Cell killing by lysosomotropic detergents

Abstract

We have studied the mechanism by which lysosomotropic detergents kill baby hamster kidney cells. Lysosomotropic detergents are lysosomotropic amines (compounds with pK between 5 and 9, such as imidazole or morpholine) containing straight-chain hydrocarbon "tails" of 9-14 carbon atoms (Firestone, R. A., J. M. Pisano, and R. J. Bonney. 1979, J. Med. Chem., 22:1130-1133). Using lucifer yellow CH as a specific fluorescent label for lysosomes, it was shown by light microscopy that N-dodecyl (C12)-imidazole acted rapidly to damage lysosomes, causing leakage of dye into the cytoplasm. This was followed at later times by vacuolization, blebbing of the plasma membrane, cell rounding, and cell death. 3H-labeled C12-imidazole rapidly diffused into cells where much of it was trapped in lysosomes as shown by its co-migration with lysosomes in Percoll gradients. Cells preincubated with C12-imidazole released it slowly into C12-imidazole-free media, permitting the cells to be killed by the preincubation dose. Cell killing by the lysosomotropic detergents exhibited strongly sigmoidal dose-response curves. The sensitivity of baby hamster kidney cells to killing by C12-imidazole was density dependent, the cells being most sensitive at lowest cell densities, and relatively resistant at confluence. The amount of 3H-C12-imidazole taken up by the cells was also density dependent, with highest specific uptake occurring at the lowest cell density. A rise in lysosomal pH, measured in fluoresceinated dextran-labeled cells, commenced immediately upon addition of C12-imidazole to cells, and continued for over an hour. This was followed after a lag of 1-2 h by inhibition of protein and RNA synthesis and by lactate dehydrogenase release. Ionophores or lysosomotropic amines, such as methylamine, that raise intralysosomal pH provided substantial protection of the cells from killing by lysosomotropic detergents. These findings provide strong support for the idea that lysosomotropic detergents kill cells by disrupting lysosomes from within.