Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.588
Filtrar
1.
Nat Commun ; 12(1): 1894, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33767165

RESUMO

Neural crest stem cells arising from caudal hindbrain (often called cardiac and posterior vagal neural crest) migrate long distances to form cell types as diverse as heart muscle and enteric ganglia, abnormalities of which lead to common congenital birth defects. Here, we explore whether individual caudal hindbrain neural crest precursors are multipotent or predetermined toward these particular fates and destinations. To this end, we perform lineage tracing of chick neural crest cells at single-cell resolution using two complementary approaches: retrovirally mediated multiplex clonal analysis and single-cell photoconversion. Both methods show that the majority of these neural crest precursors are multipotent with many clones producing mesenchymal as well as neuronal derivatives. Time-lapse imaging demonstrates that sister cells can migrate in distinct directions, suggesting stochasticity in choice of migration path. Perturbation experiments further identify guidance cues acting on cells in the pharyngeal junction that can influence this choice; loss of CXCR4 signaling results in failure to migrate to the heart but no influence on migration toward the foregut, whereas loss of RET signaling does the opposite. Taken together, the results suggest that environmental influences rather than intrinsic information govern cell fate choice of multipotent caudal hindbrain neural crest cells.


Assuntos
Sistema Nervoso Entérico/embriologia , Coração/embriologia , Células-Tronco Multipotentes/citologia , Crista Neural/citologia , Células-Tronco Neurais/citologia , Animais , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Galinhas , Proteínas Proto-Oncogênicas c-ret/genética , Receptores CXCR4/genética , Rombencéfalo/citologia , Transdução de Sinais/genética
2.
Nat Commun ; 12(1): 1714, 2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33731701

RESUMO

Advanced prostate cancer (PCa) often develops bone metastasis, for which therapies are very limited and the underlying mechanisms are poorly understood. We report that bone-borne TGF-ß induces the acetylation of transcription factor KLF5 in PCa bone metastases, and acetylated KLF5 (Ac-KLF5) causes osteoclastogenesis and bone metastatic lesions by activating CXCR4, which leads to IL-11 secretion, and stimulating SHH/IL-6 paracrine signaling. While essential for maintaining the mesenchymal phenotype and tumorigenicity, Ac-KLF5 also causes resistance to docetaxel in tumors and bone metastases, which is overcome by targeting CXCR4 with FDA-approved plerixafor. Establishing a mechanism for bone metastasis and chemoresistance in PCa, these findings provide a rationale for treating chemoresistant bone metastasis of PCa with inhibitors of Ac-KLF5/CXCR4 signaling.


Assuntos
Neoplasias Ósseas/secundário , Carcinogênese , Transição Epitelial-Mesenquimal , Fatores de Transcrição Kruppel-Like/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Acetilação , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Benzilaminas/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Ciclamos/uso terapêutico , Docetaxel/uso terapêutico , Humanos , Interleucina-11/genética , Interleucina-11/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Mutação , Osteogênese , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo
3.
J Biomed Nanotechnol ; 17(2): 263-278, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33785097

RESUMO

Pancreatic cancer is highly lethal and has a poor prognosis. The most common alteration during the formation of pancreatic tumors is the activation of KRAS (Kirsten rat sarcoma 2 viral oncogene homolog) oncogene. As a new therapeutic strategy, the C19 molecule ((2S)-N-(2,5-dichlorophenyl)-2-[(3,4-dimethoxyphenyl)-methylamine]propanamide) blocks the KRAS-membrane association in cancer cells. In addition, the chemokine receptor CXCR4 is overexpressed in pancreatic cancer. In this research, a new dendrimer-based nanoradiopharmaceutical (177Lu-DN(C19)-CXCR4L) encapsulating C19 and functionalized to target CXCR4 receptors is proposed as both, a targeted radiotherapy system (lutetium-177) and an oncotherapeutic approach by the stabilization of KRAS4b-PDESδ complex to produce dual-specific therapy in pancreatic cancer. 177The Lu-DN(C19)-CXCR4L was synthesized and characterized, C19 was encapsulated with 81% efficiency, the final nanosystem rendered a particle size of 67 nm and the specific uptake in pancreatic cell lines was demonstrated. The major cytotoxic effect was observed in the KRAS-dependent and radioresistant cell line Mia PaCa-2, which expresses a high density of CXCR4 receptors. The radiation dose of 3 Gy/Bq decreased viability to 7%, and this effect was attributed to the presence of C19. A synergistic effect (radio and chemotherapy) capable of reducing viability in pancreatic cancer cells through apoptotic mechanisms was demonstrated. Thus, 177Lu-DN(C19)-CXCR4L nanoradiopharmaceutical is efficacious in pancreatic cancer cell lines overexpressing the CXCR4 receptor.


Assuntos
Neoplasias Pancreáticas , Receptores CXCR4 , Linhagem Celular Tumoral , Humanos , Ligantes , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Receptores CXCR4/genética , Transdução de Sinais
4.
Chem Biol Interact ; 339: 109424, 2021 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-33617803

RESUMO

OBJECTIVE: To reveal the effects and related mechanism of cisatracurium on colorectal cancer (CRC) development. METHODS: HCT116 and SW480 cells were treated with various concentrations of cisatracurium or transforming growth factor-ß (TGF-ß). Chemokine C-X-C-Motif Receptor 4 (CXCR4) was overexpressed and let-7a-5p was silenced in cells by transfection with pcDNA3.1-CXCR4 or let-7a-5p inhibitor. Cell Counting Kit-8 (CCK-8) assay measured cell viability, and transwell and wound healing assays evaluated cell invasion and migration, respectively. The expression levels of let-7a-5p and CXCR4 were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Western blotting was conducted to test the levels of CXCR4, TGF-ß/SMAD2/3 signalling and metastasis-related proteins. A tumour xenograft assay was performed to assess tumour growth. RESULTS: Cisatracurium treatment suppressed the viability and metastasis of HCT116 and SW480 cells in a concentration-dependent manner, whereas activating TGF-ß/SMAD2/3 signalling significantly reversed these effects. Cisatracurium treatment markedly reduced CXCR4 expression by inhibiting TGF-ß/SMAD2/3 signalling. Besides, let-7a-5p was identified as a target of CXCR4 and could be upregulated by cisatracurium. Both CXCR4 overexpression and let-7a-5p knockdown alleviated the biological roles of cisatracurium in CRC cells. Moreover, a tumour xenograft assay further confirmed that cisatracurium inhibited tumour growth and metastasis by increasing let-7a-5p expression. CONCLUSION: Cisatracurium suppressed the viability, metastasis and tumour growth of CRC by regulating the CXCR4/let-7a-5p axis via inhibiting TGF-ß/SMAD2/3 signalling. These findings provide a theoretical basis for the role of cisatracurium in the prognosis of CRC patients.


Assuntos
Atracúrio/análogos & derivados , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , MicroRNAs/genética , Receptores CXCR4/genética , Transdução de Sinais/efeitos dos fármacos , Animais , Atracúrio/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células HCT116 , Xenoenxertos/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transdução de Sinais/genética , Proteína Smad2/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
5.
Int J Hematol ; 113(1): 5-9, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33389659

RESUMO

In recent years, multipotent mesenchymal stromal cells (MSCs) have demonstrated tremendous potential for use in regenerative medicine. CXCR4, the receptor for CXCL12, is highly expressed by bone marrow (BM) MSCs and the CXCR4/CXCL12 axis has been shown to be important for migration and homing of BM-MSCs. Typically, MSCs used for clinical applications are collected after culture expansion using enzymatic methods, such as trypsin. Here, we compared different commercially available enzymatic and non-enzymatic methods for collection and dissociation of MSCs from culture plastics and their effects on CXCR4 expression by MSCs. We found that whereas non-enzymatic dissociation buffers and methods maintained CXCR4 expression, all tested enzymatic dissociation solutions dramatically decreased expression of CXCR4. We, therefore, strongly recommend the use of non-enzymatic dissociation methods, followed by filtration through a cell strainer to obtain single cell suspensions, in order to preserve maximal CXCR4 expression and optimal homing of cells.


Assuntos
Células da Medula Óssea/metabolismo , Separação Celular/métodos , Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Tripsina , Movimento Celular , Células Cultivadas , Quimiocina CXCL12 , Ácido Edético , Humanos
6.
Hematology ; 26(1): 43-52, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33382018

RESUMO

OBJECTIVES: To investigate the role of Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in acute myeloid leukemia (AML) and analyze the potential regulatory network of MALAT1/miR-146a/ CXCR4. METHODS: The expressions of MALAT1, miR-146a and CXCR4 were performed by qRT-PCR and Western Blot. We conducted trans-well assay, CCK-8 assay and flow cytometry to evaluate the migration, proliferation and apoptosis of AML cells. Also by using luciferase reporter assay, we investigated the interaction between miR-146a and MALAT1 or CXCR4. RESULTS: Firstly, MALAT1 and CXCR4 were upregulated while miR-146a was downregulated in AML patients compared with healthy controls. We observed a negative correlation between miR-146a and MALAT1 or CXCR4, but a positive correlation between MALAT1 and CXCR4 in AML patients. MALAT1 knockdown inhibited migration and proliferation but induced apoptosis of HL-60 cells. MALAT1 restrained miR-146a expression by acting as a ceRNA. miR-146a regulated HL-60 cells migration, proliferation and apoptosis by directly targeting CXCR4 expression. Finally, we found that CXCR4 expression was downregulated by MALAT1 knockdown and partially restored by miR-146a abrogation. CONCLUSIONS: Our results showed that MALAT1 regulates migration, proliferation and apoptosis by sponging miR-146a to regulate CXCR4 expression in AML cells, providing novel insights into the role of MALAT1 as a therapeutic target in AML.


Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Receptores CXCR4/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Movimento Celular , Proliferação de Células , Feminino , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
7.
Zhonghua Fu Chan Ke Za Zhi ; 55(11): 754-759, 2020 Nov 25.
Artigo em Chinês | MEDLINE | ID: mdl-33228346

RESUMO

Objective: To observe the expression, correlation and significance of chemokine (C-X-C motif) ligand 12 (CXCL12) and chemokine (C-X-C motif) receptor 4 (CXCR4) in endometrium and myometrium of adenomyosis. Methods: Totally 38 patients were selected in this study, who underwent hysterectomy for adenomyosis at Beijing Obstetrics and Gynecology Hospital from October 2017 to December 2018 as the adenomyosis group, and, in the same period, selected 31 patients with cervical intraepithelial neoplasia Ⅲ or cervical cancer undergoing hysterectomy served as control group. The expression levels of mRNA and protein for CXCL12, CXCR4 in the endometrium and myometrium of the two groups were detected by immunohistochemistry and real-time PCR. Results: (1) The protein levels of CXCL12 and CXCR4 in endometrium in uterus with adenomyosis (0.229±0.025 and 0.226±0.016) were significantly higher than those in endometrium in uterus without adenomyosis (0.153±0.018 and 0.178±0.026); compared with each other, the differences were statistically significant (all P<0.05). And the expressions of CXCL12 and CXCR4 proteins in uterine myometrium of adenomyosis were 0.222±0.045 and 0.126±0.058, respectively, which were higher than those in the control group (0.091±0.029 and 0.099±0.020); compared with each other, the differences were statistically significant (all P<0.05). (2) The expression levels of CXCL12 and CXCR4 mRNA in endometrium of patients with adenomyosis were 6.31±0.12 and 8.49±0.21, respectively, which were higher than those in the control group (1.23±0.10 and 1.36±0.13); compared with each other, the differences were statistically significant (all P<0.05). Moreover, the expression levels of CXCL12 and CXCR4 mRNA in myometrium of patients with adenomyosis were 9.11±0.12 and 8.45±0.16, respectively, which were higher than those in the control group (1.18±0.08 and 1.46±0.13); compared with each other, the differences were statistically significant (all P<0.05). (3) In endometrium and myometrium of uterus with adenomyosis, CXCL12 and CXCR4 mRNA expression levels were positively associated (r=0.478, 0.542, all P<0.05). Conclusions: The levels of CXCL12 and CXCR4 in the endometrium and myometrium of adenomyosis are increased and positively correlated. The two chemokine may be involved in the development of adenomyosis.


Assuntos
Adenomiose/genética , Quimiocina CXCL12/genética , Endométrio/metabolismo , Miométrio/metabolismo , Receptores CXCR4/genética , Adenomiose/patologia , Adenomiose/cirurgia , Biomarcadores Tumorais/genética , Endométrio/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Histerectomia , Miométrio/patologia , Gravidez
8.
Nat Commun ; 11(1): 4820, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973160

RESUMO

Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.


Assuntos
Tirosina-tRNA Ligase/genética , Tirosina-tRNA Ligase/metabolismo , Tirosina/análogos & derivados , Tirosina/genética , Tirosina/metabolismo , Animais , Sistemas CRISPR-Cas , Quimiocinas/metabolismo , Cristalografia por Raios X , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Conformação Proteica , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Tirosina-tRNA Ligase/química
9.
Virology ; 548: 49-58, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838946

RESUMO

Human cytomegalovirus (HCMV) is a widespread herpesvirus that establishes latency in myeloid cells and persists by manipulating immune signaling. Chemokine receptor CXCR4 and its ligand CXCL12 regulate movement of myeloid progenitors into bone marrow and out into peripheral tissues. HCMV amplifies CXCL12-CXCR4 signaling through viral chemokine receptor US27 and cmvIL-10, a viral cytokine that binds the cellular IL-10 receptor (IL-10R), but precisely how these viral proteins influence CXCR4 is unknown. We used the proximity ligation assay (PLA) to examine association of CXCR4, IL-10R, and US27 in both transfected and HCMV-infected cells. CXCR4 and IL-10R colocalized to discrete clusters, and treatment with CXCL12 and cmvIL-10 dramatically increased receptor clustering and calcium flux. US27 was associated with CXCR4 and IL-10R in PLA clusters and further enhanced cluster formation and calcium signaling. These results indicate that CXCR4, IL-10R, and US27 form a novel virus-host signaling complex that enhances CXCL12 signaling during HCMV infection.


Assuntos
Infecções por Citomegalovirus/metabolismo , Citomegalovirus/metabolismo , Receptores CXCR4/metabolismo , Receptores de Quimiocinas/metabolismo , Receptores de Interleucina-10/metabolismo , Proteínas Virais/metabolismo , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Interações Hospedeiro-Patógeno , Humanos , Ligação Proteica , Receptores CXCR4/genética , Receptores de Quimiocinas/genética , Receptores de Interleucina-10/genética , Transdução de Sinais , Proteínas Virais/genética
10.
Cancer Sci ; 111(9): 3327-3337, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32639651

RESUMO

Tirabrutinib is a second-generation Bruton's tyrosine kinase inhibitor with greater selectivity than ibrutinib. Here, we conducted a multicenter, phase II study of tirabrutinib in patients with treatment-naïve (Cohort A) or with relapsed/refractory (Cohort B) Waldenström's macroglobulinemia (WM). Patients were treated with tirabrutinib 480 mg once daily. The primary endpoint was major response rate (MRR; ≥ partial response). Secondary endpoints included overall response rate (ORR; ≥ minor response), time to major response (TTMR), progression-free survival (PFS), overall survival (OS), and safety. In total, 27 patients (18 in Cohort A; 9 in Cohort B) were enrolled. The median age was 71 y, and the median serum immunoglobulin M level was 3600 mg/dL. Among the patients, 96.2% had the MYD88L265P mutation. MRR and ORR were 88.9% and 96.3%, respectively (Cohort A: MRR, 88.9%; ORR, 94.4%; Cohort B: MRR, 88.9%; ORR, 100%). Median TTMR was 1.87 mo. PFS and OS were not reached with a median follow-up of 6.5 and 8.3 mo for Cohorts A and B, respectively. The most common adverse events (AEs) were rash (44.4%), neutropenia (25.9%), and leukopenia (22.2%), with most AEs classified as grade 1 or 2. Grade ≥ 3 AEs included neutropenia (11.1%), lymphopenia (11.1%), and leukopenia (7.4%). No grade 5 AEs were noted. All bleeding events were grade 1; none were associated with drug-related atrial fibrillation or hypertension. Although the follow-up duration was relatively short, the study met the primary endpoint. Therefore, tirabrutinib monotherapy is considered to be highly effective for both untreated and relapsed/refractory WM with a manageable safety profile. (JapicCTI-173646).


Assuntos
Imidazóis/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Macroglobulinemia de Waldenstrom/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Feminino , Genótipo , Humanos , Imidazóis/administração & dosagem , Imidazóis/efeitos adversos , Masculino , Pessoa de Meia-Idade , Mutação , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Gradação de Tumores , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Pirimidinas/administração & dosagem , Pirimidinas/efeitos adversos , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Resultado do Tratamento , Macroglobulinemia de Waldenstrom/diagnóstico , Macroglobulinemia de Waldenstrom/etiologia
11.
Nat Commun ; 11(1): 3194, 2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32581241

RESUMO

Ph+ acute lymphoblastic leukemia (ALL) is characterized by the expression of an oncogenic fusion kinase termed BCR-ABL1. Here, we show that interleukin 7 receptor (IL7R) interacts with the chemokine receptor CXCR4 to recruit BCR-ABL1 and JAK kinases in close proximity. Treatment with BCR-ABL1 kinase inhibitors results in elevated expression of IL7R which enables the survival of transformed cells when IL7 was added together with the kinase inhibitors. Importantly, treatment with anti-IL7R antibodies prevents leukemia development in xenotransplantation models using patient-derived Ph+ ALL cells. Our results suggest that the association between IL7R and CXCR4 serves as molecular platform for BCR-ABL1-induced transformation and development of Ph+ ALL. Targeting this platform with anti-IL7R antibody eliminates Ph+ ALL cells including those with resistance to commonly used ABL1 kinase inhibitors. Thus, anti-IL7R antibodies may provide alternative treatment options for ALL in general and may suppress incurable drug-resistant leukemia forms.


Assuntos
Proteínas de Fusão bcr-abl/metabolismo , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Cromossomo Filadélfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Receptores CXCR4/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Feminino , Proteína Forkhead Box O1/metabolismo , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-7/farmacologia , Subunidade alfa de Receptor de Interleucina-7/antagonistas & inibidores , Subunidade alfa de Receptor de Interleucina-7/genética , Camundongos , Camundongos Mutantes , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptores CXCR4/genética , Transdução de Sinais/efeitos dos fármacos
12.
Ann Hematol ; 99(8): 1763-1769, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32577844

RESUMO

We aimed to detect the MYD88L265P and CXCR4S338X mutations in cell-free DNA (cfDNA) in patients with Waldenström macroglobulinemia (WM). We collected peripheral blood and paired bone marrow aspirates from 27 WM patients (including 16 patients with newly diagnosed WM, 3 patients with WM in relapse and 8 patients with WM during treatment). cfDNA was extracted from peripheral blood using a QIAamp Circulating Nucleic Acid Kit. The MYD88L265P and CXCR4S338X mutations were detected by real-time allele-specific PCR (AS-PCR) in cfDNA and genomic DNA (gDNA) extracted from bone marrow aspirates. The sensitivity of real-time AS-PCR for detecting MYD88L265P in cfDNA was determined using a serial dilution of 10%, 2%, 0.4% and 0.08% MYD88L265P cfDNA in wild-type cfDNA. Among the 27 patients, MYD88L265P was detected in 88.9% of them in gDNA and in 85.2% of them in cfDNA, with a concordance rate of 96.3%. The concordance rates were 93.8%, 100% and 100% in patients with newly diagnosed WM, patients with WM in relapse and patients with WM during treatment, respectively. The sensitivity of real-time AS-PCR for detecting MYD88L265P in cfDNA was 0.4%. CXCR4S338X was detected in 6.3% of the 16 newly diagnosed WM patients in both gDNA and cfDNA, with a concordance rate of 100.0%. It is feasible to apply cfDNA to detect MYD88L265P and CXCR4S338X in WM patients with a high concordance rate.


Assuntos
Ácidos Nucleicos Livres/genética , Mutação de Sentido Incorreto , Fator 88 de Diferenciação Mieloide/genética , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores CXCR4/genética , Macroglobulinemia de Waldenstrom/genética , Idoso , Idoso de 80 Anos ou mais , Substituição de Aminoácidos , Ácidos Nucleicos Livres/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fator 88 de Diferenciação Mieloide/sangue , Proteínas de Neoplasias/sangue , Receptores CXCR4/sangue , Sensibilidade e Especificidade , Macroglobulinemia de Waldenstrom/sangue , Macroglobulinemia de Waldenstrom/terapia
13.
J Vis Exp ; (159)2020 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-32538902

RESUMO

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.


Assuntos
Transferência Adotiva , Subpopulações de Linfócitos B/imunologia , Células da Medula Óssea/imunologia , Movimento Celular , Receptores CXCR4/metabolismo , Retroviridae/metabolismo , Animais , Formação de Anticorpos , Linfócitos B/imunologia , Diferenciação Celular , Imunoglobulina M/imunologia , Antígenos Comuns de Leucócito , Camundongos , Receptores CXCR4/genética , Transdução de Sinais
14.
Oncogene ; 39(28): 5112-5123, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32533098

RESUMO

HOPX is a stem cell marker in hair follicles and intestines. It was shown critical for primitive hematopoiesis. We previously showed an association between higher HOPX expression and clinical characteristics related to stemness and quiescence of leukemic cells in acute myeloid leukemia (AML) patients. To further explore its physiologic functions in hematopoietic system, we generated a mouse model with hematopoietic cell-specific knockout of Hopx (Hopx-/-). In young Hopx-/- mice, the hematopoietic stem cells (HSC) showed decreased reconstitution ability after serial transplantation. Further transcriptomic study revealed decreased HSC signatures in long-term HSCs from the Hopx-/- mice. At 18 months of age, half of the Hopx-/- mice developed cytopenia and splenomegaly. Bone marrow (BM) from the sick mice showed myeloid hyperplasia with predominant mature neutrophils, and decreased progenitor cells and lymphocytes. These phenotypes suggested critical functions of Hopx in maintaining HSC quiescence. Transcriptomic study of the Hopx-/- marrow cells showed significant downregulation of the Cxcl12-Cxcr4 axis, which is critical for maintenance of HSC quiescence. We next examined the role of Hopx in AML by using the MN1 overexpression murine leukemia model. Mice transplanted with MN1-overexpressed Hopx-/- BM cells developed AML with more aggressive phenotypes compared with those transplanted with MN1-overexpressed Hopx-wild cells. Hopx-/- MN1-overexpressed leukemia cells showed higher proliferation rate and downregulation of Cxcl12 and Cxcr4. Furthermore, in human AML, BM plasma CXCL12 levels were lower in patients with lower HOPX expression. In conclusion, our study highlights the roles of Hopx in maintenance of quiescence of the hematopoietic stem cells through CXCL12 pathway in vivo and provides implication of this protein in normal and malignant hematopoiesis.


Assuntos
Células da Medula Óssea/metabolismo , Perfilação da Expressão Gênica/métodos , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/genética , Animais , Transplante de Medula Óssea/métodos , Quimiocina CXCL12/genética , Ontologia Genética , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Camundongos Knockout , Receptores CXCR4/genética , Transdução de Sinais/genética , Transativadores/genética , Transativadores/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
15.
Anticancer Res ; 40(6): 3221-3229, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32487616

RESUMO

BACKGROUND/AIM: Chemokines are cytokines involved not only in inflammatory but also in inappropriate response of the immune system in breast cancer (BC) progression. We examined the diagnostic usefulness of CXCL12, CXCR4 and CA 15-3 in BC patients, based on ROC curve analysis. MATERIALS AND METHODS: The study group consisted of 100 patients with BC; the control group consisted of 35 women with benign breast disease and 35 healthy patients. The median concentration of chemokines was measured by ELISA and that of CA 15-3 by chemiluminescent microparticle immunoassay. RESULTS: The concentrations of CXCL12 and CXCR4 in the BC group were significantly higher than those in the control groups. The AUC value of CXCL12 (0.7502) was the highest of all the chemokines measured in the BC patients. CONCLUSION: There may be a link between CXCL12, CXCR4 and BC that can assist in the diagnosis, markedly when combined with CA 15-3.


Assuntos
Neoplasias da Mama/genética , Quimiocina CXCL12/genética , Receptores CXCR4/genética , Biomarcadores Tumorais/genética , Neoplasias da Mama/metabolismo , Quimiocina CXCL12/metabolismo , Feminino , Humanos , Receptores CXCR4/metabolismo , Transdução de Sinais
16.
Environ Toxicol ; 35(10): 1070-1081, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32420661

RESUMO

Non-small cell lung cancer (NSCLC) is often complicated by pulmonary infection, which affects treatment and prognosis. Bacterial lipopolysaccharide (LPS) is an effective stimulator of inflammatory cytokine production, and previous studies have reported that LPS promotes tumor invasion and metastasis. Mangiferin is a plant-derived C-glucosylxanthone with many biological activities, such as antioxidation and anti-inflammation. This research mainly explored the mechanism of its antitumor activities on LPS-induced A549, NCI-H460, and NCI-H520 NSCLC cells. We determined that mangiferin exhibits growth inhibiting activity against LPS-induced NSCLC cells through the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. In addition, mangiferin reversed the LPS-induced downregulation of E-cadherin (epithelial marker); conversely, it significantly inhibited the expression of raised vimentin (mesenchymal markers). Moreover, the ability of NSCLC cells to migrate, as evidenced by the wound healing and transwell migration assays, and the expression of CXCR4 increased by LPS were significantly repressed by mangiferin. In addition, mangiferin markedly mediated protein levels of PER1 and NLRP3 in LPS-induced NSCLC cells and reduced the secretion of IL-1ß. These results indicate that mangiferin is not only a remarkable anti-inflammatory compound but also an antitumor agent; thus, it has the potential for being developed into anti-inflammatory and antitumor drugs in the future.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas Circadianas Period/genética , Xantonas/farmacologia , Células A549 , Antígenos CD/genética , Caderinas/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação para Baixo , Transição Epitelial-Mesenquimal/genética , Humanos , Lipopolissacarídeos/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , RNA Interferente Pequeno/genética , Receptores CXCR4/genética , Transdução de Sinais , Vimentina/metabolismo
17.
J Leukoc Biol ; 107(6): 1123-1135, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32374043

RESUMO

Chemokines play critical roles in numerous physiologic and pathologic processes through their action on seven-transmembrane (TM) receptors. The N-terminal domain of chemokines, which is a key determinant of signaling via its binding within a pocket formed by receptors' TM helices, can be the target of proteolytic processing. An illustrative case of this regulatory mechanism is the natural processing of CXCL12 that generates chemokine variants lacking the first two N-terminal residues. Whereas such truncated variants behave as antagonists of CXCR4, the canonical G protein-coupled receptor of CXCL12, they are agonists of the atypical chemokine receptor 3 (ACKR3/CXCR7), suggesting the implication of different structural determinants in the complexes formed between CXCL12 and its two receptors. Recent analyses have suggested that the CXCL12 N-terminus first engages the TM helices of ACKR3 followed by the receptor N-terminus wrapping around the chemokine core. Here we investigated the first stage of ACKR3-CXCL12 interactions by comparing the activity of substituted or N-terminally truncated variants of CXCL12 toward CXCR4 and ACKR3. We showed that modification of the first two N-terminal residues of the chemokine (K1R or P2G) does not alter the ability of CXCL12 to activate ACKR3. Our results also identified the K1R variant as a G protein-biased agonist of CXCR4. Comparative molecular dynamics simulations of the complexes formed by ACKR3 either with CXCL12 or with the P2G variant identified interactions between the N-terminal 2-4 residues of CXCL12 and a pocket formed by receptor's TM helices 2, 6, and 7 as critical determinants for ACKR3 activation.


Assuntos
Quimiocina CXCL12/química , AMP Cíclico/química , Receptores CXCR4/química , Receptores CXCR/química , Sequência de Aminoácidos , Sítios de Ligação , Quimiocina CXCL11/química , Quimiocina CXCL11/genética , Quimiocina CXCL11/metabolismo , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , AMP Cíclico/metabolismo , Expressão Gênica , Células HEK293 , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Humanos , Simulação de Dinâmica Molecular , Mutação , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores CXCR/genética , Receptores CXCR/metabolismo , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta-Arrestinas/genética , beta-Arrestinas/metabolismo
18.
Psychiatr Danub ; 32(1): 92-96, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32303038

RESUMO

BACKGROUND: The aim of this study was to evaluate the role of polymorphisms of stromal cell-derived factor-1 (SDF-1) and chemokine receptor-4 (CXCR4) genes in dementia susceptibility in a Turkish population. SUBJECTS AND METHODS: The study group included 61 dementia patients, while the control group comprised 82 healthy individuals. Gene polymorphisms of SDF-1 3'A G801A (rs1801157) and CXCR4 C138T (rs2228014) were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. RESULTS: A significantly reduced risk for developing dementia was found for the group bearing an A allele for SDF-1 3'A polymorphism (p=0.009; χ2=6.812; OR=0.626; 95%CI= 0.429-0.913). The frequency of the CXCR4 TT and TC genotype was significantly lower in patients with dementia compared to controls (p=0.028; χ2=5.583; OR=0.215; 95%CI=0.05-0.914); (p=0.027; χ2=4.919; OR=0.484; 95% CI=0.246-0.955). Additionally, combined genotype analysis showed that the frequency of SDF1 GA-CXCR4 CC was significantly lower in patients with dementia in comparison with those of controls (p=0.049; OR=0.560; 95% CI= 0.307±1.020). CONCLUSIONS: Our study provides new evidence that SDF1 A and CXCR4 T alleles may be associated with a decreased dementia risk. The present study is important because to our knowledge, it is the first one to be conducted in a Turkish population to date, but we believe that more patients and controls are needed to obtain statistically significant results.


Assuntos
Quimiocina CXCL12/genética , Demência/genética , Polimorfismo Genético , Fatores de Proteção , Receptores CXCR4/genética , Idoso , Alelos , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Turquia
20.
J Virol ; 94(12)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32295903

RESUMO

Cell entry by HIV-1 is mediated by its principal receptor, CD4, and a coreceptor, either CCR5 or CXCR4, with viral envelope glycoprotein gp120. Generally, CCR5-using HIV-1 variants, called R5, predominate over most of the course of infection, while CXCR4-using HIV-1 variants (variants that utilize both CCR5 and CXCR4 [R5X4, or dual] or CXCR4 alone [X4]) emerge at late-stage infection in half of HIV-1-infected individuals and are associated with disease progression. Although X4 variants also appear during acute-phase infection in some cases, these variants apparently fall to undetectable levels thereafter. In this study, replication-competent X4 variants were isolated from plasma of drug treatment-naive individuals infected with HIV-1 strain CRF01_AE, which dominantly carries viral RNA (vRNA) of R5 variants. Next-generation sequencing (NGS) confirmed that sequences of X4 variants were indeed present in plasma vRNA from these individuals as a minor population. On the other hand, in one individual with a mixed infection in which X4 variants were dominant, only R5 replication-competent variants were isolated from plasma. These results indicate the existence of replication-competent variants with different coreceptor usage as minor populations.IMPORTANCE The coreceptor switch of HIV-1 from R5 to CXCR4-using variants (R5X4 or X4) has been observed in about half of HIV-1-infected individuals at late-stage infection with loss of CD4 cell count and disease progression. However, the mechanisms that underlie the emergence of CXCR4-using variants at this stage are unclear. In the present study, CXCR4-using X4 variants were isolated from plasma samples of HIV-1-infected individuals that dominantly carried vRNA of R5 variants. The sequences of the X4 variants were detected as a minor population using next-generation sequencing. Taken together, CXCR4-using variants at late-stage infection are likely to emerge when replication-competent CXCR4-using variants are maintained as a minor population during the course of infection. The present study may support the hypothesis that R5-to-X4 switching is mediated by the expansion of preexisting X4 variants in some cases.


Assuntos
Infecções por HIV/imunologia , HIV-1/genética , Receptores CCR5/genética , Receptores CXCR4/genética , Receptores de HIV/imunologia , Adulto , Idoso , Sequência de Aminoácidos , Contagem de Linfócito CD4 , Coinfecção , Progressão da Doença , Feminino , Regulação da Expressão Gênica , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , Ligação Proteica , RNA Viral/genética , RNA Viral/imunologia , Receptores CCR5/imunologia , Receptores CXCR4/imunologia , Receptores de HIV/genética , Tropismo Viral/genética , Tropismo Viral/imunologia , Ligação Viral , Internalização do Vírus
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...