The Proteolytic Activity of Separase in BCR-ABL-Positive Cells Is Increased by Imatinib

Abstract

Separase, an endopeptidase required for the separation of sister-chromatides in mitotic anaphase, triggers centriole disengagement during centrosome duplication. In cancer, separase is frequently overexpressed, pointing to a functional role as an aneuploidy promoter associated with centrosomal amplification and genomic instability. Recently, we have shown that centrosomal amplification and subsequent chromosomal aberrations are a hallmark of chronic myeloid leukemia (CML), increasing from chronic phase (CP) toward blast crisis (BC). Moreover, a functional linkage of p210BCR-ABL tyrosine kinase activity with centrosomal amplification and clonal evolution has been established in long-term cell culture experiments. Unexpectedly, therapeutic doses of imatinib (IM) did not counteract; instead induced similar centrosomal alterations in vitro. We investigated the influence of IM and p210BCR-ABL on Separase as a potential driver of centrosomal amplification in CML. Short-term cell cultures of p210BCR-ABL-negative (NHDF, UROtsa, HL-60, U937), positive (K562, LAMA-84) and inducible (U937p210BCR-ABL/c6 (Tet-ON)) human cell lines were treated with therapeutic doses of IM and analyzed by qRT-PCR, Western blot analysis and quantitative Separase activity assays. Decreased Separase protein levels were observed in all cells treated with IM in a dose dependent manner. Accordingly, in all p210BCR-ABL-negative cell lines, decreased proteolytic activity of Separase was found. In contrast, p210BCR-ABL-positive cells showed increased Separase proteolytic activity. This activation of Separase was consistent with changes in the expression levels of Separase regulators (Separase phosphorylation at serine residue 1126, Securin, CyclinB1 and PP2A). Our data suggest that regulation of Separase in IM-treated BCR-ABL-positive cells occurs on both the protein expression and the proteolytic activity levels. Activation of Separase proteolytic activity exclusively in p210BCR-ABL-positive cells during IM treatment may act as a driving force for centrosomal amplification, contributing to genomic instability, clonal evolution and resistance in CML.

Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in over/underestimation of cell viability

“…In addition to its multi-targeted mechanism of action (see Introduction), imatinib (as well as nilotinib and dasatinib) was documented to induce spindle defects, numerical and structural centrosome and chromosome alterations in human and mammalian cells in vitro in a dose-dependent manner. Also, these inhibitors affected centrosomes and spindles and induced de novo chromosomal aberrations in CML cells as well as in BCR-ABL-negative hematopoietic progenitor cells in vivo (Cohen et al, 2002;Fabarius et al, 2008Fabarius et al, , 2007Fabarius et al, , 2005Giehl et al, 2010;Haaß et al, 2012). Aditionally, the most widely used plant-based bioassays for the detection of genotoxins demonstrated that imatinib at low concentrations caused induction of micronucleuses, which are formed as a consequence of numerical and structural chromosomal damage (Pichler et al, 2014).…”

Improved FRET Biosensor for the Measurement of BCR-ABL Activity in Chronic Myeloid Leukemia Cells

“…In fact, separase, a cysteine endopeptidase involved in triggering anaphase and the maintenance of centriole numbers through cohesion hydrolysis, was highly expressed in CML cells (Haaß et al, 2012;Patel and Gordon, 2009).Two putative separase cleavage sites were identified in the CrkL sequence at amino acid positions 67-70 and 139-142 by sequencing analysis. It is therefore possible that Pickles could be cleaved by this protease, which might explain the disappearance of small bands that should be observed after cleavage at D94 by metalloproteinases.…”

“…Third, full-length proteins are cleaved and degraded. Because it was reported that the expression of BCR-ABL induces protease expression (Haaß et al, 2012;Patel and Gordon, 2009) and promotes proteasome activity (Crawford et al, 2009;Magill et al, 2004), we tested the possibility of the third scenario. To evaluate this possibility, we introduced a silent mutation into the EcoRI site in CrkL (Pickles 2.33, see also Fig.…”

“…3, the cleavage of FLAG 2.33 was enhanced in the presence of BCR-ABL. It might be accounted for by the increased expression of proteases or promoted proteasome activity by BCR-ABL (Crawford et al, 2009;Haaß et al, 2012;Magill et al, 2004;Patel and Gordon, 2009). Together, these data indicate that more than three Pickles fragments are generated through cleavage at the SH2 domain and between Y207 and CFP (Fig.…”

Separase activity distribution can be a marker of major molecular response and proliferation of CD34+ cells in TKI-treated chronic myeloid leukemia patients

“…Therefore, we have skipped use of the Cy5 label and have used this detection channel for simultaneous sorting of CD34 + cells. In our earlier work [36], we have used a lysate-based separase assay that worked with a comparable reporter peptide, but measured separase activity in protein lysates [27]. Back then, we have indeed used actin (immunostained Western blots) to normalize for separase activity.…”