Impaired 8-Hydroxyguanine Repair Activity of MUTYH Variant p.Arg109Trp Found in a Japanese Patient with Early-Onset Colorectal Cancer
Figure 3
Comparison of the activity to suppress the mutation caused by 8OHG between H1299 human cell lines inducibly expressing WT MUTYH and p.Arg81Trp variant MUTYH protein using a supF forward mutation assay. (a) Detection of MUTYH proteins in cumate-inducible stable cell lines expressing MUTYH using a Western blot analysis with an anti-MUTYH antibody. Lysates from empty vector-transposed cells and cells inducibly expressing WT type 2 MUTYH, type 2 p.Arg81Trp MUTYH variant, or p.Asp208Asn negative control in the presence of cumate were analyzed. β-Tubulin protein was also analyzed as an internal control. (b) Immunofluorescence detection of MUTYH proteins expressed in the cell lines used in (a) in the presence of cumate. The MUTYH protein (red) was stained with anti-MUTYH as the primary antibody and Alexa Fluor 594-conjugated goat anti-mouse IgG as the secondary antibody. The nuclei were counterstained with DAPI (blue). (c) Measurement of the mutation frequency of the supF gene in the pMY189 plasmid using a supF forward mutation assay in H1299 human cell lines inducibly expressing MUTYH proteins. The cell lines used in (a) in the presence of cumate were transfected with a pMY189 shuttle plasmid, and the mutation frequency of supF in these human cell lines was measured. “8OHG” indicates a pMY189 plasmid containing an 8-hydroxyguanine residue at position 159 of supF, while “G” indicates a pMY189 plasmid containing the WT supF gene. The data are shown as the means ± standard error. (d) A representative result of a supF mutation in an 8OHG-containing pMY189 replicated in empty vector-transposed H1299 cells. Sequencing electropherograms show a G to T (G:C to T:A) mutation at position 159 (marked by asterisks) of the supF gene.
