RESUMO
We tested the efficacy of a yeast cytosine deaminase::uracil phosphoribosyl transferase/5-fluorocytosine (CDU/5-FC) non-viral suicide system on eight established canine melanoma cell lines. Albeit with different degree of sensitivity 5 days after lipofection, this system was significantly efficient killing melanoma cells, being four cell lines highly, two fairly and two not very sensitive to CDU/5-FC (their respective IC50 ranging from 0.20 to 800 µM 5-FC). Considering the relatively low lipofection efficiencies, a very strong bystander effect was verified in the eight cell lines: depending on the cell line, this effect accounted for most of the induced cell death (from 70% to 95%). In our assay conditions, we did not find useful interactions either with the herpes simplex thymidine kinase/ganciclovir suicide system (in sequential or simultaneous modality) or with cisplatin and bleomycin chemotherapeutic drugs. Furthermore, only two cell lines displayed limited useful interactions of the CDU/5-FC either with interferon-ß gene transfer or the proteasome inhibitor bortezomib respectively. These results would preclude a wide use of these combinations. However, the fact that all the tested cells were significantly sensitive to the CDU/5-FC system encourages further research as a gene therapy tool for local control of canine melanoma.
Assuntos
Doenças do Cão , Melanoma , Pentosiltransferases , Animais , Cães , Citosina Desaminase/genética , Citosina Desaminase/metabolismo , Doenças do Cão/tratamento farmacológico , Flucitosina/metabolismo , Flucitosina/farmacologia , Flucitosina/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/veterinária , Pentosiltransferases/metabolismo , Timidina Quinase/genética , Uracila , Morte CelularRESUMO
In the context of comparative oncology, melanoma cells derived from companion animal tumors are good models for optimizing and predicting their in vivo response to therapeutic strategies. Here, we report that human, canine, and feline melanoma cells driven to death by bleomycin, interferon-ß gene, or herpes simplex virus thymidine kinase/ganciclovir suicide gene (SG) treatment significantly increased their internal granularity. This fact correlated with the release of a heterogeneous collection of nano- and micro-sized granules as revealed by transmission electron microscopy. While killing lipofected cells, the expressed transgenes and their derived products were incorporated into these granules that were isolated by differential centrifugation. These particulate factors (PFs) were able to transfer, in a dose- and time-dependent manner, appreciable levels of therapeutic genes, related proteins, and drugs. Thus, when recipient cells of SG-carrying PFs were exposed to ganciclovir, this prodrug was efficiently activated, eliminating them. These PFs kept the functionality of their cargo, even after being subjected to adverse conditions, such as the presence of DNase, freezing, or heating. Since our in vitro system did not include any of the immune mechanisms that could provide additional antitumor activity, the chemo-gene treatments amplified by these delivery bags of therapeutic agents offer a great clinical potential.
Assuntos
Interferon beta , Melanoma , Animais , Efeito Espectador , Gatos , Cães , Ganciclovir/farmacologia , Humanos , Interferon beta/genética , Melanoma/genética , Melanoma/terapia , Simplexvirus/genética , Timidina Quinase/genética , TransgenesRESUMO
Bovine herpevsirus 4 (BoHV-4) is a gammaherpesvirus that has been associated with different clinical conditions in cattle. In Argentina, BoHV-4 was detected in diverse bovine samples. The aim of this study was to analyze the genetic relationship of 48 field BoHV-4 strains isolated from cattle in Argentina. According to thymidine kinase (tk) gene sequences, BoHV-4 isolates belong to genotypes 1, 2 and 3. Phylogenetic analyses confirmed the presence of the three previously described viral genotypes. However, some of the studied isolates presented conflicting phylogenetic signals between the studied markers. This suggests a complex evolutionary background, that is a history of recombination, incomplete lineage sorting (deep coalescence) or a combination of these, which requires further study. These potential events make difficult the diagnosis of BoHV-4 from clinical samples of cattle and may pose a significant problem for the control of the virus in the herds.
Assuntos
Herpesvirus Bovino 4/genética , Timidina Quinase/genética , Animais , Argentina , Evolução Biológica , Bovinos/virologia , Doenças dos Bovinos/virologia , DNA Viral/genética , Evolução Molecular , Genótipo , Herpesvirus Bovino 4/isolamento & purificação , Herpesvirus Bovino 4/patogenicidade , FilogeniaRESUMO
While the induced-fit of a ligand towards an enzyme is pivotally dictated by intermolecular hydrogen bonds between the small molecule and amino acid residues in the binding site, the role of intramolecular hydrogen bond as contributing interaction for a bioactive conformation is not well understood. This work reports a theoretical conformational analysis of a thymidine kinase enzyme ligand (NMF) that is prone to experience an Fâ¯HO intramolecular hydrogen bond, inside and outside the biological binding site. This interaction stabilizes the most favorable conformations of NMF in the gas phase and, although it can be disrupted in a biological environment due to intermolecular hydrogen bonds in some cases, these interactions are competitive in other systems. Therefore, an intramolecular hydrogen bond can affect the conformational likeliness most related to the bioactivity. Moreover, isolated conformations governed by this interaction cannot be unequivocally used to generate molecular descriptors in 3D-QSAR (Quantitative Structure-Activity Relationships), since the bioactive conformation may not be determined only by intramolecular interactions.
Assuntos
Timidina Quinase , Ligação de Hidrogênio , Ligantes , Conformação MolecularRESUMO
Herpes simplex virus type 1 (HSV-1) infections affect about two-thirds of the world population, and the standard treatment consists of acyclovir (ACV) and its analogs, which interact with thymidine kinase (TK) blocking viral replication. Lately, the emergence of ACV-resistant strains has been reported, especially associated with TK mutations. In this context, ACV therapy fails against isolates encoding Y172C and Y53H/R163H TK mutants, but the molecular mechanism of drug resistance remains unclear. Thus, we examined the effects of these mutations on ACV and the cofactor ATP binding through molecular modeling approaches. We showed that Y172C prevents the anchoring of the aromatic ring of ACV through π-π stacking interactions, leading to an inversed binding mode and different interactions. On the other hand, Y53H/R163H remarkably affected the cofactor binding mode which shifted away from its binding site and also influenced the interaction network of ACV. This is likely due to the loss of polar interactions with R163 residue. Unlike what was observed in the wild-type complex, both drug and cofactor binding poses were not well positioned to allow the phosphorylation reaction which explains the resistance observed. Moreover, energy analysis corroborated the experimental data and showed lower theoretical affinity of ACV with mutant enzymes resulted from energetic loss in polar solvation in Y172C and electrostatic terms in Y53H/R163H mutant enzyme. Therefore, our study shed light on the resistance mechanism toward ACV of two mutant TKs identified in clinical HSV-1 strains and may further support the development of new anti-herpetic drugs to treat resistant infections. [Formula: see text] Communicated by Ramaswamy H. Sarma.
Assuntos
Aciclovir , Herpesvirus Humano 1 , Aciclovir/farmacologia , Antivirais/farmacologia , Farmacorresistência Viral/genética , Herpesvirus Humano 1/genética , Timidina Quinase/genética , Replicação ViralRESUMO
After 6 years of follow-up treating 364 canine melanoma patients, we present here results about the proof-of-concept, safety, and efficacy of a new surgery adjuvant combined gene therapy. The adjuvant treatment (AT) group was divided in three arms as follows: (i) complete surgery plus vaccine (CS-V), (ii) complete surgery plus combined treatment (CS-CT), and (iii) partial surgery plus combined treatment (PS-CT). Besides the genetic vaccines composed by tumor extracts and lipoplexes carrying human interleukin-2 and granulocyte-macrophage colony-stimulating factor genes, the patients were subjected to combined treatment received in the post-surgical bed injections of lipid-complexed thymidine kinase suicide gene plus ganciclovir and canine interferon-ß gene plus bleomycin. As compared with surgery-only treated controls (So), CS-CT and CS-V treatments significantly increased the fraction of local disease-free (from 20 to 89 and 74%) and distant metastases-free patients (M0: from 45 to 87 and 84%). Although less effective than CS arms, PS-CT arm demonstrated a significantly improved control of metastatic disease (M0: 80%) compared with So (M0: 44%). In addition, AT produced a significant 9.3- (CS-CT), 6.5- (CS-V), and 5.4-fold (PS-CT) increase of overall survival as compared with their respective So controls. In general terms, the AT changed a lethal disease into a chronic disease where 70% of CS-CT, 51% of CS-V, and 14% of PS-CT patients died of melanoma unrelated causes. These surgery adjuvant treatments delayed or prevented post-surgical recurrence and distant metastasis, and improved disease-free and overall survival while maintaining quality of life. These successful outcomes encourage assaying a similar scheme for human melanoma.
Assuntos
Antineoplásicos/uso terapêutico , Doenças do Cão/terapia , Terapia Genética/veterinária , Melanoma/veterinária , Neoplasias Bucais/veterinária , Procedimentos Cirúrgicos Bucais/veterinária , Animais , Bleomicina/uso terapêutico , Células Cultivadas , Quimioterapia Adjuvante , Doenças do Cão/tratamento farmacológico , Doenças do Cão/cirurgia , Cães , Feminino , Ganciclovir/uso terapêutico , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Interferon beta/uso terapêutico , Interleucina-2/uso terapêutico , Masculino , Melanoma/tratamento farmacológico , Melanoma/cirurgia , Melanoma/terapia , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/cirurgia , Neoplasias Bucais/terapia , Procedimentos Cirúrgicos Bucais/efeitos adversos , Procedimentos Cirúrgicos Bucais/métodos , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/veterinária , Timidina Quinase/genética , Timidina Quinase/metabolismo , Vacinas Sintéticas/uso terapêuticoRESUMO
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/genéticaRESUMO
TITLE: Mutacion del gen TK2 y miopatia de inicio tardio: descripcion del primer caso mexicano.
Assuntos
Doenças Musculares/genética , Mutação , Timidina Quinase/genética , Idade de Início , Feminino , Humanos , México , Adulto JovemRESUMO
BACKGROUND: Three metastatic human melanoma cell lines generated from patient removed lymph nodes and spleen metastasis were established in our laboratory. OBJECTIVE: To investigate the mechanisms enhancing the cytotoxic effects of Bleomycin (BLM), herpes simplex virus thymidine kinase/ganciclovir Suicide Gene (SG) and human interferon-ß gene (hIFNß) lipofection in early passages of these melanoma cell lines. METHODS: In these cell lines, we determined: cytotoxicity, bystander effect, lipofection efficiencies, apoptosis, necrosis, senescence, colony forming capacity and mitochondrial membrane depolarization after treatments. RESULTS: The three assayed cell lines displayed sensitivity to single and combined BLM/gene treatments. BLM improved the antitumor and anti-clonogenic effects of SG and hIFNß genes. Considering the low lipofection efficiencies (<10%), one of the main causes of the SG and hIFNß gene effectiveness was their bystander effect. In one of these cell lines, this effect eradicated up to 60% of the cells although <1% expressed the transgene. In the three cell lines, BLM alone or combined with SG or hIFNß gene significantly increased the percentage of cells exhibiting membrane compromise, DNA damage, and senescence. Interestingly, the strong BLM/hIFNß gene combination was able to generate from 73% to 98% of non-viable cells. The high proportion of senescent cells induced by BLM alone or combined with genes strongly decreased the clonogenic capacity of surviving cells. CONCLUSION: The presented results indicate that BLM improves the antitumor effects of SG and hIFNß transgene expression. Altogether, these findings strongly support the clinical potential of these combined approaches.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Bleomicina/farmacologia , Terapia Genética/métodos , Interferon beta/genética , Melanoma/terapia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Senescência Celular/efeitos dos fármacos , Técnicas de Transferência de Genes , Humanos , Melanoma/genética , Metástase Neoplásica/genética , Metástase Neoplásica/terapia , Simplexvirus/enzimologia , Simplexvirus/genética , Timidina Quinase/genéticaRESUMO
PURPOSE: To examine the effects of n-3 and n-6 polyunsaturated fatty acids (n-3 and n-6 PUFAs) in a murine model of herpetic chorioretinitis. METHODS: BALB/c mice were fed on three high fat diets, which contained: Menhaden oil (rich in n-3 PUFAs); Safflower oil (rich in n-6 PUFAs); or Corn oil (rich in saturated fatty acids) as control group, 14 days previously and until 12 days following anterior chamber (AC) HSV-1 inoculation. RESULTS: Mice fed on Menhaden oil present an early development of contralateral chorioretinitis by day 6 post-AC HSV-1 inoculation and also significant increase of RNA HSV-1 expression compared with Safflower and Corn oil groups. Furthermore, mice fed on Menhaden oil showed a significant decrease secretion of TNF-α, IFN-γ, IL-2 and IL-10 in splenic cells and both retinas. CONCLUSION: Our results showed that mice fed on Menhaden oil (n-3 PUFAs) presented an early development of contralateral chorioretinitis by day 6 post-AC HSV-1 inoculation and also a significant increase in RNA HSV-1 expression compared with animals fed on Safflower and Corn oils. This increase of HSV-1 could be associated with the higher development of chorioretinitis.