RESUMEN
Imperfect conservation of human pre-mRNA splice sites is necessary to produce alternative isoforms. This flexibility is combined with the precision of the message reading frame. Apart from intron-termini GU_AG and the branchpoint A, the most conserved are the exon-end guanine and +5G of the intron start. Association between these guanines cannot be explained solely by base-pairing with U1 snRNA in the early spliceosome complex. U6 succeeds U1 and pairs +5G in the pre-catalytic spliceosome, while U5 binds the exon end. Current U5 snRNA reconstructions by CryoEM cannot explain the conservation of the exon-end G. Conversely, human mutation analyses show that guanines of both exon termini can suppress splicing mutations. Our U5 hypothesis explains the mechanism of splicing precision and the role of these conserved guanines in the pre-catalytic spliceosome. We propose: (1) optimal binding register for human exons and U5-the exon junction positioned at U5Loop1 C39|C38; (2) common mechanism for base-pairing of human U5 snRNA with diverse exons and bacterial Ll.LtrB intron with new loci in retrotransposition-guided by base pair geometry; and (3) U5 plays a significant role in specific exon recognition in the pre-catalytic spliceosome. Statistical analyses showed increased U5 Watson-Crick pairs with the 5'exon in the absence of +5G at the intron start. In 5'exon positions -3 and -5, this effect is specific to U5 snRNA rather than U1 snRNA of the early spliceosome. Increased U5 Watson-Crick pairs with 3'exon position +1 coincide with substitutions of the conserved -3C at the intron 3'end. Based on mutation and X-ray evidence, we propose that -3C pairs with U2 G31 juxtaposing the branchpoint and the 3'intron end. The intron-termini pair, formed in the pre-catalytic spliceosome to be ready for transition after branching, and the early involvement of the 3'intron end ensure that the 3'exon contacts U5 in the pre-catalytic complex. We suggest that splicing precision is safeguarded cooperatively by U5, U6, and U2 snRNAs that stabilize the pre-catalytic complex by Watson-Crick base pairing. In addition, our new U5 model explains the splicing effect of exon-start +1G mutations: U5 Watson-Crick pairs with exon +2C/+3G strongly promote exon inclusion. We discuss potential applications for snRNA therapeutics and gene repair by reverse splicing.
RESUMEN
Type II topoisomerase enzymes are essential for resolving DNA topology problems arising through various aspects of DNA metabolism. In vertebrates two isoforms are present, one of which (TOP2A) accumulates on chromatin during mitosis. Moreover, TOP2A targets the mitotic centromere during prophase, persisting there until anaphase onset. It is the catalytically-dispensable C-terminal domain of TOP2 that is crucial in determining this isoform-specific behaviour. In this study we show that, in addition to the recently identified chromatin tether domain, several other features of the alpha-C-Terminal Domain (CTD). influence the mitotic localisation of TOP2A. Lysine 1240 is a major SUMOylation target in cycling human cells and the efficiency of this modification appears to be influenced by T1244 and S1247 phosphorylation. Replacement of K1240 by arginine results in fewer cells displaying centromeric TOP2A accumulation during prometaphase-metaphase. The same phenotype is displayed by cells expressing TOP2A in which either of the mitotic phosphorylation sites S1213 or S1247 has been substituted by alanine. Conversely, constitutive modification of TOP2A by fusion to SUMO2 exerts the opposite effect. FRAP analysis of protein mobility indicates that post-translational modification of TOP2A can influence the enzyme's residence time on mitotic chromatin, as well as its subcellular localisation.
Asunto(s)
Anafase/fisiología , Cromatina/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Metafase/fisiología , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Línea Celular , Centrómero/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Humanos , Fosforilación/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación/fisiologíaRESUMEN
Topoisomerase (topo) IIα and IIß maintain genome stability and are targets for anti-tumor drugs. In this study, we demonstrate that the decatenation checkpoint is regulated, not only by topo IIα, as previously reported, but also by topo IIß. The decatenation checkpoint is most efficient when both isoforms are present. Regulation of this checkpoint and sensitivity to topo II-targeted drugs is influenced by the C-terminal domain (CTD) of the topo II isoforms and by a conserved non-catalytic tyrosine, Y640 in topo IIα and Y656 in topo IIß. Deletion of most of the CTD of topo IIα, while preserving the nuclear localization signal (NLS), enhances the decatenation checkpoint and sensitivity to topo II-targeted drugs. In contrast, deletion of most of the CTD of topo IIß, while preserving the NLS, and mutation of Y640 in topo IIα and Y656 in topo IIß inhibits these activities. Structural studies suggest that the differential impact of the CTD on topo IIα and topo IIß function may be due to differences in CTD charge distribution and differential alignment of the CTD with reference to transport DNA. Together these results suggest that topo IIα and topo IIß cooperate to maintain genome stability, which may be distinctly modulated by their CTDs.
Asunto(s)
Antígenos de Neoplasias/química , Puntos de Control del Ciclo Celular/fisiología , Inestabilidad Cromosómica/fisiología , ADN-Topoisomerasas de Tipo II/química , Proteínas de Unión al ADN/química , Secuencia de Aminoácidos , Animales , Antígenos de Neoplasias/efectos de los fármacos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/fisiología , Línea Celular , Daño del ADN , ADN-Topoisomerasas de Tipo II/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/fisiología , ADN Complementario/genética , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Resistencia a Antineoplásicos , Fibroblastos , Células HL-60 , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Dominios Proteicos , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
We demonstrate the usefulness of synthetic lethal screening of a conditionally BCL6-deficient Burkitt lymphoma cell line, DG75-AB7, with a library of small molecules to determine survival pathways suppressed by BCL6 and suggest mechanism-based treatments for lymphoma. Lestaurtinib, a JAK2 inhibitor and one of the hits from the screen, repressed survival of BCL6-deficient cells in vitro and reduced growth and proliferation of xenografts in vivo BCL6 deficiency in DG75-AB7 induced JAK2 mRNA and protein expression and STAT3 phosphorylation. Surface IL10RA was elevated by BCL6 deficiency, and blockade of IL10RA repressed STAT3 phosphorylation. Therefore, we define an IL10RA/JAK2/STAT3 pathway each component of which is repressed by BCL6. We also show for the first time that JAK2 is a direct BCL6 target gene; BCL6 bound to the JAK2 promoter in vitro and was enriched by ChIP-seq. The place of JAK2 inhibitors in the treatment of diffuse large B-cell lymphoma has not been defined; we suggest that JAK2 inhibitors might be most effective in poor prognosis ABC-DLBCL, which shows higher levels of IL10RA, JAK2, and STAT3 but lower levels of BCL6 than GC-DLBCL and might be usefully combined with novel approaches such as inhibition of IL10RA.
Asunto(s)
Linfoma de Burkitt/tratamiento farmacológico , Carbazoles/farmacología , Subunidad alfa del Receptor de Interleucina-10/metabolismo , Janus Quinasa 2/antagonistas & inhibidores , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-6/biosíntesis , Factor de Transcripción STAT3/metabolismo , Animales , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Línea Celular Tumoral , Furanos , Humanos , Subunidad alfa del Receptor de Interleucina-10/genética , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Ratones , Ratones SCID , Proteínas Proto-Oncogénicas c-bcl-6/genética , Factor de Transcripción STAT3/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
DNA double-strand breaks (DSBs) are toxic lesions, which if improperly repaired can result in cell death or genomic instability. DSB repair is usually facilitated by the classical non-homologous end joining (C-NHEJ), or homologous recombination (HR) pathways. However, a mutagenic alternative NHEJ pathway, microhomology-mediated end joining (MMEJ), can also be deployed. While MMEJ is suppressed by C-NHEJ, the relationship between HR and MMEJ is less clear. Here, we describe a role for HR genes in suppressing MMEJ in human cells. By monitoring DSB mis-repair using a sensitive HPRT assay, we found that depletion of HR proteins, including BRCA2, BRCA1 or RPA, resulted in a distinct mutational signature associated with significant increases in break-induced mutation frequencies, deletion lengths and the annealing of short regions of microhomology (2-6 bp) across the break-site. This signature was dependent on CtIP, MRE11, POLQ and PARP, and thus indicative of MMEJ. In contrast to CtIP or MRE11, depletion of BRCA1 resulted in increased partial resection and MMEJ, thus revealing a functional distinction between these early acting HR factors. Together these findings indicate that HR factors suppress mutagenic MMEJ following DSB resection.
Asunto(s)
Proteína BRCA1/genética , Proteína BRCA2/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , ADN/metabolismo , Reparación del ADN por Recombinación , Proteína de Replicación A/genética , Proteína BRCA1/antagonistas & inhibidores , Proteína BRCA1/metabolismo , Proteína BRCA2/antagonistas & inhibidores , Proteína BRCA2/metabolismo , Secuencia de Bases , Bioensayo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Endodesoxirribonucleasas , Células Epiteliales/citología , Células Epiteliales/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Proteína Homóloga de MRE11 , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteína de Replicación A/antagonistas & inhibidores , Proteína de Replicación A/metabolismo , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , ADN Polimerasa thetaRESUMEN
Understanding the mechanisms of chromosomal double-strand break repair (DSBR) provides insight into genome instability, oncogenesis and genome engineering, including disease gene correction. Research into DSBR exploits rare-cutting endonucleases to cleave exogenous reporter constructs integrated into the genome. Multiple reporter constructs have been developed to detect various DSBR pathways. Here, using a single endogenous reporter gene, the X-chromosomal disease gene encoding hypoxanthine phosphoribosyltransferase (HPRT), we monitor the relative utilization of three DSBR pathways following cleavage by I-SceI or CRISPR/Cas9 nucleases. For I-SceI, our estimated frequencies of accurate or mutagenic non-homologous end-joining and gene correction by homologous recombination are 4.1, 1.5 and 0.16%, respectively. Unexpectedly, I-SceI and Cas9 induced markedly different DSBR profiles. Also, using an I-SceI-sensitive HPRT minigene, we show that gene correction is more efficient when using long double-stranded DNA than single- or double-stranded oligonucleotides. Finally, using both endogenous HPRT and exogenous reporters, we validate novel cell cycle phase-specific I-SceI derivatives for investigating cell cycle variations in DSBR. The results obtained using these novel approaches provide new insights into template design for gene correction and the relationships between multiple DSBR pathways at a single endogenous disease gene.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Endonucleasas/metabolismo , Hipoxantina Fosforribosiltransferasa/genética , Animales , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Ciclo Celular , Línea Celular Tumoral , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Genes Reporteros , Células HeLa , Humanos , Ratones , Mutagénesis , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The correction of disease-causing mutations by single-strand oligonucleotide-templated DNA repair (ssOR) is an attractive approach to gene therapy, but major improvements in ssOR efficiency and consistency are needed. The mechanism of ssOR is poorly understood but may involve annealing of oligonucleotides to transiently exposed single-stranded regions in the target duplex. In bacteria and yeast it has been shown that ssOR is promoted by expression of Redß, a single-strand DNA annealing protein from bacteriophage lambda. Here we show that Redß expression is well tolerated in a human cell line where it consistently promotes ssOR. By use of short interfering RNA, we also show that ssOR is stimulated by the transient depletion of the endogenous DNA mismatch repair protein MSH2. Furthermore, we find that the effects of Redß expression and MSH2 depletion on ssOR can be combined with a degree of cooperativity. These results suggest that oligonucleotide annealing and mismatch recognition are distinct but interdependent events in ssOR that can be usefully modulated in gene correction strategies.
Asunto(s)
ADN de Cadena Simple/metabolismo , Proteína 2 Homóloga a MutS/antagonistas & inhibidores , Reparación del Gen Blanco/métodos , Proteínas Virales/metabolismo , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Línea Celular , Reparación del ADN , Humanos , Oligonucleótidos/química , Oligonucleótidos/genética , Transfección , Proteínas Virales/genéticaRESUMEN
Modulating chromatin through histone methylation orchestrates numerous cellular processes. SETD2-dependent trimethylation of histone H3K36 is associated with active transcription. Here, we define a role for H3K36 trimethylation in homologous recombination (HR) repair in human cells. We find that depleting SETD2 generates a mutation signature resembling RAD51 depletion at I-SceI-induced DNA double-strand break (DSB) sites, with significantly increased deletions arising through microhomology-mediated end-joining. We establish a presynaptic role for SETD2 methyltransferase in HR, where it facilitates the recruitment of C-terminal binding protein interacting protein (CtIP) and promotes DSB resection, allowing Replication Protein A (RPA) and RAD51 binding to DNA damage sites. Furthermore, reducing H3K36me3 levels by overexpressing KDM4A/JMJD2A, an oncogene and H3K36me3/2 demethylase, or an H3.3K36M transgene also reduces HR repair events. We propose that error-free HR repair within H3K36me3-decorated transcriptionally active genomic regions promotes cell homeostasis. Moreover, these findings provide insights as to why oncogenic mutations cluster within the H3K36me3 axis.
Asunto(s)
Inestabilidad Genómica , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Reparación del ADN por Recombinación , Reparación del ADN , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Recombinación Homóloga , Humanos , Metilación , Unión Proteica , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , TransfecciónRESUMEN
BACKGROUND: Mutations in the ß-globin gene (HBB) cause haemoglobinopathies where current treatments have serious limitations. Gene correction by homologous recombination (HR) is an attractive approach to gene therapy for such diseases and is stimulated by gene-specific endonucleases, including zinc finger nucleases (ZFNs). Customised nucleases targeting HBB have previously been shown to promote HR-mediated HBB modification in 0.360% of drug-selected cells, although frequencies among unselected cells, more relevant to the goal of correcting HBB in primary stem cells, have not been reported. METHODS: ZFNs targeting HBB were tested for HBB binding (two-hybrid assay) or HBB cleavage followed by inaccurate end joining (surveyor assay)in bacteria or human cancer cell lines, respectively. ZFN-stimulated HR was measured in cell lines by a modified fluorescence-based reporter assay or by targeted insertion of a drug-resistance marker into endogenous HBB confirmed by Southern analyses. RESULTS: Although the ZFNs that we assembled in-house showed limited potential, a commercially commissioned nuclease (ZFN4) enhanced HR mediated HBB modification in up to 95% of drug-selected cells. Among unselected cells, however, this frequency was less than 0.2%. Furthermore, ZFN4 cleaved HBB at an efficiency of 12% (surveyor assay) and enhanced the HR reporter assay 20-fold less efficiently than a control endonuclease. CONCLUSIONS: With ZFN4, we achieved higher efficiencies of HR-mediated HBB modification than previously reported for drug-selected cells. Our measurements of ZFN4-induced HR in unselected cells, however, suggest that improved nucleases must be developed if therapeutic HBB correction is to be achievable in primary stem cells.
Asunto(s)
Endodesoxirribonucleasas/genética , Factores de Transcripción/genética , Dedos de Zinc/genética , Globinas beta/genética , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/terapia , Línea Celular Tumoral , Reparación del ADN por Unión de Extremidades , Endodesoxirribonucleasas/biosíntesis , Endodesoxirribonucleasas/metabolismo , Marcación de Gen , Terapia Genética , Recombinación Homóloga , Humanos , Mutación , Factores de Transcripción/biosíntesis , Factores de Transcripción/metabolismo , Talasemia beta/genética , Talasemia beta/terapiaRESUMEN
As proliferating cells transit from interphase into M-phase, chromatin undergoes extensive reorganization, and topoisomerase (topo) IIα, the major isoform of this enzyme present in cycling vertebrate cells, plays a key role in this process. In this study, a human cell line conditional null mutant for topo IIα and a derivative expressing an auxin-inducible degron (AID)-tagged version of the protein have been used to distinguish real mitotic chromosome functions of topo IIα from its more general role in DNA metabolism and to investigate whether topo IIß makes any contribution to mitotic chromosome formation. We show that topo IIß does contribute, with endogenous levels being sufficient for the initial stages of axial shortening. However, a significant effect of topo IIα depletion, seen with or without the co-depletion of topo IIß, is the failure of chromosomes to hypercompact when delayed in M-phase. This requires much higher levels of topo II protein and is impaired by drugs or mutations that affect enzyme activity. A prolonged delay at the G2/M border results in hyperefficient axial shortening, a process that is topo IIα-dependent. Rapid depletion of topo IIα has allowed us to show that its function during late G2 and M-phase is truly required for shaping mitotic chromosomes.
Asunto(s)
Antígenos de Neoplasias/fisiología , Cromosomas Humanos , ADN-Topoisomerasas de Tipo II/fisiología , Proteínas de Unión al ADN/fisiología , Mitosis/genética , Antígenos de Neoplasias/genética , Dominio Catalítico , Línea Celular , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Fase G2/genética , Humanos , Mutación , Isoformas de Proteínas/antagonistas & inhibidores , Quinolinas/farmacología , Tiazoles/farmacologíaRESUMEN
The EGF receptor (EGFR) is therapeutically targeted by antibodies and small molecules in solid tumors including lung, colorectal, and breast cancer. However, chemotherapy remains important, and efforts to improve efficacy through combination with targeted agents is challenging. This study examined the effects of short and long durations of exposure to the EGFR- and HER2-targeted tyrosine kinase inhibitors (TKI) gefitinib and lapatinib, on induction of cell death and DNA damage by topoisomerase IIα (Topo IIα) poisons, in the SK-Br-3 HER2-amplified breast cancer cell line. Short exposure to either gefitinib or lapatinib for 1 hour did not affect the induction of apoptosis by the Topo IIα poisons doxorubicin, etoposide, and m-AMSA. In contrast, cells treated for 48 hours were resistant to all three drugs. Short exposure (1 hour) to TKI did not alter the number of DNA single- or double-strand breaks (DSB) induced, whereas longer exposure (48 hours) reduced the number of DNA DSBs and the formation of γ-H2AX foci. Both gefitinib and lapatinib reduced the expression and activity of Topo IIα at 48 hours. Studies using a cell line with inducible downregulation of Topo IIα showed that expression of Topo IIα, and not Topo IIß, determined the number of DNA strand breaks induced by these chemotherapeutic agents. These results indicate that prolonged exposure to TKIs targeting EGFR and HER2 induce resistance to doxorubicin, etoposide, and m-AMSA through downregulation of Topo IIα. This may explain why their addition to chemotherapy regimens have not increased efficacy.
Asunto(s)
Antígenos de Neoplasias/genética , Antineoplásicos/farmacología , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/genética , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Amsacrina/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Roturas del ADN de Doble Cadena/efectos de los fármacos , Doxorrubicina/farmacología , Etopósido/farmacología , Gefitinib , Histonas/genética , Humanos , LapatinibRESUMEN
Type II DNA topoisomerases catalyse DNA double-strand cleavage, passage and re-ligation to effect topological changes. There is considerable interest in elucidating topoisomerase II roles, particularly as these proteins are targets for anti-cancer drugs. Here we uncover a role for topoisomerase IIα in RNA polymerase I-directed ribosomal RNA gene transcription, which drives cell growth and proliferation and is upregulated in cancer cells. Our data suggest that topoisomerase IIα is a component of the initiation-competent RNA polymerase Iß complex and interacts directly with RNA polymerase I-associated transcription factor RRN3, which targets the polymerase to promoter-bound SL1 in pre-initiation complex formation. In cells, activation of rDNA transcription is reduced by inhibition or depletion of topoisomerase II, and this is accompanied by reduced transient double-strand DNA cleavage in the rDNA-promoter region and reduced pre-initiation complex formation. We propose that topoisomerase IIα functions in RNA polymerase I transcription to produce topological changes at the rDNA promoter that facilitate efficient de novo pre-initiation complex formation.
Asunto(s)
Antígenos de Neoplasias/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , ARN Polimerasa I/genética , Transcripción Genética , ADN/metabolismo , Activación Enzimática , Hidrólisis , Regiones Promotoras GenéticasRESUMEN
Cyclin-dependent kinase 1 (Cdk1) controls cell proliferation and is inhibited by promising anticancer agents, but its mode of action and the consequences of its inhibition are incompletely understood. Cdk1 promotes S- and M-phases during the cell-cycle but also suppresses endoreduplication, which is associated with polyploidy and genome instability. The complexity of Cdk1 regulation has made it difficult to determine whether these different roles require different thresholds of kinase activity and whether the surge of activity as inhibitory phosphates are removed at mitotic onset is essential for cell proliferation. Here, we have used chemical genetics in a human cell line to address these issues. We rescued cells lethally depleted of endogenous Cdk1 with an exogenous Cdk1 conferring sensitivity to one ATP analogue inhibitor (1NMPP1) and resistance to another (RO3306). At no 1NMPP1 concentration was mitosis in rescued clones prevented without also inducing endoreduplication, suggesting that these two key roles for Cdk1 are not simply controlled by different Cdk1 activity thresholds. We also rescued RO3306-resistant clones using exogenous Cdk1 without inhibitory phosphorylation sites, indicating that the mitotic surge of Cdk1 activity is dispensable for cell proliferation. These results suggest that the basic mammalian cycle requires at least some qualitative changes in Cdk1 activity and that quantitative increases in activity need not be rapid. Furthermore, the viability of cells that are unable to undergo rapid Cdk1 activation, and the strong association between endoreduplication and impaired proliferation, may place restrictions on the therapeutic use of a Cdk1 inhibitors.
Asunto(s)
Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Proteínas Quinasas/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Proteína Quinasa CDC2/antagonistas & inhibidores , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , División Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fase G2 , Humanos , Mitosis/efectos de los fármacos , Proteínas Quinasas/genética , Pirazoles/farmacología , Pirimidinas/farmacología , Xenopus , Proteínas de Xenopus/genéticaRESUMEN
Topoisomerase II alpha (TOP2A) has a crucial role in proper chromosome condensation and segregation. Here we report the interaction of TOP2A with ataxia telangiectasia mutated (ATM) and its phosphorylation in an ATM-dependent manner after DNA damage. In vitro kinase assay and site-directed mutagenesis studies revealed that serine 1512 is the target of phosphorylation through ATM. Serine 1512 to Alanine mutation of TOP2A showed increased stability of the protein, retaining TOP2A activity at least with regard to cell survival activity. Ataxia telangiectasia-derived cell lines showed high levels of TOP2A that were associated with hypersensitivity to the TOP2 inhibitor etoposide. These findings suggest that ATM-dependent TOP2A modification is required for proper regulation of TOP2 stability and subsequently of the sensitivity to TOP2 inhibitor. In a lymphoblastoid cell line derived from a patient who developed MLL rearrangement, positive infant leukemia, defective ATM expression, and increased TOP2A expression were shown. It was intriguing that hypersensitivity to TOP2 inhibitor and susceptibility to MLL gene rearrangement were shown by low-dose etoposide exposure in this cell line. Thus, our findings have clinically important implications for the pathogenesis of infantile acute leukemia as well as treatment-associated secondary leukemia following exposure to TOP2 inhibitors.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Inhibidores de Topoisomerasa II/farmacología , Alanina/genética , Alanina/metabolismo , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/genética , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Daño del ADN , ADN-Topoisomerasas de Tipo II/biosíntesis , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Etopósido/farmacología , N-Metiltransferasa de Histona-Lisina , Humanos , Leucemia/tratamiento farmacológico , Leucemia/genética , Leucemia/metabolismo , Mutagénesis Sitio-Dirigida/métodos , Proteína de la Leucemia Mieloide-Linfoide/biosíntesis , Proteína de la Leucemia Mieloide-Linfoide/genética , Fosforilación/efectos de los fármacos , Proteínas de Unión a Poli-ADP-Ribosa , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Serina/genética , Serina/metabolismo , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BCL6 is essential for normal antibody responses and is highly expressed in germinal centre B-cells. Constitutive expression due to chromosomal translocations or mutations of cis-acting regulatory elements contributes to diffuse large B-cell lymphoma. BCL6 expression is therefore tightly regulated in a lineage- and developmental-stage-specific manner, and disruption of normal controls can contribute to lymphomagenesis. In order to discover potential cis-acting control regions we carried out DNase I-hypersensitive site mapping. Gel-shift assays and chromatin immunoprecipitation of the core region of a hypersensitive site 4.4 kb upstream of BCL6 transcription initiation (HSS-4.4) showed an E-box element-binding ZEB1 (zinc finger E-boxbinding homeobox 1) and the co-repressor CtBP (C-terminal binding protein). As compared with peripheral blood B-cells, ZEB1, a two-handed zinc finger transcriptional repressor, is expressed at relatively low levels in germinal centre cells, whereas BCL6 has the opposite pattern of expression. Transfection of ZEB1 cDNA caused a reduction in BCL6 expression and a mutated ZEB1, incapable of binding CtBP, lacked this effect. siRNA (small interfering RNA)-mediated knockdown of ZEB1 or CtBP produced an increase in BCL6 mRNA. We propose that HSS-4.4 is a distal promoter element binding a repressive complex consisting of ZEB1 and CtBP. CtBP is ubiquitously expressed and the results of the present study suggest that regulation of ZEB1 is required for control of BCL6 expression.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Oxidorreductasas de Alcohol/genética , Secuencia de Bases , Línea Celular , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Proteínas de Homeodominio/genética , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Unión Proteica/genética , Proteínas Proto-Oncogénicas c-bcl-6 , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Sitio de Iniciación de la Transcripción , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
Eukaryotic type II topoisomerases (Topo II) are implicated in a wide range of cellular processes. Cells in which Topo II protein has been specifically depleted or mutated provide powerful systems for analysing the normal in vivo functions of Topo II proteins and for assessing their roles in various chemotherapy regimens. Summarised here are the ways in which Topo II has been depleted or mutated in animal cells and the type of information gleaned. The cell lines generated are tabulated and represent a useful resource for further in vivo studies of Topo II function, one that we expect to grow in size and utility in the coming years.
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ADN-Topoisomerasas de Tipo II , Marcación de Gen/métodos , Mutación , Animales , Línea Celular , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Ratones Noqueados , Mutagénesis , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Inhibidores de Topoisomerasa IIRESUMEN
Coadministration of the iron chelator dexrazoxane reduces by 80% the incidence of heart failure in cancer patients treated with anthracyclines. The clinical application of dexrazoxane is limited, however, because its ability to inhibit topoisomerase IIα (TOP2A) is feared to adversely affect anthracycline chemotherapy, which involves TOP2A-mediated generation of DNA double-strand breaks (DSB). Here, we investigated the apoptotic effects of dexrazoxane and the anthracycline doxorubicin, alone and in combination, in a tumor cell line with conditionally regulated expression of TOP2A. Each drug caused apoptosis that was only partly dependent on TOP2A. Unexpectedly, dexrazoxane was found to cause TOP2A depletion, thereby reducing the doxorubicin-induced accumulation of DSB. Despite this latter effect, dexrazoxane showed no adverse effect on doxorubicin-induced apoptosis. This could be explained by the TOP2A-independent apoptotic effects of each drug: those of doxorubicin included TOP2A-independent DSB formation and depletion of intracellular glutathione, whereas those of dexrazoxane were caspase independent. In conclusion, both doxorubicin and dexrazoxane induce apoptosis via TOP2A-dependent and TOP2A-independent mechanisms, the latter compensating for the reduction in cell killing due to dexrazoxane-induced TOP2A depletion. These observations suggest an explanation for the absence of adverse dexrazoxane effects on clinical responses to doxorubicin.
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Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Doxorrubicina/farmacología , Razoxano/farmacología , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glutatión/análisis , Histonas/metabolismo , Humanos , Proteínas de Unión a Poli-ADP-Ribosa , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Chromosomal correction of dystrophin gene mutations is a most desirable therapeutic solution for Duchenne muscular dystrophy, as it allows production of the full-length dystrophin under the control of locus-specific promoters. Here we explored gene targeting in conditionally immortal mouse dystrophin-deficient myoblasts. We constructed an adenoviral vector for the correction of the mdx mutation, containing 6.0 kb of sequence homologous to the target locus (partial intron 21 through to exon 24 with the normal sequence of exon 23) and a neomycin expression cassette inserted in intron 23. Adenovirus-based gene targeting was previously reported to be beneficial in mouse embryonic stem cells, resulting in one targeted integration per three integration events. However, we found no targeted integration events among 144 stably transduced G418-resistant myoblast clones, reflecting efficient random integration of the adenoviral vector in myogenic cells. We found that mouse myoblasts are capable of integrating recombinant adenoviral DNA with an efficiency approaching 1%. Interestingly, dermal fibroblasts integrate adenoviral DNA up to 100 times less efficiently than myoblasts from the same mice. We also show that the efficiency of recombinant adenoviral DNA integration is influenced by preinfection cell density, possibly indicating the importance of cellular DNA replication for adenoviral integration.
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Adenoviridae , Distrofina , Marcación de Gen , Terapia Genética , Vectores Genéticos , Distrofia Muscular de Duchenne/terapia , Integración Viral , Animales , Replicación del ADN , Dermis , Células Madre Embrionarias/metabolismo , Fibroblastos , Humanos , Ratones , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Mutación , Mioblastos , Sitios de Carácter Cuantitativo/genéticaRESUMEN
SUMOylation is essential for cell-cycle regulation in invertebrates; however, its functions during the mammalian cell cycle are largely uncharacterized. Mammals express three SUMO paralogs: SUMO-1, SUMO-2, and SUMO-3 (SUMO-2 and SUMO-3 are 96% identical and referred to as SUMO-2/3). We found that SUMO-2/3 localize to centromeres and condensed chromosomes, whereas SUMO-1 localizes to the mitotic spindle and spindle midzone, indicating that SUMO paralogs regulate distinct mitotic processes in mammalian cells. Consistent with this, global inhibition of SUMOylation caused a prometaphase arrest due to defects in targeting the microtubule motor protein CENP-E to kinetochores. CENP-E was found to be modified specifically by SUMO-2/3 and to possess SUMO-2/3 polymeric chain-binding activity essential for kinetochore localization. Our findings indicate that SUMOylation is a key regulator of the mammalian cell cycle, with SUMO-1 and SUMO-2/3 modification of different proteins regulating distinct processes.
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Ciclo Celular/fisiología , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cinetocoros/metabolismo , Mitosis/fisiología , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Cisteína Endopeptidasas/metabolismo , ADN-Topoisomerasas/metabolismo , Genes Reporteros , Células HeLa , Humanos , Cinética , Metafase , Unión ProteicaRESUMEN
PURPOSE: SN 28049 (N-[2-(dimethylamino)ethyl]-2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b]-1,6-naphthyridine-4-carboxamide) is a DNA intercalating drug that binds selectively to GC-rich DNA and shows curative activity against the Colon 38 adenocarcinoma in mice. We wished to investigate the roles of topoisomerase (topo) I, topo II and RNA transcription in the action of SN 28049. METHODS: We used clonogenic assays to study the cytotoxicity of SN 28049; RNA interference and enzyme assays to examine the role of topo I in SN 28049 action; 3H uridine incorporation and reporter assays to study its effects on transcription; and RT-PCR to examine its ability to reduce endogenous h-TERT expression. RESULTS: In clonogenic assays, SN 28049 showed a biphasic cytotoxic dose response curve in H460 cells typical of acridine derivatives such as N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) although it was approximately 16-fold more potent. Down-regulation of topo IIalpha in HTETOP cells reduced the cytotoxicity of SN 28049, establishing its action as a topo IIalpha poison. Surprisingly, down-regulation of topo I in H460 cells by RNA interference sensitised them to the actions of SN 28049 and other topo II poisons. SN 28049 also inhibited topo I-mediated relaxation of supercoiled plasmid DNA. SN 28049 was also an inhibitor of transcription in HEK293 cells and was more potent at reducing luciferase expression from a GC-rich SP-1 binding promoter than from a non-GC-rich AP-1 binding promoter. The drug also reduced luciferase reporter gene expression driven by the SP-1-binding survivin promoter as well as reducing endogenous h-TERT expression in HEK293 cells whose promoter also contains SP-1 binding sites. CONCLUSION: We conclude that SN 28049 has a complex action that may involve poisoning of topo IIalpha, suppression of topo I and inhibition of gene transcription from promoters with SP-1 sites. These actions may contribute to the promising experimental solid tumour anticancer activity of SN 28049.