1: Antimicrob Agents Chemother. 2006 Apr;50(4):1213-21. 

Gentamicin causes apoptosis at low concentrations in renal LLC-PK1 cells
subjected to electroporation.

Servais H, Jossin Y, Van Bambeke F, Tulkens PM, Mingeot-Leclercq MP.

Unite de Pharmacologie Cellulaire et Moleculaire, Universite Catholique de
Louvain, 7370 avenue E. Mounier 73, B-1200 Brussels, Belgium.

Gentamicin accumulates in the lysosomes of kidney proximal tubular cells and
causes apoptosis at clinically relevant doses. Gentamicin-induced apoptosis can
be reproduced with cultured renal cells, but only at high extracellular
concentrations (1 to 3 mM; 0.4 to 1.2 g/liter) because of its low level of
uptake. We recently showed that gentamicin-induced apoptosis in LLC-PK1 cells
involves a rapid (2-h) permeabilization of lysosomes and activation of the
mitochondrial pathway of apoptosis (10 h). We now examine whether the delivery
of gentamicin to the cytosol by electroporation would sensitize LLC-PK1 cells to
apoptosis. Cells were subjected to eight pulses (1 ms) at 800 V/cm (square
waves) in the presence of gentamicin (3 microM to 3 mM; 1.2 mg/liter to 1.2
g/liter); returned to gentamicin-free medium; and examined at 8 h for their Bax
(a marker of mitochondrial pathway activation) contents by Western blotting and
competitive reverse transcriptase PCR and at 24 h for apoptosis by
4',6'-diamidino-2'-phenylindole staining (confirmed by electron microscopy) and
for necrosis (by determination of lactate dehydrogenase release).
Nonelectroporated cells were incubated with gentamicin for 8 and 24 h.
Significant increases in Bax levels (8 h) and apoptosis (24 h) were detected
with 0.03 mM (13.2 mg/liter) gentamicin in electroporated cells compared with
those achieved with 2 mM (928 mg/liter) in incubated cells. The increase in the
Bax level was not associated with an increase in the level of its mRNA but was
associated with the accumulation of ubiquitinated forms (probably as a result of
impairment of its degradation by the proteasome). Assay of cell-associated
gentamicin showed a marked, immediate, but transient accumulation in
electroporated cells, whereas a slow, steady uptake was detected in incubated
cells. The data indicate that cytosolic gentamicin triggers apoptosis.
Sequestration of gentamicin in lysosomes would, to some extent, protect against

PMID: 16569831 [PubMed - in process]