N-myc amplification in Wilms' tumor.
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Child, Preschool↗
Biomedical subjects
B W Stewart
Publications and source records attributed to B W Stewart.
Persistent and heritable structural damage induced in heterochromatic DNA from rat liver by N-nitrosodimethylamine.
Analysis, by benzoylated DEAE-cellulose chromatography, has been made of structural change in eu- and heterochromatic DNA from rat liver following administration of the carcinogen N-nitrosodimethylamine (10 mg/kg body weight). Either hepatic DNA was prelabeled with [3H]thymidine administered 2-3 weeks before injection of the carcinogen or the labeled precursor was given during regenerative hyperplasia in rats treated earlier with N-nitrosodimethylamine. Following phenol extraction of either whole liver homogenate or nuclease-fractionated eu- and heterochromatin, carcinogen-modified DNA was examined by stepwise or caffeine gradient elution from benzoylated DEAE-cellulose. In whole DNA, nitrosamine-induced single-stranded character was maximal 4-24 h after treatment, declining rapidly thereafter; gradient elution of these DNA preparations also provided short-term evidence of structural change. Following incubation of purified nuclei with micrococcal nuclease, 10-12% of labeled DNA was solubilized (eu-chromatin) by 1.0 unit of micrococcal nuclease (5 mg of DNA)-1 mL-1 after 9 min. In prelabeled animals, administration of N-nitrosodimethylamine caused a marked fall in the specific radioactivity of solubilized DNA, while that of sedimenting DNA was not affected. Caffeine gradient chromatography suggested short-term nitrosamine-induced structural change in euchromatic DNA, while increased binding of heterochromatic DNA was evident for up to 3 months after carcinogen treatment. Preparations of newly synthesized heterochromatic DNA from animals subjected to hepatectomy up to 2 months after carcinogen treatment provided evidence of heritable structural damage. Carcinogen-induced binding of heterochromatic DNA to benzoylated DEAE-cellulose was indicative of specific structural lesions whose affinity equalled that of single-stranded DNA up to 1.0 kilobase in length.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
DNA-mediated gene transfer as an indicator of DNA damage and its repair by recipient cells.
Preparations of plasmid containing the thymidine kinase gene (pHSV106) were treated with the alkylating agents methyl methanesulphonate or N-methyl-N-nitrosourea prior to transfection into thymidine kinase-deficient mouse L-cells using the DNA-calcium phosphate co-precipitation technique. Relative to transfection with unmodified plasmid, a reduced transformation efficiency was observed using alkylation-damaged plasmid, N-methyl-N-nitrosourea causing the greatest inhibition. Treatment of recipient cells with arabinosyl cytosine or dideoxythymidine during the expression period following transfection by the 'damaged' plasmid reduced transformation efficiency, suggesting that DNA repair 4-6 h post-transfection was required for gene expression.
Animals↗
Persistent structural change in replicating hepatic DNA isolated from diethylnitrosamine-treated rats.
Structural analysis, by benzoylated O-(diethylaminoethyl) (DEAE)-cellulose chromatography, was made of DNA from the livers of rats receiving 100 mg/kg diethylnitrosamine and subsequently subjected to partial hepatectomy. Under these conditions, different DNA labelling procedures permit damage to be associated with pre-existing or newly synthesised DNA. Persistent single stranded regions could be detected in DNA isolated more than 3 days after carcinogen treatment only if the animals were subjected to hepatectomy. This damage was attributable to lesions impeding DNA replication. Induction of proliferative activity up to at least 14 days after nitrosamine treatment made manifest DNA damage, the extent of which was not decreased as the interval between carcinogen treatment and surgery was increased.
Animals↗
Inhibition of poly(ADP-ribose) synthesis may affect DNA repair prior to ligation.
The effects of modification of poly(ADP-ribosyl)ation reactions have been examined in normal (F107) and ataxia telangiectasia (AT23) fibroblasts following damage by methyl methanesulphonate (MMS) and u.v. light. The technique of benzoylated DEAE (BD)-cellulose chromatography was utilized to estimate both the extent and nature of the damage to DNA induced by these agents and to examine the effects of an inhibitor of poly(ADP-ribose) synthetase, 3-aminobenzamide (3AB), on these parameters. Single strand breakage, determined by nucleoid sedimentation, and levels of poly ADP(ribose) synthesis were monitored. Increase in the proportion of DNA containing single-stranded regions, as measured by stepwise elution from BD-cellulose, was observed following MMS damage in both cell types. In the presence of 3AB, a further accumulation of DNA containing single-stranded regions occurred, with the effect being more prominent in AT23 fibroblasts. U.v. light damage did not induce increased binding to BD-cellulose in normal cells, and the increase observed in AT23 cells was much less than that seen following alkylation damage. Examination of the nature of single-stranded damage by caffeine gradient elution from BD-cellulose following MMS treatment revealed discrete structural lesions, which were enhanced in the presence of 3AB. A similar effect was exerted by arabinofuranosyl cytosine. The behaviour of these intermediates, which could be associated with repair, was not in accord with the suggestion that 3AB inhibits only the ligation stage of the repair process. Our results suggest that specific intermediate stages in DNA repair are sensitive to 3AB, and it seems likely that these stages occur prior to ligation.
Ataxia Telangiectasia↗
Long-term persistence of nitrosamine-induced structural damage to heterochromatic DNA.
Different levels of damage and repair to eu- and heterochromatic DNA from the livers of rats receiving a dose of 10 mg/kg N-nitrosodimethylamine (NDMA) were apparent. Preincorporated 3H-thymidine was lost rapidly from euchromatic DNA but persisted in the heterochromatic fraction. Persistent damage, determined as single-stranded regions binding to benzoylated DEAE-cellulose (BD-cellulose), was evident in heterochromatic DNA for up to three months. By subjecting rats treated with NDMA to partial hepatectomy, generation of single-stranded regions in the newly synthesized heterochromatic DNA could be demonstrated. Such structural defects were evident when hepatectomy was performed two months after administration of the carcinogen. These findings indicate that structural damage to nontranscribed DNA is one of the most persistent molecular lesions following exposure to nitrosamines.
Animals↗
Two-stage incorporation of thymidine triphosphate into mammalian DNA as indicated by chromatography on benzoylated DEAE-cellulose.
Replicating DNA contains single-stranded regions as indicated by the extent of its recovery in the final fraction, following stepwise elution of sheared preparations from benzoylated DEAE-cellulose using 0.3 M sodium chloride, 1.0 M sodium chloride and caffeine solutions, respectively. In preparations of hepatic DNA, isolated up to 60 min after administration of [3H]thymidine to rats subjected earlier to partial hepatectomy, the proportion of radioactivity contained in the caffeine-eluted fraction progressively decreased. This change was attributable to migration of incorporated radioactivity from replicating forms to mature (double-stranded) DNA, the latter being recovered from the column in 1.0 M sodium chloride. In terms of the same chromatograms, the relative size of the 0.3 M sodium chloride-eluted fraction also decreased, this fraction of radioactivity co-eluting from benzoylated DEAE-cellulose with thymidine triphosphate. The precursor--intermediate--product relationship between the three products separated by benzoylated DEAE-cellulose chromatography was confirmed using DNA isolated from cultured mammalian cells. Early eluting radioactivity co-chromatographed with thymidine triphosphate on thin-layer cellulose, whilst the intermediate status of caffeine-eluted radioactivity was confirmed following pulse-chase labelling procedures. Utilizing stepwise chromatography of such DNA on benzoylated DEAE-cellulose, the effect of three inhibitors could thus be described as affecting either an early or late stage of DNA synthesis. Such an approach offers a simple quantitative method of monitoring influences on DNA synthesis.
Animals↗
Digestion of repair sites in rat liver DNA by endogenous nucleases.
The proportion of sheared rat liver DNA recovered from benzoylated DEAE-cellulose in the final stage following stepwise elution with NaCl and caffeine solutions was dependent upon the DNA isolation procedure. An increase in the proportion of DNA containing single stranded regions, consequent upon delay or addition of Mg2+ prior to phenol extraction, suggested nuclease mediated degradation. Administration of methyl methanesulphonate to rats resulted in a consistent proportional increase in the caffeine-eluted fraction. The results of caffeine gradient elution of control and alkylated DNA from benzoylated DEAE-cellulose were consistent with repair-associated single stranded regions being substrates for endogenous single strand-specific exonucleases.
Animals↗
Single-strand-specific degradation of DNA during isolation of rat liver nuclei.
We have investigated structural change in rat liver DNA produced by different isolation procedures and specifically compared the integrity of DNA derived by phenol extraction from isolated and purified nuclei with preparations extracted immediately from a crude liver homogenate containing intact nuclei. As indicated by stepwise elution from benzoylated DEAE-cellulose, most structural change in DNA was evident following nuclei isolation. Damage principally involved generation of single-stranded regions in otherwise double-stranded DNA fragments; totally single-stranded DNA was not detected by hydroxylapatite chromatography. Caffeine gradient elution suggested formation of single-stranded regions extending for up to several kilobases. In neutral sucrose gradients, differences in sedimentation rates of respective DNA samples consequent upon S1 nuclease digestion could be detected after isolation of nuclei, though not in other circumstances. The observed single-strand-specific nuclease digestion of DNA could apparently be reduced if steps were taken to reduce autodigestion during nuclei isolation by reduction of temperature and covalent cation concentration. The results are discussed in terms of the use of exogenous and endogenous nucleases in chromatin fractionation studies involving isolated nuclei and possible artifactual findings that may be generated by single-strand-specific autodigestion.
Animals↗
Oncogenes. A possible role for cancer genes in human malignant disease.
A small class of normal cellular genes has the potential to transform cells malignantly. These cellular oncogenes have been identified by their similarity to the transforming genes of oncogenic RNA viruses (retroviruses), and by their ability, under certain circumstances, to transform cells into which they have been transferred. Cellular oncogenes are not normally tumorigenic and, indeed, appear to be critically involved in normal cellular processes, possibly at the level of growth regulation. However, it appears that a number of mechanisms exist whereby these oncogenes can become malignantly activated, leading to uncontrolled cellular proliferation. The evidence implicating cellular oncogenes in human malignant disease is reviewed.
Animals↗
Dose-dependent persistence of alkylation-induced single stranded regions in rat liver DNA in vivo.
Single stranded regions in DNA, presumed to be indicative of DNA repair, may be readily detected in rat liver DNA following injections of non-necrotizing single doses of methyl methanesulfonate, dimethylnitrosamine and diethylnitrosamine. The present study concerns persistence of such structural defects in vivo, as determined by benzoylated DEAE-cellulose chromatography, in relation to the dose of alkylating agent. For all these agents, the period during which structural damage may be detected is markedly dependent upon dose: prolonged persistence only occurs after the highest dose. The findings, in relation to other data, implicate reaction processes involving the final stages of repair as being critical to prolongation of this type of genomic damage in vivo.
Alkylating Agents↗
Patterns of structural change in DNA during tissue necrosis indicated by benzoylated DEAE-cellulose chromatography.
Structural change in liver DNA, isolated following administration of a necrogenic dose of carbon tetrachloride to rats, was examined by benzoylated DEAE-cellulose (BD-cellulose) chromatography. Greatest increase in the amount of DNA exhibiting single stranded character was detected 48 h after treatment, at which time massive necrosis was evident histologically. By 72 h after dosing, normal hepatic architecture had been restored and gross structural change in DNA could no longer be detected. The extent of single stranded regions in DNA isolated at various times after carbon tetrachloride intoxication was assessed by caffeine gradient elution of respective samples from BD-cellulose. By this criterion, structural damage to DNA was again most marked 48 h after treatment at which time single stranded regions extending over several kilobases were apparent. At earlier times such lesions were considerably shorter. Single stranded regions of discrete length detected 72 h after administration of the hepatotoxin appeared to be associated with cell division. Structural damage to DNA coincident with toxic injury was further characterized using BD-cellulose, by caffeine gradient elution of preparations subjected to denaturation before chromatography. The data indicate that production of single stranded regions is a major feature of DNA degradation during tissue necrosis. Whilst such degradation might otherwise confound interpretation of structural analyses based only upon strand breakage, the size of these regions suggests a basis for distinguishing between them and lesions specifically associated with repair, replication and transcription of DNA.
Animals↗
Limitations on the stability of benzoylated DEAE-cellulose.
As used for structural analysis of DNA, benzoylated DEAE-cellulose is subject to deterioration which affects chromatographic performance. This change, which specifically occurs during storage of the resin, has been characterized in terms of anomalous patterns of DNA binding. Subjecting benzoylated DEAE-cellulose to boiling in aqueous suspension, and appropriate storage, ensures maintenance of normal affinity for DNA.
Animals↗
Relationships between carcinogen metabolism, adduct binding and DNA damage in 3-methylcholanthrene-exposed lung.
The extent to which structural damage in DNA, isolated from rats receiving an intratracheal dose of 3-methylcholanthrene (3-MC), was affected by change in microsomal mixed-function oxidase activity, has been examined. Instillation of 3-MC causes a characteristic pattern of structural change in lung DNA as determined by stepwise elution from benzoylated DEAE-cellulose (BD-cellulose). The proportion of lung DNA containing single stranded regions was increased biphasically, relative maxima occurring 14 and 72 h after treatment, the first increase being proportional to the dose of 3-MC. Binding of 3-MC to lung DNA increased progressively for 24 h after treatment and decreased rapidly from 48 h onward. Cytochrome P-450 content of pulmonary, and also of hepatic microsomes from treated animals, was measured. On this basis, 3-MC metabolism was modified, carbon tetrachloride being an effective inhibitor when administered 4 h in advance of treatment whilst maximum self-induction of 3-MC metabolism required 24 h. BD-cellulose analysis of lung DNA of 3-MC-treated rats subjected to inhibiting or inducing treatment suggested that the extent of structural damage was primarily determined by the capacity of lung tissue to metabolise the carcinogen. In particular, inhibition of 3-MC metabolism by prior treatment with carbon tetrachloride prevented production of single stranded regions, whilst modifying 3-MC binding to DNA. The data indicate that structural analysis of DNA is a sensitive means of assessing levels of genomic injury by carcinogens. This procedure may be used to study the effects of complicated treatments, and specifically agents which modify carcinogen metabolism.
Animals↗
Transient structural change in hepatic DNA of rats chronically exposed to dimethylnitrosamine.
Structural analysis by chromatography on benzoylated-DEAE-cellulose (BD-cellulose) has been made of hepatic DNA from rats treated for up to 21 weeks with dimethylnitrosamine (DMN). The carcinogen (1 mg/kg body wt) was administered on a daily basis by intraperitoneal injection. By comparison with preparations from saline-treated controls, the proportion of DNA retained by benzoylated-DEAE-cellulose in the presence of 1.0 M NaCl was increased by administration of the carcinogen. Variation in the result, dependent upon the time after the final dose, suggested a complex relationship between structural damage to DNA and duration of treatment. Structural damage was confirmed by S1 nuclease digestion. The observations imply that in the course of chronic administration, increasing time is required to complete DNA repair processes operative in rat liver.
Animals↗
Structural defects in DNA from primary hepatocyte cultures.
Structural analysis of DNA from primary hepatocytes has been made by chromatography on benzoylated-DEAE-cellulose. DNA was labelled in vivo by incorporation of 3H-TdR after partial hepatectomy 2 weeks prior to animals being used to establish hepatocyte cultures. Structural change in DNA associated with the transition to primary culture was assessed by comparison of data with that obtained when DNA was isolated from the intact organ immediately after death. In terms of this comparison, DNA from primary hepatocytes was bound more extensively to benzoylated-DEAE-cellulose in the presence of 1.0 M NaCl. Thus 32% of primary hepatocyte DNA was recovered in the formamide-eluted fraction after chromatography, a result which regions. This proportion of formamide-eluted DNA in primary hepatocytes could not be further reduced by extensive shearing and could not be affected by manipulation of the isolation procedure. Analysis of DNA isolated from hepatocyte cell lines revealed structural characteristics similar to DNA isolated from intact liver. The data imply that extensive structural damage to DNA is a consequence of the primary status of the hepatocytes examined.
Animals↗
Sizing of DNA fragments by preparative, benzoylated DEAE-cellulose chromatography.
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Bacteriophage lambda↗
Injury to type-2 pneumocytes in rats exposed to cigarette smoke.
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Animals↗