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The repair of TMZinduced base damage by the BERpathway starts with all the recognition and removal of thedamaged bases by Nmethylpurine DNA glycosylase, also known as alkyladenine DNA glycosylase.7 The abasic siteproduced followingthe action of MPG is then hydrolyzed by AP endonuclease1, resulting within the incision of thedamaged DNA strand (-)-MK 801 and formation of a 3OH groupand 5deoxyribose phosphategroup in therepair gap.14 Polypolymerase 1together with PARP2 and polyglycohydrolaserecognizes the DNA strand interruptionand facilitates the recruitment of subsequent BER proteins,including the BER scaffold protein XRCC1 andDNA polymerase b.14 Polb subsequently hydrolyzesthe 5dRP moiety and inserts a single nucleotide,preparing the strand for ligation by a complex of DNAligase IIIa and XRCC1 to complete the repair method.
15Enhanced sensitivity to alkylating agents has beenobserved by modulating the BER pathway in preclinicalstudies, suggesting BER modulation is an attractivetarget for chemotherapy potentiation.16 At present,many BER proteins are under active (-)-MK 801 investigation aspotential targets for chemotherapy sensitization,including APE1,17 PARP1,18 PARG,19 and Polb.2024Methoxyamineis a little molecule that specificallyinhibits BER25 and is currently becoming evaluatedin phase I clinical trials. Methoxyamine inhibits therepair of AP internet sites by binding to and modifying the APsite, rather than directly inhibiting the enzyme APE1.AP internet sites modified by MX are refractory to APE1,preventing its processing by the ensuing steps of BER,as well as the MXmodified AP site is very cytotoxic.
26Methoxyamine potentiates a wide range of DNA damagingagents that produce AP internet sites regardless of thestatus of MMR, MGMT, and p53.17PARP1is the founding member of a largefamily of polypolymerases.2729 BI-1356 It is theprimary enzyme catalyzing the transfer of ADPriboseunits from NADto target proteins including PARP1itself. Below normal physiologic circumstances, PARP1facilitates the repair of DNA base lesions by helpingrecruit the BER proteins XRCC1 and Polb.30Inhibition of PARP1 results in decreased repair ofDNA base damage and elevated sensitivity of cells toalkylating agents, which makes it an desirable and effectivetarget HSP for chemotherapy sensitization.31 ManyPARP inhibitors happen to be developed and tested inseveral tumor types.32 They have been shown toenhance the cytotoxic effect of TMZ againstglioma,3335 leukemia,36 lung,37,38 and colon3840 carcinomacells.
Further, it has been shown lately that aPARP inhibitorTMZ has broad activityin many histologic types in subcutaneous, orthotopic,or metastatic tumor models.41 PARG BI-1356 will be the principal enzymeresponsible for the degradation of poly ADPribosein vivo via endoand exoglycosidic cleavage.28Although full ablation of PARG activity leads toearly embryonic lethality, embryonic stem cells derivedfrom a PARG null mouse42 and cells from PARG110deficient mice43 havebeen shown to be sensitive to alkylating agents andionizing radiation. Moreover, inhibition of PARGactivity was demonstrated to sensitize malignant melanomato TMZ in mouse models.19Overexpression of MPG has been reported to sensitizehuman breast cancer cells,24 osteosarcoma cells,44and ovarian cancer cells45 to the chemotherapeuticagent TMZ.
The elevated sensitivity has been shownto be the result of elevated repair initiation in the nontoxicN7methylguanine lesion,46 saturating (-)-MK 801 theratinglimiting enzyme Polb and resulting in accumulationof cytotoxic 5dRP repair intermediates.23 Sincemost BER inhibitorsinhibit the steps followingglycosylasemediated repair initiation, wehypothesize that MPG overexpression may increaseBER inhibitorinduced sensitization of glioma cells tothe alkylating agent TMZ. In this study, we show thatoverexpression of MPG sensitizes glioma cellsto MX, the PARP inhibitors PJ34 andABT888, or PARG inhibitionfollowingexposure to TMZ, demonstrating that increasedinitiation of BER combined with inhibition of theensuing repair steps gives enhanced sensitization ofglioma cells to TMZ.
Further, we show that depletionof Polb enhances the sensitization induced by the combinationof elevated repair initiation and BER inhibition,whereas elevated expression of Polb abrogates the sensitization.Further, BI-1356 we observed wide variability in mRNAexpression for MPG, Polb, and PARP1 in GBM tumors,as compared with normal brain tissue. As our functionalanalyses suggest that the expression status of both MPGand Polb might be applied to predict the effectiveness ofTMZ plus BER inhibitors within the treatment of glioma,we propose that future analyses include things like proteinexpression evaluation of crucial BER proteins andormeasurement of crucial BER enzyme activities from tumorbiopsies to aid in treatment optimization.Materials and MethodsChemicals and reagentsAlpha Eagle’s minimal important mediumwasfrom Mediatech or InVitrogen. Fetal bovine serum, heat inactivated FBS, PenStrepAmpho, glutamine,and antibioticantimycotic were fromInVitrogen. TMZ was obtained from the NationalCancer Instit