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

Wiltrud Haaß,Stefanie Nittka,Alice Fabarius,Wolfgang Seifarth,Wolf-Karsten Hofmann,Michelle Giehl,Michael Stehle,Petra Schrotz-King,Ivan Cruz Moura

doi:10.1371/journal.pone.0042863

Wiltrud Haaß, Stefanie Nittka + Show 7 more

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https://doi.org/10.1371/journal.pone.0042863

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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.

Highlights

The BCR-ABL tyrosine kinase (TK) formed by the balanced translocation t(9;22)(q34;q11) is the key player in the pathogenesis of chronic myeloid leukemia (CML)
As a continuation of our previous studies on long-term cell cultures [17], where we found that prolonged treatment with IM induced centrosomal and cytogenetic alterations in several bcr-ablnegative cell lines, we performed short-term cell culture experiments to assess the impact of therapeutic doses of IM on expression and proteolytic activity of Separase
Since the proteolytic activity of Separase is regulated in a tight cell cycle-dependent manner, treatment periods were chosen with respect to the respective cell doubling times so that less than two cell cycle rounds were completed under IM treatment (Table 1) and less than 15% of cells were apoptotic

Summary

Introduction

The BCR-ABL tyrosine kinase (TK) formed by the balanced translocation t(9;22)(q34;q11) is the key player in the pathogenesis of chronic myeloid leukemia (CML). Compromising multiple aspects of cellular behavior, including proliferation, apoptosis, cell to cell signaling and differentiation, the BCR-ABL oncoprotein triggers aberrant clonal hematopoiesis and drives disease progression from chronic phase (CP) toward the fully transformed phenotype of blast crisis (BC) [2]. Despite significant decreases in BCR-ABL mRNA levels in the bone marrow compartment under IM long-term therapy, persistance of residual CML clones with low BCR-ABL expression and insensitivity to IM treatment has been observed [3]. Clonal evolution denotes a heterogenous entity of clonal molecular changes in BCR-ABL-positive hematopoietic stem/progenitor cells and has been described in about 30% and 80% of patients in accelerated phase (AP) and BC, respectively [8]. Emergence of altered chromosome numbers, collectively termed aneuploidy, involve an additional derivative chromosome 22, chromosome 17 abnormalities, trisomy 8, and are associated with poor prognosis [9,10]

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