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. 2015 Jul;79(3):201-9.

Expression of O(6)-methylguanine-DNA Methyltransferase Causes Lomustine Resistance in Canine Lymphoma Cells

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Free PMC article

Expression of O(6)-methylguanine-DNA Methyltransferase Causes Lomustine Resistance in Canine Lymphoma Cells

Satoshi Kambayashi et al. Can J Vet Res. .
Free PMC article

Abstract

The DNA repair protein O (6)-methylguanine-DNA methyltransferase (MGMT) causes resistance to nitrosoureas in various human cancers. In this study, we analyzed the correlation between canine lymphomas and MGMT in vitro. Two of five canine lymphoma cell lines required higher concentrations of lomustine to inhibit cell growth by 50%, but their sensitivity to the drug increased when they were cultured with an MGMT inhibitor. Fluorometric oligonucleotide assay and real-time polymerase chain reaction of these cell lines revealed MGMT activity and high MGMT mRNA expression, respectively. We analyzed the methylation status of the CpG islands of the canine MGMT gene by the bisulfite-sequencing method. Unlike human cells, the canine lymphoma cell lines did not show significant correlation between methylation status and MGMT suppression levels. Our results suggest that in canine lymphoma MGMT activity may influence sensitivity to nitrosoureas; thus, inhibition of MGMT activity would benefit nitrosourea-resistant patients. Additional studies are necessary to elucidate the mechanism of regulation of MGMT expression.

La protéine de réparation O6-méthylguanine-DNA méthyltransferase (MGMT) cause de la résistance aux produits nitroso-urée dans divers cancers humains. Dans la présente étude nous avons analysé in vitro la corrélation entre les lymphomes canins et le MGMT. Deux des cinq lignées cellulaires de lymphome canin ont nécessité des concentrations plus élevées de lomustine pour inhiber de 50 % la croissance cellulaire, mais leur sensibilité au médicament augmenta lorsqu’elles furent mises en culture avec un inhibiteur de MGMT. Une épreuve fluorométrique des oligonucléotides et une épreuve d’amplification en chaîne par la polymérase en temps réel sur ces lignées cellulaires ont révélé, respectivement, une activité MGMT et une expression élevée d’ARNm de MGMT. Nous avons analysé le statut de méthylation des ilots CpG du gène MGMT canin par la méthode de séquençage au bisulfite. Contrairement aux cellules humaines, les lignées cellulaires canines de lymphome n’ont pas montré de corrélation significative entre le statut de méthylation et les niveaux de suppression de MGMT. Nos résultats suggèrent que lors de lymphome canin l’activité de MGMT peut influencer la sensibilité aux produits nitroso-urée; ainsi, l’inhibition de l’activité MGMT bénéficierait les patients résistants au nitroso-urée. Des études additionnelles sont nécessaires pour élucider le mécanisme de régulation de l’expression de MGMT.(Traduit par Docteur Serge Messier).

Figures

Figure 1
Figure 1
Mean percent viability (± standard deviation) of lymphoma cell lines pretreated with (dotted lines) or without (solid lines) O6-benzylguanine (BG), an inhibitor of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), and then incubated with various concentrations of lomustine. The experiment was done in triplicate, and the data are representative of 3 independent experiments.
Figure 2
Figure 2
Results of fluorometric oligonucleotide assay for MGMT activity. A double-stranded oligonucleotide of 45 base pairs (bp) containing a single O6-methylated guanine residue nested in a PvuII restriction site and a fluorometric 5′-hexachloro-fluorescein phosphoramidite were incubated with extracts from the 5 canine lymphoma cell lines and Jurkat cells, treated with the PvuII restriction enzyme, and then subjected to electrophoresis. If the extract contained MGMT activity, an O6-methylated guanine residue nested within the PvuII restriction site could be demethylated, and the oligonucleotide could be digested by PvuII. NC — uncleaved oligonucleotide; C — cleaved oligonucleotide.
Figure 3
Figure 3
A — Expression of mRNA by the MGMT gene in comparison with the ribosomal protein L32 (RPL32) gene with (black bars) or without (white bars) pretreatment with the methylation inhibitor 5-aza-2′-deoxycytidine (final concentration 0.5 μM) as determined by real-time polymerase chain reaction (PCR). The PCR cycle number at the threshold was represented as Ct, and the difference between Cts for the target and internal control (ΔCt) was calculated. B — Results of agarose gel electrophoresis of the PCR products. PBMC — peripheral blood mono-nuclear cell.
Figure 4
Figure 4
Scheme of CpG islands [regions of unmethylated DNA with a high frequency of dinucleotides of C (cytosine) followed immediately by G (guanine), or C plus G (CpG)] surrounding the canine MGMT gene exon 1 and the regions analyzed in this study, which were approximately 10 000 bp upstream through 7000 bp downstream of the exon 1. The islands were determined by means of Methyl Primer Express software version 1.0 (http://www.appliedbiosystems.com/absite/us/en/home/support/software-community/free-ab-software.html) with the following parameters: a CpG content greater than 55% and a ratio of observed CpG to expected CpG greater than 0.65. A — CpG islands upstream of the canine MGMT exon 1. B — Nucleotide numbers of the analyzed regions, based on the sequence of Canis lupus familiaris chromosome 28 (GenBank accession number NC_006610.2).
Figure 5
Figure 5
A — Percent methylation of 24 CpGs of region 1 obtained through bisulfite modification of DNA isolated from the canine lymphoma cells, PCR amplification, purification of the PCR products, and direct sequencing. Black bars — methylated cytosine; white bar — unmethylated cytosine; striped bars — undetermined. B to D — Amplified DNA from Ema and Nody-1 cell lines and PBMCs, respectively, was inserted into the pCR2.1 plasmid vector and transformed into bacteria. Five independent clones were analyzed and directly sequenced.
Figure 6
Figure 6
Percent methylation in regions 2-1, 2-2, 3-1, 3-2, and 4. Methods and bar identification as for Figure 5. A to C — As determined by the bisulfite-sequencing method. D to F — As determined with at least 5 clones per cell line.

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