X, 2 ug mL mouse monoclonal antibody or mouse isotype control was added and goa

Methods Patients and samples Tissue samples from 129 patients who underwent partial pancreaticoduodenectomy for primary pancreatic ductal adenocarcinoma between 1991 and 2000 were retrieved from the database of the Pathology Department of the Charité University Hospital. AP24534 臨床試験 The study was approved by the Charité University Ethics Committee, Median age of patients with pancreatic cancer was 65 years, Follow up data regarding overall survival were available for 113 patients. Within the follow up time, 89 patients died after a mean follow up time of 22. 1 months. Mean follow up time of patients still alive at the endpoint of analysis was 54. 0 months. Cases were staged according to TNM Classification of Malignant Tumours. 7th edition and were graded as recommended by the WHO, Tissue microarray construction Of all PDACs 3 um sections were cut and stained with H E.

Three representative areas from the tumor center and invasive supplier AT7519 margins were marked by a board certified pathologist, For each case three tissue cores from the selected representative tumor areas were punched out of the sample tissue blocks and embedded into a new paraffin array block using a tissue microarrayer, Immunohistochemistry For immunohistochemical detection of Sirt1 on tissue sam ples, a monoclonal rabbit antibody was used. After heat induced antigen retrieval, slides were incubated with the primary antibody at 4 degree Celsius overnight. Bound antibody was detected by a streptavidin biotin sys tem, For colour develop ment, a Fast Red system was used. Omission of the primary antibody served as negative control.

The slides were cover reversible Akt 阻害剤 slipped after counterstaining. Nuclear staining of Sirt1 was scored by applying a semi quantitative immunoreactivity scoring system, as de scribed previously. Briefly, the intensity of staining and percentage of cells stained were evaluated separately. The IRS for each individual case ranging from 0 to 12 was cal culated by multiplication of the intensity and frequency scores. Cases exhibiting an IRS from 0 6 were combined in one group, cases with an IRS of 6 were combined in a Sirt1 high group. Staining of tissue slides was evaluated by experienced pathologists blinded towards patient characteristics and outcome. Cell culture The human pancreatic cancer cell lines PANC 1 and MiaPaCa 2 were obtained from the American Type Culture Collection and cultured in Dulbeccos modified Eagles medium supplemented with 10% fetal bovine serum and P S.

For the MIA PaCa 2 cells, additionally 2. 5% horse serum and 5 ml NaHCO3 were used. These two cell lines were chosen, since PANC 1 is a proto typical Gemcitabine resistant cell line, while Mia PaCa 2 is known to retain some Gemcitabine sensitivity. Cambinol was purchased from Merck, Gefitinib was obtained from Biaffin and Nico tinamide from Sigma, Plasmids, siRNA and transfections The SIRT1 2 and GFP control expression constructs were obtained from Addgene. For SIRT1, expression of the FLAG tagged SIRT1 open reading frame was under the control of an SV40 promotor, allowing physiological levels of SIRT1 expression in cells not harbouring the Large T antigen, GFP was cloned in a pcDNA3 vec tor, allowing high protein expression controlled by CMV promotor.