RESUMO
The ability of various nucleoside triphosphate analogues of deoxyguanosine and deoxycytidine with 7-deazadeoxyadenosine (A1 ) and 5-chlorodeoxyuridine (T1 ) to serve as substrates for Taq DNA polymerase was evaluated. The triphosphate set composed of A1 , T1 , and 7-deazadeoxyguanosine with either 5-methyldeoxycytidine or 5-fluorodeoxycytidine was successfully employed in the polymerase chain reaction (PCR) of 1.5â kb fragments as well as random oligonucleotide libraries. Another effective combination of triphosphates for the synthesis of a 1â kb PCR product was A1 , T1 , deoxyinosine, and 5-bromodeoxycytidine. In vivo experiments using an antibiotic-resistant gene containing the latter set demonstrated that the bacterial machinery accepts fully modified sequences as genetic templates. Moreover, the ability of the base-modified segments to selectively protect DNA from cleavage by restriction endonucleases was shown. This approach can be used to regulate the endonuclease cleavage pattern.
Assuntos
Bromodesoxicitidina/química , Desoxiadenosinas/química , Desoxicitidina/análogos & derivados , Desoxiuridina/análogos & derivados , Taq Polimerase/química , Sequência de Bases , Biotecnologia , DNA/química , Desoxicitidina/química , Desoxiuridina/química , Biblioteca Gênica , Ácidos Nucleicos/química , Reação em Cadeia da Polimerase , Biologia SintéticaRESUMO
A double-stranded oligonucleotide, 80 base pairs in length, was multiply labeled with 5-bromo-2'-deoxycytidine (BrdC) using polymerase chain reaction (PCR). The modified oligonucleotide was irradiated with 300 nm photons and its damage was assayed by employing DHPLC, LC-MS and denaturing polyacrylamide gel electrophoresis (PAGE). Two types of damage were demonstrated, namely, single strand breaks (SSBs) and intrastrand cross-links (ICLs); the ICLs were in the form of d(G^C) and d(C^C) dimers. The former species are probably formed due to photoinduced electron transfer between the photoexcited BrdC and the ground state 2'-deoxyguanosine (dG), whereas the latter is a result of a cycloaddition reaction. Since SSBs and ICLs are potentially lethal to the cell, BrdC could be considered as a nucleoside with possible clinical applications.
Assuntos
Bromodesoxicitidina/química , DNA/química , Oligonucleotídeos/efeitos da radiação , Fármacos Fotossensibilizantes/química , Bromodesoxicitidina/efeitos da radiação , Cromatografia Líquida de Alta Pressão , Reação de Cicloadição , DNA/efeitos da radiação , Quebras de DNA de Cadeia Simples , Eletroforese em Gel de Gradiente Desnaturante , Espectrometria de Massas/métodos , Oligonucleotídeos/química , Reação em Cadeia da Polimerase , Raios UltravioletaRESUMO
Halogenated nucleotides belong to the group of radiosensitizers that sensitize solid tumors when incorporated into genomic DNA. Here, we consider the propensity of two isomeric bromocytidine derivatives, 3',5'-diphosphates of 5-bromo-2'-deoxycytidine (5BrdCDP) and 6-bromo-2'-deoxycytidine (6BrdCDP), to be damaged by electrons - one of the most abundant products formed during radiotherapy. An intranucleotide degradation mechanism leading to phosphodiester bond breakage (a model of single strand breakage in labeled DNA) and a ketone derivative formation was found for 6BrdCDP, while for 5BrdCDP a similar mechanism is sterically hindered. 5BrdCDP is, therefore, suggested to undergo electron induced degradation involving hydrogen transfer from a neighboring nucleotide or environment.
Assuntos
Bromodesoxicitidina/análogos & derivados , Bromodesoxicitidina/química , DNA/química , Elétrons , Teoria QuânticaRESUMO
We recently reported that intermittent hypoxia facilitated the proliferation of neural stem/progenitor cells (NPCs) in the subventricule zone and hippocampus in vivo. Here, we demonstrate that hypoxia promoted the proliferation of NPCs in vitro and that hypoxia-inducible factor (HIF)-1alpha, which is one of the key molecules in the response to hypoxia, was critical in this process. NPCs were isolated from the rat embryonic mesencephalon (E13.5), and exposed to different oxygen concentrations (20% O(2), 10% O(2), and 3% O(2)) for 3 days. The results showed that hypoxia, especially 10% O(2), promoted the proliferation of NPCs as assayed by bromodeoxyuridine incorporation, neurosphere formation, and proliferation index. The level of HIF-1alpha mRNA and protein expression detected by RT-PCR and western blot significantly increased in NPCs subjected to 10% O(2). To further elucidate the potential role of HIF-1alpha in the proliferation of NPCs induced by hypoxia, an adenovirus construct was used to overexpress HIF-1alpha, and the pSilencer 1.0-U6 plasmid as RNA interference vector targeting HIF-1alpha mRNA was used to knock down HIF-1alpha. We found that overexpression of HIF-1alpha caused the same proliferative effect on NPCs under 20% O(2) as under 10% O(2). In contrast, knockdown of HIF-1alpha inhibited NPC proliferation induced by 10% O(2). These results demonstrated that moderate hypoxia was more beneficial to NPC proliferation and that HIF-1alpha was critical in this process.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Animais , Bromodesoxicitidina/metabolismo , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Interferência de RNA , Ratos , Ratos WistarRESUMO
Peroxynitrite, a reactive nitrogen species generated from nitric oxide and superoxide anion radical, is an endogenous potential risk factor for human cancer. When 2'-deoxycytidine was incubated with peroxynitrite at neutral pH and 37 degrees C, the reaction was greatly enhanced by the addition of ammonium bromide. Both ammonium ion and bromide ion were required to exert the enhancing effect. In addition to ammonium ion, methylamine and dimethylamine exerted the enhancing effect in the presence of bromide ion. Two major products were identified as 5-hydroxy-2'-deoxycytidine and 5-bromo-2'-deoxycytidine. Hypochlorite solution and bromine water reacted with 2'-deoxycytidine generating 5-hydroxy-2'-deoxycytidine and 5-bromo-2'-deoxycytidine in the presence of ammonium bromide with the yields similar to those of the reaction of peroxynitrite with ammonium bromide. Fenton reaction of 2'-deoxycytidine was suppressed by the addition of ammonium bromide. Nitrogen dioxide gas did not react with 2'-deoxycytidine in the presence or the absence of ammonium bromide. These results suggest that in the presence of ammonium ion or amines, bromide ion interacts with peroxynitrous acid, which is a protonated form of peroxynitrite, but not with hydroxyl radical or nitrogen dioxide generated by homolysis of peroxynitrous acid, to form hypobromous acid. In the presence of ammonium ion or amines, bromide ion may play a role in enhancing the genotoxic effects of peroxynitrite in humans.
Assuntos
Brometos/química , Bromodesoxicitidina/síntese química , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Ácido Peroxinitroso/química , Compostos de Amônio Quaternário/química , Aminas/química , Bromodesoxicitidina/química , Desoxicitidina/síntese química , Estrutura Molecular , Fatores de TempoRESUMO
The replacement of thymidine with 5-bromo-2'-deoxyuridine (BrdU) is well-known to sensitize cells to ionizing radiation and photoirradiation. We reported here the sequence-dependent formation of intrastrand crosslink products from the UVB irradiation of duplex oligodeoxynucleotides harboring a BrdU or its closely related 5-bromo-2'-deoxycytidine (BrdC). Our results showed that two types of crosslink products could be induced from d(BrCG), d(BrUG), d(GBrU), or d(ABrU); the C(5) of cytosine or uracil could be covalently bonded to the N(2) or C(8) of its neighboring guanine, and the C(5) of uracil could couple with the C(2) or C(8) of its neighboring adenine. By using those crosslink product-bearing dinucleoside monophosphates as standards, we demonstrated, by using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), that all the crosslink products described above except d(G[N(2)-5]U) and d(G[N(2)-5]C) could form in duplex DNA. In addition, LC-MS/MS quantification results revealed that both the nature of the halogenated pyrimidine base and its 5' flanking nucleoside affected markedly the generation of intrastrand crosslink products. The yields of crosslink products were much higher while the 5' neighboring nucleoside was a dG than while it was a dA, and BrdC induced the formation of crosslink products much more efficiently than BrdU. The formation of intrastrand crosslink products from these halopyrimidines in duplex DNA may account for the photosensitizing effects of these nucleosides.
Assuntos
Bromodesoxicitidina/química , Bromodesoxiuridina/química , DNA/química , DNA/efeitos da radiação , Raios Ultravioleta , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Oligodesoxirribonucleotídeos/análise , Oligodesoxirribonucleotídeos/química , Espectrometria de Massas em TandemRESUMO
Telomerase is a ribonucleoprotein enzyme that adds telomeric sequence repeats to the ends of linear chromosomes. In vitro, telomerase has been observed to add repeats to a DNA oligonucleotide primer in a processive manner, leading to the postulation of a DNA anchor site separate from the catalytic site of the enzyme. We have substituted photoreactive 5-iododeoxypyrimidines into the DNA oligonucleotide primer d(T4G4T4G4T4G2) and, upon irradiation, obtained cross-links with the anchor site of telomerase from Euplotes aediculatus nuclear extract. No cross-linking occurred with a primer having the same 5' end and a nontelomeric 3' end. These cross-links were shown to be between the DNA primer and (i) a protein moiety of approximately 130 kDa and (ii) U51-U52 of the telomerase RNA. The cross-linked primer could be extended by telomerase in the presence of [alpha-32P]dGTP, thus indicating that the 3' end was bound in the enzyme active site. The locations of the cross-links within the single-stranded primers were 20 to 22 nucleotides upstream of the 3' end, providing a measure of the length of DNA required to span the telomerase active and anchor sites. When the single-stranded primers are aligned with the G-rich strand of a Euplotes telomere, the cross-linked nucleotides correspond to the duplex region. Consistent with this finding, a cross-link to telomerase was obtained by substitution of 5-iododeoxycytidine into the CA strand of the duplex region of telomere analogs. We conclude that the anchor site in the approximately 130-kDa protein can bind duplex as well as single-stranded DNA, which may be critical for its function at chromosome ends. Quantitation of the processivity with single-stranded DNA primers and double-stranded primers with 3' tails showed that only 60% of the primer remains bound after each repeat addition.
Assuntos
Primers do DNA , DNA , Euplotes/enzimologia , Telomerase/química , Animais , Sítios de Ligação , Bromodesoxicitidina/análogos & derivados , Reagentes de Ligações Cruzadas , Desoxicitidina/análogos & derivados , Idoxuridina , Modelos Genéticos , Peso Molecular , Conformação de Ácido Nucleico , RNA/química , RNA/metabolismo , Telomerase/isolamento & purificação , Telomerase/metabolismo , Raios UltravioletaRESUMO
Bromonucleosides constitute a significant class of molecules and are well known for their biological activity. 5-Bromouridine, 5-bromo-2'-deoxyuridine, 5-bromouridine-5'-triphosphate, and nucleotides containing 5-bromouridine have been tested and used for numerous biological studies. 8-Bromopurine nucleosides have been used as essential precursors for the synthesis of nucleosides with fluorescent properties. This unit describes protocols for the synthesis of bromonucleosides using sodium monobromoisocyanurate (SMBI) in a straightforward way. Reactions are carried out at room temperature, and aqueous solvent mixtures are used to dissolve the nucleosides. Sodium azide is used as catalyst for the bromination of pyrimidine nucleosides, and no catalyst is necessary for the bromination of purine nucleosides. Unprotected 2'-deoxy pyrimidine and 2'-deoxy purine nucleosides are treated with SMBI to afford C-5 bromo pyrimidine and C-8 bromo purine nucleosides, respectively. This methodology has been found to be efficient for the synthesis of bromonucleosides on gram scale with consistently high yields. © 2017 by John Wiley & Sons, Inc.
Assuntos
Nucleosídeos de Purina/síntese química , Nucleosídeos de Pirimidina/síntese química , Bromodesoxicitidina/síntese química , Bromodesoxicitidina/química , Bromodesoxiuridina/síntese química , Bromouracila/análogos & derivados , Técnicas de Química Sintética , Desoxiadenosinas/síntese química , Desoxiadenosinas/química , Desoxiguanosina/análogos & derivados , Desoxiguanosina/síntese química , Desoxiguanosina/química , Nucleosídeos de Purina/química , Nucleosídeos de Pirimidina/química , Uridina/análogos & derivados , Uridina/síntese química , Uridina/químicaRESUMO
3,4-Dihydro-2,2-dimethyl-2H-naptho[1,2,-b]pyran-5,6-dione (beta-lapachone) is a novel DNA repair inhibitor. It was tested for synergistic X-ray-induced lethality in combination with several halogenated pyrimidine radiosensitizers. Logarithmic-phase growing human epidermoid laryngeal carcinoma (HEp-2) cells were allowed to incorporate pyrimidine analogues for 48 h (approximately two cell doublings) and then were X-irradiated and subjected to various posttreatments. beta-Lapachone synergistically increased the dose enhancement ratios (DERs) of all analogues screened, with the exception of the 2'-chloro derivative of 5-bromodeoxyuridine. For example, following 5-bromodeoxycytidine sensitization an X-ray DER value of 1.87 +/- 0.04 at 1% survival was increased to 3.51 +/- 0.42 due to a 4-h post-X-irradiation exposure to 4 microM beta-lapachone. Do and Dq values for halogenated pyrimidine-sensitized human epidermoid laryngeal carcinoma cells were decreased 1.4- to 5.4-fold and 1.4- to 4.0-fold, respectively. beta-Lapachone had little effect upon the cytotoxicities of unirradiated human epidermoid laryngeal carcinoma cells whether or not they were previously exposed to any of the halogenated pyrimidine radiosensitizers. beta-Lapachone treatment following X-irradiation of cells that had not incorporated a pyrimidine analogue exhibited DER values of 1.38 +/- 0.05 and 1.40 +/- 0.01 at 10 and 1% survival levels, respectively. beta-Lapachone enhanced the radiosensitization of deoxycytidine analogues to a greater extent than the structurally related deoxyuridine analogues. Greater DERs and lower Do and Dq values were found for deoxycytidine than for deoxyuridine analogue radiosensitizers following beta-lapachone treatment. This agent may improve presently used radiation therapies and enhance proposed strategies which utilize deoxycytidine analogue radiosensitization together with protection of normal tissues by tetrahydrouridine to achieve tumor-selective radiotherapy.
Assuntos
Reparo do DNA/efeitos dos fármacos , Naftoquinonas/uso terapêutico , Radiossensibilizantes/farmacologia , Bromodesoxicitidina/uso terapêutico , Bromodesoxiuridina/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Linhagem Celular , Células Cultivadas , Sinergismo Farmacológico , Humanos , Neoplasias Laríngeas/tratamento farmacológicoRESUMO
Long-term, low-level, BrdUrd infusion identifies two subpopulations of GM-CFCc with quite dissimilar sensitivities to 313 nm light. The responses of these two GM-CFCc subpopulations to hydroxyurea indicate that both are rapidly proliferating at the time of the assay. However, the absolute UV-light sensitivity of the S-phase components and the effects of increasing BrdUrd concentration indicate that the two GM-CFCc subpopulations passed through the previous cell cycle at widely disparate rates. Further, those GM-CFCc originating from a parental cell with a slow turnover are associated with a lower buoyant density than those GM-CFCc that have been in rapid cycle for at least two generations. These results indicate that the resistance to 313 nm-light irradiation, shown by S-phase cells in the first cell cycle of the BrdUrd labeling, may provide evidence of the proliferative history of the cell being assayed.
Assuntos
Bromodesoxiuridina/farmacologia , Ensaio de Unidades Formadoras de Colônias/classificação , Animais , Bromodesoxicitidina/farmacologia , Ciclo Celular , Centrifugação com Gradiente de Concentração , Feminino , Hidroxiureia/farmacologia , Interfase , Cinética , Luz , CamundongosRESUMO
The kinetics of cell turnover for myeloid/monocyte cells that form colonies in agar (GM-CFC) were measured through the progressive increase in their sensitivity to 313-nm light during a period of cell labeling with BrdCyd. Two components of cell killing with distinctly separate labeling kinetics revealed both the presence of two generations within the GM-CFC compartment and the properties of the kinetics of the precursors of the GM-CFC. These precursors of the GM-CFC were not assayable in a routine GM-CFC assay when pregnant mouse uterus extract and mouse L-cell-conditioned medium were used to stimulate colony formation but were revealed by the labeling kinetics of the assayable GM-CFC. Further, these precursor cells appeared to enter the assayable GM-CFC population from a noncycling state. This was evidenced by the failure of the majority of these cells to incorporate BrdCyd during five days of infusion. The half-time for cell turnover within this precursor compartment was measured to be approximately 5.5 days. Further, these normally noncycling cells proliferated rapidly in response to endotoxin. High-proliferative-potential colony-forming cells (HPP-CFC) were tested as a candidate for this precursor population. The results of the determination of the kinetics for these cells showed that the HPP-CFC exist largely in a Go state, existing at an average rate of once every four days. The slow turnover time for these cells and their response to endotoxin challenge are consistent with a close relationship between the HPP-CFC and the Go pool of cells that is the direct precursor of the GM-CFC.
Assuntos
Células da Medula Óssea , Bromodesoxicitidina/metabolismo , Divisão Celular , Desoxicitidina/análogos & derivados , Animais , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Endotoxinas/farmacologia , Feminino , Camundongos , Monócitos/efeitos da radiação , Raios UltravioletaRESUMO
Survival patterns of burst-forming unit-erythroid (BFU-E) and cluster-forming unit-erythroid (CFU-E) were studied in B6D2F1 mice after 5'bromodeoxycytidine (BrdCyd) infusion and near-ultraviolet (near-UV) light treatment. Comparison of the kinetics of BrdCyd sensitization of these colony formers with the kinetics of granulocyte-monocyte colony-forming cells (GM-CFC) and colony-forming unit-spleen (CFU-S) demonstrated a similar, but distinguishable, pattern of labeling between BFU-E and CFU-S. BFU-E and CFU-S each contain a subpopulation of unlabeled cells that diminishes with a half-life of 35 to 40 hours. Subsequent comparisons of this close association of BFU-E and CFU-S in Sl/Sld mice, again, showed similar patterns of BrdCyd labeling without evidence of an unlabeled or G0 subpopulation. These results are discussed with respect to the likelihood of a precursor relationship between these two cell types.
Assuntos
Células Precursoras Eritroides/citologia , Eritropoese/efeitos dos fármacos , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/efeitos da radiação , Bromodesoxicitidina/toxicidade , Bromodesoxiuridina/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/efeitos da radiação , Eritropoese/efeitos da radiação , Feminino , Camundongos , Camundongos Mutantes , Raios UltravioletaRESUMO
Glomeruloid bodies (GBs), tumor-associated vascular structures with a superficial resemblance to renal glomeruli, are important histopathological features of glioblastoma multiforme, but have also been described in other types of tumors and in cerebral metastases. The purpose of this study was to elucidate the pathogenesis of these lesions in an appropriate murine model of experimental brain metastases. To do so, we injected cells from 5 different tumor lines into the internal carotid artery of mice and investigated the development, composition, and fate of GBs growing within tumor nodules. Immunohistochemical analyses and 3-dimensional reconstruction of the cerebral vasculature showed clearly that the proliferating and migrating tumor cells pull the capillaries (and the adjacent capillary branching points) into the tumor cell nest. Initially, this process lead to the appearance of simple coiled vascular structures, which later developed into chaotic and tortuous vascular aggregates with multiple narrowed afferent and efferent microvessels. Despite the absence of sprouting angiogenesis, the very low level of endothelial cell proliferation index and the ruptures of the stretched and narrowed capillary segments observed frequently between the metastatic tumor nodules, necrosis was scarce in these lesions, implying that the blood supply from the multiple afferent microvessels and from the preexistent vascular bed sufficed to provide the tumor cells with oxygen and nutrients.
Assuntos
Neoplasias Encefálicas/ultraestrutura , Artérias Cerebrais/ultraestrutura , Glioblastoma/ultraestrutura , Neoplasias Experimentais/ultraestrutura , Animais , Neoplasias Encefálicas/metabolismo , Bromodesoxicitidina/farmacocinética , Carcinoma Pulmonar de Lewis/ultraestrutura , Linhagem Celular , Artérias Cerebrais/patologia , Modelos Animais de Doenças , Glioblastoma/patologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Microscopia Eletrônica/instrumentação , Microscopia Eletrônica/métodos , Metástase Neoplásica/ultraestrutura , Transplante de Neoplasias/métodos , Neoplasias Experimentais/patologia , Neovascularização Patológica/patologia , Fatores de TempoRESUMO
Murine mammary carcinoma (FM3A TK-/HSV-1 TK+) cells, which are thymidine kinase (TK)-deficient but have been transformed with the herpes simplex virus type 1 (HSV-1) TK gene are inhibited in their growth by (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC) at 0.5, 0.5 and 0.8 ng/ml, respectively; i.e., a concentration 5000 to 20 000-fold lower than that required to inhibit the growth of the corresponding wild-type FM3A/0 cells. Hence, transformation of tumor cells with the HSV-1 TK gene makes them particularly sensitive to the cytostatic action of BVDU and related compounds.
Assuntos
Bromodesoxiuridina/análogos & derivados , Genes Virais , Neoplasias Mamárias Experimentais/enzimologia , Simplexvirus/enzimologia , Timidina Quinase/genética , Transformação Genética , Animais , Antivirais/farmacologia , Bromodesoxicitidina/análogos & derivados , Bromodesoxicitidina/farmacologia , Bromodesoxiuridina/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Feminino , Idoxuridina/análogos & derivados , Idoxuridina/farmacologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C3H , Fosforilação , Simplexvirus/genética , Timidina Quinase/metabolismoRESUMO
Infection of rat RT2 glioma cells in vitro with an adenovirus (ADV-TK) expressing herpes simplex virus (HSV) thymidine kinase (TK) and subsequent exposure to 5-bromo-2'-deoxycytidine (BrdC), which is specifically incorporated into ADV-TK-infected cell DNA as 5-bromo-2'-deoxyuridine (BrdU), results in significant radiosensitization (sensitizer enhancement ratio: 1.4-2.3) compared with Ad beta gal-infected cells. Cell killing correlated well with increased BrdU DNA incorporation and with apoptosis. Whereas radiation (4 Gy) alone was relatively ineffective in inducing apoptosis, treatment with HSV-TK/BrdC resulted in BrdC dose- (10-100 microM) and time-dependent (24-48 hours) increases, and the combination of the two treatments produced a synergistic response (1.5- to 2-fold). To investigate the effects of the ADV-TK/BrdC treatment in vivo, RT2 cells were grown as soft tissue tumors in Fischer 344 rats and conditions for virus infusion were optimized by altering the volume and rate of infusion using a rate-controlled positive pressure device. We found that relatively large volumes (100-150 microL) of virus delivered at rates of < or = 1 microL/minute were optimal and gave uniform and reproducible results. Using these optimal infusion conditions, we were able to achieve 40% adenovirus infection in the tumor. Infection of RT2 tumors with ADV-TK and continuous administration of BrdC from an osmotic pump resulted in significant (.001 < P < .009) tumor regression 6 days after radiation (30 Gy delivered as 2 x 5 Gy over 3 days) compared with controls. In situ staining of sectioned tumors with anti-BrdU antibody or by high-performance liquid chromatography analysis of extracted and hydrolyzed tumor DNA confirmed that we obtained efficient and specific incorporation of BrdU into tumor cells. These results suggest that adenovirus-mediated delivery of HSV-TK in combination with BrdC and radiation can potentially be an efficient combination modality for the treatment of gliomas.
Assuntos
Adenoviridae/genética , Neoplasias Encefálicas/terapia , Bromodesoxicitidina/uso terapêutico , Vetores Genéticos/administração & dosagem , Glioma/terapia , Neoplasias Experimentais/terapia , Radiossensibilizantes/uso terapêutico , Simplexvirus/enzimologia , Timidina Quinase/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Cromatografia Líquida de Alta Pressão , Citidina Desaminase/metabolismo , Relação Dose-Resposta à Radiação , Feminino , Células HeLa , Humanos , Marcação In Situ das Extremidades Cortadas , Transplante de Neoplasias , Ratos , Ratos Endogâmicos F344 , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Suicide gene therapy of malignant melanoma essentially requires efficient gene transfer and highly selective therapeutic gene expression. To achieve this, recombinant adeno-associated virus (rAAV) particles were constructed containing the tissue-specific promoter of the human melanoma inhibitory activity (hMIA) gene combined with four copies of the enhancer element of the murine tyrosinase gene. Three melanoma and one cervix carcinoma cell line were infected with rAAV particles carrying a reporter gene under control of the enhancer/hMIA promoter in order to determine transcriptional activity and specificity of this system. Viral particles containing the enhancer/hMIA promoter mediated reporter gene activity only in melanoma cells, whereas infection with a cytomegalovirus (CMV)-based promoter construct induced unspecific gene expression. Correspondingly, transient transduction with viral particles bearing the HSVtk gene under the control of the enhancer/MIA promoter elements followed by treatment with ganciclovir (GCV) resulted in growth inhibition only in melanoma cells, whereas the CMV promoter-based construct induced unspecific cytotoxicity. In vivo experiments in nude mice demonstrated that tumors originating from human melanoma cells disappeared after stable, but not transient transduction with vectors bearing the HSVtk gene under the control of the enhancer/hMIA promoter in response to GCV application. In face of higher transduction efficiency, these rAAV particles might therefore be a useful tool for suicide gene therapy of malignant melanoma.
Assuntos
Desoxicitidina/análogos & derivados , Terapia Genética/métodos , Melanoma/terapia , Regiões Promotoras Genéticas , Proteínas/genética , Animais , Antivirais/farmacologia , Bromodesoxicitidina/análogos & derivados , Linhagem Celular Tumoral , Separação Celular , Clonagem Molecular , Desoxicitidina/farmacologia , Dependovirus/genética , Elementos Facilitadores Genéticos , Proteínas da Matriz Extracelular , Feminino , Citometria de Fluxo , Ganciclovir/farmacologia , Técnicas de Transferência de Genes , Genes Reporter , Humanos , Imunossupressores/farmacologia , Melanoma/genética , Camundongos , Camundongos Nus , Modelos Genéticos , Monofenol Mono-Oxigenase/genética , Proteínas de Neoplasias , Transplante de Neoplasias , Plasmídeos/metabolismo , Simplexvirus/genética , Timidina Quinase/genética , Fatores de Tempo , Distribuição TecidualRESUMO
A novel radioimmunoassay of 5MedCyd is described. The assay, employing a highly specific antiserum raised in rabbits against BSA-conjugated 5MeCyd, used 5-125iodo-2'-deoxycytidine as the tracer. The measuring range for the assay was found to be 1-1000 pmol per assay of 5MedCyd. When the methods were applied to the measurement of methylation in DNA samples a good correlation between the results obtained with the radioimmunoassay and HPLC was demonstrated. The method has several advantages over the more laborious and sophisticated techniques previously available: high sensitivity, large assay range, rapidity and potential for large number of simultaneous assays, simplicity, and low cost provided that the laboratory has equipment for gamma counting.
Assuntos
Citosina/análogos & derivados , DNA/análise , Radioimunoensaio , 5-Metilcitosina , Animais , Especificidade de Anticorpos , Bromodesoxicitidina/análogos & derivados , Bovinos , Cromatografia Líquida de Alta Pressão , Reações Cruzadas , Citosina/análise , Desoxicitidina/análogos & derivados , Peixes , Metilação , CoelhosRESUMO
The halogenated pyrimidines 5-chloro-2'-deoxycytidine (CldCyd) and 5-bromo-2'-deoxycytidine (BrdCyd) can act as radiosensitizers and cytotoxic agents. It was hypothesized that tumor cells and normal cells might use different metabolic pathways to incorporate these halogenated deoxycytidines into DNA. This difference could potentially be exploited to produce selective radiosensitization and cytotoxicity of human tumor cells compared to normal human fibroblasts. This hypothesis was tested using two human melanoma cell lines and two normal fibroblast cell lines. Either CldCyd or BrdCyd alone caused both cytotoxicity and radiosensitization of tumor and normal cells. The addition of the cytidine deaminase inhibitor tetrahydrouridine (H4U) significantly protected the normal cells but had relatively little effect on the tumor cells. These data indicate that it may be possible to exploit differences between the pyrimidine metabolism of normal cells and melanoma cells to improve the therapeutic index of halogenated pyrimidines both as radiosensitizers and as cytotoxic agents.
Assuntos
Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Melanoma/patologia , Radiossensibilizantes/farmacologia , Tetra-Hidrouridina/farmacologia , Uridina/análogos & derivados , Bromodesoxicitidina/farmacologia , Linhagem Celular , Desoxicitidina/farmacologia , Fibroblastos/efeitos dos fármacos , Humanos , Técnicas In Vitro , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Treatment of 3',5'-di-O-acetyl-(E)-5-(2-bromovinyl)-2'-deoxyuridine (2) with p-chlorophenyl phosphorodichloridate and 1,2,4-triazole gave 1-(3,5-di-O-acetyl-2-deoxy-beta-D-erythro-pentofuranosyl)-(E)-5-(2-br o movinyl)- 4-(1,2,4-triazol-1-yl)pyrimidin-2(1H)-one (3). Reaction of 3 with ammonia gave (E)-5-(2-bromovinyl)-2'-deoxycytidine (1), the overall yield from 2 being 60%. A similar 4-(1,2,4-triazol-1-yl) derivative (4) was obtained from 3',5'-di-O-acetyl-thymidine by the use of phosphoryl chloride as the condensing agent. Treatment of thymidine with trimethylsilyl chloride and then with phosphoryl chloride and 1,2,4-triazole gave upon workup 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-5-methyl-4(1,2,4-triazol -1-yl) pyrimidin-2(1H)-one (5). (E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVDU) when similarly treated gave the corresponding (E)-5-(2-bromovinyl) compound 7. A minor product formed in both cases was a 4-(1,2,4-triazol-1-yl) derivative in which the nucleoside 5'-hydroxyl group had been replaced by chlorine (6 and 8). Whereas compounds 4-6 and 8 did not exhibit a selective antiviral effect, compounds 1-3 and 7 proved almost as active as the reference compound BVDU. In particular, compound 7, the 4-triazolyl derivative of BVDU, would seem worth pursuing for its potential as an inhibitor of herpes simplex virus type 1 and varicella-zoster virus.
Assuntos
Antivirais/síntese química , Bromodesoxicitidina/análogos & derivados , Bromodesoxiuridina/análogos & derivados , Bromodesoxiuridina/síntese química , Desoxicitidina/análogos & derivados , Simplexvirus/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Animais , Bromodesoxicitidina/farmacologia , Bromodesoxiuridina/farmacologia , Células Cultivadas , Indicadores e Reagentes , Rim , Coelhos , Relação Estrutura-AtividadeRESUMO
Carbocyclic analogues of 5-halocytosine nucleosides were prepared by direct halogenation of the carbocyclic analogues of cytidine, 2'-deoxycytidine, 3'-deoxycytidine, or ara-C. The 5-chloro and 5-bromo derivatives of the cytidine (carbodine) and of the 2'-deoxycytidine analogues and the 5-iodo derivatives of all four of the cytosine nucleoside analogues were prepared. All of the C-5-halocytosine nucleosides, as well as the parent C-cytosine nucleosides, were tested against a strain of herpes simplex virus type 1 (HSV-1) that induces thymidine kinase in host cells. Carbodine, 5-bromocarbodine, C-2'-deoxycytidine, C-5-bromo-2'-deoxycytidine, the four C-5-iodocytosine nucleosides, and C-ara-C inhibited replication of this strain of HSV-1 in cultured cells. Most of these compounds were tested also against the type 2 virus (HSV-2) in vitro and were active. The greatest activity observed was exerted by C-5-iodo-2'-deoxycytidine in inhibiting replication of HSV-1 in L929 cells. In tests against these DNA viruses, carbodine, a ribofuranoside analogue that had been shown previously to be highly active against human influenza A virus in vitro, was the most active compound against HSV-2 and one of the most active compounds against HSV-1 in Vero cells. 5-Bromocarbodine was active against influenza virus, but it was less active than carbodine.