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1.
Am J Respir Crit Care Med ; 210(4): 484-496, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-38717443

RESUMO

Rationale: Changes in peripheral blood cell populations have been observed, but not detailed, at single-cell resolution in idiopathic pulmonary fibrosis (IPF). Objectives: We sought to provide an atlas of the changes in the peripheral immune system in stable and progressive IPF. Methods: Peripheral blood mononuclear cells (PBMCs) from patients with IPF and control subjects were profiled using 10× chromium 5' single-cell RNA sequencing. Flow cytometry was used for validation. Protein concentrations of regulatory T cells (Tregs) and monocyte chemoattractants were measured in plasma and lung homogenates from patients with IPF and control subjects. Measurements and Main Results: Thirty-eight PBMC samples from 25 patients with IPF and 13 matched control subjects yielded 149,564 cells that segregated into 23 subpopulations. Classical monocytes were increased in patients with progressive and stable IPF compared with control subjects (32.1%, 25.2%, and 17.9%, respectively; P < 0.05). Total lymphocytes were decreased in patients with IPF versus control subjects and in progressive versus stable IPF (52.6% vs. 62.6%, P = 0.035). Tregs were increased in progressive versus stable IPF (1.8% vs. 1.1% of all PBMCs, P = 0.007), although not different than controls, and may be associated with decreased survival (P = 0.009 in Kaplan-Meier analysis; and P = 0.069 after adjusting for age, sex, and baseline FVC). Flow cytometry analysis confirmed this finding in an independent cohort of patients with IPF. The fraction of Tregs out of all T cells was also increased in two cohorts of lung single-cell RNA sequencing. CCL22 and CCL18, ligands for CCR4 and CCR8 Treg chemotaxis receptors, were increased in IPF. Conclusions: The single-cell atlas of the peripheral immune system in IPF reveals an outcome-predictive increase in classical monocytes and Tregs, as well as evidence for a lung-blood immune recruitment axis involving CCL7 (for classical monocytes) and CCL18/CCL22 (for Tregs).


Assuntos
Fibrose Pulmonar Idiopática , Leucócitos Mononucleares , Análise de Célula Única , Linfócitos T Reguladores , Humanos , Fibrose Pulmonar Idiopática/imunologia , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Análise de Célula Única/métodos , Linfócitos T Reguladores/imunologia , Leucócitos Mononucleares/imunologia , Progressão da Doença , Estudos de Casos e Controles , Citometria de Fluxo
2.
Carcinogenesis ; 41(12): 1635-1647, 2020 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-33159515

RESUMO

The p53 protein is mutated in about 50% of human cancers. Aside from losing its tumor-suppressive activities, mutant p53 may acquire pro-oncogenic activity, which is facilitated by two underlying mechanisms. The first mechanism is the inhibition of co-expressed wild-type p53 (WTp53) activity, dubbed the dominant-negative effect (DNE). The second mechanism is a neomorphic pro-oncogenic activity that does not involve the inhibition of WTp53, termed gain-of-function (GOF). Throughout the years, both mechanisms were demonstrated in a plethora of in vitro and in vivo models. However, whether both account for protumorigenic activities of mutant p53 and in which contexts is still a matter of ongoing debate. Here, we discuss evidence for both DNE and GOF in a variety of models. These models suggest that both GOF and DNE can be relevant, but are highly dependent on the specific mutation type, genetic and cellular context and even the phenotype that is being assessed. In addition, we discuss how mutant and WTp53 might not exist as two separate entities, but rather as a continuum that may involve a balance between the two forms in the same cells, which could be tilted by various factors and drugs. Further elucidation of the factors that dictate the balance between the WT and mutant p53 states, as well as the factors that govern the impact of DNE and GOF in different cancer types, may lead to the development of more effective treatment regimens for cancer patients.


Assuntos
Carcinogênese/patologia , Mutação com Ganho de Função , Genes Dominantes , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Humanos
3.
Proc Natl Acad Sci U S A ; 114(40): 10624-10629, 2017 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-28912355

RESUMO

The tumor suppressor protein p53 acts as a transcription factor, binding sequence-specifically to defined DNA sites, thereby activating the expression of genes leading to diverse cellular outcomes. Canonical p53 response elements (REs) are made of two decameric half-sites separated by a variable number of base pairs (spacers). Fifty percent of all validated p53 REs contain spacers between 1 and 18 bp; however, their functional significance is unclear at present. Here, we show that p53 forms two different tetrameric complexes with consensus or natural REs, both with long spacers: a fully specific complex where two p53 dimers bind to two specific half-sites, and a hemispecific complex where one dimer binds to a specific half-site and the second binds to an adjacent spacer sequence. The two types of complexes have comparable binding affinity and specificity, as judged from binding competition against bulk genomic DNA. Structural analysis of the p53 REs in solution shows that these sites are not bent in both their free and p53-bound states when the two half-sites are either abutting or separated by spacers. Cell-based assay supports the physiological relevance of our findings. We propose that p53 REs with long spacers comprise separate specific half-sites that can lead to several different tetrameric complexes. This finding expands the universe of p53 binding sites and demonstrates that even isolated p53 half-sites can form tetrameric complexes. Moreover, it explains the manner in which p53 binds to clusters of more than one canonical binding site, common in many natural REs.


Assuntos
DNA/química , Modelos Moleculares , Elementos de Resposta , Proteína Supressora de Tumor p53/química , DNA/metabolismo , Humanos , Ligação Proteica , Proteína Supressora de Tumor p53/metabolismo
4.
Int J Mol Sci ; 20(24)2019 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-31817996

RESUMO

The p53 protein is mutated in about 50% of human cancers. Aside from losing the tumor-suppressive functions of the wild-type form, mutant p53 proteins often acquire inherent, novel oncogenic functions, a phenomenon termed mutant p53 gain-of-function (GOF). A growing body of evidence suggests that these pro-oncogenic functions of mutant p53 proteins are mediated by affecting the transcription of various genes, as well as by protein-protein interactions with transcription factors and other effectors. In the current review, we discuss the various GOF effects of mutant p53, and how it may serve as a central node in a network of genes and proteins, which, altogether, promote the tumorigenic process. Finally, we discuss mechanisms by which "Mother Nature" tries to abrogate the pro-oncogenic functions of mutant p53. Thus, we suggest that targeting mutant p53, via its reactivation to the wild-type form, may serve as a promising therapeutic strategy for many cancers that harbor mutant p53. Not only will this strategy abrogate mutant p53 GOF, but it will also restore WT p53 tumor-suppressive functions.


Assuntos
Mutação com Ganho de Função , Neoplasias/genética , Proteína Supressora de Tumor p53/genética , Animais , Carcinogênese , Humanos , Neoplasias/metabolismo , Oncogenes
5.
medRxiv ; 2023 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-37163015

RESUMO

Rationale: Changes in peripheral blood cell populations have been observed but not detailed at single-cell resolution in idiopathic pulmonary fibrosis (IPF). Objectives: To provide an atlas of the changes in the peripheral immune system in stable and progressive IPF. Methods: Peripheral blood mononuclear cells (PBMCs) from IPF patients and controls were profiled using 10x Chromium 5' single-cell RNA sequencing (scRNA-seq). Flow cytometry was used for validation. Protein concentrations of Regulatory T-cells (Tregs) and Monocytes chemoattractants were measured in plasma and lung homogenates from patients and controls. Measurements and Main Results: Thirty-eight PBMC samples from 25 patients with IPF and 13 matched controls yielded 149,564 cells that segregated into 23 subpopulations, corresponding to all expected peripheral blood cell populations. Classical monocytes were increased in progressive and stable IPF compared to controls (32.1%, 25.2%, 17.9%, respectively, p<0.05). Total lymphocytes were decreased in IPF vs controls, and in progressive vs stable IPF (52.6% vs 62.6%, p=0.035). Tregs were increased in progressive IPF (1.8% vs 1.1%, p=0.007), and were associated with decreased survival (P=0.009 in Kaplan-Meier analysis). Flow cytometry analysis confirmed this finding in an independent cohort of IPF patients. Tregs were also increased in two cohorts of lung scRNA-seq. CCL22 and CCL18, ligands for CCR4 and CCR8 Treg chemotaxis receptors, were increased in IPF. Conclusions: The single-cell atlas of the peripheral immune system in IPF, reveals an outcome-predictive increase in classical monocytes and Tregs, as well as evidence for a lung-blood immune recruitment axis involving CCL7 (for classical monocytes) and CCL18/CCL22 (for Tregs).

6.
Nat Commun ; 13(1): 440, 2022 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-35064122

RESUMO

Dysregulated immune responses against the SARS-CoV-2 virus are instrumental in severe COVID-19. However, the immune signatures associated with immunopathology are poorly understood. Here we use multi-omics single-cell analysis to probe the dynamic immune responses in hospitalized patients with stable or progressive course of COVID-19, explore V(D)J repertoires, and assess the cellular effects of tocilizumab. Coordinated profiling of gene expression and cell lineage protein markers shows that S100Ahi/HLA-DRlo classical monocytes and activated LAG-3hi T cells are hallmarks of progressive disease and highlights the abnormal MHC-II/LAG-3 interaction on myeloid and T cells, respectively. We also find skewed T cell receptor repertories in expanded effector CD8+ clones, unmutated IGHG+ B cell clones, and mutated B cell clones with stable somatic hypermutation frequency over time. In conclusion, our in-depth immune profiling reveals dyssynchrony of the innate and adaptive immune interaction in progressive COVID-19.


Assuntos
Imunidade Adaptativa/imunologia , COVID-19/imunologia , Perfilação da Expressão Gênica/métodos , Imunidade Inata/imunologia , SARS-CoV-2/imunologia , Análise de Célula Única/métodos , Imunidade Adaptativa/efeitos dos fármacos , Imunidade Adaptativa/genética , Idoso , Anticorpos Monoclonais Humanizados/uso terapêutico , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , COVID-19/genética , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Masculino , RNA-Seq/métodos , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Tratamento Farmacológico da COVID-19
7.
J Mol Cell Biol ; 11(7): 600-604, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31318969

RESUMO

A plethora of studies suggest that the non-transformed cellular and non-cellular components of the tumor, collectively known as the tumor microenvironment, have a significant impact on the tumorigenic process. It was suggested that the microenvironment, which initially restricts tumor development, is recruited by the tumor and maintains a crosstalk that further promotes cancer progression. Indeed, many of the molecules that participate in the tumor-stroma crosstalk have been characterized. However, the crucial factors that are responsible for the initiation of this crosstalk or the 'recruitment' process remain poorly understood. We propose that oncogenes themselves may influence the 'recruitment' of the stromal cells, while focusing on mutant p53. Apart from losing its tumor-suppressing properties, mutant p53 gains novel oncogenic functions, a phenomenon dubbed mutant p53 gain of function (GOF). Here, we discuss possible ways in which mutant p53 may modulate the microenvironment in order to promote tumorigenesis. We thus propose that mutant p53 may serve as a key player in the modulation of the tumor-stroma crosstalk in a way that benefits the tumor. Further elucidation of these 'recruitment' processes, dictated by mutant p53, may be utilized for tailoring personalized therapeutic approaches for patients with tumors that harbor p53 mutation.


Assuntos
Carcinogênese , Mutação com Ganho de Função , Regulação Neoplásica da Expressão Gênica , Microambiente Tumoral/genética , Proteína Supressora de Tumor p53 , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
8.
Sci Rep ; 6: 28951, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27364600

RESUMO

Two-dimensional (2D) cell cultures growing on plastic do not recapitulate the three dimensional (3D) architecture and complexity of human tumors. More representative models are required for drug discovery and validation. Here, 2D culture and 3D mono- and stromal co-culture models of increasing complexity have been established and cross-comparisons made using three standard cell carcinoma lines: MCF7, LNCaP, NCI-H1437. Fluorescence-based growth curves, 3D image analysis, immunohistochemistry and treatment responses showed that end points differed according to cell type, stromal co-culture and culture format. The adaptable methodologies described here should guide the choice of appropriate simple and complex in vitro models.


Assuntos
Técnicas de Cocultura/métodos , Esferoides Celulares/citologia , Linhagem Celular Tumoral , Humanos , Imageamento Tridimensional , Células MCF-7 , Células Estromais/citologia
9.
Cancer Res ; 73(9): 2829-39, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23423975

RESUMO

Cellular senescence, a state of cell-cycle arrest accompanied by dramatic morphologic and metabolic changes, is a central means by which cells respond to physiologic stress and oncogene activity. Senescence is thought to play important roles in aging and in tumor suppression, yet the dynamics by which senescent cells are formed, their effects on tissue function and their eventual fate are poorly understood. To study cellular senescence within an adult tissue, we developed transgenic mice inducibly expressing p14(ARF) (human ortholog of murine p19(ARF)), a central activator of senescence. Induction of p14(ARF) in the epidermis rapidly led to widespread apoptosis and cell-cycle arrest, a stage that was transient, and was followed by p53-dependent cellular senescence. The endogenous Cdkn2a products p19(ARF) and p16(Ink4a) were activated by the transgenic p14(ARF) through p53, revealing a senescence-promoting feed-forward loop. Commitment of cells to senescence required continued p14(ARF) expression, indicating that entry into this state depends on a persistent signal. However, once formed, senescent cells were retained in the epidermis, often for weeks after transgene silencing, indicating an absence of an efficient rapidly acting mechanism for their removal. Stem cells in the hair follicle bulge were largely protected from apoptosis upon p14(ARF) induction, but irreversibly lost their ability to proliferate and initiate follicle growth. Interestingly, induction of epidermal hyperplasia prevented the appearance of senescent cells upon p14(ARF) induction. Our findings provide basic insights into the dynamics of cellular senescence, a central tumor- suppressive mechanism, and reveal the potential for prolonged retention of senescent cells within tissues.


Assuntos
Apoptose , Epiderme/metabolismo , Proteína Supressora de Tumor p14ARF/metabolismo , Animais , Ciclo Celular , Senescência Celular , Inativação Gênica , Folículo Piloso/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , RNA/metabolismo , Transgenes , Proteína Supressora de Tumor p14ARF/fisiologia , Proteína Supressora de Tumor p53/metabolismo , beta-Galactosidase/metabolismo
10.
PLoS One ; 8(4): e61353, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23630584

RESUMO

Mutations in the p53 tumor suppressor protein are highly frequent in tumors and often endow cells with tumorigenic capacities. We sought to examine a possible role for mutant p53 in the cross-talk between cancer cells and their surrounding stroma, which is a crucial factor affecting tumor outcome. Here we present a novel model which enables individual monitoring of the response of cancer cells and stromal cells (fibroblasts) to co-culturing. We found that fibroblasts elicit the interferon beta (IFNß) pathway when in contact with cancer cells, thereby inhibiting their migration. Mutant p53 in the tumor was able to alleviate this response via SOCS1 mediated inhibition of STAT1 phosphorylation. IFNß on the other hand, reduced mutant p53 RNA levels by restricting its RNA stabilizer, WIG1. These data underscore mutant p53 oncogenic properties in the context of the tumor microenvironment and suggest that mutant p53 positive cancer patients might benefit from IFNß treatment.


Assuntos
Fibroblastos/metabolismo , Interferon beta/metabolismo , Microambiente Tumoral , Proteína Supressora de Tumor p53/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Técnicas de Cocultura , Humanos , Neoplasias Pulmonares , Mutação , Proteínas Nucleares/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Estabilidade de RNA , Proteínas de Ligação a RNA , Fator de Transcrição STAT1/metabolismo , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Evasão Tumoral , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima
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