Comparison of the Effect of Mutant and Wild-Type P53 on Global Gene Expression

[CANCER RESEARCH 64, 8199–8207, November 15, 2004] 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 compared with wild-type (WT) p53 (9). Generally, mutation of residues directly contacting DNA results in a complete loss of DNA The mechanisms for “gain-of-function” phenotypes produced by mu- binding with little loss in folding of the domain, whereas mutation of tant p53s such as enhanced proliferation, resistance to transforming structural residues results in a Ͼ50% loss of folding and DNA binding growth factor-␤–mediated growth suppression, and increased tumorigenesis are not known. One theory is that these phenotypes are caused by (9). However, considering that the NH2- and COOH-terminal domains novel transcriptional regulatory events acquired by mutant p53s. Another of p53 are distinct from the compact DNA-binding domain and explanation is that these effects are a result of an imbalance of functions considerably less ordered, the mutations in the DNA-binding domain caused by the retention of some of the wild-type transcriptional regulatory are less likely to affect the integrity and function of these domains. events in the context of a loss of other counterbalancing activities. An Because p53 is found mutated in approximately 50% of all cancers, analysis of the ability of DNA-binding domain mutants A138P and R175H, p53 mutants have been rather extensively studied for their ability to ؋ 3 and wild-type p53 to regulate the expression levels of 6.9 10 genes confer “gain-of-function” phenotypes on cells. These are phenotypes revealed that the mutants retained only <5% of the regulatory activities not caused by WT p53 that are seen when mutant p53s are expressed of the wild-type protein. A138P p53 exhibited mostly retained wild-type regulatory activities and few acquired novel events. However, R175H p53 in p53 null cells. Several gain-of-function phenotypes have been possessed an approximately equal number of wild-type regulatory events found for various p53 mutants such as increased cell growth (10, 11), and novel activities. This is the first report that, after examination of the enhanced tumorigenicity (11–15) and invasiveness (11, 13, 16), dis- regulation of a large unfocused set of genes, provides data indicating that turbed spindle checkpoint (17, 18), and resistance to cytotoxic agents remaining wild-type transcriptional regulatory functions existing in the (19). Furthermore, it was shown that the expression of p53 mutants absence of counterbalancing activities as well as acquired novel events V134A and C132F caused cells to become resistant to transforming both contribute to the gain-of-function phenotypes produced by mutant growth factor (TGF)-␤–mediated growth suppression (12, 20). Addi- p53s. However, mutant p53s are likely to be distinct in terms of the extent tional evidence that mutant p53s may have the ability to antagonize to which each mechanism contributes to their gain-of-function pheno- TGF-␤ is that a decrease in the level of endogenous homozygous types. 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 INTRODUCTION suppression (21). This suggests that A138P p53 causes resistance to ␤ The protein p53 is a transcription factor that mediates several TGF- –mediated growth suppression and that these cells have devel- ␤ cellular processes including growth arrest, apoptosis, differentiation, oped a pathway that reduces the level of this mutant after TGF- and DNA damage repair (reviewed in 1–3). Because of its growth- exposure so that growth suppression can occur. Some p53 DNA- inhibiting functions as well as its mutation or loss in Ն50% of human binding domain mutants possess the ability to alter the expression of tumors (4–6), p53 is considered an important tumor suppressor pro- genes that the WT protein either does not affect or regulates in the tein. opposite fashion. For example, it has been shown that several mutants of p53 up-regulated the expression of c-myc (22), basic fibroblast The domain structure of p53 consists of an NH2-terminal transactivation domain, a core DNA-binding domain, and a COOH-terminal growth factor (23), insulin-like growth factor receptor 1 (24), and interleukin-6 (IL-6, ref. 25), whereas WT p53 repressed the expres- domain. The NH2-terminal transactivation domain seems to be a functional binding site for basal transcription factors such as p300/ sion of these genes. Also, several p53 mutants were able to increase CBP, TFIID, and TATA-binding protein (reviewed in ref. 7). These the expression of the epidermal growth factor receptor (EGFR) gene associations seem to mediate the transactivation as well as transre- to a much greater extent than WT p53 (26). These gain-of-function pression functions of p53. The central compact core of p53 forms the gene regulation events may result from the ability of these mutants to DNA-binding domain. This domain allows the protein to bind DNA in associate with different regulatory elements than WT p53 as has been a sequence-specific fashion and is where the vast majority of muta- shown with c-myc (22) and EGFR (26). Although these novel regu- tions in p53 are found in human cancer (8). The COOH-terminal latory activities may cause the gain-of-function cellular phenotypes domain contains the tetramerization region and also seems to mediate exhibited by mutant p53s, another theory is being considered. binding to basal transcription factors (2, 3). Namely, that these phenotypes arise from the retention of some of the As mentioned above, most of the naturally occurring mutations in WT-transcriptional regulatory functions in the context of a large loss p53 are found in the DNA-binding domain and, therefore, these of other counterbalancing activities. In other words, the mutations mutants have been the most extensively studied. As expected, these cause a gross imbalance of normal p53 functions that produces new mutations result in losses of various degrees of DNA-binding affinity phenotypes. Many of these retained WT functions are thought to be

the result of interactions with transcription factors via the NH2- Received 11/20/03; revised 9/7/04; accepted 9/14/04. terminal transactivation domain or COOH-terminal domain (reviewed The costs of publication of this article were defrayed in part by the payment of page in ref. 27). charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Thus far, little data have been generated addressing the relative Note: Present address for N. Dobashi is Division of Hematology and Oncology, contribution of the novel transcriptional regulatory events or the Department of Internal Medicine, Jikei Univ. School of Medicine, 3-25-8, Nishi- remaining WT activities of p53 mutants to their gain-of-function Shinbashi, Minato-ku, Tokyo, 105 Japan. Supplemental data for this article can be found at Cancer Research Online (http://[email protected]). phenotypes. We have chosen to study the DNA-binding domain Requests for reprints: Dan L. Longo, Laboratory of Immunology, Gerontology mutant A138P p53 in this regard because of our interest in its role in 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: [email protected]. the proliferation in B-cell lymphomas and evidence, stated above, ©2004 American Association for Cancer Research. suggesting that it possesses gain-of-function phenotypes. To begin to 8199

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2004 American Association for Cancer Research. MUTANT AND WILD-TYPE p53 EFFECT ON GENE EXPRESSION elucidate the mechanism of gain-of-function phenotypes caused by Microarray Analysis. Two microarrays were used in this study. Human mutant p53s, we have compared the ability of gain-of-function mu- array 1 was prepared by selecting a set of 6.9 ϫ 103 human cDNAs from a tants A138P and R175H with the ability of WT p53 to regulate the master set of approximately 15 ϫ 103 genes (Research Genetics, Inc., now expression of a large set of genes by cDNA microarray. owned by Invitrogen, Carlsbad, CA) and printed on nylon filters by a GMS417 Microarrayer (Genetic Microsystems, Woburn, MA). Human array 2 was ϫ 3 MATERIALS AND METHODS prepared by selecting a set of 6.9 10 human cDNAs from the Mammalian Gene Collection1 and printing them on nylon filters by a MicroGrid II Mi- Cell Culture. The human colon tumor (HCT) 116 p53 Ϫ/Ϫ colorectal croarrayer (Biorobotics, Cambridge, United Kingdom). The genes on these tumor cell line was a kind gift from Dr. Nikki Holbrook (Yale University, New arrays encoded proteins with a wide variety of functions including those Haven, CT). These cells were propagated in McCoy’s 5A media supplemented related to basic metabolism, cell structure, immune response, and signal with 10% fetal bovine serum and 100 units/mL penicillin/streptomycin at 37°C transduction. The procedures used for labeling cDNA and microarray hybridization were in an atmosphere of 5% CO2. The origin and culture conditions for B-cell 2 lymphoma-derived RL cells have been described previously (21). Cell densi- essentially the same as those found at the NIH internet site. ImageQuant ties were determined by mixing cells with trypan blue and counting with a software (Molecular Dynamics, Sunnyvale, CA) was used to initially view the hemocytometer. signals from the arrays and make black/gray scaling adjustments as necessary. Treatment of Cells with TGF-␤, Phorbol Myristate Acetate, and E64. Visual inspection of each array was conducted, and blank spots and those that Human TGF-␤1 (Peprotech, Inc., Rocky Hill, NJ) was obtained in solid form were affected by artifacts were not considered in the analysis. Array-Pro ␮ Analyzer software (MediaCybernetics, Carlsbad, CA) was then used to grid the and reconstituted in sterile dH2O at a concentration of 50 ng/ l. Aliquots were stored at Ϫ20°C. TGF-␤1 was added to cell culture media at a final concen- arrays and convert the hybridization signals to raw intensity values. The tration of 10 ng/␮l. Phorbol myristate acetate (PMA, Sigma, St. Louis, MO) intensity values were transferred into Microsoft Excel spreadsheets and was dissolved in ethanol at 100 ng/␮l and stored at Ϫ20°C. The final concen- matched with the corresponding gene identities. For GFP and p53/GFP ex- tration of PMA in cell culture media was 0.1 ng/mL. E64 (trans-epoxysucci- pression and array analysis, each experiment was done in duplicate. The nyl-L-leucylamido(4-guanidino)butane; BIOMOL, Plymouth Meeting, PA) duplicate experiments were conducted completely independent of each other, was dissolved in dimethyl sulfoxide at 20 mmol/L and stored at Ϫ20°C. E64 starting with separate transfections and ending with the labeled cDNA applied was added to cell culture media at a final concentration of 30 ␮mol/L. to separate arrays. Global normalization was used to compare the intensity Isolation of Cell Lysates and Immunoblotting. To collect whole cell values from each of the duplicate arrays corresponding to p53/GFP expression lysates, cell pellets were washed twice with PBS (minus calcium and magne- with each of the duplicate arrays resulting from GFP only expression as sium) and resuspended in 150 ␮L TENN [50 mmol/L Tris (pH 7.5), 5 mmol/L follows. EDTA, 150 mmol/L NaCl, 0.5% (v/v) NP40] per 3 ϫ 106 cells. Lysates were A median intensity value was found for each of the two arrays being incubated on ice for 15 minutes and pelleted in a micro-centrifuge at 14,000 compared, and the average of these values was calculated (average of medi- rpm. The supernatant was removed and saved. Immunoblotting was carried out ans). Median intensity values were used to normalize in order to attenuate by the NuPage system with its accompanying buffers (Invitrogen, Carlsbad, statistical skewing due to a large number of gene regulation events caused by CA). Lysates were applied to NuPage precast Bis-Tris gels and transferred to the expression of WT p53. Next, a normalization factor was found for each polyvinylidine fluoride membranes. Membranes were blocked with TBST [10 array by dividing the average of medians by the median intensity value of each mmol/L Tris (pH 8.0), 150 mmol/L NaCl, and 0.05% Tween 20) plus 5% array. Each intensity value on each array was then multiplied by this factor. nonfat dry milk for at least 1 hour at room temperature. Membranes were then The ratio of the normalized intensity values for each gene attributable to blotted with anti-p53 mouse monoclonal IgG Ab-2 (Oncogene, Cambridge, p53/GFP expression to that attributable to GFP expression was then calculated. MA) at 0.25 ␮g/␮l in blocking buffer described above for 1 hour at room This resulted in four ratio values for each gene that were averaged. Although temperature. This was followed by exposure to antimouse IgG-horseradish 2-fold expression level changes are often arbitrarily chosen to be the minimum peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) at 0.12 ␮g/␮lin level of significance, slightly lower ratios of Ն1.8-fold or Յ0.56 were used to blocking buffer for 1 hour at room temperature. Generally, membranes were include more genes in the analysis. Genes having a SEM of Ͼ20% of the washed 3ϫ with TBST after exposure to primary and secondary antibodies. average ratio value were not considered in the analysis. However, to include Blots were developed with ECL chemiluminescence reagents (Amersham events that had large variances but were likely to be significant, genes having Pharmacia Biotech, Buckinghamshire, England). Bands were quantitated with a SEM of Ͼ20% of the average ratio value, but all four ratios meeting the an AlphaImager Imaging System (␣ Innotech Corp., San Leandro, CA). above criteria of significance (Ն1.8-fold or Յ0.56), were included in the Construction of Wild-Type and Mutant p53 Expression Vectors. WT analysis and denoted by an asterisk. and R175H p53 cDNA were obtained as kind gifts from Dr. Bert Vogelstein Real-Time PCR. Purified RNA from HCT 116 p53 Ϫ/Ϫ cells transfected (Johns Hopkins University, Baltimore, MD). The A138P mutation was created as described above was used to make cDNA with the Taqman Reverse in the p53 cDNA by standard PCR-mediated mutagenesis with the following Transcription kit (Applied Biosystems, Foster City, CA). The specific “Re- primer (substituted nucleotide underlined): 5Ј-TTGCCAACTGCCCAAG- verse Transcription for the 18S Amplicon” protocol was used. For the real-time ACTG-3Ј. All PCR-generated DNA was found to be free of secondary muta- PCR quantification of selected genes, the following primers were used that tions by dideoxynucleotide sequencing. WT and A138P p53 cDNAs were flanked an intron(s) (indicated in parentheses) in the gene of interest to monitor cloned into the pIRES2-enhanced green fluorescent protein (EGFP) vector any possible contamination from genomic DNA: connective tissue growth (Clontech, Palo Alto, CA) via SalI and SmaI sites. The R175H p53 cDNA was factor (intron 4), 5Ј primer, 5Ј-CGAAGCTGACCTGGAAGAGAAC-3Ј and 3Ј cloned into the pIRES2-EGFP vector via EcoRI sites. These constructs allowed primer, 5Ј-ATGCTGGTGCAGCCAGAAAG-3Ј; deoxycytidine kinase (intron p53 and EGFP to be expressed from a single bicistronic mRNA. 6), 5Ј primer, 5Ј-CTTCAAGAGGTGCCTATCTTAACAC-3Ј and 3Ј primer, Transfection, Flow Cytometry, and RNA Preparation. HCT 116 p53 5Ј-GTCTTCAGCAAGATCACAAAGTACTC-3Ј; prefoldin 4 (intron 3), 5Ј Ϫ/Ϫ cells were plated in T-75 cm2 flasks at a density of 2.5 ϫ 105 cell/mL in primer, 5Ј-TGATGTCTTCATTAGCCATTCTCAAG-3Ј and 3Ј primer, 5Ј- a total of 16 mL. The following day, cells were transfected with the FuGENE GAATTGATTCCACTCTGGATTCTAAG-3Ј; and glyceraldehyde-3-phos- 6 transfection reagent (Roche Diagnostics Corp., Indianapolis, IN). A 3:1 phate dehydrogenase (introns 1 and 2), 5Ј primer, 5Ј-GTTCGACAGTCAGC- (␮l:␮g) mixture of FuGENE 6 to plasmid DNA was used. Approximately 42 CGCATC-3Ј and 3Ј primer, 5Ј-GGAATTTGCCATGGGTGGA-3Ј. hours after transfection, the cells were harvested by trypsin treatment. Cells Glyceraldehyde-3-phosphate dehydrogenase was quantified to normalize were resuspended in PBS, and green fluorescent protein (GFP)-positive cells cDNA amounts. Real-time PCR mixes were prepared containing optimized were separated from negative cells by a fluorescence cell sorter. GFP-positive concentrations of primers (150–600 nmol/L), 10 ng of cDNA, and 25 ␮Lof cells were then pelleted and washed with 5 mL of PBS. Total RNA was then 2ϫ SYBR Green PCR Master Mix (Applied Biosystems) in a total volume of purified from the cells with an Rneasy kit (Qiagen, Inc., Valencia, CA). 50 ␮L. 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 1 http://mgc.nci.nih.gov/. R175H p53/GFP-transfected cells were approximately equal. 2 http://www.grc.nia.nih.gov/branches/rrb/dna/index/protocols.htm. 8200

Reactions were incubated for 10 minutes at 95°C followed by 40 cycles of GFP-positive cells were separated from negative cells by cell sorting 95°C for 15 seconds and 60°C for 1 minute in a GeneAmp 5700 Sequence (see Materials and Methods). To simulate the conditions of a mutant Detection System or an ABI Prism 7700 Sequence Detector (Applied Biosys- p53 expressed in cells in vivo, cells were transfected and grown under tems). The PCR products were specific and free of amplified genomic DNA as nonstressed conditions: no radiation exposure or treatment with DNA indicated by agarose gel electrophoresis. Each reaction was done in duplicate damaging agents. Both A138P and WT p53 were expressed at near and the C values were averaged. The fold expression level change, 2Ϫ⌬⌬CT, T equivalent levels as determined by Western blot analysis (data not for each gene in A138P p53/GFP versus GFP expressing cells was calculated as described previously (28). 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 RESULTS cells expressing WT p53/GFP or A138P p53/GFP were compared Evidence of TGF-␤ Resistance Gain-of-Function Activity for with those expressing GFP alone (see Materials and Methods). To A138P p53. Before using A138P p53 to study the mechanisms of measure the consistency in hybridization intensity values, a regression mutant p53 gain-of-function phenotypes, it was important to provide analysis was done for replicate experiments. The variance in hybrid- additional evidence that it does have gain-of-function activity. As ization intensity values between duplicate experiments was low, as 2 noted above, we have provided data suggesting that A138P p53 causes indicated by R values ranging from 0.92 to 0.97. resistance to TGF-␤–mediated growth suppression in RL cells and As seen in Table 1, the presence of A138P p53 only altered the that these cells have developed a mechanism for down-regulating this expression level of 24 genes on human array 1 (0.4% of total). This is endogenous mutant after exposure to the cytokine (21). To provide in sharp contrast to WT p53 that affected the expression level of 702 further evidence that A138P p53 possesses this gain-of-function ac- genes as seen in Table 2. Therefore, with respect to the genes studied tivity, we partially inhibited the degradation of A138P p53 by treat- on this array, the mutant possesses only about 3% of the regulatory ment with the proteasome inhibitor E64 upon TGF-␤ exposure and activities of the WT protein. The genes regulated by A138P p53 were measured the effect on growth suppression. As seen in Fig. 1A, the not limited to a certain functional class but had a wide variety of addition of E64 upon TGF-␤ exposure increased the level of A138P functions including cell mobility, proliferation, and cell shape. Inter- p53 by 40% compared with that found with TGF-␤ exposure alone. In estingly, 71% of the regulatory activities of A138P p53 were also correlation with these results, as seen in Fig. 1B, the addition of E64 exhibited by WT p53. In other words, only 29% of the regulation blocked 60% of the TGF-␤–induced growth inhibition. These results events caused by A138P p53 were actual gain-of-function activities. further indicate that A138P p53 possesses gain-of-function properties, In fact, after closer inspection of the data, it is likely that there are very namely that of TGF-␤ resistance. few true gain-of-function transcriptional regulatory activities of Analysis of Genes Altered in Expression Level by A138P p53, A138P p53. For three of the seven gain-of-function activities, WT p53 Comparison with Wild-Type p53. To begin to address the question exhibited the same decrease in gene expression, but the fold expres- of the origin of mutant p53 gain-of-function activities, we used a sion level change, 0.57 to 0.60, was not quite robust enough to meet cDNA microarray to analyze the expression levels of ϳ6,900 genes, the arbitrary criteria of significance of Յ0.56. As mentioned above, human array 1, in response to the expression of A138P and WT p53 certain p53 mutants have been shown to confer prometastatic effects in HCT 116 p53 Ϫ/Ϫ cells. HCT 116 p53 Ϫ/Ϫ cells were transfected on cells (11, 13, 16). Interestingly, A138P p53 up-regulated the with vectors expressing GFP, WT p53/GFP, or A138P p53/GFP, and expression level of connective tissue growth factor (CTGF) 2-fold (Table 1). 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 3. 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. 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 ϳ6,900 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 Fig. 1. Partial inhibition of proteolysis of A138P p53 causes resistance to TGF-␤– equivalent levels to A138P and WT p53 as determined by Western mediated growth suppression. A, RL cells were treated with nothing (control), 0.1 ng/mL blot analysis (data not shown). In addition, R175H p53 did not seem ␮ ϩ ␤ Ϯ ␮ PMA, 30 g/mL E64, or 0.1 ng/mL PMA 10 ng/mL TGF- 30 g/mL E64 for 48 to cause growth arrest as measured by RNA yields and analysis of cell hours. Whole cell lysates were collected and immunobloted, 25 ␮g, with an antibody against p53. Nonspecific band indicates equal loading of protein lysates. B, RL cells were cycle distribution (data not shown). Again, the variance in hybridiza- treated as (in A) above. Cell densities were determined after 48 hours of exposure. tion intensity values between duplicate experiments for all samples Percentage of growth inhibition was calculated as the percentage of decrease in cell 2 density of treated cells compared with control cells. Values are the average of triplicate with human array 2 was low, as indicated by R values ranging from experiments. Error bars represent SEM. 0.93 to 0.98. 8201

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

a. Genes up-regulated Ն1.8-fold, 4 (0.1% of total). b. Genes down-regulated Ն1.8-fold (ratio of Յ0.56), 20 (0.3% of total).

Ratio of gene expression level attributable to p53/GFP versus GFP only List of genes altered in expression level by A138P p53 A138P WT

Proliferation/growth factors Connective tissue growth factor (CTGF) 2.0 Ϯ 0.3 2.7 Ϯ 0.4 Macrophage stimulating 1 (hepatocyte growth factor-like) 0.42 Ϯ 0.02 0.57 Ϯ 0.04 Signal transduction Nuclear receptor subfamily 1, group H, member 3 1.8 Ϯ 0.3 2.6 Ϯ 0.4 Metabolism Mitochondrial creatine kinase 0.39 Ϯ 0 0.31 Ϯ 0.02 Aldehyde dehydrogenase family 6, member A1 0.41 Ϯ 0.01 0.29 Ϯ 0.01 Deoxycytidine kinase 0.42 Ϯ 0.03 0.42 Ϯ 0.03 Lactate dehydrogenase A 0.52 Ϯ 0.02 0.80 Ϯ 0.06 Glucosamine-6-phosphate isomerase 0.55 Ϯ 0.03 0.44 Ϯ 0.03 Vesicle transport Kinesin family member 5B 0.41 Ϯ 0.04 0.44 Ϯ 0.06 Cell shape Villin 2 (ezrin) 0.47 Ϯ 0.05 0.57 Ϯ 0.06 Myosin, light polypeptide 6, alkali, smooth muscle and non-muscle 0.47 Ϯ 0.04 0.54 Ϯ 0.05 Cell mobility D-dopachrome tautomerase 0.51 Ϯ 0.02 0.38 Ϯ 0.01 Tumor suppressors IL-24, suppression of tumorigenicity 16 (melanoma differentiation) 0.46 Ϯ 0.02 0.37 Ϯ 0.01 Translation Heme-regulated initiation factor 2-␣ kinase 0.56 Ϯ 0.03 1.0 Ϯ 0.1 Other Zinc finger protein 22 (KOX 15) 1.9 Ϯ 0.3 3.8 Ϯ 0.6 Prefoldin 4 0.41 Ϯ 0.01 0.31 Ϯ 0.01 Galactosylceramidase 0.44 Ϯ 0.04 0.42 Ϯ 0.04 Carboxypeptidase D 0.44 Ϯ 0.06 0.60 Ϯ 0.09 Endothelin converting enzyme 1 0.56 Ϯ 0.06 0.70 Ϯ 0.07 Glutathione-S-transferase like; glutathione transferase ␻ 0.56 Ϯ 0.03 0.45 Ϯ 0.03 Ubiquitin-conjugating enzyme E2I 0.55 Ϯ 0.0 0.81 Ϯ 0.02 ESTs Clone ID 201192 1.8 Ϯ 0.2 1.9 Ϯ 0.1 Clone ID 809696 (pre-mRNA splicing?) 0.51 Ϯ 0.05 0.52 Ϯ 0.05 Clone ID 753069 0.51 Ϯ 0.04 0.45 Ϯ 0.05 NOTE: Gain of function statistics. Percentage of genes listed above altered in expression level by A138P AND WT p53, 71. Percentage of genes listed above altered in expression level by A138P p53 ONLY 29. Of the genes altered in expression level by A138P AND WT p53, percentage of genes of which the fold expression level change attributable to A138P p53 is Ͻ70% of that attributable to WT p53, 12. * Microarray contains 6.9 ϫ 103 genes in total. Gene expression level ratios of Ն1.8 and Յ0.56 were considered to be significant. Values are listed Ϯ SEM. All gene expression level ratios listed had a SEM of Յ20% of the average value (see Materials and Methods).

As seen in Table 4, A138P p53 once again altered the expression activities. Combining the data from human arrays 1 and 2, ϳ16% of level of a small percentage of genes on the array, 38 genes or 0.6% of the gene regulation events exhibited by A138P p53 are likely to be the total. In contrast, WT p53 altered the expression of 657 genes on true gain-of-function activities. The data from human arrays 1 and 2 the array (Table 2). Therefore, with respect to the genes on human suggest that gain-of-function phenotypes produced by A138P p53 are array 2, A138P p53 only retained a small amount, about 5%, of the not likely because of novel transcriptional regulatory events. Instead, regulatory activities of the WT protein. This is consistent with what these phenotypes are more likely to be the result of an imbalance of was found on human array 1. However, in contrast to what was found remaining WT regulatory events due to the retention of the ability to on human array 1, most of the regulatory events produced by A138P modulate the expression level of a small number of genes in the p53 represented increases in gene expression. Again, genes with a context of a loss of many other activities. wide variety of functions were regulated by A138P p53. Notably, As seen in Table 5, similar to A138P p53, R175H p53 modulated the A138P p53 retained the ability to up-regulate globin genes ␤ and ␥A. expression level of a small percentage of genes on human array 2: 14 This is consistent with previous studies indicating that WT p53 genes or 0.2% of the total. Therefore, R175H p53 only retained 1 to 2% up-regulates globin gene expression (31, 32). Of the regulatory activ- of the transcriptional regulatory activities of the WT protein. Similar to ities caused by A138P p53 on human array 2, 66% were categorized A138P p53, most of the regulatory events caused by R175H p53 were as novel gain-of-function activities. However, as was the case for increases in gene expression for this set of genes. Of the transcriptional human array 1, few of the regulatory activities categorized as gain- regulatory functions possessed by R175H p53, 71% were labeled as of-function are likely to be true novel events. gain-of-function activities. For reasons discussed above for A138P p53, For 19 of the 25 gain-of-function regulatory events produced by about 7 of these 10 gain-of-function regulation events are likely to A138P p53, WT p53 caused the same type of regulation, increase or represent truly novel activities. In summary, about 50% of the transcrip- decrease in expression, that was at least a 1.5-fold increase or 0.67- tional regulatory events caused by R175H p53 seem to be true gain-of- fold decrease but not robust enough to meet the arbitrary criteria of function activities. These data suggest that gain-of-function phenotypes significance. Considering experimental error, these events do not exhibited by R175H p53 are produced by a combination of novel regu- likely represent true gain-of-function activities. In summary, only latory activities and an imbalance of remaining WT functions. Indeed, about 6 of the 38, 16%, transcriptional regulatory events caused by other gain-of-function transcriptional regulatory functions for R175H p53 A138P p53 on human array 2 seem to be true gain-of-function have been described such as the up-regulation of proliferating cell nu- 8202

Table 2 Selected genes altered in expression level by WT p53 Global summary of genes altered in expression level by WT p53 Genes on human array 1: a. Genes up-regulated Ն1.8-fold, 484 (7% of total) b. Genes down-regulated Ն1.8-fold (ratio of Յ0.56), 218 (3% of total) Genes on human array 2: a. Genes up-regulated Ն1.8-fold, 168 (2% of total) b. Genes down-regulated Ն1.8-fold (ratio of Յ0.56), 489 (7% of total) Ratio of gene expression level attributable to List of genes altered in expression level by WT p53 * WT p53/GFP versus GFP only Up-regulated genes Development Mucin 9 (oviductin) 4.6 * Ϯ 1 Signal transduction Colony stimulating factor 1 (CSF-1) receptor 2.6 Ϯ 0.2 TGF-␤ receptor, type III 1.9 Ϯ 0.2 TGF-␤ receptor, type II 2.1 Ϯ 0 IL-4 receptor 2.0 Ϯ 0.1 Ribosomal S6 kinase, 70 kDa, polypeptide 2 2.4 Ϯ 0.1 Neuronal Shc 2.3 Ϯ 0.4 Pim-1 oncogene 2.3 Ϯ 0.4 Epidermal growth factor receptor 1.9 Ϯ 0.1 IL-15 receptor, ␣ 2.0 Ϯ 0 Interferon ␥ receptor 1 2.5 Ϯ 0.3 Cell shape Keratin 18 2.0 Ϯ 0.1 Vinculin 2.2 Ϯ 0.1 Cadherin 5, type 2, VE-cadherin 2.0 Ϯ 0.2 Metabolism Cytochrome P450, 51 (lanosterol 14-␣-demethylase) 3.3 Ϯ 0.6 Hemoglobin, ␤ 4.3 Ϯ 0.3 Hemoglobin, ␥ A 2.3 Ϯ 0.1 Tumor suppressors Retinoic acid receptor responder (tazarotene induced) 3 3.2 Ϯ 0.3 Down-regulated in metastasis 2.5 Ϯ 0.2 Apoptosis Requiem (apoptosis) 1.9 Ϯ 0.2 Programmed cell death 5 1.9 Ϯ 0.1 Translation Eukaryotic translation termination factor 1 2.0 Ϯ 0.1 Transcription Sp3 2.9 Ϯ 0.2 Extracellular matrix L1 cell adhesion molecule 2.3 Ϯ 0.1 Collagen, type XIV, ␣ 1 2.1 Ϯ 0.1 Collagen, type I, ␣ 2 3.0 Ϯ 0.4 Proteolysis Matrix metalloproteinase 9 (MMP-9) 2.0 Ϯ 0.1 Proliferation/growth factors Connective tissue growth factor (CTGF) 2.7 Ϯ 0.4 Mitogen-activated protein kinase kinase kinase 8 2.1 Ϯ 0.3 Growth hormone receptor 2.1 Ϯ 0.2 Fibroblast growth factor-2 (FGF-2) 1.9 Ϯ 0.2 Other Nuclear receptor subfamily 1, group H, member 3 2.6 Ϯ 0.4 Down-regulated genes Metabolism Mitochondrial creatine kinase 0.31 Ϯ 0.02 Deoxycytidine kinase 0.42 Ϯ 0.04 Aldehyde dehydrogenase family 6, A1 0.29 Ϯ 0.01 Tumor suppressors IL-24, Suppression of tumorigenicity 16 (melanoma differentiation) 0.37 Ϯ 0.01 Extracellular matrix/cell adhesion Elastin 0.51 Ϯ 0.09 Collagen, type IX, ␣ 1 0.41 Ϯ 0.04 Transcription Polymerase (RNA) II (DNA directed) polypeptide K (7.0 kDa) 0.40 Ϯ 0.03 TATA box binding protein (TBP)-associated factor, RNA polymerase II, F, 55 kDa 0.53 Ϯ 0.06 Transcription elongation factor B (SIII), polypeptide 1 0.39 Ϯ 0.01 Translation Eukaryotic translation initiation factor 2B, subunit 2 (␤) 0.51 Ϯ 0.03 Cell migration D-dopachrome tautomerase 0.38 Ϯ 0.01 Cell shape Rho GTPase activating protein 1 0.49 Ϯ 0.04 Myosin, light polypeptide 6, alkali, smooth muscle and non-muscle 0.54 Ϯ 0.05 Apoptosis p75NTR-associated cell death executor (NGFRAP1) 0.48 Ϯ 0.02 Defender against cell death 1 (DAD1) 0.49 Ϯ 0.01 Proteolysis Matrix metalloproteinase 7 (MMP-7) 0.37 Ϯ 0.06 Cathepsin B 0.56 Ϯ 0.01 Signal transduction IL-1, ␤ 0.43 Ϯ 0.01 Vesicle transport Kinesin family member 5B 0.44 Ϯ 0.06 Other Prefoldin 4 0.31 Ϯ 0.01 Galactosylceramidase 0.42 Ϯ 0.04 * An asterisk denotes genes that were consistently up-regulated and down-regulated Ն1.8-fold but with standard errors of Ͼ20% of the average value (see Materials and Methods). All other gene expression level ratios listed had standard errors of Յ20% of the average value.

8203

Table 3 Confirmation of gene regulation by A138P p53 with real-time PCR expression level change due to A138P p53 was Ͻ70% of that attrib- Microarray Real-time PCR utable to WT p53 in only 7% of these common gene regulation events. CTGF 2.0 2.3 This suggests that most of the regulation events of A138P p53 are a Deoxycytidine kinase 0.42 0.54 combination of a small amount of DNA-binding–dependent activities Prefoldin 4 0.41 0.41 not compromised by the mutation and events not mediated by direct NOTE. Fold difference in expression level attributable to A138P p53/GFP versus GFP only. 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 clear antigen, c-fos, and MDR-1 (10, 33–35). The up-regulation of cyclin compromised by the mutation in the DNA-binding domain. Consistent D3 (Table 5) may be an acquired regulatory function that contributes with this, immunoprecipitation of p53 from lysates of HCT 116 p53 to the enhanced cell growth gain-of-function phenotype of R175H p53 Ϫ/Ϫ cells expressing A138P or WT p53, followed by two-dimen- (11, 35). sional gel electrophoresis, revealed the same species and abundance of As seen in Tables 1 and 4, for most of the genes altered in their proteins associated with both forms of p53 (data not shown). R175H expression level by A138P and WT p53, the degree of regulation was p53 did not retain enough WT transcriptional regulatory events to be not significantly compromised by the mutation. In fact, the fold analyzed in this respect.

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

a. Genes up-regulated Ն1.8-fold, 33 (0.5% of total) b. Genes down-regulated Ն1.8-fold (ratio of Յ0.56), 5 (0.1% of total) Ratio of gene expression level attributable to p53/GFP versus GFP only

List of genes altered in expression level by A138P p53 A138P WT Metabolism Hemoglobin, ␤ 9.5 Ϯ 0.6 4.3 Ϯ 0.3 Hemoglobin, ␥A 4.0 Ϯ 0.2 2.4 Ϯ 0 Ribonuclease H1 2.0 Ϯ 0.2 1.9 Ϯ 0.1 2-Hydroxyphytanoyl-CoA lyase 1.9 Ϯ 0.2 1.8 Ϯ 0.1 UDP-galactose transporter related 1.9 Ϯ 0.2 1.7 Ϯ 0.1 ATP synthase, Hϩ transporting, mitochondrial F0 complex, subunit c(subunit 9), isoform 1 1.9 Ϯ 0.1 1.7 Ϯ 0.1 Aldehyde dehydrogenase 1 family, member A3 1.8 Ϯ 0.2 1.7 Ϯ 0.2 Hormone related Relaxin 1 (H1) 3.6 Ϯ 0.1 1.7 Ϯ 0.1 Transcription Inhibitor of DNA binding 3, dominant negative helix-loop-helix protein 2.3 Ϯ 0.1 2.0 Ϯ 0.1 ELF3 1.8 Ϯ 0.1 1.8 Ϯ 0.1 Hypoxia-inducible factor 1, ␣ subunit (basic helix-loop-helix transcription factor) 2.0 Ϯ 0.3 1.5 Ϯ 0.2 Signal transduction Adenylyl cyclase-associated protein 2 2.1 Ϯ 0.1 1.2 Ϯ 0 Cell motility/shape Engulfment and cell motility 1 (ced-12 homolog, Caenorhabditis elegans) 2.0 Ϯ 0.1 1.6 Ϯ 0.1 Kinesin family member 20A 1.8 Ϯ 0.3 1.4 Ϯ 0.3 ARP2 actin-related protein 2 homolog (yeast) 1.8 Ϯ 0.1 1.2 Ϯ 0.1 Cofilin 2 (muscle) 0.56 Ϯ 0.04 0.59 Ϯ 0.03 Myosin, light polypeptide 2, regulatory, cardiac, slow 0.37 Ϯ 0.02 0.30 Ϯ 0.04 DNA repair Aprataxin 2.0 Ϯ 0.2 1.8 Ϯ 0.1 Translation Mitochondrial ribosomal protein L11 1.9 Ϯ 0.1 1.7 Ϯ 0.1 Proteolysis COP9 constitutive photomorphogenic homolog subunit 3 (Arabidopsis) 1.8 Ϯ 0.2 1.4 Ϯ 0.1 Proteasome (prosome, macropain) subunit, ␣ type, 6 1.8 Ϯ 0.1 1.6 Ϯ 0 Siah-interacting protein 1.8 Ϯ 0.2 1.5 Ϯ 0.1 Cell cycle/proliferation Protein phosphatase methylesterase-1 1.8 Ϯ 0.1 1.5 Ϯ 0 Other Hypothetical protein FLJ20721 2.6 Ϯ 0.2 2.3 Ϯ 0.2 Hypothetical protein DKFZp762E1312 2.1 Ϯ 0.2 1.9 Ϯ 0 Melanoma antigen AIM1 2.1 Ϯ 0.1 2.1 Ϯ 0.1 Protein disulfide isomerase-related 2.0 Ϯ 0.1 1.6 Ϯ 0.1 Ribophorin II 1.9 Ϯ 0.1 1.6 Ϯ 0.1 Similar to Saccharomyces cerevisiae RER1 1.9 Ϯ 0.2 1.6 Ϯ 0.2 Clone NA 4–14–03 141 1.9 Ϯ 0.1 1.6 Ϯ 0.1 Structure specific recognition protein 1 1.9 Ϯ 0 1.6 Ϯ 0 Hypothetical protein MGC2654 1.8 Ϯ 0.1 1.5 Ϯ 0 Karyopherin ␣2 (RAG cohort 1, importin ␣ 1) 1.8 Ϯ 0.1 1.6 Ϯ 0 DnaJ (Hsp40) homolog, subfamily B, member 11 1.8 Ϯ 0 1.5 Ϯ 0 Hypothetical protein PP1044 0.55 Ϯ 0.09 0.48 Ϯ 0.01 Hypothetical protein MGC1203 0.51 Ϯ 0.05 1.5 Ϯ 0.1 Palate, lung and nasal epithelium carcinoma associated 0.40 Ϯ 0.03 0.29 Ϯ 0.01 KIAA1961 protein 1.8 Ϯ 0 1.3 Ϯ 0.1 NOTE. Gain of function statistics. Percentage of genes listed above altered in expression level by A138P AND WT p53, 34. Percentage of genes listed above altered in expression level by A138P p53 ONLY, 66. Of the genes altered in expression level by A138P and WT p53, percentage of genes of which the fold expression level change attributable to A138P p53 is Ͻ70% of that attributable to WT p53, 0. * Microarray contains 6.9 ϫ 103 genes in total. Gene expression level ratios of Ն1.8 and Յ0.56 were considered to be significant. Values are listed Ϯ SEM. All gene expression level ratios listed had a SEM of Յ20% of the average value (see Materials and Methods). 8204

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

a. Genes up-regulated by Ն1.8-fold, 12 (0.2% of total) b. Genes down-regulated Ն1.8-fold (ratio of Յ0.56), 2 (0.03% of total) Ratio of gene expression level attributable to p53/GFP versus GFP only

List of genes altered in expression level by R175H p53 R175H WT Metabolism NADPH-dependent FMN and FAD containing oxidoreductase 1.9 Ϯ 0.3 1.6 Ϯ 0.2 2-hydroxyphytanoyl-CoA lyase 1.8 Ϯ 0.1 1.8 Ϯ 0.1 Hydroxyacyl-CoA dehydrogenase/3-ketoacyl- CoA thiolase/enoyl-CoA 1.8 Ϯ 0.1 1.2 Ϯ 0.1 hydratase (trifunctional protein), ␣ subunit Transcription Inhibitor of DNA binding 3, dominant negative helix-loop-helix protein 1.8 Ϯ 0.1 2.0 Ϯ 0.1 Hormone related Thyroid stimulating hormone receptor 1.8 Ϯ 0.3 1.3 Ϯ 0.2 Cell motility/shape Kinesin family member 20A 1.8 Ϯ 0.3 1.4 Ϯ 0.3 Translation Mitochondrial ribosomal protein L11 1.8 Ϯ 0 1.7 Ϯ 0.1 Mitochondrial ribosomal protein S24 1.8 Ϯ 0.2 1.1 Ϯ 0.1 Cell cycle Cyclin D3 1.9 Ϯ 0.2 1.1 Ϯ 0.1 Secreted frizzled-related protein 2 0.54 Ϯ 0.01 0.86 Ϯ 0.05 Other Hypothetical protein FLJ20721 2.2 Ϯ 0.2 2.3 Ϯ 0.2 Hypothetical protein BC011880 1.9 Ϯ 0.1 1.4 Ϯ 0 Hypothetical protein MGC2603 1.9 Ϯ 0.2 1.5 Ϯ 0.2 Palate, lung and nasal epithelium carcinoma associated 0.55 Ϯ 0.02 0.29 Ϯ 0.01 NOTE. Gain of function statistics. Percentage of genes listed above altered in expression level by R175H AND WT p53, 29. Percentage of genes listed above altered in expression level by R175H p53 ONLY, 71. Of the genes altered in expression level by R175H and WT p53, percentage of genes of which the fold expression level change attributable to R175H p53 is Ͻ70% of that attributable to WT p53, 25. * Microarray contains 6.9 ϫ 103 genes in total. Gene expression level ratios of Ն1.8 and Յ0.56 were considered to be significant. Values are listed Ϯ SEM. All gene expression level ratios listed had a SEM of Յ20% of the average value (see Materials and Methods).

Wild-Type p53 Altered the Expression Level of a Large Per- nogenesis. Lastly, the expression level of mucin 9 (oviductin) was centage of Genes Studied. As shown in Table 2, WT p53 altered the increased Ͼ4-fold by WT p53. Mucin 9 is maximally expressed expression level of many genes (see supplemental Tables 1 and 2 for during ovulation (43) and is thought to mediate gamete interaction full list). In fact, 10 and 9% of the genes studied on human arrays 1 (44). Interestingly, p53 is also highly expressed during ovulation (45, and 2, respectively, exhibited altered expression levels due to WT 46) and may be a critical regulator of mucin 9 production during this p53. A significantly greater proportion of the genes regulated by WT stage of the reproduction process. 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 DISCUSSION genes reported in Table 2 have been analyzed by microarray for p53 regulation previously. Many of the gene regulation events were in This report provides data elucidating the mechanism of mutant p53 accordance with the role of p53 as a potent mediator of growth arrest gain-of-function phenotypes by comparing the effect of the expression and apoptosis. Several of the genes encoding proteins involved in the of mutant and WT p53 on the expression levels of many genes by basic cellular transcription machinery were significantly repressed by microarray analysis. The most obvious difference between the mu- WT p53, including RNA polymerase II polypeptide K (7.0 kDa) and tants, A138P and R175H, and WT p53 is the loss of the ability to transcription elongation factor B (SIII), polypeptide 1. In addition, the regulate the expression of most of the genes that the WT protein expression levels of eukaryotic translation initiation factor 2B, sub- influences. In fact, A138P and R175H p53 retained Ͻ5% of the unit 2 (␤) and eukaryotic translation termination factor 1 were re- regulatory activities of WT p53 with respect to the genes studied here. duced and increased, respectively, by WT p53. Interestingly, it was Obviously, the A138P and R175H mutations in the DNA-binding found that the transcription factor Sp3 gene was up-regulated by domain of p53 severely compromised its transcriptional regulatory almost 3-fold by WT p53. Sp3 is a potent up-regulator of the p21 activities. The ability of WT p53 to alter the expression level of ϳ10% (WAF-1) gene (36–39) and may be involved in mediating the crucial of the genes on the arrays studied here underscores the growing activation of this gene by p53. Regarding its apoptotic functions, WT number of reports indicating that it is involved in the regulation of a p53 reduced the expression level of defender against cell death 1 vast number of genes involved in many different physiologic pro- (DAD1) and up-regulated requiem and programmed cell death 5. cesses. As mentioned above, several gain-of-function cellular pheno- WT p53 also regulated the expression of two matrix metallopro- types have been found for various p53 mutants. Typically, a given teinases, MMP-7 and -9. MMP-7, down-regulated, and MMP-9, up- gain-of-function phenotype is produced by several but not all mutant regulated, are capable of degrading several extracellular matrix pro- p53s. The cause of these gain-of-function phenotypes may be related teins, and their expression has been associated with high-grade to the ability of mutant p53s to regulate the expression of genes that metastatic tumor cells (40–42). It is uncertain what specific effect WT p53 does not affect or acts on in the opposite manner. For WT p53 has on metastasis because it can up- and down-regulate genes example, it is possible that the increase in the expression of pro- that can promote or repress this process. It is thought that the net proliferative genes such as c-myc and EGFR by certain mutant p53s regulation of such metastasis-related proteins by p53, which is likely contributes to gain-of-function phenotypes such as increased rate of to be cell type dependent, determines its role in this aspect of carci- cell division and tumorigenesis. However, it is difficult to attribute 8205

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2004 American Association for Cancer Research. MUTANT AND WILD-TYPE p53 EFFECT ON GENE EXPRESSION these gain-of-function phenotypes to a few of these mutant-specific Considering that the A138P and R175H mutations are in the DNA- gene regulations alone. This is because p53 mutants also retain vari- binding domain of p53, it is likely that the large loss of regulatory ous amounts of WT p53 transcriptional regulatory events in the events exhibited by these mutants compared with WT p53 represent context of a loss of a substantial number of other such functions DNA-binding–dependent activation and repression functions. In fact, (discussed in 27). This imbalance of remaining WT functions may it was shown that R175H p53 is not able to bind the p53 consensus also cause gain-of-function phenotypes. In the experimental system DNA-binding sequence in the gadd45 promoter (9). A substantial described here, the effect of A138P and R175H p53 on the expression portion of the remaining WT regulatory events is likely due to the level of a large unfocused set of genes was determined. It was found binding of transcription cofactors through the intact NH2- (52, 53) and that A138P p53 possessed very few true novel transcriptional regu- COOH-terminal domains (54), independent of DNA binding. Sub- latory activities compared with WT p53. In other words, most of the stantiating this, it was observed that only 7% of the regulatory gene regulation events produced by A138P p53 were also properties activities possessed by A138P p53 and none of the regulatory events of WT p53. Therefore, we propose that gain-of-function phenotypes of R175H that were also produced by WT p53 were significantly possessed by A138P p53 are largely because of an imbalance of WT decreased in terms of the fold expression level change compared with transcriptional regulatory functions caused by a small amount of the corresponding WT-induced activity. Previous studies have indi- remaining WT activities existing in the absence of many other coun- cated that p53 mutants can transactivate in the absence of direct DNA terbalancing functions. Contributing to the ability of these remaining binding. In these cases, the ability of the mutants to be recruited to the regulatory activities to produce gain-of-function phenotypes is that promoters may be mediated by their interaction with Sp1 (26, 55). because mutant p53s tend to be constitutively expressed at much DNA-binding–independent transcriptional repression activities of p53 higher levels than WT p53 under nonstressed conditions, the robust- have also been described (reviewed in ref. 56). These events seem to ness of the expression level changes caused by these functions would be mediated by p53 binding and inhibiting transcription activators likely be significantly greater in cells harboring mutant versus WT such as AP-1 (57), C/EBP (52), and TFIIIB (58). p53. The high levels of mutant p53 usually found in cells are thought The data presented here indicate that novel acquired regulatory to be because of the lack of up-regulation of hdm2, which mediates functions as well as retained WT activities existing in the absence of the proteolysis of p53, normally possessed by the WT protein (47). An many counterbalancing activities are both likely to contribute to example of a regulation event that is possessed by A138P and WT p53 gain-of-function phenotypes exhibited by mutant p53s. However, p53 and could play a role in an enhanced metastatic phenotype is the mutants are likely to vary in the extent to which each mechanism increase in the expression of CTGF (Tables 1 and 2). For WT p53, this contributes to these phenotypes. Perhaps future studies will expand on up-regulation may produce a minor effect on metastasis because of its this work by examining the effect of other p53 mutants on the antimetastatic activities such as the repression of genes like basic expression of large sets of genes and begin to correlate the retention fibroblast growth factor (23) and MMP-7 (Table 2) and the activation and loss of WT transcriptional regulatory events and the acquisition of of Drg-1 (48). However, many of the antimetastatic activities of WT novel activities with particular gain-of-function phenotypes. Because p53 are likely to be absent in mutant p53s and, therefore, the up- p53 DNA-binding domain mutants retain various degrees of DNA- regulation of CTGF may contribute significantly to increased metas- binding affinity (9), it would be expected that each mutant would be tasis. distinct in terms of the number of regulatory functions it retains The most obvious difference between the two p53 mutants studied because of direct transcriptional regulation. In addition, because each here is that R175H retained less WT transcriptional regulatory events mutation can potentially produce distinct effects on the structure of than A138P. In addition, R175H p53 possessed an approximately the DNA-binding domain, p53 mutants are likely to exhibit differ- equal number of retained WT regulatory activities and acquired novel ences in acquired novel transcriptional regulatory activities. However, functions. These data suggest that novel regulatory functions as well it is likely that remaining WT-transcriptional regulatory events medi- as a small amount of retained WT functions existing in the absence of ated by binding transcription cofactors via their uncompromised NH2- balancing activities both significantly contribute to the gain-of-func- and COOH-terminal domains would be common to many of these tion phenotypes exhibited by R175H p53. It is expected that both types of mutants. For instance, as shown here with human array 2, five mechanisms are potently enhanced by the key loss of transcriptional of the six regulatory functions shared by A138P and R175H p53 are control over certain genes that regulate cell division and differentia- retained WT functions (Tables 4 and 5). In other words, for this gene tion often exhibited by p53 mutants (49, 50). The expression of set, the two mutants only share one acquired novel regulatory activity. R175H p53 is known to produce several gain-of-function phenotypes We can hypothesize that gain-of-function phenotypes often shared by including increased growth potential (11, 33), enhanced tumorigenesis p53 mutants are likely due to unbalanced retained WT functions via and metastasis (11), and drug resistance (19, 51). From the work of interaction with transcription cofactors. Conversely, gain-of-function others and that presented here, several examples of novel acquired phenotypes less commonly exhibited by mutant p53s are more likely transcriptional regulatory activities as well as remaining WT functions due to a combination of acquired novel regulatory events and an have been observed that may contribute to these gain-of-function imbalance of retained WT functions due to various losses of DNA- phenotypes. The acquired ability of R175H p53 to activate the ex- binding–dependent regulation activities. However, future studies will pression of cyclin D3, proliferating cell nuclear antigen, and c-fos be needed to test this hypothesis. (33) may contribute to enhanced proliferation, whereas the novel up-regulation of MDR-1 may cause increased drug resistance (10, 34, ACKNOWLEDGMENTS 35, 51). In addition, the retained ability of R175H p53 to activate transcription of EGFR (26) may also contribute to increased cell The authors acknowledge the adroit technical assistance of Joe Chrest, growth. It can also be noted that A138P and R175H p53 share six Kevin Becker, William Wood III, and Diane Teichberg (Gerontology Research common transcriptional regulatory events (Tables 4 and 5). Of these Center, National Institute on Aging). events, five of them represent remaining WT regulatory activities. These data suggest that transcriptional regulatory activities shared by REFERENCES mutant p53s are more likely to be retained WT functions. However, 1. Oren M. 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