RESUMO
In the field of nuclear medicine, the ß+ -emitting 43Sc and ß- -emitting 47Sc are promising candidates in cancer diagnosis and targeted radionuclide therapy (TRT) due to their favorable decay schema and shared pharmacokinetics as a true theranostic pair. Additionally, scandium is a group-3 transition metal (like 177Lu) and exhibits affinity for DOTA-based chelators, which have been studied in depth, making the barrier to implementation lower for 43/47Sc than for other proposed true theranostics. Before 43/47Sc can see widespread pre-clinical evaluation, however, an accessible production methodology must be established and each isotope's radiolabeling and animal imaging capabilities studied with a widely utilized tracer. As such, a simple means of converting an 18 MeV biomedical cyclotron to support solid targets and produce 43Sc via the 42Ca(d,n)43Sc reaction has been devised, exhibiting reasonable yields. The NatTi(γ,p)47Sc reaction is also investigated along with the successful implementation of chemical separation and purification methods for 43/47Sc. The conjugation of 43/47Sc with PSMA-617 at specific activities of up to 8.94 MBq/nmol and the subsequent imaging of LNCaP-ENZaR tumor xenografts in mouse models with both 43/47Sc-PSMA-617 are also presented.
Assuntos
Medicina Nuclear , Neoplasias da Próstata , Humanos , Animais , Camundongos , Masculino , Escândio , Medicina de Precisão , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/radioterapia , Radioisótopos/uso terapêuticoRESUMO
The development of androgen receptor signaling inhibitor (ARSI) drug resistance in prostate cancer (PC) remains therapeutically challenging. Our group has described the role of sex determining region Y-box 2 (SOX2) overexpression in ARSI-resistant PC. Continuing this work, we report that NR3C1, the gene encoding glucocorticoid receptor (GR), is a novel SOX2 target in PC, positively regulating its expression. Similar to ARSI treatment, SOX2-positive PC cells are insensitive to GR signaling inhibition using a GR modulating therapy. To understand SOX2-mediated nuclear hormone receptor signaling inhibitor (NHRSI) insensitivity, we performed RNA-seq in SOX2-positive and -negative PC cells following NHRSI treatment. RNA-seq prioritized differentially regulated genes mediating the cell cycle, including G2 checkpoint WEE1 Kinase (WEE1) and cyclin-dependent kinase 1 (CDK1). Additionally, WEE1 and CDK1 were differentially expressed in PC patient tumors dichotomized by high vs low SOX2 gene expression. Importantly, pharmacological targeting of WEE1 (WEE1i) in combination with an ARSI or GR modulator re-sensitizes SOX2-positive PC cells to nuclear hormone receptor signaling inhibition in vitro, and WEE1i combined with ARSI significantly slowed tumor growth in vivo. Collectively, our data suggest SOX2 predicts NHRSI resistance, and simultaneously indicates the addition of WEE1i to improve therapeutic efficacy of NHRSIs in SOX2-positive PC.
Assuntos
Antineoplásicos , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Transdução de Sinais , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Antagonistas de Receptores de Andrógenos/farmacologia , Receptores Citoplasmáticos e Nucleares , Linhagem Celular Tumoral , Proteínas Tirosina Quinases/metabolismo , Fatores de Transcrição SOXB1/genéticaRESUMO
Human adenovirus expresses several early proteins that control various aspects of the viral replication program, including an orchestrated expression of viral genes. Two of the earliest viral transcriptional units activated after viral genome entry into the host cell nucleus are the E1 and E4 units, which each express a variety of proteins. Chief among these are the E1A proteins that function to reprogram the host cell and activate transcription of all other viral genes. The E4 gene encodes multiple proteins, including E4orf3, which functions to disrupt cellular antiviral defenses, including the DNA damage response pathway and activation of antiviral genes. Here we report that E1A directly interacts with E4orf3 via the conserved N terminus of E1A to regulate the expression of viral genes. We show that E4orf3 indiscriminately drives high nucleosomal density of viral genomes, which is restrictive to viral gene expression and which E1A overcomes via a direct interaction with E4orf3. We also show that during infection E1A colocalizes with E4orf3 to nuclear tracks that are associated with heterochromatin formation. The inability of E1A to interact with E4orf3 has a significant negative impact on overall viral replication, the ability of the virus to reprogram the host cell, and the levels of viral gene expression. Together these results show that E1A and E4orf3 work together to fine-tune the viral replication program during the course of infection and highlight a novel mechanism that regulates viral gene expression.IMPORTANCE To successfully replicate, human adenovirus needs to carry out a rapid yet ordered transcriptional program that executes and drives viral replication. Early in infection, the viral E1A proteins are the key activators and regulators of viral transcription. Here we report, for the first time, that E1A works together with E4orf3 to perfect the viral transcriptional program and identify a novel mechanism by which the virus can adjust viral gene expression by modifying its genome's nucleosomal organization via cooperation between E1A and E4orf3.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Proteínas E4 de Adenovirus/metabolismo , Cromatina/metabolismo , Células A549 , Adenoviridae/genética , Infecções por Adenoviridae/virologia , Proteínas E1A de Adenovirus/fisiologia , Proteínas E4 de Adenovirus/fisiologia , Adenovírus Humanos/fisiologia , Linhagem Celular , Núcleo Celular/virologia , Cromatina/virologia , Citoplasma/metabolismo , Regulação Viral da Expressão Gênica/genética , Regulação Viral da Expressão Gênica/fisiologia , Genes Virais , Humanos , Proteínas Nucleares/metabolismo , Ligação Proteica , Fatores de Transcrição/metabolismo , Replicação ViralRESUMO
Adenovirus executes a finely tuned transcriptional program upon infection of a cell. To better understand the temporal dynamics of the viral transcriptional program we performed highly sensitive digital PCR on samples extracted from arrested human lung fibroblasts infected with human adenovirus 5 strain dl309. We show that the first transcript made from viral genomes is the virus associated non-coding RNA, in particular we detected abundant levels of virus associated RNA II four hours after infection. Activation of E1 and E4 occurred nearly simultaneously later in infection, followed by other early genes as well as late genes. Our study determined that genomes begin to replicate between 29 and 30 hours after infection. This study provides a comprehensive view of viral mRNA steady-state kinetics in arrested human cells using digital PCR.
Assuntos
Adenovírus Humanos/genética , Fibroblastos/virologia , Regulação Viral da Expressão Gênica , Proteínas E1 de Adenovirus/genética , Proteínas E1 de Adenovirus/metabolismo , Proteínas E4 de Adenovirus/genética , Proteínas E4 de Adenovirus/metabolismo , Proteínas Precoces de Adenovirus/genética , Proteínas Precoces de Adenovirus/metabolismo , Expressão Gênica , Genoma Viral , Humanos , Pulmão/patologia , RNA Viral/análise , RNA Viral/metabolismo , Fatores de Tempo , Ativação Transcricional , Replicação ViralRESUMO
Far-upstream element (FUSE) binding protein 1 (FUBP1) was originally identified as a regulator of the oncogene c-Myc via binding to the FUSE within the c-Myc promoter and activating the expression of the gene. Recent studies have identified FUBP1 as a regulator of transcription, translation, and splicing via its DNA and RNA binding activities. Here we report the identification of FUBP1 as a novel binding partner of E1A. FUBP1 binds directly to E1A via the N terminus (residues 1 to 82) and conserved region 3 (residues 139 to 204) of adenovirus 5 E1A. The depletion of FUBP1 via short interfering RNAs (siRNA) reduces virus growth and drives the upregulation of the cellular stress response by activating the expression of p53-regulated genes. During infection, FUBP1 is relocalized within the nucleus, and it is recruited to viral promoters together with E1A while at the same time being lost from the FUSE upstream of the c-Myc promoter. The depletion of FUBP1 affects viral and cellular gene expression. Importantly, in FUBP1-depleted cells, p53-responsive genes are upregulated, p53 occupancy on target promoters is enhanced, and histone H3 lysine 9 is hyperacetylated. This is likely due to the loss of the FUBP1-mediated suppression of p53 DNA binding. We also observed that E1A stabilizes the FUBP1-p53 complex, preventing p53 promoter binding. Together, our results identify, for the first time, FUBP1 as a novel E1A binding protein that participates in aspects of viral replication and is involved in the E1A-mediated suppression of p53 function.IMPORTANCE Viral infection triggers innate cellular defense mechanisms that have evolved to block virus replication. To overcome this, viruses have counterevolved mechanisms that ensure that cellular defenses are either disarmed or not activated to guarantee successful replication. One of the key regulators of cellular stress is the tumor suppressor p53 that responds to a variety of cellular stress stimuli and safeguards the integrity of the genome. During infection, many viruses target the p53 pathway in order to deactivate it. Here we report that human adenovirus 5 coopts the cellular protein FUBP1 to prevent the activation of the p53 stress response pathway that would block viral replication. This finding adds to our understanding of p53 deactivation by adenovirus and highlights its importance in infection and innate immunity.
Assuntos
Infecções por Adenoviridae/virologia , Adenoviridae/fisiologia , Proteínas E1A de Adenovirus/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Interações Hospedeiro-Patógeno , Proteína Supressora de Tumor p53/metabolismo , Replicação Viral , Infecções por Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/genética , Células Cultivadas , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Regulação Viral da Expressão Gênica , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas de Ligação a RNA , Ativação Transcricional , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genéticaRESUMO
Adenovirus Early 1A proteins (E1A) are crucial for initiation of the viral life cycle after infection. The E1A gene is encoded at the left end of the viral genome and consists of two exons, the first encoding 185 amino acids in the 289 residues adenovirus 5 E1A, while the second exon encodes 104 residues. The second exon-encoded region of E1A is conserved across all E1A isoforms except for the 55 residues protein, which has a unique C-terminus due to a frame shift following splicing into the second exon. This region of E1A contributes to a variety of processes including the regulation of viral and cellular gene expression, immortalization and transformation. Here we evaluated the contributions that different regions of the second exon of E1A make to the viral life cycle using deletion mutants. The region of E1A encoded by the second exon was found to be important for overall virus growth, induction of viral and cellular gene expression, viral genome replication and deregulation of the cell cycle. Efficient viral replication was found to require exon 2 and the nuclear localization signal, as loss of either resulted in severe growth deficiency. Induction of cellular DNA synthesis was also deficient with any deletion of E1A within the C-terminus even if these deletions were outside of conserved region 4. Overall, our study provides the first comprehensive insight into the contributions of the C-terminus of E1A to the replicative fitness of human adenovirus 5 in arrested lung fibroblasts.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Adenovírus Humanos/fisiologia , Replicação Viral , Proteínas E1A de Adenovirus/genética , Adenovírus Humanos/genética , Ciclo Celular , Linhagem Celular , Éxons , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Deleção de SequênciaRESUMO
The biological response to DNA double-strand breaks acts to preserve genome integrity. Individuals bearing inactivating mutations in components of this response exhibit clinical symptoms that include cellular radiosensitivity, immunodeficiency, and cancer predisposition. The archetype for such disorders is Ataxia-Telangiectasia caused by biallelic mutation in ATM, a central component of the DNA damage response. Here, we report that the ubiquitin ligase RNF168 is mutated in the RIDDLE syndrome, a recently discovered immunodeficiency and radiosensitivity disorder. We show that RNF168 is recruited to sites of DNA damage by binding to ubiquitylated histone H2A. RNF168 acts with UBC13 to amplify the RNF8-dependent histone ubiquitylation by targeting H2A-type histones and by promoting the formation of lysine 63-linked ubiquitin conjugates. These RNF168-dependent chromatin modifications orchestrate the accumulation of 53BP1 and BRCA1 to DNA lesions, and their loss is the likely cause of the cellular and developmental phenotypes associated with RIDDLE syndrome.
Assuntos
Dano ao DNA , Síndromes de Imunodeficiência/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Linhagem Celular , Histonas/metabolismo , Humanos , Síndromes de Imunodeficiência/genética , Tolerância a Radiação , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
p53 is a sequence-specific DNA-binding transcription factor and key regulator of cell cycle arrest and apoptosis. p53 is mutated in most human cancers and these mutations generally impair its ability to activate transcription. When expressed in Saccharomyces cerevisiae, p53 acts as a strong transcriptional activator allowing yeast to be used as a model system to study the effects of p53 mutations on activity. However, little is known about the exact mechanisms by which p53 functions in yeast. Using 76 mutant yeast strains, we have evaluated the effect of deleting components of the ADA, COMPASS, INO80, ISW1, Mediator, RSC, SAGA, SAS, SLIK, SWI/SNF, and SWR1 transcriptional regulatory complexes on p53-dependent transcription. In addition, we examined the role of histone H2B ubiquitylation by Rad6/Bre1 on p53 activation. Overall, our analysis indicates that there are several remarkable similarities between p53-dependent transcription in yeast and mammalian cells, suggesting that yeast can serve as a valid model system for at least some aspects of p53 function.
Assuntos
Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Proteína Supressora de Tumor p53/fisiologia , Regulação Fúngica da Expressão Gênica , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Humanos , Metilação , Ligação Proteica , Interferência de RNA/fisiologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Ubiquitinas/metabolismoRESUMO
Cells respond to DNA double-strand breaks by recruiting factors such as the DNA-damage mediator protein MDC1, the p53-binding protein 1 (53BP1), and the breast cancer susceptibility protein BRCA1 to sites of damaged DNA. Here, we reveal that the ubiquitin ligase RNF8 mediates ubiquitin conjugation and 53BP1 and BRCA1 focal accumulation at sites of DNA lesions. Moreover, we establish that MDC1 recruits RNF8 through phosphodependent interactions between the RNF8 forkhead-associated domain and motifs in MDC1 that are phosphorylated by the DNA-damage activated protein kinase ataxia telangiectasia mutated (ATM). We also show that depletion of the E2 enzyme UBC13 impairs 53BP1 recruitment to sites of damage, which suggests that it cooperates with RNF8. Finally, we reveal that RNF8 promotes the G2/M DNA damage checkpoint and resistance to ionizing radiation. These results demonstrate how the DNA-damage response is orchestrated by ATM-dependent phosphorylation of MDC1 and RNF8-mediated ubiquitination.