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1.
Methods Mol Biol ; 2206: 39-46, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754809

RESUMO

During angiogenesis, endothelial cells must undergo a coordinated set of morphological changes in order to form a new vessel. There is a need for endothelial cells to communicate with each other in order to take up different identities in the sprout and to migrate collectively as a connected chord. Endothelial cells must also interact with a wide range of other cells that contribute to vessel formation. In ischemic disease, hypoxic cells in tissue will generate proangiogenic signals that promote and guide angiogenesis. In solid tumors, this function is co-opted by tumor cells, which make a complex range of interactions with endothelial cells, even integrating into the walls of vessels. In vessel repair, cells from the immune system contribute to the promotion and remodeling of new vessels. The coculture angiogenesis assay is a long-term in vitro protocol that uses fibroblasts to secrete and condition an artificial stromal matrix for tubules to grow through. We show here how the assay can be easily adapted to include additional cell types, facilitating the study of cellular interactions during neovascularization.

2.
Pathol Res Pract ; 216(11): 153225, 2020 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-32987302

RESUMO

The in vitro 3D model established from murine pluripotential stem cells (i.e., embryoid bodies (EBs)) is a dynamic model for endothelial differentiation. The aim of the present study was to investigate whether digital image analysis (DIA) can be applied on histological sections of EBs in order to quantify endothelial differentiation over time. The EBs were established in suspension cultures for 21 days in three independent replicate experiments. At day 4, 6, 9, 14, 18, and 21, the EBs were fixed in formaldehyde, embedded in paraffin and immunohistochemically (IHC) stained for CD31. The IHC-stained slides were digitally scanned and analysed using the Visiopharm® Quantitative Digital Pathology software Oncotopix™. The EBs developed CD31+ vascular-like structures during their differentiation. The quantitative DIA of the EBs showed that the log10 values of the relative CD31+ areas increased from -0.574 ± 0.470 (mean ± SD) at day 4 to 0.093 ± 0.688 (mean ± SD) at day 21 (p < 0.001). The approach presented in this study is a fast, quantitative and reproducible alternative method for an otherwise time-consuming and observer-dependent histological investigation. The future perspectives for such a system would be implementation of a modified version of the method on different 3D cultures and IHC markers.

3.
Int J Exp Pathol ; 100(1): 12-18, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30884019

RESUMO

The aim of the present study was to characterize a patient-derived in vitro 3D model (ie tumoroid) established from colorectal adenocarcinoma. This study investigated the growth rate of tumoroids and whether the Kirsten rat sarcoma (KRAS) mutations in the parental tumour accelerate this rate. The tumoroids were established from surgical resections of primary and metastatic colorectal adenocarcinoma from 26 patients. The in vitro growth rate of these tumoroids was monitored by automated imaging and recorded as relative growth rate. The KRAS hotspot mutations were investigated on the parental tumours by Ion Torrent™ next-generation sequencing. The KRAS mutations were detected in 58% of the parental tumours, and a significantly higher growth rate was observed for tumoroids established from the KRAS-mutated tumours compared to wild-type tumours (P < 0.0001). The average relative growth rate (log10) on day 10 was 0.360 ± 0.180 (mean ± SD) for the KRAS-mutated group and 0.098 ± 0.135 (mean ± SD) for the KRAS wild-type group. These results showed that the presence of KRAS mutations in parental tumours is associated with an acceleration of the growth rate of tumoroids. The future perspective for such a model could be the implementation of chemoassays for personalized medicine.


Assuntos
Adenocarcinoma/genética , Adenocarcinoma/secundário , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA/métodos , Feminino , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Organoides , Fenótipo , Fatores de Tempo , Células Tumorais Cultivadas
4.
Mol Oncol ; 12(1): 132-147, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29130628

RESUMO

Patient-derived in vitro cultures of colorectal cancer (CRC) may help guide treatment strategies prior to patient treatment. However, most previous studies have been performed on a single biopsy per tumor. The purpose of this study was to analyze multiple spatially distinct biopsies from CRCs and see how well intratumor heterogeneity (ITH) was recapitulated in matching patient-derived spheroids. Three to five biopsies were collected from six CRC tumors. Each biopsy was split in two; one half was used for spheroid culturing, while the other half was used for DNA and RNA purification. For two patients, lymph node metastases were analyzed. Somatic mutations were called from whole exome sequencing data. Each tumor contained mutations shared across all biopsies and spheroids, including major CRC drivers such as APC, KRAS, and TP53. At the same time, all tumors exhibited ITH on both mutation and copy number level. The concordance between biopsies and spheroids ranged between 40 and 70% for coding mutations. For three patients, the biopsy and spheroid from matching areas clustered together, meaning that the spheroid resembled the area of origin more than the other areas. However, all biopsies and spheroids contained private mutations. Therefore, multiple cultures from spatially distinct sites of the tumor increase the insight into the genetic profile of the entire tumor. Molecular subtypes were called from RNA sequencing data. When based on transcripts from both cancer and noncancerous cells, the subtypes were largely independent of sampling site. In contrast, subtyping based on cancer cell transcripts alone was dependent on sample site and genetic ITH. In conclusion, all examined CRC tumors showed genetic ITH. Spheroid cultures partly reflected this ITH, and having multiple cultures from distinct tumor sites improved the representation of the genetic tumor subclones. This should be taken into account when establishing patient-derived models for drug screening.


Assuntos
Neoplasias Colorretais/genética , Heterogeneidade Genética , Esferoides Celulares/patologia , Idoso , Idoso de 80 Anos ou mais , Biópsia , Neoplasias Colorretais/patologia , Feminino , Humanos , Linfonodos/patologia , Metástase Linfática/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Filogenia , Análise de Sequência de RNA , Esferoides Celulares/citologia , Células Tumorais Cultivadas , Sequenciamento Completo do Exoma
5.
PLoS One ; 12(9): e0183074, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28877221

RESUMO

Chemotherapy treatment of cancer remains a challenge due to the molecular and functional heterogeneity displayed by tumours originating from the same cell type. The pronounced heterogeneity makes it difficult for oncologists to devise an effective therapeutic strategy for the patient. One approach for increasing treatment efficacy is to test the chemosensitivity of cancer cells obtained from the patient's tumour. 3D culture represents a promising method for modelling patient tumours in vitro. The aim of this study was therefore to evaluate how closely short-term spheroid cultures of primary colorectal cancer cells resemble the original tumour. Colorectal cancer cells were isolated from human tumour tissue and cultured as spheroids. Spheroid cultures were established with a high success rate and remained viable for at least 10 days. The spheroids exhibited significant growth over a period of 7 days and no difference in growth rate was observed for spheroids of different sizes. Comparison of spheroids with the original tumour revealed that spheroid culture generally preserved adenocarcinoma histology and expression patterns of cytokeratin 20 and carcinoembryonic antigen. Interestingly, spheroids had a tendency to resemble tumour protein expression more closely after 10 days of culture compared to 3 days. Chemosensitivity screening using spheroids from five patients demonstrated individual response profiles. This indicates that the spheroids maintained patient-to-patient differences in sensitivity towards the drugs and combinations most commonly used for treatment of colorectal cancer. In summary, short-term spheroid culture of primary colorectal adenocarcinoma cells represents a promising in vitro model for use in personalized medicine.


Assuntos
Neoplasias Colorretais/patologia , Modelos Biológicos , Medicina de Precisão , Esferoides Celulares/patologia , Adenocarcinoma/patologia , Proliferação de Células , Sobrevivência Celular , Células Epiteliais/patologia , Fibroblastos/patologia , Humanos , Queratina-20/metabolismo , Proteínas de Neoplasias/metabolismo , Células Tumorais Cultivadas
6.
Biochem J ; 441(1): 325-37, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22032326

RESUMO

Mutations in the WNK [with no lysine (K) kinase] family instigate hypertension and pain perception disorders. Of the four WNK isoforms, much of the focus has been on WNK1, which is activated in response to osmotic stress by phosphorylation of its T-loop residue (Ser382). WNK isoforms phosphorylate and activate the related SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) protein kinases. In the present study, we first describe the generation of double-knockin ES (embryonic stem) cells, where SPAK and OSR1 cannot be activated by WNK1. We establish that NKCC1 (Na+/K+/2Cl- co-transporter 1), a proposed target of the WNK pathway, is not phosphorylated or activated in a knockin that is deficient in SPAK/OSR1 activity. We also observe that activity of WNK1 and WNK3 are markedly elevated in the knockin cells, demonstrating that SPAK/OSR1 significantly influences WNK activity. Phosphorylation of another regulatory serine residue, Ser1261, in WNK1 is unaffected in knockin cells, indicating that this is not phosphorylated by SPAK/OSR1. We show that WNK isoforms interact via a C-terminal CCD (coiled-coil domain) and identify point mutations of conserved residues within this domain that ablate the ability of WNK isoforms to interact. Employing these mutants, we demonstrate that interaction of WNK isoforms is not essential for their T-loop phosphorylation and activation, at least for overexpressed WNK isoforms. Moreover, we finally establish that full-length WNK1, WNK2 and WNK3, but not WNK4, are capable of directly phosphorylating Ser382 of WNK1 in vitro. This supports the notion that T-loop phosphorylation of WNK isoforms is controlled by trans-autophosphorylation. These results provide novel insights into the WNK signal transduction pathway and provide genetic evidence confirming the essential role that SPAK/OSR1 play in controlling NKCC1 function. They also reveal a role in which the downstream SPAK/OSR1 enzymes markedly influence the activity of the upstream WNK activators. The knockin ES cells lacking SPAK/OSR1 activity will be useful in validating new targets of the WNK signalling pathway.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Células-Tronco Embrionárias , Regulação da Expressão Gênica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Mutação , Fosforilação , Isoformas de Proteínas , Estrutura Terciária de Proteína , Proteínas Serina-Treonina Quinases/genética , Simportadores de Cloreto de Sódio-Potássio/genética , Membro 2 da Família 12 de Carreador de Soluto , Fatores de Transcrição/genética , Proteína Quinase 1 Deficiente de Lisina WNK
7.
ACS Comb Sci ; 13(6): 667-75, 2011 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-21905744

RESUMO

Apoptotic induction mechanisms are of crucial importance for the general homeostasis of multicellular organisms. In cancer the apoptotic pathways are downregulated, which, at least partly, is due to an abundance of inhibitors of apoptosis proteins (IAPs) that block the apoptotic cascade by deactivating proteolytic caspases. The Smac protein has an antagonistic effect on IAPs, thus providing structural clues for the synthesis of new pro-apoptotic compounds. Herein, we report a solid-phase approach for the synthesis of Smac-derived tetrapeptide libraries. On the basis of a common (N-Me)AVPF sequence, peptides incorporating triazoloprolines and biarylalanines were synthesized by means of Cu(I)-catalyzed azide-alkyne cycloaddition and Pd-catalyzed Suzuki cross-coupling reactions. Solid-phase procedures were optimized to high efficiency, thus accessing all products in excellent crude purities and yields (both typically above 90%). The peptides were subjected to biological evaluation in a live/dead cellular assay which revealed that structural decorations on the AVPF sequence indeed are highly important for cytotoxicity toward HeLa cells.


Assuntos
Alanina/química , Proteínas Inibidoras de Apoptose/síntese química , Oligopeptídeos/síntese química , Peptidomiméticos/síntese química , Prolina/química , Técnicas de Síntese em Fase Sólida/métodos , Alanina/análogos & derivados , Alquinos/química , Azidas/química , Catálise , Cobre/química , Hidrocarbonetos Aromáticos/química , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/farmacologia , Modelos Químicos , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Paládio/química , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Prolina/análogos & derivados , Triazóis/química
8.
FEBS Lett ; 582(20): 3145-51, 2008 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-18703060

RESUMO

The c-Jun N-terminal kinase (JNK) signalling pathway has an established role in cellular stress signalling, cell survival and tumorigenesis. Here, we demonstrate that inhibition of JNK signalling results in partial delocalization of the RNA helicase DDX21 from the nucleolus to the nucleoplasm, increased nucleolar mobility of DDX21 and inhibition of rRNA processing. Furthermore, our results show that JNK signalling regulates DDX21 phosphorylation and protein expression. In conclusion, the results presented in this study reveal a previously unidentified cellular role for JNK signalling in the regulation of nucleolar functions. Based on these results, we propose that JNK-mediated effects on nucleolar homeostasis and rRNA processing should be considered when interpreting cellular phenotypes observed in JNK-deficient cell and animal models.


Assuntos
Nucléolo Celular/enzimologia , RNA Helicases DEAD-box/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Camundongos , Camundongos Knockout , Fosforilação , Biossíntese de Proteínas , RNA Ribossômico/metabolismo , Transdução de Sinais
9.
EMBO Rep ; 8(9): 839-45, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17721439

RESUMO

The oxidative-stress-responsive kinase 1 (OSR1) and the STE20/SPS1-related proline/alanine-rich kinase (SPAK) are key enzymes in a signalling cascade regulating the activity of Na(+)/K(+)/2Cl(-) co-transporters (NKCCs) in response to osmotic stress. Both kinases have a conserved carboxy-terminal (CCT) domain, which recognizes a unique peptide (Arg-Phe-Xaa-Val) motif present in OSR1- and SPAK-activating kinases (with-no-lysine kinase 1 (WNK1) and WNK4) as well as its substrates (NKCC1 and NKCC2). Here, we describe the structural basis of this recognition event as shown by the crystal structure of the CCT domain of OSR1 in complex with a peptide containing this motif, derived from WNK4. The CCT domain forms a novel protein fold that interacts with the Arg-Phe-Xaa-Val motif through a surface-exposed groove. An intricate web of interactions is observed between the CCT domain and an Arg-Phe-Xaa-Val motif-containing peptide derived from WNK4. Mutational analysis shows that these interactions are required for the CCT domain to bind to WNK1 and NKCC1. The CCT domain structure also shows how phosphorylation of a Ser/Thr residue preceding the Arg-Phe-Xaa-Val motif results in a steric clash, promoting its dissociation from the CCT domain. These results provide the first molecular insight into the mechanism by which the SPAK and OSR1 kinases specifically recognize their upstream activators and downstream substrates.


Assuntos
Ativadores de Enzimas/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
10.
Cancer Res ; 67(1): 178-85, 2007 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17210697

RESUMO

Active Ras oncogene is expressed in approximately 30% of human cancers. Yet, very little is known about the molecular mechanisms responsible for its transforming potential. Here, we show that H-Ras-mediated transformation requires isoform 2 of the c-Jun-NH(2)-terminal kinase (JNK). H-Ras-transduced JNK2-deficient (Jnk2-/-) murine embryonic fibroblasts (MEFs) were severely inhibited in colony formation and growth in soft agar in vitro as well as in tumor formation in immunodeficient mice as compared with corresponding Jnk1-/- and wild-type MEFs. Accordingly, the RNA interference-based depletion of JNK2 form wild-type MEFs also resulted in defective Ras transformation. The extra barrier against H-Ras transformation in Jnk2-/- MEFs was not due to their inability to inactivate p53 signaling because all JNK2-deficient MEF lines had lost p19(Arf). Furthermore, expression of the E6 protein of the human papilloma virus failed to overcome the transformation defect. It could, however, be overcome by coexpression of H-Ras with the SV40 large T antigen or c-Myc. Surprisingly, the H-Ras-transduced JNK2-deficient MEFs exhibited higher activity of activator protein-1 and higher levels of c-Jun expression compared with H-Ras-transduced JNK1-deficient or wild-type cells, indicating that the key target of JNK2 during Ras transformation was divergent from activator protein-1. These results clearly show that a single kinase, JNK2, could control Ras transformation and thus point out a vulnerable control point that may prove important for the tumor development in general.


Assuntos
Transformação Celular Neoplásica/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Fator de Transcrição AP-1/metabolismo , Proteínas ras/metabolismo , Animais , Transformação Celular Neoplásica/genética , Regulação para Baixo , Feminino , Genes ras , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Proteína Quinase 9 Ativada por Mitógeno/deficiência , Proteína Quinase 9 Ativada por Mitógeno/genética , Células NIH 3T3 , Fosforilação , Interferência de RNA , Transdução de Sinais , Transdução Genética , Proteínas ras/genética
11.
J Cell Biol ; 176(1): 89-100, 2007 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17190791

RESUMO

Mutations within the WNK1 (with-no-K[Lys] kinase-1) gene cause Gordon's hypertension syndrome. Little is known about how WNK1 is regulated. We demonstrate that WNK1 is rapidly activated and phosphorylated at multiple residues after exposure of cells to hyperosmotic conditions and that activation is mediated by the phosphorylation of its T-loop Ser382 residue, possibly triggered by a transautophosphorylation reaction. Activation of WNK1 coincides with the phosphorylation and activation of two WNK1 substrates, namely, the protein kinases STE20/SPS1-related proline alanine-rich kinase (SPAK) and oxidative stress response kinase-1 (OSR1). Small interfering RNA depletion of WNK1 impairs SPAK/OSR1 activity and phosphorylation of residues targeted by WNK1. Hyperosmotic stress induces rapid redistribution of WNK1 from the cytosol to vesicular structures that may comprise trans-Golgi network (TGN)/recycling endosomes, as they display rapid movement, colocalize with clathrin, adaptor protein complex 1 (AP-1), and TGN46, but not the AP-2 plasma membrane-coated pit marker nor the endosomal markers EEA1, Hrs, and LAMP1. Mutational analysis suggests that the WNK1 C-terminal noncatalytic domain mediates vesicle localization. Our observations shed light on the mechanism by which WNK1 is regulated by hyperosmotic stress.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Sorbitol/farmacologia , Sequência de Aminoácidos , Animais , Domínio Catalítico/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Clatrina/metabolismo , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/enzimologia , Ativação Enzimática/efeitos dos fármacos , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Pressão Osmótica , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/química , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK
12.
Biochem J ; 397(1): 223-31, 2006 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-16669787

RESUMO

The SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase-1) kinases interact and phosphorylate NKCC1 (Na+-K+-2Cl- co-transporter-1), leading to its activation. Recent studies indicated that SPAK and OSR1 are phosphorylated and activated by the WNK1 [with no K (lysine) protein kinase-1] and WNK4, genes mutated in humans affected by Gordon's hypertension syndrome. In the present study, we have identified three residues in NKCC1 (Thr175/Thr179/Thr184 in shark or Thr203/Thr207/Thr212 in human) that are phosphorylated by SPAK and OSR1, and have developed a peptide substrate, CATCHtide (cation chloride co-transporter peptide substrate), to assess SPAK and OSR1 activity. Exposure of HEK-293 (human embryonic kidney) cells to osmotic stress, which leads to phosphorylation and activation of NKCC1, increased phosphorylation of NKCC1 at the sites targeted by SPAK/OSR1. The residues on NKCC1, phosphorylated by SPAK/OSR1, are conserved in other cation co-transporters, such as the Na+-Cl- co-transporter, the target of thiazide drugs that lower blood pressure in humans with Gordon's syndrome. Furthermore, we characterize the properties of a 92-residue CCT (conserved C-terminal) domain on SPAK and OSR1 that interacts with an RFXV (Arg-Phe-Xaa-Val) motif present in the substrate NKCC1 and its activators WNK1/WNK4. A peptide containing the RFXV motif interacts with nanomolar affinity with the CCT domains of SPAK/OSR1 and can be utilized to affinity-purify SPAK and OSR1 from cell extracts. Mutation of the arginine, phenylalanine or valine residue within this peptide abolishes binding to SPAK/OSR1. We have identified specific residues within the CCT domain that are required for interaction with the RFXV motif and have demonstrated that mutation of these in OSR1 inhibited phosphorylation of NKCC1, but not of CATCHtide which does not possess an RFXV motif. We establish that an intact CCT domain is required for WNK1 to efficiently phosphorylate and activate OSR1. These data establish that the CCT domain functions as a multipurpose docking site, enabling SPAK/OSR1 to interact with substrates (NKCC1) and activators (WNK1/WNK4).


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Sítios de Ligação , Células Cultivadas , Ativação Enzimática , Humanos , Hiperpotassemia/genética , Hipertensão/genética , Peptídeos e Proteínas de Sinalização Intracelular , Rim/citologia , Antígenos de Histocompatibilidade Menor , Mutação , Concentração Osmolar , Fosforilação , Homologia de Sequência de Aminoácidos , Serina , Simportadores de Cloreto de Sódio-Potássio , Membro 2 da Família 12 de Carreador de Soluto , Especificidade por Substrato , Treonina , Proteína Quinase 1 Deficiente de Lisina WNK
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