RESUMO
We recently completed the total synthesis of spiruchostatin A, a depsipeptide natural product with close structural similarities to FK228, a histone deacetylase (HDAC) inhibitor (HDI) currently being evaluated in clinical trials for cancer. Here we report a detailed characterisation of the in vitro activity of spiruchostatin A. Spiruchostatin A was a potent (sub-nM) inhibitor of class I HDAC activity in vitro and acted as a prodrug, requiring reduction for activity. Spiruchostatin A was a potent (low nM) inhibitor of the growth of various cancer cell lines. Spiruchostatin A-induced acetylation of specific lysine residues within histones H3 and H4, and increased the expression of p21(cip1/waf1), but did not induce acetylation of alpha-tubulin. Spiruchostatin A also induced cell cycle arrest, differentiation and cell death in MCF7 breast cancer cells. Like FK228, spiruchostatin A was both an inducer and substrate of the ABCB1 drug efflux pump. Whereas spiruchostatin A and FK228-induced protracted histone acetylation, hydroxamate HDI-induced short-lived histone acetylation. Using a subset of HDI-target genes identified by microarray analysis, we demonstrated that these differences in kinetics of histone acetylation between HDI correlated with differences in the kinetics of induction or repression of specific target genes. Our results demonstrate that spiruchostatin A is a potent inhibitor of class I HDACs and anti-cancer agent. Differences in the kinetics of action of HDI may be important for the clinical application of these compounds.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Inibidores de Histona Desacetilases , Peptídeos Cíclicos/farmacologia , Acetilação , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Morte Celular , Diferenciação Celular , Linhagem Celular Tumoral , Depsipeptídeos/farmacologia , Depsipeptídeos/uso terapêutico , Inibidores Enzimáticos , Feminino , Humanos , Peptídeos Cíclicos/uso terapêutico , Pró-FármacosRESUMO
We measured the frequency of insertions in the Mcl-1 promoter in chronic lymphocytic leukemia (CLL) and in normal individuals. Insertions were found in 37/54 (69%) of the CLL samples. However, insertions were not associated with prognostic markers and were also detected in 38/66 (58%) of normal controls and in normal cells isolated from CLL patients. Thus, Mcl-1 insertions are not acquired during leukemogenesis and are unlikely to play an important role in this disease.
Assuntos
Elementos de DNA Transponíveis/genética , Leucemia Linfocítica Crônica de Células B/genética , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína de Sequência 1 de Leucemia de Células MieloidesRESUMO
Translational control is a key step in eukaryotic gene expression. The majority of translational control occurs at the level of initiation, thus implicating the 5' untranslated region as a major site of translational regulation. Many growth-related mRNAs have atypical 5' UTRs, which are often long and GC-rich. Such features promote formation of stable secondary structure, and many mRNAs encoding proteins involved in cell growth, proliferation and apoptosis have structured 5' UTRs, which in many cases harbour internal ribosome entry sites (IRESs) and upstream open-reading frames (uORFs). In this review we discuss how secondary structural elements in the 5' UTR can regulate translation and how mutations that perturb these secondary structural elements can have implications for disease and tumourigenesis.
Assuntos
Regiões 5' não Traduzidas/fisiologia , Doenças Genéticas Inatas/genética , Biossíntese de Proteínas , Regiões 5' não Traduzidas/química , Conformação de Ácido Nucleico , RNA/química , RNA/fisiologiaRESUMO
We have shown previously that an internal ribosome entry segment (IRES) directs the synthesis of the p36 isoform of Bag-1 and that polypyrimidine tract binding protein 1 (PTB-1) and poly(rC) binding protein 1 (PCBP1) stimulate IRES-mediated translation initiation in vitro and in vivo. Here, a secondary structural model of the Bag-1 IRES has been derived by using chemical and enzymatic probing data as constraints on the RNA folding algorithm Mfold. The ribosome entry window has been identified within this structural model and is located in a region in which many residues are involved in base-pairing interactions. The interactions of PTB-1 and PCBP1 with their cognate binding sites on the IRES disrupt many of the RNA-RNA interactions, and this creates a largely unstructured region of approximately 40 nucleotides that could permit ribosome binding. Mutational analysis of the PTB-1 and PCBP1 binding sites suggests that PCBP1 acts as an RNA chaperone to open the RNA in the vicinity of the ribosome entry window while PTB-1 is probably an essential part of the preinitiation complex.
Assuntos
Proteínas de Transporte/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Ligação a DNA , Células HeLa , Humanos , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Proteína de Ligação a Regiões Ricas em Polipirimidinas/análogos & derivados , Ligação Proteica , RNA Mensageiro/química , Proteínas de Ligação a RNA , Fatores de TranscriçãoRESUMO
The 5'-untranslated region of Bag-1 mRNA contains an internal ribosome entry segment (IRES) and the translation of Bag-1 protein can be initiated by both cap-dependent and cap-independent mechanisms. In general, cellular IRESs require non-canonical trans-acting factors for their activity, however, very few of the proteins that act on cellular IRESs have been identified. Proteins that interact with viral IRESs have also been shown to stimulate the activity of cellular IRESs and therefore the ability of a range of known viral trans-acting factors to stimulate the Bag-1 IRES was tested. Two proteins, poly r(C) binding protein 1 (PCBP1) and polypyrimidine tract binding protein (PTB), were found to increase the activity of the Bag-1 IRES in vitro and in vivo. The regions of the Bag-1 IRES RNA to which they bind have been determined, and it was shown that PCBP1 binds to a short 66 nt section of RNA, whilst PTB interacts with a number of sites over a larger area. The minimum section of the RNA that still retained activity was determined and both PCBP1 and PTB interacted with this region suggesting that these proteins are essential for Bag-1 IRES function.