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Wild-Type P53 Can Inhibit Oncogene-Mediated Focus Formation

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Wild-Type P53 Can Inhibit Oncogene-Mediated Focus Formation Proc. Natl. Acad. Sci. USA Vol. 86, pp. 8763-8767, November 1989 Cell Biology Wild-type p53 can inhibit oncogene-mediated focus formation (dominant negative mutations/tumor-suppressor genes/tumorigenesis) DANIEL ELIYAHU, DAN MICHALOVITZ, SIONA ELIYAHU, ORIT PINHASI-KIMHI, AND MOSHE OREN* Department of Chemical Immunology, Weizmann Institute of Science, Rehovot 76100, Israel Communicated by 1. Robert Lehman, July 25, 1989 ABSTRACT Mutant forms of the p53 cellular tumor an- The apparent paradox of p53 having both a positive and a tigen elicit neoplastic transformation in vitro. Recent evidence negative effect on neoplastic processes seems now to be indicated that loss of normal p53 expression is a frequent event resolved, due to the finding that wild-type (wt) p53 is devoid in certain types of tumors, raising the possibility that such loss of any demonstrable transforming activity, and the latter provides transformed cells with a selective growth advantage. activity is exhibited only by plasmids encoding certain mu- Thus, it was conceivable that the mutants might contribute to tant forms of the protein (19, 20, 22). It was therefore transformation by abrogating normal p53 function. We there- conceivable that wt p53 may indeed interfere with transfor- fore studied the effect of plasmids encoding wild-type (wt) p53 mation, and the mutant p53 could thus promote transforma- on the ability of primary rat embryo fibroblasts to be trans- tion by blocking normal p53 fufiction, acting in a dominant formed by a combination of mutant p53 and ras. It was found negative fashion (23). that wt p53 plasmids indeed caused a marked reduction in the The idea that wt p53 may be restrictive to transformation number of transformed foci. Furthermore, wt p53 plasmids was tested by studying the effect of plasmids encoding wt or also suppressed the induction of transformed foci by combi- mutant p53 on the ability of various oncogene combinations nations of bona fide oncogenes, such as myc plus ras or to elicit transformed foci in vitro. We report here that wt p53 adenovirus EIA plus ras. On the other hand, plasmids carrying plasmids exhibited a pronounced inhibitory effect in such mutations in the p53 coding region totally failed to inhibit transformation assays. The magnitude of the effect was oncogene-mediated focus induction and often even slightly directly correlated with the level of wt p53 expression and stimulated it. Hence, such mutations completely abolished the was totally abolished by point mutations in the protein. Our activity of wt p53 that is responsible for the "suppressor" findings suggest that wt p53 expression may indeed have an effect. The latter fact is of special interest, since similar adverse effect on transformed cells and that its ?nactivation mutations in p53 are often observed in human and rodent may play a crucial role in tumorigenesis. tumors. The inhibitory effect of wt p53 was most pronounced when early-passage cells were used as targets, whereas estab- lished cell lines were less sensitive. These data support the MATERIALS AND METHODS notions that wt p53 expression may be restrictive to neoplastic Plasmids. Most ofthe plasmids employed in this study have progression and that p53 inactivation may play a crucial role been described before, some of them under different names. in tumorigenesis. pLTRpS3wt encodes wt mouse p53 under the transcriptional control ofa Harvey sarcoma virus long terminal repeat (LTR) A number ofproperties ofthe p53 cellular tumor antigen have and is identical to pLTRNc9 (20). pLTRp53m is essentially led to the suggestion that it may be tightly associated with similar, except that the p53 cDNA is derived from Meth A neoplastic transformation (1-4). Based on a variety of find- fibrosarcoma cells (20, 24) and carries point mutations at ings, the p53 gene was proposed to be an oncogene of the positions corresponding to residues 168 and 234 of the "nuclear," myc-like type (5-9). protein; it is identical to pLTRc5 (20). pLTRpS3cG9 (9, 19, While there are many instances of p53 overproduction in 25) contains a chimera of mouse p53 cDNA and genomic transformed cells, there are other cases in which p53 expres- DNA; it encodes a protein with a mutation at residue 135 (19, sion is altogether absent. The most striking case is that of 20). pLTRp53dl is identical to the previously described mouse spleen tumors induced by the Friend erythroleukemia pLTRcGS, a deleted derivative of pLTRpS3cG missing the virus. The detailed analysis of those tumors, many of which bulk of the protein-coding region (25). The construction of have no detectable normal p53 at all, has led to the suggestion pCMVp53wt and pCMVpS3m will be described in detail that, in some cases, loss ofp53 function may be advantageous elsewhere. These plasmids contain p53 cDNA segments for tumorigenesis (10-13). Recent studies involving human identical to those present in pLTRp53wt and pLTRpS3m, tumors have lent strong support to this idea (14-17). A high respectively. Transcription is driven by the immediate-early proportion ofhuman osteosarcomas exhibit no p53 synthesis; enhancer-promoter of the human cytomegalovirus (CMV) this is often correlated with the presence of gross rearrange- (26). In addition, each plasmid contains a chimeric intron, ments in the p53 genes (ref. 14; C. Miller and P. Koeffler, generated by fusing the 5' part of the first CMV intron to the personal communication). The study of human colon cancer 3' part of the first p53 intron. pCMVpS3dl was prepared from has been especially revealing (16, 17). One of the two p53 pCMVpS3wt by cleaving this plasmid DNA with Xho I and alleles is completely lost in at least 75% of cases. The other Kpn I, blunting the ends, and religating. This results in a allele, in all cases studied so far, is invariably mutated. Most deletion of about 600 base pairs, removing approximately the remarkably, the mutations fall within highly conserved pro- N-terminal half of the protein. In addition, the region encod- tein domains; mutations in these regions have been shown to ing the C-terminal half, 3' to the Kpn I site, is now positioned grossly alter the biological properties of mouse p53 (18-21). out ofphase (data not shown). Consequently, the polypeptide The publication costs of this article were defrayed in part by page charge Abbreviations: CMV, cytomegalovirus; LTR, long terminal repeat; payment. This article must therefore be hereby marked "advertisement" REF, rat embryo fibroblast; wt, wild type. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 8763 Downloaded by guest on September 29, 2021 8764 Cell Biology: Eliyahu et al. Proc. Natl. Acad. Sci. USA 86 (1989) encoded by this plasmid contains only the first 15 amino acids Table 1. Effect of p53 plasmids on transformation of REFs by of authentic mouse p53. mutant p53 plus ras Cells and Transfections. Primary rat embryo fibroblasts Additional Foci per dish (REFs) were prepared from 16-day-old Fischer rat embryos cotransfected DNA (no. of dishes) and maintained as described (5). Cells were usually passaged two or three times before being transfected. Approximately None 28 (6) 8 x 105 cells growing in a 90-mm dish were transfected by the pSP6 23 (1) calcium phosphate coprecipitation method (5). Following pLTRp53dl 9 (2) transfection and glycerol shock, cells were maintained for 2-3 pLTRp53m 20 (2) days in Dulbecco's modified Eagle's medium containing 10% pLTRpS3wt 3 (2) fetal bovine serum, and then the serum concentration was pCMVp53dl 10 (2) reduced to 5%. Rat-1 cells, an established fibroblastic cell pCMVpS3m 14 (6) line, were maintained, transfected, and subjected to myco- pCMVpS3wt 0.4 (10) phenolic acid selection as described (9). Each transfection mixture contained 2 tig of pLTRpS3cG9, en- Protein Analysis. Proteins were labeled with [35S]meth- coding mutant p53 (19, 20), and 2 ,ug of pEJ6.6, carrying an activated Ha-ras gene (27), as well as 5 ,ug of additional plasmid DNA as ionine, extracted, immunoprecipitated, and analyzed by indicated. Foci were scored 3 weeks after transfection. Results are SDS/polyacrylamide gel electrophoresis as described (5, 20). given as average number of foci per transfected dish and were compiled from several independent transfections. Numbers in pa- rentheses represent the number of transfection dishes scored to RESULTS obtain the average value. pSP6 refers to pSP64 (28), which served as Effect of wt p53 on Transformation of REFs by Mutant p53 a vector control for pCMV plasmids. pLTRp53m and pLTRp53wt are Plus ras. Primary REFs can become neoplastically trans- identical to the previously described pLTRc5 and pLTRNc-9, re- spectively (20). pCMVp53m and pCMVp53wt encode mutant and wt formed through the combined action of plasmids overpro- p53, respectively, under the control of the CMV immediate-early ducing mutant p53 and activated Ha-ras (5-7, 19, 20). If the promoter (26). In addition, they contain the first noncoding CMV transforming, mutant p53 plasmids were indeed working by exon and a chimeric intron. pCMVp53dl is a deletion derivative of interfering with the activity of wt p53, then one should be able pCMVp53wt. to antagonize their transforming effects by increasing the amount of wt p53. We therefore studied the effect of various Effect of wt p53 on Transformation ofREFs by myc Plus ras. p53 plasmids on the transformation of REFs by a combina- Nonestablished REFs can be very efficiently transformed by tion of ras and pLTRp53cG9, which directs the abundant a combination of pEJ6.6 and plasmids encoding the Myc synthesis of mutant p53 (20). In addition to previously protein (30). To determine whether myc plus ras-mediated described plasmids driven by a retroviral LTR (20), we used transformation was also affected by the cellular levels of wt a new set of plasmids employing the strong CMV immediate- p53, the transfection experiments summarized in Table 2 early enhancer-promoter (26).
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