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1.
Cell ; 170(4): 774-786.e19, 2017 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-28802045

RESUMO

Conflicts between transcription and replication are a potent source of DNA damage. Co-transcriptional R-loops could aggravate such conflicts by creating an additional barrier to replication fork progression. Here, we use a defined episomal system to investigate how conflict orientation and R-loop formation influence genome stability in human cells. R-loops, but not normal transcription complexes, induce DNA breaks and orientation-specific DNA damage responses during conflicts with replication forks. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the co-directional (CD) orientation but promoting their formation in the head-on (HO) orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings connect DNA replication to R-loop homeostasis and suggest a mechanistic basis for genome instability resulting from deregulated DNA replication, observed in cancer and other disease states.


Assuntos
Replicação do DNA , Transcrição Gênica , Dano ao DNA , Período de Replicação do DNA , Instabilidade Genômica , Células HEK293 , Humanos , Plasmídeos
2.
Nat Rev Mol Cell Biol ; 18(10): 622-636, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28811666

RESUMO

One way to preserve a rare book is to lock it away from all potential sources of damage. Of course, an inaccessible book is also of little use, and the paper and ink will continue to degrade with age in any case. Like a book, the information stored in our DNA needs to be read, but it is also subject to continuous assault and therefore needs to be protected. In this Review, we examine how the replication stress response that is controlled by the kinase ataxia telangiectasia and Rad3-related (ATR) senses and resolves threats to DNA integrity so that the DNA remains available to read in all of our cells. We discuss the multiple data that have revealed an elegant yet increasingly complex mechanism of ATR activation. This involves a core set of components that recruit ATR to stressed replication forks, stimulate kinase activity and amplify ATR signalling. We focus on the activities of ATR in the control of cell cycle checkpoints, origin firing and replication fork stability, and on how proper regulation of these processes is crucial to ensure faithful duplication of a challenging genome.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Replicação do DNA , Eucariotos/metabolismo , Animais , Genoma , Genoma Humano , Humanos , Transdução de Sinais
3.
EMBO J ; 40(11): e108486, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33969907

RESUMO

USP7 inhibitors are gaining momentum as a therapeutic strategy to stabilize p53 through their ability to induce MDM2 degradation. However, these inhibitors come with an unexpected p53-independent toxicity, via an unknown mechanism. In this issue of The EMBO Journal, Galarreta et al report how inhibition of USP7 leads to re-distribution of PP2A from cytoplasm to nucleus and an increase of deleterious CDK1-dependent phosphorylation throughout the cell cycle, revealing a new regulatory mechanism for the progression of S-phase cells toward mitosis to maintain genomic integrity.


Assuntos
Ciclina B , Proteína Fosfatase 2 , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Ciclina B/genética , Mitose , Fosforilação , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo
5.
Nucleic Acids Res ; 50(20): 11492-11508, 2022 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-36318267

RESUMO

Breast cancers are known to be driven by the transcription factor estrogen receptor and its ligand estrogen. While the receptor's cis-binding elements are known to vary between tumors, heterogeneity of hormone signaling at a single-cell level is unknown. In this study, we systematically tracked estrogen response across time at a single-cell level in multiple cell line and organoid models. To accurately model these changes, we developed a computational tool (TITAN) that quantifies signaling gradients in single-cell datasets. Using this approach, we found that gene expression response to estrogen is non-uniform, with distinct cell groups expressing divergent transcriptional networks. Pathway analysis suggested the two most distinct signatures are driven separately by ER and FOXM1. We observed that FOXM1 was indeed activated by phosphorylation upon estrogen stimulation and silencing of FOXM1 attenuated the relevant gene signature. Analysis of scRNA-seq data from patient samples confirmed the existence of these divergent cell groups, with the FOXM1 signature predominantly found in ER negative cells. Further, multi-omic single-cell experiments indicated that the different cell groups have distinct chromatin accessibility states. Our results provide a comprehensive insight into ER biology at the single-cell level and potential therapeutic strategies to mitigate resistance to therapy.


Assuntos
Neoplasias da Mama , Epigênese Genética , Estrogênios , Feminino , Humanos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Estrogênios/farmacologia , Regulação Neoplásica da Expressão Gênica , Receptores de Estrogênio/metabolismo , Análise de Célula Única , RNA-Seq
6.
Am J Respir Crit Care Med ; 199(1): 83-98, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30107138

RESUMO

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by progressive narrowing of pulmonary arteries, resulting in right heart failure and death. BMPR2 (bone morphogenetic protein receptor type 2) mutations account for most familial PAH forms whereas reduced BMPR2 is present in many idiopathic PAH forms, suggesting dysfunctional BMPR2 signaling to be a key feature of PAH. Modulating BMPR2 signaling is therapeutically promising, yet how BMPR2 is downregulated in PAH is unclear. OBJECTIVES: We intended to identify and pharmaceutically target BMPR2 modifier genes to improve PAH. METHODS: We combined siRNA high-throughput screening of >20,000 genes with a multicohort analysis of publicly available PAH RNA expression data to identify clinically relevant BMPR2 modifiers. After confirming gene dysregulation in tissue from patients with PAH, we determined the functional roles of BMPR2 modifiers in vitro and tested the repurposed drug enzastaurin for its propensity to improve experimental pulmonary hypertension (PH). MEASUREMENTS AND MAIN RESULTS: We discovered FHIT (fragile histidine triad) as a novel BMPR2 modifier. BMPR2 and FHIT expression were reduced in patients with PAH. FHIT reductions were associated with endothelial and smooth muscle cell dysfunction, rescued by enzastaurin through a dual mechanism: upregulation of FHIT as well as miR17-5 repression. Fhit-/- mice had exaggerated hypoxic PH and failed to recover in normoxia. Enzastaurin reversed PH in the Sugen5416/hypoxia/normoxia rat model, by improving right ventricular systolic pressure, right ventricular hypertrophy, cardiac fibrosis, and vascular remodeling. CONCLUSIONS: This study highlights the importance of the novel BMPR2 modifier FHIT in PH and the clinical value of the repurposed drug enzastaurin as a potential novel therapeutic strategy to improve PAH.


Assuntos
Hidrolases Anidrido Ácido/genética , Hipertensão Pulmonar Primária Familiar/genética , Genes Modificadores/genética , Proteínas de Neoplasias/genética , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Modelos Animais de Doenças , Hipertensão Pulmonar Primária Familiar/metabolismo , Feminino , Humanos , Indóis/farmacologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
7.
Genes Chromosomes Cancer ; 58(5): 317-323, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30242938

RESUMO

Genome instability is an enabling characteristic of cancer that facilitates the acquisition of oncogenic mutations that drive tumorigenesis. Underlying much of the instability in cancer is DNA replication stress, which causes both chromosome structural changes and single base-pair mutations. Common fragile sites are some of the earliest and most frequently altered loci in tumors. Notably, the fragile locus, FRA3B, lies within the fragile histidine triad (FHIT) gene, and consequently deletions within FHIT are common in cancer. We review the evidence in support of FHIT as a DNA caretaker and discuss the mechanism by which FHIT promotes genome stability. FHIT increases thymidine kinase 1 (TK1) translation to balance the deoxyribonucleotide triphosphates (dNTPs) for efficient DNA replication. Consequently, FHIT-loss causes replication stress, DNA breaks, aneuploidy, copy-number changes (CNCs), small insertions and deletions, and point mutations. Moreover, FHIT-loss-induced replication stress and DNA breaks cooperate with APOBEC3B overexpression to catalyze DNA hypermutation in cancer, as APOBEC family enzymes prefer single-stranded DNA (ssDNA) as substrates and ssDNA is enriched at sites of both replication stress and DNA breaks. Consistent with the frequent loss of FHIT across a broad spectrum of cancer types, FHIT-deficiency is highly associated with the ubiquitous, clock-like mutation signature 5 occurring in all cancer types thus far examined. The ongoing destabilization of the genome caused by FHIT loss underlies recurrent inactivation of tumor suppressors and activation of oncogenes. Considering that more than 50% of cancers are FHIT-deficient, we propose that FRA3B/FHIT fragility shapes the mutational landscape of cancer genomes.


Assuntos
Hidrolases Anidrido Ácido/genética , Instabilidade Genômica , Proteínas de Neoplasias/genética , Neoplasias/genética , Hidrolases Anidrido Ácido/deficiência , Animais , Sítios Frágeis do Cromossomo , Replicação do DNA , Humanos , Proteínas de Neoplasias/deficiência
8.
Cancer Sci ; 107(4): 528-35, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26782170

RESUMO

Loss of expression of Fhit, a tumor suppressor and genome caretaker, occurs in preneoplastic lesions during development of many human cancers. Furthermore, Fhit-deficient mouse models are exquisitely susceptible to carcinogen induction of cancers of the lung and forestomach. Due to absence of Fhit genome caretaker function, cultured cells and tissues of the constitutive Fhit knockout strain develop chromosome aneuploidy and allele copy number gains and losses and we hypothesized that Fhit-deficient cells would also develop point mutations. On analysis of whole exome sequences of Fhit-deficient tissues and cultured cells, we found 300 to >1000 single-base substitutions associated with Fhit loss in the 2% of the genome included in exomes, relative to the C57Bl6 reference genome. The mutation signature is characterized by increased C>T and T>C mutations, similar to the "age at diagnosis" signature identified in human cancers. The Fhit-deficiency mutation signature also resembles a C>T and T>C mutation signature reported for human papillary kidney cancers and a similar signature recently reported for esophageal and bladder cancers, cancers that are frequently Fhit deficient. The increase in T>C mutations in -/- exomes may be due to dNTP imbalance, particularly in thymidine triphosphate, resulting from decreased expression of thymidine kinase 1 in Fhit-deficient cells. Fhit-deficient kidney cells that survived in vitro dimethylbenz(a)anthracene treatment additionally showed increased T>A mutations, a signature generated by treatment with this carcinogen, suggesting that these T>A transversions may be evidence of carcinogen-induced preneoplastic changes.


Assuntos
Hidrolases Anidrido Ácido/genética , Neoplasias Renais/genética , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Neoplasias Gástricas/genética , Animais , Carcinógenos/toxicidade , Exoma/genética , Regulação Neoplásica da Expressão Gênica , Genoma , Humanos , Neoplasias Renais/induzido quimicamente , Neoplasias Renais/patologia , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Knockout , Mutação Puntual/genética , Neoplasias Gástricas/induzido quimicamente , Neoplasias Gástricas/patologia
9.
Cell Mol Life Sci ; 71(23): 4577-87, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25283145

RESUMO

The FHIT gene at FRA3B is one of the earliest and most frequently altered genes in the majority of human cancers. It was recently discovered that the FHIT gene is not the most fragile locus in epithelial cells, the cell of origin for most Fhit-negative cancers, eroding support for past claims that deletions at this locus are simply passenger events that are carried along in expanding cancer clones, due to extreme vulnerability to DNA damage rather than to loss of FHIT function. Indeed, recent reports have reconfirmed FHIT as a tumor suppressor gene with roles in apoptosis and prevention of the epithelial-mesenchymal transition. Other recent works have identified a novel role for the FHIT gene product, Fhit, as a genome "caretaker." Loss of this caretaker function leads to nucleotide imbalance, spontaneous replication stress, and DNA breaks. Because Fhit loss-induced DNA damage is "checkpoint blind," cells accumulate further DNA damage during subsequent cell cycles, accruing global genome instability that could facilitate oncogenic mutation acquisition and expedite clonal expansion. Loss of Fhit activity therefore induces a mutator phenotype. Evidence for FHIT as a mutator gene is discussed in light of these recent investigations of Fhit loss and subsequent genome instability.


Assuntos
Hidrolases Anidrido Ácido/genética , Genes Supressores de Tumor , Instabilidade Genômica , Proteínas de Neoplasias/genética , Neoplasias/genética , Animais , Ciclo Celular , Dano ao DNA , Replicação do DNA , Transição Epitelial-Mesenquimal , Humanos , Neoplasias/patologia
10.
PLoS Genet ; 8(11): e1003077, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23209436

RESUMO

Genomic instability drives tumorigenesis, but how it is initiated in sporadic neoplasias is unknown. In early preneoplasias, alterations at chromosome fragile sites arise due to DNA replication stress. A frequent, perhaps earliest, genetic alteration in preneoplasias is deletion within the fragile FRA3B/FHIT locus, leading to loss of Fhit protein expression. Because common chromosome fragile sites are exquisitely sensitive to replication stress, it has been proposed that their clonal alterations in cancer cells are due to stress sensitivity rather than to a selective advantage imparted by loss of expression of fragile gene products. Here, we show in normal, transformed, and cancer-derived cell lines that Fhit-depletion causes replication stress-induced DNA double-strand breaks. Using DNA combing, we observed a defect in replication fork progression in Fhit-deficient cells that stemmed primarily from fork stalling and collapse. The likely mechanism for the role of Fhit in replication fork progression is through regulation of Thymidine kinase 1 expression and thymidine triphosphate pool levels; notably, restoration of nucleotide balance rescued DNA replication defects and suppressed DNA breakage in Fhit-deficient cells. Depletion of Fhit did not activate the DNA damage response nor cause cell cycle arrest, allowing continued cell proliferation and ongoing chromosomal instability. This finding was in accord with in vivo studies, as Fhit knockout mouse tissue showed no evidence of cell cycle arrest or senescence yet exhibited numerous somatic DNA copy number aberrations at replication stress-sensitive loci. Furthermore, cells established from Fhit knockout tissue showed rapid immortalization and selection of DNA deletions and amplifications, including amplification of the Mdm2 gene, suggesting that Fhit loss-induced genome instability facilitates transformation. We propose that loss of Fhit expression in precancerous lesions is the first step in the initiation of genomic instability, linking alterations at common fragile sites to the origin of genome instability.


Assuntos
Hidrolases Anidrido Ácido , Transformação Celular Neoplásica/genética , Instabilidade Genômica , Proteínas de Neoplasias , Neoplasias , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Animais , Sítios Frágeis do Cromossomo , Quebras de DNA de Cadeia Dupla , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Timidina Quinase/metabolismo
11.
Genes Chromosomes Cancer ; 52(11): 1017-29, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23929738

RESUMO

Chromosomal positions of common fragile sites differ in lymphoblasts and fibroblasts, with positions dependent on the epigenetically determined density of replication origins at these loci. Because rearrangement of fragile loci and associated loss of fragile gene products are hallmarks of cancers, we aimed to map common fragile sites in epithelial cells, from which most cancers derive. Among the five most frequently activated sites in human epithelial cells were chromosome bands 2q33 and Xq22.1, which are not among top fragile sites identified in lymphoblasts or fibroblasts. FRA16D at 16q23 was among the top three fragile sites in the human epithelial cells examined, as it is in lymphoblasts and fibroblasts, while FRA3B at 3p14.2, the top fragile locus in lymphoblasts, was not fragile in most epithelial cell lines tested. Epithelial cells exhibited varying hierarchies of fragile sites; some frequent epithelial cell fragile sites are apparently not frequently altered in epithelial cancers and sites that are frequently deleted in epithelial cancers are not necessarily among the most fragile. Since we have reported that loss of expression of the FRA3B-encoded FHIT protein causes increased replication stress-induced DNA damage, we also examined the effect of FHIT-deficiency on markers of genome instability in epithelial cells. FHIT-deficient cells exhibited increases in fragile breaks and in γH2AX and 53BP1 foci in G1 phase cells, confirming in epithelial cells that the FHIT gene and encompassing FRA3B, is a "caretaker gene" necessary for maintenance of genome stability.


Assuntos
Hidrolases Anidrido Ácido/genética , Sítios Frágeis do Cromossomo/genética , Instabilidade Genômica , Proteínas de Neoplasias/genética , Neoplasias/genética , Animais , Linhagem Celular , Cromossomos Humanos Par 2/genética , Análise Citogenética , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Fase G1/genética , Marcadores Genéticos , Humanos , Camundongos , Interferência de RNA , Origem de Replicação , Análise de Pequenas Áreas , Cromossomo X/genética
12.
bioRxiv ; 2024 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-38765978

RESUMO

Chromatin is organized into compartments enriched with functionally-related proteins driving non-linear biochemical activities. Some compartments, e.g. transcription foci, behave as liquid condensates. While the principles governing the enrichment of proteins within condensates are being elucidated, mechanisms that coordinate condensate dynamics with other nuclear processes like DNA replication have not been identified. We show that at the G1/S cell cycle transition, large transcription condensates form at histone locus bodies (HLBs) in a cyclin-dependent kinase 1 and 2 (CDK1/2)-dependent manner. As cells progress through S phase, ataxia-telangiectasia and Rad3-related (ATR) accumulates within HLBs and dissolves the associated transcription condensates. Integration of CDK1/2 and ATR signaling creates a phosphorylation code within the intrinsically-disordered region of mediator subunit 1 (MED1) coordinating condensate dynamics with DNA replication. Disruption of this code results in imbalanced histone biosynthesis, and consequently, global DNA damage. We propose the spatiotemporal dynamics of transcription condensates are actively controlled via phosphorylation and essential for viability of proliferating cells.

13.
Crit Rev Food Sci Nutr ; 52(8): 712-25, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22591342

RESUMO

Listeria monocytogenes is a Gram-positive foodborne pathogen responsible for a severe disease occurring in immuno-compromised populations. Foodborne illness caused by L. monocytogenes is a serious public health concern because of the high associated mortality. Study of the closely related, but nonpathogenic Listeria innocua has accounted for a better understanding of the behavior of L. monocytogenes in environments beyond the laboratory. Traditionally, the ecological co-habitation, genomic synteny, and physiological similarity of the two species have supported use of L. innocua for predicting the behavior of L. monocytogenes in farm and food processing environments. However, a careful review of the current literature indicates that in a given situation it may not be prudent to use L. innocua as a surrogate for L. monocytogenes without prior confirmation of their similar phenotypes, as an increasing number of studies have arisen demonstrating differences in L. monocytogenes and L. innocua stress response, and furthermore, there are differences among the L. monocytogenes subgroups. Future research should take into consideration that multiple surrogates might be required to accurately model even a single condition depending on the L. monocytogenes subgroup of interest.


Assuntos
Listeria monocytogenes/genética , Listeria monocytogenes/fisiologia , Listeria/genética , Listeria/fisiologia , Agricultura , Animais , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/genética , Manipulação de Alimentos , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Doenças Transmitidas por Alimentos/prevenção & controle , Genômica , Humanos , Listeriose/microbiologia , Modelos Biológicos , Fenótipo , Especificidade da Espécie
14.
Cancer Sci ; 102(10): 1882-8, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21707865

RESUMO

When DNA damage is detected, checkpoint signal networks are activated to stop the cell cycle, and DNA repair processes begin. Inhibitory compounds targeting components of DNA damage response pathways have been identified and are being used in clinical trials, in combination with chemotherapeutic agents, to enhance cancer therapy. Inhibitors of checkpoint kinases, Chk1 and Chk2, have been shown to sensitize tumor cells to DNA damaging agents, and treatment of BRCA1/2-deficient tumor cells, as well as triple negative breast cancers, with poly(ADP-ribose) polymerase (PARP) inhibitors has shown promise. But systematic studies to determine which tumor subtypes are likely to respond to these specific inhibitors have not been reported. The current study was designed to test sensitivity of specific breast cancer subtype-derived cells to two classes of these new inhibitory drugs, PARP and Chk1 inhibitors. Luminal, HER2 overexpressing, and triple negative breast cancer-derived cells were tested for sensitivity to killing by PARP inhibitors, ABT-888 and BSI-201, and Chk1 inhibitor, PF-00477736, alone or in combination with gemcitabine or carboplatin. Each of the triple negative breast cancer cell lines showed strong sensitivity to the Chk1 inhibitor, but only the BRCA1-deficient breast cancer cell lines showed sensitivity to the PARP inhibitors, suggesting that in vitro testing of cancer cell lines of specific subtypes, with panels of the different PARP and Chk1 inhibitors, will contribute to stratification of patients for clinical trials using these classes of inhibitors.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Proteínas Quinases/metabolismo , Proteína BRCA1/deficiência , Benzamidas/farmacologia , Benzimidazóis/farmacologia , Benzodiazepinonas/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Feminino , Humanos , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Pirazóis/farmacologia , Receptor ErbB-2/biossíntese , Receptor ErbB-2/genética , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/genética , Receptores de Progesterona/biossíntese , Receptores de Progesterona/genética
15.
J Clin Invest ; 131(11)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34060485

RESUMO

Hypoxia, a hallmark feature of the tumor microenvironment, causes resistance to conventional chemotherapy, but was recently reported to synergize with poly(ADP-ribose) polymerase inhibitors (PARPis) in homologous recombination-proficient (HR-proficient) cells through suppression of HR. While this synergistic killing occurs under severe hypoxia (<0.5% oxygen), our study shows that moderate hypoxia (2% oxygen) instead promotes PARPi resistance in both HR-proficient and -deficient cancer cells. Mechanistically, we identify reduced ROS-induced DNA damage as the cause for the observed resistance. To determine the contribution of hypoxia to PARPi resistance in tumors, we used the hypoxic cytotoxin tirapazamine to selectively kill hypoxic tumor cells. We found that the selective elimination of hypoxic tumor cells led to a substantial antitumor response when used with PARPi compared with that in tumors treated with PARPi alone, without enhancing normal tissue toxicity. Since human breast cancers with BRAC1/2 mutations have an increased hypoxia signature and hypoxia reduces the efficacy of PARPi, then eliminating hypoxic tumor cells should enhance the efficacy of PARPi therapy.


Assuntos
Dano ao DNA , Recombinação Homóloga , Neoplasias Experimentais , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
16.
EBioMedicine ; 73: 103646, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34689087

RESUMO

BACKGROUND: Senescent cells accumulate in tissues over time as part of the natural ageing process and the removal of senescent cells has shown promise for alleviating many different age-related diseases in mice. Cancer is an age-associated disease and there are numerous mechanisms driving cellular senescence in cancer that can be detrimental to recovery. Thus, it would be beneficial to develop a senolytic that acts not only on ageing cells but also senescent cancer cells to prevent cancer recurrence or progression. METHODS: We used molecular modelling to develop a series of rationally designed peptides to mimic and target FOXO4 disrupting the FOXO4-TP53 interaction and releasing TP53 to induce apoptosis. We then tested these peptides as senolytic agents for the elimination of senescent cells both in cell culture and in vivo. FINDINGS: Here we show that these peptides can act as senolytics for eliminating senescent human cancer cells both in cell culture and in orthotopic mouse models. We then further characterized one peptide, ES2, showing that it disrupts FOXO4-TP53 foci, activates TP53 mediated apoptosis and preferentially binds FOXO4 compared to TP53. Next, we show that intratumoural delivery of ES2 plus a BRAF inhibitor results in a significant increase in apoptosis and a survival advantage in mouse models of melanoma. Finally, we show that repeated systemic delivery of ES2 to older mice results in reduced senescent cell numbers in the liver with minimal toxicity. INTERPRETATION: Taken together, our results reveal that peptides can be generated to specifically target and eliminate FOXO4+ senescent cancer cells, which has implications for eradicating residual disease and as a combination therapy for frontline treatment of cancer. FUNDING: This work was supported by the Cancer Early Detection Advanced Research Center at Oregon Health & Science University.


Assuntos
Antineoplásicos/química , Proteínas de Ciclo Celular/química , Desenho de Fármacos , Fatores de Transcrição Forkhead/química , Modelos Moleculares , Peptídeos/química , Senoterapia/química , Proteína Supressora de Tumor p53/química , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Senescência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Masculino , Melanoma , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeos/farmacologia , Conformação Proteica , Senoterapia/farmacologia , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
17.
J Cell Biochem ; 109(5): 858-65, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-20082323

RESUMO

More than 12 years and >800 scientific publications after the discovery of the first gene at a chromosome fragile site, the FHIT gene at FRA3B, there are still questions to pursue concerning the selective advantage conferred to cells by loss of expression of FHIT, the most frequent target of allele deletion in precancerous lesions and cancers. These questions are considered in light of recent investigations of genetic and epigenetic alterations to the locus and in a retrospective consideration of biological roles of the Fhit protein discovered through functional studies.


Assuntos
Hidrolases Anidrido Ácido/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Hidrolases Anidrido Ácido/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Neoplasias/metabolismo
18.
Foodborne Pathog Dis ; 7(5): 499-505, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20001327

RESUMO

The goal of this study was to characterize the starvation survival response (SSR) of a wild-type Listeria monocytogenes 10403S and an isogenic DeltasigB mutant strain during multiple-nutrient starvation conditions over 28 days. This study examined the effects of inhibitors of protein synthesis, the proton motive force, substrate level phosphorylation, and oxidative phosphorylation on the SSR of L. monocytogenes 10403S and a DeltasigB mutant during multiple-nutrient starvation. The effects of starvation buffer changes on viability were also examined. During multiple-nutrient starvation, both strains expressed a strong SSR, suggesting that L. monocytogenes possesses SigB-independent mechanism(s) for survival during multiple-nutrient starvation. Neither strain was able to express an SSR following starvation buffer changes, indicating that the nutrients/factors present in the starvation buffer could be a source of energy for cell maintenance and survival. Neither the wild-type nor the DeltasigB mutant strain was able to elicit an SSR when exposed to the protein synthesis inhibitor chloramphenicol within the first 4 h of starvation. However, both strains expressed an SSR when exposed to chloramphenicol after 6 h or more of starvation, suggesting that the majority of proteins required to elicit an effective SSR in L. monocytogenes are likely produced somewhere between 4 and 6 h of starvation. The varying SSRs of both strains to the different metabolic inhibitors under aerobic or anaerobic conditions suggested that (1) energy derived from the proton motive force is important for an effective SSR, (2) L. monocytogenes utilizes an anaerobic electron transport during multiple-nutrient starvation conditions, and (3) the glycolytic pathway is an important energy source during multiple-nutrient starvation when oxygen is available, and less important under anaerobic conditions. Collectively, the data suggest that the combination of energy-dependent internal adaptation mechanisms of cells and external nutrients/factors enables L. monocytogenes to express a strong SSR.


Assuntos
Adaptação Fisiológica , Metabolismo Energético , Listeria monocytogenes/fisiologia , Viabilidade Microbiana , Estresse Fisiológico , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , Aerobiose/efeitos dos fármacos , Aerobiose/genética , Anaerobiose/efeitos dos fármacos , Anaerobiose/genética , Contagem de Colônia Microbiana , Transporte de Elétrons/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Inibidores Enzimáticos/farmacologia , Doenças Transmitidas por Alimentos/prevenção & controle , Glicólise/fisiologia , Listeria monocytogenes/genética , Listeria monocytogenes/crescimento & desenvolvimento , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/genética , Fosforilação Oxidativa/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Força Próton-Motriz/efeitos dos fármacos , Fator sigma/genética , Fator sigma/fisiologia , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Fatores de Tempo , Desacopladores/farmacologia
19.
Dev Cell ; 52(6): 675-676, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32208158

RESUMO

Replication stress underlies many genomic alterations in cancer cells. In this issue of Developmental Cell, Benedict et al. show that WAPL-dependent cohesin removal is needed to restart DNA synthesis at stalled forks and promote survival following replication stress, uncovering an unexpected link between stress and sister chromatid cohesion loss.


Assuntos
Cromátides , Proteínas Cromossômicas não Histona , Proteínas de Ciclo Celular , DNA , Replicação do DNA , Coesinas
20.
Science ; 361(6404): 806-810, 2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-30139873

RESUMO

The cell cycle is strictly ordered to ensure faithful genome duplication and chromosome segregation. Control mechanisms establish this order by dictating when a cell transitions from one phase to the next. Much is known about the control of the G1/S, G2/M, and metaphase/anaphase transitions, but thus far, no control mechanism has been identified for the S/G2 transition. Here we show that cells transactivate the mitotic gene network as they exit the S phase through a CDK1 (cyclin-dependent kinase 1)-directed FOXM1 phosphorylation switch. During normal DNA replication, the checkpoint kinase ATR (ataxia-telangiectasia and Rad3-related) is activated by ETAA1 to block this switch until the S phase ends. ATR inhibition prematurely activates FOXM1, deregulating the S/G2 transition and leading to early mitosis, underreplicated DNA, and DNA damage. Thus, ATR couples DNA replication with mitosis and preserves genome integrity by enforcing an S/G2 checkpoint.


Assuntos
Fase G2/genética , Mitose/genética , Fase S/genética , Antígenos de Superfície/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Ciclina B1/antagonistas & inibidores , Ciclina B1/metabolismo , Dano ao DNA/genética , Replicação do DNA/genética , Proteína Forkhead Box M1/metabolismo , Redes Reguladoras de Genes , Células HCT116 , Humanos , Fosforilação , Telomerase
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