Enhancing the sensitivity of the thymidine kinase assay by using DNA repair-deficient human TK6 cells

Abstract

The OECD guidelines define the bioassays of identifying mutagenic chemicals, including the thymidine kinase (TK) assay, which specifically detects the mutations that inactivate the TK gene in the human TK6 lymphoid line. However, the sensitivity of this assay is limited because it detects mutations occurring only in the TK gene but not any other genes. Moreover, the limited sensitivity of the conventional TK assay is caused by the usage of DNA repair-proficient wild-type cells, which are capable of accurately repairing DNA damage induced by chemicals. Mutagenic chemicals produce a variety of DNA lesions, including base lesions, sugar damage, crosslinks, and strand breaks. Base damage causes point mutations and is repaired by the base excision repair (BER) and nucleotide excision repair (NER) pathways. To increase the sensitivity of TK assay, we simultaneously disrupted two genes encoding XRCC1, an important BER factor, and XPA, which is essential for NER, generating XRCC1 ^-/- /XPA ^-/- cells from TK6 cells. We measured the mutation frequency induced by four typical mutagenic agents, methyl methane sulfonate (MMS), cis-diamminedichloro-platinum(II) (cisplatin, CDDP), mitomycin-C (MMC), and cyclophosphamide (CP) by the conventional TK assay using wild-type TK6 cells and also by the TK assay using XRCC1 ^-/- /XPA ^-/- cells. The usage of XRCC1 ^-/- /XPA ^-/- cells increased the sensitivity of detecting the mutagenicity by 8.6 times for MMC, 8.5 times for CDDP, and 2.6 times for MMS in comparison with the conventional TK assay. In conclusion, the usage of XRCC1 ^-/- /XPA ^-/- cells will significantly improve TK assay.

Keywords: TK assay; DNA-damaging agent; OECD guideline; TK6 cells; thymidine kinase assay.

FIGURE 1

Generation of XRCC1 ^−/− in XPA ^−/− TK6 cells. (a) Schematic representation of the human XRCC1 locus, base sequences of TALEN‐recognition sites, and structure of the targeting constructs containing genomic fragments and a selection‐marker gene (hisDR or bsrR) with loxP sites on both sides. The x‐mark indicates the TALEN‐induced DSB site. A pair of TALEN was designed to target exon 1. Schematic representation of the XRCC1 locus (upper) and configuration of the targeting construct carrying a marker gene (lower) flanked by ~1 kb of genomic sequence on either side. The closed boxes represent exon sequences. Note that the size of the schematic representation does not reflect the actual size of the DNA. (b) Western blot analysis of XRCC1 disrupted TK6 clones using the antibody against XRCC1 in XPA ^−/− human TK6 cell line

FIGURE 2

The mutation frequency (MF) of wild‐type and XRCC1 ^−/− /XPA ^−/− TK6 cells induced by CP. The percentage survival of cells following exposure of cells to the indicated concentrations of CP (a). Hundred percentage is the survival of untreated cells in (a). The average MF of cells to the indicated concentrations of CP (b). Error bars represent SD from at least three independent experiments. The statistically significant difference between spontaneously arising MFs and induced ones was calculated by the Student's t test. We defined p‐value < .05 as statistically significant and mark such difference with *. (ns) p‐value was not significant

FIGURE 3

The mutation frequency (MF) of wild‐type and XRCC1 ^−/− /XPA ^−/− TK6 cells induced by MMS. Data are shown as in Figure 2. Error bars represent SD from at least three independent experiments. The statistically significant difference between spontaneously arising MFs and induced ones was calculated by the Student's t test. We defined p‐value < .05 as statistically significant and mark such difference with *. (ns) p‐value was not significant

FIGURE 4

The mutation frequency (MF) of wild‐type and XRCC1 ^−/− /XPA ^−/− TK6 cells induced by MMC. Data are shown as in Figure 2. Error bars represent SD from at least three independent experiments. The statistically significant difference between spontaneously arising MFs and induced ones was calculated by the Student's t test. We defined p‐value < .05 as statistically significant and mark such difference with *. (ns) p‐value was not significant

FIGURE 5

The mutation frequency (MF) of wild‐type and XRCC1 ^−/− /XPA ^−/− TK6 cells induced by CDDP. Data are shown as in Figure 2. Error bars represent SD from at least three independent experiments. The statistically significant difference between spontaneously arising MFs and induced ones was calculated by the Student's t test. We defined p‐value < .05 as statistically significant and mark such difference with *. (ns) p‐value was not significant