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The gain of function of the p53 mutant Asp281Gly is dependent on its ability to form tetramers
Cancer Letters 185 (2002) 103–109 www.elsevier.com/locate/canlet
The gain of function of the p53 mutant Asp281Gly is dependent on its ability to form tetramers Annemieke Atema a, Patrick Che`ne b,* a
Dana-Farber Cancer Institute and Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA b Novartis, Oncology Department, K125 420, CH-4002 Basel, Switzerland Received 16 April 2002; received in revised form 10 May 2002; accepted 20 May 2002
Abstract The influence of tetramerisation on the properties of the p53 mutants is poorly understood. We describe here the influence of the tetramerisation on the properties of the oncogenic mutant Asp281Gly. We show that despite being both nuclear the tetrameric Asp281Gly and the monomeric Asp281GlyLeu344Pro proteins have different properties: only Asp281Gly stimulates the transcription of the multidrug resistance-1 gene promoter and induces cisplatin resistance in Saos-2 cells. Moreover, we identify a 130-kDa protein that specifically interacts with Asp281Gly but not with Asp281GlyLeu344Pro. This suggests that tetramerisation is important for the properties of the p53 mutants and that these properties might be mediated via protein-protein interactions. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: p53; Gain of function; Tetramerisation; Chemoresistance; Chemosensitivity
1. Introduction p53 is mutated in many cancers [1]. The majority of these mutations is located within its DNA binding domain (DBD) impairing the ability of the mutant proteins to associate with DNA and as a consequence to stimulate transcription. In studies where the full p53 complementary DNA is sequenced, only 4% of the mutations concern amino acids of its tetramerisation domain (TD). Different reports have shown that deletion or multiple mutations within the TD decrease both p53 DNA binding and transcriptional activity [2– 4]. Similar findings are obtained by mutating single amino acids of the TD to alanine [5,6]. These findings are further supported by the analysis of p53 mutants * Corresponding author. Tel.: 141-61-696-2050; fax: 141-61696-3835. E-mail address: [email protected] (P. Che`ne).
identified in cancers, which have a single mutation in their TD. Several of these mutants do not bind to DNA and are unable to stimulate the transcription of reporter genes under the control of p53-regulated promoters [7–11]. Altogether these published data demonstrate that single mutations in the TD can lead to mutants, which have lost the wild type function, as do proteins mutated at their DBD. Therefore, why are such mutations not identified in cancers more often? Various hypotheses can be formulated to answer this question and among them one is very attractive: proteins with a mutation in their TD would be less selected in cancers than DBD mutants because they do not show a gain of function. DBD mutants would be preferentially selected in tumours because they give an additional growth advantage when compared to TD mutants, which have lost only the wild type tumour suppressor activity.
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(02)00318-X
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Supporting this hypothesis, it has been shown that some DBD mutants inactivate wild type p53 by dominant negative effect upon heterooligomerisation [12,13] while TD mutants are not dominant negative [14]. Moreover, it has been established that the TD and/or the C-terminus regulatory region (the last 30 amino acids of p53) are necessary for the gain of function of the p53 mutants [15,16]. In this report, we compare the properties of the tetrameric oncogenic mutant Asp281Gly with the ones of its monomeric counterpart, the Asp281GlyLeu344Pro mutant. Our results indicate that the altered phenotypes observed in the presence of the Asp281Gly mutant are lost when this protein is rendered monomeric. Furthermore, our data suggest that the properties of the Asp281Gly protein might be linked to its ability to interact with other cellular proteins. 2. Material and methods 2.1. Construction of the vectors and establishment of the stable cell lines The Asp281Gly and Asp281GlyLeu344Pro genes were amplified by polymerase chain reaction (PCR) from the corresponding genes cloned in a pCite-2a vector [16]. A HindIII and an EcoRI site were introduced at the 5 0 and 3 0 end of the genes, respectively. The amplified DNA fragments were ligated into a pHMA6 vector (Boehringer Mannheim) and their sequence verified by double strand DNA sequencing. The p53 minus Saos-2 cells were transfected with the empty pHMA6 vector and the mutant expressing plasmids using the calcium-phosphate technique. The cells were then selected during 3 weeks in Dulbecco’s modified minimum essential (DMEM) containing G418 (0.8 mg/ml active concentration). Antibiotic resistant colonies were isolated with cloning rings and expanded into cell lines. Mutant expressing cells lines were selected by Western blot. 2.2. Western blotting and immunofluorescence The p53 protein was detected in Western blot as previously described [9]. For immunofluorescence, the cells were fixed with paraformaldehyde and permeabilised with Triton X-100. The DO-1 antibody
(1 mg/ml) was used to detect the p53 protein and 4,6diamino-2-phenylindole (DAPI, 0.3 mg/ml) to stain the nuclei. 2.3. [ 35S]-methionine labelling and immunoprecipitation Sub-confluent cells were starved for 45 min in methionine free DMEM and incubated for 3 h in the presence of 1.5 mCi [ 35S]-methionine (EXPRE 35S 35S protein labelling mix – NEN). The cells were then lysed and the soluble proteins were immunoprecipitated with the different antibodies and protein-G agarose beads. The bound proteins were loaded onto a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and after migration the gel was dried and exposed to a film. 2.4. Transient transfections, chloramphenicol acetyltransferase (CAT) assay and b -galactosidase measurements Sub-confluent cells in six well plates were transfected with total DNA (4 mg) and lipofectamine 2000 (GIBCO-BRL) according to the manufacturers. After (48 h) transfection, the amount of CAT protein was determined with the CAT ELISA kit (Boehringer Mannheim) according to the manufacturers. The bgalactosidase was determined as described in Sambrook et al. [17]. 2.5. Colony formation assay The cells (5000/plate) were seeded during 24 h in 10 cm plates. The medium was replaced with fresh medium containing different concentrations of cisplatin (Platinol-AQ (Bristol Laboratories) and the cells were incubated for 24 h in the presence of drug. The medium was changed and the cells were incubated for 2–3 weeks with regular medium changes every 3–4 days. After incubation, the colonies were stained with Coomassie Blue and counted. Counts were performed using the average of two equally sized, randomly determined sections from each plate. 3. Results The Asp281Gly protein is mutated in its DBD and is tetrameric. The Asp281GlyLeu344Pro protein is
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Fig. 1. Expression of the p53 mutants in Saos-2 cells analysed by Western blot. Two clones stably expressing the tetrameric mutant Asp281Gly (134.12 and 134.2) and the monomeric mutant Asp281GlyLeu344Pro (135.3 and 135.13) were analysed for their content in p53 protein by Western blot with the monoclonal antibody DO-1. One clone (152.16) stably transfected with the empty expression vector was also analysed. The molecular weights in kDa are indicated.
mutated both in its DBD and its TD and is monomeric [16]. The genes encoding for these two proteins were cloned such that they are fused at their N-terminus to a HA-tag and they were stably transfected into the p53 negative Saos-2 cells. Two stable clones expressing similar amounts of Asp281Gly and Asp281GlyLeu344Pro proteins and one clone obtained by the transfection with the empty expression vector were selected (Fig. 1). Since all the p53-expressing clones produce similar levels of this protein, the data obtained either with one or the other clone of each p53 mutant will be shown. However, the experiments have been realised with all the clones. Since the TD contains a leucine-rich nuclear export signal [18], it was important to verify that the Asp281GlyLeu344Pro protein, having been mutated at its TD, has the same sub-cellular localisation as the Asp281Gly protein. The sub-cellular localisation of Asp281Gly and Asp281GlyLeu344Pro was therefore analysed by indirect immunofluorescence (Fig. 2). The experimental data show that both mutants have
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the same localisation in the cell (Figs. 2B,C) and a nuclear stain with DAPI (Figs. 2F,G) indicates that they are predominantly located in the nucleus. The sub-cellular location of these two mutants is similar to the one of the endogenous Pro278Ala mutant found in 786-0 cells (Figs. 2D,E). No p53 is detected by immunofluorescence in the cell line expressing the empty expression vector (Figs. 2A,E). It has been previously shown that the Asp281Gly protein stimulates the transcription of reporter genes under the control of the multidrug resistance-1 gene (MDR-1) promoter in contrast to the wild type protein that represses this promoter [19]. The stable clones were therefore analysed for their ability to stimulate the expression of a CAT reporter gene under the control of the MDR-1 promoter (Fig. 3). The experimental data show that high amounts of CAT protein are detected only in the clones expressing the Asp281Gly protein. Therefore, the Asp281Gly protein stimulates the expression MDR-1 promoter only when it is in a tetrameric conformation. Several studies have shown that p53 mutants can enhance the resistance of tumour cell lines against different chemotherapeutic agents [20–22]. The cisplatin sensitivity of the stable clones was therefore investigated (Fig. 4). The experimental data show that the expression of the Asp281Gly mutant in the Saos-2 cells enhances the cisplatin resistance of these cells. This is in agreement with already published data, which have shown that p53 mutants enhance cisplatin resistance [20,22]. Very interestingly, the data show that expression of the double mutant Asp281GlyLeu344Pro does not enhance the resistance of the Saos-2 cells. Therefore, once the Asp281Gly mutant is mutated such that it becomes monomeric, it looses its ability to enhance the chemoresistance of the Saos2 cells. To identify proteins that bind to the p53 mutants, the cells from the different clones were metabolically labelled with [ 35S] methionine and their lysates were immunoprecipitated with three different antibodies: the 12CA5 antibody, which is directed against the HA-tag, located at the N-terminus of the mutant proteins, the DO-1 antibody, which is a p53 specific antibody, and the F5 antibody, which recognises the p21 Waf1/Cip1 protein and was used as a negative control antibody. The experimental data (Fig. 5 bottom) show that both the 12CA5 and the DO-1 antibodies immu-
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Fig. 2. Immunolocalisation of the p53 mutants in the Saos-2 cells. The cellular localisation of the p53 proteins was determined in the empty vector (A); the Asp281Gly mutant (B); the Asp281GlyLeu344Pro mutant (C) expressing Saos-2 cells; and in the 786-0 cells (D). The cells were incubated with the monoclonal antibody DO-1 and with a l-rhodamine conjugated anti-mouse secondary antibody. The location of the nucleus is revealed in the empty vector (E); the Asp281Gly mutant (F); the Asp281GlyLeu344Pro mutant (G) expressing Saos-2 cells; and the 786-0 cells (H) by a DAPI staining of the cells. The data presented have been realised with the clone 152.16, 134.12 and 135.3.
noprecipitate a protein with an apparent molecular weight of about 56 kDa from the lysates of the p53 mutant expressing clones. This protein is not immunoprecipitated with the F5 antibody or from the lysates from the cells transfected with the empty vector suggesting that this protein is p53. The higher apparent molecular weight of the p53 protein is due to the fact that it is expressed as a HA-tag fusion protein. The amount of Asp281Gly and Asp281GlyLeu344Pro proteins immunoprecipitated with both the DO-1 and the 12CA5 are similar, confirming that the clones are expressed at similar levels. Further examination of the autoradiographies reveals that a protein with an apparent molecular weight of about 130 kDa is immunoprecipitated with the DO-1 and the 12CA5 antibodies from the lysate of the cells expressing the tetrameric Asp281Gly mutant (Fig. 5 top). This protein is not immunoprecipitated either with the F5 antibody or from the lysate of the cells obtained with the mono-
meric Asp281GlyLeu344Pro mutant and the empty vector indicating that this protein interacts specifically with the tetrameric mutant.
4. Discussion The spectrum of the p53 mutations shows that, even when the entire p53 gene is sequenced, very few mutations are detected in the TD. However, recent publications, in which proteins identified in cancer with a single missense mutation at their TD are studied, reveal that these proteins have lost the wild type activity in fashion similar to proteins mutated at their DBD [7–11]. In this report we utilised a well-characterised oncogenic mutant, Asp281Gly, to determine the influence of the tetramerisation on the properties of this mutant. Our data reveal that the Asp281Gly protein stably
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Fig. 3. Stimulation of the MDR-1 promoter. The Saos-2 cells expressing the empty vector (clone 152.16), the Asp281Gly mutant (clone 134.2) and the Asp281GlyLeu344Pro (clone 135.13) were transiently transfected with a vector containing a CAT reporter gene under the control of the MDR-1 promoter and with a vector expressing the b-galactosidase protein. Two days after transfection the amount, of CAT protein expressed was determined. The CAT amounts were normalised to the b-galactosidase activity. The means and standard deviations of three experiments are represented.
transfected in the p53 null cells Saos-2 stimulates the MDR-1 promoter while its monomeric counterpart, the Asp281GlyLeu344Pro protein, has lost this property. We also show that the cisplatin resistance of Saos-2 cells induced by the expression of the Asp281Gly protein is lost when this protein is rendered monomeric by the mutation Leu344Pro in its TD. Finally, our results indicate that the difference in behaviour between the Asp281Gly and the Asp281GlyLeu344Pro proteins is not linked to a difference in their cellular localisation since both proteins are nuclear. Altogether these findings indicate that some of the properties of the Asp281Gly protein require a tetrameric conformation. These findings led us to the question: why the Asp281Gly mutant needs to form tetramers to show these properties? Our reasoning was as follows: p53 activation requires its accumulation in the cell, suggesting that the p53 protein must reach a certain concentration to bind to DNA and/or to the p53-binding proteins. Since the cellular concentration of the p53 mutants is high in tumour cells, these proteins should be at a concentration that allows them to
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bind to DNA and/or to the p53-binding proteins. Because most of the p53 mutants do not bind to DNA, the only type of interaction in which they could participate would be with other proteins. The constant occurrence of these protein-protein interactions in the tumour cell may deregulate the cellular machinery and induce the apparition of altered phenotypes. The so-called gain of function of the p53 mutants would then be the result of the interactions between the p53 mutants and some cellular proteins. We therefore used the tetrameric Asp281Gly and the monomeric Asp281GlyLeu344Pro mutant to identify such proteins. We have indeed identified one protein of an apparent molecular weight of 130 kDa that binds specifically to Asp281Gly and not to Asp281GlyLeu344Pro. The full characterisation and the elucidation of the biological relevance of this interaction will have to be further demonstrated. However, this finding is the first step towards a better comprehension at the molecular level of the gain of function of the p53 mutants. A full characterisation of the interaction between the 130 kDa protein and the p53 mutants may lead to the design of inhibitors of this interaction and therefore to new ways to target tumours expressing p53 mutants.
Fig. 4. Effect of a cisplatin treatment on the survival of the transfected clones. A similar number of cells of the empty vector (A – clone 152.16), the Asp281GlyLeu344Pro (X – clone 135.13) and the Asp281Gly (W – clone 134.2) expressing clones were treated with the indicated concentration of cisplatin for 24 h and grown for 2–3 weeks with regular change of medium. After growth, the colonies were stained Coomassie Blue and counted. The means and the standard deviation are given.
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Fig. 5. Identification of a protein that binds to the tetrameric Asp281Gly mutant and not to the monomeric Asp281GlyLeu344Pro protein. The Saos-2 cells were labelled with [ 35S]-methionine and after lysis, their p53 content was immunoprecipitated with the anti-HA antibody 12CA5, the anti-p53 antibody DO-1 and the anti-p21 Waf1/Cip1 antibody F5. The upper part of the figure corresponds to the region 116–180 kDa of the autoradiography of a dried gel. The location of the 130-kDa protein is marked with an arrow. The lower part of the figure corresponds to the region surrounding the 48-kDa region of the autoradiography of a dried gel. The location of the HA-p53 protein is marked with an arrow. The clones 152.16, 134.12 and 135.13 were used for this experiment.
References [1] T. Soussi, K. Dehouche, C. Beroud, p53 website and analysis of p53 gene mutations in human cancer: forging a link between epidemiology and carcinogenesis, Hum. Mutat. 15 (2000) 105–113. [2] H.W. Sturzbecher, R. Brian, C. Addison, K. Rudge, M. Remm, M. Grimaldi, E. Keenan, J.R. Jenkins, A C-terminal alpha-helix plus basic region motif is the major structural determinant of p53 tetramerisation, Oncogene 7 (1992) 1513–1523. [3] P. Balagurumoorthy, H. Sakamoto, M.S. Lewis, N. Zambrano, G.M. Clore, A.M. Gronenborn, E. Appella, R.E. Harrington, Four p53 DNA-binding domain peptides bind natural p53response elements and bend the DNA, Proc. Natl. Acad. Sci. USA 92 (1995) 8591–8595. [4] M. Tarunina, M. Grimaldi, E. Ruaro, M. Pavlenko, C. Schneider, J.R. Jenkins, Selective loss of endogenous p21waf1/cip1 induction underlies the G1 checkpoint defect of monomeric p53 proteins, Oncogene 13 (1996) 589–598. [5] J.L. Waterman, J.L. Shenk, T.D. Halazonetis, The dihedral
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symmetry of the p53 tetramerisation domain mandates a conformational switch upon DNA binding, EMBO J. 14 (1995) 512–519. P. Chene, E. Bechter, Cellular characterisation of p53 mutants with a single missense mutation in the beta-strand 326–333 and correlation of their cellular activities with in vitro properties, J. Mol. Biol. 288 (1999) 891–897. C. Ishioka, H. Shimodaira, C. Englert, A. Shimada, M. Osada, L.Q. Jia, T. Suzuki, M. Gamo, R. Kanamaru, Oligomerisation is not essential for growth suppression by p53 in p53-deficient osteosarcoma Saos-2 cells, Biochem. Biophys. Res. Commun. 232 (1997) 54–60. M.E. Lomax, D.M. Barnes, T.R. Hupp, S.M. Picksley, R.S. Camplejohn, Characterisation of p53 oligomerisation domain mutations isolated from Li-Fraumeni and Li-Fraumeni like family members, Oncogene 17 (1998) 643–649. C. Rollenhagen, P. Chene, Characterisation of p53 mutants identified in human tumors with a missense mutation in the tetramerisation domain, Int. J. Cancer 78 (1998) 372–376. T.S. Davison, P. Yin, E. Nie, C. Kay, C.H. Arrowsmith, Characterisation of the oligomerisation defects of two p53 mutants
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found in families with Li-Fraumeni and Li-Fraumeni-like syndrome, Oncogene 17 (1998) 651–656. J. Atz, P. Wagner, K. Roemer, Function, oligomerisation, and conformation of tumor-associated p53 proteins with mutated C-terminus, J. Cell. Biochem 76 (2000) 572–584. J. Milner, E.A. Medcalf, Cotranslation of activated mutant p53 with wild type drives the wild-type p53 protein into the mutant conformation, Cell 65 (1991) 765–774. P. Chene, In vitro analysis of the dominant negative effect of p53 mutants, J. Mol. Biol. 281 (1998) 205–209. P. Chene, P. Mittl, M. Grutter, In vitro structure-function analysis of the beta-strand 326–333 of human p53, J. Mol. Biol. 273 (1997) 873–881. M.W. Frazier, X. He, J. Wang, Z. Gu, J.L. Cleveland, G.P. Zambetti, Activation of c-myc gene expression by tumorderived p53 mutants requires a discrete C-terminal domain, Mol. Cell. Biol. 18 (1998) 3735–3743. P. Chene, E. Bechter, p53 mutants without a functional tetramerisation domain are not oncogenic, J. Mol. Biol. 286 (1999) 1269–1274. J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning. A
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Laboratory Manual, 2nd edition, Cold Spring Harbor, Cold Spring Harbor, NY, 1989. J.M. Stommel, N.D. Marchenko, G.S. Jimenez, U.M. Moll, T.J. Hope, G.M. Wahl, A leucine-rich nuclear export signal in the p53 tetramerisation domain: regulation of subcellular localisation and p53 activity by NES masking, EMBO J. 18 (1999) 1660–1672. D. Dittmer, S. Pati, G. Zambetti, S. Chu, A.K. Teresky, M. Moore, C. Finlay, A.J. Levine, Gain of function mutations in p53, Nat. Genet. 4 (1993) 42–46. L.H. Wang, K. Okaichi, M. Ihara, Y. Okumara, Sensitivity of anticancer drugs in Saos-2 cells transfected with mutant p53 varied with mutation point, Anticancer Res. 18 (1998) 321– 325. R. Li, P.D. Sutphin, D. Schwartz, D. Matas, N. Almog, R. Wolkowicz, N. Goldfinger, H. Pei, M. Prokocimer, V. Rotter, Mutant p53 protein expression interferes with p53-independent apoptotic pathways, Oncogene 16 (1998) 3269–3277. G. Blandino, A.J. Levine, M. Oren, Mutant p53 gain of function: differential effects of different p53 mutants on resistance of cultured cells to chemotherapy, Oncogene 18 (1999) 477–485.
The gain of function of the p53 mutant Asp281Gly is dependent on its ability to form tetramers Annemieke Atema a, Patrick Che`ne b,* a
Dana-Farber Cancer Institute and Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA b Novartis, Oncology Department, K125 420, CH-4002 Basel, Switzerland Received 16 April 2002; received in revised form 10 May 2002; accepted 20 May 2002
Abstract The influence of tetramerisation on the properties of the p53 mutants is poorly understood. We describe here the influence of the tetramerisation on the properties of the oncogenic mutant Asp281Gly. We show that despite being both nuclear the tetrameric Asp281Gly and the monomeric Asp281GlyLeu344Pro proteins have different properties: only Asp281Gly stimulates the transcription of the multidrug resistance-1 gene promoter and induces cisplatin resistance in Saos-2 cells. Moreover, we identify a 130-kDa protein that specifically interacts with Asp281Gly but not with Asp281GlyLeu344Pro. This suggests that tetramerisation is important for the properties of the p53 mutants and that these properties might be mediated via protein-protein interactions. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: p53; Gain of function; Tetramerisation; Chemoresistance; Chemosensitivity
1. Introduction p53 is mutated in many cancers [1]. The majority of these mutations is located within its DNA binding domain (DBD) impairing the ability of the mutant proteins to associate with DNA and as a consequence to stimulate transcription. In studies where the full p53 complementary DNA is sequenced, only 4% of the mutations concern amino acids of its tetramerisation domain (TD). Different reports have shown that deletion or multiple mutations within the TD decrease both p53 DNA binding and transcriptional activity [2– 4]. Similar findings are obtained by mutating single amino acids of the TD to alanine [5,6]. These findings are further supported by the analysis of p53 mutants * Corresponding author. Tel.: 141-61-696-2050; fax: 141-61696-3835. E-mail address: [email protected] (P. Che`ne).
identified in cancers, which have a single mutation in their TD. Several of these mutants do not bind to DNA and are unable to stimulate the transcription of reporter genes under the control of p53-regulated promoters [7–11]. Altogether these published data demonstrate that single mutations in the TD can lead to mutants, which have lost the wild type function, as do proteins mutated at their DBD. Therefore, why are such mutations not identified in cancers more often? Various hypotheses can be formulated to answer this question and among them one is very attractive: proteins with a mutation in their TD would be less selected in cancers than DBD mutants because they do not show a gain of function. DBD mutants would be preferentially selected in tumours because they give an additional growth advantage when compared to TD mutants, which have lost only the wild type tumour suppressor activity.
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(02)00318-X
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Supporting this hypothesis, it has been shown that some DBD mutants inactivate wild type p53 by dominant negative effect upon heterooligomerisation [12,13] while TD mutants are not dominant negative [14]. Moreover, it has been established that the TD and/or the C-terminus regulatory region (the last 30 amino acids of p53) are necessary for the gain of function of the p53 mutants [15,16]. In this report, we compare the properties of the tetrameric oncogenic mutant Asp281Gly with the ones of its monomeric counterpart, the Asp281GlyLeu344Pro mutant. Our results indicate that the altered phenotypes observed in the presence of the Asp281Gly mutant are lost when this protein is rendered monomeric. Furthermore, our data suggest that the properties of the Asp281Gly protein might be linked to its ability to interact with other cellular proteins. 2. Material and methods 2.1. Construction of the vectors and establishment of the stable cell lines The Asp281Gly and Asp281GlyLeu344Pro genes were amplified by polymerase chain reaction (PCR) from the corresponding genes cloned in a pCite-2a vector [16]. A HindIII and an EcoRI site were introduced at the 5 0 and 3 0 end of the genes, respectively. The amplified DNA fragments were ligated into a pHMA6 vector (Boehringer Mannheim) and their sequence verified by double strand DNA sequencing. The p53 minus Saos-2 cells were transfected with the empty pHMA6 vector and the mutant expressing plasmids using the calcium-phosphate technique. The cells were then selected during 3 weeks in Dulbecco’s modified minimum essential (DMEM) containing G418 (0.8 mg/ml active concentration). Antibiotic resistant colonies were isolated with cloning rings and expanded into cell lines. Mutant expressing cells lines were selected by Western blot. 2.2. Western blotting and immunofluorescence The p53 protein was detected in Western blot as previously described [9]. For immunofluorescence, the cells were fixed with paraformaldehyde and permeabilised with Triton X-100. The DO-1 antibody
(1 mg/ml) was used to detect the p53 protein and 4,6diamino-2-phenylindole (DAPI, 0.3 mg/ml) to stain the nuclei. 2.3. [ 35S]-methionine labelling and immunoprecipitation Sub-confluent cells were starved for 45 min in methionine free DMEM and incubated for 3 h in the presence of 1.5 mCi [ 35S]-methionine (EXPRE 35S 35S protein labelling mix – NEN). The cells were then lysed and the soluble proteins were immunoprecipitated with the different antibodies and protein-G agarose beads. The bound proteins were loaded onto a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and after migration the gel was dried and exposed to a film. 2.4. Transient transfections, chloramphenicol acetyltransferase (CAT) assay and b -galactosidase measurements Sub-confluent cells in six well plates were transfected with total DNA (4 mg) and lipofectamine 2000 (GIBCO-BRL) according to the manufacturers. After (48 h) transfection, the amount of CAT protein was determined with the CAT ELISA kit (Boehringer Mannheim) according to the manufacturers. The bgalactosidase was determined as described in Sambrook et al. [17]. 2.5. Colony formation assay The cells (5000/plate) were seeded during 24 h in 10 cm plates. The medium was replaced with fresh medium containing different concentrations of cisplatin (Platinol-AQ (Bristol Laboratories) and the cells were incubated for 24 h in the presence of drug. The medium was changed and the cells were incubated for 2–3 weeks with regular medium changes every 3–4 days. After incubation, the colonies were stained with Coomassie Blue and counted. Counts were performed using the average of two equally sized, randomly determined sections from each plate. 3. Results The Asp281Gly protein is mutated in its DBD and is tetrameric. The Asp281GlyLeu344Pro protein is
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Fig. 1. Expression of the p53 mutants in Saos-2 cells analysed by Western blot. Two clones stably expressing the tetrameric mutant Asp281Gly (134.12 and 134.2) and the monomeric mutant Asp281GlyLeu344Pro (135.3 and 135.13) were analysed for their content in p53 protein by Western blot with the monoclonal antibody DO-1. One clone (152.16) stably transfected with the empty expression vector was also analysed. The molecular weights in kDa are indicated.
mutated both in its DBD and its TD and is monomeric [16]. The genes encoding for these two proteins were cloned such that they are fused at their N-terminus to a HA-tag and they were stably transfected into the p53 negative Saos-2 cells. Two stable clones expressing similar amounts of Asp281Gly and Asp281GlyLeu344Pro proteins and one clone obtained by the transfection with the empty expression vector were selected (Fig. 1). Since all the p53-expressing clones produce similar levels of this protein, the data obtained either with one or the other clone of each p53 mutant will be shown. However, the experiments have been realised with all the clones. Since the TD contains a leucine-rich nuclear export signal [18], it was important to verify that the Asp281GlyLeu344Pro protein, having been mutated at its TD, has the same sub-cellular localisation as the Asp281Gly protein. The sub-cellular localisation of Asp281Gly and Asp281GlyLeu344Pro was therefore analysed by indirect immunofluorescence (Fig. 2). The experimental data show that both mutants have
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the same localisation in the cell (Figs. 2B,C) and a nuclear stain with DAPI (Figs. 2F,G) indicates that they are predominantly located in the nucleus. The sub-cellular location of these two mutants is similar to the one of the endogenous Pro278Ala mutant found in 786-0 cells (Figs. 2D,E). No p53 is detected by immunofluorescence in the cell line expressing the empty expression vector (Figs. 2A,E). It has been previously shown that the Asp281Gly protein stimulates the transcription of reporter genes under the control of the multidrug resistance-1 gene (MDR-1) promoter in contrast to the wild type protein that represses this promoter [19]. The stable clones were therefore analysed for their ability to stimulate the expression of a CAT reporter gene under the control of the MDR-1 promoter (Fig. 3). The experimental data show that high amounts of CAT protein are detected only in the clones expressing the Asp281Gly protein. Therefore, the Asp281Gly protein stimulates the expression MDR-1 promoter only when it is in a tetrameric conformation. Several studies have shown that p53 mutants can enhance the resistance of tumour cell lines against different chemotherapeutic agents [20–22]. The cisplatin sensitivity of the stable clones was therefore investigated (Fig. 4). The experimental data show that the expression of the Asp281Gly mutant in the Saos-2 cells enhances the cisplatin resistance of these cells. This is in agreement with already published data, which have shown that p53 mutants enhance cisplatin resistance [20,22]. Very interestingly, the data show that expression of the double mutant Asp281GlyLeu344Pro does not enhance the resistance of the Saos-2 cells. Therefore, once the Asp281Gly mutant is mutated such that it becomes monomeric, it looses its ability to enhance the chemoresistance of the Saos2 cells. To identify proteins that bind to the p53 mutants, the cells from the different clones were metabolically labelled with [ 35S] methionine and their lysates were immunoprecipitated with three different antibodies: the 12CA5 antibody, which is directed against the HA-tag, located at the N-terminus of the mutant proteins, the DO-1 antibody, which is a p53 specific antibody, and the F5 antibody, which recognises the p21 Waf1/Cip1 protein and was used as a negative control antibody. The experimental data (Fig. 5 bottom) show that both the 12CA5 and the DO-1 antibodies immu-
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Fig. 2. Immunolocalisation of the p53 mutants in the Saos-2 cells. The cellular localisation of the p53 proteins was determined in the empty vector (A); the Asp281Gly mutant (B); the Asp281GlyLeu344Pro mutant (C) expressing Saos-2 cells; and in the 786-0 cells (D). The cells were incubated with the monoclonal antibody DO-1 and with a l-rhodamine conjugated anti-mouse secondary antibody. The location of the nucleus is revealed in the empty vector (E); the Asp281Gly mutant (F); the Asp281GlyLeu344Pro mutant (G) expressing Saos-2 cells; and the 786-0 cells (H) by a DAPI staining of the cells. The data presented have been realised with the clone 152.16, 134.12 and 135.3.
noprecipitate a protein with an apparent molecular weight of about 56 kDa from the lysates of the p53 mutant expressing clones. This protein is not immunoprecipitated with the F5 antibody or from the lysates from the cells transfected with the empty vector suggesting that this protein is p53. The higher apparent molecular weight of the p53 protein is due to the fact that it is expressed as a HA-tag fusion protein. The amount of Asp281Gly and Asp281GlyLeu344Pro proteins immunoprecipitated with both the DO-1 and the 12CA5 are similar, confirming that the clones are expressed at similar levels. Further examination of the autoradiographies reveals that a protein with an apparent molecular weight of about 130 kDa is immunoprecipitated with the DO-1 and the 12CA5 antibodies from the lysate of the cells expressing the tetrameric Asp281Gly mutant (Fig. 5 top). This protein is not immunoprecipitated either with the F5 antibody or from the lysate of the cells obtained with the mono-
meric Asp281GlyLeu344Pro mutant and the empty vector indicating that this protein interacts specifically with the tetrameric mutant.
4. Discussion The spectrum of the p53 mutations shows that, even when the entire p53 gene is sequenced, very few mutations are detected in the TD. However, recent publications, in which proteins identified in cancer with a single missense mutation at their TD are studied, reveal that these proteins have lost the wild type activity in fashion similar to proteins mutated at their DBD [7–11]. In this report we utilised a well-characterised oncogenic mutant, Asp281Gly, to determine the influence of the tetramerisation on the properties of this mutant. Our data reveal that the Asp281Gly protein stably
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Fig. 3. Stimulation of the MDR-1 promoter. The Saos-2 cells expressing the empty vector (clone 152.16), the Asp281Gly mutant (clone 134.2) and the Asp281GlyLeu344Pro (clone 135.13) were transiently transfected with a vector containing a CAT reporter gene under the control of the MDR-1 promoter and with a vector expressing the b-galactosidase protein. Two days after transfection the amount, of CAT protein expressed was determined. The CAT amounts were normalised to the b-galactosidase activity. The means and standard deviations of three experiments are represented.
transfected in the p53 null cells Saos-2 stimulates the MDR-1 promoter while its monomeric counterpart, the Asp281GlyLeu344Pro protein, has lost this property. We also show that the cisplatin resistance of Saos-2 cells induced by the expression of the Asp281Gly protein is lost when this protein is rendered monomeric by the mutation Leu344Pro in its TD. Finally, our results indicate that the difference in behaviour between the Asp281Gly and the Asp281GlyLeu344Pro proteins is not linked to a difference in their cellular localisation since both proteins are nuclear. Altogether these findings indicate that some of the properties of the Asp281Gly protein require a tetrameric conformation. These findings led us to the question: why the Asp281Gly mutant needs to form tetramers to show these properties? Our reasoning was as follows: p53 activation requires its accumulation in the cell, suggesting that the p53 protein must reach a certain concentration to bind to DNA and/or to the p53-binding proteins. Since the cellular concentration of the p53 mutants is high in tumour cells, these proteins should be at a concentration that allows them to
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bind to DNA and/or to the p53-binding proteins. Because most of the p53 mutants do not bind to DNA, the only type of interaction in which they could participate would be with other proteins. The constant occurrence of these protein-protein interactions in the tumour cell may deregulate the cellular machinery and induce the apparition of altered phenotypes. The so-called gain of function of the p53 mutants would then be the result of the interactions between the p53 mutants and some cellular proteins. We therefore used the tetrameric Asp281Gly and the monomeric Asp281GlyLeu344Pro mutant to identify such proteins. We have indeed identified one protein of an apparent molecular weight of 130 kDa that binds specifically to Asp281Gly and not to Asp281GlyLeu344Pro. The full characterisation and the elucidation of the biological relevance of this interaction will have to be further demonstrated. However, this finding is the first step towards a better comprehension at the molecular level of the gain of function of the p53 mutants. A full characterisation of the interaction between the 130 kDa protein and the p53 mutants may lead to the design of inhibitors of this interaction and therefore to new ways to target tumours expressing p53 mutants.
Fig. 4. Effect of a cisplatin treatment on the survival of the transfected clones. A similar number of cells of the empty vector (A – clone 152.16), the Asp281GlyLeu344Pro (X – clone 135.13) and the Asp281Gly (W – clone 134.2) expressing clones were treated with the indicated concentration of cisplatin for 24 h and grown for 2–3 weeks with regular change of medium. After growth, the colonies were stained Coomassie Blue and counted. The means and the standard deviation are given.
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Fig. 5. Identification of a protein that binds to the tetrameric Asp281Gly mutant and not to the monomeric Asp281GlyLeu344Pro protein. The Saos-2 cells were labelled with [ 35S]-methionine and after lysis, their p53 content was immunoprecipitated with the anti-HA antibody 12CA5, the anti-p53 antibody DO-1 and the anti-p21 Waf1/Cip1 antibody F5. The upper part of the figure corresponds to the region 116–180 kDa of the autoradiography of a dried gel. The location of the 130-kDa protein is marked with an arrow. The lower part of the figure corresponds to the region surrounding the 48-kDa region of the autoradiography of a dried gel. The location of the HA-p53 protein is marked with an arrow. The clones 152.16, 134.12 and 135.13 were used for this experiment.
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