DNA methylation decreases in aging but not in immortal cells.

When normal diploid fibroblasts from mice, hamsters, and humans were grown in culture, the 5-methylcytosine content of their DNA's markedly decreased. The greatest rate of loss of 5-methylcytosine residues was observed in mouse cells, which survived the least number of division. Immortal mouse cell lines had more stable rates of methylation.

Induction of hepatitis B virus core gene in human cells by cytosine demethylation in the promoter.

A recombinant human cell line constructed by transfection of epithelial cells with a plasmid containing the hepatitis B virus core gene (HBc) was used to study the regulation of HBc gene expression. Methylation of a single Hpa II site 280 base pairs upstream from the structural gene was found to regulate the expression of the core gene. Expression increased in cells treated with 5'-azacytidine as a result of cytosine demethylation at this site, and there was a fivefold increase in the number of HBc gene transcripts in total cellular messenger RNA. The varied life cycle of hepatitis B virus in disease such as viral hepatitis and liver cancer may therefore be attributable to the site-specific regulation of the gene involved in replication of the viral DNA and to the cytophathic effects elicited by this gene in human cells.

Inhibition of DNA methylation in L1210 leukemic cells by 5-aza-2'-deoxycytidine as a possible mechanism of chemotherapeutic action.

The relationship between antineoplastic activity of 5-aza-2'-deoxycytidine (5-aza-dCyd) in mice with L1210 leukemia and inhibition of DNA methylation was investigated. BALB/c X DBA/2 F1 mice with L1210 leukemia were given a 15-hr i.v. infusion of 5-aza-dCyd at a total dose ranging from 0.5 mg/kg (weak antineoplastic effect) to 22 mg/kg (very potent antineoplastic effect). The DNA of L1210 leukemia cells was isolated from 5-aza-dCyd-treated mice and tested for its ability to accept methyl groups from S-adenosyl-L-methionine in a reaction catalyzed by DNA methyltransferase. The methyl-accepting ability of leukemia cell DNA was found to be dependent on the dose of 5-aza-dCyd, suggesting that this therapy induced significant changes in the level of methylation of the DNA. At the start of the 5-aza-dCyd infusion, mice were given i.p. injections of [6-3H]uridine, and the DNA of the L1210 leukemia cells was isolated at the end of therapy. Analysis of the labeled pyrimidine bases showed that 5-aza-dCyd produced a dose-dependent reduction in the 5-methylcytosine content of the DNA. Thus, there appears to be a correlation between the antileukemic activity of 5-aza-dCyd and its ability to inhibit DNA methylation.

Measurement of aflatoxin B1, its metabolites, and DNA adducts by synchronous fluorescence spectrophotometry.

Sensitive and specific methods are needed for measuring human exposure to carcinogens. Synchronous fluorescence spectrophotometry can be used to measure fmol of aflatoxins, their metabolites, and DNA adducts. Computer-assisted analysis of spectra of these agents obtained by synchronous fluorescence spectrophotometry can be displayed as contour maps which are highly specific for each agent. Individual agents in mixtures, e.g. aflatoxins B1 and M1, can be identified by fourth derivative spectral analysis. This physical method should complement immunological and other methods to measure aflatoxin B1, its metabolites, and nucleic acid adducts.

Increased sensitivity of nontumorigenic fibroblasts expressing ras or myc oncogenes to malignant transformation induced by 5-aza-2'-deoxycytidine.

The hypomethylating chemotherapeutic drug 5-aza-2'-deoxycytidine (5AzadC) has been shown to induce cell differentiation in some systems, while promoting neoplastic transformation in others. Using both in vitro and in vivo models, we have explored the relationship between oncogene expression and the susceptibility of cells to malignant transformation by 5AzadC. The study involved several nontumorigenic subclones of NIH3T3 fibroblasts, including cells transfected with deregulated c-myc, as well as phenotypic revertants expressing v-Ki-ras or long terminal repeat-activated c-Ha-ras. Transient 5AzadC treatment of the oncogene-bearing cell lines was associated with a rapid and efficient neoplastic transformation. In some cases, over 50% of the cell population exhibited loss of contact inhibition of growth within 1 week of treatment. The transformants were capable of forming s.c. tumors and experimental lung metastases in recipient nude mice. In contrast, 5AzadC failed to induce malignant properties in control 3T3 cultures transfected with the bacterial neor gene; rather, treatment of these cells was associated with differentiation into adipocytes and myotubes. The differential response to 5AzadC was also observed in vivo, in mice first inoculated s.c. with the premalignant cells and then treated with 5AzadC 24 h later. In agreement with the in vitro model, tumor development in mice correlated with the presence of cells with activated ras or myc oncogenes. Cytidine analogs that do not inhibit DNA methylation (i.e., 6-azacytidine and 1-beta-D-arabinofuranosyl cytosine) had no effect on cell phenotype. The results indicate that exposure of cells to 5AzadC can lead to tumor progression both in vitro and in vivo and suggest that preexisting alterations in oncogene expression may facilitate the evolution of cancerous growth induced by hypomethylating agents.

O6-alkyldeoxyguanosine detection by 32P-postlabeling and nucleotide chromatographic analysis.

The 32P-postlabeling procedure, developed originally by Randerath and coworkers, has been modified for the detection and analytical quantitation of O6-alkyl-2'-deoxyguanosine residues in DNA. Chromatographic techniques were developed to resolve individually the normal deoxyribonucleotide-3'-monophosphates and the O6-alkyldeoxyguanosine-3'-monophosphates by high-pressure liquid chromatography. Selective deoxyribonucleotide-3'-monophosphates (e.g., O6-alkyldeoxyguanosine-3'-monophosphates) were then converted to labeled deoxyribonucleotide-[5'-32P]monophosphates by 32P-postlabeling and nuclease P1 treatment and separated by two-dimensional thin layer chromatography. The O6-methyl- and O6-ethyl-2'-deoxyguanosine-3'-monophosphate nucleotides, and the respective 5'-monophosphates, were chemically synthesized for standardization of these quantitative procedures. The quantitation of O6-methl- and O6-ethyl-2'-deoxyguanosine was observed to be analytically accurate between one O6-alkyl-2'-deoxyguanosine residue per 10(4) and 10(7) 2'-deoxyguanosines. The limit of detection was less than one O6-alkyl-2'-deoxyguanosine in 10(7) 2'-deoxyguanosine residues in a sample size of 100 micrograms of DNA, i.e., approximately 10 pg of adduct. The quantitation of O6-methyl-2'-deoxyguanosine in the liver DNAs of rats treated with [14C-Me]N-nitrosodimethylamine compared well with values obtained by both 14C and high-pressure liquid chromatography coupled with fluorescence detection. Thus, these 32P-postlabeling and nucleotide chromatographic procedures should be useful in monitoring human exposure to methylating and ethylating carcinogens.

Combined high-performance liquid chromatography/32P-postlabeling assay of N7-methyldeoxyguanosine.

A highly sensitive and specific assay for the detection of N7-methyl-2'-deoxyguanosine (N7methyldG) has been developed by combining high-performance liquid chromatography, 32P-postlabeling, and nucleotide chromatography. Separation of normal nucleotides and adducts by high-performance liquid chromatography and then combining a portion of 2'-deoxyguanosine to the N7methyldG allows for quantitation using an internal standard. The directly determined molar ratio is not subject to errors in digestion, variable ATP-specific activity, or assumptions in relative adduct-labeling efficiency. The detection limit was one N7methyldG adduct in 10(7) unmodified 2'-deoxyguanosine bases. N7methyldG adducts have been detected in 5 human lung samples in which O6-methyl-2'-deoxyguanosine adducts had been previously determined. The mean ratio of N7methyldG to O6-methyl-2'-deoxyguanosine was determined to be approximately 10. The current assay complements the high-performance liquid chromatography/32P-postlabeling assay for O6-methyl-2'-deoxyguanosine and increases the detection sensitivity of DNA methylated by exogenous alkylating agents.

Oncogenic base substitution mutations in circulating leukocytes of normal individuals.

The background frequency of mutations in human tissues is an important issue in cancer susceptibility and genotoxic exposure determinations. Here we report the detection of rare mutant leukocytes containing oncogenic base substitutions of the Harvey-ras, N-ras, and p53 genes by the Needle-in-a-Haystack mutation assay with a sensitivity of one cell in a million. Altogether, we detected and identified 17 independent mutations of 66 separate base site analyses of peripheral blood specimens obtained from 19 apparently normal individuals. Two individuals harbored a substantially increased frequency of mutant cells, representing 9 of the 17 independent mutations found. These results suggest that up to 1 in 10 normal individuals may harbor a significant frequency of oncogenic mutations in circulating leukocytes.

Fractionation of the sequence organization of human DNA by preparative HgCl2/Cs2SO4 density gradients.

Human DNA has been fractionated on preparative HgCl2/Cs2SO4 density gradients. Repetitious sequences are found in DNA components of all base compositions but the (G + C)-rich sequences are enriched for repetitive sequences in (A + T)-rich components are tandemly arranged; those in (G + C)-rich components tend to be interspersed with single copy sequences. It is concluded that the interspersed repetitious sequences have high (G + C) compositions.

Phenylacetate in chemoprevention: in vitro and in vivo suppression of 5-aza-2'-deoxycytidine-induced carcinogenesis.

Differentiation inducers selected for their low cytotoxic and genotoxic potential could be of major value in chemoprevention and maintenance therapy. We focus here on phenylacetate, a naturally occurring plasma component recently shown to affect the growth and differentiation of established neoplasms in experimental models. The ability of phenylacetate to prevent carcinogenesis by the chemotherapeutic hypomethylating drug 5-aza-2'-deoxycytidine (5AzadC) was tested in vitro and in mice. Transient exposure of immortalized, but poorly tumorigenic ras-transformed 4C8 fibroblasts to 5AzadC resulted in neoplastic transformation manifested by loss of contact inhibition of growth, acquired invasiveness, and increased tumorigenicity in athymic mice. The latter was associated with elevation in ras expression and a decline in collagen biosynthesis. These profound phenotypic and molecular changes were prevented by a simultaneous treatment with phenylacetate. Protection from 5AzadC carcinogenesis by phenylacetate was: (a) highly efficient despite DNA hypomethylation by both drugs, (b) free of cytotoxic and genotoxic effects, (c) stable after treatment was discontinued, and (d) reproducible in vivo. Whereas athymic mice bearing 4C8 cells developed fibrosarcomas following a single i.p. injection with 5AzadC, tumor development was significantly inhibited by systemic treatment with nontoxic doses of phenylacetate. Phenylacetate and its precursor suitable for oral administration, phenylbutyrate, may thus represent a new class of chemopreventive agents, the efficacy and safety of which should be further evaluated.

Abnormal folate metabolism and mutation in the methylenetetrahydrofolate reductase gene may be maternal risk factors for Down syndrome.

BACKGROUND: Down syndrome, or trisomy 21, is a complex genetic disease resulting from the presence of 3 copies of chromosome 21. The origin of the extra chromosome is maternal in 95% of cases and is due to the failure of normal chromosomal segregation during meiosis. Although advanced maternal age is a major risk factor for trisomy 21, most children with Down syndrome are born to mothers <30 y of age. OBJECTIVE: On the basis of evidence that abnormal folate and methyl metabolism can lead to DNA hypomethylation and abnormal chromosomal segregation, we hypothesized that the C-to-T substitution at nucleotide 677 (677C-->T) mutation of the methylenetetrahydrofolate reductase (MTHFR) gene may be a risk factor for maternal meiotic nondisjunction and Down syndrome in young mothers. DESIGN: The frequency of the MTHFR 677C-->T mutation was evaluated in 57 mothers of children with Down syndrome and in 50 age-matched control mothers. Ratios of plasma homocysteine to methionine and lymphocyte methotrexate cytotoxicity were measured as indicators of functional folate status. RESULTS: A significant increase in plasma homocysteine concentrations and lymphocyte methotrexate cytotoxicity was observed in the mothers of children with Down syndrome, consistent with abnormal folate and methyl metabolism. Mothers with the 677C-->T mutation had a 2.6-fold higher risk of having a child with Down syndrome than did mothers without the T substitution (odds ratio: 2.6; 95% CI: 1.2, 5.8; P < 0.03). CONCLUSION: The results of this initial study indicate that folate metabolism is abnormal in mothers of children with Down syndrome and that this may be explained, in part, by a mutation in the MTHFR gene.

Detecting rare mutations associated with cancer risk.

For more than a decade, investigators have been searching for a means of determining the risk of individuals developing cancer by detecting rare oncogenic mutations. The accumulation of mutations and the clonal evolvement of tumors provide opportunities for monitoring disease development and intervening prior to the presentation of clinical symptoms, or determining the risk of disease relapse during remission. A number of techniques, mostly polymerase chain reaction (PCR)-based, have been developed that enable the detection of rare oncogenic mutations within the range of 10(-2) to 10(-4) wild-type cells. Only a handful of procedures enable the detection of intragenic single base mutations at one mutant in 10-6 or better. These ultra-sensitive mutation detection techniques have produced some interesting results regarding single base mutation spectra and frequencies in p53, Harvey-ras, N-ras, and other reporter genes and DNA sequences in human tissues. Although there is evidence that some individuals may harbor cells or clones expressing genomic instability, the connection with the processes of carcinogenesis is still tenuous. There remains a need for rigorous epidemiological studies employing these ultra-sensitive mutation detection procedures. Since genomic instability is considered key to tumor development, the relevance of the detection of hypermutable clones in individuals is discussed in the context of cancer risk.

Detection and quantification of 8-hydroxydeoxyguanosine adducts in peripheral blood of people exposed to ionizing radiation.

Ionizing radiation produces a variety of damaging insults to nucleic acids, including the promutagenic lesion 8-hydroxydeoxyguanosine. In the present study, the 8-hydroxydeoxyguanosine content of peripheral blood leukocyte DNA isolated from individuals exposed to therapeutic doses of ionizing radiation was determined by a HPLC-coupled 32P-postlabeling assay. Peripheral blood leukocyte DNA from individuals irradiated with 180-200 cGy were observed to contain 2-4.5 times as much 8-hydroxydeoxyguanosine as that from unexposed individuals. These results were confirmed by the use of a HPLC-coupled electrochemical detection system. Thus, human exposure to ionizing radiation significantly increased the circulating leukocyte DNA content of 8-hydroxydeoxyguanosine.

Effect of mutagen-induced cell lethality on the dose response of germline mutations.

Molecular tests for mutations require a sample of tissue from which DNA is extracted, to determine the presence or absence of one or more mutations per sample. To ensure mutation fixation each sample must consist of an equal number of cells that have had one or more DNA replications. In an in vivo test, surviving stem cells compensate to give the same number of cells per sample, leaving as the only evidence for stem cell lethality the increase in mutants of clonal origin because the mutant clone developed from a population of fewer stem cells. A problem is that an increase in mutagen dose increases stem cell death, resulting in a decreased number of surviving target cells, thus giving a downward bias of samples with one or more mutations per sample. To compare in vivo tests with molecular tests we will use as a model system the sex-linked recessive lethal (SLRL) test for germ cell mutations in Drosophila melanogaster. Spermatogonia cells in male larvae were exposed to ENU and mutations detected in sperm cells from adults. The same SLRL data were analyzed by two methods: (1) The conventional analysis of SLRL data, in which each mutation of a cluster of mutations of common origin was counted. (2) An analysis was used to simulate a sample for molecular analysis by determining mutations per male with an equal size sample of progeny per male. With this second analysis a correction factor is required based on the change in cluster size of mutants of common origin.

Inhibition of DNA methylation by 5-azacytidine.

The nucleoside analog z5C induced marked changes in the differentiated state of mouse embryo cells and inhibited the methylation of newly synthesized DNA. Other analogs of cytidine containing modifications in the 5 position (5-aza-2'-deoxycytidine, 5-fluoro-2'-deoxycytidine, and pseudoisocytidine) also induced the formation of striated muscle cells in treated cultures and inhibited DNA methylation. Together the data suggests a causative role for the methylation of specific cytosine residues in the control of gene expression. Hemimethylated duplex DNA was extracted from cultures treated with z5C and was an efficient acceptor of methyl groups from S-adenosyl-methionine in the presence of a mouse spleen methyltransferase. The ability of this hemimethylated DNA to accept methyl groups was markedly impaired if it was pretreated with several different ultimate chemical carcinogens.

Concordant p53 and mdm-2 protein expression in vulvar squamous cell carcinoma and adjacent lichen sclerosus.

To determine if carcinogenic events in vulvar skin precede the onset of morphologic atypia, the authors investigated for derangements in DNA content, cell proliferation, and cell death in vulvar carcinomas and surrounding skin in 140 samples of tumor and surrounding skin collected from 35 consecutive vulvectomy specimen for squamous cell carcinoma (SCC) or vulvar intraepithelial neoplasia (VIN) 3. Vulvar non-cancer excisions were used as controls. Investigations consisted of histologic classification and measurement of 9 variables--epidermal thickness (acanthosis and rete ridge length), immunolabeling index (LI) for 3 proteins (p53 protein, Ki-67, and mdm-2), pattern of p53 expression (dispersed vs. compact), DNA content index, and presence of aneuploidy by image analysis and apoptotic rate by Apotag labeling. Significant positive correlations were found for all nine variables studied versus increasing histologic severity in two proposed histologic stepwise models of vulvar carcinogenesis (lichen sclerosus (LS) and VIN 3 undifferentiated associated SCC groups). High p53 LI (>25) and the compact pattern of p53 expression (suspected oncoprotein) significantly correlated with LS and its associated vulvar samples compared with samples not associated with LS (P < or = 0.001). Furthermore, p53 LI, mdm-2 LI, and pattern of p53 expression were concordant between patient matched samples of LS and SCC. In addition, mdm-2 LI significantly correlated with dispersed pattern p53 LI suggesting a response to wild-type p53 protein accumulation. These findings support the hypothesis that neoplastic transformation occurs in sequential steps and compromises proteins involved in the cell cycle control. Concordance of p53 and mdm-2 protein expression in LS and adjacent SCC provides evidence that LS can act as a precursor lesion in the absence of morphologic atypia. Overexpression of mdm-2 with stabilization and inactivation of p53 protein may provide an alternate pathway for vulvar carcinogenesis.

Species-specific detection of hydrocarbon-utilizing bacteria.

Rapid detection and quantitative assessment of specific microbial species in environmental samples is desirable for monitoring changes in ecosystems and for tracking natural or introduced microbial species during bioremediation of contaminated sites. In the interests of developing rapid tests for hydrocarbon-degrading bacteria, species-specific PCR primer sets have been developed for Pseudomonas aeruginosa, Stentrophomonas (Xanthomonas) maltophilia, and Serratia marsescens. Highly variable regions of the 16S rRNA gene were used to design these primer sets. The amplification products of these primer sets have been verified and validated with hemi-nested PCR and with ligase chain reaction (LCR) techniques, and have been applied to the analyses of environmental water samples. These species-specific primer sets were also chosen to amplify in conjunction with a universal set of PCR primers chosen from highly conserved neighboring sequences in the same gene. These multiplex or competitive PCR procedures enable testing with an internal marker and/or the quantitative estimation of the relative proportion of the microbial community that any one of these species occupies. In addition, this universal PCR primer set amplified the same size amplicon from a wide spectrum of procaryotic and eucaryotic organisms and may have potential in earth biota analyses.

Tachyphylaxis to furosemide in isolated airways of guinea pigs.

This in vitro study was conducted to determine whether tachyphylaxis of guinea pig airway to furosemide occurs under conditions that produce tachyphylaxis to the beta 2-adrenoceptor agonist, salbutamol. Isometric tension was measured in tracheal rings bathed in HEPES buffer from 4-6 d newborn guinea pigs of either sex, and 6 wk old males. Paired rings were first incubated with furosemide, 30 or 300 microM, or control for 60 min, washed, then constricted with 3 microM acetylcholine. At stable contraction, relaxation to furosemide (30 microM-1 mM) was measured. For comparison, similar experiments were performed with 10 microM salbutamol incubation for 30 min. 86Rb uptake, a marker for K+ transport and Na-K-Cl cotransport activity, was also measured in these airway segments. Pre-exposure to these airway relaxants did not affect contractile force generation by acetylcholine. Tracheal desensitization to both salbutamol and furosemide was observed. Partial recovery of furosemide induced relaxation was seen one hour after desensitization. Pre-exposure to 300 microM furosemide did not inhibit the decrease in 86Rb uptake normally observed with furosemide. In summary, we found that: 1) tachyphylaxis of guinea pig airway relaxation occurred with both salbutamol and furosemide under similar experimental conditions; however 2) inhibition of 86Rb uptake by furosemide was not affected by prior exposure. Taken together, these results suggest that furosemide induced airway relaxation could be affected by repeated or prolonged exposure, but this response may not be associated with changes in furosemide-sensitive Na-K-Cl cotransporter activity.

A non-invasive method for the study of hepatic drug metabolism in rodents: antineoplastic drug effects on antipyrine metabolism in mice.

A rapid, sensitive and simple high pressure liquid chromatography (HPLC) method is described for the direct analysis of antipyrine in saliva. The detection limit was found to be 1.0 ng/ml of sample, lower than any previously reported method. Accuracy and precision were maintained with as little as 0.5 microliter of saliva. Thus the rate of elimination of antipyrine has been monitored non-invasively in rats and for the first time in mice. The antipyrine half-life was found to be 28.9 +/- 4.0 (S.E.M.) min and 111 +/- 20 min in mice and rats, respectively. In mice single i.p. doses of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) (30 mg/kg) produced increases in antipyrine half-life, up to 28 days post-treatment. The maximum effect of BCNU was observed on day 7 with an antipyrine half-life of 74.4 +/- 15.7 min. Phenobarbital induction lowered the antipyrine half-life in controls to 12.6 +/- 1.2 min. An enhanced inductive effect was observed in BCNU-treated mice: BCNU-treated, phenobarbital-induced mice displayed a half-life for antipyrine of 7.4 +/- 0.6 min on day 21 post BCNU dose. These effects could not be attributed to changes in absorption of antipyrine in BCNU-treated mice.

Temporal delineation of sequential HPRT mutations arising in vivo in a T-cell clone with a mutator phenotype.

Recurrent mutations in vivo in T-lymphocytes identify clonally restricted genomic instabilities in some individuals. Cell-based assays allow initial recognition of clones with mutator phenotypes, but genotypic selection is required to determine frequencies and temporal sequences of potentially independent mutational events isolated only as complex changes in the same allele. The present work illustrates how two single-base insertions in the HPRT gene recovered only as a double event in a cell-based assay were shown to arise as separate in vivo mutations, being individually present at frequencies of < or =10(-4) and < or =10(-5), respectively, in peripheral blood. Full characterizations of mutator clones will allow elucidation of the earliest events in the emergence of genomic instability in human somatic cells.