alkylpurine-DNA-N-glycosylase Research Articles

Mitigating temozolomide resistance in glioblastoma via DNA damage-repair inhibition.

Glioblastomas are among the most lethal cancers, with a 5 year survival rate below 25%. Temozolomide is typically used in glioblastoma treatment; however, the enzymes alkylpurine-DNA-N-glycosylase (APNG) and methylguanine-DNA-methyltransferase (MGMT) efficiently mediate the repair of DNA damage caused by temozolomide, reducing treatment efficacy. Consequently, APNG and MGMT inhibition has been proposed as a way of overcoming chemotherapy resistance. Here, we develop a mechanistic mathematical model that explicitly incorporates the effects of chemotherapy on tumour cells, including the processes of DNA damage induction, cell arrest and DNA repair. Our model is carefully parametrized and validated, and then used to virtually recreate the response of heteroclonal glioblastomas to dual treatment with temozolomide and inhibitors of APNG/MGMT. Using our mechanistic model, we identify four combination treatment strategies optimized by tumour cell phenotype, and isolate the strategy most likely to succeed in a pre-clinical and clinical setting. If confirmed in clinical trials, these strategies have the potential to offset chemotherapy resistance in patients with glioblastoma and improve overall survival.

APNG as a prognostic marker in patients with glioblastoma

AimExpression of the base excision repair enzyme alkylpurine-DNA-N-glycosylase (APNG) has been correlated to temozolomide resistance. Our aim was to evaluate the prognostic value of APNG in a population-based cohort with 242 gliomas including 185 glioblastomas (GBMs). Cellular heterogeneity of GBMs was taken into account by excluding APNG expression in non-tumor cells from the analysis.MethodsAPNG expression was evaluated using automated image analysis and a novel quantitative immunohistochemical (IHC) assay (qIHC), where APNG protein expression was evaluated through countable dots. Non-tumor cells were excluded using an IHC/qIHC double-staining. For verification, APNG was measured by a quantitative double-immunofluorescence (IF) assay. As validation APNG mRNA expression was evaluated using independent TCGA data.ResultsUsing qIHC, high levels of APNG were associated with better overall survival (OS) in univariate (HR = 0.50; P < 0.001) and multivariate analysis (HR = 0.53; P = 0.001). Patients with methylated MGMT promoters and high APNG expression demonstrated better OS, than patients with methylated MGMT promoters and low APNG expression (HR = 0.59; P = 0.08). Retesting the cohort using IF showed similar results in both univariate (HR = 0.61; P = 0.002) and multivariate analysis (HR = 0.81; P = 0.2). The results were supported by data from the TCGA database.ConclusionsUsing two different assays combined with quantitative image analysis excluding non-tumour cells, APNG was an independent prognostic factor among patients with a methylated MGMT promoter. We expect that APNG qIHC can potentially identify GBM patients who will not benefit from treatment with temozolomide.

Determining viability of using APNG status as a prognostic marker in patients with glioblastoma multiforme.

2028 Background: Expression of the enzyme alkylpurine-DNA-N-glycosylase (APNG) has been associated with poor outcome in patients with glioblastoma multiforme (GBM). APNG is part of the base excisio...

Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma.

Real-time monitoring of drug efficacy in glioblastoma multiforme (GBM) is a major clinical problem as serial re-biopsy of primary tumours is often not a clinical option. MGMT (O6-methylguanine DNA methyltransferase) and APNG (alkylpurine-DNA-N-glycosylase) are key enzymes capable of repairing temozolomide-induced DNA damages and their levels in tissue are inversely related to treatment efficacy. Yet, serial clinical analysis remains difficult, and, when done, primarily relies on promoter methylation studies of tumour biopsy material at the time of initial surgery. Here we present a microfluidic chip to analyse mRNA levels of MGMT and APNG in enriched tumour exosomes obtained from blood. We show that exosomal mRNA levels of these enzymes correlate well with levels found in parental cells and that levels change considerably during treatment of seven patients. We propose that if validated on a larger cohort of patients, the method may be used to predict drug response in GBM patients.

Malignant astrocytomas of elderly patients lack favorable molecular markers: an analysis of the NOA-08 study collective

The number of patients age >65 years with malignant gliomas is increasing. Prognosis of these patients is worse compared with younger patients. To determine biological differences among malignant gliomas of different age groups and help to explain the survival heterogeneity seen in the NOA-08 trial, the prevalence and impact of recently established biomarkers for outcome in younger patients were characterized in elderly patients. Prevalences of mutations of isocitrate dehydrogenase 1 (IDH1) and histone H3.3 (H3F3A), the glioma cytosine-phosphate-guanine island methylator phenotype (G-CIMP), and methylation of alkylpurine DNA N-glycosylase (APNG) and peroxiredoxin 1 (PRDX1) promoters were determined in a representative biomarker subset (n = 126 patients with anaplastic astrocytoma or glioblastoma) from the NOA-08 trial. IDH1 mutations (R132H) were detected in only 3/126 patients, precluding determination of an association between IDH mutation and outcome. These 3 patients also displayed the G-CIMP phenotype. None of the IDH1 wild-type tumors were G-CIMP positive. Mutations in H3F3A were absent in all 103 patients sequenced for H3F3A. MassARRAY analysis of the APNG promoter revealed generally low methylation levels and failed to confirm any predictive properties for benefit from alkylating chemotherapy. Neither did PRDX1 promoter methylation show differential methylation or association with outcome in this cohort. In a 170-patient cohort from The Cancer Genome Atlas database matched for relevant prognostic factors, age ≥65 years was strongly associated with shorter survival. Despite an age-independent stable frequency of O(6)-methylguanine-DNA methyltransferase (MGMT) promoter hypermethylation, tumors in this age group largely lack prognostically favorable markers established in younger glioblastoma patients, which likely contributes to the overall worse prognosis of elderly patients. However, the survival differences hint at fundamental further differences among malignant gliomas of different age groups.

Alkylpurine–DNA–N-glycosylase confers resistance to temozolomide in xenograft models of glioblastoma multiforme and is associated with poor survival in patients

Alkylpurine–DNA–N-glycosylase confers resistance to temozolomide in xenograft models of glioblastoma multiforme and is associated with poor survival in patients

Alkylpurine–DNA–N-glycosylase confers resistance to temozolomide in xenograft models of glioblastoma multiforme and is associated with poor survival in patients

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). O⁶-methylguanine-DNA methyltransferase (MGMT) repairs the most cytotoxic of lesions generated by TMZ, O⁶-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 N³-methyladenine and N⁷-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.

A GATA4-regulated tumor suppressor network represses formation of malignant human astrocytomas

Glioblastoma Multiforme (GBM), the most common and lethal primary human brain tumor, exhibits multiple molecular aberrations. We report that loss of the transcription factor GATA4, a negative regulator of normal astrocyte proliferation, is a driver in glioma formation and fulfills the hallmarks of a tumor suppressor gene (TSG). Although GATA4 was expressed in normal brain, loss of GATA4 was observed in 94/163 GBM operative samples and was a negative survival prognostic marker. GATA4 loss occurred through promoter hypermethylation or novel somatic mutations. Loss of GATA4 in normal human astrocytes promoted high-grade astrocytoma formation, in cooperation with other relevant genetic alterations such as activated Ras or loss of TP53. Loss of GATA4 with activated Ras in normal astrocytes promoted a progenitor-like phenotype, formation of neurospheres, and the ability to differentiate into astrocytes, neurons, and oligodendrocytes. Re-expression of GATA4 in human GBM cell lines, primary cultures, and brain tumor-initiating cells suppressed tumor growth in vitro and in vivo through direct activation of the cell cycle inhibitor P21(CIP1), independent of TP53. Re-expression of GATA4 also conferred sensitivity of GBM cells to temozolomide, a DNA alkylating agent currently used in GBM therapy. This sensitivity was independent of MGMT (O-6-methylguanine-DNA-methyltransferase), the DNA repair enzyme which is often implicated in temozolomide resistance. Instead, GATA4 reduced expression of APNG (alkylpurine-DNA-N-glycosylase), a DNA repair enzyme which is poorly characterized in GBM-mediated temozolomide resistance. Identification and validation of GATA4 as a TSG and its downstream targets in GBM may yield promising novel therapeutic strategies.

The effect of Msh2 knockdown on methylating agent induced toxicity in DNA glycosylase deficient cells

The DNA structure recognition protein MSH2 is an important protein in DNA mismatch repair due to its role in initiating the repair process. To examine the potential interactions between mismatch repair and base excision repair (BER) we have examined the effect of MSH2 knockdown on 6-thioguanine (6-TG), temozolomide (TMZ) and methylmethane sulphonate (MMS) induced toxicity in BER proficient and deficient cell lines. An shRNA expression vector containing Msh2 target sequences was designed and used to transfect mouse embryonic fibroblasts lacking either alkylpurine DNA N-glycosylase ( Mpg) or endonuclease III homologue ( Nth1). Significant knockdown of Msh2 gene expression was achieved with three different target sequences, with the highest level being shown by Msh2 283 . Clonal selection resulted in differing levels of knockdown in Mpg −/− cells: (69.0 ± 12.1% from 5 cell clones). Transfection of the Msh2 283 sequence in Mpg +/+, Nth1 +/+ and Nth1 −/− cells resulted in average knockdowns of 45.1 ± 40.5% (3 clones), 58.0 ± 21.4% (5 clones) and 74.9 ± 14.8% (3 clones), respectively. Msh2 knockdown resulted in increased resistance to 6-TG in BER (MPG and NTH1) proficient and deficient cell lines with similar levels of knockdown (84 ± 4%) but increased resistance to TMZ only in Mpg +/+ and Nth1 −/− cell lines and not Mpg −/− or Nth1 +/+ cells as assessed by an MTT assay. Msh2 knockdown had no effect on sensitivity to MMS induced toxicity. In a clonogenic assay, Msh2 silenced Mpg +/+, Mpg −/−, Nth1 +/+ and Nth1 −/− cells were more resistant to TMZ. These results confirm previous studies showing that MSH2 is a key protein in influencing 6-TG and O 6-methylguanine induced toxicity but also suggest that the effect of this protein depends upon the presence of other proteins in different DNA repair pathways.

Action of multiple base excision repair enzymes on the 2′-deoxyribonolactone

Free radical attack on the sugar-phosphate backbone generates oxidized apurinic/apyrimidinic (AP) residues in DNA. 2′-deoxyribonolactone (dL) is a C1′-oxidized AP site damage generated by UV and γ-irradiation, and certain anticancer drugs. If not repaired dL produces G → A transitions in Escherichia coli. In the base excision repair (BER) pathway, AP endonucleases are the major enzymes responsible for 5′-incision of the regular AP site (dR) and dL. DNA glycosylases with associated AP lyase activity can also efficiently cleave regular AP sites. Here, we report that dL is a substrate for AP endonucleases but not for DNA glycosylases/AP lyases. The kinetic parameters of the dL-incision were similar to those of the dR. DNA glycosylases such as E. coli formamidopyrimidine-DNA glycosylase, mismatch-specific uracil-DNA glycosylase, and human alkylpurine-DNA N-glycosylase bind strongly to dL without cleaving it. We show that dL cross-links with the human proteins 8-oxoguanine-DNA (hOGG1) and thymine glycol-DNA glycosylases (hNth1), and dR cross-links with Nth and hNth1. These results suggest that dL and dR induced genotoxicity might be strengthened by BER pathway in vivo.

Chemical rearrangement and repair pathways of 1, N6-ethenoadenine

1, N 6-Ethenoadenine (εA) is an exocyclic DNA adduct introduced to DNA by vinyl chloride and related compounds as well as in the consequence of oxidative stress and lipid peroxidation (LPO). This highly genotoxic DNA damage is chemically unstable and either depurinates or converts into pyrimidine ring-opened secondary lesions. We have studied the structures of derivatives formed during εA chemical rearrangement and identified enzymes repairing one of the rearrangement products. Rearrangement involves a water molecule addition to the C(2)N(3) bond of εA, resulting in formation of pyrimidine ring-closed B1 product, which is in equilibrium with pyrimidine ring-opened B2 compound. B2 further deformylates to yield compound C. N-Glycosidic bond of compound C is unstable and C depurinates, yielding compound D. These secondary lesions are not repaired by alkylpurine DNA N-glycosylase, which excises the parental εA. Compound B, when paired with thymine and cytosine is efficiently excised by Escherichia coli formamidopirymidine DNA N-glycosylase (Fpg), and thymine glycol DNA N-glycosylases from E. coli (Nth) and Saccharomyces cerevisiae (Ntg2). B is eliminated from B:G pair only by Nth and Ntg2 glycosylases, however none of the enzymes studied is excising B from B:A pair. This enables finishing of rearrangement, formation of AP sites and subsequently DNA strand breaks. During in vitro translesion synthesis, C is much easier bypassed by DNA polymerases, than compound B, and also than the parental εA as well as than the AP site. This bypass beyond C proceeds mainly by misinsertion of adenine and guanine, or by insertion of thymine, the latter restoring the parental A:T pair. Alternatively, looping out of adducted nucleotide alone or with adjacent one generates one- or two-nucleotide deletions. This may explain the previously reported 20-fold higher mutagenic potency of product C in comparison to εA in E. coli [Biochemistry 32 (1993) 12793].

Structural insights by molecular dynamics simulations into differential repair efficiency for ethano-A versus etheno-A adducts by the human alkylpurine-DNA N-glycosylase.

1,N6-ethenoadenine adducts (epsilonA) are formed by known environmental carcinogens and found to be removed by human alkylpurine-DNA N-glycosylase (APNG). 1,N6-ethanoadenine (EA) adducts differ from epsilonA by change of a double bond to a single bond in the 5-member exocyclic ring and are formed by chloroethyl nitrosoureas, which are used in cancer therapy. In this work, using purified recombinant human APNG, we show that EA is a substrate for the enzyme. However, the excision efficiency of EA was 65-fold lower than that of epsilonA. Molecular dynamics simulation produced similar structural motifs for epsilonA and EA when incorporated into a DNA duplex, suggesting that there are no specific conformational features in the DNA duplex which can account for the differences in repair efficiency. However, when EA was modeled into the APNG active site, based on the APNG/epsilonA-DNA crystallographic coordinates, in structures produced by 2 ns molecular dynamics simulation, we observed weakening in the stacking interaction between EA and aromatic side chains of the key amino acids in the active site. In contrast, the planar epsilonA is better stacked at the enzyme active site. We propose that the observed destabilization of the EA adduct at the active site, such as reduced stacking interactions, could account for the biochemically observed weaker recognition of EA by APNG as compared to epsilonA.

ETHANOL MODULATES RAT HEPATIC DNA REPAIR FUNCTIONS

To further explore how ethanol may act at the DNA level, studies have been made of DNA repair mechanisms in male Wistar rats given ethanol either as an acute intragastric dose (5 g/kg) or continuously in a liquid diet (5% w/v) to provide 36% of the caloric intake. These treatments generate significant levels of free radicals with evidence of damage to DNA. The acute ethanol dose significantly inhibited O(6)-alkylguanine-DNA alkyltransferase (ATase) activity by 21-32% throughout the 24-h post-treatment period and this was confirmed by immunohistochemical detection of the ATase protein in hepatic nuclei. Twelve hours after the ethanol treatment, the activities of the DNA glycosylases, alkylpurine-DNA-N-glycosylase (APNG) and 8-oxoguanine-DNA glycosylase (OXOG glycosylase) were each increased by approximately 44%. In contrast, when given chronically via the liquid diet, ethanol initially had no effect on ATase activity, but after 4 weeks ATase activity was increased by 40%. Following ethanol withdrawal, ATase activity remained elevated for at least 12 h, but, by 24 h, the activity had fallen to the uninduced control level. DNA glycosylase activities were again affected differently. After 1 week of dietary ethanol exposure, there was no effect on APNG activity but it was inhibited by 19% at 4 weeks. OXOG glycosylase activity, on the other hand, was increased by 53% after 1 week, but decreased by 40% after 4 weeks. Although some of these changes in DNA repair capacity were relatively small, over time, their potential impact on the repair of endogenous or exogenous alkylation and/or oxidation damage in DNA would be substantial. These studies indicate possible mechanisms for the co-carcinogenic effects of ethanol.

Increased susceptibility to streptozotocin-induced beta-cell apoptosis and delayed autoimmune diabetes in alkylpurine-DNA-N-glycosylase-deficient mice.

Type 1 diabetes is thought to occur as a result of the loss of insulin-producing pancreatic beta cells by an environmentally triggered autoimmune reaction. In rodent models of diabetes, streptozotocin (STZ), a genotoxic methylating agent that is targeted to the beta cells, is used to trigger the initial cell death. High single doses of STZ cause extensive beta-cell necrosis, while multiple low doses induce limited apoptosis, which elicits an autoimmune reaction that eliminates the remaining cells. We now show that in mice lacking the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG), beta-cell necrosis was markedly attenuated after a single dose of STZ. This is most probably due to the reduction in the frequency of base excision repair-induced strand breaks and the consequent activation of poly(ADP-ribose) polymerase (PARP), which results in catastrophic ATP depletion and cell necrosis. Indeed, PARP activity was not induced in APNG(-/-) islet cells following treatment with STZ in vitro. However, 48 h after STZ treatment, there was a peak of apoptosis in the beta cells of APNG(-/-) mice. Apoptosis was not observed in PARP-inhibited APNG(+/+) mice, suggesting that apoptotic pathways are activated in the absence of significant numbers of DNA strand breaks. Interestingly, STZ-treated APNG(-/-) mice succumbed to diabetes 8 months after treatment, in contrast to previous work with PARP inhibitors, where a high incidence of beta-cell tumors was observed. In the multiple-low-dose model, STZ induced diabetes in both APNG(-/-) and APNG(+/+) mice; however, the initial peak of apoptosis was 2.5-fold greater in the APNG(-/-) mice. We conclude that APNG substrates are diabetogenic but by different mechanisms according to the status of APNG activity.

Sequence-dependent repair of synthetic AP sites in 15-mer and 35-mer oligonucleotides: role of thermodynamic stability imposed by neighbor bases.

We previously reported that 15-mer oligonucleotides with a central 1, N(6)-epsilonA were cleaved by alkylpurine-DNA N-glycosylase as a function of T(m), modulated by neighbor bases [Hang, B., Sági, J., and Singer, B. (1998) J. Biol. Chem. 273, 33406-33413]. This type of investigation has now been extended to cleavage by Escherichia coli endonuclease IV of a centrally placed synthetic AP site using both 15-mer and 35-mer duplexes. In 15-mers, the triplet sequences adjunct to the central AP site greatly affected the thermodynamic stability. The repair rate paralleled the thermal stability since endonuclease IV requires a double-stranded substrate. When the AP site-containing duplexes were 35-mers, there was also a general correlation between the thermostability and cleavage efficiency. However, the difference in the cleavage rates between different sequences was much less than with the 15-mers. Since the 35-mers were more than 96% annealed, this difference presumably results from local stability and structure adjacent to the AP site. These results suggest that under enzyme limiting conditions or overproduction of AP sites, sequence-dependent differential repair could occur in vivo.

A 55-kDa protein isolated from human cells shows DNA glycosylase activity toward 3,N4-ethenocytosine and the G/T mismatch.

Etheno adducts in DNA arise from multiple endogenous and exogenous sources. Of these adducts we have reported that, 1,N6-ethenoadenine (epsilonA) and 3,N4-ethenocytosine (epsilonC) are removed from DNA by two separate DNA glycosylases. We later confirmed these results by using a gene knockout mouse lacking alkylpurine-DNA-N-glycosylase, which excises epsilonA. The present work is directed toward identifying and purifying the human glycosylase activity releasing epsilonC. HeLa cells were subjected to multiple steps of column chromatography, including two epsilonC-DNA affinity columns, which resulted in >1,000-fold purification. Isolation and renaturation of the protein from SDS/polyacrylamide gel showed that the epsilonC activity resides in a 55-kDa polypeptide. This apparent molecular mass is approximately the same as reported for the human G/T mismatch thymine-DNA glycosylase. This latter activity copurified to the final column step and was present in the isolated protein band having epsilonC-DNA glycosylase activity. In addition, oligonucleotides containing epsilonC.G or G/T(U), could compete for epsilonC protein binding, further indicating that the epsilonC-DNA glycosylase is specific for both types of substrates in recognition. The same substrate specificity for epsilonC also was observed in a recombinant G/T mismatch DNA glycosylase from the thermophilic bacterium, Methanobacterium thermoautotrophicum THF.

Alkylpurine-DNA-N-glycosylase knockout mice show increased susceptibility to induction of mutations by methyl methanesulfonate.

Alkylpurine-DNA-N-glycosylase (APNG) null mice have been generated by homologous recombination in embryonic stem cells. The null status of the animals was confirmed at the mRNA level by reverse transcription-PCR and by the inability of cell extracts of tissues from the knockout (ko) animals to release 3-methyladenine (3-meA) or 7-methylguanine (7-meG) from 3H-methylated calf thymus DNA in vitro. Following treatment with DNA-methylating agents, increased persistence of 7-meG was found in liver sections of APNG ko mice in comparison with wild-type (wt) mice, demonstrating an in vivo phenotype for the APNG null animals. Unlike other null mutants of the base excision repair pathway, the APNG ko mice exhibit a very mild phenotype, show no outward abnormalities, are fertile, and have an apparently normal life span. Neither a difference in the number of leukocytes in peripheral blood nor a difference in the number of bone marrow polychromatic erythrocytes was found when ko and wt mice were exposed to methylating or chloroethylating agents. These agents also showed similar growth-inhibitory effects in primary embryonic fibroblasts isolated from ko and wt mice. However, treatment with methyl methanesulfonate resulted in three- to fourfold more hprt mutations in splenic T lymphocytes from APNG ko mice than in those from wt mice. These mutations were predominantly single-base-pair changes; in the ko mice, they consisted primarily of AT-->TA and GC-->TA transversions, which most likely are caused by 3-meA and 3- or 7-meG, respectively. These results clearly show an important role for APNG in attenuating the mutagenic effects of N-alkylpurines in vivo.

Targeted deletion of alkylpurine-DNA-N-glycosylase in mice eliminates repair of 1,N6-ethenoadenine and hypoxanthine but not of 3,N4-ethenocytosine or 8-oxoguanine.

It has previously been reported that 1,N6-ethenoadenine (epsilonA), deaminated adenine (hypoxanthine, Hx), and 7,8-dihydro-8-oxoguanine (8-oxoG), but not 3,N4-ethenocytosine (epsilonC), are released from DNA in vitro by the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG). To assess the potential contribution of APNG to the repair of each of these mutagenic lesions in vivo, we have used cell-free extracts of tissues from APNG-null mutant mice and wild-type controls. The ability of these extracts to cleave defined oligomers containing a single modified base was determined. The results showed that both testes and liver cells of these knockout mice completely lacked activity toward oligonucleotides containing epsilonA and Hx, but retained wild-type levels of activity for epsilonC and 8-oxoG. These findings indicate that (i) the previously identified epsilonA-DNA glycosylase and Hx-DNA glycosylase activities are functions of APNG; (ii) the two structurally closely related mutagenic adducts epsilonA and epsilonC are repaired by separate gene products; and (iii) APNG does not contribute detectably to the repair of 8-oxoG.