Death activation does not stop tumor growth: quantitative evidence for concomitant activation of apoptosis in tumor growth in vitro.

A protein-independent fibrosarcoma, Gc-4 PF, grows exponentially in a protein-free medium. The doubling time (approximately 26 h) was similar to that of the serum-dependent parental clone, Gc-4 SD cultivated in the presence of fetal calf serum (FCS). We demonstrated here that the protein-free cultivation of Gc-4 PF cells concomitantly activates apoptotic phenotypes (one third of total cell population), including typical morphology, high uptake of Hoechst 33342 dye, and cleavage of DNA to large fragments, as observed in protein-deprived Gc-4 SD cell previously. Gc-4 SD cells arrested in the G0/G1-phase in response to the protein-free condition. In contrast, Gc-4 PF cells did not reach G0/G1 arrest in the protein-free condition; instead the durations of both G0/G1 and G2-phases were markedly reduced. The estimation of one cell cycle duration revealed that the cell division cycle was accelerated to 1.7 (27 h/15.4 h)-fold. Then the growth kinetics was able to be verified quantitatively by both the cell division rate and apoptotic cell loss. Protein-free cultivation resulted in slight down-regulation of c-myc protein in both cell types, while the down-regulation of p34cdc2, shown clearly in Gc-4 SD cells, was avoided in Gc-4 PF cells. Interestingly, while the expression of p53 was not affected in Gc-4 SD cells in response to the protein-free condition, the suppressor gene product expression was suppressed markedly in Gc-4 PF cells. These results suggest that Gc-4 PF cells may have acquired an ability to accelerate cell division by shortening the cell cycle duration to maintain a proper growth rate in response to intrinsic apoptosis activation with, at least in part, a suppression of p53 expression as well as an escape of down-regulation of p34cdc2.