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1.
Nat Commun ; 12(1): 5784, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34599161

RESUMO

Cardiac regeneration involves the generation of new cardiomyocytes from cycling cardiomyocytes. Understanding cell-cycle activity of pre-existing cardiomyocytes provides valuable information to heart repair and regeneration. However, the anatomical locations and in situ dynamics of cycling cardiomyocytes remain unclear. Here we develop a genetic approach for a temporally seamless recording of cardiomyocyte-specific cell-cycle activity in vivo. We find that the majority of cycling cardiomyocytes are positioned in the subendocardial muscle of the left ventricle, especially in the papillary muscles. Clonal analysis revealed that a subset of cycling cardiomyocytes have undergone cell division. Myocardial infarction and cardiac pressure overload induce regional patterns of cycling cardiomyocytes. Mechanistically, cardiomyocyte cell cycle activity requires the Hippo pathway effector YAP. These genetic fate-mapping studies advance our basic understanding of cardiomyocyte cell cycle activity and generation in cardiac homeostasis, repair, and regeneration.


Assuntos
Miócitos Cardíacos/citologia , Animais , Southern Blotting , Ciclo Celular/fisiologia , Proliferação de Células/fisiologia , Feminino , Citometria de Fluxo , Coração/fisiologia , Masculino , Camundongos , Microscopia de Fluorescência , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo
2.
Int J Mol Sci ; 22(17)2021 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-34502512

RESUMO

Primary cilia are non-motile, cell cycle-associated organelles that can be found on most vertebrate cell types. Comprised of microtubule bundles organised into an axoneme and anchored by a mature centriole or basal body, primary cilia are dynamic signalling platforms that are intimately involved in cellular responses to their extracellular milieu. Defects in ciliogenesis or dysfunction in cilia signalling underlie a host of developmental disorders collectively referred to as ciliopathies, reinforcing important roles for cilia in human health. Whilst primary cilia have long been recognised to be present in striated muscle, their role in muscle is not well understood. However, recent studies indicate important contributions, particularly in skeletal muscle, that have to date remained underappreciated. Here, we explore recent revelations that the sensory and signalling functions of cilia on muscle progenitors regulate cell cycle progression, trigger differentiation and maintain a commitment to myogenesis. Cilia disassembly is initiated during myoblast fusion. However, the remnants of primary cilia persist in multi-nucleated myotubes, and we discuss their potential role in late-stage differentiation and myofiber formation. Reciprocal interactions between cilia and the extracellular matrix (ECM) microenvironment described for other tissues may also inform on parallel interactions in skeletal muscle. We also discuss emerging evidence that cilia on fibroblasts/fibro-adipogenic progenitors and myofibroblasts may influence cell fate in both a cell autonomous and non-autonomous manner with critical consequences for skeletal muscle ageing and repair in response to injury and disease. This review addresses the enigmatic but emerging role of primary cilia in satellite cells in myoblasts and myofibers during myogenesis, as well as the wider tissue microenvironment required for skeletal muscle formation and homeostasis.


Assuntos
Centrossomo/metabolismo , Cílios/fisiologia , Músculo Esquelético/fisiologia , Animais , Axonema , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Cílios/metabolismo , Citoesqueleto , Matriz Extracelular , Humanos , Desenvolvimento Muscular/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Organelas , Transdução de Sinais
3.
Nat Commun ; 12(1): 5268, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489460

RESUMO

Developmental steps in the trypanosome life-cycle involve transition between replicative and non-replicative forms specialised for survival in, and transmission between, mammalian and tsetse fly hosts. Here, using oligopeptide-induced differentiation in vitro, we model the progressive development of replicative 'slender' to transmissible 'stumpy' bloodstream form Trypanosoma brucei and capture the transcriptomes of 8,599 parasites using single cell transcriptomics (scRNA-seq). Using this framework, we detail the relative order of biological events during asynchronous development, profile dynamic gene expression patterns and identify putative regulators. We additionally map the cell cycle of proliferating parasites and position stumpy cell-cycle exit at early G1 before progression to a distinct G0 state. A null mutant for one transiently elevated developmental regulator, ZC3H20 is further analysed by scRNA-seq, identifying its point of failure in the developmental atlas. This approach provides a paradigm for the dissection of differentiation events in parasites, relevant to diverse transitions in pathogen biology.


Assuntos
Proteínas de Protozoários/genética , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/fisiologia , Animais , Ciclo Celular/genética , Ciclo Celular/fisiologia , Regulação da Expressão Gênica , Mutação , Percepção de Quorum , Análise de Sequência de RNA/métodos , Análise de Célula Única , Trypanosoma brucei brucei/genética
4.
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
5.
Cells ; 10(9)2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34571956

RESUMO

Pulmonary arterial hypertension (PAH) is a progressive lung disease caused by thickening of the pulmonary arterial wall and luminal obliteration of the small peripheral arteries leading to increase in vascular resistance which elevates pulmonary artery pressure that eventually causes right heart failure and death. We have previously shown that transcription factor Msx1 (mainly expressed during embryogenesis) is strongly upregulated in transformed lymphocytes obtained from PAH patients, especially IPAH. Under pathological conditions, Msx1 overexpression can cause cell dedifferentiation or cell apoptosis. We hypothesized that Msx1 overexpression contributes to loss of small pulmonary vessels in PAH. In IPAH lung, MSX1 protein localization was strikingly increased in muscularized remodeled pulmonary vessels, whereas it was undetectable in control pulmonary arteries. We developed a transgenic mouse model overexpressing MSX1 (MSX1OE) by about 4-fold and exposed these mice to normoxic, sugen hypoxic (3 weeks) or hyperoxic (100% 02 for 3 weeks) conditions. Under normoxic conditions, compared to controls, MSX1OE mice demonstrated a 30-fold and 2-fold increase in lung Msx1 mRNA and protein expression, respectively. There was a significant retinal capillary dropout (p < 0.01) in MSX1OE mice, which was increased further (p < 0.03) with sugen hypoxia. At baseline, the number of pulmonary vessels in MSX1OE mice was similar to controls. In sugen-hypoxia-treated MSX1OE mice, the number of small (0-25 uM) and medium (25-50 uM) size muscularized vessels increased approximately 2-fold (p < 0.01) compared to baseline controls; however, they were strikingly lower (p < 0.001) in number than in sugen-hypoxia-treated control mice. In MSX1OE mouse lung, 104 genes were upregulated and 67 genes were downregulated compared to controls. Similarly, in PVECs, 156 genes were upregulated and 320 genes were downregulated from siRNA to MSX1OE, and in PVSMCs, 65 genes were upregulated and 321 genes were downregulated from siRNA to MSX1OE (with control in the middle). Many of the statistically significant GO groups associated with MSX1 expression in lung, PVECs, and PVSMCs were similar, and were involved in cell cycle, cytoskeletal and macromolecule organization, and programmed cell death. Overexpression of MSX1 suppresses many cell-cycle-related genes in PVSMCs but induces them in PVECs. In conclusion, overexpression of Msx1 leads to loss of pulmonary vessels, which is exacerbated by sugen hypoxia, and functional consequences of Msx1 overexpression are cell-dependent.


Assuntos
Hipóxia/metabolismo , Pulmão/metabolismo , Fator de Transcrição MSX1/metabolismo , Artéria Pulmonar/metabolismo , Animais , Apoptose/fisiologia , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Regulação para Baixo/fisiologia , Feminino , Humanos , Masculino , Camundongos , Regulação para Cima/fisiologia
6.
Molecules ; 26(18)2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34576943

RESUMO

Increased secretion of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNFα), is often associated with adipose tissue dysregulation, which often accompanies obesity. High levels of TNFα have been linked to the development of insulin resistance in several tissues and organs, including skeletal muscle and the liver. In this study, we examined the complex regulatory roles of TNFα in murine hepatocytes utilizing a combination of global proteomic and phosphoproteomic analyses. Our results show that TNFα promotes extensive changes not only of protein levels, but also the dynamics of their downstream phosphorylation signaling. We provide evidence that TNFα induces DNA replication and promotes G1/S transition through activation of the MAPK pathway. Our data also highlight several other novel proteins, many of which are regulated by phosphorylation and play a role in the progression and development of insulin resistance in hepatocytes.


Assuntos
Hepatócitos/metabolismo , Proteínas/metabolismo , Proteômica/métodos , Fator de Necrose Tumoral alfa/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Células Cultivadas , Hepatócitos/efeitos dos fármacos , Camundongos , Fosfoproteínas/metabolismo , Fosforilação , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , Espectrometria de Massas em Tandem , Fator de Necrose Tumoral alfa/farmacologia
7.
Molecules ; 26(18)2021 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-34577105

RESUMO

Granulosa cells (GCs) are essential for follicular growth, development, and atresia. The orexin-A (OXA) neuropeptide is widely involved in the regulation of various biological functions. OXA selectively binds to orexin receptor type 1 (OX1R) and mediates all its biological actions via OX1R. This study aimed to explore the expression of OXA and OX1R and their regulatory role in GCs proliferation, cell cycle progression, apoptosis, oocyte maturation, and underlying molecular mechanisms of these processes and elucidate its novel signaling pathway. Western blotting and RT-qPCR showed that OXA and OX1R were expressed during different developmental stages of GCs, and siRNA transfection successfully inhibited the expression of OX1R at the translational and transcriptional levels. Flow cytometry revealed that OX1R knockdown upregulated GCs apoptosis and triggered S-phase arrest in cell cycle progression. RT-qPCR and Western blotting showed significantly reduced expression of Bcl-2 and elevated expression of Bax, caspase-3, TNF-α, and P21 in OX1R-silenced GCs. Furthermore, the CCK-8 assay showed that knockdown of OX1R suppressed GCs proliferation by downregulating the expression of PCNA, a proliferation marker gene, at the translational and transcriptional levels. Western blotting revealed that knockdown of OX1R resulted in a considerable decrease of the phosphorylation level of the AKT and ERK1/2 proteins, indicating that the AKT/ERK1/2 pathway is involved in regulating GCs proliferation and apoptosis. In addition, OX1R silencing enhanced the mRNA expression of GDF9 and suppressed the mRNA expression of BMP15 in mouse GCs. Collectively, these results reveal a novel regulatory role of OXA in the development of GCs and folliculogenesis by regulating proliferation, apoptosis, and cell cycle progression. Therefore, OXA can be a promising therapeutic agent for female infertility.


Assuntos
Células da Granulosa/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Orexinas/fisiologia , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Regulação para Baixo/genética , Feminino , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/fisiologia , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Receptores de Orexina/genética , Receptores de Orexina/metabolismo , Orexinas/metabolismo , Folículo Ovariano/efeitos dos fármacos , Cultura Primária de Células
8.
PLoS Comput Biol ; 17(8): e1009008, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34379640

RESUMO

Modeling biochemical reactions by means of differential equations often results in systems with a large number of variables and parameters. As this might complicate the interpretation and generalization of the obtained results, it is often desirable to reduce the complexity of the model. One way to accomplish this is by replacing the detailed reaction mechanisms of certain modules in the model by a mathematical expression that qualitatively describes the dynamical behavior of these modules. Such an approach has been widely adopted for ultrasensitive responses, for which underlying reaction mechanisms are often replaced by a single Hill function. Also time delays are usually accounted for by using an explicit delay in delay differential equations. In contrast, however, S-shaped response curves, which by definition have multiple output values for certain input values and are often encountered in bistable systems, are not easily modeled in such an explicit way. Here, we extend the classical Hill function into a mathematical expression that can be used to describe both ultrasensitive and S-shaped responses. We show how three ubiquitous modules (ultrasensitive responses, S-shaped responses and time delays) can be combined in different configurations and explore the dynamics of these systems. As an example, we apply our strategy to set up a model of the cell cycle consisting of multiple bistable switches, which can incorporate events such as DNA damage and coupling to the circadian clock in a phenomenological way.


Assuntos
Ciclo Celular/fisiologia , Modelos Biológicos , Animais , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Biologia Computacional , Simulação por Computador , Dano ao DNA , Humanos , Cinética , Biologia de Sistemas
9.
J Biol Chem ; 297(3): 101068, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34375638

RESUMO

The circadian clock controls the expression of nearly 50% of protein coding genes in mice and most likely in humans as well. Therefore, disruption of the circadian clock is presumed to have serious pathological effects including cancer. However, epidemiological studies on individuals with circadian disruption because of night shift or rotating shift work have produced contradictory data not conducive to scientific consensus as to whether circadian disruption increases the incidence of breast, ovarian, prostate, or colorectal cancers. Similarly, genetically engineered mice with clock disruption do not exhibit spontaneous or radiation-induced cancers at higher incidence than wild-type controls. Because many cellular functions including the cell cycle and cell division are, at least in part, controlled by the molecular clock components (CLOCK, BMAL1, CRYs, PERs), it has also been expected that appropriate timing of chemotherapy may increase the efficacy of chemotherapeutic drugs and ameliorate their side effect. However, empirical attempts at chronochemotherapy have not produced beneficial outcomes. Using mice without and with human tumor xenografts, sites of DNA damage and repair following treatment with the anticancer drug cisplatin have been mapped genome-wide at single nucleotide resolution and as a function of circadian time. The data indicate that mechanism-based studies such as these may provide information necessary for devising rational chronochemotherapy regimens.


Assuntos
Carcinogênese/efeitos dos fármacos , Cronofarmacocinética , Relógios Circadianos/fisiologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Proteínas CLOCK/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Ciclo Celular/fisiologia , Fenômenos Cronobiológicos , Relógios Circadianos/genética , Ritmo Circadiano/fisiologia , Cisplatino/farmacocinética , Cisplatino/farmacologia , Criptocromos/genética , Criptocromos/metabolismo , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Humanos , Camundongos , Neoplasias/genética , Transcrição Genética/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
10.
J Cancer Res Clin Oncol ; 147(11): 3157-3168, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34232382

RESUMO

PURPOSE: The expression of eukaryotic translation initiation factor-2 subunit 3 (EIF2S3) in patients with non-small cell lung and colorectal cancer is lower than that in healthy individuals. However, the functions of EIF2S3 remain unclear, and its study in leukemia has not been reported. The article aims to explore the role of EIF2S3 in AML (acute myeloid leukemia) and its underlying mechanism. METHODS: Reverse transcription-quantitative PCR was performed to evaluate the expression levels of EIF2S3, and its association with patient prognosis was determined. Inducible HEL-EIF2S3 and HL-60-EIF2S3 cell lines were established by retrovirus infection. Cellular proliferation and the cell cycle were analyzed using Cell Counting Kit-8 and flow cytometric analyses. Tumorigenic ability was evaluated using xenograft nude mouse model. Gene expression profiles were analyzed in HL-60-EIF2S3 cells by next-generation sequencing, and WB analysis was performed to detect the expression of related proteins. RESULTS: The expression of EIF2S3 in patients with AML was lower than that experiencing CR (P = 0.02). Furthermore, EIF2S3 overexpression inhibited cellular proliferation, halted G0/1 to S phase cell cycle progression, and inhibited tumorigenicity (P = 0.015). 479 differentially expressed genes were identified between HL60-EIF2S3 DOX (-) and HL60-EIF2S3 DOX ( +) cells via NGS and several of them involved in MAPK/ERK signaling pathway. The phosphorylation levels of ERK decreased when EIF2S3 was overexpressed (P < 0.050). CONCLUSION: EIF2S3 overexpression may result in a decrease in cellular proliferation, cell cycle arrest, and tumorigenic inhibition via the MAPK/ERK signaling pathway in AML cells.


Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Leucemia Mieloide Aguda/metabolismo , Sistema de Sinalização das MAP Quinases , Animais , Carcinogênese , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Fator de Iniciação 2 em Eucariotos/biossíntese , Fator de Iniciação 2 em Eucariotos/genética , Células HL-60 , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima
11.
Biomolecules ; 11(7)2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201602

RESUMO

Human Periodontal Ligament Fibroblasts (hPDLF), as part of the periodontal apparatus, modulate inflammation, regeneration and bone remodeling. Interferences are clinically manifested as attachment loss, tooth loosening and root resorption. During orthodontic tooth movement (OTM), remodeling and adaptation of the periodontium is required in order to enable tooth movement. hPDLF involvement in the early phase-OTM compression side was investigated for a 72-h period through a well-studied in vitro model. Changes in the morphology, cell proliferation and cell death were analyzed. Specific markers of the cell cycle were investigated by RT-qPCR and Western blot. The study showed that the morphology of hPDLF changes towards more unstructured, unsorted filaments under mechanical compression. The total cell numbers were significantly reduced with a higher cell death rate over the whole observation period. hPDLF started to recover to pretreatment conditions after 48 h. Furthermore, key molecules involved in the cell cycle were significantly reduced under compressive force at the gene expression and protein levels. These findings revealed important information for a better understanding of the preservation and remodeling processes within the periodontium through Periodontal Ligament Fibroblasts during orthodontic tooth movement. OTM initially decelerates the hPDLF cell cycle and proliferation. After adapting to environmental changes, human Periodontal Ligament Fibroblasts can regain homeostasis of the periodontium, affecting its reorganization.


Assuntos
Apoptose/fisiologia , Ciclo Celular/fisiologia , Proliferação de Células/fisiologia , Fibroblastos/fisiologia , Ligamento Periodontal/fisiologia , Técnicas de Movimentação Dentária/métodos , Remodelação Óssea/fisiologia , Força Compressiva/fisiologia , Humanos , Ligamento Periodontal/citologia , Estresse Mecânico
12.
Biomolecules ; 11(7)2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202768

RESUMO

The extremophilic unicellular red microalga Galdieria sulphuraria (Cyanidiophyceae) is able to grow autotrophically, or mixo- and heterotrophically with 1% glycerol as a carbon source. The alga divides by multiple fission into more than two cells within one cell cycle. The optimal conditions of light, temperature and pH (500 µmol photons m-2 s-1, 40 °C, and pH 3; respectively) for the strain Galdieria sulphuraria (Galdieri) Merola 002 were determined as a basis for synchronization experiments. For synchronization, the specific light/dark cycle, 16/8 h was identified as the precondition for investigating the cell cycle. The alga was successfully synchronized and the cell cycle was evaluated. G. sulphuraria attained two commitment points with midpoints at 10 and 13 h of the cell cycle, leading to two nuclear divisions, followed subsequently by division into four daughter cells. The daughter cells stayed in the mother cell wall until the beginning of the next light phase, when they were released. Accumulation of glycogen throughout the cell cycle was also described. The findings presented here bring a new contribution to our general understanding of the cell cycle in cyanidialean red algae, and specifically of the biotechnologically important species G. sulphuraria.


Assuntos
Processos Heterotróficos/fisiologia , Microalgas/crescimento & desenvolvimento , Rodófitas/crescimento & desenvolvimento , Ciclo Celular/fisiologia , Células Cultivadas , Microalgas/citologia , Rodófitas/citologia , Temperatura
13.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203295

RESUMO

A key feature of pulmonary arterial hypertension (PAH) is the hyperplastic proliferation exhibited by the vascular smooth muscle cells from patients (HPASMC). The growth inducers FOXM1 and PLK1 are highly upregulated in these cells. The mechanism by which these two proteins direct aberrant growth in these cells is not clear. Herein, we identify cyclin-dependent kinase 1 (CDK1), also termed cell division cycle protein 2 (CDC2), as having a primary role in promoting progress of the cell cycle leading to proliferation in HPASMC. HPASMC obtained from PAH patients and pulmonary arteries from Sugen/hypoxia rats were investigated for their expression of CDC2. Protein levels of CDC2 were much higher in PAH than in cells from normal donors. Knocking down FOXM1 or PLK1 protein expression with siRNA or pharmacological inhibitors lowered the cellular expression of CDC2 considerably. However, knockdown of CDC2 with siRNA or inhibiting its activity with RO-3306 did not reduce the protein expression of FOXM1 or PLK1. Expression of CDC2 and FOXM1 reached its maximum at G1/S, while PLK1 reached its maximum at G2/M phase of the cell cycle. The expression of other CDKs such as CDK2, CDK4, CDK6, CDK7, and CDK9 did not change in PAH HPASMC. Moreover, inhibition via Wee1 inhibitor adavosertib or siRNAs targeting Wee1, Myt1, CDC25A, CDC25B, or CDC25C led to dramatic decreases in CDC2 protein expression. Lastly, we found CDC2 expression at the RNA and protein level to be upregulated in pulmonary arteries during disease progression Sugen/hypoxia rats. In sum, our present results illustrate that the increased expression of FOXM1 and PLK1 in PAH leads directly to increased expression of CDC2 resulting in potentiated growth hyperactivity of PASMC from patients with pulmonary hypertension. Our results further suggest that the regulation of CDC2, or associated regulatory proteins, will prove beneficial in the treatment of this disease.


Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteína Forkhead Box M1/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteína Quinase CDC2/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proliferação de Células/fisiologia , Proteína Forkhead Box M1/genética , Humanos , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Hipertensão Arterial Pulmonar/genética , Hipertensão Arterial Pulmonar/metabolismo , Ratos , Ratos Sprague-Dawley , Remodelação Vascular/genética , Remodelação Vascular/fisiologia
14.
J Alzheimers Dis ; 82(4): 1683-1702, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34219712

RESUMO

BACKGROUND: Aberrant cell cycle re-entry is a well-documented process occurring early in Alzheimer's disease (AD). This is an early feature of the disease and may contribute to disease pathogenesis. OBJECTIVE: To assess the effect of forced neuronal cell cycle re-entry in mice expressing humanized Aß, we crossed our neuronal cell cycle re-entry mouse model with AppNLF knock-in (KI) mice. METHODS: Our neuronal cell cycle re-entry (NCCR) mouse model is bitransgenic mice heterozygous for both Camk2a-tTA and TRE-SV40T. The NCCR mice were crossed with AppNLF KI mice to generate NCCR-AppNLF animals. Using this tet-off system, we triggered NCCR in our animals via neuronal expression of SV40T starting at 1 month of age. The animals were examined at the following time points: 9, 12, and 18 months of age. Various neuropathological features in our mice were evaluated by image analysis and stereology on brain sections stained using either immunofluorescence or immunohistochemistry. RESULTS: We show that neuronal cell cycle re-entry in humanized Aß plaque producing AppNLF KI mice results in the development of additional AD-related pathologies, namely, pathological tau, neuroinflammation, brain leukocyte infiltration, DNA damage response, and neurodegeneration. CONCLUSION: Our findings show that neuronal cell cycle re-entry enhances AD-related neuropathological features in AppNLF mice and highlight our unique AD mouse model for studying the pathogenic role of aberrant cell cycle re-entry in AD.


Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Ciclo Celular/fisiologia , Técnicas de Introdução de Genes , Camundongos Transgênicos , Neurônios/metabolismo , Neuropatologia , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Mutação
15.
J Cancer Res Clin Oncol ; 147(11): 3211-3224, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34244855

RESUMO

PURPOSE: Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are widely used for the treatment of advanced estrogen receptor (ER)-positive breast cancer. To develop a treatment strategy for cancers resistant to CDK4/6 inhibitors, here, we established palbociclib-resistant sublines and analyzed their resistance mechanisms. METHODS: Palbociclib-resistant sublines were established from T47D and MCF7 cells. Sensitivity to other drugs was assessed via the WST assay. Altered expression/phosphorylation of proteins related to signal transduction and cell cycle regulation was examined using western blotting. Copy number alterations and mutations in the retinoblastoma (RB1) gene were also analyzed. RESULTS: Although an increase in CDK6 and decrease in retinoblastoma protein (Rb) expression/phosphorylation were commonly observed in the resistant sublines, changes in other cell cycle-related proteins were heterogeneous. Upon extended exposure to palbociclib, the expression/phosphorylation of these proteins became altered, and the long-term removal of palbociclib did not restore the Rb expression/phosphorylation patterns. Consistently a copy number decrease, as well as RB1 mutations were detected. Moreover, although the resistant sublines exhibited cross-resistance to abemaciclib, their response to dinaciclib was the same as that of wild-type cells. Of note, the cell line exhibiting increased mTOR phosphorylation also showed a higher sensitivity to everolimus. However, the sensitivity to chemotherapeutic agents was unchanged in palbociclib-resistant sublines. CONCLUSION: ER-positive breast cancer cells use multiple molecular mechanisms to survive in the presence of palbociclib, suggesting that targeting activated proteins may be an effective strategy to overcome resistance. Additionally, palbociclib monotherapy induces mutations and copy number alterations in the RB1 gene.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Piperazinas/farmacologia , Piridinas/farmacologia , Receptores de Estrogênio/metabolismo , Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/biossíntese , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/biossíntese , Receptor alfa de Estrogênio/metabolismo , Humanos , Células MCF-7 , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/biossíntese , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/biossíntese , Transdução de Sinais
16.
Nat Commun ; 12(1): 4601, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326322

RESUMO

Genomic sequencing of thousands of tumors has revealed many genes associated with specific types of cancer. Similarly, large scale CRISPR functional genomics efforts have mapped genes required for cancer cell proliferation or survival in hundreds of cell lines. Despite this, for specific disease subtypes, such as metastatic prostate cancer, there are likely a number of undiscovered tumor specific driver genes that may represent potential drug targets. To identify such genetic dependencies, we performed genome-scale CRISPRi screens in metastatic prostate cancer models. We then created a pipeline in which we integrated pan-cancer functional genomics data with our metastatic prostate cancer functional and clinical genomics data to identify genes that can drive aggressive prostate cancer phenotypes. Our integrative analysis of these data reveals known prostate cancer specific driver genes, such as AR and HOXB13, as well as a number of top hits that are poorly characterized. In this study we highlight the strength of an integrated clinical and functional genomics pipeline and focus on two top hit genes, KIF4A and WDR62. We demonstrate that both KIF4A and WDR62 drive aggressive prostate cancer phenotypes in vitro and in vivo in multiple models, irrespective of AR-status, and are also associated with poor patient outcome.


Assuntos
Proteínas de Ciclo Celular/genética , Cinesina/genética , Proteínas do Tecido Nervoso/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Animais , Sistemas CRISPR-Cas , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Movimento Celular/fisiologia , Células Cultivadas , Bases de Dados Genéticas , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Cinesina/metabolismo , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica , Estadiamento de Neoplasias , Proteínas do Tecido Nervoso/metabolismo , Neoplasias da Próstata/metabolismo , Taxa de Sobrevida
17.
Nat Commun ; 12(1): 4451, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294712

RESUMO

Identifying how R-loops are generated is crucial to know how transcription compromises genome integrity. We show by genome-wide analysis of conditional yeast mutants that the THO transcription complex, prevents R-loop formation in G1 and S-phase, whereas the Sen1 DNA-RNA helicase prevents them only in S-phase. Interestingly, damage accumulates asymmetrically downstream of the replication fork in sen1 cells but symmetrically in the hpr1 THO mutant. Our results indicate that: R-loops form co-transcriptionally independently of DNA replication; that THO is a general and cell-cycle independent safeguard against R-loops, and that Sen1, in contrast to previously believed, is an S-phase-specific R-loop resolvase. These conclusions have important implications for the mechanism of R-loop formation and the role of other factors reported to affect on R-loop homeostasis.


Assuntos
DNA Fúngico/química , Estruturas R-Loop , RNA Fúngico/química , Ciclo Celular/genética , Ciclo Celular/fisiologia , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Genes Fúngicos , Instabilidade Genômica , Modelos Biológicos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estruturas R-Loop/genética , Estruturas R-Loop/fisiologia , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203322

RESUMO

BACKGROUND: In space, the reduction or loss of the gravity vector greatly affects the interaction between cells. Since the beginning of the space age, microgravity has been identified as an informative tool in biomedicine, including cancer research. The A549 cell line is a hypotriploid human alveolar basal epithelial cell line widely used as a model for lung adenocarcinoma. Microgravity has been reported to interfere with mitochondrial activity, energy metabolism, cell vitality and proliferation, chemosensitivity, invasion and morphology of cells and organelles in various biological systems. Concerning lung cancer, several studies have reported the ability of microgravity to modulate the carcinogenic and metastatic process. To investigate these processes, A549 cells were exposed to simulated microgravity (µG) for different time points. METHODS: We performed cell cycle and proliferation assays, ultrastructural analysis of mitochondria architecture, as well as a global analysis of miRNA modulated under µG conditions. RESULTS: The exposure of A549 cells to microgravity is accompanied by the generation of polynucleated cells, cell cycle imbalance, growth inhibition, and gross morphological abnormalities, the most evident are highly damaged mitochondria. Global miRNA analysis defined a pool of miRNAs associated with µG solicitation mainly involved in cell cycle regulation, apoptosis, and stress response. To our knowledge, this is the first global miRNA analysis of A549 exposed to microgravity reported. Despite these results, it is not possible to draw any conclusion concerning the ability of µG to interfere with the cancerogenic or the metastatic processes in A549 cells. CONCLUSIONS: Our results provide evidence that mitochondria are strongly sensitive to µG. We suggest that mitochondria damage might in turn trigger miRNA modulation related to cell cycle imbalance.


Assuntos
MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Células A549 , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Metabolismo Energético/fisiologia , Humanos
19.
Dev Cell ; 56(15): 2145-2159.e7, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34242584

RESUMO

In every organism, the cell cycle requires the execution of multiple processes in a strictly defined order. However, the mechanisms used to ensure such order remain poorly understood, particularly in bacteria. Here, we show that the activation of the essential CtrA signaling pathway that triggers cell division in Caulobacter crescentus is intrinsically coupled to the initiation of DNA replication via the physical translocation of a newly replicated chromosome, powered by the ParABS system. We demonstrate that ParA accumulation at the new cell pole during chromosome segregation recruits ChpT, an intermediate component of the CtrA signaling pathway. ChpT is normally restricted from accessing the selective PopZ polar microdomain until the new chromosome and ParA arrive. Consequently, any disruption to DNA replication initiation prevents ChpT polarization and, in turn, cell division. Collectively, our findings reveal how major cell-cycle events are coordinated in Caulobacter and, importantly, how chromosome translocation triggers an essential signaling pathway.


Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/metabolismo , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Caulobacter crescentus/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Divisão Celular/genética , Segregação de Cromossomos/genética , Cromossomos/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Transporte Proteico , Transdução de Sinais , Fatores de Transcrição/genética , Translocação Genética/genética
20.
Nat Commun ; 12(1): 4582, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34321470

RESUMO

SAMHD1 is a cellular triphosphohydrolase (dNTPase) proposed to inhibit HIV-1 reverse transcription in non-cycling immune cells by limiting the supply of the dNTP substrates. Yet, phosphorylation of T592 downregulates SAMHD1 antiviral activity, but not its dNTPase function, implying that additional mechanisms contribute to viral restriction. Here, we show that SAMHD1 is SUMOylated on residue K595, a modification that relies on the presence of a proximal SUMO-interacting motif (SIM). Loss of K595 SUMOylation suppresses the restriction activity of SAMHD1, even in the context of the constitutively active phospho-ablative T592A mutant but has no impact on dNTP depletion. Conversely, the artificial fusion of SUMO2 to a non-SUMOylatable inactive SAMHD1 variant restores its antiviral function, a phenotype that is reversed by the phosphomimetic T592E mutation. Collectively, our observations clearly establish that lack of T592 phosphorylation cannot fully account for the restriction activity of SAMHD1. We find that SUMOylation of K595 is required to stimulate a dNTPase-independent antiviral activity in non-cycling immune cells, an effect that is antagonized by cyclin/CDK-dependent phosphorylation of T592 in cycling cells.


Assuntos
Ciclo Celular/fisiologia , HIV-1/fisiologia , Proteína 1 com Domínio SAM e Domínio HD/genética , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Sumoilação/fisiologia , Substituição de Aminoácidos , Células HEK293 , Infecções por HIV/virologia , Humanos , Lisina , Mutação , Fosforilação , Proteína 1 com Domínio SAM e Domínio HD/química , Células U937
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