RESUMO
In classical Hodgkin lymphoma (cHL), the highly abundant CD4+ T cells in the vicinity of tumor cells are considered essential for tumor cell survival, but are ill-defined. Although they are activated, they consistently lack expression of activation marker CD26. In this study, we compared sorted CD4+CD26- and CD4+CD26+ T cells from cHL lymph node cell suspensions by RNA sequencing and T cell receptor variable gene segment usage analysis. This revealed that although CD4+CD26- T cells are antigen experienced, they have not clonally expanded. This may well be explained by the expression of exhaustion associated transcription factors TOX and TOX2, immune checkpoints PDCD1 and CD200, and chemokine CXCL13, which were amongst the 100 significantly enriched genes in comparison with the CD4+CD26+ T cells. Findings were validated in single-cell RNA sequencing data from an independent cohort. Interestingly, immunohistochemistry revealed predominant and high frequency of staining for TOX and TOX2 in the T cells attached to the tumor cells. In conclusion, the dominant CD4+CD26- T cell population in cHL is antigen experienced, polyclonal, and exhausted. This population is likely a main contributor to the very high response rates to immune checkpoint inhibitors in cHL.
Assuntos
Linfócitos T CD4-Positivos , Proteínas HMGB , Proteínas de Grupo de Alta Mobilidade , Doença de Hodgkin , Dipeptidil Peptidase 4/imunologia , Proteínas HMGB/biossíntese , Proteínas HMGB/imunologia , Proteínas de Grupo de Alta Mobilidade/biossíntese , Proteínas de Grupo de Alta Mobilidade/imunologia , Doença de Hodgkin/genética , Doença de Hodgkin/imunologia , Doença de Hodgkin/metabolismo , Humanos , Linfonodos/patologia , Fatores de Transcrição/genéticaRESUMO
OBJECTIVE: Facilitates Chromatin Transcription (FACT) complex is a histone chaperone participating in DNA repair-related and transcription-related chromatin dynamics. In this study, we investigated its oncogenic functions, underlying mechanisms and therapeutic implications in human hepatocellular carcinoma (HCC). DESIGN: We obtained HCC and its corresponding non-tumorous liver samples from 16 patients and identified FACT complex as the most upregulated histone chaperone by RNA-Seq. We further used CRISPR-based gene activation and knockout systems to demonstrate the functions of FACT complex in HCC growth and metastasis. Functional roles and mechanistic insights of FACT complex in oxidative stress response were investigated by ChIP assay, flow cytometry, gene expression assays and 4sU-DRB transcription elongation assay. Therapeutic effect of FACT complex inhibitor, Curaxin, was tested in both in vitro and in vivo models. RESULTS: We showed that FACT complex was remarkably upregulated in HCC and contributed to HCC progression. Importantly, we unprecedentedly revealed an indispensable role of FACT complex in NRF2-driven oxidative stress response. Oxidative stress prevented NRF2 and FACT complex from KEAP1-mediated protein ubiquitination and degradation. Stabilised NRF2 and FACT complex form a positive feedback loop; NRF2 transcriptionally activates the FACT complex, while FACT complex promotes the transcription elongation of NRF2 and its downstream antioxidant genes through facilitating rapid nucleosome disassembly for the passage of RNA polymerase. Therapeutically, Curaxin effectively suppressed HCC growth and sensitised HCC cell to sorafenib. CONCLUSION: In conclusion, our findings demonstrated that FACT complex is essential for the expeditious HCC oxidative stress response and is a potential therapeutic target for HCC treatment.
Assuntos
Carcinoma Hepatocelular/fisiopatologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Grupo de Alta Mobilidade/fisiologia , Chaperonas de Histonas/fisiologia , Neoplasias Hepáticas/fisiopatologia , Estresse Oxidativo/fisiologia , Fatores de Elongação da Transcrição/fisiologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carbazóis/farmacologia , Carbazóis/uso terapêutico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/prevenção & controle , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/fisiologia , Técnicas de Inativação de Genes/métodos , Proteínas de Grupo de Alta Mobilidade/antagonistas & inibidores , Proteínas de Grupo de Alta Mobilidade/biossíntese , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Hepáticas Experimentais/fisiopatologia , Neoplasias Hepáticas Experimentais/prevenção & controle , Camundongos Endogâmicos BALB C , Camundongos Nus , Estresse Oxidativo/genética , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Fatores de Elongação da Transcrição/antagonistas & inibidores , Fatores de Elongação da Transcrição/biossíntese , Fatores de Elongação da Transcrição/genética , Regulação para Cima/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
FACT, in addition to its role in transcription, is likely implicated in both transcription-coupled nucleotide excision repair and DNA double strand break repair. Here, we present evidence that FACT could be directly involved in Base Excision Repair and elucidate the chromatin remodeling mechanisms of FACT during BER. We found that, upon oxidative stress, FACT is released from transcription related protein complexes to get associated with repair proteins and chromatin remodelers from the SWI/SNF family. We also showed the rapid recruitment of FACT to the site of damage, coincident with the glycosylase OGG1, upon the local generation of oxidized DNA. Interestingly, FACT facilitates uracil-DNA glycosylase in the removal of uracil from nucleosomal DNA thanks to an enhancement in the remodeling activity of RSC. This discloses a novel property of FACT wherein it has a co-remodeling activity and strongly enhances the remodeling capacity of the chromatin remodelers. Altogether, our data suggest that FACT may acts in concert with RSC to facilitate excision of DNA lesions during the initial step of BER.
Assuntos
Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Grupo de Alta Mobilidade/genética , Histonas/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Animais , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Dano ao DNA/genética , Proteínas de Ligação a DNA/biossíntese , Células HeLa , Proteínas de Grupo de Alta Mobilidade/biossíntese , Histonas/metabolismo , Humanos , Nucleossomos/genética , Estresse Oxidativo/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Fatores de Elongação da Transcrição/biossíntese , Uracila/metabolismo , Xenopus laevisRESUMO
The Wnt signaling pathway plays a conserved role during animal development in transcriptional regulation of distinct targets in different developmental contexts but it remains unclear whether quantitative differences in the nuclear localization of effector proteins TCF and ß-catenin contribute to context-specific regulation. We investigated this question in Caenorhabditis elegans embryos by quantifying nuclear localization of fluorescently tagged SYS-1/ß-catenin and POP-1/TCF and expression of Wnt ligands at cellular resolution by time-lapse microscopy and automated lineage tracing. We identified reproducible, quantitative differences that generate a subset of Wnt-signaled cells with a significantly higher nuclear concentration of the TCF/ß-catenin activating complex. Specifically, ß-catenin and TCF are preferentially enriched in nuclei of daughter cells whose parents also had high nuclear levels of that protein, a pattern that could influence developmental gene expression. Consistent with this, we found that expression of synthetic reporters of POP-1-dependent activation is biased towards cells that had high nuclear SYS-1 in consecutive divisions. We identified new genes whose embryonic expression patterns depend on pop-1. Most of these require POP-1 for either transcriptional activation or repression, and targets requiring POP-1 for activation are more likely to be expressed in the cells with high nuclear SYS-1 in consecutive divisions than those requiring POP-1 for repression. Taken together, these results indicate that SYS-1 and POP-1 levels are influenced by the parent cell's SYS-1/POP-1 levels and this may provide an additional mechanism by which POP-1 regulates distinct targets in different developmental contexts.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Núcleo Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Grupo de Alta Mobilidade/genética , Fatores de Transcrição/genética , beta Catenina/genética , Animais , Padronização Corporal/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/biossíntese , Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Grupo de Alta Mobilidade/biossíntese , Fatores de Transcrição TCF/genética , Fatores de Transcrição TCF/metabolismo , Fatores de Transcrição/biossíntese , Via de Sinalização Wnt/genética , beta Catenina/metabolismoRESUMO
BACKGROUND: The histopathologic differentiation between Sézary syndrome (SS) and erythrodermic dermatitis may be extremely difficult. In this immunohistochemical study, it was investigated if thymocyte selection-associated high mobility group box protein (TOX) and C-MYC can be used as additional diagnostic markers to differentiate between SS and erythrodermic dermatitis. METHOD: Paraffin-embedded skin biopsies from 15 SS patients and 17 erythrodermic dermatitis patients were stained and scored for TOX or C-MYC expression. RESULTS: Strong nuclear staining for TOX in more than 50% of skin-infiltrating T cells was observed in 13 of 15 (87%) SS cases, whereas erythrodermic dermatitis cases showed weak nuclear staining in 11-50% (median: 25%) of the T cells; strong nuclear staining as found in SS was never observed in erythrodermic dermatitis. No significant differences in C-MYC expression between SS and erythrodermic dermatitis were found. In most patients of both groups, percentages of C-MYC positive-cells varied between less than 10 and 25% of skin-infiltrating T cells. CONCLUSION: Our results suggest that strong expression of TOX in more than 50% of skin-infiltrating T cells in erythrodermic skin is a useful marker in the differentiation between SS and erythrodermic dermatitis, whereas staining for C-MYC does not contribute to differential diagnosis.
Assuntos
Proteínas de Grupo de Alta Mobilidade/biossíntese , Síndrome de Sézary/metabolismo , Síndrome de Sézary/patologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Biópsia , Proteínas de Ligação a DNA/metabolismo , Dermatite Esfoliativa/diagnóstico , Dermatite Esfoliativa/metabolismo , Dermatite Esfoliativa/patologia , Diagnóstico Diferencial , Humanos , Imuno-Histoquímica , Síndrome de Sézary/diagnóstico , Pele/patologia , Fatores de Transcrição/metabolismoRESUMO
Growth factors inactivate the FOXO (forkhead box O) transcription factors through PI3K (phosphoinositide 3-kinase) and PKB (protein kinase B). By comparing microarray data from multiple model systems, we identified HBP1 (high-mobility group-box protein 1) as a novel downstream target of this pathway. HBP1 mRNA was down-regulated by PDGF (platelet-derived growth factor), FGF (fibroblast growth factor), PI3K and PKB, whereas it was up-regulated by FOXO factors. This observation was confirmed in human and murine fibroblasts as well as in cell lines derived from leukaemia, breast adenocarcinoma and colon carcinoma. Bioinformatics analysis led to the identification of a conserved consensus FOXO-binding site in the HBP1 promoter. By luciferase activity assay and ChIP, we demonstrated that FOXO bound to this site and regulated the HBP1 promoter activity in a PI3K-dependent manner. Silencing of HBP1 by shRNA increased the proliferation of human fibroblasts in response to growth factors, suggesting that HBP1 limits cell growth. Finally, by analysing a transcriptomics dataset from The Cancer Genome Atlas, we observed that HBP1 expression was lower in breast tumours that had lost FOXO expression. In conclusion, HBP1 is a novel target of the PI3K/FOXO pathway and controls cell proliferation in response to growth factors.
Assuntos
Regulação para Baixo/genética , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/antagonistas & inibidores , Proteínas de Grupo de Alta Mobilidade/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Fosfatidilinositol 3-Quinase/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Animais , Células CHO , Células Cultivadas , Sequência Conservada , Cricetinae , Cricetulus , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/biossíntese , Células HEK293 , Proteínas de Grupo de Alta Mobilidade/biossíntese , Humanos , Células MCF-7 , Masculino , Camundongos , Células NIH 3T3 , Fosfatidilinositol 3-Quinase/biossíntese , Regiões Promotoras Genéticas , Ligação Proteica/genética , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Repressoras/biossíntese , Transdução de Sinais/genéticaRESUMO
Physical inactivity-induced loss of skeletal muscle oxidative phenotype (OXPHEN), often observed in chronic disease, adversely affects physical functioning and quality of life. Potential therapeutic targets remain to be identified, since the molecular mechanisms involved in reloading-induced recovery of muscle OXPHEN remain incompletely understood. We hypothesized a role for alternative NF-κB, as a recently identified positive regulator of muscle OXPHEN, in reloading-induced alterations in muscle OXPHEN. Markers and regulators (including alternative NF-κB signaling) of muscle OXPHEN were investigated in gastrocnemius muscle of mice subjected to a hindlimb suspension/reloading (HLS/RL) protocol. Expression levels of oxidative phosphorylation subunits and slow myosin heavy chain isoforms I and IIA increased rapidly upon RL. After an initial decrease upon HLS, mRNA levels of peroxisome proliferator-activated receptor (PPAR)-γ coactivator (PGC) molecules PGC-1α and PGC-1ß and mRNA levels of mitochondrial transcription factor A (Tfam) and estrogen-related receptor α increased upon RL. PPAR-δ, nuclear respiratory factor 1 (NRF-1), NRF-2α, and sirtuin 1 mRNA levels increased during RL although expression levels were unaltered upon HLS. In addition, both Tfam and NRF-1 protein levels increased significantly during the RL period. Moreover, upon RL, IKK-α mRNA and protein levels increased, and phosphorylation of P100 and subsequent processing to P52 were elevated, reflecting alternative NF-κB activation. We conclude that RL-induced recovery of muscle OXPHEN is associated with activation of alternative NF-κB signaling.
Assuntos
Modelos Animais de Doenças , Imobilização/efeitos adversos , Músculo Esquelético/metabolismo , Transtornos Musculares Atróficos/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Fatores de Transcrição/biossíntese , Animais , Biomarcadores/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/biossíntese , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Elevação dos Membros Posteriores , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Transtornos Musculares Atróficos/etiologia , Transtornos Musculares Atróficos/reabilitação , Cadeias Pesadas de Miosina/biossíntese , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , NF-kappa B/agonistas , Fosforilação Oxidativa , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Distribuição Aleatória , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Suporte de Carga , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Neuronal nitric-oxide synthase (nNOS) has various splicing variants and different subcellular localizations. nNOS can be found also in the nucleus; however, its exact role in this compartment is still not completely defined. In this report, we demonstrate that the PDZ domain allows the recruitment of nNOS to nuclei, thus favoring local NO production, nuclear protein S-nitrosylation, and induction of mitochondrial biogenesis. In particular, overexpression of PDZ-containing nNOS (nNOSα) increases S-nitrosylated CREB with consequent augmented binding on cAMP response element consensus sequence on peroxisome proliferator-activated receptor γ co-activator (PGC)-1α promoter. The resulting PGC-1α induction is accompanied by the expression of mitochondrial genes (e.g., TFAM, MtCO1) and increased mitochondrial mass. Importantly, full active nNOS lacking PDZ domain (nNOSß) does not localize in nuclei and fails in inducing the expression of PGC-1α. Moreover, we substantiate that the mitochondrial biogenesis normally accompanying myogenesis is associated with nuclear translocation of nNOS. We demonstrate that α-Syntrophin, which resides in nuclei of myocytes, functions as the upstream mediator of nuclear nNOS translocation and nNOS-dependent mitochondrial biogenesis. Overall, our results indicate that altered nNOS splicing and nuclear localization could be contributing factors in human muscular diseases associated with mitochondrial impairment.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Núcleo Celular/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Transporte Ativo do Núcleo Celular/genética , Processamento Alternativo/genética , Animais , Proteínas de Ligação ao Cálcio/genética , Núcleo Celular/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/genética , Células HeLa , Proteínas de Grupo de Alta Mobilidade/biossíntese , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Mitocôndrias/genética , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Proteínas Musculares/genética , Doenças Musculares/genética , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Óxido Nítrico Sintase Tipo I/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Estrutura Terciária de Proteína , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
Expression of ribosomal proteins is controlled by the Target of Rapamycin (TOR) kinase. The Saccharomyces cerevisiae Forkhead-like transcription factor Fhl1 is important for this regulation, and its localization to ribosomal protein gene promoters requires the high mobility group protein HMO1. We show here that HMO1 expression is similarly controlled by TOR signaling. Reporter constructs in which lacZ is under control of the HMO1 promoter show that HMO1 promoter activity is repressed on inactivation of TOR and that HMO1 is required for this repression. Chromatin immunoprecipitation shows that Fhl1 localizes to the HMO1 promoter in an HMO1-dependent fashion and that it centers on a predicted Fhl1 site, and removal of the Fhl1 site in the HMO1 promoter attenuates the response to rapamycin. Taken together, our data show that the HMO1 promoter is regulated by TOR signaling, and that TOR can signal through an HMO1- and Fhl1-dependent mechanism, as proposed for TOR-mediated regulation of ribosomal protein expression. The shared mechanism of regulation further reinforces the central role of HMO1 in TOR-mediated regulation of ribosomal protein gene expression.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Proteínas de Grupo de Alta Mobilidade/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/genética , Regiões Promotoras Genéticas , Proteínas Ribossômicas/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Sirolimo/farmacologiaRESUMO
MicroRNAs have been implicated in regulating diverse cellular pathways. Emerging evidence indicate that the miR-17-92 cluster may have a causal role in breast cancer tumorigenesis as a novel class of oncogenes, but the role of these miRNAs in breast cancer invasion and migration remains unexplored. The aims of this study were to verify the effect of miR-17-5p (an important member of the miR-17-92 cluster) on the invasive and migratory ability of breast cancer cells. The matching of miR-17-5p and HMG box-containing protein 1 (HBP1) was predicted by TargetScan and confirmed by DNA constructs and luciferase target assay. The expression levels of miR-17-5p and its candidate target-HBP1 in MCF7 and MDA-MB-231 breast cancer cells were measured by real-time PCR and western blotting. Effects of miR-17-5p in cell cycle progression, proliferation, invasion and migration were evaluated by flow cytometry assay, 3-(4,-dimethy -lthiazol-2-yl)-2,-diphenyl -tetrazoliumbromide assay, soft-agar colony formation assay, and transwell invasive and migratory assay, respectively. The results showed that miR-17-5p was highly expressed in high-invasive MDA-MB-231 breast cancer cells but not in low-invasive MCF-7 breast cancer cells. Over-expression of miR-17-5p in MCF-7 cells rendered them the invasive and migratory abilities by targeting HBP1/ß-catenin pathway. On the other hand, down-regulation of endogenous miR-17-5p suppressed the migration and invasion of MDA-MB-231 cells in vitro. These findings suggest that miR-17-5p plays an important role in breast cancer cell invasion and migration by suppressing HBP1 and subsequent activation of Wnt/ß-catenin.
Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/fisiologia , MicroRNAs/biossíntese , Proteínas Repressoras/fisiologia , Western Blotting , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular , Feminino , Citometria de Fluxo/métodos , Proteínas de Grupo de Alta Mobilidade/biossíntese , Humanos , Invasividade Neoplásica , Proteínas Repressoras/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
A possible role for structure-specific recognition protein 1 (SSRP1) in replication-associated repair processes has previously been suggested based on its interaction with several DNA repair factors and the replication defects observed in SSRP1 mutants. In this study, we investigated the potential role of SSRP1 in association with DNA repair mediated by homologous recombination (HR), one of the pathways involved in repairing replication-associated DNA damage, in mammalian cells. Surprisingly, over-expression of SSRP1 reduced the number of hprt(+) recombinants generated via HR both spontaneously and upon hydroxyurea (HU) treatment, whereas knockdown of SSRP1 resulted in an increase of HR events in response to DNA double-strand break formation. In correlation, we found that the depletion of SSRP1 in HU-treated human cells elevated the number of Rad51 and H2AX foci, while over-expression of the wild-type SSRP1 markedly reduced HU-induced Rad51 foci formation. We also found that SSRP1 physically interacts with a key HR repair protein, Rad54 both in vitro and in vivo. Further, branch migration studies demonstrated that SSRP1 inhibits Rad54-promoted branch migration of Holliday junctions in vitro. Taken together, our data suggest a functional role for SSRP1 in spontaneous and replication-associated DNA damage response by suppressing avoidable HR repair events.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Proteínas de Grupo de Alta Mobilidade/fisiologia , Recombinação Genética , Fatores de Elongação da Transcrição/fisiologia , Animais , Western Blotting , Cricetinae , Cricetulus , Quebras de DNA de Cadeia Dupla , Dano ao DNA , DNA Helicases , DNA Cruciforme/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Genes Reporter , Proteínas de Grupo de Alta Mobilidade/biossíntese , Proteínas de Grupo de Alta Mobilidade/deficiência , Proteínas de Grupo de Alta Mobilidade/genética , Histonas/metabolismo , Humanos , Hidroxiureia , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Proteínas Nucleares/metabolismo , Fragmentos de Peptídeos , Plasmídeos , Ligação Proteica , RNA Interferente Pequeno , Rad51 Recombinase/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Elongação da Transcrição/biossíntese , Fatores de Elongação da Transcrição/deficiência , Fatores de Elongação da Transcrição/genética , TransfecçãoRESUMO
Mouse embryonic fibroblasts (MEFs) have been extensively used as feeder cells to support the in vitro propagation of human embryonic stem cells (hESCs). However, owing to the risk of cross-contamination with animal or other unknown pathogens, the use of MEFs does not meet requirements for the clinical application of hESCs. Moreover, the actual role played by the feeders in the differentiation of hESCs is still unclear. In this study, human embryonic fibroblasts (HEFs) were used as feeder cells to support the establishment and undifferentiated growth of hESCs, and the capability of HEFs to induce the differentiation of definitive endoderm (DE) was evaluated. Three new hES cell lines were derived. These cell lines exhibited and maintained the common features of traditional hESCs after prolonged culture in vitro. Furthermore, DE differentiation of the newly established hES cell lines was performed using 100 ng/ml activin A, and the effects were compared among HEFs, MEFs, and feeder-free systems. On day 5 of induction, DE (SOX17(+)) cells appeared with comparable efficiency in both human and mouse feeder systems (85.0 +/- 8.9% and 78.7 +/- 3.4%, respectively). These levels were considerably superior to that obtained in the feeder-free system (22.7 +/- 5.6%). The SOX17(+) cells tended to differentiate into an endodermal lineage in vivo and could be further induced into glucagon and C-peptide double positive islet-like clusters in vitro. Our studies suggest that, in terms of therapeutic application, HEFs can be an effective substitute for MEFs for sustaining the derivation and DE differentiation of hESCs.
Assuntos
Diferenciação Celular/fisiologia , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Fibroblastos/citologia , Ativinas/farmacologia , Animais , Peptídeo C/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Proteínas de Ligação a DNA/biossíntese , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Endoderma/citologia , Endoderma/metabolismo , Fibroblastos/metabolismo , Glucagon/farmacologia , Proteínas HMGB/biossíntese , Proteínas de Grupo de Alta Mobilidade/biossíntese , Humanos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Fatores de Transcrição SOXF , Fatores de Transcrição/biossínteseRESUMO
Pancreas extract from regenerating pancreas after partial pancreatectomy is known to contain factors that induce islet neogenesis in animals with streptozotocin (STZ)-induced diabetes [A.A. Hardikar, R.R. Bhonde, Modulating experimental diabetes by treatment with cytosolic extract from the regenerating pancreas, Diabetes Res. Clin. Pract. 46 (1999) 203-211]. In this study, we evaluate the effects of regenerating pancreas extract (RPE) from 90% partially pancreatectomized rats on induction of pancreatic differentiation of human adipose tissue-derived stem cells (hASCs). We found that undifferentiated hASCs expressed OCT-3/4, Nanog, and REX-1, markers of embryonic stem cells (ESCs). Genes involved in early pancreas development showed increased expression in RPE-treated culture. Sox17 and IPF-1 were expressed only in RPE-treated culture. Immunocytochemical analysis showed C-peptide-positive cells in RPE-treated culture but not in undifferentiated hASCs. In conclusion, hASCs have the characteristics of ESCs and the potential to differentiate into pancreas cell lineages phenotypically in response to RPE.
Assuntos
Tecido Adiposo/citologia , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Ilhotas Pancreáticas/fisiologia , Regeneração , Animais , Extratos Celulares , Proteínas de Ligação a DNA/biossíntese , Células-Tronco Embrionárias/metabolismo , Proteínas de Grupo de Alta Mobilidade/biossíntese , Proteínas de Homeodomínio/biossíntese , Humanos , Ilhotas Pancreáticas/citologia , Fatores de Transcrição Kruppel-Like/biossíntese , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/biossíntese , Fenótipo , Ratos , Fatores de Transcrição SOXF , Transativadores/biossíntese , Fatores de Transcrição/biossínteseRESUMO
BACKGROUND: Translation of preclinical studies into effective human cancer therapy is hampered by the lack of defined molecular expression patterns in mouse models that correspond to the human counterpart. We sought to generate an open source TRAMP mouse microarray dataset and to use this array to identify differentially expressed genes from human prostate cancer (PCa) that have concordant expression in TRAMP tumors, and thereby represent lead targets for preclinical therapy development. METHODS: We performed microarrays on total RNA extracted and amplified from eight TRAMP tumors and nine normal prostates. A subset of differentially expressed genes was validated by QRT-PCR. Differentially expressed TRAMP genes were analyzed for concordant expression in publicly available human prostate array datasets and a subset of resulting genes was analyzed by QRT-PCR. RESULTS: Cross-referencing differentially expressed TRAMP genes to public human prostate array datasets revealed 66 genes with concordant expression in mouse and human PCa; 56 between metastases and normal and 10 between primary tumor and normal tissues. Of these 10 genes, two, Sox4 and Tubb2a, were validated by QRT-PCR. Our analysis also revealed various dysregulations in major biologic pathways in the TRAMP prostates. CONCLUSIONS: We report a TRAMP microarray dataset of which a gene subset was validated by QRT-PCR with expression patterns consistent with previous gene-specific TRAMP studies. Concordance analysis between TRAMP and human PCa associated genes supports the utility of the model and suggests several novel molecular targets for preclinical therapy.
Assuntos
Adenocarcinoma/genética , Antígenos de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Membro 25 de Receptores de Fatores de Necrose Tumoral/biossíntese , Adenocarcinoma/patologia , Animais , Antígenos de Neoplasias/biossíntese , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Proteínas de Grupo de Alta Mobilidade/biossíntese , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/patologia , RNA Neoplásico/química , RNA Neoplásico/genética , Membro 25 de Receptores de Fatores de Necrose Tumoral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXC , Transativadores/biossíntese , Transativadores/genética , Regulação para CimaRESUMO
S100 protein is a sensitive marker for melanomas and peripheral nerve sheath tumors. It is, however, expressed by other mesenchymal and epithelial tumors. Despite its low specificity, S100 protein is valuable for the diagnosis of desmoplastic melanomas and peripheral nerve sheath tumors, for which no specific marker is available. Sox10 is a neural crest transcription factor crucial for specification, maturation, and maintenance of Schwann cells and melanocytes. Anti-Sox10 antibody was applied to a variety of neural crest-derived tumors, mesenchymal and epithelial neoplasms, and normal tissues. Sox10 nuclear expression was found in 76 of 78 melanomas (97%) and 38 of 77 malignant peripheral nerve sheath tumors (49%) whereas S100 protein was expressed in 71 melanomas (91%) and 23 malignant peripheral nerve sheath tumors (30%). Sox10 was diffusely expressed in schwannomas and neurofibromas. Sox10 reaction was seen only in sustentacular cells of pheochromocytomas/paragangliomas, and occasionally carcinoid tumors from various organs, but it was not seen in the tumor cells. In normal tissues, Sox10 was expressed in Schwann cells, melanocytes, and myoepithelial cells of salivary, bronchial, and mammary glands. Sox10 reaction was not identified in any other mesenchymal and epithelial tumors except for myoepitheliomas and diffuse astrocytomas. Sox10 was expressed by metastatic melanomas and nodal capsular nevus in sentinel lymph nodes, but not by other lymph node components such as dendritic cells. Our results indicate that Sox10 will serve as a more sensitive and specific marker for the diagnosis of melanocytic and schwannian tumors than S100 protein.
Assuntos
Biomarcadores Tumorais/análise , Proteínas de Ligação a DNA/biossíntese , Proteínas de Grupo de Alta Mobilidade/biossíntese , Melanócitos/metabolismo , Células de Schwann/metabolismo , Fatores de Transcrição/biossíntese , Humanos , Imuno-Histoquímica , Melanoma/diagnóstico , Neoplasias de Bainha Neural/diagnóstico , Proteínas S100/metabolismo , Fatores de Transcrição SOXE , Sensibilidade e Especificidade , Neoplasias Cutâneas/diagnóstico , Análise Serial de TecidosRESUMO
The transcription factor Sox2 is expressed at high levels in neural stem and progenitor cells. Here, we inactivated Sox2 specifically in the developing brain by using Cre-loxP system. Although mutant animals did not survive after birth, analysis of late gestation embryos revealed that loss of Sox2 causes enlargement of the lateral ventricles and a decrease in the number of neurosphere-forming cells. However, although their neurogenic potential is attenuated, Sox2-deficient neural stem cells retain their multipotency and self-renewal capacity. We found that expression level of Sox3 is elevated in Sox2 null developing brain, probably mitigating the effects of loss of Sox2.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/fisiologia , Células-Tronco Embrionárias/citologia , Proteínas HMGB/fisiologia , Ventrículos Laterais/embriologia , Neurônios/citologia , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/metabolismo , Inativação Gênica , Proteínas HMGB/genética , Proteínas de Grupo de Alta Mobilidade/biossíntese , Ventrículos Laterais/citologia , Ventrículos Laterais/metabolismo , Camundongos , Neurônios/metabolismo , Receptores Notch/metabolismo , Fatores de Transcrição SOXB1 , Transdução de Sinais/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
Bone morphogenetic proteins (BMPs) play critical roles at various stages in endochondral bone formation. In vitro studies have demonstrated that Smad7 regulates transforming growth factor-beta and BMP signals by inhibiting Smad pathways in chondrocytes. However, the in vivo roles of Smad7 during cartilage development are unknown. To investigate distinct effects of Smad7 at different stages during chondrocyte differentiation, we generated a series of conditional transgenic mice that overexpress Smad7 in chondrocytes at various steps of differentiation by using the Cre/loxP system. We generated Col11a2-lacZ(floxed)-Smad7 transgenic mice and mated them with three types of Cre transgenic mice to obtain Smad7(Prx1), Smad7(11Enh), and Smad7(11Prom) conditional transgenic mice. Smad7(Prx1) mice overexpressing Smad7 in condensing mesenchymal cells showed disturbed mesenchymal condensation associated with decreased Sox9 expression, leading to poor cartilage formation. Smad7(11Enh) mice overexpressing Smad7 in round chondrocytes showed decreased chondrocyte proliferation rates. Smad7(11Prom) mice overexpressing Smad7 in flat chondrocytes showed inhibited maturation of chondrocytes toward hypertrophy. Micromass culture of mesenchymal cells showed that BMP-induced cartilaginous nodule formation was down-regulated by overexpression of Smad7, but not Smad6. Overexpression of Smad7, but not Smad6, down-regulated the phosphorylation of p38 MAPKs. Our data provide in vivo evidence for distinct effects of Smad7 at different stages during chondrocyte differentiation and suggest that Smad7 in prechondrogenic cells inhibits chondrocyte differentiation possibly by down-regulating BMP-activated p38 MAPK pathways.
Assuntos
Diferenciação Celular/fisiologia , Condrócitos/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Osteogênese/fisiologia , Proteína Smad7/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/biossíntese , Proteínas Morfogenéticas Ósseas/genética , Condrócitos/citologia , Regulação para Baixo/fisiologia , Proteínas de Grupo de Alta Mobilidade/biossíntese , Proteínas de Grupo de Alta Mobilidade/genética , Camundongos , Camundongos Transgênicos , Fosforilação , Fatores de Transcrição SOX9 , Proteína Smad6/biossíntese , Proteína Smad6/genética , Proteína Smad7/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
The objective of this study was to identify differentially expressed and prognostically important genes in pediatric medulloblastoma and pediatric ependymoma by Affymetrix microarray analysis. Among the most discriminative genes, three members of the SOX transcription factor family were differentially expressed. Both SOX4 and SOX11 were significantly overexpressed in medulloblastoma (median, 11-fold and 5-fold, respectively) compared with ependymoma and normal cerebellum. SOX9 had greater expression in ependymoma (median, 16-fold) compared with normal cerebellum and medulloblastoma (p<0.001 for all comparisons). The differential expression of the SOX genes was confirmed at the protein level by immunohistochemical analysis. Survival analysis of the most discriminative probe sets for each subgroup showed that 35 and 13 probe sets were predictive of survival in patients with medulloblastoma and ependymoma, respectively. There was a trend toward better survival with increasing SOX4 expression in medulloblastoma. SOX9 expression was predictive for favorable outcome in ependymoma. The mRNA levels of BCAT1, a mediator of amino acid breakdown, were higher (median, 15-fold) in medulloblastoma patients with metastases compared with those without metastasized disease (p<0.01). However, the correlation between BCAT1 expression and metastatic medulloblastoma could not be confirmed at the protein level. The potential prognostic effect of the genes associated with outcome should be evaluated in ongoing studies using larger groups of patients. Furthermore, our findings support further analysis of the functional properties of the selected genes, especially SOX4 and BCAT1 for medulloblastoma and SOX9 for ependymoma, to evaluate the use of these genes as potential tumor markers, prognostic markers, and drug targets in pediatric brain tumors.
Assuntos
Neoplasias Encefálicas/genética , Ependimoma/genética , Proteínas de Grupo de Alta Mobilidade/genética , Meduloblastoma/genética , Análise de Sequência com Séries de Oligonucleotídeos , Transativadores/genética , Adolescente , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/mortalidade , Criança , Pré-Escolar , Ependimoma/mortalidade , Expressão Gênica , Perfilação da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/biossíntese , Humanos , Imuno-Histoquímica , Lactente , Meduloblastoma/mortalidade , Prognóstico , RNA Mensageiro/análise , Fatores de Transcrição SOX9 , Fatores de Transcrição SOXC , Transativadores/biossíntese , Transaminases/biossíntese , Transaminases/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
SOX9 is a transcription factor that plays a critical role in the development of multiple tissues. We previously reported that SOX9 in normal human adult prostate was restricted to basal epithelium. SOX9 was also expressed in a subset of prostate cancer (PCa) cells and was increased in relapsed hormone-refractory PCa. Moreover, SOX9 expression in PCa cell lines enhanced tumor cell proliferation and was beta-catenin regulated. Here we report additional in vivo results showing that SOX9 is highly expressed during fetal prostate development by epithelial cells expanding into the mesenchyme, suggesting it may contribute to invasive growth in PCa. Indeed, SOX9 overexpression in LNCaP PCa xenografts enhanced growth, angiogenesis, and invasion. Conversely, short hairpin RNA-mediated SOX9 suppression inhibited the growth of CWR22Rv1 PCa xenografts. These results support important functions of SOX9 in both the development and maintenance of normal prostate, and indicate that these functions contribute to PCa tumor growth and invasion.
Assuntos
Proteínas de Grupo de Alta Mobilidade/biossíntese , Próstata/embriologia , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Fatores de Transcrição/biossíntese , Animais , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Regulação para Baixo , Células Epiteliais/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Invasividade Neoplásica , Neovascularização Patológica/metabolismo , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Interferente Pequeno/genética , Fatores de Transcrição SOX9 , Fatores de Transcrição/genéticaRESUMO
HmgD encodes Drosophila homologue of high mobility group proteins (HMGD), which are thought to have an architectural function in chromatin organization. However, current opinions about the function of HMGD in Drosophila development are controversial. Our previous studies have shown that ubiquitous overexpression of HmgD caused the formation of melanotic tumors in the Drosophila larvae by prematurely activating the Ras-MAPK pathway. Here we report that under maternal control, the viability of flies links with overexpression of HmgD, while under ubiquitous control, ActGal4, overexpressing HmgD animals, which display prolonged larval stages around day 13, developmentally stagnate in the larva-white pupa transition. Ecdysone feeding did not rescue overexpressing HmgD animals. RT-PCR analyses show that overexpression of HmgD does not affect the temporal expression pattern of ecdysone receptor gene EcR, whereas transcriptional patterns of some key regulatory genes, such as E74A, E74B, E75A, E75B, betaFTZ-F1, are changed greatly. These results suggest that ubiquitous overexpression of HmgD results in the failure of pupariation neither by affecting the process of ecdysone synthesis and release nor by abnormal EcR transcription, but by causing expression of EcR regulatory nuclear receptors out of schedule. The results led us to postulate that overexpression of HMGD likely changes the signaling cascade of Drosophila metamorphosis by an interaction between HMGD and DNA strands, and subsequently by an error of DNA binding abilities and transcriptional activities of some nuclear receptor genes. Arch. Insect Biochem. Physiol. 2008.