Dose-response and time-course experiments for base excision-repair enzymes. (a) Dose-response and time-course experiment for AAG activity. Based on these experiments, incubations were carried out for 45 minutes with 20 µg protein in AAG buffer. (b) Dose-response and time-course experiment done for APE1 activity. Based on these experiments, incubations were carried out for 2 minutes with 0.2 µg protein APE1 buffer.
(a) Relative increase in CD68 levels when comparing normal UC colon tissues with inflamed UC colon tissues in the same individual. (b) Average change in CD68 from areas of the UC colon pathologically diagnosed as inflamed to those diagnosed as noninflamed*, noninflamed areas have significantly less CD68 than inflamed areas (Student's t test, P = 0.04). AU, arbitrary units.
(a) Pathological images obtained before laser capture microdissection (LCM) (Before) and after LCM (After), and of the cells captured (Cap). Serial frozen sections (8 _m in thickness) were prepared from each colon tissue and microdissected (55) by a PixCell II LCM System from Arcturus Engineering (Mountain View, California, USA). The sections were stained by hematoxylin and dehydrated by an ethanol-to-xylene sequence in the presence of protease inhibitors. Colon epithelium, stroma, and inflammatory cells (approximately 15,000 cells; 5,000 7-_m shots) were isolated from UC colon tissue with and without pathologically assessed inflammation. LCM cells were pooled from multiple caps, and lysed in 20 _l protein extraction buffer containing a 1:1 mixture of 2( SDS Tris-glycine sample buffer and tissue protein extraction reagent (T-PER; Pierce, Rockford, Illinois, USA). The lysed cells were heated to 65°C for 2 hours and stored at -20°C before subsequent separation by16% SDS-PAGE (Invitrogen) and electrotransfer onto a nitrocellulose membrane. The primary antibodies used for protein analysis were a rabbit polyclonal anti-human AAG antiserum (1:200 dilution) and a mouse monoclonal anti-human APE1 (1:500 dilution; Novus). MCF-7 cells and isolated AAG (1 µg; Trevigen) or APE1 (1 µg; Trevigen) enzymes were used as positive controls. (b and c) Levels of AAG (b) and APE1 (c) by western blot analysis in pathologically defined inflamed areas and in noninflamed tissue. The positive control (+) was protein extract from MCF-7 cells. Purified AAG or APE1 protein was also loaded to confirm the migration level of the specific bands (data not shown). The bottom panels represent the densitometry after adjustment for actin levels. After LCM of stroma (S), epithelium (E), and inflammatory cells (I), AAG and APE1 were undetectable in the stroma. In contrast, these enzymes were detectable at low levels in the epithelium and inflammatory cells in pathologically diagnosed noninflamed tissues. In pathologically diagnosed inflamed areas of the UC colon, levels were higher in both epithelial and inflammatory cells, indicating an 'adaptive increase' of both the AAG and APE1 enzymes. For AAG, after adjustment for actin, this adaptive increase was higher in the inflammatory cells, but still occurred in the epithelial cells. For APE1, after adjustment for actin, the adaptive increase was higher in the epithelial cells. Less than 5% of the epithelium was infiltrated by inflammatory cells, as assessed by CD68 immunohistochemistry (data not shown). This indicated that we had enriched by LCM the cell type of choice.
Percentage of tissues with MSI at given loci in relation to their rank in AAG or APE1 activity as indicated. For example, for AAG, 75% of the tissues that had MSI at a mononucleotide (Mono) repeat (BAT25 or BAT26) had AAG activity scores ranked in the bottom 1/3; 25% of the tissues that had MSI at a mononucleotide repeat (BAT25 or BAT26) had AAG activity scores ranked in the middle 1/3; 0% of the tissues that had MSI at a mononucleotide repeat (BAT25 or BAT26) had AAG activity scores ranked in the top 1/3. MSI-L, MSI-low; MSI-H, MSI-high.
Activity of AAG and APE1 in human cells. (a) Activity of AAG in K562 erythroleukemia cells expressing GFP (vector control), AAG, or AAG + APE1. Extracts (12.5 µg of each) were incubated for 24 minutes with the oligonucleotide as described in Methods. (b) Activity of APE1 in human K562 erythroleukemia cells expressing GFP (vector control), APE1, or AAG + APE1. Extracts (0.0125 µg of each) were incubated for 5 minutes with the oligonucleotide as described in Methods. *, significant difference from GFP vector control.
MSI in K562 human erythroleukemia cells. Markers assessed are indicated. Arrowheads identify where the shift occurred. Numbers under each panel represent the relative band densities of the bands designated by the arrowheads. Supplemental figure 6