Opposite cell density-dependence between spontaneous and oxidative stress-induced apoptosis in mouse fibroblast L-cells

Abstract

Background/aims: Spontaneous apoptosis in culture is cell density-dependent. Yet, little is known about the density-dependence of apoptosis induced by oxidative stress and the underlying signaling mechanisms.

Methods: We compared apoptosis occurring spontaneously and induced by H2O2 in mouse fibroblast L-cells seeded at three different densities, using ELISA and TUNEL to measure DNA fragmentation, Western blot and pharmacological probes to evaluate the roles of signaling molecules.

Results: The high-density (HD, 2.5 x 10(5)/cm2) culture had approximately 1.5- and approximately 3-times more spontaneous apoptosis than medium-density (MD, 5 x 10(4)/cm2) and low-density (LD, 1 x 10(4)/cm2) cultures, respectively. The opposite was seen with H2O2 (500 microM)-induced apoptosis: more apoptosis in LD than in MD and HD. Caspase inhibitor (Z-VAD-fmK) diminished the density-dependence of both spontaneous and H2O2-induced apoptosis. Activation of caspase 3 by H2O2 was more pronounced in LD than in MD and HD. Inhibition of PI3K/Akt increased spontaneous apoptosis more in LD and H2O2-induced apoptosis more in HD, diminishing the density-dependence of apoptosis. p38 MAPK inhibition reduced H2O2-induced apoptosis more in LD, diminishing the density-dependence of H2O2-induced apoptosis without altering that of spontaneous apoptosis. Akt level and activity and Hsp72 level were increased in HD, but decreased in MD and LD cells, by H2O2, and the active p38 MAPK was enhanced by H2O2 in all groups.

Conclusion: Spontaneous apoptosis is cell density-dependent whereas H2O2-induced apoptosis is reverse density-dependent. PI3K/Akt, p38 MAPK and Hsp72 pathways are critical for the opposite density-dependence of apoptosis.