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independent with the molecularbeacon and cell line. Five minutes wasselected to eliminate the variable measurements and tofacilitate valid comparisons between trials and circumstances.The mean of 3 separate trials was plotted,with error bars representing the common error of themean.DNA extraction and MSP assay for human MGMTpromoterDNA was purified Celecoxib from 5106 LN428 cells and T98Gcells working with the DNeasy tissue kitaccording tothe manufacturer’s instruction, and methylation of theMGMT promoter was determined by methylationspecificPCR, as we've described previously.54The sense and antisense primers for the methylatedhuman MGMT promoters were 5TTTCGACGTTCGTAGGTTTTCGC3and 5GCACTCTTCCGAAAACGAAACG3, respectively, along with the primers applied todetect the unmethylated human MGMT promoterswere 5TTTGTGTTTTGATGTTTGTA GGTTTTTGT3and 5AACTCCACACTCTTCCAAAAACAAAACA3, respectively.
54 The PCR productswere analyzed Celecoxib by 4agarose gel electrophoresisusing Universal Alogliptin unmethylatedDNAas a damaging controlDNA and Universal methylated DNAas a optimistic control DNA.Cloning and expression of human MGMTThe human MGMT cDNAwas amplifiedby PCR working with primers hMGMTFand hMGMTR. MGMT cDNA wasthen cloned through a topoisomerase cloning procedure intothe pENTRD cloning plasmid, as per themanufacturer’s protocol. The human MGMT openreading framewas transferred frompENTRhMGMT to a Gateway modified pIRESPuroplasmid through LR recombination reaction, as per the manufacturer.ResultsMXinduced potentiation of TMZ is enhanced byoverexpression of MPGTo test our hypothesis that elevated repair initiation byMPG will further sensitize glioma cells exposed to BERinhibitors, we stably overexpressed WT MPG in theLN428 glioma cell line.
Overexpression of MPG wasconfirmed at the protein and mRNA levels working with immunoblotand qRTPCR analyses, HSP respectively, with an approximate 40fold increase ofmRNA.To confirm the elevated glycosylase activity in theMPG overexpressing cells, we developeda realtime, quantitative fluorescent MPG activity assayusing a modified form of molecular beacons, similar tothose previously reported for oxidative damage.55,56However, instead of incorporating many baselesions into the stem,55,56 we developed a BER beaconwith a single base lesion to additional accuratelyand quantitatively decide lesion repair rates.This special BERbeacon comprises a single DNA oligodeoxynucleotidedesigned to type a stemloop structureand contains a 5fluorophoreand a 3quencheron either end with the oligonucleotide.
A 1,N6ethenoadenine lesion, a substrate ofMPG,57 was positioned within the stem region of theBERbeacon at base5 from the 5end Alogliptin and is applied toprobe for MPG activity. The same BERbeacon structurewith a typical adenine was applied as the control substrate.Following removal of 1A byMPG and subsequent DNAstrand excision by APE1 5to the AP internet site, the fluorophore6FAM is separated from the quencherand the increase in fluorescence signalis proportionalto the degree of MPG activity. TheLN428 lysate incubated using the control beaconhad a minimalincrease in fluorescence, indicating the control beaconis largely intact. The LN428 lysate had small or noendogenous MPG activity, because when incubated withthe beacon containing the MPGspecific substrate 1A,there was no observable adjust in fluorescence.
The LN428MPG lysatealso did not have a negligible increase in fluorescencewhen incubated using the control beacon, indicating that MPG overexpressiondoes not increase cleavage of typical DNA.Nonetheless, the LN428MPG lysate exhibited robustMPG activity Celecoxib visible with a large increase in fluorescencewhen incubated using the molecular beacon containingthe MPG substrate 1A.This corresponded to an overall 7.9fold increase inMPG activity, as compared withthe LN428 cells and an estimated rate of repairof107.00 AUmin, whereas the background rate ofrepair within the LN428 cell lysate was similar towards the backgroundsignal working with the control beacon. This demonstrates that the LN428MPG cell linehas elevated functional MPG and does not recognizenormal DNA as a substrate.
These data are in linewith our earlier report showing that overexpression ofMPG final results in an increase in DNA glycosylaseactivity.23Using Alogliptin a shortterm cell survival assay, we next assayed the potentiation of TMZ byMX within the LN428 cells, with or without MPGoverexpression. MX sensitized both cell lines to TMZ,but sensitization with the LN428 cells was minimal. Within the LN428 cells, MXinduced a 1.5fold increase in sensitivity to TMZ. Nonetheless, the potentiation ofTMZ induced by MX was considerably greater in theLN428MPG cells, decreasing the half maximal inhibitoryconcentrationin the combined treatment4fold, as compared using the LN428 cells. To confirm that MPG overexpressioninducedpotentiation can be a result of elevated glycosylase activity,we overexpressed a mutant MPGin theglioma cell line LN428. This activesite mutant hasbeen shown to have 100fold much less glycosylase activitythan WT MPG.58 Overexpression with the mutantMPG did not sensitize LN428 cells to a combinedtreatment of MX an