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[Cancer Research 64, 8199-8207, November 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Comparison of the Effect of Mutant and Wild-Type p53 on Global Gene Expression

Thomas J. O’Farrell, Paritosh Ghosh, Nobuaki Dobashi, Carl Y. Sasaki and Dan L. Longo

Laboratory of Immunology, Gerontology Research Center, National Institute on Aging, NIH, Baltimore, Maryland


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanisms for "gain-of-function" phenotypes produced by mutant p53s such as enhanced proliferation, resistance to transforming growth factor-ß–mediated growth suppression, and increased tumorigenesis are not known. One theory is that these phenotypes are caused by novel transcriptional regulatory events acquired by mutant p53s. Another explanation is that these effects are a result of an imbalance of functions caused by the retention of some of the wild-type transcriptional regulatory events in the context of a loss of other counterbalancing activities. An analysis of the ability of DNA-binding domain mutants A138P and R175H, and wild-type p53 to regulate the expression levels of 6.9 x 103 genes revealed that the mutants retained only <5% of the regulatory activities of the wild-type protein. A138P p53 exhibited mostly retained wild-type regulatory activities and few acquired novel events. However, R175H p53 possessed an approximately equal number of wild-type regulatory events and novel activities. This is the first report that, after examination of the regulation of a large unfocused set of genes, provides data indicating that remaining wild-type transcriptional regulatory functions existing in the absence of counterbalancing activities as well as acquired novel events both contribute to the gain-of-function phenotypes produced by mutant p53s. However, mutant p53s are likely to be distinct in terms of the extent to which each mechanism contributes to their gain-of-function phenotypes.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The protein p53 is a transcription factor that mediates several cellular processes including growth arrest, apoptosis, differentiation, and DNA damage repair (reviewed in 1, 2, 3 ). Because of its growth-inhibiting functions as well as its mutation or loss in ≥50% of human tumors (4, 5, 6) , p53 is considered an important tumor suppressor protein.

The domain structure of p53 consists of an NH2-terminal transactivation domain, a core DNA-binding domain, and a COOH-terminal domain. The NH2-terminal transactivation domain seems to be a functional binding site for basal transcription factors such as p300/CBP, TFIID, and TATA-binding protein (reviewed in ref. 7 ). These associations seem to mediate the transactivation as well as transrepression functions of p53. The central compact core of p53 forms the DNA-binding domain. This domain allows the protein to bind DNA in a sequence-specific fashion and is where the vast majority of mutations in p53 are found in human cancer (8) . The COOH-terminal domain contains the tetramerization region and also seems to mediate binding to basal transcription factors (2 , 3) .

As mentioned above, most of the naturally occurring mutations in p53 are found in the DNA-binding domain and, therefore, these mutants have been the most extensively studied. As expected, these mutations result in losses of various degrees of DNA-binding affinity compared with wild-type (WT) p53 (9) . Generally, mutation of residues directly contacting DNA results in a complete loss of DNA binding with little loss in folding of the domain, whereas mutation of structural residues results in a >50% loss of folding and DNA binding (9) . However, considering that the NH2- and COOH-terminal domains of p53 are distinct from the compact DNA-binding domain and considerably less ordered, the mutations in the DNA-binding domain are less likely to affect the integrity and function of these domains.

Because p53 is found mutated in approximately 50% of all cancers, p53 mutants have been rather extensively studied for their ability to confer "gain-of-function" phenotypes on cells. These are phenotypes not caused by WT p53 that are seen when mutant p53s are expressed in p53 null cells. Several gain-of-function phenotypes have been found for various p53 mutants such as increased cell growth (10 , 11) , enhanced tumorigenicity (11, 12, 13, 14, 15) and invasiveness (11 , 13 , 16) , disturbed spindle checkpoint (17 , 18) , and resistance to cytotoxic agents (19) . Furthermore, it was shown that the expression of p53 mutants V134A and C132F caused cells to become resistant to transforming growth factor (TGF)-ß–mediated growth suppression (12 , 20) . Additional evidence that mutant p53s may have the ability to antagonize TGF-ß is that a decrease in the level of endogenous homozygous mutant A138P p53 was observed in a B-cell lymphoma cell line, RL, after exposure to the cytokine that coincided with the onset of growth suppression (21) . This suggests that A138P p53 causes resistance to TGF-ß–mediated growth suppression and that these cells have developed a pathway that reduces the level of this mutant after TGF-ß exposure so that growth suppression can occur. Some p53 DNA-binding domain mutants possess the ability to alter the expression of genes that the WT protein either does not affect or regulates in the opposite fashion. For example, it has been shown that several mutants of p53 up-regulated the expression of c-myc (22) , basic fibroblast growth factor (23) , insulin-like growth factor receptor 1 (24) , and interleukin-6 (IL-6, ref. 25 ), whereas WT p53 repressed the expression of these genes. Also, several p53 mutants were able to increase the expression of the epidermal growth factor receptor (EGFR) gene to a much greater extent than WT p53 (26) . These gain-of-function gene regulation events may result from the ability of these mutants to associate with different regulatory elements than WT p53 as has been shown with c-myc (22) and EGFR (26) . Although these novel regulatory activities may cause the gain-of-function cellular phenotypes exhibited by mutant p53s, another theory is being considered. Namely, that these phenotypes arise from the retention of some of the WT-transcriptional regulatory functions in the context of a large loss of other counterbalancing activities. In other words, the mutations cause a gross imbalance of normal p53 functions that produces new phenotypes. Many of these retained WT functions are thought to be the result of interactions with transcription factors via the NH2-terminal transactivation domain or COOH-terminal domain (reviewed in ref. 27 ).

Thus far, little data have been generated addressing the relative contribution of the novel transcriptional regulatory events or the remaining WT activities of p53 mutants to their gain-of-function phenotypes. We have chosen to study the DNA-binding domain mutant A138P p53 in this regard because of our interest in its role in the proliferation in B-cell lymphomas and evidence, stated above, suggesting that it possesses gain-of-function phenotypes. To begin to elucidate the mechanism of gain-of-function phenotypes caused by mutant p53s, we have compared the ability of gain-of-function mutants A138P and R175H with the ability of WT p53 to regulate the expression of a large set of genes by cDNA microarray.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
The human colon tumor (HCT) 116 p53 –/– colorectal tumor cell line was a kind gift from Dr. Nikki Holbrook (Yale University, New Haven, CT). These cells were propagated in McCoy’s 5A media supplemented with 10% fetal bovine serum and 100 units/mL penicillin/streptomycin at 37°C in an atmosphere of 5% CO2. The origin and culture conditions for B-cell lymphoma-derived RL cells have been described previously (21) . Cell densities were determined by mixing cells with trypan blue and counting with a hemocytometer.

Treatment of Cells with TGF-ß, Phorbol Myristate Acetate, and E64.
Human TGF-ß1 (Peprotech, Inc., Rocky Hill, NJ) was obtained in solid form and reconstituted in sterile dH2O at a concentration of 50 ng/µl. Aliquots were stored at –20°C. TGF-ß1 was added to cell culture media at a final concentration of 10 ng/µl. Phorbol myristate acetate (PMA, Sigma, St. Louis, MO) was dissolved in ethanol at 100 ng/µl and stored at –20°C. The final concentration of PMA in cell culture media was 0.1 ng/mL. E64 (trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane; BIOMOL, Plymouth Meeting, PA) was dissolved in dimethyl sulfoxide at 20 mmol/L and stored at –20°C. E64 was added to cell culture media at a final concentration of 30 µmol/L.

Isolation of Cell Lysates and Immunoblotting.
To collect whole cell lysates, cell pellets were washed twice with PBS (minus calcium and magnesium) and resuspended in 150 µL TENN [50 mmol/L Tris (pH 7.5), 5 mmol/L EDTA, 150 mmol/L NaCl, 0.5% (v/v) NP40] per 3 x 106 cells. Lysates were incubated on ice for 15 minutes and pelleted in a micro-centrifuge at 14,000 rpm. The supernatant was removed and saved. Immunoblotting was carried out by the NuPage system with its accompanying buffers (Invitrogen, Carlsbad, CA). Lysates were applied to NuPage precast Bis-Tris gels and transferred to polyvinylidine fluoride membranes. Membranes were blocked with TBST [10 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, and 0.05% Tween 20) plus 5% nonfat dry milk for at least 1 hour at room temperature. Membranes were then blotted with anti-p53 mouse monoclonal IgG Ab-2 (Oncogene, Cambridge, MA) at 0.25 µg/µl in blocking buffer described above for 1 hour at room temperature. This was followed by exposure to antimouse IgG-horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) at 0.12 µg/µl in blocking buffer for 1 hour at room temperature. Generally, membranes were washed 3x with TBST after exposure to primary and secondary antibodies. Blots were developed with ECL chemiluminescence reagents (Amersham Pharmacia Biotech, Buckinghamshire, England). Bands were quantitated with an AlphaImager Imaging System ({alpha} Innotech Corp., San Leandro, CA).

Construction of Wild-Type and Mutant p53 Expression Vectors.
WT and R175H p53 cDNA were obtained as kind gifts from Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD). The A138P mutation was created in the p53 cDNA by standard PCR-mediated mutagenesis with the following primer (substituted nucleotide underlined): 5'-TTGCCAACTGCCCAAGACTG-3'. All PCR-generated DNA was found to be free of secondary mutations by dideoxynucleotide sequencing. WT and A138P p53 cDNAs were cloned into the pIRES2-enhanced green fluorescent protein (EGFP) vector (Clontech, Palo Alto, CA) via SalI and SmaI sites. The R175H p53 cDNA was cloned into the pIRES2-EGFP vector via EcoRI sites. These constructs allowed p53 and EGFP to be expressed from a single bicistronic mRNA.

Transfection, Flow Cytometry, and RNA Preparation.
HCT 116 p53 –/– cells were plated in T-75 cm2 flasks at a density of 2.5 x 105 cell/mL in a total of 16 mL. The following day, cells were transfected with the FuGENE 6 transfection reagent (Roche Diagnostics Corp., Indianapolis, IN). A 3:1 (µl:µg) mixture of FuGENE 6 to plasmid DNA was used. Approximately 42 hours after transfection, the cells were harvested by trypsin treatment. Cells were resuspended in PBS, and green fluorescent protein (GFP)-positive cells were separated from negative cells by a fluorescence cell sorter. GFP-positive cells were then pelleted and washed with 5 mL of PBS. Total RNA was then purified from the cells with an Rneasy kit (Qiagen, Inc., Valencia, CA). Integrity of the purified RNA was monitored by agarose gel electrophoresis. Yields of RNA per cell from GFP-, WT p53/GFP-, A138P p53/GFP-, and R175H p53/GFP-transfected cells were approximately equal.

Microarray Analysis.
Two microarrays were used in this study. Human array 1 was prepared by selecting a set of 6.9 x 103 human cDNAs from a master set of approximately 15 x 103 genes (Research Genetics, Inc., now owned by Invitrogen, Carlsbad, CA) and printed on nylon filters by a GMS417 Microarrayer (Genetic Microsystems, Woburn, MA). Human array 2 was prepared by selecting a set of 6.9 x 103 human cDNAs from the Mammalian Gene Collection1 and printing them on nylon filters by a MicroGrid II Microarrayer (Biorobotics, Cambridge, United Kingdom). The genes on these arrays encoded proteins with a wide variety of functions including those related to basic metabolism, cell structure, immune response, and signal transduction.

The procedures used for labeling cDNA and microarray hybridization were essentially the same as those found at the NIH internet site.2 ImageQuant software (Molecular Dynamics, Sunnyvale, CA) was used to initially view the signals from the arrays and make black/gray scaling adjustments as necessary. Visual inspection of each array was conducted, and blank spots and those that were affected by artifacts were not considered in the analysis. Array-Pro Analyzer software (MediaCybernetics, Carlsbad, CA) was then used to grid the arrays and convert the hybridization signals to raw intensity values. The intensity values were transferred into Microsoft Excel spreadsheets and matched with the corresponding gene identities. For GFP and p53/GFP expression and array analysis, each experiment was done in duplicate. The duplicate experiments were conducted completely independent of each other, starting with separate transfections and ending with the labeled cDNA applied to separate arrays. Global normalization was used to compare the intensity values from each of the duplicate arrays corresponding to p53/GFP expression with each of the duplicate arrays resulting from GFP only expression as follows.

A median intensity value was found for each of the two arrays being compared, and the average of these values was calculated (average of medians). Median intensity values were used to normalize in order to attenuate statistical skewing due to a large number of gene regulation events caused by the expression of WT p53. Next, a normalization factor was found for each array by dividing the average of medians by the median intensity value of each array. Each intensity value on each array was then multiplied by this factor. The ratio of the normalized intensity values for each gene attributable to p53/GFP expression to that attributable to GFP expression was then calculated. This resulted in four ratio values for each gene that were averaged. Although 2-fold expression level changes are often arbitrarily chosen to be the minimum level of significance, slightly lower ratios of ≥1.8-fold or ≤0.56 were used to include more genes in the analysis. Genes having a SEM of >20% of the average ratio value were not considered in the analysis. However, to include events that had large variances but were likely to be significant, genes having a SEM of >20% of the average ratio value, but all four ratios meeting the above criteria of significance (≥1.8-fold or ≤0.56), were included in the analysis and denoted by an asterisk.

Real-Time PCR.
Purified RNA from HCT 116 p53 –/– cells transfected as described above was used to make cDNA with the Taqman Reverse Transcription kit (Applied Biosystems, Foster City, CA). The specific "Reverse Transcription for the 18S Amplicon" protocol was used. For the real-time PCR quantification of selected genes, the following primers were used that flanked an intron(s) (indicated in parentheses) in the gene of interest to monitor any possible contamination from genomic DNA: connective tissue growth factor (intron 4), 5' primer, 5'-CGAAGCTGACCTGGAAGAGAAC-3' and 3' primer, 5'-ATGCTGGTGCAGCCAGAAAG-3'; deoxycytidine kinase (intron 6), 5' primer, 5'-CTTCAAGAGGTGCCTATCTTAACAC-3' and 3' primer, 5'-GTCTTCAGCAAGATCACAAAGTACTC-3'; prefoldin 4 (intron 3), 5' primer, 5'-TGATGTCTTCATTAGCCATTCTCAAG-3' and 3' primer, 5'-GAATTGATTCCACTCTGGATTCTAAG-3'; and glyceraldehyde-3-phosphate dehydrogenase (introns 1 and 2), 5' primer, 5'-GTTCGACAGTCAGCCGCATC-3' and 3' primer, 5'-GGAATTTGCCATGGGTGGA-3'.

Glyceraldehyde-3-phosphate dehydrogenase was quantified to normalize cDNA amounts. Real-time PCR mixes were prepared containing optimized concentrations of primers (150–600 nmol/L), 10 ng of cDNA, and 25 µL of 2x SYBR Green PCR Master Mix (Applied Biosystems) in a total volume of 50 µL.

Reactions were incubated for 10 minutes at 95°C followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute in a GeneAmp 5700 Sequence Detection System or an ABI Prism 7700 Sequence Detector (Applied Biosystems). The PCR products were specific and free of amplified genomic DNA as indicated by agarose gel electrophoresis. Each reaction was done in duplicate and the CT values were averaged. The fold expression level change, 2–{Delta}{Delta}CT, for each gene in A138P p53/GFP versus GFP expressing cells was calculated as described previously (28) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Evidence of TGF-ß Resistance Gain-of-Function Activity for A138P p53.
Before using A138P p53 to study the mechanisms of mutant p53 gain-of-function phenotypes, it was important to provide additional evidence that it does have gain-of-function activity. As noted above, we have provided data suggesting that A138P p53 causes resistance to TGF-ß–mediated growth suppression in RL cells and that these cells have developed a mechanism for down-regulating this endogenous mutant after exposure to the cytokine (21) . To provide further evidence that A138P p53 possesses this gain-of-function activity, we partially inhibited the degradation of A138P p53 by treatment with the proteasome inhibitor E64 upon TGF-ß exposure and measured the effect on growth suppression. As seen in Fig. 1ACitation , the addition of E64 upon TGF-ß exposure increased the level of A138P p53 by 40% compared with that found with TGF-ß exposure alone. In correlation with these results, as seen in Fig. 1BCitation , the addition of E64 blocked 60% of the TGF-ß–induced growth inhibition. These results further indicate that A138P p53 possesses gain-of-function properties, namely that of TGF-ß resistance.



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  		Fig. 1. Partial inhibition of proteolysis of A138P p53 causes resistance to TGF-ß–mediated growth suppression. A, RL cells were treated with nothing (control), 0.1 ng/mL PMA, 30 µg/mL E64, or 0.1 ng/mL PMA + 10 ng/mL TGF-ß ± 30 µg/mL E64 for 48 hours. Whole cell lysates were collected and immunobloted, 25 µg, with an antibody against p53. Nonspecific band indicates equal loading of protein lysates. B, RL cells were treated as (in A) above. Cell densities were determined after 48 hours of exposure. Percentage of growth inhibition was calculated as the percentage of decrease in cell density of treated cells compared with control cells. Values are the average of triplicate experiments. Error bars represent SEM.

 
Analysis of Genes Altered in Expression Level by A138P p53, Comparison with Wild-Type p53.
To begin to address the question of the origin of mutant p53 gain-of-function activities, we used a cDNA microarray to analyze the expression levels of ~6,900 genes, human array 1, in response to the expression of A138P and WT p53 in HCT 116 p53 –/– cells. HCT 116 p53 –/– cells were transfected with vectors expressing GFP, WT p53/GFP, or A138P p53/GFP, and GFP-positive cells were separated from negative cells by cell sorting (see Materials and Methods). To simulate the conditions of a mutant p53 expressed in cells in vivo, cells were transfected and grown under nonstressed conditions: no radiation exposure or treatment with DNA damaging agents. Both A138P and WT p53 were expressed at near equivalent levels as determined by Western blot analysis (data not shown). Expression of WT or mutant p53 did not seem to cause growth arrest in these cells as measured by RNA yields and analysis of cell cycle distribution (data not shown). Message levels of genes in cells expressing WT p53/GFP or A138P p53/GFP were compared with those expressing GFP alone (see Materials and Methods). To measure the consistency in hybridization intensity values, a regression analysis was done for replicate experiments. The variance in hybridization intensity values between duplicate experiments was low, as indicated by R2 values ranging from 0.92 to 0.97.

As seen in Table 1Citation , the presence of A138P p53 only altered the expression level of 24 genes on human array 1 (0.4% of total). This is in sharp contrast to WT p53 that affected the expression level of 702 genes as seen in Table 2Citation . Therefore, with respect to the genes studied on this array, the mutant possesses only about 3% of the regulatory activities of the WT protein. The genes regulated by A138P p53 were not limited to a certain functional class but had a wide variety of functions including cell mobility, proliferation, and cell shape. Interestingly, 71% of the regulatory activities of A138P p53 were also exhibited by WT p53. In other words, only 29% of the regulation events caused by A138P p53 were actual gain-of-function activities. In fact, after closer inspection of the data, it is likely that there are very few true gain-of-function transcriptional regulatory activities of A138P p53. For three of the seven gain-of-function activities, WT p53 exhibited the same decrease in gene expression, but the fold expression level change, 0.57 to 0.60, was not quite robust enough to meet the arbitrary criteria of significance of ≤0.56. As mentioned above, certain p53 mutants have been shown to confer prometastatic effects on cells (11 , 13 , 16) . Interestingly, A138P p53 up-regulated the expression level of connective tissue growth factor (CTGF) 2-fold (Table 1)Citation . Several studies have indicated that CTGF is a potent stimulator of angiogenesis in certain cell lines (29 , 30) . The up-regulation of this gene by mutant p53s may contribute to their prometastatic effects. Because of this possible relationship and to provide a general verification of the gene regulation events found by microarray, real-time PCR was used to corroborate the modulations in expression levels of CTGF, deoxycytidine kinase, and prefoldin 4 as shown in Table 3Citation . This method confirmed that A138P p53 up-regulated CTGF by 2-fold and down-regulated deoxycytidine kinase and prefoldin 4 by 50 to 60%, indicating a good correlation between the microarray and real-time PCR data.


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  		Table 1 Summary of genes altered in expression level by A138P p53, human array1

 

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  		Table 2 Selected genes altered in expression level by WT p53

 

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  		Table 3 Confirmation of gene regulation by A138P p53 with real-time PCR

 
Analysis of Genes Altered in Expression Level by R175H and A138P p53 on Human Array 2, Comparison with Wild-Type p53.
To determine whether the transcriptional regulatory activities of other p53 mutants are also mostly remaining WT functions, we analyzed the regulatory activities of another known gain-of-function p53 mutant, R175H. Because human array 1 was not available for use at the time, another cDNA microarray was used containing ~6,900 genes, human array 2, to analyze the transcriptional regulatory activities of R175H, A138P, and WT p53. The expression system used was identical to that described above for human array 1. R175H p53 was expressed at near equivalent levels to A138P and WT p53 as determined by Western blot analysis (data not shown). In addition, R175H p53 did not seem to cause growth arrest as measured by RNA yields and analysis of cell cycle distribution (data not shown). Again, the variance in hybridization intensity values between duplicate experiments for all samples with human array 2 was low, as indicated by R2 values ranging from 0.93 to 0.98.

As seen in Table 4Citation , A138P p53 once again altered the expression level of a small percentage of genes on the array, 38 genes or 0.6% of the total. In contrast, WT p53 altered the expression of 657 genes on the array (Table 2)Citation . Therefore, with respect to the genes on human array 2, A138P p53 only retained a small amount, about 5%, of the regulatory activities of the WT protein. This is consistent with what was found on human array 1. However, in contrast to what was found on human array 1, most of the regulatory events produced by A138P p53 represented increases in gene expression. Again, genes with a wide variety of functions were regulated by A138P p53. Notably, A138P p53 retained the ability to up-regulate globin genes ß and {gamma}A. This is consistent with previous studies indicating that WT p53 up-regulates globin gene expression (31 , 32) . Of the regulatory activities caused by A138P p53 on human array 2, 66% were categorized as novel gain-of-function activities. However, as was the case for human array 1, few of the regulatory activities categorized as gain-of-function are likely to be true novel events.


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  		Table 4 Summary of genes altered in expression level by A138P p53, human array 2

 
For 19 of the 25 gain-of-function regulatory events produced by A138P p53, WT p53 caused the same type of regulation, increase or decrease in expression, that was at least a 1.5-fold increase or 0.67-fold decrease but not robust enough to meet the arbitrary criteria of significance. Considering experimental error, these events do not likely represent true gain-of-function activities. In summary, only about 6 of the 38, 16%, transcriptional regulatory events caused by A138P p53 on human array 2 seem to be true gain-of-function activities. Combining the data from human arrays 1 and 2, ~16% of the gene regulation events exhibited by A138P p53 are likely to be true gain-of-function activities. The data from human arrays 1 and 2 suggest that gain-of-function phenotypes produced by A138P p53 are not likely because of novel transcriptional regulatory events. Instead, these phenotypes are more likely to be the result of an imbalance of remaining WT regulatory events due to the retention of the ability to modulate the expression level of a small number of genes in the context of a loss of many other activities.

As seen in Table 5Citation , similar to A138P p53, R175H p53 modulated the expression level of a small percentage of genes on human array 2: 14 genes or 0.2% of the total. Therefore, R175H p53 only retained 1 to 2% of the transcriptional regulatory activities of the WT protein. Similar to A138P p53, most of the regulatory events caused by R175H p53 were increases in gene expression for this set of genes. Of the transcriptional regulatory functions possessed by R175H p53, 71% were labeled as gain-of-function activities. For reasons discussed above for A138P p53, about 7 of these 10 gain-of-function regulation events are likely to represent truly novel activities. In summary, about 50% of the transcriptional regulatory events caused by R175H p53 seem to be true gain-of-function activities. These data suggest that gain-of-function phenotypes exhibited by R175H p53 are produced by a combination of novel regulatory activities and an imbalance of remaining WT functions. Indeed, other gain-of-function transcriptional regulatory functions for R175H p53 have been described such as the up-regulation of proliferating cell nuclear antigen, c-fos, and MDR-1 (10 , 33, 34, 35) . The up-regulation of cyclin D3 (Table 5)Citation may be an acquired regulatory function that contributes to the enhanced cell growth gain-of-function phenotype of R175H p53 (11 , 35) .


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  		Table 5 Summary of genes altered in expression level by R175H p53, human array 2

 
As seen in Tables 1Citation and 4Citation , for most of the genes altered in their expression level by A138P and WT p53, the degree of regulation was not significantly compromised by the mutation. In fact, the fold expression level change due to A138P p53 was <70% of that attributable to WT p53 in only 7% of these common gene regulation events. This suggests that most of the regulation events of A138P p53 are a combination of a small amount of DNA-binding–dependent activities not compromised by the mutation and events not mediated by direct DNA binding but by its ability to interact with transcription cofactors through its NH2-terminal transactivation domain and COOH-terminal domain (2 , 3 , 7) . These two domains were not likely to have been compromised by the mutation in the DNA-binding domain. Consistent with this, immunoprecipitation of p53 from lysates of HCT 116 p53 –/– cells expressing A138P or WT p53, followed by two-dimensional gel electrophoresis, revealed the same species and abundance of proteins associated with both forms of p53 (data not shown). R175H p53 did not retain enough WT transcriptional regulatory events to be analyzed in this respect.

Wild-Type p53 Altered the Expression Level of a Large Percentage of Genes Studied.
As shown in Table 2Citation , WT p53 altered the expression level of many genes (see supplemental Tables 1 and 2 for full list). In fact, 10 and 9% of the genes studied on human arrays 1 and 2, respectively, exhibited altered expression levels due to WT p53. A significantly greater proportion of the genes regulated by WT p53 on human array 1 were up- rather than down-regulated. However, the opposite result was obtained on human array 2. Very few of the genes reported in Table 2Citation have been analyzed by microarray for p53 regulation previously. Many of the gene regulation events were in accordance with the role of p53 as a potent mediator of growth arrest and apoptosis. Several of the genes encoding proteins involved in the basic cellular transcription machinery were significantly repressed by WT p53, including RNA polymerase II polypeptide K (7.0 kDa) and transcription elongation factor B (SIII), polypeptide 1. In addition, the expression levels of eukaryotic translation initiation factor 2B, subunit 2 (ß) and eukaryotic translation termination factor 1 were reduced and increased, respectively, by WT p53. Interestingly, it was found that the transcription factor Sp3 gene was up-regulated by almost 3-fold by WT p53. Sp3 is a potent up-regulator of the p21 (WAF-1) gene (36, 37, 38, 39) and may be involved in mediating the crucial activation of this gene by p53. Regarding its apoptotic functions, WT p53 reduced the expression level of defender against cell death 1 (DAD1) and up-regulated requiem and programmed cell death 5.

WT p53 also regulated the expression of two matrix metalloproteinases, MMP-7 and -9. MMP-7, down-regulated, and MMP-9, up-regulated, are capable of degrading several extracellular matrix proteins, and their expression has been associated with high-grade metastatic tumor cells (40, 41, 42) . It is uncertain what specific effect WT p53 has on metastasis because it can up- and down-regulate genes that can promote or repress this process. It is thought that the net regulation of such metastasis-related proteins by p53, which is likely to be cell type dependent, determines its role in this aspect of carcinogenesis. Lastly, the expression level of mucin 9 (oviductin) was increased >4-fold by WT p53. Mucin 9 is maximally expressed during ovulation (43) and is thought to mediate gamete interaction (44) . Interestingly, p53 is also highly expressed during ovulation (45 , 46) and may be a critical regulator of mucin 9 production during this stage of the reproduction process.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This report provides data elucidating the mechanism of mutant p53 gain-of-function phenotypes by comparing the effect of the expression of mutant and WT p53 on the expression levels of many genes by microarray analysis. The most obvious difference between the mutants, A138P and R175H, and WT p53 is the loss of the ability to regulate the expression of most of the genes that the WT protein influences. In fact, A138P and R175H p53 retained <5% of the regulatory activities of WT p53 with respect to the genes studied here. Obviously, the A138P and R175H mutations in the DNA-binding domain of p53 severely compromised its transcriptional regulatory activities. The ability of WT p53 to alter the expression level of ~10% of the genes on the arrays studied here underscores the growing number of reports indicating that it is involved in the regulation of a vast number of genes involved in many different physiologic processes. As mentioned above, several gain-of-function cellular phenotypes have been found for various p53 mutants. Typically, a given gain-of-function phenotype is produced by several but not all mutant p53s. The cause of these gain-of-function phenotypes may be related to the ability of mutant p53s to regulate the expression of genes that WT p53 does not affect or acts on in the opposite manner. For example, it is possible that the increase in the expression of pro-proliferative genes such as c-myc and EGFR by certain mutant p53s contributes to gain-of-function phenotypes such as increased rate of cell division and tumorigenesis. However, it is difficult to attribute these gain-of-function phenotypes to a few of these mutant-specific gene regulations alone. This is because p53 mutants also retain various amounts of WT p53 transcriptional regulatory events in the context of a loss of a substantial number of other such functions (discussed in 27 ). This imbalance of remaining WT functions may also cause gain-of-function phenotypes. In the experimental system described here, the effect of A138P and R175H p53 on the expression level of a large unfocused set of genes was determined. It was found that A138P p53 possessed very few true novel transcriptional regulatory activities compared with WT p53. In other words, most of the gene regulation events produced by A138P p53 were also properties of WT p53. Therefore, we propose that gain-of-function phenotypes possessed by A138P p53 are largely because of an imbalance of WT transcriptional regulatory functions caused by a small amount of remaining WT activities existing in the absence of many other counterbalancing functions. Contributing to the ability of these remaining regulatory activities to produce gain-of-function phenotypes is that because mutant p53s tend to be constitutively expressed at much higher levels than WT p53 under nonstressed conditions, the robustness of the expression level changes caused by these functions would likely be significantly greater in cells harboring mutant versus WT p53. The high levels of mutant p53 usually found in cells are thought to be because of the lack of up-regulation of hdm2, which mediates the proteolysis of p53, normally possessed by the WT protein (47) . An example of a regulation event that is possessed by A138P and WT p53 and could play a role in an enhanced metastatic phenotype is the increase in the expression of CTGF (Tables 1Citation and 2Citation ). For WT p53, this up-regulation may produce a minor effect on metastasis because of its antimetastatic activities such as the repression of genes like basic fibroblast growth factor (23) and MMP-7 (Table 2)Citation and the activation of Drg-1 (48) . However, many of the antimetastatic activities of WT p53 are likely to be absent in mutant p53s and, therefore, the up-regulation of CTGF may contribute significantly to increased metastasis.

The most obvious difference between the two p53 mutants studied here is that R175H retained less WT transcriptional regulatory events than A138P. In addition, R175H p53 possessed an approximately equal number of retained WT regulatory activities and acquired novel functions. These data suggest that novel regulatory functions as well as a small amount of retained WT functions existing in the absence of balancing activities both significantly contribute to the gain-of-function phenotypes exhibited by R175H p53. It is expected that both mechanisms are potently enhanced by the key loss of transcriptional control over certain genes that regulate cell division and differentiation often exhibited by p53 mutants (49 , 50) . The expression of R175H p53 is known to produce several gain-of-function phenotypes including increased growth potential (11 , 33) , enhanced tumorigenesis and metastasis (11) , and drug resistance (19 , 51) . From the work of others and that presented here, several examples of novel acquired transcriptional regulatory activities as well as remaining WT functions have been observed that may contribute to these gain-of-function phenotypes. The acquired ability of R175H p53 to activate the expression of cyclin D3, proliferating cell nuclear antigen, and c-fos (33) may contribute to enhanced proliferation, whereas the novel up-regulation of MDR-1 may cause increased drug resistance (10 , 34 , 35 , 51) . In addition, the retained ability of R175H p53 to activate transcription of EGFR (26) may also contribute to increased cell growth. It can also be noted that A138P and R175H p53 share six common transcriptional regulatory events (Tables 4Citation and 5Citation ). Of these events, five of them represent remaining WT regulatory activities. These data suggest that transcriptional regulatory activities shared by mutant p53s are more likely to be retained WT functions. However, future studies will be needed to test this hypothesis.

Considering that the A138P and R175H mutations are in the DNA-binding domain of p53, it is likely that the large loss of regulatory events exhibited by these mutants compared with WT p53 represent DNA-binding–dependent activation and repression functions. In fact, it was shown that R175H p53 is not able to bind the p53 consensus DNA-binding sequence in the gadd45 promoter (9) . A substantial portion of the remaining WT regulatory events is likely due to the binding of transcription cofactors through the intact NH2- (52 , 53) and COOH-terminal domains (54) , independent of DNA binding. Substantiating this, it was observed that only 7% of the regulatory activities possessed by A138P p53 and none of the regulatory events of R175H that were also produced by WT p53 were significantly decreased in terms of the fold expression level change compared with the corresponding WT-induced activity. Previous studies have indicated that p53 mutants can transactivate in the absence of direct DNA binding. In these cases, the ability of the mutants to be recruited to the promoters may be mediated by their interaction with Sp1 (26 , 55) . DNA-binding–independent transcriptional repression activities of p53 have also been described (reviewed in ref. 56 ). These events seem to be mediated by p53 binding and inhibiting transcription activators such as AP-1 (57) , C/EBP (52) , and TFIIIB (58) .

The data presented here indicate that novel acquired regulatory functions as well as retained WT activities existing in the absence of many counterbalancing activities are both likely to contribute to gain-of-function phenotypes exhibited by mutant p53s. However, p53 mutants are likely to vary in the extent to which each mechanism contributes to these phenotypes. Perhaps future studies will expand on this work by examining the effect of other p53 mutants on the expression of large sets of genes and begin to correlate the retention and loss of WT transcriptional regulatory events and the acquisition of novel activities with particular gain-of-function phenotypes. Because p53 DNA-binding domain mutants retain various degrees of DNA-binding affinity (9) , it would be expected that each mutant would be distinct in terms of the number of regulatory functions it retains because of direct transcriptional regulation. In addition, because each mutation can potentially produce distinct effects on the structure of the DNA-binding domain, p53 mutants are likely to exhibit differences in acquired novel transcriptional regulatory activities. However, it is likely that remaining WT-transcriptional regulatory events mediated by binding transcription cofactors via their uncompromised NH2- and COOH-terminal domains would be common to many of these types of mutants. For instance, as shown here with human array 2, five of the six regulatory functions shared by A138P and R175H p53 are retained WT functions (Tables 4Citation and 5Citation ). In other words, for this gene set, the two mutants only share one acquired novel regulatory activity. We can hypothesize that gain-of-function phenotypes often shared by p53 mutants are likely due to unbalanced retained WT functions via interaction with transcription cofactors. Conversely, gain-of-function phenotypes less commonly exhibited by mutant p53s are more likely due to a combination of acquired novel regulatory events and an imbalance of retained WT functions due to various losses of DNA-binding–dependent regulation activities. However, future studies will be needed to test this hypothesis.


   ACKNOWLEDGMENTS
 
The authors acknowledge the adroit technical assistance of Joe Chrest, Kevin Becker, William Wood III, and Diane Teichberg (Gerontology Research Center, National Institute on Aging).


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Present address for N. Dobashi is Division of Hematology and Oncology, Department of Internal Medicine, Jikei Univ. School of Medicine, 3-25-8, Nishi-Shinbashi, Minato-ku, Tokyo, 105 Japan. Supplemental data for this article can be found at Cancer Research Online (http://cancerres@aacrjournals.org).

Requests for reprints: Dan L. Longo, Laboratory of Immunology, Gerontology Research Center, National Institute on Aging, NIH, 5600 Nathan Shock Dr., Box 21, Baltimore, MD 21224. Phone: 410-558-8110; Fax: 410-558-8137; E-mail: LongoD{at}nih.gov

1 http://mgc.nci.nih.gov/. Back

2 http://www.grc.nia.nih.gov/branches/rrb/dna/index/protocols.htm. Back

Received 11/20/03. Revised 9/ 7/04. Accepted 9/14/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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