Journal: Carcinogenesis

Article Title: Altered expression of the human base excision repair gene NTH1 in gastric cancer.

doi: 10.1093/carcin/bgp108

Figure Lengend Snippet: Fig. 1. Downregulation of NTH1 expression in gastric cancer cell lines. (A) Measurement of the level of expression of NTH1 transcripts in gastric cancer cell lines by QRT-PCR with a LightCycler instrument. The amounts of NTH1 transcripts normalized to the amount of transcripts of a housekeeping gene, PBGD, in eight gastric cancer cell lines are shown in the graph. The average expression level of four normal gastric mucosa samples was measured as a control and set equal to 1.0. (B) Measurement of the expression level of NTH1 protein in gastric cancer cell lines by western blot analysis. The levels of NTH1 protein expression in eight gastric cancer cell lines were measured by western blot analysis with anti-NTH1 monoclonal antibody. AGS cells transiently transfected with NTH1 expression vector or empty pcDNA3.1 vector were also used, and 8 lg (corresponding to one-fifth the weight applied in other lanes) of the total cell extract of these AGS cells was applied for sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Expression of b-tubulin protein was analyzed as an internal control. (C) Examination of the effect of 5-aza-dC and trichostatin A (TSA) on the NTH1 mRNA expression level in six gastric cancer cell lines showing a low level of NTH1 expression. Cells were treated or not treated with 2 lM 5-aza-dC for 48 h or 330 nM TSA for 24 h, and NTH1 mRNA expression was measured by QRT- PCR. The amount of NTH1 transcripts normalized to the amount of PBGD transcripts is shown in the graph. The expression level in the cells not treated with 5-aza-dC or TSA was set equal to 1.0 in each cell line. Expression of HAND1 was also measured as a positive control with reference to a previous paper (23).

Article Snippet: Next, the slides were incubated with an anti-NTH1 polyclonal antibody (Novus Biologicals, Littleton, CO) at a dilution of 1:800 for 30 min at room temperature and then with dextran polymer conjugated with goat anti-rabbit immunoglobulin G and horseradish peroxidase (ChemMate Envision Kit, DAKO, Kyoto, Japan) for 30 min at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Transfection, Plasmid Preparation, Polyacrylamide Gel Electrophoresis, Positive Control

Journal: Carcinogenesis

Article Title: Altered expression of the human base excision repair gene NTH1 in gastric cancer.

doi: 10.1093/carcin/bgp108

Figure Lengend Snippet: Fig. 2. Low level of Tg DNA glycosylase activity in AGS cells. (A) Western blotting of FLAG-NTH1 protein in parental AGS cells, empty vector- transfected AGS clones and FLAG-NTH1 stably expressing AGS clones with anti-NTH1 or anti-FLAG antibodies. Expression of b-tubulin protein was also analyzed as an internal control. (B and C) Measurement of Tg DNA glycosylase activity in AGS cells. Cleavage activity against double-strand DNA containing base pair Tg:A was measured in empty vector-transfected AGS clones and in stably FLAG-NTH1-expressing AGS clones. Cell extracts and a 32P-labeled double-stranded oligonucleotide containing or not containing a single 5R,6S-Tg:A mispair were incubated and subjected to 20% polyacrylamide gel electrophoresis. A 32P-labeled marker oligonucleotide was used as a size marker for the cleaved products. Representative results are shown in the panel in (B). The intact 30mer oligonucleotides and cleavage products are indicated by the arrows. The amount of cleaved products as percentages of the total were calculated and shown in a bar graph of (C). Values are means ± SDs of three independent experiments. P values were calculated by the two-tailed Student’s t-test, P , 0.01. Activity in the empty vector-transfected clones was significantly lower than in the FLAG-NTH1-expressing clones.

Article Snippet: Next, the slides were incubated with an anti-NTH1 polyclonal antibody (Novus Biologicals, Littleton, CO) at a dilution of 1:800 for 30 min at room temperature and then with dextran polymer conjugated with goat anti-rabbit immunoglobulin G and horseradish peroxidase (ChemMate Envision Kit, DAKO, Kyoto, Japan) for 30 min at room temperature.

Techniques: Activity Assay, Western Blot, Plasmid Preparation, Transfection, Clone Assay, Stable Transfection, Expressing, Control, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Marker, Two Tailed Test

Journal: Carcinogenesis

Article Title: Altered expression of the human base excision repair gene NTH1 in gastric cancer.

doi: 10.1093/carcin/bgp108

Figure Lengend Snippet: Fig. 3. Low level of NTH1 expression and abnormal cellular localization of NTH1 in primary gastric cancers. (A) Comparison of NTH1 mRNA levels in the cancerous tissue of 50 primary gastric cancers and corresponding non-cancerous gastric tissue by QRT-PCR analysis. After the relative amounts of NTH1 transcripts were normalized to those of the GAPD transcript, T/N values were calculated by dividing the normalized transcript amounts in gastric cancer tissue by the amounts in the corresponding non-cancerous tissue. The ‘N’ and ‘C’ under each case number indicate the nuclear localization pattern of NTH1 protein and the cytoplasmic localization pattern of NTH1 protein, respectively, which were identified by the immunohistochemical analysis in (B). The results of genotyping NTH1 promoter polymorphisms are also shown under the NTH1 localization pattern row. The ‘D’ indicates the heterozygote for the c.-241_-221del polymorphism and wild-type. The c.-163C.G polymorphism was not found in any of the cases. Cases no. 18, 25, 28, 35, 42, 45 and 49 were not genotyped for these polymorphisms because of the lack of a genomic DNA sample. (B) Determination of the NTH1 localization pattern in primary gastric cancer tissues by immunohistochemical analysis with anti-NTH1 polyclonal antibody. Nuclear localization of NTH1 protein in normal gastric mucosa is shown in the left panels. Gastric cancers showing NTH1 expression predominantly in the nucleus and in the cytoplasm are shown in the middle panels and in the right panels, respectively. The lower panels are a higher magnification of the area enclosed by the square in upper panels. The scale bars in the upper panels and lower panels represent 100 lm and 25 lm, respectively. The immunohistochemical findings in the 50 gastric cancer cases are denoted at the row of NTH1 localization in (A).

Article Snippet: Next, the slides were incubated with an anti-NTH1 polyclonal antibody (Novus Biologicals, Littleton, CO) at a dilution of 1:800 for 30 min at room temperature and then with dextran polymer conjugated with goat anti-rabbit immunoglobulin G and horseradish peroxidase (ChemMate Envision Kit, DAKO, Kyoto, Japan) for 30 min at room temperature.

Techniques: Expressing, Comparison, Quantitative RT-PCR, Immunohistochemical staining

Journal: Carcinogenesis

Article Title: Altered expression of the human base excision repair gene NTH1 in gastric cancer.

doi: 10.1093/carcin/bgp108

Figure Lengend Snippet: Fig. 4. Measurement of NTH1 promoter activity and identification of NTH1 promoter polymorphisms associated with reductions in promoter activity. (A) Measurement of luciferase reporter activity in the upstream region of the NTH1 gene. Luciferase reporter activity was measured 24 h after transfection with reporter plasmids, and the firefly luciferase activity was normalized to the Renilla luciferase activity. The luciferase activity values are means ± SDs of three independent experiments. The luciferase activity of the cells transfected with each luciferase reporter plasmid is shown relative to the activity when transfected with the empty vector, which has been set equal to 1. (B) Identification of the c.-163C.G and c.-241_-221del polymorphisms in the NTH1 promoter region. The left panels show the results of the PCR–SSCP analysis. Arrows point to abnormally shifted bands obtained by PCR–SSCP analysis. The middle panels and right panels show the results of direct sequencing analysis of the PCR product covering the polymorphic sites. The middle panels show the wild-type allele, and the right panels show both the wild-type and polymorphism alleles. (C) Reduction of promoter activity in the NTH1 promoter sequence containing the c.-163C.G or c.-241_-221del polymorphism revealed by the luciferase reporter assay. Luciferase reporter activity was measured 24 h after transfection with the reporter plasmids, and the firefly luciferase activity was normalized to the Renilla luciferase activity. The luciferase activity values are means ± SDs of three independent experiments. The luciferase activity of the cells transfected with reporter plasmid containing each polymorphism is shown relative to that of the cells transfected with wild-type reporter plasmid, which has been set equal to 1. P values were calculated by the two-tailed Student’s t-test, P , 0.05.

Article Snippet: Next, the slides were incubated with an anti-NTH1 polyclonal antibody (Novus Biologicals, Littleton, CO) at a dilution of 1:800 for 30 min at room temperature and then with dextran polymer conjugated with goat anti-rabbit immunoglobulin G and horseradish peroxidase (ChemMate Envision Kit, DAKO, Kyoto, Japan) for 30 min at room temperature.

Techniques: Activity Assay, Luciferase, Transfection, Plasmid Preparation, Sequencing, Reporter Assay, Two Tailed Test