RESUMO
(1) Background: A premature termination codon (PTC) can be induced by a type of point mutation known as a nonsense mutation, which occurs within the coding region. Approximately 3.8% of human cancer patients have nonsense mutations of p53. However, the non-aminoglycoside drug PTC124 has shown potential to promote PTC readthrough and rescue full-length proteins. The COSMIC database contains 201 types of p53 nonsense mutations in cancers. We built a simple and affordable method to create different nonsense mutation clones of p53 for the study of the PTC readthrough activity of PTC124. (2) Methods: A modified inverse PCR-based site-directed mutagenesis method was used to clone the four nonsense mutations of p53, including W91X, S94X, R306X, and R342X. Each clone was transfected into p53 null H1299 cells and then treated with 50 µM of PTC124. (3) Results: PTC124 induced p53 re-expression in H1299-R306X and H1299-R342X clones but not in H1299-W91X and H1299-S94X clones. (4) Conclusions: Our data showed that PTC124 more effectively rescued the C-terminal of p53 nonsense mutations than the N-terminal of p53 nonsense mutations. We introduced a fast and low-cost site-directed mutagenesis method to clone the different nonsense mutations of p53 for drug screening.
RESUMO
The fate of proteins is determined by the addition of various forms of polyubiquitin during ubiquitin-mediated proteasomal degradation. Cylindromatosis (CYLD), a K63-specific deubiquitinase, is enriched in postsynaptic density fractions of the rodent central nervous system (CNS), but the synaptic role of CYLD in the CNS is poorly understand. Here we show that CYLD deficiency (Cyld-/-) results in reduced intrinsic hippocampal neuronal firing, a decrease in the frequency of spontaneous excitatory postsynaptic currents and a decrease in the amplitude of field excitatory postsynaptic potentials. Moreover, Cyld-/- hippocampus shows downregulated levels of presynaptic vesicular glutamate transporter 1 (vGlut1) and upregulated levels of postsynaptic GluA1, a subunit of the AMPA receptor, together with an altered paired-pulse ratio (PPR). We also found increased activation of astrocytes and microglia in the hippocampus of Cyld-/- mice. The present study suggests a critical role for CYLD in mediating hippocampal neuronal and synaptic activity.
Assuntos
Hipocampo , Transmissão Sináptica , Camundongos , Animais , Hipocampo/fisiologia , Transmissão Sináptica/fisiologia , Neurônios , Potenciais Pós-Sinápticos Excitadores/fisiologia , Plasticidade Neuronal , Enzima Desubiquitinante CYLDRESUMO
The development of targeted tyrosine kinase inhibitors (TKIs) has succeeded in altering the course of chronic myeloid leukemia (CML). However, a number of patients have failed to respond or experienced disease relapse following TKI treatment. Proviral integration site for moloney murine leukemia virus1 (PIM1) is a serine/threonine kinase that participates in regulating apoptosis, cell cycle, signal transduction and transcriptional pathways, which are associated with tumor progression, and poor prognosis. SMI4a is a selective PIM1 kinase inhibitor that inhibits PIM1 kinase activity in vivo and in vitro. The present study aimed to explore the mechanism underlying the antitumor effect of SMI4a in K562 and imatinibresistant K562 (K562/G) cell lines. It was demonstrated that SMI4a inhibited the proliferation of K562 and K562/G cells using a WST8 assay. The Annexin Vpropidium iodide assay demonstrated that SMI4a induced apoptosis of K562 and K562/G cells in a dose, and timedependent manner. Furthermore, Hoechst 33342 staining was used to verify the apoptosis rate. The clone formation assay revealed that SMI4a significantly inhibited the colony formation capacity of K562 and K562/G cells. Western blot analysis demonstrated that SMI4a decreased phosphorylated (p)Ser9glycogen synthase kinase (GSK) 3ß/pGSK3ß and inhibited the translocation of ßcatenin. In addition, the downstream gene expression of apoptosis regulator Bax and poly(ADPribose) polymerase1 was upregulated, and apoptosis regulator Bcl2 and Myc protooncogene protein expression levels were downregulated. Immunofluorescence results demonstrated changes in the expression level of ßcatenin in the plasma and nucleus. The results of the present study suggest that SMI4a is an effective drug to use in combination with current chemotherapeutics for the treatment of imatinib-resistant CML.