a systematic study using traditional cell culture systems representing a panel of thoroughly characterized CML cell lines. This approach offers a much more homogenous genetic background than clinical samples and provides the appropriate controls to evaluate Separase in the absence of IM or after RNAi silencing. We investigated the influence of IM on Separase protein levels and Separase proteolytic activity in a panel of 12 / 18 Separase Activity in CML six well characterized human cell lines with focus on cells expressing the b3a2 and b2a2 fusion type of p210BCR-ABL. These experiments confirmed and expanded our recently published data where solely b3a2 cell lines have been investigated. We were able to confirm our recent data on K562 and LAMA-84 cell lines where IM treatment led to considerable decreases in Separase protein levels and unexpected activation of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19703425 Separase proteolytic activity. The approximately 5.4-fold and 2.5-fold increase in Separase activity in LAMA-84 and K562 cells, respectively, confirms that Separase proteolytic activity is controlled on posttranslational level and independent of p210BCR-ABL tyrosine kinase activity. In contrast, in b2a2 fusion type cells and in non-malignant UROtsa cells we found slightly decreased or unchanged Separase protein levels. This becomes apparent in the calculated quotients of Separase activity /Separase protein level . Quotients ranging around 1.0 point to a constant activity/protein ratio. Slight alterations compared to corresponding untreated cells are probably due to the antiproliferative effects of IM in BCR-ABL-positive cells as IM treatment resulted in slight decreases in the proportion of G2/M and S phase cells while the amount of apoptotic cells was stable below 12%. When we tried to mimic the observed posttranslational upregulation of Separase proteolytic activity in b3a2 cells by RNAi-related espl1 silencing 
we observed a striking increase in Separase activation in both b3a2 and b2a2 cells. Overall, our results point to the existence of two corresponding mechanisms contributing to the observed Separase regulation in p210BCR-ABL positive cells. i) Selective downregulation of Separase protein levels by IM treatment exclusively in b3a2 cells, but not in b2a2 cells. ii) Posttranslational hyperactivation of the remaining Separase buy PTK/ZK molecules to functionally compensate for the dropped Separase protein levels. This latter mechanism is p210BCR-ABL-dependent as it was not observed in NHDF cells but it does not rely on the kinase activity of p210BCR-ABL because hyperactivation occurred in face of IM treatment. Since hyperactivation of Separase proteolytic activity can be induced also in b2a2 fusion type cells by siRNA-mediated downregulation of Separase protein levels, the responsible actuator seems to be solely associated with the diverging response of b3a2 and b2a2 to IM treatment. Biochemically, it may be traced back to the additional 25 amino acid residues within the b3a2 p210BCR-ABL oncoprotein. Studies from the pre-IM era have proposed that a sequential arrangement of six hydrophobic amino acid residues encoded by bcr exon e14 may alter juxtaposition of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19703425 p210BCR-ABL domains and subsequently, may change the binding kinetics for interacting protein partners. Therefore, we speculate that differential protein interacting capabilities of the b3a2 and b2a2 fusion proteins may alter either expression or stability of Separase in IM treated CML cells. So far, the corresponding regulatory prot