scholarly journals Carcinogen treatment leads to mitotic defects and arrest in cancer and noncancer cells

2018 ◽  
Author(s):  
Rebecca G Hartling ◽  
Christian J Pacheco ◽  
Emily A Bystrak ◽  
Nicholas J Quintyne
Keyword(s):  
Estrogen Receptor ◽  
Mitotic Index ◽  
Cell Lines ◽  
Mitotic Spindle ◽  
Chromosomal Instability ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Chromosomal Rearrangements ◽  
Anaphase Bridges ◽  
Multipolar Spindles

Genomic instability can manifest due to both chromosomal rearrangements and gain and loss of entire chromosomes. One mechanism by which a carcinogen acts is by increasing the rate of mitotic spindle defects during proliferation. These defects can lead to chromosomal instability that manifest as lagging chromosomes, anaphase bridges, or multipolar spindles. While several mechanisms exist to rectify these errors prior to completion of mitosis, some cells will escape repair, while others will prematurely exit mitosis. Here we examine the effects of two carcinogenic molecules: Fulvestrant, a chemotherapeutic that functions as a selective estrogen receptor degrader, and vinyl chloride, a hydrocarbon used to produce PVC. We exposed two cancer lines, A549 and UPCI:SCC103, and one noncancer line, GM03349, to increasing concentrations of the carcinogen for increasing durations, up to 48 hours exposure. We found that exposure to the carcinogen lowered the mitotic index in the cancer cell lines, while raising it in the noncancer line. Concurrently, we observed massive increases in the frequency of mitotic defects, with the most significant increases seen in prevalence of lagging chromosomes in prometaphase and metaphase and anaphase bridges. Live cell imaging showed that the occurrence of either of these defects had the strongest correlation with the likelihood that the cell would fail to complete mitosis. We also show that washing out the carcinogen decreases the frequency of mitotic defects in all three cell lines, but the mitotic index does not recover in the cancer cells. These findings demonstrate that carcinogen-induced mitotic defects have marked effects on the proliferative population of cells in terms of potential for contributing to chromosomal instability or removal from that population.

scholarly journals Carcinogen treatment leads to mitotic defects and arrest in cancer and noncancer cells

2018 ◽  
Author(s):  
Rebecca G Hartling ◽  
Christian J Pacheco ◽  
Emily A Bystrak ◽  
Nicholas J Quintyne
Keyword(s):  
Estrogen Receptor ◽  
Mitotic Index ◽  
Cell Lines ◽  
Mitotic Spindle ◽  
Chromosomal Instability ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Chromosomal Rearrangements ◽  
Anaphase Bridges ◽  
Multipolar Spindles

Genomic instability can manifest due to both chromosomal rearrangements and gain and loss of entire chromosomes. One mechanism by which a carcinogen acts is by increasing the rate of mitotic spindle defects during proliferation. These defects can lead to chromosomal instability that manifest as lagging chromosomes, anaphase bridges, or multipolar spindles. While several mechanisms exist to rectify these errors prior to completion of mitosis, some cells will escape repair, while others will prematurely exit mitosis. Here we examine the effects of two carcinogenic molecules: Fulvestrant, a chemotherapeutic that functions as a selective estrogen receptor degrader, and vinyl chloride, a hydrocarbon used to produce PVC. We exposed two cancer lines, A549 and UPCI:SCC103, and one noncancer line, GM03349, to increasing concentrations of the carcinogen for increasing durations, up to 48 hours exposure. We found that exposure to the carcinogen lowered the mitotic index in the cancer cell lines, while raising it in the noncancer line. Concurrently, we observed massive increases in the frequency of mitotic defects, with the most significant increases seen in prevalence of lagging chromosomes in prometaphase and metaphase and anaphase bridges. Live cell imaging showed that the occurrence of either of these defects had the strongest correlation with the likelihood that the cell would fail to complete mitosis. We also show that washing out the carcinogen decreases the frequency of mitotic defects in all three cell lines, but the mitotic index does not recover in the cancer cells. These findings demonstrate that carcinogen-induced mitotic defects have marked effects on the proliferative population of cells in terms of potential for contributing to chromosomal instability or removal from that population.

The Spindle Checkpoint Protein BubR1 Is Differentially Expressed in the Lymphatic and Myeloid Lineage and Is a Determinator of Response to Spindle Poisons

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1407-1407
Author(s):  
Dominik Schnerch ◽  
Marie Follo ◽  
Andrea Schmidts ◽  
Julika Krohs ◽  
Julia Felthaus ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mitotic Spindle ◽  
Cancer Biology ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cpg Island ◽  
Mitotic Checkpoint ◽  
Live Cell ◽  
Cyclin B ◽  
Upstream Region

Abstract Abstract 1407 Introduction: Spindle poisons disrupt the mitotic spindle thus leading to activation of the spindle assembly checkpoint (SAC). The SAC is a mitotic surveillance mechanism that can interfere with anaphase-promoting complex/cyclosome- (APC/C-) dependent proteolysis of key cell cycle regulators, such as securin and cyclin B, to delay cells at the metaphase to anaphase transition. The SAC protein BubR1 is a tumor suppressor with critical functions during mitosis and has also been shown to be important for the stablilization of cyclin B during interphase. We found this critical player to be downregulated in AML cell lines, primary AML blast populations and myeloblast-like murine 32D cells when compared to ALL cell lines and lymphoblast-like murine BaF3 cells. While myeloblast-like 32D cells are untransformed cells but also exhibit BubR1 repression we speculate that there might be a physiological role for a reduced BubR1 expression level in healthy myelopoiesis. However, in highly proliferative AML cells BubR1 repression might be a source of genetic instability due to a less efficient SAC-mediated interference with APC/C-dependent proteolysis in the presence of inaccuracies during mitosis. Methods and results: Since repression of BubR1 is known to shorten the metaphase duration, we performed live-cell imaging of leukemia cells and found myeloblastic Kasumi-1 cells, expressing histone H2-GFP, to proceed faster through mitosis as compared to lymphoblastic DG-75 cells. While DG-75 cells exhibited a stable metaphase arrest upon spindle disruption using the spindle poison nocodazole, Kasumi-1 cells showed only a transient arrest, degraded securin and cyclin B and underwent sister-chromatid separation in the absence of a functional mitotic spindle. These findings suggest that the mitotic checkpoint is unable to properly interfere with APC/C-dependent proteolysis to prevent mitotic progression in myeloblastic leukemia upon treatment with spindle poisons. By using inducible retroviral reexpression of BubR1 and its downstream effector cyclin B we could enhance the ability of Kasumi-1 cells to accumulate in mitosis upon spindle disruption. Moreover, restoration of BubR1 led to higher cyclin B levels. Live-cell imaging analyses of Kasumi-1 cells, which expressed doxycyclin-inducible BubR1, revealed a prolonged metaphase, suggesting a more stringent control by the mitotic checkpoint when BubR1 expression is restored. Prolonged metaphase delays were also detected after reexpression of BubR1 when we challenged the cells with lower doses of spindle poisons suggesting that BubR1 is an important sensitizer for antimitotic therapies. Therefore, our finding of low BubR1 expression in AML provides an explanation for the poor response of myeloid leukemia to spindle poisons as compared to lymphoblastic leukemia. BubR1 has also been reported to be inactivated through promoter hypermethylation in various malignancies. The existence of a CpG island in the upstream region of the Bub1b locus (BubR1 coding sequence) tempted us to treat Kasumi-1 cells with the demethylating agent decitabine. Promotor demethylation led to an upregulation of BubR1 in mitotic AML cells providing evidence that BubR1 is a druggable target to enhance mitotic surveillance in AML cells. Conclusions: Because mitotic therapies are widely used in the treatment of different malignancies, a further understanding of these processes might lead to a better understanding of cancer biology and improved therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

Analysis of Mitosis In Acute Myeloid Leukemia Using Live-Cell Imaging.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3363-3363
Author(s):  
Dominik Schnerch ◽  
Julia Felthaus ◽  
Lara Mentlein ◽  
Monika Engelhardt ◽  
Ralph M. Waesch
Keyword(s):  
Single Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Genetic Instability ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cyclin B ◽  
Single Cell Level ◽  
Cell Level ◽  
Mitotic Control

Abstract Abstract 3363 Proper mitotic control is a prerequisite to guarantee the equal distribution of the genetic material onto the two developing daughter cells. A mitotic key regulator is cyclin B. High levels of cyclin B facilitate entry into mitosis whereas its controlled degradation coordinates chromosome separation and cytokinesis. The latter events are coordinated by the anaphase- promoting complex / cyclosome (APC/C), a ubiquitin ligase that couples ubiquitin chains to cyclin B, mediating its proteasomal degradation. The regulation of the APC/C-activity by complex protein networks, such as the spindle assembly checkpoint, therefore presents the basis for an accurate mitosis. Mitotic errors give rise to daughter cells with an aberrant set of chromosomes and contribute to genetic instability. Genetic instability is a hallmark of cancer cells and plays an important role in the onset and progression of acute myeloid leukemia (AML). In rare cases, de novo AMLs present with multiple cytogenetic aberrations (complex karyotype). However, a larger number of patients develop karyotype deviations in the course of the disease, sometimes even under therapy, which comes along with an adverse prognosis. Understanding the biology that drives the gain and loss of genetic material therefore bears the potential of identifying new therapeutic targets. We compared a number of lymphoblastic and myeloid cell lines and found AML cell lines to be deficient in arresting at metaphase in the presence of the microtubule-disrupting agent nocodazole. Cyclin B was expressed at much lower levels in the AML cell line Kasumi-1 and did not accumulate following spindle disruption as observed in the lymphoblastic cell line DG-75. We could show that Kasumi-1 cells, when challenged with nocodazole, were not capable of properly maintaining chromatid-cohesion and underwent premature sister chromatid separation. These findings suggest that mitotic control mechanisms do not work tightly enough in AML cells to prevent chromosome separation in the presence of spindle disruption. We applied live-cell imaging to exactly characterize mitotic timing in Kasumi-1 cells at a single cell level. The expression of a GFP-tagged derivative of histone H2 served to visualize the nuclear envelope breakdown and anaphase onset. Detection of the latter events allowed the faithful measurement of mitotic timing. We could find a significant shortening of mitosis in Kasumi-1 cells as compared to the lymphoblastic cell line DG-75. In both AML cell lines and primary AML blasts we identified the spindle assembly checkpoint components BubR1 and Bub1 to be downregulated. Interestingly, re-expression of BubR1 in Kasumi-1 cells led to a significant stabilization of cyclin B on western blots. To address the question whether an increased expression of cyclin B leads to a more pronounced mitotic delay in the presence of spindle-disruption in AML cells is subject of current experiments. It was reported that different cell types can escape from a mitotic block as a consequence of cyclin B degradation. In the literature, this phenomenon was referred to as mitotic slippage and is known to drive genetic instability. To monitor cyclin B turnover and localization at a single cell level, we generated a chimeric cyclin B-molecule, SNAP-cyclin B, which can couple to a suitable fluorochrome in a self-labeling reaction after addition to the growth medium. In this system, the fluorescence intensity reflects the amount of chimeric cyclin B and allows the monitoring of APC/C-dependent proteolysis. In our current approaches we aim at studying cyclin B-turnover at a single cell level in AML cell lines as well as primary leukemia cells by using live-cell imaging before and after BubR1- and Bub1-rescue. An aberrant cell cycle control is found in most human malignancies and might be an important driving force in leukemogenesis. We hypothesize that BubR1, in concert with different other regulators, might lead to inaccuracies in mitotic control. This hypothesis is underlined by the shortened time to anaphase in Kasumi-1 cells and a decreased expression of cyclin B, both of which are characteristics of BubR1-depletion. Mitotic regulators are already targets in AML therapy and a deeper understanding of mitotic processes in AML might lead to improved approaches. Disclosures: No relevant conflicts of interest to declare.

O-154 First mitotic spindle formation led by sperm centrosome-dependent microtubule organising centres may cause high incidence of zygotic division errors in humans

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Y Kai ◽  
H Kawano ◽  
N Yamashita
Keyword(s):  
Mitotic Spindle ◽  
High Frequency ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Immunofluorescence Assay ◽  
Live Cell ◽  
Microtubule Assembly ◽  
Cell Stage ◽  
Spindle Formation

Abstract Study question Why do multinucleated blastomeres appear at high frequency in two-cell-stage embryos in humans? Summary answer Failure in microtubule assembly during the first mitotic spindle body formation by sperm centrosome-dependent microtubule organising centres (MTOCs) may lead to chromosomal instability. What is known already Unlike that in mice, multinucleated blastomeres appear at high frequency in two-cell-stage embryos in humans. However, the underlying mechanism remains elusive. In mice, multiple acentriolar MTOCs appear around the male and female pronuclei after pronuclear disappearance and contribute to dual-spindle formation, engulfing each parental chromosome. This spindle formation may ensure an error-free division, keeping the chromosomes stable during the first cleavage, as observed in mice, but it is unclear whether a similar mechanism exists in humans. Study design, size, duration To examine how sperm centrosomes contribute to MTOC formation in humans, two types of 3PN zygotes derived fromeither conventional in vitro fertilization (c-IVF, n = 30) or intracytoplasmic sperm injection (ICSI, n = 10) were used. The zygotes were collected from October 2018 to January 2020. MTOC and mitotic spindle formation at consecutive stages of development during the first cleavage were analysed under static and dynamic conditions using immunofluorescence assay and fluorescent live-cell imaging. Participants/materials, setting, methods Under ethics approval, 3PN zygotes were donated by infertile couples undergoing c-IVF or ICSI cycles at the Yamashita Shonan Yume Clinic in Japan. All participants provided informed consent. Immunofluorescence assay was performed using antibodies against α-tubulin, pericentrin, and H3K9me3 after fixation with MTSB-XF solution. Fluorescent live-cell imaging was performed using TagGFP2-H2B mRNA (chromosome marker) and FusionRed-MAP4 mRNA (microtubule marker). Main results and the role of chance Immunofluorescence revealed that while 3PN zygotes derived from c-IVF showed four pericentrin dots, those derived from ICSI exhibited two pericentrin dots. In pro-metaphase, an independent group of chromosomes derived from each pronucleus and MTOCs were formed by the sperm centrosome at the core. Microtubules from each MTOC extended toward the chromosomes in the early metaphase; a quadrupolar spindle was formed in the c-IVF-derived zygotes, and a bipolar spindle was formed in the ICSI-derived zygotes by the MTOCs at the zygote apex after chromosome alignment. In pro-metaphase, the microtubules extended from the MTOCs to the nearest chromosome. Since microtubule assembly was found on oocyte-derived chromosomes, we hypothesised that whether a chromosome is surrounded by microtubules depends on the location of the MTOCs, irrespective of its origin. Live-cell imaging of histone H2B and MAP4 revealed that four MTOCs appeared around the three pronuclei just before the disappearance of the pronuclear membrane; microtubules then extended from the MTOCs toward the chromosomes, beginning to form a mitotic spindle as the chromosomes moved to the centre of the oocyte. Interestingly, one of the three assembled chromosome groups showed no microtubule assembly in the pro-metaphase. Similar results were obtained in all six 3PN zygotes subjected. Limitations, reasons for caution We demonstrated the high risk of developing bare chromosomes not surrounded by microtubules during the formation of the first mitotic spindle, using human tripronuclear zygotes. However, owing to unavailability of normal fertilized oocytes for this study because of the clinical use, we were unable to confirm this in normal zygotes. Wider implications of the findings Although two sperm centrosome-dependent MTOCs are expected to be formed in normal fertilized oocytes, these MTOCs are not sufficient to completely enclose physically separated female and male chromosomes with the microtubules. This explains the high frequency of zygotic division errors that lead to unstable human chromosomes. Trial registration number not applicable

Time-lapse Live Cell Imaging and Flow Analysis of Multidrug Resistance Reversal by Verapamil in Bladder Cancer Cell Lines

Urology ◽  
2009 ◽  
Vol 74 (2) ◽  
pp. 378-384 ◽  
Author(s):  
Jonathan M. Featherstone ◽  
Bashir A. Lwaleed ◽  
Alan G. Speers ◽  
Matthew C. Hayes ◽  
Brian R. Birch ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Multidrug Resistance ◽  
Cell Lines ◽  
Cancer Cell ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Flow Analysis ◽  
Time Lapse ◽  
Bladder Cancer Cell ◽  
Resistance Reversal

scholarly journals Kinesin-5 regulates the growth of the axon by acting as a brake on its microtubule array

2007 ◽  
Vol 178 (6) ◽  
pp. 1081-1091 ◽  
Author(s):  
Kenneth A. Myers ◽  
Peter W. Baas
Keyword(s):  
Growth Rates ◽  
Mitotic Spindle ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Motor Protein ◽  
Live Cell ◽  
Cultured Neurons ◽  
Wild Type ◽  
Microtubule Array ◽  
Inverse Effect

Kinesin-5 is a homotetrameric motor protein that interacts with adjacent microtubules in the mitotic spindle. Kinesin-5 is also highly expressed in developing postmitotic neurons. Axons of cultured neurons experimentally depleted of kinesin-5 grow up to five times longer than controls and display more branches. The faster growth rates are accompanied by a doubling of the frequency of transport of short microtubules, suggesting a major role for kinesin-5 in the balance of motor-driven forces on the axonal microtubule array. Live-cell imaging reveals that the effects on axonal length of kinesin-5 depletion are caused partly by a lower propensity of the axon and newly forming branches to undergo bouts of retraction. Overexpression of wild-type kinesin-5, but not a rigor mutant of kinesin-5, has the inverse effect on axonal length. These results indicate that kinesin-5 imposes restrictions on the growth of the axon and does so at least in part by generating forces on the axonal microtubule array.

scholarly journals Label-free live cell imaging by Confocal Raman Microscopy identifies CHO host and producer cell lines

2016 ◽  
Vol 12 (1) ◽  
pp. 1600037 ◽  
Author(s):  
Batirtze Prats Mateu ◽  
Eva Harreither ◽  
Markus Schosserer ◽  
Verena Puxbaum ◽  
Elisabeth Gludovacz ◽  
...  
Keyword(s):  
Cell Lines ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Raman Microscopy ◽  
Live Cell ◽  
Confocal Raman Microscopy ◽  
Label Free ◽  
Producer Cell ◽  
Confocal Raman

scholarly journals C. elegans chromosomes connect to centrosomes by anchoring into the spindle network

2016 ◽  
Author(s):  
Stefanie Redemann ◽  
Johannes Baumgart ◽  
Norbert Lindow ◽  
Sebastian Fürthauer ◽  
Ehssan Nazockdast ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Mitotic Spindle ◽  
Live Cell Imaging ◽  
Large Scale ◽  
Cell Imaging ◽  
Electron Tomography ◽  
Mitotic Spindles ◽  
C Elegans ◽  
Microtubule Growth ◽  
Segregation Of Chromosomes

AbstractThe mitotic spindle ensures the faithful segregation of chromosomes. To discover the nature of the crucial centrosome-to-chromosome connection during mitosis, we combined the first large-scale serial electron tomography of whole mitotic spindles in early C. elegans embryos with live-cell imaging. Using tomography, we reconstructed the positions of all microtubules in 3D, and identified their plus- and minus-ends. We classified them as kinetochore (KMTs), spindle (SMTs), or astral microtubules (AMTs) according to their positions, and quantified distinct properties of each class. While our light microscopy and mutant studies show that microtubules are nucleated from the centrosomes, we find only a few KMTs are directly connected to the centrosomes. Indeed, by quantitatively analysing several models of microtubule growth, we conclude that minus-ends of KMTs have selectively detached and depolymerized from the centrosome. In toto, our results show that the connection between centrosomes and chromosomes is mediated by an anchoring into the entire spindle network and that any direct connections through KMTs are few and likely very transient.

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