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2.
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
3.
Molecules ; 26(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34443613

RESUMO

Adipogenesis is a complex process in which cell commitment and mitotic clonal expansion (MCE) are in-sequence crucial events leading to terminal adipocyte differentiation. The molecules able to block some key signals in this cascade can hamper adipogenesis becoming promising agents to counteract hyperplasia and hypertrophy of adipose tissue. Mono- and di-caffeoylquinic acid isomers are biologically active polyphenols, displaying in vitro and in vivo antioxidant, hepatoprotective, anti-diabetic and anti-obesity properties. Among these isomers, 3,5-dicaffeoylquinic acid (DCQA) has been reported to inhibit lipid accumulation in adipose cells more successfully than others. Thus, we investigated DCQA effects and molecular mechanisms on 3T3-L1 pre-adipocytes induced to differentiate with a hormonal cocktail (MDI). Oil Red O incorporation assessed that DCQA pre-treatment inhibited lipid accumulation in 3T3-L1 cells induced to differentiate for 10 days. At this time, an increased phosphorylation of both AMP-activated kinase and acetyl-CoA carboxylase, as well as a strong decrease in fatty acid synthase protein level, were registered by immunoblotting, thereby suggesting that DCQA treatment can reduce fatty acid anabolism in 3T3-L1 adipocytes. Furthermore, BrdU incorporation assay, performed 48 h after hormonal stimulation, revealed that DCQA treatment was also able to hinder the 3T3-L1 cell proliferation during the MCE, which is an essential step in the adipogenic process. Thus, we focused our attention on early signals triggered by the differentiation stimuli. In the first hours after hormonal cocktail administration, the activation of ERK1/2 and Akt kinases, or CREB and STAT3 transcription factors, was not affected by DCQA pre-treatment. Whereas 24 h after MDI induction, DCQA pre-treated cells showed increased level of the transcription factor Nrf2, that induced the expression of the antioxidant enzyme heme oxygenase 1 (HO-1). In control samples, the expression level of HO-1 was reduced 24 h after MDI induction in comparison with the higher amount of HO-1 protein found at 2 h. The HO-1 decrease was functional by allowing reactive oxygen species to boost and allowing cell proliferation induction at the beginning of MCE phase. Instead, in DCQA-treated cells the HO-1 expression was maintained at high levels for a further 24 h; in fact, its expression decreased only 48 h after MDI stimulation. The longer period in which HO-1 expression remained high led to a delay of the MCE phase, with a subsequent inhibition of both C/EBP-α expression and adipocyte terminal differentiation. In conclusion, DCQA counteracting an excessive adipose tissue expansion may become an attractive option in obesity treatment.


Assuntos
Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Ácido Clorogênico/análogos & derivados , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Mitose/efeitos dos fármacos , Células 3T3-L1 , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Animais , Ácido Clorogênico/farmacologia , Camundongos
4.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445496

RESUMO

Post-translational modification of the DNA replication machinery by ubiquitin and SUMO plays key roles in the faithful duplication of the genetic information. Among other functions, ubiquitination and SUMOylation serve as signals for the extraction of factors from chromatin by the AAA ATPase VCP. In addition to the regulation of DNA replication initiation and elongation, we now know that ubiquitination mediates the disassembly of the replisome after DNA replication termination, a process that is essential to preserve genomic stability. Here, we review the recent evidence showing how active DNA replication restricts replisome ubiquitination to prevent the premature disassembly of the DNA replication machinery. Ubiquitination also mediates the removal of the replisome to allow DNA repair. Further, we discuss the interplay between ubiquitin-mediated replisome disassembly and the activation of CDK1 that is required to set up the transition from the S phase to mitosis. We propose the existence of a ubiquitin-CDK1 relay, where the disassembly of terminated replisomes increases CDK1 activity that, in turn, favors the ubiquitination and disassembly of more replisomes. This model has important implications for the mechanism of action of cancer therapies that induce the untimely activation of CDK1, thereby triggering premature replisome disassembly and DNA damage.


Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitina/metabolismo , Animais , Replicação do DNA , Humanos , Mitose , Processamento de Proteína Pós-Traducional
5.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34445214

RESUMO

Deubiquitinating enzymes play key roles in the precise modulation of Aurora B-an essential cell cycle regulator. The expression of Aurora B increases before the onset of mitosis and decreases during mitotic exit; an imbalance in these levels has a severe impact on the fate of the cell cycle. Dysregulation of Aurora B can lead to aberrant chromosomal segregation and accumulation of errors during mitosis, eventually resulting in cytokinesis failure. Thus, it is essential to identify the precise regulatory mechanisms that modulate Aurora B levels during the cell division cycle. Using a deubiquitinase knockout strategy, we identified USP48 as an important candidate that can regulate Aurora B protein levels during the normal cell cycle. Here, we report that USP48 interacts with and stabilizes the Aurora B protein. Furthermore, we showed that the deubiquitinating activity of USP48 helps to maintain the steady-state levels of Aurora B protein by regulating its half-life. Finally, USP48 knockout resulted in delayed progression of cell cycle due to accumulation of mitotic defects and ultimately cytokinesis failure, suggesting the role of USP48 in cell cycle regulation.


Assuntos
Aurora Quinase B/metabolismo , Citocinese , Mitose , Proteases Específicas de Ubiquitina/metabolismo , Aurora Quinase B/genética , Estabilidade Enzimática , Células HEK293 , Células HeLa , Humanos , Proteases Específicas de Ubiquitina/genética
6.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360748

RESUMO

Research on the budding yeast Saccharomyces cerevisiae has yielded fundamental discoveries on highly conserved biological pathways and yeast remains the best-studied eukaryotic cell in the world. Studies on the mitotic cell cycle and the discovery of cell cycle checkpoints in budding yeast has led to a detailed, although incomplete, understanding of eukaryotic cell cycle progression. In multicellular eukaryotic organisms, uncontrolled aberrant cell division is the defining feature of cancer. Some of the most successful classes of anti-cancer chemotherapeutic agents are mitotic poisons. Mitotic poisons are thought to function by inducing a mitotic spindle checkpoint-dependent cell cycle arrest, via the assembly of the highly conserved mitotic checkpoint complex (MCC), leading to apoptosis. Even in the presence of mitotic poisons, some cancer cells continue cell division via 'mitotic slippage', which may correlate with a cancer becoming refractory to mitotic poison chemotherapeutic treatments. In this review, knowledge about budding yeast cell cycle control is explored to suggest novel potential drug targets, namely, specific regions in the highly conserved anaphase-promoting complex/cyclosome (APC/C) subunits Apc1 and/or Apc5, and in a specific N-terminal region in the APC/C co-factor cell division cycle 20 (Cdc20), which may yield molecules which block 'mitotic slippage' only in the presence of mitotic poisons.


Assuntos
Antineoplásicos/farmacologia , Apoptose , Pontos de Checagem do Ciclo Celular , Mitose , Neoplasias , Saccharomyces cerevisiae , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Apoptose/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Mitose/efeitos dos fármacos , Mitose/genética , Neoplasias/genética , Neoplasias/metabolismo , Venenos/química , Venenos/farmacologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
7.
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
8.
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
9.
Elife ; 102021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34388088

RESUMO

Neuronal cell fate determinants establish the identities of neurons by controlling gene expression to regulate neuronal morphology and synaptic connectivity. However, it is not understood if neuronal cell fate determinants have postmitotic functions in synapse pattern formation. Here we identify a novel role for UNC-4 homeobox protein and its corepressor UNC-37/Groucho, in tiled synaptic patterning of the cholinergic motor neurons in Caenorhabditis elegans. We show that unc-4 is not required during neurogenesis but is required in the postmitotic neurons for proper synapse patterning. In contrast, unc-37 is required in both developing and postmitotic neurons. The synaptic tiling defects of unc-4 mutants are suppressed by bar-1/ß-catenin mutation, which positively regulates the expression of ceh-12/HB9. Ectopic ceh-12 expression partly underlies the synaptic tiling defects of unc-4 and unc-37 mutants. Our results reveal a novel postmitotic role of neuronal cell fate determinants in synapse pattern formation through inhibiting the canonical Wnt signaling pathway.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Expressão Gênica , Genes Homeobox , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Correpressoras/genética , Proteínas de Homeodomínio/metabolismo , Mitose , Neurônios/fisiologia , Sinapses/fisiologia , Fatores de Transcrição/metabolismo
10.
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
11.
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
12.
Nat Commun ; 12(1): 5157, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34453048

RESUMO

During mitosis, transcription is globally attenuated and chromatin architecture is dramatically reconfigured. We exploited the M- to G1-phase progression to interrogate the contributions of the architectural factor CTCF and the process of transcription to genome re-sculpting in newborn nuclei. Depletion of CTCF during the M- to G1-phase transition alters short-range compartmentalization after mitosis. Chromatin domain boundary re-formation is impaired upon CTCF loss, but a subset of boundaries, characterized by transitions in chromatin states, is established normally. Without CTCF, structural loops fail to form, leading to illegitimate contacts between cis-regulatory elements (CREs). Transient CRE contacts that are normally resolved after telophase persist deeply into G1-phase in CTCF-depleted cells. CTCF loss-associated gains in transcription are often linked to increased, normally illegitimate enhancer-promoter contacts. In contrast, at genes whose expression declines upon CTCF loss, CTCF seems to function as a conventional transcription activator, independent of its architectural role. CTCF-anchored structural loops facilitate formation of CRE loops nested within them, especially those involving weak CREs. Transcription inhibition does not significantly affect global architecture or transcription start site-associated boundaries. However, ongoing transcription contributes considerably to the formation of gene domains, regions of enriched contacts along gene bodies. Notably, gene domains emerge in ana/telophase prior to completion of the first round of transcription, suggesting that epigenetic features in gene bodies contribute to genome reconfiguration prior to transcription. The focus on the de novo formation of nuclear architecture during G1 entry yields insights into the contributions of CTCF and transcription to chromatin architecture dynamics during the mitosis to G1-phase progression.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Mitose , Transcrição Genética , Animais , Fator de Ligação a CCCTC/genética , Divisão Celular , Linhagem Celular , Cromatina/genética , Montagem e Desmontagem da Cromatina , Elementos Facilitadores Genéticos , Camundongos , Regiões Promotoras Genéticas
13.
Cancer Sci ; 112(10): 4355-4364, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34375487

RESUMO

Mitosis is a prognostic factor for cutaneous melanoma (CM), but accurate mitosis detection in CM tissues is difficult. Therefore, the 8th Edition of the American Joint Committee on Cancer staging system has removed the mitotic rate as a category criterion of the tumor T-category, based on the evidence that the mitotic rate was not an independent prognostic factor for melanoma survival. As single-nucleotide polymorphisms (SNPs) have been shown to be potential predictors for cutaneous melanoma-specific survival (CMSS), we investigated the potential prognostic value of SNPs in mitosis-related pathway genes in CMSS by analyzing their associations with outcomes of 850 CM patients from The University of Texas MD Anderson Cancer Center in a discovery dataset and validated the findings in another dataset of 409 CM patients from the Harvard University Nurses' Health Study and Health Professionals Follow-up Study. In both datasets, we identified two SNPs (SDCCAG8 rs10803138 G>A and MAGI2 rs3807694 C>T) as independent prognostic factors for CMSS, with adjusted allelic hazards ratios of 1.49 (95% confidence interval = 1.17-1.90, P = .001) and 1.45 (1.13-1.86, P = .003), respectively. Furthermore, their combined unfavorable alleles also predicted a poor survival in both discovery and validation datasets in a dose-response manner (Ptrend  = .0006 and .0001, respectively). Additional functional analysis revealed that both SDCCAG8 rs10803138 A and MAGI2 rs3807694 T alleles were associated with elevated mRNA expression levels in normal tissues. Therefore, these findings suggest that SDCCAG8 rs10803138 G>A and MAGI2 rs3807694 C>T are independent prognostic biomarkers for CMSS, possibly by regulating the mRNA expression of the corresponding genes involved in mitosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Autoantígenos/genética , Variação Genética , Guanilato Quinases/genética , Melanoma/mortalidade , Mitose/genética , Proteínas de Neoplasias/genética , Neoplasias Cutâneas/mortalidade , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Intervalos de Confiança , Feminino , Humanos , Masculino , Melanoma/genética , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Curva ROC , Neoplasias Cutâneas/genética , Adulto Jovem
14.
Biomolecules ; 11(7)2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34356684

RESUMO

The maintenance of genome integrity in the cell is an essential process for the accurate transmission of the genetic material. BRCA2 participates in this process at several levels, including DNA repair by homologous recombination, protection of stalled replication forks, and cell division. These activities are regulated and coordinated via cell-cycle dependent modifications. Pathogenic variants in BRCA2 cause genome instability and are associated with breast and/or ovarian cancers. BRCA2 is a very large protein of 3418 amino acids. Most well-characterized variants causing a strong predisposition to cancer are mutated in the C-terminal 700 residues DNA binding domain of BRCA2. The rest of the BRCA2 protein is predicted to be disordered. Interactions involving intrinsically disordered regions (IDRs) remain difficult to identify both using bioinformatics tools and performing experimental assays. However, the lack of well-structured binding sites provides unique functional opportunities for BRCA2 to bind to a large set of partners in a tightly regulated manner. We here summarize the predictive and experimental arguments that support the presence of disorder in BRCA2. We describe how BRCA2 IDRs mediate self-assembly and binding to partners during DNA double-strand break repair, mitosis, and meiosis. We highlight how phosphorylation by DNA repair and cell-cycle kinases regulate these interactions. We finally discuss the impact of cancer-associated variants on the function of BRCA2 IDRs and more generally on genome stability and cancer risk.


Assuntos
Proteína BRCA2/química , Proteína BRCA2/metabolismo , Reparo do DNA/fisiologia , Proteína BRCA2/genética , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Feminino , Humanos , Interfase/fisiologia , Espectroscopia de Ressonância Magnética , Mitose , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo
15.
J Cell Sci ; 134(2)2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-34432036

RESUMO

The budding yeast phosphatase Cdc14 has a central role in mitotic exit and cytokinesis. Puzzlingly, a uniform picture for the three human CDC14 paralogues CDC14A, CDC14B and CDC14C in cell cycle control has not emerged to date. Redundant functions between the three CDC14 phosphatases could explain this unclear picture. To address the possibility of redundancy, we tested expression of CDC14 and analysed cell cycle progression of cells with single and double deletions in CDC14 genes. Our data suggest that CDC14C is not expressed in human RPE1 cells, excluding a function in this cell line. Single- and double-knockouts (KO) of CDC14A and CDC14B in RPE1 cells indicate that both phosphatases are not important for the timing of mitotic phases, cytokinesis and cell proliferation. However, cycling CDC14A KO and CDC14B KO cells show altered ciliogenesis compared to wild-type cells. The cilia of cycling CDC14A KO cells are longer, whereas CDC14B KO cilia are more frequent and disassemble faster. In conclusion, this study demonstrates that the cell cycle functions of CDC14 proteins are not conserved between yeast and human cells.


Assuntos
Fosfatases de Especificidade Dupla , Proteínas de Saccharomyces cerevisiae , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Divisão Celular , Citocinese/genética , Fosfatases de Especificidade Dupla/genética , Humanos , Mitose , Proteínas Tirosina Fosfatases/genética
16.
Artigo em Inglês | MEDLINE | ID: mdl-34454691

RESUMO

BACKGROUND: 5-aza-2'-deoxycytidine (5azadC, decitabine) is a DNA hypomethylating agent used in the treatment of myelodysplastic syndromes. Due to cytotoxic side effects dose optimization is essential. The aim of this study was to define and quantify the effects of 5azadC on biomarkers of chromosomal stability, and telomere length, in human lymphoblastoid cell line, WIL2-NS, at clinically relevant dosages. METHODS: Human WIL2-NS cells were maintained in complete medium containing 0, 0.2 or 1.0 µM 5azadC for four days, and analysed daily for telomere length (flow cytometry), chromosomal stability (cytokinesis-block micronucleus cytome (CBMN-cyt) assay), and global methylation (%5me-C). RESULTS: DNA methylation decreased significantly in 1.0 µM 5azadC, relative to control (p < 0.0001). Exposure to 1.0 µM 5azadC resulted in 1.7-fold increase in telomere length (p < 0.0001), in parallel with rapid increase in biomarkers of DNA damage; (micronuclei (MN, 6-fold increase), nucleoplasmic bridges (NPB, a 12-fold increase), and nuclear buds (NBud, a 13-fold increase) (all p < 0.0001). Fused nuclei (FUS), indicative of mitotic dysfunction, showed a 5- and 13-fold increase in the 0.2 µM and 1.0 µM conditions, respectively (p = 0.001) after 4 days. CONCLUSIONS: These data show that (i) clinically relevant concentrations of 5azadC are highly genotoxic; (ii) hypomethylation was associated with increased TL and DNA damage; and (iii) longer TL was associated with chromosomal instability. These findings suggest that lower doses of 5azdC may be effective as a hypomethylating agent, while potentially reducing DNA damage and risk for secondary disease.


Assuntos
Dano ao DNA/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Decitabina/farmacologia , Telômero/efeitos dos fármacos , Biomarcadores/metabolismo , Linhagem Celular , Instabilidade Cromossômica/efeitos dos fármacos , Citocinese/efeitos dos fármacos , Humanos , Linfócitos/efeitos dos fármacos , Testes para Micronúcleos/métodos , Mitose/efeitos dos fármacos
17.
Methods Mol Biol ; 2287: 127-148, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270027

RESUMO

The completely homozygous genetic background of doubled haploids (DHs) has many applications in breeding programs and research studies. Haploid induction and chromosome doubling of induced haploids are the two main steps of doubled haploid creation. Both steps have their own complexities. Chromosome doubling of induced haploids may happen spontaneously, although usually at a low rate. Therefore, artificial/induced chromosome doubling of haploid cells/plantlets is necessary to produce DHs at an acceptable level. The most common method is using some mitotic spindle poisons that target the organization of the microtubule system. Colchicine is a well-known and widely used antimitotic. However, there are substances alternative to colchicine in terms of efficiency, toxicity, safety, and genetic stability, which can be applied in in vitro and in vivo pathways. Both pathways have their own advantages and disadvantages. However, in vitro-induced chromosome doubling has been much preferred in recent years, maybe because of the dual effect of antimitotic agents (haploid induction and chromosome doubling) in just one step, and the reduced generation of chimeras. Plant genotype, the developmental stage of initial haploids, and type-concentration-duration of application of antimitotic agents, are top influential parameters on chromosome doubling efficiency. In this review, we highlight different aspects related to antimitotic agents and to plant parameters for successful chromosome doubling and high DH yield.


Assuntos
Cromossomos de Plantas , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/genética , Melhoramento Vegetal/métodos , Mitose/genética
18.
Molecules ; 26(14)2021 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-34299490

RESUMO

In this study, we designed, synthesized and evaluated, in vitro, novel chalcone analogs containing dialkylamino pharmacophores in the cervical cancer cell line, OV2008. The compound, DML6 was selective and significantly decreased the proliferation of OV2008 and HeLa cells in sub-micromolar concentrations, compared to prostate, lung, colon, breast or human embryonic kidney cell line (HEK293). DML6, at 5 µM, arrested the OV2008 cells in the G2 phase. Furthermore, DML6, at 5 µM, increased the levels of reactive oxygen species and induced a collapse in the mitochondrial membrane potential, compared to OV2008 cells incubated with a vehicle. DML6, at 5 µM, induced intrinsic apoptosis by significantly (1) increasing the levels of the pro-apoptotic proteins, Bak and Bax, and (2) decreasing the levels of l the anti-apoptotic protein, Bcl-2, compared to cell incubated with a vehicle. Furthermore, DML6, at 5 and 20 µM, induced the cleavage of caspase-9, followed by subsequent cleavage of the executioner caspases, caspase-3 and caspase-7, which produced OV2008 cell death. Overall, our data suggest that DML6 is an apoptosis-inducing compound that should undergo further evaluation as a potential treatment for cervical cancer.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Chalconas/farmacologia , Mitose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Neoplasias do Colo do Útero/tratamento farmacológico , Células A549 , Animais , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Cricetulus , Feminino , Células HEK293 , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Neoplasias do Colo do Útero/metabolismo
19.
Cancer Sci ; 112(9): 3669-3681, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34212455

RESUMO

Overcoming cisplatin (CDDP) resistance is a major issue in urothelial cancer (UC), in which CDDP-based chemotherapy is the first-line treatment. WEE1, a G2 /M checkpoint kinase, confers chemoresistance in response to genotoxic agents. However, the efficacy of WEE1 blockade in UC has not been reported. MK-1775, a WEE1 inhibitor also known as AZD-1775, blocked proliferation of UC cell lines in a dose-dependent manner irrespective of TP53 status. MK-1775 synergized with CDDP to block proliferation, inducing apoptosis and mitotic catastrophe in TP53-mutant UC cells but not in TP53-WT cells. Knocking down TP53 in TP53-WT cells induced synergism of MK-1775 and CDDP. In UMUC3 cell xenografts and two patient-derived xenograft lines with MDM2 overexpression, in which the p53/cell cycle pathway was inactivated, AZD-1775 combined with CDDP suppressed tumor growth inducing both M-phase entry and apoptosis, whereas AZD-1775 alone was as effective as the combination in RT4 cell xenografts. Drug susceptibility assay using an ex vivo cancer tissue-originated spheroid system showed correlations with the in vivo efficacy of AZD-1775 alone or combined with CDDP. We determined the feasibility of the drug susceptibility assay using spheroids established from UC surgical specimens obtained by transurethral resection. In conclusion, WEE1 is a promising therapeutic target in the treatment of UC, and a highly specific small molecule inhibitor is currently in early phase clinical trials for cancer. Differential antitumor efficacy of WEE1 blockade alone or combined with CDDP could exist according to p53/cell cycle pathway activity, which might be predictable using an ex vivo 3D primary culture system.


Assuntos
Antineoplásicos/administração & dosagem , Proteínas de Ciclo Celular/antagonistas & inibidores , Cisplatino/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/administração & dosagem , Pirimidinonas/administração & dosagem , Neoplasias da Bexiga Urinária/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , Mitose/efeitos dos fármacos , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Transfecção , Resultado do Tratamento , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Nat Commun ; 12(1): 4113, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226540

RESUMO

Tri-methylation on lysine 40 of α-tubulin (α-TubK40me3) is a recently identified post-translational modification involved in mitosis and cytokinesis. However, knowledge about α-TubK40me3 in microtubule function and post-mitotic cells remains largely incomplete. Here, we report that α-TubK40me3 is required for neuronal polarization and migration by promoting microtubule formation. α-TubK40me3 is enriched in mouse cerebral cortex during embryonic day (E)14 to E16. Knockdown of α-tubulin methyltransferase SETD2 at E14 leads to the defects in neuronal migration, which could be restored by overexpressing either a cytoplasm-localized SETD2 truncation or α-TubK40me3-mimicking mutant. Furthermore, α-TubK40me3 is preferably distributed on polymerized microtubules and potently promotes tubulin nucleation. Downregulation of α-TubK40me3 results in reduced microtubule abundance in neurites and disrupts neuronal polarization, which could be rescued by Taxol. Additionally, α-TubK40me3 is increased after losing α-tubulin K40 acetylation (α-TubK40ac) and largely rescues α-TubK40ac function. This study reveals a critical role of α-TubK40me3 in microtubule formation and neuronal development.


Assuntos
Movimento Celular , Microtúbulos/metabolismo , Neurônios/metabolismo , Tubulina (Proteína)/metabolismo , Acetilação , Animais , Córtex Cerebral , Citocinese , Técnicas de Silenciamento de Genes , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Lisina/metabolismo , Metilação , Camundongos , Mitose , Neurogênese , Paclitaxel , Processamento de Proteína Pós-Traducional
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