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New aspects on retinoic acid induced HL-60 granulocytic differentiation
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The underlying mechanisms of myeloid cell differentiation are still not entirely elucidated. It is therefore an urgent task to extent the basis for a comprehensive model of granulocytes differentiation. A primary goal for studies has been the identification of differentiation markers. These often correspond to genes that are expressed at defined steps during the differentiation process or in the fully differentiated phenotype. Here is demonstrated that the program of phenotype formation entails dramatic changes in long-term tyrosine phosphorylation of some cytoplasmic proteins during retinoic acid (RA)-induced HL-60 cell differentiation to granulocytes. A significant increase in the phosphotyrosine content of a kinase (Erk2) in the commitment and terminal stages of HL-60 cell differentiation (48-120 h) was related to promotion of cell survival and triggering of apoptosis. In addition, some cytosolic proteins of apoptotic origin were delineated in the differentiating population. The pattern of tyrosine phosphorylations of specific cytosolic proteins in maturing and apoptotic granulocytes should be a powerful tool for further elucidation of differentiation mechanisms. We developed a new approach based on cytosolic protein tagging and nuclear import analysis combined with assessment of specific protein tyrosine phosphorylations by two-dimensional gel electrophoresis. Many of the genes involved in the differentiation of multipotent stem cells to specific cellular lineages are still unknown. Accordingly, we searched for additional tyrosine phosphorylated, RA-induced transcription regulators that are translocated into the nucleus during commitment of HL-60 cells to become granulocyte-like cells. It is shown, that transcription factors required for lineage commitment of differentiating (C/EBPß, c-myb) and for survival of differentiated cells (STATs, NFKB) co-operatively promoted signaling in the HL-60 cell line in response to RA. Identification of the tyrosine-phosphorylated proteins being translocated into the nucleus during HL-60 cell differentiation was our further goaL This way, we show for the first time that human gamma~dystrobrevin is expressed in promyelocytic HL-60 cell line. We detected gamma-dystrobrevin in the cytosol and the nuclei of these cells, and, in the latter location; it was modified by tyrosine phosphorylation soon after the onset of the RA-triggered differentiation. Varying patterns of distribution of gamma-dystrobrevin could be discerned in HL-60 promyelocytes, RA-differentiated mature granulocytes, and hwnan neutrophils. Moreover, we found gamma-dystrobrevin in association with actin and myosin light chain. Our results provide new information about potential involvement of gamma-dystrobrevin in signal transduction in myeloid cells during induction of granulocytic differentiation and/or at the commitment stage of differentiation or phagocytic cells. In addition, our study confirms and extends knowledge about the genes involved in granulocytic differentiation and growth arrest. This shuiy corroborates the value of cDNA RDA to isolate factors involved in myeloid maturation. In conclusion, the more detailed analysis of granulocytic differentiation of HL-60 cells presented here provides new data that should allow further refinement of models of myeloid differentiation.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2002. , 119 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 723
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-26659Local ID: 11225ISBN: 91-7373-165-X (print)OAI: oai:DiVA.org:liu-26659DiVA: diva2:247208
Public defence
2002-04-11, Berzeliussalen, Universitetssjukhuset, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-09-17Bibliographically approved
List of papers
1. Tyrosine phosphorylation of cytoplasmic proteins in proliferating, differentiating, apoptotic HL-60 cells and blood neutrophils
Open this publication in new window or tab >>Tyrosine phosphorylation of cytoplasmic proteins in proliferating, differentiating, apoptotic HL-60 cells and blood neutrophils
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2000 (English)In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 57, no 13-14, 1997-2008 p.Article in journal (Refereed) Published
Abstract [en]

Two-dimensional electrophoretic analysis was used to assess quantitative and qualitative changes in the expression and tyrosine phosphorylation of cytoplasmic proteins of proliferating, differentiating HL-60 cells and mature human blood neutrophils. The total tyrosine phosphorylation level of cytoplasmic proteins appeared approximately constant during the pre-commitment period, i.e., 6-24 h after induction of differentiation by 700 nM all-trans retinoic acid. At the time of granulocytic phenotype formation (48-120 h), the total level of tyrosine phosphorylation of cytoplasmic proteins increased significantly. Tyrosine phosphorylation of cytoplasmic proteins in matured blood neutrophils was significantly lower than that of cytoplasmic proteins of HL-60 cells differentiated for 96 h with retinoic acid. Immunoblotting with anti-Erk2 and anti-phosphotyrosine monoclonal IgG2bk antibodies showed that Erk2 was expressed and tyrosine-phosphorylated at different levels in HL-60 proliferating cells and in cells at all stages of differentiation. Our data showed that tyrosine phosphorylation of cytoplasmic proteins in differentiating HL-60 cells changes dramatically during the period of phenotype formation and is accompanied by increasing activity of Erk2. An increasing number of apoptotic cells appeared in the differentiating HL-60 cell population during the granulocyte maturation stage (48-120 h of differentiation). The appearance at this time of differentiation of a new set of tyrosine-phosphorylated cytoplasmic proteins (also distinctive for apoptotic HL-60 cells mediated by etoposide) together with an increasing number of apoptotic cells in the differentiating population strongly suggests that these proteins are associated with the apoptotic process.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26006 (URN)10.1007/PL00000681 (DOI)10458 (Local ID)10458 (Archive number)10458 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
2. Parallel assessment of tyrosine phosphorylation and nuclear targeting of proteins
Open this publication in new window or tab >>Parallel assessment of tyrosine phosphorylation and nuclear targeting of proteins
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2001 (English)In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 31, no 3, 508-517 p.Article in journal (Refereed) Published
Abstract [en]

Phosphotyrosine signaling plays a vital role in cell regulation - from receptor activation, through stimulation of signal networks and nuclear targeting, to final cellular responses. Here, we propose a new approach to monitor the spatial and temporal aspects of tyrosine phosphorylation and dephosphorylation. The method can be used to determine whether protein tyrosine phosphorylations and dephosphorylations occur in the cytosol or the nucleus and to ascertain whether such modifications are associated with nuclear traffic. Promyelocytic leukemia (HL-60) cells are used as the experimental model. Biotinylated cytosolic proteins from donor cells are used to trace nuclear transport in permeabilized recipient cells. Thereafter, 2-D gel electrophoresis is applied to fractionate the cytosolic and nuclear proteins of the recipient cells, which are subsequently blotted onto polyvinylidene difluoride membranes. The membranes are developed with streptavidin and then reprobed with anti-phosphotyrosine antibodies. The major advantages of the protocol are that it is simple to perform, and reproducible results are obtained by overlaying the patterns of biotinylated and/or tyrosine-phosphorylated proteins. Moreover, several hundred cytosolic and nuclear proteins can be analyzed in parallel. Thus, by comparing the 2-D gel electrophoresis maps of biotinylated and tyrosine-phosphorylated proteins, it is possible to determine the involvement of trafficking of the latter proteins in cell signaling.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26005 (URN)11570494 (PubMedID)10457 (Local ID)10457 (Archive number)10457 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
3. Assessment of transcription regulators and translocation of these proteins into the nucleus during granulocyte lineage commitment of haematopoietis HL-60 cells
Open this publication in new window or tab >>Assessment of transcription regulators and translocation of these proteins into the nucleus during granulocyte lineage commitment of haematopoietis HL-60 cells
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Expression of transcription factors required for lineage commitment of differentiating cells (C/EBPß and c-myb) and for survival of differentiated cells (STATs and NFκB) was examined in the HL-60 cell line. Differentiation was induced by treating the cells with retinoic acid (RA). c-Myb expression in the nucleus restored at the precommitment stage (18 h) what concurred with the highest nuclear level of C/EBPß, which suggests a combinatorial interaction of these transcription factors in granulocytic signalling pathway. Expression of STAT5a and STAT5b varied during differentiation; there were no significant changes in STAT3 levels. Increased cytosolic level of NFκB p65 during precommitment and commitment stages of granulocytic differentiation coincides with augmentation of STAT5a protein level what could be an evidence of their possible co-operation during granulocyticlineage commitment of HL-60 cells. Our results suggest that the studied proteins cooperatively promote signalling in differentiating promyelocytic HL-60 cell line in response to retinoic acid.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-81476 (URN)
Available from: 2012-09-17 Created: 2012-09-17 Last updated: 2012-09-17Bibliographically approved
4. Characterization of Human Alpha-Dystrobrevin Isoforms in HL-60 Human Promyelocytic Leukemia Cells Undergoing Granulocytic Differentiation
Open this publication in new window or tab >>Characterization of Human Alpha-Dystrobrevin Isoforms in HL-60 Human Promyelocytic Leukemia Cells Undergoing Granulocytic Differentiation
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2002 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 13, no 12, 4195-4205 p.Article in journal (Refereed) Published
Abstract [en]

The biochemical properties and spatial localization of the protein alpha-dystrobrevin and other isoforms were investigated in cells of the human promyelocytic leukemia line HL-60 granulocytic differentiation as induced by retinoic acid (RA). Alpha-dystrobrevin was detected both in the cytosol and the nuclei of these cells, and a short isoform (gamma-dystrobrevin) was modified by tyrosine phosphorylation soon after the onset of the RA-triggered differentiation. Varying patterns of distribution of alpha-dystrobrevin and its isoforms could be discerned in HL-60 promyelocytes, RA-differentiated mature granulocytes, and human neutrophils. Moreover, the gamma-dystrobrevin isoform was found in association with actin and myosin light chain. The results provide new information about potential involvement of alpha-dystrobrevin and its splice isoforms in signal transduction in myeloid cells during induction of granulocytic differentiation and/or at the commitment stage of differentiation or phagocytic cells.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26511 (URN)10.1091/mbc.E02-03-0128 (DOI)11068 (Local ID)11068 (Archive number)11068 (OAI)
Note

On the day of the defence day the status of this article was submitted.

Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved

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