RESUMO
The development of resistance and the activation of bypass pathway signalling represents a major problem for the clinical application of protein kinase inhibitors. While investigating the effect of either a c-Rel deletion or RelAT505A phosphosite knockin on the Eµ-Myc mouse model of B-cell lymphoma, we discovered that both NF-κB subunit mutations resulted in CHK1 inhibitor resistance, arising from either loss or alteration of CHK1 activity, respectively. However, since Eµ-Myc lymphomas depend on CHK1 activity to cope with high levels of DNA replication stress and consequent genomic instability, it was not clear how these mutant NF-κB subunit lymphomas were able to survive. To understand these survival mechanisms and to identify potential compensatory bypass signalling pathways in these lymphomas, we applied a multi-omics strategy. With c-Rel-/- Eµ-Myc lymphomas we observed high levels of Phosphatidyl-inositol 3-kinase (PI3K) and AKT pathway activation. Moreover, treatment with the PI3K inhibitor Pictilisib (GDC-0941) selectively inhibited the growth of reimplanted c-Rel-/- and RelAT505A, but not wild type (WT) Eµ-Myc lymphomas. We also observed up-regulation of a RHO/RAC pathway gene expression signature in both Eµ-Myc NF-κB subunit mutation models. Further investigation demonstrated activation of the RHO/RAC effector p21-activated kinase (PAK) 2. Here, the PAK inhibitor, PF-3758309 successfully overcame resistance of RelAT505A but not WT lymphomas. These findings demonstrate that up-regulation of multiple bypass pathways occurs in CHK1 inhibitor resistant Eµ-Myc lymphomas. Consequently, drugs targeting these pathways could potentially be used as either second line or combinatorial therapies to aid the successful clinical application of CHK1 inhibitors.
Assuntos
Linfoma , Fosfatidilinositol 3-Quinases , Animais , Inositol , Linfoma/tratamento farmacológico , Linfoma/genética , Linfoma/metabolismo , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Regulação para Cima , Quinases Ativadas por p21/genéticaRESUMO
HIF1α (hypoxia inducible factor 1α) is the central regulator of the cellular response to low oxygen and its activity is deregulated in multiple human pathologies. Consequently, given the importance of HIF signaling in disease, there is considerable interest in developing strategies to modulate HIF1α activity and down-stream signaling events. In the present study we find that under hypoxic conditions, activation of the PERK branch of the unfolded protein response (UPR) can suppress the levels and activity of HIF1α by preventing efficient HIF1α translation. Activation of PERK inhibits de novo HIF1α protein synthesis by preventing the RNA-binding protein, YB-1, from interacting with the HIF1α mRNA 5'UTR. Our data indicate that activation of the UPR can sensitise tumor cells to hypoxic stress, indicating that chemical activation of the UPR could be a strategy to target hypoxic malignant cancer cells.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteína 1 de Ligação a Y-Box/metabolismo , eIF-2 Quinase/metabolismo , Regiões 5' não Traduzidas , Regulação para Baixo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células PC-3 , Biossíntese de Proteínas , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Tapsigargina/farmacologia , Hipóxia Tumoral/genética , Resposta a Proteínas não Dobradas , eIF-2 Quinase/antagonistas & inibidoresRESUMO
BACKGROUND: Thermal regulation of gene expression occurs in many microorganisms, and is mediated via several typical mechanisms. Yersinia pestis is the causative agent of the plague and spreads by zoonotic transfer from fleas to mammalian blood with a concomitant rapid temperature change, from ambient to 37 °C, which induces the expression of capsular antigen (Caf1) that inhibits phagocytosis. Caf1 is formed into long polymeric fimbriae by a periplasmic chaperone (Caf1M) and outer membrane usher (Caf1A). All three are encoded on an operon regulated by an AraC-type transcription factor Caf1R. The aim of this study was to determine the role of Caf1R in the thermal control of caf1 operon gene expression. RESULTS: PCR analysis of cDNA demonstrated that the genes of the operon are transcribed as a single polycistronic mRNA. Bioinformatic analysis, supported by deletion mutagenesis, then revealed a region containing the promoter of this polycistronic transcript that was critical for Caf1 protein expression. Caf1R was found to be essential for Caf1 protein production. Finally, RT-PCR analysis and western blot experiments showed large, Caf1R dependent increases in caf1 operon transcripts upon a shift in temperature from 25 °C to 35 °C. CONCLUSIONS: The results show that thermal control of Caf1 polymer production is established at the transcriptional level, in a Caf1R dependent manner. This gives us new insights into how a virulent pathogen evades destruction by the immune system by detecting and responding to environmental changes.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Temperatura , Fatores de Transcrição/genética , Yersinia pestis/genética , Regulação Bacteriana da Expressão Gênica , Evasão da Resposta Imune , ÓperonRESUMO
The cellular response to hypoxia is characterised by a switch in the transcriptional program, mediated predominantly by the hypoxia inducible factor family of transcription factors (HIF). Regulation of HIF1 is primarily controlled by post-translational modification of the HIF1α subunit, which can alter its stability and/or activity. This study identifies an unanticipated role for the X-linked inhibitor of apoptosis (XIAP) protein as a regulator of Lys63-linked polyubiquitination of HIF1α. Lys63-linked ubiquitination of HIF1α by XIAP is dependent on the activity of E2 ubiquitin conjugating enzyme Ubc13. We find that XIAP and Ubc13 dependent Lys63-linked polyubiquitination promotes HIF1α nuclear retention leading to an increase in the expression of HIF1 responsive genes. Inhibition of the Lys63-linked polyubiquitination pathway leads to reduced levels of nuclear HIF1α, promoter occupancy, HIF-dependent gene expression and cell viability. Our data reveals an additional and significant level of control of the HIF1 by XIAP, with important implications in understanding the role of HIF1 and XIAP in human disease.
Assuntos
Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lisina/metabolismo , Ubiquitinação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Hipóxia Celular , Linhagem Celular , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Regiões Promotoras Genéticas , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/metabolismo , Regulação para CimaRESUMO
Damaged DNA can be repaired by removal and re-synthesis of up to 30 nucleotides during base or nucleotide excision repair. An important question is what happens when many more nucleotides are removed, resulting in long single-stranded DNA (ssDNA) lesions. Such lesions appear on chromosomes during telomere damage, double strand break repair or after the UV damage of stationary phase cells. Here, we show that long single-stranded lesions, formed at dysfunctional telomeres in budding yeast, are re-synthesized when cells are removed from the telomere-damaging environment. This process requires Pol32, an accessory factor of Polymerase δ. However, re-synthesis takes place even when the telomere-damaging conditions persist, in which case the accessory factors of both polymerases δ and ε are required, and surprisingly, salt. Salt added to the medium facilitates the DNA synthesis, independently of the osmotic stress responses. These results provide unexpected insights into the DNA metabolism and challenge the current view on cellular responses to telomere dysfunction.
Assuntos
DNA Polimerase III/metabolismo , DNA Polimerase II/metabolismo , Reparo do DNA , Cloreto de Sódio/farmacologia , Telômero/enzimologia , Proliferação de Células/efeitos dos fármacos , Cromossomos Fúngicos/efeitos dos fármacos , Cromossomos Fúngicos/enzimologia , Cromossomos Fúngicos/metabolismo , DNA Polimerase I/fisiologia , DNA Fúngico/biossíntese , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fleomicinas/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Telômero/efeitos dos fármacos , Telômero/metabolismo , Homeostase do Telômero , Fatores de Transcrição/metabolismoRESUMO
The nuclear factor-κB (NF-κB) family of transcription factors can directly or indirectly regulate many important areas of biology, including immunity, inflammation and cell survival. One intriguing aspect of NF-κB crosstalk with other cell signalling pathways is its regulation of mitochondrial biology, including biogenesis, metabolism and apoptosis. In addition to regulating the expression of mitochondrial genes encoded in the nucleus, NF-κB signalling components are also found within mitochondria themselves and associated with mitochondrial DNA. However, complete biochemical analysis of mitochondrial and sub-mitochondrial localisation of all NF-κB subunits has not been undertaken. Here, we show that only the RelA NF-κB subunit and its inhibitor IκBα reside within mitochondria, whilst p50 is found in the endoplasmic reticulum (ER). Fractionation of mitochondria revealed that only RelA was found in the mitoplast, the location of the mtDNA. We demonstrate that hypoxia leads to a very rapid but transient accumulation of RelA and IκBα in mitochondria. This effect required reactive oxygen species (ROS) but was not dependent on the hypoxia sensing transcription factor subunit HIF1α or intracellular Ca2+ release. We also observed rapid mitochondrial localisation of transcription factor STAT3 following hypoxia. Inhibition of STAT3 blocked RelA and IκBα mitochondrial localisation revealing a previously unknown aspect of crosstalk between these key cellular regulators.
Assuntos
Mitocôndrias/genética , Inibidor de NF-kappaB alfa/genética , NF-kappa B/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição RelA/genética , Cálcio/metabolismo , Hipóxia Celular/genética , DNA Mitocondrial/genética , Retículo Endoplasmático/genética , Células HEK293 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células MCF-7 , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Hypoxia-Inducible Factors (HIFs) play essential roles in the physiological response to low oxygen in all multicellular organisms, while their deregulation is associated with human diseases. HIF levels and activity are primarily controlled by the availability of the oxygen-sensitive HIFα subunits, which is mediated by rapid alterations to the rates of HIFα protein production and degradation. While the pathways that control HIFα degradation are understood in great detail, much less is known about the targeted control of HIFα protein synthesis and what role this has in controlling HIF activity during the hypoxic response. This review will focus on the signalling pathways and RNA binding proteins that modulate HIFα mRNA half-life and/or translation rate, and their contribution to hypoxia-associated diseases.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Oxigênio/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Hipóxia Celular/efeitos dos fármacos , Humanos , Proteínas de Ligação a RNA/metabolismo , Ubiquitina/metabolismoRESUMO
Telomere attrition is linked to cancer, diabetes, cardiovascular disease and aging. This is because telomere losses trigger further genomic modifications, culminating with loss of cell function and malignant transformation. However, factors regulating the transition from cells with short telomeres, to cells with profoundly altered genomes, are little understood. Here, we use budding yeast engineered to lack telomerase and other forms of telomere maintenance, to screen for such factors. We show that initially, different DNA damage checkpoint proteins act together with Exo1 and Mre11 nucleases, to inhibit proliferation of cells undergoing telomere attrition. However, this situation changes when survivors lacking telomeres emerge. Intriguingly, checkpoint pathways become tolerant to loss of telomeres in survivors, yet still alert to new DNA damage. We show that Rif1 is responsible for the checkpoint tolerance and proliferation of these survivors, and that is also important for proliferation of cells with a broken chromosome. In contrast, Exo1 drives extensive genomic modifications in survivors. Thus, the conserved proteins Rif1 and Exo1 are critical for survival and evolution of cells with lost telomeres.
Assuntos
Exodesoxirribonucleases/metabolismo , Instabilidade Genômica , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Proliferação de Células/genética , Senescência Celular/genética , Cromossomos Fúngicos/metabolismo , Quebras de DNA de Cadeia Dupla , Endonucleases/metabolismo , Deleção de Genes , Viabilidade Microbiana/genética , Modelos Biológicos , Fenótipo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Replicative senescence is a permanent cell cycle arrest in response to extensive telomere shortening. To understand the mechanisms behind a permanent arrest, we screened for factors affecting replicative senescence in budding yeast lacking telomere elongation pathways. Intriguingly, we found that DNA polymerase epsilon (Pol ε) acts synergistically with Exo1 nuclease to maintain replicative senescence. In contrast, the Pol ε-associated checkpoint and replication protein Mrc1 facilitates escape from senescence. To understand this paradox, in which DNA-synthesizing factors cooperate with DNA-degrading factors to maintain arrest, whereas a checkpoint protein opposes arrest, we analyzed the dynamics of double- and single-stranded DNA (ssDNA) at chromosome ends during senescence. We found evidence for cycles of DNA resection, followed by resynthesis. We propose that resection of the shortest telomere, activating a Rad24(Rad17)-dependent checkpoint pathway, alternates in time with an Mrc1-regulated Pol ε resynthesis of a short, double-stranded chromosome end, which in turn activates a Rad9(53BP1)-dependent checkpoint pathway. Therefore, instead of one type of DNA damage, different types (ssDNA and a double-strand break-like structure) alternate in a "vicious circle," each activating a different checkpoint sensor. Every time resection and resynthesis switches, a fresh signal initiates, thus preventing checkpoint adaptation and ensuring the permanent character of senescence.