The gene (SAM- and SH3-domain containing 1) has originally been identified
The gene (SAM- and SH3-domain containing 1) has originally been identified as a candidate tumour suppressor gene in breast cancer. of a member of the (was mapped to chromosome 6q24.3, loss of heterozygosity (LOH) of this region (occurring in 30% of primary breast cancers) was associated with poor survival and increase in tumour size. Moreover, a strong reduction of expression was observed in the majority of breast tumours when compared to normal mammary epithelia (Zeller deregulation in human cancer has not been determined previously. The domain structure and strong sequence similarities places in the (shows ubiquitous expression in human tissue (Zeller constitutes an independent prognostic parameter in colon cancer. PATIENTS, MATERIALS AND METHODS Patients Informed, written consent regarding the use of the tissue samples was obtained from each subject before the study. Tissue samples were obtained from 113 patients admitted to our Department of Surgery with the diagnosis colon carcinoma. The group consisted of 69 male and 44 female patients, mean age was 64 years. None of the patients suffered of a known second neoplastic disease; only complete resected tumours (R0) were included in the study. Median survival after surgery was 91 months (range: 44C131 months). During this period, 38 patients died owing to tumour-related causes. Disease recurred in 15 patients, 34 patients developed metachronous distant metastases, and 23 patients showed disease progression. Tumour localisation was: ascending colon (41 cases), Z-LEHD-FMK supplier transverse colon (12 cases), descending colon (18 cases) and sigmoid colon (42 cases). Tumour grading was: G1 (three cases), G2 (74 cases), G3 (33 cases) and G4 (three cases). Tumour stages according UICC classification were: stage I (12 cases), stage II (45 cases), stage III (23 cases), and stage IV (33 cases). Sixty-five patients had no Z-LEHD-FMK supplier adjuvant treatment, and 48 patients received systemic chemotherapy. As a control, we examined normal colon tissue (15 patients), benign colonic adenomas (nine patients), and liver metastases from 10 patients. Samples were frozen in liquid nitrogen immediately after surgery and stored at ?80C. RNA Isolation from cell lines and tissue samples To establish and validate the quantification of expression the following human cell lines were used: HEK293 (embryonic kidney epithelial cells), HeLa (cervical carcinoma), SKOV-3 (ovarian adenocarcinoma), CaCo2 (grade II colorectal adenocarcinoma), HT29 (grade I colorectal adenocarcinoma), Jurkat (T-lymphocyte from acute T-cell leukaemia), and Ramos (B-lymphocyte from Burkitt’s lymphoma). For RNA isolation from tissue, we used 40 sections of 12?and transcripts was determined by real-time reverse transcriptaseCpolymerase chain reaction (RTPCR) using the ABI PRISM 7300 sequence detection system (Applied Biosystems, Foster City, CA, USA) with the dye SYBRGreen I. Expression of the housekeeping gene was used as internal reference. ??HPRT-F:?5-GCT TTC CTT GGT CAG GCA GTA TAA T-3??HPRT-R:?5-AAG GGC ATA TCC TAC AAC AAA CTT G-3??SASH1-F:?5- CGG GAA Z-LEHD-FMK supplier AGC GTC AAG TCG GA-3??SASH1-R:?5- ATC TCC TTT CTT GAG CTT GAG-3??SLY1-F:?5- TCC AGC AGC TTC AAG GAT TT-3??SLY1-R:?5- CAT CTT GCC CAT CTT CCT GT-3 Statistical analysis Analyses were performed using SPSS version 9.0 (SPSS, Munich, Germany). Statistical significance was defined as was assessed in terms of survival by the Cox proportional hazards model using univariate and multivariate analysis. Significance was tested by analysis. Preparation of protein lysates Resected tumours and normal colon tissue samples (as certified by an FLJ20285 experienced pathologist) were snap-frozen in liquid nitrogen in lactate buffered Ringer’s solution, and.