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Human myeloid leukemia cells (HL-60/S4) exposed to hyperosmotic stress with sucrose undergo dehydration and cell shrinkage. Interphase chromatin and mitotic chromosomes congeal, exhibiting altered phase separation (demixing) of chromatin proteins. To investigate changes in the transcriptome, we exposed HL-60/S4 cells to hyperosmotic sucrose stress (~600 milliOsmolar) for 30 and 60 minutes. We employed RNA-Seq of polyA mRNA to identify genes with increased or decreased transcript levels relative to untreated control cells (i.e., differential gene expression). These genes were examined for over-representation of Gene Ontology (GO) terms. In stressed cells, multiple GO terms associated with transcription, translation, mitochondrial function and proteosome activity, as well as "replication-dependent histones", were over-represented among genes with increased transcript levels; whereas, genes with decreased transcript levels were over-represented with transcription repressors. The transcriptome profiles of hyperosmotically-stressed cells suggest acquisition of cellular rebuilding, a futile homeostatic response, as these cells are ultimately doomed to a dehydrated death. [ABSTRACT FROM AUTHOR]

Abstract Background Mammalian blood neutrophilic granulocytes are terminally differentiated cells, possessing extensive heterochromatin and lobulated (or ring-shaped) nuclei. Despite the extensive amount of heterochromatin, neutrophils are capable of increased gene expression, when activated by bacterial infection. Understanding the mechanisms of transcriptional repression and activation in neutrophils requires detailing the chromatin epigenetic markers, which are virtually undescribed in this cell type. Much is known about the heterochromatin epigenetic markers in other cell-types, permitting a basis for comparison with those of mature normal neutrophilic granulocytes. Results Immunostaining and immunoblotting procedures were employed to study the presence of repressive histone modifications and HP1 proteins in normal human and mouse blood neutrophils, and in vitro differentiated granulocytes of the mouse promyelocytic (MPRO) system. A variety of repressive histone methylation markers were detectable in these granulocytes (di- and trimethylated H3K9; mono-, di- and trimethyl H3K27; di- and trimethyl H4K20). However, a paucity of HP1 proteins was noted. These granulocytes revealed negligible amounts of HP1 α and β, but exhibited detectable levels of HP1 γ. Of particular interest, mouse blood and MPRO undifferentiated cells and granulocytes revealed clear co-localization of trimethylated H3K9, trimethylated H4K20 and HP1 γ with pericentric heterochromatin. Conclusion Mature blood neutrophils possess some epigenetic heterochromatin features that resemble those of well-studied cells, such as lymphocytes. However, the apparent paucity of HP1 proteins in neutrophils suggests that heterochromatin organization and binding to the nuclear envelope may differ in this cell-type. Future investigations should follow changes in epigenetic markers and levels of HP1 proteins during granulopoiesis and bacterial activation of neutrophils.

Abstract Background During granulopoiesis in the bone marrow, the nucleus differentiates from ovoid to lobulated shape. Addition of retinoic acid (RA) to leukemic HL-60 cells induces development of lobulated nuclei, furnishing a convenient model system for nuclear differentiation during granulopoiesis. Previous studies from our laboratory have implicated nuclear envelope composition as playing important roles in nuclear shape changes. Specifically noted were: 1) a paucity of lamins A/C and B1 in the undifferentiated and RA treated cell forms; 2) an elevation of lamin B receptor (LBR) during induced granulopoiesis. Results The present study demonstrates that perturbation of cytoskeletal elements influences nuclear differentiation of HL-60 cells. Because of cytotoxicity from prolonged exposure to cytoskeleton-modifying drugs, most studies were performed with a Bcl-2 overexpressing HL-60 subline. We have found that: 1) nocodazole prevents RA induction of lobulation; 2) taxol induces lobulation and micronuclear formation, even in the absence of RA; 3) cytochalasin D does not inhibit RA induced nuclear lobulation, and prolonged exposure induces nuclear shape changes in the absence of RA. Conclusions The present results, in the context of earlier data and models, suggest a mechanism for granulocytic nuclear lobulation. Our current hypothesis is that the nuclear shape change involves factors that increase the flexibility of the nuclear envelope (reduced lamin content), augment connections to the underlying heterochromatin (increased levels of LBR) and promote distortions imposed by the cytoskeleton (microtubule motors creating tension in the nuclear envelope).