PEROXIDE-GENERATED APOPTOSIS INDUCTION IN HEPATOCYTES FROM LONG LIVING AMES DWARF MOUSE
M.A. Kennedy*, S.G. Rakoczy, H.M. Brown-Borg
University of North Dakota School of Medicine and Health Sciences
Department of Pharmacology, Physiology and Therapeutics
501 North Columbia Road Grand Forks, North Dakota 58203
The Ames dwarf mouse is a mammalian model of extended life span, living
approximately 50% longer than its wild type siblings. It exhibits
upregulated antioxidant defenses and lower oxidative DNA and protein
damage compared to age-matched wild type littermates. The purpose of
this experiment was to investigate the response of dwarf versus wild
type mice to oxidative stress in culture. We examined the induction of
apoptosis in hepatocytes from 6-month-old dwarf and wild type mice
following a 30-minute treatment with hydrogen peroxide (H2O2) or
saline. Hepatocytes were isolated following collagenase perfusion and
cultured at 37 degrees C in 5% CO2 for 18 hours following peroxide
challenge. Cells were collected and assayed for
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
conversion to formazan, DNA fragmentation, and caspase-3 activity. In
addition, protein levels of procaspase-3 and Bcl-2 were determined.
The MTT assay showed a reduction in cell numbers following H2O2
treatment. DNA laddering was evident in treated cells indicating the
induction of apoptosis in both dwarf and wild type hepatocytes. Levels
of caspase-3 activity showed a sixfold increase in dwarf cells treated
with H2O2 versus those treated with saline, whereas a fourfold increase
in caspase-3 activity was observed in wild type cells treated with H2O2
over those treated with saline alone. Untreated dwarf hepatocytes
consistently exhibited higher procaspase-3 protein levels when compared
to corresponding untreated wild type hepatocytes. These data suggest
that hepatocytes from the dwarf mouse may be more readily induced to
undergo apoptosis; therefore, these mice may eliminate damaged cells
more efficiently than their age-matched wild type counterparts.
Key words:
dwarf, apoptosis, hydrogen peroxide
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