RESUMEN
The participation of the main caspases in the cytotoxic effects induced by monoclonal antibody 14G2a specific against tumor-associated ganglioside GD2 was studied in the EL-4 cells. It has been found constitutive expression ofprocaspases genes in the EL-4 cells; incubation of the cells with 14G2a antibodies didnot result in increasing of the procaspases expression. Weak enzymatic activity of caspases has been shown using fluorescent labeled substrates. At the same cell death level, activity of caspase-3 and caspase-9 in the cells incubated with 14G2a was about 7.5- and 3-fold lower than in cells after incubation with staurosporine. Pan caspase inhibitor Z-VAD-FMK, and caspase-3 inhibitor reduced the cytotoxic effects induced by 14G2a at 9-16 and 6-13%, respectively. At the same conditions, pan caspase inhibitor decreased staurosporine-induced apoptosis at 55-65%. Inhibitors of other caspases had no effect on the cell death triggered by the antibodies. Inhibition analysis demonstrated also that caspases did not involved in the cell volume decreasing and permeabilization of the cell plasma membrane, which were the first stages of anti-GD2-mAb-induced cell death in the EL-4 cells. Thus, despite the slight activation of caspases during the cell death induced by antibodies directed to GD2, they do not play a key role and do not determine the mechanism of cell death triggered through the tumor-associated ganglioside GD2.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasa 3/biosíntesis , Caspasa 9/biosíntesis , Neuroblastoma/genética , Anticuerpos Monoclonales/administración & dosificación , Caspasa 3/genética , Caspasa 9/genética , Inhibidores de Caspasas/administración & dosificación , Línea Celular Tumoral , Gangliósidos/biosíntesis , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neuroblastoma/enzimología , Neuroblastoma/patología , Estaurosporina/administración & dosificaciónRESUMEN
Myelopeptide-4 (MP-4) (Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro), inducing the terminal differentiation of HL-60 leukemia cells, was labeled with fluorescein isothiocyanate. The specific binding of this modified peptide to the surface of HL-60 cells and its ability to penetrate into the cells were studied. It was shown by cytometry and confocal microscopy to be bound on the HL-60 cell surface, to penetrate into their cytoplasm, and finally to concentrate around the cell nucleus. These phenomena are probably necessary for the exhibition of MP-4 differentiating activity.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Citoplasma/metabolismo , Oligopéptidos/farmacología , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceína-5-Isotiocianato/farmacología , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Colorantes Fluorescentes/farmacología , Células HL-60 , Humanos , Microscopía Confocal , Microscopía Fluorescente , Oligopéptidos/química , Oligopéptidos/metabolismo , Transporte de Proteínas/efectos de los fármacosRESUMEN
An experimental model system involving the modification of carbohydrate composition of the target cell surface with neoglycolipids was developed for studying the role of surface carbohydrates of target cells in the NK-cell-mediated cytotoxicity. The polymeric glycoconjugates of the Glyc-PAA-PEA and Glyc-PAA(Flu)-PEA types (where Glyc was an oligosaccharide residue, PAA poly(acrylamide) polymer, and PEA the phosphatidylethanolamine residue, and Flu fluorescein residue) capable of incorporation into the cell membrane were synthesized. The optimum structures of neoglycoconjugates and conditions for their incorporation into K562 and Raji cell lines, which differ in their sensitivity to the NK-cell-mediated lysis were selected. The mechanism of association of glycoconjugates with the plasma cell membrane and the kinetics of their elimination from the cell surface were investigated using the fluorescent-labeled Glyc-PAA(Flu)-PEA derivatives. The spatial accessibility of the carbohydrate ligands for the interaction with human NK cells was demonstrated. The target cells modified with the Le(x) trisaccharide were shown to be more sensitive to the cytotoxic effect of human NK cells than the intact cells. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.
Asunto(s)
Membrana Celular/metabolismo , Citotoxicidad Inmunológica , Glicoconjugados/metabolismo , Glucolípidos/metabolismo , Células Asesinas Naturales/inmunología , Línea Celular , Membrana Celular/química , Glicoconjugados/síntesis química , Glicoconjugados/química , Glucolípidos/química , Humanos , Antígeno Lewis X/análisis , Antígeno Lewis X/química , Antígeno Lewis X/metabolismoRESUMEN
Six-membered peptide fragment TGENHR (HLDF-6) was identified in the HL-60 cell culture of human promyelocyte leukemia treated with retinoic acid when studying the differentiation factor HLDF of this cell line. HLDF-6 retains the ability of the full-size factor to induce the differentiation and arrest the proliferation of the starting HL-60 cells. It was shown that the synthetic peptide HLDF-6 has no specific receptors on the surface of the HL-60 cells but can affect the binding of interleukin IL-1 beta, a cytokine involved in proliferation, to the cell surface. It was found on a model of transplantable NSO myeloma that HLDF-6 has an antitumor activity.
Asunto(s)
Antineoplásicos/química , Proteínas de Neoplasias/química , Fragmentos de Péptidos/química , Animales , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Diferenciación Celular , División Celular/efectos de los fármacos , Células HL-60 , Humanos , Interferón-alfa/metabolismo , Interleucina-1/metabolismo , Masculino , Fluidez de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Unión Proteica , Proteínas Recombinantes/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A structural homology between the endogenous differentiation factor of the HL-60 cell line of promyelocyte leukemia (HLDF) and several DNA/RNA-binding and DNA/RNA-hydrolyzing proteins was revealed, and expression of the hldf gene in prokaryotic systems was studied. On the basis of these experiments, the amino acid sequence of an 8-membered fragment of HLDF with potential nuclease activity was identified. The synthetic octapeptide RRWHRLKE was shown to be capable of the cleavage of RNA, linear DNA from phage lambda, and all forms of plasmid DNA. We established that treatment of the HL-60 cell culture with this peptide (10(-6) M) results in an increase in the number of apoptotic cells and suggested that HLDF is involved in processes of apoptosis.
Asunto(s)
Desoxirribonucleasas/metabolismo , Células HL-60/enzimología , Linfocinas/metabolismo , Ribonucleasas/metabolismo , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Secuencia de Bases , ADN Complementario/análisis , ADN Complementario/genética , Desoxirribonucleasas/genética , Células HL-60/patología , Humanos , Linfocinas/genética , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/metabolismo , Péptidos/farmacología , Ribonucleasas/genéticaRESUMEN
The concentration of intracellular free calcium ions in the follicle wall cells and in the follicle cells of Rana temporaria in Ringer solution is 150 +/- 10 and in the follicle wall cells of Xenopus laevis, 220 +/- 10 nM. In a chloride-free saline, its concentration in the same cells is 2.5-3 times that in Ringer solution. Voltage-dependent Ca(2+)-channel blockers diltiazem and verapamil (100 microM) reduce the level of intracellular free calcium ions in R. temporaria follicle wall cells cultivated in a chloride-free saline to 170 +/- 20 nM, which practically does not differ from the level in Ringer solution. Inhibitors (100 microM) decrease the rate of "spontaneous" maturation of R. temporaria follicle-enclosed oocytes both in chloride-free and Ringer solutions. It was concluded that an increased level of intracellular free calcium ions in the follicle cells, among other factors, may determine the stimulating effect of the medium (Ringer or chloride-free solution) on "spontaneous" maturation of follicle-enclosed amphibian oocytes. Voltage-dependent calcium channels appear to be involved in Ca2+ influx into the cells.