A novel fluorometric oligonucleotide assay to measure O 6-methylguanine DNA methyltransferase, methylpurine DNA glycosylase, 8-oxoguanine DNA glycosylase and abasic endonuclease activities: DNA repair status in human breast carcinoma cells overexpressing methylpurine DNA glycosylase

DNA repair status plays a major role in mutagenesis, carcinogenesis and resistance to genotoxic agents. Because DNA repair processes involve multiple enzymatic steps, understanding cellular DNA repair status has required several assay procedures. We have developed a novel in vitro assay that allows quantitative measurement of alkylation repair via O⁶-methylguanine DNA methyltransferase (MGMT) and base excision repair (BER) involving methylpurine DNA glycosylase (MPG), human 8-oxoguanine DNA glycosylase (hOGG1) and yeast and human abasic endonuclease (APN1 and APE/ref-1, respectively) from a single cell extract. This approach involves preparation of cell extracts in a common buffer in which all of the DNA repair proteins are active and the use of fluorometrically labeled oligonucleotide substrates containing DNA lesions specific to each repair protein. This method enables methylation and BER capacities to be determined rapidly from a small amount of starting sample. In addition, the stability of the fluorometric oligonucleotides precludes the substrate variability caused by continual radiolabeling. In this report this technique was applied to human breast carcinoma MDA-MB231 cells overexpressing human MPG in order to assess whether up-regulation of the initial step in BER alters the activity of selected other BER (hOGG1 and APE/ref-1) or direct reversal (MGMT) repair activities.     

Formation and subsequent removal of O6-methylguanine from deoxyribonucleic acid in rat liver and kidney after small doses of dimethylnitrosamine

1. The amounts of 7-methylguanine and O⁶-methylguanine present in the DNA of liver and kidney of rats 4h and 24h after administration of low doses of dimethylnitrosamine were measured. 2. O⁶-Methylguanine was rapidly removed from liver DNA so that less than 15% of the expected amount (on the basis of 7-methylguanine found) was present within 4h after doses of 0.25mg/kg body wt. or less. Within 24h of administration of dimethylnitrosamine at doses of 1mg/kg or below, more than 85% of the expected amount of O⁶-methylguanine was removed. Removal was most efficient (defined in terms of the percentage of the O⁶-methylguanine formed that was subsequently lost within 24h) after doses of 0.25–0.5mg/kg body wt. At doses greater or less than this the removal was less efficient, even though the absolute amount of O⁶-methylguanine lost during 24h increased with the dose of dimethylnitrosamine over the entire range of doses from 0.001 to 20mg/kg body wt. 3. Alkylation of kidney DNA after intraperitoneal injections of 1–50μg of dimethylnitrosamine/kg body wt. occurred at about one-tenth the extent of alkylation of liver DNA. Removal of O⁶-methylguanine from the DNA also took place in the kidney, but was slower than in the liver. 4. After oral administration of these doses of dimethylnitrosamine, the alkylation of kidney DNA was much less than after intraperitoneal administration and represented only 1–2% of that found in the liver. 5. Alkylation of liver and kidney DNA was readily detectable when measured 24h after the final injection in rats that received daily injections of 1μg of [³H]dimethylnitrosamine/kg for 2 or 3 weeks. After 3 weeks, O⁶-methylguanine contents in the liver DNA were about 1% of the 7-methylguanine contents. The amount of 7-methylguanine in the liver DNA was 10 times that in the kidney DNA, but liver O⁶-methylguanine contents were only twice those in the kidney. 6. Extracts able to catalyse the removal of O⁶-methylguanine from alkylated DNA in vitro were isolated from liver and kidney. These extracts did not lead to the loss of 7-methylguanine from DNA. 7. The possible relevance of the formation and removal of O⁶-methylguanine in DNA to the risk of tumour induction by exposure to low concentrations of dimethylnitrosamine is discussed.     

Repair of O6-methyl-guanine residues in DNA takes place by a similar mechanism in extracts from hela cells,human liver,and rat liver

Extracts from HeLa S₃ cells, human liver, and rat liver were found to contain an activity that transfers the methyl group from O⁶-methyl-guanine residues in DNA to a cysteine residue of an acceptor protein. The molecular weights of the acceptor proteins in HeLA cells and human liver are 24,000 ± 1,000 and 23,000 ± 1,000. respectively. Assuming that each acceptor molecule is used only once, the average number of acceptor molecules in HeLa cells was calculated to be about 50,000. The extracts also contained 3-methyl-adenine-DNA glycosylase activity and 7-methyl-guanine-DNA glycosylase activity, although the latter activity was not detected in extracts from human liver in our assay system. Thus, the three major alkylation products resulting from the effect of methylating agents, such as N-methyl-N-nitroso urea, can all be repaired in animal cells. Pretreatment of HeLa cells with N-methyl-N′-nitro-N-nitrosoguanidinc (0.1 μg/ml) strongly reduced the capacity of HeLa cell extracts to repair O⁶-methyl-guanine residues, while the activity of three DNA-N-glycosylases was essentially unaltered. This inactivation was not caused by a direct methylation of the enzyme by the carcinogen. The results demonstrate that the mechanism of repair of O⁶-methyl-guaninc residues, in DNA is strikingly similar in E coli and animal cells, including humans.     

Development of a new application of the comet assay to assess levels of O6-methylguanine in genomic DNA (CoMeth)

O⁶-methylguanine (O⁶meG) is one of the most premutagenic, precarcinogenic, and precytotoxic DNA lesions formed by alkylating agents. Repair of this DNA damage is achieved by the protein MGMT, which transfers the alkyl groups from the O⁶ position of guanine to a cysteine residue in its active center. Because O⁶meG repair by MGMT is a stoichiometric reaction that irreversibly inactivates MGMT, which is subsequently degraded, the repair capacity of O⁶meG lesions is dependent on existing active MGMT molecules. In the absence of active MGMT, O⁶meG is not repaired, and during replication, O⁶meG:T mispairs are formed. The MMR system recognizes these mispairs and introduces a gap into the strand. If O⁶meG remains in one of the template strands the futile MMR repair process will be repeated, generating more strand breaks (SBs). The toxicity of O⁶meG is, therefore, dependent on MMR and DNA SB induction of cell death. MGMT, on the other hand, protects against O⁶meG toxicity by removing the methyl residue from the guanine. Although removal of O⁶meG makes MGMT an important anticarcinogenic mechanism of DNA repair, its activity significantly decreases the efficacy of cancer chemotherapeutic drugs that aim at achieving cell death through the action of the MMR system on unrepaired O⁶meG lesions. Here, we report on a modification of the comet assay (CoMeth) that allows the qualitative assessment of O⁶meG lesions after their conversion to strand breaks in proliferating MMR-proficient cells after MGMT inhibition. This functional assay allows the testing of compounds with effects on O⁶meG levels, as well as on MGMT or MMR activity, in a proliferating cell system. The expression of MGMT and MMR genes is often altered by promoter methylation, and new epigenetically active compounds are being designed to increase chemotherapeutic efficacy. The CoMeth assay allows the testing of compounds with effects on O⁶meG, MGMT, or MMR activity. This proliferating cell system complements other methodologies that look at effects on these parameters individually through analytical chemistry or in vitro assays with recombinant proteins.     

Development of an O(6)-alkylguanine-DNA alkyltransferase assay based on covalent transfer of the benzyl moiety from [benzene-3H]O(6)-benzylguanine to the protein

Although it is known that (i) O⁶-alkylguanine-DNA alkyltransferase (AGT) confers tumor cell resistance to guanine O⁶-targeting drugs such as cloretazine, carmustine, and temozolomide and that (ii) AGT levels in tumors are highly variable, measurement of AGT activity in tumors before treatment is not a routine clinical practice. This derives in part from the lack of a reliable clinical AGT assay; therefore, a simple AGT assay was devised based on transfer of radioactive benzyl residues from [benzene-³H]O⁶-benzylguanine ([³H]BG) to AGT. The assay involves incubation of intact cells or cell homogenates with [³H]BG and measurement of radioactivity in a 70% methanol precipitable fraction. Approximately 85% of AGT in intact cells was recovered in cell homogenates. Accuracy of the AGT assay was confirmed by examination of AGT levels by Western blot analysis with the exception of false-positive results in melanin-containing cells due to [³H]BG binding to melanin. Second-order kinetic constants for human and murine AGT were 1100 and 380 M⁻¹ s⁻¹, respectively. AGT levels in various human cell lines ranged from less than 500 molecules/cell (detection limit) to 45,000 molecules/cell. Rodent cell lines frequently lacked AGT expression, and AGT levels in rodent cells were much lower than in human cells.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators: I. Development of a short term in vitro microcytotoxicity assay

We describe a short term in vitro microcytotoxicity assay to study the killing by macrophages of adhering tumor cells prelabeled with [³H]proline. With this assay, killing of line 1 hepatoma cells can be demonstrated within 6 hr of cocultivation with normal macrophages activated in vitro with the lymphocyte mediator macrophage activating factor (MAF).The data show that the decrease in residual adhering radioactivity, on which the calculations of percent kill are based, results from the lysis as well as from the detachment of tumor cells. However, detached tumor cells fail to exclude trypan blue and are no longer capable of DNA and protein synthesis. This suggests that the detachment of intact but nonviable tumor cells precedes actual target cell lysis in this system.     

O~6-甲基鸟嘌呤受体蛋白的定量测定

本文介绍了一种细胞提取液O~6—甲基鸟嘌呤(O~6—MeGua)受体蛋白测定及其底物O~6-[~8H-Me]Gua DNA的制备方法。废物与受体蛋白反应后,甲基从O~6-[~3H-Me]Gua DNA转移到受体蛋白,生成甲基-S-半胱氨酸(Me-S-Cys)。经盐酸水解后,直接测定酸不溶部分的受体蛋白沉淀。方法简便、快速、准确。     

Effects of inhibition of protein synthesis on DNA replication in cultured mammalian cells

We have investigated the effects of inhibiting protein synthesis on the overall rate of DNA synthesis and on the rate of replication fork movement in mammalian cells. In order to test the validity of using [³H]thymidine incorporation as a measure of the overall rate of DNA synthesis during inhibition of protein synthesis, we have directly measured the size and specific radioactivity of the cells' [³H]dTTP pool. In three different mammalian cell lines (mouse L, Chinese hamster ovary, and HeLa) nearly complete inhibition of protein synthesis has little effect on pool size (±26%) and even less effect on its specific radioactivity (±11%). Thus [³H]thymidine incorporation can be used to measure accurately changes in rate of DNA synthesis resulting from inhibition of protein synthesis.Using the assay of [³H]thymidine incorporation to measure rate of DNA synthesis, and the assay of [¹⁴C]leucine or [¹⁴C]valine incorporation to measure rate of protein synthesis, we have found that eight different methods of inhibiting protein synthesis (cycloheximide, puromycin, emetine, pactamycin, 2,4-dinitrophenol, the amino acid analogs canavanine and 5-methyl tryptophan, and a temperature-sensitive leucyl-transfer tRNA synthetase) all cause reduction in rate of DNA synthesis in mouse L, Chinese hamster ovary, or HeLa cells within two hours to a fairly constant plateau level which is approximately the same as the inhibited rate of protein synthesis.We have used DNA fiber autoradiography to measure accurately the rate of replication fork movement. The rate of movement is reduced at every replication fork within 15 minutes after inhibiting protein synthesis. For the first 30 to 60 minutes after inhibiting protein synthesis, the decline in rate of fork movement (measured by fiber autoradiography) satisfactorily accounts for the decline in rate of DNA synthesis (measured by [³H]thymidine incorporation). At longer times after inhibiting protein synthesis, inhibition of fork movement rate does not entirely account for inhibition of overall DNA synthesis. Indirect measurements by us and direct measurements suggest that the additional inhibition is the result of decline in the frequency of initiation of new replicons.     

Enzymatic removal of O6-methylguanine from DNA by mammalian cell extracts

Extracts from various rat tissues were incubated with [³H]methylated DNA or chromatin in order to compare their abilities to catalyze the removal of labeled $\text{O}{}^6$-methylguanine from acid precipitable DNA. Liver extracts had the greatest activity. Kidney extracts had about 35% of the activity in liver and extracts from lung, colon, small intestine and brain were much less active. The enzyme responsible for this reaction does not appear to be an $\text{N}$-glycosidase because no labeled $\text{O}{}^6$-methylguanine could be detected in the supernatant fraction even though more than 50% of this base was lost from the DNA. The released radioactivity was present as methanol which is consistent with the possibility that the reaction may involve a demethylase action on either the DNA substrate or an oligonucleotide derived from it.     

Enzymatic degradation of heparin-related mucopolysaccharides from the surface of endothelial cell cultures

When cultures of endothelial cells prelabeled with H₂^{3 5}SO₄ are exposed to a purified preparation from induced Flavobacterium heparinum containing heparinase and heparitinase activities, radioactivity accumulates in the supernatant medium. After further treatment in vitro with crude enzyme this material migrates, in part, as glucosamine (N, O-disulfated glucosamine), a breakdown product characteristic of heparin and heparin-related mucopolysaccharides. After exposure of the cultures to the purified enzyme, the amount of acid-insoluble ^{3 5}S radioactivity that can be removed with EDTA is decreased compared to that that can be removed from control cultures. Since the amount of radioactivity that is released as breakdown products is much higher than the amount of radioactivity that is secreted into the supernatant medium as intact (non-dialysable) mucopolysaccharide chains in control plates, the action of the enzyme appears to be on the cell itself. The data presented support previous studies suggesting that chains of heparitin sulfate that are accessible to the action of the enzyme are present at the surface of endothelial cells.     

The metabolism of [3−3H]oleanolic acid-3-O-mono-[14C]glucoside in isolated cells from Calendula officinalis leaves

The radioactive precursor, [3?³H]oleanolic acid-3-O-mono-[¹⁴C]glucoside was administrated to isolated cells obtained from the leaves of Calendula officinalis. The radioactivity of the precursor was incorporated into fractions containing free oleanolic acid, individual glucosides, glucuronide F and other glucuronides. The ratio of ³H: ¹⁴C radioactivity in these fractions indicated that glucosides were formed in a process involving direct glycosylation of the precursor, whereas the glucuronides were formed from oleanolic acid released by hydrolysis of the precursor. Dynamics curves showed that glucoside II formed by direct glycosylation of the precursor was intensively transformed to other derivatives.     

Enzymes Induced in a Bacterium by Growth on Sodium Dodecyl Sulfate

Alkyl sulfatase was induced by growth on nutrient broth plus sodium dodecyl sulfate (SDS) in a bacterium we have designated Pseudomonas C12B. Measurement of the radioactivity of S³⁵O₄⁼ released from SDS³⁵ by the enzyme in cell-free extracts provided an effective assay technique. The barium chloranilate assay for release of SO₄⁼ from SDS was somewhat less sensitive but effective if analyzed at 332 mμ. This test was only approximately 55% as sensitive if analyzed at 530 mμ. The activity of the glyoxylate bypass enzymes, isocitrate lyase and malate synthetase, was significantly stimulated by growth of the bacteria on SDS as the sole carbon source, but not by growth on nutrient broth or nutrient broth plus SDS.     

De Novo Biosynthesis of Deoxyribonucleic Acid Polymerase during Wheat Embryo Germination

An 8-fold enhancement in the activity of a DNA-dependent DNA polymerase was found in extracts from germinating wheat (Triticum vulgare var. Florence) embryos, as compared to the activity found in extracts from ungerminated embryos. The enhancement of this activity during the first hours of germination is concomitant to the increase of a Dnase activity. The two activities could be separated and the increased level of the DNA polymerase upon germination was observed in an enzymatic fraction which contains very low DNase activity. Addition of the protein synthesis inhibitor, blasticidin S, to germinating wheat embryos, reduced the increase in DNA polymerase. Incorporation of radioactive amino acids into a phosphocellulose preparation, which contains the DNA polymerase starts during the first 6 hours of germination. The amount of radioactivity incorporated is doubled in the next 6 hours, and the incorporation is continued between 12 and 18 hours of germination.     

A simple real-time assay for in vitro translation

A high-throughput assay for real-time measurement of translation rates in cell-free protein synthesis (SNAP assay) is described. The SNAP assay enables quantitative, real-time measurement of overall translation rates in vitro via the synthesis of O⁶-alkylguanine DNA O⁶-alkyltransferase (SNAP). SNAP production is continuously detected by fluorescence produced by the reaction of SNAP with a range of quenched fluorogenic substrates. The capabilities of the assay are exemplified by measurements of the activities of Escherichia coli MRE600 ribosomes and fluorescently labeled E. coli mutant ribosomes in the PURExpress translation system and by determination of the 50% inhibitory concentrations (IC₅₀) of three common macrolide antibiotics.     

Oxidation of C1-compounds in Pseudomonas C

Extracts of Pseudomonas C grown on methanol as sole carbon and energy source contain a methanol dehydrogenase activity which can be coupled to phenazine methosulfate. This enzyme catalyzes two reactions namely the conversion of methanol to formaldehyde (phenazine methosulfate coupled) and the oxidation of formaldehyde to formate (2,6-dichloroindophenol-coupled). Activities of glutathione-dependent formaldehyde dehydrogenase (NAD⁺) and formate dehydrogenase (NAD⁺) were also detected in the extracts.The addition of d-ribulose 5-phosphate to the reaction mixtures caused a marked increase in the formaldehyde-dependent reduction of NAD⁺ or NADP⁺. In addition, the oxidation of [¹⁴C]formaldehyde to CO₂, by extracts of Pseudomonas C, increased when d-ribulose 5-phosphate was present in the assay mixtures.The amount of radioactivity found in CO₂, was 6.8-times higher when extracts of methanol-grown Pseudomona C were incubated for a short period of time with [1-¹⁴C]glucose 6-phosphate than with [U-¹⁴C]glucose 6-phosphate.These data, and the presence of high specific activities of hexulose phosphate synthase, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase indicate that in methanol-grown Pseudomonas C, formaldehyde carbon is oxidized to CO₂ both via a cyclic pathway which includes the enzymes mentioned and via formate as an oxidation intermediate, with the former predominant.     

Isolation and properties of naphthoate synthetase from Mycobacterium phlei

Cell-free extracts obtained by sonication of Mycobacterium phlei cells contain an important enzyme of the menaquinone (= vitamin K₂) biosynthetic pathway. This enzyme, naphthoate synthetase (1,4-dihydroxy-2-naphthoate synthetase), was partially purified by chromatography on Sepharose 6BCL. Conversion of o-succinylbenzoate to 1,4-dihydroxy-2-naphthoate was followed by a radioactivity assay using o-[2,3-¹⁴C₂]succinylbenzoate, or by a spectrophotofluorometric assay. o-[1-¹³C]Succinylbenzoate was converted intact by the extracts to dihydroxynaphthoate containing ¹³C only in the carboxyl carbon atom. For maximum activity, the enzyme requires ATP, Mg²⁺, and coenzyme A. The pH optimum is 6.9 and the molecular weight approximately 44,000. In the presence of farnesyl pyrophosphate, the extracts convert o-[2,3-¹⁴C₂]succinylbenzoate to ¹⁴C-containing menaquinone.     

Repair and replication of DNA in rat and mouse tissues in relation to cancer induction by N-nitroso-N-alkyl-ureas

The high susceptibility of certain organs, for example rat brain, to induction of cancer by N-nitroso-N-alkyl-ureas, has been related to a low ability to remove O⁶-alkylguanine (O⁶AG) from DNA. It is therefore reasonable to ask why mouse brain, in which there is also a slow disappearance of O⁶AG from DNA after treatment with nitroso-alkyl-ureas, is not susceptible and why, in mice, thymus and lung are the main target organs. The explanation of the species difference could lie in the fact that replication of alkylated DNA is an essential event in initiation. If nitroso-alkyl-ureas had a greater inhibitory effect in some organs than in others, replication might be inhibited until after the O⁶AG had been removed, so preventing replication of DNA while still alkylated. This concept was tested by comparing the effect of N-nitroso-N-methyl-urea (NMU) on incorporation of [³H]TdR into DNA of relevant organs in Wistar rats and C57BL mice, and by determining ability to remove O⁶AG from DNA by measuring the alkyl acceptor protein (AAP) concentrations in these organs. No evidence was obtained that the AAP content was lower or inhibition of replication was less extensive in the organ of the species more susceptible to carcinogenesis than in the same organ of the less susceptible species.     

Development of a rapid,small-scale DNA repair assay for use on clinical samples

Double-strand breaks (DSBs) are the most lethal form of DNA damage. They can be repaired by one of two pathways, homologous recombination and non-homologous end joining (NHEJ). A NHEJ assay has previously been reported which measures joining using cell-free extracts and a linearised plasmid as DNA substrate. This assay was designed for 3 × 10⁹ cells grown in vitro and utilised radioactively labelled substrate. We have scaled down the method to use smaller cell numbers in a variety of cell lines. Altering the cellular extraction procedure decreased background DNA contamination. The cleaner preparations allowed us to use SYBR Green I staining to identify joined products, which was as sensitive as ³²P-end-labelled DNA. NHEJ was found in established tumour cell lines from different originating tissues, though actual levels and fidelity of repair differed. This method also allowed end joining to be assessed in clinical specimens (human blood, brain and bladder tumours) within 24 h of receiving samples. The application of this method will allow investigation of the role of DSB DNA repair pathways in human tumours.     

The formation of methylated bases in DNA by dimethylnitrosamine and its relation to differences in the formation of tumours in the livers of GR and C3HF mice

Metabolism of and DNA methylation by dimethylnitrosamine (DMNA) were measured in the livers of GR male and C3Hf male and female mice which showed widely different susceptibilities to tumour formation by this hepatocarcinogen.It was previously shown that continuous DMNA administration results in vascular tumours in the livers of C3Hf female mice, whereas C3Hf males develop a high incidence of hepatomas both after continuous treatment and after a single injection of DMNA to adult animals. GR males showed a low susceptibility to the formation of liver tumours under these conditions.N-demethylation of DMNA by liver microsomes showed similar activity for both C3Hf sexes; but GR males were significantly more active.At 5 and 48 h after a single injection of [¹⁴C]DMNA, the amounts of O⁶-methylguanine (O⁶-MeGua), 7-methylguanine (7-MeGua), 1-methyladenine (1-MeAde) and 3-methyladenine (3-MeAde) were similar for C3Hf males and females, with the possible exception of 7-MeGua which seemed to be slightly higher in the female. O⁶ MeGua disappeared from C3Hf liver DNA with an apparent half-life time of about 24 h. Especially at 48 h after injection, GR liver DNA was methylated to a higher extent than was C3Hf liver DNA. This result, which antiparallels the tumour incidences, may be explained by the differences in rate of N-demethylation of DMNA. where higher 7-MeGua values were found for fasted animals under otherwise identical conditions.The general conclusior to be drawn is that neither the metabolism of DMNA nor DNA methylation by this carcinogen in the livers of male GR and C3Hf male and female mice correlates With the formation of hepatomas after DMNA administration. A possible explanation of the absence of such a correlation between DNA methylation and tumour formation might be that there exists no causal relationship between both events. However, a complicating factor is that the eventual development of a tumour may be influenced by a number of—sometimes decisive—secondary factors like hormonal²⁵ or immunological²⁶ status or the presence of cellular proliferation in target organs^27,28. Evidence from other systems suggests a relationship between inactivating, mutagenic or carcinogenic effects of alkylating agents and their ability to interact with nucleic acids, especially DNA^29,30.