Methods (supplementary material)
Cytosolic and mitochondrial protein extraction. Cytosolic and mitochondrial fractions were
isolated from mock, 100MOI of Ad-SV and Ad-MMP-2 infected cells as described elsewhere
(Li et al., 2003). Cross-contamination of cytosol and mitochondria was determined on the
basis of the amount of activity of marker enzymes for cytosol (lactate dehydrogenase [LDH]
and
mitochondria (succinate dehydrogenase [SDH]. Protein levels in the samples were
determined with Bio-Rad protein assay kit and analyzed by immunoblot using anticytochrome-C antibody.
FACS analysis. Annexin V expression was determined as per the manufacturer’s instructions.
A549 lung cancer cells were cultured in 100-mm dishes and infected as described above.
Cells were trypsinized 36 h after infection, washed three times with cold PBS, resuspended in
500µL of 1X binding buffer, and incubated with 5µL of Annexin V-FITC and 5 µL of
propidium iodide (PI) at room temperature for 5min in the dark. Flow cytometric analysis
was performed on a FACS Calibur Flow cytometer (Becton Dickinson Biosciences, San Jose,
CA) with an excitation wavelength of 488 nm and emission wavelength of 530 nm. Data
acquisition and analysis were performed using CellQuest (BD Biosciences) software.
Immunocyto- and Immunohisto-chemical analysis. A549 cells (5103) were cultured in
chamber slides and infected with mock, 100MOI Ad-SV or the indicated MOI of Ad-MMP-2
for 48h. Cells were washed with PBS and fixed for 30 min in cold methanol and
permeabilized in 0.1% Triton-X100 in PBS. Non-specific binding was blocked by 3% BSA
in PBS for 1h, followed by incubation with 1:100 dilution of anti-Fas, anti-Fas-L. Mouse
IgG was used as a negative control. The excess antibody was removed by washing at least
three times with PBS, and then incubated with FITC/ Texas Red - conjugated secondary
antibody for 1h. The cells were washed and mounted with 4', 6-diamidino-2-phenylindole
(DAPI) staining solution. For detection of TIMP-3 expression, after primery anti-body, HRP-
conjugated secondary antibody at 1:200 dilution and 3,3-diaminobenzidine (DAB peroxidase
substrate) solution were used.
The slides were counterstained with hematoxylin and
mounted. The bright field images were captured with an Olympus BX 60 research
fluorescence microscope attached with CCD camera. For immunohistochemical analysis,
tissue sections (4-5 µm-thick) were deparaffinized in xylene, rehydrated in graded ethanol
solutions, washed with PBS and permeabilized in 0.1% Triton X-100 and incubated overnight
with primary antibodies for Fas, Fas-L, TIMP-3 and cleaved Bid and processed as described
above.
Extraction of extracellular matrix (ECM) protein. A549 lung cancer cells were infected as
mentioned above for 48h. The cells were washed with PBS three times and then lysed with
PBS containing 0.5% Triton X-100 (v/v) for 20 min at room temperature. After aspirating the
Triton X-100, the remaining ECM was washed thrice with PBS followed by two washes with
20 mM Tris-HCl (pH 7.4) containing 100mM NaCl and 0.1% (v/v) Tween 20. The cells were
observed under a light microscope to ensure that no un-lysed cells were left in the culture
dish. Sample buffer (200L) was added to each culture dish and cells were agitated for 20
min on a rocker at room temperature. Using 25L of these extracts immunoblot analysis for
TIMP-3 was performed as mentioned above.
Terminal deoxynucleotidyl transferase-mediated nick labeling (TUNEL) assay. We detected
apoptosis in Ad-MMP-2-treated cells as well as the subcutaneous tumor tissue sections of
Ad-MMP-2-treated mice using terminal deoxynucleotidyl transferase dUTP nick-end labeling
(TUNEL) enzyme reagent and 4,6-diamidno-2-phenylindole according to the manufacturer’s
instructions. Briefly, 5103 cells were plated in 8-well chamber slides and infected as
described above.
After 48h, cells were washed three times with PBS, fixed 4%
paraformaldehyde in PBS for 1h at 15-25°C, washed with PBS and permeabilized in 0.1%
Triton-X100 in 0.1% sodium citrate in PBS for 2 min (for cells) or 8 min (for tissue sections)
on ice. Slides were then washed with PBS and incubated in TUNEL reaction mixture in a
humidified atmosphere for 1h at 37°C in the dark, washed and mounted onto slides with
GEL/MOUNT Aqueous Mounting Medium with Anti-Fading Agents. The cells were also
counter stained with 4’, 6-diamidino-2-phenylindole (DAPI) nuclear staining mounting
solution. To determine the number of apoptotic cells in the tissue sections, paraffin-embedded
tissue sections were deparaffinize and rehydrated. Slides were imaged with an Olympus BX
60 research fluorescence microscope attached with CCD camera. The apoptotic index was
defined as follows: apoptotic index (%) = 100 × (apoptotic cells/total cells).
Results (supplementary material)
Neutralization of Fas-L with anti-Fas-L antibody reverses Fas-mediated apoptosis. To
confirm whether Fas and Fas-L were involved in mediating this increase in apoptosis, A549
cells were incubated for 1h with neutralizing antibodies against Fas-L prior to infection with
Ad-MMP-2. This pretreatment of A549 cells reduced TUNEL staining (Fig. 4A) in AdMMP-2-infected cells by >45% from an average 705% to 303% apoptotic cells (Fig. 4B).
This result was confirmed by immunoblotting for the downstream mediators of Fas-mediated
apoptosis, caspase-8 and PARP-1 cleavage. As shown in Figure 4C, caspase-8 and PARP-1
cleavage were inhibited when cells were pretreated with anti-Fas-L. The Ad-MMP-2 induced
apoptosis was not reverted back in non-specific antibody treated A549 cells (Fig. 4D). These
results strongly suggest that the Ad-MMP-2-induced apoptosis is mediated by the Fas/Fas-L
pathway.
Pancaspase inhibitors and caspase-8 specific inhibitor blocked Ad-MMP-2-induced
apoptosis.
To further demonstrate the activation of caspases during Ad-MMP-2-induced
apoptosis, the apoptotic effect of pancaspase inhibitors, as well as specific caspase inhibitors
for caspase-8 and caspase-9, was examined. The specific caspase inhibitors were Z-VADFMK for pancaspase, Z-IETD-FMK for caspase-8, and Z-LEHD-FMK for caspase-9. The
various concentrations of these caspase inhibitors did not affect the viability of the A549 cells
(data not shown). First, as shown in Fig. 2D, we observed that the general caspase antagonist,
Z-VAD-FMK inhibited Ad-MMP-2-induced apoptosis as determined by inhibition of
cleavage of PARP and caspases-8, and -9. Pretreatment of cultured A549 cells with 25µM of
the caspase-8-specific inhibitor, Z-IETD-FMK clearly suppressed the activation of caspases-9
and blocked PARP cleavage. However pretreatment with caspase-9 inhibitor, Z-LEHD-FMK
inhibited caspase-9 cleavage but did not affect caspase-8 cleavage, suggesting that caspase-8
functions upstream of caspase 9. These findings constitute the first evidence that the mostupstream change in the caspase cascade that occurs in Ad-MMP-2 apoptotic cells is the
activation of caspase-8. We further demonstrate that MMP-2 inhibitor 1, a specific inhibitor
of MMP-2 also induced apoptosis as determined by cleavage of caspase-8 and PARP-1 (Fig.
2E).