Volume 117, Issue 2, Pages 350-358 (August 1999) Reduced expression of cyclooxygenase 2 proteins in hereditary nonpolyposis colorectal cancers relative to sporadic cancers  Frank A. Sinicrope, Michael Lemoine, Lixuan Xi, Patrick M. Lynch, Karen R. Cleary, Yu Shen, Marsha L. Frazier  Gastroenterology  Volume 117, Issue 2, Pages 350-358 (August 1999) DOI: 10.1053/gast.1999.0029900350

1 Fig. 1. Expression and localization of COX-2 proteins in (A and B) human CRC cells and (C-K) neoplastic tissues in representative photomicrographs. Brown in the cells and tissue sections indicates COX-2 staining detected with the chromogen diaminobenzidine. As a control for immunostaining, HCA-7 cells, known to constitutively express COX-2, are shown in the (B) presence and (A) absence of the anti–COX-2 MAb. As an additional control, sections were incubated with the anti–COX-2 antibody plus a sequence-specific COX-2 blocking peptide (D and E). In a COX-2 immunopositive carcinoma (E), this peptide completely suppressed COX-2 staining (D). COX-2 staining was granular (C) and localized to the cytoplasm of dysplastic and malignant tumor cells (C, E-J). (F-I) Histologically normal mucosa stained negative for COX-2. COX-2 staining intensity is reduced in a carcinoma from an (G) HNPCC [weak, 1+] vs. (H) a sporadic [strong, 3+] patient. (I) COX-2 staining is shown in a diminutive sporadic adenoma. In some cases, COX-2 staining was detected in (J) interstitial mononuclear cells and (K) fibroblasts adjacent to malignant glands. Gastroenterology 1999 117, 350-358DOI: (10.1053/gast.1999.0029900350)

2 Fig. 2. Immunohistochemical analysis of COX-2 staining intensity in colorectal carcinomas from patients with HNPCC (●) and sporadic CRC (○). A comparison of frequency and staining intensity (0, absent; 1+, weak; 2+, medium; 3+, strong) in malignant epithelial cells is shown. COX-2 expression was significantly reduced in HNPCCs compared with sporadics. 1Reference group; 2P = 0.035; 3P = 0.030. Gastroenterology 1999 117, 350-358DOI: (10.1053/gast.1999.0029900350)

3 Fig. 3. Immunoblot showing COX-2 protein levels (72-kilodalton band) in representative paired tissues from sporadic CRCs (T) and normal mucosa (N) (left panel). Increased COX-2 expression was seen in tumors compared with normal tissue in which a low level of COX-2 was detected. HCA-7 colon cancer cells constitutively express a high level of COX-2 and served as a positive control.22 The human colon cancer cell line HCT-116, which carries a mutation in the hMLH1 gene,31 was negative for COX-2 proteins as was the mismatch repair–deficient RKO cell line44 (right panel). Gastroenterology 1999 117, 350-358DOI: (10.1053/gast.1999.0029900350)

4 Fig. 4. Mutations were detected in the (A)10 tract of the TGF-β RII gene in colorectal carcinomas from HNPCC patients (1-4). DNA was extracted from tumor (T) and peripheral blood leukocytes (B), which serve as controls, and subjected to PCR using radiolabeled primers. Tumors from patients 1-3 and 5 show extra bands consistent with 1– or 2–base pair deletions. The tumor from patient 4 has wild-type RII alleles. Gastroenterology 1999 117, 350-358DOI: (10.1053/gast.1999.0029900350)