Effects of c-myc expression on proliferation, quiescence, and the G0 to G1 transition in nontransformed cells.

The relative contributions of deregulation versus high level expression of the c-myc gene to malignant transformation are not clear. To investigate the effects of subtle perturbations in c-myc expression on normal cellular physiology, we isolated cell lines with one c-myc gene copy disrupted by targeted homologous recombination. The heterozygous cell lines were further modified by introducing a c-myc transgene expressed 4-fold above the normal diploid level. During exponential growth, heterozygous cells expressed c-Myc mRNA at approximately 50% of the level found in diploid cells, resulting in a slower growth rate. The c-myc transgene reversed the growth defect and accelerated growth relative to diploid cells. Serum deprivation of transgene-expressing cells caused a fraction of the culture to undergo apoptosis. After an initial wave of apoptosis, the remainder of the culture successfully entered Go. Transgene mRNA continued to be constitutively expressed in quiescent cells, but c-Myc protein was not detectable. During the G0 to G1 transition, heterozygous cells induced c-myc expression with normal kinetics, but levels throughout the time course were consistently at least 50% lower than those in diploid cells. The reduction in c-myc expression was correlated with a 3-4-h delay in entry into S phase. The presence of the transgene, which was expressed constitutively throughout the G0 to G1 transition, reversed the delay but did not further accelerate entry into S phase. Our results show that even small perturbations in c-myc expression cause changes in the proliferative status of cells and thus argue that the natural regulation patterns of the gene are crucial for the maintenance of normal cellular physiology.