Abstract
BackgroundInteractions between genes and environment are critical factors for causing cancer in humans. The genotoxicity of environmental chemicals can be enhanced via the modulation of susceptible genes in host human cells. DNA polymerase kappa (Pol κ) is a specialized DNA polymerase that plays an important role in DNA damage tolerance through translesion DNA synthesis. To better understand the protective roles of Pol κ, we previously engineered two human cell lines either deficient in expression of Pol κ (KO) or expressing catalytically dead Pol κ (CD) in Nalm-6-MSH+ cells and examined cytotoxic sensitivity against various genotoxins. In this study, we set up several genotoxicity assays with cell lines possessing altered Pol κ activities and investigated the protective roles of Pol κ in terms of genotoxicity induced by mitomycin C (MMC), a therapeutic agent that induces bulky DNA adducts and crosslinks in DNA.ResultsWe introduced a frameshift mutation in one allele of the thymidine kinase (TK) gene of the KO, CD, and wild-type Pol κ cells (WT), thereby establishing cell lines for the TK gene mutation assay, namely TK+/- cells. In addition, we formulated experimental conditions to conduct chromosome aberration (CA) and sister chromatid exchange (SCE) assays with cells. By using the WT TK+/- and KO TK+/- cells, we assayed genotoxicity of MMC. In the TK gene mutation assay, the cytotoxic and mutagenic sensitivities of KO TK+/- cells were higher than those of WT TK+/- cells. MMC induced loss of heterozygosity (LOH), base pair substitutions at CpG sites and tandem mutations at GpG sites in both cell lines. However, the frequencies of LOH and base substitutions at CpG sites were significantly higher in KO TK+/- cells than in WT TK+/- cells. MMC also induced CA and SCE in both cell lines. The KO TK+/- cells displayed higher sensitivity than that displayed by WT TK+/- cells in the SCE assay.ConclusionsThese results suggest that Pol κ is a modulating factor for the genotoxicity of MMC and also that the established cell lines are useful for evaluating the genotoxicity of chemicals from multiple endpoints in different genetic backgrounds of Pol κ.
Highlights
Interactions between genes and environment are critical factors for causing cancer in humans
Establishment of thymidine kinase (TK) gene mutation assay in Nalm-6-MSH + cells and Their DNA polymerase (Pol) κ derivatives To establish the TK gene mutation assay, a +1-bp frameshift mutation was introduced at exon 4 of one allele of the TK1 in wild-type Pol κ cells (WT), KO and catalytically dead Pol κ (CD) cells (Fig. 1a)
The resulting cell lines were named as WT TK+/, KO TK+/, and CD TK+/- based on the different expression profile of Pol κ
Summary
Interactions between genes and environment are critical factors for causing cancer in humans. DNA polymerase kappa (Pol κ) is a specialized DNA polymerase that plays an important role in DNA damage tolerance through translesion DNA synthesis. TLS is performed by specialized DNA polymerases (Pols). These Pols take over primer DNA from replicative Pols, e.g., Pol δ and Pol ε, at or before lesions and insert dNMPs opposite lesions in an error-free or error-prone manner [1,2,3]. The replicative Pols return to the primer DNA and continue whole chromosome replication [4]. TLS is regarded as a mode of DNA damage tolerance that may cause mutations in compensation for cell survival [5]
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