Metaphase Cells Research Articles

Application of Interphase Fluorescent in Situ Hybridization: a Screening Tool for the Diagnosis of Microdeletion Syndrome.

Most laboratories adopt the results of metaphase fluorescent in situ hybridization (FISH) for the diagnosis of microdeletion syndromes. To investigate the discrepancy between the results of interphase and metaphase, we compared the quantitative results of FISH for 5 kinds of microdeletion syndrome and gender determination disorders (SDD). A total of 282 (135 for DiGeorge syndrome, 20 for Kalmann syndrome, 7 for Miller-Dieker syndrome, 38 for Prader Willi/Angelman syndrome, 62 for Williams syndrome, and 20 for SDD (SRY FISH)) were enrolled. For SRY FISH, we artificially mixed fresh blood of male and female with various ratios and then compared the results of metaphase and interphase SRY FISH. Using a bio-cell chip, we performed interphase FISH in 168 patients with microdeletion syndromes and compared the results with manual interphase. The concordance rate between the results of metaphase and interphase was 100% in microdeletion syndrome. In the disorders of gender development, SRY FISH showed 100% concordance between interphase and metaphase when we counted 50 metaphase cells and 100 interphase cells. Comparison with mixtures of male and female blood at various ratios also showed 100% concordance. The results of bio-cell chip showed 100% concordance between previous interphase FISH results. Considering the complete concordance between interphase and metaphase in microdeletion syndrome, the application of interphase FISH without performing metaphase FISH can be a screening test for microdeletion syndrome. Confirmation by metaphase FISH can be performed only in cases with abnormal results by interphase FISH.

ВИКОРИСТАННЯ ЕКСПЕРИМЕНТІВ З МІКРОЯДРАМИ ДЛЯ ВИЯВЛЕННЯ РИЗИКІВ РАКУ ЛЮДИНИ: КОРОТКИЙ ОГЛЯД

Introduction. Micronuclei (MN) are small extranuclear DNA-containing structures that are formed as a consequence of structural and numerical chromosomal aberrations. The advantage of MN experiments compared to conventional chromosomal analyses in metaphase cells is that the scoring is by far less time consuming and laborious. MN experiments are currently widely used for the routine screening of chemicals in vitro and in vivo but also for environmental control and human biomonitoring Objectives. The purpose of this review was to collect data on the use of MN experiments for the detection of increased cancer risks as a consequence of environmental, lifestyle and occupational exposures and the detection/diagnosis of different forms of cancer. Methods. Analysis of the literature on methods for MN experiments with humans; as well as the use of this technique in different areas of research. Results. To date, a wide range of protocols for human biomonitoring studies has been developed for the measurement of MN formation in peripheral blood cells and in epithelial from different organs (buccal and nasal cavity, cervix and bladder). In addition to MN, other nuclear anomalies can be scored which reflect genetic instability as well as acute toxicity and the division of target cells. Conclusions. The evidence is accumulating that MN can be used as a diagnostic tool for the detection of increased cancer risks as well as for the early diagnosis of cervical and bladder cancer

DIAPH1 facilitates paclitaxel-mediated cytotoxicity of ovarian cancer cells

The chemotherapeutic agent paclitaxel (PTX) selectively binds to and stabilizes microtubule (MTs). Also, the activated formin Diaphanous Related Formin 1 (DIAPH1) binds to MTs and increases its stability. In a recent study, we found that high DIAPH1 levels correlated with increased survival of ovarian cancer (Ovca) patients. A possible explanation for this finding is that Ovca cells with high DIAPH1 levels are more sensitive to PTX. To examine this assumption, in this study the effect of DIAPH1 depletion on PTX-mediated cytotoxicity of OVCAR8 and OAW42 cells was analyzed. Our data showed that down-regulation of DIAPH1 expression decreased PTX sensitivity in both cell lines by reducing apoptosis or necrosis. Analysis of MT stability by Western blotting revealed a decreased concentration of stable, detyrosinated MTs in PTX-treated DIAPH1 knock-down compared to control cells. Also, in fixed metaphase cells the level of stable, detyrosinated spindle MTs decreased in cells with reduced DIAPH1 expression. In vitro analysis with recombinant DIAPH1 protein showed that PTX and DIAPH1 exhibited additive effects on MT-polymerization, showing that also in a cell-free system DIAPH1 increased the effect of PTX on MT-stability. Together, our data strongly indicate that DIAPH1 increases the response of Ovca cells to PTX by enhancing PTX-mediated MT-stability.

A Cytogenetic and Morphological Study on Nannospalax ehrenbergi in Adana (Ceyhan) Province

This study was conducted to explain the karyological analysis and morphological characteristics of blind mole rat samples collected from Ceyhan, Adana in Turkey. The chromosomes of the blind mole rat samples were stained and analysed using conventional methods. The chromosomes were defined according to centromere positions by processing photographs of metaphase cells. The karyotype of Nannospalax ehrenbergi showed 53 chromosomes (2n=53) and fundamental number of chromosomal arms NF=66 and the number of autosomal arms NFa= 62. The karyotype showed a hybrid individual. X and Y chromosomes were determined as metacentric and acrocentric, respectively. The autosomal set had 5 (+1 single homologous) pairs of metacentric/submetacentric and 20 pairs of acrocentric. Morphological studies were carried out on two male Nannospalax ehrenbergi skulls from Adana province. Lengths of three external (height, hindfoot length and weights (gr)) and 24 cranial morphological points on skulls were measured using an electronic caliper. This was the first study in which 2n=53 NF=66 population was determined from Ceyhan. This form was described as a hybrid form. The portion behind the palate (os palatinum) has no a sharply defined styloid process.

Characterization of a recently synthesized microtubule-targeting compound that disrupts mitotic spindle poles in human cells

We reveal the effects of a new microtubule-destabilizing compound in human cells. C75 has a core thienoisoquinoline scaffold with several functional groups amenable to modification. Previously we found that sub micromolar concentrations of C75 caused cytotoxicity. We also found that C75 inhibited microtubule polymerization and competed with colchicine for tubulin-binding in vitro. However, here we found that the two compounds synergized suggesting differences in their mechanism of action. Indeed, live imaging revealed that C75 causes different spindle phenotypes compared to colchicine. Spindles remained bipolar and collapsed after colchicine treatment, while C75 caused bipolar spindles to become multipolar. Importantly, microtubules rapidly disappeared after C75-treatment, but then grew back unevenly and from multiple poles. The C75 spindle phenotype is reminiscent of phenotypes caused by depletion of ch-TOG, a microtubule polymerase, suggesting that C75 blocks microtubule polymerization in metaphase cells. C75 also caused an increase in the number of spindle poles in paclitaxel-treated cells, and combining low amounts of C75 and paclitaxel caused greater regression of multicellular tumour spheroids compared to each compound on their own. These findings warrant further exploration of C75’s anti-cancer potential.

Non-age-related neoplastic loss of sex chromosome correlated with prolonged survival in real-world CBF-AML patients.

In this real-world clinical study, in which we determined eligibility for allogenic hematopoietic stem cell transplantation by prognostic factors and minimal residual disease status, we retrospectively evaluated cytogenetic, genetic, and clinical features in 96 patients with core-binding factor acute myeloid leukemia (CBF-AML) including 62 patients with RUNX1/RUNX1T1 and 34 patients with CBFβ/MYH11. Multivariate analyses for 5-year overall survival (OS) in CBF-AML patients revealed that age of 50years or older (HR: 3.46, 95% CI 1.47-8.11, P = 0.004) and receiving 2 or more induction cycles (HR: 3.55, 95% CI 1.57-8.05, P = 0.002) were independently associated with worse OS and that loss of sex chromosome (LOS) was independently associated with better OS (HR: 0.09, 95% CI 0.01-0.71, P = 0.022). At the time of complete remission, all 21 karyotyped patients with LOS had a normal karyotype. Furthermore, in all 9 patients with LOS who had a mosaic of metaphase cells with and without t(8;21) or inv(16), the metaphase cells without t(8;21)/inv(16) showed a normal karyotype. These results proved that LOS was not age-related and physiological, but rather a neoplastic chromosomal abnormality.

R-Loop Associated Mitotic Stress Confers Vulnerabilities in Splicing Factor Mutant Leukemia

The RNA splicing factor SF3B1 is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL). Co-expression of mutant Sf3b1 and Atm deletionin murine B cellsresults in CLL development, in which CLL cells exhibit RNA splicing dysregulation, increased DNA damage and recurrent chromosomal amplification. These findings suggest a strong link between splicing factormutation, chromosomal instability (CIN), and oncogenesis, although the mechanism has yet to be elucidated.CIN in cells with defective RNA processing is triggered, at least in part, by excessive R-loop (RNA:DNA hybrid with displaced single strand DNA). Using dotblot assay, we observed R-loop augmentation in SF3B1 mutant (MT) compared to wild-type (WT) cells in both human cell lines and murine CLL cells. Along with R-loop accumulation, SF3B1 MT increased DNA double strand breaks (DSB) (measured by comet assay) and mis-segregated chromosomes during mitosis (quantified by immunofluorescence using anti-phospho-histone H3 and anti-αtubulin antibodies). Overexpression of R-loop resolving enzyme RNaseH1 in SF3B1 MT cell lines reduced DSB (t-test p-value <0.0001) and mitotic stress (t-test p-value <0.0001), confirming the causative role of R-loop in CIN.To elucidate the mechanisms underlying SF3B1 MT-associated CIN, we used DNA:RNA hybrid immunoprecipitation (DRIP) coupled with sequencing to map R-loop in SF3B1 MT and WT Nalm6 cells. Although SF3B1 MT caused minimal changes in overall R-loop distribution, it led to increased R-loop formation at centromeric regions. Proper centromeric R-loop (cen-R-loop) formation is essential for accurate chromosome segregation. Those R-loop are coated by phosphorylated replication protein A (p-RPA). p-RPA and centromere immunofluorescence (IF) co-staining revealed increased cen-R-loop in SF3B1 MT cells. Intriguingly, overexpression of RNaseH1 reduced both levels of centromeric p-RPA and percentage of cells with defective mitosis, suggesting SF3B1 MT associated cen-R-loop impacts CIN.To further discern the molecular underpinnings of R-loop mediated CIN, we investigated the effect of their accumulation on spindle geometry in metaphase cells. By analyzing the total body of chromosomes two-dimensional area, we observed one fold greater magnitude of chromosome oscillations in SF3B1 Nalm6 MT cells. Accordingly, compared to WT cells, SF3B1 MT cells displayed longer and wider spindles to accommodate an aneuploid chromosomal content. Removal of R-loops rescued mitotic spindle architecture and CIN in SF3B1 MT cells, highlighting a direct contribution of R-loop to kinetochore-microtubule organization and stability.As there is no evidence of strong binding of SF3B1 to centromeres, we tested the possibility that SF3B1 MT contributes to cen-R-loop formation through alternative splice variants. Through overlapping conserved SF3B1 MT-associated splice variants across isogenic cell lines and murine model with available R-loop interactome data, we identified SERBP1, which encodes an RNA-binding protein, as a target candidate . SF3B1 MT induces loss-of-function in SERBP1 through alternative splicing. Knockdown of SERBP1 triggered R-loop accumulation, increased centromeric p-RPA coating, and aberrant mitosis, which recapitulated the SF3B1 MT phenotype. Importantly, overexpression of SERBP1 WT alleviated both DSB and R-loop accumulation in SF3B1 MT cells. These observations strongly suggest that SF3B1 MT augments R-loop formation through dysregulated splicing of SERBP1 and possibly other genes involved in R-loop metabolism.We also investigated the possibility that the impact of RNA splicing defect on R-loop biology is a generalizable phenomenon. Examing a panel of leukemia-associated splicing factors (U2AF1, SRSF2, ZRSR2, RNU1), we found that these mutations all display R-loop dependent mitotic stress. Thus, mitotic stress caused by aberrant R-loop formation could be a convergent mechanism in splicing factor mutated leukemia cells.Altogether, we demonstrated that SF3B1 mutation promotes CIN by destabilizing mitotic spindles through aberrant R-loop accumulation at the centromere, via dysregulated splicing in genes such as SERBP1. Our study highlights an unrecognized role of cen-R-loop as a critical link between RNA splicing dysregulation and CIN, providing an opportunity for therapeutic targeting of R-loop in splicing factor mutant leukemias. DisclosuresWu: Pharmacyclics: Research Funding; BioNTech: Current equity holder in publicly-traded company.

Non-Age-Related Neoplastic Loss of Sex Chromosomecorrelated to Prolonged Survival in CBF-AML Patients in Real-World Clinical Setting

IntroductionSince Core-binding factor acute myeloid leukemia (CBF-AML) is categorized as a favorable cytogenetic risk group, allogenic hematopoietic stem cell transplantation (HSCT) is generally not recommended during the first complete remission (CR1). However, approximately 30-50% of CBF-AML patients relapse, and about 50% of relapsed patients remain incurable. Therefore, in real-world clinical setting, the indication of HSCT even in CR1 is considered based on prognosis prediction and minimal residual disease (MRD) status. With respect to prognosis prediction by chromosomal abnormalities, the prognostic impact of loss of sex chromosome (LOS) in the patients with CBF-AML is still controversial. Previous studies suggest that the impact of LOS in predicting the prognosis of CBF-AML may depend on treatment strategies including HSCT. In addition, although CBF-AML develops LOS at a high rate of 20-40%, it is still unclear whether LOS is age-related or neoplastic.MethodsHokkaido Leukemia Net (HLN) is prospective cohort study collecting AML samples from hospitals of North Japan Hematology Study Group (NJHSG).Cases of CBF-AML that were registered in HLN from January 2010 to December 2019 were used for analysis in the present study. All the patients received either intensive or low-intensity chemotherapy. HSCT was performed at the discretion of the attending physicians even in CR1. Conventional cytogenetic analysis was performed at the time of diagnosis and after each treatment. FLT3-ITD and KIT exon17 mutations were analyzed using genome DNA template at the central laboratory. The results of genetic analysis were returned to physicians so that they could reflect the genetic result in their treatment choice. We evaluated the impacts of several factors including advanced age, poor response to chemotherapy, LOS, and KIT exon17 mutation on overall survival (OS) and relapse-free survival (RFS) in these patients. This study was conducted in compliance with ethical principles based on the Declaration of Helsinki and was approved by the institutional review board of Hokkaido University Hospital.ResultsA total 96 CBF-AML patients including 62 patients with RUNX1/RUNX1T1 and 34 patients with CBFβ/MYH11 were enrolled in the present study. Intensive induction chemotherapy using '7+3' cytarabine plus anthracycline and low-intensity induction chemotherapy were performed in 90 patients and 6 patients, respectively. In 90 patients who underwent intensive induction chemotherapy, the CR rate after one cycle of induction therapy was 73.3%. Total CR rate as the best response was 91.7% for all CBF-AML patients. HSCT in CR1 was performed in 15 patients (15.6%) at the discretion of the attending physician based on genetic alterations, poor response to chemotherapy, and sustained detection of MRD after chemotherapy. There was no significant difference in 5-year OS (RUNX1/RUNX1T1 64.9% vs CBFβ/MYH11 52.9%; P=0.546) or 5-year RFS (63.6% vs 53.3%; P=0.364) between the CBF subtypes. LOS was significantly more frequent in patients with RUNX1/RUNX1T1 than in patients with CBFβ/MYH11 (35.5% vs 5.9%; P=0.001). Multivariate analyses for 5-year OS in CBF-AML patients revealed that age of 50 years or older (HR: 3.46, 95% CI: 1.47-8.11, P=0.004) and receiving 2 or more induction cycles (HR: 3.55, 95% CI: 1.57-8.05, P=0.002) were independently associated with worse OS and that LOS was independently associated with better OS (HR: 0.09, 95% CI: 0.01-0.71, P=0.022). We next sought to clarify whether LOS was age-related or neoplastic based on the transition of chromosome results. All the 24 patients with LOS achieved CR, and chromosome analysis at CR showed a normal karyotype in all of the 21 patients for whom chromosome analysis was performed. In the remaining 3 patients, only FISH analysis for RUNX1/RUNX1T1 or CBFβ/MYH11 was performed. Furthermore, chromosome analysis showed mosaics of metaphase cells with and without t(8;21) in 9 patients at diagnosis. In all of those patients, metaphase cells without t(8;21) showed a normal karyotype.ConclusionIn our real-world clinical setting in which '7+3' induction was performed and the indication of HSCT was determined by prognostic factors and MRD status, LOS correlated to prolonged survival in CBF-AML patients. Furthermore, the transition of chromosome results revealed that LOS was not age-related physiological loss but part of neoplastic chromosomal abnormalities. [Display omitted] DisclosuresTeshima: CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Janssen Pharmaceutical K.K.: Other; Novartis International AG: Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Gentium/Jazz Pharmaceuticals: Consultancy; Takeda Pharmaceutical Company: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin Co.,Ltd.: Honoraria, Research Funding; Astellas Pharma Inc.: Research Funding; Merck Sharp & Dohme: Membership on an entity's Board of Directors or advisory committees; TEIJIN PHARMA Limited: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Bristol Myers Squibb: Honoraria; Fuji pharma CO.,Ltd: Research Funding; Pfizer Inc.: Honoraria; Sanofi S.A.: Research Funding. Kondo: Astellas Pharma Inc.: Consultancy, Honoraria; Otsuka Pharmaceutical: Honoraria, Research Funding; Novartis Pharma KK: Honoraria; Bristol-Myers Squibb Company: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Sanwa Kagaku Kenkyusho CO.,LTD: Consultancy.

COMPARISON BETWEEN MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION (MLPA) AND CYTOGENETICS FOR VALIDATION OF CHROMOSOMAL ABERRATIONS IN CHRONIC LYMPHOCYTIC LEUKEMIA

Recently, a multiplex ligation-dependent probe amplification (MLPA®) has emerged as a method that is reported to be fast, sensitive, and cost-effective that can detect copy number variation. In the present study, MLPA analysis was compared with chromosome banding analysis (CBA) data to determine the efficiency of MLPA in patients with chronic lymphocytic leukemia (CLL). CBA karyotyped 65 patients. Chromosomal analysis was performed on G-banded metaphase cells from cultured lymphocytes in a culture medium at 37°C for 72 hours. Numerical, as well as structural abnormalities were reported on at least 20 well-spread and well-banded metaphases. Genomic DNA was extracted from peripheral blood samples and subjected to MLPA through the probemix, kit SALSA®MLPA®P037 containing 54 MLPA probes, including 41 probes for 2p, 6q 8p/q, 9p21, 11q, 12p/q, 13q, and 17p chromosomal regions. The PCR products were separated by capillary electrophoresis using an ABI 3500 DNA analyzer. Cytogenetics resulted in 41 normal karyotypes, and 25 karyotype aberrations (13 tris12; 3 del13; 2 del11; 4 del17; others 3). Genetic abnormalities observed by MLPA were 45 (14 tris12; 26 del13q; 2 del11q; 3 TP53 deletion), and 22 were normal by MLPA. Of the 41 normal cytogenetics, MLPA was concordant in 20 patients and discordant in 21 patients. Of those 21, MLPA identified 19 del13q, 1 delq11, and 1 tris12. Regarding abnormalities, cytogenetics and MLPA were concordant in 12 tris12, 2 del13, 3 del17, and 1 del11. Statistical difference was seen when comparing both methodologies in favor of MLPA ( p= 0.003; chi-square test). In this study, we aimed to determine the usefulness of the MLPA probemix P037-B1 as a routine for detecting clinically relevant chromosome abnormalities in CLL in laboratories without cytogenetic routine. MLPA identified more abnormalities than cytogenetic (44%). We observed that MLPA was consistent with the cytogenetic results in 20 of 41 normal patients (48%). The main discordant results identified by MLPA were 13q deletion, the most common cytogenetic change in chronic lymphoblastic leukemia (CLL). In one patient, results of the MLPA did not show deletion in the TP53 gene, while isochromosome 17 was observed by cytogenetic. A low proportion of malignant cells with the relevant abnormality could be responsible for the discrepancies in the MLPA technique. Besides that pitfall, our findings highlight the advantage of MLPA compared with conventional cytogenetics. In conclusion, MLPA is a reliable high-throughput technique to detect CNVs and a cost-effective one that can be included in routine diagnostic protocols in laboratories that lack conventional cytogenetics.

Genomic anatomy of male-specific microchromosomes in a gynogenetic fish.

Unisexual taxa are commonly considered short-lived as the absence of meiotic recombination is supposed to accumulate deleterious mutations and hinder the creation of genetic diversity. However, the gynogenetic gibel carp (Carassius gibelio) with high genetic diversity and wide ecological distribution has outlived its predicted extinction time of a strict unisexual reproduction population. Unlike other unisexual vertebrates, males associated with supernumerary microchromosomes have been observed in gibel carp, which provides a unique system to explore the rationales underlying male occurrence in unisexual lineage and evolution of unisexual reproduction. Here, we identified a massively expanded satellite DNA cluster on microchromosomes of hexaploid gibel carp via comparing with the ancestral tetraploid crucian carp (Carassius auratus). Based on the satellite cluster, we developed a method for single chromosomal fluorescence microdissection and isolated three male-specific microchromosomes in a male metaphase cell. Genomic anatomy revealed that these male-specific microchromosomes contained homologous sequences of autosomes and abundant repetitive elements. Significantly, several potential male-specific genes with transcriptional activity were identified, among which four and five genes displayed male-specific and male-biased expression in gonads, respectively, during the developmental period of sex determination. Therefore, the male-specific microchromosomes resembling common features of sex chromosomes may be the main driving force for male occurrence in gynogenetic gibel carp, which sheds new light on the evolution of unisexual reproduction.

Highly Efficient Microcell-Mediated Transfer of HACs Containing a Genomic Region of Interest into Mammalian Cells.

Human artificial chromosomes (HACs) are considered promising tools for gene delivery, functional analyses, and gene therapy. HACs have the potential to overcome many of the problems caused by the use of viral-based gene transfer systems, such as limited cloning capacity, lack of copy number control, and insertional mutagenesis during integration into host chromosomes. The recently developed alphoidtetO -HAC has an advantage over other HAC vectors because it can be easily eliminated from dividing cells by inactivation of its conditional kinetochore. This provides a unique control mechanism to study phenotypes induced by a gene or genes carried on the HAC. The alphoidtetO -HAC has a single gene acceptor loxP site that allows insertion of an individual gene of interest or a cluster of genes of up to several Mb in size in Chinese hamster ovary (CHO) hybrid cells. The HACs carrying chromosomal copies of genes can then be transferred from these donor CHO cells to different recipient cells of interest via microcell-mediated chromosome transfer (MMCT). Here, we describe a detailed protocol for loading a gene of interest into the alphoidtetO -HAC vector and for the subsequent transfer of the HAC to recipient cells using an improved MMCT protocol. The original MMCT protocol includes treatment of donor cells with colcemid to induce micronucleation, wherein the HAC becomes surrounded with a nuclear membrane. That step is followed by disarrangement of the actin cytoskeleton using cytochalasin B to help induce microcell formation. The updated MMCT protocol, described here, features the replacement of colcemid and cytochalasin B with TN16 + griseofulvin and latrunculin B, respectively, and the use of collagen/laminin surface coating to promote attachment of metaphase cells to plates during micronuclei induction. These modifications increase the efficiency of HAC transfer to recipient cells ten fold. The improved MMCT protocol has been successfully tested on several recipient cell lines, including human mesenchymal stem cells and mouse embryonic stem cells. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Insertion of a BAC containing a gene of interest into a single loxP loading site of alphoidtetO -HAC in hamster CHO cells Basic Protocol 2: Microcell-mediated chromosome transfer from donor hamster CHO cells to mammalian cells.

The Anaphase Promoting Complex/Cyclosome: A New Promising Target in Mantle Cell Lymphoma and Diffuse Large B Cell Lymphoma

Despite current therapy improvements for mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL), most MCL patients and ± 40% of the DLBCL patients experience relapses. Both MCL and DLBCL are aggressive B cell lymphomas, characterized with a higher proliferation rate. The anaphase promoting complex/cyclosome (APC/C) is an important checkpoint in mitosis, regulating the onset of the anaphase and cell cycle. This E3 ubiquitin ligase becomes activated upon interaction with the co-factor CDC20. CDC20 is frequently overexpressed in different human cancers (prostate cancer, breast cancer, multiple myeloma, ...). This overexpression is associated with an aggressive course of the disease and a shorter overall survival. So far little is known about CDC20 in B cell lymphomas. Here, we investigated the inhibition of APC/C-CDC20 in MCL and DLBCL.Using publicly available gene expression profiling data, we found that the majority of the DLBCL patients (>77%) exhibits a high CDC20 mRNA expression and that this overexpression is correlated with a worse prognosis, indicating that CDC20 is also involved in DLBCL. Next, we evaluated the anti-lymphoma activity of the APC/C-CDC20 inhibitor proTAME (pT) using human MCL (Jeko-1, Mino and Rec-1) and DLBCL (OCI-Ly1, OCI-Ly7, SU-DHL-6 and OCI-Ly3) cell lines. The pT treatment significantly increased the percentage of cells in metaphase. This metaphase arrest was accompanied by a strong reduction in MCL and DLBCL viability and a significantly increase in apoptosis. The induced apoptosis was found mediated by phosphorylation of the anti-apoptotic proteins Bcl-2/Bcl-XL and degradation/cleavage of the anti-apoptotic protein Mcl-1. Finally, APC/C-CDC20 inhibition significantly enhanced the sensitivity of MCL and DLBCL cells to the BH3 mimetic ABT-199, as evidenced by a strong increase in caspase 3-dependent apoptosis compared to single agents.In conclusion, we identified APC/C-CDC20 as a promising new target in the treatment of the aggressive B cell lymphomas MCL and DLBCL. In the future, the potential of specific CDC20 targeting, either alone or in combinations with currently available anti-cancer therapies, will continue to be investigated. DisclosuresNo relevant conflicts of interest to declare.

RSK Inhibition Induces Metaphase Arrest and Apoptosis in Acute Myeloid Leukemia Cells

The 90 kDa Ribosomal S6 Kinase (RSK) drives cell proliferation and survival in several cancers, although its oncogenic mechanism has not been well characterized. To examine the role of RSK in acute myeloid leukemia (AML), we analyzed apoptosis, DNA-damage, and the cell cycle profile following treatment with BI-D1870, a potent inhibitor of RSK. BI-D1870 treatment (5 µM, 12 h) of HL-60 and KG-1 cells increased the G2/M population and induced apoptosis. Previously, RSK has been shown to activate CDC2 for G2/M progression by dephosphorylating the negative regulatory Tyr14 through activation of CDC25 or inhibition of MYT1. Phosphorylation of CDC2 at Tyr14 was increased transiently during G2 phase following treatment with BI-D1870, while phosphorylation of the positive regulatory Ser198 of CDC25C was inhibited in G2 and mitotic populations. However, BI-D1870 treatment did not decrease CDC2/Cyclin B kinase activity, suggesting that inhibition of CDC2 is not a critical regulatory mechanism in BI-D1870-induced mitotic arrest. The mitotic population was further characterized by evaluation of expression levels of Cyclin A and Cyclin B with flow cytometry. Approximately 70% of mitotic cells accumulated in Cyclin A-negative/Cyclin B-high metaphase at 12 hours after 5 µM BI-D1870 treatment. Metaphase arrest was confirmed using a thymidine/nocodazole block to achieve synchronization at metaphase. Our results show that the metaphase/anaphase transition is significantly stalled by RSK inhibition as assessed by degradation of Cyclin B and Securin following release from mitotic arrest (Metaphase cells in mitotic population (%), control vs. BI-D1870, 0 h post-release: 80.53 ± 0.09% vs. 88.03 ± 0.67%; 2 h post-release: 56.27 ± 1.42% vs. 83.37 ± 0.32%; 4 h post-release: 23.53 ± 0.52% vs. 62.90 ± 0.62%, mean ± SEM, n=3, p< 0.01). We also assessed the efficacy of combination treatment of BI-D1870 with standard AML chemotherapeutic drugs cytarabine and daunorubicin. The combination of BI-D1870 with cytarabine or daunorubicin in HL-60 cells showed an additive effect. Furthermore, BI-D1870 induced apoptosis and metaphase arrest in primary AML cells and significantly suppressed the CD34+ subpopulation containing leukemia stem cells. Mitotic progression is controlled by the sequential degradation of cell cycle regulators including Cyclin A, Cyclin B, and Securin by the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. Since proper association of CDC20 with APC/C is critical to destroy Cyclin B and Securin for metaphase/anaphase progression, we examined the effect of BI-D1870 on the complex formation of CDC20 with APC/C. Co-IP experiments showed that treatment of BI-D1870 impeded the association of activator CDC20 with APC/C, but increased binding of inhibitor MAD2 to APC/C, preventing metaphase/anaphase progression. These data show that BI-D1870 inhibits metaphase/anaphase progression by inactivating APC/C, resulting in apoptosis of AML cells. We are currently studying the effects of RSK knockdown on AML cells. In summary, our study demonstrates that RSK plays an important role in maintaining AML cell survival and proliferation, and RSK inhibitors could be used therapeutically to treat AML by inducing apoptosis and cell cycle arrest through the inhibition of APC/C in AML cells. DisclosuresNo relevant conflicts of interest to declare.

Transplacental transfer of congenital B‐cell acute lymphoblastic leukemia to the maternal vasculature

To the Editor: Monozygotic twin infants with KMT2A-rearranged (KMT2Ar) acute lymphoblastic leukemia (ALL) demonstrate near-uniform disease concordance due to prenatal transfer of pre-leukemic cells via placental vascular anastomoses.1-4 However, despite several historical reports recognizing the presence of fetal nucleated cells in the maternal circulation during pregnancy,5 fetal–maternal dissemination of leukemic clones has not been reported. We present a neonate with congenital KMT2Ar-ALL with documentation of metastasis to the maternal decidual placental vessels without clinical consequence. A newborn infant born at 37 4/7 weeks gestation was found to have leukocytosis (white blood cells 72.1 × 103/μl) with 62% blasts characterized by convoluted nuclear contour, prominent nucleoli, and scant deep-blue cytoplasm. Flow cytometry revealed expression of HLA-DR, CD15, CD19, CD22, CD34, cytoplasmic TdT, and cCD79a, consistent with B-cell ALL. G-banding karyotyping analysis revealed t(4;11)(q21;q23) with a dominant clone (five metaphase cells) containing a (4;11)(q21;q23) translocation and a subclone (one cell) containing an additional copy of der(4). Placental microscopy demonstrated clusters of blasts in both chorionic surface (fetal) vessels and decidual (maternal) vessels (Figure 1). Fluorescence in situ hybridization (FISH) analysis showed these clusters to be positive for the KMT2A rearrangement in both fetal and maternal vessels. These findings indicate that malignant fetal cells had infiltrated the maternal placental vasculature. Unlike in older children and adolescents, the prognosis of ALL in the neonatal age group is dismal.6 Despite intensive therapy, our patient experienced a highly complicated clinical course with eventual decompensation from Enterococcus faecalis bacteremia and infectious rotavirus leading to death on day 47 of life. Notably, throughout the patient's nearly 7-week clinical course, his mother did not demonstrate any clinically apparent findings to suggest sequelae of transplacental leukemia cell transfer. The discovery of leukemia cells of shared clonal lineage between monozygotic twins with ALL supports the so-called “intraplacental metastasis hypothesis”1 and confirms the capacity for pre-leukemic cells to spread from one twin to the other.7 These pre-leukemic cells are thought to transfuse via monochorionic placental vascular anastomoses rather than across the placental membrane barrier itself. Although placental involvement of congenital neuroblastoma has been reported,8 numerous reports of pregnant patients with hematologic malignancies demonstrate a noteworthy absence of clinically apparent transplacental “metastases” to exposed neonates.9-12 For example, in a report of prenatally diagnosed maternal ALL with leukemia cells detected in the cord blood, these cells were efficiently cleared and did not result in leukemic engraftment in the neonate.13 In this report, numerous lymphoblasts characterized by the KMT2A rearrangement were observed in the decidual (maternal) placental blood vessels, suggesting substantial cross-placental infiltration or local proliferation following infiltration. Our findings suggest the capacity for pre-leukemic cells to circumvent the placental membrane barrier as an additional means of transplacental penetration. The absence of a clinical impact on maternal health suggests that infiltrated malignant fetal cells undergo extraplacental maternal immune clearance and do not result in clinical consequence to the exposed mother. The authors declare that there is no conflict of interest.

Cytogenetic analysis of an amniotic sample with X chromosome abnormality signaled by non-invasive prenatal testing

To determine the chromosomal karyotype of a fetus with copy number variation (CNV) of the X chromosome signaled by non-invasive prenatal testing (NIPT). NIPT was performed on the peripheral blood sample taken from the pregnant women. Amniotic fluid and cord blood samples were subjected to conventional G banded karyotyping, and were further analyzed by high-throughput sequencing for chromosome microdeletion/microduplication. The results were then verified by fluorescence in situ hybridization (FISH) on metaphase cells. The NIPT test of pregnant women suggested low risk for 21-trisomy, 18-trisomy, and 13-trisomy, whilst indicated the number of chromosome X to be low. The G banded karyotype of the amniotic fluid and cord blood cells was 46,XX. The result of high-throughput sequencing chromosome microdeletion/microduplication detection was seq[hg19](X)× 1, (Y)× 2. FISH showed a clear red signal at each end of a whole chromosome, and a green signal on the other chromosome, with a karyotype of 46,X,ish idic(Y) (q11.23) (SRY++, DXZ1+). C banding showed that there is a dense and a slightly loose centromere at both ends of the Y chromosome, and the parachromatin region was missing. The karyotype of amniotic fluid and cord blood cells was finally determined to be 46,X, pus idic(Y) (q11.23). For chromosome anomalies suggested by auxiliary report of NIPT, conventional karyotyping combined with high-throughput sequencing for chromosome microdeletion/microduplication should be adopted for the prevention and reduction of the rate of chromosome microdeletion/microduplication syndromes.

Polymorphism of nucleolar organizer regions in different Ukrainian cattle breeds

Aim. To study the activity of nucleolar organizer regions (NORs) in different Ukrainian cattle breeds in terms of their apparent activity status in silver stain and possible relation with milk productivity. Methods. Chromosome prepara- tions using lymphocytes from the peripheral blood of 90 cows of different breeds were used in the study. NOR activity was determined by visual evaluation of concentrations of silver precipitation on NORs in individual chromosomes. A 50 % silver nitrate solution was used to stain chromosome preparations. NORs were detected as dark spots on telomeres of the corresponding chromosomes. Results. The cytological analysis of chromosome preparations from lymphocytes of first lactation cows detected NOR polymorphism in Ukrainian Red-and-Motley dairy cattle (URM), Ukrainian Black-and-White dairy breed (UBW), and hybrid cows, obtained by crossing Ukrainian Red-and-Motley dairy breed and Montbeliarde bulls (URM × M). First lactation cows of URM and UBM had higher or the same inci- dence of cells with four (29.8 and 30 %) and five (17.1 and 19.5 %) NORs, while in URM × M cows the incidence of cells with the same number of NORs was almost twice lower; cells with 7 and 8 NORs exceeded a similar index for other investigated breeds almost twice (2.5 against 4.5 % and 2.0 against 4.2 %). The highest level of chromosomal aberrations (CA) was observed in the group of animals with medium number (2 to 3 NORs per cell), and the lowest – in the group with a high number of NORs (from 6 to 7) with a reliable intergroup difference (p &lt; 0.01). NOR activity was the highest in the group of animals of local origin (URM × M) with a milk yield over 7,000 kg in 305 days of the first lactation and the lowest in the UBW cows with a milk yield of 4–5,000 kg during the first lactation. Conclusions. We determined the differences in the activity of nucleolar organizers between the investigated groups of cows of dairy breed. URM × M hybrids reliably (р ≤ 0.05) exceeded dairy UBW cows by this index. No statistically significant different was found between other investigated groups of animals by this trait. Higher dairy productivity was found in the animals with higher frequency of NORs in the chromosomes of metaphase cells. In our opinion, the number of active NORs demonstrates relative variability between their number and the rate of protein synthesis, required to implement the productivity traits of the investigated animals.

Chromosomal anomalies of the fetus of pregnant women from settlements prone to technogenic pollution

The article presents the analysis results of the spectrum and frequency of distribution of the fetus chromosomal set of 590 pregnant women living in the city of Aktobe and various districts of the Aktobe region in Kazakhstan. Biological material was obtained by biopsy of the chorion and placenta, amniocentesis and cordocentesis. For the preparation of chromosome formations, the conventional “direct” method and the classical method of cell cultivation were used. Metaphase cells were stained with GTG and FISH methods. In total, 64 (10.9%) cases of fetal karyotype abnormalities were detected. Of these, genomic abnormalities accounted for 59 (10.0%), and structural – 5 (8.5%). Among the genomic abnormalities in 39 (66.1%) cases, the fetus had a Down syndrome karyotype. The frequency of chromosomal abnormalities in the fetus of pregnant women in Aktobe, where industrial facilities of ferrous and nonferrous metallurgy are located, was 7.9%. The frequency of fetal chromosomal abnormalities in 3 districts of Aktobe region (fetal karyotypes of 39 pregnant women were studied) without large industrial facilities was 12.8%, and with the presence of large industrial complexes in 5 districts (fetal karyotypes of 131 pregnant women were studied) – 19,8%, which is 1.5 times more. Thus, it can be assumed that technogenic pollution by large industrial complexes (mining, cement, chemical, oil and gas processing industries) of places of residence contributes to the appearance of genomic and structural disorders of the genetic apparatus of human cells. Key words: prenatal diagnosis, fetal karyotype, chromosomal abnormalities, technogenic factor. &nbsp;

A Cytogenetic Study on Spermophilus Xanthoprymnus (Bennett, 1835) in Isparta Province

This study was conducted to explain the karyological characteristics of S. xanthoprymnus from Gencali in Turkey. The chromosomes of the specimens were traditionally stained and examined according to standard procedures. The chromosomes were determined according to centromere positions by examining the photographs of metaphase cells. The karyotype of S. xanthoprymnus contained 42 chromosomes (2n=42) and fundamental number of chromosomal arms NF=81 and the number of autosomal arms NFa=78. The karyotype includes four metacentric chromosomes ranging from large to small, three pairs of large and medium-sized submetacentric autosome, twelve ranging from large to medium size subtelocentric, and a pair of acrocentric autosomes. The X chromosome was determined as large subtelocentric and Y chromosome as small acrocentric. Due to there is no karyological information and geographical distribution of existentence cytotypes in Turkey, revelation of chromosomal structure in Anatolian ground squirrel populations will provide properly assessment of taxonomy of those rodents in further studies.

Intercomparison of Conventional and QuickScan Dicentric Scoring for the Validation of Individual Biodosimetry Analysis in Taiwan

Purpose The dicentric chromosome assay (DCA), the gold standard for radiation biodosimetry, evaluates an individual absorbed radiation dose by the analysis of DNA damage in human lymphocytes. The conventional (C-DCA) and QuickScan (QS-DCA) scoring methods are sensitive for estimating radiation dose. The Biodosimetry Laboratory at Institute of Nuclear Energy Research (INER), Taiwan, participated in intercomparison exercises conducted by Health Canada (HC) in 2014, 2015 and 2018 to validate the laboratory’s accuracy and performance. Material and Methods Blood samples for the conventional dose response curve for Taiwan were irradiated with 0, 0.25, 0.5, 1, 2, 3, 4 and 5 Gy. Ten blind blood samples were provided by HC. Either or both of two methods of conventional (C) or QuickScan (QS) scoring could be chosen for the HC’s intercomparison. For C-DCA triage scoring, only cells with 46 centromeres were counted and each scorer recorded the number of dicentrics in the first 50 metaphases or stopped scoring when 30 dicentrics were reached. Scorers also recorded how much time it took to analyze 10, 20, and 50 cells. Subsequently, the data were entered into the Dose Estimate software (DoseEstimate_v5.1) and dose estimates were calculated. With QS-DCA scoring, a minimum of 50 metaphase cells (or 30 dicentrics) were scored in apparently complete metaphases without verification of exactly 46 centromeres. Results For the blinded blood samples irradiated at HC and shipped to INER, the mean absolute deviation (MAD) derived after scoring 50 cells for C-DCA and QS-DCA was <0.5 Gy for all three intercomparisons, meeting the criteria for acceptance. Conclusion The results indicated that the Biodosimetry Laboratory at INER can provide reliable dose estimates in the case of a large-scale radiation accident.

ТРЁХЦВЕТНЫЙ FISH-МЕТОД: КРИВЫЕ ДОЗА-ЭФФЕКТ ДЛЯ ТРАНСЛОКАЦИЙ В КУЛЬТУРАХ ЛИМФОЦИТОВ ПЕРИФЕРИЧЕСКОЙ КРОВИ ПОСЛЕ ГАММА-ОБЛУЧЕНИЯ IN VITRO

Purpose: Plotting dose-effect curves for translocations identified using the tricolor FISH method based on the results of cytogenetic analysis of cultures of peripheral blood lymphocytes of healthy donors after in vitro gamma irradiation. &#x0D; Material and methods: Venous blood was obtained from three donors (2 men and 1 woman aged from 28 to 41 years) and subjected to in vitro gamma irradiation from a 60Co source at doses of 0.10; 0.15; 0.25; 0.35; 0.50; 0.75; 1.00; 1.50; 2.00 and 3.00 Gy at 37 ° C (dose rate 0.5 Gy / min). For tricolor FISH staining, two different sets of DNA probes were used for chromosome pairs 1, 4, 12 and 2, 3, 8. Metaphases with a quasi-diploid number of chromosomes (40-46) and a complete set of all FISH-stained chromosomes, taking into account their total length, were selected for analysis. Differentiation of stable and unstable cells was also carried out. In the cytogenetic analysis, traditional terminology was used with the designation of translocations as reciprocal (complete, two-sided), non-reciprocal (terminal, incomplete, or unilateral), or interstitial.&#x0D; Results: The obtained numerical data were used to statistically compare the frequencies of FISH-recorded translocations when using different sets of DNA probes, when calculating of chromosome aberrations were in all (unstable and stable) and stable metaphase cells, when comparing of the frequencies of FISH-recorded translocations and dicentrics, and assessing of the contribution of the level of translocations between FISH-stained chromosome pairs in the total translocation frequency. The plotted dose-effect curves generally corresponded to the linear-quadratic form.&#x0D; Conclusion: Dose dependences obtained for translocations using two different selected tricolor sets of DNA probes did not differ statistically significantly. At the same time, cytogenetic analysis of only stable metaphase cells revealed a tendency to register lower levels of translocations than when analyzing all cells (unstable and stable ), at the highest doses of 2 and 3 Gy. The levels of dicentrics formed with the participation of FISH-stained chromosomes were significantly lower than the number of observed translocations. The quantitative contribution of translocations between FISH-stained pairs of chromosomes turned out to be very low, which clearly does not contribute to an increase in the sensitivity of the FISH method of retrospective dose estimation as compared to its one-color version. At the same time, the three-color FISH-staining makes it possible to identify such variants of chromosomal rearrangements that are not recorded using the one-color FISH method.