c-MYB is a transcriptional regulator of ESPL1/Separase in BCR-ABL-positive chronic myeloid leukemia

Background: Genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML). Recently, we have shown that clonal evolution and blast crisis correlate with altered expression and activity of Separase, a cysteine endopeptidase that is a mitotic key player in chromosomal segregation and centriole duplication. Hyperactivation of Separase in human hematopoietic cells has been linked to a feedback mechanism that posttranslationally stimulates Separase proteolytic activity after imatinib therapy-induced reduction of Separase protein levels. Methods and Results: In search for potential therapy-responsive transcriptional mechanisms we have investigated the role of the transcription factor c-MYB for Separase expression in CML cell lines (LAMA-84, K562, BV-173) and in clinical samples. Quantitative RT-PCR and Western blot immunostaining experiments revealed that c-MYB expression levels are decreased in an imatinib-dependent manner and positively correlate with Separase expression levels in cell lines and in clinical CML samples. RNA silencing of c-MYB expression in CML cell lines resulted in reduced Separase protein levels. Gelshift and ChIP assays confirmed that c-MYB binds to a putative c-MYB binding sequence located within the ESPL1 promoter. Conclusions: Our data suggest that ESPL1/Separase is a regulatory target of c-MYB. Therefore, c-MYB, known to be required for BCR-ABL-dependent transformation of hematopoietic progenitors and leukemogenesis, may also control the Separase-dependent fidelity of mitotic chromosomal segregation and centriole duplication essential for maintenance of genomic stability

Формирование экологической компетентности как показатель качества выпускника в техническом вузе

Объект исследования: процесс формирования экологической компетентности студентов технического вуза. Цель работы: теоретическое обоснование и создание модели образовательного процесса, направленного на формирование экологической компетентности специалиста технического профиля. Методы, которые использовались для выполнения поставленной цели: анализ литературы по теме, изучение и обобщение передового опыта, сравнительно - сопоставительный, анкетирование, моделирование.Object of research: process of formation of ecological competence of students of technical college. Work purpose: theoretical justification and creation of model of the educational process directed on formation of ecological competence of the expert of a technical profile. Methods which were used for performance of a goal: the analysis of literature on a subject, studying and generalization of the best practices, rather - comparative, questioning, modeling

Measurement of separase proteolytic activity in single living cells by a fluorogenic flow cytometry assay.

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

Increased separase activity and occurrence of centrosome aberrations concur with transformation of MDS.

ESPL1/separase, a cysteine endopeptidase, is a key player in centrosome duplication and mitotic sister chromatid separation. Aberrant expression and/or altered separase proteolytic activity are associated with centrosome amplification, aneuploidy, tumorigenesis and disease progression. Since centrosome alterations are a common and early detectable feature in patients with myelodysplastic syndrome (MDS) and cytogenetic aberrations play an important role in disease risk stratification, we examined separase activity on single cell level in 67 bone marrow samples obtained from patients with MDS, secondary acute myeloid leukemia (sAML), de novo acute myeloid leukemia (AML) and healthy controls by a flow cytometric separase activity assay. The separase activity distribution (SAD) value, a calculated measure for the occurrence of cells with prominent separase activity within the analyzed sample, was tested for correlation with the centrosome, karyotype and gene mutation status. We found higher SAD values in bone marrow cells of sAML patients than in corresponding cells of MDS patients. This concurred with an increased incidence of aberrant centrosome phenotypes in sAML vs. MDS samples. No correlation was found between SAD values and the karyotype/gene mutation status. During follow-up of four MDS patients we observed increasing SAD values after transformation to sAML, in two patients SAD values decreased during azacitidine therapy. Cell culture experiments employing MDS-L cells as an in vitro model of MDS revealed that treatment with rigosertib, a PLK1 inhibitor and therapeutic drug known to induce G2/M arrest, results in decreased SAD values. In conclusion, the appearance of cells with unusual high separase activity levels, as indicated by increased SAD values, concurs with the transformation of MDS to sAML and may reflect separase dysregulation potentially contributing to clonal evolution during MDS progression. Separase activity measurement may therefore be useful as a novel additional molecular marker for disease monitoring

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

Separase, a cysteine endopeptidase, is a key player in mitotic sister chromatid separation, replication fork dynamics, and DNA repair. Aberrant expression and/or altered separase proteolytic activity are associated with aneuploidy, tumorigenesis, and disease progression. Since genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML), we have comparatively examined separase proteolytic activity in TKI-treated chronic phase CML. Separase proteolytic activity was analyzed on single cell level in 88 clinical samples and in 14 healthy controls by a flow cytometric assay. In parallel, BCR-ABL1 gene expression and replication fork velocity were measured by qRT-PCR and DNA fiber assays, respectively. The separase activity distribution (SAD) value indicating the occurrence of MNCs with elevated separase proteolytic activity within samples was found to positively correlate with BCR-ABL1 gene expression levels and loss of MMR (relapse) throughout routine BCR-ABL1 monitoring. Analyses of CD3

Gene Expression Pattern of <i>ESPL1</i>, <i>PTTG1</i> and <i>PTTG1IP</i> Can Potentially Predict Response to TKI First-Line Treatment of Patients with Newly Diagnosed CML

The achievement of major molecular response (MMR, BCR::ABL1 ≤ 0.1% IS) within the first year of treatment with tyrosine kinase inhibitors (TKI) is a milestone in the therapeutic management of patients with newly diagnosed chronic myeloid leukemia (CML). We analyzed the predictive value of gene expression levels of ESPL1/Separase, PTTG1/Securin and PTTG1IP/Securin interacting protein for MMR achievement within 12 months. Relative expression levels (normalized to GUSB) of ESPL1, PTTG1 and PTTG1IP in white blood cells of patients (responders n = 46, non-responders n = 51) at the time of diagnosis were comparatively analyzed by qRT-PCR. 3D scatter plot analysis combined with a distance analysis performed with respect to a commonly calculated centroid center resulted in a trend to larger distances for non-responders compared to the responder cohort (p = 0.0187). Logistic regression and analysis of maximum likelihood estimates revealed a positive correlation of distance (cut-off) with non-achieving MMR within 12 months (p = 0.0388, odds ratio 1.479, 95%CI: 1.020 to 2.143). Thus, 10% of the tested non-responders (cut-off ≥ 5.9) could have been predicted already at the time of diagnosis. Future scoring of ESPL1, PTTG1 and PTTG1IP transcript levels may be a helpful tool in risk stratification of CML patients before initiation of TKI first = line treatment

From Watsons Bay, New South Wales, ca. 1880 [picture] /

Part of collection: Collection of photographs of New South Wales, ca. 1876-1897.; Title devised by cataloguer based on inscription on image.; "C. Bayliss Photo Sydney" -- photographer's blind stamp lower left.; Inscriptions: "P.S. 'Captain Cook' I"--In pen on verso.; Condition: Edge tear upper left.; Also available in an electronic version via the Internet at: http://nla.gov.au/nla.pic-vn4193075