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Cellular & Molecular Immunology Aug 2020It has been reported that neutrophil extracellular traps (NETs) impair wound healing in diabetes and that inhibiting NET generation (NETosis) improves wound healing in...
It has been reported that neutrophil extracellular traps (NETs) impair wound healing in diabetes and that inhibiting NET generation (NETosis) improves wound healing in diabetic mice. Gonadotropin-releasing hormone (GnRH) agonists are associated with a greater risk of diabetes. However, the role of GnRH in diabetic wound healing is unclear. We determined whether GnRH-promoted NETosis and induced more severe and delayed diabetic wound healing. A mouse model of diabetes was established using five injections with streptozotocin. Mice with blood glucose levels >250 mg/dL were then used in the experiments. GnRH agonist treatment induced delayed wound healing and increased NETosis at the skin wounds of diabetic mice. In contrast, GnRH antagonist treatment inhibited GnRH agonist-induced delayed wound healing. The expression of NETosis markers PAD4 and citrullinated histone H3 were increased in the GnRH-treated diabetic skin wounds in diabetic mice and patients. In vitro experiments also showed that neutrophils expressed a GnRH receptor and that GnRH agonist treatment increased NETosis markers and promoted phorbol myristate acetate (PMA)-induced NETosis in mouse and human neutrophils. Furthermore, GnRH antagonist treatment suppressed the expression of NETosis markers and PMA-induced NETosis, which were increased by GnRH treatment. These results indicated that GnRH-promoted NETosis and that increased NETosis induced delayed wound healing in diabetic skin wounds. Thus, inhibition of GnRH might be a novel treatment of diabetic foot ulcers.
Topics: Animals; Citrullination; Diabetes Mellitus, Experimental; Disease Models, Animal; Extracellular Traps; Gonadotropin-Releasing Hormone; HL-60 Cells; Histones; Humans; Mice, Inbred C57BL; Neutrophils; Protein-Arginine Deiminase Type 4; Receptors, LHRH; Wound Healing
PubMed: 31217526
DOI: 10.1038/s41423-019-0252-y -
Blood Mar 2021
Topics: Blood Cell Count; Child; Erythrocytes; Female; Granulocyte Precursor Cells; Humans; Leukemia, Myeloid, Acute
PubMed: 33734334
DOI: 10.1182/blood.2020009744 -
The American Journal of Pathology Nov 2021Myelodysplastic syndromes (MDS) are clonal neoplasms of the hematopoietic stem cell that result in aberrant differentiation of hematopoietic lineages caused by a wide...
Myelodysplastic syndromes (MDS) are clonal neoplasms of the hematopoietic stem cell that result in aberrant differentiation of hematopoietic lineages caused by a wide range of underlying genetic, epigenetic, and other causes. Despite the myriad origins, a recognizable MDS phenotype has been associated with miRNA aberrant expression. A model of aberrant myeloid maturation that mimics MDS was generated using a stable knockdown of miR-378-3p. This model exhibited a transcriptional profile indicating aberrant maturation and function, immunophenotypic and morphologic dysplasia, and aberrant growth that characterizes MDS. Moreover, aberrant signal transduction in response to stimulation specific to the stage of myeloid maturation as indicated by CyTOF mass cytometry was similar to that found in samples from patients with MDS. The aberrant signaling, immunophenotypic changes, cellular growth, and colony formation ability seen in this myeloid model could be reversed with azacytidine, albeit without significant improvement of neutrophil function.
Topics: Adult; Aged; Aged, 80 and over; Female; Gene Knockdown Techniques; HL-60 Cells; Humans; Male; MicroRNAs; Middle Aged; Myelodysplastic Syndromes
PubMed: 34364880
DOI: 10.1016/j.ajpath.2021.07.006 -
Journal of Cellular and Molecular... Sep 2022Acute promyelocytic leukaemia (APL) occurs in approximately 10% of acute myeloid leukaemia patients. Arsenic trioxide (ATO) has been for APL chemotherapy, but recently...
Acute promyelocytic leukaemia (APL) occurs in approximately 10% of acute myeloid leukaemia patients. Arsenic trioxide (ATO) has been for APL chemotherapy, but recently several ATO-resistant cases have been reported worldwide. Cisplatin (CDDP) enhances the toxicity of ATO in ovarian, lung cancer, chronic myelogenous leukaemia, and HL-60 cells. Hence, the goal of this study was to investigate a novel target of CDDP action in APL cells, as an alternate option for the treatment of ATO-resistant APL patients. We applied biochemical, molecular, confocal microscopy and advanced gene editing (CRISPR-Cas9) techniques to elucidate the novel target of CDDP action and its functional mechanism in APL cells. Our main findings revealed that CDDP activated p53 in APL cells through stress signals catalysed by ATM and ATR protein kinases, CHK1 and CHK2 phosphorylation at Ser 345 and Thr68 residues, and downregulation and dissociation of MDM2-DAXX-HAUSP complex. Our functional studies confirmed that CDDP-induced repression of MDM2-DAXX-HAUSP complex was significantly reversed in both nutilin-3-treated KG1a and p53-knockdown NB4 cells. Our findings also showed that CDDP stimulated an increased number of promyelocytes with dense granules, activated p53 expression, and downregulated MDM2 in liver and bone marrow of APL mice. Principal conclusion of our study highlights a novel mode of action of CDDP targeting p53 expression which may provide a basis for designing new anti-leukaemic compounds for treatment of APL patients.
Topics: Animals; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Cisplatin; Female; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Mice; Ovarian Neoplasms; Tumor Suppressor Protein p53
PubMed: 35946055
DOI: 10.1111/jcmm.17502 -
Nature Structural & Molecular Biology Jun 2024Despite the importance of citrullination in physiology and disease, global identification of citrullinated proteins, and the precise targeted sites, has remained...
Despite the importance of citrullination in physiology and disease, global identification of citrullinated proteins, and the precise targeted sites, has remained challenging. Here we employed quantitative-mass-spectrometry-based proteomics to generate a comprehensive atlas of citrullination sites within the HL60 leukemia cell line following differentiation into neutrophil-like cells. We identified 14,056 citrullination sites within 4,008 proteins and quantified their regulation upon inhibition of the citrullinating enzyme PADI4. With this resource, we provide quantitative and site-specific information on thousands of PADI4 substrates, including signature histone marks and transcriptional regulators. Additionally, using peptide microarrays, we demonstrate the potential clinical relevance of certain identified sites, through distinct reactivities of antibodies contained in synovial fluid from anti-CCP-positive and anti-CCP-negative people with rheumatoid arthritis. Collectively, we describe the human citrullinome at a systems-wide level, provide a resource for understanding citrullination at the mechanistic level and link the identified targeted sites to rheumatoid arthritis.
Topics: Humans; Citrullination; Protein-Arginine Deiminase Type 4; Arthritis, Rheumatoid; Citrulline; HL-60 Cells; Proteomics; Protein-Arginine Deiminases; Substrate Specificity; Synovial Fluid
PubMed: 38321148
DOI: 10.1038/s41594-024-01214-9 -
Biomolecules Nov 2022In this study, we have tested the hypothesis that the expression and secretion of galectins are driven through mechanisms globally impacted by homeostatic regulation...
In this study, we have tested the hypothesis that the expression and secretion of galectins are driven through mechanisms globally impacted by homeostatic regulation involving the post-translational modification of intracellular proteins with -linked N-acetylglucosamine (-GlcNAc). We showed that neutrophilic differentiation of HL-60 cells induced by all- retinoic acid (ATRA) and 6-diazo-5-oxo-L-norleucine (DON) was associated with a significant drop of cellular -GlcNAc levels in serum-contained and serum-free cell culture media. Galectin gene and protein expression profiles in HL-60 cells were specifically modified by ATRA and by inhibitors of -GlcNAc cycle enzymes, however overall trends for each drug were similar between cells growing in the presence or absence of serum except for and . The secretion of four galectins (-1, -3, -9, and -10) by HL-60 cells in a serum-free medium was stimulated by -GlcNAc-reducing ATRA and DON while -GlcNAc-elevating thiamet G (-GlcNAcase inhibitor) failed to change the basal levels of extracellular galectins. Taken together, these results demonstrate that -GlcNAc homeostasis is essential not only for regulation of galectin expression in cells but also for the secretion of multiple members of this protein family, which can be an important novel aspect of unconventional secretion mechanisms.
Topics: Humans; Acetylglucosamine; Cell Differentiation; Galectins; HL-60 Cells; N-Acetylglucosaminyltransferases; Protein Processing, Post-Translational; Neutrophils
PubMed: 36551191
DOI: 10.3390/biom12121763 -
Scientific Reports Jan 2022CUX1, encoding a homeodomain-containing transcription factor, is recurrently deleted or mutated in multiple tumor types. In myeloid neoplasms, CUX1 deletion or mutation...
CUX1, encoding a homeodomain-containing transcription factor, is recurrently deleted or mutated in multiple tumor types. In myeloid neoplasms, CUX1 deletion or mutation carries a poor prognosis. We have previously established that CUX1 functions as a tumor suppressor in hematopoietic cells across multiple organisms. Others, however, have described oncogenic functions of CUX1 in solid tumors, often attributed to truncated CUX1 isoforms, p75 and p110, generated by an alternative transcriptional start site or post-translational cleavage, respectively. Given the clinical relevance, it is imperative to clarify these discrepant activities. Herein, we sought to determine the CUX1 isoforms expressed in hematopoietic cells and find that they express the full-length p200 isoform. Through the course of this analysis, we found no evidence of the p75 alternative transcript in any cell type examined. Using an array of orthogonal approaches, including biochemistry, proteomics, CRISPR/Cas9 genomic editing, and analysis of functional genomics datasets across a spectrum of normal and malignant tissue types, we found no data to support the existence of the CUX1 p75 isoform as previously described. Based on these results, prior studies of p75 require reevaluation, including the interpretation of oncogenic roles attributed to CUX1.
Topics: Animals; Genomics; HL-60 Cells; Hematopoietic Stem Cells; Homeodomain Proteins; Humans; K562 Cells; MCF-7 Cells; Mice; NIH 3T3 Cells; Protein Isoforms; RNA Processing, Post-Transcriptional; Repressor Proteins; Transcription Factors; Transcription Initiation Site; Transcription, Genetic; Transcriptional Activation; U937 Cells
PubMed: 34997000
DOI: 10.1038/s41598-021-03930-4 -
The EMBO Journal Feb 2021The ability of cells to polarize and move toward external stimuli plays a crucial role in development, as well as in normal and pathological physiology. Migrating cells...
The ability of cells to polarize and move toward external stimuli plays a crucial role in development, as well as in normal and pathological physiology. Migrating cells maintain dynamic complementary distributions of Ras activity and of the phospholipid phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2). Here, we show that lagging-edge component PI(3,4)P2 also localizes to retracting leading-edge protrusions and nascent macropinosomes, even in the absence of phosphatidylinositol 3,4,5-trisphosphate (PIP3). Once internalized, macropinosomes break up into smaller PI(3,4)P2-enriched vesicles, which fuse with the plasma membrane at the rear of the cell. Subsequently, the phosphoinositide diffuses toward the front of the cell, where it is degraded. Computational modeling confirms that this cycle gives rise to stable back-to-front gradient. These results uncover a surprising "reverse-fountain flow" of PI(3,4)P2 that regulates polarity.
Topics: Cell Membrane; Cell Movement; Dictyostelium; HL-60 Cells; Humans; Microtubules; Phosphatidylinositol Phosphates
PubMed: 33586225
DOI: 10.15252/embj.2020105094 -
Scientific Reports Jan 2021The human leukemia cell line (HL-60) is an alternative to primary neutrophils in research studies. However, because HL-60 cells proliferate in an incompletely...
The human leukemia cell line (HL-60) is an alternative to primary neutrophils in research studies. However, because HL-60 cells proliferate in an incompletely differentiated state, they must undergo differentiation before they acquire the functional properties of neutrophils. Here we provide evidence of swarming and chemotaxis in differentiated HL-60 neutrophil-like cells (dHL-60) using precise microfluidic assays. We found that dimethyl sulfoxide differentiated HL-60 cells (DdHL-60) have a larger size, increased length, and lower ability to squeeze through narrow channels compared to primary neutrophils. They migrate through tapered microfluidic channels slower than primary neutrophils, but faster than HL-60s differentiated by other protocols, e.g., using all-trans retinoic acid. We found that dHL-60 can swarm toward zymosan particle clusters, though they display disorganized migratory patterns and produce swarms of smaller size compared to primary neutrophils.
Topics: Antineoplastic Agents; Cell Differentiation; Chemotactic Factors; Chemotaxis; Cryoprotective Agents; Dimethyl Sulfoxide; HL-60 Cells; Humans; Leukocytes, Mononuclear; Neutrophils; Tretinoin
PubMed: 33436661
DOI: 10.1038/s41598-020-78854-6 -
Cell Death & Disease Dec 2021Acute myeloid leukemia (AML) is an aggressive and heterogeneous clonal hematologic malignancy for which novel therapeutic targets and strategies are required. Emerging...
Acute myeloid leukemia (AML) is an aggressive and heterogeneous clonal hematologic malignancy for which novel therapeutic targets and strategies are required. Emerging evidence suggests that WTIP is a candidate tumor suppressor. However, the molecular mechanisms of WTIP in leukemogenesis have not been explored. Here, we report that WTIP expression is significantly reduced both in AML cell lines and clinical specimens compared with normal controls, and low levels of WTIP correlate with decreased overall survival in AML patients. Overexpression of WTIP inhibits cell proliferation and induces apoptosis both in vitro and in vivo. Mechanistic studies reveal that the apoptotic function of WTIP is mediated by upregulation and nuclear translocation of FOXO3a, a member of Forkhead box O (FOXO) transcription factors involved in tumor suppression. We further demonstrate that WTIP interacts with FOXO3a and transcriptionally activates FOXO3a. Upon transcriptional activation of FOXO3a, its downstream target PUMA is increased, leading to activation of the intrinsic apoptotic pathway. Collectively, our results suggest that WTIP is a tumor suppressor and a potential target for therapeutic intervention in AML.
Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Case-Control Studies; Cell Proliferation; Co-Repressor Proteins; Cytoskeletal Proteins; Forkhead Box Protein O3; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Mice; Proto-Oncogene Proteins; Signal Transduction; THP-1 Cells; Transcriptional Activation; Transfection; Tumor Burden; Tumor Suppressor Proteins; U937 Cells; Up-Regulation; Xenograft Model Antitumor Assays
PubMed: 34930905
DOI: 10.1038/s41419-021-04467-0