RESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1007253.].
RESUMO
Epstein-Barr virus (EBV) is a ubiquitous oncogenic virus that induces many cancers. N6-Methyladenosine (m6A) modification regulates many cellular processes. We explored the role of m6A in EBV gene regulation and associated cancers. We have comprehensively defined m6A modification of EBV latent and lytic transcripts. Furthermore, m6A modification demonstrated a functional role in regulation of the stability of viral transcripts. The methyltransferase METTL14 was induced at the transcript and protein levels, and knock-down of METTL14 led to decreased expression of latent EBV transcripts. METTL14 was also significantly induced in EBV-positive tumors, promoted growth of EBV-transformed cells and tumors in Xenograft animal models. Mechanistically, the viral-encoded latent oncoprotein EBNA3C activated transcription of METTL14, and directly interacted with METTL14 to promote its stability. This demonstrated that EBV hijacks METTL14 to drive EBV-mediated tumorigenesis. METTL14 is now a new target for development of therapeutics for treatment of EBV-associated cancers.
Assuntos
Transformação Celular Viral , Infecções por Vírus Epstein-Barr/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/metabolismo , Metiltransferases/biossíntese , Proteínas de Neoplasias/biossíntese , Neoplasias/metabolismo , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Infecções por Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Células HEK293 , Humanos , Masculino , Metiltransferases/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Neoplasias/virologiaRESUMO
EBV latent antigen 3C (EBNA3C) is essential for EBV-induced primary B-cell transformation. Infection by EBV induces hypermethylation of a number of tumor suppressor genes, which contributes to the development of human cancers. The Ras association domain family isoform 1A (RASSF1A) is a cellular tumor suppressor, which regulates a broad range of cellular functions, including apoptosis, cell-cycle arrest, mitotic arrest, and migration. However, the expression of RASSF1A is lost in many human cancers by epigenetic silencing. In the present study, we showed that EBNA3C promoted B-cell transformation by specifically suppressing the expression of RASSF1A. EBNA3C directly interacted with RASSF1A and induced RASSF1A degradation via the ubiquitin-proteasome-dependent pathway. SCFSkp2, an E3-ubiquitin ligase, was recruited by EBNA3C to enhance RASSF1A degradation. Moreover, EBNA3C decreased the transcriptional activity of RASSF1A promoter by enhancing its methylation through EBNA3C-mediated modulation of DNMTs expression. EBNA3C also inhibited RASSF1A-mediated cell apoptosis, disrupted RASSF1A-mediated microtubule and chromosomal stability, and promoted cell proliferation by upregulating Cyclin D1 and Cyclin E expression. Our data provides new details, which sheds light on additional mechanisms by which EBNA3C can induce B-cell transformation. This will also facilitate the development of novel therapeutic approaches through targeting of the RASSF1A pathway.
Assuntos
Infecções por Vírus Epstein-Barr/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Proteínas Supressoras de Tumor/genética , Antígenos Virais/genética , Apoptose , Linfócitos B/metabolismo , Linfócitos B/virologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Transformação Celular Viral/genética , Metilação de DNA/genética , Regulação para Baixo , Epigênese Genética/genética , Infecções por Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos , Ativação Linfocitária/genética , Regiões Promotoras Genéticas/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Kaposi's sarcoma associated herpesvirus (KSHV), like all herpesviruses maintains lifelong persistence with its host genome in latently infected cells with only a small fraction of cells showing signatures of productive lytic replication. Modulation of cellular signaling pathways by KSHV-encoded latent antigens, and microRNAs, as well as some level of spontaneous reactivation are important requirements for establishment of viral-associated diseases. Hypoxia, a prominent characteristic of the microenvironment of cancers, can exert specific effects on cell cycle control, and DNA replication through HIF1α-dependent pathways. Furthermore, hypoxia can induce lytic replication of KSHV. The mechanism by which KSHV-encoded RNAs and antigens regulate cellular and viral replication in the hypoxic microenvironment has yet to be fully elucidated. We investigated replication-associated events in the isogenic background of KSHV positive and negative cells grown under normoxic or hypoxic conditions and discovered an indispensable role of KSHV for sustained cellular and viral replication, through protection of critical components of the replication machinery from degradation at different stages of the process. These include proteins involved in origin recognition, pre-initiation, initiation and elongation of replicating genomes. Our results demonstrate that KSHV-encoded LANA inhibits hypoxia-mediated degradation of these proteins to sustain continued replication of both host and KSHV DNA. The present study provides a new dimension to our understanding of the role of KSHV in survival and growth of viral infected cells growing under hypoxic conditions and suggests potential new strategies for targeted treatment of KSHV-associated cancer.
Assuntos
Antígenos Virais/metabolismo , Respiração Celular/fisiologia , Herpesvirus Humano 8/metabolismo , Proteínas Nucleares/metabolismo , Antígenos Virais/genética , Antígenos Virais/imunologia , Linhagem Celular Tumoral , Herpesvirus Humano 8/patogenicidade , Humanos , Hipóxia/metabolismo , Proteínas Nucleares/imunologia , Sarcoma de Kaposi/virologia , Microambiente Tumoral , Latência Viral/genética , Replicação Viral/genéticaRESUMO
BACKGROUND: Herpes Simplex Virus type I (HSV-1) is a large double-stranded DNA virus that enters productive infection in epithelial cells and reorganizes the host nucleus. Cohesin, a major constituent of interphase and mitotic chromosomes comprised of SMC1, SMC3, and SCC1 (Mcd1/Rad21), SCC3 (SA1/SA2), have diverse functions, including sister chromatid cohesion, DNA double-stranded breaks repair, and transcriptional control. Little is known about the role of cohesin in HSV-1 lytic infection. METHODS: We measured the effect on HSV-1 transcription, genome copy number, and viral titer by depleting cohesin components SMC1 or Rad21 using RNAi, followed by immunofluorescence, qPCR, and ChIP experiments to gain insight into cohesin's function in HSV-1 transcription and replication. RESULTS: Here, we report that cohesion subunits SMC1 and Rad21 are recruited to the lytic HSV-1 replication compartment. The knockdown results in decreased viral transcription, protein expression, and maturation of viral replication compartments. SMC1 and Rad21 knockdown leads to the reduced overall RNA pol II occupancy level but increased RNA pol II ser5 phosphorylation binding on viral genes. Consistent with this, the knockdown increased H3K27me3 modification on these genes. CONCLUSIONS: These results suggest that cohesin facilitates HSV-1 lytic transcription by promoting RNA Pol II transcription activity and preventing chromatin's silencing on the viral genome.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Fibroblastos/virologia , Herpesvirus Humano 1/genética , RNA Polimerase II/metabolismo , Transcrição Gênica , Proteínas de Ciclo Celular/classificação , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proteínas Cromossômicas não Histona/classificação , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/metabolismo , Genoma Viral , Células HeLa , Interações entre Hospedeiro e Microrganismos , Humanos , Proteínas Nucleares/metabolismo , Ligação Proteica , CoesinasRESUMO
Shugoshin-1 (Sgo1) protects the integrity of the centromeres, and H2A phosphorylation is critical for this process. The mitotic checkpoint kinase Bub1, phosphorylates H2A and ensures fidelity of chromosome segregation and chromosome number. Oncogenic KSHV induces genetic alterations through chromosomal instability (CIN), and its essential antigen LANA regulates Bub1. We show that LANA inhibits Bub1 phosphorylation of H2A and Cdc20, important for chromosome segregation and mitotic signaling. Inhibition of H2A phosphorylation at residue T120 by LANA resulted in dislocation of Sgo1, and cohesin from the centromeres. Arrest of Cdc20 phosphorylation also rescued degradation of Securin and Cyclin B1 at mitotic exit, and interaction of H2A, and Cdc20 with Bub1 was inhibited by LANA. The N-terminal nuclear localization sequence domain of LANA was essential for LANA and Bub1 interaction, reversed LANA inhibited phosphorylation of H2A and Cdc20, and attenuated LANA-induced aneuploidy and cell proliferation. This molecular mechanism whereby KSHV-induced CIN, demonstrated that the NNLS of LANA is a promising target for development of anti-viral therapies targeting KSHV associated cancers.
Assuntos
Aneuploidia , Antígenos Virais/genética , Antígenos Virais/metabolismo , Proteínas de Ciclo Celular/metabolismo , Herpesvirus Humano 8/imunologia , Herpesvirus Humano 8/patogenicidade , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Antígenos Virais/química , Proteínas Cdc20/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Centrômero/metabolismo , Instabilidade Cromossômica , Ciclina B1/metabolismo , Herpesvirus Humano 8/genética , Histonas/metabolismo , Humanos , Mitose , Modelos Biológicos , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Proteínas Nucleares/química , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Securina/metabolismoRESUMO
Kaposi's sarcoma associated herpesvirus (KSHV) infection stabilizes hypoxia inducible factors (HIFs). The interaction between KSHV encoded factors and HIFs plays a critical role in KSHV latency, reactivation and associated disease phenotypes. Besides modulation of large-scale signaling, KSHV infection also reprograms the metabolic activity of infected cells. However, the mechanism and cellular pathways modulated during these changes are poorly understood. We performed comparative RNA sequencing analysis on cells with stabilized hypoxia inducible factor 1 alpha (HIF1α) of KSHV negative or positive background to identify changes in global and metabolic gene expression. Our results show that hypoxia induces glucose dependency of KSHV positive cells with high glucose uptake and high lactate release. We identified the KSHV-encoded vGPCR, as a novel target of HIF1α and one of the main viral antigens of this metabolic reprogramming. Bioinformatics analysis of vGPCR promoter identified 9 distinct hypoxia responsive elements which were activated by HIF1α in-vitro. Expression of vGPCR alone was sufficient for induction of changes in the metabolic phenotype similar to those induced by KSHV under hypoxic conditions. Silencing of HIF1α rescued the hypoxia associated phenotype of KSHV positive cells. Analysis of the host transcriptome identified several common targets of hypoxia as well as KSHV encoded factors and other synergistically activated genes belonging to cellular pathways. These include those involved in carbohydrate, lipid and amino acids metabolism. Further DNA methyltranferases, DNMT3A and DNMT3B were found to be regulated by either KSHV, hypoxia, or both synergistically at the transcript and protein levels. This study showed distinct and common, as well as synergistic effects of HIF1α and KSHV-encoded proteins on metabolic reprogramming of KSHV-infected cells in the hypoxia.
Assuntos
Linfócitos B/virologia , Herpesvirus Humano 8/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Linfócitos B/metabolismo , Western Blotting , Regulação Viral da Expressão Gênica , Glucose/metabolismo , Herpesvirus Humano 8/genética , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Ácido Láctico/metabolismo , Leucócitos Mononucleares/virologia , Metaboloma , Microscopia Confocal , Fenótipo , Regiões Promotoras Genéticas , RNA Viral/química , Espécies Reativas de Oxigênio/análise , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Análise de Sequência de RNA , Ativação TranscricionalRESUMO
CTCF is an essential epigenetic regulator mediating chromatin insulation, long-range regulatory interactions, and the organization of large topological domains in the nucleus. Phenotypes of CTCF haploinsufficient mutations in humans, knockout in mice, and depletion in cells are often consistent with impaired genome stability, but a role of CTCF in genome maintenance has not been fully investigated. Here, we report that CTCF maintains genome stability, is recruited to sites of DNA damage, and promotes homologous recombination repair of DNA double-strand breaks (DSBs). CTCF depletion increased chromosomal instability, marked by chromosome breakage and end fusions, elevated genotoxic stress-induced genomic DNA fragmentation, and activated the ataxia telangiectasia mutated (ATM) kinase. We show that CTCF could be recruited to drug-induced 53BP1 foci and known fragile sites, as well as to I-SceI endonuclease-induced DSBs. Laser irradiation analysis revealed that this recruitment depends on ATM, Nijmegen breakage syndrome (NBS), and the zinc finger DNA-binding domain of CTCF. We demonstrate that CTCF knockdown impaired homologous recombination (HR) repair of DSBs. Consistent with this, CTCF knockdown reduced the formation of γ-radiation-induced Rad51 foci, as well as the recruitment of Rad51 to laser-irradiated sites of DNA lesions and to I-SceI-induced DSBs. We further show that CTCF is associated with DNA HR repair factors MDC1 and AGO2, and directly interacts with Rad51 via its C terminus. These analyses establish a direct, functional role of CTCF in DNA repair and provide a potential link between genome organization and genome stability.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Dano ao DNA , Instabilidade Genômica , Osteossarcoma/genética , Reparo de DNA por Recombinação , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Fator de Ligação a CCCTC/antagonistas & inibidores , Fator de Ligação a CCCTC/genética , Humanos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , RNA Interferente Pequeno/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Células Tumorais CultivadasRESUMO
Cell cycle regulation is one of the hallmarks of virus-mediated oncogenesis. Epstein-Barr virus (EBV)-induced lymphomas express a repertoire of essential viral latent proteins that regulate expression of cell cycle-related proteins to dysregulate this process, thereby facilitating the proliferation of infected cells. We now demonstrate that the essential EBV latent protein 3C (EBNA3C) stabilizes cyclin D2 to regulate cell cycle progression. More specifically, EBNA3C directly binds to cyclin D2 and they colocalize together in nuclear compartments. We show that EBNA3C regulates the promoter of cyclin D2 through cooperation with master transcription factor Bcl6 and enhances its stability by inhibiting its ubiquitin-dependent degradation. EBNA3C also promoted cell proliferation in the presence of cyclin D2, suggesting that cyclin D2 contributes to EBNA3C-mediated cell cycle progression. These results provide new clues as to the role of this essential viral latent protein and its ability to regulate expression of cellular factors, which drives the oncogenic process.IMPORTANCE Epstein-Barr virus (EBV) is the first identified human tumor virus and is associated with a range of human cancers. During EBV-induced lymphomas, the essential viral latent proteins modify the expression of cell cycle-related proteins to disturb the cell cycle process, thereby facilitating the proliferative process. The essential EBV nuclear antigen 3C (EBNA3C) plays an important role in EBV-mediated B-cell transformation. Here we show that EBNA3C stabilizes cyclin D2 to regulate cell cycle progression. More specifically, EBNA3C directly binds to cyclin D2, and they colocalize together in nuclear compartments. EBNA3C enhances cyclin D2 stability by inhibiting its ubiquitin-dependent degradation and significantly promotes cell proliferation in the presence of cyclin D2. Our results provide novel insights into the function of EBNA3C on cell progression by regulating the cyclin D2 protein and raise the possibility of the development of new anticancer therapies against EBV-associated cancers.
Assuntos
Proliferação de Células/genética , Ciclina D2/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Regulação da Expressão Gênica , Linfócitos B/virologia , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Transformação Celular Viral , Herpesvirus Humano 4/fisiologia , HumanosRESUMO
Herpesvirus-induced disease is one of the most lethal factors which leads to high mortality in HIV/AIDS patients. EBV, also known as human herpesvirus 4, can transform naive B cells into immortalized cells in vitro through the regulation of cell cycle, cell proliferation, and apoptosis. EBV infection is associated with several lymphoma and epithelial cancers in humans, which occurs at a much higher rate in immune deficient individuals than in healthy people, demonstrating that the immune system plays a vital role in inhibiting EBV activities. EBV latency infection proteins can mimic suppression cytokines or upregulate PD-1 on B cells to repress the cytotoxic T cells response. Many malignancies, including Hodgkin Lymphoma and non-Hodgkin's lymphomas occur at a much higher frequency in EBV positive individuals than in EBV negative people during the development of HIV infection. Importantly, understanding EBV pathogenesis at the molecular level will aid the development of novel therapies for EBV-induced diseases in HIV/AIDS patients.
Assuntos
Síndrome da Imunodeficiência Adquirida , Herpesvirus Humano 4 , Neoplasias , Síndrome da Imunodeficiência Adquirida/fisiopatologia , Síndrome da Imunodeficiência Adquirida/virologia , Carcinogênese , Coinfecção/virologia , Infecções por Vírus Epstein-Barr/fisiopatologia , Infecções por Vírus Epstein-Barr/virologia , Infecções por HIV/fisiopatologia , Infecções por HIV/virologia , Herpesvirus Humano 4/fisiologia , Humanos , Hospedeiro Imunocomprometido , Linfoma Relacionado a AIDS/fisiopatologia , Linfoma Relacionado a AIDS/virologia , Neoplasias/fisiopatologia , Neoplasias/virologiaRESUMO
UNLABELLED: Studies of herpes simplex virus (HSV) infections of humans are limited by the use of rodent models such as mice, rabbits, and guinea pigs. Tree shrews (Tupaia belangeri chinensis) are small mammals indigenous to southwest Asia. At behavioral, anatomical, genomic, and evolutionary levels, tree shrews are much closer to primates than rodents are, and tree shrews are susceptible to HSV infection. Thus, we have studied herpes simplex virus 1 (HSV-1) infection in the tree shrew trigeminal ganglion (TG) following ocular inoculation. In situ hybridization, PCR, and quantitative reverse transcription-PCR (qRT-PCR) analyses confirm that HSV-1 latently infects neurons of the TG. When explant cocultivation of trigeminal ganglia was performed, the virus was recovered after 5 days of cocultivation with high efficiency. Swabbing the corneas of latently infected tree shrews revealed that tree shrews shed virus spontaneously at low frequencies. However, tree shrews differ significantly from mice in the expression of key HSV-1 genes, including ICP0, ICP4, and latency-associated transcript (LAT). In acutely infected tree shrew TGs, no level of ICP4 was observed, suggesting the absence of infection or a very weak, acute infection compared to that of the mouse. Immunofluorescence staining with ICP4 monoclonal antibody, and immunohistochemistry detection by HSV-1 polyclonal antibodies, showed a lack of viral proteins in tree shrew TGs during both acute and latent phases of infection. Cultivation of supernatant from homogenized, acutely infected TGs with RS1 cells also exhibited an absence of infectious HSV-1 from tree shrew TGs. We conclude that the tree shrew has an undetectable, or a much weaker, acute infection in the TGs. Interestingly, compared to mice, tree shrew TGs express high levels of ICP0 transcript in addition to LAT during latency. However, the ICP0 transcript remained nuclear, and no ICP0 protein could be seen during the course of mouse and tree shrew TG infections. Taken together, these observations suggest that the tree shrew TG infection differs significantly from the existing rodent models. IMPORTANCE: Herpes simplex viruses (HSVs) establish lifelong infection in more than 80% of the human population, and their reactivation leads to oral and genital herpes. Currently, rodent models are the preferred models for latency studies. Rodents are distant from primates and may not fully represent human latency. The tree shrew is a small mammal, a prosimian primate, indigenous to southwest Asia. In an attempt to further develop the tree shrew as a useful model to study herpesvirus infection, we studied the establishment of latency and reactivation of HSV-1 in tree shrews following ocular inoculation. We found that the latent virus, which resides in the sensory neurons of the trigeminal ganglion, could be stress reactivated to produce infectious virus, following explant cocultivation and that spontaneous reactivation could be detected by cell culture of tears. Interestingly, the tree shrew model is quite different from the mouse model of HSV infection, in that the virus exhibited only a mild acute infection following inoculation with no detectable infectious virus from the sensory neurons. The mild infection may be more similar to human infection in that the sensory neurons continue to function after herpes reactivation and the affected skin tissue does not lose sensation. Our findings suggest that the tree shrew is a viable model to study HSV latency.
Assuntos
Herpesvirus Humano 1/fisiologia , Transcrição Gênica , Gânglio Trigeminal/virologia , Tupaiidae/virologia , Latência Viral , Replicação Viral , Animais , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Herpesviridae , Camundongos Endogâmicos BALB C , Proteínas Virais/biossíntese , Eliminação de Partículas ViraisRESUMO
Herpes Simplex Virus type I (HSV-1) latently infects peripheral nervous system (PNS) sensory neurons, and its reactivation leads to recurring cold sores. The reactivated HSV-1 can travel retrograde from the PNS into the central nervous system (CNS) and is known to be causative of Herpes Simplex viral encephalitis. HSV-1 infection in the PNS is well documented, but little is known on the fate of HSV-1 once it enters the CNS. In the murine model, HSV-1 genome persists in the CNS once infected through an ocular route. To gain more details of HSV-1 infection in the CNS, we characterized HSV-1 infection of the tree shrew (Tupaia belangeri chinensis) brain following ocular inoculation. Here, we report that HSV-1 enters the tree shrew brain following ocular inoculation and HSV-1 transcripts, ICP0, ICP4, and LAT can be detected at 5 days post-infection (p.i.), peaking at 10 days p.i. After 2 weeks, ICP4 and ICP0 transcripts are reduced to a basal level, but the LAT intron region continues to be expressed. Live virus could be recovered from the olfactory bulb and brain stem tissue. Viral proteins could be detected using anti-HSV-1 antibodies and anti-ICP4 antibody, during the acute stage but not beyond. In situ hybridization could detect LAT during acute infection in most brain regions and in olfactory bulb and brain stem tissue well beyond the acute stage. Using a homogenate from these tissues' post-acute infection, we did not recover live HSV-1 virus, supporting a latent infection, but using a modified explant cocultivation technique, we were able to recover reactivated virus from these tissues, suggesting that the HSV-1 virus latently infects the tree shrew CNS. Compared to mouse, the CNS acute infection of the tree shrew is delayed and the olfactory bulb contains most latent virus. During the acute stage, a portion of the infected tree shrews exhibit symptoms similar to human viral encephalitis. These findings, together with the fact that tree shrews are closely related to primates, provided a valuable alternative model to study HSV-1 infection and pathogenesis in the CNS.
Assuntos
Encefalite por Herpes Simples/virologia , Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/genética , Gânglio Trigeminal/virologia , Ativação Viral , Latência Viral , Animais , Tronco Encefálico/metabolismo , Tronco Encefálico/patologia , Tronco Encefálico/virologia , Modelos Animais de Doenças , Encefalite por Herpes Simples/genética , Encefalite por Herpes Simples/metabolismo , Encefalite por Herpes Simples/patologia , Feminino , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/patogenicidade , Especificidade de Hospedeiro , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/genética , MicroRNAs/metabolismo , Bulbo Olfatório/metabolismo , Bulbo Olfatório/patologia , Bulbo Olfatório/virologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Técnicas de Cultura de Tecidos , Gânglio Trigeminal/metabolismo , Gânglio Trigeminal/patologia , Tupaiidae , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: Replication of viral genome is the central event during the lytic infectious cycle of herpes simplex virus 1 (HSV-1). However, the details of HSV-1 replication process are still elusive due to the limitations of current molecular and conventional fluorescent microscopy methods. Stimulated emission depletion (STED) microscopy is one of the recently available super-resolution techniques allowing observation at sub-diffraction resolution. METHODS: To gain new insight into HSV-1 replication, we used a combination of stimulated emission depletion microscopy, fluorescence in situ hybridization (FISH) and immunofluorescence (IF) to observe the HSV-1 replication process. RESULTS: Using two colored probes labeling the same region of HSV-1 genome, the two probes highly correlated in both pre-replication and replicating genomes. In comparison, when probes from different regions were used, the average distance between the two probes increased after the virus enters replication, suggesting that the HSV-1 genome undergoes dynamic structure changes from a compact to a relaxed formation and occupies larger space as it enters replication. Using FISH and IF, viral single strand binding protein ICP8 was seen closely positioned with HSV-1 genome. In contrast, ICP8 and host RNA polymerase II were less related. This result suggests that ICP8 marked regions of DNA replication are spatially separated from regions of active transcription, represented by the elongating form of RNA polymerase II within the viral replication compartments. Comparing HSV-1 genomes at early stage of replication with that in later stage, we also noted overall increases among different values. These results suggest stimulated emission depletion microscopy is capable of investigating events during HSV-1 replication. CONCLUSION: 1) Replicating HSV-1 genome could be observed by super-resolution microscopy; 2) Viral genome expands spatially during replication; 3) Viral replication and transcription are partitioned into different sub-structures within the replication compartments.
Assuntos
Herpesvirus Humano 1/fisiologia , Microscopia/métodos , Replicação Viral , Células Cultivadas , Fibroblastos/virologia , HumanosRESUMO
BACKGROUND: The cell cycle is tightly regulated by checkpoints, which play a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anticancer treatments, allowing for DNA repair prior to cell division. Manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies. METHODS: In this study, we conducted a forward genome-wide CRISPR/Cas9 screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress. RESULTS: Our findings revealed that canonical DNA repair pathways, including the Ataxia-telangiectasia mutated (ATM)/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of PKMYT1, in ensuring cell survival. Depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between alkylating agents and a Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice. CONCLUSIONS: Collectively, our findings highlight the potential of targeting cell cycle timing through Myt1 inhibition as an effective strategy to enhance the efficacy of current standard cancer therapies, potentially leading to improved disease outcomes.
Assuntos
Antineoplásicos Alquilantes , Dano ao DNA , Pontos de Checagem da Fase G2 do Ciclo Celular , Temozolomida , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Temozolomida/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Camundongos , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sistemas CRISPR-Cas , Camundongos Nus , Linhagem Celular Tumoral , Células Tumorais Cultivadas , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Reparo do DNA/efeitos dos fármacosRESUMO
In this study, nanoindentation tests at three different strain rates within 100 nm indentation depth were conducted on an a-plane (112¯0) ZnO single crystal to investigate the effect of strain rate on its nano-scale mechanical behavior. The load-indentation-depth curves, pop-in events, hardness and Young's moduli of an a-plane (112¯0) ZnO single crystal at different strain rates were investigated at the nano-scale level. The results indicated that, with the indentation depth increasing, the load increased gradually at each maximum indentation depth, hma, during the loading process. A distinct pop-in event occurred on each loading curve except that corresponding to the hmax of 10 nm. The applied load at the same indentation depth increased with the increasing strain rate during the nanoindentation of the a-plane (112¯0) ZnO single crystal. The higher strain rate deferred the pop-in event to a higher load and deeper indentation depth, and made the pop-in extension width larger. The hardness showed reverse indentation size effect (ISE) before the pop-in, and exhibited normal ISE after the pop-in. Both the hardness and the Young's modulus of the a-plane (112¯0) ZnO single crystal increased with the increasing strain rate, exhibiting the positive strain-rate sensitivity.
RESUMO
PURPOSE: Mutations of the isocitrate dehydrogenase (IDH) gene are common genetic mutations in human malignancies. Increasing evidence indicates that IDH mutations play critical roles in malignant transformation and progression. However, the therapeutic options for IDH-mutated cancers remain limited. In this study, the investigation of patient cohorts revealed that the PI3K/protein kinase B (AKT) signaling pathways were enhanced in IDH-mutated cancer cells. EXPERIMENTAL DESIGN: In this study, we investigated the gene expression profile in IDH-mutated cells using RNA sequencing after the depletion of AKT. Gene set enrichment analysis (GSEA) and pathway enrichment analysis were used to discover altered molecular pathways due to AKT depletion. We further investigated the therapeutic effect of the AKT inhibitor, ipatasertib (Ipa), combined with temozolomide (TMZ) in cell lines and preclinical animal models. RESULTS: GSEA and pathway enrichment analysis indicated that the PI3K/AKT pathway significantly correlated with Nrf2-guided gene expression and ferroptosis-related pathways. Mechanistically, AKT suppresses the activity of GSK3ß and stabilizes Nrf2. Moreover, inhibition of AKT activity with Ipa synergizes with the genotoxic agent TMZ, leading to overwhelming ferroptotic cell death in IDH-mutated cancer cells. The preclinical animal model confirmed that combining Ipa and TMZ treatment prolonged survival. CONCLUSIONS: Our findings highlighted AKT/Nrf2 pathways as a potential synthetic lethality target for IDH-mutated cancers.
Assuntos
Neoplasias Encefálicas , Ferroptose , Glioma , Animais , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Isocitrato Desidrogenase/genética , Fator 2 Relacionado a NF-E2/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ferroptose/genética , Linhagem Celular Tumoral , Glioma/tratamento farmacológico , Glioma/genética , Glioma/patologia , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Mutação , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismoRESUMO
Cancer stem cells (CSCs) are a small population of cells in human malignancies that resemble the biology of human pluripotent stem cells. CSCs are closely related to the critical hallmarks in human cancers, ranging from oncogenesis to disease progression, therapeutic resistance, and overall outcome. Mutations in isocitrate dehydrogenase (IDH) were recently identified as founder mutations for human cancers. An increasing amount of evidence indicates that IDH mutations are closely related to the establishment and maintenance of CSCs. Biosynthesis of oncometabolite, metabolic reprogramming, and epigenetic shifts establish distinctive molecular signatures in IDH-mutated CSCs. Additionally, IDH mutation and IDH-related pathways could be valuable molecular targets to impact the CSC components in human cancers and to improve the disease outcome.
Assuntos
Isocitrato Desidrogenase , Neoplasias , Humanos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Mutação , Neoplasias/genética , Células-Tronco Neoplásicas/metabolismoRESUMO
The mechanical properties of hybrid composite interfaces are critical in determining the overall properties of composite materials. To investigate the mechanical performance of hybrid composite interfaces, an accurate and efficient method must be developed. In this work, nanoindentation is used in this work to investigate the mechanical performance of the carbon/glass interface and the influence of the distance between carbon and the glass fibers on the modulus of the thermoset matrix. The results show that the interface sizes around the carbon and glass fibers are around 1.5 and 2.0 µm, respectively. The modulus around the carbon fibers is 5-11 GPa without the fiber effect, while that around the glass fibers is 4-10 GPa. The modulus of the matrix is not affected by the two types of fibers when the distance between them is greater than 4.5 µm.
RESUMO
Nuclear factor-erythroid 2-related factor 2 (NRF2) is a master regulator of a series of cytoprotective genes, which protects cells from stress conditions such as reactive oxygen species (ROS) and electrophiles. Besides its pivotal role in cellular physiology and stress response, several recent advances revealed that NRF2-governed pathways are extensively exploited in cancer cells, and play critical roles in granting growth advantage, supporting cancer metabolism, and promoting malignant transformation. In the present review, we focus on the regulatory mechanisms of NRF2 and its roles in cancer biology. We also discuss the value of targeting NRF2-associated pathways as a means of developing future cancer therapeutics.
Assuntos
Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias/fisiopatologia , Carcinógenos/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias/genética , Oncogenes/fisiologia , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismoRESUMO
Glioma is one of the most common and lethal brain tumors. Surgical resection followed by radiotherapy plus chemotherapy is the current standard of care for patients with glioma. The existence of resistance to genotoxic therapy, as well as the nature of tumor heterogeneity greatly limits the efficacy of glioma therapy. DNA damage repair pathways play essential roles in many aspects of glioma biology such as cancer progression, therapy resistance, and tumor relapse. O6-methylguanine-DNA methyltransferase (MGMT) repairs the cytotoxic DNA lesion generated by temozolomide (TMZ), considered as the main mechanism of drug resistance. In addition, mismatch repair, base excision repair, and homologous recombination DNA repair also play pivotal roles in treatment resistance as well. Furthermore, cellular mechanisms, such as cancer stem cells, evasion from apoptosis, and metabolic reprogramming, also contribute to TMZ resistance in gliomas. Investigations over the past two decades have revealed comprehensive mechanisms of glioma therapy resistance, which has led to the development of novel therapeutic strategies and targeting molecules.