Parathyroid Hormone-Related Protein Gene Expression in Human Squamous Carcinoma Cells Is Repressed by Mutant Isoforms of P53' John Foley,2John J

ICANCER RESEARCH56. 4056-4062. September 1. 19961 Parathyroid Hormone-related Protein Gene Expression in Human Squamous Carcinoma Cells Is Repressed by Mutant Isoforms of p53' John Foley,2John J. Wysolmerski,ArthurE. Broadus,and WilliamM. Philbrick Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06520

ABSTRACT Accordingly, squamous cell tumors (commonly of the skin, lung, head, neck, and urogenital tract) have been uniformly reported to be Parathyroid hormone-related protein (PTHrP) is a normal secretory the most common HHM-associated tumors (10). Despite the relatively product of a variety of squamous epithelia, including epidermal kerati high eutopic levels of PTHrP gene expression in the tissues that give nocytes. Only a subset of squamous carcinomas, however, express the gene at levels sufficient to cause humoral hypercalcemia. In the present study, rise to squamous carcinomas, only a minority of these tumors ulti comparison of PTHrP expression levels with p53 functional status In a mately cause HHM (1 1). At this point, it is unclear why some tumors series of squamous carcinoma lines has revealed an association between produce high levels of PTHrP and cause HHM and others do not. expression ofspeciflc mutant isoforms ofp53 and very low levels of PTHrP In all examples of malignant cells studied to date, PIHrP appears mRNA. Evaluation of p53 isoforms with mutations in codons 248 and 273 to be constitutively secreted, so that its rate of secretion is a direct showed them to be capable of repressing PTHrP gene expression in a function of its rate of synthesis (12). Furthermore, the capacity of a high-expressing, p53-negative squamous line by approximately 50%. Con given tumor to cause HHM has been found to correlate with the level versely, inactivation of an endogenous mutant p53 with E1B proteins of PTHrP gene expression (13). Analysis of a number of squamous resulted in an Increase in PTHrP expression in a low-expressing cell line. tumors and tumor-derived squamous carcinoma cell lines has revealed Subsequent analysis of promoter-specific PTHrP transcripts in a p53- negative squamous line transfected with mutant p53 isoforms suggested that PTHrP mRNA levels vary widely and that only those tumors with that down-regulation occurred primarily at the two TATA-based promot the highest levels of PTHrP gene expression are capable of inducing eN. Direct testing of a murine PTHrP reporter construct in transient hypercalcemia in athymic mice (14). Thus, the question of why only transfection assays confirmed the capacity of the 248 and 273 mutants to a fraction of squamous cell tumors cause hypercalcemia reduces repress this TATA-based promoter, although only about halfas effectively fundamentally to a question of the mechanisms governing PTHrP as wild-type p53. expression in malignant cells. Mutations in the tumor suppressor, p53, and disruption of p53 function by viral oncogenes are very common INTRODUCTION findings in human squamous carcinomas (15â€”18)and are thought to play a major role in the malignant transformation of these cells (19). PIHrP3 was first recognized as the causative factor of the paraneo We therefore explored the possibility of a relationship between p53 plastic syndrome HHM (1). Purification of the HHM factor from function and PTHrP gene expression in squamous carcinoma cells. tumors revealed a 17-kDa peptide with homology to V1'H over the first 13 N-terminal amino acids (2). Subsequently, it has been recog nized that the PTHrP gene is widely expressed in normal adult and MATERIALS AND METHODS fetal tissues (1). Although the physiological function of PTHrP re mains unclear, mounting experimental evidence suggests that the Plasmids. The following p53 plasmids were obtained from A. Levine at peptide acts to regulate the rate of programmed differentiation during Princeton University (Princeton, NJ): pC53-SN3 is the wild-type p53 expres organogenesis. In developing tissues, PTHrP is expressed principally sion plasmid; pS3-248@ is the p53 Arg248 to lip mutant expression plasmid, in the epithelial layers while the classical PTH/PTHrP receptor is and p53-4.2N3 is the p53 Arg273to His mutant expression plasmid (20). EIB plasmids were obtained from E. White at Rutgers University (Piscataway, NJ): typically expressed in the neighboring mesenchyme, suggesting that pCMVE1B, pCMV5SK, and pCMV19K are expression vectors encoding both PTHrP might be involved in epithelial-mesenchymal interactions (4). E1B proteins, E1B 55-kDa protein and E1B 19-kDaprotein, respectively (21). A number of recent experiments using targeted overexpression or pLJ-Neo was used in cotransfections and to generate G418-resistant control targeted disruption of the PTHrP gene in mice support this idea and lines. indicate that PTHrP is capable of influencing processes as diverse as Cells and Culture Conditions. The SqCC/Yl line was developedat Yale, hair follicle development, branching morphogenesis of the mammary the CE-48 line was a gift from Dr. C-P. Hu (Taipei, Taiwan) and A253, A43l, gland, and linear growth and mineralization of endochondral bone C4-l, FaDu, CaLu, and CaSki cells were obtained from American Type (5â€”7). Culture Collection. All human tumor cell lines were grown in DMEM (Life Since PTHrP is an endogenous product of many types of epithelia, Technologies, Inc., Gaithersburg, MD) with 10% fetal bovine serum (Sigma, including epidermal keratinocytes and the lining cells of the bronchi, St. Louis, MO) and 1% L-glutamine (Sigma). oral epithelium, cervix, and vulva (8), most squamous neoplasms Stable Transfection and Selection. Approximately 1 X 106 A253 and presumably develop from cells which eutopically express the gene (9). A43l cells were transfected with 5 @xgofthe appropriate mutant p53 or E1B expression plasmid using a calcium phosphate method with a glycerol shock. Forty h after transfection, the cells were trypsinized and placed in fresh Received 3/28/96; accepted 7/2/96. medium containing 0.75 mg/mi G418 (Geneticin; Life Technologies, Inc., 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 Gaithersburg, MD). Three to 5 weeks later, colonies were isolated using a 18 U.S.C. Section 1734 solely to indicate this fact. modified ring cloning procedure and placed into 24-well plates for growth and I This work was supported by American Cancer Society Grant BE-152D (W. M. P.), evaluation of protein expression. The presence of p53 and E1B isoforms was The Ruth Esthn Goldberg Memorial for Cancer Research (W. M. P.), and NIH National evaluated by insmunohistochemistry using the methods detailed elsewhere Research Service Award CA63765 (J. F.) and CIA Grant CA60498 (J. J. W.). 2 To whom requests for reprints should be addressed, at Section of Endocrinology and (22). The mab DO-i (concentration 1: 1000; Santa Cruz Biotech, Inc., Santa Metabolism, Department of Internal Medicine, Yale University School of Medicine, P. 0. Cruz, CA) was used to label p53. The mab 13D2 (concentration 1:1500) and Box 3333, New Haven, CT 06520-8020. Phone: (203) 785-6425, (203) 785-5488; Fax: the rabbit polyclonal ab p21 (concentration I:4500, courtesy of E. White, (203)785-6015. Rutgers) were used to label E1B 55 and E1B 19, respectively. Because there 3 The abbreviations used are: PTHrP, parathyroid hormone-related protein; PTH, parathyroid hormone; HHM, humoral hypercalcemia of malignancy; HTLV-I, human was some degree of heterogeneity in the transfected cells with respect to I-cell leukemia virus 1; ab, antibody; mab, monoclonal antibody; CMV, cytomegalovirus. mutant p53 expression and El B expression, we chose to perform experiments 4056

p53 status in these cell lines (Fig. 1), it became apparent that those â€˜- a lines expressing moderate to high levels of PTHrP mRNA were all @ â€˜-c@)O â€¢@,:@o(v) @@U)>- LC) functionally p53 negative due to either deletions in the p.53 gene or â€˜@1-(@ W c@ CO 0 destabilization of p53 protein by the human papilloma virus E6 gene mutation in codon 273, causing a histidine to be substituted for an arginine, M M M N E6 N N whereas the isoform in the FaDu line has a point mutation at codon Fig. 1. RNase protection analysis of PTHrP mRNA expression in a panel of squamous 248, causing a leucine to arginine substitution (25). Both represent carcinoma cell lines. Each lane represents 40 @sgoftotal RNA hybridized with the human common, well-characterized mutations which induce a stabilized p53 PTHrP-coding region probe. A253 and SqCC/Y 1 expressed high levels of PTHrP mRNA. CaLu and CaSki expressed intermediate levels of PTHrP mRNA, and A431, FaDu, and protein without producing the conformational alterations common to CE-48 all contained levels of PTHrP that were barely detectable in this assay. f3-actin the majority of mutant p53 isoforms (26). mRNA levels varied widely among cell types; therefore, loading was confirmed by Repression by Mutant p53 Isoforms. We next evaluated the running equivalent amounts of RNA on an agarose gel and staining with ethidium bromide (data not shown). p53 status is indicated below the various lanes: N, p53-negative due to effect of mutant p53 isoforms on PTHrP gene expression by devel deletions of p53 genes and/or silent p53 alleles; E6, lack of p53 protein due to rapid oping stably transfected clonal lines that expressed specific isoforms. protein turnover induced by the HPV 16 or 18 E6 oncoprotein; M, mutant p53. As a recipient cell, we chose the A253 squamous carcinoma line, which is functionally p53 negative (25) and expresses abundant only in lines having >95% immunopositivity for p53 and >70% positivity for PTHrP iTIRNA (Fig. 1). A253 cells were transfected with a neomycin EIB, respectively. resistance gene (Neo) linked to a CMV-driven expression vector that Transient Transfection. Transient transfection of A253 cells was per contained one of the following mutant p53 isoforms: 273 His (the formed using 1.5 X 106cells plated on 100-mm plates. Five @.tgeachof the mutation found in the CE-48 and A431 cell lines) or 248 Irp (an 1.2-kb mouse 5' PTHrP luciferase construct and p53/vector constructs (vector alternate codon 248 mutation). Ultimately, selected colonies were consisted ofpC53-SN3 in which p53 sequences had been removed by a BamHl evaluated for p53 protein expression using immunohistochemistry, restriction digest) were precipitated and applied to A253 cells for 8 h before and lines in which the population of p53-expressing cells was more glycerol shock. After 40 h, cells were washed twice in PBS and extracted in than 95% (Fig. 3, B and C) were used for the analysis of PTHrP 400 pAof luciferase lysis buffer (Promega, Madison, WI). Twenty @lofcell mRNA expression. extract were used in a reaction mixture that contained beetle luciferin (Pro We grew the various lines to confluence and harvested RNA for mega), and light activity/luciferase activity was measured in a Berthold lumi nometer. Luciferase activity was normalized to total protein in the cell extract. analysis by RNase protection using human PTHrP-coding region and Protein content of cell extracts was measured by a Lowry-based kit (Bio-Rad, @-actin probes. As shown in Fig. 2, PTHrP mRNA levels were Hercules, CA). reduced in the A253 lines expressing the 248 and 273 p53 isoforms as RNase Protection. Total cellular RNA was prepared from confluent flasks compared to A253 cells transfected with a Neo resistance vector of cultured cell lines using a modification of the guanidium isothiocyanate alone. Densitometric scanning using a f3-actin standard revealed that cesium chloride procedure (23). RNase protection analysis for the human PTHrP mRNA levels were reduced by approximately 50% in the 248 PTHrP coding region was performed as described previously (24) using and 273 lines. To rule out effects on gene expression due to nonphysi 2 X l0@ cpm ofa labeled antisense probe corresponding to a 308-bp PvuII-SacI ological overexpression of p53 protein in the transfected lines, we fragment from the coding region of the human PTHrP cDNA; a 2 X i04 cpm evaluated p53 protein levels using Western blotting and found that the of a labeled antisense probe corresponding to a 207-bp Hinfl-TaqI fragment of p53 protein levels in lines transfected with 248 or 273 constructs were the human f3-actinexon 4 was used as an internal control. RNase protection roughly equivalent to the level expressed by A431 cells, which have analysis of the promoter-specific transcripts was performed as described pre viously (24) using 2 x l0@cpm of a labeled antisense probe corresponding to a single copy of the p53 gene with a mutation at codon 273 (Fig. 3D). a 803-bp Bam-Nsi fragment of the 5' flanking region of the human PTHrP E1B Reverses p53-induced Repression. We next undertook a set gene. of experiments to determine whether disruption of the function of an Western Blots. Cell lines were grown to confluence on 100-mm tissue endogenous mutant p53 isoform would increase PTHrP gene expres culture dishes. Protein extracts were made by adding 200 @lofcell lysis buffer sion in a low-expressing line. Here, we chose the A43 1 line, in which [10 ms@iTris(pH 7.5), 0.25 M sucrose, 0.16 M KC1,0.5 mMEDTA, 50 mM the p53 gene contains a His for Arg substitution at codon 273 and 6-aminocaproic acid, 0.5% NP4O, 10 j.tg/ml aprotinin, and 0.1 mrvi phenyl methylsulfonyl fluoride), and 67 j.d of 3X Laemmli sample buffer were added and the samples boiled for 2 mm. For each sample, 15 @goftotal protein were loaded onto a 7.5% SDS-polyacrylamide gel and electroblotted onto Immo bilon (Millipore, Bedford, MA) using standard methods. The Western blot was incubated with the anti-p53 mab DO-I (Santa Cruz Biotech, Inc.) and visual @ @@d)c@) @ ized with an ECL system (Kirkegaard & Perry, Gaithersburg, MD). F@-N@ C@C%JCsJC@J RESULTS @@@ PThrP _U PTHrP mRNA Expression in a Panel of Human Squamous Carcinoma Cell Lines with Alterations in the p53 Gene. We ex @ amined a series of squamous carcinoma lines that previously had been @3-actinâ€¢ â€” characterized with regard to p53 functional status (25); cell lines which were found to be incapable of PTHrP expression due to Fig. 2. RNase protection analysis of PTHrP mRNA expression in A253 cell lines acquired methylation of the gene were excluded. Seven cell lines were permanently transfected with mutant p53 isoforms. All lanes represent 20 @sgoftotal RNA grown to confluence under identical culture conditions, and RNA was hybridized with the human PTHrP-coding region probe and the human @-actinprobe. A253-Neo represents a line transfected with vector alone: 248- and 273-designated clones harvested for RNase protection analysis with a human PTHrP-coding represent A253 cells transfected with the p53 expression vectors expressing these region probe. When PTHrP mRNA levels were compared with the isoforms. 4057

REPRESSION OF PTHrP BY p53 MUTANTS

A B C e (Fig. 5). None of the A43 1 lines transfected with the Neo resistance a vector alone were found to express increased levels of PTHrP mRNA @ . .(data TranscriptsfromthePTHrPTATAPromotersAreReducedin not shown). A253LinesExpressingthe 248and 273Mutant p53 Isoforms. The human PTHrP gene has a complex-controlling region that encom I passes three promoters: two that contain consensus TATA elements .0 A B C

, @ D\ \ f'@@

:4 Fig. 4. EIB immunohistochemistry in A43l cell lines. Calcium phosphate cotransfec tion was used to introduce the three El B expression vectors (driven by the CMV promoter) and a Neo resistance gene into A43 I squamous carcinoma cells. These cells express the 273 mutant p53 isoform and very low levels of PTHrP mRNA (Fig. I). Cells Fig. 3. p53 immunochemistry in A253 cell lines. Calcium phosphate transfection was were labeled with E1B antibodies: A, native A431 labeled with anti-EIB 55-kDa ab; B, used to introduce three Neo-linked mutant p53 expression vectors (driven by the CMV E1B 55-kDa transfected cells labeled with anti-EIB 55-kDa ab; and C. E1B 19-kDa promoter) into p53-negative A253 squamous carcinoma cells: A, A253-Neo; B, 248 transfected cells labeled with anti-EIB l9-kDa ab. Cells were grown on coverslips. transfected; and C. 273 transfected. Cells were grown on coverslips, methanol fixed, paraformaldehyde fixed, permeabilized in 3% Triton X-lOO. labeled with abs. visualized labeled with the p53 ab D0-l, visualized with peroxidase immunohistochemistry, and using peroxidase immunohistochemistry, and counterstained with nuclear fast red. The counterstained with nuclear fast red. Bar, 1 p.m. Note that A253-Neo cells do not express E1B 55-kDa ab labeled pockets along the nucleus (B. arrows), and the EIB 19 ab labeled detectable levels of p53 protein, whereas mutant p53-transfected lines express high levels what apparently is the nuclear membrane and occasionally filamentous structures in the of p53 protein. In general, the expression of p53 in these cells did not appear to provide cytoplasm (C, arrows). The native A431 cells did not label with either of the abs. EIB any growth advantage in culture. Only 4 of 40 of the 248 clones and 3 of 40 of the 273 expression did not appear to be advantageous for growth in culture. Only 4 of 20 El B, 2 clones were >95% p53 positive. D. relative p53 protein levels in protein extracts derived of 20 E IB 55, and 5 of 13 E IB I9 clones had >70% E IB-expressing cells. Also. the E IB- from mutant p53-transfected lines detected with Western blotting. A431 cells have a and EIB 55-expressing lines were very unstable. with the number of E1B-expressing cells single p53 gene that has a mutation at codon 273, and protein extracts from these cells declining precipitously after several passages. served as an indicator of p53 protein levels in a cell line bearing an endogenous p53 mutation. 248-21 and 273-3 represent protein extracts made from individual A253 lines that were permanently transfected with expression vectors that express these specific mutant p53 isoforms. A253 represents extracts made from parental A253 cells. Equivalent amounts of total protein ( 15 ag) from each line were loaded onto 7.5% Laemmli gels, electroblotted, probed with the DO-I mab, and visualized using ECL. Note that the .0 @

which expresses PTHrP mRNA at levelsjust above the detection limit of the assay. As a vehicle for disrupting p53. we used the adenoviral E1B oncoproteins. The E1B proteins are derived from a single gene @@@@@ which is translated from two reading frames. One of the resulting @-actinWs@1 products is a 55-kDa protein that binds directly to the NH2 terminus of p53 and has been reported to bind to mutant p53 isoforms in vivo Fold 3.0 1.8 â€” 3.8 1.8 â€” 3@4 11 (27, 28). The other E1B product is a 19-kDa protein that does not bind I nduct ion to p53 but disrupts the ability of wild-type p53 to repress certain Fig. 5. RNase protection analysis of PTHrP mRNA expression in A43 I cell lines promoters (29). We used three distinct E1B vectors, two of which permanently transfected with El B isoforms. Using a human PTHrP-coding region probe expressed either the l9-kDa or 55-kDa isoform and one of which and a human @3-actinprobe.RNase protection assays were performed on 25 @sgoftotal RNA from the various El B-expressing lines and native A43 I cells. One expression vector expressed both. We permanently cotransfected A43l cells with both a produced both EIB proteins. whereas point mutations in the other constructs limited Neo resistance vector and expression vectors in which the El B expression to either the E1B 55-kDa protein or l9-kDa protein. As shown, when isoforms were placed under the control of the CMV promoter. After compared to native A43 I cells, a clear increase in PTHrP mRNA expression was observed in the transfected lines that expressed both El B proteins (lines b-) as well as those that selection, colonies were isolated and evaluated for expression of the expressed the 55-kDa or 19-kDa isoforms alone (lines 55 and 19. respectively). Fold E1B 55-kDa or l9-kDa proteins (Fig. 4, B and C). The lines selected induction of each of the EIB lanes relative to the A431 lanes was determined by for study were at least 70% E1B positive. densitometric scanning using the f3-actin standard from each lane. The variable induction of PTHrP mRNA was probably due in part to the clonal variability of E1B isoform The E1B-expressing A431 cell lines were grown to confluence, expression. Preliminary experiments using E1B expression plasmids and 5' PTHrP RNA was harvested, and PTHrP gene expression was evaluated by constructs transiently transfected into the p53-negative cell line A253 suggested that El B did not directly activate PTHrP gene expression: these results indicate that El B can RNase protection. As compared to native A431 cells, all of the relieve the repression of PTHrP gene expression. implicating p53 as the agent of that E1B-expressing lines contained higher levels of PTHrP mRNA repression. 4058

@@ BAM HI 250â€” @ 240â€” @.

Protected Fragments 160â€”

GC 250 150â€” P1 @i@i,9 240 11 P3 I I 160

Fig. 6. PTHrP mRNA expression in A253 lines permanently transfected with mutant p53 evaluated by promoter-specific RNase protection. In A, the schematic represents the 5' flanking region of the human PTHrP gene, where boxes represent exons and lines represent intronic sequences. The expected fragments generated from each of the three promoters in a RNase protection assay with the Bam-Nsi probe are illustrated below the schematic. Transcripts produced from the GC promoter initiate 10 bp upstream of exon 3 and therefore generate a 250-bp protected fragment. Transcripts produced from the P1 TATA include exon 3 and therefore generate a 240-bp fragment. Transcripts produced from the P3 TATA initiate at exon 4 and are the only transcripts that include this exon and generate a series of fragments centered on 160 bp. In B, each lane represents 40 @xgoftotal RNA hybridized with the human Bam-Nsi 5' PTHrP probe. The lane representing parental A253 cells was exposed for 5 days; however, a 13-day exposure was required to detect bands in lanes representing 248 and 273 lines. In native A253 cells, the bands at 240 bp and 160 bp were more intense than the band at 250 bp, indicating relatively greater levels of PTHrP gene transcription from the TATA promoters than the GC-rich promoter. In A253 lines permanently transfected with the 248 and 273 isoforms, the 240-bp and 160-bp bands appeared to have the same relative intensity as the 250-bp band, indicating that the 248 and 273 isoforms more effectively repressed PTHrP gene transcription derived from TAIA promoters.

(the upstream P1 and downstream P3 promoters) plus a third mid 87% in the l00-bp region immediately surrounding the TATA ele region GC-rich element (24). Although wild-type p53 has been shown ment). We therefore cotransfected a 1.2-kb mouse 5 â€˜PTHrP-lucifer to efficiently repress TATA-based promoters, it appears to be inef ase construct along with expression plasmids containing either the 248 fective at repressing initiator elements with characteristics similar to or 273 mutant p53 isoforms into the p53-negative A253 cell line. As the PTHrP GC-rich promoter (30â€”32).We therefore wanted to de seen in Fig. 7, the mutant 248 and 273 isoforms repressed reporter termine whether the decreased PTHrP mRNA expression levels ob activity by 65% and 55%, respectively, indicating that the capacity to served in the mutant p53-bearing A253 lines were due to down mediate repression lies within the 5' flanking region of the gene. Not regulation of transcripts from all three promoters in parallel or from surprisingly, the wild-type isoform proved even more effective, re one promoter specifically. To approach this question, we used a pressing reporter activity by 90%. Although progressive deletion of RNase protection assay with a human Bam-Nsi 5' PTHrP probe that sequences 5' to the promoter (reduction to 500 bp or 200 bp) greatly protects three distinct fragments, each derived from a different pro diminished overall reporter activity, the relative degree of repression moter. Transcripts from the GC promoter produce a 250-bp fragment, induced by both the wild-type and mutant isoforms remained the whereas transcripts from the P1 upstream TATA and P3 downstream same, as would be expected for a TATA-based promoter (data not TATA give rise to a 240-bp fragment and to a series of bands shown). representing multiple initiation sites centered around 160 bp, respec tively (Fig. 6A). The use of a single probe for all three classes of transcripts thus allows direct quantitative comparison of relative pro 120000 moter usage both within and between cell lines. As shown in Fig. 6B, the band at 240 bp and the cluster at 160 bp are clearly reduced in intensity in the A253 lines transformed with the 248 and 273 mutant 5) 0 p53 isoforms as compared with native A253 cells. Thus, the attenu 0@ 05 ation of PTHrP mRNA expression levels observed in the cell lines E containing mutant p53 appeared to result primarily from a reduction in transcripts derived from the IATA promoters. 8 248 and 273 Mutant p53 Isoforms Repress the Activity of a 5' 5) PTHrP Reporter Gene. The reduction in the levels of steady-state

mRNA derived from the TATA-based PTHrP promoters suggested C) that the transcriptional activities of these promoters were reduced in the cell lines expressing mutant p53 isoforms. Although the most straightforward way to test this hypothesis would presumably have .11 been to use transient transfection assays with reporter gene constructs Vector 1.2 +wtp53 Ã·248 +273 controlled by each of the two human TATA promoters, our experience with an extensive series of human PTHrP promoter deletion con Fig. 7. The 248 and 273 isoforms repress activity of a 5' PTHrP reporter gene in transient transfections. Columns, comparison of normalized luciferase activity from p53- structs has shown both the TATA-based promoters to be capable of negative A253 cells transiently cotransfected with the mouse 1.2-kb 5' PTHrP and only poor levels of aberrantly initiated transcription when cloned in contained vector or various p53 expression vectors. Reporter gene activity was repressed isolation and tested in this manner. In contrast, the murine PTHrP by wild-type p53 and the 248 and 273 mutant isoforms. Wild-type p53 repressed the activity of the 1.2-kb construct by 90%, whereas the 248 and 273 mutant p53 isoforms promoter region, which contains only a single functional TATA repressed the activity by 65% and 55%, respectively. In these experiments. transfection promoter, displays readily detectable levels of reporter activity and is efficiency was normalized by dividing the luciferase activity of a protein extract from a transfected plate by total protein in the cell extract. Data represent the mean of two largely homologous to its human counterpart (approximately 73% independent experiments in which each cotransfection was performed on two parallel sequence identity over the 1.2-kb 5 â€˜flanking region and more than plates. Bars, SE. 4059

DISCUSSION versely, inactivation of an endogenous p53 mutant (273) with E1B proteins resulted in increased PTHrP expression in a low-expressing The syndrome of humoral hypercalcemia of malignancy provides a cell line. The fact that either the 55-kDa or 19-kD E1B product could classic and common example of the pathophysiological consequences mediate this effect suggests that both p53 N-terminal activity and of dysregulated hormone production by tumors. As predicted by interaction with other proteins in the transcription apparatus may be Baylin and Mendelsohn (33) 16 years ago, the production of hor involved in the mechanism of repression (27, 39). Subsequent analysis mones or growth factors by tumors has become, in most instances, a of promoter-specific PTHrP transcripts in a p53-negative squamous question of dysregulated gene expression, and there is a growing line transfected with mutant p53 isoforms suggested that down-regu literature on the mechanisms involved. Specific examples include lation occurred primarily at the two TAIA-based promoters. This is in alterations in the stability of mRNA, methylation of control regions, keeping with the previous identification of these promoters as the and level or activity of trans-acting factors (33). The PTHrP gene is principle source of PTHrP transcripts in squamous lines (14); in a complex transcriptional unit which is driven by three promoters, contrast, it is the GC-rich element which serves as the most active gives rise to multiple alternatively spliced 5' transcripts, and termi promoter in renal carcinoma lines (24). Direct testing of a murine nates in three independent 3' exons, each containing AUUUA motifs PTHrP reporter construct confirmed the capacity of the 248 and 273 that likely confer the rapid turnover of PTHrP mRNAs. Therefore, mutants to repress this TATA-based promoter, although only about dysregulation of PTHrP gene expression in tumor cells could result half as effectively as wild-type p53. from a number of different mechanisms, and several have been Although repression of gene expression is a hallmark of wild-type already described. In renal carcinoma cell lines, differential methyl p53 function, mutant p53 isoforms are generally considered to be ation of specific sites within the 5' flanking region of the PTHrP gene deficient in this process (26). Only the bcl-2 gene, which plays a appears to be the defining mechanism (34), whereas in adult I-cell major role in the inhibition of apoptosis, has thus far been shown to leukemia cells, high levels of PIHrP result from transactivation of the be directly repressed by both mutant and wild-type p53 isoforms (40, PTHrP gene by the viral tax gene product (35, 36). 41). This appears to be mediated by a l95-bp stretch found in the 5' In the case of squamous carcinoma, the cells of origin of this untranslated region of the bcl-2 gene (42). Transient assays have been neoplasm normally express relatively high levels of PTHrP. There used successfully to test the effects of both wild-type and mutant p53 fore, the production of PTHrP by squamous carcinomas is presumably isoforms (including the 143, 175, 248, 273, and 281) on a thymidine eutopic and most likely involves modulation of gene expression rather kinase, minimal TATA promoter (43). In this experiment, wild-type than inappropriate activation. Consistent with this idea, initial surveys p53 repressed reporter activity by 99%, whereas the mutant constructs indicated that squamous carcinomas (both tumors and tumor-derived achieved repression levels of 35â€”85%(43). It appears, therefore, that cell lines) displayed a wide range of PTHrP mRNA levels and did not mutant p53 isoforms retain some capacity to repress TATA-based appear to be simply â€œonoroff' with respect to PTHrP gene expres promoters in transient transfection assays, although the mechanism sion, as had been found to be the case in renal carcinomas (14). remains to be defined. It is also possible, however, that the repression Subsequent studies in vivo using athymic mice as hosts for injected observed for the PTHrP gene may result from a particular sensitivity tumor cells clearly demonstrated that only those cell lines producing of the PTHrP promoters or, alternatively, may be due to interactions PTHrP mRNA (and consequently protein) in amounts exceeding an with squamous cell-specific factors. In this case, promoter repression effective threshold were capable of inducing hypercalcemia ( 14). One would not represent a universal property of these mutant p53 possible explanation for these observations would be the presence of isoforms. a common derangement within the cell lines expressing low levels of These observations raise the question of whether wild-type p53 PTHrP, perhaps as a consequence of the transformation process itself. plays a role in repressing PTHrP expression in untransformed cells in Although any number of candidates could be considered, mutations vivo. Although this idea holds some appeal, supportive evidence is in the tumor suppressor p.53 certainly represent the most prevalent currently lacking. Wild-type p53 protein has a rapid turnover rate genetic lesions in human cancers, these mutations encompass: (a) (<30 mm; Ref. 44) and, unless stabilized by mutations which induce chromosomal rearrangements and localized deletions (which result in conformational changes, would be unlikely to be present at levels gene loss); (b) nonsense and splice site mutations (which truncate the sufficient to affect PTHrP gene regulation. Also, although transient protein); and (c) missense mutations (which alter its conformation; transfection of the wild-type isoform generates profound effects on Ref. 37). Viral infection can also contribute to p53 dysfunction; the E6 PTHrP reporter gene expression, these circumstances undoubtedly gene product from the human papilloma virus, often found in cervical represent gross overexpression of p53 relative to endogenous levels. carcinoma, binds to p53 and promotes its degradation (38). Missense Finally, obtaining evidence for activation of PTHrP expression above mutations, which give rise to mutant isoforms of p53, are quite endogenous levels, concurrent with the loss of p53 function, is made common in squamous carcinomas from a variety of tissues, occurring difficult by the lack of an appropriate standard. Untransformed squa in 48 and 64% of invasive tumors of the skin and of the head and neck, mous epithelial cell types, including epidermal keratinocytes, typi respectively (15, 16). These mutations typically stabilize the protein, cally display relatively high levels of PTHrP expression, yet may not thereby increasing its effective concentration, and inactivate protypi accurately reflect levels in vivo (45). In any case, the finding of cal properties of the wild-type molecule including DNA binding, gene p53-negative status in tumor lines with unequivocal overexpression of transactivation, promoter repression, specific protein binding, and the PTHrP would constitute only circumstantial evidence and would not ability to suppress cell growth (26). It has begun to appear, however, rule out independent transactivation mechanisms, which have recently that not all mutations inactivate these properties to the same extent. shown to be operative in these cell types (14). We have attempted to In the present study, comparison of PTHrP expression levels with address these issues using permanently transfected lines, but have p53 functional status in a series of squamous carcinoma lines has been hampered by the cell death which results from high levels of revealed an association between the expression of certain mutant wild-type p53. It is possible that the question of whether wild-type isoforms of p53 and very low levels of PTHrP mRNA. Evaluation of p53 plays a role in repressing PTHrP gene expression could be p53 isoforms with mutations in codons 248 and 273 demonstrated addressed by examining PTHrP expression in epithelial tissues of them to be capable of repressing PTHrP gene expression in a high PS3â€”'â€” mice relative to those of p53+1+ littermates. We are cur expressing, p53-negative squamous line by approximately 50%. Con rently pursuing this and other experimental approaches. 4060

Finally, to generalize these findings, it will be necessary to test a 8. Turzynski, A., Baumgart, S., Bauch, B., and Dietel, M. Morphological characteristics number of different mutant p53 isoforms to see whether repression of of tumors with humoral hypercalcemia of malignancy: functional morphology of PTHrP. Recent Results Cancer Res., 137: 76â€”97,1994. PTHrP expression constitutes a commonly held property. Evidence is 9. Yuspa. S. H. The pathogenesis of of squamous cell cancer: lessons learned from continuing to emerge that indicates that the class of mutant p53 studies of skin carcinogenesis. Cancer Res., 54: 1178â€”1189, 1994. 10. Stewardt, A. F., Horst, R., Deftos, L. J., Cadman. E. C.. Lang. R.. and Broadus, isoforms represented by the 248 and 273 mutations may represent A. E. Biochemical evaluation of patients with cancer associated hypercalcemia: only partial inactivation of wild-type functions (46â€”49), whereas evidence for humoral and non-humoral groups. N. EngI. J. Med.. 303: 1377â€”1383, classical p53 mutants are characterized by a profound derangement of 1980. I I. Bender, R.. and Hansen, H. Hypercalcemia in bronchogenic carcinoma. a prospective those functions. These latter isoforms can be immunoprecipitated by study of 200 patients. Ann. lntem. Med., 322: 1106â€”1111. 1990. a mab that recognizes epitopes accessible only when the protein is in I2. Henderson, J., Sebagi, M.. Rhim, R., Goltzman, D., and Kremer, R. Dysregulation of a denatured conformation; neither wild-type p53 nor the 248 and 273 parathyroid hormone-like peptide expression and secretion in a keratinocyte model of tumor progression. Cancer Res., 51: 6521â€”6528, 1991. isoforms are recognized by this ab (26). In this regard, preliminary 13. Lowik. C. W. G. M., Hoekman, K., and Offringa, R. Regulation of parathyroid experiments with selected classical mutant p53 isoforms suggest that hormone-like protein production in cultured normal and malignant keratinocytes. these proteins are ineffective at repressing PTHrP gene expression, J. Invest. Dermatol., 98: 198â€”203,1992. 14. Wysolmerski, J. J., Vasavada, R., Foley, J., Weir, E. C., Burtis, W. J., Kukreja. S. C.. which would be consistent with their lack of activity in the repression Guise, I. A., Broadus, A. E., and Philbrick, W. M. Transactivation of the PTHrP gene of promoter activity in general. Thus, the repression of PTHrP gene in squamous carcinomas predicts the occurrence of hypercalcemia in athymic mice. Cancer Res., 56: 1043â€”1049,1996. expression may represent a unique property of a specific subset of 15. Boyle, J. 0., Hakim, J., Koch, W.. van der Reit, P., Hruban, R. H., Roa, R. A., Correo. mutant p53 isoforms which retain some wild-type functions. R.. Eby, Y. J., Ruppert, M., and Sidransky, D. 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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1996 American Association for Cancer Research. Parathyroid Hormone-related Protein Gene Expression in Human Squamous Carcinoma Cells Is Repressed by Mutant Isoforms of p53

John Foley, John J. Wysolmerski, Arthur E. Broadus, et al.

Cancer Res 1996;56:4056-4062.

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