Methylation of cytosines in DNA is important for the regulation of expression of many genes. During carcinogenesis, normal patterns of gene methylation can be altered. Oxygen radical injury, shown to damage DNA in a variety of ways associated with cancer development and other conditions, has been suggested to affect DNA methylation, but a mechanism has not been demonstrated. Using oligonucleotides containing the common oxygen radical adduct 8-hydroxyguanine to replace guanine, we found that the enzymatic methylation of adjacent cytosines is profoundly altered. Furthermore, there is a high degree of positional specificity with respect to this effect. Thus, free radical injury may explain some of the altered methylation observed during carcinogenesis. MATERIALS AND METHODSFour synthetic oligonucleotides containing 8-hydroxyguanine residues were kindly provided by Francis Johnson (23). Two of the four 8-hydroxyguanine-containing oligomers also contained 5-methylcytosines at the CpG site. Sequences of the oligomers were as follows:Endogenously generated reactive oxygen species, such as peroxides and oxygen free radicals, may play an important role in carcinogenesis (reviewed in refs. 1-3). These substances induce DNA strand breaks (4) and a number of specific types of adducts to DNA bases (1, 5, 6). These oxidants also produce a variety of other effects characteristic of carcinogens, including induction of malignant transformation in tissue culture (7,8), chromosomal changes, mutations, and gene amplification. In addition, we recently observed alterations in specific DNA sequences in oxidant-transformed cells suggestive of altered cytosine methylation (9, 10). Cytosine methylation is a covalent modification of DNA that is important in gene regulation; it may have other functions as well (11)(12)(13)(14). Most 5-methylcytosines occur at CpG sites. There is evidence that tissue-specific patterns of methylation are established during embryonic development and are faithfully maintained from cell generation to generation. In recent years, alterations in cytosine methylation, most commonly hypomethylation, have been associated with the development of cancer (15,16). With the recognition that 8-hydroxyguanine is a common oxygen radical adduct of DNA (5, 6, 17-22), we constructed a model system to investigate the possible interrelationship between oxidants and DNA methylation. This model system consisted of a DNA methylase (Hpa II methylase), S-adenosylmethionine (as a source of methyl groups), and a series of synthetic deoxynucleotide oligomers containing complementary CCGG sites (the Hpa II methylase recognition site). We found that substitution of either of the guanines of the CCGG recognition site with 8-hydroxyguanine, a common oxygen radical-induced guanine derivative, dramatically altered binding of the methylase to the oligomer and could dramatically inhibit methylation. Thus, oxygen radical injury to DNA may influence gene expression by affecting DNA methylation. OH D, 5'-GTACCCG6TGACACACC-3'Complementary s...
8-Hydroxyl-2'-deoxyguanosine (also referred to as 8-hydroxyguanine [8-OH-dG] or 7,8-dihydro-8-oxoguanine), a common DNA adduct resulting from injury to DNA via reactive oxygen species, affects the in vitro methylation of nearby cytosine moieties by the human DNA methyltransferase. The exact position of 8-OH-deoxyguanosine relative to a CpG dinucleotide appears important to this effect. Our data indicate that 8-OH-deoxyguanosine diminishes the ability of the methyltransferase to methylate a target cytosine when the 8-OH-deoxyguanosine is one or two nucleotides 3' from the cytosine, on the same strand. On the other hand 8-OH-deoxyguanosine does not diminish the ability of the enzyme to respond to a methyl director (5-methylcytosine) when the 8-OH-deoxyguanosine is on the same strand but one or two nucleotides 3' from the methyl director. Differences in methylation rates as great as 13-fold have been detected using various 8-OH-deoxyguanosine-containing oligonucleotides as substrates in methylation assays. Our findings suggest that oxidative damage of parental strand guanines would permit normal copying of methylation patterns through maintenance methylation, while oxidative damage of guanines in the nascent strand DNA would inhibit such methylation.
Laminin-5, the major extracellular matrix protein produced by mammary epithelial cells, is composed of three chains (designated ␣3A, 3, and ␥2), each encoded by a separate gene. Laminin-5 is markedly down-regulated in breast cancer cells. Little is known about the regulation of laminin gene transcription in normal breast cells, nor about the mechanism underlying the down-regulation seen in cancer. In the present study, we cloned the promoter of the gene for the human laminin ␣3A chain (LAMA3A) and investigated its regulation in functionally normal MCF10A breast epithelial cells and several breast cancer cell lines. Using site-directed mutagenesis of promoter-reporter constructs in transient transfection assays in MCF10A cells, we find that two binding sites for Kruppel-like factor 4 (KLF4/GKLF/EZF) are required for expression driven by the LAMA3A promoter. Electrophoretic mobility shift assays reveal absence of KLF4 binding activity in extracts from T47D, MDA-MB 231, ZR75-1, MDA-MB 436, and MCF7 breast cancer cells. Transient transfection of a plasmid expressing KLF4 activates transcription from the LAMA3A promoter in breast cancer cells. A reporter vector containing duplicate KLF4-binding sites in its promoter is expressed at high levels in MCF10A cells but at negligible levels in breast cancer cells. Thus, KLF4 is required for LAMA3A expression and absence of laminin ␣3A in breast cancer cells appears, at least in part, attributable to the lack of KLF4 activity.The laminin family of extracellular matrix glycoproteins is heterotrimeric proteins consisting of three distinct subunits, designated ␣, , and ␥ that are encoded by the LAMA, LAMB, and LAMC genes, respectively. To date there are five ␣ chains, three  chains, and two ␥ chains that assemble into 12 laminins. Laminin-5 (␣3A, 3, and ␥2) is the major extracellular matrix protein produced in mammary epithelial cells. In these cells laminin-5 functions as a ligand for the ␣ 3  1 and ␣ 6  4 integrins to regulate adhesion, migration, and morphogenesis The murine LAMA3A promoter has been studied and found to contain three binding sites for the complex dimeric transcription factor, AP-1, one site of which is essential for basal expression of LAMA3A in keratinocytes (4). Mutation of this single key AP-1 site reduced promoter activity by ϳ90% while mutation of the other two sites had much less effect (4). In the present study we analyze the human LAMA3A promoter in the MCF10A mammary epithelial cell line and the T47D breast cancer cell line. We sought to find a mechanism that would explain the LAMA3A down-regulation in the nonexpressing cells. In doing this, we demonstrated a key role for the transcription factor Kruppel-like factor 4 (KLF4)
We examined expression of N-methylpurine-DNA glycosylase (MPG), a DNA repair enzyme that removes Nalkylpurine damage, in normal, malignant, and immortalized breast epithelial cells, and breast cancer cell lines (MDA-MB-231, MCF7, T47D). Northern analysis showed increased expression in cancer versus normal breast epithelial cells (2^24-fold). Southern blots revealed no gene amplification or polymorphisms. Immunofluorescence, immunohistochemistry, and Western blot analysis demonstrated increased MPG protein expression in the tumor cells that correlated with elevated glycosylase activity. Since MPG overexpression has been shown to be paradoxically associated with increased susceptibility to DNA damage, up-regulation of this gene may suggest a functional role in breast carcinogenesis.z 1998 Federation of European Biochemical Societies.
8-OH-deoxyguanosine can diminish the ability of the restriction endonucleases Hpa II and Msp I to cleave DNA. The exact position of the adduct within the recognition site appears to determine the extent of the effect.
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