RESUMO
The clonogenic assay is widely considered to be the most valid test for predicting tumor cell sensitivity to cytostatic drugs. In this study it was compared with early growth curves of human leukemic cell lines (HL-60, K562, Reh) after treatment with different types of cytostatic drugs (Adriamycin,cis-diamminedichloroplatinum(II):,1-beta-D-arabinofuranosyl- cytosine, and 5-fluorouracil) for 1 and 24 h. Following drug treatment two parallel cultures were started: a soft agar culture for the clonogenic assay; and a liquid suspension culture for vital cell counting by measuring esterase activity with fluorescein diacetate at different time points. The latter was recorded using flow cytometry during the following 3 days in 12-h intervals. For each drug concentration a survival factor was calculated from the growth curve between 24 and 72 h. This survival factor takes into account both the y intercept of the extrapolated growth curve and the slope of the growth curve. The dose-response curves resulting from either the survival factors or the clonogenic assay were always nearly identical. The results demonstrate that in established cell lines flow cytometric determination of vital cell increase rates provides a convenient alternative to the clonogenic assay for drug testing.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Citarabina/farmacologia , Doxorrubicina/farmacologia , Citometria de Fluxo , Fluoruracila/farmacologia , HumanosRESUMO
The tumor-suppressor p53 inhibits cell cycle progression by direct transactivation of the p21WAF1/CIP1/SDI1 gene, which encodes a universal inhibitor of cyclin dependent kinases (cdk). The proto-oncogene product c-Myc induces cell cycle progression and, in the absence of survival factors, apoptosis. However, a direct link between the cell cycle machinery and c-Myc has not yet been established. We show that c-Myc has not yet been established. We show that c-Myc abrogates a p53-induced G1-arrest without elevating the expression of cdks or cyclins involved in the G1/S-transition. Instead, the results suggest that c-Myc interferes with the inhibitory action of p21 on cdk/cyclin-complexes by inducing a heat-labile inhibitor of p21. The inactivation of p21 and related cdk-inhibitors may explain several of the oncogenic actions of c-Myc, including the induction of proliferation, immortalisation and the inhibition of differentiation. Modulation of cdk activity by the induction of an inhibitor of cdk-inhibitors represents a novel mechanism of cell cycle regulation in mammalian cells.
Assuntos
Ciclinas/antagonistas & inibidores , Fase G1/genética , Genes p53 , Proteínas Proto-Oncogênicas c-myc/fisiologia , Células 3T3 , Animais , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Ativação Enzimática , Genes myc , Camundongos , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
Cell membrane damage by ultrasound was studied in human nucleated cells in vitro at various concentrations. Suspensions of human blood cells, cells of a human leukemic cell line (Reh) and mixtures of nucleated cells with erythrocytes were exposed to continuous ultrasound of 782 kHz at a SPTA intensity of 15 W/cm2. The surviving nucleated cells without membrane damage were counted on the basis of exclusion of ethidium bromide using a flow cytometer. At high cell concentrations as present in whole blood, we observed no cell death, whereas below 5 X 10(7) cells/ml most of the granulocytes, stimulated lymphocytes and Reh cells were damaged. The concentration threshold below which cells were damaged seemed not to depend on the size of the cells, rather on the concentration of particles in the suspension.
Assuntos
Contagem de Células , Membrana Celular/ultraestrutura , Ultrassom/efeitos adversos , Agregação Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Índices de Eritrócitos , Eritrócitos/efeitos dos fármacos , Eritrócitos/ultraestrutura , Fixadores/farmacologia , Citometria de Fluxo , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/ultraestruturaRESUMO
In order to test a mathematical model of G1/S-phase transition, the proliferative response of the murine myeloid interleukin 3 (IL-3) dependent cell line NFS-78 to graded reduction of IL-3 levels was measured. Exponentially growing cells were exposed to bromodeoxyuridine (BUdR), which replaces thymidine (TdR) in the DNA double strands during DNA synthesis. After incubation periods ranging from 3 to 36 h the cells were fixed and stained with a fluorescence dye mixture of Hoechst 33258 and ethidium bromide (EB) and subsequently analyzed in a two-parametrical flow cytometer. The BUdR-quenched TdR-specific Hoechst 33258 fluorescence of each cell provides information on the cell cycle location at the start of incubation and on whether or not a cell has divided. The DNA-specific EB fluorescence provides information on the actual cell cycle location at the end of the incubation period. From the 2-dimensional fluorescence distributions the efflux from G1-phase was calculated. Upon IL-3 reduction the cells showed accumulation in the G1-phase along with a reduction in the progression rate through the other phases of the cell cycle. By staining with the vital dye Hoechst 33342 as well as with propidium iodide (PI) it was further possible to show that cell death after IL-3 withdrawal occurred in all phases of the cell cycle.
Assuntos
Fase G1/fisiologia , Interleucina-3/metabolismo , Modelos Biológicos , Animais , Ciclo Celular/fisiologia , Linhagem Celular , DNA/biossíntese , Citometria de Fluxo , CamundongosRESUMO
A bivariate flow cytometric technique has been developed in which Hoechst 33342 stained vital cells are discriminated from early damaged cells by differences in their fluorescence emission spectra. A discrete population of cells with intact cell membranes passing from vital to dead could be identified by using propidium iodide exclusion in combination with a concentration-independent spectrum shift of Hoechst fluorescence to longer wavelengths. The appearance after injury of two subsets of cells with intact membranes representing vital and early damaged cells, respectively, indicates two types of binding of Hoechst 33342 to these cells, presumably resulting from differences in chromatin structure. The method was tested in murine and human hemopoietic cell lines by cytotoxic treatment with cytosine arabinoside and interleukin 3 deprivation in factor-dependent lines. The power of the method was demonstrated by a cell cycle analysis of the early damaged cells after both cytotoxic treatment and growth factor deprivation.
Assuntos
Benzimidazóis , Sobrevivência Celular , Citometria de Fluxo/métodos , Coloração e Rotulagem , Animais , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Citarabina/farmacologia , DNA/análise , Dano ao DNA , Interleucina-3/farmacologia , Camundongos , Propídio , Espectrometria de Fluorescência , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Suspended cells of a human lymphoblastic cell line were exposed to pulsed ultrasound of 775 kHz. The pulse lengths were varied between 16 and 1000 microseconds. The mark/space ratio was always kept at 1:1. Two ultrasound intensity levels were used: 3.6 and 6.4 W/cm2 spatial peak and temporal peak. After an exposure time of 5 min, cell membrane damage was measured cytometrically by a dye exclusion test. No membrane damage was observable at 16 microseconds, whereas, at pulse lengths of 1000 microseconds, about one-third of the cells were damaged.
Assuntos
Células Tumorais Cultivadas/patologia , Ultrassom , Ultrassonografia/efeitos adversos , Linhagem Celular , Membrana Celular , HumanosRESUMO
Permeabilization of L1210 cells by lithotripter shock waves in vitro was monitored by evaluating the accumulation of fluorescein-labeled dextrans with a relative molecular mass ranging from 3,900-2,000,000. Incubation with labeled dextran alone caused a dose- and time-dependent increase in cellular fluorescence as determined by flow cytometry, with a vesicular distribution pattern in the cells consistent with endocytotic uptake. Shock wave exposure prior to incubation with labeled dextran revealed similar fluorescence intensities compared to incubation with labeled dextran alone. When cells were exposed to shock waves in the presence of labeled dextran, mean cellular fluorescence was further increased, indicating additional internalization of the probe. Confocal laser scanning microscopy confirmed intracellular fluorescence of labeled dextran with a diffuse distribution pattern. Fluorescence-activated cell sorting with subsequent determination of proliferation revealed that permeabilized cells were viable and able to proliferate. Permeabilization of the membrane of L1210 cells by shock waves in vitro allowed loading of dextrans with a relative molecular mass up to 2,000,000. Permeabilization of tumor cells by shock waves provides a useful tool for introducing molecules into cells which might be of interest for drug targeting in tumor therapy in vivo.
Assuntos
Permeabilidade da Membrana Celular , Membrana Celular/fisiologia , Leucemia L1210/fisiopatologia , Litotripsia , Animais , Dextranos , Citometria de Fluxo , Fluoresceína-5-Isotiocianato/análogos & derivados , Cinética , Camundongos , Fatores de Tempo , Células Tumorais CultivadasRESUMO
We assume the existence of a specific G1 protein which is an initiator of DNA replication. This initiator is supposed to be synthesized according to Michaelis-Menten kinetics. In order to start DNA replication, it is assumed that this G1 specific protein must be produced in a required amount. Intra-cellular growth inhibitors and extra-cellular growth factors control the production of the initiator. This model allows to calculate the average G1 phase time as a function of the various chemical concentrations of nutrients, enzymes, growth inhibitors and growth factors. This model is compared to cell kinetics experiments on a leukemic cell line responding to Interleukin 3 deprivation. The curves giving the experimental average G1 phase times with respect to Interleukin-3 concentrations are fitted by the mathematical model with a quite good agreement.
Assuntos
Fase G1/fisiologia , Interleucina-3/metabolismo , Leucemia Experimental/fisiopatologia , Modelos Biológicos , Células Cultivadas , DNA/biossíntese , CinéticaRESUMO
We investigated the ultrastructural effects of recombinant tumor necrosis factor alpha (TNF) on primary leukemia cells of 8 patients (4 cases of acute myelogenous leukemia and 4 cases of chronic myelogenous leukemia) as well as on bone marrow cells of 1 normal control. The cells were kept in liquid culture for up to 92 h in the presence of up to 10,000 U/ml of recombinant human TNF without adding colony-stimulating factors. Under these conditions the concentration of viable leukemic cells decreased by 14 to 53%, compared to control cultures. In acute myelogenous leukemia, all cases to some degree developed an enlargement of mitochondria; in 2 cases prominent cytoplasmic processes, and in 2 cases cytoplasmic vacuoles were observed. In chronic myelogenous leukemia, an enlargement and deformation of all cell types was observed to varying degrees. In the normal bone marrow sample only minor cytoplasmic changes occurred. In all cultures apoptotic changes were rarely observed and--if present--were observed also in cultures without TNF. When the DNA of leukemic cells treated with TNF was separated on agarose, no fragments characteristic of apoptosis were visible. Our results demonstrate that TNF does not induce direct cytotoxicity or apoptosis in acute or chronic myeloid leukemias and are compatible with the notion that some leukemic cells may be activated or stimulated by TNF. The mitochondrion appears to be one of the primary targets of TNF. Electron microscopy is useful for monitoring the changes induced by TNF.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mieloide Aguda/patologia , Fator de Necrose Tumoral alfa/farmacologia , Apoptose , Divisão Celular , Sobrevivência Celular , Citoplasma/ultraestrutura , DNA de Neoplasias/isolamento & purificação , Eletroforese em Gel de Ágar , Humanos , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/administração & dosagem , Vacúolos/ultraestruturaRESUMO
Human plasmacytoid precursor dendritic cells (ppDC) are a major source of type I IFN upon exposure to virus and bacteria, yet the stimulus causing their maturation into DCs is unknown. After PBMC activation with immunostimulatory bacterial DNA sequences (CpG-DNA) we found that ppDC are the primary source of IFN-alpha. In fact, either CpG-DNA or dsRNA (poly(I:C)) induced IFN-alpha from purified ppDC. Surprisingly, only CpG-DNA triggered purified ppDC survival, maturation, and production of TNF, GM-CSF, IL-6, and IL-8, but not IL-10 or IL-12. Known DC activators such as CD40 ligation triggered ppDC maturation, but only IL-8 production, while bacterial LPS was negative for all activation criteria. An additional finding was that only CpG-DNA could counteract IL-4-induced apoptosis in ppDC. Therefore, CpG-DNA represents a pathogen-associated molecular pattern for ppDC. In contrast to these finding, CpG-DNA, like LPS, caused TNF, IL-6, and IL-12 release from PBMC and purified monocytes; however, differentiation of monocytes into DCs with GM-CSF and IL-4 unexpectedly resulted in refractoriness to CpG-DNA, but not LPS. Taken together, these results suggest that within a DC subset a multiplicity of responses can be generated by distinct environmental stimuli and that responses to a given stimulus may be dissimilar between DC subsets.
Assuntos
Adjuvantes Imunológicos/farmacologia , Ilhas de CpG/imunologia , DNA Bacteriano/farmacologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Integrina alfaXbeta2 , Oligodesoxirribonucleotídeos/farmacologia , Receptores de Interleucina-3/biossíntese , Células 3T3 , Animais , Diferenciação Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Células Cultivadas , Citocinas/biossíntese , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Escherichia coli/imunologia , Humanos , Integrina alfaXbeta2/biossíntese , Interferon Tipo I/biossíntese , Subunidade alfa de Receptor de Interleucina-3 , Isoantígenos/fisiologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Monócitos/citologia , Monócitos/imunologia , Plasmócitos/citologia , Plasmócitos/imunologia , Plasmócitos/metabolismo , Plasmócitos/microbiologia , Poli I-C/farmacologia , Células-Tronco/citologia , Células-Tronco/imunologia , Células-Tronco/metabolismo , Células-Tronco/microbiologia , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Human CMV has evolved multiple strategies to interfere with immune recognition of the host. A variety of mechanisms target Ag presentation by MHC class I molecules resulting in a reduced class I cell-surface expression. This down-regulation of class I molecules is expected to trigger NK cytotoxicity, which would have to be counteracted by the virus to establish long-term infection. Here we describe that the human CMV open reading frame UL40 encodes a canonical ligand for HLA-E, identical with the HLA-Cw03 signal sequence-derived peptide. Expression of UL40 in HLA-E-positive target cells conferred resistance to NK cell lysis via the CD94/NKG2A receptor. Generation of the UL40-derived HLA-E ligand was also observed in TAP-deficient cells. The presence of a functional TAP-independent HLA-E ligand in the UL40 signal sequence implicates this viral gene as an important negative regulator of NK activity.
Assuntos
Citomegalovirus/imunologia , Citotoxicidade Imunológica/imunologia , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunossupressores/farmacologia , Células Matadoras Naturais/imunologia , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Células Cultivadas , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Humanos , Células K562 , Ligantes , Dados de Sequência Molecular , Oligopeptídeos , Fases de Leitura Aberta/imunologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/isolamento & purificação , Peptídeos/imunologia , Processamento de Proteína Pós-Traducional/imunologia , Transfecção , Proteínas Virais/genética , Antígenos HLA-ERESUMO
Treatment of cells with DNA damaging agents leads to induction of a variety of genes involved in different cellular processes. We have applied a lacZ-based gene trap strategy to search for new mammalian genes induced by genotoxic stress. A population of 32 x 10(3) neo r clones stably transfected with a gene trap vector was obtained, stained with fluorescein di-beta-d-galactopyranoside and analyzed by flow activated cell sorting and replica plating. This strategy allowed isolation of 30 neo r 'putative inducible' cell lines expressing lacZ only after a DNA damaging treatment. For three clones the site of integration and the degree of inducibility after UV treatment were determined by Southern blot and beta-galactosidase measurement respectively. One cell line (clone VI) showed a single integration site and a reproducible 3-fold induction of beta-galactosidase activity following UV irradiation. Fused transcripts were isolated from induced cells and a portion of the trapped gene was amplified by rapid amplification of cDNA ends. Sequence analysis and comparison with available gene and protein databanks revealed that the gene was novel.
Assuntos
Dano ao DNA , Técnicas Genéticas , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Células CHO , Clonagem Molecular , Cricetinae , DNA/genética , DNA/isolamento & purificação , Vetores Genéticos , Hibridização in Situ Fluorescente , Óperon Lac , Dados de Sequência Molecular , Análise de Sequência de DNA , Transfecção , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
Bacterial DNA and immunostimulatory (i.s.) synthetic CpG-oligodeoxynucleotides (ODN) act as adjuvants for Th1 responses and cytotoxic T cell responses to proteinaceous antigens. Dendritic cells (DC) can be referred to as "nature's adjuvant" since they display the unique capacity to sensitize naive T cells. Here, we demonstrate that bacterial DNA or i.s. CpG-ODN cause simultaneous maturation of immature DC and activation of mature DC to produce cytokines. These events are associated with the acquisition of professional antigen-presenting cell (APC) function. Unfractionated murine bone marrow-derived DC and FACS-fractionated MHC class IIlow (termed immature DC) or MHC class IIhigh populations (termed mature DC) were stimulated with bacterial DNA or i.s. CpG-ODN. Similar to lipopolysaccharide, i.s. CpG-ODN caused up-regulation of MHC class II, CD40 and CD86, but not CD80 on immature and mature DC. In parallel both DC subsets were activated to produce large amounts of IL-12, IL-6 and TNF-alpha. CpG-ODN-activated DC displayed professional APC function in allogeneic mixed lymphocyte reaction and in staphylococcal enterotoxin B-driven naive T cell responses. We interpret these findings to mean that bacterial DNA and i.s. CpG-ODN cause maturation (first step) and activation (second step) of DC to bring about conversion of immature DC into professional APC.
Assuntos
Ilhas de CpG/imunologia , DNA Bacteriano/imunologia , Células Dendríticas/imunologia , Oligodesoxirribonucleotídeos/imunologia , Adjuvantes Imunológicos , Animais , Células da Medula Óssea , Antígenos CD40/metabolismo , Células Cultivadas , Indução Enzimática , Feminino , Antígeno HLA-B7/metabolismo , Hematopoese , Antígenos de Histocompatibilidade Classe II/metabolismo , Interleucina-12/biossíntese , Interleucina-6/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T Citotóxicos/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Regulação para Cima , beta-Galactosidase/metabolismoRESUMO
Homing behavior and function of autoimmune CD4+ T cells in vivo was analyzed before and during EAE, using MBP-specific T cells retrovirally engineered to express the gene of green fluorescent protein. The cells migrate from parathymic lymph nodes to blood and to the spleen. Preceding disease onset, large numbers of effector cells invade the CNS, with only negligible numbers left in the periphery. In early EAE, most (>90%) infiltrating CD4+ cells were effector cells. Migratory effector cells downregulate activation markers (CD25, OX-40) but upregulate several chemokine receptors and adsorb MHC class II on their membranes. Within the CNS, the effector cells are reactivated, with upregulated proinflammatory cytokines and downmodulated T cell receptor-associated structures, presumably reflecting autoantigen recognition in situ.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Movimento Celular , Encefalomielite Autoimune Experimental/imunologia , Animais , Sistema Nervoso Central/imunologia , Proteínas de Fluorescência Verde , Antígenos de Histocompatibilidade Classe II/imunologia , Injeções Intraperitoneais , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Ativação Linfocitária/imunologia , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/metabolismo , Ratos , Ratos Endogâmicos LewRESUMO
Infection of primary B-lymphocytes by Epstein-Barr virus (EBV) leads to growth transformation of these B-cells in vitro. EBV nuclear antigen 2 (EBNA2), one of the first genes expressed after EBV infection of B-cells, is a transcriptional activator of viral and cellular genes and is essential for the transforming potential of the virus. We generated conditional EBV mutants by expressing EBNA2 as chimeric fusion protein with the hormone binding domain of the estrogen receptor on the genetic background of the virus. Growth transformation of primary normal B-cells by mutant virus resulted in estrogen-dependent lymphoblastoid cell lines expressing the chimeric EBNA2 protein. In the absence of estrogen about half of the cells enter a quiescent non-proliferative state whereas the others die by apoptosis. EBNA2 is thus required not only for initiation but also for maintenance of transformation. Growth arrest occurred at G1 and G2 stages of the cell cycle, indicating that functional EBNA2 is required at different restriction points of the cell cycle. Growth arrest is reversible for G1/G0 cells as indicated by the sequential accumulation and modification of cell cycle regulating proteins. EBV induces the same cell cycle regulating proteins as polyclonal stimuli in primary B-cells. These data suggest that EBV is using a common pathway for B-cell activation bypassing the requirement for antigen, T-cell signals and growth factors.
Assuntos
Antígenos Virais/genética , Linfócitos B/virologia , Ciclo Celular , Transformação Celular Viral , Proteínas de Ligação a DNA/genética , Herpesvirus Humano 4/genética , Antígenos Virais/metabolismo , Ciclo Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr , Estrogênios/farmacologia , Citometria de Fluxo , Fase G1 , Fase G2 , Herpesvirus Humano 4/crescimento & desenvolvimento , Humanos , Hibridização in Situ Fluorescente , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão , Transdução de SinaisRESUMO
APO-1 is a cell surface molecule that induces apoptosis when ligated with the monoclonal antibody anti-APO-1. Expression of APO-1 and response to anti-APO-1 was investigated in a number of Epstein-Barr virus (EBV)-positive and -negative Burkitt lymphoma (BL) cell lines, in EBV-immortalized lymphoblastoid cell lines, and in cells from fresh BL biopsies. APO-1 was not expressed in EBV-negative cell lines and in EBV-positive BL cell lines with a phenotype corresponding to BL tumor biopsy cells (CD10+, CD21-, CD23-, CD30-, CD39-, CDw70-, CD77+). Accordingly, fresh BL cells obtained from three BL biopsies were APO-1 negative. EBV-positive BL cell lines that had acquired a lymphoblastoid phenotype (CD10-, CD21+, CD23+, CD30+, CD39+, CDw70+, CD77-) upon prolonged in vitro cultivation, as well as normal B-lymphoblastoid cell lines, expressed a high density of APO-1. APO-1 may, therefore, be regarded as a B-cell activation marker. APO-1 expression is not the only prerequisite for anti-APO-1-induced apoptosis because 6 of 7 APO-1-expressing EBV-positive BL cell lines were not sensitive to anti-APO-1, whereas all lymphoblastoid cell lines were killed by anti-APO-1. The sensitivity of lymphoblastoid cell lines to anti-APO-1-mediated apoptosis may open a new therapeutic approach for the treatment of EBV-induced lymphoproliferative lesions in immunocompromised individuals, because these are composed of cells with a lymphoblastoid phenotype.