THE HELIX-LOOP-HELIX PROTEIN ID-1 AND A RETINOBLASTOMA PROTEIN-BINDING MUTANT OF SV40 T-ANTIGEN SYNERGIZE TO REACTIVATE DNA-SYNTHESIS IN SENESCENT HUMAN FIBROBLASTS

Normal somatic cells of higher organisms do not divide indefinitely. A fter a finite number of divisions, normal cells irreversibly cease pro liferation by a process termed replicative or cellular senescence. Rep licative senescence is controlled by multiple, dominant-acting genes a bout which very little is known. The only genes known to reactivate DN A synthesis in senescent cells are viral oncogenes encoding proteins t hat bind and inactivate the p53 and retinoblastoma (pRb) tumor suppres sor proteins. SV40 T antigen is the best studied of these viral oncopr oteins. T[K1] is a T antigen point mutant that selectively is defectiv e in binding pRb and the pRb-related proteins p107 and p130. We show t hat T[K1] stimulated quiescent human fibroblasts to synthesize DNA nea rly as well as wild-type T but was incapable of stimulating senescent cells. We tested several growth regulatory genes that are repressed in senescent cells for ability to restore activity to T[K1]. These inclu ded c-fos, c-jun, Id-1, Id-2, E2F-1, and cdc2. Only the helix-loop-hel ix (HLH) protein, Id-1, restored the ability of T[K1] to reactivate DN A synthesis in senescent cells. This activity of Id-1 was not shared b y Id-2, a related protein, and depended on an intact HLH domain. It di d not appear that Id-1 interacted directly with pRb or p107. Constitut ive Id-1 expression failed to rescue proliferating cells from growth i nhibition by pRb, p107, or p130, and failed to interact with pRb in th e yeast two hybrid system. Because Id proteins negatively regulate bas ic-HLH (bHLH) transcription factors, we suggest that senescent cells e xpress one or more bHLH factor that cooperates with pRb, or pRb-relate d proteins, to suppress proliferation. (C) 1996 Wiley-Liss, Inc.