RESUMO
BACKGROUND: Ability to adapt to temperature changes trough the Heat Shock Response (HSR) pathways is one of the most fundamental and clinically relevant cellular response systems. Heat Shock (HS) affects the signalling and gene expression responses of the Nuclear Factor κB (NF-κB) transcription factor, a critical regulator of proliferation and inflammation, however, our quantitative understanding of how cells sense and adapt to temperature changes is limited. METHODS: We used live-cell time-lapse microscopy and mathematical modelling to understand the signalling of the NF-κB system in the human MCF7 breast adenocarcinoma cells in response to pro-inflammatory Interleukin 1ß (IL1ß) and Tumour Necrosis Factor α (TNFα) cytokines, following exposure to a 37-43 °C range of physiological and clinical temperatures. RESULTS: We show that exposure to 43 °C 1 h HS inhibits the immediate NF-κB signalling response to TNFα and IL1ß stimulation although uptake of cytokines is not impaired. Within 4 h after HS treatment IL1ß-induced NF-κB responses return to normal levels, but the recovery of the TNFα-induced responses is still affected. Using siRNA knock-down of Heat Shock Factor 1 (HSF1) we show that this stimulus-specificity is conferred via the Inhibitory κB kinase (IKK) signalosome where HSF1-dependent feedback regulates TNFα, but not IL1ß-mediated IKK recovery post HS. Furthermore, we demonstrate that through the temperature-dependent denaturation and recovery of IKK, TNFα and IL1ß-mediated signalling exhibit different temperature sensitivity and adaptation to repeated HS when exposed to a 37-43 °C temperature range. Specifically, IL1ß-mediated NF-κB responses are more robust to temperature changes in comparison to those induced by TNFα treatment. CONCLUSIONS: We demonstrate that the kinetics of the NF-κB system following temperature stress is cytokine specific and exhibit differential adaptation to temperature changes. We propose that this differential temperature sensitivity is mediated via the IKK signalosome, which acts as a bona fide temperature sensor trough the HSR cross-talk. This novel quantitative understanding of NF-κB and HSR interactions is fundamentally important for the potential optimization of therapeutic hyperthermia protocols. Video Abstract.
Assuntos
Expressão Gênica/efeitos dos fármacos , Resposta ao Choque Térmico , Inflamação/metabolismo , Interleucina-1beta/farmacologia , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Humanos , Células MCF-7RESUMO
Modern immunotherapy together with targeted therapy has revolutionized the treatment of advanced melanoma. Inhibition of immune checkpoints significantly improved the median overall survival and gave hope to many melanoma patients. However, this treatment has three serious drawbacks: high cost, serious side effects, and an effectiveness limited only to approximately 50% of patients. Some patients do not derive any or short-term benefit from this treatment due to primary or secondary resistance. The response to immunotherapy depends on many factors that fall into three main categories: those associated with melanoma cells, those linked to a tumor and its microenvironment, and those classified as individual ontogenic and physiological features of the patient. The first category comprises expression of PD-L1 and HLA proteins on melanoma cells as well as genetic/genomic metrics such as mutational load, (de)activation of specific signaling pathways and epigenetic factors. The second category is the inflammatory status of the tumor: "hot" versus "cold" (i.e., high versus low infiltration of immune cells). The third category comprises metabolome and single nucleotide polymorphisms of specific genes. Here we present up-to-date data on those biological factors influencing melanoma response to immunotherapy with a special focus on signaling pathways regulating the complex process of anti-tumor immune response. We also discuss their potential predictive capacity.
Assuntos
Biomarcadores Tumorais , Inibidores de Checkpoint Imunológico/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/etiologia , Terapia de Alvo Molecular , Animais , Apresentação de Antígeno/imunologia , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Suscetibilidade a Doenças , Resistencia a Medicamentos Antineoplásicos , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Imunomodulação/efeitos dos fármacos , Imunoterapia , Melanoma/diagnóstico , Melanoma/metabolismo , Mutação , Prognóstico , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Resultado do TratamentoRESUMO
Elevated temperature induces the heat shock (HS) response, which modulates cell proliferation, apoptosis, the immune and inflammatory responses. However, specific mechanisms linking the HS response pathways to major cellular signaling systems are not fully understood. Here we used integrated computational and experimental approaches to quantitatively analyze the crosstalk mechanisms between the HS-response and a master regulator of inflammation, cell proliferation, and apoptosis the Nuclear Factor κB (NF-κB) system. We found that populations of human osteosarcoma cells, exposed to a clinically relevant 43°C HS had an attenuated NF-κB p65 response to Tumor Necrosis Factor α (TNFα) treatment. The degree of inhibition of the NF-κB response depended on the HS exposure time. Mathematical modeling of single cells indicated that individual crosstalk mechanisms differentially encode HS-mediated NF-κB responses while being consistent with the observed population-level responses. In particular "all-or-nothing" encoding mechanisms were involved in the HS-dependent regulation of the IKK activity and IκBα phosphorylation, while others involving transport were "analogue". In order to discriminate between these mechanisms, we used live-cell imaging of nuclear translocations of the NF-κB p65 subunit. The single cell responses exhibited "all-or-nothing" encoding. While most cells did not respond to TNFα stimulation after a 60 min HS, 27% showed responses similar to those not receiving HS. We further demonstrated experimentally and theoretically that the predicted inhibition of IKK activity was consistent with the observed HS-dependent depletion of the IKKα and IKKß subunits in whole cell lysates. However, a combination of "all-or-nothing" crosstalk mechanisms was required to completely recapitulate the single cell data. We postulate therefore that the heterogeneity of the single cell responses might be explained by the cell-intrinsic variability of HS-modulated IKK signaling. In summary, we show that high temperature modulates NF-κB responses in single cells in a complex and unintuitive manner, which needs to be considered in hyperthermia-based treatment strategies.
Assuntos
Resposta ao Choque Térmico/fisiologia , Modelos Biológicos , NF-kappa B/metabolismo , Linhagem Celular , Biologia Computacional , Simulação por Computador , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Análise de Célula Única , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Spermatocytes are among the most heat-sensitive cells and the exposure of testes to heat shock results in their Heat Shock Factor 1 (HSF1)-mediated apoptosis. Several lines of evidence suggest that pleckstrin-homology-like domain family A, member 1 (PHLDA1) plays a role in promoting heat shock-induced cell death in spermatogenic cells, yet its precise physiological role is not well understood. Aiming to elucidate the hypothetical role of PHLDA1 in HSF1-mediated apoptosis of spermatogenic cells we characterized its expression in mouse testes during normal development and after heat shock. We stated that transcription of Phlda1 is upregulated by heat shock in many adult mouse organs including the testes. Analyzes of the Phlda1 expression during postnatal development indicate that it is expressed in pre-meiotic or somatic cells of the testis. It starts to be transcribed much earlier than spermatocytes are fully developed and its transcripts and protein products do not accumulate further in the later stages. Moreover, neither heat shock nor expression of constitutively active HSF1 results in the accumulation of PHLDA1 protein in meiotic and post-meiotic cells although both conditions induce massive apoptosis of spermatocytes. Furthermore, the overexpression of PHLDA1 in NIH3T3 cells leads to cell detachment, yet classical apoptosis is not observed. Therefore, our findings indicate that PHLDA1 cannot directly contribute to the heat-induced apoptosis of spermatocytes. Instead, PHLDA1 could hypothetically participate in death of spermatocytes indirectly via activation of changes in the somatic or pre-meiotic cells present in the testes.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Fatores de Transcrição de Choque Térmico/farmacologia , Espermatócitos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Clonagem Molecular , Resposta ao Choque Térmico/fisiologia , Masculino , Camundongos , Células NIH 3T3 , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Testículo/metabolismo , Testículo/patologia , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: The cellular response to ionizing radiation involves activation of p53-dependent pathways and activation of the atypical NF-κB pathway. The crosstalk between these two transcriptional networks include (co)regulation of common gene targets. Here we looked for novel genes potentially (co)regulated by p53 and NF-κB using integrative genomics screening in human osteosarcoma U2-OS cells irradiated with a high dose (4 and 10 Gy). Radiation-induced expression in cells with silenced TP53 or RELA (coding the p65 NF-κB subunit) genes was analyzed by RNA-Seq while radiation-enhanced binding of p53 and RelA in putative regulatory regions was analyzed by ChIP-Seq, then selected candidates were validated by qPCR. RESULTS: We identified a subset of radiation-modulated genes whose expression was affected by silencing of both TP53 and RELA, and a subset of radiation-upregulated genes where radiation stimulated binding of both p53 and RelA. For three genes, namely IL4I1, SERPINE1, and CDKN1A, an antagonistic effect of the TP53 and RELA silencing was consistent with radiation-enhanced binding of both p53 and RelA. This suggested the possibility of a direct antagonistic (co)regulation by both factors: activation by NF-κB and inhibition by p53 of IL4I1, and activation by p53 and inhibition by NF-κB of CDKN1A and SERPINE1. On the other hand, radiation-enhanced binding of both p53 and RelA was observed in a putative regulatory region of the RRAD gene whose expression was downregulated both by TP53 and RELA silencing, which suggested a possibility of direct (co)activation by both factors. CONCLUSIONS: Four new candidates for genes directly co-regulated by NF-κB and p53 were revealed.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Ósseas/genética , Regulação Neoplásica da Expressão Gênica , Osteossarcoma/genética , Radiação Ionizante , Sítios de Ligação , Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/radioterapia , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , L-Aminoácido Oxidase/genética , L-Aminoácido Oxidase/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Osteossarcoma/patologia , Osteossarcoma/radioterapia , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativação Transcricional , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
SPEN (spen family transcription repressor) is a nucleic acid-binding protein putatively involved in repression of gene expression. We hypothesized that SPEN could be involved in general downregulation of the transcription during the heat shock response in mouse spermatogenic cells through its interactions with chromatin. We documented predominant nuclear localization of the SPEN protein in spermatocytes and round spermatids, which was retained after heat shock. Moreover, the protein was excluded from the highly condensed chromatin. Chromatin immunoprecipitation experiments clearly indicated interactions of SPEN with chromatin in vivo However, ChIP-Seq analyses did not reveal any strong specific peaks both in untreated and heat shocked cells, which might suggest dispersed localization of SPEN and/or its indirect binding to DNA. Using in situ proximity ligation assay we found close in vivo associations of SPEN with MTA1 (metastasis-associated 1), a member of the nucleosome remodeling complex with histone deacetylase activity, which might contribute to interactions of SPEN with chromatin.
Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animais , Núcleo Celular/química , Cromatina/química , Proteínas de Ligação a DNA , Histona Desacetilases/metabolismo , Temperatura Alta , Masculino , Camundongos , Camundongos Endogâmicos , Proteínas Nucleares/análise , Proteínas de Ligação a RNA , Proteínas Repressoras , Espermátides/ultraestrutura , Espermatócitos/ultraestrutura , Espermatogênese , Testículo/citologia , Transativadores , Fatores de Transcrição/metabolismoRESUMO
Heat shock transcription factors (HSFs), as regulators of heat shock proteins (HSPs) expression, are well known for their cytoprotective functions during cellular stress. They also play important yet less recognized roles in gametogenesis. All HSF family members are expressed during mammalian spermatogenesis, mainly in spermatocytes and round spermatids which are characterized by extensive chromatin remodeling. Different HSFs could cooperate to maintain proper spermatogenesis. Cooperation of HSF1 and HSF2 is especially well established since their double knockout results in meiosis arrest, spermatocyte apoptosis, and male infertility. Both factors are also involved in the repackaging of the DNA during spermatid differentiation. They can form heterotrimers regulating the basal level of transcription of target genes. Moreover, HSF1/HSF2 interactions are lost in elevated temperatures which can impair the transcription of genes essential for spermatogenesis. In most mammals, spermatogenesis occurs a few degrees below the body temperature and spermatogenic cells are extremely heat-sensitive. Pro-survival pathways are not induced by heat stress (e.g., cryptorchidism) in meiotic and postmeiotic cells. Instead, male germ cells are actively eliminated by apoptosis, which prevents transition of the potentially damaged genetic material to the next generation. Such a response depends on the transcriptional activity of HSF1 which in contrary to most somatic cells, acts as a proapoptotic factor in spermatogenic cells. HSF1 activation could be the main trigger of impaired spermatogenesis related not only to elevated temperature but also to other stress conditions; therefore, HSF1 has been proposed to be the quality control factor in male germ cells.
Assuntos
Proteínas de Choque Térmico/metabolismo , Espermatócitos/metabolismo , Espermatogênese/fisiologia , Animais , Resposta ao Choque Térmico/fisiologia , Humanos , Infertilidade Masculina/metabolismo , Masculino , Mamíferos/metabolismo , Mamíferos/fisiologiaRESUMO
Heat shock inhibits NF-κB signaling, yet the knowledge about its influence on the regulation of NF-κB-dependent genes is limited. Using genomic approaches, i.e., expression microarrays and ChIP-Seq, we aimed to establish a global picture for heat shock-mediated impact on the expression of genes regulated by TNFα cytokine. We found that 193 genes changed expression in human U-2 osteosarcoma cells stimulated with cytokine (including 77 genes with the κB motif in the proximal promoters). A large overlap between sets of genes modulated by cytokine or by heat shock was revealed (86 genes were similarly affected by both stimuli). Binding sites for heat shock-induced HSF1 were detected in regulatory regions of 1/3 of these genes. Furthermore, pre-treatment with heat shock affected the expression of 2/3 of cytokine-modulated genes. In the largest subset of co-affected genes, heat shock suppressed the cytokine-mediated activation (antagonistic effect, 83 genes), which genes were associated with the canonical functions of NF-κB signaling. However, subsets of co-activated and co-repressed genes were also revealed. Importantly, pre-treatment with heat shock resulted in the suppression of NF-κB binding in the promoters of the cytokine-upregulated genes, either antagonized or co-activated by both stimuli. In conclusion, we confirmed that heat shock inhibited activation of genes involved in the classical cytokine-mediated functions of NF-κB. On the other hand, genes involved in transcription regulation were over-represented in the subset of genes upregulated by both stimuli. This suggests the replacement of NF-κB-mediated regulation by heat shock-mediated regulation in the latter subset of genes, which may contribute to the robust response of cells to both stress conditions.
Assuntos
Citocinas/metabolismo , Febre/metabolismo , Regulação da Expressão Gênica/fisiologia , Resposta ao Choque Térmico , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Linhagem Celular Tumoral , Humanos , Transcrição GênicaRESUMO
BACKGROUND: Elevated temperatures induce activation of the heat shock transcription factor 1 (HSF1) which in somatic cells leads to heat shock proteins synthesis and cytoprotection. However, in the male germ cells (spermatocytes) caspase-3 dependent apoptosis is induced upon HSF1 activation and spermatogenic cells are actively eliminated. RESULTS: To elucidate a mechanism of such diverse HSF1 activity we carried out genome-wide transcriptional analysis in control and heat-shocked cells, either spermatocytes or hepatocytes. Additionally, to identify direct molecular targets of active HSF1 we used chromatin immunoprecipitation assay (ChIP) combined with promoter microarrays (ChIP on chip). Genes that are differently regulated after HSF1 binding during hyperthermia in both types of cells have been identified. Despite HSF1 binding to promoter sequences in both types of cells, strong up-regulation of Hsps and other genes typically activated by the heat shock was observed only in hepatocytes. In spermatocytes HSF1 binding correlates with transcriptional repression on a large scale. HSF1-bound and negatively regulated genes encode mainly for proteins required for cell division, involved in RNA processing and piRNA biogenesis. CONCLUSIONS: Observed suppression of the transcription could lead to genomic instability caused by meiotic recombination disturbances, which in turn might induce apoptosis of spermatogenic cells. We propose that HSF1-dependent induction of cell death is caused by the simultaneous repression of many genes required for spermatogenesis, which guarantees the elimination of cells damaged during heat shock. Such activity of HSF1 prevents transmission of damaged genetic material to the next generation.
Assuntos
Apoptose/genética , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Resposta ao Choque Térmico/genética , Hepatócitos/citologia , Espermatócitos/citologia , Fatores de Transcrição/metabolismo , Animais , Imunoprecipitação da Cromatina , DNA/metabolismo , Fatores de Transcrição de Choque Térmico , Hepatócitos/metabolismo , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Espermatócitos/metabolismo , Espermatogênese/genética , Transcrição GênicaRESUMO
BACKGROUND: Heat Shock Transcription Factor 1 (HSF1) is activated under stress conditions. In turn, it induces expression of Heat Shock Proteins (HSPs), which are well-known regulators of protein homeostasis. Elevated levels of HSF1 and HSPs were observed in many types of tumors. The aim of the present study was to determine whether HSF1 could have an effect on the survival of cancer cells treated with chemotherapeutic cytotoxic agents. METHODS: We constructed mouse (B16F10) and human (1205Lu, WM793B) melanoma cells overexpressing full or mutant form of human HSF1: a constitutively active one with a deletion in regulatory domain or a dominant negative one with a deletion in the activation domain. The impact of different forms of HSF1 on the expression of HSP and ABC genes was studied by RT-PCR and Western blotting. Cell cultures were treated with increasing amounts of doxorubicin, paclitaxel, cisplatin, vinblastine or bortezomib. Cell viability was determined by MTT, and IC50 was calculated. Cellular accumulation of fluorescent dyes and side population cells were studied using flow cytometry. RESULTS: Cells overexpressing HSF1 and characterized by increased HSPs accumulation were more resistant to doxorubicin or paclitaxel, but not to cisplatin, vinblastine or bortezomib. This resistance correlated with the enhanced efflux of fluorescent dyes and the increased number of side population cells. The expression of constitutively active mutant HSF1, also resulting in HSPs overproduction, did not reduce the sensitivity of melanoma cells to drugs, unlike in the case of dominant negative form expression. Cells overexpressing a full or dominant negative form of HSF1, but not a constitutively active one, had higher transcription levels of ABC genes when compared to control cells. CONCLUSIONS: HSF1 overexpression facilitates the survival of melanoma cells treated with doxorubicin or paclitaxel. However, HSF1-mediated chemoresistance is not dependent on HSPs accumulation but on an increased potential for drug efflux by ABC transporters. Direct transcriptional activity of HSF1 is not necessary for increased expression of ABC genes, which is probably mediated by HSF1 regulatory domain.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Ligação a DNA/genética , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica , Melanoma/genética , Paclitaxel/farmacologia , Fatores de Transcrição/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica , Fatores de Transcrição de Choque Térmico , Humanos , Concentração Inibidora 50 , Melanoma/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos , Mutação , Fatores de Transcrição/metabolismoRESUMO
HSF1 is a well-known heat shock protein expression regulator in response to stress. It also regulates processes important for growth, development or tumorigenesis. We studied the HSF1 influence on the phenotype of non-tumorigenic human mammary epithelial (MCF10A and MCF12A) and several triple-negative breast cancer cell lines. MCF10A and MCF12A differ in terms of HSF1 levels, morphology, growth in Matrigel, expression of epithelial (CDH1) and mesenchymal (VIM) markers (MCF10A are epithelial cells; MCF12A resemble mesenchymal cells). HSF1 down-regulation led to a reduced proliferation rate and spheroid formation in Matrigel by MCF10A cells. However, it did not affect MCF12A proliferation but led to CDH1 up-regulation and the formation of better organized spheroids. HSF1 overexpression in MCF10A resulted in reduced CDH1 and increased VIM expression and the acquisition of elongated fibroblast-like morphology. The above-mentioned results suggest that elevated levels of HSF1 may direct mammary epithelial cells toward a mesenchymal phenotype, while a lowering of HSF1 could reverse the mesenchymal phenotype to an epithelial one. Therefore, HSF1 may be involved in the remodeling of mammary gland architecture over the female lifetime. Moreover, HSF1 levels positively correlated with the invasive phenotype of triple-negative breast cancer cells, and their growth was inhibited by the HSF1 inhibitor DTHIB.
RESUMO
NF-κB transcription factor regulates numerous genes important for inflammation, immune responses and cell survival. HSF1 is the primary transcription factor activated under stress conditions that is responsible for induction of genes encoding heat shock proteins. Previous studies have shown that the NF-κB activation pathway is blocked by heat shock possibly involving heat shock proteins. Here, we investigate whether active HSF1 inhibited this pathway in the absence of stress conditions. Activation of the NF-κB pathway and expression of NF-κB-dependent genes were analyzed in TNFα-stimulated U-2 OS human osteosarcoma cells that were either heat-shocked or engineered to express a constitutively active form of HSF1 in the absence of heat shock. As expected, heat shock resulted in a general blockade in the degradation of the IκBα inhibitor, nuclear translocation of NF-κB and expression of NF-κB-dependent target genes. In marked contrast, the presence of constitutively active HSF1 did not block TNFα-induced activation of the NF-κB pathway or expression of a set of the NF-κB-dependent genes. We conclude that in the absence of heat shock, the NF-κB activation pathway is inhibited by neither active HSF1 transcription factor nor by increased levels of HSF1-induced heat shock proteins.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Proteínas de Choque Térmico/metabolismo , NF-kappa B/metabolismo , Osteossarcoma/metabolismo , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Western Blotting , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico/fisiologia , Temperatura Alta , Humanos , NF-kappa B/genética , Osteossarcoma/genética , Osteossarcoma/patologia , Plasmídeos , Reação em Cadeia da Polimerase , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fatores de Transcrição/genética , Transfecção , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The binding of capacitated spermatozoa to the egg's extracellular coat and induction of acrosome reaction are necessary for successful fertilization in mammals. Biogenesis of acrosome is complicated, and not all proteins involved in this process are known. In this study, we have cloned a novel mouse gene, Spaca7, that is expressed exclusively in the testes. During the postnatal development, transcripts of the gene could be detected at a very low level in 18-day-old mouse testes and at a higher level in 21-day-old mouse testes and later, which corresponds to an expansion of round spermatids. In the stably transfected PT67 cells, SPACA7 fused with EGFP was predominantly localized in the Golgi apparatus. In transgenic mouse testes, the fusion protein was found in acrosome (starting from the first stages of acrosome formation in late pachytene spermatocytes and finally in spermatozoa isolated from caput and cauda of epididymis). Confocal microscopy studies revealed an intra-acrosomal not membrane-bound localization of SPACA7/EGFP, which suggests that the protein can be released during acrosome reaction and involved in fertilization. Acrosomal localization of endogenous SPACA7 protein was also found in human spermatozoa.
Assuntos
Reação Acrossômica/genética , Proteínas de Plasma Seminal/isolamento & purificação , Animais , Células Cultivadas , Clonagem Molecular , Fertilização/genética , Fertilização/fisiologia , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Células NIH 3T3 , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Plasma Seminal/genética , Proteínas de Plasma Seminal/metabolismo , Espermatogênese/genética , Espermatogênese/fisiologia , Espermatozoides/química , Espermatozoides/metabolismo , TransfecçãoRESUMO
Heat Shock Factor 1 (HSF1), a transcription factor frequently overexpressed in cancer, is activated by proteotoxic agents and participates in the regulation of cellular stress response. To investigate how HSF1 level affects the response to proteotoxic stress, we integrated data from functional genomics analyses performed in MCF7 breast adenocarcinoma cells. Although the general transcriptional response to heat shock was impaired due to HSF1 deficiency (mainly chaperone expression was inhibited), a set of genes was identified, including ATF3 and certain FOS and JUN family members, whose stress-induced activation was stronger and persisted longer than in cells with normal HSF1 levels. These genes were direct HSF1 targets, suggesting a dual (activatory/suppressory) role for HSF1. Moreover, we found that heat shock-induced inflammatory response could be stronger in HSF1-deficient cells. Analyses of The Cancer Genome Atlas data indicated that higher ATF3, FOS, and FOSB expression levels correlated with low HSF1 levels in estrogen receptor-positive breast cancer, reflecting higher heat shock-induced expression of these genes in HSF1-deficient MCF7 cells observed in vitro. However, differences between the analyzed cancer types were noted in the regulation of HSF1-dependent genes, indicating the presence of cell-type-specific mechanisms. Nevertheless, our data indicate the existence of the heat shock-induced network of transcription factors (associated with the activation of TNFα signaling) which includes HSF1. Independent of its chaperone-mediated cytoprotective function, HSF1 may be involved in the regulation of this network but prevents its overactivation in some cells during stress.
Assuntos
Proteínas de Ligação a DNA , Genes fos , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição de Choque Térmico/genética , Resposta ao Choque Térmico/genética , Humanos , Inflamação/genética , Fatores de Transcrição/metabolismoRESUMO
Highly evolutionary conserved heat shock proteins (HSPs) act as molecular chaperones in regulation of cellular homeostasis and promoting survival. Generally they are induced by a variety of stressors whose effect could be disastrous on the organism, but they are also widely constitutively expressed in the absence of stress. Varied HSP expressions seem to be very essential in the critical steps of embryonic and extra-embryonic structures formation and may correspond to cell movements, proliferation, morphogenesis and apoptosis, which occur during embryonic development. While our knowledge of detailed HSP expression patterns is in constant progress, their functions during embryonic development are not yet fully understood. In the paper, we review available data on HSP expression and discuss their role during vertebrate development.
Assuntos
Desenvolvimento Embrionário , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/fisiologia , Vertebrados/embriologia , Animais , Apoptose , Movimento Celular , Proliferação de Células , Feminino , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico/genética , Mamíferos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Morfogênese , Gravidez , Xenopus , Peixe-ZebraRESUMO
The protein-DNA interactions play a crucial role in many cellular processes such as DNA replication, recombination, repair, or gene transcription. Here we describe a chromatin immunoprecipitation (ChIP) technique, a tool for identifying DNA regions, which are in vivo associated with protein of interest. The main steps of ChIP are: fixation, chromatin fragmentation, immunoprecipitation with a specific antibody, and analysis of the immunoprecipitated DNA. Different analytical tools combined with ChIP (ChIP-chip, ChIP-seq) enable researchers to map protein binding in a truly genome-wide manner with high resolution.
Assuntos
Imunoprecipitação da Cromatina/métodos , Fatores de Transcrição/metabolismo , Cromatina/metabolismo , DNA/metabolismo , Mapeamento de Peptídeos , Ligação ProteicaRESUMO
Next-generation sequencing (NGS) in liquid biopsies may contribute to the diagnosis, monitoring, and personalized therapy of cancer through the real-time detection of a tumor's genetic profile. There are a few NGS platforms offering high-sensitivity sequencing of cell-free DNA (cfDNA) samples. The aim of this study was to evaluate the Ion AmpliSeq HD Technology for targeted sequencing of tumor and liquid biopsy samples from patients with fourth-stage melanoma. Sequencing of 30 samples (FFPE tumor and liquid biopsy) derived from 14 patients using the Oncomine™ Pan-Cancer Cell-Free Assay was performed. The analysis revealed high concordance between the qPCR and NGS results of the BRAF mutation in FFPE samples (91%), as well as between the FFPE and liquid biopsy samples (91%). The plasma-tumor concordance of the non-BRAF mutations was 28%. A total of 17 pathogenic variants in 14 genes (from 52-gene panel), including TP53, CTNNB1, CCND1, MET, MAP2K1, and GNAS, were identified, with the CTNNB1S45F variant being the most frequent. A positive correlation between the LDH level and cfDNA concentration as well as negative correlation between the LDH level and time to progression was confirmed in a 22-patient cohort. The analysis showed both the potential and limitations of liquid biopsy genetic profiling using HD technology and the Ion Torrent platform.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biópsia Líquida/métodos , Melanoma/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Ácidos Nucleicos Livres/análise , Ácidos Nucleicos Livres/genética , Feminino , Formaldeído , Humanos , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Inclusão em ParafinaRESUMO
The mouse 3110001I22Rik gene located in the first intron of Bfar is considered as a Bfar variant coding for the BFARv3 protein. However, it differs from other BFAR isoforms and resembles periphilin 1 (PPHLN1) due to its two (Lge1 and serine-rich) conserved domains. We identified the BFARv3/EGFP-interacting proteins by co-immunoprecipitation coupled to mass spectrometry, which revealed 40S ribosomal proteins (RPS3, RPS14, RPS19, RPS25, RPS27), histones (H1.2, H1.4, H3.3C), proteins involved in RNA processing and splicing (SFPQ, SNRPA1, HNRNPA3, NONO, KHDRBS3), calcium signaling (HPCAL1, PTK2B), as well as HSD17B4, GRB14, POSTN, and MYO10. Co-immunoprecipitation revealed that both Lge1 and Ser-rich domains of BFARv3 were necessary for binding to RNA-interacting factors NONO and SFPQ, known to be components of paraspeckles. Reciprocal co-immunoprecipitation and the proximity ligation assay confirmed that both BFARv3 and PPHLN1 could interact with NONO and SFPQ, suggesting a new function for PPHLN1 as well. BFARv3 and its Lge1 or Ser-rich-deficient mutants preferentially localize in the nucleus. We found an accumulation of BFARv3/EGFP (but not its mutated forms) in the nuclear granules, which was enhanced in response to arsenite treatment and ionizing radiation. Although Bfar v3 is expressed ubiquitously in mouse tissues, its expression is the highest in metaphase II oocytes. The BFARv3 interactome suggests its role in RNA metabolism, which is critical for the transcriptionally silent MII oocyte. Mouse BFARv3 has no ortholog in the human genome, thus it may contribute to the differences between these two species observed in oocyte maturation and early embryonic development.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas de Membrana/genética , Oócitos/metabolismo , RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Células Cultivadas , Feminino , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos EndogâmicosRESUMO
Heat shock factor 1 (HSF1), a key regulator of transcriptional responses to proteotoxic stress, was linked to estrogen (E2) signaling through estrogen receptor α (ERα). We found that an HSF1 deficiency may decrease ERα level, attenuate the mitogenic action of E2, counteract E2-stimulated cell scattering, and reduce adhesion to collagens and cell motility in ER-positive breast cancer cells. The stimulatory effect of E2 on the transcriptome is largely weaker in HSF1-deficient cells, in part due to the higher basal expression of E2-dependent genes, which correlates with the enhanced binding of unliganded ERα to chromatin in such cells. HSF1 and ERα can cooperate directly in E2-stimulated regulation of transcription, and HSF1 potentiates the action of ERα through a mechanism involving chromatin reorganization. Furthermore, HSF1 deficiency may increase the sensitivity to hormonal therapy (4-hydroxytamoxifen) or CDK4/6 inhibitors (palbociclib). Analyses of data from The Cancer Genome Atlas database indicate that HSF1 increases the transcriptome disparity in ER-positive breast cancer and can enhance the genomic action of ERα. Moreover, only in ER-positive cancers an elevated HSF1 level is associated with metastatic disease.
About 70% of breast cancers rely on supplies of a hormone called estrogen which is the main hormone responsible for female physical characteristics to grow. Breast cancer cells that are sensitive to estrogen possess proteins known as estrogen receptors and are classified as estrogen-receptor positive. When estrogen interacts with its receptor in a cancer cell, it stimulates the cell to grow and migrate to other parts of the body. Therefore, therapies that decrease the amount of estrogen the body produces, or inhibit the receptor itself, are widely used to treat patients with estrogen receptor-positive breast cancers. When estrogen interacts with an estrogen receptor known as ERα it can also activate a protein called HSF1, which helps cells to survive under stress. In turn, HSF1 regulates several other proteins that are necessary for ERα and other estrogen receptors to work properly. Previous studies have suggested that high levels of HSF1 may worsen the outcomes for patients with estrogen receptor-positive breast cancers, but it remains unclear how HSF1 acts in breast cancer cells. Vydra, Janus, Kus et al. used genetics and bioinformatics approaches to study HSF1 in human breast cancer cells. The experiments revealed that breast cancer cells with lower levels of HSF1 also had lower levels of ERα and responded less well to estrogen than cells with higher levels of HSF1. Further experiments suggested that in the absence of estrogen, HSF1 helps to keep ERα inactive. However, when estrogen is present, HSF1 cooperates with ERα and enhances its activity to help cells grow and migrate. Vydra, Janus, Kus et al. also found that cells with higher levels of HSF1 were less sensitive to two drug therapies that are commonly used to treat estrogen receptor-positive breast cancers. These findings reveal that the effect HSF1 has on ERα activity depends on the presence of estrogen. Therefore, cancer therapies that decrease the amount of estrogen a patient produces may have a different effect on estrogen receptor-positive tumors with high HSF1 levels than tumors with low HSF1 levels.
Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Estrogênios/metabolismo , Fatores de Transcrição de Choque Térmico/genética , Transdução de Sinais , Adulto , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/metabolismo , Feminino , Fatores de Transcrição de Choque Térmico/metabolismo , Humanos , Pessoa de Meia-Idade , Adulto JovemRESUMO
Heat shock can induce either cytoprotective mechanisms or cell death. We found that in certain human and mouse cells, including spermatocytes, activated heat shock factor 1 (HSF1) binds to sequences located in the intron(s) of the PMAIP1 (NOXA) gene and upregulates its expression which induces apoptosis. Such a mode of PMAIP1 activation is not dependent on p53. Therefore, HSF1 not only can activate the expression of genes encoding cytoprotective heat shock proteins, which prevents apoptosis, but it can also positively regulate the proapoptotic PMAIP1 gene, which facilitates cell death. This could be the primary cause of hyperthermia-induced elimination of heat-sensitive cells, yet other pro-death mechanisms might also be involved.