 23). In addition, Carragher et al. (1) demonstrated
that the association of FAK with other focal adhesion complex proteins such as paxillin and p130cas was markedly diminished by
degradation of FAK. Therefore, the degradation of focal
adhesion complex proteins may trigger apoptosis via the loss of
their integrity of focal adhesions as well as antiapoptotic
signals. On the other hand, cleavage of focal adhesion complex proteins
prevents the cells from adhering to the dish. It is therefore not
surprising that all the apoptotic cells at the first phase were
found to stick to collagen gel, instead of remaining on the dish.
Furthermore, during 24-48-h collagen gel overlay, massive apoptosis was observed. The apoptosis of this phase was observed in cells that formed lumen and directly contacted with collagen gel. To examine whether direct contact with collagen gel may trigger apoptosis, we cultured cells in or on collagen gel. In our previous report, we discovered that MDCK cells developed apoptosis when cultured in collagen gel (19). In addition, our current study has shown that epithelial cells, but not fibroblasts or tumor cells, developed apoptosis when seeded on collagen gel (6). It is therefore likely that epithelial cells may develop apoptosis on direct contact with collagen gel, regardless of membrane site. Although those with intact focal adhesion complex proteins may form lumen with each other and escape the first phase of apoptosis, they may undergo apoptosis during the second phase due to direct contact with collagen gel. During collagen gel overlay-induced apoptosis of the second phase, ZVAD-fmk, but not YVAD-CHO or DEVD-fmk, significantly attenuated collagen gel overlay-induced apoptosis and proteolysis of FAK. It is thus concluded that activation of caspase(s) other than caspase-1, -3, or -7 plays an important role in triggering apoptosis of the second phase. The underlying mechanisms involved in collagen gel overlay-induced caspase activation remain to be elucidated.
The study reported here demonstrates that collagen gel overlay-induced
proteolysis of focal adhesion complex proteins is not the cause of cell
remodeling but is associated with collagen gel overlay-induced
apoptosis. The temporal and spatial changes of collagen gel
overlay-induced cell remodeling and the two phases of apoptosis
are summarized in Fig. 9. Collagen gel
overlay initiates a series of proteolysis that selectively degrades the
focal adhesion complex proteins in a small proportion of cells, the
results of which may trigger drastic morphological events. Cells that
are depleted of intact focal adhesion complex proteins can neither form
lumen structures nor adhere to the culture dish. However, they tend to
stick to collagen gel and develop apoptosis due to loss of
survival signals from focal adhesion complex proteins during the first
phase of apoptosis. In contrast, cells exhibiting intact focal
adhesion complex proteins tend to develop lumen structure within
24 h of collagen gel overlay. Between 24 and 48 h, cells that
form lumen start to develop massive apoptosis where they directly contact collagen gel. The apoptosis initiated during the first phase is dependent on the activation of protease(s) other
than caspases, whereas the apoptosis observed during the second
phase results from the activation of caspases. The nature of
protease(s) that selectively degrade the focal adhesion complex proteins in collagen gel overlay-induced apoptosis of the fist phase is still unknown, as is the identity of the caspase(s) involved in apoptosis of the second phase. It should be of interest to explore further the mechanisms involved in collagen gel overlay-induced activation of protease(s) as well as caspases.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. W.-J. Chuang for the gift of protease inhibitor cocktail and Dr. H.-C. Chen for anti-src and anti-p130cas antibodies.
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FOOTNOTES |
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* Y.-K. Wang and H.-H. Lin contributed equally to this work.
This work was supported by National Science Council of Taiwan Grant 89-2320-B0309-005 (to H. H. Lin) and National Health Research Institute Grant NHRI-EX90-9031-SL (to M.-J. Tang).
Address for reprint requests and other correspondence: M.-J. Tang, Dept. of Physiology, National Cheng-Kung Univ. Medical College, Tainan, Taiwan (E-mail: mjtang1{at}mail.ncku.edu.tw).
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.
Received 13 July 2000; accepted in final form 9 January 2001.
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