Alterations in contact and density-dependent arrest state in senescent WI-38 cells

Normal human WI-38 fibroblast-like cells in culture undergo a process of senescence, one feature of which is a gradual decline in proliferative capacity. As these cells reach the end of their replicative life span they exhibit decreases in the fraction of cells able to synthesize DNA, in the number of doublings per passage (constant seeding density), and in the cell harvest and saturation densities. They also display increased average cell cycle times, largely at the expense of longer G₁ intervals. These alterations are accompanied by morphologic changes, including cell enlargement. Before the end of the replicative life span or phase-out, there is a highly reproducible (55/58 sublines) cell loss of approximately 50%; however, a stable population survives that can exist in a viable yet nonproliferative state for many months. This stable population maintains an extremely low saturation density, representing <5% of that achieved by early passage cultures. Further, we show that maximum harvest densities achieved by senescent cells are lower, irrespective of seeding densities, i.e. when placed at cell densities higher than those normally achieved by senescent cultures they display a net decline in cell number. This decline continues until the cell density approximates the density that would have been achieved had the cultures been seeded at standard density (1×10⁴ cells/cm²). By measuring the accumulation of an mRNA species, EPC-1, that is expressed when early passage cultures reach a growth-arrested state via density-dependent contact inhibition, we also show that senescent cells are unable to produce this transcript at either their normal confluent density or at high cell density obtained by overseeding. The above results suggest that there are significant alterations in cell-to-cell contact sensitivity and arrest state of senescent cultures. These changes result in both a cytotoxic response to crowding and failure to express at least one molecular marker which is induced as young cells approach growth arrest by contact inhibition.