A critical role of redox state in determining HL-60 cell granulocytic differentiation and apoptosis via involvement of PKC and NF-kappaB.

The modifications of intracellular redox balance leads to important cellular changes in many cell types. Here, a causal relationship among redox state, granulocytic differentiation induced by all-trans retinoic acid (RA) or dibutyryl cAMP (dbcAMP) and apoptosis have been studied in the human acute promyelocytic leukaemia HL-60 cells. The modulation of intracellular reactive oxygen species levels by D: , L: -buthionine-(S, R) sulfoximide (BSO), and N: -acetyl-L: -cysteine (NAC) caused inducer- and time-dependent or stage-specific effects on HL-60 cell growth inhibition, differentiation and subsequent apoptosis.

Alterations in protein expression in HL-60 cells during etoposide-induced apoptosis modulated by the caspase inhibitor ZVAD.fmk.

DNA topoisomerase inhibitors induce a specific signaling cascade that promotes an active apoptotic caspase-dependent cell death process. However, little is known about the initial signals elicited by these agents. In the present study, we compared apoptosis in HL-60 cells treated either with the chemotherapeutic drug etoposide (VP16) alone or combined with the broad caspase inhibitor ZVAD.

Novel human testis-specific histone H2B encoded by the interrupted gene on the X chromosome.

Testis-specific histones are synthesized and accumulated at specific stages of mammalian spermatogenesis. Their proposed functions range from facilitation of the replacement of somatic histones by protamines to epigenetic control of gene transcription. Several testis histone variants were characterized in mouse and rat; however, few are known in humans.

[Surgical approach to lung tuberculosis].

The article describes treatment results of 1228 patients operated on because of different forms of pulmonary tuberculosis. According to spreading of tuberculosis and developed complications in spite of the medicamental treatment the patients were divided in to two clinical groups. The first group included 417 patients with direct indications for surgery.

Identification of apoptotic tyrosine-phosphorylated proteins after etoposide or retinoic acid treatment.

A main shortcoming of using HL-60 cells as a model of granulocyte-macrophage differentiation is that some cells in the differentiating population undergo apoptosis. To address this issue, we have identified which tyrosine-phosphorylated proteins are involved in apoptosis and differentiation, respectively. HL-60 cells were induced specifically to undergo apoptosis with 68 microM etoposide, and to undergo granulocytic differentiation with 1 microM retinoic acid (RA).

Translocation of transcription regulators into the nucleus during granulocyte commitment of HL-60 cells.

Expression of transcription factors required for lineage commitment of differentiating cells (C/EBPbeta and c-Myb) and for survival of differentiated cells (STATs and NFkappaB) was examined in the HL-60 cell line. Differentiation was induced by treating the cells with retinoic acid. c-Myb expression in the nucleus restored at the precommitment stage (18 h) what concurred with the highest nuclear level of C/EBPbeta, which suggests a combinatorial interaction of these transcription factors in the granulocytic signalling pathway.

3-Deazauridine triggers dose-dependent apoptosis in myeloid leukemia cells and enhances retinoic acid-induced granulocytic differentiation of HL-60 cells.

Therapeutic nucleoside analogue 3-deazauridine (DU) exerts cytotoxic activity against cancer cells by disruption of DNA synthesis resulting in cell death. The present study evaluates whether DU alone at doses 2.5-15 microM or in combination with all trans retinoic acid (RA) or dibutyryl cAMP (dbcAMP) is effective against myelogenous leukemia.

[The surgical treatment of lung tuberculosis].

Unlabelled: The article informs about the treatment results of 1036 patients operated on because of different forms of pulmonary tuberculosis. According to spreading of tuberculosis and developed complications in spite of the medicamentical treatment the patients were divided into two clinical groups. The first group included 387 patients with direct and relative indications for surgery.

Characterization of human alpha-dystrobrevin isoforms in HL-60 human promyelocytic leukemia cells undergoing granulocytic differentiation.

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.

Human testis/sperm-specific histone H2B (hTSH2B). Molecular cloning and characterization.

Human sperm, unlike the sperm of other mammals, contain replacement histones with unknown biological functions. Here, we report the identification of the novel human gene coding for a testis/sperm-specific histone H2B (hTSH2B). This variant histone is 85% homologous to somatic H2B and has over 93% homology with the testis H2B of rodents.

Parallel assessment of tyrosine phosphorylation and nuclear targeting of proteins.

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.

Serum levels of elastin-derived peptides in patients with ruptured and asymptomatic abdominal aortic aneurysms.

Objective: to determine whether serum elastin-derived peptides (S-EDP), are lower in patients with ruptured abdominal aortic aneurysms (rAAA) than asymptomatic (aAAA).

Materials And Methods: serum samples were collected preoperatively from 45 consecutive patients with aAAA and 15 haemodynamically stable patients with rAAA. S-EDP (ng/ml) was measured by a competitive enzyme-linked immunosorbent assay (ELISA).

Tyrosine phosphorylation of cytoplasmic proteins in proliferating, differentiating, apoptotic HL-60 cells and blood neutrophils.

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.

Human sperm telomere-binding complex involves histone H2B and secures telomere membrane attachment.

Telomeres are unique chromatin domains located at the ends of eukaryotic chromosomes. Telomere functions in somatic cells involve complexes between telomere proteins and TTAGGG DNA repeats. During the differentiation of germ-line cells, telomeres undergo significant reorganization most likely required for additional specific functions in meiosis and fertilization.

Activity of matrix metalloproteinase-2 and -9 in abdominal aortic aneurysms. Relation to size and rupture.

Objectives: to investigate the activity of matrix metalloproteinase (MMP)-2 and -9 in asymptomatic abdominal aortic aneurysms (aAAAs) and ruptured abdominal aortic aneurysms (rAAAs).

Design: cross-sectional study.

Materials And Methods: MMP-2 and MMP-9 activity was estimated in biopsies from the anterior wall of 60 AAAs using gelatin zymography.

Modulation of apoptosis of proliferating and differentiating HL-60 cells by protein kinase inhibitors: suppression of PKC or PKA differently affects cell differentiation and apoptosis.

The relationship between RA- or dbcaMP-mediated differentiation and subsequent apoptosis in HL-60 cells was assessed by modulating the levels of differentiation suppressing the activity of PKC and PKA with calphostin C or GF 109203X and H89, respectively. Results demonstrated that (1) RA and dbcAMP caused a dose-dependent increase in apoptosis concomitant with progressive differentiation; (2) the suppression of PKC activity resulted in an increase of apoptosis unrelated to the modulated levels of differentiation; (3) the inhibition of PKA decreased granulocytic differentiation, but did not significantly affect apoptosis; (4) the pretreatment of cells with dbcAMP strongly potentiated RA-mediated differentiation without apparent changes in apoptosis; (5) cell differentiation and apoptosis were associated with cell cycle arrest in G1 phase and G2/M phases, respectively. Our findings indicate that the functional maturity of differentiating cells is not directly related to the apoptotic programme, and suggest that induction of cell differentiation and apoptosis are regulated by separate mechanisms in which PKC and PKA are involved.

Long-term changes in tyrosine phosphorylation of the abundant nuclear proteins during granulocytic differentiation of HL-60 cells.

The two-dimensional electrophoretic patterns of nuclear proteins and their tyrosine phosphorylation were compared for HL-60 cells before and after differentiation induction to granulocytes by dimethyl sulfoxide, all-trans retinoic acid and N6,O2-dibutyryl adenosine 3':5'-cyclic monophosphate. Regardless of the inducer used, some nuclear proteins, which are tyrosine-phosphorylated in proliferating HL-60 cells, undergo gradual dephosphorylation 12-72 h after induction of differentiation, followed by drastic dephosphorylation during maturation to granulocytes. At least 13 nuclear proteins with a molecular mass of 35-110 kDa are dephosphorylated, and 6 nuclear proteins undergo tyrosine phosphorylation.

Two-dimensional high-resolution electrophoresis of elastin-derived peptides.

Degradation of human aortic elastin in vivo yields a restricted number of differentially sized and charged peptides. Elastin-derived peptides (EDP) can be analyzed by two-dimensional electrophoresis after their extraction from human abdominal aortic tissue by 0.2 M sodium chloride.

Turnover of histone acetyl groups during sea urchin early development is not required for histone, heat shock and actin gene transcription.

Histone acetylation is an extremely complex, reversible and specific process. In order to evaluate the importance of this modification for gene expression during sea urchin development, acetyl group turnover of histone lysine residues was blocked by sodium butyrate. The continuous presence of 15 Mm sodium butyrate in the incubation medium from the onset of development blocked gastrulation and resulted in chromatin containing hyperacetylated histone molecules in amounts usually not found in nature.

Relationship between differentiation mechanisms involving cAMP-dependent protein kinase and protein kinase C in uninduced and differentiating HL-60 cells.

The influence of protein kinases on the differentiation of a human promyelocytic leukemia-cell line HL-60 to granulocytes was studied by using H8 and staurosporine as inhibitors of PKA and PKC respectively. In order to determine the significance of these protein kinases of uninduced and differentiating cells for the final differentiation, the cells were treated with the inhibitors before (-24 hours-0 hours) and after induction (0 hour-96 hours) of differentiation. To elucidate potential "cross-talking" between PKA and PKC of uninduced and differentiating HL-60 cells, the effects of H8 and staurosporine on differentiation were further examined by exposing the cells, pretreated with inhibitors for approximately one cell cycle time before induction, to the same or a different inhibitor.

Protein kinase inhibitors exert stage specific and inducer dependent effects on HL-60 cell differentiation.

The human promyelocytic leukemia cell line HL-60 was induced to differentiate to mature granulocytic cells by dbcAMP or RA. The influence of distinct protein kinases during different stages of this differentiation was studied by the use of H8, staurosporine and genistein as inhibitors of PKA, PKC and PTK respectively. In dbcAMP-mediated differentiation, the PKA activity of uninduced cells is crucial for the induction of differentiation, but therefore its significance drastically declines and a more important role is played by PKC and PTK.

[Nucleosomes of active chromatin from sea urchin embryo cells are rich in early histone variants].

By means of selective micrococcal nuclease digestion chromatin from early stages of the sea urchin St. droebachiensis embryogenesis was divided into fractions differing by their transcriptional activity. The electrophoretic analysis of histones at the gastrula stage showed that the transcriptionally active chromatin fraction was enriched with early variants of histone H2A and H1.

Distribution of histone variants in the sea urchin chromatin fractions obtained by selective micrococcal nuclease digestion.

Chromatin fractions differing in their transcriptional activity were isolated by selective micrococcal nuclease digestion of nuclei from sea urchin embryos (Strongylocentrotus droebachiensis) at the gastrula and pluteus stage. The electrophoretic analysis of the chromatin proteins at the gastrula stage showed that a soluble, transcriptionally active fraction of chromatin was enriched with early variants of histones H1 and H2A. The early and late variants of histone H2A at the pluteus stage were distributed randomly between chromatin fractions.

Dissociation and isolation of chromatin proteins in salt solutions by an aqueous two-phase system.

An aqueous two-phase system containing 7% Dextran T 500-5% polyethylene glycol (PEG) 6000 has been adopted for rapid selective stepwise extractions of high-mobility-group proteins and histones from both isolated chromatin and intact nuclei of calf thymus. After the dissociated proteins in the PEG phase were precipitated with 20% trichloroacetic acid at 4 degrees C, proteins were recovered from this phase by solubilization of PEG with acidified acetone at room temperature. This method allows preparation of nuclei depleted of histone H1.