TAT-RasGAP Requiresp53andPUMAto ... · TAT-RasGAP 317-326 Requiresp53andPUMAto SensitizeTumorCellstoGenotoxins DavidMichodandChristianWidmann DepartmentofPhysiologyandDepartmentofCellBiologyandMorphology

TAT-RasGAP317-326 Requires p53 and PUMA toSensitize Tumor Cells to Genotoxins

David Michod and Christian Widmann

Department of Physiology and Department of Cell Biology and Morphology,Lausanne University, Lausanne, Switzerland

AbstractAlthough chemotherapy has revolutionized cancer

treatment, the associated side effects induced by lack of

specificity to tumor cells remain a challenging problem.

We have previously shown that TAT-RasGAP317-326,

a cell-permeable peptide derived from RasGAP,

specifically sensitizes cancer cells to the action of

genotoxins. The underlying mechanisms of this

sensitization were not defined however. Here, we report

that TAT-RasGAP317-326 requires p53, but not the Ras

effectors Akt and extracellular signal-regulated kinase,

to mediate its tumor sensitization abilities. The

TAT-RasGAP317-326 peptide, although not modulating

the transcriptional activity of p53 or its phosphorylation

and acetylation status, nevertheless requires a

functional p53 cellular status to increase the sensitivity

of tumor cells to genotoxins. Genes regulated by p53

encode proapoptotic proteins, such as PUMA, and cell

cycle control proteins, such as p21. The ability of

TAT-RasGAP317-326 to sensitize cancer cells was found

to require PUMA but not p21. TAT-RasGAP317-326 did

not affect PUMA levels, however, but increased

genotoxin-induced mitochondrial depolarization and

caspase-3 activation. These results indicate that

TAT-RasGAP317-326 sensitizes tumor cells by activating

signals that intersect with the p53 pathway downstream

of, or at the level of, proapoptotic p53 target gene

products to increase the activation of the mitochondrial

death pathway. (Mol Cancer Res 2007;5(5):497–507)

IntroductionSince its introduction as a cancer therapyf50 years ago (1),

chemotherapy has remained a widely used and efficient

antitumor procedure. Most chemotherapeutic agents kill tumor

cells by inducing DNA damage and are therefore called

genotoxins. Ideally, genotoxins should only target cancer cells

to induce their demise with minimal collateral damage to

normal cells. In reality, however, the effectiveness of chemo-

therapy has suffered from a range of confounding factors,

including systemic toxicity due to a lack of specificity, rapid

drug metabolism, and both intrinsic and acquired drug

resistance. The efficiency of chemotherapy would therefore

be ameliorated by increasing the specificity of chemotherapeu-

tic agents toward cancer cells.

We have recently described a peptide derived from the

RasGAP protein that increases the sensitivity of cancer cells

to genotoxins. RasGAP, a regulator of Ras, bears two caspase-

3 cleavage sites (2, 3). At low levels of caspase activity,

RasGAP is cleaved into two fragments (4). The NH2-terminal

fragment (fragment N) seems to be a general blocker of

apoptosis downstream of caspase activation and is in fact

crucially required for the survival of stressed cells (5). At

higher levels of caspase activity, fragment N is further cleaved

into fragments N1 and N2, abrogating its antiapoptotic

activity (4, 6). Fragment N2, in contrast to fragment N,

potently sensitizes cancer cells toward genotoxin-induced

apoptosis (4, 6). A minimal sequence within fragment N2 that

can still sensitize tumor cells to the action of genotoxins has

been identified (7). This sequence has been rendered cell

permeable by linking it to the TAT48-57 peptide. This construct,

called TAT-RasGAP317-326, efficiently sensitizes cancer cells to

genotoxin-induced apoptosis. Importantly, TAT-RasGAP317-326does not sensitize nontumor cells (7). However, the molecular

mechanisms underlying the sensitizing properties of TAT-

RasGAP317–326 are still poorly understood.

Genotoxin-induced DNA damage leads to apoptosis mainly

in a p53-dependent manner (8, 9). At the top of the signaling

networks induced by DNA damage lie three related protein

kinases, DNA-dependent protein kinase, ataxia-telangiectasia

mutated, and ataxia-telangiectasia mutated and Rad3-related,

which orchestrate the damage response, sometimes in concert

and sometimes separately, by activating kinases that phosphor-

ylate p53 (8). The p53 protein transmits the apoptotic signal by a

complex mechanism involving, at least in part, its ability to

transactivate proapoptotic target genes, such as PUMA (10).

Here, we have assessed the role of the p53 pathway in the ability

of TAT-RasGAP317-326 to sensitize cancer cells to genotoxins.

ResultsTAT-RasGAP317-326 Does Not Modulate the Activity of Aktor the Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase PathwayRasGAP, depending on the circumstances, can regulate Ras

either negatively or positively (11, 12). Conceivably, therefore,

the TAT-RasGAP317-326 peptide could have an effect on Ras

activity. However, this peptide does not affect the Ras effector

pathways leading to nuclear factor-nB, c-Jun NH2-terminal

Received 8/15/06; revised 2/22/07; accepted 3/6/07.Grant support: Oncosuisse and Swiss National Science Foundation.The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Requests for reprints: Christian Widmann, Department of Physiology, LausanneUniversity, Rue du Bugnon 7/9, 1005 Lausanne, Switzerland. Phone: 41-21-692-5123; Fax: 41-21-692-5255. E-mail: [email protected] D 2007 American Association for Cancer Research.doi:10.1158/1541-7786.MCR-06-0257

Mol Cancer Res 2007;5(5). May 2007 497

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kinase, and p38 mitogen-activated protein kinase activation (7).

But as it has been shown that fragment N activates Akt (13) and

because the 317-326 sequence of RasGAP is born by fragment

N, we determined if the TAT-RasGAP317-326 peptide also

modulates the activity of Akt. Figure 1A shows that neither the

control HIV-TAT48-57 peptide nor the TAT-RasGAP317-326–

sensitizing peptide induced the phosphorylation of Akt.

Moreover, these peptides did not affect the ability of cisplatin

FIGURE 1. Akt and ERKactivation is not modulated byTAT-RasGAP317-326. U2OScells were treated for 16 h withthe indicated combinationsof 20 Amol/L HIV-TAT48-57,20 Amol/L TAT-RasGAP317-326,and 30 Amol/L cisplatin. Theextent of Akt (A) and ERK (B)activation was detected byWestern blot using antibodiesrecognizing the phosphorylat-ed (phospho ) forms of Akt andERK. The total levels of eachprotein were assessed usingantibodies recognizing allforms of Akt and ERK. Quan-titations of the bands are pre-sented below the Westernblots. Columns, mean of threeindependent experiments nor-malized to the values obtainedin untreated cells; bars, SD.NS, not significant.

FIGURE 2. TAT-RasGAP317-326 re-quires p53 to sensitize cancer cell linesto cisplatin-induced apoptosis. A. SAOSand U2OS cells were incubated withincreasing concentrations of cisplatin inthe presence or absence of 20 Amol/L ofHIV-TAT48-57 or TAT-RasGAP317-326. Theextent of apoptosis was scored 22 h later.B. HCT116 cells expressing or not p53were treated as in A. C and D. H1299cells encoding a p53 construct whoseexpression is negatively regulated bytetracycline were cultured with (+ tet ) orwithout (� tet) 1 Amol/L tetracycline. C.Cells were then treated as described in A.D. Expression of p53 was determined byWestern blot. E. The p53-repressibleH1299 cells cultured in the presence orabsence of tetracycline were transfectedwith an empty vector (pcDNA3) or a vectorexpressing the N2 fragment of RasGAP.The cells were then treated with increas-ing concentrations of cisplatin. The per-centage of cells undergoing apoptosiswas scored 22 h later. Points, mean ofthree independent determinations; bars,SD. Asterisks, significant differences be-tween the genotoxin-treated cells incubat-ed with TAT-RasGAP317-326 and those leftuntreated or incubated with HIV-TAT48-57.

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to decrease the phosphorylation of Akt. These results indicate

that Akt is not a target of TAT-RasGAP317-326 and, conse-

quently, that Akt is probably not involved in the ability of this

peptide to sensitize cancer cells to genotoxins.

The extracellular signal-regulated kinase (ERK) mitogen-

activated protein kinase pathway is one of the main Ras effector

pathways (14-16) implicated in the increased proliferative rate

of tumor cells (17). As shown in Fig. 1B, neither the control

HIV-TAT48-57 peptide nor the TAT-RasGAP317-326–sensitizing

peptide activated the phosphorylation of ERKs. They were also

not able to modulate the effect of cisplatin on ERK

phosphorylation. These and earlier results (7) indicate that

TAT-RasGAP317-326–mediated enhancement of genotoxin-

induced apoptosis does not depend on Ras.

TAT-RasGAP317-326 Enhances Cisplatin-Induced Apopto-sis in a p53-Dependent MannerThe p53 transcription factor is activated in response to

genotoxic stress. It can induce cell cycle arrest, DNA repair, and

apoptosis (10, 18). Several approaches were used to determine

whether p53 was required for the TAT-RasGAP317-326 peptide

to favor genotoxin-induced apoptosis. First, the U2OS and

SAOS osteosarcoma cell lines were analyzed. The former

expresses p53, whereas the latter is deficient in this protein.1

Figure 2A shows that the p53-negative SAOS cells were not

sensitized by the TAT-RasGAP317-326 peptide to undergo

cisplatin-induced apoptosis, in contrast to the U2OS p53-

positive cells. Despite their common origin, these osteosarcoma

cell lines could differ in other aspects, and therefore, a second

approach was undertaken that used the wild-type p53-contain-

ing HCT116 colorectal carcinoma cell line and its p53-negative

variant obtained by somatic homologous recombination (19).

Figure 2B shows that the parental HCT116 cells were

efficiently sensitized by the TAT-RasGAP317-326 peptide to

cisplatin-induced death but that the variant lacking p53

remained unaffected by the peptide. To confirm these results,

we used the H1299 lung carcinoma cell line in which p53 can

be repressed by tetracycline (20). As shown in Fig. 2C, this

repressible cell line expresses p53 in the absence of tetracycline

but is totally devoid of p53 in the presence of the drug. Figure

2D shows that only in the condition allowing p53 expression

was this cell line sensitized by the TAT-RasGAP317-326 peptide

to apoptosis induced by cisplatin. These results indicate that

p53 is required for the sensitization property of the TAT-

RasGAP317-326 peptide. Consistent with this notion is the

observation that, in several tumor cell types, the TAT-

RasGAP317-326 peptide does not sensitize cell death induced

by staurosporine (Fig. 3), a stimulus known to kill cells

independently of p53 (21).

The TAT-RasGAP317-326 peptide is derived from the N2

fragment of RasGAP that is physiologically produced in cells

undergoing apoptosis. To determine whether this N2 fragment

also required p53 to sensitize tumor cells, the repressible

H1299 cells were transfected with a plasmid encoding frag-

ment N2 and subjected to increasing concentrations of

cisplatin. Figure 2E shows that fragment N2 requires p53 to

sensitize this cancer cell line to genotoxin-induced apoptosis.

This indicates that the TAT-RasGAP317-326 peptide and

fragment N2 from which it is derived regulate genotoxin-

induced death in a similar manner.

TAT-RasGAP317-326 Does Not Modulate p53 Transcrip-tional Activity and StabilizationIn wild-type p53-expressing cells, genotoxins induce stabi-

lization of p53 through inhibition of interaction between p53 and

Mdm2, an E3 ligase targeting p53 to proteasomal degradation.

This leads to an up-regulation of p53 cellular levels (compare

also lanes 1 and 2 in Fig. 4A; refs. 22-25). The increase in p53

levels and activity is accompanied by an increased transcription

of the p21 gene and a concomitant augmentation of p21 protein

levels (compare also lanes 1 and 2 in Fig. 4A; ref. 26). We

therefore investigated if TAT-RasGAP317-326 could modulate the

expression levels of p53 or alter its transcriptional activity. As

shown in Fig. 4A-C, cisplatin induced an increased expression

of p53 and its target gene product p21 independently of the

presence of the TAT-RasGAP317-326 peptide. This indicates that

the TAT-RasGAP317-326 peptide does not affect the stability

or the transcriptional activity of p53. This latter point was

FIGURE 3. TAT-RasGAP317-326 does not modulate staurosporine-induced tumor cell death. The cancer cell lines used in Fig. 2 were treatedwith increasing concentrations of staurosporine in the presence of20 Amol/L of HIV-TAT48-57 or TAT-RasGAP317-326 during 22 h. The extentof apoptosis was then scored. Points, mean of three independentdeterminations; bars, SD.

1 http://www.lgcpromochem.com/atcc/

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confirmed using a p53 transcription reporter assay. As shown in

Fig. 4D, TAT-RasGAP317-326 was unable to modulate the

transcriptional activity of p53 whether the cells were treated

with cisplatin or not.

TAT-RasGAP317-326 – Induced Sensitization of TumorCells Requires a Functional p53 StatusTo evaluate which functions of p53 were required for the

sensitization ability of the TAT-RasGAP317-326 peptide,

HCT116 p53�/� cells were reconstituted with various p53

mutants (see Materials and Methods). First, p53 forms bearing

mutations abrogating their activity were used (R175H and

L22Q/W23S). As expected, the 175H and 22Q/23S mutants did

not allow cells to up-regulate their p53 and p21 cellular levels

in response to genotoxins (Fig. 5A). In contrast to wild-type

p53-expressing cells, cells expressing these mutants were not

sensitized by the TAT-RasGAP317-326 peptide (Fig. 5B).

Therefore, despite the fact that TAT-RasGAP317-326 peptide

does not influence p53 transcriptional activity, it nevertheless

requires functional p53 proteins to mediate its sensitization

property.

We next used p53 proteins bearing various alanine

substitutions at sites known to be phosphorylated following

DNA damage (27), including some (e.g., Ser15 and Ser20) that

have been shown in vivo to play important role in p53-induced

apoptosis and tumor suppression (28). Cells expressing p53

mutants with one or two of these point mutations (at position

33-37, 15, and 20) were less sensitive to cisplatin than wild-

type p53-expressing cells (Fig. 5C), showing that these

mutations impaired, but did not abrogate, p53 functions. Cells

expressing the mutant bearing four point mutations were even

less sensitive to intermediate cisplatin concentrations than the

other mutants, but this difference was not observed at higher

cisplatin concentrations (Fig. 5C). We next examined how these

different mutants responded to TAT-RasGAP317-326 in the

presence of low (20 Amol/L) or intermediate (50 Amol/L)cisplatin concentrations. As shown in Fig. 5D, none of the

mutant p53-expressing HCT116 cells underwent increased

cisplatin-induced cell death in the presence of TAT-Ras-

GAP317-326. These results indicate that the ability of TAT-

RasGAP317-326 to sensitize tumor cells is drastically affected

when the functionality of p53 to mediate genotoxin-induced

apoptosis is affected (even to limited extents).

To further characterize the resistance of the cells expressing

the p53 forms bearing the point mutations to the action of the

TAT-RasGAP317-326 peptide, their ability to modulate p53 and

p21 cellular levels in response to cisplatin was assessed in the

presence or absence of the peptide. In wild-type p53-expressing

cells, cisplatin induced an f20-fold increase in p53 levels. Incontrast, this induction was decreased to 3- to 5-fold in cells

expressing the p53 mutants (Fig. 6). Moreover, although the

p21 levels were increased by cisplatin in wild-type p53-

expressing cells, they remained unchanged in mutants 15,

33-37, and 9-15-33-37 or minimally increased in mutant 20

(Fig. 6). In none of the mutants were the levels of p53 and p21

modulated by the TAT-RasGAP317-326 peptide (Fig. 6).

Taken together, the results presented in Figs. 5 and 6 indicate

that the p53 mutants used here, despite being still more

F IGURE 4 . TAT -Ra s -GAP317-326 does not modulatethe expression and the tran-scriptional activity of p53 inU2OS cells. A. Western blotanalysis of p53 and p21 expres-sion in U2OS cell treated with15 Amol/L cisplatin in the pres-ence or absence of 20 Amol/Lof HIV-TAT48-57 or TAT-Ras-GAP317-326 for 16 h. Thecorresponding quantitations arepresented in B and C. Theywere done on three independentWestern blots and normalizedagainst the positive controls(cells without treatment). D.U2OS cells were transfectedwith 0.1 Ag of a firefly lucifer-ase reporter plasmid bearingp53-binding elements and with0.5 Ag of a plasmid encodingthe Renilla luciferase. Thecells were treated 2 d later with15 Amol/L cisplatin in the pres-ence or absence of 20 Amol/Lof the HIV-TAT48-57 or TAT-RasGAP317-326 peptides during16 h. Firefly luciferase activitynormalized to the Renilla lucif-erase activity and expressed asfold increase of the basal p53activity obtained in control un-treated cells. Columns, meanof three independent determi-nations; bars, SD; NS, not sig-nificant.

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sensitive to cisplatin-induced apoptosis compared with p53�/�

cells, are strongly affected in their ability to regulate the trans-

cription of at least one of its target genes (p21) in response to

DNA damage. This could explain why TAT-RasGAP317-326 was

not able to sensitize the mutant p53-expressing cells to cisplatin.

TAT-RasGAP317-326 Does Not Affect p53 Phosphorylationand Acetylation

The results described above indicate that TAT-RasGAP317-326does not modulate p53 transcriptional activity. The peptide

might, however, affect p53 posttranslational modifications that

could modulate p53 activity in ways that could not have been

detected in the experimental conditions used above. The p53

protein can be phosphorylated on about 15 serine and

threonine residues and acetylated on 4 lysine residues (29).

We therefore assessed, using commercially available anti-

bodies, the capacity of TAT-RasGAP317-326 to modulate the

phosphorylation of p53 at some of the serine residues, as well

as its acetylation on Lys382. Figure 7 shows that the increase

in p53 phosphorylation at Ser15, Ser20, Ser37, and Ser46

induced by cisplatin was not affected by TAT-RasGAP317-326.

Similarly, the acetylation of p53 at position 382 induced by

the genotoxin was not changed in the presence of the peptide

(Fig. 7, bottom). This suggests that TAT-RasGAP317-326 does

not modulate the posttranslational modifications occurring on

p53, although we cannot exclude that phosphorylation and

FIGURE 5. Cells expressing p53 mutants are not sensitized by TAT-RasGAP317-326. A. HCT116 p53+/+ or HCT116 p53�/� cells stably infected withlentiviruses encoding wild-type (WT) or the indicated mutant forms of p53 were lysed, and the expression of p53 and p21 was assessed by Western blotanalysis. B. Alternatively, these cells were treated with a low (20 Amol/L) or a high (100 Amol/L) concentration of cisplatin in the presence or absenceof 20 Amol/L of HIV-TAT48-57 or TAT-RasGAP317-326 for 22 h. The percentage of cells undergoing apoptosis was then scored. C. HCT116 p53+/+ or HCT116p53�/� cells stably infected with lentiviruses encoding wild-type or mutant forms of p53 bearing alanine substitutions at the indicated phosphorylation sites weresubmitted to increasing concentrations of cisplatin for 22 h, and the percentage of apoptosis was then scored. Asterisks, statistically significant differencebetween the p53 9-15-33-37 mutant-expressing cells and the cells expressing the other p53 mutants. The difference between the p53 mutant-expressing cellsand either wild-type p53-expressing cells or p53�/� cells was statistically significant at cisplatin concentrations of z30 Amol/L (not indicated on the figure).D. Alternatively, these cells were treated with low (20 Amol/L) and intermediate (50 Amol/L) cisplatin concentrations in the presence or absence of theHIV-TAT48-57 or TAT-RasGAP317-326 peptides as indicated in Fig. 4B. Columns, mean of three independent determinations; bars, SD. Asterisks, significantdifferences between the genotoxin-treated cells incubated with TAT-RasGAP317-326 and those left untreated or incubated with the HIV-TAT48-57 peptide. NS,not significant.



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acetylation sites other than those tested here are regulated by

the RasGAP peptide.

TAT-RasGAP317-326 – Induced Sensitization of TumorCells Requires PUMA but not p21The results presented above suggest that some p53 target

genes may be required for the sensitization of tumor cells

by TAT-RasGAP317-326. We therefore assessed the implica-

tion of two important p53 target genes, the cell cycle inhi-

bitor p21cip1 and the Bcl2 family member PUMA , in this

response. Figure 8A shows that HCT116 cells lacking p21

were still sensitized to cisplatin-induced apoptosis by the

TAT-RasGAP317-326 peptide. As p21 is a critical mediator of

p53-induced cell cycle arrest (30), this suggests that the TAT-

RasGAP317-326 peptide does not mediate its effect through

modulation of the cell cycle. In contrast, HCT116 cells devoid

of PUMA were completely resistant to the sensitization

mediated by TAT-RasGAP317-326 (Fig. 8B). We next assessed

if TAT-RasGAP317-326 was able to modulate the expression of

PUMA. As shown in Fig. 9A and B, PUMA expression was

up-regulated by cisplatin. However, the TAT-RasGAP317-326peptide did not further modulate PUMA protein levels in cells

whether treated with the genotoxin or not. These results show

that TAT-RasGAP317-326 requires the expression of PUMA to

FIGURE 6. p53 and p21 expression in HCT116 cells expressing p53 mutants bearing alanine substitutions at NH2-terminal p53 phosphorylation sites. A.Western blot analysis of p53 and p21 expression of the cells described in Fig. 5 after treatment with 50 Amol/L cisplatin (+) in the presence or absence of20 Amol/L of the HIV-TAT48-57 or TAT-RasGAP317-326 peptides during 16 h. B. Corresponding quantitative analyses done on three independent Westernblots and normalized against the values obtained in untreated control cells. Columns, mean; bars, SD.

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have its effect but does not modulate its expression. We also

tested whether Mcl-1, an antiapoptotic Bcl2 family member

that can be degraded after DNA damage (31, 32), could be

negatively affected by TAT-RasGAP317-326, which could

explain the sensitization induced by the peptide on geno-

toxin-treated cells. In our experimental conditions, however,

cisplatin did not induce reduction in Mcl-1 levels (Fig. 9C,

compare lanes 1 and 2). Surprisingly, TAT-RasGAP317-326induced an f2-fold induction of Mcl-1 in HCT116 cells

(Fig. 9C). The reason for this increase is not known at the

present time. However, the combination of cisplatin and TAT-

RasGAP317-326 led to a down-regulation of Mcl-1 protein levels

(Fig. 9C). This could be the result of increased caspase activity

(see Fig. 10B) as Mcl-1 is a caspase substrate (33-36).

Nevertheless, the fact that TAT-RasGAP317-326 by itself does

not induce a decrease of Mcl-1 suggests that Mcl-1 is not

playing a direct role in the ability of the peptide to sensitize

cancer cells to genotoxin-induced death.

As PUMA is a BH3-only Bcl-2 family member that favors

mitochondrial-dependent apoptotic responses (37), the results

described above indicate that TAT-RasGAP317-326 increases

the sensitivity of tumor cells to genotoxin by enhancing

the intrinsic, mitochondrial-dependent, apoptotic pathway.

Consistent with this interpretation is the observation that the

FIGURE 7. p53 phosphorylation (at Ser15,Ser20, Ser37, and Ser46) and acetylation are notmodulated by the TAT-RasGAP317-326 peptide.HCT116 cells treated with 20 Amol/L cisplatin inthe presence or absence of 20 Amol/L of HIV-TAT48-57 or TAT-RasGAP317-326 for 16 h. The cellswere then lysed, and the extent of phosphorylationat the indicated serine sites, as well as the extent ofacetylation, was assessed by Western blot analy-sis. The quantitative analyses were done on threeindependent Western blots and normalized againstthe values obtained in untreated cells. Columns,mean; bars, SD; NS, not significant.



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TAT-RasGAP317-326 peptide allowed cisplatin to induce a

stronger drop in mitochondrial membrane potential (Fig. 10A)

and an increased activation of caspase-3 (Fig. 10B).

DiscussionRasGAP has first been identified as a Ras regulator (38), but

this protein has also been shown to bear two caspase-3 cleavage

sites and cleaved during apoptosis (2, 3, 5). The NH2-terminal

fragment resulting from the first cleavage event on RasGAP

(called fragment N; ref. 4) displays strong antiapoptotic

properties by activating the Ras/phosphatidylinositol 3-kinase

Akt pathway (13). The ability of fragment N to stimulate Akt

does not result in nuclear factor-nB activation (13) and neitherdoes the TAT-RasGAP317-326 peptide require nuclear factor-nBto mediate its sensitization effects (7). Here, we have extended

these observations by showing that TAT-RasGAP317-326 does

not modulate Akt or ERK activity, which indicates that Ras

effector pathways are not involved in the tumor sensitization

properties of the TAT-RasGAP317-326 peptide.

On the other hand, our results clearly show that TAT-

RasGAP317-326 requires p53 to increase the genotoxin sensitivity

of tumor cells but does not so by modulating its transcriptional

activity or its phosphorylation and acetylation status. The TAT-

RasGAP317-326 peptide does, however, require that p53 is fully

functional in its ability to sense DNA damage via phosphory-

lation of its NH2 terminus by kinases, such as ataxia-

telangiectasia mutated, ataxia-telangiectasia mutated and

Rad3-related, or DNA-dependent protein kinase (34). Indeed,

all the p53 mutants we have used in the present study that bore

mutations at these phosphorylation sites were incapable of

allowing TAT-RasGAP317-326 to mediate its sensitization effect.

Presumably, the signals initiated by the TAT-RasGAP317-326peptide integrate with the p53 pathway downstream of one or

several of the p53 target genes. Consistent with this notion is the

observation that the p53 target PUMA is crucially required for

the TAT-RasGAP317-326 peptide to induce tumor cell sensitiza-

tion (Fig. 8B). PUMA levels were not modulated by the peptide,

suggesting that neither the stability of PUMA nor the

transcription rate of its gene or the translation rate of its mRNA

was affected by the peptide. Although we did not detect any

change in the migration pattern of PUMA on polyacrylamide

gels that sometimes happen as a result of posttranslational modi-

fications, it cannot be ruled out that the TAT-RasGAP317-326peptide induced such modifications on PUMA. To our knowl-

edge, however, posttranslational modifications of PUMA have

yet to be described. If the peptide does not directly affect

PUMA levels, it might regulate its ability to stimulate

apoptosis. In this context, it is interesting to note that PUMA

can displace p53 from Bcl-XL, allowing p53 to induce

mitochondrial permeabilization and apoptosis (39). It could

therefore be anticipated that TAT-RasGAP317-326 modulates this

concerted action of p53 and PUMA at the mitochondria level

to increase the sensitivity of tumor cells to the action of

genotoxins. This is consistent with our observation that TAT-

RasGAP317-326 augmented mitochondrial depolarization and

the ensuing caspase activation induced by cisplatin (Fig. 10).

Our results also show that the TAT-RasGAP317-326 peptide

does not facilitate tumor cell death by modulating p21 levels, as

cells bearing deletions of the p21 gene are as sensitive to

cisplatin in the presence of TAT-RasGAP317-326 as p21-positive

cells (Fig. 8A). Recently, the rapamycin derivative mammalian

target of rapamycin inhibitor everolimus has been shown to

sensitize tumor cells to genotoxins in a manner that required

inhibition of p53-induced p21 expression (40). The mechanism

by which TAT-RasGAP317-326 sensitizes tumor cells is therefore

different from those resulting from the inhibition of the

mammalian target of rapamycin pathway. This study and ours

point out to the central role of p53 in sensitizing tumor cells to

the action of genotoxins. They also indicate that there are more

than one p53 target that can be modulated to enhance the p53-

dependent cell death of cancer cells, which may eventually lead

to the development of parallel strategies to increase the efficacy

of genotoxic drugs to specifically target tumors.

Materials and MethodsCells and TransfectionThe U2OS and SAOS osteosarcoma cell lines (LGC Pro-

mochem; American Type Culture Collection) were maintained in

FIGURE 8. TAT-RasGAP317-326 requires PUMA, but not p21, tosensitize HCT116 cells to cisplatin-induced apoptosis. A. HCT116 p21+/+

or HCT116 p21�/� cells were treated with 20 Amol/L cisplatin in thepresence or absence of 20 Amol/L of HIV-TAT48-57 or TAT-RasGAP317-326for 22 h, and the extent of apoptosis was scored. Columns, mean of threeindependent determinations; bars, SD. B. HCT116 PUMA+/+ or HCT116PUMA�/� cells were treated and analyzed as described in A. Asterisks,significant differences between the genotoxin-treated cells incubated withTAT-RasGAP317-326 and those left untreated or incubated with the HIV-TAT48-57 peptide.

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DMEM (Sigma) containing 10% FCS (Sigma) at 37jC and 5%CO2. HCT116 p53

+/+, HCT116 p53�/�, HCT116 PUMA�/�,and HCT116 p21�/� have been described earlier (41, 42) andwere cultured as described above. TheH1299 cell line encoding a

p53 construct under the control of a tetracycline-repressible

promoter (Tet-off system) was described earlier (20). Experi-

ments using cells were done in 2 mL in six-well plates con-

taining 1.5 � 105 cells per well for U2OS, SAOS, and H1299

cells or 4� 105 cells per well for HCT116 cells. H1299 cells weretransfected using LipofectAMINE 2000 (Invitrogen) with 0.5 Aggreen fluorescent protein–expressing plasmid and with 1 Agcontrol plasmid or 1 Ag HA-N2–expressing plasmid. Briefly,1.5 � 105 cells, plated the previous day, were incubated for

5 h with a DNA (1.5 Ag)-LipofectAMINE 2000 (2 AL) mixturein 1 mL of DMEM without serum at 37jC in 5% CO2. When

cisplatin was used, it was added at the time the medium with

serum was added back to the transfected cells. The cells were

split the day before the treatment at a concentration of 1.5 � 105

per well. U2OS cells were transfected using the calcium/phos-

phate precipitation procedure (7, 43) using 0.1 Ag pRL-TK, avector encoding the Renilla reniformis luciferase from Promega,

and 0.5 Ag p53.luc, a reporter plasmid bearing the firefly luci-ferase cDNA under the control of p53-responsive elements (44).

Lentiviral InfectionThe lentiviral vectors expressing wild-type and mutant p53

proteins have been described previously (44). Lentiviruses were

produced as described (5). Lentivirus-containing supernatants

were collected 36 h after transfection, 0.45 Am filtered, and

frozen at �80jC. The amount of virus leading to infection of30% of the HCT116 cells was chosen. The cells were further

selected in puromycin-containing medium to ensure that each

cell expressed the lentivirus-encoded proteins.

Peptide SynthesisThe HIV-TAT48-57 (GRKKRRQRRR) and TAT-RasGAP317-326

(GRKKRRQRRRGGWMWVTNLRTD) peptides were synthe-

sized at the Institute of Biochemistry (University of Lausanne,

Lausanne, Switzerland) using N-(9-fluorenyl)methoxycar-

bonyl technology, purified by high-performance liquid chro-

matography, and tested by mass spectrometry. Peptides were

diluted at a concentration of 1 mmol/L in H2O and stored

at �20jC.

ChemicalsCisplatin was from Sigma and was diluted in PBS at a final

concentration of 1 mmol/L and stored at �80jC. Hoechst

FIGURE 9. TAT-RasGAP317-326 does not modulate the expression of PUMA to sensitize the HCT116 cancer cell line to cisplatin-induced apoptosis.A. HCT116 PUMA+/+ or HCT116 PUMA�/� cells were treated with 20 Amol/L cisplatin in the presence or absence of 20 Amol/L of HIV-TAT48-57 or TAT-RasGAP317-326. The cells were lysed 16 h later, and the expression of PUMA was assessed by Western blot analysis. B. Corresponding quantitativeanalyses done on three independent Western blots and normalized against the values obtained in untreated control cells. Columns, mean; bars, SD.C. U2OS cells were treated with 15 Amol/L cisplatin in the presence or absence of 20 Amol/L of HIV-TAT48-57 or TAT-RasGAP317-326. The cells were lysed16 h later, and the expression of Mcl-1 was assessed by Western blot analysis. Quantitations of the bands are presented below the Western blots. Columns,mean of three independent experiments normalized to the values obtained in untreated cells; bars, SD.



505



33342 was from Roche. It was diluted in water at a final

concentration of 10 mg/mL and stored at 4jC in the dark.

Tetracycline was from Sigma and was diluted in H2O at a final

concentration of 1 mmol/L and stored at �80jC.

Apoptosis MeasurementsApoptosis was determined by scoring the number of cells

displaying pyknotic nuclei (a pyknotic nucleus is condensed

and reduced in size and usually displays increased staining

capacity). Nuclei of live cells were labeled with Hoechst

33342 (10 Ag/mL final concentration) for f5 min, and thecells (at least 400 per condition) were then analyzed using

an inverted Zeiss Axiovert 25 microscope equipped with

fluorescence (HBO/AC) and transmitted light optics and a

40� objective (Zeiss 440865; LD Achroplan 40�/0.60 KorrPh2; 1/0-2).

Luciferase Reporter AssayLuciferase assay was done using the Dual-Luciferase

Reporter Assay from Promega as per the manufacturer’s

instructions. For each measurement, light emission was

quantified during 12 s using a Lumat LB 9501 luminometer

(Berthold Technologies).

Western Blot AnalysisWestern blots were done as described (7). The primary

antibodies were detected by Alexa Fluor 680–conjugated

secondary antibodies (Molecular Probes) diluted 1:5,000 in

TBS buffer [18 mmol/L HCl, 130 mmol/L NaCl, 20 mmol/L

Tris-HCl (pH 7.2), 0.1% Tween 20, 5% nonfat dry milk] and

subsequently visualized with the Odyssey IR imaging system

(LI-COR). The antibodies specific for phosphorylated Akt

(Cell Signaling Technology), total Akt (Santa Cruz Biotech-

nology), phosphorylated ERK (Cell Signaling Technology),

and total ERK (Cell Signaling Technology) were diluted

1:1,000 in 5% bovine serum albumin in TBS. The mono-

clonal antibody against p53 DO1 (gift from Dr. Richard

Iggo, Bute Medical School, University of St. Andrews, Fife,

Scotland), the polyclonal antibody against p53 (Cell

Signaling Technology), the antibody against p21 (Cell

Signaling Technology), and the antibody against PUMA

(Axxora) were diluted 1:1,000 in 5% nonfat dry milk in

TBS. The antibody recognizing the active, cleaved, form of

caspase-3 was from Cell Signaling Technology. It was used

at a 1:1,000 dilution in 5% milk in TBS. The antibody

specific for Mcl-1 was from Sigma-Aldrich. It was used at a

1:1,000 dilution in 5% milk in TBS. The antibodies specific

for the various p53 serine phosphorylation sites and for the

acetylated form of p53 were from Cell Signaling Technology.

They were used at a 1:1,000 dilution in 5% bovine serum

albumin in TBS. Quantitation was done using the Odyssey IR

imaging software.

Mitochondrial Membrane Potential MeasurementMitochondrial membrane depolarization was assessed

using the JC-1 mitochondrial membrane potential sensor

(Sigma). This compound exhibits potential-dependent accu-

mulation in mitochondria. On excitation at 488 nm, JC-1

fluoresces at 529 nm (green) in the cytoplasm and at 590 nm

(red) in the mitochondria. Mitochondrial depolarization is

indicated by a decrease in the red/green fluorescence

intensity ratio. The cells were processed as follows. The

medium of the cell culture (with its associated floating cells)

was collected. The remaining adherent cells were washed

with 1 mL PBS and collected. The adherent cells were

detached with 500 AL of a trypsin/EDTA solution (5 g/L

FIGURE 10. TAT-RasGAP317-326 increases the apoptotic signalsdownstream of PUMA. U2OS cells were treated with 20 Amol/L cisplatin inthe presence or absence of 20 Amol/L HIV-TAT48-57 or TAT-RasGAP317-326for 16 h. They were then processed as follows. A. The mitochondrialpotential of the cells was measured as described in Materials and Methods.Insets, mean F SD (from three independent experiments) of the percen-tage of cells having lost their mitochondrial potential. The difference betweencells treated with cisplatin alone or in combination with HIV-TAT48-57 withcells treated with cisplatin and TAT-RasGAP317-326 was statisticallysignificant. B. The cells were lysed, and the cleavage of caspase-3 wasassessed by Western blot analysis. The experiment was done two moretimes with similar results.

Michod and Widmann


506



porcine trypsin and 2 g/L EDTA; Sigma-Aldrich) that was

pooled with the collected medium and PBS. The cells were

pelleted by centrifugation and resuspended in 1 mL culture

medium and incubated with 2 Amol/L JC-1 for 30 min at

37jC in the dark. The cells were then kept on ice before

being analyzed with a FACScan apparatus (BD Biosciences).

In the experiments shown, FL-1 corresponds to the green

channel and FL-2 to the red channel.

Statistical AnalysisAll the statistical analyses were done with Microsoft Excel

(XP edition) using the two-tailed unpaired Student’s t test.

Significance is indicated by an asterisk when P < 0.05/n , where

P is the probability derived from the t test analysis and n is the

number of comparisons done (Bonferroni correction).

AcknowledgmentsWe thank Dr. Bert Vogelstein for the gift of the various HCT116 cell lines, Dr.Richard Iggo and Dr. Mathias Kaeser for suggestions and comments and for thegift of the plasmids encoding p53 and its mutants, and Dr. Peter Clarke for hiscritical reading of the manuscript.

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2007;5:497-507. Mol Cancer Res David Michod and Christian Widmann Tumor Cells to Genotoxins

Requires p53 and PUMA to Sensitize317-326TAT-RasGAP

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TAT-RasGAP Requiresp53andPUMAto ... · TAT-RasGAP 317-326 Requiresp53andPUMAto SensitizeTumorCellstoGenotoxins DavidMichodandChristianWidmann DepartmentofPhysiologyandDepartmentofCellBiologyandMorphology