RESUMO
The capacity of imatinib mesylate to reverse established pulmonary arterial hypertension (PAH) has been attributed to a reduction in pulmonary arterial muscularization via inhibition of platelet-derived growth factor receptor-ß on vascular smooth muscle cells. However, there is also a significant immunomodulatory component to the action of imatinib that may account for its efficacy in PAH. We found that monocrotaline-induced pulmonary hypertension was associated with a significant decrease in pulmonary natural killer (NK) cells and T lymphocytes and the accumulation of macrophages in the lungs of F344 rats. The prevention of pulmonary hypertension by imatinib blocked these changes in pulmonary leukocyte content and induced elevations in pulmonary interferon-γ, tumor necrosis factor α, and IL-10, corresponding to the enhanced activity of splenic NK cells ex vivo. Treatment with anti-asialo GM1 antiserum (ASGM1), which ablated circulating NK cells and depleted T cells, eliminated the therapeutic benefit of imatinib. ASGM1-treated animals also exhibited significant pulmonary arteriolar muscularization in response to monocrotaline challenge compared with immunocompetent controls despite daily imatinib administration to both groups. In the athymic rat, imatinib decreased right ventricular hypertrophy and pulmonary arteriolar muscularization in monocrotaline-challenged animals versus saline-treated controls but did not prevent pulmonary macrophage accumulation or the development of pulmonary hypertension. These data demonstrate that the immunomodulatory effects of imatinib are critical to its therapeutic action in experimental PAH.
Assuntos
Benzamidas/uso terapêutico , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/patologia , Linfócitos/metabolismo , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Citocinas/metabolismo , Modelos Animais de Doenças , Hipertensão Pulmonar/induzido quimicamente , Hipertrofia Ventricular Direita/patologia , Mesilato de Imatinib , Imunomodulação/efeitos dos fármacos , Contagem de Leucócitos , Depleção Linfocítica , Linfócitos/efeitos dos fármacos , Masculino , Monocrotalina , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Piperazinas/farmacologia , Pirimidinas/farmacologia , Ratos , Ratos Endogâmicos F344 , Ratos NusRESUMO
A defective response to DNA damage is observed in several human autosomal recessive ataxias with oculomotor apraxia, including ataxia-telangiectasia. We report that senataxin, defective in ataxia oculomotor apraxia (AOA) type 2, is a nuclear protein involved in the DNA damage response. AOA2 cells are sensitive to H2O2, camptothecin, and mitomycin C, but not to ionizing radiation, and sensitivity was rescued with full-length SETX cDNA. AOA2 cells exhibited constitutive oxidative DNA damage and enhanced chromosomal instability in response to H2O2. Rejoining of H2O2-induced DNA double-strand breaks (DSBs) was significantly reduced in AOA2 cells compared to controls, and there was no evidence for a defect in DNA single-strand break repair. This defect in DSB repair was corrected by full-length SETX cDNA. These results provide evidence that an additional member of the autosomal recessive AOA is also characterized by a defective response to DNA damage, which may contribute to the neurodegeneration seen in this syndrome.
Assuntos
Dano ao DNA , Estresse Oxidativo , RNA Helicases/fisiologia , Apraxias/etiologia , Apraxias/patologia , Ataxia/etiologia , Ataxia/patologia , Células Cultivadas , Quebras de DNA de Cadeia Dupla , DNA Helicases , Reparo do DNA , Humanos , Peróxido de Hidrogênio/farmacologia , Enzimas MultifuncionaisRESUMO
Using a chemical genetics screen, we have identified ent-15-oxokaurenoic acid (EKA) as a chemical that causes prolonged mitotic arrest at a stage resembling prometaphase. EKA inhibits the association of the mitotic motor protein centromeric protein E with kinetochores and inhibits chromosome movement. Unlike most antimitotic agents, EKA does not inhibit the polymerization or depolymerization of tubulin. To identify EKA-interacting proteins, we used a cell-permeable biotinylated form that retains biological activity to isolate binding proteins from living cells. Mass spectrometric analysis identified six EKA-binding proteins, including Ran-binding protein 2, a kinetochore protein whose depletion by small interfering RNA causes a similar mitotic arrest phenotype.
Assuntos
Cromossomos/ultraestrutura , Diterpenos do Tipo Caurano/química , Diterpenos do Tipo Caurano/farmacologia , Cinetocoros/metabolismo , Mitose , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Biotinilação , Linhagem Celular Tumoral , Química/métodos , Células HeLa , Humanos , Imageamento Tridimensional , Espectrometria de Massas , Polímeros/química , Ligação Proteica , Fuso Acromático , Tubulina (Proteína)/químicaRESUMO
A phenotypic cell-based assay for inhibitors of the G (2) DNA damage checkpoint was used to screen plant extracts from the US National Cancer Institute Natural Products Repository. It revealed activity in a methanol extract from the common ragweed Ambrosia artemisiifolia. Assay-guided fractionation led to the identification of the sesquiterpene lactones psilostachyins A and C as novel checkpoint inhibitors. Elimination of their alpha,beta-unsaturated carbonyl group caused a loss of activity, suggesting that the compounds can bind covalently to target proteins through Michael addition. Psilostachyins A and C also blocked cells in mitosis and caused the formation of aberrant microtubule spindles. However, the compounds did not interfere with microtubule polymerization in vitro. The related sesquiterpene lactones psilostachyin B, paulitin and isopaulitin were also isolated from the same extract but showed no checkpoint inhibition. The identification of the target(s) of psilostachyins A and C may provide further insight into the signalling pathways involved in cell cycle arrest and mitotic progression.