We’ve investigated the mutagenicity induced in the locus in Chinese hamster ovary (CHO) cells treated with increasing concentrations of Me-lex, a minor groove selective methylating agent that efficiently generates more than 90-95% of 3-MeA DNA adducts. result in recombination and genomic deletions, therefore representing a severe threat for genome integrity. and in mammalian cells, and a correlation between the level of 3-MeA and cell death has been shown [5-7]. Studies in mouse embryonic stem (Sera) cells showed that 3-MeA is also the major DNA foundation adduct created by Me-lex in cultured cells and that unrepaired 3-MeA lesions induced sister chromatid exchanges, chromosome aberrations, S-phase arrest, p53 induction and VER-50589 manufacture apoptosis . More recently, the cytotoxic potential of Me-lex has been shown in mismatch restoration deficient leukemic cells and in human being glioma cell lines [9-11]. In those reports, the use of 3-MeA inducing compounds was suggested like a encouraging VER-50589 manufacture pharmacological strategy for the treatment of tumors resistant to classical wide-spectrum methylating providers. Although VER-50589 manufacture Me-lex cytotoxicity has been documented in several experimental systems, its mutagenicity has been extensively analyzed only in candida [4,12,13]. The Me-lex induced mutation spectrum was identified with a functional assay after treatment of a plasmid manifestation vector harbouring the human being crazy type p53 cDNA, followed by transformation in candida strains comprising the ADE2 gene controlled by a p53 response element. In parallel, the Me-lex induced methylation pattern was identified at the same human being p53 cDNA sequence in order to make a correlation between sites of methylation and sites of mutation . The results acquired with this combined approach showed that Me-lex with low rate of recurrence induced mutations that consisted primarily in AT-targeted foundation pair changes, with AT>TA transversions becoming the predominant class of foundation pair substitutions. The methylation analysis elegantly confirmed the almost unique reactivity of Me-lex at adenines within or adjacent A/T rich sequences. However, with the exception of few hot places, there was minimal overlap between methylated and mutated bases indicating that, with this experimental system, greatly adducted sites are not necessarily converted into foundation pair substitutions . Other results acquired in restoration deficient candida strains shown that Me-lex toxicity and mutagenicity are dependent on the DNA restoration background. Foundation excision restoration (BER) deficient strains, lacking 3-methyladenine DNA glycosylase or both AP endonucleases, are significantly more sensitive to Me-lex toxicity with respect to the parental strain. However, only the removal of AP endonucleases induced a significant increase in mutagenicity . Furthermore, with the same approach, we have recently demonstrated an involvement of candida translesions Rabbit polyclonal to IL29 synthesis (TLS) polymerases, such as Pol and REV1, in the mutation fixation process of Me-lex induced lesions . The evidence gathered to day shows that Me-lex is definitely a molecule with low mutagenic activity that is 3-MeA derived, but there have been no studies dealing with its mutagenicity in mammalian cells. Furthermore, the Me-lex mutagenic potential in higher eukaryotic cell system is unfamiliar. Herein, we investigated the cytotoxicity and mutagenicity of Me-lex in the X-linked hypoxanthine-guanine phosphoribosyltransferase (gene has been widely used like a selectable genetic marker for studies on chemical- and radiation-induced mutations in a number of mammalian cell systems [15-19]. The large size of the gene, with nine exons dispersed over 34 kb, allows the detection of various types of mutations ranging from solitary foundation substitutions to large rearrangements, deletions and insertions . Moreover, mutations arising in splice junctions or intron sequences that may impact mRNA splicing can be recognized . Compared to the analysis conducted on a target gene harboured on a plasmid, the use of an endogenous gene allows a comprehensive evaluation of the activity of the compound. Our data confirm the low mutagenic potential of Me-lex. However, in contrast with earlier yeast-based studies that demonstrated foundation pair substitutions, the mutation spectrum acquired in mammalian VER-50589 manufacture cells exposed a high percentage of genomic deletions. Based upon these data, we hypothesize that a large proportion of the rare mutations induced by Me-lex result from the processing of 3-MeA within A/T rich sequences in non-coding and regulatory regions of the gene. The clustering of these VER-50589 manufacture lesions in A-rich sequences could represent a strong impediment to the progression of DNA replication fork, therefore advertising genomic rearrangements and/or large deletions leading to cell death. 2. Materials and Methods 2.1. Dangerous procedures Me-lex should be considered a toxic compound, and was dealt with accordingly. 2.2. Compounds Reagents of the highest purity were purchased from Sigma Aldrich (Milano, Italy) unless normally stated. Me-lex.