RESUMO
The protein methyltransferase Set7/9 was recently shown to regulate p53 activity in cancer cells. However, the impact of Set7/9 on p53 function in vivo is unclear. To explore these issues, we created a null allele of Set7/9 in mice. Cells from Set7/9 mutant mice fail to methylate p53 K369, are unable to induce p53 downstream targets upon DNA damage, and are predisposed to oncogenic transformation. Importantly, we find that methylation of p53 by Set7/9 is required for the binding of the acetyltransferase Tip60 to p53 and for the subsequent acetylation of p53. We provide the first genetic evidence demonstrating that lysine methylation of p53 by Set7/9 is important for p53 activation in vivo and suggest a mechanistic link between methylation and acetylation of p53 through Tip60.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Proteínas Metiltransferases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Acetiltransferases/metabolismo , Animais , Ciclo Celular/fisiologia , Linhagem Celular , Linhagem Celular Transformada , Transformação Celular Viral , Dano ao DNA/genética , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Dosagem de Genes , Glutationa Transferase/metabolismo , Células HCT116 , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/genética , Humanos , Rim/citologia , Lisina/metabolismo , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Proteínas Metiltransferases/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
The role of P-glycoprotein (P-gp) on the distribution of the benzodiazepine triazolam (TRZ) and the azole antifungal agent ketoconazole (KET), and on the TRZ-KET interaction, was studied using mdr1a(-) or mdr1a/b(-/-) mice (P-gp-deficient mice) and matched controls. TRZ and KET also were studied in Caco-2 cells in Transwell culture. After single i.p. injections of TRZ or KET in separate groups of control mice, brain concentrations of TRZ exceeded those in serum [brain/serum area under the concentration curve (AUC) ratio, 5.0], whereas brain/serum AUC ratios for KET were approximately 0.5. On the basis of single time points, brain concentrations of TRZ, or brain/serum ratios, were similar in P-gp-deficient animals compared with controls, whereas P-gp-deficient animals had significantly higher KET brain concentrations and brain/serum ratios. Coadministration of KET with TRZ increased TRZ concentrations in serum, liver, and brain, both in controls and in P-gp-deficient animals, probably attributable to impairment by KET of CYP3A-mediated clearance of TRZ. However, KET did not increase brain/serum ratios of TRZ in either group. In Caco-2 cells, basal-to-apical flux of TRZ was higher than apical-to-basal flux. However, verapamil (100 microM) did not alter flux in either direction. KET inhibited basal-to-apical transport of rho-damine-123, with a 50% inhibitory concentration of 2.7 microM. Thus, TRZ does not appear to undergo measurable blood-brain barrier efflux transport by P-gp in this animal model. KET impairs clearance of TRZ but does not increase tissue uptake. However, KET itself may be a substrate for efflux transport at the blood-brain barrier.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/deficiência , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Cetoconazol/farmacocinética , Triazolam/farmacocinética , Animais , Células CACO-2 , Relação Dose-Resposta a Droga , Interações Medicamentosas , Humanos , Masculino , Camundongos , Camundongos KnockoutRESUMO
Antiretroviral agents may participate in drug interactions that influence the efficacy and toxicity of other antiretrovirals, as well as pharmacologic treatments of coincident or complicating diseases. The viral protease inhibitor, ritonavir, may cause drug interactions by inhibiting the activity of cytochrome P450-3A (CYP3A) isoforms. In a single-dose, blinded, four-way crossover study, 10 healthy volunteer subjects received 50 mg of trazodone hydrochloride or matching placebo concurrent with low-dose ritonavir (four doses of 200 mg each) or with placebo. Compared to the control condition, ritonavir significantly reduced apparent oral clearance of trazodone (155 +/- 23 vs. 75 +/- 12 ml/min, p < 0.001), prolonged elimination half-life (6.7 +/- 0.7 vs. 14.9 +/- 3.9 h, p < 0.05), and increased peak plasma concentrations (842 +/- 64 vs. 1125 +/- 111 ng/ml, p < 0.05) (mean +/- SE). Coadministration of trazodone with ritonavir increased sedation, fatigue, and performance impairment compared to trazodone plus placebo; differences reached significance only for the digitsymbol substitution test. Three subjects experienced nausea, dizziness, or hypotension when trazodone was given with ritonavir; 1 of these subjects also experienced syncope. Thus short-term low-dose administration of ritonavir impairs oral clearance of trazodone and increases the occurrence of adverse reactions. The findings are consistent with impairment of CYP3A-mediated trazodone metabolism by ritonavir.
Assuntos
Inibidores da Protease de HIV/administração & dosagem , Inibidores da Protease de HIV/farmacologia , Ritonavir/administração & dosagem , Ritonavir/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacocinética , Trazodona/farmacocinética , Administração Oral , Adulto , Área Sob a Curva , Cromatografia Líquida de Alta Pressão , Estudos Cross-Over , Interações Medicamentosas , Quimioterapia Combinada , Feminino , Meia-Vida , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores Seletivos de Recaptação de Serotonina/efeitos adversos , Inibidores Seletivos de Recaptação de Serotonina/sangue , Fatores de Tempo , Trazodona/efeitos adversos , Trazodona/sangueRESUMO
The effect of microsomal protein concentration on the inhibitory potency of a series of CYP3A inhibitors was assessed in vitro using diazepam 3-hydroxylation (yielding temazepam) as an index of CYP3A activity. With diazepam concentrations fixed at 100 micro M, inhibition of temazepam formation by fixed concentrations of ritonavir, ketoconazole, itraconazole, OH-itraconazole, norfluoxetine, and fluvoxamine decreased substantially as active protein concentrations increased from 0.0625 to 3.0 mg/ml. However protein concentration had only a small effect on the inhibitory activity of fluconazole. Equilibrium dialysis indicated extensive microsomal binding of all inhibitors except fluconazole; binding increased with higher protein concentrations. Based on the CYP3A content of liver microsomes, decrements in inhibitory potency of stronger inhibitors (ketoconazole and ritonavir) could be explained by specific binding, whereas nonspecific binding is anticipated to account for the effect on weaker inhibitors (norfluoxetine and fluvoxamine). Thus, microsomal binding (specific, nonspecific, or a combination of both) may have a major effect on estimation of inhibitory potency of p450 inhibitors and may contribute to variations among laboratories. The effect can be minimized by use of the lowest possible microsomal protein concentration for in vitro studies of metabolic inhibition.
Assuntos
Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Microssomos Hepáticos/enzimologia , Oxirredutases N-Desmetilantes/antagonistas & inibidores , Oxirredutases N-Desmetilantes/metabolismo , Citocromo P-450 CYP3A , Relação Dose-Resposta a Droga , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Proteínas/química , Proteínas/metabolismoRESUMO
Interactions of midazolam and ketoconazole were studied in vivo and in vitro in rats. Ketoconazole (total dose of 15 mg/kg intraperitoneally) reduced clearance of intravenous midazolam (5 mg/kg) from 79 to 55 ml/min/kg (p < 0.05) and clearance of intragastric midazolam (15 mg/kg) from 1051 to 237 ml/min/kg (p < 0.05), increasing absolute bioavailability from 0.11 to 0.36 (p < 0.05). Presystemic extraction occurred mainly across the liver as opposed to the gastrointestinal tract mucosa. Midazolam increased electroencephalographic (EEG) amplitude in the beta-frequency range. Ketoconazole shifted the concentration-EEG effect relationship rightward (increase in EC(50)), probably because ketoconazole is a neutral benzodiazepine receptor ligand. Ketoconazole competitively inhibited midazolam hydroxylation by rat liver and intestinal microsomes in vitro, with nanomolar K(i) values. At a total serum ketoconazole of 2 microg/ml (3.76 microM) in vivo, the predicted reduction in clearance of intragastric midazolam by ketoconazole (to 6% of control) was slightly greater than the observed reduction in vivo (to 15% of control). However, unbound serum ketoconazole greatly underpredicted the observed clearance reduction. Although the in vitro and in vivo characteristics of midazolam in rats incompletely parallel those in humans, the experimental model can be used to assess aspects of drug interactions having potential clinical importance.