Individual transforming events in long-term cell culture of NIH 3T3 cells as products of epigenetic induction.

The NIH 3T3 line of mouse fibroblasts undergoes "spontaneous" transformation in culture, exhibited in the development of foci of transformed cells overgrowing the confluent monolayer. Evidence is provided here to support the proposition that the spontaneous generation of individual transformed variants is a product of epigenetic induction by various types of growth inhibition. Novel transformed variants generally arise after prolonged confluence and cessation of net growth, with these new types of foci appearing during a second round of confluence, although not in the first round. Few or no transformed variants exist in the cultures prior to growth constraint. The susceptibility of NIH 3T3 cells to induction of transformation is itself shown to be subject to epigenetic influence, with capacity for transformation reflecting passage histories. Cell sublines maintained in long-term subconfluent passage that allows unimpeded growth become more refractory with time to spontaneous transformation, while sublines passaged in more growth-restricting conditions maintain their sensitivity. The differing capacities of the sublines are reflected in the degree of growth inhibition required to induce individual events of spontaneous transformation, and in the frequency at which such new variants arise. Thus growth inhibition not only induces individual transforming events, but even increases cell susceptibility to further induction of transformation. These phenomena are consistent with the progressive state selection model of heritable change, which postulates a self-regulating selection of better adapted states from among those made available to a biological system during heterogeneous fluctuations in its total pattern of chemical equilibria.