scholarly journals The cyclin Cln1 controls polyploid titan cell formation following a stress-induced G2 arrest in Cryptococcus

2021 ◽  
Author(s):  
Sophie Altamirano ◽  
Zhongming Li ◽  
Man Shun Fu ◽  
Minna Ding ◽  
Sophie R Fulton ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Formation ◽  
Genomic Diversity ◽  
G2 Arrest ◽  
Pathogenic Yeast ◽  
Cell Cycles ◽  
Unique Cell ◽  
Key Aspects

The pathogenic yeast Cryptococcus neoformans produces polyploid titan cells in response to the host lung environment that are critical for host adaptation and subsequent disease. We analyzed the in vivo and in vitro cell cycles to identify key aspects of the C. neoformans cell cycle that are important for the formation of titan cells. We identified unbudded 2C cells, referred to as a G2 arrest, produced both in vivo and in vitro in response to various stresses. Deletion of the non-essential cyclin Cln1 resulted in over-production of titan cells in vivo, and transient morphology defects upon release from stationary phase in vivo. Using a copper-repressible promoter PCTR4-CLN1 strain and a two-step in vitro titan cell formation assay, our in vitro studies revealed Cln1 functions after the G2 arrest. These studies highlight unique cell cycle alterations in C. neoformans that ultimately promote genomic diversity and virulence in this important fungal pathogen.

scholarly journals Excess histone H3 is a Chk1 inhibitor that controls embryonic cell cycle progression

2020 ◽  
Author(s):  
Yuki Shindo ◽  
Amanda A. Amodeo
Keyword(s):  
Cell Cycle ◽  
Cell Fate ◽  
Cell Cycle Progression ◽  
Histone H3 ◽  
Embryonic Cell ◽  
Cycle Progression ◽  
Cell Cycles ◽  
Embryonic Cell Cycle

AbstractThe early embryos of many species undergo a switch from rapid, reductive cleavage divisions to slower, cell fate-specific division patterns at the Mid-Blastula Transition (MBT). The maternally loaded histone pool is used to measure the increasing ratio of nuclei to cytoplasm (N/C ratio) to control MBT onset, but the molecular mechanism of how histones regulate the cell cycle has remained elusive. Here, we show that excess histone H3 inhibits the DNA damage checkpoint kinase Chk1 to promote cell cycle progression in the Drosophila embryo. We find that excess H3-tail that cannot be incorporated into chromatin is sufficient to shorten the embryonic cell cycle and reduce the activity of Chk1 in vitro and in vivo. Removal of the Chk1 phosphosite in H3 abolishes its ability to regulate the cell cycle. Mathematical modeling quantitatively supports a mechanism where changes in H3 nuclear concentrations over the final cell cycles leading up to the MBT regulate Chk1-dependent cell cycle slowing. We provide a novel mechanism for Chk1 regulation by H3, which is crucial for proper cell cycle remodeling during early embryogenesis.

scholarly journals The p21 dependent G2 arrest of the cell cycle in epithelial tubular cells links to the early stage of renal fibrosis

2019 ◽  
Author(s):  
Takayuki Koyano ◽  
Masumi Namba ◽  
Tomoe Kobayashi ◽  
Kyomi Nakakuni ◽  
Daisuke Nakano ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Renal Fibrosis ◽  
Kinase Inhibitor ◽  
Early Stage ◽  
G2 Arrest ◽  
Tubular Cells ◽  
Initial Stage ◽  
Deficient Mice

AbstractRenal fibrosis is accompanied with the progression of chronic kidney disease (CKD). Despite a number of past and ongoing studies, our understanding of the underlying mechanisms remains elusive. Here we explored the progression of renal fibrosis by using a mouse model, unilateral ureter obstruction (UUO). We found that in the initial stage of the progression where extracellular matrix did not deposit yet, the proximal tubular cells arrested at the G2 of the cell cycle. This G2 arrest was induced prior to activation of both DNA damage checkpoint and Wnt/β-Catenin pathway. Further analyses in vivo and in vitro indicated the cyclin dependent kinase inhibitor p21 is involved in the G2 arrest after the damage. The newly produced monoclonal antibody against p21 revealed that the p21 levels were sharply upregulated in response to the damage during the initial stage, but dropped down toward the later stage. To examine the function of p21 in the progression of renal fibrosis, we constructed the novel p21 deficient mice by i-GONAD. Compared with wild-type mice, p21 deficient mice showed the exacerbation of the fibrosis. Thus we propose that during the initial stage of the fibrosis following the renal damage, tubular cells arrest in the G2 phase depending on p21, thereby safeguarding the kidney functions.

scholarly journals The cell cycle of glial cells grown in vitro: an immunocytochemical method of analysis.

1992 ◽  
Vol 40 (9) ◽  
pp. 1405-1411 ◽  
Author(s):  
P E Knapp
Keyword(s):  
Cell Cycle ◽  
Glial Cells ◽  
Average Length ◽  
Proliferating Cells ◽  
Cell Cycles ◽  
Mouse Cerebral Cortex ◽  
Immunocytochemical Method ◽  
Autoradiographic Technique

Studies of cell cycles have traditionally employed [3H]- and [14C]-thymidine to label the DNA of proliferating cells and autoradiography to reveal the thymidine label. The development of antibodies to the thymidine analogue 5-bromodeoxyuridine (BrdU) has allowed the development of an immunocytochemical method analogous to the thymidine autoradiographic technique. In direct comparisons, we found that the immunocytochemical method consistently detected a larger number of proliferating cells. This suggests that it may be a more sensitive index of proliferation than thymidine autoradiography in some systems. We used the BrdU method to analyze the cycle of astroglia cultured from neonatal mouse cerebral cortex. Cells were exposed to BrdU for 1 hr to label a discrete subpopulation of proliferating cells. At 2-36 hr after the pulse, a combination of anti-BrdU immunocytochemistry and counterstaining with propidium iodide was used to identify proliferating cells. The length of the cell cycle was determined by charting the percent of BrdU-labeled mitotic cells vs time after the pulse. We found the average length of the cell cycle of astrocytes grown in vitro to be 20.5 hr. The combined G2 + M phases were 2-3 hr. These values are virtually identical with those found for glial cells in vivo, suggesting that the culture environment does not interfere with the normal control of cell cycle length.

scholarly journals Candida albicans Cyclin Clb4 Carries S-Phase Cyclin Activity

2010 ◽  
Vol 9 (9) ◽  
pp. 1311-1319 ◽  
Author(s):  
Ayala Ofir ◽  
Daniel Kornitzer
Keyword(s):  
Cell Cycle ◽  
Candida Albicans ◽  
Cell Cycle Progression ◽  
Sequence Similarity ◽  
Eukaryotic Cell ◽  
S Phase ◽  
Pathogenic Yeast ◽  
Content Type

ABSTRACT Cyclin-dependent kinases (CDKs) are key regulators of eukaryotic cell cycle progression. The cyclin subunit activates the CDK and also imparts to the complex, at least in some cases, substrate specificity. Saccharomyces cerevisiae, an organism in which the roles of individual cyclins are best studied, contains nine cyclins (three G1 cyclins and six B-type cyclins) capable of activating the main cell cycle CDK, Cdc28. Analysis of the genome of the pathogenic yeast Candida albicans revealed only two sequences corresponding to B-type cyclins, C. albicans Clb2 (CaClb2) and CaClb4. Notably, no homolog of the S. cerevisiae S-phase-specific cyclins, Clb5/Clb6, could be detected. Here, we performed an in vitro analysis of the activity of CaClb2 and CaClb4 and of three G1 cyclins, as well as an analysis of the phenotype of S. cerevisiae cells expressing CaClb2 or CaClb4 instead of Clb5. Remarkably, replacement of CLB5 by CaCLB4 caused rapid diploidization of S. cerevisiae. In addition, both in vivo and in vitro analyses indicate that, in spite of the higher sequence similarity of CaClb2 to Clb5/Clb6, CaClb4 is the functional homolog of Clb5/Clb6. The activity of a CaClb2/CaClb4 cyclin hybrid suggests that the cyclin box domain of CaClb4 carries the functional specificity of the protein. These results have implications for our understanding of the evolution of specificity of the cell cycle cyclins.

Mechanisms specifying area fate in cortex include cell-cycle-dependent decisions and the capacity of progenitors to express phenotype memory

Development ◽  
1997 ◽  
Vol 124 (8) ◽  
pp. 1623-1630 ◽  
Author(s):  
K.L. Eagleson ◽  
L. Lillien ◽  
A.V. Chan ◽  
P. Levitt
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Cell Cycles ◽  
Complete Cell ◽  
Pass Through ◽  
Cycle Dependent ◽  
Functional Areas ◽  
Inductive Signals

Progenitor cells in the early developing cerebral cortex produce neurons destined for discrete functional areas in response to specific inductive signals. Using lineage analysis, we show that cortical progenitor cells at different fetal ages retain the memory of an area-specific inductive signal received in vivo, even though they may pass through as many as two cell cycles in the absence of the signal in culture. When exposed to inductive signals in vitro, only those progenitors that progress through at least one complete cell cycle alter their areal phenotype. Our findings suggest that induction of an areal phenotype is linealy inherited, with the phenotype specified prior to the final cell cycle.

Involvement of P-TEFb/CDK9 in Cooperative Transcriptional Activation of Megakaryocytic Genes by GATA-1 and RUNX1 and in Programming of Megakaryocytic Development In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1227-1227
Author(s):  
Kamaleldin E. Elagib ◽  
Ivailo S. Mihaylov ◽  
Lorrie L. Delehanty ◽  
Grant C. Bullock ◽  
Kevin D. Ouma ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
Program Analysis ◽  
Dominant Negative ◽  
In Vivo Model ◽  
Megakaryocytic Differentiation ◽  
Human Cd34 ◽  
Unique Cell

Abstract Programming of megakaryocytic differentiation requires precise coordination of multiple signal transduction and transcription pathways. Previous in vivo and in vitro studies have implicated RUNX1 and GATA-1 as transcription factors that collaborate in the execution of this program. Analysis of the mechanism for the synergy of these two factors revealed induction of RUNX1 hyperphosphorylation by GATA-1 coexpression. A pharmacologic screen identified roscovitine as an inhibitor of the transcriptional cooperation, implicating a cyclin-dependent kinase (Cdk). A screen employing a panel of dominant-negative Cdk mutants identified Cdk9 as a critical component of the GATA-1-RUNX1 cooperation. In addition, HEXIM1, an endogenous Cdk9 inhibitor, similarly blocked transcriptional synergy. Furthermore, two kinase inhibitory compounds, DRB and flavopiridol, also blocked GATA-1-RUNX1 cooperation at concentrations specific for Cdk9 inhibition. Regarding the mechanism for GATA-1 induction of RUNX1 phosphorylation, coimmunoprecipitation experiments showed GATA-1 binding to both Cdk9 and cyclinT1. To examine the role of P-TEFb in primary megakaryocytic differentiation, human CD34+ cells in megakaryocytic cultures underwent treatment with 50 nM flavopiridol, a dose selective for Cdk9 inhibition. This treatment blocked megakaryocytic polyploidization while having no effect on the cell cycle properties of the non-megakaryocytic cells in the cultures. The treatment also impaired upregulation of CD41. Extending these findings to an in vivo model system, mice underwent treatment with daily low dose flavopiridol (5–7 mg/kg/day), a regimen previously shown to have no toxicity. Wild type C57BL/6 (wt BL/6) mice were compared with the ΔneoΔHS strain (GATA-1Lo) which has diminished GATA-1 expression in megakaryocytes. After only 1 week of treatment, the GATA-1Lo mice developed worsening thrombocytopenia associated with new-onset anemia, with several dying after 2 weeks of treatment. Flow cytometry on marrow from the treated GATA-1Lo mice revealed a marked expansion of abnormal megakaryocytes showing coexpression of CD71 plus CD41 and loss of polyploidization. Marrow and spleen histology showed extensive replacement by immature-appearing megakaryocytes with hypolobulated nuclei, as well as frequent pyknotic megakaryocytes. The control mice, flavopiridol treated wt BL/6 and saline treated GATA-1Lo, displayed none of these abnormalities. Additional experiments determined the flavopiridol effect on the GATA-1Lo mice to be completely reversible, with normalization of all parameters 2 weeks after ending treatment. In aggregate, these data implicate P-TEFb recruitment by GATA-1 in mediating cooperative activation of megakaryocytic promoters with RUNX1. This pathway may depend in part on the direct phosphorylation of RUNX1 by Cdk9. In mice, a synthetic lethal relationship between megakaryocytic GATA-1 deficiency and Cdk9 inhibition exists, manifesting as a fulminant but reversible megakaryocytic proliferative disorder reminiscent of the Down syndrome-associated megakaryocyte proliferations. A model is proposed in which P-TEFb, as a component of GATA-1-RUNX1 transcriptional complexes, plays an integral role in the specific programming of megakaryocytic differentiation, with particular importance in the unique cell cycle changes associated with this lineage.

350 COMPARATIVE TIME-LAPSE STUDIES ON PORCINE EMBRYOS FERTILIZED IN VITRO WITH FLOW CYTOMETRICALLY SEX SORTED SPERMATOZOA

2006 ◽  
Vol 18 (2) ◽  
pp. 282
Author(s):  
D. Rath ◽  
S. Schulze
Keyword(s):  
Cell Cycle ◽  
In Vitro Fertilization ◽  
Time Lapse ◽  
Culture Period ◽  
Similar Data ◽  
Vitro Fertilization ◽  
Cell Cycles ◽  
Cell Numbers

The objective of the present time-lapse studies was to compare developmental characteristics of porcine embryos after in vitro fertilization with flow cytometrically sexed spermatozoa during an in vitro culture period. Immature oocytes were matured (n = 851) and fertilized in vitro using 50 spermatozoa of either sex per oocyte. Additionally, in vivo-matured oocytes (n = 700) were derived from hormonally stimulated (eCG/hCG) prepuberal gilts, which were slaughtered 38 h after hCG treatment. Potential zygotes were introduced into the time-lapse system (50 µL-microdrops, NCSU-23) 18 h after the onset of in vitro fertilization. The onset and duration of cell cycles and blastomere rotation as well as collapses and re-expansion of cytoplasm were investigated. At the end of the culture period (168 h), embryos were labeled with Hoechst 33342 to identify the number of cell nuclei. The time-lapse system consisted of an incubation chamber installed on an inverted phase-contrast microscope and gassed with 5% CO2 in maximally humidified air. Computer controlled positioning of the chamber allowed the capture and storage of digital images of individual embryos every 30 min over a 7-day period. Converted time values were tested by ANOVA or ANOVA on ranks. In total, 700 in vivo-matured oocytes were fertilized in vitro (Y-sperm: 342; X-sperm: 358). Cleavage rates were 45.6% for male and 40.2% for female embryos. Out of these, 45.5% developed to male and 62.5% to female blastocysts, respectively. The onset and duration of cell cycles differed significantly at the 2-cell and morula stages (P < 0.01). The onset and number of rotations as well as collapses and re-expansion of cytoplasm were not different. Mean cell numbers of blastocysts were equal for both sexes (male: 35.2; female: 38.8). In parallel, 851 in vitro-matured oocytes were fertilized in vitro (Y-sperm: 431; X-sperm: 422). Cleavage rates were 45.5% for male and 49.3% for female embryos. Out of these, 54.1% developed to male and 56.7% to female blastocysts, respectively. The onset and duration of cell cycles and rotations as well as collapses were not significantly different between sexes. Mean cell numbers of blastocysts were equal for both sexes (male: 29.5; female: 27.7). Comparison between male embryos derived from in vivo (VV) or in vitro (VT)-matured oocytes showed a delay of the onset of the second cell cycle for VV (P < 0.001) and at the blastocyst stage for VT (P < 0.001). Accordingly, in these stages the duration of the cell cycle was shortened (P < 0.001). The onset and duration of rotations as well as collapses and re-expansion of cytoplasm were not different. Similar data were obtained for the female embryos. The data suggest slight sex related differences (onset of cell cycles), but these might be masked in embryos produced from in vitro-matured oocytes due to their higher individual variability. The experiment also shows the advantages of a time-lapse system to identify dynamic cell processes. It might also be a useful tool to precisely correlate cell cycle events to activation of certain marker genes.

Probing the ultrastructure of the mitotic and meiotic spindle in eggs: An expeditious approach based on semi-thin (0.25 μm) serial sections

1983 ◽  
Vol 41 ◽  
pp. 552-555
Author(s):  
Conly L. Rieder ◽  
S. Bowser ◽  
R. Nowogrodzki ◽  
K. Ross ◽  
G. Sluder
Keyword(s):  
Small Sample Size ◽  
Small Sample ◽  
Meiotic Spindle ◽  
Serial Sections ◽  
Mitotic Apparatus ◽  
Thin Sections ◽  
Cell Cycles ◽  
Ultrastructural Studies

Eggs have long been a favorite material for studying the mechanism of karyokinesis in-vivo and in-vitro. They can be obtained in great numbers and, when fertilized, divide synchronously over many cell cycles. However, they are not considered to be a practical system for ultrastructural studies on the mitotic apparatus (MA) for several reasons, the most obvious of which is that sectioning them is a formidable task: over 1000 ultra-thin sections need to be cut from a single 80-100 μm diameter egg and of these sections only a small percentage will contain the area or structure of interest. Thus it is difficult and time consuming to obtain reliable ultrastructural data concerning the MA of eggs; and when it is obtained it is necessarily based on a small sample size.We have recently developed a procedure which will facilitate many studies concerned with the ultrastructure of the MA in eggs. It is based on the availability of biological HVEM's and on the observation that 0.25 μm thick serial sections can be screened at high resolution for content (after mounting on slot grids and staining with uranyl and lead) by phase contrast light microscopy (LM; Figs 1-2).

A Novel Camptothecin Derivative 3j Inhibits Nsclc Proliferation Via Induction of Cell Cycle Arrest By Topo I-Mediated DNA Damage

2019 ◽  
Vol 19 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Yang Liu ◽  
Jingyin Zhang ◽  
Shuyun Feng ◽  
Tingli Zhao ◽  
Zhengzheng Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Dna Damage ◽  
Cyclin D ◽  
Dna Relaxation ◽  
Cycle Arrest ◽  
H460 Cell ◽  
H1975 Cell

Objective: The aim of this study is to investigate the inhibitory effect of camptothecin derivative 3j on Non-Small Cell Lung Cancer (NSCLCs) cells and the potential anti-tumor mechanisms. Background: Camptothecin compounds are considered as the third largest natural drugs which are widely investigated in the world and they suffered restriction because of serious toxicity, such as hemorrhagic cystitis and bone marrow suppression. Methods: Using cell proliferation assay and S180 tumor mice model, a series of 20(S)-O-substituted benzoyl 7- ethylcamptothecin compounds were screened and evaluated the antitumor activities in vitro and in vivo. Camptothecin derivative 3j was selected for further study using flow cytometry in NSCLCs cells. Cell cycle related protein cyclin A2, CDK2, cyclin D and cyclin E were detected by Western Blot. Then, computer molecular docking was used to confirm the interaction between 3j and Topo I. Also, DNA relaxation assay and alkaline comet assay were used to investigate the mechanism of 3j on DNA damage. Results: Our results demonstrated that camptothecin derivative 3j showed a greater antitumor effect in eleven 20(S)-O-substituted benzoyl 7-ethylcamptothecin compounds in vitro and in vivo. The IC50 of 3j was 1.54± 0.41 µM lower than irinotecan with an IC50 of 13.86±0.80 µM in NCI-H460 cell, which was reduced by 8 fold. In NCI-H1975 cell, the IC50 of 3j was 1.87±0.23 µM lower than irinotecan (IC50±SD, 5.35±0.38 µM), dropped by 1.8 fold. Flow cytometry analysis revealed that 3j induced significant accumulation in a dose-dependent manner. After 24h of 3j (10 µM) treatment, the percentage of NCI-H460 cell in S-phase significantly increased (to 93.54 ± 4.4%) compared with control cells (31.67 ± 3.4%). Similarly, the percentage of NCI-H1975 cell in Sphase significantly increased (to 83.99 ± 2.4%) compared with control cells (34.45 ± 3.9%) after treatment with 10µM of 3j. Moreover, increased levels of cyclin A2, CDK2, and decreased levels of cyclin D, cyclin E further confirmed that cell cycle arrest was induced by 3j. Furthermore, molecular docking studies suggested that 3j interacted with Topo I-DNA and DNA-relaxation assay simultaneously confirmed that 3j suppressed the activity of Topo I. Research on the mechanism showed that 3j exhibited anti-tumour activity via activating the DNA damage response pathway and suppressing the repair pathway in NSCLC cells. Conclusion: Novel camptothecin derivative 3j has been demonstrated as a promising antitumor agent and remains to be assessed in further studies.

scholarly journals BRD4 PROTAC degrader ARV-825 inhibits T-cell acute lymphoblastic leukemia by targeting 'Undruggable' Myc-pathway genes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shuiyan Wu ◽  
You Jiang ◽  
Yi Hong ◽  
Xinran Chu ◽  
Zimu Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Rna Seq ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bet Protein

Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL. Methods Expression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP). Results BRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo. Conclusions BRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

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