In systems used to express transformation using focus formation as the end point, nontransformed cells generally express a down-regulation of cell growth and division made evident by the formation of a monolayer of cells that completely covers the growth surface. In C3H 10T1/2 cells, down-regulation is thought to be progressively effected principally by cell-to-cell communication via gap junctions. Starting with a sparse population in asynchronous growth-e.g. containing cells in all phases of the growth cycle-as the area density increases, cells are progressively lost from the distribution in the order M phase, G2 phase, S phase and G1 phase, leading to the accumulation of viable cells out of cycle in so-called G0 phase. We have measured the progressive phase transitions as a function of inoculum size and time. The influence of a promoter and an antipromoter was also examined as well as the expression of the cyclin/cyclin-dependent kinase inhibitors p21 Waf1/ Cip1 and p27 Kip1 as the cells grew into confluence. Using cells synchronized in mitosis, we found that with increasing cell density the expression of p27 increased and concomitantly p21 decreased.

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