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Molecular Biology and Genetics |
Department of Molecular Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195 [K. V. G., A. V. B., L. G. B., P. M. C., A. V. G.], and Department of Pathology, University of Iowa, Iowa City, Iowa 52242 [O. W. R., M. B. C.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nevertheless, the wild-type alleles of p53 are frequently lost during further tumor progression, indicating that the dominant-negative effect of p53 mutants is insufficient for complete inactivation of the wild-type function and that they might have some additional properties contributing to cancer development. In fact, some of the p53 mutants exhibit gain-of-function and can modulate tumor cell phenotype, even in the absence of the wild-type allele, by increasing clonogenic potential, tumorigenicity, and invasiveness (5, 6, 7, 8) . In addition, some p53 mutants are capable of (a) further increasing genetic instability by abrogating the mitotic spindle checkpoint (9) ; (b) changing differentiation status of cells (10) ; and (c) increasing resistance to certain chemotherapeutic drugs (11, 12, 13) .
The majority among p53 mutant tumors express a single mutant allele of p53 (see p53 mutation database).4 However, p53 mutation databases contain several cases in which two different p53 mutations are coexpressed in one tumor, presumably resulting in the formation of mixed proteins built from all possible combinations of mutant p53 monomers. The biological significance of coexpression of two different mutants in one tumor has not been analyzed and became the subject of the present work.
As a model, we chose prostate carcinoma cell line DU145, which carries different mutations in two alleles of p53 (14) none of which has been characterized before. To understand the biological sense of their coexistence in one tumor, we analyzed the functionality of these mutants. Our approach was to introduce them into p53-deficient cells of prostate and nonprostate origin and to determine phenotypic changes associated with their separate or combined expression. We found that the properties of the p53 protein complex resulting from the coexpression in one cell of two DU145 mutants was different from what could be expected from a simple sum of the properties of two mutants. One of the most remarkable new functions of the new p53 was its ability to confer resistance to Fas-mediated apoptosis, a new gain-of-function activity that presumably reflects the potential mechanism behind the selection of this combination of p53 mutants within a tumor.
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MATERIALS AND METHODS |
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Transfection and Retroviral Transduction.
Packaging cells plated in 60-mm plates were transfected with 10 µg of retroviral vector DNA using standard calcium phosphate procedure. The medium was changed after 8 h. Virus-containing media supplied with 8 µg/ml of Polybrene (Sigma) were collected at 24 and 48 h posttransfection and were used for infection. Virus-transduced cells were selected for the resistance to an appropriate selective agent (G418, hygromycin, or puromycin, depending on the vector) up to a complete death of noninfected cells. Transfections of other cells were carried out with LipofectAMINE Plus reagent (Life Technologies, Inc.) according to the manufacturer’s recommendations.
Isolation and Cloning of Mutant p53 from DU145 Cells.
cDNA was synthesized from total RNA of DU145 cells using SuperScript II (Life Technologies, Inc.) reverse transcriptase from oligo(dT) primers. Primers flanking the coding region of the human p53 gene (left, 5'-acgcttccctggattgg; right, 5'-ggcaggggagggagaga) were used for PCR amplification with synthesized cDNA as a template. The PCR product of 1287 bp was cloned to pCR2.1 vector using TA Cloning kit (Invitrogene). Because it was known that Du145 cells contain different point mutations in each allele of the p53 gene (223 codon CCT to CTT in one, and codon 274 GTT to TTT in the other; Ref. 14
), we screened resulting clones by hybridization with the mutant-specific 17-mer oligonucleotide probes synthesized to contain mutant nucleotides in the middle. Four different 17-mer probes were used for hybridization: two corresponding to the wild-type sequence of codons 223 (5'-TGAGCCGCCTGAGGTTG) and 274 (5'-AGGTGCAGTTTTGTGCC), and two corresponding to the mutated sequence of the same codons: 223, 5'-TGAGCCGCTTGAGGTTG; 274, 5'-AGGTGCATTTTTGTGCC. Hybridization was carried out under conditions calculated to distinguish between perfectly matched hybrids and hybrids with a single mismatch (at 48°C in 4x SSC). Each variant of mutant p53 was recloned in retroviral-expressing vectors with different selective markers; pPSH (15)
transgene expression was regulated by LTR5
and hygromycin resistance gene by SV40 promoter; and pLPCX (Clontech) transgene expression was driven by CMV and puromycin resistance gene by LTR.
Colony, Cell Growth, and Apoptotic Assays.
For colony assay, subconfluent cultures in 60-mm plates were transfected or infected with the tested constructs and selected with an appropriate antibiotic (or combination of antibiotics in case of infection or transfection with two different vectors). In parallel, the same transfection solutions or virus-containing media were transferred on Rat1 cells, known to be resistant to transduction with wild-type p53, for estimation of transfection efficiency or virus titer. Cell growth after treatment with apoptosis-inducing agents was estimated by methylene blue staining of methanol-fixed cells attached to the plate with subsequent dissolving of the stain in 0.1 N HCl followed by measurement of absorbance values using ELISA-reader, 
= 600 nm, and calculated as a percentage of absorbance values of untreated cells.
Apoptosis in cell cultures was monitored by 4',6-diamidino-2-phenylindole staining of formalin/glutaraldehyde-fixed cultures, calcein, and DNA fragmentation assays as described previously (16) . All of the assays were done in 4–6 parallels and repeated at least two times. The activation of caspases 2, 8, and 9 was monitored by Western immunoblotting as described previously (16 , 17) using antibodies from Upstate (anti-caspase 2 and 8) and from PharMingen (anti-caspase 9).
FACS Analysis.
Staining of DNA with PI for the estimation of DNA content was done as described previously (18)
. For green fluorescent protein expression analysis, cells were lifted by treatment with 0.5 mM EDTA and washed with PBS, cell pellets were resuspended in PBS with 0.5% of BSA and 1 µg/ml of PI. Results were analyzed using FACSorter (Becton Dickinson) and CellQuest software.
Immunoprecipitation and Western Blotting.
For immunoprecipitation, cells in 150-mm plates were lysed in 2 ml of NP40 buffer [50 mM Tris HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, and 2% NP40) containing 1 mM phenylmethylsulfyl fluoride (Sigma), 10 µg/ml aprotinin (Sigma), 10 µg/ml leupeptin (Sigma), 10 mM sodium orthovanadate (Sigma), and 1 mM NaF (Sigma). Cell lysates were incubated 30 min on ice and centrifuged at 15,000 x g for 20 min. Then lysates were precleaned for 1 h at 4°C with the mixture of nonspecific antibodies corresponding to the isotype of the primary antibodies together with protein A/G agarose (Santa Cruz Biotechnology). Ten µg of one of four antibodies (Pab 240 (Santa Cruz), Pab 1620 (Calbiochem), DO1 (Santa Cruz), and nonspecific mouse IgG mixture) were added to 0.5 ml of the lysate and incubated overnight at 4°C, after which 20 µL of protein A/G agarose (Santa Cruz) were added for 1 h. After washings in lyses buffer, agarose pellets were boiled in Laemmli sample buffer, used for Western immunoblotting (see below) and detected with biotinylated anti-p53 antibodies (Boehringer Mannheim) and streptavidin-conjugated horseradish peroxidase. For Western blotting, cells were lysed in radioimmunoprecipitation assay buffer [25 mM Tris HCl (pH 7.2), 125 mM NaCl, 1% NP40, 1% sodium deoxycholate, and 1 mM EDTA] containing 1 mM phenylmethylsulfyl fluoride (Sigma), 10 µg/ml aprotinin (Sigma), and 10 µg/ml of leupeptin (Sigma). Protein concentrations were determined with Bio-Rad Dc protein assay kit. Equal protein amounts were run on gradient 4–20% precast gels (Novex) and blotted onto polyvinylidene difluoride membranes (Amersham). The following antibodies were used: anti-p53–monoclonal mouse DO1 (Santa Cruz) and biotinylated polyclonal rabbit (Boehringer Mannheim); anti-p21–polyclonal rabbit (Santa-Cruz); anti-mdm2–monoclonal mouse (Santa Cruz); anti-CD95–monoclonal mouse (Transduction Laboratories); anti-actin–polyclonal goat (Santa-Cruz). Horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz. Quantitation of the data were performed using Quantity One software from Bio-Rad.
Transactivation Assay.
Cells in subconfluent cultures were transfected with a combination of the plasmids carrying wild-type p53 or p53 mutants with reporter construct containing reporter CAT DNA under the minimal thymidine kinase promoter and p53-binding sequence from the promoter of p21/Waf1 gene (19)
. A control CMV-LacZ vector expressing ß-galactosidase from the CMV promoter was included in all transfections. Parental vector, pBasicCAT, lacking p53 consensus element, was used as a control in the assays. Forty-eight-h posttransfection cell extracts were prepared, normalized according to protein concentrations, and used for the determination of CAT and ß-galactosidase (Promega) activities.
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RESULTS |
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even after immunoprecipitation (data not shown). Thus, PC3 cells can also be considered p53-deficient.
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As shown in Fig. 1
, expression of wild-type p53 was toxic for all of the cell lines tested, as judged by the inhibition of the colony growth of retrovirus-transduced cells. Surprisingly, the effects of the DU145-derived mutants were cell type-dependent. Neither p53–223Leu nor p53–274Phe affected the growth of PC3 cells, whereas the expression of both proteins was confirmed by Western immunoblotting (Fig. 1a)
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Both mutants caused a growth-suppressive effect on mouse 10(1) cells, although less dramatic than wild-type p53. Consistently, cells, expanded from rare colonies, appeared after the hygromycin selection of virus-transduced 10(1) cells expressed a barely detectable level of mutated p53 protein (Fig. 1b)
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Saos-2 cells showed the third pattern of sensitivity to DU145-derived mutants. p53–223Leu mutant was as active as wild-type p53 in the inhibition of colony growth of Saos-2, whereas p53–274Phe had no growth-inhibitory effect in these cells (Fig. 1c)
. The inhibition of colony growth in Saos-2 cells by p53–223Leu correlated with the induction of apoptosis by this mutant in a transient-transfection assay. The proapoptotic effect of the p53–223Leu mutant in these experiments was similar to that of wild-type p53, whereas transfection of p53–274Phe did not induce apoptosis (Fig. 1d)
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Thus, the analysis of biological effects of p53–223Leu and p53–274Phe mutants on p53-deficient cells indicates that both of them clearly differed from wild-type p53. This difference was more pronounced in the case of p53–274Phe mutant, which showed minor growth inhibition in only one cell line and was incapable of inducing apoptosis. Another mutant, p53–223Leu, displays both of these activities in two cell lines. Remarkably, none of the mutants showed any wild-type function in human prostate PC3 cells, suggesting that some unknown tissue-specific factors might modulate p53 activity in different cell contexts.
DU145-derived p53 Mutants Do Not Possess Dominant-Negative Activity against Wild-Type p53.
Some tumor-derived p53 mutants, acting as inhibitors of wild-type p53 (Ref. 21
, and references therein), display a dominant-negative effect. We used two approaches to estimating the potential dominant-negative activity of DU145-derived mutants: (a) testing the toxicity of wild-type p53 transduced into the cells expressing the studied mutants; and (b) the suppression of p53-dependent apoptosis by transduction of the mutants.
The first type of assay was done using PC3 cells, stably expressing p53–223Leu or p53–274Phe. They were transduced with a retrovirus carrying human wild-type p53 cDNA, and the growth of virus-transduced colonies was analyzed. The results of these experiments (Fig. 2a)
show that neither of the DU145-derived mutants protected PC3 cells from growth inhibition by wild-type p53. Cells expressing previously isolated dominant-negative mutant GSE56 (22)
were protected from wild-type p53 activity under these conditions (Fig. 2a)
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) of either of the tested mutants without any detectable changes in cell growth or morphology (not shown). Moreover, C8 cells expressing DU145-derived mutants are still capable of inducing p21waf1 on DNA damage (UV). However, neither p53–223Leu nor p53–274Phe protected C8 cells from p53-dependent apoptosis caused by serum withdrawal or by DNA-damaging drug treatment (etoposide, doxorubicin), as opposed to control dominant-negative p53 mutants p53–175His or GSE56 (Refs. 24
, 25
; Fig. 2b
). Thus, DU145 p53 mutants do not show dominant-negative effect against endogenous wild-type p53 in C8 cells.
p53–274Phe Can Abrogate Growth-suppressive Activity of p53–223Leu.
We then tested whether p53–223Leu and p53–274Phe could affect each other’s properties if coexpressed within the same p53-deficient cells, thus imitating the situation existing in DU145 cells. p53-deficient cells were infected with the combination of two retroviruses, carrying different selectable markers, followed by the selection of cells that coexpress both constructs. Combinations used included each of the DU145-derived p53 mutants coexpressed with either the insert-free vector or with another mutant.
In PC3 cells, coexpression of both mutants did not have any detectable effect on colony growth (Fig. 1a)
. In 10(1) cells, previously shown to be sensitive to each mutant separately, their combination did not show any toxicity (Fig. 1b)
. In Saos-2 cells, which were sensitive to p53–223Leu, expression of p53–274Phe almost completely neutralized the growth suppressive effect of p53–223Leu (Fig. 1c)
. In transient transfection assays, coexpression of p53–274Phe with p53–223Leu resulted in 
3-fold less proportion of apoptotic cells than in the population transduced with p53–223Leu alone (Fig. 1d)
, indicating that the p53–274Phe mutant can abrogate the residual proapoptotic activity of p53–223Leu.
Thus, we have an interesting example of two tumor-derived p53 mutants that do not possess dominant-negative activity against wild-type p53 but are capable of neutralizing each other’s growth inhibitory activity when expressed together. This observation suggests that the DU145-derived mutants could functionally interact with each other, presumably creating p53 protein with new properties.
Conformation of DU145-derived Mutants.
Biological activity of p53 largely depends on conformation of DNA-binding domain. Proper folding of DNA-binding domain of p53 can be estimated experimentally by the potency of interaction with the conformation-sensitive antibody capable of distinguishing between wild-type and mutant forms of the protein. We analyzed conformation of DU145-derived p53 mutants expressed in PC3 cells, separately and in combination, by immunoprecipitation with conformation-specific antibodies (Pab1620 and Pab240 recognizing preferentially wild-type and mutant p53, respectively) followed by p53 detection by biotinylated polyclonal antibodies on Western blots. As seen in Fig. 3
, p53–223Leu mutant retained wild-type conformation as confirmed by its ability to bind with Pab1620 antibodies. However, p53–274Phe was precipitated well by both antibodies. Interestingly, combined expression of both mutants generated p53 protein with predominantly denatured-form DNA-binding domain preferentially binding to Pab 240.
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and doxorubicin-induced (data not shown) DNA damage in PC3 cells (Fig. 4a)
. Hence, although the two mutant proteins studied differed in their conformation, their ability to be accumulated in response to DNA damage makes them similar to wild-type p53. Interestingly, cells expressing a combination of both mutants did not show any changes in the amount of p53 protein after DNA damage (Fig. 4a)
, suggesting that properties of the mixed p53 protein complex formed by a combination of two DU145-derived mutants may be different from the properties of the proteins formed by each of the subunits expressed separately.
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. Similar results were obtained with Hdm2 expression. Detectable levels of Hdm2 were observed only in the case of PC3 expressing p53–223Leu mutant after treatment with UV (Fig. 4a)
, again indicating that p53–274Phe as well as the combination of mutants were incapable of visible induction of a p53-responsive gene.
Transactivation abilities of DU145-derived mutants were also tested in a direct transactivation assay using transient transfection of different p53-expressing constructs in combination with a CAT reporter construct driven by the promoter with p53-binding site from the p21waf1 gene (26)
. In these experiments p53–223Leu was as active as wild-type p53 in transactivation of the reporter construct, whereas p53–274Phe had no detectable activity (Fig. 4b)
. Coexpression of p53–274Phe inhibited transactivation by p53–223Leu, thus correlating with the above-described effects of this mutant on growth-inhibitory activity of p53–223Leu. The lack of detectable transactivation activity of p53–274Phe suggests that the toxicity of this protein to 10(1) cells is mediated not through p21.
DU145-derived p53 Mutants and Fas Sensitivity of PC3 Cells.
PC3 cells and DU145 cells differ dramatically in their sensitivity to Fas-mediated apoptosis (16)
. Whereas PC3 cells are highly sensitive, DU145 are completely resistant even to a high dose of anti-Fas antibodies. We compared the sensitivity to Fas-mediated apoptosis of parental PC3 cells and PC3 cells expressing p53–223Leu, p53–274Phe, or their combination. Fig. 5
demonstrated the dose dependence of these variants of PC3 cells to the Fas ligand agonist, anti-Fas mAb. Cell death was estimated by calcein and DNA fragmentation assays. Both methods revealed strong protection of PC3 from anti-Fas mAb-induced apoptosis by the combination of two DU145-derived mutants (results of a representative calcein assay are shown in Fig. 5
). The results of cellular assays were confirmed by the analysis of caspase activation in p53 mutant-transduced PC3 cell variants (Fig. 5
, bottom panel). Activated forms of caspases 8, -2, and -9 were detected in cell lysates as early as 4 h after treatment of PC3 cells, transduced with empty vectors or with p53–223Leu mutant. p53–274Phe mutant decreased the level of activated caspases, whereas a combination of both mutants prevented the appearance of activated forms of all of the studied caspases at the 4th and 8th h after adding anti-Fas mAb (Fig. 5)
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). We found that both p53–223Leu and p53–274Phe induced Fas resistance in a dose-dependent manner; however, neither of them alone was as powerful in protection from apoptosis as their combination suggesting that the mutants act in an additive way.
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. This phenomenon, however, can hardly be attributed to cycloheximide-mediated reduction of the p53-mutant proteins because prolonged (up to 31 h) treatment with cycloheximide showed no dramatic changes in the levels of the mutant p53 (Fig. 7b)
. Thus, if Fas resistance is, in fact, mediated by some unstable protein factor, it is unlikely to be mutant p53 itself but, rather, it could be one of the proteins induced by the p53 mutants.
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demonstrate that p53–223Leu expression is associated with a slight decrease in the amount of Fas. The expression of p53–274Phe results in a more remarkable (2-fold) reduction of Fas expression as compared with that in control vector-transduced PC3. The combination of both mutants has a synergistic effect on the reduction of Fas, exceeding the effect of a "double dose" of p53–274Phe reached by coexpression of two retroviral vectors (pPS-hygro and pLPC) in the same cells.
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DISCUSSION |
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Inactivation of both p53 and Fas signaling are frequent events in cancer that play an important role in cancer progression. Whereas the suppression of p53 results in genomic instability and rapid progression (27) , the blocking of Fas sensitivity contributes to tumor escape from host immune response (28) . In addition, both may increase tumor resistance to anticancer treatment through the suppression of apoptosis (23 , 29) . Thus far, there have been two mechanistic links between these two pathways. In some cells, Fas behaves as a p53-responsive gene (30) and Fas sensitivity could be modulated in a p53-dependent manner after DNA-damaging treatments in vitro and in vivo (31) . p53 protein was also shown to be involved in intracellular transport of Fas (32) . In both cases, p53 cooperates with Fas expression. Therefore, p53 deficiency is expected to attenuate Fas-mediated apoptosis. In fact, a correlation between p53 status of the tumor and its sensitivity to Fas has been experimentally confirmed by the analysis of a series of isogenic tumor cell lines differing in their p53 status (31) . Presumably, attenuation of sensitivity to Fas is one of the driving forces behind the selection of tumor cell variants with inactivated p53.
Although the inactivation of p53 may cause a reduction in cell sensitivity to Fas-induced apoptosis, it, nevertheless, cannot make the tumor cell completely Fas resistant; Fas sensitivity of p53-deficient PC3 cells (16) is an illustration for this statement. However, transduction with p53 mutants derived from Fas-resistant cells of prostate origin, DU145, converts PC3 into Fas resistance, indicating a new type of Fas-p53 interaction, in which altered p53 protein plays the role of an active inhibitor of Fas-mediated apoptosis, which causes a strong down-regulation of Fas expression. This phenomenon demonstrates a new gain-of-function activity of mutant p53.
Gain-of-function has been reported for many tumor-derived p53 mutants (5 , 13 , 21) . Mutant p53 can acquire alterations in their transactivation capabilities (13 , 33) and increase cell resistance to cytotoxic drugs even when transduced on a p53-deficient background. Down-regulation of Fas expression accompanied by resistance to Fas-mediated apoptosis is a new type of gain-of-function by p53 mutants. It is likely that mutants with such unusual properties were picked by natural selection because they provide additional selective advantages to tumor cells in vivo. Although each of the DU145-derived mutants possesses this anti-Fas property, their combined expression generates p53 protein with much stronger anti-Fas activity, providing an unusual example of gain-of-function that presumably results from the combination of two mutant p53 subunits in one protein.
Functional interaction of structurally different p53 monomers within one protein has been known for a long time as a phenomenon associated with dominant-negative mutants. It is believed that incorporation of a mutant subunit into a p53 homotetramer may result in its functional inactivation (21) . In this case, one allele (inactive) clearly dominates over another one (wild-type). DU145-derived mutants, however, represent another more complicated form of functional interaction of two altered proteins. Although none of them alone showed dominant-negative activity against wild-type p53, in combination they can modify each other’s properties, presumably by creating a mixed protein complex consisting of two mutant forms of p53 protein that is not functionally equivalent to the homogeneous complexes formed by any of the subunits.
In principle, another mechanism could be responsible for the observed phenomenon. Instead of the formation of a putative "mixed" protein complex with new properties, Fas resistance may result from combined changes in gene expression caused by each of the mutants. It this scenario, DU145-derived mutants would cooperate indirectly through their responsive genes. Although we cannot unequivocally choose between the two models, altered biochemical properties of p53 protein, resulting from the coexpression of the studied mutants, argues in favor of the first possibility.
Fas-suppressing p53 mutants can be viewed as therapeutic targets to develop compounds that would cause antitumor effect by sensitization to Fas. However, it remains unclear how common is the anti-Fas activity of tumor-derived p53 mutants; this question is the subject of our ongoing study.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grants CA75179 and CA60730 (to A. V. G.) and CA76673 (to M. B. C.). 
2 K. V. G. and O. W. R. contributed equally to the work and are listed alphabetically.
3 To whom requests for reprints should be addressed, at Department of Molecular Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. Phone: (216) 445-1205; Fax: (216) 444-0512; E-mail: gudkov{at}ccf.org
4 Internet address: http://www.iarc.fr/p53/Index.html.
5 The abbreviations used are: LTR, long terminal repeat; CAT, chloramacetyltransferase; CMV, cytomegalovirus; FACS, fluorescence-activated cell sorter; PI, propidium iodide; mAb, monoclonal antibody.
Received 11/25/02. Accepted 3/31/03.
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REFERENCES |
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-induced apoptosis in the human prostatic carcinoma cell line LNCaP. Oncogene, 19: 1959-1968, 2000.[Medline]
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