Volume 119, Issue 4, Pages 929-942 (October 2000) p16INK4a expression begins early in human colon neoplasia and correlates inversely with markers of cell proliferation  Charlotte Y. Dai, Emma E. Furth, Rosemarie Mick, Jim Koh, Tetsuji Takayama, Yoshiro Niitsu, Greg H. Enders  Gastroenterology  Volume 119, Issue 4, Pages 929-942 (October 2000) DOI: 10.1053/gast.2000.17952 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Detection of increased p16INK4a expression in carcinomas by 3 techniques: immunoprecipitation followed by Western blotting, RT-PCR, and IHC. (A) Specificity of anti-p16INK4a monoclonal Ab JC2. Extracts from human osteogenic sarcoma cells transfected with either p16INK4a (p16+)28 or the empty vector (−) and SaOS2 cells that express endogenous p16INK4a were subjected to Western blotting with JC2. Load, actin. (B) Detection of elevated p16INK4a in a carcinoma by immunoprecipitation and Western blotting. Extracts from a matched pair of colon carcinoma and stripped normal mucosa (CA1 and NL1; lanes 3–6), and another stripped normal mucosa (NL2; lanes 7 and 8) were subjected to immunoprecipitation with either a rabbit anti-p16INK4a polyclonal Ab or nonspecific IgG, followed by Western blotting with pooled p16INK4a monoclonal Abs JC2 and JC4. Cell extracts from U2-OS and OSp16.1 cells (lanes 1 and 2) were used as negative and positive controls, respectively. Load, actin. (C) Tumor-associated p16INK4a binds Cdk6. Extracts prepared from CA.1 and Nl.1 were subjected to immunoprecipitation by using the designated Abs, followed by Western blotting with JC2 and JC4 Abs. (D) Absent and elevated p16INK4a RNA, respectively, in 2 carcinomas, detected by RT-PCR. RT-PCR was performed on total RNA extracted from 2 normal colon and carcinoma pairs (NL.3 and CA.3, NL.4 and CA.4), by using p16INK4a exon 1'-specific primers. PCR product of 36B4 cDNA was used as a loading control. +C, positive control, PCR product from a p16INK4a expression vector. Facing page: (E) Increased p16INK4a expression in a single carcinoma detected by 3 techniques. Protein extracts, RNA, and formalin-fixed tissue blocks were prepared from a colorectal carcinoma (CA.5) and matched normal colon and subjected to immunoprecipitation and Western blotting, RT-PCR, and IHC. The former 2 techniques were performed as described in B and D. IHC was performed by using JC2. Gastroenterology 2000 119, 929-942DOI: (10.1053/gast.2000.17952) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Detection of increased p16INK4a expression in carcinomas by 3 techniques: immunoprecipitation followed by Western blotting, RT-PCR, and IHC. (A) Specificity of anti-p16INK4a monoclonal Ab JC2. Extracts from human osteogenic sarcoma cells transfected with either p16INK4a (p16+)28 or the empty vector (−) and SaOS2 cells that express endogenous p16INK4a were subjected to Western blotting with JC2. Load, actin. (B) Detection of elevated p16INK4a in a carcinoma by immunoprecipitation and Western blotting. Extracts from a matched pair of colon carcinoma and stripped normal mucosa (CA1 and NL1; lanes 3–6), and another stripped normal mucosa (NL2; lanes 7 and 8) were subjected to immunoprecipitation with either a rabbit anti-p16INK4a polyclonal Ab or nonspecific IgG, followed by Western blotting with pooled p16INK4a monoclonal Abs JC2 and JC4. Cell extracts from U2-OS and OSp16.1 cells (lanes 1 and 2) were used as negative and positive controls, respectively. Load, actin. (C) Tumor-associated p16INK4a binds Cdk6. Extracts prepared from CA.1 and Nl.1 were subjected to immunoprecipitation by using the designated Abs, followed by Western blotting with JC2 and JC4 Abs. (D) Absent and elevated p16INK4a RNA, respectively, in 2 carcinomas, detected by RT-PCR. RT-PCR was performed on total RNA extracted from 2 normal colon and carcinoma pairs (NL.3 and CA.3, NL.4 and CA.4), by using p16INK4a exon 1'-specific primers. PCR product of 36B4 cDNA was used as a loading control. +C, positive control, PCR product from a p16INK4a expression vector. Facing page: (E) Increased p16INK4a expression in a single carcinoma detected by 3 techniques. Protein extracts, RNA, and formalin-fixed tissue blocks were prepared from a colorectal carcinoma (CA.5) and matched normal colon and subjected to immunoprecipitation and Western blotting, RT-PCR, and IHC. The former 2 techniques were performed as described in B and D. IHC was performed by using JC2. Gastroenterology 2000 119, 929-942DOI: (10.1053/gast.2000.17952) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 p16INK4a staining is detected by IHC in rare cells of normal colon crypts and, at increased frequency, in benign lesions of human colonic epithelium. (A–D) Normal colonic mucosa. (A) Normal colonic mucosa showing no staining in the absence of primary Ab. (B) Typical normal mucosa, showing no staining with JC2. (C) Exceptional normal mucosa showing a few crypt base cells staining with JC2. (D) Higher magnification of the boxed area in C showing a few cells with typical preferentially nuclear staining with JC2. (E–H) ACF, showing increased p16INK4a staining compared with normal mucosa. Note the irregular contour and large size of many crypts, with p16INK4a expression at their bases. (I–L) Increased p16INK4a expression in adenoma. (I) Markedly heterogeneous p16INK4a staining in an adenoma. (J) Increased p16INK4a expression at the base of the neoplastic crypts, bordering on normal tissue. (K) Markedly increased p16INK4a expression in an adenoma compared with adjacent normal mucosa. (L) Higher magnification of region in K, showing cells staining strongly for p16INK4a intermingled within the same epithelial sheet with unstained cells. Gastroenterology 2000 119, 929-942DOI: (10.1053/gast.2000.17952) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 p16INK4a expression is detected by IHC in most primary and metastatic human colonic carcinomas. (A–D) Primary carcinoma. (A) Increased p16INK4a expression in an adenocarcinoma (CA) arising within an adenoma (AD). (B) Increased p16INK4a expression in neoplastic cells at the border of a carcinoma (CA) and normal tissue (NL). (C) p16INK4a staining in a poorly differentiated colon carcinoma. (D) Intermingling of carcinoma cells with strongly positive and negative p16INK4a staining. (E–G) Metastases. (E) p16INK4a staining was detected in colon carcinoma cells metastatic (Met) to a lymph node (LN). (F and G) p16INK4a staining in colon carcinoma cells metastatic to liver. Gastroenterology 2000 119, 929-942DOI: (10.1053/gast.2000.17952) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Higher fraction of p16INK4a-positive cells in 3 carcinomas compared with 3 adenomas. IHC was performed on 6 representative colon neoplasms. Eight to 10 random high-power images were collected from each specimen. There were, on average, 306 cells per field with a total of 2000–3000 per specimen. The percent of p16INK4a-positive cells was determined in each field (symbols). For each field, the precision was defined as the width of the 95% confidence interval (a function of observed percent and number of cells counted). For 42 of 53 fields (79%), the precision was within 6%. A higher fraction of cells were p16INK4a-positive in the carcinomas than in the adenomas (P < 0.001, by a Poisson random effects model; see Materials and Methods). Gastroenterology 2000 119, 929-942DOI: (10.1053/gast.2000.17952) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 p16INK4a expression is inversely correlated with markers of cell proliferation. (A–C) Coimmunofluorescence staining of pRb and Ki67 in a single section of normal colonic mucosa. (A) pRb staining (Ab directed against amino acids 300–380) in green. (B) Ki67 staining in red. (C) Merged image. Epithelial cells located at the base of the crypt displayed nuclear staining for both antigens. The presence of yellow cells in the merged image indicates substantial overlap between pRb and Ki67 staining. (D–I) Contrasting staining patterns of p16INK4a (D, green) and pRb (G, green) with 3-μm serial sections of a colon carcinoma costained with Ki67 (E and H, red). We estimate that 70% of nuclei in such sections are identical (D–F are from one section, G–I from the next). The absence of yellow cells in the merged image (F) suggests mutually exclusive expression of p16INk4a and Ki67. p16INK4a and pRb Abs stained spatially distinct areas (D vs. G). This difference was reinforced by the finding that p16INK4a and Ki67 staining was mutually exclusive, whereas pRb and Ki67 staining overlapped substantially. (J–O) p16INK4a expression is negatively correlated with cyclin A. (J and K) Cells stained positive for cyclin A were observed at the base of the crypts in normal colon by IF (J) and IHC (K). (L) Heterogeneous cyclin A staining was detected in a colon carcinoma. (M–O) Coimmunofluorescence staining of p16INK4a (M, green) and cyclin A (N, red). The lack of yellow cells in the merged image (O) suggests a mutually exclusive pattern of expression, consistent with results obtained with Ki67. Nuclear DNA was stained with Hoechst (blue). Gastroenterology 2000 119, 929-942DOI: (10.1053/gast.2000.17952) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Quantitative analysis of the negative correlation between p16INK4a and Ki67 staining in neoplasms. Percentage of Ki67-positive cells in p16INK4a-positive and p16INK4a-negative cell populations was determined in 4 carcinomas (CA) and 1 adenoma (AD), based on randomly captured images. The total cell number determined for each tumor ranged from 1600 to 3300. The results are shown as a paired scatter plot of the 5 tumors analyzed, with results from a single field connected by a line. There was a statistically significant difference in the fraction of Ki67-positive cells between p16INK4a-positive and -negative cells in the 5 tumors (P < 0.001 by a Poisson random effects model; this P value was not altered by exclusion of the lone adenoma and/or CAlll). Gastroenterology 2000 119, 929-942DOI: (10.1053/gast.2000.17952) Copyright © 2000 American Gastroenterological Association Terms and Conditions