irANCER RESEARCH 55, 1894-1901, May 1, 1995] Breaks in Genomic DNA and within the p53 Gene Are Associated with Hypomethylation in Livers of Folate/methyl-deficient Rats1 Igor P. Pogribny, Alexei G. Basnakian, Barbara J. Miller, Nadejda G. Lopatina, Lionel A. Poirier, and S. Jill James2 National Center for Toxicological Research, Fond and Drug Administration, Division of Nutritional Toxicology, Jefferson, Arkansas 72079 ABSTRACT Male weanling Fischer 344 rats were fed either a semipurified diet deficient in the methyl donors methionine, choline, and folie acid or a supplemented control diet for a period of 9 weeks. At intervals of 2,5, and 7 days, 3 weeks, and 9 weeks after initiation of the respective diets, the relative level of DNA strand breaks and the degree of cytosine methylation were quantified in high molecular weight DNA and also within the p53 gene in liver samples from these rats. Genome-wide strand break accu mulation was associated with progressive genomic hypomethylation and increased DNA methyltransferase activity. With the use of quantitative PCR as a gene-specific DNA strand break assay, unique DNA strand breaks were detected in exon 5 but not in exons 6-8 of the/»53gene, and were accompanied by significant p53 gene hypomethylation. DNA hypo methylation has been shown to alter the conformation and stability of the chromatin structure, rendering affected regions more accessible to DNA- damaging agents. To determine whether methylation status alters the sensitivity of DNA to strand breakage, DNA in isolated nuclei was meth ylated in vitro and exposed to endogenous calcium/magnesium-dependent endonuclease activated under defined conditions. The incidence of en zyme-induced DNA strand breaks was decreased significantly with in creased DNA methylation. In nuclei isolated from livers of methyl-defi cient rats, the hypomethylated DNA was found to be more sensitive to enzyme- and oxidant-induced DNA strand break induction. Taken to gether, these results provide evidence that DNA strand breaks are induced in high molecular weight DNA and also within the p53 gene in liver tissue from methyl-deficient rats. The increased incidence of these strand breaks in DNA from methyl-deficient rats may be related to alterations in chro matin accessibility associated with DNA hypomethylation. INTRODUCTION Chronic dietary insufficiency of the lipotropic nutrients choline and methionine has been reproducibly shown to be hepatocarcinogenic in the rat and in certain mouse strains with or without chemical initiation (1-3). Several hypotheses have emerged over the years in attempt to understand the biochemical and molecular basis for this nutritional model of multistage hepatocarcinogenesis. While none of these is mutually exclusive, the major hypotheses that have been proposed implicate: (a) DNA hypomethylation leading to inappropriate expres sion of growth regulatory oncogenes (4-6); (¿>) oxidative free radical DNA damage secondary to early lipid accumulation and peroxidation (1, 7, 8); (c) aberrant membrane phospholipid metabolism, receptor expression, and protein kinase C-mediated signal transduction (3, 9, 10); (d) abnormal deoxynucleotide metabolism promoting DNA base misincorporation transition mutations (11, 12); and (e) oncogene and tumor suppressor gene aberrations (4, 13, 14). The one common factor in all these hypotheses is the absolute requirement for regenerative proliferation to promote the heritable genetic and/or epigenetic Received 1/25/95; accepted 3/7/95. 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 Financial support for this work was provided by American Cancer Society Research Grant CN-73C (to S. J. J.) and by an appointment to the Postgraduate Research Program administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the United States Department of Energy and the Food and Drug Administration (A. G. B., N. G. L.). 2 To whom requests for reprints should be addressed. changes that lead to neoplastic transformation. In fact, several of these hypotheses are difficult to separate from, and may be partially ex plained by, events that are secondary to the regenerative proliferation stimulated by the cytotoxic effects of the diet. The global DNA hypomethylation associated with methyl defi ciency has been causally linked to a diet-induced depletion of SAM,3 the substrate for the methyltransferase (6). Hepatic levels of S-adeno- sylmethionine in rats are reduced by 25% within the first week of consuming a methyl-deficient diet (15) and continue to decrease progressively in subsequent weeks (16). Maintenance methyltrans ferase activity is stimulated in the livers of methyl-deficient rats in order to maintain the methylation status of newly synthesized DNA strands during diet-induced regenerative proliferation. However, in the presence of reduced SAM availability, progressive DNA hypo methylation is promoted (5). Genome-wide alterations in DNA methylation, as well as regional variations in gene-specific methylation patterns, have been observed in a variety of preneoplastic and transformed cell models (17). How ever, a mechanistic relationship between DNA methylation and ma lignant transformation has proven difficult to define since both global hypomethylation and regional hypermethylation may occur simulta neously during various stages of tumor progression (18, 19). The implicit hypothesis in many studies of DNA methylation and carci- nogenesis is that hypomethylation of growth-promoting proto-onco- genes and/or hypermethylation of tumor suppressor genes will alter transcription factor binding and expression of these genes to promote a selective growth advantage for the initiated cell (6). An alternative consideration is that DNA hypomethylation may promote malignant transformation by inducing regional alterations in DNA conformation and chromatin structure. Local conformational changes can promote genomic instability by increasing the accessibility of specific se quences to DNA-damaging agents (20, 21). Loss of methylated cy- tosines alters the conformation and stability of the chromatin structure presumably by decreasing binding sites for methyl-specific proteins. In the absence of methyl-directed protein binding, affected DNA sequences are rendered more accessible to oxidant and/or enzyme- induced DNA strand breakage (22-24). Thus, dysregulation of DNA methylation patterns and associated changes in DNA-protein binding may promote neoplasia not only by altering the transcription of cancer-related genes but also by altering local DNA structure and sequence accessibility to DNA-damaging agents. In light of these considerations, we examined the kinetics of DNA strand breakage as related to methylation status of the DNA in livers from folate/methyl-deficient rats at intervals from 2 days to 9 weeks after diet initiation. Increased expression of the p53 gene is a DNA damage response that is initiated by the presence of DNA strand breaks (25). The p53 gene product also appears to function as a competence factor that is normally up-regulated during cellular pro liferation (25, 26). Both genome-wide and p53 gene-specific hypo methylation and DNA strand break accumulation were evaluated in the present study. The results are consistent with the hypothesis that hypomethylation induces regional alterations in chromatin/DNA 3 The abbreviations used are: SAM, S-adenosylmethionine; qPCR, quantitative PCR. 1894 on June 2, 2015. © 1995 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from