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1.
Zoolog Sci ; 38(5): 436-443, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34664918

RESUMO

Here, we report that the gross morphology of the testes changes under 'non-mating' or 'mating' conditions in medaka (Oryzias latipes). During these conditions, an efferent duct expands and a histological unit of spermatogenesis, the lobule, increases its number under 'non-mating' conditions. Based on BrdU labeling experiments, lower mitotic activity occurs in gonial cells under 'non-mating' conditions, which is consistent with the reduced number of germ cell cysts. Interestingly, the total number of type A spermatogonia was maintained, regardless of the mating conditions. In addition, the transition from mitosis to meiosis may have been retarded under the 'non-mating' conditions. The minimum time required for germ cells to become sperm, from the onset of commitment to spermatogenesis, was approximately 14 days in vivo. The time was not found to significantly differ between 'non-mating' and 'mating' conditions. The collective data suggest the presence of a mechanism wherein the homeostasis of spermatogenesis is altered in response to the mating conditions.


Assuntos
Oryzias/fisiologia , Espermatogênese/fisiologia , Testículo/fisiologia , Animais , Copulação , Feminino , Masculino , Meiose , Mitose , Oryzias/anatomia & histologia , Testículo/anatomia & histologia
2.
Nat Commun ; 12(1): 5931, 2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34635673

RESUMO

The chromatin remodeler RSF1 enriched at mitotic centromeres is essential for proper chromosome alignment and segregation and underlying mechanisms remain to be disclosed. We here show that PLK1 recruitment by RSF1 at centromeres creates an activating phosphorylation on Thr236 in the activation loop of Aurora B and this is indispensable for the Aurora B activation. In structural modeling the phosphorylated Thr236 enhances the base catalysis by Asp200 nearby, facilitating the Thr232 autophosphorylation. Accordingly, RSF1-PLK1 is central for Aurora B-mediated microtubule destabilization in error correction. However, under full microtubule-kinetochore attachment RSF1-PLK1 positions at kinetochores, halts activating Aurora B and phosphorylates BubR1, regardless of tension. Spatial movement of RSF1-PLK1 to kinetochores is triggered by Aurora B-mediated phosphorylation of centromeric histone H3 on Ser28. We propose a regulatory RSF1-PLK1 axis that spatiotemporally controls on/off switch on Aurora B. This feedback circuit among RSF1-PLK1-Aurora B may coordinate dynamic microtubule-kinetochore attachment in early mitosis when full tension yet to be generated.


Assuntos
Aurora Quinase B/genética , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos , Mitose , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/genética , Transativadores/genética , Ácido Aspártico/metabolismo , Aurora Quinase B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Proteínas Nucleares/deficiência , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Serina/metabolismo , Transativadores/deficiência
3.
PLoS One ; 16(10): e0258156, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34624021

RESUMO

Telomeres are nucleoprotein complexes that protect the ends of eukaryotic linear chromosomes from degradation and fusions. Telomere dysfunction leads to cell growth arrest, oncogenesis, and premature aging. Telomeric RNAs have been found in all studied species; however, their functions and biogenesis are not clearly understood. We studied the mechanisms of development disorders observed upon overexpression of telomeric repeats in Drosophila. In somatic cells, overexpression of telomeric retrotransposon HeT-A is cytotoxic and leads to the accumulation of HeT-A Gag near centrosomes. We found that RNA and RNA-binding protein Gag encoded by the telomeric retrotransposon HeT-A interact with Polo and Cdk1 mitotic kinases, which are conserved regulators of centrosome biogenesis and cell cycle. The depletion of proteins Spindle E, Ccr4 or Ars2 resulting in HeT-A overexpression in the germline was accompanied by mislocalization of Polo as well as its abnormal stabilization during oogenesis and severe deregulation of centrosome biogenesis leading to maternal-effect embryonic lethality. These data suggest a mechanistic link between telomeric HeT-A ribonucleoproteins and cell cycle regulators that ensures the cell response to telomere dysfunction.


Assuntos
Centrossomo/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Desenvolvimento Embrionário , Oogênese , Proteínas Serina-Treonina Quinases/metabolismo , Telômero/metabolismo , Animais , Morte Celular , Centríolos/metabolismo , Embrião não Mamífero/metabolismo , Mitose , Ligação Proteica , RNA/metabolismo , Retroelementos/genética , Ribonucleoproteínas/metabolismo , Zigoto/metabolismo
4.
Elife ; 102021 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-34677125

RESUMO

Retinal progenitor cells (RPCs) divide in limited numbers to generate the cells comprising vertebrate retina. The molecular mechanism that leads RPC to the division limit, however, remains elusive. Here, we find that the hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) in an RPC subset by deletion of tuberous sclerosis complex 1 (Tsc1) makes the RPCs arrive at the division limit precociously and produce Müller glia (MG) that degenerate from senescence-associated cell death. We further show the hyperproliferation of Tsc1-deficient RPCs and the degeneration of MG in the mouse retina disappear by concomitant deletion of hypoxia-induced factor 1-alpha (Hif1a), which induces glycolytic gene expression to support mTORC1-induced RPC proliferation. Collectively, our results suggest that, by having mTORC1 constitutively active, an RPC divides and exhausts mitotic capacity faster than neighboring RPCs, and thus produces retinal cells that degenerate with aging-related changes.


Assuntos
Células Ependimogliais/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Retina/patologia , Células-Tronco/patologia , Proteína 1 do Complexo Esclerose Tuberosa/genética , Animais , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Mitose , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo
5.
FEBS Lett ; 595(21): 2675-2690, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34626438

RESUMO

14-3-3 proteins are conserved, dimeric, acidic proteins that regulate multiple cellular pathways. Loss of either 14-3-3ε or 14-3-3γ leads to centrosome amplification. However, we find that while the knockout of 14-3-3ε leads to multipolar mitoses, the knockout of 14-3-3γ results in centrosome clustering and pseudo-bipolar mitoses. 14-3-3γ knockouts demonstrate compromised desmosome function and a decrease in keratin levels, leading to decreased cell stiffness and an increase in centrosome clustering. Restoration of desmosome function increased multipolar mitoses, whereas knockdown of either plakoglobin or keratin 5 led to decreased cell stiffness and increased pseudo-bipolar mitoses. These results suggest that the ability of the desmosome to anchor keratin filaments maintains cell stiffness, thus inhibiting centrosome clustering, and that phenotypes observed upon 14-3-3 loss reflect the dysregulation of multiple pathways.


Assuntos
Proteínas 14-3-3 , Centrossomo , Desmossomos , Mitose , Células HCT116 , Humanos , Fuso Acromático
6.
Nat Cell Biol ; 23(10): 1095-1104, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34616022

RESUMO

BRCA2-mutant cells are defective in homologous recombination, making them vulnerable to the inactivation of other pathways for the repair of DNA double-strand breaks (DSBs). This concept can be clinically exploited but is currently limited due to insufficient knowledge about how DSBs are repaired in the absence of BRCA2. We show that DNA polymerase θ (POLθ)-mediated end joining (TMEJ) repairs DSBs arising during the S phase in BRCA2-deficient cells only after the onset of the ensuing mitosis. This process is regulated by RAD52, whose loss causes the premature usage of TMEJ and the formation of chromosomal fusions. Purified RAD52 and BRCA2 proteins both block the DNA polymerase function of POLθ, suggesting a mechanism explaining their synthetic lethal relationships. We propose that the delay of TMEJ until mitosis ensures the conversion of originally one-ended DSBs into two-ended DSBs. Mitotic chromatin condensation might further serve to juxtapose correct break ends and limit chromosomal fusions.


Assuntos
Proteína BRCA2/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Polimerase Dirigida por DNA/metabolismo , Recombinação Homóloga , Mitose , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteína BRCA2/genética , Ciclo Celular , DNA Polimerase Dirigida por DNA/genética , Células HeLa , Humanos , Proteína Rad52 de Recombinação e Reparo de DNA/genética
7.
Life Sci ; 285: 120006, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34606852

RESUMO

Neurodegenerative diseases (NDDs) are the most common life-threatening disease of the central nervous system and it cause the progressive loss of neuronal cells. The exact mechanism of the disease's progression is not clear and thus line of treatment for NDDs is a baffling issue. During the progression of NDDs, oxidative stress and DNA damage play an important regulatory function, and ultimately induces neurodegeneration. Recently, aberrant cell cycle events have been demonstrated in the progression of different NDDs. However, the pertinent role of signaling mechanism, for instance, post-translational modifications, oxidative stress, DNA damage response pathway, JNK/p38 MAPK, MEK/ERK cascade, actively participated in the aberrant cell cycle reentry induced neuronal cell death. Mounting evidence has demonstrated that aberrant cell cycle re-entry is a major contributing factor in the pathogenesis of NDDs rather than a secondary phenomenon. In the brain of AD patients with mild cognitive impairment, post miotic cell division can be seen in the early stage of the disease. However, in the brain of PD patients, response to various neurotoxic signals, the cell cycle re-entry has been observed that causes neuronal apoptosis. On contrary, the contributing factors that leads to the induction of cell cycle events in mature neurons in HD and ALS brain pathology is remain unclear. Various pharmacological drugs have been developed to reduce the pathogenesis of NDDs, but they are still not helpful in eliminating the cause of these NDDs.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Mitose , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Neurônios/patologia , Acetilação , Animais , Histona Desacetilases/metabolismo , Humanos , Ubiquitina-Proteína Ligases/metabolismo
9.
BMC Cancer ; 21(1): 981, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34470602

RESUMO

BACKGROUND: Paclitaxel (Taxol) is a microtubule-stabilizing drug used to treat several solid tumors, including ovarian, breast, non-small cell lung, and pancreatic cancers. The current treatment of ovarian cancer is chemotherapy using paclitaxel in combination with carboplatin as a frontline agent, and paclitaxel is also used in salvage treatment as a second line drug with a dose intensive regimen following recurrence. More recently, a dose dense approach for paclitaxel has been used to treat metastatic breast cancer with success. Paclitaxel binds to beta tubulin with high affinity and stabilizes microtubule bundles. As a consequence of targeting microtubules, paclitaxel kills cancer cells through inhibition of mitosis, causing mitotic catastrophes, and by additional, not yet well defined non-mitotic mechanism(s). RESULTS: In exploring methods to modulate activity of paclitaxel in causing cancer cell death, we unexpectedly found that a brief exposure of paclitaxel-treated cells in culture to low intensity ultrasound waves prevented the paclitaxel-induced cytotoxicity and death of the cancer cells. The treatment with ultrasound shock waves was found to transiently disrupt the microtubule cytoskeleton and to eliminate paclitaxel-induced rigid microtubule bundles. When cellular microtubules were labelled with a fluorescent paclitaxel analog, exposure to ultrasound waves led to the disassembly of the labeled microtubules and localization of the signals to perinuclear compartments, which were determined to be lysosomes. CONCLUSIONS: We suggest that ultrasound disrupts the paclitaxel-induced rigid microtubule cytoskeleton, generating paclitaxel bound fragments that undergo degradation. A new microtubule network forms from tubulins that are not bound by paclitaxel. Hence, ultrasound shock waves are able to abolish paclitaxel impact on microtubules. Thus, our results demonstrate that a brief exposure to low intensity ultrasound can reduce and/or eliminate cytotoxicity associated with paclitaxel treatment of cancer cells in cultures.


Assuntos
Neoplasias da Mama/patologia , Microtúbulos/patologia , Mitose , Neoplasias Ovarianas/patologia , Paclitaxel/farmacologia , Ondas Ultrassônicas , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/radioterapia , Proliferação de Células , Citoesqueleto/metabolismo , Feminino , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/efeitos da radiação , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/radioterapia , Tubulina (Proteína)/metabolismo , Células Tumorais Cultivadas
10.
FASEB J ; 35(10): e21923, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34551143

RESUMO

Our recent studies have shown that haspin, a protein kinase imperative for mitosis, is engaged in the interphase progression of HeLa and U2OS cancer cells. In this investigation, we employed the Fucci reporter system and time-lapse imaging to examine the impact of haspin gene silencing on cell cycle progressions at a single-cell level. We found that the loss of haspin induced multiple cell cycle defects. Specifically, the S/G2 duration was greatly prolonged by haspin gene depletion or inhibition in synchronous HeLa cells. Haspin gene depletion in asynchronous HeLa and U2OS cells led to a similarly protracted S/G2 phase, followed by mitotic cell death or postmitotic G1 arrest. In addition, haspin deficiency resulted in robust induction of the p21CIP1/WAF1 checkpoint protein, a target of the p53 activation. Also, co-depleting haspin with either p21 or p53 could rescue U2OS cells from postmitotic G1 arrest and partially restore their proliferation. These results substantiate the haspin's capacity to regulate interphase and mitotic progression, offering a broader antiproliferative potential of haspin loss in cancer cells.


Assuntos
Ciclo Celular , Proliferação de Células , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/deficiência , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Corantes Fluorescentes , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Humanos , Interfase/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitose/efeitos dos fármacos , Neoplasias/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Fase S/efeitos dos fármacos , Tubercidina/análogos & derivados , Tubercidina/farmacologia , Proteína Supressora de Tumor p53/genética , Ubiquitinação , Regulação para Cima/efeitos dos fármacos
11.
Cells ; 10(9)2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34572033

RESUMO

The Dictyostelium centrosome is a nucleus-associated body with a diameter of approx. 500 nm. It contains no centrioles but consists of a cylindrical layered core structure surrounded by a microtubule-nucleating corona. At the onset of mitosis, the corona disassembles and the core structure duplicates through growth, splitting, and reorganization of the outer core layers. During the last decades our research group has characterized the majority of the 42 known centrosomal proteins. In this work we focus on the conserved, previously uncharacterized Cep192 protein. We use superresolution expansion microscopy (ExM) to show that Cep192 is a component of the outer core layers. Furthermore, ExM with centrosomal marker proteins nicely mirrored all ultrastructurally known centrosomal substructures. Furthermore, we improved the proximity-dependent biotin identification assay (BioID) by adapting the biotinylase BioID2 for expression in Dictyostelium and applying a knock-in strategy for the expression of BioID2-tagged centrosomal fusion proteins. Thus, we were able to identify various centrosomal Cep192 interaction partners, including CDK5RAP2, which was previously allocated to the inner corona structure, and several core components. Studies employing overexpression of GFP-Cep192 as well as depletion of endogenous Cep192 revealed that Cep192 is a key protein for the recruitment of corona components during centrosome biogenesis and is required to maintain a stable corona structure.


Assuntos
Centrossomo/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Dictyostelium/metabolismo , Microtúbulos/metabolismo , Mitose , Proteínas de Protozoários/metabolismo , Proteínas Cromossômicas não Histona/genética , Dictyostelium/crescimento & desenvolvimento , Proteínas de Protozoários/genética , Fuso Acromático
12.
Cells ; 10(9)2021 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-34571889

RESUMO

Aberrant centrosome activities in mutants of Dictyostelium discoideum result in anomalies of mitotic spindles that affect the reliability of chromosome segregation. Genetic instabilities caused by these deficiencies are tolerated in multinucleate cells, which can be produced by electric-pulse induced cell fusion as a source for aberrations in the mitotic apparatus of the mutant cells. Dual-color fluorescence labeling of the microtubule system and the chromosomes in live cells revealed the variability of spindle arrangements, of centrosome-nuclear interactions, and of chromosome segregation in the atypical mitoses observed.


Assuntos
Segregação de Cromossomos , Dictyostelium/genética , Instabilidade Genômica , Mitose , Mutação , Fuso Acromático/genética , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Fuso Acromático/patologia , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo
13.
Cell Mol Life Sci ; 78(21-22): 6775-6795, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34476544

RESUMO

The centrosome is a tiny cytoplasmic organelle that organizes and constructs massive molecular machines to coordinate diverse cellular processes. Due to its many roles during both interphase and mitosis, maintaining centrosome homeostasis is essential to normal health and development. Centrosome instability, divergence from normal centrosome number and structure, is a common pathognomonic cellular state tightly associated with cancers and other genetic diseases. As novel connections are investigated linking the centrosome to disease, it is critical to understand the breadth of centrosome functions to inspire discovery. In this review, we provide an introduction to normal centrosome function and highlight recent discoveries that link centrosome instability to specific disease states.


Assuntos
Centrossomo/fisiologia , Instabilidade Cromossômica/genética , Animais , Doenças Genéticas Inatas/genética , Humanos , Interfase/genética , Mitose/genética , Neoplasias/genética , Organelas/genética
14.
Cell Prolif ; 54(11): e13110, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34592789

RESUMO

OBJECTIVES: Besides its role in regulating phosphatidylinositol-3 kinase (PI3K) signalling in the cytosol, PTEN also has a nuclear function. In this study, we attempted to understand the mechanism of chromatin PTEN in suppressing chromosomal instability during cell division. MATERIALS AND METHODS: Immunocoprecipitation, ectopic expression, and deletional analyses were used to identify the physical interaction between Chromobox Homolog protein 8 (CBX8) and PTEN, as well as the functional domain(s) of PTEN mediating the interaction. Cell synchronization followed by immunoblotting was employed to study cell cycle regulation of CBX8 and the functional interaction between chromatin PTEN and CBX8. Small interfering RNAs (siRNAs) were used to study the role of PTEN and CBX8 in modulating histone epigenetic markers during the cell cycle. RESULTS: Polycomb group (PcG) proteins including CBXs function to repress gene expression in a wide range of organisms including mammals. We recently showed that PTEN interacted with CBX8, a component of Polycomb Repressing Complex 1 (PRC1), and that CBX8 co-localized with PTEN in the nucleus. CBX8 levels were high, coinciding with its phosphorylation in mitosis. Phosphorylation of CBX8 was associated with monoubiquitinated PTEN and phosphorylated-BubR1 on chromatin. Moreover, CBX8 played an important role in cell proliferation and mitotic progression. Significantly, downregulation of either PTEN or CBX8 induced H3K27Me3 epigenetic marker in mitotic cells. CONCLUSION: CBX8 is a new component that physically interacts with chromatin PTEN, playing an important role in regulating mitotic progression.


Assuntos
Proliferação de Células/fisiologia , Mitose/fisiologia , PTEN Fosfo-Hidrolase/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Ciclo Celular/fisiologia , Cromatina/metabolismo , Histonas/metabolismo , Humanos , Fosforilação , Complexo Repressor Polycomb 1/genética , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo
15.
J Cell Sci ; 134(19)2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34518877

RESUMO

At mitotic exit the cell cycle engine is reset to allow crucial processes, such as cytokinesis and replication origin licensing, to take place before a new cell cycle begins. In budding yeast, the cell cycle clock is reset by a Hippo-like kinase cascade called the mitotic exit network (MEN), whose activation is triggered at spindle pole bodies (SPBs) by the Tem1 GTPase. Yet, MEN activity must be extinguished once MEN-dependent processes have been accomplished. One factor contributing to switching off the MEN is the Amn1 protein, which binds Tem1 and inhibits it through an unknown mechanism. Here, we show that Amn1 downregulates Tem1 through a dual mode of action. On one side, it evicts Tem1 from SPBs and escorts it into the nucleus. On the other, it promotes Tem1 degradation as part of a Skp, Cullin and F-box-containing (SCF) ubiquitin ligase. Tem1 inhibition by Amn1 takes place after cytokinesis in the bud-derived daughter cell, consistent with its asymmetric appearance in the daughter cell versus the mother cell. This dual mechanism of Tem1 inhibition by Amn1 may contribute to the rapid extinguishing of MEN activity once it has fulfilled its functions.


Assuntos
Proteínas Monoméricas de Ligação ao GTP , Proteínas de Saccharomyces cerevisiae , Transporte Ativo do Núcleo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citocinese , Regulação para Baixo/genética , Humanos , Mitose , Proteínas Monoméricas de Ligação ao GTP/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo
16.
Molecules ; 26(18)2021 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-34577149

RESUMO

Aurora-A kinase, a key mitosis regulator, is expressed in a cell cycle-dependent manner and has an essential role in maintaining chromosomal stability and the normal progression of the cell through mitosis. Aurora-A kinase is overexpressed in many malignant solid tumors, such as breast, ovarian, colon, and pancreatic cancers. Thus, inhibiting Aurora-A kinase activity is a promising approach for cancer treatment. Here, new triazole derivatives were designed as bioisosteric analogues of the known inhibitor JNJ-7706621. The new compounds showed interesting inhibitory activity against Aurora-A kinase, as attested by IC50s in the low to submicromolar range.


Assuntos
Aurora Quinase A , Inibidores de Proteínas Quinases , Triazóis , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células HeLa , Humanos , Mitose/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(4): 1050-1055, 2021 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-34362481

RESUMO

OBJECTIVE: To investigate the effect of sulforaphane (SFN) on G2/M phase arrest of acute myeloid leukemia cells and its molecular mechanism. METHODS: KG1a and KG1cells were treated by different concentrations of SFN for 48 h. Flow cytometry (FCM) was used to analyze the phase distribution of cell cycle. High-throughput sequencing was used to detect the effect of SFN on the expression of cell cycle related genes in KG1a cells. The mRNA expression of P53, P21, CDC2 and CyclinB1 were detected by qPCR. The protein expression of P53, CDC2, P-CDC2 and CyclinB1 were detected by Western blot. RESULTS: Cells in the G2/M phase were increased from 11.9% to 54.0% in KG1a cells and 18.5% to 83.3% in KG1 cells after treated by SFN (8 µ mol / L) for 48 hours(P<0.001). KEGG analysis indicated that P53 pathway was enriched in KG1a cells after treated by SFN. The heat-map graph showed that SFN could change the relevant genes of the cell cycle in KG1a cells. After SFN treatment, the mRNA level of P53 and P21 were significantly increased in KG1 and KG1a cells(P<0.05 or P<0.01). The mRNA level of CDC2 showed a decrease trend with the increasing dose of SFN. At the dosage of 8 µmol /L, the mRNA expression levels of CDC2 was significantly lower than that in control group(P<0.05). At the same time, the protein level of P53 was significantly increased in KG1 and kG1a cells after treated by SFN(P<0.05). The protein level of CDC2 showed a decrease trend with the increasing dose of SFN in a dose manner(r=0.9482 and r=0.8977). The protein levels of CDC2 in SFN 8 and 12 µ mol/L groups were significantly lower than that in control group(P<0.05, P<0.01). The protein levels of P-CDC2 was increased. But the change of mRNA and protein level of CyclinB1 was not significant. CONCLUSION: SFN induces leukemia cells to block in G2/M phase by activating P53 signaling pathway, which can inhibit the expression of CDC2 and the activity of CDC2/cyclinB1.


Assuntos
Isotiocianatos , Leucemia Mieloide Aguda , Ciclo Celular , Humanos , Isotiocianatos/farmacologia , Mitose , Sulfóxidos
18.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(4): 1129-1135, 2021 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-34362492

RESUMO

OBJECTIVE: To investigate the effect of CDK1 interference regulation of PLK1, Aurora B and TRF1 on the proliferation of leukemia cells. METHODS: The human myelogenous leukemia cell line HL-60 was selected as the research object, and the effect of TRF1 expression and its changes on cell proliferation and cycle was investigated by regulating intracellular CDK1 expression. The objects were divided into 5 groups, including control group, shRNA-NC group, CDK1-shRNA group, pcDNA group and pcDNA-CDK1 group. RT-PCR was used to detect the CDK1 expression of cells in each group; colony formation was used to detect the proliferation of the cells. Western blot was used to detect the expression of CDK1, PLK1, Aurora B, TRF1, and cyclin p53, p27, cyclinA. RESULTS: The phosphorylation level of PLK1, Aurora B and the expression of TRF1 in the CDK1-shRNA group were significantly down-regulated as compared with those in the control group (P<0.05). Compared with the control group, the cells in CDK1-shRNA group showed lower clone formation rate, the increasing of cycle-associated proteins p53 and p27 and the decreasing of cyclinA expression (P<0.05). It was shown that interfered CDK1 expression could inhibit the proliferation of HL-60 cells and prolong the time that they enter mitosis, thereby extending the cell cycle. Compared with the control group, the overexpressed CDK1 in the pcDNA-CDK1 group made the phosphorylation level of PLK1, Aurora B, and TRF1 expression increase significantly (P<0.05), also the colony formation rate (P<0.05). The cycle-related proteins p53 and p27 was down-regulated, while cyclinA expression was up-regulate significantly (P<0.05). The results indicted that overexpressed CDK1 could stimulate adverse reactions, thereby promoting the proliferation of HL-60 cells and shortening the cell cycle. CONCLUSION: Knocking out CDK1 can inhibit the phosphorylation of PLK1 and Aurora B and negatively regulate TRF1, thereby inhibiting the proliferation of leukemia cells.


Assuntos
Proteínas de Ciclo Celular , Leucemia , Proteína Quinase CDC2 , Proteínas de Ciclo Celular/genética , Proliferação de Células , Humanos , Mitose , Fosforilação , Proteínas Proto-Oncogênicas/genética
19.
Toxicol Lett ; 350: 240-248, 2021 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-34333065

RESUMO

Certain medicines including anticancer drugs, NSAIDs and antiepileptic drugs are known to cause drug-induced nephropathy. For example, antiepileptic drugs such as carbamazepine (CBZ) and valproic acid have been reported to cause damage to the proximal tubular cells. Although there has been a great deal of research concerning the nephrotoxicity of CBZ, little is known about that of oxcarbazepine (OXC), a derivative of CBZ. To investigate the molecular mechanism underlying renal proximal tubular cell death caused by OXC, we examined alterations in the gene expression profile of NRK-52E proximal tubular cells during OXC exposure. DNA microarray analysis revealed that the levels of genes related to mitotic processes including chromosomal and cytoplasmic segregation, progression to G2/M phase, and formation of the mitotic spindle are increased after exposure to 50 µM OXC for 6 h. Cell cycle analysis by flow cytometry showed that OXC at concentrations between 25 and 100 µM induces G2/M arrest. We also found that OXC significantly increases histone H3 phosphorylation, indicative of mitotic cells. These results imply that OXC induces cell cycle arrest at the mitotic phase. Immunofluorescence analysis showed monopolar spindles, which are formed in response to centrosome separation defects, in OXC-treated cells. We also show that OXC suppresses the phosphorylation of PLK1, which is involved not only in the activation of the kinesin family of motor proteins for centrosome separation and bipolar spindle assembly, but also in the cleavage of centrosomal proteins. Thus, our results indicate that OXC inhibits centrosome separation by reducing the activation of PLK1, which leads to the formation of an abnormal spindle and induces mitotic catastrophe and apoptosis in NRK-52E cells.


Assuntos
Anticonvulsivantes/efeitos adversos , Anticonvulsivantes/uso terapêutico , Apoptose/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Epilepsia/tratamento farmacológico , Túbulos Renais Distais/efeitos dos fármacos , Mitose/efeitos dos fármacos , Oxcarbazepina/toxicidade , Animais , Humanos , Modelos Animais , Ratos
20.
Cytogenet Genome Res ; 161(5): 249-256, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34433167

RESUMO

B chromosomes occur in different species of the small characid fishes of the genus Moenkhausia. These supernumerary elements, that do not recombine with chromosomes of the standard A complement and follow their own evolutionary mechanism vary in number, morphology, and distribution. Here, we show karyotypic data of individuals of 2 populations of Moenkhausia oligolepis of the Brazilian Amazon (Pedro Correia and Taboquinha streams, Tocantins river basin), both with a diploid number of 50 chromosomes and karyotypic formula of 10m + 32sm + 8a. In addition to the normal complement, we also observed the occurrence of B chromosomes in the 2 populations with intra- and interindividual variation ranging from 0 to 10 Bs, independent of sex. The C-banding pattern evidenced heterochromatic blocks located mainly in the pericentromeric region of the chromosomes, while the B chromosomes appeared euchromatic. Silver-stained nucleolus organizer regions were identified in multiples sites, and some of these blocks were positive when stained with chromomycin A3. The karyotype analysis and the application of whole-chromosome painting in populations of M. oligolepis reinforce the conservation of the basal diploid number for the genus, as well as the evolutionary tendency in these fishes to carry B chromosomes. Both populations turned out to be in different stages of stability and expansion of their B chromosomes. We further suggest that the origin of these chromosomes is due to the formation of isochromosomes. Here, we identified a pair of complement A chromosomes involved in this process.


Assuntos
Characidae/genética , Instabilidade Cromossômica , Cromossomos/química , Cariotipagem/métodos , Animais , Brasil , Cromomicina A3/química , Bandeamento Cromossômico , Mapeamento Cromossômico , Feminino , Corantes Fluorescentes/química , Hibridização in Situ Fluorescente , Cariótipo , Masculino , Mitose , Ploidias
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