DNA polymerase kappa protects human cells against MMC-induced genotoxicity through error-free translesion DNA synthesisReport as inadecuate




DNA polymerase kappa protects human cells against MMC-induced genotoxicity through error-free translesion DNA synthesis - Download this document for free, or read online. Document in PDF available to download.

Genes and Environment

, 39:6

First Online: 07 January 2017Received: 27 July 2016Accepted: 08 December 2016

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.

KeywordsTranslesion DNA synthesis DNA polymerase κ Nalm-6-MSH+ Genotoxicity assay Mitomycin C AbbreviationsBPDEBenzoapyrene-7,8-dihydrodiol-9,10-epoxide

CAChromosome aberration

CDNalm-6-MSH+ cell expressing catalytically dead Pol κ

KOPol κ-deficient Nalm-6-MSH+ cell

LOHLoss of heterozygosity

MMCMitomycin C

NERNucleotide excision repair

PEPlating efficiency

PolDNA polymerase

SCESister chromatid exchange

TFTTrifluorothymidine

TKThymidine kinase

TLSTranslesion DNA synthesis

WTWild-type Nalm-6-MSH+ cell

Electronic supplementary materialThe online version of this article doi:10.1186-s41021-016-0067-3 contains supplementary material, which is available to authorized users.

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Author: Yuki Kanemaru - Tetsuya Suzuki - Akira Sassa - Kyomu Matsumoto - Noritaka Adachi - Masamitsu Honma - Satoshi Numazawa - Tak

Source: https://link.springer.com/



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