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1.
Am J Physiol Renal Physiol ; 324(1): F75-F90, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36454702

RESUMO

Induction of SRY box transcription factor 9 (SOX9) has been shown to occur in response to kidney injury in rodents, where SOX9-positive cells proliferate and regenerate the proximal tubules of injured kidneys. Additionally, SOX9-positive cells demonstrate a capacity to differentiate toward other nephron segments. Here, we characterized the role of SOX9 in normal and injured human kidneys. SOX9 expression was found to colocalize with a proportion of so-called scattered tubular cells in the uninjured kidney, a cell population previously shown to be involved in kidney injury and regeneration. Following injury and in areas adjacent to inflammatory cell infiltrates, SOX9-positive cells were increased in number. With the use of primary tubular epithelial cells (PTECs) obtained from human kidney tissue, SOX9 expression was spontaneously induced in culture and further increased by transforming growth factor-ß1, whereas it was suppressed by interferon-γ. siRNA-mediated knockdown of SOX9 in PTECs followed by analysis of differential gene expression, immunohistochemical expression, and luciferase promoter assays suggested lamin B receptor (LBR), high mobility group AT-hook 2 (HMGA2), and homeodomain interacting protein kinase 3 (HIPK3) as possible target genes of SOX9. Moreover, a kidney explant model was used to demonstrate that only SOX9-positive cells survive the massive injury associated with kidney ischemia and that the surviving SOX9-positive cells spread and repopulate the tubules. Using a wound healing assay, we also showed that SOX9 positively regulated the migratory capacity of PTECs. These findings shed light on the functional and regulatory aspects of SOX9 activation in the human kidney during injury and regeneration.NEW & NOTEWORTHY Recent studies using murine models have shown that SRY box transcription factor 9 (SOX9) is activated during repair of renal tubular cells. In this study, we showed that SOX9-positive cells represent a proportion of scattered tubular cells found in the uninjured human kidney. Furthermore, we suggest that expression of LBR, HMGA2, and HIPK3 is altered by SOX9 in the kidney tubular epithelium, suggesting the involvement of these gene products in kidney injury and regeneration.


Assuntos
Rim , Receptores Citoplasmáticos e Nucleares , Humanos , Camundongos , Animais , Rim/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Túbulos Renais Proximais/metabolismo , Fatores de Transcrição/metabolismo , Túbulos Renais/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fatores de Transcrição SOX9/metabolismo , Receptor de Lamina B
2.
J Pathol ; 252(4): 384-397, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32815150

RESUMO

Clear cell renal cell carcinoma (ccRCC) is the most common form of renal cancer. Due to inactivation of the von Hippel-Lindau tumour suppressor, the hypoxia-inducible transcription factors (HIFs) are constitutively activated in these tumours, resulting in a pseudo-hypoxic phenotype. The HIFs induce the expression of genes involved in angiogenesis and cell survival, but they also reset the cellular metabolism to protect cells from oxygen and nutrient deprivation. ccRCC tumours are highly vascularized and the cytoplasm of the cancer cells is filled with lipid droplets and glycogen, resulting in the histologically distinctive pale (clear) cytoplasm. Intratumoural heterogeneity may occur, and in some tumours, areas with granular, eosinophilic cytoplasm are found. Little is known regarding these traits and how they relate to the coexistent clear cell component, yet eosinophilic ccRCC is associated with higher grade and clinically more aggressive tumours. In this study, we have for the first time performed RNA sequencing comparing histologically verified clear cell and eosinophilic areas from ccRCC tissue, aiming to analyse the characteristics of these cell types. Findings from RNA sequencing were confirmed by immunohistochemical staining of biphasic ccRCC. We found that the eosinophilic phenotype displayed a higher proliferative drive and lower differentiation, and we confirmed a correlation to tumours of higher stage. We further identified mutations of the tumour suppressor p53 (TP53) exclusively in the eosinophilic ccRCC component, where mTORC1 activity was also elevated. Also, eosinophilic areas were less vascularized, yet harboured more abundant infiltrating immune cells. The cytoplasm of clear cell ccRCC cells was filled with lipids but had very low mitochondrial content, while the reverse was found in eosinophilic tissue. We herein suggest possible transcriptional mechanisms behind these phenomena. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.


Assuntos
Carcinoma de Células Renais/patologia , Eosinofilia/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/patologia , Carcinoma de Células Renais/genética , Proliferação de Células/genética , Eosinofilia/genética , Humanos , Neoplasias Renais/genética , Mutação , Análise de Sequência de RNA , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética
3.
Am J Pathol ; 189(10): 1933-1944, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31404540

RESUMO

The polymeric Ig receptor (PIgR) constitutes an important part of the immune system by mediating transcytosis of dimeric IgA into mucosal fluids. Although well studied in organs such as the intestine, the regulation and localization of PIgR in human kidney are incompletely characterized. Herein, using immunohistochemistry, we show that in healthy human kidneys, PIgR is expressed by the progenitor-like tubular scattered cells of the proximal tubules and by parietal epithelial cells of glomeruli. We further show that proximal tubular expression of PIgR becomes widespread during kidney disease, correlating to elevated levels of urinary secretory IgA. Urinary secretory IgA levels also correlated to the degree of tubular fibrosis, plasma creatinine, and urea levels. In addition, primary tubular cells were cultured to study the function and regulation of PIgR in vitro. Cellular PIgR expression was induced by conditioned medium from activated human leukocytes, as well as by inflammatory cytokines, whereas transforming growth factor-ß1 caused decreased expression. Furthermore, interferon-γ increased the transcytosis of dimeric IgA in cultured tubular cells. Finally, a correlation study of mRNA data from the Genotype-Tissue Expression portal indicated that PIGR mRNA expression in kidney correlates to the expression of TNFSF13, a cytokine involved in plasma cell class switching to IgA. These results indicate that PIgR induction is an integral part of the injury phenotype of renal tubular cells.


Assuntos
Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Nefropatias/metabolismo , Rim/metabolismo , Receptores de Imunoglobulina Polimérica/metabolismo , Adulto , Idoso , Apoptose , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Feminino , Seguimentos , Humanos , Nefropatias/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Receptores de Imunoglobulina Polimérica/genética , Adulto Jovem
4.
Lab Invest ; 97(11): 1296-1305, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28759013

RESUMO

Papillary renal cell carcinoma (pRCC) is the second most common type of renal cell carcinoma. The only curative treatment available for pRCC is radical surgery. If the disease becomes widespread, neither chemo- nor radiotherapy will have therapeutic effect, hence further research on pRCC is of utmost importance. Histologically, pRCC is characterized by a papillary growth pattern with focal aggregation of macrophages of the foam cell phenotype. In other forms of cancer, a clear role for tumor-associated macrophages during cancer growth and progression has been shown. Although the presence of foamy macrophages is a histological hallmark of pRCC tumors, little is known regarding their role in pRCC biology. In order to study the interaction between pRCC tumor and myeloid cells, we established primary cultures from pRCC tissue. We show that human pRCC cells secrete the chemokines IL-8, CXCL16, and chemerin, and that these factors attract primary human monocytes in vitro. RNAseq data from The Cancer Genome Atlas confirmed a high expression of these factors in pRCC tissue. Conditioned medium from pRCC cultures induced a shift in human monocytes toward the M2 macrophage phenotype. In extended cultures, these macrophages became enlarged and loaded with lipids, adopting the foam cell morphology found in pRCC tissue. These results show for the first time that pRCC primary tumor cells secrete factors that attract and differentiate monocytes into anti-inflammatory tumor-associated macrophages with foam cell histology.


Assuntos
Carcinoma de Células Renais/metabolismo , Quimiocinas CXC/metabolismo , Quimiocinas/metabolismo , Células Espumosas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interleucina-8/metabolismo , Neoplasias Renais/metabolismo , Monócitos/metabolismo , Receptores Depuradores/metabolismo , Idoso , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/cirurgia , Transdiferenciação Celular , Células Cultivadas , Quimiocina CXCL16 , Quimiotaxia de Leucócito , Técnicas de Cocultura , Meios de Cultivo Condicionados , Células Espumosas/imunologia , Células Espumosas/patologia , Humanos , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/patologia , Gradação de Tumores , Proteínas de Neoplasias/metabolismo , Nefrectomia , Carga Tumoral , Células Tumorais Cultivadas , Microambiente Tumoral
5.
BMC Nephrol ; 18(1): 320, 2017 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-29065889

RESUMO

BACKGROUND: Caveolae are membrane invaginations measuring 50-100 nm. These organelles, composed of caveolin and cavin proteins, are important for cellular signaling and survival. Caveolae play incompletely defined roles in human kidneys. Induction of caveolin-1/CAV1 in diseased tubules has been described previously, but the responsible mechanism remains to be defined. METHODS: Healthy and atrophying human kidneys were stained for caveolar proteins, (caveolin 1-3 and cavin 1-4) and examined by electron microscopy. Induction of caveolar proteins was studied in isolated proximal tubules and primary renal epithelial cells. These cells were challenged with hypoxia or H2O2. Primary tubular cells were also subjected to viral overexpression of megakaryoblastic leukemia 1 (MKL1) and MKL1 inhibition by the MKL1 inhibitor CCG-1423. Putative coregulators of MKL1 activity were investigated by Western blotting for suppressor of cancer cell invasion (SCAI) and filamin A (FLNA). Finally, correlative bioinformatic studies of mRNA expression of caveolar proteins and MKL1 were performed. RESULTS: In healthy kidneys, caveolar proteins were expressed by the parietal epithelial cells (PECs) of Bowman's capsule, endothelial cells and vascular smooth muscle. Electron microscopy confirmed caveolae in the PECs. No expression was seen in proximal tubules. In contrast, caveolar proteins were expressed in proximal tubules undergoing atrophy. Caveolar proteins were also induced in cultures of primary epithelial tubular cells. Expression was not enhanced by hypoxia or free radical stress (H2O2), but proved sensitive to inhibition of MKL1. Viral overexpression of MKL1 induced caveolin-1/CAV1, caveolin-2/CAV2 and SDPR/CAVIN2. In kidney tissue, the mRNA level of MKL1 correlated with the mRNA levels for caveolin-1/CAV1, caveolin-2/CAV2 and the archetypal MKL1 target tenascin C (TNC), as did the MKL1 coactivator FLNA. Costaining for TNC as readout for MKL1 activity demonstrated overlap with caveolin-1/CAV1 expression in PECs as well as in atrophic segments of proximal tubules. CONCLUSIONS: Our findings support the view that MKL1 contributes to the expression of caveolar proteins in healthy kidneys and orchestrates the induction of tubular caveolar proteins in renal injury.


Assuntos
Injúria Renal Aguda/metabolismo , Caveolina 1/biossíntese , Túbulos Renais Proximais/metabolismo , Proteínas de Ligação a RNA/biossíntese , Transativadores/fisiologia , Injúria Renal Aguda/induzido quimicamente , Cavéolas/efeitos dos fármacos , Cavéolas/metabolismo , Cavéolas/ultraestrutura , Caveolina 1/genética , Células Cultivadas , Expressão Gênica , Humanos , Peróxido de Hidrogênio/toxicidade , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Túbulos Renais/ultraestrutura , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/ultraestrutura , Proteínas de Ligação a RNA/genética
6.
Cancer Cell ; 10(5): 413-23, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17097563

RESUMO

In neuroblastoma specimens, HIF-2alpha but not HIF-1alpha is strongly expressed in well-vascularized areas. In vitro, HIF-2alpha protein was stabilized at 5% O2 (resembling end capillary oxygen conditions) and, in contrast to the low HIF-1alpha activity at this oxygen level, actively transcribed genes like VEGF. Under hypoxia (1% O2), HIF-1alpha was transiently stabilized and primarily mediated acute responses, whereas HIF-2alpha protein gradually accumulated and governed prolonged hypoxic gene activation. Knockdown of HIF-2alpha reduced growth of neuroblastoma tumors in athymic mice. Furthermore, high HIF-2alpha protein levels were correlated with advanced clinical stage and high VEGF expression and predicted poor prognosis in a clinical neuroblastoma material. Our results demonstrate the relevance of HIF-2alpha in neuroblastoma progression and have general tumor biological implications.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neuroblastoma/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Criança , Feminino , Perfilação da Expressão Gênica , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Transplante de Neoplasias , Neuroblastoma/genética , Neuroblastoma/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Oxigênio/metabolismo , Fenótipo , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismo , RNA Mensageiro/metabolismo , Ativação Transcricional , Células Tumorais Cultivadas
7.
Mol Clin Oncol ; 16(5): 101, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35463211

RESUMO

Renal cell carcinoma (RCC) is a tumour type with an indolent growth pattern and rather vague symptoms. The present study developed a platform for liquid biopsy of RCC based upon the isolation of circulating tumour cells (CTCs). Founded on the observation that RCC tumour cells are considerably larger than leucocytes, the present study employed a microfluidics-based system for isolation of RCC CTCs from whole blood. Using this system, it was revealed that 66% of spiked-in RCC tumour cells could be retrieved using this approach. Furthermore, it was demonstrated that these cells could be molecularly detected with digital PCR using RCC-specific genes down to one tumour cell, whilst avoiding detection in samples lacking tumour cells. Finally, subtype specific transcripts were identified to distinguish the different subtypes of RCC, which were then validated in patient tumours. The present study established a novel workflow for the isolation of RCC CTCs from whole blood, with the potential to detect these cells irrespective of subtype.

8.
Cell Rep ; 40(6): 111177, 2022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-35947955

RESUMO

Acute myeloid leukemia (AML) is a heterogeneous disease with variable patient responses to therapy. Selinexor, an inhibitor of nuclear export, has shown promising clinical activity for AML. To identify the molecular context for monotherapy sensitivity as well as rational drug combinations, we profile selinexor signaling responses using phosphoproteomics in primary AML patient samples and cell lines. Functional phosphosite scoring reveals that p53 function is required for selinexor sensitivity consistent with enhanced efficacy of selinexor in combination with the MDM2 inhibitor nutlin-3a. Moreover, combining selinexor with the AKT inhibitor MK-2206 overcomes dysregulated AKT-FOXO3 signaling in resistant cells, resulting in synergistic anti-proliferative effects. Using high-throughput spatial proteomics to profile subcellular compartments, we measure global proteome and phospho-proteome dynamics, providing direct evidence of nuclear translocation of FOXO3 upon combination treatment. Our data demonstrate the potential of phosphoproteomics and functional phosphorylation site scoring to successfully pinpoint key targetable signaling hubs for rational drug combinations.


Assuntos
Leucemia Mieloide Aguda , Proteína Supressora de Tumor p53 , Apoptose , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Hidrazinas , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Proteoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Triazóis , Proteína Supressora de Tumor p53/metabolismo
9.
Commun Biol ; 1: 37, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30271923

RESUMO

In the cell, γ-tubulin establishes a cellular network of threads named the γ-string meshwork. However, the functions of this meshwork remain to be determined. We investigated the traits of the meshwork and show that γ-strings have the ability to connect the cytoplasm and the mitochondrial DNA together. We also show that γ-tubulin has a role in the maintenance of the mitochondrial network and functions as reduced levels of γ-tubulin or impairment of its GTPase domain disrupts the mitochondrial network and alters both their respiratory capacity and the expression of mitochondrial-related genes. By contrast, reduced mitochondrial number or increased protein levels of γ-tubulin DNA-binding domain enhanced the association of γ-tubulin with mitochondria. Our results demonstrate that γ-tubulin is an important mitochondrial structural component that maintains the mitochondrial network, providing mitochondria with a cellular infrastructure. We propose that γ-tubulin provides a cytoskeletal element that gives form to the mitochondrial network.

10.
Clin Cancer Res ; 23(8): 2105-2115, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-27663598

RESUMO

Purpose: Renal cell carcinoma (RCC) is derived from a tissue with a remarkable capacity for vectorial transport. We therefore performed an unbiased exploration of transporter proteins in normal kidney and kidney cancer to discover novel clinical targets.Experimental Design: Using The Cancer Genome Atlas (TCGA) database, we investigated differences in membrane transporter expression in clear cell RCC (ccRCC) and normal kidney. We identified the dopamine transporter SLC6A3 as a specific biomarker for ccRCC. To investigate the functionality of SLC6A3, we used a [3H]-dopamine uptake assay on ccRCC cells. We further explored the effect of hypoxia-inducible factor (HIF) proteins on SLC6A3 expression by introducing siRNA in ccRCC cells and by hypoxic treatment of nonmalignant cells.Results: We show that ccRCC expresses very high transcript levels of SLC6A3 in contrast to normal kidney tissue and other tumor types, which do not express appreciable levels of this transporter. Importantly, we demonstrate that the elevated expression of SLC6A3 in ccRCC cells is associated with specific uptake of dopamine. By targeting the expression of HIF-1α and HIF-2α, we could show that SLC6A3 expression is primarily influenced by HIF-2α and that hypoxia can induce SLC6A3 expression in normal renal cells.Conclusions: We conclude that the dopamine transporter SLC6A3 constitutes a novel biomarker that is highly specific for ccRCC. We further postulate that the protein can be exploited for diagnostic or therapeutic purposes for detection or treatment of ccRCC. Clin Cancer Res; 23(8); 2105-15. ©2016 AACR.


Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Renais/patologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/biossíntese , Neoplasias Renais/patologia , Western Blotting , Carcinoma de Células Renais/metabolismo , Análise por Conglomerados , Humanos , Neoplasias Renais/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma , Regulação para Cima
11.
Cell Rep ; 20(6): 1476-1489, 2017 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-28793269

RESUMO

Comprehensive transcriptome studies of cancers often rely on corresponding normal tissue samples to serve as a transcriptional reference. In this study, we performed in-depth analyses of normal kidney tissue transcriptomes from the TCGA and demonstrate that the histological variability in cellularity, inherent in the kidney architecture, lead to considerable transcriptional differences between samples. This should be considered when comparing expression profiles of normal and cancerous kidney tissues. We exploited these differences to define renal-cell-specific gene signatures and used these as a framework to analyze renal cell carcinoma (RCC) ontogeny. Chromophobe RCCs express FOXI1-driven genes that define collecting duct intercalated cells, whereas HNF-regulated genes, specific for proximal tubule cells, are an integral part of clear cell and papillary RCC transcriptomes. These networks may be used as a framework for understanding the interplay between genomic changes in RCC subtypes and the lineage-defining regulatory machinery of their non-neoplastic counterparts.


Assuntos
Carcinoma de Células Renais/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/metabolismo , Néfrons/metabolismo , Carcinoma de Células Renais/classificação , Carcinoma de Células Renais/genética , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Neoplasias Renais/classificação , Neoplasias Renais/genética , Néfrons/citologia , Transcriptoma
12.
Cell Rep ; 15(8): 1822-36, 2016 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-27184840

RESUMO

Metabolic reprogramming is a hallmark of clear cell renal cell carcinoma (ccRCC) progression. Here, we used genome-scale metabolic modeling to elucidate metabolic reprogramming in 481 ccRCC samples and discovered strongly coordinated regulation of glycosaminoglycan (GAG) biosynthesis at the transcript and protein levels. Extracellular GAGs are implicated in metastasis, so we speculated that such regulation might translate into a non-invasive biomarker for metastatic ccRCC (mccRCC). We measured 18 GAG properties in 34 mccRCC samples versus 16 healthy plasma and/or urine samples. The GAG profiles were distinctively altered in mccRCC. We derived three GAG scores that distinguished mccRCC patients with 93.1%-100% accuracy. We validated the score accuracies in an independent cohort (up to 18 mccRCC versus nine healthy) and verified that the scores normalized in eight patients with no evidence of disease. In conclusion, coordinated regulation of GAG biosynthesis occurs in ccRCC, and non-invasive GAG profiling is suitable for mccRCC diagnosis.


Assuntos
Carcinoma de Células Renais/sangue , Carcinoma de Células Renais/urina , Glicosaminoglicanos/sangue , Glicosaminoglicanos/urina , Neoplasias Renais/sangue , Neoplasias Renais/urina , Idoso , Biomarcadores Tumorais/metabolismo , Vias Biossintéticas/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Regulação Neoplásica da Expressão Gênica , Glicosaminoglicanos/biossíntese , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes
14.
Oncotarget ; 7(3): 2837-54, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26701207

RESUMO

The Myc oncoprotein is tightly regulated at multiple levels including ubiquitin-mediated protein turnover. We recently demonstrated that inhibition of Cdk2-mediated phosphorylation of Myc at Ser-62 pharmacologically or through interferon (IFN)-γ-induced expression of p27(Kip1) (p27) repressed Myc's activity to suppress cellular senescence and differentiation. In this study we identified an additional activity of p27 to interfere with Myc independent of Ser-62 phosphorylation. p27 is required and sufficient for IFN-γ-induced turnover of Myc. p27 interacted with Myc in the nucleus involving the C-termini of the two proteins, including Myc box 4 of Myc. The C-terminus but not the Cdk2 binding fragment of p27 was sufficient for inducing Myc degradation. Protein expression data of The Cancer Genome Atlas breast invasive carcinoma set revealed significantly lower Myc protein levels in tumors with highly expressed p27 lacking phosphorylation at Thr-157--a marker for active p27 localized in the nucleus. Further, these conditions correlated with favorable tumor stage and patient outcome. This novel regulation of Myc by IFN-γ/p27(KIP1) potentially offers new possibilities for therapeutic intervention in tumors with deregulated Myc.


Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Interferon gama/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Senescência Celular/fisiologia , Chlorocebus aethiops , Quinase 2 Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Fosforilação , Ligação Proteica
15.
Cancer Lett ; 197(1-2): 145-50, 2003 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-12880974

RESUMO

Hypoxia in solid tumors is associated with aggressive behavior and poor outcome. We recently discovered that hypoxia alters the expression of differentiation marker genes in neuroblastoma cells, in that the tumor cells adjust to the hypoxic environment by down-regulating genes associated with a neuronal and upregulating genes associated with a neural crest-like phenotype. As there is a correlation in neuroblastoma between low stage of differentiation and high (aggressive) clinical stage, we propose that dedifferentiation of neuroblastoma cells in hypoxic tumor regions contribute to the malignancy of the tumor.


Assuntos
Diferenciação Celular , Hipóxia/metabolismo , Neuroblastoma/patologia , Hipóxia Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Neuroblastoma/genética , Neuroblastoma/metabolismo , Sistema Nervoso Simpático/metabolismo , Fatores de Transcrição , Células Tumorais Cultivadas
16.
J Biotechnol ; 192 Pt A: 62-5, 2014 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-25277986

RESUMO

Over the past years, massive progress has been made in the ability to collect large-scale gene expression data from a limited sample size. Combined with improvements in multiplex flow cytometry-based techniques, this has made it possible to isolate and characterize specific cellular subtypes within heterogeneous populations, with a great impact on our understanding of different biological processes. However, sorting based on intracellular markers requires fixation and permeabilization of samples, and very often the integrity of RNA molecules is compromised during this process. Many attempts have been made to improve the quality of nucleic acids from such samples, but RNA degradation still remains a limiting factor for downstream analyses. Here we present a method to isolate high quality RNA from cells that have been fixed, permeabilized, intracellularly labeled and sorted. By performing all incubation steps in the presence of a high salt buffer, RNA degradation was avoided and samples with remarkable integrity were obtained. This procedure offers a straightforward and very affordable technique to retrieve high quality RNA from isolated cell populations, which increases the possibilities to characterize gene expression profiles of subpopulations from mixed samples, a technique with implications in a broad range of research fields.


Assuntos
Estabilidade de RNA/efeitos dos fármacos , RNA/isolamento & purificação , Cloreto de Sódio/farmacologia , Coloração e Rotulagem/métodos , Anticorpos/farmacologia , Soluções Tampão , Carbocianinas/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Citometria de Fluxo , Corantes Fluorescentes/farmacologia , Formaldeído/farmacologia , Humanos , Queratina-7/imunologia , Queratina-8/imunologia , Metanol/farmacologia , Polímeros/farmacologia
17.
Eur J Obstet Gynecol Reprod Biol ; 181: 10-4, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25126978

RESUMO

OBJECTIVE: The objective of this study was to evaluate the protective effects of a new device for reducing perineal tears during vaginal childbirth. STUDY DESIGN: A multicenter open randomized controlled trial (RCT) was performed in Helsingborg, Lund and Malmö, Sweden consisting of 1148 women. Women anticipating a vaginal delivery were either randomized to the intervention group (n=574 in which the perineal protection device was used, or a control group (n=574), in which the perineal protection device was not used. The main outcome measurements were incidence of vaginal and perineal tears (1st to 4th degree tears) and adverse effects on the parturient and newborn. RESULTS: The incidences of first- and second-degree tears of the vagina (p=0.018) and perineum (p=0.005) were significantly reduced in the intervention group compared with the controls. In the intervention- and control group, 184 women (34.9%) and 142 (26.6%) showed no perineal tearing, respectively (p=0.034). Numbers needed to treat to avoid any perianal tearing was 12. The incidence of anal sphincter rupture (ASR) was the same in both groups (n=19; 3.4%). No negative effects on mother or child from using the device were observed. CONCLUSIONS: The perineal protective device significantly reduced the incidence of first- and second-degree tears in the vagina and perineum during vaginal birth and also significantly increased the number of parturients with a fully intact posterior commissure. No significant reduction of ASR and no negative effects of the device were observed.


Assuntos
Canal Anal/lesões , Lacerações/prevenção & controle , Complicações do Trabalho de Parto/prevenção & controle , Períneo/lesões , Equipamentos de Proteção , Adolescente , Adulto , Feminino , Humanos , Trabalho de Parto , Pessoa de Meia-Idade , Gravidez , Equipamentos de Proteção/efeitos adversos , Ruptura/prevenção & controle , Índices de Gravidade do Trauma , Adulto Jovem
18.
EMBO Mol Med ; 5(7): 1067-86, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23776131

RESUMO

SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCF(FBXO28) activity and stability are regulated during the cell cycle by CDK1/2-mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F-box mutant unable to support MYC ubiquitylation results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCF(FBXO28) plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Mama/patologia , Proteína Quinase CDC2/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Sequência de Aminoácidos , Mama/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Fosforilação , Prognóstico , Regiões Promotoras Genéticas , Proteólise , Proteínas Ligases SKP Culina F-Box/análise , Proteínas Ligases SKP Culina F-Box/genética , Transdução de Sinais , Análise de Sobrevida , Ativação Transcricional , Ubiquitinação
19.
Int J Cancer ; 119(3): 624-9, 2006 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-16506218

RESUMO

Deletions of the short arm of chromosome 3 are often observed in a specific subset of aggressive neuroblastomas (NBs) with loss of distal 11q and without MYCN amplification. The critical deleted region encompasses the locus of the von Hippel-Lindau gene (VHL, 3p25). Constitutional loss of function mutations in the VHL gene are responsible for the VHL syndrome, a dominantly inherited familial cancer syndrome predisposing to a variety of neoplasms, including pheochromocytoma. Pheochromocytomas are, like NB, derived from neural crest cells, but, unlike NB, consist of more mature chromaffin cells instead of immature neuroblasts. Further arguments for a putative role of VHL in NB are its function as oxygen sensitizer and the reported relation between hypoxia and dedifferentiation of NB cells, leading to a more aggressive phenotype. To test the possible involvement of VHL in NB, we did mRNA expression analysis and sought evidence for VHL gene inactivation. Although no evidence for a classic tumor suppressor role for VHL in NB could be obtained, a strong correlation was observed between reduced levels of VHL mRNA and low patient survival probability (p=0.013). Furthermore, VHL appears to have predictive power in NTRK1 (TRKA) positive tumor samples with presumed favorable prognosis, which makes it a potentially valuable marker for more accurate risk assessment in this subgroup of patients. The significance of the reduced VHL expression levels in relation to NB tumor biology remains unexplained, as functional analysis demonstrated no clear effect of the reduction in VHL mRNA expression on protein stability of its downstream target hypoxia-inducible factor alpha.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neuroblastoma/patologia , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Western Blotting , Linhagem Celular Tumoral , Criança , Pré-Escolar , Metilação de DNA , Análise Mutacional de DNA , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lactente , Recém-Nascido , Mutação , Neuroblastoma/genética , Neuroblastoma/metabolismo , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sobrevida , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
20.
Genes Chromosomes Cancer ; 45(2): 107-17, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16235245

RESUMO

Despite oncogene amplification being a characteristic of many tumor types, the mechanisms leading to amplicon formation have remained largely unresolved. In this study, we used a combinatorial approach of fluorescence in situ hybridization and single-nucleotide polymorphism chip gene copy number analyses to unravel the mechanism leading to nonsyntenic coamplification of MYC and ATBF1 in SJNB-12 cells. To explain our findings, we propose a complex series of events consisting of multiple double-strand breaks, accompanied (or triggered) by the formation of a reciprocal translocation t(8;16), as well as excisions and deletions near the translocation breakpoints. This study provides evidence for a translocation-excision-deletion-amplification sequence of events rather than a breakage-fusion-bridge model, which has been more frequently proposed to explain proto-oncogene amplification. Furthermore, it illustrates the power of presently available tools for detailed analysis of the complex rearrangements that accompany amplicon formation.


Assuntos
Amplificação de Genes , Deleção de Genes , Genes myc , Proteínas de Homeodomínio/genética , Translocação Genética , Linhagem Celular Tumoral , Cromossomos Humanos Par 16 , Cromossomos Humanos Par 8 , Humanos , Hibridização in Situ Fluorescente , Proto-Oncogene Mas
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