An in vitro model, based on normal (primary) human astrocytes (NHAs), was used to investigate the nature of the selection pressures for events that occur during the progression of astrocyte-derived tumors and, in particular, the potential role of proliferative life span barriers (PLBs). As with fibroblasts, NHAs senesced with elevated p21(WAF1) and senescence-associated beta-galactosidase activities. Unlike fibroblasts, replicative senescence (M1) occurred much earlier, after approximately 20 pd and was not bypassed by hTERT expression. Abrogation of p53 function, by expression of human papillomavirus type 16 E6, led to an extension of life span, implying that replicative senescence in NHAs was p53-dependent but telomere-independent. human papillomavirus type16 E6 expression promoted additional growth of up to 12 pd, until a second telomere-independent PLB (termed M(INT)) was imposed associated with elevated p16(INK4A) levels. A proportion of cells escaped from M(INT) lost p16(INK4A) expression and achieved approximately an additional 25 pd until a crisis-like third PLB (M2) was reached. Expression of hTERT in post-M(INT) cells allowed these cells to become immortal and bypass this third PLB. The in vitro PLBs appear, in order of occurrence, dependent upon p53, p16(INK4A), and telomere erosion, a situation that mirrors an equivalent order of mutational events during tumor progression in vivo. This study describes a model that provides a plausible explanation for the selective pressures driving mutational events in this tumor type and provides direct evidence of a p53-dependent, telomere-independent PLB.