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1.
Cell ; 150(4): 725-37, 2012 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-22901805

RESUMO

Tissue-specific transcription patterns are preserved throughout cell divisions to maintain lineage fidelity. We investigated whether transcription factor GATA1 plays a role in transmitting hematopoietic gene expression programs through mitosis when transcription is transiently silenced. Live-cell imaging revealed that a fraction of GATA1 is retained focally within mitotic chromatin. ChIP-seq of highly purified mitotic cells uncovered that key hematopoietic regulatory genes are occupied by GATA1 in mitosis. The GATA1 coregulators FOG1 and TAL1 dissociate from mitotic chromatin, suggesting that GATA1 functions as platform for their postmitotic recruitment. Mitotic GATA1 target genes tend to reactivate more rapidly upon entry into G1 than genes from which GATA1 dissociates. Mitosis-specific destruction of GATA1 delays reactivation selectively of genes that retain GATA1 during mitosis. These studies suggest a requirement of mitotic "bookmarking" by GATA1 for the faithful propagation of cell-type-specific transcription programs through cell division.


Assuntos
Células Eritroides/metabolismo , Fator de Transcrição GATA1/metabolismo , Hematopoese , Mitose , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Código das Histonas , Camundongos , Proteínas Nucleares/metabolismo , Especificidade de Órgãos , Proteínas Proto-Oncogênicas/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fatores de Transcrição/metabolismo
2.
Blood Adv ; 7(15): 4049-4063, 2023 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-36763539

RESUMO

Golgi membrane protein 1 (GOLM1) is aberrantly expressed in many types of solid tumors and contributes to cancer development; however, its role in hematopoietic and lymphoid neoplasms remains unknown. Here, we report that GOLM1 was significantly upregulated in anaplastic large cell lymphoma (ALCL), particularly in anaplastic lymphoma kinase-positive (ALK+) ALCL. Mechanistically, the expression of GOLM1 was induced by nucleophosmin-ALK in both ALK-transformed T cells and ALCL cell lines through AKT/mTOR pathway. Knockdown of GOLM1 expression led to a reduction in the growth and viability of ALCL cells with increased spontaneous apoptosis, whereas ectopic expression of GOLM1 protected ALCL cells from apoptosis induced by staurosporine treatment. Moreover, GOLM1 directly interacted with B-cell lymphoma-extra large protein (a crucial anti-apoptosis regulator) and significantly prolonged its stability. Introduction of GOLM1 promoted ALK+ ALCL cells colony formation in vitro and tumor growth in a murine xenograft model. Taken together, our findings demonstrate, to our knowledge, for the first time that GOLM1 plays a critical role in suppressing apoptosis and promoting the progression of ALK+ ALCL and provide evidence that GOLM1 is a potential biomarker and therapeutic target in ALK-induced hematological malignancies.


Assuntos
Linfoma Anaplásico de Células Grandes , Receptores Proteína Tirosina Quinases , Humanos , Camundongos , Animais , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Quinase do Linfoma Anaplásico , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma Anaplásico de Células Grandes/patologia , Linhagem Celular Tumoral , Estaurosporina , Proteínas de Membrana/genética
3.
Cancer Prev Res (Phila) ; 16(3): 163-173, 2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36534786

RESUMO

Chronic hepatitis C can lead to cirrhosis and hepatocellular carcinoma. We studied the safety and immunogenicity of a novel therapeutic hepatitis C virus (HCV) genotype 1a/1b consensus DNA vaccine, INO-8000, encoding HCV NS3, NS4A, NS4B, and NS5A proteins alone or co-administered with DNA-encoding IL12 (INO-9012), a human cytokine that stimulates cellular immune function, in individuals with chronic hepatitis C. This was a phase I, multisite dose-escalation trial with an expansion cohort evaluating doses of 0, 0.3, 1.0, and 3.0 mg of INO-9012 (IL12 DNA) as an addition to 6.0 mg of (INO-8000; HCV DNA vaccine). Vaccines were administered by intramuscular injection followed by electroporation at study entry and at weeks 4, 12, and 24. HCV-specific CD4+ and CD8+ T-cell immune responses were measured by IFNγ ELISpot and flow cytometry-based assays. Transient, mild-to-moderate injection site reactions unrelated to IL12 DNA dose were common. Increases in HCV-specific IFNγ production occurred in 15/20 (75%) participants. Increases in the frequency of HCV-specific CD4+ and CD8+ T cells occurred at all dose levels, with the greatest increases seen at 1.0 mg of INO-9012. HCV-specific CD8+ and CD4+ T-cell activities increased in 16/18 (89%) and 14/17 (82%) participants with available data, respectively. The vaccine regimen was safe and induced HCV-specific CD4+ and CD8+ cellular immune responses of modest magnitude in most HCV-infected participants. The addition of 1.0 mg of IL12 DNA provided the best enhancement of immune responses. The vaccine regimen had little effect on controlling HCV viremia. PREVENTION RELEVANCE: The administration of IL12 DNA along with a hepatitis C viral antigen DNA vaccine enhanced the HCV-specific immune responses induced by the vaccine in individuals with chronic hepatitis C, an important cause of hepatocellular carcinoma. IL12 could be an effective adjuvant in vaccines targeting HCV and other oncogenic viruses.


Assuntos
Carcinoma Hepatocelular , Hepatite C Crônica , Hepatite C , Neoplasias Hepáticas , Vacinas de DNA , Humanos , Vacinas de DNA/efeitos adversos , Vacinas de DNA/genética , Hepatite C Crônica/complicações , Hepatite C Crônica/tratamento farmacológico , Carcinoma Hepatocelular/prevenção & controle , Proteínas não Estruturais Virais/genética , Neoplasias Hepáticas/prevenção & controle , Hepatite C/prevenção & controle , Hepacivirus/genética , DNA , Interleucina-12
4.
J Immunother Cancer ; 9(7)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34230114

RESUMO

BACKGROUND: Human telomerase reverse transcriptase (hTERT) is frequently classified as a 'universal' tumor associated antigen due to its expression in a vast number of cancers. We evaluated plasmid DNA-encoded hTERT as an immunotherapy across nine cancer types. METHODS: A phase 1 clinical trial was conducted in adult patients with no evidence of disease following definitive surgery and standard therapy, who were at high risk of relapse. Plasmid DNA encoding one of two hTERT variants (INO-1400 or INO-1401) with or without plasmid DNA encoding interleukin 12 (IL-12) (INO-9012) was delivered intramuscularly concurrent with the application of the CELLECTRA constant-current electroporation device 4 times across 12 weeks. Safety assessments and immune monitoring against native (germline, non-mutated, non-plasmid matched) hTERT antigen were performed. The largest cohort of patients enrolled had pancreatic cancer, allowing for additional targeted assessments for this tumor type. RESULTS: Of the 93 enrolled patients who received at least one dose, 88 had at least one adverse event; the majority were grade 1 or 2, related to injection site. At 18 months, 54.8% (51/93) patients were disease-free, with median disease-free survival (DFS) not reached by end of study. For patients with pancreatic cancer, the median DFS was 9 months, with 41.4% of these patients remaining disease-free at 18 months. hTERT immunotherapy induced a de novo cellular immune response or enhanced pre-existing cellular responses to native hTERT in 96% (88/92) of patients with various cancer types. Treatment with INO-1400/INO-1401±INO-9012 drove hTERT-specific IFN-γ production, generated hTERT-specific CD4+ and CD8+ T cells expressing the activation marker CD38, and induced hTERT-specific activated CD8 +CTLs as defined by cells expressing perforin and granzymes. The addition of plasmid IL-12 adjuvant elicited higher magnitudes of cellular responses including IFN-γ production, activated CD4+ and CD8+ T cells, and activated CD8+CTLs. In a subset analysis of pancreatic cancer patients, the presence of immunotherapy-induced activated CD8+ T cells expressing PD-1, granzymes and perforin correlated with survival. CONCLUSIONS: Plasmid DNA-encoded hTERT/IL-12 DNA immunotherapy was well-tolerated, immune responses were noted across all tumor types, and a specific CD8+ phenotype increased by the immunotherapy was significantly correlated with survival in patients with pancreatic cancer.


Assuntos
DNA/genética , Imunoterapia/métodos , Interleucina-12/metabolismo , Neoplasias/genética , Plasmídeos/metabolismo , Telomerase/genética , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
5.
Cancer Res ; 81(12): 3241-3254, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33619116

RESUMO

Fusion genes including NPM-ALK can promote T-cell transformation, but the signals required to drive a healthy T cell to become malignant remain undefined. In this study, we introduce NPM-ALK into primary human T cells and demonstrate induction of the epithelial-to-mesenchymal transition (EMT) program, attenuation of most T-cell effector programs, reemergence of an immature epigenomic profile, and dynamic regulation of c-Myc, E2F, and PI3K/mTOR signaling pathways early during transformation. A mutant of NPM-ALK failed to bind several signaling complexes including GRB2/SOS, SHC1, SHC4, and UBASH3B and was unable to transform T cells. Finally, T-cell receptor (TCR)-generated signals were required to achieve T-cell transformation, explaining how healthy individuals can harbor T cells with NPM-ALK translocations. These findings describe the fundamental mechanisms of NPM-ALK-mediated oncogenesis and may serve as a model to better understand factors that regulate tumor formation. SIGNIFICANCE: This investigation into malignant transformation of T cells uncovers a requirement for TCR triggering, elucidates integral signaling complexes nucleated by NPM-ALK, and delineates dynamic transcriptional changes as a T cell transforms.See related commentary by Spasevska and Myklebust, p. 3160.


Assuntos
Desdiferenciação Celular , Transformação Celular Neoplásica/patologia , Reprogramação Celular , Linfoma Anaplásico de Células Grandes/patologia , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Apoptose , Proliferação de Células , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/metabolismo , Humanos , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/imunologia , Linfoma Anaplásico de Células Grandes/metabolismo , Fosforilação , Proteínas Tirosina Quinases/genética , Receptores de Antígenos de Linfócitos T/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais Cultivadas
6.
EClinicalMedicine ; 31: 100689, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33392485

RESUMO

BACKGROUND: A vaccine against SARS-CoV-2 is of high urgency. Here the safety and immunogenicity induced by a DNA vaccine (INO-4800) targeting the full length spike antigen of SARS-CoV-2 are described. METHODS: INO-4800 was evaluated in two groups of 20 participants, receiving either 1.0 mg or 2.0 mg of vaccine intradermally followed by CELLECTRA® EP at 0 and 4 weeks. Thirty-nine subjects completed both doses; one subject in the 2.0 mg group discontinued trial participation prior to receiving the second dose. ClinicalTrials.gov identifier: NCT04336410. FINDINGS: The median age was 34.5, 55% (22/40) were men and 82.5% (33/40) white. Through week 8, only 6 related Grade 1 adverse events in 5 subjects were observed. None of these increased in frequency with the second administration. No serious adverse events were reported. All 38 subjects evaluable for immunogenicity had cellular and/or humoral immune responses following the second dose of INO-4800. By week 6, 95% (36/38) of the participants seroconverted based on their responses by generating binding (ELISA) and/or neutralizing antibodies (PRNT IC50), with responder geometric mean binding antibody titers of 655.5 [95% CI (255.6, 1681.0)] and 994.2 [95% CI (395.3, 2500.3)] in the 1.0 mg and 2.0 mg groups, respectively. For neutralizing antibody, 78% (14/18) and 84% (16/19) generated a response with corresponding geometric mean titers of 102.3 [95% CI (37.4, 280.3)] and 63.5 [95% CI (39.6, 101.8)], in the respective groups. By week 8, 74% (14/19) and 100% (19/19) of subjects generated T cell responses by IFN-É£ ELISpot assay with the median SFU per 106 PBMC of 46 [95% CI (21.1, 142.2)] and 71 [95% CI (32.2, 194.4)] in the 1.0 mg and 2.0 mg groups, respectively. Flow cytometry demonstrated a T cell response, dominated by CD8+ T cells co-producing IFN-É£ and TNF-α, without increase in IL-4. INTERPRETATION: INO-4800 demonstrated excellent safety and tolerability and was immunogenic in 100% (38/38) of the vaccinated subjects by eliciting either or both humoral or cellular immune responses. FUNDING: Coalition for Epidemic Preparedness Innovations (CEPI).

7.
J Clin Invest ; 126(7): 2642-60, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27294527

RESUMO

Programmed death ligand-1 (PD-L1) interaction with PD-1 induces T cell exhaustion and is a therapeutic target to enhance immune responses against cancer and chronic infections. In murine bone marrow transplant models, PD-L1 expression on host target tissues reduces the incidence of graft-versus-host disease (GVHD). PD-L1 is also expressed on T cells; however, it is unclear whether PD-L1 on this population influences immune function. Here, we examined the effects of PD-L1 modulation of T cell function in GVHD. In patients with severe GVHD, PD-L1 expression was increased on donor T cells. Compared with mice that received WT T cells, GVHD was reduced in animals that received T cells from Pdl1-/- donors. PD-L1-deficient T cells had reduced expression of gut homing receptors, diminished production of inflammatory cytokines, and enhanced rates of apoptosis. Moreover, multiple bioenergetic pathways, including aerobic glycolysis, oxidative phosphorylation, and fatty acid metabolism, were also reduced in T cells lacking PD-L1. Finally, the reduction of acute GVHD lethality in mice that received Pdl1-/- donor cells did not affect graft-versus-leukemia responses. These data demonstrate that PD-L1 selectively enhances T cell-mediated immune responses, suggesting a context-dependent function of the PD-1/PD-L1 axis, and suggest selective inhibition of PD-L1 on donor T cells as a potential strategy to prevent or ameliorate GVHD.


Assuntos
Antígeno B7-H1/metabolismo , Doença Enxerto-Hospedeiro/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T/metabolismo , Animais , Apoptose , Células da Medula Óssea/citologia , Transplante de Medula Óssea , Citocinas/metabolismo , Feminino , Glucose/imunologia , Glutamina/metabolismo , Glicólise , Humanos , Inflamação , Leucócitos Mononucleares/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Oxigênio , Fosforilação , Transdução de Sinais , Linfócitos T/citologia , Resultado do Tratamento
8.
Trends Cell Biol ; 20(1): 52-61, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20004579

RESUMO

The expression of protein-coding genes is enhanced by the exquisite coupling of transcription by RNA polymerase II with pre-messenger RNA processing reactions, such as 5'-end capping, splicing and 3'-end formation. Integration between cotranscriptional processing events extends beyond the nucleus, as proteins that bind cotranscriptionally can affect the localization, translation and degradation of the mature messenger RNA. MicroRNAs are RNA polymerase II transcripts with crucial roles in the regulation of gene expression. Recent data demonstrate that processing of primary microRNA transcripts might be yet another cotranscriptional event that is woven into this elaborate nuclear network. This review discusses the extensive molecular interactions that couple the earliest steps in gene expression and therefore influence the final fate and function of the mature messenger RNA or microRNA produced.


Assuntos
Núcleo Celular/genética , Redes Reguladoras de Genes , MicroRNAs/genética , Precursores de RNA/genética , Transcrição Gênica , Animais , Humanos , Splicing de RNA
9.
Cell Cycle ; 8(3): 345-56, 2009 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-19177009

RESUMO

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate expression of their target messenger RNAs. We recently demonstrated that primary miRNA transcripts (pri-miRNAs) retained at transcription sites are processed with enhanced efficiency, suggesting that pri-miRNA processing is coupled to transcription in mammalian cells. We also observed that transiently expressed pri-miRNAs accumulate in nuclear foci with splicing factor SC35 and Microprocessor components, Drosha and DGCR8. Here, we show that pri-miRNAs containing a self-cleaving hepatitis delta ribozyme accumulate in the nucleoplasm after release from their transcription sites, but are not efficiently processed. Pri-miRNAs with ribozyme-generated 3' ends do not localize to SC35-containing foci, whereas cleaved and polyadenylated pri-miRNA transcripts with or without the pre-miRNA hairpin do. Pri-miRNA/SC35 foci contain a number of proteins normally associated with SC35 domains, including ASF/SF2, PABII, and the prolyl isomerase, Pin1. In contrast, RNA polymerase II and PM/Scl-100 do not strongly colocalize with pri-miRNAs in SC35-containing foci. These data argue that pri-miRNA/SC35-containing foci are not major sites of pri-miRNA processing and that pri-miRNA processing is coupled to transcription. We discuss the implications of our findings relative to recent insights into miRNA biogenesis, mRNA metabolism, and the nuclear organization of gene expression.


Assuntos
Núcleo Celular , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , Processamento Pós-Transcricional do RNA , Ribonucleoproteínas/metabolismo , Animais , Fracionamento Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células HeLa , Humanos , MicroRNAs/química , MicroRNAs/genética , Proteínas Nucleares/genética , Conformação de Ácido Nucleico , RNA Catalítico/metabolismo , Ribonucleoproteínas/genética , Fatores de Processamento de Serina-Arginina , Transdução de Sinais/fisiologia , Transcrição Gênica
10.
J Cell Biol ; 182(1): 61-76, 2008 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-18625843

RESUMO

MicroRNAs (miRNAs) are noncoding RNAs with important roles in regulating gene expression. In studying the earliest nuclear steps of miRNA biogenesis, we observe that primary miRNA (pri-miRNA) transcripts retained at transcription sites due to the deletion of 3'-end processing signals are converted more efficiently into precursor miRNAs (pre-miRNAs) than pri-miRNAs that are cleaved, polyadenylated, and released. Flanking exons, which also increase retention at transcription sites, likewise contribute to increased levels of intronic pri-miRNAs. Consistently, efficiently processed endogenous pri-miRNAs are enriched in chromatin-associated nuclear fractions. In contrast, pri-miRNAs that accumulate to high nuclear levels after cleavage and polyadenylation because of the presence of a viral RNA element (the ENE of the Kaposi's sarcoma-associated herpes virus polyadenylated nuclear RNA) are not efficiently processed to precursor or mature miRNAs. Exogenous pri-miRNAs unexpectedly localize to nuclear foci containing splicing factor SC35; yet these foci are unlikely to represent sites of miRNA transcription or processing. Together, our results suggest that pri-miRNA processing is enhanced by coupling to transcription.


Assuntos
MicroRNAs/biossíntese , MicroRNAs/genética , Transcrição Gênica , Cromatina/genética , Éxons/genética , Células HeLa , Humanos , Íntrons/genética , Proteínas Nucleares/metabolismo , Poliadenilação , RNA Polimerase III/metabolismo , Processamento Pós-Transcricional do RNA , Transporte de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , Sequências Reguladoras de Ácido Nucleico/genética , Ribonucleoproteínas/metabolismo , Deleção de Sequência , Fatores de Processamento de Serina-Arginina , Frações Subcelulares , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
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