Our results suggest that the up-regulation of Necl-4 protein was dependent on Rap1 and afadin

ls depends on the ordered centriole duplication strictly limited to once per cell cycle. Disengagement of mother and daughter centriole PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19712481 is a prerequisite for semiconservative centriole duplication and is provided by proteolytic cleavage of cohesin, a “glue” multi-protein complex that is also responsible for sister-chromatide MedChemExpress MEK 162 cohesion. Separase, a cysteine endopeptidase encoded by the gene espl1, conducts cleavage of cohesin. Ectopic activation of Separase proteolytic activity causes premature sister-chromatide separation and centriole disengagement and overexpression of Separase has been reported to induce centrosome amplification, aneuploidy and tumorigenesis. Recently, striking functional evidence for the oncogenic activity of deregulated Separase was provided by Pati and coworkers who have generated a transgenic MMTV-Espl1 mouse model that overexpresses Separase protein in the mammary gland. These mice developed aggressive and highly aneuploid mammary carcinomas with high levels of CIN and cell cycle defects including multiple centrosomes and multinucleated cells. Moreover, Separase overexpression has been considered as potential predictor of progression-free survival and relapse in glioblastoma. The proteolytic activity of Separase is tightly regulated by multiple inhibitory mechanisms combining Securin binding, specific serine residue phosphorylation by CyclinB1/ Cdk1, autocatalytic cleavage and PP2A-dependent stabilization of Separase-bound Securin. The finding that espl1/Separase acts as an oncogene/-protein in various cancers including CML renders this protease a key target PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19713490 to unravel the molecular mechanisms involved in the development of centrosome amplification and clonal evolution in IM-treated CML. In this study, we used the cytogenetic data from CML study IV to investigate incidences of newly arising unbalanced ACA and blast crises under IM long-term treatment with regard to the p210BCR-ABL breakpoint variants b2a2 and b3a2. In search for potential underlying molecular mechanisms we performed protein biochemical studies on Separase expression and activity profiles in a panel of b2a2 and b3a2 CML cell lines. Our data point to the existence of a p210BCR-ABL-dependent feedback mechanism that posttranslationally stimulates Separase proteolytic activity after an IM-induced decrease in Separase protein levels exclusively in b3a2 cells. As a known promoter of aneuploidy and clonal evolution hyperactive Separase is an excellent candidate for explaining the cytogenetic in vivo data. Materials and Methods Cell lines and culture conditions Six human cell lines were investigated. NHDF was derived from Promocell GmbH. KCL-22, BV-173, LAMA-84 and K562 were obtained from the DSMZ. The immortalized human urothelial cell line UROtsa was a gift of the Department of Urology, Mannheim Medical Center, Mannheim Germany and cultured as previously described. All cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 2% glutamine and 1% penicillin-streptomycin at 37C in 5% CO2 atmosphere. Exponentially growing cells were used in at least triplicate experiments. Patients and ethics statement Clinical and cytogenetic data of 1151 patients with Ph+ and BCR-ABL+ CP CML randomized to the German CML-Study IV were investigated. Mean observation time was 5.6 years. There were 459 female 3 / 18 Separase Activity in CML and 692 male patients with a median age of 53 years; median age was lower in ACA patients. The definitions of CML phase