Since Separase is one of the master key players in centriole duplication, and overexpression has been associated with formation of supernumerary centrosomes in cancers including CML 
, we investigated the influence of BCR-ABL TK on separase in the therapeutic context of IM. We analyzed Separase on multiple regulatory levels of expression, i.e. transcriptional, translational and post-translational levels, in a panel of six well characterized and widely accepted human cell lines. Of these, K562, LAMA-84 and U937p210BCR-ABL/c6 displayed different levels of p210BCR-ABL protein and, therefore, mimic the different stages of CML () 
. Since each cell line is unique with respect to karyotype, BCR-ABL copy number, cell cycling (doubling) time and IM sensitivity, each cell line was treated individually according to its unique growth and sensitivity behaviour. A distinct IM dose and time schedule was applied, where lower IM doses and incubation times were applied for fast-growing, BCR-ABL growth-dependent, cells (K562 and LAMA-84) than for BCR-ABL-positive slow-growing cells (U937p210BCR-ABL/c6-On) and BCR-ABL-negative cells (NHDF, UROtsa, HL-60, U937). This treatment schedule allowed for preparation of RNA and protein lysates in sufficient amounts and quality to perform the presented qRT-PCR, Western Blot experiments and Separase activity assays.
We found that regulation of separase in IM-treated BCR-ABL-positive cells is complex and occurs on both protein expression and proteolytic activity levels. i) Treatment of BCR-ABL-negative cells (NHDF, UROtsa, HL-60, U937) with IM strongly pointed to a regulation of Separase protein expression on levels of translation and/or protein stability rather than transcription, as transcript and protein level changes did not coincide upon IM application (). This may also be true for BCR-ABL-positive cells, although concomitant transcript and protein level decreases were observed after IM application (). We surmise that this coincidence may be due to the antiproliferative and proapoptotic effect of IM in BCR-ABL-positive cells (K562 and LAMA-84) as supported by the observed cell cycle profiles of IM-treated and untreated cell (). IM treatment resulted in considerable decreases in the proportion of G2/M and S phase cells, whereas the amount of apoptotic cells increased.
ii) Post-translational regulation on the proteolytic activity level becomes evident when all untreated cell lines under investigation were compared with respect to BCR-ABL TK activity, Separase protein levels and Separase proteolytic activity (). While Separase protein expression correlated positively with p210BCR-ABL TK activity as reported by others 
, and was in fact highest in K562 and LAMA-84, all exponentially growing cells displayed about the same proportion of Separase proteolytic activity (). This strongly suggests that regulation of Separase proteolytic activity is independent of p210BCR-ABL whereas Separase protein expression is linked to BCR-ABL TK activity. Our experiments demonstrate that IM application can affect both levels of Separase regulation.
Decreased Separase protein levels were observed in all investigated cell lines after IM application. This effect is BCR-ABL-independent as it was equally observed in both BCR-ABL-positive and negative cells. Except for BCR-ABL-positive cells, decreased Separase proteolytic activity levels were observed in all p210BCR-ABL-negative cell lines. FACS analyses revealed that the parallel changes in Separase protein and activity levels are not associated with changes in the proportion of G2/M cells. Decreased Separase protein level may be related to decreased translation and/or enhanced degradation of Separase protein. Reduced Separase proteolytic activity may be best explained by a reduced proportion of cells entering mitotic anaphase, where the protease is regularly activated by the anaphase-promoting complex/cyclosome (APC/C) 
. Since our FACS analyses revealed no changes (NHDF, UROtsa, HL-60), or an 6% increase in G2/M cells after IM treatment (U937, ), we assume that the majority of cells were on hold at the G2/M check point before the transition to M phase 
. An IM-induced G2/M arrest has been reported previously for various cancer cells 
The second level of regulation (proteolytic activity level) was exclusively affected by IM in p210BCR-ABL-positive cells (K562, LAMA-84, U937p210BCR-ABL/c6-On). We observed increased Separase proteolytic activities despite lowered Separase protein levels after IM application. This unexpected activation (LAMA-84, 5.4-fold; K562, 2.5-fold; U937p210BCR-ABL/c6-On, 2.9-fold; calculated as a ratio [Separase protein/Separase proteolytic activity] from data given in , suggests a BCR-ABL-dependent compensatory mechanism that is able to counterbalance the inhibitory effect of IM on Separase protein expression by raising the proteolytic activity of Separase.
For a mechanistic explanation we conclude that Separase was activated regularly via the APC/C, a large multi-subunit complex operating as a specific E3 ubiquitin ligase 
, because in all cell lines with increased Separase proteolytic activity (K562, U937, U937p210BCR-ABL/c6-On), we measured decreased protein levels of Securin, pSer1126 and CyclinB1 ( and ). APC/C promotes the metaphase/anaphase transition by ubiquitizing and degrading Securin, the main inhibitor of Separase proteolytic activity. Moreover, APC/C also ubiquinates CyclinB1 and accelerates its degradation during late mitotic phase, which results in activation of Separase and mitotic exit 
. Dysregulation of APC/C-dependent proteolysis of these substrates is considered to contribute to mitotic catastrophe and tumorigenesis 
. The activity of APC/C is regulated by a complex network of antagonistic phosphorylating events of its subunits resulting in CDC20 binding, one of its main activating subunits. We hypothesize that IM targets one or more phosphoproteins of the APC/C, thereby activating the E3 ubiquitin ligase function. This may favor the degradation of Securin and CyclinB1, and selective dephosphorylation of Separase at serine residue 1126 (pSER1126). Finally, this may lead to activation of Separase. The explanation of why Separase activation is exclusively observed in BCR-ABL-positive cells remains elusive. However, a potential mechanistic link is provided by a previous microarray study reporting that BCR-ABL expression promotes overexpression of CDC20 
and thereby enables activation of the APC/C 
. We further suggest that this Separase activating effect, observed exclusively in BCR-ABL-positive cells, is not attributed to BCR-ABL TK activity, but to the protein itself as we consider the applied IM concentrations high enough for almost complete inhibition of ABL-related TK activity in our experiments (IC50 in cellular assays: 280 nM) 
. Therefore, protein-protein interaction rather than ABL-related TK activity may be responsible for the observed effects. This might be favored by the coiled-coil domain of the BCR protein that remains in the BCR-ABL fusion protein and promotes dimerization of p210BCR-ABL or perhaps binding to other proteins 
There is a potential clinical impact of our observation. We hypothesize that the increased proteolytic activity of Separase may be a trigger for unscheduled centriole duplication and subsequent centrosomal amplification that probably contributes to chromosomal missegregation and the development of genomic instability during further cell cycles. This assumption is concordant with the molecular pathology of CML and also with our earlier observations 
. Clonal evolution with consistent chromosomal aberrations, in addition to the t(9;22)(q34;q11), is frequently detected in 30% of patients with AP and about 80% patients in BC 
. Development of resistance in patients undergoing IM therapy frequently concurs with clonal evolution, which points to clonal evolution as a mechanism of resistance 
. Furthermore, under IM, the outcome of patients with clonal evolution is significantly inferior compared to those without 
; suggesting a close conditional interrelationship to IM treatment. It is therefore tempting to speculate that the IM-related upregulation of Separase proteolytic activity in BCR-ABL-positive cells may play a role as a promoting mechanism for the development of tumor heterogeneity. Even in dormant BCR-ABL- low-expressing clones, such as quiescent stem cells 
, this may eventually create descendant cell populations with enhanced fidelity to escape therapeutic pressure.
In summary, we found that the regulation of Separase in IM-treated BCR-ABL-positive cells occurs on both protein expression and enzyme activity levels. Furthermore, we established a mechanistic link between IM treatment, BCR-ABL expression and increased Separase proteolytic activity. Our in vitro study has provided a hypothesis of how BCR-ABL-positive cells undergoing IM therapy may trigger centrosomal amplification and genomic instability. In CML patients during IM treatment, enhanced Separase proteolytic activity in bcr-abl-positive stem and progenitor cells with residual BCR-ABL protein expression may promote tumor heterogeneity, clonal evolution and development of resistance. We believe that future studies on the Separase regulatory network in CML may give rise to new concepts in carcinogenesis and leukemia therapy.