Search

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 O6-alkylguanines (O6-AlkGs) in DNA and applied it to human DNA samples. The method exploits the ability of the Schizosaccharomyces pombe alkyltransferase-like protein (Atl1) to recognise and bind to a wide range of O6-AlkGs in DNA. We established an Atl1-based slot-blot (ASB) assay and validated it using calf thymus DNA alkylated in vitro with a range of alkylating agents and both calf thymus and human placental DNA methylated in vitro with temozolomide (TMZ). ASB signals were directly proportional to the levels of O6-meG in these controls. Pre-treatment of DNA with the DNA repair protein O6-methylguanine–DNA methyltransferase (MGMT) reduced binding of Atl1, confirming its specificity. In addition, MCF 10A cells were treated with 500 μM TMZ and the extracted DNA, analysed using the ASB, was found to contain 1.34 fmoles O6 -meG/μg DNA. Of six human breast tumour DNA samples assessed, five had detectable O6-AlkG levels (mean ± SD 1.24 ± 0.25 O6-meG equivalents/μg DNA. This study shows the potential usefulness of the ASB assay to detect and quantify total O6-AlkGs in human DNA samples. [ABSTRACT FROM AUTHOR]

DNA alkyltransferase and alkyltransferase-like family proteins are responsible for the repair of highly mutagenic and cytotoxic O6-alkylguanine and O4-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. In contrast, alkyltransferase-like proteins recruit nucleotide excision repair components for O6-alkylguanine elimination. One or more of these proteins are found in all kingdoms of life, and where this has been determined, their overall DNA repair mechanism is strictly conserved between organisms. Nevertheless, between species, subtle as well as more extensive differences that affect target lesion preferences and/or introduce additional protein functions have evolved. Examining these differences and their functional consequences is intricately entwined with understanding the details of their DNA repair mechanism(s) and their biological roles. In this review, we will present and discuss various aspects of the current status of knowledge on this intriguing protein family. [ABSTRACT FROM AUTHOR]

O6-methylguanine-DNA methyltransferase (MGMT) constitutes an important cellular mechanism for repairing potentially cytotoxic DNA damage induced by guanine O6-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 [3H]-methylated-DNA-based MGMT inactivation assays and calculated the IC50 values. Using the results of 370 compounds, we performed quantitative structure–activity relationship (QSAR) modeling to identify the correlation between the chemical structure and MGMT-inactivating ability. Modeling was based on subdividing the sorted pIC50 values or on chemical structures or was random. A total of nine molecular descriptors were presented in the model equation, in which the mechanistic interpretation indicated that the status of nitrogen atoms, aliphatic primary amino groups, the presence of O-S at topological distance 3, the presence of Al-O-Ar/Ar-O-Ar/R..O..R/R-O-C=X, the ionization potential and hydrogen bond donors are the main factors responsible for inactivation ability. The final model was of high internal robustness, goodness of fit and prediction ability (R2pr = 0.7474, Q2Fn = 0.7375–0.7437, CCCpr = 0.8530). After the best splitting model was decided, we established the full model based on the entire set of compounds using the same descriptor combination. We also used a similarity-based read-across technique to further improve the external predictive ability of the model (R2pr = 0.7528, Q2Fn = 0.7387–0.7449, CCCpr = 0.8560). The prediction quality of 66 true external compounds was checked using the "Prediction Reliability Indicator" tool. In summary, we defined key structural features associated with MGMT inactivation, thus allowing for the design of MGMT inactivators that might improve clinical outcomes in cancer treatment. [ABSTRACT FROM AUTHOR]

Introduction The most common primary adult human brain tumors are gliomas, with grade IV glioblastoma multiforme (GBM) astrocytoma being most common and malignant (1-3). With the current standard of treatment [...], Glioblastoma multiforme (GBM) is the most common and lethal of all gliomas. The current standard of care includes surgery followed by concomitant radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). [O.sup.6]-methylguanine-DNA methyltransferase (MGMT) repairs the most cytotoxic of lesions generated by TMZ, [O.sup.6]-methylguanine. Methylation of the MGMT promoter in GBM correlates with increased therapeutic sensitivity to alkylating agent therapy. However, several aspects of TMZ sensitivity are not explained by MGMT promoter methylation. Here, we investigated our hypothesis that the base excision repair enzyme alkylpurine-DNA-N-glycosylase (APNG), which repairs the cytotoxic lesions [N.sup.3]-methyladenine and [N.sup.7]-methylguanine, may contribute to TMZ resistance. Silencing of APNG in established and primary TMZ-resistant GBM cell lines endogenously expressing MGMT and APNG attenuated repair of TMZ-induced DNA damage and enhanced apoptosis. Reintroducing expression of APNG in TMZ-sensitive GBM lines conferred resistance to TMZ in vitro and in orthotopic xenograft mouse models. In addition, resistance was enhanced with coexpression of MGMT. Evaluation of APNG protein levels in several clinical datasets demonstrated that in patients, high nuclear APNG expression correlated with poorer overall survival compared with patients lacking APNG expression. Loss of APNG expression in a subset of patients was also associated with increased APNG promoter methylation. Collectively, our data demonstrate that APNG contributes to TMZ resistance in GBM and may be useful in the diagnosis and treatment of the disease.

Alkyltransferase-like proteins (ATLs) share functional motifs with the cancer chemotherapy target [O.sup.6]- alkylguanine-DNA alkyltransferase (AGT) and paradoxically protect cells from the biological effects of DNA alkylation damage, despite lacking the reactive cysteine and alkyltransferase activity of AGT. Here we determine Schizosaccharomyces pombe ATL structures without and with damaged DNA containing the endogenous lesion [O.sup.6]-methylguanine or cigarette-smoke-derived [O.sup.6]-4-(3-pyridyl)-4-oxobutylguanine. These results reveal non-enzymatic DNA nucleotide flipping plus increased DNA distortion and binding pocket size compared to AGT. Our analysis of lesion-binding site conservation identifies new ATLs in sea anemone and ancestral archaea, indicating that ATL interactions are ancestral to present-day repair pathways in all domains of life. Genetic connections to mammalian XPG (also known as ERCC5) and ERCC1 in S. pombe homologues Rad13 and Swi10 and biochemical interactions with Escherichia coli UvrA and UvrC combined with structural results reveal that ATLs sculpt alkylated DNA to create a genetic and structural intersection of base damage processing with nucleotide excision repair., DNA [O.sup.6]-alkylguanine lesions are mutagenic and cytotoxic: they mispair during replication with thymine, resulting in G*C to A*T transition mutations (1-4). Human [O.sup.6]-alkylguanine DNA lesions are repaired by [O.sup.6]-alkylguanine-DNA alkyltransferase [...]

[O.sup.6]-Methylguanine ([O.sup.6]-MeG), a procarcinogenic DNA adduct that arises from exposure to methylating agents, has been detected in human colorectal DNA at levels comparable to those that cause adverse effects in model systems. [O.sup.6]-MeG levels vary within the colon, being higher in the cancer-prone regions of the large bowel. In rats and mice, [O.sup.6]-MEG persistence in colon DNA is associated with the induction of colon tumors after treatment with methylating agents. These tumors frequently contain K-ras GC [right arrow] AT transition mutations, which is consistent with the mutagenic properties of [O.sup.6]-MEG: such mutations are also commonly found in human colorectal cancers. [O.sup.6]-Alkylguanine adducts are removed by the DNA repair protein, [O.sup.6]-alkylguanine DNA-alkyltransferase (MGMT). MGMT overexpression in transgenic mice reduces the formation of K-ras GC [right arrow] AT mutations and tumors induced by methylating agents. Interindividual variations in human colon MGMT activity are large and large bowel tumors can occur in regions of low activity. Low MGMT activity in normal mucosa has been associated with the occurrence of K-ras GC [right arrow] AT mutations, whereas reduced MGMT expression and an increased frequency of K-ras GC[right arrow]AT mutations in colorectal cancers have been linked to MGMT promoter methylation. MGMT activity is also lower in adenomas than in adjacent normal tissue but only in those adenomas with this specific mutation. These results are entirely consistent with the hypothesis that GC [right arrow] AT mutations in the K-ras oncogene result from the formation and persistence of [O.sup.6]-alkylguanine lesions in colorectal DNA. Human exposure to endogenous or exogenous alkylating agents may thus be an environmental determinant of colorectal cancer risk. J. Nutr. 132: 3518S-3521S, 2002. KEY WORDS: * [O.sup.6]-methylguanine * [O.sup.6]-alkylguanine DNA-alkyltransferase * MGMT * K-ras * colorectal cancer