Evidence for cellular aging in long-term confluent cultures: heritable impairment of proliferation, accumulation of age pigments and their loss in neoplastic transformation.

Previous experiments had shown that repeated rounds of prolonged growth constraint at confluence of NIH 3T3 sublines result in persistent changes in the growth behavior that are characteristic of cellular aging. These changes, which include an enduring decrease in the rate of proliferation in low density subcultures and a marked increase in neoplastic transformation, are here reproduced cumulatively over a 6 week period during which cultures are maintained in a single, continuous round of constraint at confluence. By testing multiple cultures at weekly intervals we show that the persistent reduction in exponential growth in low density subcultures is a property of the entire treated cell population that is first demonstrable in the cell population used here within a few days after the constraint of confluence is imposed. There is also a reduction in saturation density of cells subcultured from this early confluence which is reversed in longer term confluence when the cells become transformed. The reduction in exponential growth rate in serial subcultures becomes more pronounced in cells after longer periods of confluence. It is strongly manifest at 6 weeks when most of the cells have undergone neoplastic transformation. The transformation initially involves only a very small fraction of cells in a confluent culture, and is only detectable after 3 weeks of confluence. Beyond that time there is selective overgrowth of the transformed cells so they become the dominant element at 6 weeks. The very same cells from the 6 week cultures that have a reduced rate of growth when subcultured at low density, grow to higher saturation densities at confluence. The reduced growth rates are heterogeneously distributed among clones derived from the 6 week confluent cultures. Typical age pigment bodies appear in the cytoplasm of the cells after 3-4 days of confluence, and fill the cytoplasm at 2 weeks. They tend to enlarge into residual bodies at 3 weeks but largely disappear at 6 weeks when most of the cells are transformed. The results reinforce the conclusion that the prolonged constraint of confluence of these cells reproduces the major growth and morphological effects of cellular aging in the body.