Genetic consequences of tolerance to methylation DNA damage in mammalian cells

Abstract

We previously characterized a clone of CHO cells, clone B, that displayed tolerance to the cytotoxic effects of N-methylnitrosourea (MNU) and 6-thioguanine (6-TG). To determine whether this phenotype affected the mutagenic response of the cells, MNU-induced mutation to 8-azaadenine resistance (8-AAr) was measured in the parental and clone B cells. Comparable mutation frequencies were found in the two cell lines up to 0.5 mM MNU, while at higher MNU concentrations mutations could be reproducibly measured only in clone B cells. Similar amounts of DNA methylated bases were found in the two cell lines after a 30 min treatment with different concentrations of [3H]MNU and the same linear relationship was observed when mutation induction by MNU was plotted as a function of the amount of O6-methylguanine (O6-MeGua) in DNA, indicating that mutation induction in both cell lines was related to the presence of this methylated base. Fifteen MNU-induced 8-AAr mutants were isolated from each cell line and the sequences of the adenine phosphoribosyltransferase (aprt) mutations determined. The type (in 90% of the cases, GC to AT transitions), the sequence context and the strand localization of the mutations indicated that all mutations were targeted at O6-MeGua in DNA and no difference was found between the two lines. These results are consistent with a mechanism of tolerance of O6-MeGua that does not alter the processing of this methylated base into a mutation. Growth in 6-TG induced point mutations in clone B but not in the parental cells. A model is proposed in which the alkylation tolerant variant is altered in a mismatch correction pathway responsible for the cytotoxicity of the methylated base.

Development and Validation of a New, Sensitive Immunochemical Assay for O6-Methylguanine in DNA and Its Application in a Population Study

“…3A and B). The adduct levels measured in these samples are in good agreement with those previously reported by other studies (24,33). The accuracy of the assay was confirmed by direct comparison of adduct levels measured in the same samples by ELISA and by HPLC using [ Figure 5.…”

Mouse embryonic stem cells are hypersensitive to apoptosis triggered by the DNA damage O6-methylguanine due to high E2F1 regulated mismatch repair

“…24,27 If this does not occur, cells will survive and O 6 MeG adducts will give rise to mutations. 12 Interestingly, MMR is upregulated in ES cells since all ES cell lines showed a significantly higher MSH2 and MSH6 protein level than differentiated cells. This resulted in a higher G-T DNA binding activity of MutSa.…”

“…10 When O 6 MeG is not repaired by MGMT, it will mispair with thymine during DNA replication 11 and, therefore, in the absence of mismatch repair (MMR), and a second round of replication, indues GC to AT point mutations. 12 In MMR competent cells, the O 6 MeG/thymine mismatch is bound by MutSa, 13 a heterodimer comprised of the proteins MSH2 and MSH6. 14 The MMR protein binding has been shown to be required for O 6 MeG-triggered apoptosis.…”

Generating mutations but providing chemosensitivity: the role of O6-methylguanine DNA methyltransferase in human cancer

“…Avoidance of the mutagenic effect is directly related to the presence of a functional MGMT protein (Pegg et al, 1995). In vitro assays show that endogenous MGMT expression protects mammalian cell lines from spontaneous G : C to A : T transitions in the aprt gene (Aquilina et al, 1992). Animal models also show that transgenic mice overexpressing MGMT are protected against O 6 -methylguanine-DNA adducts caused by (Dumenco et al, 1993) and against G to A mutations in K-ras in aberrant colorectal crypt foci and lung tumors (Zaidi et al, 1995;Liu et al, 1999).…”