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By studying the oligomeric state, chiroptical properties and the reactivity of sulfhydryl and phenolic groups, we compared the properties of human, horse, bovine, canine, rabbit and rat globins in solution. The results indicate that, in spite of the differences in the amino acid sequence, all the mammalian globins studied exhibit analogical properties suggesting similar conformation. [ABSTRACT FROM AUTHOR]

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. At subtoxic concentrations (0.5-1 microM), SAHA increased the cell adhesivity to fibronectin (FN) in all leukemia/lymphoma-derived cell lines but not in normal lymphocytes. This increase was accompanied by an enhanced expression of integrin beta1 and paxillin, an essential constituent of focal adhesion complexes, both at the protein and mRNA level. On the other hand, the inhibition of ROCK protein, an important regulator of cytoskeleton structure, had no consistent effect on SAHA-induced increase in the cell adhesivity. The promotion of cell adhesivity to FN seems to be specific for SAHA as we observed no such effects with other HDAC inhibitors (trichostatin A and sodium butyrate).

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. The observed 14-3-3 modifications were partially blocked by caspase-3 inhibition. In addition to caspases, a non-caspase protease is likely to contribute to 14-3-3's cleavage in an isoform-specific manner. While 14-3-3 gamma seems to be cleaved mainly by caspase-3, the alternative mechanism is essentially involved in the case of 14-3-3 tau, and a combined effect was observed for the isoforms epsilon, beta, and zeta. We suggest that the processing of 14-3-3 proteins could form an integral part of the programmed cell death or at least of some apoptotic pathways.

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 effects of unlabeled caspase inhibitors z-DEVD-fmk and z-VAD-fmk on FLICA staining were analyzed to evaluate the contribution of caspase-bound FLICA to the fluorescent signal. Covalent binding of inhibitors to caspase-3 subunit was revealed by Western blotting., Results: Although the unlabeled inhibitors irreversibly bind to caspase-3, completely inhibit its activity, and prevent FLICA binding to caspase-3 even at concentrations lower than 5 muM, they have no effect on FLICA staining of apoptotic cells., Conclusions: Fluorescent signal of FLICA is characteristic for apoptotic cells but originates mainly from yet unspecified site(s) that differ from the caspase active site. This finding puts in doubt the specificity of staining by various FLICAs with regard to individual caspases and shows the need for an extreme care in the interpretation of data obtained using these labels., (Copyright 2007 International Society for Analytical Cytology.)

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.e., in myeloblasts and promyelocytes enlarged AgNORs were translocated in the nucleolar peripheral part. In addition, the number of translocated AgNORs at the nucleolar periphery was significantly smaller. Such translocation of a reduced number of AgNORs was easily produced by experimental aging, i.e., starving of cultured leukemic early granulocytic precursors (HL-60 and K562 cells) in vitro and seems to be reversible. Similar translocation of a reduced number of AgNORs was also produced by aging of leukemic plasmacytic precursors. Thus, the translocation of the reduced number of AgNORs to the nucleolar periphery in some blastic leukemic hematopoietic cells might be an useful marker of their aging at the single cell level. However, more studies in this direction are required in the future.

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. In JURL-MK1 cells, the apoptosis is faster in comparison with K562 cells: the caspase-3 activity reaches the peak value (20 to 30 fold of the control) after about 40 h and the apoptosis proceeds to its culmination point, the DNA fragmentation, within 48 h following 1 microM Imatinib addition. Unlike K562 cells, JURL-MK1 cells possess a probably functional p53 protein inducible by TPA (tetradecanoyl phorbol acetate) or UV-B irradiation. However, no increase in p53 expression was observed in Imatinib-treated JURL-MK1 cells indicating that the difference in the apoptosis rate between the two cell lines is not due to the lack of p53 in K562 cells. Imatinib also triggers erythroid differentiation both in JURL-MK1 and K562 cells. Glycophorin A expression occurred simultaneously with the apoptosis, even at the single cell level. In K562 cells, but not in JURL-MK1 cells, the differentiation process involved increased hemoglobin synthesis. However, during spontaneous evolution of JURL-MK1 cells in culture, the effects produced by Imatinib progressively changed from the fast apoptosis to the more complete erythroid differentiation. We suggest that the apoptosis and the erythroid differentiation are parallel effects of Imatinib and their relative contributions, kinetics and completeness are related to the differentiation stage of the treated cells.

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). TUNEL and propidium iodide assays were used for additional control of the incidence of apoptotic and necrotic cells., Results: In contrast to HL-60 cells, PDT did not induce apoptosis in K562 cells. However, after PDT some K562 cells exhibited major alterations, expressed by nuclear and cell swelling, reflecting a necrotic process, confirmed by propidium iodide. In addition, PDT produced a reduction of AgNORs, though smaller than that previously described in apoptotic HL-60 cells., Conclusions: PDT produced only necrotic changes in some K562 cells under conditions which induced in apoptosis in HL-60 cells. Thus the induction of apoptosis, nuclear and nucleolar changes in individual cells does not depend on the inducer--PDT--but on the cell properties.

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., (Copyright 2004 Elsevier Inc.)