Abstract
ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML). Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110)-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110) as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90–180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic activity in leukemic cell lines and peripheral blood samples from leukemia patients.
Highlights
Introduction[4] The intratumor heterogeneity is a major obstacle for efficient diagnosis, prognosis and therapy of human malignancies as tumor subclones with distinct aneuploidies feature an extreme phenotypic plasticity and can evolve depending on the selective pressure of the cancer-specific environment
[4] The intratumor heterogeneity is a major obstacle for efficient diagnosis, prognosis and therapy of human malignancies as tumor subclones with distinct aneuploidies feature an extreme phenotypic plasticity and can evolve depending on the selective pressure of the cancer-specific environment. [5,6,7,8,9] One of the major paths to aneuploidy is chromosomal missegregation caused by multipolar mitotic spindle formation due to supernumerary centrosomes (= centrosome amplification). [10,11,12] Centrosome amplification, in particular, the accumulation of additional centrosomes (n>2), is frequently detected in solid and hematological human cancers and has already been found in pre-neoplastic lesions i.e. early stages of carcinogenesis. [13,14,15]
[30] As shown in Fig 1 the acetyl group was replaced by the fluorophore Cy5 (Exmax = 635 nm, Emmax = 650 nm) resulting in the final dual fluorophore substrate [Cy5-D-R-E-I-M-R]2-rhodamine 110 (Rh110) where two Cy5-conjugated peptides were linked to one Rh110 molecule
Summary
[4] The intratumor heterogeneity is a major obstacle for efficient diagnosis, prognosis and therapy of human malignancies as tumor subclones with distinct aneuploidies feature an extreme phenotypic plasticity and can evolve depending on the selective pressure of the cancer-specific environment. A cysteine endopeptidase, is a key player in chromosomal segregation during mitosis. It performs proteolytic cleavage of the cohesin protein Rad during the metaphase to anaphase transition. Once activated prior anaphase onset, Separase cleaves first the centrosomal pool of cohesin thereby promoting centriole disengagement that is the licensing step for centriole duplication in the S phase. Multiple inhibitory mechanisms combining Securin binding, specific serine residue phosphorylation (pSer1126) by CyclinB1/Cdk, autocatalytic cleavage, and PP2A-dependent stabilization of Separase-bound Securin work together to prevent unscheduled activation of intracellular Separase. [20,21,22]
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