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It has been proposed that ATR kinase senses the completion of DNA replication to initiate the S/G2 transition. In contrast to this model, we show here that the TRESLIN-MTBP complex prevents a premature entry into G2 from early S-phase independently of ATR/CHK1 kinases. TRESLIN-MTBP acts transiently at pre-replication complexes (preRCs) to initiate origin firing and is released after the subsequent recruitment of CDC45. This dynamic behavior of TRESLIN-MTBP implements a monitoring system that checks the activation of replication forks and senses the rate of origin firing to prevent the entry into G2. This system detects the decline in the number of origins of replication that naturally occurs in very late S, which is the signature that cells use to determine the completion of DNA replication and permit the S/G2 transition. Our work introduces TRESLIN-MTBP as a key player in cell-cycle control independent of canonical checkpoints.
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Proteínas de Ciclo Celular , Replicação do DNA , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem/genética , Proteínas de Ligação a DNA/genéticaRESUMO
The PBRM1 subunit of the PBAF (SWI/SNF) chromatin remodeling complex is mutated in â¼40% of clear cell renal cancers. PBRM1 loss has been implicated in responses to immunotherapy in renal cancer, but the mechanism is unclear. DNA damage-induced inflammatory signaling is an important factor determining immunotherapy response. This response is kept in check by the G2/M checkpoint, which prevents progression through mitosis with unrepaired damage. We found that in the absence of PBRM1, p53-dependent p21 up-regulation is delayed after DNA damage, leading to defective transcriptional repression by the DREAM complex and premature entry into mitosis. Consequently, DNA damage-induced inflammatory signaling pathways are activated by cytosolic DNA. Notably, p53 is infrequently mutated in renal cancer, so PBRM1 mutational status is critical to G2/M checkpoint maintenance. Moreover, we found that the ability of PBRM1 deficiency to predict response to immunotherapy correlates with expression of the cytosolic DNA-sensing pathway in clinical samples. These findings have implications for therapeutic responses in renal cancer.
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A versatile division of apicomplexan parasites and a dearth of conserved regulators have hindered the progress of apicomplexan cell cycle studies. While most apicomplexans divide in a multinuclear fashion, Toxoplasma gondii tachyzoites divide in the traditional binary mode. We previously identified five Toxoplasma CDK-related kinases (Crk). Here, we investigated TgCrk4 and its cyclin partner TgCyc4. We demonstrated that TgCrk4 regulates conventional G2 phase processes, such as repression of chromosome rereplication and centrosome reduplication, and acts upstream of the spindle assembly checkpoint. The spatial TgCyc4 dynamics supported the TgCrk4-TgCyc4 complex role in the coordination of chromosome and centrosome cycles. We also identified a dominant TgCrk4-TgCyc4 complex interactor, TgiRD1 protein, related to DNA replication licensing factor CDT1 but played no role in licensing DNA replication in the G1 phase. Our results showed that TgiRD1 also plays a role in controlling chromosome and centrosome reduplication. Global phosphoproteome analyses identified TgCrk4 substrates, including TgORC4, TgCdc20, TgGCP2, and TgPP2ACA. Importantly, the phylogenetic and structural studies suggest the Crk4-Cyc4 complex is limited to a minor group of the binary dividing apicomplexans.
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Proteínas de Protozoários , Toxoplasma , Toxoplasma/metabolismo , Toxoplasma/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Fase G2/genética , Centrossomo/metabolismo , Divisão Celular , Ciclinas/metabolismo , Ciclinas/genéticaRESUMO
Cytosolic caspase-8 is a mediator of death receptor signaling. While caspase-8 expression is lost in some tumors, it is increased in others, indicating a conditional pro-survival function of caspase-8 in cancer. Here, we show that tumor cells employ DNA-damage-induced nuclear caspase-8 to override the p53-dependent G2/M cell-cycle checkpoint. Caspase-8 is upregulated and localized to the nucleus in multiple human cancers, correlating with treatment resistance and poor clinical outcome. Depletion of caspase-8 causes G2/M arrest, stabilization of p53, and induction of p53-dependent intrinsic apoptosis in tumor cells. In the nucleus, caspase-8 cleaves and inactivates the ubiquitin-specific peptidase 28 (USP28), preventing USP28 from de-ubiquitinating and stabilizing wild-type p53. This results in de facto p53 protein loss, switching cell fate from apoptosis toward mitosis. In summary, our work identifies a non-canonical role of caspase-8 exploited by cancer cells to override the p53-dependent G2/M cell-cycle checkpoint.
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Caspase 8/metabolismo , Núcleo Celular/enzimologia , Proliferação de Células , Pontos de Checagem da Fase G2 do Ciclo Celular , Neoplasias/enzimologia , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina Tiolesterase/metabolismo , Antineoplásicos/farmacologia , Apoptose , Caspase 8/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Núcleo Celular/patologia , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Masculino , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Células PC-3 , Estabilidade Proteica , Transdução de Sinais , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Ubiquitina Tiolesterase/genéticaRESUMO
To maintain genome stability, cells need to replicate their DNA before dividing. Upon completion of bulk DNA synthesis, the mitotic kinases CDK1 and PLK1 become active and drive entry into mitosis. Here, we have tested the hypothesis that DNA replication determines the timing of mitotic kinase activation. Using an optimized double-degron system, together with kinase inhibitors to enforce tight inhibition of key proteins, we find that human cells unable to initiate DNA replication prematurely enter mitosis. Preventing DNA replication licensing and/or firing causes prompt activation of CDK1 and PLK1 in S phase. In the presence of DNA replication, inhibition of CHK1 and p38 leads to premature activation of mitotic kinases, which induces severe replication stress. Our results demonstrate that, rather than merely a cell cycle output, DNA replication is an integral signaling component that restricts activation of mitotic kinases. DNA replication thus functions as a brake that determines cell cycle duration.
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Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fase S , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Ativação Enzimática , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinase 1 Polo-LikeRESUMO
Accurate and complete DNA duplication is critical for maintaining genome integrity. Multiple mechanisms regulate when and where DNA replication takes place, to ensure that the entire genome is duplicated once and only once per cell cycle. Although the bulk of the genome is copied during the S phase of the cell cycle, increasing evidence suggests that parts of the genome are replicated in G2 or mitosis, in a last attempt to secure that daughter cells inherit an accurate copy of parental DNA. Remaining unreplicated gaps may be passed down to progeny and replicated in the next G1 or S phase. These findings challenge the long-established view that genome duplication occurs strictly during the S phase, bridging DNA replication to DNA repair and providing novel therapeutic strategies for cancer treatment.
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Replicação do DNA , Mitose , Humanos , Fase S/genética , Ciclo Celular/genética , Replicação do DNA/genética , Mitose/genética , DNARESUMO
The involvement of secreted phospholipase A2s in respiratory diseases, such as asthma and respiratory viral infections, is well-established. However, the specific role of secreted phospholipase A2 group IIE (PLA2G2E) during influenza virus infection remains unexplored. Here, we investigated the role of PLA2G2E during H1N1 influenza virus infection using a targeted mouse model lacking Pla2g2e gene (Pla2g2e-/-). Our findings demonstrated that Pla2g2e-/- mice had significantly lower survival rates and higher viral loads in lungs compared to wild-type mice following influenza virus infection. While Pla2g2e-/- mice displayed comparable innate and humoral immune responses to influenza virus challenge, the animals showed impaired influenza-specific cellular immunity and reduced T cell-mediated cytotoxicity. This indicates that PLA2G2E is involved in regulating specific T cell responses during influenza virus infection. Furthermore, transgenic mice expressing the human PLA2G2E gene exhibited resistance to influenza virus infection along with enhanced influenza-specific cellular immunity and T cell-mediated cytotoxicity. Pla2g2e deficiency resulted in perturbation of lipid mediators in the lung and T cells, potentially contributing to its impact on the anti-influenza immune response. Taken together, these findings suggest that targeting PLA2G2E could hold potential as a therapeutic strategy for managing influenza virus infections.IMPORTANCEThe influenza virus is a highly transmissible respiratory pathogen that continues to pose a significant public health concern. It effectively evades humoral immune protection conferred by vaccines and natural infection due to its continuous viral evolution through the genetic processes of antigenic drift and shift. Recognition of conserved non-mutable viral epitopes by T cells may provide broad immunity against influenza virus. In this study, we have demonstrated that phospholipase A2 group IIE (PLA2G2E) plays a crucial role in protecting against influenza virus infection through the regulation of T cell responses, while not affecting innate and humoral immune responses. Targeting PLA2G2E could therefore represent a potential therapeutic strategy for managing influenza virus infection.
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Vírus da Influenza A Subtipo H1N1 , Pulmão , Infecções por Orthomyxoviridae , Animais , Camundongos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Vírus da Influenza A Subtipo H1N1/imunologia , Pulmão/virologia , Pulmão/imunologia , Pulmão/patologia , Humanos , Fosfolipases A2 do Grupo II/genética , Fosfolipases A2 do Grupo II/imunologia , Linfócitos T/imunologia , Camundongos Knockout , Imunidade Celular , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Carga Viral , Modelos Animais de Doenças , Imunidade Humoral , Imunidade Inata , Influenza Humana/imunologia , Influenza Humana/virologia , FemininoRESUMO
Microspherule protein 1 (Mcrs1) is a component of the nonspecific lethal (NSL) complex and the chromatin remodeling INO80 complex, which participates in transcriptional regulation during mitosis. Here, we investigate the roles of Mcrs1 during female meiosis in mice. We demonstrate that Mcrs1 is a novel regulator of the meiotic G2/M transition and spindle assembly in mouse oocytes. Mcrs1 is present in the nucleus and associates with spindle poles and chromosomes of oocytes during meiosis I. Depletion of Mcrs1 alters HDAC2-mediated H4K16ac, H3K4me2, and H3K9me2 levels in nonsurrounded nucleolus (NSN)-type oocytes, and reduces CDK1 activity and cyclin B1 accumulation, leading to G2/M transition delay. Furthermore, Mcrs1 depletion results in abnormal spindle assembly due to reduced Aurora kinase (Aurka and Aurkc) and Kif2A activities, suggesting that Mcrs1 also plays a transcription-independent role in regulation of metaphase I oocytes. Taken together, our results demonstrate that the transcription factor Mcrs1 has important roles in cell cycle regulation and spindle assembly in mouse oocyte meiosis.
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Meiose , Fuso Acromático , Feminino , Camundongos , Animais , Fuso Acromático/metabolismo , Metáfase , Oócitos/metabolismo , Pontos de Checagem do Ciclo Celular , Proteínas Repressoras/metabolismo , Cinesinas/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Confining the activity of a designed protein to a specific microenvironment would have broad-ranging applications, such as enabling cell type-specific therapeutic action by enzymes while avoiding off-target effects. While many natural enzymes are synthesized as inactive zymogens that can be activated by proteolysis, it has been challenging to redesign any chosen enzyme to be similarly stimulus responsive. Here, we develop a massively parallel computational design, screening, and next-generation sequencing-based approach for proenzyme design. For a model system, we employ carboxypeptidase G2 (CPG2), a clinically approved enzyme that has applications in both the treatment of cancer and controlling drug toxicity. Detailed kinetic characterization of the most effectively designed variants shows that they are inhibited by â¼80% compared to the unmodified protein, and their activity is fully restored following incubation with site-specific proteases. Introducing disulfide bonds between the pro- and catalytic domains based on the design models increases the degree of inhibition to 98% but decreases the degree of restoration of activity by proteolysis. A selected disulfide-containing proenzyme exhibits significantly lower activity relative to the fully activated enzyme when evaluated in cell culture. Structural and thermodynamic characterization provides detailed insights into the prodomain binding and inhibition mechanisms. The described methodology is general and could enable the design of a variety of proproteins with precise spatial regulation.
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Desenho Assistido por Computador , Desenho de Fármacos , Precursores Enzimáticos , Engenharia de Proteínas , gama-Glutamil Hidrolase , Domínio Catalítico , Desenho de Fármacos/métodos , Precursores Enzimáticos/química , Precursores Enzimáticos/farmacologia , Humanos , Células PC-3 , Engenharia de Proteínas/métodos , gama-Glutamil Hidrolase/química , gama-Glutamil Hidrolase/farmacologiaRESUMO
BACKGROUND: Juvenile hormones (JH) play crucial role in regulating development and reproduction in insects. The most common form of JH is JH III, derived from MF through epoxidation by CYP15 enzymes. However, in the higher dipterans, such as the fruitfly, Drosophila melanogaster, a bis-epoxide form of JHB3, accounted most of the JH detected. Moreover, these higher dipterans have lost the CYP15 gene from their genomes. As a result, the identity of the P450 epoxidase in the JH biosynthesis pathway in higher dipterans remains unknown. RESULTS: In this study, we show that Cyp6g2 serves as the major JH epoxidase responsible for the biosynthesis of JHB3 and JH III in D. melanogaster. The Cyp6g2 is predominantly expressed in the corpus allatum (CA), concurring with the expression pattern of jhamt, another well-studied gene that is crucial in the last steps of JH biosynthesis. Mutation in Cyp6g2 leads to severe disruptions in larval-pupal metamorphosis and exhibits reproductive deficiencies, exceeding those seen in jhamt mutants. Notably, Cyp6g2-/-::jhamt2 double mutants all died at the pupal stage but could be rescued through the topical application of JH analogs. JH titer analyses revealed that both Cyp6g2-/- mutant and jhamt2 mutant lacking JHB3 and JH III, while overexpression of Cyp6g2 or jhamt caused a significant increase in JHB3 and JH III titer. CONCLUSIONS: These findings collectively established that Cyp6g2 as the major JH epoxidase in the higher dipterans and laid the groundwork for the further understanding of JH biosynthesis. Moreover, these findings pave the way for developing specific Cyp6g2 inhibitors as insect growth regulators or insecticides.
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Sistema Enzimático do Citocromo P-450 , Drosophila melanogaster , Hormônios Juvenis , Animais , Corpora Allata/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Hormônios Juvenis/biossíntese , Hormônios Juvenis/metabolismo , Larva/crescimento & desenvolvimento , Larva/genética , Metamorfose Biológica/genética , Oxirredutases , Pupa/crescimento & desenvolvimento , Pupa/genética , Pupa/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is a highly lethal cancer, and proteomic studies have shown increased protein diversity and abundance in HCC tissues, whereas the role of protein translation has not been extensively explored in HCC. Our research focused on key molecules in the translation process to identify a potential contributor in HCC. We discovered that EIF4G2, a crucial translation initiation factor, is significantly upregulated in HCC tissues and associated with poor prognosis. This study uniquely highlights the impact of EIF4G2 deletion, which suppresses tumor growth and metastasis both in vitro and in vivo. Furthermore, polysome analysis and nascent protein synthesis assays revealed EIF4G2's role in regulating protein translation, specifically identifying PLEKHA1 as a key translational product. This represents a novel mechanistic insight into HCC malignancy. RNA immunoprecipitation (RIP) and Dual-luciferase reporter assays further revealed that EIF4G2 facilitates PLEKHA1 translation via an IRES-dependent manner. Importantly, the synergistic effects of EIF4G2 depletion and PLEKHA1 reduction in inhibiting cell migration and invasion underscore the therapeutic potential of targeting this axis. This study not only advances our understanding of translational regulation in HCC but also identifies the EIF4G2-PLEKHA1 axis as a promising therapeutic target, offering new avenues for intervention in HCC treatment.
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INTRODUCTION: Estrogen receptor (ER) positive patients compromise about 70% of breast cancers. Tamoxifen, an antagonist of ERα66 (the classic ER), is the most effective and the standard first-line drug. However, its efficacy is limited by the development of acquired resistance. METHODS: A specific inhibitor of Hsp70-Bim protein-protein interaction (PPI), S1g-2, together with an inhibitor of Hsp70-Bag3 PPI, MKT-077 and an ATP-competitive inhibitor VER155008, were used as chemical tools. Cell viability assays, co-immunoprecipitation and gene knockdown were used to investigate the role of Hsp70 in tamoxifen resistance. A xenograft model was established in which tamoxifen-resistant breast cancer (MCF-7/TAM-R) cells maintained in the presence of 5 µM tamoxifen were subcutaneously inoculated. The anti-tumor efficiency of S1g-2 was measured after a daily injection of 0.8 mg/kg for 14 days. RESULTS: It was revealed that Hsp70-Bim PPI protects ERα-positive breast cancer from tamoxifen-induced apoptosis through binding and stabilizing ERα36, rather than ERα66, resulting in sustained EGFR mRNA and protein expression. Disruption of Hsp70-Bim PPI and downregulation of ERα36 expression in tumor samples are consistent with the in vitro functions of S1g-2, resulting in about a three-fold reduction in tumor volume. CONCLUSIONS: The in vivo activity and safety of S1g-2 illustrated that it is a potential strategy for Hsp70-Bim disruption to overcome tamoxifen-resistant ER-positive breast cancer.
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Neoplasias da Mama , Tamoxifeno , Humanos , Feminino , Tamoxifeno/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteína 11 Semelhante a Bcl-2/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão GênicaRESUMO
The 2022 global mpox outbreak was driven by human-to-human transmission, but modes of transmission by sexual relationship versus sexual contact remain unclear. We evaluated sexual transmission of mpox by using monkeypox virus (MPXV) G2R-mRNA as a marker of ongoing viral replication through in vitro experiments. We analyzed clinical samples of 15 MPXV-positive patients in Italy from different biological regions by using the setup method. The presence of MPXV DNA, MPXV G2R-mRNA, or both in all analyzed lesion swab samples, independent of viral load, confirmed a higher infectivity risk from skin lesions. Positivity for MPXV G2R-mRNA in nasopharyngeal swabs was associated with high MPXV load, whereas positive results for MPXV G2R-mRNA were obtained only in the 2 semen samples with the lowest MPXV loads. Our results suggest that close or skin-to-skin contact during sexual intercourse is the main route of sexual transmission and that semen is a minor driver of infection, regardless of MPXV load.
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Monkeypox virus , Mpox , Humanos , Itália/epidemiologia , Masculino , Feminino , Mpox/transmissão , Mpox/epidemiologia , Mpox/virologia , Monkeypox virus/genética , Carga Viral , Adulto , Pessoa de Meia-Idade , Replicação Viral , Comportamento Sexual , RNA Viral , Sêmen/virologia , DNA ViralRESUMO
Non-small cell lung cancer (NSCLC) stands prominent among the prevailing and formidable oncological entities. The immune and metabolic-related molecule Phospholipase A2, group IID (PLA2G2D) exerts promotional effects on tumor progression. However, its involvement in cancer angiogenesis remains elusive. Therefore, this investigation delved into the functional significance of PLA2G2D concerning angiogenesis in NSCLC. This study analyzed the expression and enriched pathways of PLA2G2D in NSCLC tissues through bioinformatics analysis, and measured the expression of PLA2G2D in NSCLC cells using qRT-PCR and western blot (WB). Subsequently, the viability and angiogenic potential of NSCLC cells were assessed employing CCK-8 and angiogenesis assays, respectively. The expression profile of angiogenic factors was analyzed through WB. Finally, the expression of glycolysis pathway-related genes, extracellular acidification rate and oxygen consumption rate, and the levels of pyruvate, lactate, citrate, and malate were analyzed in NSCLC cells using qRT-PCR, Seahorse XF 96, and related kits. Bioinformatics analysis revealed the upregulation of PLA2G2D in NSCLC tissues and its association with VEGF and glycolysis signaling pathways. Molecular and cellular experiments demonstrated that upregulated PLA2G2D promoted the viability, angiogenic ability, and glycolysis pathway of NSCLC cells. Rescue assays revealed that the effects of high expression of PLA2G2D on the viability, angiogenic ability, and glycolysis of NSCLC cells were weakened after the addition of the glycolysis inhibitor 2-DG. In summary, PLA2G2D plays a key role in NSCLC angiogenesis through aerobic glycolysis, displaying great potential as a target for anti-angiogenesis therapy.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Neovascularização Patológica , Humanos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/irrigação sanguínea , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/genética , Neovascularização Patológica/metabolismo , Linhagem Celular Tumoral , Glicólise , Fosfolipases A2 do Grupo II/metabolismo , Fosfolipases A2 do Grupo II/genética , Regulação Neoplásica da Expressão Gênica , Proliferação de Células , Transdução de Sinais , AngiogêneseRESUMO
Cell cycle regulation is critical to blood vessel formation and function, but how the endothelial cell cycle integrates with vascular regulation is not well-understood, and available dynamic cell cycle reporters do not precisely distinguish all cell cycle stage transitions in vivo. Here we characterized a recently developed improved cell cycle reporter (PIP-FUCCI) that precisely delineates S phase and the S/G2 transition. Live image analysis of primary endothelial cells revealed predicted temporal changes and well-defined stage transitions. A new inducible mouse cell cycle reporter allele was selectively expressed in postnatal retinal endothelial cells upon Cre-mediated activation and predicted endothelial cell cycle status. We developed a semi-automated zonation program to define endothelial cell cycle status in spatially defined and developmentally distinct retinal areas and found predicted cell cycle stage differences in arteries, veins, and remodeled and angiogenic capillaries. Surprisingly, the predicted dearth of S-phase proliferative tip cells relative to stalk cells at the vascular front was accompanied by an unexpected enrichment for endothelial tip and stalk cells in G2, suggesting G2 stalling as a contribution to tip-cell arrest and dynamics at the front. Thus, this improved reporter precisely defines endothelial cell cycle status in vivo and reveals novel G2 regulation that may contribute to unique aspects of blood vessel network expansion.
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Senescence is an irreversible withdrawal from cell proliferation that can be initiated after DNA damage-induced cell cycle arrest in G2 phase to prevent genomic instability. Senescence onset in G2 requires p53 (also known as TP53) and retinoblastoma protein (RB, also known as RB1) family tumour suppressors, but how they are regulated to convert a temporary cell cycle arrest into a permanent one remains unknown. Here, we show that a previously unrecognised balance between the cyclin-dependent kinase (CDK) inhibitor p21 and the checkpoint kinase Chk1 controls cyclin D-CDK activity during G2 arrest. In non-transformed cells, p21 activates RB in G2 by inhibiting cyclin D1 complexed with CDK2 or CDK4. The resulting G2 exit, which precedes the appearance of senescence markers, is associated with a mitotic bypass, Chk1 downregulation and reduction in the number of DNA damage foci. In p53/RB-proficient cancer cells, a compromised G2 exit correlates with sustained Chk1 activity, delayed p21 induction, untimely cyclin E1 re-expression and genome reduplication. Conversely, Chk1 depletion promotes senescence by inducing p21 binding to cyclin D1- and cyclin E1-CDK complexes and downregulating CDK6, whereas knockdown of the checkpoint kinase Chk2 enables RB phosphorylation and delays G2 exit. In conclusion, p21 and Chk2 oppose Chk1 to maintain RB activity, thus promoting the onset of senescence induced by DNA damage in G2.
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Ciclina D1 , Proteína Supressora de Tumor p53 , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo , Fosforilação , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Cancer cells exhibit high glycolytic activity, metabolizing glucose as their primary energy substrate. Toxic metabolites produced during glycolysis, such as methylglyoxal, induce carbonyl stress (CS), promoting inflammation and oxidative stress. The elevated glucose metabolism in cancer cells creates this toxic environment. However, little research has focused on the molecules mediating these reactions and stresses, and their role in selecting and enriching apoptosis-resistant cells. This study investigated the impact of constitutively suppressing oxidized lipid receptor G2A (GPR132) expression on the relationship between CS and oxidative stress in glucose-loaded cancer cells. G2A has recently attracted attention as a tumor promoter. However, our study shows that G2A suppression under glucose loading significantly reduces CS and associated oxidative stress, thereby enhancing cancer cell survival. This suggests a new mechanism contrary to conventional thinking, involving the acute induction of glyoxalase 1 (Glo1). G2A may thus play a role in selecting and enriching apoptosis-resistant cell populations under high glucose conditions by regulating Glo1 expression. These findings improve our understanding of the adaptive capacity of cancer cells to glucose toxicity.
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BACKGROUND: ZNF468 is a relatively unexplored gene that has been implicated in potential oncogenic properties in various cancer types. However, the exact role of ZNF468 in radiotherapy resistance of esophageal squamous cell carcinomas (ESCCs) is not well understood. METHODS: Bioinformatic analysis was performed using the TCGA database to assess ZNF468 expression and prognostic significance in pan-cancer and ESCC. Functional experiments were conducted using ZNF468 overexpressing and knockdown cell lines to assess its impact on cell survival, DNA damage response, cell cycle, and apoptosis upon radiation. A luciferase reporter assay was utilized to validate ZNF468 binding to the AURKA promoter. RESULTS: ZNF468 was significantly upregulated in diverse cancer types, including ESCC, and its high expression correlated with adverse prognosis in specific tumors. In the ESCC cohort, ZNF468 exhibited substantial upregulation in post-radiotherapy tissues, indicating its potential role in conferring radiotherapy resistance. Functional experiments revealed that ZNF468 enhances cell viability and facilitates DNA damage repair in radiotherapy-treated ESCC cells, while dampening the G2/M cell cycle arrest and apoptosis induced by radiation. Moreover, ZNF468 facilitated AURKA transcription, resulting in upregulated Aurora A expression, and subsequently inhibited P53 expression, unveiling key molecular mechanisms underlying radiotherapy resistance in ESCC. CONCLUSION: ZNF468 plays an oncogenic role in ESCC and contributes to radiotherapy resistance. It enhances cell survival while dampening radiation-induced G2/M cell cycle arrest and apoptosis. By modulating AURKA and P53 expression, ZNF468 represents a promising therapeutic target for enhancing radiotherapy efficacy in ESCC.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Humanos , Apoptose/genética , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/radioterapia , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/radioterapia , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Tolerância a Radiação/genética , Proteína Supressora de Tumor p53RESUMO
Cyanobacteria represent a rich resource of a wide array of unique bioactive compounds that are proving to be potent sources of anticancer drugs. Selenium nanoparticles (SeNPs) have shown an increasing potential as major therapeutic platforms and led to the production of higher levels of ROS that can present desirable anticancer properties. Chitosan-SeNPs have also presented antitumor properties against hepatic cancer cell lines, especially the Cht-NP (Chitosan-NPs), promoting ROS generation and mitochondria dysfunction. It is proposed that magnetic fields can add new dimensions to nanoparticle applications. Hence, in this study, the biosynthesis of SeNPs using Alborzia kermanshahica and chitosan (CS) as stabilizers has been developed. The SeNPs synthesis was performed at different cyanobacterial cultivation conditions, including control (without magnetic field) and magnetic fields of 30 mT and 60 mT. The SeNPs were characterized by uv-visible spectroscopy, Fourier-transform infrared spectroscopy (FT-IR), Dynamic light scattering (DLS), zeta potential, and TEM. In addition, the antibacterial activity, inhibition of bacterial growth, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC), as well as the antifungal activity and cytotoxicity of SeNPs, were performed. The results of uv-visible spectrometry, DLS, and zeta potential showed that 60 mT had the highest value regarding the adsorption, size, and stabilization in compared to the control. FTIR spectroscopy results showed consistent spectra, but the increased intensity of peaks indicates an increase in bond number after exposure to 30 mT and 60 mT. The results of the antibacterial activity and the inhibition zone diameter of synthesized nanoparticles showed that Staphylococcus aureus was more sensitive to nanoparticles produced under 60 mT. Se-NPs produced by Alborzia kermanshahica cultured under a 60 mT magnetic field exhibit potent antimicrobial and anticancer properties, making them a promising natural agent for use in the pharmaceutical and biomedical industries.
Assuntos
Quitosana , Campos Magnéticos , Selênio , Selênio/química , Selênio/farmacologia , Quitosana/química , Quitosana/farmacologia , Humanos , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/biossíntese , Testes de Sensibilidade Microbiana , Nanopartículas/química , Antineoplásicos/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/química , Nanopartículas Metálicas/químicaRESUMO
It is urgent to explore factors affecting immunotherapy efficacy to benefit non-small cell lung cancer (NSCLC) patient survival. Bioinformatics predicted genes associated with programmed cell death ligand 1 (PD-L1) expression and analysed phospholipase A2 group IID (PLA2G2D) expression in NSCLC. BODIPY 493/503 dye staining and kits detected lipids, triglycerides, and phospholipids in H1299 cells, respectively. Extracellular vesicles (EVs) were extracted for morphology and size assessment using electron microscopy. Western blot assayed CD9, CD63, HSP90, EVs-PD-L1, PD-L1, and PLA2G2D expression. CCK-8, LDH, and ELISA tested proliferation and toxicity of CD8+ T cells, interleukin-2, and interferon-gamma secretion, respectively. PLA2G2D, PD-L1, and Ki67 expression was detected by immunohistochemistry. Immunofluorescence assayed PLA2G2D localisation and CD8+ T cell content. Flow cytometry assessed PD-L1 and CD8 expression. In NSCLC, upregulated EVs-PD-L1 and clinical characteristics showed a strong correlation. H1299 cells with overexpression PD-L1 significantly reduced proliferation, toxicity of CD8+ T cells, and interleukin-2 and interferon-gamma levels. Bioinformatics revealed positive correlations between PLA2G2D and overexpressed PD-L1. PLA2G2D was expressed in macrophages and dendritic cells in NSCLC tissue. Overexpression PLA2G2D (oe-PLA2G2D) increased lipids, triglycerides, and phospholipids contents in H1299 cells. oe-PLA2G2D significantly reduced proliferation, toxicity of CD8+ T cells, and interleukin-2 and interferon-gamma levels. si-PD-L1 restored inhibition of oe-PLA2G2D on CD8+ T cells. oe-PLA2G2D significantly increased mice tumour volume and weight, upregulated expression of blood EVs-PD-L1 and tissue PD-L1, PLA2G2D, Ki67, and decreased CD8+ T cell content. PLA2G2D facilitated immune escape in NSCLC by regulating CD8+ T cell immune function by upregulating EVs-PD-L1.