Three separate groups of mice were used as controls: one group of mice was
maintained with regular water (n = 16 WT; n = 10 Tg), a second group of mice was injected with AOM and given water for the remainder of the experiment (n = 9 WT; n = 7 Tg), and the third group of mice was treated with DSS as described above without AOM injection (n = 20 WT; n = 8 Tg). AOM alone represents treatment with the alkylating agent in the absence of inflammation and is expected to result in no tumor formation in WT mice at the dose used (). DSS alone represents repeated cycles of inflammation and was included to assess whether inflammation alone caused dysplasia in these mice. The study was powered in such a fashion that the control arms required less mice as death before the end of the study was not anticipated for these treatment groups. Body weight, stool consistency, and stool occult blood was monitored during the DSS treatment and recovery phases. Upon sacrifice, colon was excised from the ileocecal junction to anus, cut open longitudinally, and prepared for histologic evaluation.
To assess mortality, the same protocol was followed with the exception of extending through 126 days or until mice developed rectal prolapse and/or more than 20% body weight loss. Colons were assessed macroscopically for polyps using a dissecting microscope.
Clinical score assessment
Assessment of body weight loss, stool consistency, and the presence of occult/gross blood by a guaiac test (Hemoccult SENSA; Beckman Coulter) were determined daily during DSS administration to generate a clinical activity score as described previously
(). During recovery periods, body weight was measured every week for each group. Endoscopic assessment of polyp formation and polyp count
Colonoscopy was done to assess polyp formation in mice by using the Coloview (Karl Storz Veterinary Endoscopy). Mice were euthanized with 1.5% to 2% isoflurane and approximately 3 cm of the colon proximal to the anus was visualized after inflation of the colon with air. Mice were then sacrificed by cervical dislocation after euthanizing with isoflurane. Colon was excised from ileocecal junction to anus, washed with 0.9% NaCl, cut open longitudinally and preserved in 10% neutral-buffered formalin. The number and size of the polyps were determined using a Jenko dissecting microscope. Histopathology scoring
Colons fixed in 10% neutral buffered formalin were Swiss-rolled, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin stain for
histopathologic examination of polyps and adenocarcinoma (neoplasia). Scoring was carried out in a blind fashion by 2 qualified pathologists. Score was given on the basis of these criteria: normal (score 0), low-grade dysplasia (score 1), high-grade dysplasia
(score 2), intramucosal adenocarcinoma (score 3), and invasive adenocarcinoma (score 4).
Immunohistochemistry
Paraffin-embedded sections (5 μm) of colon were analyzed for Ki67 staining as a marker of cell proliferation as previously described (). A minimum of 15 crypts with normal morphology were counted for Ki67-positive cells per section. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining
Immunofluorescent terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was carried out to measure apoptosis from
paraffin-embedded sections using the In Situ Cell Death Detection Kit as described by the manufacturer (Roche). Nuclei were stained with 4′, 6-diamidino-2-phenylindole to count total cells per crypt. A minimum of 10 crypts with normal morphology were counted per section.
p53 mutational analyses
Tumor tissue was microdissected from paraffin-embedded sections (7 μm) of
Swiss-rolled mouse colon. Genomic DNA was isolated from microdissected tissues using the QIAamp DNA FFPE Tissue purification kit (Qiagen). The PCR amplicons were generated and sequenced as previously described (). See the Supplementary Materials and Methods section for primer sequences.
Human tissue samples
Matching normal and tumor tissues were obtained at the time of surgical resection from patients with one or more UC-associated colorectal neoplasms, consisting of adenocarcinomas or dysplasias. Normal control samples consisted of colonic normal mucosa adjacent to tumors or ileal mucosa. Inflamed UC tissues were obtained from patients without evident dysplasia. All tissues were grossly dissected free of normal surrounding tissue, and parallel sections were used for histologic characterization. Tissue collection was approved by patients according to Institutional Review Board guidelines.
Total RNA extraction and quantitative real-time PCR
Total RNA was extracted for human tissues using the RNeasy kit (Qiagen).
Quantitative real-time PCR was carried out as described previously (). See the Supplementary Materials and Methods section for primer sequences.
Cell culture and transfection
Caco2-BBE cells were used to assess the interaction of PHB with STAT3. As Caco2-BBE cells have mutated p53, WT HCT116 human CRC cells were used to assess PHB interaction with p53. All cell lines were obtained from the American Type Culture Collection. Cells were grown and transfected as previously described (Kathiria and colleagues; submitted for publication).
γ-Irradiation
γ-Irradiation was used to induce DNA damage in WT and p53−/− HCT116 cells () after 72 hours of transfection with pEGFPN1 vector or pEGFPN1-PHB. Cells were irradiated by using 137Cs γ-irradiator at 6.5 cGy/s for 32 seconds for a total of 209.4 cGy. Cells were harvested 24 hours after γ-irradiation for subsequent assays. Protein extraction, Western blot analysis, and immunoprecipitation Mucosal strippings from Tg and WT mice were obtained for Western blot analysis as described previously (). Total protein was isolated from cultured cells as described previously (Kathiria and colleagues; submitted for publication). Antibodies used were mouse monoclonal PHB (Thermo Fisher), mouse monoclonal GFP, p53, Bcl-2, Bcl-xL and Bad and rabbit polyclonal BAX, p-Bad (ser 155), STAT3 (Santa Cruz Biotechnology), rabbit polyclonal proliferating cell nuclear antigen (PCNA) antibody (Abcam), rabbit polyclonal caspase-3, p53, pSTAT3 (Cell Signaling Technology), rabbit polyclonal p53 upregulated modulator of apoptosis (PUMA), and mouse monoclonal anti-β-actin (Sigma-Aldrich).
PHB was immunoprecipitated from 0.6 mg total protein lysates from HCT116 or Caco2-BBE cells or 0.20 mg total protein lysates from mouse mucosa with 1 μg mouse anti-PHB, anti-p53, or anti-pSTAT3 antibody and 30 μL 50% protein A
sepharose beads (GE Healthcare). Blots were incubated with the rabbit p53 antibody or PHB antibody, respectively. Omission of primary antibody during the
immunoprecipitation was carried out as a negative control.
Statistical analysis
Values are expressed as mean ± SEM. Statistical analysis was conducted using 2-way ANOVA and subsequent pairwise comparisons using Bonferroni post hoc tests. A P value less than 0.05 was considered statistically significant in all analyses. Results
IEC-specific PHB overexpression decreases colonic tumorigenesis in a mouse model of CAC
Previous studies have shown that PHB has an antitumorigenic role in gastric, prostate, and liver
cancers (). We used the AOM DSS mouse model to study the role of PHB in CAC. Body weight was measured weekly as one parameter to assess the severity of disease. WT and Tg mice given water only throughout the experiment and mice injected with AOM followed by water alone showed similar body weight gain over the 8-week protocol (Supplementary Fig. S1A) and did not develop polyps as previously reported for this dose of AOM (). WT mice given 2 cycles of DSS without AOM injection lost more body weight following DSS administration and recovered less weight compared with Tg mice over the 8-week protocol (Supplementary Fig. S1B), similar to our previous findings (). Although both WT and Tg AOM DSS-treated mice lost weight after the administration of DSS, Tg mice gained body weight more rapidly during the recovery phase and maintained body weight better than WT mice throughout the remainder of the study ().
Three separate groups of mice were used as controls: one group of mice was
maintained with regular water (n = 16 WT; n = 10 Tg), a second group of mice was injected with AOM and given water for the remainder of the experiment (n = 9 WT; n = 7 Tg), and the third group of mice was treated with DSS as described above without AOM injection (n = 20 WT; n = 8 Tg). AOM alone represents treatment with the alkylating agent in the absence of inflammation and is expected to result in no tumor formation in WT mice at the dose used (). DSS alone represents repeated cycles of inflammation and was included to assess whether inflammation alone caused dysplasia in these mice. The study was powered in such a fashion that the control arms required less mice as death before the end of the study was not anticipated for these treatment groups. Body weight, stool consistency, and stool occult blood was monitored during the DSS treatment and recovery phases. Upon sacrifice, colon was excised from the ileocecal junction to anus, cut open longitudinally, and prepared for histologic evaluation.
To assess mortality, the same protocol was followed with the exception of extending through 126 days or until mice developed rectal prolapse and/or more than 20% body weight loss. Colons were assessed macroscopically for polyps using a dissecting microscope.
Clinical score assessment
Assessment of body weight loss, stool consistency, and the presence of occult/gross blood by a guaiac test (Hemoccult SENSA; Beckman Coulter) were determined daily during DSS administration to generate a clinical activity score as described previously
(). During recovery periods, body weight was measured every week for each group. Endoscopic assessment of polyp formation and polyp count
Colonoscopy was done to assess polyp formation in mice by using the Coloview (Karl Storz Veterinary Endoscopy). Mice were euthanized with 1.5% to 2% isoflurane and approximately 3 cm of the colon proximal to the anus was visualized after inflation of the colon with air. Mice were then sacrificed by cervical dislocation after euthanizing with isoflurane. Colon was excised from ileocecal junction to anus, washed with 0.9% NaCl, cut open longitudinally and preserved in 10% neutral-buffered formalin. The number and size of the polyps were determined using a Jenko dissecting microscope. Histopathology scoring
Colons fixed in 10% neutral buffered formalin were Swiss-rolled, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin stain for
histopathologic examination of polyps and adenocarcinoma (neoplasia). Scoring was carried out in a blind fashion by 2 qualified pathologists. Score was given on the basis of these criteria: normal (score 0), low-grade dysplasia (score 1), high-grade dysplasia
(score 2), intramucosal adenocarcinoma (score 3), and invasive adenocarcinoma (score 4).
Immunohistochemistry
Paraffin-embedded sections (5 μm) of colon were analyzed for Ki67 staining as a marker of cell proliferation as previously described (). A minimum of 15 crypts with normal morphology were counted for Ki67-positive cells per section. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining
Immunofluorescent terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was carried out to measure apoptosis from
paraffin-embedded sections using the In Situ Cell Death Detection Kit as described by the manufacturer (Roche). Nuclei were stained with 4′, 6-diamidino-2-phenylindole to count total cells per crypt. A minimum of 10 crypts with normal morphology were counted per section.
p53 mutational analyses
Tumor tissue was microdissected from paraffin-embedded sections (7 μm) of
Swiss-rolled mouse colon. Genomic DNA was isolated from microdissected tissues using the QIAamp DNA FFPE Tissue purification kit (Qiagen). The PCR amplicons were generated and sequenced as previously described (). See the Supplementary Materials and Methods section for primer sequences.
Human tissue samples
Matching normal and tumor tissues were obtained at the time of surgical resection from patients with one or more UC-associated colorectal neoplasms, consisting of adenocarcinomas or dysplasias. Normal control samples consisted of colonic normal mucosa adjacent to tumors or ileal mucosa. Inflamed UC tissues were obtained from patients without evident dysplasia. All tissues were grossly dissected free of normal surrounding tissue, and parallel sections were used for histologic characterization. Tissue collection was approved by patients according to Institutional Review Board guidelines.
Total RNA extraction and quantitative real-time PCR
Total RNA was extracted for human tissues using the RNeasy kit (Qiagen).
Quantitative real-time PCR was carried out as described previously (). See the Supplementary Materials and Methods section for primer sequences.
Cell culture and transfection
Caco2-BBE cells were used to assess the interaction of PHB with STAT3. As Caco2-BBE cells have mutated p53, WT HCT116 human CRC cells were used to assess PHB interaction with p53. All cell lines were obtained from the American Type Culture Collection. Cells were grown and transfected as previously described (Kathiria and colleagues; submitted for publication).
γ-Irradiation
γ-Irradiation was used to induce DNA damage in WT and p53−/− HCT116 cells () after 72 hours of transfection with pEGFPN1 vector or pEGFPN1-PHB. Cells were irradiated by using 137Cs γ-irradiator at 6.5 cGy/s for 32 seconds for a total of 209.4 cGy. Cells were harvested 24 hours after γ-irradiation for subsequent assays. Protein extraction, Western blot analysis, and immunoprecipitation Mucosal strippings from Tg and WT mice were obtained for Western blot analysis as described previously (). Total protein was isolated from cultured cells as described previously (Kathiria and colleagues; submitted for publication). Antibodies used were mouse monoclonal PHB (Thermo Fisher), mouse monoclonal GFP, p53, Bcl-2, Bcl-xL and Bad and rabbit polyclonal BAX, p-Bad (ser 155), STAT3 (Santa Cruz Biotechnology), rabbit polyclonal proliferating cell nuclear antigen (PCNA) antibody (Abcam), rabbit polyclonal caspase-3, p53, pSTAT3 (Cell Signaling Technology), rabbit polyclonal p53 upregulated modulator of apoptosis (PUMA), and mouse monoclonal anti-β-actin (Sigma-Aldrich).
PHB was immunoprecipitated from 0.6 mg total protein lysates from HCT116 or Caco2-BBE cells or 0.20 mg total protein lysates from mouse mucosa with 1 μg mouse anti-PHB, anti-p53, or anti-pSTAT3 antibody and 30 μL 50% protein A
sepharose beads (GE Healthcare). Blots were incubated with the rabbit p53 antibody or PHB antibody, respectively. Omission of primary antibody during the
immunoprecipitation was carried out as a negative control.
Statistical analysis
Values are expressed as mean ± SEM. Statistical analysis was conducted using 2-way ANOVA and subsequent pairwise comparisons using Bonferroni post hoc tests. A P value less than 0.05 was considered statistically significant in all analyses. Results
IEC-specific PHB overexpression decreases colonic tumorigenesis in a mouse model of CAC
Previous studies have shown that PHB has an antitumorigenic role in gastric, prostate, and liver
cancers (). We used the AOM DSS mouse model to study the role of PHB in CAC. Body weight was measured weekly as one parameter to assess the severity of disease. WT and Tg mice given water only throughout the experiment and mice injected with AOM followed by water alone showed similar body weight gain over the 8-week protocol (Supplementary Fig. S1A) and did not develop polyps as previously reported for this dose of AOM (). WT mice given 2 cycles of DSS without AOM injection lost more body weight following DSS administration and recovered less weight compared with Tg mice over the 8-week protocol (Supplementary Fig. S1B), similar to our previous findings (). Although both WT and Tg AOM DSS-treated mice lost weight after the administration of DSS, Tg mice gained body weight more rapidly during the recovery phase and maintained body weight better than WT mice throughout the remainder of the study ().