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1.
J Clin Pharmacol ; 62(3): 376-384, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34554584

RESUMO

Identification of a pharmacodynamic (PD) biomarker, which is predictive of the efficacy outcome, is of ultimate interest in drug development. The objectives of the current analyses are to develop the pharmacokinetic (PK)/PD model for biomarkers (thymidine kinase 1 [TK1] in serum and phosphor-retinoblastoma protein [pRb] and Ki67 in skin tissues) related to cyclin-dependent kinase (CDK) 4/6 inhibition by palbociclib and to explore the relationship of the biomarker response with the efficacy end point (progression-free survival). The data used for analysis consisted of extensive sampling of palbociclib PK and longitudinal rich sampling for the PD biomarkers TK1, pRb, and Ki67 in 26 patients. A 2-compartment model was used to describe the PK of palbociclib. A precursor-dependent indirect response PD model was developed to describe the pRb time course, whereas a similar PD model with an additional transit compartment to model the delayed effect on Ki67 and TK1 response was used to describe the Ki67 and TK1 time course. Palbociclib effect on biomarkers was modeled as a maximum inhibition model. A Cox proportional hazard model was used to assess the relationship of progression-free survival with the biomarker response. The PK/PD models adequately described the observed PK of palbociclib and the longitudinal change of pRb, Ki67, and TK1. Palbociclib exposure significantly correlated with the reduction of all 3 biomarkers, and the estimated concentration to achieve 50% inhibition of the synthesis rate values were 45.2, 42.4, 50.2 ng/mL, respectively, for pRb, Ki67, and TK1. The exploratory biomarker-response analyses showed that a longer PFS was associated with lower baseline TK1 and simulated minimum TK1. Such results may warrant further confirmation from future large-scale study. Clinical Trial Registration: NCT02499146.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Antígeno Ki-67/sangue , Piperazinas/farmacologia , Piridinas/farmacologia , Proteína do Retinoblastoma/sangue , Timidina Quinase/sangue , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais , Neoplasias da Mama/patologia , China , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Feminino , Humanos , Antígeno Ki-67/efeitos dos fármacos , Taxa de Depuração Metabólica , Modelos Biológicos , Estadiamento de Neoplasias , Piperazinas/farmacocinética , Piperazinas/uso terapêutico , Intervalo Livre de Progressão , Piridinas/farmacocinética , Piridinas/uso terapêutico , Proteína do Retinoblastoma/efeitos dos fármacos , Timidina Quinase/efeitos dos fármacos
2.
Antiviral Res ; 195: 105166, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34419483

RESUMO

Acyclovir (ACV) and penciclovir and their prodrugs are recommended for therapy or prophylaxis of Herpes simplex virus 1 (HSV-1) infections. Their administration, however, can lead to the emergence of resistant strains with altered viral thymidine kinase (TK) function, especially in immunocompromised patients. Furthermore, amino acid (aa) changes of the viral deoxyribonucleic acid polymerase (POL) may contribute to resistance to the aforementioned nucleoside analogues. Given this, treatment with foscarnet (FOS) or cidofovir (CDV) may represent an important alternative. Both drugs directly affect POL activity. Several aa changes of POL, such as L49I, E70K, L359I, E421V, P829S, T1121M, and M1226I, have been observed in ACV-resistant clinical strains which also carried relevant aa changes in their TK. Their contribution to ACV, FOS, and CDV resistance is not fully understood. In this study, these seven aa changes with unknown significance for ACV, FOS and CDV resistance were introduced separately into the POL of a recombinant HSV-1 strain rHSV-1(17+)Lox, equipped with or without information for expression of green fluorescent protein (GFP). The GFP-expressing variants were tested for susceptibility to ACV, FOS and CDV. An rHSV-1(17+)Lox GFP strain with the S724N change conferring resistance to ACV and FOS was generated and included as a control. Only the S724N change was confirmed to induce ACV and FOS resistance, whereas the other changes did not contribute to resistance. The underlying nucleotide substitutions of the POL gene should be therefore considered as natural polymorphism. These data will improve sequence-based prediction of antiviral susceptibility.


Assuntos
Antivirais/farmacologia , DNA Polimerase Dirigida por DNA/efeitos dos fármacos , Herpes Simples/tratamento farmacológico , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/genética , Aciclovir/farmacologia , Animais , Chlorocebus aethiops , Cidofovir/farmacologia , Farmacorresistência Viral/efeitos dos fármacos , Foscarnet/farmacologia , Guanina/farmacologia , Humanos , Hospedeiro Imunocomprometido , Testes de Sensibilidade Microbiana , Timidina Quinase/efeitos dos fármacos , Células Vero
3.
Mutagenesis ; 32(1): 193-202, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27658822

RESUMO

Nowadays engineered nanomaterials (ENMs) are increasingly used in a wide range of commercial products and biomedical applications. Despite this, the knowledge of human potential health risk as well as comprehensive biological and toxicological information is still limited. We have investigated the capacity of two frequently used metallic ENMs, nanosilver and magnetite nanoparticles (MNPs), to induce thymidine kinase (Tk +/-) mutations in L5178Y mouse lymphoma cells and transformed foci in Bhas 42 cells. Two types of nanosilver, spherical nanoparticles (AgNM300) and fibrous (AgNM302) nanorods/wires, and MNPs differing in surface modifications [MNPs coated with sodium oleate (SO-MNPs), MNPs coated with SO + polyethylene glycol (SO-PEG-MNPs) and MNPs coated with SO + PEG + poly(lactide-co-glycolic acid) SO-PEG-PLGA-MNPs] were included in this study. Spherical AgNM300 showed neither mutagenic nor carcinogenic potential. In contrast, silver nanorods/wires (AgNM302) increased significantly the number of both gene mutations and transformed foci compared with the control (untreated) cells. Under the same treatment conditions, neither SO-MNPs nor SO-PEG-PLGA-MNPs increased the mutant frequency compared with control cells though an equivocal mutagenic effect was estimated for SO-PEG-MNPs. Although SO-MNPs and SO-PEG-MNPs did not show any carcinogenic potential, SO-PEG-PLGA-MNPs increased concentration dependently the number of transformed foci in Bhas 42 cells compared with the control cells. Our results revealed that fibrous shape underlies the mutagenic and carcinogenic potential of nanosilver while surface chemistry affects the biosafety of MNPs. Considering that both nanosilver and MNPs are prospective ENMs for biomedical applications, further toxicological evaluations are warranted to assess comprehensively the biosafety of these nanomaterials.


Assuntos
Nanopartículas Metálicas/toxicidade , Mutação , Prata/toxicidade , Timidina Quinase/efeitos dos fármacos , Animais , Carcinógenos/farmacologia , Carcinógenos/toxicidade , Compostos Férricos/farmacologia , Compostos Férricos/toxicidade , Nanopartículas Metálicas/química , Camundongos , Testes de Mutagenicidade , Mutagênicos/farmacologia , Mutagênicos/toxicidade , Prata/farmacologia , Timidina Quinase/genética
4.
Appl Radiat Isot ; 106: 251-5, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26282567

RESUMO

In this report we describe studies with N5-2OH, a carboranyl thymidine analog (CTA), which is a substrate for thymidine kinase 1 (TK1), using the F98 rat glioma model. In vivo BNCT studies have demonstrated that intracerebral (i.c.) osmotic pump infusion of N5-2OH yielded survival data equivalent to those obtained with i.v. administration of boronophenylalanine (BPA). The combination of N5-2OH and BPA resulted in a modest increase in MST of F98 glioma bearing rats compared to a statistically significant increase with the RG2 glioma model, as has been previously reported by us (Barth et al., 2008). This had lead us to synthesize a second generation of CTAs that have improved in vitro enzyme kinetics and in vivo tumor uptake (Agarwal et al., 2015).


Assuntos
Terapia por Captura de Nêutron de Boro , Neoplasias Encefálicas/radioterapia , Timidina Quinase/efeitos dos fármacos , Timidina/administração & dosagem , Animais , Ratos , Timidina/análogos & derivados
5.
Hum Gene Ther ; 21(7): 843-54, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20132050

RESUMO

Human papillomavirus type 16 (HPV16) is associated with the development of anogenital cancers and their precursor lesions, intraepithelial neoplasia. Treatment strategies against HPV-induced intraepithelial neoplasia are not HPV specific and mostly consist of physical removal or ablation of lesions. We had previously designed an HPV-specific approach to kill HPV-infected cells by the herpes simplex virus type 1 thymidine kinase (TK) gene driven by HPV E2 binding to E2-binding sites (E2BS) in the native HPV16 long control region. E2-induced TK expression renders the cells sensitive to the prodrug ganciclovir. To optimize this therapeutic approach, we modified the native long control region by adding variable numbers of E2BS adjacent to E2BS4, resulting in greatly increased cell death in HPV-positive cell lines with variable levels of E2 protein expression and no reduction in HPV specificity. Our results showed maximum increase in TK expression and cell killing when one additional E2BS was added adjacent to E2BS. As HPV-infected patients also exhibit variable E2 expression across lesions and within a lesion, this approach may potentiate the clinical utility of the herpes simplex virus type 1 TK/ganciclovir therapeutic approach.


Assuntos
Proteínas de Ligação a DNA/genética , Terapia Genética/métodos , Herpesvirus Humano 1/efeitos dos fármacos , Proteínas Oncogênicas Virais/genética , Regiões Promotoras Genéticas , Timidina Quinase/efeitos dos fármacos , Timidina Quinase/metabolismo , Sequência de Bases , Sítios de Ligação , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/farmacologia , Regulação da Expressão Gênica , Herpesvirus Humano 1/genética , Humanos , Dados de Sequência Molecular , Proteínas Oncogênicas Virais/metabolismo , Proteínas Oncogênicas Virais/farmacologia , Plasmídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timidina Quinase/genética , Regulação para Cima
6.
Food Chem Toxicol ; 48(3): 831-8, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20035819

RESUMO

Rev7 is an indigestible gum polymer used for the manufacturing of chewing gum. It allows for the formulation of chewing gum with low adhesion; thus can be readily removed from surfaces such as sidewalks, clothing, carpets and furniture. In a toxicological safety assessment, Rev7 was found to be non-mutagenic in the AMES assay. The highest concentration tested in a mouse lymphoma thymidine kinase locus gene mutation assay induced a slight but biologically relevant increase in mutations under non-metabolic activation conditions after 24h. Because of this finding, a mouse micronucleus assay was performed, and the test article was found to be negative for inducing chromosomal damage. A 28-day repeated oral toxicity study resulted in a NOAEL of 80,000 ppm; the highest concentration tested. Rev7 was found to be free from contaminants such as heavy metals, monomers, and solvents. Lastly, Rev7 did not demonstrate skin-sensitizing properties in the murine local lymph node assay.


Assuntos
Goma de Mascar/toxicidade , Polímeros/uso terapêutico , Succinatos/uso terapêutico , Animais , Hidroxitolueno Butilado/análise , Eritrócitos/efeitos dos fármacos , Eritrócitos/ultraestrutura , Feminino , Ensaio Local de Linfonodo , Masculino , Metais Pesados/análise , Camundongos , Testes para Micronúcleos , Testes de Mutagenicidade , Ratos , Ratos Sprague-Dawley , Segurança , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Solventes/análise , Timidina Quinase/efeitos dos fármacos , Timidina Quinase/genética
7.
Transplant Proc ; 40(8): 2665-9, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18929831

RESUMO

OBJECTIVE: Our aim was to study whether herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV)-mediated lentiviral gene transfer diminished graft-versus-host disease (GVHD) in a mouse model of allogeneic bone marrow transplantation (BMT). MATERIALS AND METHODS: Donor splenic lymphocytes infected with lentiviral vectors carrying HSV-TK were mixed with donor bone marrow cells before cotransplantation into recipient mice irradiated with 60Co gamma-ray. GCV (25 mg/kg/d) was intraperitoneally administered beginning on days 0, 7, and 12 after transplantation for 7 days. The survival time, severity and incidence of GVHD, T-lymphocyte immune reconstitution, and percentage of allogeneic chimeras were observed after allo-BMT. RESULTS: The average survival times of mice in the TK/GCV 0 day, TK/GCV 7 day, and TK/GCV 12 day groups were 30.10 +/- 5.21, 36.40 +/- 5.28, and 28.20 +/- 4.82 days, respectively, all of which were longer than that of the control group (P < .05). The effect at 7 days was best: it was significantly different from the 0 and 12 day groups. The incidence of grade III to IV GVHD after allo-BMT in the control group was 100%, whereas there were pathological changes of grade II to III GVHD in the experimental groups. After allo-BMT, CD4+ and CD4+/CD8+ T lymphocytes in the experimental groups were all higher than in the control group (P < .05), but CD8+ T lymphocyte percentages were all lower than the latter. CONCLUSIONS: HSV-TK/GCV expression mediated by lentiviral transduction plays a role to prevent and treat GVHD after allo-BMT. The effect to control GVHD was most pronounced when GCV was administered on day 7 after transplantation.


Assuntos
Transplante de Medula Óssea/patologia , Ganciclovir/uso terapêutico , Doença Enxerto-Hospedeiro/prevenção & controle , Lentivirus/efeitos dos fármacos , Simplexvirus/enzimologia , Timidina Quinase/metabolismo , Animais , Antivirais/uso terapêutico , Transplante de Medula Óssea/imunologia , Raios gama , Sobrevivência de Enxerto/imunologia , Doença Enxerto-Hospedeiro/patologia , Lentivirus/fisiologia , Linfócitos/imunologia , Linfócitos/virologia , Camundongos , Simplexvirus/efeitos dos fármacos , Baço/imunologia , Baço/virologia , Timidina Quinase/efeitos dos fármacos , Transplante Homólogo
8.
Clin Cancer Res ; 14(11): 3416-26, 2008 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-18519772

RESUMO

PURPOSE: Inhibition of the protein kinase mammalian target of rapamycin (mTOR) is being evaluated for treatment of a variety of malignancies. However, the effects of mTOR inhibitors are cytostatic and standard size criteria do not reliably identify responding tumors. The aim of this study was to evaluate whether response to mTOR inhibition could be assessed by positron emission tomography (PET) imaging of tumor metabolism. EXPERIMENT DESIGN: Glucose, thymidine, and amino acid utilization of human glioma cell lines with varying degrees of sensitivity to mTOR inhibition were assessed by measuring in vitro uptake of [18F]fluorodeoxyglucose ([18F]FDG), [18F]fluorothymidine ([18F]FLT), and [3H]l-tyrosine before and after treatment with the mTOR inhibitor rapamycin. The tumor metabolic activity in vivo was monitored by small-animal PET of tumor-bearing mice. The mechanisms underlying changes in metabolic activity were analyzed by measuring expression and functional activity of enzymes and transporters involved in the uptake of the studied imaging probes. RESULTS: In sensitive cell lines, rapamycin decreased [18F]FDG and [18F]FLT uptake by up to 65% within 24 hours after the start of therapy. This was associated with inhibition of hexokinase and thymidine kinase 1. In contrast, [3H]l-tyrosine uptake was unaffected by rapamycin. The effects of rapamycin on glucose and thymidine metabolism could be imaged noninvasively by PET. In sensitive tumors, [18F]FDG and [18F]FLT uptake decreased within 48 hours by 56 +/- 6% and 52 +/- 8%, respectively, whereas there was no change in rapamycin-resistant tumors. CONCLUSIONS: These encouraging preclinical data warrant clinical trials evaluating [18F]FDG and [18F]FLT-PET for monitoring treatment with mTOR inhibitors in patients.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Glioblastoma/diagnóstico por imagem , Glioblastoma/metabolismo , Tomografia por Emissão de Pósitrons , Proteínas Quinases/efeitos dos fármacos , Sirolimo/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Radioisótopos de Flúor/farmacocinética , Fluordesoxiglucose F18/farmacocinética , Glucose/metabolismo , Humanos , Camundongos , Proteínas Quinases/metabolismo , Serina-Treonina Quinases TOR , Timidina/metabolismo , Timidina Quinase/efeitos dos fármacos , Timidina Quinase/metabolismo , Tirosina/efeitos dos fármacos , Tirosina/metabolismo
9.
Antiviral Res ; 78(3): 268-74, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18325606

RESUMO

Feline herpesvirus-1 (FHV-1) is the causative agent of a severe ocular disease in cats for which a safe potent antiviral chemotherapeutic agent is highly demanded. The sensitivity of FHV-1 to inhibition by three anti-herpetic nucleoside analogues [acyclovir (ACV), penciclovir (PCV) and cidofovir (CDV)] was tested by means of yield reduction assay. ACV showed very poor ability to inhibit FHV-1 replication. At low multiplicity of infection (MOI), both PCV and CDV were nearly equally effective with IC50 values ranging between 6 and 8 microg/ml. However, when the MOI was raised to 3PFU/cell, the activity of CDV was markedly reduced (IC50 25 microg/ml), while that of PCV remained relatively low (IC50 10 microg/ml). Although FHV-1 is normally insensitive to ACV, it exhibited >1000-fold increase in sensitivity when the thymidine kinase (TK) encoded by herpes simplex virus-1 (HSV-1) was supplied in trans. Furthermore, three PCV-resistant FHV-1 variants selected in vitro were shown to carry mutations in the TK gene. Taken together, these data provided direct evidence that PCV is a potent selective inhibitor of FHV-1 and that the virus-encoded TK is an important determinant of the virus susceptibility to nucleoside analogues.


Assuntos
Aciclovir/análogos & derivados , Antivirais/farmacologia , Herpesvirus Humano 1 , Timidina Quinase , Aciclovir/farmacologia , Animais , Gatos , Linhagem Celular , Farmacorresistência Viral/genética , Guanina , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Concentração Inibidora 50 , Rim/citologia , Rim/virologia , Testes de Sensibilidade Microbiana/métodos , Mutação , Timidina Quinase/efeitos dos fármacos , Timidina Quinase/genética , Timidina Quinase/metabolismo , Replicação Viral/efeitos dos fármacos
10.
Am J Pathol ; 170(3): 865-74, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17322372

RESUMO

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-gamma. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity.


Assuntos
Mitocôndrias/efeitos dos fármacos , Inibidores da Transcriptase Reversa/toxicidade , Timidina Quinase/efeitos dos fármacos , Timidina Quinase/metabolismo , Transgenes , Animais , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/metabolismo , Ecocardiografia , Humanos , Immunoblotting , Imuno-Histoquímica , Isoenzimas/efeitos dos fármacos , Isoenzimas/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Miocárdio/enzimologia , Peptídeos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Succinato Desidrogenase/efeitos dos fármacos , Regulação para Cima
11.
J Neurooncol ; 82(3): 239-48, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17102907

RESUMO

OBJECTIVE: To study on the antitumor effect of combining wt-p53 gene with suicide gene therapy (HSV-tk+GCV) for malignant gliomas. METHODS: AdCMV-p53 was transfected into C6 glioma cells at MOI of (Multiplicity of infection) 0(G100), 10(TPG1), 100(TPG2), then AdCMV-tk was transducted to C6 glioma cells of G100, TPG1 and TPG2, respectively, at MOI of 100. The C6 glioma cells tranfected with both AdCMV-p53 and AdCMV-tk were exposed to various concentration of GCV. The cell survival rate was measured by MTT assay in vitro. Rat glioma model was established by injecting 5 x 10(5) C6 glioma cells into right caudate nucleus of SD rats. AdCMV-p53 and AdCMV-tk were injected into glioma on day 5 and 6, respectively. On day 7, ganciclovir (GCV) was administrated intraperitoneally at 15 mg/kg/day for 14 days. The survival time of all rats was observed. The growth of intracerebral tumors was monitored dynamically by enhanced MRI. Cell apoptosis was evaluated by TUNEL method. Expression of HSV-tk gene was identified by in situ hybridization and expression of exogenous p53 gene was detected with Western blotting. RESULTS: In vitro, wt-p53 significantly enhanced antitumor effect of HSV-tk/GCV. The concentration of GCV for ID50 of TPG2 cells (0.001 microg/ml GCV) was 10 times lower than that for the cells of tk-GCV group (MOI = 100), while the concentration of GCV for ID100 of TPG2 (0.01 microg/ml GCV) and TPG1(0.1 microg/ml GCV) was 100 and 10 times lower than that for the cells of tk-GCV group (MOI = 100), respectively. Apoptosis of C6 glioma cells also could be induced by transfection with wt-p53 gene slightly. For in vivo study, the survival time of tumor-bearing rats treated with HSV-TK/GCV or wt-p53 combined with HSV-TK/GCV was significantly prolonged and the intracerebral tumors were regressed and disappeared earlier in the combined gene therapy group than those in the HSV-TK/GCV therapy group as shown in enhanced MRI. However, only half dose of GCV for the rats treated with both wt-p53 and HSV-TK/GCV was needed to obtain the same efficacy as those rats treated with HSV-TK/GCV alone. These results indicate that the transfection of wt-p53 potentiates the effect of HSV-TK/GCV therapy. CONCLUSIONS: The combination of HSV-tk/GCV system with wt-p53 gene transduction is optimal for clinical therapeutic trials of suicide gene therapy for malignant gliomas.


Assuntos
Genes Transgênicos Suicidas , Terapia Genética/métodos , Glioma/terapia , Neoplasias Experimentais/terapia , Timidina Quinase/genética , Proteína Supressora de Tumor p53/genética , Adenoviridae/genética , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Southern Blotting , Western Blotting , Linhagem Celular Tumoral , Ganciclovir/metabolismo , Ganciclovir/farmacologia , Vetores Genéticos , Humanos , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Ratos , Ratos Sprague-Dawley , Timidina Quinase/efeitos dos fármacos , Timidina Quinase/metabolismo , Transfecção
12.
J Med Chem ; 49(17): 5377-81, 2006 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-16913728

RESUMO

Synthesis of three novel 2'-deoxy-2'-[18F]fluoro-1-beta-D-arabinofuranosyluracil derivatives [18F]FPAU, [18F]FBrVAU, and [18F]FTMAU is reported. The compounds were synthesized by coupling of 1-bromo-2-deoxy-2-fluoro sugars with corresponding silylated uracil derivatives. In vitro cell uptake indicated that all three compounds are taken up selectively in RG2TK+ cells with negligible uptake in RG2 cells. The results indicate that [18F]FBrVAU and [18F]FTMAU have better uptake profiles in comparison to [18F]FPAU and have potential as PET probes for imaging HSV1-tk gene expression.


Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Herpesvirus Humano 1/enzimologia , Tomografia por Emissão de Pósitrons/métodos , Nucleosídeos de Pirimidina/síntese química , Nucleosídeos de Pirimidina/farmacocinética , Timidina Quinase/genética , Animais , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Radioisótopos de Flúor , Herpesvirus Humano 1/efeitos dos fármacos , Técnicas In Vitro , Estrutura Molecular , Nucleosídeos de Pirimidina/química , Ratos , Estereoisomerismo , Timidina Quinase/efeitos dos fármacos , Fatores de Tempo
14.
Liver Int ; 26(3): 352-61, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16584399

RESUMO

UNLABELLED: The effect of serotonin receptor 2 blockade (5-HT(2)) on liver regeneration after 30-34% and 60-70% partial hepatectomy in the rat liver was investigated. MATERIALS AND METHODS: Male Wistar rats were subjected to 60-70% (group I) and 30-34% (group II) partial hepatectomy. Serotonin receptor 2 blockade was exerted by intraperitoneal administration of ketanserin at different doses and time points after partial hepatectomy. The rats of all groups were killed at different time points until 96 h after partial hepatectomy. The rate of liver regeneration was evaluated by the mitotic index in hematoxylin and eosin sections, the immunochemical detection of Ki67 and proliferating cell nuclear antigens, the rate of [(3)H]-thymidine incorporation into hepatic DNA and liver thymidine kinase enzymatic activity. RESULTS: Liver regeneration peaked at 24 and 32 h after partial hepatectomy in 60-70% hepatectomized rats. In 30-34% hepatectomized rats liver regeneration peaked at 60 h, whereas low rates of regenerative activity were observed between 24 and 72 h after partial hepatectomy. Ketanserin administration arrested liver regeneration only when administered at 16 h after 60-70% partial hepatectomy. Ketanserin also abrogated the observed peak of regenerative activity at 60 h in 30-34% hepatectomized rats when administered at 52 h after partial hepatectomy. All indices of liver regeneration were affected by ketanserin administration. CONCLUSIONS: Serotonin receptor 2 blockade can arrest liver regeneration only when administered close to G1/S transition point, and that while serotonin may be a cofactor for DNA synthesis, it does not play a role in initiation of liver regeneration.


Assuntos
Hepatectomia/métodos , Ketanserina/farmacologia , Regeneração Hepática/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/fisiopatologia , Receptores 5-HT2 de Serotonina/efeitos dos fármacos , Antagonistas da Serotonina/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , DNA/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ketanserina/administração & dosagem , Antígeno Ki-67/efeitos dos fármacos , Antígeno Ki-67/metabolismo , Fígado/metabolismo , Masculino , Índice Mitótico , Antígeno Nuclear de Célula em Proliferação/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos Wistar , Antagonistas da Serotonina/administração & dosagem , Timidina Quinase/efeitos dos fármacos , Timidina Quinase/metabolismo , Fatores de Tempo
15.
In Vivo ; 19(4): 695-704, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15999536

RESUMO

BACKGROUND: The mechanism of cadmium-induced liver regeneration arrest in relation to hepatic stimulator substance (HSS) biological activity was investigated. MATERIALS AND METHODS: In Wistar rats subjected to 65 - 70% partial hepatectomy, saline, cadmium, cadmium and HSS were administered. The rats were also subjected to 30 - 34% partial hepatectomy. Mitotic index, immunochemistry for PCNA, 3[H]-thymidine incorporation into DNA and thymidine kinase activity were used as indices of liver regeneration. HSS biological activity was evaluated in all groups of rats using a bioassay. RESULTS: Liver regeneration and HSS activity were arrested by cadmium during the first 24 h after partial hepatectomy. Both in normal and in cadmium-treated rats, the HSS activity was increased and liver regeneration coincided. HSS activity was stable in 30 - 34% hepatectomized rats. HSS administration was able to restore liver regeneration arrest induced by cadmium. CONCLUSION: The biological activity of HSS increased at the time of G1/S transition of hepatocytes in the cell cycle and no increase was observed with asynchronous G1/S transition (30 - 34% partial hepatectomy). The suppression of HSS biological activity by cadmium seems to represent an important factor for liver regeneration arrest induced by the metal and HSS administration is able to restore liver regeneration.


Assuntos
Cádmio/toxicidade , Substâncias de Crescimento/metabolismo , Regeneração Hepática/efeitos dos fármacos , Mitógenos/metabolismo , Peptídeos/metabolismo , Animais , DNA/biossíntese , DNA/efeitos dos fármacos , Substâncias de Crescimento/farmacologia , Hepatectomia , Imuno-Histoquímica , Injeções Intraperitoneais , Peptídeos e Proteínas de Sinalização Intercelular , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/patologia , Masculino , Mitógenos/farmacologia , Índice Mitótico , Peptídeos/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos Wistar , Timidina Quinase/efeitos dos fármacos , Timidina Quinase/metabolismo
16.
Arch Toxicol ; 79(8): 466-74, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15995853

RESUMO

Acetaminophen-induced toxicity has been attributed to cytochrome P-450-generated metabolites, which covalently modify target proteins. However, the mechanism of liver injury pathogenesis needs to be further elucidated. Platelet-activating factor (PAF) is one of the mediators involved in inflammatory tissue alterations associated with acute liver failure. In this study, alterations in blood PAF levels and the serum activity of PAF-acetylhydrolase (PAF-AH) were investigated over the time course of liver injury and regeneration induced by acetaminophen treatment in rats. The administration of a toxic dose of acetaminophen (3.5 g/kg) in rats caused acute hepatic injury, as evident by alterations of biochemical (serum enzymes: ALT, AST and ALP) and liver histopathological (degree of inflammation and apoptosis) indices between 20 and 40 h post-treatment. The hepatic damage was followed by liver regeneration, made evident by three independent indices ([3H]thymidine incorporation into hepatic DNA, liver thymidine kinase activity and hepatocyte mitotic index), presenting a peak at 72 h. The PAF levels were elevated at 24 and 28 h, presenting a remarkable peak at 32 h post-treatment. PAF-AH activity presented different kinetics to that of PAF. The enzyme activity was relatively low at all time points examined before the rise in PAF activity, peaking later, at 72, 84 and 96 h. Our data demonstrate that PAF is involved in the pathogenesis of acute liver failure and in augmented compensatory liver tissue repair post-acetaminophen treatment. However, the putative role of PAF during liver toxicity and regeneration remains to be established.


Assuntos
Acetaminofen/toxicidade , Analgésicos não Narcóticos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Regeneração Hepática/efeitos dos fármacos , Fator de Ativação de Plaquetas/metabolismo , Acetiltransferases/sangue , Animais , Apoptose/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , DNA/biossíntese , DNA/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Hepatopatias/metabolismo , Hepatopatias/patologia , Masculino , Índice Mitótico , Ratos , Ratos Wistar , Timidina Quinase/efeitos dos fármacos , Timidina Quinase/metabolismo , Fatores de Tempo
17.
Cell Res ; 14(4): 295-302, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15353126

RESUMO

Ionizing radiation is one of the most effective tools in cancer therapy. In a previous study, we reported that protein tyrosine kinase (PTK) inhibitors modulate the radiation responses in the human chronic myelogenous leukemia (CML) cell line K562. The receptor tyrosine kinase inhibitor, genistein, delayed radiation-induced cell death, while non-recepter tyrosine kinase inhibitor, herbimycin A (HMA) enhances radiation-induced apoptosis. In this study, we focused on the modulation of radiation-induced cell death by genistein and performed PCR-select suppression subtractive hybridization (SSH) to understand its molecular mechanism. We identified human thymidine kinase 1 (TK1), which is cell cycle regulatory gene and confirmed expression of TK1 mRNA by Northern blot analysis. Expression of TK1 mRNA and TK1 enzymatic activity were parallel in their increase and decrease. TK1 is involved in G1-S phase transition of cell cycle progression. In cell cycle analysis, we showed that radiation induced G2 arrest in K562 cells but it was not able to sustain. However, the addition of genistein to irradiated cells sustained a prolonged G2 arrest up to 120 h. In addition, the expression of cell cycle-related proteins, cyclin A and cyclin B1, provided the evidences of G1/S progression and G2-arrest, and their relationship with TK1 in cells treated with radiation and genistein. These results suggest that the activation of TK1 may be critical to modulate the radiation-induced cell death and cell cycle progression in irradiated K562 cells.


Assuntos
Morte Celular/fisiologia , Genes cdc/fisiologia , Genisteína/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Timidina Quinase/metabolismo , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Ciclina A/metabolismo , Ciclina B/metabolismo , Ciclina B1 , Ativação Enzimática , Fase G1/efeitos dos fármacos , Fase G1/genética , Fase G1/efeitos da radiação , Fase G2/efeitos dos fármacos , Fase G2/genética , Fase G2/efeitos da radiação , Genes cdc/efeitos dos fármacos , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/radioterapia , Hibridização de Ácido Nucleico/métodos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , RNA Mensageiro/efeitos da radiação , Fase S/efeitos dos fármacos , Fase S/genética , Fase S/efeitos da radiação , Timidina Quinase/efeitos dos fármacos , Timidina Quinase/genética , Fatores de Tempo
18.
AIDS Res Hum Retroviruses ; 20(7): 701-3, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15307913

RESUMO

It has been demonstrated that prolonged in vivo or in vitro treatment with some nucleosides analogs may favor the selection of cells with a reduced activity of enzymes involved in the phosphorylation of these drugs leading to a reduced sensitivity to their antiretroviral action. The aim of this study was to evaluate the effect, in vivo, of zidovudine and stavudine treatment on thymidine kinase (TK) activity. The results obtained showed that TK levels in PBMC from naive patients and stavudine-treated patients did not significantly differ (naive TK = 4.16 +/- 1.19 U/mg protein; stavudine TK = 3.65 +/- 1.73 U/mg protein; p = 0.42), suggesting that the treatment with this nucleoside analog is not associated to a defect of TK activity. On the contrary, PBMC from zidovudine-treated patients showed a significant reduction in TK activity compared to naive patients (naive TK = 4.16 +/- 1.19 U/mg protein; zidovudine TK = 2.70 +/- 1.54; p = 0.014. Although the clinical significance of these results has to be established, we can speculate that stavudine and zidovudine, which are presumably phosphorylated by the same cellular kinases, might display a different ability to in vivo select cells with a resistant phenotype.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Estavudina/uso terapêutico , Timidina Quinase/sangue , Infecções por HIV/sangue , Infecções por HIV/enzimologia , Humanos , Leucócitos Mononucleares/enzimologia , Timidina Quinase/efeitos dos fármacos , Zidovudina/uso terapêutico
19.
Nutr Cancer ; 46(1): 66-72, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12925306

RESUMO

Inositol hexaphosphate (IP6) or phytic acid, contained in most mammalian cells, has been shown to have anticancer and anti-cell-proliferative effects in several experimental models of carcinogenesis. We investigated the effect of topical application of IP6 on 7,12-dimethylbenzanthracene (DMBA)-induced complete carcinogenesis and on selective critical events of proliferation, differentiation, or apoptosis after DMBA exposure. IP6 inhibited skin tumor development significantly in a dose-dependent manner. IP6 induced the DMBA-inhibited transglutaminase activity. DNA synthesis, as determined by [3H]thymidine incorporation, was suppressed by IP6 in a dose-dependent manner. IP6 also inhibited thymidine kinase enzyme, which is responsible for [3H]thymidine incorporation into DNA. Our results show that topical application of IP6 inhibits DMBA-induced mouse skin tumor development and that IP6 exerts its tumor inhibitory effect probably by modulating proliferation, differentiation, or apoptosis. It seems that IP6 is an effective and potential chemopreventive agent for management of skin tumorigenesis.


Assuntos
9,10-Dimetil-1,2-benzantraceno/administração & dosagem , Ácido Fítico/uso terapêutico , Neoplasias Cutâneas/prevenção & controle , Animais , Testes de Carcinogenicidade , Divisão Celular/efeitos dos fármacos , DNA/biossíntese , DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Epiderme/enzimologia , Epiderme/patologia , Feminino , Camundongos , Ácido Fítico/farmacologia , Neoplasias Cutâneas/induzido quimicamente , Timidina Quinase/efeitos dos fármacos , Transglutaminases/efeitos dos fármacos
20.
Mol Pharmacol ; 64(2): 289-97, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12869633

RESUMO

Selective killing of tumor cells is an important goal for cancer therapeutics. The tumor suppressor transcription factor p53 is absent or mutated in more than 50% of human tumors. Thus, determining approaches that use p53 status to regulate therapy may be an important strategy for attaining cancer selectivity. We have shown previously that a designed transcriptional repressor, K2-5F, strongly and selectively reduces the expression of its target gene MDR1. In this study, we exploited p53 status and the strong repressor activity of K2-5F to establish a system for preferential killing of p53-negative cells. In this system, the expression of K2-5F is induced by p53 in normal cells, and the K2-5F repressor then inhibits the expression of herpes simplex virus thymidine kinase (HSV-TK) driven by an MDR1 minipromoter. In p53-deficient cells, little K2-5F is expressed, and thus HSV-TK is expressed, allowing the cells to be killed by ganciclovir (GCV). K2-5F induced by exogenous p53 dramatically reduced the expression of HSV-TK in human embryonic kidney 293 cells, and it subsequently increased cell survival in response to GCV. To further evaluate this approach in a uniform genetic background, we developed Saos-2 cells stably expressing physiological levels of p53 and paired them with wild-type p53-negative Saos-2 cells. Stable expression of moderate levels of p53 in Saos-2 cells was able to induce the expression of K2-5F and reduce HSV-TK expression and resulted in a modest but distinct protection from GCV toxicity. Thus, this system may be suitable for further development as an approach to selective cancer therapy.


Assuntos
Apoptose/fisiologia , Ganciclovir/farmacologia , Pró-Fármacos/farmacologia , Timidina Quinase/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Antivirais/metabolismo , Antivirais/farmacologia , Ganciclovir/metabolismo , Inativação Gênica , Humanos , Pró-Fármacos/metabolismo , Proteínas Repressoras/metabolismo , Simplexvirus/enzimologia , Timidina Quinase/efeitos dos fármacos , Timidina Quinase/genética , Células Tumorais Cultivadas
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