1: Toxicol Appl Pharmacol. 2005 Aug 15;206(3):321-33. 

Gentamicin-induced apoptosis in LLC-PK1 cells: Involvement of lysosomes and

Servais H, Van Der Smissen P, Thirion G, Van der Essen G, Van Bambeke F, Tulkens
PM, Mingeot-Leclercq MP.

Unite de pharmacologie cellulaire et moleculaire, Universite catholique de
Louvain, UCL 73.70 Avenue E. Mounier 73, B-1200 Brussels, Belgium.

Gentamicin accumulates in lysosomes and induces apoptosis in kidney proximal
tubules and renal cell lines. Using LLC-PK1 cells, we have examined the
concentration- and time-dependency of the effects exerted by gentamicin (1-3 mM;
0-3 days) on (i) lysosomal stability; (ii) activation of mitochondrial pathway;
(iii) occurrence of apoptosis (concentrations larger than 3 mM caused extensive
necrosis as assessed by the measurement of lactate dehydrogenase release).
Within 2 h, gentamicin induced a partial relocalization [from lysosomes to
cytosol] of the weak organic base acridine orange. We thereafter observed (a) a
loss of mitochondrial membrane potential (as from 10 h, based on
spectrophotometric and confocal microscopy using JC1 probe) and (b) the release
of cytochrome c from granules to cytosol, and the activation of caspase-9 (as
from 12 h; evidenced by Western blot analysis). Increase in caspase-3 activity
(assayed with Ac-DEVD-AFC in the presence of z-VAD-fmk]) and appearance of
fragmented nuclei (DAPI staining) was then detected as from 16 to 24 h together
with nuclear fragmentation. Gentamicin produces a fast (within 4 h) release of
calcein from negatively-charged liposomes at pH 5.4, which was slowed down by
raising the pH to 7.4, or when phosphatidylinositol was replaced by cardiolipin
(to mimic the inner mitochondrial membrane). The present data provide temporal
evidence that gentamicin causes apoptosis in LLC-PK1 with successive alteration
of the permeability of lysosomes, triggering of the mitochondrial pathway, and
activation of caspase-3.

PMID: 16039943 [PubMed - in process]