BMC Cancer 2009, 9:251 http://www.biomedcentral.com/1471-2407/9/251 of animals, a total of 60 accounted for most of the well- oriented crypts. The number of colonocytes per crypt was also determined in at least 60 well-oriented crypts per ani- mal. B-catenin was evaluated for cellular localization and its nuclear, cytoplasmic, and membranous was recorded. The intensity of staining and its distribution was recorded in 60 well-oriented crypts using semiquantitative scale (0- negative, 1-weak membranous staining, and 2-strong membranous staining), as well as the anatomical location of the positive cells in the mucosa (crypt base, crypt body, and colonic plate). Statistical analysis Descriptive statistics were used to summarize data. Initial descriptive analyses were done to determine whether to use parametric t-test or non-parametric Mann-Whitney test for mean comparisons and whether to assume equal variance or unequal variance. A Student's t-test was used to examine the differences for continuous variable under assuming either equal or unequal variance. Association between variables were determined using Spearman corre- lation coefficient analysis. Linear regression model were also used after checking assumption of linearity, normal- ity and constant variance of independent error. A p-value of less than 0.05 was considered statistically significant. The analysis was performed using the SPSS – 15 software. Results Correlation between the number of cells per crypt and the length of the crypt was significant (p < 0.0001, Spearman Correlation). The coefficient of correlation was 0.86. This result indicated that the increased length of the crypts was due to hyperplasia, rather than hypertrophy or other phe- nomena like edema or various artifacts. All available well-oriented crypts were evaluated for the number of cells per crypt as well as for the absolute length of the crypt. The number of observations (N) per animal varied from 60 to 246 per animal depending on the number of available well-oriented crypts. The results are summarized in Tables 1 and 2 and Figure 1, 2, 3, 4, 5, 6, and 7. The length of the crypts in CyCAP mice and WT mice (r = 0.723, p = 0.018, Spearman Correlation) as well as the number of colonocytes per crypt (r = 0.863, p = 0.001, Spearman Correlation) in non-tumorous areas of the colon were positively associated with total number of tumors induced by azoxymethane (Figure 1 and 2). Also, the length of the crypts (r = 0.782, p = 0.008, Spearman Correlation) and the number of colonocytes per crypt (r = 0.927, p < 0.0001, Spearman Correlation) in non-tumor- ous areas of the colon of CyCAP mice and WT mice correlated with the total size of the tumors. In addition, untreated 8-week old CyCAP mice had significantly thicker mucosa and significantly larger numbers of colonocytes per crypt (hyperplasia) (p < 0.0001, T-test), as well as higher proliferation fraction as measured by Ki-67 immunohistochemical test (p = 0.032, Mann-Whitney Test) even before azoxymethane treatment was initiated (Figure 1, 2A, and 3) indicating spontaneous develop- ment of mucosal hyperplasia. The difference in diffuse mucosal thickness was even more pronounced after the treatment with carcinogen (p < 0.0001, T-test, Figure 2A, 3, 4, and 5). Similarly, the mice had larger numbers of cells per crypt both before (p = 0.032, Linear Regres- sion; plots not shown) and after treatment with carcino- gen (p = 0.012, Linear Regression; plots not shown) compared with WT animals. CyCAP mice had significantly larger numbers of ACF and tumors per animal than control WT animals (p = 0.016, T-Test, Figure 2B). The mean tumor size was larger in CyCAP mice (p = 0.031, T-Test) as well as cumula- tive tumor size per animal (p = 0.003, T-Test, Figure 2C). Spectrum of histologic changes is shown in Figure 6. Beta-catenin was not detected in the nuclei of the colono- cytes either in WT or KO mice. Membranous β-catenin was overall less expressed in WT mice than in KO mice (p < 0.0001, Mann-Whitney Test). In WT mice, β-catenin was very weakly expressed in the colonic crypts with more notable expression in the colonic plate/luminal surface of the mucosa (Figure 7A). Membranous β-catenin in CyCAP KO mice is not only overall stronger, but also there is strong expression not only in the colonic plate, but focally also in an irregular fashion including the base of the crypts (Figure 7B). The difference between WT and KO mice in the distribution of β-catenin expression was also highly significant (p < 0.0001, Mann-Whitney Test). This overex- pression of the membranous β-catenin is highly variable from one crypt to another. No definite difference in c-myc expression between WT and KO mice was noted. Cyclin D1 expression was significantly higher in KO mice (p < 0.0001, Mann-Whitney Test) and also positively corre- lated with Ki-67 expression (p = 0.019, Linear-by-Linear Association). Discussion Historically, it has been claimed that the transition from hyperplasia to neoplasia is frequent [54,55]. Human stud- ies and experimental data from animals suggest that high rates of colonic epithelial cell replication enhance the development of colon cancer [56-58]. Exaggerated mucosal proliferative activity is in some way associated with the development of large-bowel neoplasia. Individu- als with inflammatory and proliferative diseases of the bowel such as Crohn's disease, ulcerative colitis, and familial polyposis suffer an unusually high risk and early onset of colorectal cancer. However, as mentioned above, this has never been shown in a very common form of colonic mucosal hyperplasia, i.e. hyperplastic polyps [59] Page 4 of 11 (page number not for citation purposes)