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Research in Pharmaceutical Sciences 2023Previous studies highlighted that chemoprevention curcumin analog-1.1 (CCA-1.1) demonstrated an antitumor effect on breast, leukemia, and colorectal cancer cells. By...
Chemoprevention curcumin analog 1.1 promotes metaphase arrest and enhances intracellular reactive oxygen species levels on TNBC MDA-MB-231 and HER2-positive HCC1954 cells.
BACKGROUND AND PURPOSE
Previous studies highlighted that chemoprevention curcumin analog-1.1 (CCA-1.1) demonstrated an antitumor effect on breast, leukemia, and colorectal cancer cells. By utilizing immortalized MDA-MB-231 and HCC1954 cells, we evaluated the anticancer properties of CCA-1.1 and its mediated activity to promote cellular death.
EXPERIMENTAL APPROACH
Cytotoxicity and anti-proliferation were assayed using trypan blue exclusion. The cell cycle profile after CCA-1.1 treatment was established through flow cytometry. May-Grünwald-Giemsa and Hoechst staining were performed to determine the cell cycle arrest upon CCA-1.1 treatment. The involvement of CCA-1.1 in mitotic kinases (aurora A, p-aurora A, p-PLK1, and p-cyclin B1) expression was investigated by immunoblotting. CCA-1.1-treated cells were stained with the X-gal solution to examine the effect on senescence. ROS level and mitochondrial respiration were assessed by DCFDA assay and mitochondrial oxygen consumption rate, respectively.
FINDINGS/RESULTS
CCA-1.1 exerted cytotoxic activity and inhibited cell proliferation with an irreversible effect, and the flow cytometry analysis demonstrated that CCA-1.1 significantly halted during the G2/M phase, and further assessment revealed that CCA-1.1 caused metaphase arrest. Immunoblot assays confirmed CCA-1.1 suppressed aurora A kinase in MDA-MB-231 cells. The ROS level was elevated after treatment with CCA-1.1, which might promote cellular senescence and suppress basal mitochondrial respiration in MDA-MB-231 cells.
CONCLUSION AND IMPLICATIONS
Our data suggested the proof-of-concept that supports the involvement in cell cycle regulation and ROS generation as contributors to the effectiveness of CCA-1.1 in suppressing breast cancer cell growth.
PubMed: 37614620
DOI: 10.4103/1735-5362.378083 -
Animals : An Open Access Journal From... Dec 2022This brief review is focused on the viviparous lizard (Lichtenstein, 1823), of the family Lacertidae, which possesses female heterogamety and multiple sex chromosomes... (Review)
Review
A Brief Review of Meiotic Chromosomes in Early Spermatogenesis and Oogenesis and Mitotic Chromosomes in the Viviparous Lizard (Squamata: Lacertidae) with Multiple Sex Chromosomes.
This brief review is focused on the viviparous lizard (Lichtenstein, 1823), of the family Lacertidae, which possesses female heterogamety and multiple sex chromosomes (male 2 = 36, ZZZZ/ZZW, female 2 = 35, with variable W sex chromosome). Multiple sex chromosomes and their changes may influence meiosis and the female meiotic drive, and they may play a role in reproductive isolation. In two cryptic taxa of with different W sex chromosomes, meiosis during early spermatogenesis and oogenesis proceeds normally, without any disturbances, with the formation of haploid spermatocytes, and in female meiosis with the formation of synaptonemal complexes (SCs) and the lampbrush chromosomes. In females, the SC number was constantly equal to 19 (according to the SC length, 16 SC autosomal bivalents plus three presumed SC sex chromosome elements). No variability in the chromosomes at the early stages of meiotic prophase I, and no significant disturbances in the chromosome segregation at the anaphase-telophase I stage, have been discovered, and haploid oocytes ( = 17) at the metaphase II stage have been revealed. There should be a factor/factors that maintain the multiple sex chromosomes, their equal transmission, and the course of meiosis in these cryptic forms of
PubMed: 36611629
DOI: 10.3390/ani13010019 -
The Journal of Cell Biology Dec 2021The dynein motor performs multiple functions in mitosis by engaging with a wide cargo spectrum. One way to regulate dynein's cargo-binding selectivity is through the...
The dynein motor performs multiple functions in mitosis by engaging with a wide cargo spectrum. One way to regulate dynein's cargo-binding selectivity is through the C-terminal domain (CTD) of its light intermediate chain 1 subunit (LIC1), which binds directly with cargo adaptors. Here we show that mitotic phosphorylation of LIC1-CTD at its three cdk1 sites is required for proper mitotic progression, for dynein loading onto prometaphase kinetochores, and for spindle assembly checkpoint inactivation in human cells. Mitotic LIC1-CTD phosphorylation also engages the prolyl isomerase Pin1 predominantly to Hook2-dynein-Nde1-Lis1 complexes, but not to dynein-spindly-dynactin complexes. LIC1-CTD dephosphorylation abrogates dynein-Pin1 binding, promotes prophase centrosome-nuclear envelope detachment, and impairs metaphase chromosome congression and mitotic Golgi fragmentation, without affecting interphase membrane transport. Phosphomutation of a conserved LIC1-CTD SP site in zebrafish leads to early developmental defects. Our work reveals that LIC1-CTD phosphorylation differentially regulates distinct mitotic dynein pools and suggests the evolutionary conservation of this phosphoregulation.
Topics: Animals; Cell Line, Tumor; Centrosome; Cytoplasmic Dyneins; Dynactin Complex; Evolution, Molecular; Golgi Apparatus; Humans; Interphase; Kinetochores; Metaphase; Microtubule-Associated Proteins; Mitosis; Mutant Proteins; NIMA-Interacting Peptidylprolyl Isomerase; Nuclear Envelope; Phosphorylation; Protein Binding; Protein Subunits; Rats; Zebrafish
PubMed: 34709360
DOI: 10.1083/jcb.202005184 -
Journal of Cell Science Jan 2021Errors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic...
Errors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic chromosomes must align at the metaphase plate with stable amphitelic attachments to microtubules emanating from opposing spindle poles. The astrin-kinastrin (astrin is also known as SPAG5 and kinastrin as SKAP) complex, also containing DYNLL1 and MYCBP, is a spindle and kinetochore protein complex with important roles in bipolar spindle formation, chromosome alignment and microtubule-kinetochore attachment. However, the molecular mechanisms by which astrin-kinastrin fulfils these diverse roles are not fully understood. Here, we characterise a direct interaction between astrin and the mitotic kinase Plk1. We identify the Plk1-binding site on astrin as well as four Plk1 phosphorylation sites on astrin. Regulation of astrin by Plk1 is dispensable for bipolar spindle formation and bulk chromosome congression, but promotes stable microtubule-kinetochore attachments and metaphase plate maintenance. It is known that Plk1 activity is required for effective microtubule-kinetochore attachment formation, and we suggest that astrin phosphorylation by Plk1 contributes to this process.
Topics: Alcian Blue; Cell Cycle Proteins; Chromosome Segregation; HeLa Cells; Humans; Kinetochores; Metaphase; Microtubule-Associated Proteins; Microtubules; Mitosis; Phenazines; Phenothiazines; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Resorcinols; Spindle Apparatus; Polo-Like Kinase 1
PubMed: 33288550
DOI: 10.1242/jcs.251025 -
Cellular and Molecular Life Sciences :... Oct 2023Induction of DNA damage response (DDR) to ensure accurate duplication of genetic information is crucial for maintaining genome integrity during DNA replication. Cellular...
Induction of DNA damage response (DDR) to ensure accurate duplication of genetic information is crucial for maintaining genome integrity during DNA replication. Cellular senescence is a DDR mechanism that prevents the proliferation of cells with damaged DNA to avoid mitotic anomalies and inheritance of the damage over cell generations. Human WWOX gene resides within a common fragile site FRA16D that is preferentially prone to form breaks on metaphase chromosome upon replication stress. We report here that primary Wwox knockout (Wwox) mouse embryonic fibroblasts (MEFs) and WWOX-knockdown human dermal fibroblasts failed to undergo replication-induced cellular senescence after multiple passages in vitro. Strikingly, by greater than 20 passages, accelerated cell cycle progression and increased apoptosis occurred in these late-passage Wwox MEFs. These cells exhibited γH2AX upregulation and microsatellite instability, indicating massive accumulation of nuclear DNA lesions. Ultraviolet radiation-induced premature senescence was also blocked by WWOX knockdown in human HEK293T cells. Mechanistically, overproduction of cytosolic reactive oxygen species caused p16 promoter hypermethylation, aberrant p53/p21 signaling axis and accelerated p27 protein degradation, thereby leading to the failure of senescence induction in Wwox-deficient cells after serial passage in culture. We determined that significantly reduced protein stability or loss-of-function A135P/V213G mutations in the DNA-binding domain of p53 caused defective induction of p21 in late-passage Wwox MEFs. Treatment of N-acetyl-L-cysteine prevented downregulation of cyclin-dependent kinase inhibitors and induced senescence in Wwox MEFs. Our findings support an important role for fragile WWOX gene in inducing cellular senescence for maintaining genome integrity during DDR through alleviating oxidative stress.
Topics: Animals; Humans; Mice; Cellular Senescence; DNA; Fibroblasts; Genomic Instability; HEK293 Cells; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Ultraviolet Rays; WW Domain-Containing Oxidoreductase
PubMed: 37897534
DOI: 10.1007/s00018-023-04950-1 -
Essays in Biochemistry Sep 2020While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of... (Review)
Review
While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of homolog segregation during meiosis I (MI). CENP-C is a critical component of the centromere for kinetochore assembly in mitosis. Recent work, however, has highlighted the unique features of meiotic CENP-C. Centromere establishment and stability require CENP-C loading at the centromere for CENP-A function. Pre-meiotic loading of proteins necessary for homolog recombination as well as cohesion also rely on CENP-C, as do the main scaffolding components of the kinetochore. Much of this work relies on new technologies that enable in vivo analysis of meiosis like never before. Here, we strive to highlight the unique role of this highly conserved centromere protein that loads on to centromeres prior to M-phase onset, but continues to perform critical functions through chromosome segregation. CENP-C is not merely a structural link between the centromere and the kinetochore, but also a functional one joining the processes of early prophase homolog synapsis to late metaphase kinetochore assembly and signaling.
Topics: Animals; Centromere; Chromosomal Proteins, Non-Histone; Drosophila; HeLa Cells; Humans; Kinetochores; Meiosis; Mice
PubMed: 32794572
DOI: 10.1042/EBC20190080 -
Cell Cycle (Georgetown, Tex.) Jul 2023Tightly controlled fluctuations in kinase and phosphatase activity play important roles in regulating M-phase transitions. Protein Phosphatase 1 (PP1) is one of these...
Tightly controlled fluctuations in kinase and phosphatase activity play important roles in regulating M-phase transitions. Protein Phosphatase 1 (PP1) is one of these phosphatases, with oscillations in PP1 activity driving mitotic M-phase. Evidence from a variety of experimental systems also points to roles in meiosis. Here, we report that PP1 is important for M-phase transitions through mouse oocyte meiosis. We employed a unique small-molecule approach to inhibit or activate PP1 at distinct phases of mouse oocyte meiosis. These studies show that temporal control of PP1 activity is essential for the G2/M transition, metaphase I/anaphase I transition, and the formation of a normal metaphase II oocyte. Our data also reveal that inappropriate activation of PP1 is more deleterious at the G2/M transition than at prometaphase I-to-metaphase I, and that an active pool of PP1 during prometaphase is vital for metaphase I/anaphase I transition and metaphase II chromosome alignment. Taken together, these results establish that loss of oscillations in PP1 activity causes a range of severe meiotic defects, pointing to essential roles for PP1 in female fertility, and more broadly, M-phase regulation.
Topics: Female; Mice; Animals; Meiosis; Oocytes; Metaphase; Anaphase; Mitosis; Protein Phosphatase 1; Mammals
PubMed: 37340734
DOI: 10.1080/15384101.2023.2225924 -
Scientific Reports Mar 2023Gloriosine, the predominant metabolite of Gloriosa superba L., shares chemical properties with colchicine. We analyze the microtubule-binding affinity of gloriosine at...
Gloriosine, the predominant metabolite of Gloriosa superba L., shares chemical properties with colchicine. We analyze the microtubule-binding affinity of gloriosine at the colchicine binding site (CBS) using an in silico-in vivo approach. The In silico docking of gloriosine showed a binding score of (-) 7.5 kcal/Mol towards β-tubulin at CBS and was validated by overlapping the coupling pose of the docked ligand with co-crystallized colchicine. 2D plots (Ligplot +) showed > 85% overlap between gloriosine and colchicine. The ADMET profile of gloriosine was in accordance with Lipinski's rule of five. Gloriosine belongs to class II toxicity with anLD value of 6 mg/kg. In vivo and transmission electron microscopy studies revealed that gloriosine induces abnormalities in cell division such as condensed chromosomes in C-metaphase and enlarged nucleus with increased nuclear material. Gloriosine treated cells exhibited mitotic index of about 14% compared to control of 24% and high anti-proliferative activity i.e. 63.94% cell viability at a low concentration (0.0004 mg/ml). We conclude that gloriosine has a strong affinity for β-tubulin at CBS and thus can be used as a colchicine alternative in cytology and other clinical conditions.
Topics: Colchicine; Tubulin; Microtubules; Binding Sites; Protein Binding
PubMed: 36964265
DOI: 10.1038/s41598-023-31187-6 -
Cells Jul 2021Glioblastoma multiforme (GBM) is characterized by several genetic abnormalities, leading to cell cycle deregulation and abnormal mitosis caused by a defective...
BACKGROUND
Glioblastoma multiforme (GBM) is characterized by several genetic abnormalities, leading to cell cycle deregulation and abnormal mitosis caused by a defective checkpoint. We previously demonstrated that arecaidine propargyl ester (APE), an orthosteric agonist of M2 muscarinic acetylcholine receptors (mAChRs), arrests the cell cycle of glioblastoma (GB) cells, reducing their survival. The aim of this work was to better characterize the molecular mechanisms responsible for this cell cycle arrest.
METHODS
The arrest of cell proliferation was evaluated by flow cytometry analysis. Using immunocytochemistry and time-lapse analysis, the percentage of abnormal mitosis and aberrant mitotic spindles were assessed in both cell lines. Western blot analysis was used to evaluate the modulation of Sirtuin2 and acetylated tubulin-factors involved in the control of cell cycle progression.
RESULTS
APE treatment caused arrest in the M phase, as indicated by the increase in p-HH3 (ser10)-positive cells. By immunocytochemistry, we found a significant increase in abnormal mitoses and multipolar mitotic spindle formation after APE treatment. Time-lapse analysis confirmed that the APE-treated GB cells were unable to correctly complete the mitosis. The modulated expression of SIRT2 and acetylated tubulin in APE-treated cells provides new insights into the mechanisms of altered mitotic progression in both GB cell lines.
CONCLUSIONS
Our data show that the M2 agonist increases aberrant mitosis in GB cell lines. These results strengthen the idea of considering M2 acetylcholine receptors a novel promising therapeutic target for the glioblastoma treatment.
Topics: Acetylation; Arecoline; Cell Line, Tumor; Cell Nucleus; Glioblastoma; Humans; Metaphase; Mitosis; Receptor, Muscarinic M2; Sirtuin 2; Spindle Apparatus; Time-Lapse Imaging; Tubulin
PubMed: 34359896
DOI: 10.3390/cells10071727 -
BMC Genetics Dec 2020Anastrepha fraterculus is recognized as a quarantine pest in several American countries. This fruit fly species is native to the American continent and distributed...
BACKGROUND
Anastrepha fraterculus is recognized as a quarantine pest in several American countries. This fruit fly species is native to the American continent and distributed throughout tropical and subtropical regions. It has been reported as a complex of cryptic species, and at least eight morphotypes have been described. Only one entity of this complex, formerly named Anastrepha fraterculus sp. 1, is present in Argentina. Previous cytogenetic studies on this morphotype described the presence of sex chromosome variation identified by chromosomal size and staining patterns. In this work, we expanded the cytological study of this morphotype by analyzing laboratory strains and wild populations to provide information about the frequency and geographic distribution of these sex chromosome variants. We analyzed the mitotic metaphases of individuals from four laboratory strains and five wild populations from the main fruit-producing areas of Argentina, including the northwest (Tucumán and La Rioja), northeast (Entre Ríos and Misiones), and center (Buenos Aires) of the country.
RESULTS
In wild samples, we observed a high frequency of XX (0.94) and XY (0.93) karyomorphs, whereas XX and XY were exclusively found at a low frequency in Buenos Aires (0.07 and 0.13, respectively), Entre Ríos (0.16 and 0.14, respectively) and Tucumán (0.03 and 0.04, respectively). XX and XY karyomorphs were not found in wild populations but were detected at a low frequency in laboratory strains. In fact, karyomorph frequencies differed between wild populations and laboratory strains. No significant differences among A. fraterculus wild populations were evidenced in either karyotypic or chromosomal frequencies. However, a significant correlation was observed between Y chromosomal frequency and latitude.
CONCLUSIONS
We discuss the importance of cytogenetics to understand the possible route of invasion and dispersion of this pest in Argentina and the evolutionary forces acting under laboratory conditions, possibly driving changes in the chromosomal frequencies. Our findings provide deep and integral genetic knowledge of this species, which has become of relevance to the characterization and selection of valuable A. fraterculus sp. 1 strains for mass rearing production and SIT implementation.
Topics: Animals; Argentina; Chromosomes, Insect; Female; Genetics, Population; Geography; Karyotyping; Male; Polymorphism, Genetic; Sex Chromosomes; Tephritidae
PubMed: 33339514
DOI: 10.1186/s12863-020-00944-1