Alkyltransferase-like proteins (ATLs) share functional motifs with the cancer chemotherapy target O6-alkylguanine DNA-alkyltransferase (AGT) and paradoxically protect cells from the biological effects of DNA alkylation damage, despite lacking the AGT reactive cysteine and alkyltransferase activity. Here we determine S. pombe ATL structures without and with damaged DNA containing endogenous lesion O6-methylguanine or cigarette smoke-derived O6-4-(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 ATL interactions are ancestral to present-day repair pathways in all domains of life. Genetic connections to XPG and ERCC1 in S. pombe homologs Rad13 and Swi10 and biochemical interactions with 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.
We have analyzed a panel of 14 cases of childhood adrenocortical tumors unselected for family history and have identified germline TP53 mutations in >80%, making this the highest known incidence of a germline mutation in a tumor-suppressor gene in any cancer. The spectrum of germline TP53 mutations detected is remarkably limited. Analysis of tumor tissue for loss of constitutional heterozygosity, with respect to the germline mutant allele and the occurrence of other somatic TP53 mutations, indicates complex sequences of genetic events in a number of tumors. None of the families had cancer histories that conformed to the criteria for Li-Fraumeni syndrome, but, in some families, we were able to demonstrate that the mutation had been inherited. In these families there were gene carriers unaffected in their 40s and 50s, and there were others with relatively late-onset cancers. These data provide evidence that certain TP53 alleles confer relatively low penetrance for predisposition to the development of cancer, and they imply that deleterious TP53 mutations may be more frequent in the population than has been estimated previously. Our findings have considerable implications for the clinical management of children with andrenocortical tumors and their parents, in terms of both genetic testing and the early detection and treatment of tumors.
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 6 -methylguanine-DNA methyltransferase (MGMT) repairs the most cytotoxic of lesions generated by TMZ, O 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 3 -methyladenine and N 7 -methylguanine, may contribute to TMZ resistance. Silencing of APNG in established and primary TMZresistant 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.
Purpose: Temozolomide, a DNA methylating agent used to treat melanoma, induces DNA damage, which is repaired by O 6 -alkylguanine alkyltransferase (ATase) and poly(ADP-ribose) polymerase-1 (PARP-1)^dependent base excision repair. The current study was done to define the effect of temozolomide on DNA integrity and relevant repair enzymes as a prelude to a phase I trial of the combination of temozolomide with a PARP inhibitor. Experimental Design: Temozolomide (200 mg/m 2 oral administration) was given to 12 patients with metastatic malignant melanoma. Peripheral blood lymphocytes (PBL) were analyzed for PARP activity, DNA single-strand breakage, ATase levels, and DNA methylation. PARP activity was also measured in tumor biopsies from 9 of 12 patients and in PBLs from healthy volunteers. Results: Temozolomide pharmacokinetics were consistent with previous reports. Temozolomide therapy caused a substantial and sustained elevation of N 7 -methylguanine levels, a modest and sustained reduction in ATase activity, and a modest and transient increase in DNA strand breaks and PARP activity in PBLs. PARP-1activity in tumor homogenates was variable (828 F 599 pmol PAR monomer/mg protein) and was not consistently affected by temozolomide treatment. Conclusions:The effect of temozolomide reported here are consistent with those documented in previous studies with temozolomide and similar drug, dacarbazine, demonstrating that a representative patient population was investigated. Furthermore, PARP activity was not inhibited by temozolomide treatment and this newly validated pharmacodynamic assay is therefore suitable for use in a proof-of-principle phase I trial a PARP-1inhibitor in combination with temozolomide.Intrinsic or acquired resistance remains a major limitation in the use of cytotoxic chemotherapy. A variety of cancer cell resistance mechanisms have been described or proposed, including decreased drug uptake into cells, increased drug efflux intracellular drug inactivation, alteration of the cellular target, repair of drug-induced damage, or development of tolerance to drug-induced lesions (1). Strategies directed at overcoming these mechanisms of drug resistance are being evaluated, including the targeting of DNA repair pathways, in an attempt to improve on the efficacy of existing cytotoxic drugs.Temozolomide is an orally available monofunctional DNA alkylating agent used to treat gliomas and malignant melanoma (2). Temozolomide is rapidly absorbed and undergoes spontaneous breakdown to form the active monomethyl triazene, 5-(3-methyl-1-triazeno)imidazole-4-carboxamide. Monomethyl triazene forms several DNA methylation products, the predominate species being N 7 -methylguanine (70%), N 3 -methyladenine (9%), and O system signals G 2 -M cell cycle arrest and the initiation of apoptosis (5 -7). The quantitatively more important N 7 -methylguanine and N 3 -methyladenine lesions formed by temozolomide are rapidly repaired by base excision repair.The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) plays a ...
We have studied a total of 36 tumours from 28 patients with germline mutations to the TP53 gene for loss of heterozygosity at TP53 using techniques of both direct sequencing and restriction fragment length polymorphism analysis. All patients were from families conforming to the de®nition of classical Li ± Fraumeni syndrome (LFS) or were Li ± Fraumeni-like (LFL). The data we have obtained show that loss of the wild-type TP53 gene is observed in under half (44%) of all tumours, and that the pattern of LOH at TP53 may be mutation speci®c. LOH has been observed in premalignant as well as invasive tumours. Two tumours (6%) show loss of the mutant allele and retention of the wild-type. To con®rm that TP53 is indeed the target for LOH events on chromosome 17, we have used additional microsatellite repeats to examine patterns of allelic imbalance along the length of chromosome 17. Data from this analysis indicate that TP53 is the target of loss, but reveal some other interesting patterns of allelic imbalance at other loci on chromosome 17.
The repair of specific types of DNA alkylation damage by O6-alkylguanine-DNA alkyltransferase (MGMT) is a major mechanism of resistance to the carcinogenic and chemotherapeutic effects of certain alkylating agents. MGMT expression levels vary widely between individuals but the underlying causes of this variability are not known. To address this, we used an expressed single nucleotide polymorphism (SNP) and demonstrated that the MGMT alleles are frequently expressed at different levels in peripheral blood mononuclear cells (PBMC). This suggests that there is a genetic component of inter-allelic variation of MGMT levels that maps close to or within the MGMT locus. We then used quantitative trait locus (QTL) analysis using intragenic SNPs and found that there are at least two sites influencing inter-individual variation in PBMC MGMT activity. One is characterized by an SNP at the 3' end of the first intron and the second by two SNPs in the last exon. The latter are in perfect disequilibrium and both result in amino acid substitutions-one of them, Ile143Val, affecting an amino acid close to the Cys145 residue at the active site of MGMT. Using in vitro assays, we further showed that while the Val143 variant did not affect the activity of the protein on methylated DNA substrate, it was more resistant to inactivation by the MGMT pseudosubstrate, O6-(4-bromothenyl)guanine. These findings suggest that further investigations of the potential epidemiological and clinical significance of inherited differences in MGMT expression and activity are warranted.
Background:Poly adenosine diphosphate (ADP)-ribose polymerase (PARP) is essential in cellular processing of DNA damage via the base excision repair pathway (BER). The PARP inhibition can be directly cytotoxic to tumour cells and augments the anti-tumour effects of DNA-damaging agents. This study evaluated the optimally tolerated dose of olaparib (4-(3--4-fluorophenyl) methyl-1(2H)-one; AZD2281, KU0059436), a potent PARP inhibitor, with dacarbazine and assessed safety, toxicity, clinical pharmacokinetics and efficacy of combination treatment.Patients and methods:Patients with advanced cancer received olaparib (20–200 mg PO) on days 1–7 with dacarbazine (600–800 mg m−2 IV) on day 1 (cycle 2, day 2) of a 21-day cycle. An expansion cohort of chemonaive melanoma patients was treated at an optimally tolerated dose. The BER enzyme, methylpurine-DNA glycosylase and its substrate 7-methylguanine were quantified in peripheral blood mononuclear cells.Results:The optimal combination to proceed to phase II was defined as 100 mg bd olaparib with 600 mg m−2 dacarbazine. Dose-limiting toxicities were neutropaenia and thrombocytopaenia. There were two partial responses, both in patients with melanoma.Conclusion:This study defined a tolerable dose of olaparib in combination with dacarbazine, but there were no responses in chemonaive melanoma patients, demonstrating no clinical advantage over single-agent dacarbazine at these doses.
We present an extended family with LiFraumeni syndrome characterised by gastric and breast carcinoma, glioma, sarcoma, and leukaemia. This family showed strong evidence of linkage to TP53, and three of four tumours analysed showed loss of the wild type allele. A codon 175 missense mutation was identified in exon S in all available affected subjects. Counselling, screening, and issues surrounding presymptomatic testing are discussed. (J Med Genet 1995;32:942-945)
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