N-methylpurine DNA glycosylase and DNA polymerase beta modulate BER inhibitor potentiation of glioma cells to temozolomide. Neuro Oncol

Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
Neuro-Oncology (Impact Factor: 5.56). 03/2011; 13(5):471-86. DOI: 10.1093/neuonc/nor011
Source: PubMed


Temozolomide (TMZ) is the preferred chemotherapeutic agent in the treatment of glioma following surgical resection and/or radiation. Resistance to TMZ is attributed to efficient repair and/or tolerance of TMZ-induced DNA lesions. The majority of the TMZ-induced DNA base adducts are repaired by the base excision repair (BER) pathway and therefore modulation of this pathway can enhance drug sensitivity. N-methylpurine DNA glycosylase (MPG) initiates BER by removing TMZ-induced N3-methyladenine and N7-methylguanine base lesions, leaving abasic sites (AP sites) in DNA for further processing by BER. Using the human glioma cell lines LN428 and T98G, we report here that potentiation of TMZ via BER inhibition [methoxyamine (MX), the PARP inhibitors PJ34 and ABT-888 or depletion (knockdown) of PARG] is greatly enhanced by over-expression of the BER initiating enzyme MPG. We also show that methoxyamine-induced potentiation of TMZ in MPG expressing glioma cells is abrogated by elevated-expression of the rate-limiting BER enzyme DNA polymerase β (Polβ), suggesting that cells proficient for BER readily repair AP sites in the presence of MX. Further, depletion of Polβ increases PARP inhibitor-induced potentiation in the MPG over-expressing glioma cells, suggesting that expression of Polβ modulates the cytotoxic effect of combining increased repair initiation and BER inhibition. This study demonstrates that MPG overexpression, together with inhibition of BER, sensitizes glioma cells to the alkylating agent TMZ in a Polβ-dependent manner, suggesting that the expression level of both MPG and Polβ might be used to predict the effectiveness of MX and PARP-mediated potentiation of TMZ in cancer treatment.

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Available from: Robert W Sobol
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    • "In vertebrates, Nam conversion to NMN is catalyzed by NAMPT [23] [24]. NAD + is a critical factor for energy metabolites, as a messenger and for protein modifications such as PARP1/ARTD1 mediated PAR (degraded by PARG, ARH3 and TARG1) and is required for the activity of sirtuins, among other proteins [16]. the PAR-degrading enzyme PARG [8]. However, in addition to its role in BER, ARTD1 participates in additional DNA repair pathways such as NHEJ [9], NER [10], in sensing and repairing DNA doublestrand breaks [11] and is suggested to participate in the excision step during mismatch repair [12]. "

    Full-text · Article · Nov 2014 · DNA Repair
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    • "Temozolomide is an imidazoterazine-class chemotherapeutic alkylating agent that is currently used for the treatment of anaplastic astrocytoma and newly diagnosed glioblastoma [60]. It causes cancer cell death by inducing DNA base lesions, including N7-MeG (>70%), N3-MeA (9.2%) and O6-MeG (5%), through methylation at the N7 position of guanine, the N3 position of adenine, and the O6 position of guanine [61]. It has been found that the majority of temozolomide-induced base lesions, N7-MeG and N3-MeA, are subjected to the BER pathway [62], [63], and this has been demonstrated as one of the major mechanisms responsible for temozolomide chemotherapeutic resistance [64]. "
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    ABSTRACT: Expansion of GAA·TTC repeats within the first intron of the frataxin gene is the cause of Friedreich's ataxia (FRDA), an autosomal recessive neurodegenerative disorder. However, no effective treatment for the disease has been developed as yet. In this study, we explored a possibility of shortening expanded GAA repeats associated with FRDA through chemotherapeutically-induced DNA base lesions and subsequent base excision repair (BER). We provide the first evidence that alkylated DNA damage induced by temozolomide, a chemotherapeutic DNA damaging agent can induce massive GAA repeat contractions/deletions, but only limited expansions in FRDA patient lymphoblasts. We showed that temozolomide-induced GAA repeat instability was mediated by BER. Further characterization of BER of an abasic site in the context of (GAA)20 repeats indicates that the lesion mainly resulted in a large deletion of 8 repeats along with small expansions. This was because temozolomide-induced single-stranded breaks initially led to DNA slippage and the formation of a small GAA repeat loop in the upstream region of the damaged strand and a small TTC loop on the template strand. This allowed limited pol β DNA synthesis and the formation of a short 5'-GAA repeat flap that was cleaved by FEN1, thereby leading to small repeat expansions. At a later stage of BER, the small template loop expanded into a large template loop that resulted in the formation of a long 5'-GAA repeat flap. Pol β then performed limited DNA synthesis to bypass the loop, and FEN1 removed the long repeat flap ultimately causing a large repeat deletion. Our study indicates that chemotherapeutically-induced alkylated DNA damage can induce large contractions/deletions of expanded GAA repeats through BER in FRDA patient cells. This further suggests the potential of developing chemotherapeutic alkylating agents to shorten expanded GAA repeats for treatment of FRDA.
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    • "APE1 activity was measured using a modification of our recently described BER molecular assay [57]–[59]. All oligonucleotides were purchased from Sigma-Aldrich (Saint Louis, MO): Top-FAM-5′-GAGAAΦATAGTCGCC-3′ Bottom-DAB-3′-CTCTTGTATCAGCGC-5′ [Φ; Tetrahydrofuran (THF)]. "
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