Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.703
Filtrar
1.
Vet Microbiol ; 237: 108370, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31585643

RESUMO

Caprine alphaherpesvirus 1 (CpHV-1) is a pathogen associated with systemic infection and respiratory disease in kids and subclinical infection or reproductive failure and abortions in adult goats. The enzyme thymidine kinase (TK) is an important viral product involved in nucleotide synthesis. This property makes the tk gene a common target for herpesvirus attenuation. Here we deleted the tk gene of a CpHV-1 isolate and characterized the recombinant CpHV-1ΔTKin vitro and in vivo. In vitro characterization revealed that the recombinant CpHV-1ΔTK replicated to similar titers and produced plaques of similar size to the parental CpHV-1 strain in BT and CRIB cell lines. Upon intranasal inoculation of young goats, the parental virus replicated more efficiently and for a longer period than the recombinant virus. In addition, infection with the parental virus resulted in mild systemic and respiratory signs whereas the kids inoculated with the recombinant CpHV-1ΔTK virus remained healthy. Goats inoculated with the parental virus also developed higher neutralizing antibody titers when compared to CpHV-1ΔTK inoculated animals. Dexamethasone (Dx) administration on days 35-39 post-inoculation did not result in virus shedding in nasal secretions, indicating lack of reactivation from latency. However, viral DNA was detected in the trigeminal ganglia of animals euthanized at 14 days post-Dx, indicating that both viruses successfully established latent infection. Our results show that the recombinant CpHV-1ΔTK presents an attenuated phenotype when compared to the parental virus, and hence may represent a promising vaccine candidate to prevent CpHV-1 disease in goats.


Assuntos
Alphaherpesvirinae/genética , Deleção de Genes , Doenças das Cabras/virologia , Timidina Quinase/genética , Alphaherpesvirinae/patogenicidade , Animais , Bovinos , Linhagem Celular , DNA Viral/isolamento & purificação , Dexametasona/farmacologia , Regulação Enzimológica da Expressão Gênica , Regulação Viral da Expressão Gênica , Cabras , Muco/virologia , Proteínas Virais , Eliminação de Partículas Virais
2.
PLoS Genet ; 15(10): e1008439, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31589613

RESUMO

Metabolic alterations that are critical for cancer cell growth and metastasis are one of the key hallmarks of cancer. Here, we show that thymidine kinase 1 (TK1) is significantly overexpressed in tumor samples from lung adenocarcinoma (LUAD) patients relative to normal controls, and this TK1 overexpression is associated with significantly reduced overall survival and cancer recurrence. Genetic knockdown of TK1 with short hairpin RNAs (shRNAs) inhibits both the growth and metastatic attributes of LUAD cells in culture and in mice. We further show that transcriptional overexpression of TK1 in LUAD cells is driven, in part, by MAP kinase pathway in a transcription factor MAZ dependent manner. Using targeted and gene expression profiling-based approaches, we then show that loss of TK1 in LUAD cells results in reduced Rho GTPase activity and reduced expression of growth and differentiation factor 15 (GDF15). Furthermore, ectopic expression of GDF15 can partially rescue TK1 knockdown-induced LUAD growth and metastasis inhibition, confirming its important role as a downstream mediator of TK1 function in LUAD. Collectively, our findings demonstrate that TK1 facilitates LUAD tumor and metastatic growth and represents a target for LUAD therapy.


Assuntos
Adenocarcinoma de Pulmão/genética , Fator 15 de Diferenciação de Crescimento/metabolismo , Neoplasias Pulmonares/genética , Recidiva Local de Neoplasia/genética , Timidina Quinase/metabolismo , Adenocarcinoma de Pulmão/mortalidade , Adenocarcinoma de Pulmão/patologia , Adulto , Idoso , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Proteínas de Ligação a DNA/metabolismo , Conjuntos de Dados como Assunto , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Pulmão/patologia , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Prognóstico , Análise de Sobrevida , Timidina Quinase/genética , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
3.
BMC Cancer ; 19(1): 921, 2019 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-31521130

RESUMO

BACKGROUND: Free flap-mediated gene therapy in the tumor bed following surgical resection is a promising approach in cancer targeted treatment of residual disease. We investigated the selective killing efficacy of a lentivirus-mediated cytosine deaminase-thymidine kinase (CDglyTK) gene in transplanted breast cancer delivered into a free flap by intra-artery perfusion. METHODS: Proliferation, apoptosis, and cell cycle of rat SHZ-88 breast cancer cells transfected with a lentivirus-mediated CD/TK gene were measured following treatment with ganciclovir and 5-flucytosine in vitro. A model of residual disease of breast cancer in a rat superficial inferior epigastric artery (SIEA) flap model was used to study the therapeutic potential of a double suicide CD/TK and prodrug system in vivo. RESULTS: Killing efficacy of the double suicide CD/TK and prodrug system on SHZ-88 cells was mediated by increased apoptosis and cell cycle arrest at the G1 phase with significant bystander effect. Following recombinant lentivirus transfection of rat SIEA flap by intra-artery perfusion, CD/TK gene expression was limited to the flap, and the volume and weight of transplanted tumors were significantly reduced without observable toxicity. CONCLUSIONS: SIEA flaps transfected with a lentivirus-mediated CDglyTK gene by intra-artery perfusion effectively suppress transplanted breast tumor growth without obvious systemic toxic effects in rats.


Assuntos
Neoplasias da Mama/genética , Citosina Desaminase/genética , Retalhos de Tecido Biológico , Terapia Genética , Vetores Genéticos/genética , Lentivirus/genética , Proteínas Recombinantes de Fusão , Timidina Quinase/genética , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Efeito Espectador , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Terapia Combinada , Modelos Animais de Doenças , Feminino , Ganciclovir/farmacologia , Genes Transgênicos Suicidas , Vetores Genéticos/administração & dosagem , Humanos , Perfusão , Ratos , Transgenes , Resultado do Tratamento
4.
EBioMedicine ; 46: 356-367, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31383553

RESUMO

BACKGROUND: TK2 is a nuclear gene encoding the mitochondrial matrix protein thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial nucleotide salvage pathway. Deficiency of TK2 activity causes mitochondrial DNA (mtDNA) depletion, which in humans manifests predominantly as a mitochondrial myopathy with onset typically in infancy and childhood. We previously showed that oral treatment of the Tk2 H126N knock-in mouse model (Tk2-/-) with the TK2 substrates, deoxycytidine (dCtd) and thymidine (dThd), delayed disease onset and prolonged median survival by 3-fold. Nevertheless, dCtd + dThd treated Tk2-/- mice showed mtDNA depletion in brain as early as postnatal day 13 and in virtually all other tissues at age 29 days. METHODS: To enhance mechanistic understanding and efficacy of dCtd + dThd therapy, we studied the bioavailability of dCtd and dThd in various tissues as well as levels of the cytosolic enzymes, TK1 and dCK that convert the deoxynucleosides into dCMP and dTMP. FINDINGS: Parenteral treatment relative to oral treatment produced higher levels of dCtd and dThd and improved mtDNA levels in liver and heart, but did not ameliorate molecular defects in brain or prolong survival. Down-regulation of TK1 correlated with temporal- and tissue-specificity of response to dCtd + dThd. Finally, we observed in human infant and adult muscle expression of TK1 and dCK, which account for the long-term efficacy to dCtd + dThd therapy in TK2 deficient patients. INTERPRETATIONS: These data indicate that the cytosolic pyrimidine salvage pathway enzymes TK1 and dCK are critical for therapeutic efficacy of deoxynucleoside therapy for Tk2 deficiency. FUND: National Institutes of Health P01HD32062.


Assuntos
Desoxirribonucleosídeos/farmacologia , Timidina Quinase/deficiência , Animais , Disponibilidade Biológica , Barreira Hematoencefálica/metabolismo , DNA Mitocondrial , Desoxirribonucleosídeos/farmacocinética , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Especificidade de Órgãos , Fosforilação Oxidativa , Fenótipo , Timidina Quinase/genética , Timidina Quinase/metabolismo
5.
Breast Cancer Res Treat ; 178(1): 57-62, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31346846

RESUMO

PURPOSE: Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) improve progression-free survival (PFS) in patients with hormone receptor-positive (HR+) advanced breast cancer. However, a better knowledge of predictive biomarkers of response and resistance to CDK4/6i is needed. Therefore, the present article addresses the role of the mRNA expression of thymidine kinase 1 (TK1), CDK4, 6 and 9 in plasma-derived exosomes and their relevance in the pharmacologic activity of CDK4/6i. METHODS: Blood samples of 40 HR+/HER2- advanced breast cancer patients were collected before (T0) the administration of palbociclib plus hormonal therapy and after 3 months (T1). RNA was isolated from exosomes and analysed for the expression of TK1, CDK 4, 6 and 9 by digital droplet PCR (ddPCR). RESULTS: A higher value of TK1 copies/ml at baseline (T0) was significantly associated with the number of previous lines of chemotherapy (p = 0.009). In patients with PD, a significant increase was observed in the number of copies/ml of TK1 (p = 0.01) and CDK9 (p = 0.03) comparing T1 vs. T0 values. No significant correlations between response to treatment and clinical parameters were found at univariate analysis. High baseline CDK4 expression was significantly correlated with longer PFS in patients treated with fulvestrant + palbociclib (low versus high: 6.45 months vs. not reached, p = 0.01). CONCLUSIONS: The present study demonstrates that, in plasma-derived exosomes, high baseline CDK4 mRNA levels are associated with response to palbociclib plus hormonal therapy, while the increase in TK1 and CDK9 mRNA copies/ml is associated with clinical resistance.


Assuntos
Neoplasias da Mama/genética , Quinase 9 Dependente de Ciclina/genética , Resistencia a Medicamentos Antineoplásicos , Exossomos/genética , Timidina Quinase/genética , Regulação para Cima , Adulto , Idoso , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica , Piperazinas/uso terapêutico , Piridinas/uso terapêutico , Resultado do Tratamento
6.
EBioMedicine ; 46: 342-355, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31351931

RESUMO

BACKGROUND: Thymidine kinase 2 (TK2) catalyses the phosphorylation of deoxythymidine (dThd) and deoxycytidine (dCtd) within mitochondria. TK2 deficiency leads to mtDNA depletion or accumulation of multiple deletions. In patients, TK2 mutations typically manifest as a rapidly progressive myopathy with infantile onset, leading to respiratory insufficiency and encephalopathy in the most severe clinical presentations. TK2-deficient mice develop the most severe form of the disease and die at average postnatal day 16. dThd+dCtd administration delayed disease progression and expanded lifespan of a knockin murine model of the disease. METHODS: We daily administered TK2 knockout mice (Tk2KO) from postnatal day 4 with equimolar doses of dThd+dCtd, dTMP+dCMP, dThd alone or dCtd alone. We monitored body weight and survival and studied different variables at 12 or 29 days of age. We determined metabolite levels in plasma and target tissues, mtDNA copy number in tissues, and the expression and activities of enzymes with a relevant role in mitochondrial dNTP anabolism or catabolism. FINDINGS: dThd+dCtd treatment extended average lifespan of Tk2KO mice from 16 to 34 days, attenuated growth retardation, and rescued mtDNA depletion in skeletal muscle and other target tissues of 12-day-old mice, except in brain. However, the treatment was ineffective in 29-day-old mice that still died prematurely. Bioavailability of dThd and dCtd markedly decreased during mouse development. Activity of enzymes catabolizing dThd and dCtd increased with age in small intestine. Conversely, the activity of the anabolic enzymes decreased in target tissues during mouse development. We also found that administration of dThd alone had the same impact on survival to that of dThd+dCtd, whereas dCtd alone had no influence on lifespan. INTERPRETATION: dThd+dCtd treatment recruits alternative cytosolic salvage pathways for dNTP synthesis, suggesting that this therapy would be of benefit for any Tk2 mutation. dThd accounts for the therapeutic effect of the combined treatment in mice. During the first weeks after birth, mice experience marked tissue-specific metabolic regulations and ontogenetic changes in dNTP metabolism-related enzymes that limit therapeutic efficacy to early developmental stages. FUND: This study was funded by grants from the Spanish Ministry of Industry, Economy and Competitiveness, the Spanish Instituto de Salud Carlos III, the Fundación Inocente, Inocente, AFM Téléthon and the Generalitat de Catalunya. The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


Assuntos
Desoxirribonucleosídeos/farmacologia , Metabolismo Energético/efeitos dos fármacos , Timidina Quinase/deficiência , Fatores Etários , Animais , Biomarcadores , Ativação Enzimática , Expressão Gênica , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Timidina Quinase/genética , Timidina Quinase/metabolismo
7.
Orphanet J Rare Dis ; 14(1): 100, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31060578

RESUMO

BACKGROUND: TK2 gene encodes for mitochondrial thymidine kinase, which phosphorylates the pyrimidine nucleosides thymidine and deoxycytidine. Recessive mutations in the TK2 gene are responsible for the 'myopathic form' of the mitochondrial depletion/multiple deletions syndrome, with a wide spectrum of severity. METHODS: We describe 18 patients with mitochondrial myopathy due to mutations in the TK2 gene with absence of clinical symptoms until the age of 12. RESULTS: The mean age of onset was 31 years. The first symptom was muscle limb weakness in 10/18, eyelid ptosis in 6/18, and respiratory insufficiency in 2/18. All patients developed variable muscle weakness during the evolution of the disease. Half of patients presented difficulty in swallowing. All patients showed evidence of respiratory muscle weakness, with need for non-invasive Mechanical Ventilation in 12/18. Four patients had deceased, all of them due to respiratory insufficiency. We identified common radiological features in muscle magnetic resonance, where the most severely affected muscles were the gluteus maximus, semitendinosus and sartorius. On muscle biopsies typical signs of mitochondrial dysfunction were associated with dystrophic changes. All mutations identified were previously reported, being the most frequent the in-frame deletion p.Lys202del. All cases showed multiple mtDNA deletions but mtDNA depletion was present only in two patients. CONCLUSIONS: The late-onset is the less frequent form of presentation of the TK2 deficiency and its natural history is not well known. Patients with late onset TK2 deficiency have a consistent and recognizable clinical phenotype and a poor prognosis, due to the high risk of early and progressive respiratory insufficiency.


Assuntos
Miopatias Mitocondriais/enzimologia , Timidina Quinase/deficiência , Adolescente , Adulto , Criança , DNA Mitocondrial/genética , Feminino , Humanos , Transtornos de Início Tardio/enzimologia , Transtornos de Início Tardio/metabolismo , Transtornos de Início Tardio/patologia , Masculino , Pessoa de Meia-Idade , Miopatias Mitocondriais/genética , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Doenças Musculares/enzimologia , Doenças Musculares/genética , Mutação/genética , Estudos Retrospectivos , Timidina Quinase/genética , Adulto Jovem
8.
Anal Chim Acta ; 1057: 114-122, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30832910

RESUMO

Organic-inorganic hybrid nanomaterial has gained much attention due to its excellent performances in bioanalysis and biomedicine. However, the preparation of DNA-inorganic hybrid nanomaterial with suitable size for cell uptake remains a huge challenge. Herein, a moderate biomineralization strategy for synthesis of Y-DNA@Cu3(PO4)2 (Y-DNA@CuP) hybrid nanoflowers is reported. Y-DNA with a loop structure is used as both the biomineralization template and the recognition unit for thymidine kinase 1 (TK1) mRNA. The Y-DNA probe can linearly response to TK1 mRNA target sequence in a range from 2 nM to 150 nM with the limit of detection as low as 0.56 nM. Interestingly, the presence of Y-DNA significantly decreases the size of Cu3(PO4)2 (CuP) particles, which allows them suitable for intracellular applications as gene nanocarriers. Once inside the cells, the hybrid nanoflowers dissolve and release the Y-DNA probes. Then, the intracellular TK1 mRNA hybridizes with the loop region of Y-DNA, which dissociates the Cy3-labeled loop strand and turns on the red fluorescence. Through the real-time imaging of the intracellular TK1 mRNA, the assessment of tumor cells before and after the treatment of drugs including ß-estradiol and tamoxifen is achieved.


Assuntos
DNA/química , Portadores de Fármacos/química , Nanoestruturas/química , Imagem Óptica/métodos , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Espaço Intracelular/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Timidina Quinase/genética
9.
Virus Res ; 264: 56-67, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30796929

RESUMO

Feline herpesvirus-1 (FHV-1) infection occurs worldwide and is a leading cause of respiratory and ocular diseases in cats. Current vaccines reduce the severity of symptoms but do not prevent infection and, therefore, do not provide defense against an establishment of latency and reactivation. We hypothesize that immunomodulation of FHV-1 is the cause of lack in protection and that deletion of virulence/immune modulatory genes of FHV-1 will enhance safety and immunogenicity. Our objective was to use feline respiratory epithelial cell (FREC) cultures to define in vitro growth characteristics and immunomodulation resulting from infection of FRECs with the virulent FHV-1 strain C27 (WT) and glycoprotein C-deletion (gC-), glycoprotein E-deletion (gE-), serine/threonine protein kinase-deletion (PK-), as well as gE and thymidine kinase-double-deletion (gE-TK-) mutants generated by bacterial artificial chromosome mutagenesis. Differentiated FRECs were mock inoculated or inoculated with WT, gC-, gE-, PK-, or gE-TK- mutants. Virus titration and real-time quantitative PCR assays were performed on samples collected at 1 hpi followed by 24 h intervals between 24 and 96 hpi to determine growth kinetics. Real-time PCR was used to quantitate IFNα, TNFα, IL-1ß, IL-10, and TGFß-specific mRNA levels. Immunoassays were performed to measure the protein levels of subsets of cytokines/chemokines secreted by FRECs. Inoculation of FRECs with gE-TK- resulted in significantly lower end-point titers than inoculation with WT or gE-. Both PK- and gC- inoculated FRECs also produced significantly lower end-point titers at 96 hpi than WT. Overall, intracellular virus titers were higher than those of extracellular virus. PCR results for viral DNA paralleled the virus titration results. Further, in contrast to WT inoculation, an increase in IFNα and IL-10 mRNA expression was not observed following inoculation with gE-TK- and PK-, but inoculation with gE-TK- and PK- did result in increased TGFß expression in FRECs compared to responses following infection with WT. Moreover, gE-TK- and PK- blocked the inhibition of IL-8 and neutrophil chemoattractant (KC), which was observed following inoculation with WT. In summary, the results obtained in FRECs may be used to predict the safety and immunogenicity characteristics of these mutants in vivo. Our study highlights the value of the FREC system for studying replication kinetics/immune modulation factors of FHV-1 and screening prospective vaccine candidates before their use in experimental cats.


Assuntos
Células Epiteliais/imunologia , Imunidade Inata , Varicellovirus/fisiologia , Replicação Viral , Animais , Gatos , Linhagem Celular , Citocinas/genética , Citocinas/imunologia , Células Epiteliais/virologia , Deleção de Genes , Glicoproteínas de Membrana/genética , Mutação , Reação em Cadeia da Polimerase , Estudos Prospectivos , Timidina Quinase/genética , Timidina Quinase/imunologia , Varicellovirus/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologia , Virulência/genética
10.
Int J Mol Sci ; 20(4)2019 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-30769780

RESUMO

Human induced pluripotent stem cells (iPSCs) hold enormous promise for regenerative medicine. The major safety concern is the tumorigenicity of transplanted cells derived from iPSCs. A potential solution would be to introduce a suicide gene into iPSCs as a safety switch. The herpes simplex virus type 1 thymidine kinase (HSV-TK) gene, in combination with ganciclovir, is the most widely used enzyme/prodrug suicide system from basic research to clinical applications. In the present study, we attempted to establish human iPSCs that stably expressed HSV-TK with either lentiviral vectors or CRISPR/Cas9-mediated genome editing. However, this task was difficult to achieve, because high-level and/or constitutive expression of HSV-TK resulted in the induction of cell death or silencing of HSV-TK expression. A nucleotide metabolism analysis suggested that excessive accumulation of thymidine triphosphate, caused by HSV-TK expression, resulted in an imbalance in the dNTP pools. This unbalanced state led to DNA synthesis inhibition and cell death in a process similar to a "thymidine block", but more severe. We also demonstrated that the Tet-inducible system was a feasible solution for overcoming the cytotoxicity of HSV-TK expression. Our results provided a warning against using the HSV-TK gene in human iPSCs, particularly in clinical applications.


Assuntos
Terapia Genética , Células-Tronco Pluripotentes Induzidas/enzimologia , Simplexvirus/enzimologia , Timidina Quinase/genética , Apoptose/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Ganciclovir/farmacologia , Edição de Genes , Regulação Enzimológica da Expressão Gênica/genética , Regulação Viral da Expressão Gênica/genética , Genes Transgênicos Suicidas/genética , Vetores Genéticos/uso terapêutico , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Lentivirus/genética , Nucleotídeos/biossíntese , Nucleotídeos/genética , Simplexvirus/genética
11.
J Gen Virol ; 100(4): 642-655, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30230443

RESUMO

Koi herpesvirus (KHV, Cyprinidherpesvirus 3) causes a fatal disease of koi and common carp. To obtain safe and efficacious live vaccines, we generated deletion mutants of KHV lacking the nonessential genes encoding two enzymes of nucleotide metabolism, thymidine kinase (TK, ORF55) and deoxyuridine-triphosphatase (DUT, ORF123). Since single-deletion mutants based on a KHV isolate from Israel (KHV-I) only exhibited partial attenuation (Fuchs W, Fichtner D, Bergmann SM, Mettenleiter TC. Arch Virol 2011;156 : 1059-1063), a corresponding double mutant was generated and tested in vivo, and shown to be almost avirulent but still protective. To overcome the low in vitro virus titres of KHV-I (≤105 p.f.u. ml-1), single and double TK and DUT deletions were also introduced into a cell culture-adapted KHV strain from Taiwan (KHV-T). The deletions did not affect in vitro virus replication, and all KHV-T mutants exhibited wild-type-like plaque sizes and titres exceeding 107 p.f.u. ml-1, as a prerequisite for economic vaccine production. Compared to wild-type and revertant viruses, the single-deletion mutants of KHV-T were significantly attenuated in vivo, and immersion of juvenile carp in water containing high doses of the double mutant caused almost no fatalities. Nevertheless, the deletion mutants induced similar levels of KHV-specific serum antibodies to the parental wild-type virus, and conferred solid protection against disease after challenge with wild-type KHV. For the convenient differentiation of DNA samples prepared from gill swabs of carp infected with wild-type and TK-deleted KHV we developed a triplex real-time PCR. Thus, KHV-TΔDUT/TK might be suitable as a genetic DIVA vaccine in the field.


Assuntos
Herpesviridae/genética , Herpesviridae/imunologia , Pirofosfatases/genética , Pirofosfatases/imunologia , Timidina Quinase/genética , Timidina Quinase/imunologia , Animais , Carpas/imunologia , Carpas/virologia , Células Cultivadas , DNA Viral/genética , DNA Viral/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Israel , Deleção de Sequência/genética , Deleção de Sequência/imunologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Replicação Viral/genética , Replicação Viral/imunologia
12.
Plant J ; 97(3): 430-446, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30317699

RESUMO

Nucleotide biosynthesis proceeds through a de novo pathway and a salvage route. In the salvage route, free bases and/or nucleosides are recycled to generate the corresponding nucleotides. Thymidine kinase (TK) is the first enzyme in the salvage pathway to recycle thymidine nucleosides as it phosphorylates thymidine to yield thymidine monophosphate. The Arabidopsis genome contains two TK genes -TK1a and TK1b- that show similar expression patterns during development. In this work, we studied the respective roles of the two genes during early development and in response to genotoxic agents targeting the organellar or the nuclear genome. We found that the pyrimidine salvage pathway is crucial for chloroplast development and genome replication, as well as for the maintenance of its integrity, and is thus likely to play a crucial role during the transition from heterotrophy to autotrophy after germination. Interestingly, defects in TK activity could be partially compensated by supplementation of the medium with sugar, and this effect resulted from both the availability of a carbon source and the activation of the nucleotide de novo synthesis pathway, providing evidence for a compensation mechanism between two routes of nucleotide biosynthesis that depend on nutrient availability. Finally, we found differential roles of the TK1a and TK1b genes during the plant response to genotoxic stress, suggesting that different pools of nucleotides exist within the cells and are required to respond to different types of DNA damage. Altogether, our results highlight the importance of the pyrimidine salvage pathway, both during plant development and in response to genotoxic stress.


Assuntos
Arabidopsis/genética , Genoma de Planta/genética , Pirimidinas/metabolismo , Timidina Quinase/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Cloroplastos/metabolismo , Dano ao DNA , Nucleotídeos/metabolismo , Timidina/metabolismo , Timidina Quinase/genética
13.
Biotech Histochem ; 94(1): 60-64, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30317880

RESUMO

Although angiogenesis plays a crucial role in cancer growth and progression, no reliable method for assessing angiogenesis in tumor tissue sections currently is available. Using biomarkers with high specificity for proliferating endothelial cells could help quantify angiogenic activity. Thymidine kinase-1 (TK1) is an enzyme involved in the salvage pathway of DNA synthesis and its activity is correlated with cell proliferation. We investigated the use of double immunostaining for TK1 and CD31 for identifying activated tumor vessels. Differences in TK1/CD31 positive vessel rates (PVRs) between tumor and adjacent normal tissues were evaluated in 39 colorectal carcinoma (CRC) samples and compared with those of Ki67/CD31 double stained tissues. Mean TK1/CD31 PVR (23.6%) in CRCs was 13.9 fold greater than in adjacent normal tissues (1.7%)). By comparison, mean Ki67/CD31 PVR in CRCs was 20.0%, i.e. only 4.8 fold greater than in normal tissues (4.2%). Also, mean TK1/CD31 PVR in normal tissues was significantly less than mean Ki67/CD31 PVR. Our findings indicate that double immunostaining for TK1/CD31 can detect activated tumor vessels more accurately than staining for Ki67/CD31 and potentially could identify tumors that will respond to anti-angiogenic therapy.


Assuntos
Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Timidina Quinase/metabolismo , Biomarcadores Tumorais , Proliferação de Células , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Timidina Quinase/genética
14.
J Med Microbiol ; 68(1): 77-80, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30451652

RESUMO

The efficacy of iclaprim against Staphylococcus aureus ATCC 25923 and its corresponding isogenic TK-deficient mutant S. aureus strain AH 1246 mixed with cytodex beads was studied in a mouse abscess infection model. Iclaprim (2-80 mg kg-1) administered as a single dose via the subcutaneous route (2 h post-infection) was efficacious against the TK-deficient mutant with 1 and 2 log10 c.f.u. reductions at the 24 h post initiation of treatment time point, at doses of 14.4 and 30 mg kg-1, respectively. In contrast, poor antibacterial activity was observed against corresponding wild-type (TK-competent) S. aureus strain, ATCC 25923, at all doses tested. The PK/PD parameter which appeared to correlate best with efficacy was AUC/MIC (R2=0.91). This study showed that TK-deficient mutants may be used to evaluate DHFRi activity and PK/PD relationship in a mouse abscess model.


Assuntos
Antibacterianos/farmacocinética , Pirimidinas/farmacocinética , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Abscesso/genética , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Mutação , Infecções Estafilocócicas/microbiologia , Timidina Quinase/genética
15.
Virus Genes ; 55(1): 76-86, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30478778

RESUMO

The UL24 homologous genes are conserved in alphaherpesviruses. However, the proximity of the UL24 gene and the UL23 gene encoding for thymidine kinase (TK) in the genome of suid herpesvirus 1 (SuHV-1) makes it difficult to mutate UL24 without affecting the expression of the TK gene, and thus functional studies of the UL24 gene have lagged behind. In this study, CRISPR/Cas9 and homologous recombination were adopted to generate UL24 and TK mutant viruses. Deletion of either the UL24 or the TK gene resulted in significantly reduced SuHV-1 replication and spread capacity in Vero cells. However, UL24-deleted virus still maintained a certain degree of lethality in mice, while TK-deleted viruses completely lost their lethality in mice. Similarly, neurovirulence of UL24-deleted virus in mice was not significantly affected compared to parental virus. In comparison, infection with the TK-deleted viruses resulted in significantly reduced neurovirulence and complete loss of lethality. In addition, and for the first time, viral UL24 protein was found to be expressed late during SuHV-1 infection; enhanced green fluorescence protein (eGFP) labeled UL24 protein was shown to be localized in the nucleus via heterologous expression. In conclusion, the UL24 gene of SuHV-1 encodes a nuclear-localized viral protein and acts as a minor virulence-associated factor compared to the TK gene.


Assuntos
Herpesvirus Suídeo 1/fisiologia , Proteínas Virais/metabolismo , Animais , Sistemas CRISPR-Cas , DNA Viral , Feminino , Células HeLa , Recombinação Homóloga , Humanos , Camundongos , Mutação , Transporte Proteico , Pseudorraiva/metabolismo , Pseudorraiva/virologia , RNA Guia/genética , Timidina Quinase/genética , Timidina Quinase/metabolismo , Núcleo Espinal do Trigêmeo/virologia , Células Vero , Carga Viral , Proteínas Virais/genética , Virulência
16.
PLoS Biol ; 16(12): e2005907, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30592710

RESUMO

Metastatic dissemination employs both the blood and lymphatic vascular systems. Solid tumors dynamically remodel and generate both vessel types during cancer progression. Lymphatic vessel invasion and cancer cells in the tumor-draining lymph nodes (LNs) are prognostic markers for breast cancer metastasis and patient outcome, and tumor-induced lymphangiogenesis likely influences metastasis. Deregulated tumor tissue fluid homeostasis and immune trafficking associated with tumor lymphangiogenesis may contribute to metastatic spreading; however, the precise functional characterization of lymphatic endothelial cells (LECs) in tumors is challenged by the lack of specific reagents to decipher their rate-limiting role in metastasis. Therefore, we generated novel transgenic mice (PDPN promoter-driven Cre recombinase transgene [PDPN-Cre] and PDPN promoter-driven thymidine kinase transgene [PDPN-tk]) that allow for the identification and genetically controlled depletion of proliferating podoplanin (Pdpn)-expressing LECs. We demonstrate that suppression of lymphangiogenesis is successfully achieved in lymphangioma lesions induced in the PDPN-tk mice. In multiple metastatic breast cancer mouse models, we identified distinct roles for LECs in primary and metastatic tumors. Our findings support the functional contribution of primary tumor lymphangiogenesis in controlling metastasis to axillary LNs and lung parenchyma. Reduced lymphatic vessel density enhanced primary tumor lymphedema and increased the frequency of intratumoral macrophages but was not associated with a significant impact on primary tumor growth despite a marked reduction in metastatic dissemination. Our findings identify the rate-limiting contribution of the breast tumor lymphatic vessels for lung metastasis.


Assuntos
Neoplasias da Mama/metabolismo , Glicoproteínas de Membrana/fisiologia , Animais , Neoplasias da Mama/fisiopatologia , Movimento Celular , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Feminino , Humanos , Linfonodos/patologia , Linfangiogênese/genética , Linfangiogênese/fisiologia , Sistema Linfático/fisiologia , Vasos Linfáticos/patologia , Macrófagos , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Metástase Neoplásica/fisiopatologia , Timidina Quinase/genética
17.
Nature ; 563(7733): 701-704, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30429614

RESUMO

Human pluripotent cell lines hold enormous promise for the development of cell-based therapies. Safety, however, is a crucial prerequisite condition for clinical applications. Numerous groups have attempted to eliminate potentially harmful cells through the use of suicide genes1, but none has quantitatively defined the safety level of transplant therapies. Here, using genome-engineering strategies, we demonstrate the protection of a suicide system from inactivation in dividing cells. We created a transcriptional link between the suicide gene herpes simplex virus thymidine kinase (HSV-TK) and a cell-division gene (CDK1); this combination is designated the safe-cell system. Furthermore, we used a mathematical model to quantify the safety level of the cell therapy as a function of the number of cells that is needed for the therapy and the type of genome editing that is performed. Even with the highly conservative estimates described here, we anticipate that our solution will rapidly accelerate the entry of cell-based medicine into the clinic.


Assuntos
Proteína Quinase CDC2/genética , Divisão Celular/genética , Terapia Baseada em Transplante de Células e Tecidos/métodos , Genes Transgênicos Suicidas/genética , Segurança do Paciente , Animais , Proliferação de Células , Terapia Baseada em Transplante de Células e Tecidos/normas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Ganciclovir/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simplexvirus/enzimologia , Simplexvirus/genética , Timidina Quinase/genética , Timidina Quinase/metabolismo
18.
Plant Physiol ; 178(4): 1643-1656, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30305373

RESUMO

Thymidine kinase (TK) is a key enzyme of the salvage pathway that recycles thymidine nucleosides to produce deoxythymidine triphosphate. Here, we identified the single TK of maize (Zea mays), denoted CPTK1, as necessary in the replication of the plastidial genome (cpDNA), demonstrating the essential function of the salvage pathway during chloroplast biogenesis. CPTK1 localized to both plastids and mitochondria, and its absence resulted in an albino phenotype, reduced cpDNA copy number and a severe deficiency in plastidial ribosomes. Mitochondria were not affected, indicating they are less reliant on the salvage pathway. Arabidopsis (Arabidopsis thaliana) TKs, TK1A and TK1B, apparently resulted from a gene duplication after the divergence of monocots and dicots. Similar but less-severe effects were observed for Arabidopsis tk1a tk1b double mutants in comparison to those in maize cptk1 TK1B was important for cpDNA replication and repair in conditions of replicative stress but had little impact on the mitochondrial phenotype. In the maize cptk1 mutant, the DNA from the small single-copy region of the plastidial genome was reduced to a greater extent than other regions, suggesting preferential abortion of replication in this region. This was accompanied by the accumulation of truncated genomes that resulted, at least in part, from unfaithful microhomology-mediated repair. These and other results suggest that the loss of normal cpDNA replication elicits the mobilization of new replication origins around the rpoB (beta subunit of plastid-encoded RNA polymerase) transcription unit and imply that increased transcription at rpoB is associated with the initiation of cpDNA replication.


Assuntos
Replicação do DNA/genética , Genomas de Plastídeos/genética , Proteínas de Plantas/metabolismo , Timidina Quinase/metabolismo , Zea mays/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cloroplastos/genética , DNA de Cloroplastos/genética , DNA de Cloroplastos/metabolismo , Duplicação Gênica , Regulação da Expressão Gênica de Plantas , Ribossomos Mitocondriais/metabolismo , Mutação , Proteínas de Plantas/genética , Biossíntese de Proteínas , Timidina Quinase/genética
19.
Arch Toxicol ; 92(12): 3585-3595, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30328498

RESUMO

The Thymidine kinase (Tk) gene forward mutation assay, known as the mouse lymphoma assay (MLA), has been widely used for evaluating the genotoxicity of chemical agents. A striking morphological feature of Tk mutant colonies is the bimodal distribution of their sizes, with cells from the large colonies growing at a normal rate and cells from the small colonies growing at a slower rate than normal. To understand the molecular distinction for the different growth rates, we performed whole genome sequencing (WGS) analysis of the large and small colony mutants generated from the MLA. Three large colony and three small colony mutants generated from cells treated with 4-nitroquinoline 1-oxide (4-NQO) or the vehicle control were selected for analysis. The WGS data were analyzed for loss of heterozygosity (LOH) and chromosome copy number along chromosome 11, where the Tk gene is located. Although there were LOH alterations in both large and small colony mutants, copy number changes near Tk locus were found only in small colony mutants produced by the vehicle control and 4-NQO treatments. The chromosome copy number in the regions near the Tk locus increased from two to three or four in the spontaneous small colony mutants and decreased from two to one in the 4-NQO-induced small colony mutants. These results suggest that chromosome damage was repaired differently in the large and small colony mutants, resulting in significant chromosome alterations in the small colony mutants, but not in the large colony mutants. Thus, chromosome alterations near the Tk locus may play a major role in the inhibition of cell growth in the Tk small colony mutants.


Assuntos
Aberrações Cromossômicas , Leucemia L5178/genética , Timidina Quinase/genética , Sequenciamento Completo do Genoma/métodos , 4-Nitroquinolina-1-Óxido/toxicidade , Animais , Variações do Número de Cópias de DNA/genética , Camundongos , Testes de Mutagenicidade/métodos , Mutagênicos/toxicidade , Mutação
20.
Sci Rep ; 8(1): 14581, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30275449

RESUMO

Bone morphogenetic protein 2 (BMP-2) is considered an effective growth factor for bone formation, and is used for making osteo-inductive scaffolds, but the related clinical investigations have shown low success rates. In this study, we genetically manipulated teratoma-derived fibroblast (TDF) cells by simultaneous introduction of BMP-2 and herpes simplex virus-thymidine kinase (HSV-tk) encoding genes. Self-production of BMP-2 in TDF cells strongly enhanced the alkaline phosphatase (ALP) activity, calcium content, and elevated the mRNA expression of osteogenic marker genes during in vitro osteogenesis. The bone formation volume was also remarkably enhanced in calvarial and femoral critical-size defect models. Ganciclovir (GCV) treatment induced apoptosis in TDF cells co-expressing HSV-tk and BMP-2, implying that HSV-tk suicide gene can modulate the side-effects of stem cell therapy, e.g., development of uncontrollable teratoma and tumor formation. Altogether, our findings revealed a safe and highly efficient technique with potential therapeutic applications for bone regeneration.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Osteogênese , Proteínas Recombinantes/metabolismo , Animais , Doenças Ósseas/terapia , Proteína Morfogenética Óssea 2/genética , Linhagem Celular , Modelos Animais de Doenças , Expressão Gênica , Humanos , Camundongos Endogâmicos BALB C , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Crânio/patologia , Timidina Quinase/genética , Timidina Quinase/metabolismo , Tíbia/patologia , Resultado do Tratamento
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA