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PubMed1995

A monoclonal antibody that recognizes alkali-stabilized melphalan-DNA adducts and its application in immunofluorescence microscopy.

Monoclonal antibodies were produced that recognized alkali-stabilized modifications of DNA formed by the anticancer drug melphalan in order to permit measurement of melphalan-DNA adducts in individual cells by immunofluorescent staining. Antibody Amp4/42 did not bind to alkali-treated control DNA or to DNA that had been alkylated with melphalan but not exposed to alkali. In a competitive enzyme-linked immunoadsorbent assay using DNA that had been reacted with radioactive melphalan in simple solution a 50% reduction in assay signal was caused by approximately 100 fmol total melphalan-DNA adducts/assay well. This sensitivity was only slightly influenced by heat denaturation of the DNA before alkylation or by the frequency of alkylated sites on DNA. The heat stability of the adducts recognized by Amp4/42 was greatly increased by the alkali-induced change which, in 0.1 M NaOH at 37 degrees C, was complete by 30 min. Amp4/42 appears to recognize a ring-opened structure resulting from alkaline hydrolysis of 7-alkyldeoxyguanosine. Melphalan-DNA adducts formed in mammalian cells showed an alkali-induced increase in immunoreactivity which occurred at a similar rate to that seen in DNA that had been alkylated in simple solution, but their maximum overall immunoreactivity was approximately 10-fold lower. This indicated that in cells the adducts recognized by Amp4/42 were formed or persisted as a smaller proportion of total adducts compared with alkylation of pre-purified DNA in simple solution. This antibody permitted immunofluorescent detection of melphalan-DNA adducts in single cells.

Animals↗
PubMed1999

Purification and characterization of a recombinant Haemophilus influenzae outer membrane phosphomonoesterase e (P4).

Haemophilus influenzae is a common inhabitant of the upper respiratory tract and can cause serious infections of mucosal surfaces. Results from recent studies indicate that this pathogen possesses copious amounts of surface-localized phosphomonoesterase activity mediated by the bacterial lipoprotein e (P4). While the enzyme has previously been purified to apparent homogeneity, purification of large amounts of protein has been prevented by presence of N-terminal lipid modification. Recombinant DNA technology was employed to simultaneously replace the N-terminal lipid modification signal sequence with one for protein secretion without such modification and to place expression of the protein under the control of the T7-inducible promoter. Results from this work show that high levels of phosphomonoesterase activity were achieved after IPTG induction and purified to apparent homogeneity after two chromatography steps. Consistent with loss of the N-terminal lipid modification, the recombinant enzyme was easily extracted from the bacterial membrane and partitioned within the matrix of gel filtration chromatography resin while retaining a denatured molecular weight similar to that of wild-type e (P4). Results from physicochemical characterization suggest that the recombinant protein was similar to wild-type protein in SDS-PAGE-derived molecular weight, primary structure, substrate specificity, pH optimum, and sensitivity or resistance to various inhibitors. Acquisition of sufficient amounts of recombinant P4 was a prelude for studies to elucidate the structure and function of this unusual phosphomonoesterase.

Bacterial Outer Membrane Proteins↗
PubMed2006

Oxidative stress and DNA damage induced by a drinking-water chlorination disinfection byproduct 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in mice.

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a water chlorine disinfection byproduct, can induce DNA damage (e.g., modification of nucleotides and DNA strand breaks) and subsequent DNA repair in vitro. However, the underlying mechanism(s) how DNA damage is induced by MX is unknown. We hypothesized that MX may cause oxidative stress that leads to DNA damage in vivo. In the present study, we exposed groups of mice to MX at concentrations of 0 (solvent control), 11 (low), 33 (medium) and 99 (high) mg/kg b.w. by single intraperitoneal injection. After treating the mice for 3h, we detected cellular levels of malondialdehyde (MDA) and glutathione (GSH) to assess oxidative stress in the target cells. In addition, we also evaluated DNA damage using single cell gel electrophoresis (SCGE or Comet assay). We found that the levels of DNA damage in all cell types were correlated positively with levels of MDA but negatively with levels of GSH (P<0.05 for all). Also, there were negative correlations between levels of MDA and GSH (r=-0.995 for liver cells, -0.916 for kidney cells, -0.975 for intestine cells, respectively; P<0.05 for all but kidney cells). Our findings suggest that MX may induce DNA damage by the mechanism of causing cellular oxidative stress as measured by increased MDA and decreased GSH, at least in mice.

Animals↗
PubMed1999

Accumulation of DNA damage in pre- and posthepatectomized liver of aged rats.

Although the majority of the literature supports the concept that an accumulation of DNA damage or a modification in the DNA structure of postmitotic cells occurs with increasing age, there are also several reports that show no DNA changes in these cells with increasing age. In the study reported here, two components of the DNA damage hypothesis of aging were tested. Young (4-6 months) and old (18-20 month) unirradiated or irradiated, pre- and posthepatectomized male Fisher 344 rats were killed, and the posterior lateral lobe of the liver removed. Single cell/nuclei suspensions were made, and the DNA damage accumulated with age or remaining at various times after irradiation was measured using the alkaline elution technique. The results demonstrate that, 1) DNA damage accumulates in rat liver cells with age, and 2) liver cells repair their radiation-induced DNA damage slower in posthepatectomized, but not prehepatectomized aged rats.

Aging↗
PubMed2002

Intercenter reproducibility of binary typing for Staphylococcus aureus.

The reproducibility of the binary typing (BT) protocol developed for epidemiological typing of Staphylococcus aureus was analyzed in a biphasic multicenter study. In a Dutch multicenter pilot study, 10 genetically unique isolates of methicillin-resistant S. aureus (MRSA) were characterized by the BT assay as presented by van Leeuwen et al. [J. Clin. Microbiol. 2001 39 (1) 328]. The BT assay, including a standardized DNA extraction protocol was performed in duplicate in eleven medical microbiology laboratories. Two different hybridization detection procedures were applied and a prelabeled DNA sample as process control was included. Only three laboratories accurately identified all strains. Divergence in technical procedures resulted in misinterpretation due to an increasing number of faint or absent hybridization signals in combination with high background staining. The binary type of the process control was determined correctly by all participating laboratories. The feasibility of the BT protocol was related directly to the skill of the laboratory personnel. On the basis of the national study, we concluded that the DNA extraction protocol needed modification to improve DNA yield and purity. Subsequently, seven European laboratories participated in an international study to determine the reproducibility of the modified BT protocol. Each center was asked to analyze 10 DNA samples previously extracted from 10 MRSA strains (phase 1) and, additionally, to analyze 10 MRSA strains, using the standardized or their in-house DNA isolation protocol (phase 2). A prelabeled DNA process control sample was included again. The binary types of all DNA samples were identified correctly by all but one laboratories. This latter laboratory diverged from the protocol by adding an excess of labeled DNA to the hybridization mixture, resulting in a high background and, therefore, noninterpretable BT results. All centers produced identical BT results for the process control. Five of the seven centers correctly identified the binary types of all 10 MRSA strains in phase 2 of the international study. Three of these centers used their in-house DNA extraction protocol. Divergence from the standard BT protocol in the remaining two centers resulted in no interpretable BT data for the 10 MRSA strains. The study demonstrated that each center that followed the BT protocol to the letter could generate reproducible results, irrespective whether or not an in-house DNA isolation protocol was used. The current BT protocol thus represents a simple method generating robust, reproducible genotype data for S. aureus strains.

Bacterial Typing Techniques↗
PubMed2005

Messenger RNA expression patterns in bovine embryos derived from in vitro procedures and their implications for development.

The preimplantation bovine embryo is initially under the control of maternal genomic information that is accumulated during oogenesis. The genetic programme of development soon becomes dependent on new transcripts derived from activation of the embryonic genome. The early steps in development, including the timing of the first cleavage, activation of the embryonic genome, compaction and blastocyst formation, can be affected by the culture media and conditions, as well as the production procedure itself. These perturbations can possibly result in a marked decrease in the quality of the resulting blastocysts and may even affect the viability of offspring born after transfer. In vitro procedures such as in vitro production and somatic nuclear transfer of bovine embryos have been shown to be correlated with significant up- or downregulation, de novo induction or silencing of genes critical for undisturbed fetal and neonatal development. These alterations are likely to be caused by epigenetic modifications, such as DNA methylation and histone modifications. Analysis of perturbed epigenetic reprogramming and of the related phenomena, such as genomic imprinting and X-chromosome inactivation, in bovine embryos is promising for understanding the underlying mechanisms of developmental abnormalities, such as large offspring syndrome.

Animals↗
PubMed1981

In vitro reactions of aflatoxin B1-adducted DNA.

The chemical stability of aflatoxin B1 bound to calf thymus DNA was studied over a 48-hour exposure to phosphate buffers at pH 6.8-8.0 (37 degrees C). During this time, aliquots of the aflatoxin B1-modified DNA were acid-hydrolyzed and analyzed for the presence of 2,3-dihydro-2-(N7-guanyl)-3-hydroxyflatoxin B1, 2,3-dihydro-2,3-dihydroxy-aflatoxin B1, and the tentatively identified, 2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'-oxo-N5-pyrimidyl-3-hydroxyflatoxin B1 and 2,3-dihydro-2-(8,9-dihydro-8-hydroxy-N7-guanyl)-3-hydroxyaflatoxin B1. Initial experiments determined the stability of 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 in DNA at levels of modification of one residue per 60 and 1500 nucleotides. The acid-hydrolysis products obtained from these modified nucleic acids were qualitatively similar, but their proportional concentrations were different. These quantitative differences were dependent upon both pH and the initial level of modification of the DNA. During the first 6 hr of incubation, under all conditions examined, the formation of 2,3-dihydro-2,3-dihydroxyaflatoxin B1 was responsible for the initial decrease of the 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 adduct in DNA. After 48 hr of incubation at pH 7.0, the major reaction of the modified DNA was depurination of the 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 adduct. However, at pH 8.0, the predominant reaction product formed in 48 hr was the putative 2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'-oxo-N5-pyrimidyl)-3-hydroxy-aflatoxin B1. The putative DNA-bound products resulting from the elimination of the positive charge in the imidazole ring of the aflatoxin-N7-guanine adduct [2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'-oxo-N5-pyrimidyl)-3-hydroxy-aflatoxin B1 and 2,3-dihydro-2-(8,9-dihydro-8-hydroxy-N7-guanyl)-3-hydroxyaflatoxin B1] were found to be stable in DNA for at least 24 hr at both pH 6.8 and 7.4. Taken together with observed patterns of stability of aflatoxin B1 adducts in vivo, these observations strongly suggest the involvement of enzymatic repair processes in removal of the N7-guanyl adduct and also emphasize the possible biological significance of the stable imidazole ring-opened adduct.

Aflatoxin B1↗
PubMed1983

Evidence for a restriction/modification-like system in Anacystis nidulans infected by cyanophage AS-1.

Anacystis nidulans infected by AS-1 cyanophage contains an endonuclease (AS-1 endonuclease) which splits host DNA but not AS-1 phage DNA [Szekeres, M. (1981) Virology, 111, 1-10]. AS-1 phage DNA proved to be resistant not only to AS-1 endonuclease but also to a number of restriction endonucleases the recognition sites of which contain a central dG-dC dinucleotide. Since an unmodified 5'dG-dC dinucleotide was shown to be present at the sites at which DNA is cleaved by AS-1 endonuclease, the results suggest that the sites attacked preferentially by the AS-1 endonuclease are specifically protected on the AS-1 DNA molecule. The modification of AS-1 DNA was shown to occur specifically in infected Anacystis because AS-1 DNA fragments which are normally resistant to AS-1 endonuclease became susceptible to this enzyme if inserted into pBR322 plasmid and cloned in Escherichia coli. AS-1 DNA was shown to contain about 5% of a modified nucleotide which was not 5-methyldeoxycytidylic acid. Results presented and our earlier data suggest that in Anacystis infected by AS-1 phage, a restriction/modification-like system operates which is able to eliminate 'unwanted' (host) DNA selectively.

Base Sequence↗
PubMed2006

Reactive oxygen species: role in the development of cancer and various chronic conditions.

Oxygen derived species such as superoxide radical, hydrogen peroxide, singlet oxygen and hydroxyl radical are well known to be cytotoxic and have been implicated in the etiology of a wide array of human diseases, including cancer. Various carcinogens may also partly exert their effect by generating reactive oxygen species (ROS) during their metabolism. Oxidative damage to cellular DNA can lead to mutations and may, therefore, play an important role in the initiation and progression of multistage carcinogenesis. The changes in DNA such as base modification, rearrangement of DNA sequence, miscoding of DNA lesion, gene duplication and the activation of oncogenes may be involved in the initiation of various cancers. Elevated levels of ROS and down regulation of ROS scavengers and antioxidant enzymes are associated with various human diseases including various cancers. ROS are also implicated in diabetes and neurodegenerative diseases. ROS influences central cellular processes such as proliferation a, apoptosis, senescence which are implicated in the development of cancer. Understanding the role of ROS as key mediators in signaling cascades may provide various opportunities for pharmacological intervention.

Journal Article↗
PubMed2025

Advances in tumor subclone formation and mechanisms of growth and invasion.

Tumor subclones refer to distinct cell populations within the same tumor that possess different genetic characteristics. They play a crucial role in understanding tumor heterogeneity, evolution, and therapeutic resistance. The formation of tumor subclones is driven by several key mechanisms, including the inherent genetic instability of tumor cells, which facilitates the accumulation of novel mutations; selective pressures from the tumor microenvironment and therapeutic interventions, which promote the expansion of certain subclones; and epigenetic modifications, such as DNA methylation and histone modifications, which alter gene expression patterns. Major methodologies for studying tumor subclones include single-cell sequencing, liquid biopsy, and spatial transcriptomics, which provide insights into clonal architecture and dynamic evolution. Beyond their direct involvement in tumor growth and invasion, subclones significantly contribute to tumor heterogeneity, immune evasion, and treatment resistance. Thus, an in-depth investigation of tumor subclones not only aids in guiding personalized precision therapy, overcoming drug resistance, and identifying novel therapeutic targets, but also enhances our ability to predict recurrence and metastasis risks while elucidating the mechanisms underlying tumor heterogeneity. The integration of artificial intelligence, big data analytics, and multi-omics technologies is expected to further advance research in tumor subclones, paving the way for novel strategies in cancer diagnosis and treatment. This review aims to provide a comprehensive overview of tumor subclone formation mechanisms, evolutionary models, analytical methods, and clinical implications, offering insights into precision oncology and future translational research.

Humans↗
PubMed1985

Polyclonal antibodies to quantitate cis-diamminedichloroplatinum(II)--DNA adducts in cancer patients and animal models.

cis-Diamminedichloroplatinum (II) (cis-DDP), the antitumor drug, is cytotoxic in vitro primarily by binding to DNA and disrupting its normal functions. We have studied cis-DDP modification of DNA in nucleated peripheral blood cells (buffy coat cells) of testicular and ovarian cancer patients receiving cis-DDP chemotherapy, and of untreated controls. Using a highly sensitive enzyme-linked immunosorbent assay (ELISA) with an antiserum specific for the bidentate intrastrand N7-deoxyguanosine adduct, blood cell DNA was assayed at multiple times during courses of cis-DDP treatment. A total of 138 samples were analyzed from 54 individuals. Of these, all samples from 18 untreated controls were negative, while 44 out of 120 samples from cis-DDP patients were positive. Testicular and ovarian cancer patients receiving chemotherapy on the first course, and given cis-DDP in 21- or 28-day cycles (five days of drug infusion followed by two or three drug-free weeks) accumulated cis-DDP-DNA adducts in blood cell DNA as a function of dose. Patients receiving their first course of cis-DDP on 56-day cycles and those given high doses of this drug after failing other chemotherapy showed much slower adduct accumulation than patients receiving their first course on 21- or 28-day cycles. Adduct accumulation, in positive patients, occurred both as a function of total cumulative dose and with increasing cycle number, suggesting that adduct removal took at least a month in these patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Methylation-specific oligonucleotide microarray.

The methylation-specific oligonucleotide (MSO) microarray is a high-throughput approach capable of detecting DNA methylation in genes across several CpG sites. Based on the bisulfite modification of DNA that converts unmethylated cytosines to uracil but leaves the 5'methylcytosine intact, the method utilizes short oligonucleotides corresponding to the methylated and unmethylated alleles as probes affixed on solid support and products amplified from bisulfite-treated DNA as targets for hybridization. MSO is suitable for examining a panel of genes across multiple clinical samples. This approach can generate a robust dataset for discovering profiles of gene methylation in cancer with aberrant DNA methylation in the neoplastic genome and widespread hypermethylation in tumor suppressor genes. MSO and other oligonucleotide-based arrays have been applied successfully for analyses of single genes and have been useful in delineating and predicting tumor subgroups using clustering methods. Here we focus on design criteria important to the interrogation of multiple CpG sites across several genes.

DNA Methylation↗
PubMed1991

Inactivation of DNA polymerase beta by in vitro phosphorylation with protein kinase C.

The Mr = 38,300 polypeptide of the purified recombinant rat DNA polymerase beta served as an excellent substrate for protein kinase C (PKC) in vitro but not for the catalytic subunit of cAMP-dependent protein kinase. The phosphorylation by PKC resulted in inactivation of DNA polymerase beta activity, and recovery was achieved by dephosphorylation with alkaline phosphatase. Since the phosphorylated DNA polymerase beta was retained with use of a single-stranded DNA-cellulose column, inactivation might occur at a site different from that for the DNA binding. Amino acid sequence analysis of the phosphopeptides revealed that the phosphorylated sites were 2 serine residues at positions 44 and 55 from the NH2 terminus, either or both of which might be involved in the catalytic activity of DNA polymerase beta. Thus, the inactivation of the DNA repair enzyme, DNA polymerase beta, by PKC may be an important process in the modification of DNA metabolism in the nucleus through signal transduction processes.

Alkaline Phosphatase↗
PubMed1994

Recognition of platinum-induced DNA damage by nuclear proteins: screening for mechanisms.

The association of high mobility group (HMG) proteins with cisplatin-damaged DNA has attracted considerable interest because of a possible relationship with drug-acquired resistance and the repair of DNA damage caused by this important chemotherapeutic agent. We have further characterized the binding of HMG proteins to cisplatin-damaged DNA using a modification of the damaged DNA affinity precipitation assay (DDAP) and proteins isolated from the nuclei of V79 cells. HMG proteins recognized cisplatin adducts only in double-stranded DNA sequences. Pre-treatment of cells with cisplatin (1 microM) prior to protein extraction decreased the apparent yield of HMG proteins. However, the pre-treatment of isolated protein did not prevent recognition of the DNA adducts. To investigate the possible role of HMG proteins in toxicity and resistance, we have extended the DDAP method to study other platinum agents, some of which are active in cisplatin-resistant cells. A comparison of trans- and cis-[PtCl2(NH3)quinoline] is presented as an example. HMG proteins recognized DNA damage caused by the cis, but not the trans isomer. However, the trans isomer is known to be significantly more toxic and is highly active in cisplatin-resistant cells, suggesting a mechanism of action different from cisplatin. Therefore, the toxicity of trans-[PtCl2(NH3)quinoline] appears to be unrelated to the recognition of damage by HMG proteins. The DDAP assay may provide an additional screen for new mechanisms of cytotoxic platinum agents.

Animals↗
PubMedDate not supplied

S-Adenosyl-L-methionine-dependent restriction enzymes.

Restriction-modification (R-M) enzymes are classified into type I, II, III, and IV, based on their recognition sequence, subunit composition, cleavage position, and cofactor requirements. While the role of S-Adenosyl-L-methionine (AdoMet) as the methyl group donor in the methylation reaction is undisputed, its requirement in DNA cleavage reaction has been subject to intense study. AdoMet is a prerequisite for the DNA cleavage by most type I enzymes known so far, with the exception of R.EcoR124I. A number of new type II restriction enzymes belonging to the type IIB and IIG family were found to show AdoMet dependence for their cleavage reaction. The type III enzymes have been found to require AdoMet for their restriction function. AdoMet functions as an allosteric effector of the DNA cleavage reaction and has been shown to bring about conformational changes in the protein upon binding.

DNA Modification Methylases↗
PubMed1994

Restriction enzymes in cells, not eppendorfs.

Restriction enzymes are essential reagents to molecular biologists, but their relevance to bacterial populations is less obvious. Most bacteria encode restriction and modification systems and these are commonly considered to be a barrier to phage infection. Current evidence also supports a more general role for them in genetic recombination.

Bacteria↗
PubMed1976

Alterations in the transcriptional capacity of hepatic DNA-dependent RNA polymerase I and II by N-hydroxy-2-acetylaminofluorene.

Using hepatic RNA polymerases I and II, acetylaminofluorene modification of DNA in vitro results in reduction of RNA elongation but not in an altered frequency of incorporation of [alpha32P]ribonucleotides. In contrast, in vivo modification of RNA polymerases by a single dose of N-2-hydroxy-2-acetylaminofluorene results in an alteration of their capacity for transcribing a normal RNA product but does not affect the product size transcribed from normal or carcinogen-modified templates.

Animals↗
PubMed1986

Photodynamic guanine modification by hematoporphyrin is specific for single-stranded DNA with singlet oxygen as a mediator.

Photodynamic modification of DNA by hematoporphyrin (Hp) was characterized by the DNA sequencing technique using 32P-labeled DNA fragments, and the reaction mechanism was investigated by ESR spectroscopy. Mild photodynamic treatment of single-stranded DNA with Hp induced an alteration of guanine residues, and subsequent treatment with piperidine led to chain cleavages at each guanine residue. On the other hand, methylene blue plus light modified the guanine residues in both single-stranded and double-stranded DNA. ESR studies using 2,2,6,6-tetramethylpiperidine and 2,2,6,6-tetramethyl-4-piperidone as singlet oxygen traps demonstrated that Hp plus light produced almost the same amount of singlet oxygen as methylene blue plus light and that the photochemically generated singlet oxygen reacts significantly with guanylate but only slightly with other mononucleotides. An ESR spin destruction method revealed that photoexcited Hp generated porphyrin radical, but guanylate did not react with this radical. These results indicate that photoexcited Hp reacts with oxygen to generate singlet oxygen which oxidizes the guanine residues of single-stranded DNA and that the difference in photoreactivities of DNA with Hp and methylene blue may be explained in terms of the structural difference in their intercalating abilities.

DNA Restriction Enzymes↗