RESUMEN
In the presence of sustained DNA damage occurring in S-phase or G2, normal cells arrest before mitosis and eventually become senescent. The checkpoint kinases Chk1/Chk2 and the CDK inhibitor p21 are known to have important complementary roles in this process, in G2 arrest and cell cycle exit, respectively. However, additional checkpoint roles have been reported for these regulators and it is not clear to what extent their functions are redundant. Here we compared the respective roles of Chk1, Chk2 and p21 in DNA damage-induced G2 arrest in normal human fibroblasts, normal epithelial cells and frequently used p53 proficient cancer cells. We show that in normal cells, Chk1, but not Chk2, is involved in G2 arrest whereas neither are essential. In contrast, p21 is required. However, Chk1, but not Chk2, becomes necessary for arrest in U2OS osteosarcoma cells. We find that their ATM/p53/p21 response in G2 phase is defective, like in other cancer cells with wild-type p53, and conclude that cross-talk between the Chk1 and p21 pathways allows them to switch dependency for G2 arrest onto Chk1. Using the specific ATM inhibitor KU-55933 we confirm the essential role of ATM in the induction of p21 for G2 arrest of normal cells. Efficient p21 induction is required for nuclear sequestration of inactive cyclin B1-Cdk1 complexes preceding irreversible cell cycle exit in G2. Our results demonstrate that p21 is able to fulfill the Chk1 functions in G2 arrest under continuous genotoxic stress, which has important implications for cancer chemotherapy.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Puntos de Control de la Fase G2 del Ciclo Celular/fisiología , Proteínas Quinasas/metabolismo , Transducción de Señal/fisiología , Antineoplásicos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada , Bleomicina/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Células Cultivadas , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasa de Punto de Control 2 , Ciclina B1/genética , Ciclina B1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dicetopiperazinas , Femenino , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Células HCT116 , Células HeLa , Humanos , Immunoblotting , Masculino , Morfolinas/farmacología , Fosforilación , Piperazinas/farmacología , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Pironas/farmacología , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
P27kip is a key inhibitory protein of the cell-cycle progression, which is rapidly downregulated in early G1 phase by a post-translational mechanism involving the proteosomal degradation. In this study, using a wounding model that induces cell-cycle entry of human dermal fibroblasts, we demonstrate that p27mRNA is downregulated when cells progress into the G1 phase, and then it returns to its basal level when cells approach the S phase. By using a quantitative polymerase chain reaction screening we identified inhibitors of differentiation (Id3), a bHLH transcriptional repressor, as a candidate mediator accounting for p27 mRNA decrease. Id3 silencing, using an small interfering RNA approach, reversed the injury mediated p27 downregulation demonstrating that Id3 is involved in the transcriptional repression of p27. Reporter gene experiments and a chromatin immunoprecipitation assay showed that Id3 likely exerts its repressive action through ELK1 inhibition. By inhibiting early p27 downregulation, Id3 depletion blocked (i) the G1-phase progression as assessed by the inhibition of pRb phosphorylation and p130 degradation and (ii) the G1/S transition as observed by the inhibition of cyclin A induction, demonstrating that p27 mRNA decrease is required for cell proliferation. Apart from its effect on the early p27 diminution, Id3 appears also involved in the control of the steady-state level of p27 at the G1/S boundary. In conclusion, this study identifies a novel mechanism of p27 regulation which besides p27 protein degradation also implicates a transcriptional mechanism mediated by Id3.
Asunto(s)
Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Fase G1/fisiología , Regulación de la Expresión Génica , Proteínas Inhibidoras de la Diferenciación/fisiología , Proteínas de Neoplasias/fisiología , ARN Mensajero/metabolismo , Fase S/fisiología , Animales , Células Cultivadas , Inmunoprecipitación de Cromatina , Dermis/lesiones , Dermis/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Immunoblotting , Luciferasas , Ratones , Células 3T3 NIH , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Transfección , Cicatrización de Heridas/fisiologíaRESUMEN
In most tumor cells a chromosomal instability leads to an abnormal chromosome number (aneuploidy). The mitotic checkpoint is essential for ensuring accurate chromosome segregation by allowing mitotic delay in response to a spindle defect. This checkpoint delays the onset of anaphase until all the chromosomes are correctly aligned on the mitotic spindle. When unattached kinetochores are present, the metaphase/anaphase transition is not allowed and the time available for chromosome-microtubule capture increases. Genes required for this delay were first identified in Saccharomyces cerevisiae (the MAD, BUB and MPS1 genes) and subsequently, homologs have been identified in higher eucaryotes showing that the spindle checkpoint pathway is highly conserved. The checkpoint functions by preventing an ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC) from ubiquitinylating proteins whose destruction is required for anaphase onset.
Asunto(s)
División Celular , Cromosomas/fisiología , Aneuploidia , Animales , Segregación Cromosómica , Humanos , Cinetocoros , Mitosis , Neoplasias/genética , Saccharomyces cerevisiae/genética , Huso AcromáticoRESUMEN
BACKGROUND/AIMS: Limited information is available on the expression and role of C/EBP factors in human liver and hepatocytes. We investigated the expression and DNA-binding activity of C/EBPalpha and C/EBPbeta in human liver needle biopsies, surgical lobectomies and differentiated cultured hepatocytes derived from lobectomies. METHODS: RNA and protein extracts were analyzed by RNAse protection, immunoblot and gel shift assays. RESULTS: C/EBP mRNAs, isoforms and DNA-binding activities were low/undetectable in lobectomies. In contrast, several C/EBPalpha (47, 45, 35 and 33 kDa) and C/EBPbeta isoforms (47, 43, 40, 35 and 21 kDa) were observed in needle biopsies. In cultured hepatocytes, the C/EBP expression pattern dramatically changed with time. C/EBPalpha mRNA and the 45 kDa isoform increased in parallel, reaching a maximum after 3-4 weeks coincident with weak DNA-binding activity. C/EBPbeta mRNA and isoform expression increased rapidly reaching a plateau within 1-2 weeks; all C/EBPbeta isoforms were phosphorylated. C/EBPbeta exhibited greater DNA-binding activity than C/EBPalpha, and this activity paralleled C/EBPbeta isoform expression. CONCLUSIONS: C/EBP isoforms exhibit markedly different expression patterns in lobectomies, needle biopsies and cultured hepatocytes. Stress stimuli during and/or after surgery for lobectomy resections may account for this difference. The pattern of C/EBP isoform expression in long-term highly differentiated cultured hepatocytes is close to that observed in needle biopsies.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , ADN/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Adulto , Anciano , Animales , Diferenciación Celular , Células Cultivadas , Femenino , Expresión Génica , Hepatocitos/citología , Humanos , Técnicas In Vitro , Macaca , Masculino , Persona de Mediana Edad , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Irreversible G(1) arrest in senescent human fibroblasts is mediated by two inhibitors of cyclin-dependent kinases (Cdks), p21(Cip1/SDI1/WAF1) and p16(Ink4A). To determine the physiological and molecular events that specifically require p21, we studied senescence in human diploid fibroblasts expressing the human papillomavirus type 16 E6 oncogene, which confers low p21 levels via enhanced p53 degradation. We show that in late-passage E6 cells, high Cdk activity drives the cell cycle, but population expansion is slowed down by crisis-like events, probably owing to defective cell cycle checkpoints. At the end of lifespan, terminal-passage E6 cells exhibited several aspects of the senescent phenotype and accumulated unphosphorylated pRb and p16. However, both replication and cyclin-Cdk2 kinase activity were still not blocked, demonstrating that phenotypic and replicative senescence are uncoupled in the absence of normal p21 levels. At this stage, E6 cells also failed to upregulate p27 and inactivate cyclin-Cdk complexes in response to serum deprivation. Eventually, irreversible G(1) arrest occurred coincident with inactivation of cyclin E-Cdk2 owing to association with p21. Similarly, when p21(-/-) mouse embryo fibroblasts reached the end of their lifespan, they had the appearance of senescent cells yet, in contrast to their wild-type counterparts, they were deficient in downregulating bromodeoxyuridine incorporation, cyclin E- and cyclin A-Cdk2 activity, and inhibiting pRb hyperphosphorylation. These data support the model that the critical event ensuring G(1) arrest in senescence is p21-dependent Cdk inactivation, while other aspects of senescent phenotype appear to occur independently of p21.
Asunto(s)
Quinasas CDC2-CDC28 , Senescencia Celular , Ciclinas/fisiología , Proteínas Represoras , Animales , Ciclo Celular , Células Cultivadas , Ciclina D1/metabolismo , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/genética , Inhibidores Enzimáticos , Fibroblastos/citología , Fibroblastos/metabolismo , Fase G1 , Expresión Génica , Humanos , Ratones , Mitosis , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Fenotipo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Bcl-xL, a member of the Bcl-2 family, inhibits apoptosis, and its expression is regulated at the transcriptional level, yet nothing is known about the transcription factors specifically activating this promoter. The bcl-x promoter contains potential Ets binding sites, and we show that the transcription factor, Ets2, first identified by its sequence identity to v-ets of the E26 retrovirus, can transactivate the bcl-x promoter. Transient expression of Ets2 results in the upregulation of Bcl-xL but not of Bcl-xS, an alternatively spliced gene product which induces apoptosis. Ets2 is ubiquitously expressed at low levels in a variety of cell types and tissues but is specifically induced to abundant levels during macrophage differentiation. Since Bcl-xL is also upregulated during macrophage differentiation, we asked whether the bcl-x could be a direct downstream target gene of Ets2 in macrophages. BAC1.2F5 macrophages, which are dependent on macrophage colony-stimulating factor 1 (CSF-1) for their growth and survival, were used in these studies. We show that CSF-1 stimulation of BAC1.2F5 macrophages results in the upregulation of expression of ets2 and bcl-xL with similar kinetics of induction. In the absence of CSF-1, these macrophages undergo cell death by apoptosis, whereas constitutive expression of Ets2 rescues these cells from cell death, and bcl-xL is upregulated. These results strongly suggest a novel role of Ets2 in affecting apoptosis through its regulation of Bcl-xL transcription.
Asunto(s)
Apoptosis/fisiología , Proteínas de Unión al ADN , Factor Estimulante de Colonias de Macrófagos/deficiencia , Macrófagos/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras , Transactivadores/genética , Factores de Transcripción , Empalme Alternativo , División Celular , Macrófagos/citología , Fosforilación , Proteína Proto-Oncogénica c-ets-2 , Proteína de Retinoblastoma/metabolismo , Transcripción Genética , Activación Transcripcional , Regulación hacia Arriba , Proteína bcl-XRESUMEN
The irreversible G1 arrest in senescent human diploid fibroblasts is probably caused by inactivation of the G1 cyclin-cyclin-dependent kinase (Cdk) complexes responsible for phosphorylation of the retinoblastoma protein (pRb). We show that the Cdk inhibitor p21(Sdi1,Cip1,Waf1), which accumulates progressively in aging cells, binds to and inactivates all cyclin E-Cdk2 complexes in senescent cells, whereas in young cells only p21-free Cdk2 complexes are active. Furthermore, the senescent-cell-cycle arrest occurs prior to the accumulation of the Cdk4-Cdk6 inhibitor p16(Ink4a), suggesting that p21 may be sufficient for this event. Accordingly, cyclin D1-associated phosphorylation of pRb at Ser-780 is lacking even in newly senescent fibroblasts that have a low amount of p16. Instead, the cyclin D1-Cdk4 and cyclin D1-Cdk6 complexes in these cells are associated with an increased amount of p21, suggesting that p21 may be responsible for inactivation of both cyclin E- and cyclin D1-associated kinase activity at the early stage of senescence. Moreover, even in the late stage of senescence when p16 is high, cyclin D1-Cdk4 complexes are persistent, albeit reduced by
Asunto(s)
Quinasas CDC2-CDC28 , Senescencia Celular/fisiología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/fisiología , Ciclinas/fisiología , Proteínas Proto-Oncogénicas , Diferenciación Celular , Línea Celular , Ciclina A/metabolismo , Ciclina D1/metabolismo , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Fibroblastos/citología , Humanos , Fosforilación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Retinoblastoma/metabolismo , Serina/metabolismoRESUMEN
Dimethylsulfoxide (DMSO) was shown to inhibit the proliferation of several B cell lines including Raji, Daudi, and SKW6-CL4 but the mechanisms involved in this growth arrest are still unclear. We show that in 7TD1 mouse hybridoma cells a DMSO-induced reversible G1 arrest involves inactivation of Rb kinases, cyclin D2/CDK4 and cyclin E/CDK2. This occurs by at least three distinct mechanisms. Inhibition of cyclin D2 neosynthesis leads to a dramatic decrease of cyclinD2/CDK4 complexes. This in turn enables the redistribution of p27[KIP1] from cyclin D2/CDK4 to cyclin E/CDK2 complexes. In addition, the simultaneous accumulation of p21[CIP1] entails increasing association with cyclin D3/CDK4 and cyclin E/CDK2. Thus, p21[CIP1] and p27[KIP1], act in concert to inhibit cyclin E/CDK2 activity which, together with CDK4 inactivation, confers a G1-phase arrest.
Asunto(s)
Antiinflamatorios/farmacología , Linfocitos B/efectos de los fármacos , Quinasas CDC2-CDC28 , Proteínas de Ciclo Celular , Ciclinas/antagonistas & inhibidores , Ciclinas/efectos de los fármacos , Dimetilsulfóxido/farmacología , Fase G1/fisiología , Proteínas Proto-Oncogénicas , Proteínas Supresoras de Tumor , Administración Tópica , Animales , Linfocitos B/citología , Linfocitos B/fisiología , División Celular/fisiología , Ciclina A/antagonistas & inhibidores , Ciclina A/metabolismo , Ciclina D2 , Ciclina E/antagonistas & inhibidores , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Hibridomas/citología , Hibridomas/efectos de los fármacos , Hibridomas/fisiología , Ratones , Proteínas Asociadas a Microtúbulos/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/genética , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Retinoblastoma/metabolismoRESUMEN
Normal human diploid fibroblasts (HDF) have a finite proliferative life-span at the end of which they are arrested with a G1 phase DNA content regardless of the culture conditions. Serum stimulated senescent HDF fail to phosphorylate their retinoblastoma protein (pRb) and consequently do not express a large cohort of late G1 phase genes whose products are necessary for entry into S phase. Because pRb is believed to be phosphorylated sequentially in G1 phase by cyclin D-CDK4/6 and cyclin E-CDK2 complexes, we and others have investigated the status of these complexes in senescent HDF. There is little or no cyclin E-associated kinase activity in senescent IMR90 even though potentially active cyclin E-CDK2 complexes are present, suggesting the presence of an inhibitor. Likewise, cyclin D is complexed with its catalytic partners CDK4 and CDK6 in senescent HDF, but it is not known whether these complexes are active. p21Sdi1,Cip1,Waf1, a ubiquitous inhibitor of the activity of cyclin-CDK complexes, increases progressively throughout the life-span of HDF, but then declines again after the cells become senescent. In contrast, p16Ink4a, which binds monomeric CDK4 and CDK6 thereby preventing their binding to cyclin D, is increased dramatically at the time of senescence and remains high for at least 2 mo. Thus, it is possible that increased p21 initiates the senescent cell cycle arrest in normal cells, but p16 is important for the long-term maintenance of that arrest.
Asunto(s)
Ciclo Celular/fisiología , Senescencia Celular/fisiología , Quinasas Ciclina-Dependientes/fisiología , Ciclinas/fisiología , Animales , Ciclina D , Regulación del Desarrollo de la Expresión Génica/fisiología , HumanosRESUMEN
Cell cycle arrest in G1 in response to ionizing radiation or senescence is believed to be provoked by inactivation of G1 cyclin-cyclin-dependent kinases (Cdks) by the Cdk inhibitor p21(Cip1/Waf1/Sdi1). We provide evidence that in addition to exerting negative control of the G1/S phase transition, p21 may play a role at the onset of mitosis. In nontransformed fibroblasts, p21 transiently reaccumulates in the nucleus near the G2/M-phase boundary, concomitant with cyclin B1 nuclear translocation, and associates with a fraction of cyclin A-Cdk and cyclin B1-Cdk complexes. Premitotic nuclear accumulation of cyclin B1 is not detectable in cells with low p21 levels, such as fibroblasts expressing the viral human papillomavirus type 16 E6 oncoprotein, which functionally inactivates p53, or in tumor-derived cells. Moreover, synchronized E6-expressing fibroblasts show accelerated entry into mitosis compared to wild-type cells and exhibit higher cyclin A- and cyclin B1-associated kinase activities. Finally, primary embryonic fibroblasts derived from p21-/- mice have significantly reduced numbers of premitotic cells with nuclear cyclin B1. These data suggest that p21 promotes a transient pause late in G2 that may contribute to the implementation of late cell cycle checkpoint controls.
Asunto(s)
Núcleo Celular/metabolismo , Ciclinas/metabolismo , Fase G2 , Mitosis , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Fibroblastos/citología , Fibroblastos/metabolismo , HumanosRESUMEN
Human diploid fibroblasts have a finite proliferative lifespan in culture, at the end of which they are arrested with G1 phase DNA contents. Upon serum stimulation, senescent cells are deficient in carrying out a subset of early signal transduction events such as activation of protein kinase C and induction of c-fos. Later in G1, they uniformly fail to express late G1 genes whose products are required for DNA synthesis, implying that they are unable to pass the R point. Failure to pass the R point may occur because senescent cells are unable to phosphorylate the retinoblastoma protein, owing to the accumulation of inactive complexes of cyclin E/Cdk2 and possibly cyclin D/Cdk4. Senescent cells contain high amounts of p21, a potent cyclin-dependent kinase inhibitor whose levels are also elevated in cells arrested in G1 following DNA damage, suggesting that both arrests might share a common mechanism. Cell aging is accompanied by a progressive shortening of chromosomal telomeres, which could be perceived by the cells as a form of DNA damage that gives rise to the signals that inactive the cell cycle machinery.
Asunto(s)
Ciclo Celular , Senescencia Celular , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Fase G1 , ADN/biosíntesis , ADN/metabolismo , Daño del ADN , Fibroblastos/citología , Fibroblastos/fisiología , Humanos , Modelos BiológicosRESUMEN
Cyclin-dependent kinases (Cdks) previously have been shown to drive the major cell cycle transitions in eukaryotic organisms ranging from yeast to humans. We report here the identification of a 28-kDa protein, p28Ick (inhibitor of cyclin-dependent kinase), that binds to and inhibits the kinase activity of preformed Cdk/cyclin complexes from human cells. p28 inhibitory activity fluctuates during the cell cycle with maximal levels in G1 and accumulates in G1- and G0-arrested cells. These results suggest that control of the G1/S transition may be influenced by a family of Cdk inhibitors that include p28Ick and the recently described inhibitors p21Cip1/Waf1/Cap20 and p16Ink4.
Asunto(s)
Quinasas CDC2-CDC28 , Ciclo Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes , Inhibidores de Proteínas Quinasas , Proteínas Serina-Treonina Quinasas , Quinasa 2 Dependiente de la Ciclina , Ciclinas/metabolismo , Células HeLa , Humanos , Lovastatina/farmacología , Masculino , Proteínas Quinasas/metabolismoRESUMEN
gamma-Irradiation of human diploid fibroblasts in the G1 interval caused arrest of the cell cycle prior to S phase. This cell cycle block was correlated with a lack of activation of both cyclin E-Cyclin-dependent kinase 2 (Cdk2) and cyclin A-Cdk2 kinases and depended on wild-type p53. Although the accumulation of cyclin A was strongly inhibited in gamma-irradiated cells, cyclin E accumulated and bound Cdk2 at normal levels but remained in an inactive state. We found that both whole-cell lysates and inactive cyclin E-Cdk2 complexes prepared from irradiated cells contained an activity capable of inactivating cyclin E-Cdk2 complexes. The protein responsible for this activity was shown to be p21CIP1/WAF1, recently described as a p53-inducible Cdk inhibitor. Our data suggest a model in which ionizing radiation confers G1 arrest via the p53-mediated induction of a Cdk inhibitor protein.
Asunto(s)
Fibroblastos/citología , Fase G1/fisiología , Fase G1/efectos de la radiación , Proteínas Quinasas/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Secuencia de Bases , Células Cultivadas , Ciclina D1 , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Ciclinas/fisiología , Activación Enzimática , Fibroblastos/enzimología , Fibroblastos/efectos de la radiación , Rayos gamma , Humanos , Masculino , Datos de Secuencia Molecular , Proteínas Oncogénicas/metabolismo , Inhibidores de Proteínas Quinasas , Proteína de Retinoblastoma/metabolismoRESUMEN
Cyclin-dependent kinases (Cdks) control the major cell cycle transitions in eukaryotic cells. On the basis of a variety of experiments where cyclin function either is impaired or enhanced, D-type cyclins as well as cyclins E and A have been linked to G1 and G1/S phase roles in mammalian cells. We therefore sought to determine if agents that block the G1/S phase transition do so at the level of regulating the Cdk activities associated with these cyclins. A variety of conditions that lead to G1 arrest were found to correlate with accumulation of G1-specific Cdk inhibitors, including treatment of fibroblasts with ionizing radiation, treatment of epithelial cells with TGF-beta, treatment of HeLa cells with the drug lovastatin, and removal of essential growth factors from a variety of different cell types. Mechanistically, inhibition of Cdks was found to involve the stoichiometric binding of Cdk inhibitor proteins. p21Waf1/Cip1 was associated with DNA damage induced arrest while p27Kip1/p28Ick1 accumulated under a variety of antiproliferative conditions.
Asunto(s)
Quinasas Ciclina-Dependientes/fisiología , Ciclinas/fisiología , Fase G1 , Animales , Ciclo Celular/fisiología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , HumanosRESUMEN
Senescent human diploid fibroblasts are unable to enter S phase in response to mitogenic stimulation. One of the key deficiencies in mitogen-stimulated senescent cells is their failure to phosphorylate the retinoblastoma protein, which acts as an inhibitor of entry into S phase in its unphosphorylated form. Recent data suggest that cyclin-dependent kinases (Cdks) regulated by G1 cyclins (D type and E) are responsible for the primary phosphorylation of the retinoblastoma protein prior to the G1/S boundary. Surprisingly, we found 10- to 15-fold higher constitutive amounts of both cyclin E and cyclin D1 in senescent cells compared to quiescent early-passage cells. Nevertheless, cyclin E-associated kinase activity in senescent cells was very low and did not increase significantly upon mitogenic stimulation even though cyclin E-Cdk2 complexes were abundant. In contrast to early-passage cells in late G1 phase, senescent cells contained mainly underphosphorylated cyclin E and proportionally more unphosphorylated and inactive Cdk2, perhaps accounting for the low kinase activity. We also show that a majority of the Cdk2 in senescent cells, but not in early-passage cells, was complexed with cyclin D1. Cyclin D1-Cdk2 complexes, severalfold enriched in senescent cells, contained exclusively unphosphorylated Cdk2. Amounts of cyclin A, which ordinarily accumulates in S and G2 phases, were extremely low in stimulated senescent cells. We suggest that the failure to activate cyclin E-Cdk2 kinase activity in senescent cells may account for the inability of these cells to phosphorylate the retinoblastoma protein in late G1 phase, which in turn may block the expression of late G1 genes such as cyclin A that are required for entry into S phase.
Asunto(s)
Quinasas CDC2-CDC28 , Ciclo Celular , Senescencia Celular , Quinasas Ciclina-Dependientes , Ciclinas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Quinasa 2 Dependiente de la Ciclina , Expresión Génica , Humanos , Sustancias Macromoleculares , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteínas/metabolismo , ARN Mensajero/genética , Fosfatasas cdc25RESUMEN
Cyclin E is classified as a putative G1 cyclin on the basis of its cyclic pattern of mRNA expression, with maximal levels being detected near the G1/S boundary. We report here that cyclin E is found associated with the transcription factor E2F in a temporally regulated fashion. E2F is known to be a critical transcription factor for the expression of some S phase-specific proteins and is thought to be important for a series of others. Antisera specific for cyclin E were raised and used to demonstrate an association between cyclin E and E2F. This cyclin E/E2F complex was seen in a variety of human cell lines from various tissues, but its appearance was detected primarily during the G1 phase of the cell cycle. The cyclin E/E2F association decreased as cells entered S phase, just as the association of E2F with cyclin A became detectable. We characterized the cyclin E-E2F complex further to show that both the cyclin-dependent kinase-2 (cdk2) and p107 were present. Therefore, the p107/E2F complex is associated with two different cdk2 kinase complexes--one containing cyclin A and the other containing cyclin E--and the appearance of these complexes is temporally regulated during the cell cycle. The presence of cyclin E/E2F complexes in the G1 phase suggests a role for cyclin E in the control of genes required for the G1-to-S transition.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas Portadoras , Proteínas de Ciclo Celular , Quinasas Ciclina-Dependientes , Ciclinas/metabolismo , Proteínas de Unión al ADN , Proteínas Nucleares , Proteínas Serina-Treonina Quinasas , Factores de Transcripción/metabolismo , Secuencia de Bases , Western Blotting , Línea Celular , Quinasa 2 Dependiente de la Ciclina , Ciclinas/genética , Ciclinas/inmunología , Factores de Transcripción E2F , Electroforesis en Gel de Poliacrilamida , Fase G1/genética , Humanos , Sueros Inmunes , Datos de Secuencia Molecular , Oligonucleótidos , Pruebas de Precipitina , Proteínas Quinasas/metabolismo , Proteínas/metabolismo , ARN Mensajero/metabolismo , Proteína 1 de Unión a Retinoblastoma , Proteína p107 Similar a la del Retinoblastoma , Factor de Transcripción DP1RESUMEN
The retinoblastoma susceptibility gene (RB) product, the retinoblastoma protein (pRb), functions as a regulator of cell proliferation. Introduction of the RB gene into SAOS-2 osteosarcoma cells, which lack functional pRb, prevents cell cycle progression. Such growth-suppressive functions can be modulated by phosphorylation of pRb, which occurs via cell cycle-regulated kinases. We show that constitutively expressed cyclins A and E can overcome pRb-mediated suppression of proliferation. pRb becomes hyperphosphorylated in cells overexpressing these cyclins, and this phosphorylation is essential for cyclin A- and cyclin E-mediated rescue of pRb-blocked cells. This suggests that G1 and S phase cyclins can act as regulators of pRb function in the cell cycle by promoting pRb phosphorylation.
Asunto(s)
Ciclinas/fisiología , Proteína de Retinoblastoma/fisiología , Núcleo Celular , Ciclinas/metabolismo , Fase G1/fisiología , Expresión Génica , Vectores Genéticos , Humanos , Mutación , Fenotipo , Fosforilación , Proteínas Quinasas , Proteínas Recombinantes , Transfección , Células Tumorales CultivadasRESUMEN
G1 cyclins control the G1 to S phase transition in the budding yeast, Saccharomyces cerevisiae. Cyclin E was discovered in the course of a screen for human complementary DNAs that rescue a deficiency of G1 cyclin function in budding yeast. The amounts of both the cyclin E protein and an associated protein kinase activity fluctuated periodically through the human cell cycle; both were maximal in late G1 and early S phases. Cyclin E-associated kinase activity was correlated with the appearance of complexes containing cyclin E and the cyclin-dependent kinase Cdk2. Thus, the cyclin E-Cdk2 complex may constitute a human G1-S phase-specific regulatory protein kinase.
Asunto(s)
Quinasas CDC2-CDC28 , Ciclo Celular , Quinasas Ciclina-Dependientes , Ciclinas/metabolismo , Protamina Quinasa/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Quinasa 2 Dependiente de la Ciclina , Células HeLa , Humanos , Técnicas Inmunológicas , Sustancias Macromoleculares , Unión ProteicaRESUMEN
Cyclins and cyclin-associated cdc kinases are key regulators of oocyte maturation (Maller, J. L. (1990) in The Biology and Medicine of Signal Transduction (Nishizuka, Y., Endo, M., and Tanaka, C., eds) pp. 323-328, Raven Press, New York), yeast cell cycles (Nurse, P. (1990) Nature 344, 503-508), DNA replication in cell-free systems (D'Urso, F., Marraccino, R. L., Marshak, R. R., and Roberts, J. M. (1990) Science 250, 786-791), and amphibian cell proliferative transitions (Hunt, T. (1991) Nature 350, 462-463). The extent to which these regulatory molecules participate in the growth control of differentiated epithelial cells like hepatocytes is unknown. Therefore, we investigated the expression of "G1" (E, C, and D) and "G2/M" (A, B1, and B2) cyclin mRNAs, the relative levels of cyclin A- and B1-associated histone H1-kinase activity, and the appearance of cyclin-associated kinases (p32/p33cdk2 and p33/p34cdc2) in regenerating rat liver and in control tissues from sham hepatectomized rats. To do this, we exploited a battery of human cyclin cDNAs and cyclin antisera that recognize rat molecules. The results suggest an apparent sequence of regeneration-specific changes: 1) elevated and induced expression of cyclins E (2.1 kilobases (kb)) and C (4 kb), and D mRNAs (4 kb), within 12 h, respectively; 2) induction of cyclins A (3.4 and 1.8 kb), B1 (2.5 and 1.8 kb), and B2 (1.9 kb) mRNAs at 24 h; 3) induction of cyclin A- and B1-associated nuclear histone H1 kinase at 24 h; and 4) enhanced levels of PSTAIRE-containing proteins of Mr approximately 32-33 and 33-34 kDa in nuclear extracts from 24-h regenerating liver that co-immunoprecipitate with cyclin A and B1 antisera, respectively. These observations provide an intellectual framework that unifies the biology of hepatocyte mitogenesis, proto-oncogene expression, and the machinery of the cell cycle.
Asunto(s)
Ciclinas/metabolismo , Regeneración Hepática , Protamina Quinasa/biosíntesis , ARN Mensajero/biosíntesis , Animales , Proteína Quinasa CDC2/metabolismo , Ciclo Celular , Ciclinas/genética , Cinética , Hígado/citología , Hígado/fisiología , Proto-Oncogenes Mas , RatasRESUMEN
In budding yeast, Saccharomyces cerevisiae, the cell cycle is controlled at the G1/S phase transition by regulating the activity of the CDC28 protein kinase. This is the budding yeast homologue of the cdc2 protein kinase associated in most organisms with control of mitosis. In budding yeast CDC28 controls both the G1/S phase transition and the G2/M phase transition by being differentially activated by two distinct classes of positive regulatory subunits known as G1 cyclins or CLNs and B-type cyclins or CLBs, respectively. To establish whether a similar dual role for Cdc2-related kinases exists in animal cells, we and others have sought human homologues of yeast G1 cyclins. Of several candidates, cyclin E is the most promising in that it accumulates prior to S phase and is associated with a pre-S phase protein kinase activity. The kinetics of accumulation of cyclin E-associated protein kinase activity is consistent with a role at the mammalian cell cycle restriction point.