Confluence is an agent that promotes the progressive acquisition of the focus forming phenotype in clones of cells within NIH-3T3 populations. This conclusion is based on four results. (a) Even in cultures which have been confluent for more than 2 weeks without making foci, some cells in those cultures have been affected by the confluent state and will make foci if replated and allowed to grow into new saturated cultures. Because replicate dishes are very similar in the number and type of foci formed after replating, we conclude that the progression toward focus formation is substantially completed before replating, while the cells are in their original confluent cultures. (b) Different NIH-3T3 populations were produced by expansion of small or large numbers of starting cells. When plated without exposure to confluence there was little difference in focus production among cells from these different sized starter populations. However, confluence caused foci to arise more frequently in platings of 105 cells derived from large starting numbers than from platings of 105 cells derived from small starting numbers of cells. This implies that the confluent state successfully promotes the acquisition of the focus forming phenotype in a limited percentage of cells in an NIH-3T3 culture and that those cells are absent from many small starting populations. (c) There is a progressive temporal effect of the confluent state on focus formation; the number and density of foci that emerge from replated confluent cultures increase with the length of time the cells spend in the confluent state. (d) There is heterogeneity among different batches of NIH-3T3 cells in the ability of the confluent state to induce acquisition of the focus forming phenotype. Also, the morphology of the foci that do arise after confluent treatment differs substantially among cell populations. Nonetheless, the foci formed from a single batch of cells are typically similar in morphology, indicating that those foci arose from one clone, or very few clones, of cells.

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This research was supported by U.S. Public Health Service Grant CA 15744 and the Council for Tobacco Research Grant 1948.

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