elevated expression of ribosomal protein genes l37, rpp-1...

Elevated Expression of Ribosomal Protein Genes L37, RPP-1, and S2 inthe Presence of Mutant p531

W. Troy Loging 2 and David Reisman3

Department of Biological Sciences, University of South Carolina, Columbia,South Carolina 29208

AbstractThe wild-type p53 protein is a DNA-binding transcriptionfactor that activates genes such asp21, MDM2, GADD45,and Bax that are required for the regulation of cell cycleprogression or apoptosis in response to DNA damage.Mutant forms of p53, which are transforming oncogenesand are expressed at high levels in tumor cells, generallyhave a reduced binding affinity for the consensus DNAsequence. Interestingly, some p53 mutants that are nolonger effective at binding to the consensus DNAsequence and transactivating promoters containing thistarget site have acquired the ability to transform cells inculture, in part through their ability to transactivatepromoters of a number of genes that are not targets ofthe wild-type protein. Certain p53 mutants are thereforeconsidered to be gain-of-function mutants and appear tobe promoting proliferation or transforming cells throughtheir ability to alter the expression of novel sets of genes.Our goal is to identify genes that have altered expressionin the presence of a specific mutant p53 (Arg to Trpmutation at codon 248) protein. Through examiningdifferential gene expression in cells devoid of p53expression and in cells that express high levels of mutantp53 protein, we have identified three ribosomal proteingenes that have elevated expression in response to mutantp53. Consistent with these findings, the overexpression ofa number of ribosomal protein genes in human tumorsand evidence for their contribution to oncogenictransformation have been reported previously, althoughthe mechanism leading to this overexpression hasremained elusive. We show results that indicate thatexpression of these specific ribosomal protein genes isincreased in the presence of the R248W p53 mutant,which provides a mechanism for their overexpression inhuman tumors.

IntroductionThe wt4 p53gene encodes a DNA-binding transcription factorthat functions as a negative growth regulator (1, 2) that controlsentry into the G1-S and G2-M phases of the cell cycle. Condi-tions of cell stress, such as DNA damage and hypoxia (3, 4),lead to p53 induction. p53 transactivates a number of differentgenes and, depending on which genes are induced, the expres-sion of p53 can have a number of different effects on the cellcycle. For example, G1 arrest is thought to occur in part throughthe induction of p21, DNA damage repair is thought to occurthrough the induction of GADD45, and apoptosis is thought tooccur through the induction of Bax (5).

Mutations in the DNA-binding domain of p53 have beenfound in more than 60% of all human tumors examined, makingmutation in p53 the most commonly observed mutation inhuman cancers. One of the most commonly occurring muta-tions in p53 is the Arg to Trp mutation at codon 248 (R248W),and recent findings using X-ray crystallography suggest thatthis, as well as other mutations, affects DNA binding withoutaffecting the overall structure of the protein. The positivelycharged side chain of Arg248on the wt protein interacts with thenegatively charged phosphodiester backbone of the target DNAsequence. Other amino acids, such as Arg175 and Asp281, pro-vide conformational stability through interactions with otheramino acids on p53 (6). Therefore, p53 mutants can be placedinto two general categories: (a) those affecting DNA contact;and (b) those affecting the the conformation required for DNAcontact to take place.

Recent findings have suggested that in some cases, theprocess by which mutant p53 leads to cell transformation isthrough a gain of function. It appears that loss or inactivation ofp53 often may not necessarily be sufficient for transformation,because the majority of common tumor types are found tocontain missense mutations rather than premature terminationsor deletions in the gene (7). The presence of mutant p53 maycoincide with the expression of novel sets of downstream genes(8). Expression of a number of mutant p53 proteins has in factbeen shown to alter the transcription of genes such as themultidrug resistance gene (MDR1; Ref. 9), c-myc(10), the HIVtype 1 long terminal repeat (11, 12), and the human epidermalgrowth factor receptor (13). We hypothesize that the gain-of-function activity of mutant p53 seen in a variety of tumor cellsexpressing mutant p53 is due to its ability to alter the expressionof genes that may contribute to increased proliferation or to thetransformed phenotype.

Because altered gene expression is known to occur inpresence of mutant p53, and these changes may play a role inthe process of oncogenic transformation, it is important toidentify genes that may be targets of mutant p53. We therefore

Received 5/10/99; revised 8/18/99; accepted 9/5/99.The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby markedadvertisementinaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.1 Supported by a University of South Carolina Research and Productive Schol-arship Award, NIH, and American Cancer Society.2 Present address: Duke University Medical Center, Department of Pathology,Room 189 MSRB, Duke University, Durham, NC 27710.3 To whom requests for reprints should be addressed. Phone: (803) 777-8108;Fax: (803) 777-4002; E-mail: [email protected].

4 The abbreviations used are: wt, wild-type; CMV, cytomegalovirus; RPP-1,ribosomal phosphoprotein P1; FBS, fetal bovine serum.

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sought to develop a system in which genes that are expressed inthe presence of mutant p53 could be isolated. Through subtrac-tive screening of a cDNA library derived from cells expressingthe R248W p53 mutant, we observed elevated expression ofseveral mRNAs transcripts. Three cDNAs that we have isolatedcontain high homology to mRNAs encoding ribosomal proteinsL37, RPP-1, and S2. Interestingly, a number of recent studieshave shown that these ribosomal protein genes are expressed athigh levels in colon tumors. Our results indicate that the ex-pression of these specific ribosomal protein genes is increasedin the presence of the R248W p53 mutant and thus may providea mechanism for their overexpression in human tumors.

Materials and MethodsCell Culture and Transfections. 10(3) cells (provided by Dr.Arnold Levine, Princeton, University, Princeton, NJ) are mu-rine fibroblasts that are devoid of endogenous p53 expressiondue to point mutations in the gene that introduce a prematuretranslation stop codon at amino acid 173. 10(3) cells andderivatives of 10(3) expressing mutant p53 were maintained inDMEM containing 10% FBS, 2 mM glutamine, 100mg/mlstreptomycin, and 100mg/ml penicillin. All Burkitt’s lympho-ma-derived cell lines and early-passage EBV-nontransformedlymphoblastoid cell lines (LCLs; provided by Dr. Bill Sugden,University of Wisconsin, Madison, WI) were grown in RPMI1640 supplemented with 10% FBS, 2 mM glutamine, 100mg/mlstreptomycin, and 100mg/ml penicillin.

The human colon carcinoma-derived R248W p53 mutantwas placed under the control of the CMV promoter on aplasmid that also expresses resistance to the drug G418. Cellsat 30–40% confluence were transfected with this expressionvector by calcium phosphate coprecipitation. Twenty-four hafter transfection, the cells were shifted to media containing500mg/ml G418. After approximately 2–3 weeks of selection,viable clones of cells were picked and expanded into cell linesby continual growth in selective media. For chloramphenicolacetyltransferase assays, extracts were prepared at 48 h aftertransfection and assayed for chloramphenicol acetyltransferaseactivity using equivalent amounts of protein in a 1-h reaction at37°C in the presence of acetyl-CoA and [14C]chloramphenicol.The products were separated by TLC and subjected to autora-diography.Western Blot Analysis/Western Transfer. Cells werewashed twice in PBS, and total cell extracts were prepared byresuspending the pellet in 100–150ml of sample buffer [70 mMTris-HCl (pH 6.8), 10% glycerol, 5%b-mercaptoethanol, 3%SDS, and 0.01% bromphenol blue]. Equal amounts of proteinfrom each sample were separated by electrophoresis and trans-ferred to Hybond-enhanced chemiluminescence nitrocellulosemembrane (Amersham). Human p53 was detected by incuba-tion of the membrane with anti-p53 pAb421 monoclonal anti-body for 1 h, followed by washing and subsequent incubationwith horseradish peroxidase-conjugated sheep antimouse im-munoglobulin. Proteins were visualized by chemiluminescence(Amersham) and exposure to Kodak X-ray film from 15 s to1 h. The molecular weights were determined by prestainedstandards (Bio-Rad). Equivalent protein loading was verifiedby staining the gel with Coomassie Blue after transfer.cDNA Library Formation and cDNA Recovery. Total RNAwas prepared from CM248.2 cell pellets by acid-guanidiumthiocyanate/phenol-chloroform extraction using TRI-Reagent(Molecular Research Center, Inc.). mRNA was isolated byoligo(dT) cellulose column chromatography. cDNA was gen-erated by reverse transcription using an oligo(dT) primer and

Moloney murine leukemia virus reverse transcriptase (LifeTechnologies, Inc.). After ligation ofEcoRI adapters, thecDNA was ligated into a Lambda Zap vector (Stratagene) toobtain 6.03 105 independent cDNA clones. The Lambda ZAPvector used to created the cDNA library contained T3/T7primer regions flanking the cDNA cloning site. Therefore, weexploited this feature to isolate the cDNA inserts by PCR usingT3/T7 primers. This allowed for amplification of the insertsfrom the vector and their subsequent purification by agarose gelelectrophoresis.Subtractive Hybridization. cDNA probes enriched forCM248.2-specific sequences were obtained through subtractivehybridization. Subtracted cDNA probes were obtained by pho-tobiotinylation of 10 mg of polyadenylated mRNA isolatedfrom the p53-null cell line 10(3). Onemg of single-strandedcDNA generated from polyadenylated mRNA and isolatedfrom the mutant p53-expressing cells, CM248.2, was hybrid-ized to 10mg of biotinylated 10(3) mRNA for 24 h at 65°C. ThecDNA-mRNA hybrid was eliminated after the addition ofstreptavidin by phenol-chloroform extraction. The remainingcDNA, calculated to be 4–5% of the initial input mRNA, waslabeled witha-32P by random priming.Northern Blot Analysis/Northern Transfer Analysis. TotalRNA was prepared from cell pellets by acid-guanidium thio-cyanate/phenol-chloroform extraction using TRI-Reagent (Mo-lecular Research Center, Inc.). Tenmg of RNA were denaturedwith formaldehyde-formamide and separated in a 1% agarose/formaldehyde gel. RNA was then transferred to BioTrace NTmembrane (Gelman Sciences). The blot was hybridized with[a-32P]dATP-labeled probes. After multiple washings at 55°Cwith 23 saline-sodium phosphate-EDTA, signals were detectedusing a Storm 860 PhosphorImager (General Dynamics).

ResultsWe sought to isolate genes that are overexpressed in thepresence of the R248W p53 mutant through subtractivescreening of a cDNA library generated from cells expressingthis p53 mutant. The parental cell line that was used in theseexperiments is an immortalized, p53-null, murine fibroblastcell line known as 10(3). The 10(3) cells are ideally suitedfor these experiments because, although they are immortal-ized, they do not express a transformed phenotype (14). Toestablish derivatives of the 10(3) cells expressing mutantp53, we carried out stable transfections with a mammalianexpression vector containing p53 with an Arg to Trp muta-tion at codon 248 expressed from the CMV promoter. Aftertransfection and selection for G418 resistance, 32 G418-resistant clones were obtained. The p53 expression in thesecells was assayed by Western blot analysis using anti-p53antibodies. Of the 32 clones generated, 12 were found toexpress mutant p53 (Inset, Fig. 1). Three of the mutantp53-expressing clones, labeled CM248.2, CM248.6, andCM248.9, were used in growth assays along with the paren-tal 10(3) line. All three p53-expressing cell lines showed aconsiderable enhancement in growth rates when comparedwith 10(3). The doubling time of the 10(3) cell line wasfound to be approximately 36 h at logarithmic growth ascompared with 18 h for CM248.2 and CM248.9 clones (Fig.1). The CMV248.6 cells had an intermediate doubling timeof approximately 24 h. The number of cells at saturationdensity was found to be higher in the CM248.2 and CM248.9clones than in the 10(3) cell line. These results indicate that,consistent with previously reported results, the 10(3) cells

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expressing high levels of mutant p53 have increased growthpotential.

The ability of mutant but not wt p53 to alter gene expres-sion of the multidrug resistance gene (MDR1) represents a gain-of-function activity that appears to be essential for transforma-tion by certain mutants of p53. Therefore, to determine whetherthe mutant p53 expressed in the CM248.2 cells coincided withexpression of theMDR1 gene, the CM248.2 cell line wasassayed forMDR1expression with the pMDR-CAT reporter. Incells lacking p53 [parental 10(3) cells], there was a very lowlevel of MDR1 expression. In contrast, in the CM248.2 cells,which express the mutant p53 protein, transcription of theMDR1 gene was enhanced approximately 5–7-fold (data notshown), indicating that these cells have altered gene expressionattributable to the mutant p53-dependent gain-of-functionactivity.

Having established altered gene expression in cell linesthat express the R248W mutant p53 allele, we screened forother cellular genes whose expression may be altered in thepresence of this mutant. To do this, we prepared a cDNA libraryin bacteriophage lambda having approximately 600,000 inde-pendent cDNA clones from mRNA derived from the CM248.2cell line. We used this cell line because it was found to expressthe highest levels of mutant p53 protein and demonstrated agrowth advantage over the parental 10(3) cells. This cDNAlibrary, which we predicted would contain cDNA clones rep-resenting genes induced in the presence of mutant p53, wasthen screened by hybridization to a subtracted cDNA probeenriched for cDNAs specific for the CM248.2 cells. This sub-tracted cDNA probe was prepared by generating single-strandcDNA from the CM248.2 cells and then hybridizing this cDNAto an excess ofin vitro biotinylated mRNA from parentalp53-negative 10(3) cells. The hybrids were treated with strepta-vidin and removed by phenol-chloroform extraction. By thismethod, we routinely eliminated approximately 95% of thestarting cDNA material that represents transcripts common toboth cell lines. The remaining single-stranded cDNA moleculestherefore represent a population that is highly enriched forcDNAs that are unique to the cells expressing the p53 mutant.This material was labeled and used as a probe to screen thecDNA library. Twenty individual plaques that showed hybrid-ization were picked and plaque-purified. During the final roundof plaque purification, duplicate filters were probed with la-beled cDNA derived from 10(3) and CM248.2 cells. Only thosecandidates that showed elevated levels of hybridization to

cDNA derived from CM248.2 cells were picked for furtheranalysis.

The plaque-purified cDNA clones were isolated by PCR,labeled with32P, and used to probe Northern blots containingRNA isolated from the parental 10(3) cells and the p53-expressing CM248.2 cells. Three of the cDNA clones isolatedshowed up to 5-fold elevated expression in CM248.2 cells (Fig.2, A andC). These clones, labeled Bri4, Bri5, and Bri8, weresubcloned, sequenced, and compared to known sequenceswithin the Genetics Computer Group (GCG) databases. Bri4was found to be close to 100% homologous to the acidic RPP-1mRNA from mouse (accession number U29402), Bri5 wasfound to be homologous to ribosomal protein L37 mRNA fromrat (accession number S79981), and Bri8 was found to behomologous to ribosomal protein S2 mRNA from rat (acces-sion number X57432). Interestingly, all three of these riboso-mal protein genes have been shown in previous studies to beoverexpressed in transformed cells derived from human tumorbiopsies (15–18). As a control to ask whether the elevatedexpression of these three genes was due to a general elevatedexpression of numerous ribosomal protein genes, we testedwhether the gene encoding the large 60S ribosomal subunitprotein L24 was also expressed at an elevated level in CM248.2cells. As shown in Fig. 2B, L24 mRNA was expressed atapproximately equal levels in 10(3) and CM248.2 cells. Theseresults indicate that the elevated expression of RPP-1, L37, andS2 is not due to an overall general increase in the expression ofribosomal protein genes.

To test whether RPP-1, L37, and S2 genes are expressedat elevated levels in cells derived from human tumors thatexpress mutant p53, we carried out Northern transfer analysison RNA derived from a small panel of immortalized nontu-morigenic human B-cell lines and Burkitt’s lymphoma celllines. Our previous work has determined that mutant p53 isexpressed at elevated levels in a number of Burkitt’s lymphomacell lines and that the elevatedp53 gene expression is due inpart to elevated transcription of the mutantp53 gene (19). Asshown in Fig. 3, all three mRNAs were in fact present atelevated levels in the Burkitt’s lymphoma cell lines (Namalwaand BL-30) when compared with nontransformed EBV-immor-talized B-cell lines (LCL-3 and LCL-4). These results areconsistent with the results presented above that the RPP-1, L37,and S2 genes have increased gene expression in the presence ofmutant p53 protein.

Fig. 1. Growth curve of 10(3), CM248.2, CM248.6, andCM248.9 cells in 10% FBS. Cells were plated at 13 106

and harvested on the days indicated. Cells were allowed togrow to confluence.Inset, Western blot analysis showingthe expression of mutant p53 in the murine 10(3) cell lineand in cell lines derived by the introduction of the CMV-derived vector expressing mutant p53. The cell lines aredesignated with the prefix CM248. The anti-p53 antibodypAb421 was used to detect p53 protein.

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DiscussionWe have initiated a search for genes that are activated by anoncogenic form of the p53 tumor suppressor. Mutant p53clearly contributes to the transformed phenotype because it cantransform primary rat embryo fibroblasts in cooperation withthe activatedras oncogene (20–22) and because it is tumori-genic in transgenic mice when expressed at high levels (23).However, this transforming activity in tissue culture assays maybe due to the ability of mutant p53 to function as atrans-dominant negative repressor of wt p53 functions (24). A gain oftransforming function by mutant p53, on the other hand, hasbeen observed by introducing mutant p53 into immortalizedcell lines lacking p53. A number of groups have shown that theintroduction of mutant p53 into p53-negative immortalizedcells can increase their growth rates in culture and convertedthem into cells that are tumorigenicin vivo (14, 25). Oneparticular p53 mutant at codon 172 can cooperate with theneu/ERBB2 oncogene to promote mammary tumorigenesis intransgenic mice (26). These findings indicate that at least someforms of mutant p53 acquire novel functions that may contrib-ute to oncogenic transformation.

The ability of mutant p53 to enter the nucleus and trans-activate gene expression has been shown to be required for its

transforming activity. Missense mutations are rarely if everobserved in either the NH2-terminal transactivation domain ofthe protein or the nuclear localization signals. In fact, mutationsin the nuclear localization signals destroy the transformingactivity of mutant p53 (27). Here we report that three ribosomalprotein genes, L37, RPP-1, and S2, are expressed at elevatedlevels in cells expressing the R248W p53 allele. The expressionof these mRNAs was shown to be elevated 3–5-fold in com-parison to the p53-null parental cell line. Furthermore, all threewere found to be expressed at elevated levels in Burkitt’slymphoma cell lines overexpressing mutant p53. We are now inthe process of determining the mechanism of induction of thesegenes by p53.

Interestingly, this is not the first report that these riboso-mal protein genes are overexpressed in human tumors. L37overexpression was first identified in colon carcinomas (15), atumor type in which there is a high frequency of expression ofthe mutant (R248W) p53. In the same study, the acidic ribo-somal phosphoprotein P0, a gene having high homology to theacidic RPP-1, was found to be overexpressed predominantly inboth colonic and hepatocellular carcinomas. Furthermore, usingthe newly described SAGE technique to examine gene expres-sion in human colon tumors, Vogelstein and colleagues found

Fig. 2. A, Northern transfer analy-sis showing elevated expression inCM248.2 cells of mRNAs identifiedthrough differential screening. ThecDNA inserts were PCR-amplified,labeled, and used to probe transferscontaining 10(3) and CM248.2RNA. cDNA clones designated asBri4, Bri5, and Bri8 are shown.Mouseb-actin was used as a controlto test for equal loading.B, to testwhether or not increased ribosomalgene expression was a general phe-nomenon, ribosomal L24 cDNAwas used as a probe on blots con-taining 10(3) and CM248.2 totalRNA. L24 mRNA was expressed atapproximately equal levels.C,graphical representation of L37, S2,RPP-1, and L24 expression in 10(3)and CM248.2 cells. Quantificationof the hybridization signals was per-formed on a Storm 860 PhosphorIm-ager. Data are presented as expres-sion relative to the level of the signalobtained withb-actin in 10(3) cells.h, 10(3) cells.■, CM248.2.




the L37 and RPP-1 genes to be among 20 transcripts showingthe greatest increase in expression when compared to normaltissue (18). Our results indicate that the expression of thesespecific ribosomal protein genes is increased in the presence ofthe Arg248 p53 mutant and may thus provide a mechanism fortheir overexpression in human tumors. Because a large propor-tion of colon tumors express R248W mutant p53, we proposethat altered expression of these particular ribosomal genesoccurs, either directly or indirectly, in the presence of mutantp53.

In addition to serving as biomarkers for tumorgenesis, thehigh level of expression of these genes may be linked to thetransformed phenotype, because other specific ribosomal geneshave been found to be connected to oncogenic transformation.The involvement of ribosomal protein genes in oncogenic trans-formation is supported, for example, by the finding that anactivated oncogene isolated from a human breast cancer con-sisted of a fusion between thetrk proto-oncogene and theribosomal protein L7 gene (28). To date, it is unclear how theseribosomal genes contribute to the transformation phenotype.Because some ribosomal proteins appear to have multiple ac-tivities in the cell, their role in transformation may be quitecomplex. Examples can be seen in reports showing that certainribosomal proteins inDrosophilaexhibit DNA damage repairactivities (29, 30). A connection between ribosomal genes andp53 has previously been shown to exist, because p53 was foundto be covalently linked to 5.8S rRNA (31) and to be part of aribonucleoprotein complex consisting of p53, 5S rRNA, andribosomal protein L5 (32, 33). Furthermore, wt p53 has alsobeen shown to regulate the levels of some ribosomal proteingenes, because the expression of certain ribosomal proteins isdecreased in the presence of wt p53 (34). Thus, by means of anumber of different approaches, p53 has been shown to belinked to ribosomal components and possibly to the regulationof ribosome activity. Our findings that mutant p53 may be

linked to ribosomal protein gene expression are intriguing andsuggest that p53 may be linked to ribosome activity or regula-tion in human tumors through the modulation of a subset ofribosome-related genes. The mechanism by which mutant p53contributes to activation of ribosomal protein genes is currentlybeing addressed.

AcknowledgmentsWe thank Drs. Bob Lawther, Mike Felder, and Mike Dewey for comments on themanuscript and Ginger Foley and Michael Brown for technical support.

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Fig. 3. Northern transfer analysis of ribosomal gene expression in humanlymphoid cell lines. Expression of L37, RPP-1, and S2 transcripts was tested inB-lymphoid cell lines expressing either wt p53 (EBV-immortalized LCL3 orLCL4) or mutant p53 (Burkitt’s lymphomas: Namalwa; codon 248; BL-30, codon246). Quantification of the hybridization signals was performed on a Storm 860PhosphorImager. The results showed an elevated expression of these genes in theBurkitt’s lymphoma derived cell lines. Glyceraldehyde-3-phosphate dehydrogen-ase (GAPDH) was used as a control to test for equal loading.

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1999;8:1011-1016. Cancer Epidemiol Biomarkers Prev W. Troy Loging and David Reisman and S2 in the Presence of Mutant p53Elevated Expression of Ribosomal Protein Genes L37, RPP-1,

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