RESUMEN
Addition of serum to mitogen-starved cells activates the cellular immediate-early gene (IEG) response. Serum response factor (SRF) contributes to such mitogen-stimulated transcriptional induction of many IEGs during the G0-G1 cell cycle transition. SRF is also believed to be essential for cell cycle progression, as impairment of SRF activity by specific antisera or antisense RNA has previously been shown to block mammalian cell proliferation. In contrast, Srf(-/-) mouse embryos grow and develop up to E6.0. Using the embryonic stem (ES) cell system, we demonstrate here that wild-type ES cells do not undergo complete cell cycle arrest upon serum withdrawal but that they can mount an efficient IEG response. This IEG response, however, is severely impaired in Srf(-/-) ES cells, providing the first genetic proof that IEG activation is dependent upon SRF. Also, Srf(-/-) ES cells display altered cellular morphology, reduced cortical actin expression, and an impaired plating efficiency on gelatin. Yet, despite these defects, the proliferation rates of Srf(-/-) ES cells are not substantially altered, demonstrating that SRF function is not required for ES cell cycle progression.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Genes Inmediatos-Precoces , Proteínas Inmediatas-Precoces , Proteínas Nucleares/metabolismo , Animales , Secuencia de Bases , Ciclo Celular , Ensayo de Unidades Formadoras de Colonias , Cartilla de ADN/genética , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario y Fetal/genética , Genes fos , Ratones , Ratones Noqueados , Microscopía Electrónica de Rastreo , Proteínas Nucleares/genética , Factor de Respuesta Sérica , Transducción de Señal , Células Madre/citología , Células Madre/metabolismo , Factores de Transcripción/genéticaRESUMEN
Senescence is now understood to be the final phenotypic state adopted by a cell in response to several distinct cell physiological processes, including proliferation, oncogene activation and oxygen free radical toxicity. The role of telomere maintenance in immortalization and the roles of p16(INK4A), p19(ARF), p53 and other genes in senescence are being further elucidated. Significant progress continues to be made in our understanding of cellular senescence and immortalization.
Asunto(s)
Senescencia Celular/genética , Animales , División Celular/genética , Regulación de la Expresión Génica , Genes p16/genética , Humanos , Ratones , Oncogenes/genética , Estrés Oxidativo/genética , Proteínas/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/genética , Telómero/genética , Telómero/metabolismo , Proteína p14ARF Supresora de TumorRESUMEN
During malignant transformation, cancer cells acquire genetic mutations that override the normal mechanisms controlling cellular proliferation. Primary rodent cells are efficiently converted into tumorigenic cells by the coexpression of cooperating oncogenes. However, similar experiments with human cells have consistently failed to yield tumorigenic transformants, indicating a fundamental difference in the biology of human and rodent cells. The few reported successes in the creation of human tumour cells have depended on the use of chemical or physical agents to achieve immortalization, the selection of rare, spontaneously arising immortalized cells, or the use of an entire viral genome. We show here that the ectopic expression of the telomerase catalytic subunit (hTERT) in combination with two oncogenes (the simian virus 40 large-T oncoprotein and an oncogenic allele of H-ras) results in direct tumorigenic conversion of normal human epithelial and fibroblast cells. These results demonstrate that disruption of the intracellular pathways regulated by large-T, oncogenic ras and telomerase suffices to create a human tumor cell.
Asunto(s)
Antígenos Transformadores de Poliomavirus/fisiología , Transformación Celular Neoplásica , ARN , Telomerasa/fisiología , Animales , Adhesión Celular , División Celular , Línea Celular , Transformación Celular Neoplásica/genética , Células Cultivadas , Proteínas de Unión al ADN , Células Epiteliales , Fibroblastos , Genes ras , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Telomerasa/genética , TelómeroRESUMEN
The retinoblastoma protein (pRb) acts to constrain the G1-S transition in mammalian cells. Phosphorylation of pRb in G1 inactivates its growth-inhibitory function, allowing for cell cycle progression. Although several cyclins and associated cyclin-dependent kinases (cdks) have been implicated in pRb phosphorylation, the precise mechanism by which pRb is phosphorylated in vivo remains unclear. By inhibiting selectively either cdk4/6 or cdk2, we show that endogenous D-type cyclins, acting with cdk4/6, are able to phosphorylate pRb only partially, a process that is likely to be completed by cyclin E-cdk2 complexes. Furthermore, cyclin E-cdk2 is unable to phosphorylate pRb in the absence of prior phosphorylation by cyclin D-cdk4/6 complexes. Complete phosphorylation of pRb, inactivation of E2F binding, and activation of E2F transcription occur only after sequential action of at least two distinct G1 cyclin kinase complexes.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas Portadoras , Ciclina E/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Proteína de Retinoblastoma/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Ciclina D , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción E2F , Fase G1 , Humanos , Fosforilación , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1 , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
Cyclin E is critical for the advance of cells through the G1 phase of their growth cycle. Transcription of the cyclin E gene is known to be cell cycle-dependent. We have shown previously that mRNA levels of cyclin E are regulated positively by mitogens and negatively by TGF-beta. Much circumstantial evidence implicates both E2F transcription factors and the retinoblastoma protein (pRB) in the control of cyclin E expression. However, the molecular basis of this control has remained unclear. We report here the cloning of the cyclin E promoter and the identification of several putative E2F binding sites within the promoter sequence. We have found that cell cycle regulation of cyclin E transcription is mediated by E2F binding sites present in the promoter. The activity of this promoter can be regulated negatively by pRB. Our results suggest the operation of a positive-feedback loop in late G1 that functions to ensure continued cyclin E expression and pRB inactivation.