Evidence for macromolecular crowding as a direct apoptotic stimulus

Potassium loss and persistent shrinkage have both been implicated in apoptosis but their relationship and respective roles remain controversial. We approached this problem by clamping intracellular sodium and potassium in HeLa or MDCK cells using a combination of ionophores. Although ionophore treatment caused significant cell swelling, the initial volume could be restored and further reduced by application of sucrose. The swollen cells treated with ionophores remained viable for at least 8 h without any signs of apoptosis. Application of sucrose and the resulting shrinkage caused volume-dependent intrinsic apoptosis with all its classical features: inversion of phosphatidylserine, caspase activation and Bcl-2-dependent release of cytochrome c from mitochondria. In other experiments, apoptosis was induced by addition of the protein kinase inhibitor staurosporine at various degrees of swelling. Our results show that: (1) persistent shrinkage can cause apoptosis regardless of intracellular sodium or potassium composition or of the state of actin cytoskeleton; (2) strong potassium dependence of caspase activation is only observed in swollen cells with a reduced density of cytosolic proteins. We conclude that macromolecular crowding can be an important factor in determining the transition of cells to apoptosis.