|22 June 2000, Volume 19, Number 27, Pages 3095-3100|
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|Identification of a tumor-derived p53 mutant with novel transactivating selectivity|
|Yi Pan and Dale S Haines|
The Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 N. Broad Street, Philadelphia, Pennsylvania, PA 19140, USA
Correspondence to: D S Haines, The Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 N. Broad Street, Philadelphia, Pennsylvania, PA 19140, USA
MDM2 is a p53-responsive molecule that when overexpressed, can alter growth control pathways via p53-dependent and independent mechanisms. We have identified a mutant p53 containing line that expresses high levels of transcripts that are regulated by the p53-responsive promoter of the MDM2 gene. Analysis of cloned product obtained from these tumor cells revealed that they harbor a mutant p53 protein (possessing an Arg to Gln substitution at codon 213) that is a potent transactivator of MDM2 expression. Consistent with this activity, the R213Q mutant was found to have the ability to interact with DNA sequences located within the MDM2 promoter. In contrast to previously described tumor-derived p53 mutants which retain MDM2 transactivation function and possess partial growth suppressive activity, the R213Q mutant is severely compromised in its ability to induce p53-regulated transcripts that encode for proteins involved in cell-cycle arrest and apoptosis. The R213Q mutant can also be expressed at high levels in stably transfected cells and cells that harbor this mutant possess elevated levels of MDM2 protein. The R213Q mutant was also found to be able to up-regulate MDM2 during a genotoxic stress response. R213Q is the first described tumor-derived p53 mutant that is deficient at up-regulating both cell cycle arrest and apoptotic factors, but is highly proficient at inducing the growth-promoting molecule MDM2. Oncogene (2000) 19, 3095-3100
p53; MDM2; p53 target genes
The functional activity of the p53 tumor suppressor protein is inactivated by mutation in a large percentage of human tumors (Hainaut et al., 1998). It is highly probable that the most important biochemical activity of p53 is to function as a transcription factor. p53 is capable of binding to DNA in a sequence specific manner and influencing gene expression (el-Deiry et al., 1992; Funk et al., 1992; Zambetti et al., 1992). The majority of tumor derived p53 mutants harbor amino acid changes in the DNA binding domain (Cho et al., 1994) and many mutants are unable to bind to specific promoter elements (el-Deiry et al., 1992; Funk et al., 1992). Thus, mutational inactivation of p53 binding function for sequences that are recognized by wild-type (wt) p53 is likely to be a key mechanism which contributes to the development of a tumor cell.
It is now becoming apparent that mutations in the p53 gene do not necessarily give rise to an inert protein. Considering that p53 functions as a tetramer (Wang et al., 1995), it is not surprising that p53 mutant proteins can block the transcriptional activity of wt p53 (Kern et al., 1992). Tumor-derived p53 mutants have also been identified that are able to induce cell cycle arrest, but not apoptosis (Friedlander et al., 1996; Rowan et al., 1996), and this appears to be associated with the inability of these mutants to bind to a subset of p53-response elements (Friedlander et al., 1996; Ludwig et al., 1996). Interestingly, some tumor-derived p53 mutants which have lost wt p53 function appear to have acquired the ability to provide a selective growth advantage to the tumor cell (Roemer, 1999). This 'gain of function' activity is thought to be due to their ability to up-regulate the expression of growth promoting molecules that are not targets of wt p53. In support of this theory, the promoter elements of myc (Frazier et al., 1998), BAG-1 (Yang et al., 1999) and the multidrug resistant genes (Lin et al., 1995) have been shown to be mutant p53-responsive.
The MDM2 gene is a transcriptional target of wt p53 (Barak et al., 1993; Wu et al., 1993). The protein product of the MDM2 gene is a natural negative regulator of p53 activity (Montes de Oca Luna et al., 1995; Jones et al., 1995) and is oncogenic when overexpressed (Fakharzadeh et al., 1991). The functional properties of MDM2 are therefore unique when compared to other transcriptional targets of p53, since it is involved in the promotion and not the inhibition of cellular proliferation. It is generally accepted that p53 function is compromised in MDM2 overexpressing cells, and there is now good evidence which suggests that the activity of multiple growth control molecules may be de-regulated in addition to p53 in MDM2 overproducing tumors. MDM2 has been shown to interact with a number of growth control molecules (Xiao et al., 1995; Martin et al., 1995). Moreover, MDM2 overexpression can promote cell growth via p53-independent mechanisms (Dubs-Poterszman et al., 1995; Sigalas et al., 1996; Sun et al., 1998; Lundgren et al., 1997; Jones et al., 1998) and p53 mutations have been detected in MDM2 overexpressing tumors (Marks et al., 1996; Pan and Haines, 1999; Eischen et al., 1999). This genetic analysis, in combination with the MDM2 functional studies described above, are good indicators that MDM2 overexpression can provide a growth advantage to a p53 mutant tumor cell.
The MDM2-P2 promoter possesses two p53-response elements and it controls the expression of transcripts (termed MDM2-P2 transcripts) in a p53-dependent manner (Wu et al., 1993; Zauberman et al., 1995). In a previous study, we demonstrated that the levels of MDM2-P2 transcripts can be used to predict p53 gene status in human leukemic cell lines (Bull et al., 1998). In a later survey of more human leukemic cell lines, we noticed one mutant p53 containing line (Duthu et al., 1992) that expresses elevated levels of MDM2-P2 RNA. Figure 1 shows that the level of MDM2-P2 transcripts present in the Raji cell line (R) is much higher than that measured in other p53 mutant lines. These results raise the possibility that p53-independent mechanisms are responsible for the production of MDM2-P2 transcripts in Raji cells. Alternatively, this cell line may harbor a p53 mutant that is proficient at up-regulating MDM2 gene expression. To investigate this possibility, full-length p53 cDNAs were cloned from this line and their sequence determined. Sequence analysis revealed that the Raji line harbors two different mutant alleles; one of these alleles encodes for a mutant protein (the R213Q mutant) that possesses an Arg to Gln amino acid change at codon 213, while the other allele encodes for a mutant protein (the Y234H mutant) that contains a Tyr to His substitution at codon 234 of p53. These mutations are identical to those previously reported to be present in the Raji line (Duthu et al., 1992). To determine if one or both of these mutants possesses the ability to up-regulate MDM2 expression, the R213Q and Y234H mutants were cloned into a eukaryotic expression vector (pCEP, Invitrogen) and transfected into the p53 null H1299 cell line. Protein extracts were prepared from transiently transfected cells and the level of MDM2 protein was measured by a Western blot. Figure 2a shows that the R213Q mutant is able to up-regulate MDM2 protein in transiently transfected H1299 cells. No apparent induction of MDM2 protein was observed in cells that had been transfected with the Y234H mutant. Surprisingly, the fold induction observed with the R213Q mutant was similar to that measured with a pCEP wt p53 expression construct at all amounts of input DNA tested. These results strongly suggest that MDM2-P2 production in Raji cells is due to the presence of a p53 mutant that is able to up-regulate the expression of these transcripts.
We next wanted to determine if the R213Q mutant can bind to DNA sequences located within the MDM2-P2 promoter. Assessment of binding to the endogenous MDM2 promoter by transfected R213Q was assessed by a chromatin immunoprecipitation assay (ChIP) (Boyd et al., 1998). This approach was chosen over traditional in vitro DNA binding assays because it allows for the study of protein-promoter interactions in the context of the endogenous genomic environment. wt p53, the R213Q mutant and a MDM2 transactivating deficient mutant control (Y234H) were again transfected into H1299 cells. DNA-protein interactions were first stabilized in transfected cells by chemical cross-linking. After preparation of nuclear extracts and DNA sonication, p53-DNA complexes were immunopurified using polyclonal p53 antibodies and protein A/DNA sepharose. DNA was further purified and PCR using this DNA and primers complementary to sequences flanking the p53-binding site of the MDM2 promoter was performed. Figure 2b shows that there was a greater amount of PCR product present in samples that were derived from p53 immunoprecipitates of R213Q and wt p53 transfected cells when compared to the control samples (i.e. samples derived from p53 immunoprecipitates of vector control and R234H transfected cells and in the control immunoprecipitations ('-Ab' lanes) from all of the transfections). Since equivalent amount of product was observed in PCR reactions using DNA that was prepared from a fraction of the extract prior to immunoprecipitation ('C' lanes), it is unlikely that the signal observed in the R213Q or wt p53 samples is simply due to a higher amount of input DNA. The presence of signals in samples derived from wt p53 and R213Q transfections does not appear to be due to a general higher affinity of these proteins for DNA since no PCR product was observed in PCR reactions using primers complementary to a control sequence (i.e. the coding region of the TGF-B type II receptor, data not shown). These results suggest that the R213Q mutant can bind to DNA sequences that are located within the MDM2-P2 promoter. Consistent with the binding data, elevated amounts of MDM2-P2 transcripts were present in R213Q transfected cells (see Figure 2C). These results strongly suggest that R213Q is able to induce MDM2 protein expression by binding to sequences located within the MDM2-P2 promoter and up-regulating the expression of transcripts that are under the control of this promoter.
The results presented above show that the R213Q mutant retains transactivation function on the MDM2-P2 promoter. To determine if the R213Q mutant possesses the ability to up-regulate growth suppressive molecules that are induced by wt p53, we next measured the levels of wt p53-responsive transcripts p21 (el-Deiry et al., 1993), BTG-2 (Rouault et al., 1996), PIG3 (Polyak et al., 1997) and PIG11 (Polyak et al., 1997) in transfected cells. These were chosen because like MDM2, they are very strongly induced by wt p53 (Zhu et al., 1998) and they encode for molecules that have been implicated in distinct p53-controlled responses. p21 and BTG-2 are known to play important roles in controlling cell cycle progression (Montagnoli et al., 1996; Rouault et al., 1996; Waldman et al., 1995). In contrast, PIG3 and PIG11 have been postulated to participate in p53-induced apoptosis (Polyak et al., 1997). We also included two other p53 mutants (R175P and R181L) in this analysis (these were generated by site-directed mutagenesis using pCEP-wt p53 as the template). These mutants possess the ability to up-regulate MDM2 and p21, and can induce cell cycle arrest (Crook et al., 1994; Ludwig et al., 1996; Rowan et al., 1996). They are, however, defective in their ability to induce apoptosis (Ludwig et al., 1996; Rowan et al., 1996). RNA was isolated following transfection and Northern analysis for p21, BTG-2, PIG3 and PIG11 was performed. Figure 3a shows high levels of MDM2 protein in cells that had been transfected with the R213Q, R175P and R181L mutants. However, in contrast to cells transfected with the R213Q mutant, cells transfected with the R175P and R181L mutants express much higher levels of p21, PIG3, PIG11 and BTG-2 RNA (Figure 3b). These results suggest that the R213Q mutant is defective at inducing the expression of putative growth suppressive proteins but is proficient at inducing MDM2 protein expression. They also indicate that the R213Q mutant is unique when compared to previously described mutants that retain MDM2 transactivation function.
Because MDM2 can promote cell growth via p53-independent mechanisms when overexpressed in vitro (Dubs-Poterszman et al., 1995; Sigalas et al., 1996; Sun et al., 1998) and in vivo (Lundgren et al., 1997; Jones et al., 1998), we next wanted to determine if MDM2 protein could be further up-regulated by this mutant in cells exposed to a genotoxic stress. A pool of clones derived from R213Q stably transfected H1299 cells were treated with the DNA cross-linking agent cisplatin. The levels of MDM2 protein were then measured in treated and untreated cells. Figure 4 shows an induction of MDM2 protein in cisplatin treated cells that had been transfected with the R213Q mutant. No induction of MDM2 protein was observed in cells that had been transfected with another p53 mutant that is unable to up-regulate MDM2 (the Y234H mutant). This result shows that the R213Q mutant is able to up-regulate MDM2 during a genotoxic stress response. We also performed a Northern blot and found that the levels of bax or p21 RNA do not increase to a greater extent in R213Q transfected cells versus Y234H or vector alone transfected cells (data not shown). Because we do not have a good positive control for these downstream targets in stably transfected cells (wt p53 is not tolerated in stably transfected cells - see Figure 5), we do not know if the R213Q mutant is not functioning on these promoters during a genotoxic stress response or if the signal generated in these cells is not sufficient to activate R213Q binding function on these promoters.
The results presented above raise the possibility that we have identified a tumor-derived p53 mutant of novel function; one that is deficient in its growth inhibitory activities but is able to induce the expression of the growth promoting molecule MDM2. It remains entirely possible however that this mutant is still a potent growth suppressor due to its ability to up-regulate the expression of growth inhibitory targets other than the ones analysed here or perhaps via growth inhibitory activities of p53 that are independent of sequence specific transactivation. To get an initial indication if this mutant retains growth inhibitory activity, we first determined if transfection of this mutant into cells reduces their growth capacity using a colony formation assay. H1299 cells were transfected with R213Q, two p53 mutants that are devoid of detectable transactivation function (H163C and Y234H), R175P, R181L and wt p53. After transfection, cells were grown in the presence of hygromycin (selectable agent used to maintain the presence of the p53 expression constructs in transfected cells) for 2 weeks and the number of visible colonies were counted. When compared to two other tumor-derived p53 mutants that possess no detectable transactivation function, there is a reduction in the number of hygromycin colonies in R213Q transfected cells. The number of colonies in R213Q transfected cells is however much greater when compared to wt p53 transfected cells, or other p53 mutants (R175P and R181L) with partial growth inhibitory activity (Ludwig et al., 1996; Rowan et al., 1996). These results suggest that the R213Q mutant can inhibit the proliferative capacity of cells when overexpressed at super-physiological levels, although not to the same extent as wt p53.
To get an indication if this mutant retains growth inhibitory activity by another method, we employed a p53 toleration assay. It has been known for some time that mutant p53, but not wt p53, can be expressed at elevated levels in stably transfected tumor cell lines (Baker et al., 1990; Johnson et al., 1991). This is presumably due to the fact that transfected mutant p53 has very little negative effect on cell growth and its expression can be maintained at high levels. In contrast, because wt p53 is a potent growth suppressor, transfection of tumor cells with p53 selects for events which lower the expression of this molecule to a level that is no longer growth inhibitory. Because toleration is assessed under conditions of stable expression when the amount of p53 protein produced in the cells is much less and more at a 'physiological' level than during transient expression, it is likely to be a better indicator of the growth suppressive activity of a molecule when compared to a colony formation assay. To determine if transfected cells can tolerate high levels of the R213Q mutant, two cell lines (one null (H1299) and one with a mis-sense mutation (ASPC-1)) were transfected with this mutant, two other p53 mutants that are devoid of detectable transactivation activity (Y163C and Y234H), the R175P and R181L mutants and a wt p53 expression construct. After transfection, cells were grown in the presence of hygromycin for 2 weeks. Protein extracts were prepared from pooled colonies of transfected cells and the relative amount of p53 in these extracts was determined by a Western blot. Figure 5 shows a high amount of p53 protein in R213Q transfected cells. The amount of p53 protein measured in both of these cell lines is comparable to that measured in cells that had been transfected with the other p53 mutant constructs that do not possess any detectable transactivation function. Consistent with a deficiency in p53 transactivation function (Ludwig et al., 1996), the R175P mutant was found to be expressed at higher levels than wt p53 in stably transfected cells. The amount of p53 protein measured in R175P stably transfected cells was however not as high as that measured in R213Q stably transfected cells. As expected, the levels of MDM2 protein were also found to be high in R213Q stably transfected cells (Figure 5). These results show that the R213Q mutant can be stably expressed at high levels in transfected cells and suggests that the R213Q mutant lacks growth suppressive activity under conditions of stable expression.
Because the amount of p53 measured in R213Q transfected cells was the same as that measured in p53 mutant lines that are unable to up-regulate MDM2, we did perform some experiments to determine if this mutant is resistant to MDM2-mediated degradation. However, it appears that this mutant is just as susceptible to MDM2 induced ubiquitination and degradation when compared to wt p53 or other p53 mutants (data not shown). This result was not surprising considering that the level of p53 produced from the CMV promoter is probably in dramatic excess to MDM2 (even when being induced by p53). Thus, it is highly likely that the difference in levels measured between the various p53 constructs is more a reflection of their dissimilar growth control activities and not to their differences in the ability to be degraded by MDM2.
The reasons for why the R213Q mutant is unable to up-regulate the expression of genes analysed here is unclear and obviously requires investigation. We plan to initiate studies that will determine if this mutant can bind to the promoters of genes that it is unable to up-regulate. The p53 binding elements located in the MDM2 promoter is different in both sequence and structure when compared to p53 binding elements that are located in other p53-responsive genes (Zaubermann et al., 1995). It is therefore entirely possible that this mutation is altering the binding of p53 to these promoters and not to the MDM2 promoter. Interestingly, it has been demonstrated that the MDM2-P2 exists in a nucleosome-free state (Xiao et al., 1998) and investigators have speculated that efficient p53 transactivation of some downstream targets may require chromatin remodeling (Zhu et al., 1999). Thus, it is possible that the R213Q p53 mutant may be able to bind to p53 binding elements but is deficient in its ability to recruit proteins that are required for relieving chromatin-mediated repression and/or gene specific transactivation. Determining the reason(s) for why the R213Q is unable to up-regulate the expression of growth suppressive genes may improve our understanding of the mechanisms controlling p53 transactivation specificity.
The p53 growth control pathway can be perturbed by multiple mechanisms in human tumor cells. p53 gene mutations do give rise to proteins that have no detectable DNA binding activity or transactivation function (el-Deiry et al., 1992; Funk et al., 1992). Interestingly, it is now becoming apparent that mutations can generate a protein with transcriptional activities that are not only distinct from wt p53, but may provide a growth advantage to the tumor cell (so called 'gain of function' mutations). p53 mutants have been identified that can function on the myc (Frazier et al., 1998), BAG-1 (Yang et al., 1999) and multidrug resistant gene (Lin et al., 1995) promoters. Considering that MDM2 can alter growth control pathways via p53 independent mechanism when overexpressed (Lundgren et al., 1997; Jones et al., 1998; Sigalas et al., 1996; Sun et al., 1998), it is possible that the R213Q mutant may provide a growth advantage to transformed cells via its ability to up-regulate MDM2 expression alone or in combination with other growth promoting molecules.
The studies presented here and published previously (Crook et al., 1994) show that retention of MDM2 transactivation is not unique to the R213Q mutant. Two tumor derived mutants (R175P and R181L) that possess partial growth suppressive activity are also potent inducers of MDM2 expression. Although these types of mutations may not appear to alter the ratio of MDM2 to negative growth regulators to the same degree as the R213Q mutant, they may do so under certain conditions. For example, both the R175P and R181L mutants have been shown to be functional at inducing cell cycle arrest but not apoptosis and this has been shown to be associated with its ability to up-regulate only a subset of p53-responsive genes (Ludwig et al., 1996; Rowan et al., 1996). These results have led to the speculation that a specific subset of genes are induced by p53 under certain situations and depending on the type of genes that are induced, a p53-mediated cell-cycle arrest or apoptotic-response is initiated. Therefore, signals which induce a p53-apoptotic response in tumors which harbor an apoptotic-defective p53 mutant could result in a dramatic distortion in the ratio of MDM2 to negative growth regulators. Because MDM2 is involved in the negative regulation of p53 function, we hypothesize that retention of MDM2 promoter transactivation will be a universal feature of tumor derived mutants that retain partial transcriptional activity on growth suppressive targets. If this is the case, MDM2-transactivating p53 mutants may occur in a significant percentage of human cancers and their functional properties need further elucidation.
The authors would like to thank W el-Deiry for helpful discussions and D George and M Murphy for critically reviewing the manuscript and M Murphy for help with ChIP. This work was supported in part by grants from NIH (CA70165) and the US Army (DAMD17-97-1-7169).
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Figure 1 MDM2-P2 RNA levels are elevated in the p53 mutant Raji cell line. Measurement of MDM2-P2 transcript was performed by RNase protection as described (Bull et al., 1998) with cellular RNA isolated from the p53 mutant lines Molt4 (M), HEL (H), CEM (C), Jurkat (J), and Raji (R). All hybridization reactions included a smaller probe complementary to the glyceraldehyde-phosphate-dehydrogenase (GAPDH) `housekeeping' gene to control for potential variability in sample processing
Figure 2 R213Q retains MDM2 transactivation function. (a) H1299 cells were transfected with varying amounts of wt p53 and the R213Q mutant and 3 g of the Y234H mutant and the pCEP Vector as described previously (Haines et al., 1994) (V). Forty-eight hours after transfection, protein extract was prepared and the amount of MDM2 and p53 protein was measured by a Western blot as described previously (Pan and Haines, 1999). Ponceau S. staining of blots after transfer revealed equivalent loading of total protein (data not shown). (b) Cells were transfected with the indicated plasmids and ChIP was performed as described previously (Murphy et al., 1999). PCR was performed using primers complementary to sequences (available upon request) flanking the p53 response elements located within the MDM2 promoter and DNA (at two different dilutions) isolated after immunoprecipitation with the rabbit p53 polyclonal (+Ab) or the beads alone as control (-Ab). 'C' denotes PCR reactions using the total chromatin control. PCR products were separated on 1.5% agarose gels and stained with Ethidium Bromide. Presented here are pictures of PCR products derived from non-diluted DNA (in the case of the immunoprecipitations) or the least diluted DNA (in the case of the total chromatin controls). Reactions containing the least amount of DNA either gave no signal (for all control immunoprecipitations and for p53 immunoprecipitations from pCEP and pY234H transfected cells) or signal that was too faint to see after the scanning of pictures (for the total chromatin control reactions and for p53 immunoprecipitations from wt p53 and R213Q transfected cells). Two different dilutions were used to verify that PCR was performed under conditions that did not exceed the linear range of amplification. (c) H1299 cells were transfected with 5 g each of vector (pCEP), the Y234H mutant, the R213Q mutant and wt p53. Protein and RNA was prepared 48 h after transfection. The levels of p53 protein in transfected cells was measured by a Western blot and the levels of MDM2-P2 transcripts by RNase protection
Figure 3 Induction of p53-responsive genes by the R213Q, R175P and R181L p53 mutants. H1299 cells were transfected with 5 g each of vector (pCEP), the R213Q mutant, wt p53, the R175P mutant, and the R181L mutant. Cells were harvested 48 h after transfection for protein and RNA preparation. (a) p53 and MDM2 protein levels were measured by a Western blot. Equivalent blotting of protein for this Western blot was verified by Ponceau S. staining (data not shown). (b) The levels of p21, BTG-2, PIG3 and PIG11 in transfected cells was determined by Northern analysis. Also depicted is the ethidium stain of the agarose gel prior to transfer to show equivalent loading of RNA
Figure 4 Cisplatin induces MDM2 in stably transfected R213Q p53 mutant cells. Cells from stably transfected R213Q and Y234H p53 mutant expressing cells were either treated with media (-) or media containing 4 g/ml cisplatin for 16 hours (+). After treatment, protein extract was prepared and the level of MDM2 was determined by Western blotting. Equivalent blotting of protein for the Western blots was verified by Ponceau S. staining (data not shown)
Figure 5 High levels of p53 protein and MDM2 protein are present in R213Q stably transfected cells. H1299 and ASPC-1 cells were transfected with 5 g each of vector (pCEP), the p53 mutants Y163C, Y234H, R213Q, R175P, R181L and wt p53. H1299 cells were transfected by the calcium phosphate method while ASPC-1 cells were transfected with Lipofectamine. After transfection, cells were cultured in the presence of hygromycin until visible colonies were seen. Cells were then trypsinized and protein extract was prepared from hygromycin resistant cells. The amount of p53 and MDM2 protein in these extracts was measured by a Western blot. Ponceau S. staining of the blot after transfer revealed equivalent loading of protein (data not shown)
Table 1 The R213Q mutant is less toxic to transfected H1299 cells than wt p53 or the R175P and R181L mutants
|Received 19 April 2000; revised 27 April 2000; accepted 4 May 2000|
|22 June 2000, Volume 19, Number 27, Pages 3095-3100|
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