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1.
Elife ; 102021 09 29.
Article in English | MEDLINE | ID: mdl-34586063

ABSTRACT

Lung epithelial progenitors differentiate into alveolar type 1 (AT1) and type 2 (AT2) cells. These cells form the air-blood interface and secrete surfactant, respectively, and are essential for lung maturation and function. Current protocols to derive and culture alveolar cells do not faithfully recapitulate the architecture of the distal lung, which influences cell fate patterns in vivo. Here, we report serum-free conditions that allow for growth and differentiation of mouse distal lung epithelial progenitors. We find that Collagen I promotes the differentiation of flattened, polarized AT1 cells. Using these organoids, we performed a chemical screen to investigate WNT signaling in epithelial differentiation. We identify an association between Casein Kinase activity and maintenance of an AT2 expression signature; Casein Kinase inhibition leads to an increase in AT1/progenitor cell ratio. These organoids provide a simplified model of alveolar differentiation and constitute a scalable screening platform to identify and analyze cell differentiation mechanisms.


Subject(s)
Cell Differentiation , Pulmonary Alveoli/cytology , Stem Cells/cytology , Animals , Casein Kinases/antagonists & inhibitors , Casein Kinases/metabolism , Cells, Cultured , Collagen Type I/metabolism , Culture Media, Serum-Free , Epithelial Cells/cytology , Epithelial Cells/metabolism , Genetic Markers , Mice , Mice, Inbred C57BL , Pulmonary Alveoli/embryology , Pulmonary Alveoli/enzymology , Pulmonary Alveoli/metabolism , Transcription, Genetic , Wnt Signaling Pathway
2.
Hematol Oncol ; 38(1): 3-11, 2020 Feb.
Article in English | MEDLINE | ID: mdl-31782972

ABSTRACT

The knowledge accumulated over the last decade on B-cell-derived non-Hodgkin lymphoma (B-NHL) pathogenesis has led to the identification of several molecular abnormalities, opening new perspectives in the design of novel therapies. Indeed, drugs targeting specific biochemical pathways critical for B-NHL cell survival, proliferation, and fitness within the malignant microenvironment are now available to the clinician: the B-cell receptor signaling inhibitors of BTK, PI3Kδ, ζ, γ, and SYK or the pro-apoptotic BH3-mimetics are clear examples of it. Moreover, it is emerging that malignant B-cell growth is sustained not only by mutations in oncogenes/tumor suppressors but also by the "addiction" to nononcogene (ie, nonstructurally altered) molecules. In this regard, a consistent body of data has established that the Ser/Thr kinases CK1, CK2, and GSK3 are involved in malignant lymphocyte biology and act as pro-survival and signaling-boosting molecules, both in precursor and mature B-cell tumors. Currently, an experimental and clinical groundwork is available, upon which to design CK1-, CK2-, and GSK3-directed antilymphoma/leukemia therapies. In this review, we have examined the main features of CK1, CK2, and GSK3 kinases, summarized the data in B-NHL supporting them as suitable therapeutic targets, and proposed a perspective on potential future research development.


Subject(s)
Casein Kinases/antagonists & inhibitors , Glycogen Synthase Kinase 3/antagonists & inhibitors , Lymphoma, B-Cell/drug therapy , Lymphoma, B-Cell/enzymology , Molecular Targeted Therapy , Protein Kinase Inhibitors/therapeutic use , Animals , Humans , Prognosis
3.
Am J Physiol Renal Physiol ; 316(5): F807-F813, 2019 05 01.
Article in English | MEDLINE | ID: mdl-30759025

ABSTRACT

Circadian rhythms govern physiological functions and are important for overall health. The molecular circadian clock comprises several transcription factors that mediate circadian control of physiological function, in part, by regulating gene expression in a tissue-specific manner. These connections are well established, but the underlying mechanisms are incompletely understood. The overall goal of this study was to examine the connection among the circadian clock protein Period 1 (Per1), epithelial Na+ channel (ENaC), and blood pressure (BP) using a multipronged approach. Using global Per1 knockout mice on a 129/sv background in combination with a high-salt diet plus mineralocorticoid treatment, we demonstrated that loss of Per1 in this setting is associated with protection from hypertension. Next, we used the ENaC inhibitor benzamil to demonstrate a role for ENaC in BP regulation and urinary Na+ excretion in 129/sv mice. We targeted Per1 indirectly using pharmacological inhibition of Per1 nuclear entry in vivo to demonstrate altered expression of known Per1 target genes as well as a BP-lowering effect in 129/sv mice. Finally, we directly inhibited Per1 via genetic knockdown in amphibian distal nephron cells to demonstrate, for the first time, that reduced Per1 expression is associated with decreased ENaC activity at the single channel level.


Subject(s)
Blood Pressure , Circadian Rhythm , Epithelial Sodium Channels/metabolism , Hypertension/prevention & control , Nephrons/metabolism , Period Circadian Proteins/metabolism , Amiloride/analogs & derivatives , Amiloride/pharmacology , Animals , Antihypertensive Agents/pharmacology , Blood Pressure/drug effects , Casein Kinases/antagonists & inhibitors , Casein Kinases/metabolism , Circadian Rhythm/drug effects , Desoxycorticosterone/analogs & derivatives , Disease Models, Animal , Epithelial Sodium Channel Blockers/pharmacology , Epithelial Sodium Channels/drug effects , Epithelial Sodium Channels/genetics , Hypertension/genetics , Hypertension/metabolism , Hypertension/physiopathology , Male , Mice, 129 Strain , Mice, Knockout , Mineralocorticoids , Natriuresis , Nephrons/drug effects , Period Circadian Proteins/antagonists & inhibitors , Period Circadian Proteins/deficiency , Period Circadian Proteins/genetics , Pyrimidines/pharmacology , Sodium Chloride, Dietary , Time Factors , Xenopus
4.
Cell Physiol Biochem ; 39(3): 939-49, 2016.
Article in English | MEDLINE | ID: mdl-27513568

ABSTRACT

BACKGROUND/AIMS: The echinocandin antifungal agent caspofungin has been shown to trigger apoptosis of fungal cells. Beyond that, caspofungin is toxic for host mitochondria. Even though lacking mitochondria, erythrocytes may enter apoptosis-like suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, caspase activation and/or activation of p38 kinase, protein kinase C, and casein kinase. The present study explored, whether caspofungin induces eryptosis and, if so, to shed some light on the cellular mechanisms involved. METHODS: Flow cytometry was employed to determine phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified from hemoglobin concentration in the supernatant. RESULTS: A 48 hours exposure of human erythrocytes to caspofungin (≥ 30 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly enhanced hemolysis, but did not significantly increase Fluo3-fluorescence, DCFDA fluorescence or ceramide abundance. The effect of caspofungin on annexin-V-binding was not significantly blunted by removal of extracellular Ca2+, by inhibition of caspases with pancaspase inhibitor zVAD (10 µM), or by addition of the antioxidant N-acetyl-cysteine (1 mM), p38 kinase inhibitor SB203580 (2 µM) or protein kinase C inhibitor staurosporine (1 µM). The effect of caspofungin on annexin-V-binding was, however, significantly blunted in the presence of casein kinase inhibitor D4476 (10 µM). CONCLUSIONS: Caspofungin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect possibly involving activation of casein kinase.


Subject(s)
Antifungal Agents/pharmacology , Calcium/metabolism , Echinocandins/pharmacology , Eryptosis/drug effects , Erythrocytes/drug effects , Lipopeptides/pharmacology , Reactive Oxygen Species/metabolism , Acetylcysteine/pharmacology , Aniline Compounds , Annexin A5 , Casein Kinases/antagonists & inhibitors , Casein Kinases/genetics , Casein Kinases/metabolism , Caspases/genetics , Caspases/metabolism , Caspofungin , Cells, Cultured , Ceramides/metabolism , Erythrocytes/chemistry , Erythrocytes/cytology , Flow Cytometry , Fluoresceins , Fluorescent Dyes , Gene Expression , Hemolysis/drug effects , Humans , Oligopeptides/pharmacology , Oxidative Stress , Phosphatidylserines/metabolism , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/genetics , Protein Kinase C/metabolism , Protein Kinase Inhibitors/pharmacology , Xanthenes , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolism
5.
Neurobiol Dis ; 79: 1-13, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25862939

ABSTRACT

14-3-3 proteins are key regulators of cell survival. We have previously demonstrated that 14-3-3 levels are decreased in an alpha-synuclein (αsyn) mouse model of Parkinson's disease (PD), and that overexpression of certain 14-3-3 isoforms is protective in several PD models. Here we examine whether changes in 14-3-3 phosphorylation may contribute to the neurodegenerative process in PD. We examine three key 14-3-3 phosphorylation sites that normally regulate 14-3-3 function, including serine 58 (S58), serine 184 (S184), and serine/threonine 232 (S/T232), in several models of PD and in human PD brain. We observed that an increase in S232 phosphorylation is observed in rotenone-treated neuroblastoma cells, in cells overexpressing αsyn, and in human PD brains. Alterations in S58 phosphorylation were less consistent in these models, and we did not observe any phosphorylation changes at S184. Phosphorylation at S232 induced by rotenone is reduced by casein kinase inhibitors, and is not dependent on αsyn. Mutation of the S232 site affected 14-3-3θ's neuroprotective effects against rotenone and 1-methyl-4-phenylpyridinium (MPP(+)), with the S232D mutant lacking any protective effect compared to wildtype or S232A 14-3-3θ. The S232D mutant partially reduced the ability of 14-3-3θ to inhibit Bax activation in response to rotenone. Based on these findings, we propose that phosphorylation of 14-3-3s at serine 232 contributes to the neurodegenerative process in PD.


Subject(s)
14-3-3 Proteins/metabolism , Parkinson Disease/metabolism , Parkinsonian Disorders/metabolism , 1-Methyl-4-phenylpyridinium , 14-3-3 Proteins/genetics , Animals , Casein Kinases/antagonists & inhibitors , Casein Kinases/metabolism , Cell Line, Tumor , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Hippocampus/metabolism , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Mice, Inbred C57BL , Mice, Transgenic , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/metabolism , Rotenone , Temporal Lobe/metabolism , alpha-Synuclein/genetics , alpha-Synuclein/metabolism , bcl-2-Associated X Protein/metabolism
6.
BMC Cancer ; 9May 13, 2009. tab
Article in English | CUMED | ID: cum-39785

ABSTRACT

BACKGROUND: Cervical cancer is now considered the second leading cause of death among women worldwide, and its incidence has reached alarming levels, especially in developing countries. Similarly, high grade squamous intraepithelial lesion (HSIL), the precursor stage for cervical cancer, represents a growing health problem among younger women as the HSIL management regimes that have been developed are not fully effective. From the etiological point of view, the presence of Human Papillomavirus (HPV) has been demonstrated to play a crucial role for developing cervical malignancies, and viral DNA has been detected in 99.7 percent of cervical tumors at the later stages. CIGB-300 is a novel cyclic synthetic peptide that induces apoptosis in malignant cells and elicits antitumor activity in cancer animal models. CIGB-300 impairs the Casein Kinase (CK2) phosphorylation, by targeting the substrate's phosphoaceptor domain. Based on the perspectives of CIGB-300 to treat cancer, this first-in-human study investigated its safety and tolerability in patients with cervical malignancies. METHODS: Thirty-one women with colposcopically and histologically diagnosed microinvasive or pre-invasive cervical cancer were enrolled in a dose escalating study. CIGB-300 was administered sequentially at 14, 70, 245 and 490 mg by intralesional injections during 5 consecutive days to groups of 7 - 10 patients. Toxicity was monitored daily until fifteen days after the end of treatment, when patients underwent conization. Digital colposcopy, histology, and HPV status were also evaluated. RESULTS: No maximum-tolerated dose or dose-limiting toxicity was achieved. The most frequent local events were pain, bleeding, hematoma and erythema at the injection site....(AU)


Subject(s)
Humans , Female , Adolescent , Adult , Middle Aged , Aged , Casein Kinases/antagonists & inhibitors , Uterine Cervical Dysplasia/drug therapy , Peptides, Cyclic/administration & dosage , Protein Kinase Inhibitors/administration & dosage , Uterine Cervical Neoplasms/drug therapy
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