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Molecular Cancer Research 5, 497-507, May 1, 2007. doi: 10.1158/1541-7786.MCR-06-0257
© 2007 American Association for Cancer Research
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Signaling and Regulation

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

David Michod and Christian Widmann

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

Requests for reprints: Christian Widmann, Department of Physiology, Lausanne University, Rue du Bugnon 7/9, 1005 Lausanne, Switzerland. Phone: 41-21-692-5123; Fax: 41-21-692-5255. E-mail: Christian.Widmann{at}unil.ch


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Although 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;16(1):497–507)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Since its introduction as a cancer therapy ~50 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 chemotherapy 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 chemotherapeutic 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-RasGAP317-326 does 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 phosphorylate 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.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
TAT-RasGAP317-326 Does Not Modulate the Activity of Akt or the Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase Pathway
RasGAP, 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-{kappa}B, c-Jun NH2-terminal 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 to decrease the phosphorylation of Akt. These results indicate that Akt is not a target of TAT-RasGAP317-326 and, consequently, that Akt is probably not involved in the ability of this peptide to sensitize cancer cells to genotoxins.


Figure 1
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  		FIGURE 1. Akt and ERK activation is not modulated by TAT-RasGAP317-326. U2OS cells were treated for 16 h with the indicated combinations of 20 µmol/L HIV-TAT48-57, 20 µmol/L TAT-RasGAP317-326, and 30 µmol/L cisplatin. The extent of Akt (A) and ERK (B) activation was detected by Western blot using antibodies recognizing the phosphorylated (phospho) forms of Akt and ERK. The total levels of each protein were assessed using antibodies recognizing all forms of Akt and ERK. 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. NS, not significant.

 
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 Apoptosis in a p53-Dependent Manner
The 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-containing 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).


Figure 2
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  		FIGURE 2. TAT-RasGAP317-326 requires p53 to sensitize cancer cell lines to cisplatin-induced apoptosis. A. SAOS and U2OS cells were incubated with increasing concentrations of cisplatin in the presence or absence of 20 µmol/L of HIV-TAT48-57 or TAT-RasGAP317-326. The extent of apoptosis was scored 22 h later. B. HCT116 cells expressing or not p53 were treated as in A. C and D. H1299 cells encoding a p53 construct whose expression is negatively regulated by tetracycline were cultured with (+ tet) or without (– tet) 1 µmol/L tetracycline. C. Expression of p53 was determined by Western blot. D. Cells were then treated as described in A. E. The p53-repressible H1299 cells cultured in the presence or absence of tetracycline were transfected with an empty vector (pcDNA3) or a vector expressing the N2 fragment of RasGAP. The cells were then treated with increasing concentrations of cisplatin. The percentage of cells undergoing apoptosis was scored 22 h later. Points, mean of three independent determinations; bars, SD. Asterisks, significant differences between the genotoxin-treated cells incubated with TAT-RasGAP317-326 and those left untreated or incubated with HIV-TAT48-57.

 

Figure 3
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  		FIGURE 3. TAT-RasGAP317-326 does not modulate staurosporine-induced tumor cell death. The cancer cell lines used in Fig. 2 were treated with increasing concentrations of staurosporine in the presence of 20 µmol/L of HIV-TAT48-57 or TAT-RasGAP317-326 during 22 h. The extent of apoptosis was then scored. Points, mean of three independent determinations; bars, SD.

 
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 fragment 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 Transcriptional Activity and Stabilization
In wild-type p53-expressing cells, genotoxins induce stabilization 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 ref. lanes 1 and 2 in Fig. 4A 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 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.


Figure 4
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  		FIGURE 4. TAT-RasGAP317-326 does not modulate the expression and the transcriptional activity of p53 in U2OS cells. A. Western blot analysis of p53 and p21 expression in U2OS cell treated with 15 µmol/L cisplatin in the presence or absence of 20 µmol/L of HIV-TAT48-57 or TAT-RasGAP317-326 for 16 h. The corresponding quantitations are presented in B and C. They were done on three independent Western blots and normalized against the positive controls (cells without treatment). D. U2OS cells were transfected with 0.1 µg of a firefly luciferase reporter plasmid bearing p53-binding elements and with 0.5 µg of a plasmid encoding the Renilla luciferase. The cells were treated 2 d later with 15 µmol/L cisplatin in the presence or absence of 20 µmol/L of the HIV-TAT48-57 or TAT-RasGAP317-326 peptides during 16 h. Firefly luciferase activity normalized to the Renilla luciferase activity and expressed as fold increase of the basal p53 activity obtained in control untreated cells. Columns, mean of three independent determinations; bars, SD; NS, not significant.

 
TAT-RasGAP317-326–Induced Sensitization of Tumor Cells Requires a Functional p53 Status
To 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.


Figure 5
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  		FIGURE 5. Cells expressing p53 mutants are not sensitized by TAT-RasGAP317-326. A. HCT116 p53+/+ or HCT116 p53–/– cells stably infected with lentiviruses encoding wild-type (WT) or the indicated mutant forms of p53 were lysed, and the expression of p53 and p21 was assessed by Western blot analysis. B. Alternatively, these cells were treated with a low (20 µmol/L) or a high (100 µmol/L) concentration of cisplatin in the presence or absence of 20 µmol/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 HCT116 p53–/– cells stably infected with lentiviruses encoding wild-type or mutant forms of p53 bearing alanine substitutions at the indicated phosphorylation sites were submitted to increasing concentrations of cisplatin for 22 h, and the percentage of apoptosis was then scored. Asterisks, statistically significant difference between the p53 9-15-33-37 mutant-expressing cells and the cells expressing the other p53 mutants. The difference between the p53 mutant-expressing cells and either wild-type p53-expressing cells or p53–/– cells was statistically significant at cisplatin concentrations of ≥30 µmol/L (not indicated on the figure). D. Alternatively, these cells were treated with low (20 µmol/L) and intermediate (50 µmol/L) cisplatin concentrations in the presence or absence of the HIV-TAT48-57 or TAT-RasGAP317-326 peptides as indicated in Fig. 4B. Columns, mean of three independent determinations; bars, SD. Asterisks, significant differences 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.

 
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 µmol/L) or intermediate (50 µmol/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-RasGAP317-326. 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 ~20-fold increase in p53 levels. In contrast, 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).


Figure 6
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  		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 µmol/L cisplatin (+) in the presence or absence of 20 µmol/L of the HIV-TAT48-57 or TAT-RasGAP317-326 peptides during 16 h. B. Corresponding quantitative analyses done on three independent Western blots and normalized against the values obtained in untreated control cells. Columns, mean; bars, SD.

 
Taken together, the results presented in Figs. 5 and 6 indicate that the p53 mutants used here, despite being still more sensitive to cisplatin-induced apoptosis compared with p53–/– cells, are strongly affected in their ability to regulate the transcription 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 Phosphorylation and Acetylation
The results described above indicate that TAT-RasGAP317-326 does 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 antibodies, 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 acetylation sites other than those tested here are regulated by the RasGAP peptide.


Figure 7
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  		FIGURE 7. p53 phosphorylation (at Ser15, Ser20, Ser37, and Ser46) and acetylation are not modulated by the TAT-RasGAP317-326 peptide. HCT116 cell treated with 20 µmol/L cisplatin in the presence or absence of 20 µmol/L of HIV-TAT48-57 or TAT-RasGAP317-326 for 16 h. The cells were then lysed, and the extent of phosphorylation at the indicated serine sites, as well as the extent of acetylation, was assessed by Western blot analysis.

 
TAT-RasGAP317-326–Induced Sensitization of Tumor Cells Requires PUMA but not p21
The 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 implication of two important p53 target genes, the cell cycle inhibitor 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-RasGAP317-326 peptide 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 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 genotoxin-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-RasGAP317-326 induced an ~2-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.


Figure 8
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  		FIGURE 8. TAT-RasGAP317-326 requires PUMA, but not p21, to sensitize HCT116 cells to cisplatin-induced apoptosis. A. HCT116 p21+/+ or HCT116 p21–/– cells were treated with 20 µmol/L cisplatin in the presence or absence of 20 µmol/L of HIV-TAT48-57 or TAT-RasGAP317-326 for 22 h, and the extent of apoptosis was scored. Columns, mean of three independent determinations; bars, SD. B. HCT116 PUMA+/+ or HCT116 PUMA–/– cells were treated and analyzed as described in A. Asterisks, significant differences between the genotoxin-treated cells incubated with TAT-RasGAP317-326 and those left untreated or incubated with the HIV-TAT48-57 peptide.

 

Figure 9
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  		FIGURE 9. TAT-RasGAP317-326 does not modulate the expression of PUMA to sensitize HCT116 cancer cell line to cisplatin-induced apoptosis. A. HCT116 PUMA+/+ or HCT116 PUMA–/– cells were treated with 20 µmol/L cisplatin in the presence or absence of 20 µmol/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 quantitative analyses 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 µmol/L cisplatin in the presence or absence of 20 µmol/L of HIV-TAT48-57 or TAT-RasGAP317-326. The cells were lysed 16 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.

 

Figure 10
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  		FIGURE 10. TAT-RasGAP317-326 increases the apoptotic signals downstream of PUMA. U2OS cells were treated with 20 µmol/L cisplatin in the presence or absence of 20 µmol/L HIV-TAT48-57 or TAT-RasGAP317-326 for 16 h. They were then processed as follows. A. The mitochondrial potential of the cells was measured as described in Materials and Methods. Insets, mean ± SD (from three independent experiments) of the percentage of cells having lost their mitochondrial potential. The difference between cells treated with cisplatin, alone or in combination with HIV-TAT48-57, with cells treated with cisplatin and TAT-RasGAP317-326 was statistically significant. B. The cells were lysed, and the cleavage of caspase-3 was assessed by Western blot analysis. The experiment was done two more times with similar results.

 
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 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).


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
RasGAP 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-{kappa}B activation (13) and neither does the TAT-RasGAP317-326 peptide require nuclear factor-{kappa}B to 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 phosphorylation 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-RasGAP317-326 peptide 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 sensitization (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 modifications, it cannot be ruled out that the TAT-RasGAP317-326 peptide induced such modifications on PUMA. To our knowledge, 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 Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cells and Transfection
The U2OS and SAOS osteosarcoma cell lines (LGC Promochem; American Type Culture Collection) were maintained in DMEM (Sigma) containing 10% FCS (Sigma) at 37°C and 5% CO2. HCT116 p53+/+, HCT116 p53–/–, HCT116 PUMA–/–, and HCT116 p21–/– have been described earlier (41, 42) and were cultured as described above. The H1299 cell line encoding a p53 construct under the control of a tetracycline-repressible promoter (Tet-off system) was described earlier (20). Experiments using cells were done in 2 mL in six-well plates containing 1.5 x 105 cells per well for U2OS, SAOS, and H1299 cells or 4 x 105 cells per well for HCT116 cells. H1299 cells were transfected using LipofectAMINE 2000 (Invitrogen) with 0.5 µg green fluorescent protein–expressing plasmid and with 1 µg control plasmid or 1 µg HA-N2–expressing plasmid. Briefly, 1.5 x 105 cells, plated the previous day, were incubated for 5 h with a DNA (1.5 µg)-LipofectAMINE 2000 (2 µL) mixture in 1 mL of DMEM without serum at 37°C 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 x 105 per well. U2OS cells were transfected using the calcium/phosphate precipitation procedure (7, 43) using 0.1 µg pRL-TK, a vector encoding the Renilla reniformis luciferase from Promega, and 0.5 µg p53.luc, a reporter plasmid bearing the firefly luciferase cDNA under the control of p53-responsive elements (44).

Lentiviral Infection
The 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 µm filtered, and frozen at –80°C. The amount of virus leading to infection of 30% 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 Synthesis
The HIV-TAT48-57 (GRKKRRQRRR) and TAT-RasGAP317-326 (GRKKRRQRRRGGWMWVTNLRTD) peptides were synthesized at the Institute of Biochemistry (University of Lausanne, Lausanne, Switzerland) using N-(9-fluorenyl)methoxycarbonyl technology, purified by high-performance liquid chromatography, and tested by mass spectrometry. Peptides were diluted at a concentration of 1 mmol/L in H2O and stored at –20°C.

Chemicals
Cisplatin was from Sigma and was diluted in PBS at a final concentration of 1 mmol/L and stored at –80°C. Hoechst 33342 was from Roche. It was diluted in water at a final concentration of 10 mg/mL and stored at 4°C in the dark. Tetracycline was from Sigma and was diluted in H2O at a final concentration of 1 mmol/L and stored at –80°C.

Apoptosis Measurements
Apoptosis 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 µg/mL final concentration) for ~5 min, and the cells (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 40x objective (Zeiss 440865; LD Achroplan 40x/0.60 Korr Ph2; {infty}/0-2).

Luciferase Reporter Assay
Luciferase 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 Analysis
Western 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 Biotechnology), phosphorylated ERK (Cell Signaling Technology), and total ERK (Cell Signaling Technology) were diluted 1:1,000 in 5% bovine serum albumin in TBS. The monoclonal 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 Measurement
Mitochondrial membrane depolarization was assessed using the JC-1 mitochondrial membrane potential sensor (Sigma). This compound exhibits potential-dependent accumulation 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 µL of a trypsin/EDTA solution (5 g/L 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 µmol/L JC-1 for 30 min at 37°C 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 Analysis
All 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).


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We 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 the gift of the plasmids encoding p53 and its mutants, and Dr. Peter Clarke for his critical reading of the manuscript.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Grant support: Oncosuisse and Swiss National Science Foundation.

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.

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

Received 8/15/06; revised 2/22/07; accepted 3/ 6/07.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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