RESUMEN
Introduction of simian virus 40 T antigen into rodent fibroblasts gives rise to cells that can proliferate indefinitely but are dependent upon it for maintenance of their growth once the normal mitotic life span has elapsed. Inactivation of T antigen in these immortalized cells causes rapid and irreversible cessation of growth. To determine whether this growth arrest is associated with entry into senescence, we have undertaken a genetic and biological analysis of conditionally immortal (tsa) cell lines derived by immortalizing rat embryo fibroblasts with the thermolabile tsA58 T antigen. This analysis has identified the following parallels between the tsa cells after inactivation of T antigen and senescent rat embryo fibroblasts: (i) growth arrest is irreversible; (ii) it occurs in G1 as well as G2; (iii) the G1 block can be partially overcome by stimulation with 20% fetal calf serum, but the G2 block cannot be overcome; (iv) 20% fetal calf serum induces c-fos, but c-myc is unaltered; and (v) fibronectin and p21(Waf1/Cip1/Sdi1) are upregulated upon growth arrest. These results suggest that T-antigen-immortalized fibroblasts are committed to undergo senescence but are prevented from undergoing this process by T antigen. Inactivation of T antigen removes this block and results in senescence of the cells. Thus, these cell lines may represent a powerful system for study of the molecular basis of entry into senescence.
Asunto(s)
Antígenos Virales de Tumores/fisiología , Transformación Celular Viral , Senescencia Celular/fisiología , Fibroblastos/citología , Virus 40 de los Simios/genética , Animales , Antígenos Virales de Tumores/genética , Bovinos , División Celular , Línea Celular Transformada , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Ciclinas/genética , Replicación del ADN , Embrión de Mamíferos/citología , Sangre Fetal/fisiología , Fibronectinas/biosíntesis , Fibronectinas/genética , Fase G1 , Fase G2 , Regulación Viral de la Expresión Génica , Genes Inmediatos-Precoces , Genes fos , Genes myc , Ratas , Ratas Sprague-Dawley , Virus 40 de los Simios/fisiologíaRESUMEN
CpG islands were identified and localized to chromosome 1p36 by means of pulsed-field gel blot hybridization with 1p36-specific microclone probes. Five CpG islands, designated CpG17, CpG28, CpG60, CpG112a, and CpG112b, were molecularly cloned from corresponding cosmids. All five islands are associated with transcribed sequences, as shown by RNA blot hybridizations. Screening of cDNA libraries with the island-specific genomic probes led to the isolation of two cDNA clones to date. These encode the human transcription factor E2F-2 and the dominant-negative helix-loop-helix gene ID3, respectively. Pulsed-field gel electrophoresis analysis also revealed that these two genes are located next to each other at a distance of about 25 kb.
