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[Cancer Research 64, 4749-4754, July 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Ser392 Phosphorylation Regulates the Oncogenic Function of Mutant p53

Damian B. S. Yap1, Jung-Kuang Hsieh1, Shan Zhong1, Vicky Heath2, Barry Gusterson2, Tim Crook1 and Xin Lu1

1 Ludwig Institute for Cancer Research, St. Mary’s Branch, London, United Kingdom, and 2 Department of Pathology, University of Glasgow, Glasgow, United Kingdom


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite the wealth of information on the regulation of wild-type p53 function by phosphorylation, nothing is known about the biological effect of phosphorylation on mutant p53. Here we show that p53H175 is phosphorylated like wild-type p53 in cells of the same background. Ser392 nonphosphorylatable p53 mutants p53H175A392 and p53W248A392 more potently transformed rat embryo fibroblasts in cooperation with the ras oncogene than p53H175S392 and p53W248S392. p53H175A392 also had an enhanced ability to confer cellular resistance to the cytotoxic effect of cisplatin and UV radiation. This correlated with p53H175A392 being a more potent dominant negative mutant than p53H175 in inhibiting the apoptotic functions of wild-type p53. Moreover, p53H175E392, which mimics the phosphorylated form of p53H175, was less able to confer cellular resistance to DNA-damaging agents. p53H175 and p53W248 are phosphorylated like wild-type p53 in cells of the same background. Ser392 nonphosphorylated p53 was present in human breast tumors expressing mutant p53 including p53H175. Together, these results demonstrated a novel function of Ser392 phosphorylation in regulating the oncogenic function of mutant p53.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In around 50% of human cancers (1) , mutations abolish the ability of p53 to induce apoptosis, inhibit growth, or suppress transformation (2) . Most mutations occur in the central DNA binding domain of p53 at mutational hot spots that render p53 incapable of binding to DNA (3 , 4) . The Arg to His or Trp mutation at codon 175 (p53H175) or 248 (p53W248) is found in a wide variety of human cancers. Residue 248 of p53 contacts DNA, whereas residue 175 controls the conformational integrity of p53. Hence, mutations at codons 175 and 248 disrupt the conformation of p53 and make it incapable of binding DNA, activating transcription, or inducing apoptosis (5) .

In response to DNA damage, the p53 protein accumulates as a result of posttranslational modifications (6, 7, 8) , of which phosphorylation is one of the best studied. Phosphorylation on NH2-terminal residues, especially Ser15, Thr18, Ser20, and Ser37 (9, 10, 11, 12) , is believed to affect interaction with the negative regulator mdm2 and hence contribute to the stabilization of p53 (9 , 12) . Phosphorylation on COOH-terminal Ser315 and Ser392 in particular is believed to enhance the specific DNA binding of p53 in vitro (13 , 14) .

In contrast to the plethora of information about phosphorylation of wild-type p53, nothing is known about the modulation of mutant p53 activity by phosphorylation. It was found recently that mutant p53 can be phosphorylated on various sites including Ser392 (15) in vitro and in human tumors in vivo. Most tumor treatments cause DNA damage, which consequently results in the phosphorylation of wild-type p53 (16 , 17) . These observations prompted us to investigate whether the phosphorylation of mutant p53 could be regulated by DNA damage and whether phosphorylation of mutant p53 modulates its oncogenic function. We focused our present study on the Ser392 phosphorylation site. This site on wild-type p53 is phosphorylated by complexes containing casein kinase II (18 , 19) . In response to DNA damage, there is an increase in the phosphorylation of wild-type p53 on this site (16 , 20 , 21) . In addition, phosphorylation of p53 at Ser392 enhances the DNA binding activity of wild-type p53 in vitro (22) . It was then predicted that phosphorylation might activate the transactivation or apoptotic function of wild-type p53. However, abolishing the phosphorylation of p53 at Ser392 (by a Ser to Ala mutation) in wild-type p53 failed to show a significant effect on the stability or transcriptional activities of wild-type p53 (2 , 23, 24, 25) . Hence, the biological role of Ser392 phosphorylation of wild-type p53 remains unknown, despite the fact that Ser392 is one of the most conserved phosphorylation sites in the COOH terminus of p53, and phosphorylation on this site of wild-type p53 is induced by DNA damage signals. Perhaps the phosphorylation on Ser392 may have other biological functions that are independent of the DNA binding activity of p53. To address this issue, we investigated the biological importance of Ser392 phosphorylation in regulating the oncogenic function of p53H175 and p53W248, two tumor-derived hot spot mutants of p53 that are defective in binding DNA (26) .


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Reagents.
Primary rat embryo fibroblasts (REFs; BioWhittaker), p53-null H1299 non-small cell lung carcinoma cells, and p53-null osteosarcoma Soas-2 cells were maintained in DMEM (Life Technologies, Inc.) supplemented with 10% head-inactivated FCS, 2 mM L-glutamine, and 200 units/ml penicillin/streptomycin. Cisplatin (1 mg/ml) was from David Bull Laboratories. G418 (neomycin) used for selection of resistant colonies was obtained from Life Technologies, Inc., whereas FP3.2 phospho-specific anti-phosphorylated Ser392 p53 (16) was purchased from Serotech. To detect proliferating cell nuclear antigen, supernatant from the hybridoma PC-10 was used. Plasmids expressing mutant p53 were constructed by PCR mutagenesis and sequenced to confirm their identity. All plasmids (CMV Bam Neo or pcDNA3) express human proteins under the control of the cytomegalovirus promoter.

Immunoblotting.
Cells were scraped into an Eppendorf tube, washed, and then lysed in 50 mM Tris, 1 mM EDTA, 100 mM NaCl, 0.1% NP40, 5 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, 1 mM Na3VO4, 10 mM ß-glyceryophosphate, 1 mM benzamidine, and 120 nM okadaic acid. Unless otherwise stated, equal amounts of protein were loaded on gels and blotted as described previously (27) . For quantification of the signal, the Genome machine was used to digitally capture the image, and quantification was carried out using Syngene GeneSnap software.

Transfections and Construction of Cell Lines Expressing p53 Mutants.
Saos-2 or H1299 cells were plated at the required densities and transfected using the calcium phosphate method (28) . For transient assays, cells were harvested 24–48 h after transfection. For construction of stable cell lines, H1299 cells were transfected with plasmids (CMV Bam Neo or pcDNA3) expressing mutant p53 and then selected for 3 weeks in 1 mg/ml G418 (neomycin). Resistant colonies were then ring cloned and stained for mutant p53. At least two to three positive clones were pooled for each stable cell line expressing the same p53 mutant. Clone B231 is a wild-type p53-inducible H1299 cell line under the control of a tetracycline-responsive promoter that can be induced by 2 µg/ml doxycyclin.

Immunocytochemistry.
Cells were grown in 3-cm tissue culture dishes. After 24 h, the medium was removed, and the cells were washed with PBS, fixed using methanol/acetone, and probed with DO-13 mouse serum followed by an antimouse antibody conjugated with FITC. The samples were mounted with Citiflour shielding agent (Citiflour, Kent, United Kingdom) and observed under a fluorescence microscope (Nikon).

Transformation Assays.
Primary REFs were transfected with 2 µg of p53 mutants or vector only and 5 µg of EJ6.6 Ras (encoding activated human Ha-ras gene) as described previously (2) . After transfection, cells were selected in 400 µg/ml G418 for 2 weeks. Transformed colonies were scored by morphology under a light microscope and stained using Giemsa stain, and representative plates were photographed.

Drug Treatment and Colony Assays.
Cells were plated 24 h before any treatment at 1.5 x 106 cells/10-cm dish. For UV treatment, the medium was removed, the cells were irradiated using 10–20 J/m2 UV radiation, and the medium was replaced. For cisplatin treatment, the medium was removed, and fresh medium with 2.5 µg/ml cisplatin was added to the dishes. If lysates were to be used, the cells were harvested 12–15 h after drug treatment. For colony assays 48–72 h after treatment, both floating cells in the medium and adherent cells removed using trypsin were pooled and washed with PBS. Medium and cells from duplicate dishes were also pooled and split (1:4) into 10-cm dishes. Dishes were incubated to allow formation of colonies for 7–21 days. These colonies were fixed with methanol:acetone (1:1) and stained with Giemsa stain, and then representative plates were photographed. The untreated dishes were confluent, showing that the handling procedures themselves did not result in a reduction in colony number (data not shown).

Flow Cytometry Analysis.
The method was carried as described previously (29) . Briefly, 1.0–1.5 x 106 cells were transfected with the respective amounts of plasmid [wild-type p53 (3 µg), p73{alpha} (5 µg), and p53 mutants p53H175 (15 µg), p53H175A392 (20 µg), and p53H175E392 (15 µg)] and empty vector to make the total amount of DNA constant. Cells were harvested using 4 mM EDTA, stained with FITC-conjugated anti-CD20 antibody (Becton Dickinson), and then fixed with 70% methanol. This was replaced with 1x PBS containing propidium iodide (50 µg/ml) and RNase (100 µg/ml; Sigma) for analysis by flow cytometry. The DNA content of all of the cells was analyzed by fluorescence-activated cell sorting (FACSort, Becton Dickinson) as described previously (30) . The sub-G1 DNA content is indicative of apoptosis and is shown in the bar graphs.

Immunohistochemistry.
The anti-p53 antibody DO.1 was used to detect expression levels of p53, whereas the antibody FP3.2 was used to detect Ser392-phosphorylated p53. Immunohistochemistry was scored using the "Quickscore" method (31) . Briefly, two scores were given. First, the a score is the number of nuclei staining positive as a proportion of the total number of nuclei: 1 = 0–4%; 2 = 5–19%; 3 = 20–39%; 4 = 40–59%; 5 = 60–79%; and 6 = 80–100%. Second, the b score represents the intensity of staining: 0 = negative; 1 = weak; 2 = intermediate; and 3 = strong. The product of a x b = total score, which can range from 0 to 18.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The Mutant p53 p53H175 Is Phosphorylated on Ser392 like Wild-Type p53.
Previous studies have shown that Ser392 on wild-type p53 was phosphorylated in response to DNA-damaging agents. To test whether the mutant p53 was similarly phosphorylated under those conditions, we treated cells with UV radiation and cisplatin, a commonly used chemotherapeutic drug. To eliminate any variations caused by different cell contexts, we constructed H1299 cell lines that stably express mutant p53H175 or wild-type p53 under the induction of doxycyclin (clone B231).3 As a control to demonstrate the specificity of the antibody for the phosphorylated form of Ser392, a p53H175 mutant was generated in which Ser392 was changed to alanine, p53H175A392. This was also stably transfected into H1299 cells. After treatment with cisplatin or UV radiation, the phosphorylation status of p53 was analyzed with the anti-p53 Ser392-phospho-specific antibody (FP3.2; Ref. 16 ). The total p53 levels were assessed using a non-phospho-specific antibody (DO-13). The phospho-specific antibody recognized the Ser392-phosphorylated form of the p53 protein but not p53H175A392, in which the Ser to Ala mutation prevents phosphorylation at this site, even though similar levels of p53 protein were expressed (Fig. 1)Citation . Phosphorylation of p53H175 was also evident in untreated cells. Finally, DNA-damaging agents such as UV radiation and cisplatin increased phosphorylation at Ser392 of both wild-type p53 and p53H175. This shows that the mutant p53H175 can be phosphorylated in vivo and also respond to DNA-damaging agents like wild-type p53.



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  		Fig. 1. p53 mutants are phosphorylated like wild-type p53 on Ser392. Immunoblot shows lysates from H1299 non-small cell lung carcinoma cells that stably express p53 mutants as labeled. B231 is a clone that inducibly expresses wild-type p53. The antibody FP3.2 was used to detect Ser392-phosphorylated p53 (top panel), whereas DO-13 was used to probe for total p53 (bottom panel).

 
Ser392 Phosphorylation Regulates the Transforming Activity of Mutant p53.
In addition to the loss of wild-type function of p53, certain hot spot mutants such as p53H175 are also able to transform REFs in cooperation with an activated ras oncogene (2 , 32) . We tested the effect of phosphorylation at Ser392 on the transforming activity of p53H175 and p53W248. Because the oncogenic activity of mutant p53 differs according to the polymorphic R/P codon 72, we also tested this aspect in the REF transformation assay. REFs were transfected with p53R72H175S392, p53R72H175A392, p53P72H175S392, p53P72H175A392, p53R72W248S392, p53R72W248A392, p53P72W248S392, or p53P72W248A392 together with the ras oncogene. Consistent with previous findings, the R72 polymorphic p53 mutants were more active than P72 in transformation of REFs. Interestingly, we also observed that all Ala392 p53 mutants were far more active than their counterparts in cooperation with the ras oncogene at transforming REFs (Fig. 2A)Citation . For both R72 or P72 polymorphic p53H175 or p53W248, a point mutation of Ser392 to Ala392 enhanced the transforming activity of these p53 mutants by 57% and 90%, respectively (Fig. 2B)Citation . Moreover, we also tested the transforming activity of p53P72H175E392, a mutant that is designed to mimic the Ser392-phosphorylated form of p53H175. The number of transformed foci produced by p53H175E392 was similar to that produced by p53H175, even though far more transforming colonies were produced by the nonphosphorylatable p53 mutant p53H175A392 (Fig. 2C)Citation . These results demonstrated that Ser392 phosphorylation plays a very important role in regulating the oncogenic function of mutant p53 irrespective of its polymorphism and mutation sites.



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  		Fig. 2. Preventing phosphorylation at Ser392 of p53H175 or p53W248 enhances their transformation potential. Transformed colonies of rat embryo fibroblasts (BioWhittaker) were transfected with 2 µg of p53 mutant DNA and 5 µg of EJ6.6 (activated) Ras as described previously (2) . Graphs show the number of transformed rat embryo fibroblast colonies after transfection with each of the respective p53 mutants.

 
Increased Inhibition of Wild-Type p53 and p73 by the Nonphosphorylatable p53 Mutant p53H175A392.
One mechanism by which mutant p53 transforms cells is by overcoming the tumor suppression function of wild-type p53 (and perhaps p73) in a dominant negative fashion (32 , 33) . Because one of the most important tumor suppression functions of wild-type p53 is its ability to induce apoptosis, we tested the ability of different phosphorylation mutants of p53H175 to inhibit the apoptotic function of p53 and p73. In agreement with the REF assays of the p53H175 mutants, p53H175A392 was the most potent inhibitor of the apoptotic function of wild-type p53 (Fig. 3A)Citation . There was little difference in the ability of the three p53H175 mutants to inhibit the apoptotic function of p73 (Fig. 3B)Citation . It is also important to note that the expression level of p53H175A392 was consistently the lowest among the three mutants (Fig. 3C)Citation . Therefore, p53H175A392 appears to be the most active inhibitor of apoptosis induced by p53. The results suggested that phosphorylation at Ser392 of p53H175 has a significant effect on its ability to inhibit the apoptotic function of p53. Preventing phosphorylation of p53H175 increases its potency in acting as a dominant negative inhibitor with respect to wild-type p53. This may be the molecular explanation of the enhanced transformation potential of p53H175A392 in REFs.



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  		Fig. 3. The p53 phosphorylation mutants exhibit a dominant negative effect on p53- and p73-induced apoptosis. A and B, flow cytometric assays measuring the effect of p53 mutants on apoptosis induced by transiently transfecting Saos-2 cells with 3 µg of p53 (A) or 5 µg of p73 (B) in the presence or absence of 15 µg of the p53 mutants p53H175 and p53H175E392 or 20 µg of p53H175A392 as described previously (27 , 30) . C, expression levels of p53 mutants when transiently transfected in Saos-2 cells blotted by DO-13.

 
p53H175A392 Enhances Cellular Resistance to the Cytotoxic Effect of UV Radiation and Cisplatin.
Most tumor therapies in use today cause DNA damage. At the cellular level this activates various p53-dependent and -independent pathways that, in most cases, trigger apoptosis (34) , but some tumors are relatively resistant to cancer therapies. The hypothesis that this is due to dominant negative activity of mutant p53 is supported by data showing that p53H175 can protect H1299 cells from p53-independent apoptosis induced by cisplatin and etoposide (35) . If the phosphorylation of mutant p53 at Ser392 plays an important role in regulating the activities of p53H175, mutations mimicking or preventing phosphorylation at Ser392 of p53H175 might also alter the antiapoptotic function of mutant p53 and hence cellular resistance to DNA damage. Tumor cells commonly express only mutant p53 because of loss of heterozygosity (invariably, the loss of the wild-type p53). Therefore, to mimic tumor cells in vivo, the p53-null human lung carcinoma cell line H1299 was engineered to stably express only p53H175, p53H175A392, or p53H175E392. To minimize clonal effects, several clones positive for expression were pooled for each mutant. Fig. 4ACitation shows cell staining in which all of the p53 mutant proteins were expressed and appeared to be predominantly nuclear.



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  		Fig. 4. Mimicking phosphorylation at Ser392 of p53 mutant, p53H175 by a Glu mutation abolishes oncogenic function, whereas preventing phosphorylation by an Ala mutation enhances it. A, immunostaining of pooled H1299 stable clones expressing p53 mutants using anti-p53 (DO-13). B and C, survival assay showing colonies after treatment with chemotherapeutic drug cisplatin (1.25 µg/ml) or UV (20 J/m2) after 7 (+UV) or 14 (+cis-) days. D, Western blot showing the total p53 levels (determined using the mouse monoclonal antibody DO-13). Quantification was performed using the Syngene GeneSnap program, and values are shown below the figure. E, graph depicting the potency of p53H175A392. This is shown by the number of colonies per unit protein of the respective p53 mutant and is calculated by dividing colony numbers in C over the protein levels in E.

 
To test the effect of the phosphorylation mutants of p53H175, stable cell lines expressing these mutants were treated with UV radiation or cisplatin. Both DNA-damaging agents can trigger the phosphorylation of Ser392 in wild-type p53 (16 , 18) . The colony forming assays (Fig. 4, B and C)Citation are consistent with published data that apoptosis induced by cisplatin in H1299 cells is inhibited by expression of p53H175 (35) . In addition, we observed that although H1299 cell lines stably expressing p53H175 and p53H175A392 were much less sensitive to cell killing caused by treatment with cisplatin or UV radiation in comparison with parental or vector stably transfected H1299 cells, p53H175E392 lost its protective ability. Although similar numbers of colonies were obtained in H1299 cells expressing p53H175 and p53H175A392, both of which are resistant to cisplatin and UV radiation, the expression levels of the two proteins are dramatically different (Fig. 4D)Citation . To reflect the potency of p53H175 and p53H175A392, the number of colonies (Fig. 4C)Citation was divided by the protein levels (Fig. 4D)Citation of the two p53 mutants for each treatment. The result shown in Fig. 4ECitation suggested that the potency of p53H175A392 is much greater than that of p53H175. This implies that dephosphorylation of p53H175 at Ser392 enhances the ability of p53H175 to confer radioresistance and chemoresistance.

p53H175E392 Remains Transcriptionally Inactive in Vivo.
Remarkably, p53H175E392 lost its ability to protect cells from the killing effect of cisplatin or UV radiation (Fig. 4, B and C)Citation . This was not due to a lack of protein expression of p53H175E392 (Fig. 5A)Citation . With cisplatin, p53H75E392 gave fewer colonies than the control vector. A previous report demonstrated that a peptide corresponding to the COOH terminus of p53 (residues 361–382) could reactivate the DNA binding activity of p53H175 (36) . Furthermore, a similar p53 peptide (residues 369–383) could reactivate latent DNA binding of wild-type p53, as could phosphorylation of the casein kinase II site (Ser392) of wild-type p53 (13) . Thus, replacing Ser392 with Glu392, p53H175E392 should mimic the phosphorylated form of p53H175 that might reactivate the DNA binding activity of p53H175. If this was the case, it may explain the failure of p53H175E392 to protect cells from the killing effect of UV radiation and cisplatin. To test this, we measured the transactivation function of all three mutants of p53H175. If the DNA binding activity of p53H175E392 was reactivated in vivo, then it should transactivate p53 target genes such as mdm2 and Bax in a manner similar to wild-type p53 because its transactivation domain is exactly the same as that of wild-type p53. Fig. 5BCitation shows that p53H175E392 has very little transcriptional activity on either the bax or mdm2 promoters compared with wild-type p53. In fact, the transcriptional activity of p53H175E392 is very similar to that of mutant p53 p53H175 or p53H175A392. Thus, p53H175E392 retains the properties of mutant p53 with respect to transactivation, yet it is impaired in its ability to confer cellular resistance to UV radiation and cisplatin. Therefore, the failure of p53H175E392 to protect cells from the killing effect of UV radiation and cisplatin suggested that phosphorylation at Ser392 inhibits the ability of p53H175 to confer cellular resistance to DNA damage. This is complementary to the observation on p53H175A392 that the ability of p53H175 to confer cellular resistance to cisplatin and UV radiation can be enhanced by dephosphorylation at Ser392.



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  		Fig. 5. p53H175E392 is an impotent mutant, but nonetheless a mutant. A, Western blots showing total p53 (blotted by DO-13) of p53H175 and p53H175E392 on treatment with various DNA-damaging agents. B, transactivation assays were performed as described elsewhere (27 , 29) . Graph represents fold of activation the respective reporters (averaged from duplicate experiments). Western blot above the graph shows expression level of p53 (top band), whereas PCNA (bottom band) is used as a loading control.

 
Some Breast Tumors Express Ser392 Unphosphorylated Mutant p53 in Vivo.
Knowing that nonphosphorylatable p53 mutants were more oncogenic, we examined the phosphorylation status of Ser392 in a panel of 60 human breast tumors. The expression levels of total p53 and Ser392-phosphorylated p53 were also compared within the same tumor samples. The expression levels of total p53 and Ser392-phosphorylated p53 were detected with antibodies DO.1 and FP3.2, respectively. Of the 60 tumors, 12 express mutant p53, and 2 of them contain p53H175. The majority of the mutant p53-containing tumors expressed low levels of p53 (7 of 12 tumors). Interestingly, in the remaining five mutant p53-expressing tumors, we observed a significant reduction in the levels of Ser392-phosphorylated p53, even though their total p53 expression levels were high (Table 1Citation , cases 19, 32, 33, 48, and 53). Moreover, two of the tumors expressing p53H175 belong to this group. This result demonstrates that some tumors with mutant p53 express unphosphorylated Ser392 p53 in vivo. Perhaps dephosphorylation at Ser392 may confer a growth advantage for these tumors.


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  		Table 1 Immunohistochemistry staining of 60 breast tumor cases with anti-p53 antibodies. DO.1 and FP3.2 was used to measure the expression levels of p53 and Ser392-phosphorylated p53, respectively. The intensity and proportion of total p53 (DO.1) or Ser392-phosphorylated p53 (Ser392) staining were scored as described in "Materials and Methods." Lack of p53 expression was scored as 0, whereas the highest p53 expression in 100% of the cells was scored as 18. The mutated nucleotide is shown in lowercase, and the affected p53 residues are indicated with numbers and letters. Some breast tumors expressing mutant p53 have high levels of p53 (DO.1), and the majority of them are unphosphorylated at Ser392.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we presented a novel biological function of p53 phosphorylation: modulating the oncogenicity of mutant p53. Unlike what we learned from wild-type p53, phosphorylation at Ser392 has a clear effect on the antiapoptotic function of mutant p53, and this is independent of the DNA binding activity of p53. We have used a genetic approach by using mutants that mimic phosphorylation and dephosphorylation, and the effects we observed underscore the importance of this posttranslational modification. Even though we have chosen to focus on one phosphorylation site (Ser392) of two p53 mutants, p53H175 and p53W248, the conclusions drawn from this study demonstrated the biological importance of Ser392 phosphorylation in regulating the oncogenic function of mutant p53. Enhanced transformation potential of p53H175A392 correlates with increased dominant negative activity over wild-type p53; the ability of p53H175A392 to inhibit p53-induced apoptosis is clearly better than that of p53H175 in all of the experiments we performed. Under the same experimental conditions, the difference in their ability to inhibit p73-induced apoptosis was minimal. Only when the expression levels of p53H175 and p53H175A392 are taken into consideration can the suggestion that p53H175A392 is more potent than p53H175 in inhibiting p73-induced apoptosis be given credence. Therefore, the main mechanism of transformation by this p53 mutant in primary cells (in which wild-type p53 is present) is by acting in a dominant negative manner with respect to p53, even though it can also inhibit apoptosis induced by the p53 family member p73. All of these observations agree with the role of p53 as a tumor suppressor (37) , whereas p73 is more involved in development (38) . This may also explain why mutant p53H175E392 that mimics the phosphorylated form of p53 was not more active than p53H175 at transforming REFS in cooperation with the ras oncogene. Unphosphorylated p53 at Ser392 is expressed in human breast tumors containing mutant p53, in agreement with the observation that the nonphosphorylatable p53 mutants, p53H175A392 and p53W248A392, were more transforming. Moreover, most of the mutant p53 expressed in tumors containing p53H175 was unphosphorylated at Ser392. Thus, dephosphorylation of p53H175 or p53W248 is likely to play an important role in the initial stages of tumor development. In tumor cells with only mutant p53, the unphosphorylated form of mutant p53 is likely to be more potent at protecting cells from the cytotoxic effect of radiotherapy and chemotherapy. The phosphorylated form of p53H175, on the other hand, is correspondingly less able to confer resistance. DNA damage can result in phosphorylation at Ser392 of mutant p53. This may explain in part why some tumors expressing mutant p53 are still sensitive to treatment. The factors that influence the phosphorylation status of mutant p53 at Ser392 are likely to play an important role in determining tumor responses to various cancer treatments. More than 10 phosphorylation sites have been identified in wild-type p53 (39) ; we do not yet know whether phosphorylation at any of these other sites would be as important as Ser392 in regulating the oncogenic functions of mutant p53. Understanding the effects of phosphorylation on mutant p53 function may allow us to predict and perhaps even influence the responsiveness to chemotherapy of tumors harboring mutant p53.


   ACKNOWLEDGMENTS
 
We thank Prof. P. Farrell for reading the manuscript and Daniele Bergamaschi for technical support.


   FOOTNOTES
 
Grant support: Ludwig Institute for Cancer Research.

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.

Note: D. B. S. Yap and J-K. Hsieh contributed equally to this work.

Requests for reprints: Xin Lu, Ludwig Institute for Cancer Research, St. Mary’s Branch, Norfolk Place, London, W2 1PG, United Kingdom. Phone: 442075637710; Fax: 442077248586; E-mail: x.lu{at}ic.ac.uk

3 L. Fallis, unpublished data. Back

Received 5/14/02. Revised 5/ 5/04. Accepted 5/12/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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