Matrix Metalloproteinases 9 and 10 Inhibit Protein Kinase

CPotentiated, p53-Mediated Apoptosis

Eric Meyer, Jean-Yves Vollmer, Raymonde Bovey, and Ivan Stamenkovic
Division of Experimental Pathology, Institut Universitaire de Pathologie, Universite de Lausanne, Lausanne, Switzerland
Abstract
p53, a major sensor of DNA damage, is a transcription factor
that, depending on its phosphorylation status, regulates the
cell cycle, DNA repair, or apoptosis. The protein kinase C
(PKC) family of isozymes is also implicated in cell cycle and
programmed cell death (PCD) control and has recently been
shown to influence p53 function. Using three human colon
adenocarcinoma cell lines SW480, EB-1, and HCT116 that
either lack p53 function and were engineered to express
inducible wild-type p53 (wt p53), or that constitutively express
wt p53, we show that phorbol estermediated PKC activation
potentiates p53-induced PCD. Despite the effectiveness of
PKC/p53 synergy in inducing SW480 tumor cell death,
however, a fraction of the cells invariably survive. To address
the putative mechanisms that underlie resistance to PKC/p53-
induced cell death, we generated a phorbol 12-myristate 13-
acetate/p53resistant SW480 subline and compared the gene
expression profile of resistant and parental cells by DNA
microarray analysis. The results of these experiments show
that PKC/p53-resistant cells express a higher level of several
matrix metalloproteinases (MMP), including MMP-9, MMP-10,
and MMP-12, and corresponding real-time PCR assays
indicate that p53 is a negative regulator of MMP-9 gene
expression. Using MMP inhibitors and MMP-specific small
interfering RNA, we show that MMP function confers
protection from PKC/p53-induced apoptosis and identify the
protective MMPs as MMP-9 and MMP-10. Taken together,
these observations provide evidence that MMPs are implicat-
ed in tumor cell resistance to the synergistic proapoptotic
effect of PKC and p53. (Cancer Res 2005; 65(10): 4261-72)
Introduction
Resistance to apoptosis is a hallmark of cancer. Although
numerous molecular events can enable cells to survive in the face
of proapoptotic stimuli, the full repertoire of mechanisms used by
cancer cells to resist programmed cell death has yet to be
established. In the physiologic setting, the transcription factor p53
plays a major role in regulating cell survival, in addition to several
other cellular processes that include cell cycle checkpoint control
(1), DNA repair and senescence (2, 3). It is therefore not surprising
that p53 is among the most commonly affected molecules in
cancer, >50% of malignant human tumors bearing mutations in the
TP53 gene. p53 is activated in response to a multitude of signals,
including oncogene activation, hypoxia (4), and DNA damage (5),
and when required, induces a cell cycle checkpoint blockade and/
or cell death through modification of the expression level of several
target genes. p53 target genes include, among many others, Bax (6),
Mdm2 (7), Apaf1 (8), Fas (9), p21
WAF1
, and DR5 (10). The
consequences of the induction of these target genes are a function
of the cellular context.
Recently, a new mode of action has been proposed for the
proapoptotic activity of p53 (11). According to this view, p53 may
function in a transcription-independent manner and may induce
cell death by modifying mitochondrial membrane permeability,
thereby activating the intrinsic apoptotic pathway. Regulation of
p53 activity occurs at several levels, including post-translational
modification (phosphorylation and acetylation), induction of the
Mdm2 protein, which in turn induces ubiquitin-mediated p53
degradation (12), and intracellular localization. To act as a
transcription factor, p53 must translocate from the cytosol to the
nucleus. In contrast, its transcription-independent activity
requires its presence at the mitochondrial surface where it can
associate with the mitochondrial regulators of apoptosis Bax and
Bcl-X
L
(13). Localization is regulated in part by Mdm2, which
sequesters p53 and forces its translocation back toward the
cytosol, augmenting its availability for mitochondrial association
(14, 15).
Recent evidence suggests that protein kinase C (PKC) isozymes
are involved in the regulation of the p53 DNA-binding activity by
directly phosphorylating p53 on the Ser
378
residue (16). It has also
been proposed that PKCy regulates p53 expression at the
transcriptional level and contributes to the accumulation of the
p53 protein (17). PKC family members are serine/threonine
protein kinases that are divided into three subfamilies depending
on their structure and mode of activation. The classic PKC
subfamily includes the a, h
I
, h
II
, and g isotypes, which are
activated by diacylglycerol and phosphatidylserine in a calcium-
dependent manner. The atypical PKCs, which constitute the
second subfamily, include the ~ and E isotypes that are activated
by phosphatidylserine alone. Finally, the novel PKC subfamily is
composed of the y, q, D, and u, isotypes, which are activated by
diacylglycerol and phosphatidylserine in a calcium-independent
manner. These kinases play a role in multiple cellular functions,
one of them being apoptosis. However, the precise role played by
each of the different isotypes in programmed cell death (PCD)
remains to be fully elucidated. For example, the novel PKC, PKCy,
is considered proapoptotic and is cleaved by caspase-3 (18, 19),
one of the effector caspases whose activation is common to
several apoptotic pathways. The resulting cleaved fragment is
proapoptotic and initiates an apoptotic amplification loop by
targeting its own activator, caspase-3. On the other hand, the full-
size PKCy can mediate apoptosis at the mitochondrial level
(20, 21). In contrast, the classic PKCs, PKCa, PKCh
I
, and PKCh
II
are believed to exert antiapoptotic activity. PKCa, for example, is
able to phosphorylate and stabilize the antiapoptotic molecule
Bcl-2 (22). Several other reports show data suggesting that classic
Note: E. Meyer and J-Y. Vollmer contributed equally to this work.
Requests for reprints: Ivan Stamenkovic, Institute of Pathology, Centre
Hospitalier Universitaire Vaudois, Rue du Bugnon 25, 1011 Lausanne, Switzerland.
Phone: 121-314-7136; Fax: 121-314-7110; E-mail: ivan.stamenkovic@chuv.hospvd.ch.
I2005 American Association for Cancer Research.
www.aacrjournals.org 4261 Cancer Res 2005; 65: (10). May 15, 2005
Research Article
PKCs may display proapoptotic activity. The overall picture
emerging from these observations is that even if the role played
by PKC family members in apoptosis differs from isotype to
isotype, the consequences of their activation are most likely
dependent on the molecular and cellular context and the type of
apoptotic stimulus.
In the present study, we sought to determine the effect of PKC
activity on p53-induced PCD and the physiologic mechanisms that
underlie resistance to p53-mediated apoptosis in tumor cells. To
this end, we engineered the SW480 human colon adenocarcinoma
cell line, which contains an endogenous mutant p53 (23), to
overexpress wild-type p53 (wt p53) under the regulation of a
tetracycline (Tet-ON) conditional system. Using this model system
as well as additional colon carcinoma cell lines that express
inducible exogenous or constitutive endogenous p53, we show that
PKC activation and p53 expression function in synergy to induce
apoptosis in colon carcinoma cells and that resistance to the
combined proapoptotic effect of these molecules is conferred, at
least in part, by matrix metalloproteinase 9 (MMP-9) and MMP-10
expression.
Materials and Methods
Cell Culture and Reagents
The SW480 parental human colon adenocarcinoma cell line was
obtained from European Collection of Animal Cell Cultures (Porton Down,
United Kingdom) and the HCT p53
+/+
, HCT p53
/
, HCT Bax
+/
control,
and HCT Bax
/
cells were a gift from Bert Vogelstein (The Johns Hopkins
University Medical Institutions, Baltimore, MD). The p53-inducible EB-1
colon adenocarcinoma cell line was a gift from Phil Shaw (Department
of Pathology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzer-
land; ref. 24). Cell stocks were maintained in DMEM (Life Technologies,
Basel, Switzerland) supplemented with penicillin, streptomycin, and 10%
fetal bovine serum (FBS, Life Technologies). Tetracyclin (Calbiochem,
Schwalbach, Germany) and ZnCl
2
(Sigma Co., Buchs, Switzerland) were
dissolved in water, whereas 5-fluorouracil (5-FU) and phorbol 12-myristate
13-acetate (PMA, both from Sigma) were dissolved in DMSO. z-VAD-fmk
(BIOMOL Res. Lab., Butler Pike, PA) and 1,10-phenanthroline monohydrate
(Sigma) were dissolved in DMSO. The metalloproteinase inhibitor GM6001
and its negative control (Calbiochem) were dissolved in DMSO, as were the
PKC inhibitors rottlerin/mallotoxin, bisindolylmaleimide I, and Go6976
(Calbiochem).
Tetracycline-Regulated Expression System Construction
The human wt p53 cDNA sequence was subcloned as a 1,200-bp BamHI-
EcoRI fragment into the pcDNA4/TO/myc-His A vector (Invitrogen, Basel,
Switzerland). The resulting vector (p4TO-hwtp53) allowed expression of wt
p53 under the control of a tetracycline operator 2 sequencecontaining
cytomegalovirus promoter. The SW480 cell line was first stably transfected
with the pcDNA6/TR vector (Invitrogen), containing sequences encoding
the tetracycline repressor protein, using LipofectAMINE 2000 (Invitrogen)
according to the manufacturers recommendations. The bulk population
was then stably transfected with the p4TO-hwtp53 plasmid. After selection
with Zeocin and Blasticidin (Invitrogen), a monoclonal population was
isolated and named SMF1.
Isolation of a PKC/p53-Induced Apoptosis-Resistant Cell
Population
SMF1 cells were treated with tetracycline and PMA (2 and 0.16 Amol/L,
respectively) for 24 hours. Resistant cells were rinsed with PBS and cultured
in DMEM/10% FBS for 2 weeks. The resulting population was subjected to
additional rounds of tetracycline + PMA stimulation and selection. After the
fifth round of selection, surviving cells were tested for p53/PKC resistance
and preservation of tetracycline-inducible wt p53 expression. The resulting
SMF1-derived cell line was named S1R6.
cDNA Array Analysis
Total RNA was isolated from SMF1 or S1R6 cells after 8 hours of DMSO
or PMA/tetracycline stimulation using the RNeasy RNA extraction kit
(QIAGEN, Basel, Switzerland) according to the manufacturers recommen-
dations. The isolated RNA was linear-amplified using the Arcturus
(Mountain View, CA) riboamp RNA amplification kit. For each experiment,
6 Ag of sample 1 mRNA were labeled with dCTP-Cy3 and 6 Ag of sample 2
mRNA were labeled with dCTP-Cy5. Labeled samples were pooled and
hybridized in duplicate to human cDNA microarray (Hu10K) chips from
NCCR/ISREC containing 11,552 spotted elements corresponding to 9,500
unique human cDNAs ( for an accurate description see http://intranet.
isrec.ch/microarrays/nccr_isrec.html). Hybridization was done in hybrid-
ization chambers (Corning, Baar, Switzerland) in a 64jC water bath for
16 hours. After several careful washing steps, the chips were scanned in a
Perkin-Elmer/GSI Lumonics ScanArray 4000 scanner and scanned slide
images were converted to a tagged image file format. Spot intensity was
analyzed with the ScanAlyze software (http://rana.lbl.gov/EisenSoftware.
htm) and further primary data analysis was done using com.braju.sma
routines in the R statistical package (http://www.maths.lth.se/help/R/ and
http://www.r-project.org/). We used the nonlinear Scaled print-tip normal-
ization method (25). Quality control of slide hybridization was done using
variables described on the corresponding web site: http://intranet.isrec.ch/
microarrays/nccr_isrec.html.
When comparing SMF1-DMSO and SMF1-PMA/tetracyclinetreated
cells, we considered spots with an average ratio value superior to 1.85 to
correspond to positively regulated genes; a ratio of 1.00 was used for
comparisons between PMA/tetracycline-treated SMF1 and S1R6 cells. The
average ratio was determined for each set of experiments as the experiment
background intensity plus two SDs.
Real-time PCR
Total RNA was isolated with the RNeasy extraction kit (QIAGEN) and
cDNAs were produced by reverse transcription with the Moloney murine
leukemia virus Reverse Transcriptase RNase H minus, point mutant
(Promega, Wallisellen, Switzerland) and Random primers (Promega). Real-
time PCR was done in an Applied Biosystems 7900HT Sequence Detection
System using the ABI Taqman Master mix chemistry. The sets of primers
and Taqman probes were as follows: cyclophilin A, ABI human PPIA
endogenous control [VIC/minor groove binder (MGB) Taqman probe]; large
ribosomal protein, ABI human RLPO endogenous control (VIC/MGB
Taqman probe); MMP-9 (26): forward primer 5V-CCTGGAGACCTGAGAAC-
CAATCT-3V, reverse primer 5V-TGCCACCCGAGTGTAACCA-3V, FAM/
TAMRA-Taqman probe 5V-CAGCTGGCAGAGGAATACCTGTACCGCT-3V;
MMP-10, ABI Assay on Demand Ref. Hs00233987_m1 (FAM/MGB Taqman
probe); MMP-12, ABI Assay on Demand Ref. Hs00159178_m1 (FAM/MGB
Taqman probe).
The resulting data were analyzed with the comparative C
t
method for
relative gene expression quantification.
Cell Death Analysis
Propidium iodide sub-G
1
peak quantification (Nicoletti stain).
Following stimulation, dead cellcontaining supernatants were collected
and pooled with trypsinized living cells. Samples were then centrifuged and
washed with PBS. After a second centrifugation step, cells were permeabilized
with 70% ethanol, washed in PBS, and treated for 15 minutes at 37jC with
RNase A (Sigma, 1 mg/mL in PBS) and for 15 additional minutes at room
temperature with propidium iodide (PI, Sigma, 100 Ag/mL in PBS). Samples
where then analyzed on a FACS analyzer (Becton Dickinson, San Jose, CA).
The percentage of specific cell death was calculated using the following
formula:
S C=100 C 100 %specific cell death; where
S sample cell death and C control sample cell death:
Annexin Vstaining. The Annexin V-Biotin Apoptosis Detection Kit from
Oncogene Research Products (Lucerne, Switzerland) was used according to
the manufacturers protocol. Briefly, dead and living cells were pooled and
washed once with PBS. The Annexin V-Biotin conjugate was added to the
Cancer Research
Cancer Res 2005; 65: (10). May 15, 2005 4262 www.aacrjournals.org
sample in binding buffer [10 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl,
2.5 mmol/L CaCl
2
, 1 mmol/L MgCl
2
, 4% bovine serum albumin] and
incubated at room temperature in the dark for 15 minutes, centrifuged, and
resuspended in 0.5 mL binding buffer supplemented with 15 AL
streptavidin-FITC (eBioscience, San Diego, CA; 15 Ag/mL) and 10 AL PI
(Sigma, 1 Ag/mL). Cells were kept at 4jC in the dark and analyzed
immediately by flow cytometry.
Terminal deoxynucleotidyl transferasemediated nick-end labeling
staining. Terminal deoxynucleotidyl transferasemediated nick-end label-
ing (TUNEL) assays were done using the In situ Cell Death Detection Kit,
AP from Roche Diagnostics (Rotkreuz, Switzerland), according to the
manufacturers instructions. Briefly, cells were grown on glass chamber
slides. Following stimulation, the slides were washed in PBS, air-dried, and
fixed in 4% paraformaldehyde in PBS (pH 7,4). After a second washing
step, cells were permeabilized in 0.1% Triton X-100 with 0.1% sodium
citrate. Samples were labeled by adding fluorescein-dUTP conjugate and
terminal deoxynucleotidyl transferase and incubating for 1 hour at 37jC
in the dark. After several washes in PBS, the cells were examined under a
fluorescence microscope. The signal was converted using an AP-
conjugated anti-fluorescein antibody. Phosphatase activity was detected
by immersion of the slides in AP substratecontaining staining solution
[nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate mix in 0.1
mol/L Tris (pH 9,5), 0.05 mol/L MgCl
2
, and 0.1 mol/L NaCl] for 45 minutes
in the dark. The reaction was stopped by addition of double distilled
water.
Mitochondrial transmembrane potential (#W
m
) analysis. Briefly,
DMSO- or PMA/tetracycline-stimulated cells (5 10
5
cells per sample) were
washed twice with cold PBS containing 1% serum. For each sample, two
tubes were prepared, one containing cells stained with the carbocyanine dye
3,3V-dihexyloxacarbocyanine iodide (Molecular Probes, Eugene, OR) and the
other containing unstained cells that served as negative controls for
fluorescence quantification. After 15 minutes of incubation at 37jC, the
samples were washed in PBS containing 1% serum and resuspended in a
final volume of 200 AL PBS/1% FBS. Samples were kept at 4jC in the dark
and analyzed immediately by flow cytometry.
Crystal violet staining. DMSO-, PMA-, 5-FU-, or 5-FU/PMA-treated cells
were washed with PBS. Cells were stained with 0.5% Crystal violet solution
(25% methanol) for 15 minutes. After two washes with distilled water, cells
were lysed in 1% SDS and absorbance at 620 nm was measured. All 620-nm
absorbance values were expressed as a percentage of values obtained from
lysates of DMSO-treated cells, giving the percentage of cell viability. Cell
death rate was calculated by subtracting this percentage from 1.
Western Blotting
SDS-PAGE protein electrophoresis and Western blotting were done
according to standard procedures. For immunodetection of the human p53
protein, we used the DO-1 mouse monoclonal IgG2a antibody (Santa Cruz
Biotechnology, Santa Cruz, CA). For caspase-3/Yama/Apopain/CPP32, we
used the Ab-1 mouse monoclonal IgG1 antibody (Oncogene Research
Products), which recognizes both the zymogen and the cleaved form of the
caspase-3. For human h-actin, we used the AC-74 mouse monoclonal IgG2
a
antibody (Sigma). The secondary antibody was a horseradish peroxidase
conjugated antimouse IgG and the signal was revealed using the Enhanced
Chemiluminescence PLUS substrate solutions (Amersham Biosciences.,
Otelfingen, Switzerland).
mRNA Interference
For specific gene silencing, we used the small interfering RNA (siRNA)
technology. Cells grown to 30% to 40% confluence in 24-well culture plates
were transfected with siRNA solution consisting of oligofectamine reagent
and the Opti-MEM (both from Invitrogen). Following transfection, cells
were maintained in DMEM/10% FCS without antibiotics for 48 hours. We
used the following siRNA: control, sense sequence 5V-AAAGGAAACUG-
GAAAAAUGtt-3Vand antisense sequence 5V-CAUUUUUCCAGUUUCCUUUtt-3V;
antiMMP-9, sense sequence 5V-CAUCACCUAUUGGAUCCAAtt-3V and
antisense sequence 5V-UUGGAUCCAAUAGGUGAUGtt-3V; antiMMP-10,
sense sequence 5V-GGAGGACUCCAACAAGGAUtt-3Vand antisense sequence
5V-AUCCUUGUUGGAGUCCUCCtc-3V; antiMMP-12, sense sequence 5V-
GGAAACAUUAUAUCACCUAtt-3Vand antisense sequence 5V-UAGGUGAU-
AUAAUGUUUCCtc-3V.
All siRNAs were synthesized by Ambion, Inc. (Huntingdon, United
Kingdom).
Gelatin Zymography
After cell stimulation, the serum-free supernatant was concentrated with
centricon column with a 30-kDa cutoff value (Millipore, Volketswil,
Switzerland). The concentrated solution was mixed with Laemmli sample
buffer, without h-mercaptoethanol, and loaded onto a gelatin (1 mg/mL)
containing 10% SDS-PAGE gel. Following the completion of electrophoresis,
the gel was washed in Triton X-100 solution and the proteases were
activated overnight at 37jC in 5 mmol/L CaCl
2
and 50 mmol/L Tris (pH 8).
The gel was then stained with Coomassie blue.
Results
To address the mechanisms that underlie tumor cell resistance to
apoptosis and to elucidate the effect of PKC activation on p53-
induced proapoptotic signaling in human cancer cells, we selected
the SW480 cell line established from a primary human colon
adenocarcinoma. These cells express a p53 protein containing two
distinct mutations: G273A (ref. 23; converting an arginine to a
histidine residue) andC309T(converting a proline toa serine residue;
ref. 27), which result in p53 inactivation. To study the effect of wt p53
overexpressioninSW480 cells, we applieda tetracycline-regulatedwt
p53 expression system (28). PKC activation was achieved by
exogenous administration of the phorbol ester PMA, which is a
major activator of PKCs as well as of other signaling pathways,
including mitogen-activated protein kinases and nuclear factor nh.
SMF1 cells overexpress wild-type human p53 under the
control of tetracycline (Tet-ON system) and initiate a cell
death programme accelerated by phorbol 12-myristate 13-
acetate stimulation. SMF1 cells were generated by genetically
modifying SW480 cells to stably express the tetracycline repressor
protein allowing conditional expression of wt p53. Expression of
exogenous p53 was achieved by addition of tetracycline to the
culture medium (Fig. 1A). DMSO and PMA stimulation (Fig. 1A,
lanes 1 and 3, respectively) had no effect on endogenous p53
expression in SMF1 cell lysates, whereas lysates from SMF1 cells
stimulated with either tetracycline alone or tetracycline and PMA
show expression of both the endogenous and the wild-type
exogenous protein (Fig. 1A, lanes 2 and 4, respectively). Twenty-
four hours following induction of expression, p53-dependent cell
death was almost negligible (mean 1%, Fig. 1B). Stimulation with
PMA alone was not accompanied by significant induction of cell
death, but costimulation with tetracycline and PMA resulted in 73%
cell death at 24 hours (Fig. 1B). Observations by light microscopy
indicated the presence of detectable cell death 12 hours following
combined p53 induction and PMA stimulation in SMF1 cells (data
not shown). Thus, PMA treatment sensitized SMF1 cells to p53-
dependent cell death. The notion that this effect was p53 dependent
and not related to some property of the tetracycline molecule itself
is supported by the observation that PMA-induced sensitization did
not occur in tetracycline-treated parental SW480 cells that do not
express wt p53 (Fig. 1B). In addition, tetracycline stimulation of
SW480 cells stably transfected with tetracycline repressor and the
p53-independent gene PLSCR1 did not result in a higher ratio of cell
death than in the parental SW480 cells (data not shown).
To ensure that the observed sensitization of p53-mediated cell
death by PMA was not a cell typespecific event, we tested the
effect of p53 induction in the presence and absence of PMA in the
EB-1 colon carcinoma cell line (24) engineered to express wt p53
MMP-9 and MMP-10 Inhibit p53/PKC-Induced Apoptosis
www.aacrjournals.org 4263 Cancer Res 2005; 65: (10). May 15, 2005
gene under the control of the metallothionein MT-1 promoter.
Unlike SMF1 cells, which contain mutated p53, EB-1 cells lack
endogenous p53 expression. Treatment of EB-1 cells with ZnCl
2
led to a robust induction of p53 (Fig. 1C) accompanied by 20%
cell death at 24 hours, whereas costimulation with ZnCl
2
and
PMA resulted in 40% cell death at the same time point (Fig. 1C).
To determine whether the observed effect of p53/PMA
costimulation occurs in a more physiologic context, we stimulated
HCT116 colon carcinoma cells, which constitutively express wt p53
(HCT
+/+
), and their p53
/
derivatives (HCT
/
) with the
anticancer drug 5-FU, a known p53 inducer. Treatment of HCT
+/+
cells with 5-FU augmented endogenous p53 expression and
induced 34% cell death at 24 hours (Fig. 1D). Consistent with the
observations using SMF1 and EB-1 cells, 5-FUinduced cell death
was potentiated by costimulation with PMA (Fig. 1D). 5-FU
cytotoxicity was due, in part, to p53-independent events, as 5-FU
treatment of HCT p53
/
cells resulted in 15% to 19% cell death
(Fig. 1D). Importantly, however, PMA costimulation did not
enhance 5-FUmediated death of HCT
/
cells (Fig. 1D).
Cell death observed in phorbol 12-myristate 13-acetate/
tetracycline stimulated cells shows features of apoptosis,
including DNA cleavage, phosphatidylserine externalization,
and mitochondrial transmembrane potential #W
m
alteration.
Differentiation between apoptotic and necrotic cell death is
based on several morphologic and functional criteria. We
therefore applied a series of approaches to determine the type
of cell death that was induced by the combination of p53 and
PMA. Analysis of genomic DNA from dying cells revealed a DNA
ladder (data not shown) which is a hallmark of apoptosis. p53/
PMA costimulation was observed to induce several other
apoptosis-specific molecular and cellular features. Thus, TUNEL
staining (Fig. 2A) showed the presence of nick-containing DNA
in the PMA/tetracycline-stimulated cells, which occurs in most
forms of apoptosis as a result of endonuclease activation.
Annexin V-biotin conjugate reactivity revealed externalization of
phosphatidylserine (29, 30), another characteristic feature of
programmed cell death (Fig. 2B). Similar phosphatidyl serine
externalization was observed in EB-1 and HCT
+/+
cells in
response to ZnCl
2
and 5-FU stimulation, respectively (data not
shown). Finally, using a mitochondrial membrane carbocyanine
dye (Fig. 2C), we could show that p53/PMA altered the
mitochondrial transmembrane potential DW
m
(31, 32), in SMF1
cells, suggesting that activation of the intrinsic mitochondrial
apoptotic pathway may contribute to cell death induced by the
combined effect of p53 and PMA.
Selection of S1R6 cells that are partially resistant to p53/
phorbol 12-myristate 13-acetate costimulation induced apo-
ptosis. Because all three cell types displayed a similar response to
p53/PMA costimulation, we selected SMF1 cells for subsequent
experiments. Although most of the SMF1 cells treated with
tetracycline and PMA underwent apoptosis, a small fraction
(<10%) survived. To address the mechanisms that underlie tumor
cell resistance to the synergistic cell death inducing effect of PMA
and p53, we subjected the surviving cells to several rounds of
Figure 1. Tetracycline-inducible p53 expression in SMF1 cells and PMA
potentiation of p53-induced cell death. A, Western blot analysis of SMF1
cell lysates using anti-human p53 and anti-human h-actin antibodies. SMF1
cells were stimulated with DMSO (lane 1), DMSO + 2 Amol/L tetracycline
(lane 2), 0.16 Amol/L PMA (lane 3) or PMA + tetracyclin (lane 4) for 24 hours.
B and C, sub-G
1
apoptotic DNA quantification by flow cytometry. B, SMF1
cells (black bars) and SW480 parental cells (white columns) were stimulated
with DMSO (D), PMA (P), tetracycline (T), or PMA + tetracycline (PT) for
24 hours. C, EB-1 cells were stimulated with DMSO (D), PMA (P), 100 Amol/L
ZnCl
2
(Z), or 100 Amol/L ZnCl
2
+ PMA (Z + P) for 24 hours. Inset, Western blot
analysis of p53 induction using the anti-p53 monoclonal antibody DO-1.
D, crystal violet staining: HCT 116 p53
+/+
(black columns) or HCT 116 p53
/
(white columns) were stimulated with DMSO (D), PMA (P), 50 Ag/mL 5-FU,
or 50 Ag/mL 5-FU + PMA (5-FU + P) for 24 hours. Inset, Western blot analysis
of p53 induction in HCT
+/+
and HCT
/
cells using the anti-p53 monoclonal
antibody DO-1. Columns, mean of experiments done in triplicate; bars, SD.
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Cancer Res 2005; 65: (10). May 15, 2005 4264 www.aacrjournals.org
tetracycline/PMA treatment and selection. The resulting S1R6 cell
line displayed markedly different sensitivity to PMA/p53 compared
with the parental SMF1 cells (Fig. 2). Thus, in the TUNEL assay
(Fig. 2A), PMA/tetracycline stimulation induced only weak staining
in S1R6 cells, whereas the majority of the SMF1 cells were strongly
positive (DMSO treated SMF1 and S1R6 cells, used as controls,
were TUNEL negative). Similarly, phosphatidylserine externaliza-
tion in S1R6 was weaker than in SMF1 (18% compared with 45%,
Fig. 2B). In addition, whereas there was little relevant mitochon-
drial transmembrane potential difference between DMSO- and
PMA/tetracycline-treated S1R6 cells (DMSO: 14% and PMA/
tetracycline: 16%), a major difference was observed in SMF1 cells
(DMSO: 9% and PMA/tetracycline: 49%, Fig. 2C). A cell death time
course (Fig. 2D) indicates that at 24 and 48 hours, the cell death
observed in S1R6 represents 21% and 46%, respectively, of that
induced in SMF1 cells.
Phorbol 12-myristate 13-acetate/tetracyclineinduced apo-
ptosis is caspase dependent. Most of the apoptotic signaling
pathways described thus far are mediated by the caspase family of
cysteine-proteases (33). The activated forms of these enzymes
cleave diverse targets that are important for several key cellular
functions, including DNA repair and cell structure maintenance,
and are also able to activate other caspases. We therefore
addressed their implication in PMA/p53-induced apoptosis.
Caspase-3 (Apopain/YAMA/CPP32), a member of the effector
caspase subfamily, was observed to be activated by PMA/p53, as
shown by the cleavage of the 32-kDa zymogen (Fig. 3). The active
17-kDa fragment was detectable only in the PMA/tetracycline-
stimulated SMF1 cell lysates after 24 hours (Fig. 3A). Cleavage was
not detected in SMF1 cell lysates at 8 hours or in S1R6 cell lysates
at any time. The 116-kDa form of poly(ADP-ribose) polymerase
(PARP), a nuclear protein implicated in DNA repair and known to
Figure 2. S1R6 cells display augmented resistance to PMA/tetracycline-induced apoptosis. A, TUNEL staining of SMF1 and S1R6 cells treated with DMSO or
0.16 Amol/L PMA + 2 Amol/L tetracycline (PT) for 24 hours. B, Annexin V staining of phosphatidylserine externalization. SMF1 (black column) and S1R6 (white column)
cells were stimulated with DMSO (D) or PMA + tetracycline (P + T) for 24 hours. Columns, mean of triplicate experiments; bars, SD. C, mitochondrial transmembrane
potential analysis by flow cytometry. SMF1 and S1R6 cells were treated with DMSO or PMA + tetracycline (PT) for 20 hours. Solid arrows, peaks corresponding
to cells with normal mitochondria; hatched arrows, peaks corresponding to cells with damaged mitochondria. D, sub-G
1
apoptotic DNA quantification by flow cytometry.
SMF1 cells (n) or S1R6 cells (.) were stimulated with PMA + tetracycline for 24 or 48 hours. Points, mean of three different experiments; bars, SD.
MMP-9 and MMP-10 Inhibit p53/PKC-Induced Apoptosis
www.aacrjournals.org 4265 Cancer Res 2005; 65: (10). May 15, 2005
be a target of at least caspase-3 and caspase-7 was also found to be
cleaved in SMF1 cells after PMA + tetracycline stimulation (data
not shown). Finally, using the generic caspase inhibitor, z-VAD-fmk
(34), which inactivates most members of the family, we observed
complete inhibition of PMA/tetracycline-induced apoptosis in S1R6
cells but incomplete inhibition in SMF1 cells (Fig. 3B).
Because activation of Bax, which leads to the release of
cytochrome c from mitochondria and caspase activation, is a key
mechanism underlying p53-mediated cell death, we tested the
effect of 5-FU in the presence and absence of PMA on HCT 116
Bax
/
cells. Cell death induced by 5-FU and 5-FU/PMA was lower
in HCT Bax
/
cells than in HCT Bax
+/
counterparts (Fig. 3C).
The observed cell death in HCT Bax
/
cells most likely cor-
responds to p53-independent cytotoxicity of 5-FU consistent with
the notion that Bax is implicated in apoptosis induced by p53 and
p53/PMA.
Phorbol 12-myristate 13-acetate sensitization to p53-
induced apoptosis is protein kinase C mediated. Because the
phorbol ester PMA is known to activate several members of the
PKC family in addition to other signaling pathways, we attempted
to identify the PKC family members implicated in the observed
PMA/p53-induced apoptosis using specific PKCs inhibitors,
including rottlerin (35), a novel PKC subfamily inhibitor, Go6976
(36, 37), a specific inhibitor of the classic PKCs a and h, and bis-
indolylmaleimide I (38), a broad classic PKC inhibitor. When
pretreated with either classic PKC inhibitor, both SMF1 and S1R6
cells displayed a marked reduction in cell death following PMA/
tetracycline stimulation (Fig. 4). In contrast, the novel PKC
inhibitor rottlerin enhanced PMA/tetracycline sensitivity of both
cell lines. These observations suggest that the potentiation of p53-
induced cell death by PMA in SW480 cells is mediated by PKCs of
the classic subfamily and antagonized by PKCs of the novel
subfamily.
S1R6 expresses higher levels of matrix metalloproteinases
after phorbol 12-myristate 13-acetate/tetracycline stimula-
tion. To address the mechanisms responsible for the observed
resistance of S1R6 cells to PKC/p53-induced apoptosis, we did a
microarray gene expression analysis of SMF1 and S1R6 cells.
First, we compared gene expression in DMSO- and PMA/
tetracycline-stimulated SMF1 cells. Among 474 genes that
displayed a >3-fold induction in cells treated with PMA/
tetracycline, were several p53 target genes, PKC-related genes,
and genes implicated in apoptosis (data not shown). P53 target
genes included PIG11, CDKN1A (encoding p21), and MDM2. The
gene encoding p53 itself, TP53, was up-regulated, and as
expected, genes encoding PKC family members, including PRKCA
and PRKCZ, and genes encoding proapoptotic proteins,
including BIK, CASP10, and APAF1 were also induced by PMA/
p53 (data not shown).
A second round of microarray analysis was done to compare gene
expression in SMF1- versus S1R6-PKC/p53stimulated cells. Ninety-
seven genes were found to display a >2 fold induction in S1R6 cells
(Table 1 and data not shown). At least two groups of genes of
potential interest were up-regulated. The first includes genes
encoding proteins involved in cell adhesion and cell-cell commu-
nication. The second includes genes encoding matrix metal-
loproteinases (MMP), and more specifically, MMP-9, MMP-10, and
MMP-12. Because of their implication in the regulation of cell
Figure 3. PMA/tetracycline-dependent apoptosis is mediated by caspase-3. A, Western blot analysis of SMF1 and S1R6 cell lysates using antihuman caspase-3
antibody. SMF1 and S1R6 cells were stimulated for 8 or 24 hours with DMSO (D) or 0.16 Amol/L PMA + 2 Amol/L tetracycline (PT). Representative of two independent
experiments. B, sub-G
1
apoptotic DNA quantification by flow cytometry. SMF1 cells (white columns) and S1R6 cells (black columns) were stimulated for 15 hours with
DMSO or 0.16 Amol/L PMA + 2 Amol/L tetracycline. z-VAD-fmk was added 3 hours before stimulation at a concentration of 100 Amol/L. Representative of three
independent experiments. C, crystal violet staining: HCT 116 Bax
+/
(black columns) or HCT 116 Bax
/
(white columns) cells were stimulated with DMSO (D), PMA
(P), 50 Ag/mL 5-FU, or 50 Ag/mL 5-FU + PMA (5-FU + P) for 24 hours. Columns, mean of experiments performed in triplicate; bars, SD.
Cancer Research
Cancer Res 2005; 65: (10). May 15, 2005 4266 www.aacrjournals.org
survival, we chose to address the functional role played by the
MMPs in response to PMA/p53 stimulation.
Matrix metalloproteinase activity plays a role in protein
kinase C/p53-induced apoptosis. To test the potential role of
MMP-mediated proteolysis in colon carcinoma cell response to
PMA/p53 signals, we first blocked MMP proteolytic activity using
two broad spectrum MMP inhibitors: the metal chelating agent
o-phenanthroline (PHE), a molecule known to interfere with the
MMP-zinc ion fixation (39), and GM6001 (40), a hydroxamic acid
isomer known to be a potent collagenase inhibitor. Figure 5A
illustrates the effect of pretreatment of SMF1 or S1R6 cells with
these drugs. PMA/tetracycline-induced apoptosis was augmented
in both cell lines following PHE compared with DMSO treatment
(64% and 36% for SMF1 and 53% and 24% for S1R6,
respectively). Similarly, treatment with GM6001 increased the
fraction of cells undergoing apoptosis in response to PMA/
tetracycline to 75% and 35% among SMF1 and S1R6 cells,
respectively. Thus, MMP activity seems to provide the tumor
cells with partial protection against apoptosis induced by p53/
PKC costimulation. To make sure that these effects were not
specific to the DNA degradation marker of apoptosis, we did
Annexin V binding analysis and found that both inhibitors affect
phosphatidylserine externalization in a similar way (data not
shown).
Matrix metalloproteinase 9 and matrix metalloproteinase
10specific small interfering RNAs sensitize S1R6 cells
towards phorbol 12-myristate 13-acetate/tetracyclinein-
duced cell death. To identify the MMPs that confer protection
to S1R6 cells from PKC/p53-induced cell death, we did siRNA-
transient transfection experiments (41) to specifically silence MMP-
9, MMP-10, and MMP-12 transcripts. Each siRNA induced a
reduction of the corresponding mRNA expression as detected by
real-time PCR analysis (Fig. 5B-D). MMP-9 and MMP-10 gene
silencing was observed to be followed by corresponding cell
sensitization to PMA/tetracycline-induced cell death (Fig. 5B
and C). MMP-9 and MMP-10specific siRNAs induced 257% and
161% sensitization, respectively. In contrast, MMP-12 siRNA failed
to modify S1R6 cell sensitivity to PKC/p53-induced apoptosis
(Fig. 5D). Thus, consistent with the results obtained using chemical
MMP inhibitors, MMP-9 and MMp-10 expression may play
a protective role in colon cancer against PKC/p53-induced
apoptosis.
This notion is supported by the observation that sensitization of
EB-1 cells, which express MMP-9, to ZnCl
2
/PMA-mediated
apoptosis could also be achieved by MMP-9specific siRNA, with
a 30% increase in cell death (data not shown). By contrast, HCT
+/+
cells, which are negative for MMP-9 expression, as assessed by
gelatin zymography, displayed no sensitization to p53-mediated
cell death following transfection with MMP-9 siRNA (data not
shown).
p53 down-regulates expression of matrix metalloproteinase
9 at the transcriptional level. p53 is known to induce the
expression of several MMPs, but its effect on MMP-9 has not been
fully explored. To determine the effect of p53 on MMP-9
expression, we quantified the mRNA expression of the gene
encoding MMP-9 by real-time PCR in response to p53 induction.
Surprisingly, p53 was observed to be a negative regulator of the
Figure 4. PMA sensitization to p53-induced apoptosis is PKC mediated. Sub-G
1
apoptotic DNA quantification by flow cytometry. A, SMF1 cells were stimulated
for 24 hours with DMSO or 0.16 Amol/L PMA + 2 Amol/L tetracycline. PKC
inhibitors bisindolylmaleimide I (1 Amol/L), Go 6976 (1 Amol/L), or rottlerin
(25 Amol/L) were added 3 hours before stimulation (white columns, no
pretreatment with PKC inhibitors; black columns, pretreatment with Go 6976;
dotted columns, pretreatment with bisindolylmaleimide I; hatched columns,
pretreatment with rottlerin). B, S1R6 cells were treated as in (A). Representative
of three independent experiments.
Table 1. S1R6 cells display augmented MMP expression
compared with SMF1 cells
Cluster Overexpression Gene Description
MMPs 4 MMP-9 MMP-9
(gelatinase B)
4 MMP-10 MMP-10
(stromelysin 2)
3 MMP-12 MMP-12
(macrophage
elastase)
Cell adhesion 8 CLDN7 Claudin 7
5 CDH1 E-cadherin
4 OCLN Occludin
2 CLDN1 Claudin1
2 CLDN11 Claudin 11
NOTE: SMF1 and S1R6 cells were treated for 8 h with 0.16 Amol/L
PMA + 2 Amol/L tetracyclin, and mRNA was extracted and amplified
for microarray analysis. Clusters of genes that were upregulated in
PMA/tetracyclin-stimulated S1R6 cells compared with PMA/tetracy-
clin-treated SMF1 cells. The fold increase was rounded off to the
nearest whole number.
MMP-9 and MMP-10 Inhibit p53/PKC-Induced Apoptosis
www.aacrjournals.org 4267 Cancer Res 2005; 65: (10). May 15, 2005
MMP-9 gene. Figure 6A shows relative quantification using the
cyclophilin A mRNA as endogenous control. As expected, PMA
induced MMP-9 (15.6-fold), but the addition of tetracycline
together with PMA reduced this induction to 6.07-fold. It is
important to note that the mRNA was extracted from PMA +
tetracycline stimulated SMF1 cells before the onset of cell death.
To eliminate the possibility of an effect due to intrinsic properties
of the tetracycline molecule (independent of p53 induction),
we did the same experiment in the SW480 parental cells. We
observed a similar induction by PMA, but the addition of
tetracycline failed to down-regulate MMP-9 (data no shown).
These observations suggest that p53 is a negative regulator of
MMP-9 mRNA expression. Accordingly, MMP-9 protein levels, as
assessed by gelatin zymography, were decreased by p53 (Fig. 6B).
Discussion
Inactivation of p53-dependent cell death by mutation or
deletion of the TP53 gene is one of the key events in
carcinogenesis. Nevertheless, many malignant tumors retain p53
expression and function, and an understanding of the mecha-
nisms that potentiate p53-mediated tumor cell death as well as
those that tumor cells deploy to survive in the face of p53
signals may have important implications in cancer treatment.
One of the principal observations of the present work is that
PMA treatment of three different colon carcinoma cell lines
expressing inducible exogenous or constitutive endogenous p53
accelerates and enhances cell death induced by p53 expression
alone. Importantly, cell death triggered as a result of 5-FU
induced endogenous p53 expression, which reflects a more
physiologic situation than tetracycline- or ZnCl
2
-mediated
induction of exogenously introduced p53, was augmented in
the presence of PMA.
Several criteria support the notion that the observed cell death
was apoptotic. First, phosphatidylserine, normally present in the
inner layer of the cytoplasmic membrane, was externalized in all
three cell types. Second, SMF1 cells were positive for TUNEL
staining. Third, chromatolysis and DNA fragmentation occurred in
a nonstochastic manner. Fourth, caspases were involved in this
process as shown by the cleavage of PARP and caspase-3. However,
z-VAD-fmk was not able to completely abrogate SMF1 cell death
even at a high concentration (100 Amol/L), suggesting that the cell
death we observed is predominantly but not exclusively caspase
dependent. Interestingly, mitochondria were also affected as shown
by the modification of the mitochondrial transmembrane potential,
suggesting that the intrinsic mitochondrial apoptotic pathway is
also involved here. As might be expected, the use of HCT116 cells
Figure 5. PKC/p53-induced apoptosis is inhibited by MMP inhibitors.
A, sub-G
1
apoptotic DNA quantification by flow cytometry. SMF1 and
S1R6 cells were stimulated for 24 hours with 0.16 Amol/L PMA + 2 Amol/L
tetracycline. No inhibitors (white columns) or MMP inhibitors, 100 Amol/L
PHE (black columns), 100 Amol/L GM

(dotted columns), and 100 Amol/L GM

+
(hatched columns) were added 3 hours before stimulation. Representative
of three independent experiments. B, sub-G
1
apoptotic DNA quantification
by flow cytometry (% specific cell death) as a function of MMP expression.
S1R6 cells were transfected with control siRNA (V) or MMP-9specific
siRNA (M) and 48 hours later, stimulated with 0.16 Amol/L PMA + 2 Amol/L
tetracycline for 24 hours. Expression was determined by quantification of
MMP-9 mRNA by real-time PCR 48 hours post-transfection in S1R6
cells (relative expression with respect to control siRNA transfected cells).
C and D, experiment was performed as in (B) but using MMP-10 siRNA
(C) and MMP-12 siRNA (D). Representative of three independent experiments.
Cancer Research
Cancer Res 2005; 65: (10). May 15, 2005 4268 www.aacrjournals.org
with homozygous deletion of Bax showed that Bax is involved in
p53/PMA-mediated apoptosis.
The mechanism that underlies PMA-mediated potentiation of
p53-induced cell death is consistent with PKC involvement.
Phorbol esters activate the PKC family whose members
phosphorylate and thereby regulate the activity of numerous
cellular substrates, possibly including p53 itself. p53 activity is
regulated, at least in part, by phosphorylation at several levels.
First, p53 stability is increased by the activity of several kinases,
including ATM, ATR, Chk1, and Chk2 (42), which directly
phosphorylate p53 and/or its negative regulator MDM2 (43).
Second, by phosphorylating sites at its NH
2
terminus, kinases can
inactivate a nuclear export signal and thereby regulate the cellular
localization of p53 (44). Third, the activity itself of p53 is prone to
regulation by kinases. Thus, phosphorylation of p53 has been
shown to control both its DNA binding ability and its
transcriptional activity. In addition, p53 can be phosphorylated
by the c-Jun NH
2
-terminal protein kinases, known to be activated
by PMA-induced PKC activity (45), and by the CAK (46) complex
(CDK7, cyclin H, and p36MAT1). Changes in p53 phosphorylation
may therefore provide at least one mechanism to explain the
PKC-mediated sensitization of p53-dependent cell death observed
in this study.
Members of the PKC family have been shown to interact with
p53 and modulate p53-induced apoptosis and cell growth arrest.
The classic PKCs are able to induce p53 translocation to the
nucleus and the G
1
cell cycle control checkpoint (47) and PKCy has
been shown to induce an accumulation of p53 (17). Moreover, p53
contains a PKC binding site in the central oligomerization domain
and can be directly phosphorylated by PKCs at Ser
378
and by PKCa
and PKC~ at Ser
371
(48). In addition, a recent study has shown
activation by cyclin-dependent kinase 2/cyclin A and PKC of the
p53-specific DNA sequence binding ability (16).
Because all novel PKCs and classic PKCs contain a phorbol ester
binding motif, it would seem reasonable to expect that either or
both of these subfamilies are implicated in the potentiation of p53-
mediated cell death. Consistent with this notion, both classic PKC
and novel PKC inhibitors affected p53-dependent apoptosis but in
opposite directions. Thus, bis-indolylmaleimide I, which inhibits
PKCa, PKCh
I
, PKCh
II
, PJCg, PKCy, and PKCq isozymes, was the
strongest inhibitor of PMA-dependent cell death potentiation, with
Go6976, which inhibits PKCa and PKCh
I
isozymes, displaying a
lesser degree of inhibition. Based on these findings, it would seem
that the observed PMA-associated cell death enhancement was
mediated by the PKCa, PKCh
I
, PKCh
II
, PKCg, and PKCq isozymes.
In contrast, PKCy and PKCu seemed to have an antiapoptotic effect
in p53-stimulated SW480 cells.
PKC targets independent of the p53 may contribute to the
observed synergistic proapoptotic effect of PKC activation and p53
induction (Fig. 7). The function of PKCy, which is both a substrate
and an activator of caspase-3, has been suggested to be linked to
the mitochondrial release of cytochrome c during apoptosis.
Consistent with these observations, the phorbol ester 12-O-
tetradecanoylphorbol-13-acetate induces the release of cytochrome
c and activation of caspase-3. Our observation that inhibition
of PKCy by rottlerin sensitized colon carcinoma cells to p53 in
our in vitro system may therefore be somewhat surprising and
would seem to contradict the evidence suggesting that PKCy is
proapoptotic. However, rottlerin has been suggested to inhibit
the intrinsic cell death pathway but to potentiate extrinsic cell
death pathways (49). Thus, depending on the type of proapoptotic
stimulus, rottlerin may be expected to exert a stimulatory or
inhibitory effect. In the case of our model system, the observed
effect of rottlerin is consistent with the possibility that even if there
was activation of the intrinsic pathway, as suggested by changes
in the mitochondrial transmembrane potential, the extrinsic pro-
apoptotic pathway was likely to be dominant.
Selection of SMF1 cells that were resistant to PMA/p53-induced
cell death (termed S1R6), allowed the identification of potential
mechanisms that underlie the resistance. S1R6 cells were observed
to retain tetracycline-regulated p53 expression, such that their
resistance could not be attributed to loss of p53 induction.
However, comparison of the gene expression profiles of S1R6 and
SMF1 cells showed up-regulation of a relatively small number of
genes among which two groups seemed of immediate interest:
MMPs and cell adhesion molecules.
Members of the MMP family, also called matrixins, are cysteine
proteases with zinc iondependent proteolytic activity (reviewed in
refs. 5052). Thus far, >20 MMPs have been identified in mammals
and are currently grouped according to structural features (51). The
majority of MMPs are secreted into the extracellular matrix (ECM),
a subset being membrane bound by virtue of a transmembrane or
PI-linked domain (51). All are produced as inactive zymogens and
are activated by proteolysis. MMPs were originally described as
being primarily involved in the proteolysis of ECM components
and therefore believed to primarily mediate tissue remodeling.
However, it is becoming increasingly clear that these enzymes play
a critical role in a broad range of physiologic and pathologic
processes. Thus, MMPs can cleave and regulate the activity of a
host of growth factors, chemokines, and cytokines as well as cell
surface adhesion receptors and proteoglycans involved in intercel-
lular communication and cell migration that are directly implicat-
ed in tumor progression and metastasis (5052).
Functional analysis in the present study showed that
expression of MMP-9, and to a lesser extent that of MMP-10
but not MMP-12, confer partial resistance to PMA/p53-mediated
Figure 6. p53 is a negative regulator of MMP-9 mRNA and protein expression.
A, relative quantification of MMP-9 mRNA expression by real-time PCR. SMF1
cells were stimulated with DMSO (D), 0.16 Amol/L PMA (P), 2 Amol/L
tetracycline (T), or PMA + tetracycline (PT) for 24 hours. The 2DDCT value
represents MMP-9 expression relative to that in DMSO treated cells. B, MMP-9
activity as illustrated by gelatin zymography. SMF1 cells were treated as
indicated for 24 hours. Columns, mean of experiments performed in triplicate;
bars, SD.
MMP-9 and MMP-10 Inhibit p53/PKC-Induced Apoptosis
www.aacrjournals.org 4269 Cancer Res 2005; 65: (10). May 15, 2005
cell death. A potential issue in our approach is the use of
tetracycline to induce p53, because tetracycline and its derivative
doxycyline have been shown to inhibit MMP activity and MMP-2
gene expression. However, inhibition occurs with an IC
50
of 30 to
300 Amol/L, whereas we used a concentration of 2 Amol/L, well
below the reported inhibitory levels. Accordingly, gelatin
zymography assays showed no difference between lysate and
supernatant MMP activity from cells treated or not with
tetracycline. The presence of tetracycline therefore seems to
have no effect on the activity of MMP-9 in our system at the
concentration used.
Similar to PKC, MMP activity may have both proapoptotic and
antiapoptotic effects. Proapoptotic effects can be due to the
proteolytic degradation of ECM proteins, such as laminin, that
serve as ligands of the integrin class of cell surface adhesion
receptors (5052). In the absence of ligand, specific subclasses of
integrins can no longer trigger signals necessary for normal
epithelial and endothelial cell survival, resulting in a form of
apoptosis known as anoikis. Conversely, ECM degradation can
result in the release and augmented bioactivity of potent survival
factors, such as insulin-like growth factor I (IGF-I) that may
directly promote tumor cell resistance to proapoptotic signals and
favor tumor growth. MMP-mediated cleavage of the integral
membrane ligand of the death-inducing receptor Fas (FasL) can
result in increased or reduced apoptosis depending on the
physiologic context (5052). The proapoptotic or antiapoptotic
effect of any given MMP may therefore depend on the local cellular
production and relative availability of proteolytically released and
activated death-inducing and/or growth and survival factors.
There are several potential mechanisms whereby MMP-9 may
promote tumor cell resistance to proapoptotic stimuli. MMP-9
can release and activate transforming growth factor h (TGF-h; ref.
50), cleave several chemokines and activate other MMPs including
MMP-1 (4951). TGF-h can promote survival in transformed cells,
as can the chemokine IL-8, whose function is potentiated by
MMP-9mediated proteolysis (5052). Activation of MMP-1 leads
to the release of IGF-I from ECM-sequestered IGF binding
proteins (5052). However, the full range of MMP substrates is far
from elucidated, and it is quite possible that proteolytic
activation or inactivation of other regulators of cell survival
may be implicated in the observed antiapoptotic effect of MMP-9
expression. High expression of MMP-9 is often associated with
poor prognosis in cancer patients and, in at least some tumor
types, correlates with increased angiogenesis and metastatic
proclivity (5052).
Interestingly, regulation of MMP expression is reported to be
subjected, at least in part, to p53 control. Thus, p53 has been
shown to induce transcriptional activation of the MMP-2 (53) gene
promoter and to attenuate the production of MMP-1 (54) and
MMP-13 (55). Consistent with earlier work by others (53), we have
found that MMP-2 expression is enhanced by p53 (data not shown).
In the present study, we observed that p53 represses the human
MMP-9 gene, which in and of itself may contribute to p53-induced
cell death. It is also interesting to note that the two MMPs (MMP-2
Figure 7. Schematic representation of the effect of PKC isoforms and MMP-9 on p53-mediated cell death. PKCs directly affect p53 phosphorylation, localization,
and expression at the protein level. Classic PKCs such as the a and h isoforms synergize with p53 in inducing apoptosis, by directly influencing p53 activity and/or
promoting nonp53-mediated proapoptotic events, whereas PKCy has an antagonistic effect. In addition to inducing intracellular proapoptotic pathways, p53 inhibits
transcription of MMP-9, which in its active form, promotes cell survival by proteolytically activating and/or inhibiting extracellular survival and proapoptotic factors,
respectively. These factors may function in an autocrine and/or paracrine manner.
Cancer Research
Cancer Res 2005; 65: (10). May 15, 2005 4270 www.aacrjournals.org
and MMP-9) that form the gelatinase subfamily of MMPs, based on
structural similarities and shared substrate specificity (5052), are
differentially regulated by p53.
The second group of genes of potential interest found to be
overexpressed in S1R6 cells encode proteins involved in cell-cell
communication and/or adhesion. Several are components of tight
junctions (zonula occludens) and contribute to the epithelial
barrier function (claudin and occludin family of proteins),
whereas E-cadherin is part of the zonula adherens junctions that
generate signals sensed by p53 (56). Although further work will be
required to address their role, it is conceivable that elevated
expression of these proteins contributes to resistance to PKC/p53-
dependent apoptosis.
Taken together, our observations provide evidence that PKCs are
able to potentiate p53-induced apoptosis and that expression of
MMP-9 and MMP-10 confers partial protection against the proapop-
totic PKC/p53 signals. Understanding of the precise mechanism of
PKC/p53 synergy and identification of the relevant PKC targets may
help develop therapeutic strategies that, in combination with selective
MMP inhibitors, could augment the effectiveness of tumor cell
elimination.
Acknowledgments
Received 8/16/2004; revised 12/15/2004; accepted 1/4/2005.
Grant support: National Center for Competence in Research Molecular Oncology,
Swiss National Scientific Foundation grant 31-65090.01, and Oncosuisse grant 1267-08-
2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Josiane Wyniger and Otto Hagenbuchle for providing the cDNA
microarrays, Bert Vogelstein for the HCT cell lines, Phil Shaw for EB-1 cells, Richard
Iggo for the wt human p53 cDNA, and Nathalie Mayran for her advice regarding siRNA
experiments.
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