ucn-1028-c and Leukemia--Erythroblastic--Acute

The effect of alpha- and beta-tocopherol on human erythroleukemia cell (HEL) adhesion induced by phorbol 12-myristate 13-acetate (PMA) has been studied. Adhesion induced by PMA stimulation was prevented by 44.5% by physiological concentrations of alpha-tocopherol. Under the same experimental conditions, beta-tocopherol, an analogue of alpha-tocopherol, produced 11% inhibition of adhesion. Cell response gradually increased from 0 to 24 h of alpha-tocopherol treatment. Only a slight time dependency of beta-tocopherol inhibition was observed. Another human erythroleukemia cell line (K562) and the human monocyte tumor cell line U937 showed 5.0 and 11.2% inhibition, respectively. Similar to alpha-tocopherol, the protein kinase C inhibitor, Calphostin C, and the MAPK inhibitor, PD98059, prevented PMA-induced cell adhesion. An inhibition of ERK-1 phosphorylation was observed for alpha-tocopherol only in HEL, implying that MAP kinase pathway is involved in this cell line. Fluorescence-activated cell sorting (FACS), by using various integrin-specific monoclonal antibodies, has shown that alpha (1-6), beta1, and alphav integrins are less expressed at the cell surface after alpha-tocopherol treatment. Beta-tocopherol treatment was less effective.

Topics: Antibodies, Monoclonal; Cell Adhesion; Down-Regulation; Flavonoids; Flow Cytometry; Humans; Integrins; Isoenzymes; K562 Cells; Leukemia, Erythroblastic, Acute; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Naphthalenes; Neoplasm Proteins; Phosphorylation; Protein Kinase C; Protein Kinase C-alpha; Protein Processing, Post-Translational; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; U937 Cells; Vitamin E

Resistance of tumor cells to lysis by complement is generally attributed to several protective mechanisms. The relative impact of these mechanisms in the same tumor cell, however, has not been assessed yet. We have analyzed the interaction of the human erythroleukemia tumor cell line K562 with human complement. K562 cells express the membrane complement regulatory proteins CD59, CD55 and CD46. As shown here for the first time, K562 also spontaneously release the soluble regulators C1 inhibitor, factor H, and soluble CD59. Complement resistance of K562 cells is augmented upon treatment with PMA, TNF or even with sublytic complement. Unlike TNF and sublytic complement, PMA enhanced the expression of membrane-bound CD55 and CD59 and led to increased secretion of soluble CD59. In addition, we show that complement-resistant K562 cells express a membrane-associated proteolytic activity, higher than the complement-sensitive K562/S cells. Treatment of complement-resistant K562 cells with serine protease inhibitors enhance their sensitivity to complement-mediated lysis. Inhibitors of protein kinase C (PKC) also sensitize K562 cells to complement lysis, implicating PKC-mediated signaling in cell resistance to complement. Neutralization of the CD55 and CD59 but not of CD46 regulatory activity with specific antibodies significantly increases complement-mediated K562 cell lysis. Treatment of K562 cells with a mixture of inhibitory reagents results in a significant additive enhancing effect on complement-mediated lysis of K562. In conclusion, K562 cells resist a complement attack by concomitantly using multiple molecular evasion strategies. Future attempts in antibody-based tumor therapy should include strategies to interfere with those resistance mechanisms.

Topics: alpha-Amylases; Antigens, CD; CD55 Antigens; CD59 Antigens; Cell Membrane; Complement Activation; Complement System Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Erythrocytes; Flow Cytometry; Humans; K562 Cells; Leukemia, Erythroblastic, Acute; Membrane Cofactor Protein; Membrane Glycoproteins; Naphthalenes; Neutrophils; Plant Proteins; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Trypsin Inhibitors; Tumor Necrosis Factor-alpha

Sphingosine kinase functions in both the catabolism of sphingosine and in signal transduction pathways utilizing sphingosine-1-phosphate. The regulation of sphingosine kinase activity in human erythroleukemia (HEL) cells was investigated by treatment with several bioactive agents. Treatment of HEL cells with phorbol 12-myristate 13-acetate (PMA) caused a time- and concentration-dependent increase in sphingosine kinase activity measured in vitro. Sphingosine kinase activity increased in a phorbol ester- and diacylglycerol-specific manner. Staurosporine and calphostin C, protein kinase C (PKC) inhibitors, blocked the increased in sphingosine kinase activity, suggesting a PKC-dependent regulation. The effects of PMA on sphingosine kinase were dependent on transcription and translation. Purified PKC had no direct effect on sphingosine kinase activity. However, these studies led to the observation that HEL cell sphingosine kinase activity is stimulated in vitro by phosphatidylserine. Interestingly, other inducers of HEL cell differentiation, dimethylsulfoxide and retinoic acid, did not affect sphingosine kinase activity. These results indicate a separate and distinct pathway of PKC-dependent sphingosine kinase activation, and suggest a role for sphingosine kinase in regulation of cell function.

Topics: Cell Differentiation; Dimethyl Sulfoxide; Enzyme Activation; Enzyme Inhibitors; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Naphthalenes; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The plasma protein fibronectin is an important opsonin in wound repair and host defense. To better understand the process of fibronectin-mediated phagocytosis, we have transfected K562 cells, which endogenously express alpha 5 beta 1, with alpha v beta 3. In these transfectants, antibodies to alpha v beta 3 block phagocytosis of fibronectin-opsonized beads completely, even though half the ingestion occurs through endogenous alpha 5 beta 1 receptors. alpha 5 beta 1-mediated adhesion to fibronectin-coated surfaces is unaffected by alpha v beta 3 ligation. Neither alpha v beta 5 nor alpha M beta 2 ligation affects alpha 5 beta 1 phagocytic function in transfectants expressing these receptors. Pharmacologic data suggest that alpha v beta 3 ligation suppresses the phagocytic competence of high affinity alpha 5 beta 1 receptors through a signal transduction pathway, perhaps involving protein kinase C. In addition to its significance for phagocytosis, alpha v beta 3 regulation of alpha 5 beta 1 function may be significant for its roles in cell migration, metastasis, and angiogenesis.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Benzoquinones; Cell Adhesion; Cell Line; Cloning, Molecular; Fibronectins; Flow Cytometry; Genistein; Humans; Integrins; Isoflavones; Isoquinolines; Kinetics; Lactams, Macrocyclic; Leukemia, Erythroblastic, Acute; Naphthalenes; Phagocytosis; Piperazines; Polycyclic Compounds; Protein Kinase C; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Quinones; Receptors, Cytoadhesin; Receptors, Fibronectin; Receptors, Vitronectin; Rifabutin; Signal Transduction; Transfection; Tumor Cells, Cultured