Bracken Fern Carcinogen, Ptaquiloside, Forms a Guanine -Adduct in DNA.

Bracken fern ( sp.) is a viable and vigorous plant with invasive potential, ingestion of which causes chronic illness and cancers in farm animals. Bracken is a suspected human carcinogen, and exposure can result from ingestion of bracken-contaminated water, dairy products, or meat derived from livestock grazing on bracken fern.

Development and Application of a Slot-Blot Assay Using the Damage Sensing Protein Atl1 to Detect and Quantify -Alkylated Guanine Bases in DNA.

Humans are unavoidably exposed to numerous different mutagenic DNA alkylating agents (AAs), but their role in the initiation of cancers is uncertain, in part due to difficulties in assessing human exposure. To address this, we have developed a screening method that measures promutagenic -alkylguanines (-AlkGs) in DNA and applied it to human DNA samples. The method exploits the ability of the alkyltransferase-like protein (Atl1) to recognise and bind to a wide range of -AlkGs in DNA.

The DNA Alkyltransferase Family of DNA Repair Proteins: Common Mechanisms, Diverse Functions.

DNA alkyltransferase and alkyltransferase-like family proteins are responsible for the repair of highly mutagenic and cytotoxic O-alkylguanine and O-alkylthymine bases in DNA. Their mechanism involves binding to the damaged DNA and flipping the base out of the DNA helix into the active site pocket in the protein. Alkyltransferases then directly and irreversibly transfer the alkyl group from the base to the active site cysteine residue.

Mass Spectrometric Analysis of the Active Site Tryptic Peptide of Recombinant -Methylguanine-DNA Methyltransferase Following Incubation with Human Colorectal DNA Reveals the Presence of an -Alkylguanine Adductome.

Human exposure to DNA alkylating agents is poorly characterized, partly because only a limited range of specific alkyl DNA adducts have been quantified. The human DNA repair protein, -methylguanine -methyltransferase (MGMT), irreversibly transfers the alkyl group from DNA -alkylguanines (-alkGs) to an acceptor cysteine, allowing the simultaneous detection of multiple -alkG modifications in DNA by mass spectrometric analysis of the MGMT active site peptide (ASP). Recombinant MGMT was incubated with oligodeoxyribonucleotides (ODNs) containing different -alkGs, Temozolomide-methylated calf thymus DNA (Me-CT-DNA), or human colorectal DNA of known -MethylG (-MeG) levels.

QSAR and Chemical Read-Across Analysis of 370 Potential MGMT Inactivators to Identify the Structural Features Influencing Inactivation Potency.

-methylguanine-DNA methyltransferase (MGMT) constitutes an important cellular mechanism for repairing potentially cytotoxic DNA damage induced by guanine -alkylating agents and can render cells highly resistant to certain cancer chemotherapeutic drugs. A wide variety of potential MGMT inactivators have been designed and synthesized for the purpose of overcoming MGMT-mediated tumor resistance. We determined the inactivation potency of these compounds against human recombinant MGMT using [H]-methylated-DNA-based MGMT inactivation assays and calculated the IC values.

Synthesis of oligodeoxyribonucleotides containing a tricyclic thio analogue of -methylguanine and their recognition by MGMT and Atl1.

Promutagenic -alkylguanine adducts in DNA are repaired in humans by -methylguanine-DNA-methyltransferase (MGMT) in an irreversible reaction. Here we describe the synthesis of a phosphoramidite that allows the preparation of oligodeoxyribonucleotides (ODNs) containing a novel tricyclic thio analogue of -methylguanine in which the third ring bridges the 6-thio group and C7 of a 7-deazapurine. These ODNs are very poor substrates for MGMT and poorly recognised by the alkyltransferase-like protein, Atl1.

PON1 increases cellular DNA damage by lactone substrates.

Paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated enzyme that by hydrolysing exogenous and endogenous substrates can provide protection against substrate induced toxicity. To investigate the extent to which PON1 provides protection against lactone induced DNA damage, DNA damage was measured in HepG2 cells using the neutral Comet assay following lactone treatment in the presence and absence of exogenous recombinant PON1 (rPON1). Low dose lactones (10 mM) caused little or no damage while high doses (100 mM) induced DNA damage in the following order of potency: α-angelica lactone > γ-butyrolactone ~ γ-hexalactone > γ-heptalactone ~ γ-octaclactone ~ γ-furanone ~ γ-valerolactone > γ-decalactone.

-methylguanine-induced transcriptional mutagenesis reduces p53 tumor-suppressor function.

Altered protein function due to mutagenesis plays an important role in disease development. This is perhaps most evident in tumorigenesis and the associated loss or gain of function of tumor-suppressor genes and oncogenes. The extent to which lesion-induced transcriptional mutagenesis (TM) influences protein function and its contribution to the development of disease is not well understood.

L-β-N-methylamino-l-alanine (BMAA) nitrosation generates a cytotoxic DNA damaging alkylating agent: An unexplored mechanism for neurodegenerative disease.

Background: L-β-N-methylamino-l-alanine (BMAA) is a non-proteinic amino acid, that is neurotoxic in vitro and in animals, and is implicated in the causation of amyotrophic lateral sclerosis and parkinsonism-dementia complex (ALS-PDC) on Guam. Given that natural amino acids can be N-nitrosated to form toxic alkylating agents and the structural similarity of BMAA to other amino acids, our hypothesis was that N-nitrosation of BMAA might result in a toxic alkylating agent, providing a novel mechanistic hypothesis for BMAA action.

Findings: We have chemically nitrosated BMAA with sodium nitrite to produce nitrosated BMAA (N-BMAA) which was shown to react with the alkyl-trapping agent, 4-(p-nitrobenzyl)pyridine, cause DNA strand breaks in vitro and was toxic to the human neuroblastoma cell line SH-SY5Y under conditions in which BMAA itself was minimally toxic.

IGF-1R inhibition induces schedule-dependent sensitization of human melanoma to temozolomide.

Prior studies implicate type 1 IGF receptor (IGF-1R) in mediating chemo-resistance. Here, we investigated whether IGF-1R influences response to temozolomide (TMZ), which generates DNA adducts that are removed by O6-methylguanine-DNA methyltransferase (MGMT), or persist causing replication-associated double-strand breaks (DSBs). Initial assessment in 10 melanoma cell lines revealed that TMZ resistance correlated with MGMT expression (r = 0.

MGMT Expression Predicts PARP-Mediated Resistance to Temozolomide.

Melanoma and other solid cancers are frequently resistant to chemotherapies based on DNA alkylating agents such as dacarbazine and temozolomide. As a consequence, clinical responses are generally poor. Such resistance is partly due to the ability of cancer cells to use a variety of DNA repair enzymes to maintain cell viability.

The bacterial alkyltransferase-like (eATL) protein protects mammalian cells against methylating agent-induced toxicity.

In both pro- and eukaryotes, the mutagenic and toxic DNA adduct O(6)-methylguanine (O(6)MeG) is subject to repair by alkyltransferase proteins via methyl group transfer. In addition, in prokaryotes, there are proteins with sequence homology to alkyltransferases, collectively designated as alkyltransferase-like (ATL) proteins, which bind to O(6)-alkylguanine adducts and mediate resistance to alkylating agents. Whether such proteins might enable similar protection in higher eukaryotes is unknown.

Increased N7-methyldeoxyguanosine DNA adducts after occupational exposure to pesticides and influence of genetic polymorphisms of paraoxonase-1 and glutathione S-transferase M1 and T1.

There are concerns about genetic risks associated with long-term exposure to pesticides as these compounds may damage DNA, resulting in mutations that eventually lead to cancer, neurological, and reproductive adverse health effects. This study assessed DNA damage in intensive agricultural workers exposed to pesticides by determining the levels of N7-methyldeoxyguanosine (N7-MedG), an adduct known to be a robust biomarker of recent exposure to chemical methylating agents. A cohort of 39 plastic greenhouse workers was assessed for changes in lymphocyte DNA N7-MedG levels between low level and high level exposures during the course of a spraying season.

Differential chemosensitivity to antifolate drugs between RAS and BRAF melanoma cells.

Background: The importance of the genetic background of cancer cells for the individual susceptibility to cancer treatments is increasingly apparent. In melanoma, the existence of a BRAF mutation is a main predictor for successful BRAF-targeted therapy. However, despite initial successes with these therapies, patients relapse within a year and have to move on to other therapies.

O⁶-carboxymethylguanine in DNA forms a sequence context-dependent wobble base-pair structure with thymine.

N-Nitrosation of glycine and its derivatives generates potent alkylating agents that can lead to the formation of O(6)-carboxymethylguanine (O(6)-CMG) in DNA. O(6)-CMG has been identified in DNA derived from human colon tissue and its occurrence has been linked to diets high in red and processed meats, implying an association with the induction of colorectal cancer. By analogy to O(6)-methylguanine, O(6)-CMG is expected to be mutagenic, inducing G-to-A mutations that may be the molecular basis of increased cancer risk.

Repair and removal of azoxymethane-induced O6-methylguanine in rat colon by O6-methylguanine DNA methyltransferase and apoptosis.

Azoxymethane (AOM) is an alkylating agent that generates mutagenic and carcinogenic O(6)-methylguanine (O(6)meG) adducts in DNA. O(6)meG has been detected in human colonic DNA; hence, understanding the innate cellular events occurring in response to the formation of O(6)meG is important in developing preventive strategies for colorectal cancer. We explored the time-course, dose-response, and kinetics of O(6)meG formation and its removal by the DNA repair protein, O(6)-methylguanine DNA methyltransferase (MGMT), and apoptosis.

Bayesian inference supports a location and neighbour-dependent model of DNA methylation propagation at the MGMT gene promoter in lung tumours.

We exploit model-based Bayesian inference methodologies to analyse lung tumour-derived methylation data from a CpG island in the O6-methylguanine-DNA methyltransferase (MGMT) promoter. Interest is in modelling the changes in methylation patterns in a CpG island in the first exon of the promoter during lung tumour development. We propose four competils of methylation state propagation based on two mechanisms.

Topographical study of O(6)-alkylguanine DNA alkyltransferase repair activity and N7-methylguanine levels in resected lung tissue.

Background: Tobacco specific nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are genotoxic alkylating agents found within cigarette smoke that induce lung adenocarcinomas in animal models. In humans, adenocarcinomas originate most frequently in the lung periphery. The aim of this study was to determine whether peripheral lung has increased susceptibility to the genotoxic effects of alkylating agents by comparing DNA alkylation damage (N7-methylguanine: N7-meG) and repair (O(6)-alkylguanine DNA alkyltransferase: MGMT) in peripheral relative to central lung tissue.

Structures of DNA duplexes containing O6-carboxymethylguanine, a lesion associated with gastrointestinal cancer, reveal a mechanism for inducing pyrimidine transition mutations.

N-nitrosation of glycine and its derivatives generates potent alkylating agents that can lead to the formation of O(6)-carboxymethylguanine (O(6)-CMG) in DNA. O(6)-CMG has been identified in DNA derived from human colon tissue, and its occurrence has been linked to diets high in red and processed meats. By analogy to O(6)-methylguanine, O(6)-CMG is expected to be highly mutagenic, inducing G to A mutations during DNA replication that can increase the risk of gastrointestinal and other cancers.

Triazene compounds in the treatment of acute myeloid leukemia: a short review and a case report.

Acute myeloid leukemia (AML) is a highly lethal disease, especially in old patients. Chemoresistance and the absence of host immune responses against autochthonous malignancy play a major role in the poor prognosis of AML. The triazene compounds Dacarbazine and Temozolomide are monofunctional alkylators that donate methyl groups to many sites in DNA, including the O(6)-position of guanine producing O(6)-methylguanine (O(6)-MeG).

The nitrosated bile acid DNA lesion O6-carboxymethylguanine is a substrate for the human DNA repair protein O6-methylguanine-DNA methyltransferase.

The consumption of red meat is a risk factor in human colorectal cancer (CRC). One hypothesis is that red meat facilitates the nitrosation of bile acid conjugates and amino acids, which rapidly convert to DNA-damaging carcinogens. Indeed, the toxic and mutagenic DNA adduct O(6)-carboxymethylguanine (O(6)-CMG) is frequently present in human DNA, increases in abundance in people with high levels of dietary red meat and may therefore be a causative factor in CRC.

Alkyltransferase-like protein (Atl1) distinguishes alkylated guanines for DNA repair using cation-π interactions.

Alkyltransferase-like (ATL) proteins in Schizosaccharomyces pombe (Atl1) and Thermus thermophilus (TTHA1564) protect against the adverse effects of DNA alkylation damage by flagging O(6)-alkylguanine lesions for nucleotide excision repair (NER). We show that both ATL proteins bind with high affinity to oligodeoxyribonucleotides containing O(6)-alkylguanines differing in size, polarity, and charge of the alkyl group. However, Atl1 shows a greater ability than TTHA1564 to distinguish between O(6)-alkylguanine and guanine and in an unprecedented mechanism uses Arg69 to probe the electrostatic potential surface of O(6)-alkylguanine, as determined using molecular mechanics calculations.

Synthesis of oligodeoxyribonucleotides containing a conformationally-locked anti analogue of O6-methyl-2'-deoxyguanosine and their recognition by MGMT and Atl1.

We show that DNA containing a conformationally-locked anti analogue of O(6)-alkylguanine is a poor substrate for human O(6)-methylguanine-DNA methyltransferase (MGMT) and the alkyltransferase-like protein, Atl1. This highlights the requirement for the syn conformation and rationalises why certain O(6)-alkylguanines are poor MGMT substrates.

Molecular characterization of an adaptive response to alkylating agents in the opportunistic pathogen Aspergillus fumigatus.

An adaptive response to alkylating agents based upon the conformational change of a methylphosphotriester (MPT) DNA repair protein to a transcriptional activator has been demonstrated in a number of bacterial species, but this mechanism appears largely absent from eukaryotes. Here, we demonstrate that the human pathogen Aspergillus fumigatus elicits an adaptive response to sub-lethal doses of the mono-functional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We have identified genes that encode MPT and O(6)-alkylguanine DNA alkyltransferase (AGT) DNA repair proteins; deletions of either of these genes abolish the adaptive response and sensitize the organism to MNNG.

Atl1 regulates choice between global genome and transcription-coupled repair of O(6)-alkylguanines.

Nucleotide excision repair (NER) has long been known to remove DNA lesions induced by chemical carcinogens, and the molecular mechanism has been partially elucidated. Here we demonstrate that in Schizosaccharomyces pombe a DNA recognition protein, alkyltransferase-like 1 (Atl1), can play a pivotal role in selecting a specific NER pathway, depending on the nature of the DNA modification. The relative ease of dissociation of Atl1 from DNA containing small O(6)-alkylguanines allows accurate completion of global genome repair (GGR), whereas strong Atl1 binding to bulky O(6)-alkylguanines blocks GGR, stalls the transcription machinery, and diverts the damage to transcription-coupled repair.