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1.
Int J Tuberc Lung Dis ; 12(2): 146-51, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18230246

RESUMO

SETTING: A tertiary care and research institution in Italy. BACKGROUND: Small DNA fragments from cells dying throughout the body have been detected in urine (transrenal DNA [Tr-DNA]). OBJECTIVE: To test the hypothesis that Mycobacterium tuberculosis Tr-DNA could be detected in the urine of pulmonary tuberculosis (TB) patients. DESIGN: We studied 43 patients with culture-confirmed pulmonary TB with no evidence of extra-pulmonary involvement, 10 patients with pulmonary diseases other than TB and 13 healthy controls. DNA was extracted from urine and analysed by semi-nested polymerase chain reaction (PCR). RESULTS: M. tuberculosis-specific sequences were found in the urine of 34 of 43 (79%) TB patients studied, whereas all controls were negative. The transrenal nature of M. tuberculosis DNA was demonstrated by two lines of evidence: first, separate analysis of supernatants and sediments from eight of the study patients found seven positive supernatants but only two matched positive sediments. Second, M. tuberculosis-specific sequences were amplified by semi-nested PCR with primers designed for short but not large amplicons. CONCLUSION: Small M. tuberculosis DNA fragments may be detected in the urine of a significant proportion of patients with pulmonary TB. If these observations are confirmed by larger studies, Tr-DNA technology could represent a new approach for detecting pulmonary M. tuberculosis infection.


Assuntos
DNA Bacteriano/análise , Mycobacterium tuberculosis/genética , Tuberculose Pulmonar/urina , Urina/microbiologia , Adulto , Humanos , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase , Tuberculose Pulmonar/diagnóstico
2.
Ann N Y Acad Sci ; 945: 239-49, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11708486

RESUMO

It is well documented that plasma contains DNA from tissues throughout the body, including developing fetuses, and tumors. A portion of this DNA crosses the kidney barrier and appears in urine (i.e., transrenal DNA). However, molecular, cellular, and physiological mechanisms of the circulating DNA phenomenon and renal clearance are in an early phase of investigation. Here, we discuss possible forms of circulating DNA, factors affecting representation of different tissues and genomic sequences in plasma DNA, possible mechanisms of renal DNA clearance, and technical problems encountered in DNA isolation from urine. We suggest that apoptotic cells are an important source of DNA in both plasma and urine. Further analysis of the data has led us to propose that a significant portion of circulating DNA can be represented in apoptotic bodies.


Assuntos
Apoptose , DNA/sangue , DNA/urina , DNA/isolamento & purificação , DNA/metabolismo , Genoma , Humanos , Rim/metabolismo
3.
Nucleic Acids Res ; 29(17): E90-0, 2001 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-11522846

RESUMO

The enriched PCR widely used for detection of mutant K-RAS in either tumor tissues or circulating DNA was modified so that abundant wild-type K-RAS alleles are cleaved prior to PCR. We took advantage of an AluI recognition site located immediately upstream of the K-RAS codon 12. The site was reconstituted upon DNA denaturation followed by annealing with a 'stencil', a 16-bp synthetic oligonucleotide complementary to the wild-type sequence. As opposed to normal K-RAS, the mutant allele forms, upon annealing with the stencil, a mismatch at the codon 12 which lies within the AluI enzyme binding site and partially inhibits its activity. The mismatch also lowers the melting temperature of the stencil-mutant K-RAS double helix as compared to stencil-wild-type duplex, so that only the latter is double stranded and selectively digested by AluI at elevated temperatures. The proposed method of stencil-aided mutation analysis (SAMA) based on selective pre-PCR elimination of wild-type sequences can be highly advantageous for detection of mutant K-RAS due to: (i) an enhanced sensitivity because of reduced competition with a great excess of normal K-RAS, and (ii) a decrease in a number of false-positive results from Taq polymerase errors. Application of SAMA for generalized detection of DNA mutations is discussed.


Assuntos
Genes ras/genética , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Sítios de Ligação/genética , Primers do DNA , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Genótipo , Humanos , Mutação , Oligonucleotídeos/genética , Células Tumorais Cultivadas
4.
Drug Chem Toxicol ; 24(3): 287-99, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11452400

RESUMO

Peroxysomicine A1, a novel potential anticancer compound induced cell death in established cell lines and in a primary culture of rat neonatal cardiomyocytes. Non-transformed cells are less sensitive to the compound than transformed cell lines. Fluorescent microscopy of dying cells stained with DNA-specific dyes revealed chromatin condensation and nuclear fragmentation as well as membrane blebbing characteristic of apoptosis. Flow cytometry of cells treated with peroxysomicine A1, demonstrated appearance of cells containing less than 2C DNA, that indicated degradation of nuclear DNA, another hallmark of apoptotic cell death. Z-VAD, a nonspecific caspase inhibitor, prevented DNA fragmentation but not cell death registered by permeabilization of cell outer membrane. Peroxysomicine A1 also inhibited proliferation of various cell lines. Flow cytometry analysis showed significant accumulation of dividing cells in G2/M phases of cell cycle indicating, most likely delay in G2. These results provide initial insight into the mechanisms of action of peroxysomicine A1 and suggest that peroxysomicine A1 is a potent inhibitor of cell proliferation and inducer of apoptosis and may be a useful antineoplastic chemotherapeutic agent.


Assuntos
Antracenos/toxicidade , Antineoplásicos Fitogênicos/toxicidade , Fragmentação do DNA/efeitos dos fármacos , Clorometilcetonas de Aminoácidos/farmacologia , Inibidores de Caspase , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , Relação Dose-Resposta a Droga , Citometria de Fluxo , Células HeLa/efeitos dos fármacos , Células HeLa/patologia , Humanos , Células Jurkat/efeitos dos fármacos , Células Jurkat/patologia
5.
Ann N Y Acad Sci ; 946: 160-8, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11762984

RESUMO

Cardiomyopathies are observed with increasing frequency in association with AIDS and HIV infection. Although indirect evidence exists suggesting an association between apoptosis regulation and HIV infection, there is yet no direct evidence that HIV-associated cardiomyopathies involve increased level of apoptosis in the heart. However, since it is now known that apoptosis plays a significant role in heart injury associated with other conditions such as ischemia/reperfusion and heart failure, there is a possibility that dysregulation of apoptosis plays a similarly important role in HIV-associate cardiomyopathies. Here we will briefly review the evidence that apoptotic death of cardiomyocytes occurs and what novel therapeutic strategies may be suggested.


Assuntos
Apoptose , Cardiomiopatias/patologia , Miocárdio/patologia , Cardiomiopatias/tratamento farmacológico , Morte Celular , Humanos , Miocárdio/metabolismo , Transdução de Sinais
6.
Neurol Clin ; 16(3): 735-45, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9666047

RESUMO

The phenomenon of aging is distinct from processes associated with advanced age known to increase risk of diseases, such as cancer. Furthermore, the process of aging is not necessarily related to phenomena such as in vitro replicative senescence; however, any unifying hypothesis of aging must account for all age-dependent phenomena, including senescence. It is proposed that apoptosis forms the ultimate protective process for preservation of phenotypic fidelity in multicellular organisms since it is the process by which the organism detects damage and replaces the defective cell. Time-dependent degeneration of apoptosis control is the rate-limiting step in the process of aging.


Assuntos
Envelhecimento/fisiologia , Apoptose/fisiologia , Idoso , Idoso de 80 Anos ou mais , Humanos
7.
Am J Pathol ; 151(5): 1257-63, 1997 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9358751

RESUMO

Involvement of ceramide signaling in the initiation of apoptosis induction in myocardial cells by in vitro and in vivo ischemia and reperfusion was analyzed. Synthetic cell permeable C2-ceramide induced apoptotic death of rat neonatal cardiomyocytes in vitro. In vitro ischemia (oxygen/serum/glucose deprivation) led to a progressive accumulation of ceramide in cardiomyocytes. After 16 hours of simulated in vitro reperfusion (readdition of oxygen, serum and glucose), the level of ceramide in surviving cells was found to have returned to baseline, whereas, levels in nonadherent dead cells remained high. In the rat heart left coronary artery occlusion model, ischemia with the subsequent reperfusion, but not ischemia alone, induced apoptosis in myocardial cells as demonstrated by DNA electrophoresis and measurement of soluble chromatin degradation products. The content of ceramide in ischemic area was elevated to 155% baseline levels at 30 minutes, and to 330% after 210 minutes of ischemia. Ischemia (30 minutes) followed by reperfusion (180 minutes) increased the ceramide level to 250% in the ischemic area. The combination of results obtained in both in vitro and animal models demonstrate for the first time that ceramide signaling can be involved in ischemia/reperfusion death of myocardial cells.


Assuntos
Apoptose/fisiologia , Ceramidas/fisiologia , Isquemia Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Animais , Células Cultivadas , Ceramidas/metabolismo , Isquemia Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Ratos , Ratos Sprague-Dawley , Esfingosina/análogos & derivados , Esfingosina/farmacologia
8.
J Biol Chem ; 272(26): 16351-7, 1997 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-9195941

RESUMO

Interferon (IFN)-gamma increases the sensitivity of tumor cell lines, many of which are p53 mutants, to tumor necrosis factor-alpha-mediated and anti-Fas antibody-mediated cell death. To better understand the mechanism of IFN-gamma action in modulating the cell death response independently of p53 function, we analyzed the death of the human colon adenocarcinoma cell line, HT-29, following treatment with IFN-gamma and various cytotoxic agents. Here we show that IFN-gamma modulates cell death by sensitizing the cells to killing by numerous pro-apoptotic stimuli but not pro-necrotic stimuli. Furthermore, we show that select genes from several important apoptosis-related gene families are induced by IFN-gamma, including the apoptosis-signaling receptors CD95 (Fas/APO-1) and TNFR 1 and interleukin-1beta-converting enzyme (Ice) family members Ice, CPP32 (Yama, apopain), ICErel-II (TX, Ich-2), Mch-3 (ICE-LAP3, CMH-1), Mch-4, and Mch-5 (MACH, FLICE). Of the bcl-2 family members, IFN-gamma directly induced bak but notably not bax, which is activated by p53. The IFN-responsive transcriptional activator interferon regulatory factor-1 was also strongly induced and translocated into the nucleus following IFN-gamma treatment. We propose that IFN-gamma modulates a p53-independent apoptotic pathway by both directly and indirectly inducing select apoptosis-related genes.


Assuntos
Apoptose/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon gama/farmacologia , Proteína Supressora de Tumor p53/fisiologia , Cicloeximida/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HT29 , Humanos , Fator Regulador 1 de Interferon , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/análise
9.
Cancer Res ; 57(9): 1758-68, 1997 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-9135020

RESUMO

Of six prostatic carcinoma cell lines examined (ALVA31, DU145, JCA1, LNCaP, ND1, and PC3) by flow cytometric analysis, all were found to be positive for Fas antigen. Furthermore, of the prostate tissue specimens studied (six cases), all revealed Fas expression in benign and malignant epithelial cells. The agonistic anti-Fas monoclonal antibody (IPO-4) induced apoptosis in only two of six cell lines investigated, PC3 and ALVA31. PCR analysis indicated that all cell lines expressed normal transmembrane and death domains of Fas antigen. Using Western blot analysis, we found abundant expression of p53 in the cytoplasm of two Fas-resistant cell lines, DU145 and ND1, and did not find p53 in two Fas-sensitive cell lines, PC3 and ALVA31. Western blot and PCR analysis did not show consistent differences between cell lines examined in the expression of Bcl-2, Bcl-X(L), Bcl-X(S), and Bak. In contrast, Bax protein was not detected in two Fas-resistant cell lines, DU145 and ND1. We also showed that three Fas-resistant cell lines, DU145, ND1, and JCA1, expressed CD40, whereas the two Fas-sensitive cell lines, PC3 and ALVA31, were CD40 negative. Fas-sensitive cell lines were transfected with the cDNA encoding CD40, and the CD40-positive transfectant became more resistant to growth inhibition mediated by treatment with TNF-alpha and anti-Fas monoclonal antibody. Treatment with cycloheximide converted the phenotype of resistant cell lines from Fas resistant to Fas sensitive. Moreover, anti-Fas treatment of both resistant and sensitive cell lines induced rapid tyrosine phosphorylation or dephosphorylation of multiple proteins. These results suggest that the apoptotic machinery involved in DNA fragmentation is already in place in Fas-resistant cell lines, and thus, Fas-mediated apoptosis could be a target for therapeutic intervention.


Assuntos
Apoptose , Neoplasias da Próstata/patologia , Receptor fas/fisiologia , Reações Antígeno-Anticorpo , Antígenos CD40/genética , Antígenos CD40/metabolismo , Divisão Celular , Cicloeximida/farmacologia , Fragmentação do DNA , Citometria de Fluxo , Humanos , Masculino , Fosfotirosina/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2 , Proteína bcl-X
11.
Proc Natl Acad Sci U S A ; 94(25): 13636-41, 1997 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-9391078

RESUMO

Quiescent mouse embryonic C3H/10T1/2 cells are more resistant to different proapoptotic stimuli than are these cells in the exponential phase of growth. However, the exponentially growing 10T1/2 cells are resistant to inhibitors of RNA or protein synthesis, whereas quiescent cells die upon these treatments. Conditioned medium from quiescent 10T1/2 cells possesses anti-apoptotic activity, suggesting the presence of protein(s) that function as an inhibitor of the apoptotic program. Using differential display technique, we identified and cloned a cDNA designated sarp1 (secreted apoptosis-related protein) that is expressed in quiescent but not in exponentially growing 10T1/2 cells. Hybridization studies with sarp1 revealed two additional family members. Cloning and sequencing of sarp2 and sarp3 revealed 38% and 40% sequence identity to sarp1, respectively. Human breast adenocarcinoma MCF7 cells stably transfected with sarp1 or infected with SARP1-expressing adenovirus became more resistant, whereas cells transfected with sarp2 displayed increased sensitivity to different proapoptotic stimuli. Expression of sarp family members is tissue specific. sarp mRNAs encode secreted proteins that possess a cysteine-rich domain (CRD) homologous to the CRD of frizzled proteins but lack putative membrane-spanning segments. Expression of SARPs modifies the intracellular levels of beta-catenin, suggesting that SARPs interfere with the Wnt-frizzled proteins signaling pathway.


Assuntos
Apoptose/fisiologia , Transativadores , Sequência de Aminoácidos , Animais , Apoptose/genética , Divisão Celular/genética , Linhagem Celular , Clonagem Molecular , Meios de Cultivo Condicionados , Proteínas do Citoesqueleto/metabolismo , Receptores Frizzled , Expressão Gênica , Humanos , Interfase/genética , Camundongos , Dados de Sequência Molecular , Família Multigênica , Receptores Acoplados a Proteínas G , Receptores de Neurotransmissores/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Distribuição Tecidual , Transfecção , beta Catenina
12.
Cell Death Differ ; 4(7): 608-16, 1997 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14555974

RESUMO

Apoptosis, or programmed cell death, is an active metabolic response to physiological signals or exposure to cytotoxic agents. Recent evidence has shown that the cell death response can be modified by agents presumed to be unrelated to the initial signal, but capable of interfering with the molecular mechanisms of the apoptotic pathway progression. Here we show the results of investigations on the use of a phospholipid-based pharmaceutical preparation for suppression of myocardial damage. First, we show that serum or serum/glucose deprivation, in vitro ischemia with subsequent simulated reperfusion, inhibition of protein synthesis, and treatment with ceramide, staurosporine, adriamycin, cis-platinum and menadione induce apoptotic death in a primary culture of rat neonatal cardiomyocytes. Then we demonstrate that a mixture of specific phospholipids, which has been originally purified from soy flour on the basis of its anti-apoptotic activity, prevents cardiomyocyte death induced by serum or serum/glucose deprivation, by ischemia with subsequent simulated reperfusion, and by ceramide, but not by other cytotoxic treatments. This suggests that ceramide, a lipid secondary messenger which triggers apoptosis induced by some cytotoxic agents, may be involved in the process of signaling ischemia/reperfusion induced apoptotic death of cardiomyocytes. These results further demonstrate that an active pharmaceutical preparation for the suppression of cardiomyocyte death can be formulated based upon a novel strategy of apoptosis modification.

13.
Cell Death Differ ; 2(4): 235-41, 1995 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17180028

RESUMO

Despite the clinical importance of cardiomyocyte death following ischemia and reperfusion, little is known about the nature of the process. In primary rat neonatal cardiomyocyte cultures, cell death was induced by ischemia (deprivation of oxygen, serum and glucose) and reperfusion. We report here that ischemia induced primarily necrosis, whereas subsequent reperfusion induced apoptosis. Apoptosis of rat neonatal cardiomyocytes could not be prevented by protein synthesis inhibitors, suggesting that molecular components of the apoptotic pathway pre-exist in these cells. IGFs and calpain inhibitors had no effect on necrotic death during ischemia, but they significantly reduced apoptotic death during reperfusion. These results support the concept that inhibition of post-ischemic apoptotic death in the myocardium may provide a valuable new therapeutic strategy for the treatment of acute myocardial ischemia.

14.
Int J Radiat Biol ; 68(3): 277-80, 1995 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-7561387

RESUMO

Elevated levels of hsp70 mRNA in cells of lung, spleen and small intestine have been observed upon chronic in vivo gamma-irradiation of mice at a dose-rate of 3 cGy/day for 30 days. Changes in hsp70 gene expression in lung cells were analysed in more detail. Chronic irradiation with an accumulated dose of 48 cGy increased the amount of hsp70 mRNA at dose rates of 3 and 6 cGy/day and had no effect on hsp70 mRNA level at dose-rates of 1.2 and 12 cGy/day. An increased level of hsp70 mRNA was observed by day 7 of irradiation at a dose-rate of 3 cGy/day, was high by day 30 but by the end of the second month the amount of mRNA dropped to control values. Western blotting revealed a higher level of hsp70 protein as well, although the amount of protein increased to a lesser degree than that of mRNA.


Assuntos
Proteínas de Choque Térmico HSP70/genética , Pulmão/efeitos da radiação , Animais , Relação Dose-Resposta à Radiação , Raios gama , Expressão Gênica/efeitos da radiação , Proteínas de Choque Térmico HSP70/metabolismo , Intestino Delgado/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Baço/efeitos da radiação , Fatores de Tempo
15.
Nature ; 374(6524): 736-9, 1995 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-7715731

RESUMO

Members of the Bcl-2 family of proteins are characterized by their ability to modulate cell death. Bcl-2 and some of its homologues inhibit apoptosis, whereas other family members, such as Bax, will accelerate apoptosis under certain conditions. Here we describe the identification and characterization of a complementary DNA that encodes a previously unknown Bcl-2 homologue designated Bak. Like Bax, the bak gene product primarily enhances apoptotic cell death following an appropriate stimulus. Unlike Bax, however, Bak can inhibit cell death in an Epstein-Barr-virus-transformed cell line. The widespread tissue distribution of Bak messenger RNA, including those containing long-lived, terminally differentiated cell types, suggests that cell-death-inducing activity is broadly distributed, and that tissue-specific modulation of apoptosis is controlled primarily by regulation of molecules that inhibit apoptosis.


Assuntos
Apoptose/fisiologia , Proteínas de Membrana/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Linhagem Celular Transformada , DNA Complementar , Humanos , Células Híbridas , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/análise , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Proteína Killer-Antagonista Homóloga a bcl-2
16.
Eur J Biochem ; 215(3): 893-901, 1993 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-8354294

RESUMO

Three nuclease activities have been found and characterized in rat thymocyte nuclear extracts. A Mn(2+)-dependent nuclease is loosely bound to nuclear components and can be extracted with 0.35 M NaCl. The enzyme is activated by Mn2+ but not by Mg2+, Ca2+, or both. Its molecular mass is 36-40 kDa when measured by gel filtration and 37 kDa by SDS/PAGE. An acidic nuclease is independent of divalent ions, produces DNA strand breaks with 5'-OH ends, its molecular mass is about 37 kDa. Two fractions of Ca2+/Mn(2+)-dependent nuclease, differing in binding to CM-Sepharose but identical in other respects, are active in the presence of Mn2+ but can be additionally activated by Ca2+. They are inactive in the presence of Mg2+ or Ca2+ but cleave DNA in Ca2+/Mg(2+)-containing medium. The molecular mass of the enzyme is 22 kDa as determined by both gel filtration and electrophoresis. The dependence of nuclease activities on pH, ions, and sulfhydryl reagents is described. Cycloheximide injection to both control and irradiated animals strongly inhibits the activities of Ca2+/Mn(2+)-dependent nuclease from thymocyte nuclei separated by chromatography on CM-Sepharose and does not change the activities of Mn(2+)-dependent and acidic nucleases. Nuclease activity in thymocyte nuclei from irradiated rats is increased in Ca2+/Mg(2+)-containing and Ca2+/Mn(2+)-containing media whereas there is no change in the activity of acidic nuclease. Ca2+/Mn(2+)-dependent nuclease is extracted from thymocyte nuclei of irradiated rats with 0.35 M NaCl but from control nuclei only with 0.5 M NaCl. Possible reasons of labilization of Ca2+/Mn(2+)-dependent-nuclease binding to the nuclear structures in dying thymocytes are discussed.


Assuntos
Apoptose , Núcleo Celular/enzimologia , Desoxirribonucleases/metabolismo , Timo/enzimologia , Animais , Apoptose/efeitos da radiação , Cálcio/metabolismo , Núcleo Celular/efeitos da radiação , Células Cultivadas , Cromatografia em Gel , Cromatografia por Troca Iônica , Cicloeximida/farmacologia , DNA/metabolismo , Desoxirribonucleases/antagonistas & inibidores , Magnésio/metabolismo , Masculino , Manganês/metabolismo , Ratos , Ratos Wistar , Timo/citologia
17.
Biochem Biophys Res Commun ; 191(3): 1309-18, 1993 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-8466507

RESUMO

The mode and the kinetics of differentiation and death of murine N1E115 neuroblastoma cells induced by dimethyl sulfoxide and other nonspecific factors in vitro were investigated. After morphological differentiation neuroblastoma cells die by apoptosis which is indicated by characteristic morphological features and by internucleosomal DNA fragmentation. Durations of both differentiation and apoptosis are dependent on the nature of stimuli used. Protein synthesis inhibitor cycloheximide does not prevent differentiation and apoptosis of neuroblastoma cells induced by dimethyl sulfoxide and even accelerates both processes. The relationship between cell death and differentiation is discussed.


Assuntos
Apoptose , Diferenciação Celular , Neurônios/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Dano ao DNA , Dimetil Sulfóxido/farmacologia , Técnicas In Vitro , Camundongos , Neuroblastoma/patologia , Células Tumorais Cultivadas
18.
Cytometry ; 14(6): 603-9, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-8404366

RESUMO

Flow cytometry is more and more widely used for investigations of cell death, predominantly in the study of DNA degradation in cells dying by apoptosis. There are different interpretations of changes observed in DNA histograms of these cells. We describe an approach based on extraction of chromatin degradation products from fixed cells and subsequent staining with DNA specific dyes. Apoptotic cells containing fragmented DNA are observed in < 2C DNA region of DNA histograms. DNA histograms of irradiated thymocytes dying in vitro and stained without extraction of fragmented DNA do not differ from control. Under the same staining conditions DNA histograms of lymphocytes dying in thymus of irradiated animals reveal fluorescent material in < 2C DNA region, most likely due to formation of apoptotic bodies (cell fragments, some of them contain fragments of nuclei). Similar changes are observed in thymocytes dying upon glucocorticoid treatment. Our present results and other data indicate that reduced amount of DNA in dying cells is the main reason for changes of DNA histograms. Examples of application of the method described for the investigations of cell death modifiers are presented.


Assuntos
Morte Celular/fisiologia , Citometria de Fluxo/métodos , Timo/citologia , Animais , Morte Celular/efeitos da radiação , Permeabilidade da Membrana Celular/fisiologia , Núcleo Celular/química , Núcleo Celular/ultraestrutura , Células Cultivadas , Cromatina/química , Cromatina/ultraestrutura , DNA/análise , Ácido Edético/farmacologia , Glucocorticoides/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Wistar , Timo/fisiologia , Timo/efeitos da radiação
19.
J Cell Physiol ; 148(2): 267-73, 1991 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-1880154

RESUMO

The mode and the kinetics of the death of T-thymoma cells upon dexamethasone treatment and gamma-irradiation (10Gy) have been studied using flow cytometry and biochemical analysis. It has been shown that the hormone and gamma-irradiation induce cell death by apoptosis. In both cases the cells are initially blocked in G2/M and die only after overcoming the blockage and cytokinesis. A short exposure to dexamethasone results in a cytostatic effect, whereas a cytotoxic effect is absent. Reducing serum concentration to 2% causes more rapid death both following gamma-irradiation and dexamethasone. These results are discussed in relation to cell death and proliferation.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Dexametasona/farmacologia , Timoma/patologia , Animais , Sangue , Sobrevivência Celular/efeitos da radiação , Meios de Cultura , DNA de Neoplasias/metabolismo , Fase G2/efeitos dos fármacos , Fase G2/efeitos da radiação , Raios gama , Mitose/efeitos dos fármacos , Mitose/efeitos da radiação , Células Tumorais Cultivadas
20.
J Immunol Methods ; 134(2): 201-5, 1990 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-2254666

RESUMO

A new assay for the evaluation of cell viability is described. It is based on the staining of dead cells and subsequently the whole cell population with ethidium bromide (EtBr). The method makes it possible to measure cytotoxic and cytostatic effects simultaneously and cell cultures grown both in suspension as well as by adherence may be assessed. The advantages and disadvantages of this assay are discussed.


Assuntos
Sobrevivência Celular/fisiologia , Técnicas Citológicas , Animais , Automação , Permeabilidade da Membrana Celular/efeitos dos fármacos , Digitonina/farmacologia , Etídio , Masculino , Micromanipulação , Ratos , Ratos Endogâmicos , Espectrometria de Fluorescência , Linfócitos T/efeitos dos fármacos , Linfócitos T/efeitos da radiação , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia
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