ArticlePloS one2015
Measurement of separase proteolytic activity in single living cells by a fluorogenic flow cytometry assay.
Article in PloS one, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
The trial behind it
Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
8 citing papers in PubMed, 12 citations in OpenAlex.
- Separase activity distribution can be a marker of major molecular response and proliferation of CD34Annals of hematology · 2020Trial
- Synthesis of Rhodamines and Rosamines Using 3,6-Difluoroxanthone as a Common Intermediate.The Journal of organic chemistry · 2021Article
- Structure and Function of the Separase-Securin Complex.Sub-cellular biochemistry · 2021Review
- NudC-like protein 2 restrains centriole amplification by stabilizing HERC2.Cell death & disease · 2019Article
- Cell penetrating peptides in preclinical and clinical cancer diagnosis and therapy.Oncotarget · 2018Review
- Increased separase activity and occurrence of centrosome aberrations concur with transformation of MDS.PloS one · 2018Article
- Kinase-templated abiotic reaction.Chemical science · 2017Article
- Serum ESPL1 Can Be Used as a Biomarker for Patients With Hepatitis B Virus-Related Liver Cancer: A Chinese Case-Control Study.Technology in cancer research & treatmentArticle
Corrections and comments
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Authors and funding
6 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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