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...

Chronically instrumented dogs underwent 2- or 5-h regional reductions in coronary flow that were followed, respectively, by balanced reductions in myocardial contraction and O(2) consumption ("hibernation") and persistently reduced contraction despite normal myocardial O(2) consumption ("stunning"). Previously unidentified myofibrillar disruption developed during flow reduction in both experimental models and persisted throughout the duration of reperfusion (2-24 h). Aberrant perinuclear aggregates that resembled thick filaments and stained positively with a monoclonal myosin antibody were present in 34 +/- 3.8% (SE) and 68 +/- 5.9% of "hibernating" and "stunned" subendocardial myocytes in areas subjected to flow reduction and in 16 +/- 2.5% and 44 +/- 7.4% of subendocardial myocytes in remote areas of the same ventricles. Areas of myofibrillar disruption also showed glycogen accretion and unusual heterochromatin clumping adjacent to the inner nuclear envelope. The degrees of flow reduction employed were sufficient to reduce regional myofibrillar creatine kinase activity by 25-35%, but troponin I degradation was not evident. The observed changes may reflect an early, possibly reversible, phase of the myofibrillar loss characteristic of hypocontractile myocardium in patients undergoing revascularization.

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The fluorescent proteinase transition-state analog inhibitor, dansyl-L-argininal (DnsArgH), may be a selective probe of cysteine and serine-type proteinases in a fibrosarcoma tumor cell line (HSDM1C1). DnsArgH binds with high affinity to proteinases because of its transition-state analog properties, and on association it gives a dramatically increased fluorescent yield. The DnsArgH binding is inhibited by the serine proteinase inhibitor diisopropyl fluorophosphate and by the cysteine proteinase inhibitor p-chloromercuribenzoate. Significant evidence exists for the role of proteolytic enzymes in malignant transformation, tumor cell invasion, and tumor cell metastasis (1-3). For example, treatment of normal cells with proteolytic enzymes can lead to the appearance of phenotypic characteristics in the untransformed cells that are similar to those of transformed cells (1, 4-7). In addition, proteolytic enzymes secreted from transformed cells may degrade host tissues into which the tumor cells invade, and may, through the degradation of surrounding tissue, promote tumor cell metastasis (1-3). Particular proteinases are reported to be secreted in larger amounts by transformed cells than their normal counterparts. These identified proteinases secreted by transformed cells include the serinetype proteinase plasminogen activator (1, 2, 8, 9), the lysosomal cysteine-type proteinase cathepsin-B (1, 10), and collagenolytic proteinases (1,3). The existence of a role for proteolytic enzymes in tumorigenesis and metastasis is supported by experiments in which these processes are shown to be inhibited by inhibitors of proteinase enzymes (1, 2).Whereas a large amount of evidence exists for secretion into the extracellular environment of relatively high concentrations of proteinases by tumor cells, recent reports also document the existence of proteinase activity either associated with the outer cell membrane or membrane fractions within transformed cells (1,2,(11)(12)(13)(14)(15)(16)(17)(18)(19). Similar to the types of proteinases reported to be found secreted into extracellular fluid, both a neutral trypsin-like seine proteinase, which may be plasminogen activator (1,2,(11)(12)(13)(14)(15)(16), and a cathepsin-B cysteine proteinase (1, 17) have been reported. A high concentration of cell surface proteinases may provide an intimate mechanism by which the membrane of the transformed cell can both maintain malignant traits within the tumor and promote the degradation of surrounding tissue during tumor cell invasion and metastasis.In this communication we report the development of a general proteinase fluorescent probe, dansyl-L-argininal (DnsArgH), which is capable of detecting both serine and cysteine proteinases with a primary site specificity toward arginine. Thus, both the neutral trypsin-like serine proteinases and the cathepsin-B-like cysteinyl proteinase, previously reported to be associated with transformed cells should be detected with DnsArgH. The fluorescent aldehyde binds to proteinases with a high affinity an...