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ELife Jun 2024Cohesin is a multi-subunit protein that plays a pivotal role in holding sister chromatids together during cell division. Sister chromatid cohesion 3 (SCC3), constituents...
Cohesin is a multi-subunit protein that plays a pivotal role in holding sister chromatids together during cell division. Sister chromatid cohesion 3 (SCC3), constituents of cohesin complex, is highly conserved from yeast to mammals. Since the deletion of individual cohesin subunit always causes lethality, it is difficult to dissect its biological function in both mitosis and meiosis. Here, we obtained weak mutants using CRISPR-Cas9 system to explore its function during rice mitosis and meiosis. The weak mutants displayed obvious vegetative defects and complete sterility, underscoring the essential roles of SCC3 in both mitosis and meiosis. SCC3 is localized on chromatin from interphase to prometaphase in mitosis. However, in meiosis, SCC3 acts as an axial element during early prophase I and subsequently situates onto centromeric regions following the disassembly of the synaptonemal complex. The loading of SCC3 onto meiotic chromosomes depends on REC8. shows severe defects in homologous pairing and synapsis. Consequently, SCC3 functions as an axial element that is essential for maintaining homologous chromosome pairing and synapsis during meiosis.
Topics: Chromosome Pairing; Meiosis; Cell Cycle Proteins; Oryza; Chromosomal Proteins, Non-Histone; Cohesins; Mitosis; Synaptonemal Complex; CRISPR-Cas Systems
PubMed: 38864853
DOI: 10.7554/eLife.94180 -
Medicine Sep 2023Hepatocellular carcinoma (HCC) poses a global health challenge. Effective biomarkers are required for early diagnosis to improve survival rates of patients with HCC....
Hepatocellular carcinoma (HCC) poses a global health challenge. Effective biomarkers are required for early diagnosis to improve survival rates of patients with HCC. Spindle and kinetochore-associated complex subunits 1 (SKA1) is essential for proper chromosome segregation in the mitotic cell cycle. Previous studies have shown that overexpression of SKA1 is associated with a poor prognosis in various cancers. The expression, prognostic value, and clinical functions of SKA1 in HCC were evaluated with several bioinformatics web portals. Additionally, we identified target long non-coding RNAs (lncRNAs) and microRNAs by analyzing messenger RNA (mRNA)-miRNA and miRNA-lncRNA interaction data and elucidated the potential competing endogenous RNA (ceRNA) mechanism associated with SKA1. High SKA1 expression was associated with poor prognosis in patients with HCC. Furthermore, multivariate Cox regression analysis revealed that SKA1 expression was an independent prognostic factor for HCC. GO and KEGG analyses showed that SKA1 is related to the cell cycle checkpoints, DNA replication and repair, Rho GTPases signaling, mitotic prometaphase, and kinesins. Gene set enrichment analysis revealed that high levels of SKA1 are associated with cancer-promoting pathways. DNA methylation of SKA1 in HCC tissues was lower than that in normal tissues. Ultimately, the following 9 potential ceRNA-based pathways targeting SKA1 were identified: lncRNA: AC026401.3, Small Nucleolar RNA Host Gene 3 (SNHG3), and AC124798.1-miR-139-5p-SKA1; lncRNA: AC26356.1, Small Nucleolar RNA Host Gene 16 (SNHG16), and FGD5 Antisense RNA 1-miR-22-3p-SKA1; lncRNA: Cytoskeleton Regulator RNA (CYTOR), MIR4435-2 Host Gene, and differentiation antagonizing non-protein coding RNA-miR-125b-5p-SKA1. SKA1 expression levels significantly correlated with immune cell infiltration and immune checkpoint genes in the HCC tissues. SKA1 is a potential prognostic biomarker for HCC. This study provides a meaningful direction for research on SKA1-related mechanisms, which will be beneficial for future research on HCC-related molecular biological therapies and targeted immunotherapy.
Topics: Humans; Carcinoma, Hepatocellular; RNA, Long Noncoding; RNA, Small Nucleolar; Liver Neoplasms; MicroRNAs; Computational Biology; Immunoassay; Chromosomal Proteins, Non-Histone
PubMed: 37746945
DOI: 10.1097/MD.0000000000034826 -
Development (Cambridge, England) Jun 2024The RNA-binding protein cytoplasmic polyadenylation element binding 1 (CPEB1) plays a fundamental role in regulating mRNA translation in oocytes. However, the specifics...
The RNA-binding protein cytoplasmic polyadenylation element binding 1 (CPEB1) plays a fundamental role in regulating mRNA translation in oocytes. However, the specifics of how and which protein kinase cascades modulate CPEB1 activity are still controversial. Using genetic and pharmacological tools, and detailed time courses, we have re-evaluated the relationship between CPEB1 phosphorylation and translation activation during mouse oocyte maturation. We show that both the CDK1/MAPK and AURKA/PLK1 pathways converge on CPEB1 phosphorylation during prometaphase of meiosis I. Only inactivation of the CDK1/MAPK pathway disrupts translation, whereas inactivation of either pathway alone leads to CPEB1 stabilization. However, CPEB1 stabilization induced by inactivation of the AURKA/PLK1 pathway does not affect translation, indicating that destabilization and/or degradation is not linked to translational activation. The accumulation of endogenous CCNB1 protein closely recapitulates the translation data that use an exogenous template. These findings support the overarching hypothesis that the activation of translation during prometaphase in mouse oocytes relies on a CDK1/MAPK-dependent CPEB1 phosphorylation, and that translational activation precedes CPEB1 destabilization.
Topics: Animals; Oocytes; Meiosis; mRNA Cleavage and Polyadenylation Factors; Phosphorylation; Mice; Protein Biosynthesis; Female; CDC2 Protein Kinase; Transcription Factors; Aurora Kinase A; Cyclin B1; Cell Cycle Proteins; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Signal Transduction
PubMed: 38785133
DOI: 10.1242/dev.202712 -
BMC Cancer Aug 2023HeberFERON is a co-formulation of α2b and γ interferons, based on their synergism, which has shown its clinical superiority over individual interferons in basal cell...
BACKGROUND
HeberFERON is a co-formulation of α2b and γ interferons, based on their synergism, which has shown its clinical superiority over individual interferons in basal cell carcinomas. In glioblastoma (GBM), HeberFERON has displayed promising preclinical and clinical results. This led us to design a microarray experiment aimed at identifying the molecular mechanisms involved in the distinctive effect of HeberFERON compared to the individual interferons in U-87MG model.
METHODS
Transcriptional expression profiling including a control (untreated) and three groups receiving α2b-interferon, γ-interferon and HeberFERON was performed using an Illumina HT-12 microarray platform. Unsupervised methods for gene and sample grouping, identification of differentially expressed genes, functional enrichment and network analysis computational biology methods were applied to identify distinctive transcription patterns of HeberFERON. Validation of most representative genes was performed by qPCR. For the cell cycle analysis of cells treated with HeberFERON for 24 h, 48 and 72 h we used flow cytometry.
RESULTS
The three treatments show different behavior based on the gene expression profiles. The enrichment analysis identified several mitotic cell cycle related events, in particular from prometaphase to anaphase, which are exclusively targeted by HeberFERON. The FOXM1 transcription factor network that is involved in several cell cycle phases and is highly expressed in GBMs, is significantly down regulated. Flow cytometry experiments corroborated the action of HeberFERON on the cell cycle in a dose and time dependent manner with a clear cellular arrest as of 24 h post-treatment. Despite the fact that p53 was not down-regulated, several genes involved in its regulatory activity were functionally enriched. Network analysis also revealed a strong relationship of p53 with genes targeted by HeberFERON. We propose a mechanistic model to explain this distinctive action, based on the simultaneous activation of PKR and ATF3, p53 phosphorylation changes, as well as its reduced MDM2 mediated ubiquitination and export from the nucleus to the cytoplasm. PLK1, AURKB, BIRC5 and CCNB1 genes, all regulated by FOXM1, also play central roles in this model. These and other interactions could explain a G2/M arrest and the effect of HeberFERON on the proliferation of U-87MG.
CONCLUSIONS
We proposed molecular mechanisms underlying the distinctive behavior of HeberFERON compared to the treatments with the individual interferons in U-87MG model, where cell cycle related events were highly relevant.
Topics: Humans; Glioblastoma; Apoptosis; Cell Line, Tumor; G2 Phase Cell Cycle Checkpoints; Interferon-alpha; Anaphase; Interferon-gamma; Skin Neoplasms
PubMed: 37644431
DOI: 10.1186/s12885-023-11330-2 -
Research Square Feb 2024The Survivin protein has roles in repairing incorrect microtubule-kinetochore attachments at prometaphase, and the faithful execution of cytokinesis, both as part of the...
The Survivin protein has roles in repairing incorrect microtubule-kinetochore attachments at prometaphase, and the faithful execution of cytokinesis, both as part of the (CPC) (1). In this context, errors frequently lead to aneuploidy, polyploidy and cancer (1). Adding to these well-known roles of this protein, this paper now shows for the first time that Survivin is required for cancer cells to enter mitosis, and that, in its absence, HeLa cells accumulate at early prophase, or prior to reported before (2, 3). This early prophase blockage is demonstrated by the presence of an intact nuclear lamina and low Cdk1 activity (4). Importantly, escaping the arrest induced by Survivin abrogation leads to multiple mitotic defects, or , and eventually cell death. Mechanistically, Cdk1 does not localize at the centrosome in the absence of Survivin pointing at an impairment in signaling through the Cdc25B-Cdk1 axis. In agreement, even though Survivin directly interacts with Cdc25B, both and , in its absence, an inactive cytosolic Cdc25B-Cdk1-Cyclin B1 complex accumulates. This flaw in Cdc25B activation can however be reversed in Survivin-depleted HeLa cell extracts to which the recombinant Survivin protein is added back. Finally, a role for Survivin in the Cdc25B-mediated activation of Cdk1 is confirmed by overriding the early prophase blockage induced in cells lacking Survivin through the expression of a gain-of-function Cdc25B mutant.
PubMed: 38464014
DOI: 10.21203/rs.3.rs-3949429/v1 -
BioRxiv : the Preprint Server For... Jan 2024Genetic screens for recessive alleles induce mutations, make the mutated chromosomes homozygous, and then assay those homozygotes for the phenotype of interest. When...
Genetic screens for recessive alleles induce mutations, make the mutated chromosomes homozygous, and then assay those homozygotes for the phenotype of interest. When screening for genes required for female meiosis, the phenotype of interest has typically been nondisjunction from chromosome segregation errors. As this requires that mutant females be viable and fertile, any mutants that are lethal or sterile when homozygous cannot be recovered by this approach. To overcome these limitations, our lab has screened the VALIUM22 collection produced by the Harvard TRiP Project, which contains RNAi constructs targeting genes known to be expressed in the germline in a vector optimized for germline expression. By driving RNAi with GAL4 under control of a germline-specific promoter ( or , we can test genes that would be lethal if knocked down in all cells, and by examining unfertilized metaphase-arrested mature oocytes, we can identify defects associated with genes whose knockdown results in sterility or causes other errors besides nondisjunction. We screened this collection to identify genes that disrupt either of two phenotypes when knocked down: the ability of meiotic chromosomes to congress to a single mass at the end of prometaphase, and the sequestration of Mps1-GFP to ooplasmic filaments in response to hypoxia. After screening >1450 lines of the collection, we obtained multiple hits for both phenotypes, identified novel meiotic phenotypes for genes that had been previously characterized in other processes, and identified the first phenotypes to be associated with several previously uncharacterized genes.
PubMed: 38293152
DOI: 10.1101/2024.01.12.575435 -
BioRxiv : the Preprint Server For... Jan 2024The RNA-binding protein cytoplasmic polyadenylation element binding 1 (CPEB1) plays a fundamental role in the regulation of mRNA translation in oocytes. However, the...
The RNA-binding protein cytoplasmic polyadenylation element binding 1 (CPEB1) plays a fundamental role in the regulation of mRNA translation in oocytes. However, the nature of protein kinase cascades modulating the activity of CPEB1 is still a matter of controversy. Using genetic and pharmacological tools and detailed time courses, here we have reevaluated the relationship between CPEB1 phosphorylation and the activation of translation during mouse oocyte maturation. We show that both the CDK1/MAPK and AURKA/PLK1 pathways converge on the phosphorylation of CPEB1 during prometaphase. Only inactivation of the CDK1/MAPK pathway disrupts translation, while inactivation of either pathway leads to CPEB1 stabilization. However, stabilization of CPEB1 induced by inactivation of the AURKA/PLK1 does not affect translation, indicating that destabilization/degradation can be dissociated from translational activation. The accumulation of the endogenous CCNB1 protein closely recapitulates the translation data. These findings support the overarching hypothesis that the activation of translation in prometaphase in mouse oocytes relies on a CDK1-dependent CPEB1 phosphorylation, and this translational activation precedes CPEB1 destabilization.
PubMed: 38293116
DOI: 10.1101/2024.01.17.575938 -
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 -
Saudi Pharmaceutical Journal : SPJ :... Jan 2024Pentagamavunone-1 (PGV-1), an analog of curcumin, has been studied for its cytotoxic effects in 4T1, MCF7, MCF7/HER2, and T47D breast cancer cells. Its antiproliferative...
Pentagamavunone-1 (PGV-1), an analog of curcumin, has been studied for its cytotoxic effects in 4T1, MCF7, MCF7/HER2, and T47D breast cancer cells. Its antiproliferative effect is partly mediated through G2/M arrest; however, its molecular mechanism during cell cycle progression remains unknown. In this study, we aimed to determine whether PGV-1 has any anticancer effects on highly aggressive breast cancer cells, with a focus on cell cycle regulatory activity, reactive oxygen species (ROS) generation, and their mediated effects on cancer cells. MDA-MB-231 (triple-negative) and HCC1954 (overexpressed HER2) immortalized human breast cancer cells were used in the study. PGV-1 exhibited cytotoxic activity with an irreversible antiproliferative impact on treated cells and had good selectivity when tested in fibroblast cells. Oral PGV-1 administration suppressed tumor growth in a cell-derived xenograft mouse model. PGV-1 induced the phosphorylation of Aurora A kinase and PLK1 in MDA-MB-231 cells, while PLK1 and cyclin B1 phosphorylation were enhanced in the PGV-1-treated HCC1954 cells during prometaphase arrest. Intracellular ROS production was substantially higher upon PGV-1 treatment following mitotic arrest, and this activity caused impairment of mitochondrial respiration, induced senescence, and subsequently triggered early-to-late apoptosis. Collectively, these results suggest that the molecular mechanism of PGV-1 involves the regulation of mitotic kinases to cause cell cycle arrest and the enhancement of ROS production to impair mitochondrial activity and induce cellular senescence. The therapeutic activities demonstrated by PGV-1 in this study show its potential as an appealing candidate for chemotherapy in breast cancer treatment.
PubMed: 38146327
DOI: 10.1016/j.jsps.2023.101892 -
Scientific Reports Feb 2024Centrosome amplification is a hallmark of cancer and PLK4 is one of the responsible factors for cancer associated centrosome amplification. Increased PLK4 levels was...
Centrosome amplification is a hallmark of cancer and PLK4 is one of the responsible factors for cancer associated centrosome amplification. Increased PLK4 levels was also shown to contribute to generation of cells with centriole amplification in mammalian tissues as olfactory neuron progenitor cells. PLK4 overexpression generates centriole rosette (CR) structures which harbor more than two centrioles each. Long term PLK4 overexpression results with centrosome amplification, but the maturation of amplified centrioles in CRs and linking of PLK4 induced amplified centrosomes has not yet been investigated in detail. Here, we show evidence for generation of large clustered centrosomes which have more than 2 centriole rosettes and define these structures as centriole rosette clusters (CRCs) in cells that have high PLK4 levels for 2 consecutive cell cycles. In addition, we show that PLK4 induced CRs follow normal centrosomal maturation processes and generate CRC structures that are inter-connected with canonical centrosomal linker proteins as C-Nap1, Rootletin and Cep68 in the second cell cycle after PLK4 induction. Increased PLK4 levels in cells with C-Nap1 and Rootletin knock-out resulted with distanced CRs and CRCs in interphase, while Nek2 knock-out inhibited separation of CRCs in prometaphase, providing functional evidence for the binding of CRC structures with centrosomal linker proteins. Taken together, these results suggest a cell cycle dependent model for PLK4 induced centrosome amplification which occurs in 2 consecutive cell cycles: (i) CR state in the first cell cycle, and (ii) CRC state in the second cell cycle.
Topics: Animals; Humans; Centrioles; Centrosomal Associated Proteins; Cell Cycle Proteins; Centrosome; Neoplasms; Mammals; Protein Serine-Threonine Kinases
PubMed: 38388511
DOI: 10.1038/s41598-024-53985-2