Matrix Metalloproteinases 9 and 10 Inhibit Protein Kinase C–Potentiated, p53-Mediated Apoptosis

8/12/2019 Matrix Metalloproteinases 9 and 10 Inhibit Protein Kinase CPotentiated, p53-Mediated Apoptosis

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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 theprotective 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

TP53gene. 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), p21WAF1 , 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 p53degradation (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-XL (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 Ser378 residue (16). It has also

been proposed that PKCy regulates p53 expression at thetranscriptional 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, hI, hII, 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 Eisotypes 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 byeach 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, PKChI, and PKChIIare 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: [email protected] American Association for Cancer Research.

www.aacrjournals.org 4261 Cancer Res 2005; 65: (10). May 15, 2005

Research Article


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sample in binding buffer [10 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl,

2.5 mmol/L CaCl2, 1 mmol/L MgCl2, 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 MgCl2, 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 (#Wm) analysis. Briefly,

DMSO- or PMA/tetracycline-stimulated cells (5 105 cells per sample) werewashed 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% Cr ystal 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-nmabsorbance 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 BlottingSDS-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 humanh-actin, we used the AC-74 mouse monoclonal IgG2aantibody (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 InterferenceFor 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-3V and antisense sequence 5V-UAGGUGAU-

AUAAUGUUUCCtc-3V.

All siRNAs were synthesized by Ambion, Inc. (Huntingdon, United

Kingdom).

Gelatin ZymographyAfter 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 CaCl2and 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

histidineresidue)and C309T (converting a prolineto a serineresidue;ref. 27), which result in p53 inactivation. To study the effect of wt p53

overexpression in SW480cells, we applied a tetracycline-regulated wt

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 celldeath 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 PLSCR1did 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



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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 ZnCl2led to a robust induction of p53 (Fig. 1C) accompanied by 20%

cell death at 24 hours, whereas costimulation with ZnCl2 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-FUtreatment 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#Wmalteration.

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 ZnCl2 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 DWm (31, 32), in SMF1

cells, suggesting that activation of the intrinsic mitochondrialapoptotic 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

(


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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 stainingin 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 enzymescleave 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 or0.16Amol/L PMA + 2Amol/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 transmembranepotential analysis by flow cytometry. SMF1 and S1R6 cells were treated with DMSO or PMA + tetracycline (PT) for 20 hours. Solid arrows, peaks correspondingto cells with normal mitochondria;hatched arrows, peaks corresponding to cells with damaged mitochondria. D, sub-G1apoptotic 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.




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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 observedcomplete 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 PRKCAand 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-3antibody. SMF1 and S1R6 cells were stimulated for 8 or 24 hours with DMSO ( D) or 0.16Amol/L PMA + 2Amol/L tetracycline (PT). Representative of two independentexperiments.B, sub-G1apoptotic DNA quantification by flow cytometry. SMF1 cells (white columns) and S1R6 cells (black columns) were stimulated for 15 hours withDMSO 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 threeindependent 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

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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 5Aillustrates 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 % a nd 36% for S MF1 and 53% an d 2 4% for S1R 6,

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 areduction 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. 5Band 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 ofEB-1 cells, which express MMP-9, to ZnCl2/PMA-mediatedapoptosis could also be achieved by MMP-9specific siRNA, witha 30% increase in cell death (data not shown). By contrast, HCT+/+

cells, which are negative for MMP-9 expression, as assessed bygelatin zymography, displayed no sensitization to p53-mediatedcell death following transfection with MMP-9 siRNA (data notshown).

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-G1apoptotic DNA quantification by flow cytometry. A, SMF1 cells were stimulatedfor 24 hours with DMSO or 0.16 Amol/L PMA + 2 Amol/L tetracycline. PKCinhibitors bisindolylmaleimide I (1 Amol/L), Go6976 (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 Go6976;dotted columns, pretreatment with bisindolylmaleimide I; hatched columns,pretreatment with rottlerin). B, S1R6 cells were treated as in ( A). Representativeof 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 75 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.




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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. Weobserved 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 ofMMP-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 incarcinogenesis. Nevertheless, many malignant tumors retain p53expression and function, and an understanding of the mecha-

nisms that potentiate p53-mediated tumor cell death as well asthose that tumor cells deploy to survive in the face of p53signals 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

physi ologic situation than tetracycline- or ZnCl2-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 theinner 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-G1 apoptotic DNA quantification by flow cytometry. SMF1 andS1R6 cells were stimulated for 24 hours with 0.16 Amol/L PMA + 2 Amol/Ltetracycline. No inhibitors (white columns) or MMP inhibitors, 100 Amol/LPHE (black columns), 100Amol/L GM (dotted columns), and 100Amol/L GM+

(hatched columns) were added 3 hours before stimulation. Representativeof three independent experiments. B, sub-G1apoptotic DNA quantificationby flow cytometry (% specific cell death) as a function of MMP expression.S1R6 cells were transfected with control siRNA (V) or MMP-9specificsiRNA (M) and 48 hours later, stimulated with 0.16 Amol/L PMA + 2 Amol/Ltetracycline for 24 hours. Expression was determined by quantification ofMMP-9 mRNA by real-time PCR 48 hours post-transfection in S1R6cells (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.

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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 directlyphosphorylate p53 and/or its negative regulator MDM2 (43).

Second, by phosphorylating sites at its NH2 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 NH2-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 G1cell cycle control checkpoint (47) and PKCyhas

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 Ser378 and by PKCa

and PKC~ at Ser371 (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, PKChI, PKChII, PJCg, PKCy, and PKCq isozymes, was the

strongest inhibitor of PMA-dependent cell death potentiation, with

Go6976, which inhibits PKCa and PKChI isozymes, displaying a

lesser degree of inhibition. Based on these findings, it would seem

that the observed PMA-associated cell death enhancement wasmediated by the PKCa, PKChI, PKChII, PKCg, and PKCqisozymes.

In contrast, PKCyand PKCuseemed 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 andwould 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 theirresistance 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 asbeing 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. SMF1cells were stimulated with DMSO (D), 0.16Amol/L PMA (P), 2 Amol/Ltetracycline (T), or PMA + tetracycline (PT) for 24 hours. The 2DDCT value

represents MMP-9 expression relative to that in DMSO treated cells.B, MMP-9activity as illustrated by gelatin zymography. SMF1 cells were treated asindicated for 24 hours. Columns, mean of experiments performed in triplicate;bars, SD.




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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 IC50 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 adhesionreceptors (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 integralmembrane 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 withpoor 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-9gene, 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/orpromoting nonp53-mediated proapoptotic events, whereas PKCy has an antagonistic effect. In addition to inducing intracellular proapoptotic pathways, p53 inhibitstranscription 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.

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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-cellcommunication 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 thatgenerate signals sensed by p53 (56). Although further work will berequired 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 ofMMP-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 mayhelp 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 pagecharges. This article must therefore be hereby marked advertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Josiane Wyniger and Otto Hagenbuchle for providing the cDNAmicroarrays, Bert Vogelstein for the HCT cell lines, Phil Shaw for EB-1 cells, RichardIggo for the wt human p53 cDNA, and Nathalie Mayran for her advice regarding siRNAexperiments.

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Matrix Metalloproteinases 9 and 10 Inhibit Protein Kinase C–Potentiated, p53-Mediated Apoptosis

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