A new approach for the electrophoretic detection of apoptosis
A new approach for the electrophoretic detection of apoptosis
Basil A.
Eldadah
,
Alexander G.
Yakovlev
and
Alan I.
Faden*
Department of Neurology and the Georgetown Institute for Cognitive and
Computational Sciences, Georgetown University Medical Center, NRB Room EP20,
3970 Reservoir Road, NW,
Washington
, DC 20007,
USA
Received July 21, 1996
;
Revised and Accepted September 2, 1996
ABSTRACT
Apoptotic cell death is often characterized by internucleosomal cleavage of genomic DNA, which exhibits a distinctive ladder upon electrophoresis. However, techniques used for the
isolation and detection of DNA to demonstrate laddering may not be sufficiently
sensitive, particularly when cleaved DNA is present at modest levels. We
propose a new approach for isolating total cellular DNA using a silica-based resin that improves the resolution of DNA laddering. In addition, we
introduce a rapid DNA labeling method that can increase the sensitivity of
detecting DNA laddering. Each of these methods can be used for DNA from cell
cultures or tissues.
Apoptosis is a form of cell death distinct from coagulative necrosis in which
cells are deleted in a focal, type-specific manner under certain physiological or pathological conditions (
1
,
2
). This process is characterized morphologically by cell shrinkage, membrane blebbing, and nuclear condensation, after which cells are fragmented and phagocytosed by neighboring cells (
3
). During apoptosis, these morphological changes are often accompanied by
internucleosomal cleavage of genomic DNA, which when electrophoresed shows a characteristic ladder consisting of 180 bp multimeric bands (
4
).
Although in some experimental models apoptotic DNA fragmentation is widespread
and easily detected, in other cases apoptosis may be found in only a limited
number of cells and, consequently, DNA fragmentation may be masked by necrotic and/or undegraded DNA. In the latter case, conventional ethidium bromide staining of DNA may not be sufficiently sensitive to reveal a classical
DNA ladder. Southern blot hybridization has been used in such cases to detect
low levels of apoptotic DNA fragmentation; however, hybridization is time-consuming, requires the use of specific DNA probes, and may give rise to high background
signals (
5
). Alternatively, the sensitivity of DNA electrophoretic analysis can be increased by enzymatic radiolabeling of DNA fragments by terminal deoxynucleotidyl transferase (TdT) or the Klenow
fragment of DNA polymerase I (
6
,
7
).
Here we present a new approach to detect internucleosomal DNA cleavage in both
in vitro
and
in vivo
models of apoptosis. This method involves the gentle isolation of genomic DNA
with a silica-based resin, and, where greater sensitivity is required, the labeling of
internucleosomal fragments by
Taq
DNA polymerase.
To demonstrate the utility of this method in an
in vitro
model of apoptosis, we used primary cultures of cerebellar granule cells, in
which apoptosis can be readily induced by withdrawal of serum (
8
) and/or deprivation of depolarizing concentrations of potassium (
9
). Cerebellar granule cells were prepared from 8 day old rat pups as described
previously (
10
), except that cells were plated onto poly-lysine coated 60 mm tissue culture dishes at a density of 1.25 * 10
6
cells per ml and at a volume of 3 ml per dish. On
in vitro
day 7, the culture medium was replaced with fresh medium devoid of serum or supplemented potassium, and incubated at 37oC for 14 h.
To isolate total DNA, the cells of one dish were washed with 3 ml PBS and lysed
in situ
with 2 ml of 7 M guanidine hydrochloride. The lysate was transferred to a 10 ml
culture tube containing 1 ml Wizard Minipreps DNA Purification Resin (Promega),
mixed gently, and centrifuged at 2000
g
for 3 min. The resin-DNA pellet was resuspended in 3 ml of washing solution (90 mM NaCl, 9 mM Tris-HCl pH 7.4, 2.25 mM EDTA, 55% ethanol) and drawn by vacuum through
a 3cc syringe attached to a Wizard Minicolumn (Promega). The column was washed
twice with 3 ml of washing solution and dried by centrifugation over a
microfuge tube at 5000
g
for 5 min. To elute the DNA, 50 [mu]l TE buffer pH 8.0 were added, followed by incubation for 5 min at room
temperature and centrifugation over a new microfuge tube at 5000
g
for 5 min. Residual RNA was removed by addition of 1 [mu]g RNase A and incubation at 37oC for 30 min.
We compared the electrophoretic ladder of resin-isolated DNA with that of DNA isolated by two other commonly used methods
(
11
,
12
). After ethidium bromide staining, all three samples exhibited the characteristic banding pattern of internucleosomally-cleaved DNA (Fig.
1
); however, the most prominent ladder was observed in the resin-extracted sample. This result may be due to the omission of a phenolic
extraction and agitation step which can shear DNA and cause background
electrophoretic smearing. In addition, our technique does not include a
precipitation step in which low molecular weight DNA fragments may be lost.
Finally, because of the apparent differences in the elution rates of different
molecular weights, resin-isolated samples may show an enriched fraction of internucleosomally-cleaved DNA.
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