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Plant Physiology and Biochemistry : PPB Feb 2024Like other heavy metals, Cr (VI) is a powerful carcinogen and mutagen agent. Its toxic effects on plants are well considered. In order to elucidate its adverse effects,...
Like other heavy metals, Cr (VI) is a powerful carcinogen and mutagen agent. Its toxic effects on plants are well considered. In order to elucidate its adverse effects, the present work aims to study the mitosis aberrations of Cr (VI) on the Vicia faba root-cells and its molecular docking analysis to understand the genotoxicity mechanisms. In-vivo, Vicia faba plants were exposed to 50 and 100 μM Cr (VI) for 48 h. In-silico, molecular docking and molecular dynamics simulation were used to study the interactions between dichromate and tubulin tyrosine ligase T2R-TTL (PDBID: 5XIW) with reference to Colchicine (microtubule inhibitor). According to our results, Cr (VI) affects growth and cell division and also induces many mitosis aberrations such as chromosome sticking, anaphase/telophase bridges, lagging chromosomes and fragmentation during all phases of mitosis. On the one hand, Cr (VI) reduces mitotic index and promotes micronuclei induction. The in-silico results showed that dichromate establishes very strong bonds at the binding site of the tubulin tyrosine ligase T2R-TTL, with a binding affinity of -5.17 Kcal/Mol and an inhibition constant of 163.59 μM. These interactions are similar to those of colchicine with this protein, so dichromate could be a very potent inhibitor of this protein's activity. TTL plays a fundamental role in the tyrosination/detyrosination of tubulin, which is crucial to the regulation of the microtubule cytoskeleton. Its inhibition leads to the appearance of many morphogenic abnormalities such as mitosis aberrations. In conclusion, our data confirm the highest genotoxicity effects of Cr (VI) on Vicia faba root-cells.
Topics: Vicia faba; Molecular Docking Simulation; Tubulin; Chromium; Fabaceae; Mitosis; DNA Damage; Colchicine; Tyrosine; Ligases; Chromosome Aberrations
PubMed: 38237423
DOI: 10.1016/j.plaphy.2024.108361 -
Journal of Cell Science Feb 2024The mammalian cell cycle alternates between two phases - S-G2-M with high levels of A- and B-type cyclins (CycA and CycB, respectively) bound to cyclin-dependent kinases...
The mammalian cell cycle alternates between two phases - S-G2-M with high levels of A- and B-type cyclins (CycA and CycB, respectively) bound to cyclin-dependent kinases (CDKs), and G1 with persistent degradation of CycA and CycB by an activated anaphase promoting complex/cyclosome (APC/C) bound to Cdh1 (also known as FZR1 in mammals; denoted APC/C:Cdh1). Because CDKs phosphorylate and inactivate Cdh1, these two phases are mutually exclusive. This 'toggle switch' is flipped from G1 to S by cyclin-E bound to a CDK (CycE:CDK), which is not degraded by APC/C:Cdh1, and from M to G1 by Cdc20-bound APC/C (APC/C:Cdc20), which is not inactivated by CycA:CDK or CycB:CDK. After flipping the switch, cyclin E is degraded and APC/C:Cdc20 is inactivated. Combining mathematical modelling with single-cell timelapse imaging, we show that dysregulation of CycB:CDK disrupts strict alternation of the G1-S and M-G1 switches. Inhibition of CycB:CDK results in Cdc20-independent Cdh1 'endocycles', and sustained activity of CycB:CDK drives Cdh1-independent Cdc20 endocycles. Our model provides a mechanistic explanation for how whole-genome doubling can arise, a common event in tumorigenesis that can drive tumour evolution.
Topics: Animals; Cell Cycle; Anaphase-Promoting Complex-Cyclosome; Cell Cycle Proteins; Cyclins; Cyclin-Dependent Kinases; Mitosis; Cdc20 Proteins; Mammals
PubMed: 38206091
DOI: 10.1242/jcs.261364 -
The Plant Journal : For Cell and... Apr 2024In eukaryotes, double-strand breaks (DSBs) are either repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). In somatic plant cells, HR is very...
Deficiency of both classical and alternative end-joining pathways leads to a synergistic defect in double-strand break repair but not to an increase in homology-dependent gene targeting in Arabidopsis.
In eukaryotes, double-strand breaks (DSBs) are either repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). In somatic plant cells, HR is very inefficient. Therefore, the vast majority of DSBs are repaired by two different pathways of NHEJ. The classical (cNHEJ) pathway depends on the heterodimer KU70/KU80, while polymerase theta (POLQ) is central to the alternative (aNHEJ) pathway. Surprisingly, Arabidopsis plants are viable, even when both pathways are impaired. However, they exhibit severe growth retardation and reduced fertility. Analysis of mitotic anaphases indicates that the double mutant is characterized by a dramatic increase in chromosome fragmentation due to defective DSB repair. In contrast to the single mutants, the double mutant was found to be highly sensitive to the DSB-inducing genotoxin bleomycin. Thus, both pathways can complement for each other efficiently in DSB repair. We speculated that in the absence of both NHEJ pathways, HR might be enhanced. This would be especially attractive for gene targeting (GT) in which predefined changes are introduced using a homologous template. Unexpectedly, the polq single mutant as well as the double mutant showed significantly lower GT frequencies in comparison to wildtype plants. Accordingly, we were able to show that elimination of both NHEJ pathways does not pose an attractive approach for Agrobacterium-mediated GT. However, our results clearly indicate that a loss of cNHEJ leads to an increase in GT frequency, which is especially drastic and attractive for practical applications, in which the in planta GT strategy is used.
Topics: Arabidopsis; DNA-Binding Proteins; DNA Repair; Gene Targeting; DNA End-Joining Repair
PubMed: 38179887
DOI: 10.1111/tpj.16604 -
Environmental Science and Pollution... Jan 2024Disinfection by-products (DBPs) are a concern due to their presence in chlorinated wastewater, sewage treatment plant discharge, and surface water, and their potential...
Disinfection by-products (DBPs) are a concern due to their presence in chlorinated wastewater, sewage treatment plant discharge, and surface water, and their potential for environmental toxicity. Despite some attention to their ecotoxicity, little is known about the phytotoxicity of DBPs. This study aimed to evaluate the individual and combined phytotoxicity of four trihalomethanes (THMs: trichloromethane (TCM), bromodichloromethane (BDCM), dibromochloromethane (DBCM), and tribromomethane (TBM) and their mixture (THM4)), and trichloroacetic acid (TCAA) using genotoxic and cytotoxic assays. The analysis included seed germination tests using Vigna radiata and root growth tests, mitosis studies, oxidative stress response, chromosomal aberrations (CA), and DNA laddering using Allium cepa. The results showed a progressive increase in root growth inhibition for both plant species as the concentration of DBPs increased. High concentrations of mixtures of four THMs resulted in significant (p < 0.05) antagonistic interactions. The effective concentration (EC) value for V. radiata was 5655, 3145, 2690, 1465, 3570, and 725 mg/L for TCM, BDCM, DBCM, TBM, THM4, and TCAA, respectively. For A. cepa, the EC for the same contaminants was 700, 400, 350, 250, 450, and 105 mg/L, respectively. DBP cytotoxicity was observed through CAs, including C-metaphase, unseparated anaphase, lagging chromosome, sticky metaphase, and bridging. Mitotic depression (MD) increased with dose, reaching up to 54.4% for TCAA (50-500 mg/L). The electrophoresis assay showed DNA fragmentation and shearing, suggesting genotoxicity for some DBPs. The order of phytotoxicity for the tested DBPs was TCAA > TBM > DBCM > BDCM > THM4 > TCM. These findings underscore the need for further research on the phytotoxicity of DBPs, especially given their common use in agricultural practices such as irrigation and the use of sludge as manure.
Topics: Trichloroacetic Acid; Onions; Vigna; Trihalomethanes; Disinfection; Chloroform; Water Pollutants, Chemical
PubMed: 38110686
DOI: 10.1007/s11356-023-31419-2 -
The Journal of Biological Chemistry Jan 2024Bub1 is a conserved mitotic kinase involved in signaling of the spindle assembly checkpoint. Multiple phosphorylation sites on Bub1 have been characterized, yet it is...
Bub1 is a conserved mitotic kinase involved in signaling of the spindle assembly checkpoint. Multiple phosphorylation sites on Bub1 have been characterized, yet it is challenging to understand the interplay between the multiple phosphorylation sites due to the limited availability of phosphospecific antibodies. In addition, phosphoregulation of Bub1 in Schizosaccharomyces pombe is poorly understood. Here we report the identification of a new Mph1/Mps1-mediated phosphorylation site, i.e., Ser532, of Bub1 in Schizosaccharomyces pombe. A phosphospecific antibody against phosphorylated Bub1-Ser532 was developed. Using the phosphospecific antibody, we demonstrated that phosphorylation of Bub1-Ser352 was mediated specifically by Mph1/Mps1 and took place during early mitosis. Moreover, live-cell microscopy showed that inhibition of the phosphorylation of Bub1 at Ser532 impaired the localization of Bub1, Mad1, and Mad2 to the kinetochore. In addition, inhibition of the phosphorylation of Bub1 at Ser532 caused anaphase B lagging chromosomes. Hence, our study constitutes a model in which Mph1/Mps1-mediated phosphorylation of fission yeast Bub1 promotes proper kinetochore localization of Bub1 and faithful chromosome segregation.
Topics: Anaphase; Antibodies, Phospho-Specific; Cell Cycle Proteins; Chromosome Segregation; Kinetochores; Mitosis; Phosphorylation; Phosphoserine; Protein Serine-Threonine Kinases; Protein Transport; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Signal Transduction; Spindle Apparatus
PubMed: 38097187
DOI: 10.1016/j.jbc.2023.105559 -
European Journal of Medical Research Dec 2023Hepatocellular carcinoma (HCC) is one of the most prevalent forms of cancer and poses a threat to the health and survival of humans. Mitochondrial ribosomal protein L48...
BACKGROUND
Hepatocellular carcinoma (HCC) is one of the most prevalent forms of cancer and poses a threat to the health and survival of humans. Mitochondrial ribosomal protein L48 (MRPL48) belongs to the mitochondrial ribosomal protein family, which participates in energy production. Studies have shown that MRPL48 can predict osteosarcoma incidence and prognosis, as well as promotes colorectal cancer progression. However, the role of MRPL48 in HCC remains unknown.
METHODS
TCGA, GEO, HCCDB, CPTAC, SMART, UALCAN, Kaplan-Meier plotter, cBioPortal, and MethSurv were performed for bioinformatics purposes. Quantitative RT-PCR, immunoblotting, and functional studies were conducted to validate the methodology in vitro.
RESULTS
MRPL48 was greatly overexpressed in HCC tissues, compared with healthy tissue, which was subsequently demonstrated in vitro as well. The survival and regression analyses showed that MRPL48 expression is of significant clinical prognostic value in HCC. The ROC curve and nomogram analysis indicated that MRPL48 is a powerful predictor of HCC. MRPL48 methylation was adversely associated with the expression of MRPL48, and patients with a low level of methylation had poorer overall survival than those with a high level of methylation. GSEA showed that the expression of the MRPL48 was correlated with Resolution of Sister Chromatid Cohesion, Mitotic Prometaphase, Retinoblastoma Gene in Cancer, RHO Gtpases Activate Formins, Mitotic Metaphase and Anaphase, and Cell Cycle Checkpoints. An analysis of immune cell infiltration showed a significant association between MRPL48 and immune cell infiltration subsets, which impacted the survival of HCC patients. Additionally, MRPL48 knockdown reduced HCC cell proliferation, migration, and invasion in vitro.
CONCLUSIONS
We demonstrated that MRPL48 expression may be associated with HCC development and prognosis. These findings may open up new research directions and opportunities for the development of HCC treatments.
Topics: Humans; Prognosis; Carcinoma, Hepatocellular; Liver Neoplasms; Biomarkers; Ribosomal Proteins
PubMed: 38093387
DOI: 10.1186/s40001-023-01571-z -
Stem Cell Research & Therapy Dec 2023Limbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in...
BACKGROUND
Limbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications.
METHODS
After in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Using a clinically applicable protocol, the data collected on LSCs at passage 1 were summarized, including: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency); expression of putative stem cell markers through flow cytometry, immunofluorescence, and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined.
RESULTS
Expression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n = 873) was performed, and only a small fraction < 2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype.
CONCLUSION
This manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans.
Topics: Humans; Stem Cells; Limbal Stem Cells; Limbus Corneae; Epithelium, Corneal; Spindle Apparatus; Endotoxins
PubMed: 38093301
DOI: 10.1186/s13287-023-03586-z -
Cancer Biology & Therapy Dec 2023Cyclin-dependent Kinase 2 (CDK2) inhibition prevents supernumerary centrosome clustering. This causes multipolarity, anaphase catastrophe and apoptotic death of...
Cyclin-dependent Kinase 2 (CDK2) inhibition prevents supernumerary centrosome clustering. This causes multipolarity, anaphase catastrophe and apoptotic death of aneuploid cancers. This study elucidated how CDK2 antagonism affected centrosome stoichiometry. Focused ion beam scanning electron microscopy (FIB-SEM) and immunofluorescent imaging were used. Studies interrogated multipolar mitosis after pharmacologic or genetic repression of CDK2. CDK2/9 antagonism with CYC065 (Fadraciclib)-treatment disordered centrosome stoichiometry in aneuploid cancer cells, preventing centrosome clustering. This caused ring-like chromosomes or multipolar cancer cells to form before onset of cell death. Intriguingly, CDK2 inhibition caused a statistically significant increase in single centrioles rather than intact centrosomes with two centrioles in cancer cells having chromosome rings or multipolarity. Statistically significant alterations in centrosome stoichiometry were undetected in other mitotic cancer cells. To confirm this pharmacodynamic effect, CDK2 but not CDK9 siRNA-mediated knockdown augmented cancer cells with chromosome ring or multipolarity formation. Notably, engineered gain of CDK2, but not CDK9 expression, reversed emergence of cancer cells with chromosome rings or multipolarity, despite CYC065-treatment. In marked contrast, CDK2 inhibition of primary human alveolar epithelial cells did not confer statistically significant increases of cells with ring-like chromosomes or multipolarity. Hence, CDK2 antagonism caused differential effects in malignant versus normal alveolar epithelial cells. Translational relevance was confirmed by CYC065-treatment of syngeneic lung cancers in mice. Mitotic figures in tumors exhibited chromosome rings or multipolarity. Thus, CDK2 inhibition preferentially disorders centrosome stoichiometry in cancer cells. Engaging this disruption is a strategy to explore against aneuploid cancers in future clinical trials.
Topics: Humans; Animals; Mice; Cyclin-Dependent Kinase 2; Centrosome; Anaphase; Mitosis; Aneuploidy; Neoplasms
PubMed: 38031910
DOI: 10.1080/15384047.2023.2279241 -
Cellular and Molecular Life Sciences :... Nov 2023Faithful chromosome segregation requires correct attachment of kinetochores with the spindle microtubules. Erroneously-attached kinetochores recruit proteins to activate...
Faithful chromosome segregation requires correct attachment of kinetochores with the spindle microtubules. Erroneously-attached kinetochores recruit proteins to activate Spindle assembly checkpoint (SAC), which senses the errors and signals cells to delay anaphase progression for error correction. Temporal control of the levels of SAC activating-proteins is critical for checkpoint activation and silencing, but its mechanism is not fully understood. Here, we show that E3 ubiquitin ligase, SCF-FBXW7 targets BubR1 for ubiquitin-mediated degradation and thereby controls SAC in human cells. Depletion of FBXW7 results in prolonged metaphase arrest with increased stabilization of BubR1 at kinetochores. Similar kinetochore stabilization is also observed for BubR1-interacting protein, CENP-E. FBXW7 induced ubiquitination of both BubR1 and the BubR1-interacting kinetochore-targeting domain of CENP-E, but CENP-E domain degradation is dependent on BubR1. Interestingly, Cdk1 inhibition disrupts FBXW7-mediated BubR1 targeting and further, phospho-resistant mutation of Cdk1-targeted phosphorylation site, Thr 620 impairs BubR1-FBXW7 interaction and FBXW7-mediated BubR1 ubiquitination, supporting its role as a phosphodegron for FBXW7. The results demonstrate SCF-FBXW7 as a key regulator of spindle assembly checkpoint that controls stability of BubR1 and its associated CENP-E at kinetochores. They also support that upstream Cdk1 specific BubR1 phosphorylation signals the ligase to activate the process.
Topics: Humans; Cell Cycle Proteins; F-Box-WD Repeat-Containing Protein 7; HeLa Cells; Kinetochores; Mitosis; Protein Serine-Threonine Kinases; Spindle Apparatus; Ubiquitin; Ubiquitin-Protein Ligases
PubMed: 38008853
DOI: 10.1007/s00018-023-05019-9 -
BioRxiv : the Preprint Server For... Feb 2024Genomic information must be faithfully transmitted into two daughter cells during mitosis. To ensure the transmission process, interphase chromatin is further condensed...
Genomic information must be faithfully transmitted into two daughter cells during mitosis. To ensure the transmission process, interphase chromatin is further condensed into mitotic chromosomes. Although protein factors like condensins and topoisomerase IIα are involved in the assembly of mitotic chromosomes, the physical bases of the condensation process remain unclear. Depletion force/macromolecular crowding, an effective attractive force that arises between large structures in crowded environments around chromosomes, may contribute to the condensation process. To approach this issue, we investigated the "chromosome milieu" during mitosis of living human cells using orientation-independent-differential interference contrast (OI-DIC) module combined with a confocal laser scanning microscope, which is capable of precisely mapping optical path differences and estimating molecular densities. We found that the molecular density surrounding chromosomes increased with the progression from prometaphase to anaphase, concurring with chromosome condensation. However, the molecular density went down in telophase, when chromosome decondensation began. Changes in the molecular density around chromosomes by hypotonic or hypertonic treatment consistently altered the condensation levels of chromosomes. , native chromatin was converted into liquid droplets of chromatin in the presence of cations and a macromolecular crowder. Additional crowder made the chromatin droplets stiffer and more solid-like, with further condensation. These results suggest that a transient rise in depletion force, likely triggered by the relocation of macromolecules (proteins, RNAs and others) via nuclear envelope breakdown and also by a subsequent decrease in cell-volumes, contributes to mitotic chromosome condensation, shedding light on a new aspect of the condensation mechanism in living human cells.
PubMed: 37986866
DOI: 10.1101/2023.11.11.566679