Low Incidence of Point Mutations Detected in the p53 Tumor Suppressor Gene from Chemically Induced Rat Renal Mesenchymal Tumors I

Previous studies have shown renal mesenchymal tumors (RMTs) induced in rats by a single intrarenal injection of nickel subsulfide and iron are more pleomorphic and metastatically aggressive than RMTs induced by a single ip injection of methyl(methoxymethyl)nitrosamine (DMNOMe). While both RMT types contain high levels of K-ras activation, the specific mutational spectra within codon 12 of K-ras are quite different. Nickel subsulfide and iron-induced tumors exhibited codon 12 GGT-*GTr transversions exclusively, while DMN-OMe RMTs showed a wide array of codon 12 mutations, as well as mutations within codons 61 and 63 [K. G. Higinbotham, J. M. Rice, B. A. Diwan, K. S. Kasprzak, C. D. Reed, and A. O. Perantoni, Cancer Res., 52: 4747-4751, 1992; K. G. Higinbotham, J. M. Rice, and A. O. Perantoni, Mol. Carcinog., 5:136-139, 1992]. In an effort to further correlate carcinogen-specific molecular events in renal tumors, we investigated the p53 tumor suppressor gene in RMTs induced by these two carcinogens for the presence of point mutations. The evolutionarily conserved portion of the coding region of the gene, including part of exon 4 through exon 10, was surveyed for point mutations utilizing single-strand conformation polymorphism and chemical cleavage of mismatches analyses. None (0 of 10) of the nickel subsulfide and iron-induced RMTs and only I of 10 DMN-OMe-induced tumors that were evaluated contained point mutations within this portion of the p53 gene. Direct sequencing of the one single-strand conformation polymorphism and chemical cleavage of mismatches-"positive" DMN-OMeinduced RMT revealed a GCC--*GTC (Ala--*Val) transition in codon 345 within exon 10. These results suggest that the different tumorigenic phenotypes exhibited by these two RMTs are not the result of specific mutations or patterns of mutations within the portion of the p53 gene examined and that the mutated p53 tumorigenic pathway, whereby p53 plays a major role in many human neoplasms, does not function in RMTs induced by either agent. I N T R O D U C ~ O N Much attention has been placed recently on understanding the role of p53 in carcinogenesis. The p53 gene codes for a nuclear protein whose functionality appears to be very important in negative regulation of the cell cycle (1, 2). p53 also seems to influence other critical cellular functions, including cellular differentiation, signal transduction, apoptosis, transcription, transformation, and tumorigenesis (17). In normal growing cells, the p53 protein has a half-life of approximately 6 to 20 min (8, 9). In contrast, some point mutated forms of the protein can exhibit half-lives of 1.5 to 12 h (8, 10) and subsequently accumulate intracellularly. The mutational events seem to cluster predominantly around five highly conserved amino acid domains which include portions of exons 1-7 (reviewed in Ref. 11). Deletions or insertions causing frame-shifts result in nonfunctional, truncated forms of the p53 protein. Mutations which destroy or create splicing Received 4/14/93; accepted 11/1/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. 2 To whom requests for reprints should be addressed, at NIH, National Cancer InstituteFCRDC, Bldg. 538, Rm. 206, Frederick, MD 21702. sites in the gene can have similar protein-inactivating effects. The functionality of the wild-type p53 can also be altered by its interaction with binding proteins, such as heat shock protein 70 (12, 13) and the mdm-2 gene product (14, 15). To date, the loss of wild-type p53 function, or gain of a new function due to mutation, is one of the most commonly reported genetic changes in human cancer with an overall frequency of 50% (3, 16). In contrast, little is known about the role of p53 inactivation in experimental tumors from animal models. The presence of one or more p53 pseudogenes (~b-genes) in the mouse (17) and the rat (18, 41) genomes complicates analysis of sequence changes at the DNA level in these species. Recently, a report based on genomic DNA analysis has suggested that inactivation of p53 plays an important role in chemically induced renal tumorigenesis in the rat (19). RMTs 3 induced in Wistar rats with N-nitrosodimethylamine reportedly exhibited a high incidence (75%) of point mutations within the p53 gene. The majority of these mutations were detected within the evolutionarily conserved regions of exon 6 and were predominantly at two specific nucleotides, 610 (G--,A) and 638 (G->A). Unexpectedly, in two of the eight tumors studied, both mutations were detected coincidentally, suggesting possible RMT p53 mutational "hot spots" similar to the codon 249 "hot spot" found in human hepatocellular carcinomas from persons living in certain high cancer risk areas (20-22). In an effort to further correlate specific molecular events with individual carcinogens, the present study was designed to compare the mutational spectra evoked in the rat p53 gene by DMN-OMe and Ni3S 2 + Fe ~ in RMTs as detected by SSCP (23-25) and CCM (26-28). RMTs induced by these carcinogens differ in their K-ras mutational spectra as well as their metastatic potential. RMTs induced in rats with Ni3S2 + Fe ~ exhibited point mutations in exon 1 of K-ras which consisted exclusively of GGT---~GTT transversions within codon 12 (29). In contrast, DMN-OMe-induced rat RMTs showed a wider range of mutations within exon 1 (codons 12 and 13) as well as within exon 2 (codons 61 and 63) (30) . 4 In addition to the dissimilar patterns of K-ras mutations demonstrated by these two initiating protocols, the biological behavior of the tumors induced was remarkably different. RMTs induced with Ni3S2 + Fe ~ were biologically very aggressive tumors which frequently metastasized to the lung. In contrast, RMTs induced by DMN-OMe rarely metastasized. These experimental circumstances, therefore, provide opportunities both to compare the spectrum of p53 mutations caused by carcinogens possessing different patterns of mutational activities and to establish to what degree specific p53 mutational events correlate with and perhaps contribute to the metastatic potential of these neoplasms. 3 The abbreviations used are: RMT, renal mesenchymal tumor; Ni3S2 + Fe ~ nickel subsulfide and iron; DMN-OMe, methyl(methoxymethyl)nitrosamine; SSCP, singlestrand conformation polymorphism; CCM, chemical cleavage of mismatches; HA, hydroxylamine; NMU, N-nitrosomethylurea; OT, osmium tetroxide; PCR, polymerase chain reaction; PIP, piperidine; RT-PCR, reverse transcription-polymerase chain reaction; TBE, tris-borate-EDTA; @genes, pseudogenes; cDNA, complementary DNA. 4 A. O. Perantoni, K. G. Higinbotham, C. D. Reed, M. Watatani, T. Enomoto, L. M. Anderson, and J. M. Rice. Heterogeneity and multiple mutational activation of the K-ras oncogene in renal mesenchymal tumors induced in newborn F344 rats by methyl(methoxymethyl)nitrosamine, manuscript in preparation.