Spontaneous formation of morphologically altered foci of types II and III (neoplastic transformation) was examined in populations of C3H 10T½ (10T½) cells. Initial surviving cell densities ranged from 3 to 3 × 105 cells/100-mm dish and the final cell density was approximately 2 × 106 cells/dish, yielding widely differing numbers of population doublings but similar numbers of cell births from the time of cell plating to the attainment of confluence. Spontaneous formation of foci was independent of the initial surviving cell densities (and, therefore, of the number of population doublings) but was related to the number of cell divisions (cell births) between the time the cell population was plated and when suppression of proliferation of wild-type cells occurred in confluent cultures. In 418 pooled asynchronously proliferating cultures in 100-mm dishes the 95% confidence limits for the fraction of dishes containing foci was 0.041–0.089 for type II foci and 0.008–0.036 for type III foci; for cell populations in 2041 pooled cultures in 100-mm dishes, the proliferation of which was synchronized by release from confluence-induced arrest of proliferation, the 95% confidence limits for the fraction of dishes containing foci were 0.150–0.166 for type II foci and 0.017–0.032 for type III foci. Using the Poisson method, the 95% confidence limits for rates of spontaneous transformation in asynchronously proliferating populations of 10T½ cells were 1.4–3.2 × 10-8/cell/division for type II foci and 0.28 to 1.3 × 10-8/cell/division for type III foci; in populations in which proliferation was initially synchronized by release from confluence-induced arrest, spontaneous transformation rates were 5.6–6.3 × 10-8/cell/division for type II foci and 0.59–1.1 × 10-8/cell/division for type III foci. Spontaneous transformation occurred in populations of wild-type 10T½ cells at the rates and with the characteristics expected of the mutation of a single gene locus.

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Supported by NIH Grant CA-32036.

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