To the Large Nucleolar Bodies in Apoptotic Leukaemic Granulocytic Progenitors without Further Differentiation. Are Large Nucleoli Always Present in Proliferating Cells?

Large nucleoli have generally been believed to be present in less differentiated and proliferating cells including the malignant ones. Such nucleoli have also been considered to be active in the biosynthetic process and major cell developmental activities. In contrast, after cytostatic treatment, apoptotic leukaemic progenitors still containing nuclei did not exhibit substantial reduction of the nucleolar size but displayed decreased nucleolar biosynthetic activity.

Changes in cell adhesivity and cytoskeleton-related proteins during imatinib-induced apoptosis of leukemic JURL-MK1 cells.

The fusion protein Bcr-Abl, which is the molecular cause of chronic myelogenous leukemia (CML) interacts in multiple points with signaling pathways regulating the cellular adhesivity and cytoskeleton architecture and dynamics. We explored the effects of imatinib mesylate, an inhibitor of Bcr-Abl protein used in front-line CML therapy, on the adhesivity of JURL-MK1 cells to fibronectin and searched for underlying changes in the cell proteome. As imatinib induces apoptosis of JURL-MK1 cells, we used three different caspase inhibitors to discriminate between direct consequences of Bcr-Abl inhibition and secondary changes related to the apoptosis.

Suberoylanilide hydroxamic acid (SAHA) at subtoxic concentrations increases the adhesivity of human leukemic cells to fibronectin.

Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) which is being introduced into clinic for the treatment of hematological diseases. We studied the effect of this compound on six human hematopoietic cell lines (JURL-MK1, K562, CML-T1, Karpas-299, HL-60, and ML-2) as well as on normal human lymphocytes and on leukemic primary cells. SAHA induced dose-dependent and cell type-dependent cell death which displayed apoptotic features (caspase-3 activation and apoptotic DNA fragmentation) in most cell types including the normal lymphocytes.

Isoform-specific cleavage of 14-3-3 proteins in apoptotic JURL-MK1 cells.

The proteins of 14-3-3 family are substantially involved in the regulation of many biological processes including the apoptosis. We studied the changes in the expression of five 14-3-3 isoforms (beta, gamma, epsilon, tau, and zeta) during the apoptosis of JURL-MK1 and K562 cells. The expression level of all these proteins markedly decreased in relation with the apoptosis progression and all isoforms underwent truncation, which probably corresponds to the removal of several C-terminal amino acids.

Rho-signaling pathways in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is a hematological malignancy that is characteristic by as expansion of myeloid cells and their premature release into the circulation. The molecular cause of CML is the fusion oncoprotein Bcr-Abl whose constitutive tyrosine-kinase (TK) activity maintains enhanced signaling through multiple signal transduction pathways and confers proliferative and survival advantage to CML cells. These effects can be largely suppressed by TK inhibitor Imatinib mesylate, currently the leading drug in CML treatment.

To the density and distribution of heterochromatin in differentiating, maturing and apoptotic cells represented by granulocytic, lymphocytic and erythrocytic precursors.

The present study was undertaken to provide more information on the density and distribution of heterochromatin in early and advanced stages of the granulocytic, lymphocytic and erythroid development. Heterochromatin was visualized using a simple cytochemical method for the demonstration of DNA followed by computer-assisted densitometry of the digitized images. The largest heterochromatin density in early proliferating stages of all studied blood cell lineages was noted in the perinucleolar region and centrally located chromocentres.

Mean diameter of nucleolar bodies in cultured human leukemic myeloblasts is mainly related to the S and G2 phase of the cell cycle.

Mean diameter of nucleolar bodies (nucleoli without the perinucleolar chromatin) per cell was studied in human leukemic myeloblasts represented by K 562 and Kasumi 1 cell lines which originated from chronic and acute myeloid leukaemia. The measurement of mean diameter of nucleolar bodies in specimens stained for RNA was very simple. Such approach eliminated the variability of the perinucleolar chromatin discontinuous shell which might influence the measured nucleolar size as suggested by earlier studies.

A karyometric study on ageing and butyrate or imatinib treated human leukemic myeloblasts represented by K562 cells originated from chronic myeloid leukaemia.

The present study was undertaken to provide more information on nuclear diameter in leukemic granulocytic early precursors myeloblasts. These cells represented by K562 myeloblasts originated from the blastic phase of the chronic myeloid leukaemia (CML) carry characteristic bcr-abl fusion gene. They represent a convenient model for in vitro studies of CML myeloblasts and are sensitive to various agents which may induce ageing, differentiation and cell death.

Labeling of apoptotic JURL-MK1 cells by fluorescent caspase-3 inhibitor FAM-DEVD-fmk occurs mainly at site(s) different from caspase-3 active site.

Background: Fluorochrome-labeled inhibitors of caspases (FLICA) have been designed as an alternative tool for the detection of caspase activation in whole cells. They should label the active site of the corresponding caspase through a covalent attachment to the reactive cysteine residue.

Methods: One of the FLICAs, FAM-DEVD-fmk, was used to monitor apoptosis progression in leukemic JURL-MK1 cells by means of flow cytometry.

The proteomic study of sodium butyrate antiproliferative/cytodifferentiation effects on K562 cells.

Employing methods of cell biology and proteome analysis tools, we examined effects of an inhibitor of histone deacetylases, sodium butyrate (SB), on the proliferation/differentiation characteristics of chronic myelogenous leukemia (CML)-derived cells K562. SB suppressed proliferation of K562 cells by inducing cell cycle arrest in G1 phase, which was followed by their transition to G0 phase (decrease of Ki-67 antigen-positive cells) and erythroid differentiation (increased glycophorin A expression and synthesis of hemoglobins). Neither terminal apoptosis (low counts of TUNEL-positive cells) nor necrosis (moderate counts of propidium iodide-positive cells) occurred.

On the nucleolar size and density in human early granulocytic progenitors, myeloblasts.

Human myeloblasts were studied in bone marrow of patients suffering from chronic phase of chronic myeloid leukaemia to provide more information on the nucleolar diameter in these early granulocytic progenitors. These cells are a convenient model for such study since the number of myeloblasts in diagnostic bone marrow smears of investigated patients is larger than in not-leukemic persons because of the increased granulopoiesis. The nucleolar diameter was measured in myeloblasts after various cytochemical procedures such as methods for visualisation of RNA, DNA and proteins of AgNORs using digitized images and image processing.

Photodynamic treatment (ALA-PDT) suppresses the expression of the oncogenic Bcr-Abl kinase and affects the cytoskeleton organization in K562 cells.

K562 is the chronic myelogenous leukemia (CML)-derived cell line that expresses high levels of chimeric oncoprotein Bcr-Abl. The deregulated (permanent) kinase activity of Bcr-Abl leads to continuous proliferation of K562 cells and their resistance to the apoptosis promotion by conventional drugs. The photodynamic treatment (PDT) based on the application of 5-aminolevulinic acid (ALA) and irradiation with blue light (ALA-PDT) resulted in the suppression of K562 cells proliferation.

Intranucleolar translocation of AgNORs in early granulocytic precursors in chronic myeloid leukaemia and K 562 cells.

The present study was undertaken to provide missing information on the distribution of AgNORs in large nucleoli of human leukaemic early granulocytic precursors in vivo as well as in vitro. In vivo, the distribution of AgNORs was studied in early granulocytic precursors of patients suffering from chronic myeloid leukaemia who were both untreated and treated with imatinib mesylate. AgNORs were visualized by silver reaction under conditions which facilitated to see their distribution by light microscopy.

To the intranucleolar translocation of AgNORs in leukemic early granulocytic and plasmacytic precursors.

Early leukemic granulocytic and plasmacytic precursors were studied in vitro and in vivo to provide an information on the intranucleolar distribution of AgNORs (silver stained nucleolus organizer regions). In most of these cells AgNORs appeared as clusters of silver stained particles distributed in the whole nucleolar body. On the other hand, in some leukemic early granulocytic precursors, i.

Fast apoptosis and erythroid differentiation induced by imatinib mesylate in JURL-MK1 cells.

We compare the effects of Imatinib mesylate (Glivec) on chronic myeloid leukemia derived cell lines K562 and JURL-MK1. In both cell lines, the cell cycle arrests in G(1)/G(0) phase within 24 h after the addition of 1 microM Imatinib. This is followed by a decrease of Ki-67 expression and the induction of apoptosis.

The effect of 5-aminolevulinic acid-based photodynamic treatment (PDT) on nucleoli of leukemic granulocytic precursors represented by K562 blastic cells in vitro.

Background: Since photodynamic treatment (PDT) induces apoptosis in individual HL-60 cells originating from early granulocytic precursors of acute myeloid leukemia, the present study was undertaken to provide information on such treatment of K562 cells, which originate from early granulocytic precursors of chronic myeloid leukemia (blastic phase) and carry the bcr/abl fusion gene with anti-apoptotic properties.

Material/methods: PDT was based on the 5-aminolevulinic acid treatment of K562 cells, followed by blue light irradiation under conditions which in HL-60 cells induce an apoptotic process without previous terminal maturation. Nuclei and nucleoli were visualized by cytochemical procedures to demonstrate DNA, RNA and silver stained proteins of nucleolus organizer regions (AgNORs).

A short note on the nucleolar size and density in apoptotic leukemic granulocytic precursors (HL-60 cells).

The present study was undertaken to provide more information on the nucleolar size and density in mononuclear blastic granulocytic precursors represented by HL-60 cells the proliferation of which was blocked by photodynamic treatment (PDT) which induced apoptotic process without preceding terminal maturation. Both the nucleolar size and density did not change in apoptotic cells in comparison with controls. Thus, large and dense nucleoli in apoptotic cells are not necessarily related to the nucleolar biosynthetic or cell proliferation activity.

A note on the decreased number and loss of fibrillar centres in nucleoli of apoptotic HL-60 leukaemic granulocytic precursors produced by 5-aminolaevulinic acid-based photodynamic treatment.

The nuclear and nucleolar ultrastructure was studied by means of conventional transmission electron microscopy to provide more and complementary information on nucleolar changes accompanying the apoptotic process in leukaemic granulocytic precursors (HL-60 cells) produced by PDT without previous terminal differentiation. PDT induced the apoptotic process using BL irradiation and ALA as a precursor of the photosensitizer protoporphyrin IX. PDT produced marked changes of the nucleolar ultrastructure in apoptotic cells, such as reduction of the number and loss of fibrillar centres surrounding dense fibrillar components.

Interferon-alpha suppresses proliferation of chronic myelogenous leukemia cells K562 by extending cell cycle S-phase without inducing apoptosis.

We examined the effects of interferon-alpha (IFN-alpha) treatment on the growth, cell cycle, proliferation, and apoptotic parameters as well as adhesive properties and proteome of chronic myelogenous leukemia (CML)-derived K562 cells. IFN-alpha treatment (200 to 600 U/ml, 24 to 72 h) suppressed growth and caused accumulation of K562 cells in the S-phase of cell cycle (increase in S-phase cells by up to 52% in comparison with the untreated controls) at the expenses of cells in G1-phase. No transition of cells to G0-phase occurred as followed from Ki-67 protein determination.

Early apoptotic features of K562 cell death induced by 5-aminolaevulinic acid-based photodynamic therapy.

5-Aminolaevulinic acid-based photodynamic therapy (ALA-PDT) is used to eliminate cancerous cells through photoactivation of endogenously formed protoporphyrin IX (PPIX) following the administration of PPIX precursor, 5-aminolaevulinic acid (ALA). We report on the kinetics of PPIX accumulation and the mechanism of cytotoxic effects of ALA-PDT studied in the chronic myelogenous leukaemia derived cell line K562. The PPIX distribution and, consequently, cytotoxic effects were found to be heterogenous.

On the way to functional genomics.

Proteomics, the global comprehensive analysis of cellular proteins, will contribute to our understanding of gene function in the post-genomic era. The strategies of proteome analysis include the protein expression proteomics dealing with the comparison of cellular or tissue protein levels in the control and affected state (e.g.

Nucleoli in large (giant) bi- and multinucleate cells after apoptosis-inducing photodynamic treatment.

The present experimental study was undertaken to provide information on nucleolar changes accompanying the apoptotic process in large or giant binucleate and multinucleate cells (LBMNCs). Such cells were present in a small but constant percentage in cultures of HL-60 cells. The apoptotic process was induced by photodynamic treatment (PDT) by means of 5-aminolaevulinic acid (ALA) as the precursor of the photosensitizer protoporphyrin IX and irradiation with broad spectrum blue light (BL).

Mitochondrial and endoplasmic reticulum stress-induced apoptotic pathways are activated by 5-aminolevulinic acid-based photodynamic therapy in HL60 leukemia cells.

We studied the mechanism of the cytotoxic effects of 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT; induction with 1 mM ALA for 4 h followed by a blue light dose of 18 J/cm(2)) on the human promyelocytic leukemia cell line HL60 using biochemical and electron microscopy methods. The disruption of mitochondrial membrane potential, deltapsi(m), was paralleled by a decrease in ATP level, unmasking of the mitochondrial antigen 7A6, release of cytochrome c into the cytoplasm, activation of caspases 9 and 3 and cleavage of poly(ADP-ribose) polymerase (PARP). This was followed by DNA fragmentation.

[Use of photodynamic therapy for elimination of residual leukemic cells in autologous transplants of hematopoietic progenitor cells].

Background: The increasing use of autologous hematopoietic cell support in various malignancies including leukemia and lymphoma currently bears the problem of tumor contamination of the graft with tumor cells which after re-infusion contribute to the disease relapses. It is therefore desirable to eradicate the cancer cell fraction of the graft without causing damage to the normal stem cell fraction. The purging processes based on photodynamic treatments appear to be perspective means for this purpose.

Nucleolar asynchrony observed in HL-60 leukemic granulocytic precursors resistant to 5-aminolaevulinic acid-based photodynamic treatment.

To provide more information on the 5-aminolaevulinic acid (ALA)-induced photodynamic effect on nucleoli, morphologically expressed nucleolar asynchrony (the presence of 'active' large nucleoli with an uniform distribution of RNA and 'resting' ring-shaped nucleoli in one and the same nucleus) was studied in cultured HL-60 leukemic granulocytic precursors using a simple cytochemical procedure for the demonstration of RNA. Nucleolar asynchrony was mainly expressed in cells which were apparently resistant to ALA-based photodynamic treatment (PDT) since most of them (about 75%) exhibited this phenomenon.