RESUMO
Cellular senescence, a stable form of cell cycle arrest, accompanied by pronounced secretory activity, has functional roles in both physiological and pathological conditions. Although senescence has been linked for a long time with cancer and ageing, recent studies have revealed a functional role of senescence in development, regeneration and reprogramming. Notably, the transient presence of senescent cells may be beneficial, in contrast to the potential deleterious effects of persistent senescence in aged or chronically damaged tissues. We will discuss how senescence contributes to embryonic development, cell plasticity and tissue regeneration, as a highly coordinated and programmed cellular state.
Assuntos
Plasticidade Celular , Neoplasias , Humanos , Idoso , Senescência Celular/genética , Envelhecimento/genética , Pontos de Checagem do Ciclo Celular , Neoplasias/genética , Neoplasias/metabolismoRESUMO
The placenta is an autonomous organ that maintains fetal growth and development. Its multinucleated syncytiotrophoblast layer, providing fetal nourishment during gestation, exhibits characteristics of cellular senescence. We show that in human placentas from pregnancies with intrauterine growth restriction, these characteristics are decreased. To elucidate the functions of pathways regulating senescence in syncytiotrophoblast, we used dynamic contrast-enhanced MRI in mice with attenuated senescence programs. This approach revealed an altered dynamics in placentas of p53-/- , Cdkn2a-/- , and Cdkn2a-/- ;p53-/- mice, accompanied by histopathological changes in placental labyrinths. Human primary syncytiotrophoblast upregulated senescence markers and molecular pathways associated with cell-cycle inhibition and senescence-associated secretory phenotype. The pathways and components of the secretory phenotype were compromised in mouse placentas with attenuated senescence and in human placentas from pregnancies with intrauterine growth restriction. We propose that molecular mediators of senescence regulate placental structure and function, through both cell-autonomous and non-autonomous mechanisms.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Retardo do Crescimento Fetal/genética , Redes Reguladoras de Genes , Placenta/diagnóstico por imagem , Proteína Supressora de Tumor p53/genética , Animais , Senescência Celular , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Humanos , Imageamento por Ressonância Magnética , Camundongos , Fenótipo , Placenta/metabolismo , Gravidez , Transdução de Sinais , Trofoblastos/metabolismoRESUMO
Mammalian cells mostly rely on extracellular molecules to transfer signals to other cells. However, in stress conditions, more robust mechanisms might be necessary to facilitate cell-cell communications. Cellular senescence, a stress response associated with permanent exit from the cell cycle and the development of an immunogenic phenotype, limits both tumorigenesis and tissue damage. Paradoxically, the long-term presence of senescent cells can promote tissue damage and aging within their microenvironment. Soluble factors secreted from senescent cells mediate some of these cell-nonautonomous effects. However, it is unknown whether senescent cells impact neighboring cells by other mechanisms. Here we show that senescent cells directly transfer proteins to neighboring cells and that this process facilitates immune surveillance of senescent cells by natural killer (NK) cells. We found that transfer of proteins to NK and T cells is increased in the murine preneoplastic pancreas, a site where senescent cells are present in vivo. Proteomic analysis and functional studies of the transferred proteins revealed that the transfer is strictly dependent on cell-cell contact and CDC42-regulated actin polymerization and is mediated at least partially by cytoplasmic bridges. These findings reveal a novel mode of intercellular communication by which senescent cells regulate their immune surveillance and might impact tumorigenesis and tissue aging.
Assuntos
Senescência Celular/fisiologia , Pâncreas/citologia , Actinas/metabolismo , Animais , Comunicação Celular/fisiologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Camundongos , Pâncreas/fisiologia , Polimerização , Transporte Proteico , Linfócitos T/citologia , Linfócitos T/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Cellular senescence is a permanent state of cell cycle arrest that protects the organism from tumorigenesis and regulates tissue integrity upon damage and during tissue remodeling. However, accumulation of senescent cells in tissues during aging contributes to age-related pathologies. A deeper understanding of the mechanisms regulating the viability of senescent cells is therefore required. Here, we show that the CDK inhibitor p21 (CDKN1A) maintains the viability of DNA damage-induced senescent cells. Upon p21 knockdown, senescent cells acquired multiple DNA lesions that activated ataxia telangiectasia mutated (ATM) and nuclear factor (NF)-κB kinase, leading to decreased cell survival. NF-κB activation induced TNF-α secretion and JNK activation to mediate death of senescent cells in a caspase- and JNK-dependent manner. Notably, p21 knockout in mice eliminated liver senescent stellate cells and alleviated liver fibrosis and collagen production. These findings define a novel pathway that regulates senescent cell viability and fibrosis.
Assuntos
Caspases/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Regulação da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Animais , Linhagem Celular , Sobrevivência Celular , Humanos , CamundongosRESUMO
Cellular senescence limits proliferation of potentially detrimental cells, preventing tumorigenesis and restricting tissue damage. However, the function of senescence in nonpathological conditions is unknown. We found that the human placental syncytiotrophoblast exhibited the phenotype and expressed molecular markers of cellular senescence. During embryonic development, ERVWE1-mediated cell fusion results in formation of the syncytiotrophoblast, which serves as the maternal/fetal interface at the placenta. Expression of ERVWE1 caused cell fusion in normal and cancer cells, leading to formation of hyperploid syncytia exhibiting features of cellular senescence. Infection by the measles virus, which leads to cell fusion, also induced cellular senescence in normal and cancer cells. The fused cells activated the main molecular pathways of senescence, the p53- and p16-pRb-dependent pathways; the senescence-associated secretory phenotype; and immune surveillance-related proteins. Thus, fusion-induced senescence might be needed for proper syncytiotrophoblast function during embryonic development, and reuse of this senescence program later in life protects against pathological expression of endogenous fusogens and fusogenic viral infections.
Assuntos
Senescência Celular/fisiologia , Produtos do Gene env/metabolismo , Vírus do Sarampo/fisiologia , Proteínas da Gravidez/metabolismo , Fusão Celular , Linhagem Celular , Linhagem Celular Tumoral , Senescência Celular/genética , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Regulação da Expressão Gênica , Produtos do Gene env/genética , Humanos , Sarampo/fisiopatologia , Placenta/citologia , Gravidez , Proteínas da Gravidez/genética , Trofoblastos/metabolismoRESUMO
The T-cell receptor (TCR) repertoire is formed by random recombinations of genomic precursor elements; the resulting combinatorial diversity renders unlikely extensive TCR sharing between individuals. Here, we studied CDR3ß amino acid sequence sharing in a repertoire-wide manner, using high-throughput TCR-seq in 28 healthy mice. We uncovered hundreds of public sequences shared by most mice. Public CDR3 sequences, relative to private sequences, are two orders of magnitude more abundant on average, express restricted V/J segments, and feature high convergent nucleic acid recombination. Functionally, public sequences are enriched for MHC-diverse CDR3 sequences that were previously associated with autoimmune, allograft, and tumor-related reactions, but not with anti-pathogen-related reactions. Public CDR3 sequences are shared between mice of different MHC haplotypes, but are associated with different, MHC-dependent, V genes. Thus, despite their random generation process, TCR repertoires express a degree of uniformity in their post-genomic organization. These results, together with numerical simulations of TCR genomic rearrangements, suggest that biases and convergence in TCR recombination combine with ongoing selection to generate a restricted subset of self-associated, public CDR3 TCR sequences, and invite reexamination of the basic mechanisms of T-cell repertoire formation.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Motivos de Nucleotídeos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Análise de Sequência de RNA/métodos , Animais , Linfócitos T CD4-Positivos/imunologia , Feminino , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Recombinação V(D)JRESUMO
p73 has been identified as a structural and functional homolog of the tumor suppressor p53. The transcriptional coactivator Yes-associated protein (YAP) has been demonstrated to interact with and to enhance p73-dependent apoptosis in response to DNA damage. Here, we show the existence of a proapoptotic autoregulatory feedback loop between p73, YAP, and the promyelocytic leukemia (PML) tumor suppressor gene. We demonstrate that PML is a direct transcriptional target of p73/YAP, and we show that PML transcriptional activation by p73/YAP is under the negative control of the proto-oncogenic Akt/PKB kinase. Importantly, we find that PML and YAP physically interact through their PVPVY and WW domains, respectively, causing PML-mediated sumoylation and stabilization of YAP. Hence, we determine a mechanistic pathway in response to DNA damage that could have relevant implications for the treatment of human cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Proteínas de Ligação a DNA/metabolismo , Retroalimentação Fisiológica , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular , Linhagem Celular , Cisplatino/farmacologia , Retroalimentação Fisiológica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Modelos Biológicos , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteína da Leucemia Promielocítica , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética , Ubiquitina/metabolismo , Proteínas de Sinalização YAPRESUMO
The adaptive arm of the immune system has been suggested as an important factor in brain function. However, given the fact that interactions of neurons or glial cells with T lymphocytes rarely occur within the healthy CNS parenchyma, the underlying mechanism is still a mystery. Here we found that at the interface between the brain and blood circulation, the epithelial layers of the choroid plexus (CP) are constitutively populated with CD4(+) effector memory cells with a T-cell receptor repertoire specific to CNS antigens. With age, whereas CNS specificity in this compartment was largely maintained, the cytokine balance shifted in favor of the T helper type 2 (Th2) response; the Th2-derived cytokine IL-4 was elevated in the CP of old mice, relative to IFN-γ, which decreased. We found this local cytokine shift to critically affect the CP epithelium, triggering it to produce the chemokine CCL11 shown to be associated with cognitive dysfunction. Partial restoration of cognitive ability in aged mice, by lymphopenia-induced homeostasis-driven proliferation of memory T cells, was correlated with restoration of the IL-4:IFN-γ ratio at the CP and modulated the expression of plasticity-related genes at the hippocampus. Our data indicate that the cytokine milieu at the CP epithelium is affected by peripheral immunosenescence, with detrimental consequences to the aged brain. Amenable to immunomodulation, this interface is a unique target for arresting age-related cognitive decline.
Assuntos
Envelhecimento/imunologia , Envelhecimento/patologia , Encéfalo/imunologia , Encéfalo/patologia , Plexo Corióideo/imunologia , Plexo Corióideo/patologia , Células Th2/imunologia , Células Th2/patologia , Imunidade Adaptativa , Animais , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/patologia , Proliferação de Células , Epitélio/imunologia , Epitélio/patologia , Hipocampo/imunologia , Hipocampo/patologia , Memória Imunológica , Linfopenia/imunologia , Linfopenia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuroimunomodulação , Receptores de Interferon/deficiência , Receptores de Interferon/genética , Receptor de Interferon gamaRESUMO
MOTIVATION: The clonal theory of adaptive immunity proposes that immunological responses are encoded by increases in the frequency of lymphocytes carrying antigen-specific receptors. In this study, we measure the frequency of different T-cell receptors (TcR) in CD4 + T cell populations of mice immunized with a complex antigen, killed Mycobacterium tuberculosis, using high throughput parallel sequencing of the TcRß chain. Our initial hypothesis that immunization would induce repertoire convergence proved to be incorrect, and therefore an alternative approach was developed that allows accurate stratification of TcR repertoires and provides novel insights into the nature of CD4 + T-cell receptor recognition. RESULTS: To track the changes induced by immunization within this heterogeneous repertoire, the sequence data were classified by counting the frequency of different clusters of short (3 or 4) continuous stretches of amino acids within the antigen binding complementarity determining region 3 (CDR3) repertoire of different mice. Both unsupervised (hierarchical clustering) and supervised (support vector machine) analyses of these different distributions of sequence clusters differentiated between immunized and unimmunized mice with 100% efficiency. The CD4 + TcR repertoires of mice 5 and 14 days postimmunization were clearly different from that of unimmunized mice but were not distinguishable from each other. However, the repertoires of mice 60 days postimmunization were distinct both from naive mice and the day 5/14 animals. Our results reinforce the remarkable diversity of the TcR repertoire, resulting in many diverse private TcRs contributing to the T-cell response even in genetically identical mice responding to the same antigen. However, specific motifs defined by short stretches of amino acids within the CDR3 region may determine TcR specificity and define a new approach to TcR sequence classification. AVAILABILITY AND IMPLEMENTATION: The analysis was implemented in R and Python, and source code can be found in Supplementary Data. CONTACT: b.chain@ucl.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Regiões Determinantes de Complementaridade/química , Receptores de Antígenos de Linfócitos T/química , Sequência de Aminoácidos , Animais , Análise por Conglomerados , Imunização , Camundongos , Mycobacterium tuberculosis/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/química , Análise de Sequência de Proteína , Máquina de Vetores de SuporteRESUMO
T cells play fundamental roles in adaptive immunity, relying on a diverse repertoire of T-cell receptor (TCR) α and ß chains. Diversity of the TCR ß chain is generated in part by a random yet intrinsically biased combinatorial rearrangement of variable (Vß), diversity (Dß), and joining (Jß) gene segments. The mechanisms that determine biases in gene segment use remain unclear. Here we show, using a high-throughput TCR sequencing approach, that a physical model of chromatin conformation at the DJß genomic locus explains more than 80% of the biases in Jß use that we measured in murine T cells. This model also predicts correctly how differences in intersegment genomic distances between humans and mice translate into differences in Jß bias between TCR repertoires of these two species. As a consequence of these structural and other biases, TCR sequences are produced with different a priori frequencies, thus affecting their probability of becoming public TCRs that are shared among individuals. Surprisingly, we find that many more TCR sequences are shared among all five mice we studied than among only subgroups of three or four mice. We derive a necessary mathematical condition explaining this finding, which indicates that the TCR repertoire contains a core set of receptor sequences that are highly abundant among individuals, if their a priori probability of being produced by the recombination process is higher than a defined threshold. Our results provide evidence for an expanded role of chromatin conformation in VDJ rearrangement, from control of gene accessibility to precise determination of gene segment use.
Assuntos
Montagem e Desmontagem da Cromatina/imunologia , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/fisiologia , Loci Gênicos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/imunologia , Animais , Montagem e Desmontagem da Cromatina/genética , Camundongos , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
Cellular senescence is a permanent growth arrest mechanism triggered by various forms of stress. Senescent cells accumulate in the mammalian organism with age and are present at sites of tissue damage and age related pathologies. However, the characterization of senescence cells in vivo is currently limited and the need for new technologies to detect and monitor the senescence state in vivo has greatly increased. Here we demonstrate the use of the ImageStreamX as a powerful method for detection and quantification of senescent cells at distinct tissues and cell subpopulations. The identification of senescent cells using ImageStreamX enables the use of a combination of several senescence-related markers, together with the commonly used senescence-associated beta-galactosidase assay. These can be combined with the use of other molecular features typical of senescence cells, such as the γH2AX foci, indicating the activation of DNA damage response. This novel method offers a feasible solution to quantify senescent cells in vivo, in a comprehensive manner. Such quantification is necessary in order to understand the role of cellular senescence in aging and disease.
Assuntos
Bioensaio/métodos , Biomarcadores/análise , Senescência Celular , Dano ao DNA , Proteína HMGB1/metabolismo , Histonas/metabolismo , beta-Galactosidase/metabolismo , Animais , Colo/citologia , Colo/metabolismo , Pulmão/citologia , Pulmão/metabolismo , CamundongosRESUMO
BACKGROUND: Early-onset preeclampsia (EOPE; <34 weeks' gestation) usually has more severe morbidity for the mother and fetus compared to late-onset preeclampsia (LOPE). Telomere homeostasis is disrupted in preeclampsia (PE) and senescence markers are increased. The pathophysiologic differences between early and LOPE are not fully unraveled yet. METHODS: We studied placental biopsies from 7 pregnancies with EOPE, 6 pregnancies with LOPE, and 13 healthy gestational age-matched controls. Telomere length and aggregate formation were assessed using qualitative fluorescence in situ hybridization and electronic quantitative methods. Senescence markers were evaluated including senescence-associated heterochromatin foci, ß-galactosidase (SAß-Gal), and P16 staining, as was the expression of P16 complementary DNA (cDNA) using real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: There were no differences in maternal age, gravidity, parity, body mass index, and mode of conception between the study and the control groups. The percentage of trophoblasts with short telomeres was higher in placental samples from EOPE (52.61% [12.27%]) versus LOPE (28.72% [10.14%]); both were higher compared to controls (7.53% [5.14%], P = .03). Aggregate formation was enhanced in EOPE (8.72% [2.49%]) compared to LOPE (4.54% [1.45%]); both were higher than in healthy controls (2.72% [1.08%], P = .03). Trophoblasts from EOPE versus LOPE were more likely to stain positive for SAß-Gal and P16 compared to controls (P < .001). P16 cDNA expression assayed by RT-qPCR was 7.51 times higher in EOPE compared to controls and 5.86 times higher than in LOPE. CONCLUSIONS: Impaired telomere homeostasis and senescence markers are more prominent in EOPE versus LOPE. These findings may contribute to our understanding of the pathophysiology and explain their different clinical presentations and outcomes.
Assuntos
Senescência Celular/fisiologia , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Homeostase do Telômero/fisiologia , Adulto , Biomarcadores/metabolismo , Feminino , Idade Gestacional , Humanos , Gravidez , Fatores de Tempo , Trofoblastos/metabolismo , Adulto JovemRESUMO
We examined the microRNA profiles of Glioblastoma stem (CD133+) and non-stem (CD133-) cell populations and found up-regulation of several miRs in the CD133- cells, including miR-451, miR-486, and miR-425, some of which may be involved in regulation of brain differentiation. Transfection of GBM cells with the above miRs inhibited neurosphere formation and transfection with the mature miR-451 dispersed neurospheres, and inhibited GBM cell growth. Furthermore, transfection of miR-451 combined with Imatinib mesylate treatment had a cooperative effect in dispersal of GBM neurospheres. In addition, we identified a target site for SMAD in the promoter region of miR-451 and showed that SMAD3 and 4 activate such a promoter-luciferase construct. Transfection of SMAD in GBM cells inhibited their growth, suggesting that SMAD may drive GBM stem cells to differentiate to CD133- cells through up-regulation of miR-451 and reduces their tumorigenicity. Identification of additional miRs and target genes that regulate GBM stem cells may provide new potential drugs for therapy.
Assuntos
Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , MicroRNAs/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Piperazinas/farmacologia , Pirimidinas/farmacologia , Antígeno AC133 , Antígenos CD/análise , Benzamidas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glicoproteínas/análise , Humanos , Mesilato de Imatinib , Células-Tronco Neoplásicas/metabolismo , Peptídeos/análise , Regiões Promotoras Genéticas , Proteína Smad3/metabolismo , Proteína Smad4/metabolismoRESUMO
OBJECTIVE: Placenta percreta (PP) is an abnormal condition of trophoblast maturation and terminal differentiation through the uterine wall. We opted to study telomere homeostasis and senescence expression in trophoblasts from PP, the most severe subgroup of placenta accreta. STUDY DESIGN: Paraffin-embedded placental biopsies from pregnancies with percreta and normal placentation, matched by gestational age at delivery, were assessed for telomere length, aggregates, and senescence-associated heterochromatin foci using quantitative fluorescence in situ hybridization. Cyclin-dependent kinase inhibitors p21, p15, p16, and the tumor suppressor protein p53, known senescence-related markers, were assessed using immunohistochemical staining. RESULTS: Short telomeres were found more often in trophoblasts from the samples of PP (n = 9) compared to controls (n = 8; 54% ± 20% vs 2.3% ± 1.16%, respectively; P < .05). More cells with telomere aggregates (18.3% ± 6.9%) were observed in the PP than in the control group (4.8% ± 5.4%; P = .0005). The percentage of nucleic senescence-associated heterochromatin foci in the PP and control samples was similar (10.9% ± 10.4% vs 10.7% ± 15%, respectively; P = .97). Immunohistochemistry of senescence markers was expressed differently in PP compared to the controls: higher p15 expression (46.42% ± 15.2% vs 36.63% ± 12.2%, P = .004), higher p21 expression (59.8% ± 22.1% vs 47.5% ± 21.9%, P = .011), lower p16 expression (54.8% ± 26.3% vs 73.4% ± 18.9%, P = .000), and lower p53 expression (24.4% ± 33.8% vs 34% ± 14.4%, P = .000). CONCLUSION: Placenta percreta exhibits telomere alterations and changes in expression of several senescence markers. These might be related to altered trophoblast invasion maturation and placental detachment postpartum.
Assuntos
Senescência Celular , Placenta Acreta/fisiopatologia , Homeostase do Telômero , Adulto , Feminino , Humanos , Placenta Acreta/metabolismo , Gravidez , Trofoblastos/metabolismo , Trofoblastos/fisiologiaRESUMO
Cellular senescence is a stress response that imposes stable cell-cycle arrest in damaged cells, preventing their propagation in tissues. However, senescent cells accumulate in tissues in advanced age, where they might promote tissue degeneration and malignant transformation. The extent of immune-system involvement in regulating age-related accumulation of senescent cells, and its consequences, are unknown. Here we show that Prf1-/- mice with impaired cell cytotoxicity exhibit both higher senescent-cell tissue burden and chronic inflammation. They suffer from multiple age-related disorders and lower survival. Strikingly, pharmacological elimination of senescent-cells by ABT-737 partially alleviates accelerated aging phenotype in these mice. In LMNA+/G609G progeroid mice, impaired cell cytotoxicity further promotes senescent-cell accumulation and shortens lifespan. ABT-737 administration during the second half of life of these progeroid mice abrogates senescence signature and increases median survival. Our findings shed new light on mechanisms governing senescent-cell presence in aging, and could motivate new strategies for regenerative medicine.
Assuntos
Senescência Celular , Imunossenescência , Perforina/fisiologia , Animais , Compostos de Bifenilo/farmacologia , Compostos de Bifenilo/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Feminino , Inflamação/etiologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitrofenóis/farmacologia , Nitrofenóis/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Progéria/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêuticoRESUMO
Epidermal growth factor receptor (EGFR) mutations identify patients with lung cancer who derive benefit from kinase inhibitors. However, most patients eventually develop resistance, primarily due to the T790M second-site mutation. Irreversible inhibitors (e.g., osimertinib/AZD9291) inhibit T790M-EGFR, but several mechanisms, including a third-site mutation, C797S, confer renewed resistance. We previously reported that a triple mixture of monoclonal antibodies, 3×mAbs, simultaneously targeting EGFR, HER2, and HER3, inhibits T790M-expressing tumors. We now report that 3×mAbs, including a triplet containing cetuximab and trastuzumab, inhibits C797S-expressing tumors. Unlike osimertinib, which induces apoptosis, 3×mAbs promotes degradation of the three receptors and induces cellular senescence. Consistent with distinct mechanisms, treatments combining 3×mAbs plus sub-inhibitory doses of osimertinib synergistically and persistently eliminated tumors. Thus, oligoclonal antibodies, either alone or in combination with kinase inhibitors, might preempt repeated cycles of treatment and rapid emergence of resistance.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Cetuximab/farmacologia , Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/terapia , Piperazinas/farmacologia , Trastuzumab/farmacologia , Acrilamidas , Compostos de Anilina , Apoptose , Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/genética , Humanos , Imunoterapia , Neoplasias Pulmonares/genética , Mutação , Piperazinas/administração & dosagem , Inibidores de Proteínas QuinasesRESUMO
To gain insight into the transformation of epidermal cells into squamous carcinoma cells (SCC), we compared the response to ultraviolet B radiation (UVB) of normal human epidermal keratinocytes (NHEK) versus their transformed counterpart, SCC, using biological and molecular profiling. DNA microarray analyses (Affymetrix), approximately 12000 genes) indicated that the major group of upregulated genes in keratinocytes fall into three categories: (i). antiapoptotic and cell survival factors, including chemokines of the CXC/CC subfamilies (e.g. IL-8, GRO-1, -2, -3, SCYA20), growth factors (e.g. HB-EGF, CTGF, INSL-4), and proinflammatory mediators (e.g. COX-2, S100A9), (ii). DNA repair-related genes (e.g. GADD45, ERCC, BTG-1, Histones), and (iii). ECM proteases (MMP-1, -10). The major downregulated genes are DeltaNp63 and PUMILIO, two potential markers for the maintenance of keratinocyte stem cells. NHEK were found to be more resistant than SCC to UVB-induced apoptosis and this resistance was mainly because of the protection from cell death by secreted survival factors, since it can be transferred from NHEK to SCC cultures by the conditioned medium. Whereas the response of keratinocytes to UVB involved regulation of key checkpoint genes (p53, MDM2, p21(Cip1), DeltaNp63), as well as antiapoptotic and DNA repair-related genes - no or little regulation of these genes was observed in SCC. The effect of UVB on NHEK and SCC resulted in upregulation of 251 and 127 genes, respectively, and downregulation of 322 genes in NHEK and 117 genes in SCC. To further analyse these changes, we used a novel unsupervised coupled two-way clustering method that allowed the identification of groups of genes that clearly partitioned keratinocytes from SCC, including a group of genes whose constitutive expression levels were similar before UVB. This allowed the identification of discriminating genes not otherwise revealed by simple static comparison in the absence of UVB irradiation. The implication of the changes in gene profile in keratinocytes for epithelial cancer is discussed.
Assuntos
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/radioterapia , Genoma Humano , Queratinócitos/efeitos da radiação , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/radioterapia , Terapia Ultravioleta , Apoptose/genética , Apoptose/efeitos da radiação , Carcinoma de Células Escamosas/patologia , Transformação Celular Neoplásica , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Cutâneas/patologiaRESUMO
Reduction in T cell receptor (TCR) diversity in old age is considered as a major cause for immune complications in the elderly population. Here, we explored the consequences of aging on the TCR repertoire in mice using high-throughput sequencing (TCR-seq). We mapped the TCRß repertoire of CD4+ T cells isolated from bone marrow (BM) and spleen of young and old mice. We found that TCRß diversity is reduced in spleens of aged mice but not in their BM. Splenic CD4+ T cells were also skewed toward an effector memory phenotype in old mice, while BM cells preserved their memory phenotype with age. Analysis of Vß and Jß gene usage across samples, as well as comparison of CDR3 length distributions, showed no significant age dependent changes. However, comparison of the frequencies of amino-acid (AA) TCRß sequences between samples revealed repertoire changes that occurred at a more refined scale. The BM-derived TCRß repertoire was found to be similar among individual mice regardless of their age. In contrast, the splenic repertoire of old mice was not similar to those of young mice, but showed an increased similarity with the BM repertoire. Each old-mouse had a private set of expanded TCRß sequences. Interestingly, a fraction of these sequences was found also in the BM of the same individual, sharing the same nucleotide sequence. Together, these findings show that the composition and phenotype of the CD4+ T cell BM repertoire are relatively stable with age, while diversity of the splenic repertoire is severely reduced. This reduction is caused by idiosyncratic expansions of tens to hundreds of T cell clonotypes, which dominate the repertoire of each individual. We suggest that these private and abundant clonotypes are generated by sporadic clonal expansions, some of which correspond to pre-existing BM clonotypes. These organ- and age-specific changes of the TCRß repertoire have implications for understanding and manipulating age-associated immune decline.