RESUMO
The primary objective of this study was to determine the sequence of biochemical signaling events that occur after modulation of the cellular redox state in the B cell lymphoma line, PW, with emphasis on the role of mitochondrial signaling. L-Buthionine sulphoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), was used to modulate the cellular redox status. The sequence and role of mitochondrial events and downstream apoptotic signals and mediators was studied. After BSO treatment, there was an early decline in cellular glutathione (GSH), followed by an increase in reactive oxygen species (ROS) production, which induced a variety of apoptotic signals (detectable at different time points) in the absence of any external apoptotic stimuli. The sequence of biochemical events accompanying apoptosis included a 95% decrease in total GSH and a partial (25%) preservation of mitochondrial GSH, without a significant increase in ROS production at 24h. Early activation and nuclear translocation of the nuclear factor kappa B subunit Rel A was observed at approximately 3h after BSO treatment. Cytochrome c release into the cytosol was also seen after 24h of BSO treatment. p53 protein expression was unchanged after redox modulation for up to 72 h, and p21waf1 independent loss of cellular proliferation was observed. Surprisingly, a truncated form of p53 was expressed in a time-dependent manner, beginning at 24h after BSO incubation. Irreversible commitment to apoptosis occurred between 48 and 72 h after BSO treatment when mitochondrial GSH was depleted, and there was an increase in ROS production. Procaspase 3 protein levels showed a time-dependent reduction following incubation with BSO, notably after 48 h, that corresponded with increasing ROS levels. At 96 h, caspase 3 cleavage products were detectable. The pan-caspase inhibitor zVADfmk, partially blocked the induction of apoptosis at 48 h, and was ineffective after 72 h. PW cells could be rescued from apoptosis by removing them from BSO after up to 48, but not 72 h incubation with BSO. Mitochondrial transmembrane potential (DeltaPsi(m)) remained intact in most of the cells during the 72 h observation period, indicating that DeltaPsi(m) dissipation is not an early signal for the induction of redox dependent apoptosis in PW cells. These data suggest that a decrease in GSH alone can act as a potent early activator of apoptotic signaling. Increased ROS production following mitochondrial GSH depletion, represents a crucial event, which irreversibly commits PW cells to apoptosis.
Assuntos
Apoptose/fisiologia , Butionina Sulfoximina/farmacologia , Glutationa/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Citosol/metabolismo , Ditiotreitol/farmacologia , Glutationa/deficiência , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Mitocôndrias/fisiologia , NF-kappa B/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologia , Fator de Transcrição RelA , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/biossínteseRESUMO
Concentrations of rotenone (ROT) that block electron flow through mitochondrial complex I (100 nM) did not significantly alter either cell viability or the growth of PW cells. However, 10- to 50-fold higher concentrations (1-5 microM) were found to induce a dose-dependent cell cycle arrest predominantly at the G2/M stage of the cycle and apoptosis. Apoptosis was dependent on the cell cycle arrest, since apoptosis but not the G2/M arrest was prevented with the broad spectrum caspase inhibitor zVADfmk. Biochemical features of apoptosis included mitochondrial cytochrome c release, reactive oxygen species generation, and the activation of procaspase 3. Thus, ROT inhibition of mitochondrial electron transport may be insufficient to induce apoptosis in PW cells. Instead, apoptosis in these cells occurs as a consequence of disruption of the cell cycle and is only indirectly dependent upon mitochondrial electron transport.
Assuntos
Apoptose/efeitos dos fármacos , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Rotenona/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Linfócitos B/metabolismo , Inibidores de Caspase , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Grupo dos Citocromos c/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Humanos , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais Cultivadas , Desacopladores/farmacologiaRESUMO
Neonatal human retina cells transformed by the SV40 tumor antigens were shown to leave the cell cycle and differentiate following treatment with agents that raise intracellular levels of cyclic AMP. This was true for both precrisis and immortal cell lines. However, with time, some of the differentiated retinoblasts withdrew neurites and returned to the cell cycle. Attempts to inhibit this process by developing cell lines transformed using SV tsA 58 with a temperature-sensitive phenotype for growth did not enhance but inhibited retinoblast-differentiating capacity. Growth restriction at the nonpermissive temperature was found to activate a senescence pathway. We propose that at the nonpermissive temperature, stable SV40 T-ag-p53 complexes fragment releasing p53, which transactivates p21waf1/cip1/sdi1 with the subsequent accumulation of p21 culminating in growth inhibition and senescence.
Assuntos
Transformação Celular Viral/fisiologia , Senescência Celular/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2 , Retina/citologia , Vírus 40 dos Símios/fisiologia , Antígenos Transformantes de Poliomavirus/fisiologia , Apoptose , Ciclo Celular , Morte Celular , Diferenciação Celular , Linhagem Celular Transformada , AMP Cíclico/fisiologia , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/análise , Ciclinas/genética , Humanos , Recém-Nascido , Proteínas Proto-Oncogênicas/análise , RNA Mensageiro/análise , Temperatura , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2RESUMO
The life span of normal human cells in culture is extended by two to four total life spans following retrovirus-mediated transfer of the adenovirus type 12 E1B 54,000-molecular-weight protein (54K protein). This extension of the in vitro growth potential was accomplished without any of the obvious changes in morphology or growth properties that are usually associated with viral transformation. These 54K+ cells escape the normal senescence checkpoint (M1) and show a very extended secondary growth phase. The 54K+ human cells eventually enter crisis (M2), which does not appear to be due to either telomere attrition or the activation of the senescence-associated proteins p21SdilCipIWaf1 and p16INK4A. Even in the absence of telomerase activity, high-molecular-weight heterogeneous telomeres are produced and maintained in both 54K+ adult dermal fibroblasts and embryo kidney cells, indicating that the 54K protein may interfere with the normal metabolism of telomeric structures during cell division. These findings are discussed with reference to the known ability of the 54K protein to influence p53 function.
Assuntos
Proteínas E1B de Adenovirus/fisiologia , Adenovírus Humanos/fisiologia , Sobrevivência Celular , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Adenovírus Humanos/genética , Adulto , Animais , Proteínas de Transporte/biossíntese , Divisão Celular , Células Cultivadas , Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Humanos , Masculino , Mamíferos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Telômero , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/metabolismoRESUMO
The cellular protein MDM2 can bind to the tumor suppressor gene product p53 and abrogate its transcriptional activity. In addition, p53 can regulate expression of the mdm2 gene. We and others have previously shown that p53 is present at high levels in adenovirus-transformed cells which express the larger E1B protein. In view of these observations the expression of MDM2 in a panel of adenovirus transformed human cell lines has been examined. Two major species (98K and 80K) were detected, together with a number of minor species of higher and lower molecular weight. While there was little variation in levels of 98K protein between cell lines, appreciable differences in the expression of the 80K component were apparent. There was no correlation between MDM2 and p53 expression in any of the adenovirus transformants, nor with the viral proteins expressed. The pattern and level of MDM2 detected was similar to that seen in human tumor cell lines and in human fetal tissue. Northern blot analysis suggested that MDM2 expression was regulated at the transcriptional level. Stable interactions were observed between p53 and MDM2 in the adenovirus-transformed cell lines and in Ad5 E1 HEK 293 cells a ternary complex of p53, MDM2, and the Ad5 E1B 58K protein was demonstrated. In view of the lack of correlation between the level of p53 and MDM2 in adenovirus E1-transformed cells, the capacity of p53 to cause transcriptional activation was assessed using transfected CAT constructs linked to p53 responsive elements. p53 transcriptional activity was similar in all of the cell lines examined and did not correlate with protein expression. It is concluded, on the basis of all of these data, that the high concentrations of p53 found in adenovirus transformants are not transcriptionally active and have no influence on MDM2 expression. However, when expression of p53 was increased following infection with mutant adenoviruses, which do not express the larger E1B proteins, there was an appreciable increase in p53 transcriptional activity and in the levels of all of the MDM2 components.
Assuntos
Adenovírus Humanos/genética , Transformação Celular Viral , Proteínas de Neoplasias/biossíntese , Proteínas Nucleares , Proteínas Proto-Oncogênicas/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Proteínas E1B de Adenovirus/biossíntese , Proteínas E1B de Adenovirus/metabolismo , Fracionamento Celular , Linhagem Celular Transformada , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Citoplasma/metabolismo , Feto , Humanos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , RNA Mensageiro/biossíntese , Transcrição Gênica , Ativação Transcricional , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Regulação para CimaRESUMO
The expression of p53 in a large panel of adenovirus (Ad) 2/5- and 12-transformed human, rat, and mouse cells has been examined. In all cases, in the absence of the larger Ad E1B protein, the level of p53 is very low. In human and rat cells when the Ad 12 E1B 54K polypeptide is expressed, p53 is much more abundant, although this is not the case in Ad 12 E1-transformed mouse cells. We conclude that expression of p53 is determined by virus serotype, host cell type, and viral proteins expressed. p53 in Ad 12 E1-transformed human cells is wild type but has an extended half-life. Stabilization is not through protein-protein interaction with the Ad E1B protein. The level of expression of c-Myc is also elevated in Ad-transformed human cells but this does not correlate with the presence of the E1B protein or with p53. However, Northern blot analysis indicates a direct correlation between mRNA and protein levels. We conclude that c-Myc is regulated at the transcriptional level, whereas p53 is regulated at the post-translational level in adenovirus transformants.