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American Journal of Pathology, Vol. 139, No. 2, August 1991
Copyight © American Association of Pathologists
Rapid Communication
Analysis of TNFot-induced DNA Strand
Breaks at the Single Cell Level
Karin Fehsel, Victoria Kolb-Bachofen, and
Hubert Kolb
From the Diabetes Research Institute, University of
ganization of metaphase chromosomes related to banding patterns either by DNAse or restriction enzyme
digestion.13-16
Dusseldorf, Dusseldorf Federal Republic of Germany.
Materials and Methods
Treatment ofL929 cells with TNFac initiates apoptosis
and subsequent cell death. The authors have visualized sites of DNA damage in situ by using DNA polymerase to synthesize new strands from the DNA
strands breaks as starting point. Biotin-dUTP was incorporated into the newly synthesized strand and visualized by immunocytochemistry. DNA strand
breaks were first observed 3 to 4 hours after contact
with TNFa andpreceded cell death. Limiting doses of
TNFa caused DNA strand breaks only in a subpopulation of L929 cell& At a low dose, TNFa led to DNA
damage without any subsequent loss of cell viability.
The new assay also detects DNase-induced single
strand breaks and thus is able to visualize apoptotic
as well as non-apoptotic types of DNA damage. (Am
JPathol 1991, 139:251-254)
Apoptosis is a suicide process of programmed cell death
in response to specific stimuli characterized by extensive
cleavage of chromatin. The latter is believed to be due to
the activation of an endogeneous endonuclease, which
results in DNA cleavage.1 Apoptosis occurs physiologically in several tissues-7 and in cells that are attacked by
cytotoxic lymphocytes.810 Also TNFa induces apoptosis
in HeLa- and L929 cells.1" The production of oligonucleosome-length DNA fragments can either be resolved by
agarose gel electrophoresis or by the determination of
the amount of previously incorporated 3H-thymidine in
the 27,000 xg supernatant of lysed cells.12 For both methods 105 to 107 cells are needed.
In this study, we have adapted the method of in situ
nick translation of visualize DNA strand breaks during
apoptosis at the single cell level. In situ nick translation is
a usual procedure to study the higher-order regional or-
The mouse fibroblast cell line L929 was grown on glass
slides (Chamber Tecs, Nunc, Roskilde, Denmark) in
RPMI 1640 medium with 10% FCS at 37C in an atmosphere of 7% CO2 in air in a humidified incubator. Per
chamber 2000 cells were allowed to adhere over night
followed by the addition of different concentrations of recombinant murine TNFa (Genzyme, Boston, MA). The
cells were incubated for 1, 2, 3, 4, 5, 16, and 24 hours.
The TNFa-treatment was stopped by washing the slides
in TNFa-free medium and air drying. On parallel slides,
the viability of the cells was determined by exclusion of
Trypan blue dye. Subsequently, the cells were fixed in
acetone for 10 minutes. Endogenous peroxidase activity
was inhibited by incubating the slides in methanol plus
0.3% H202 for 30 minutes.
In situ nick translation was done as described.14 The
nick translation mixture (all components from Boehringer,
Mannheim, Germany) contained 3 p.M biotinin-dUTP; 4
U/1 00 ,ul Kornberg polymerase, 3 p.M each dGTP, dCTP,
dATP; 50 mM Tris-HCI pH 7.5; 5 mM MgCI2; 0.1 mM
dithiothreitol. The reaction was performed in a volume of
50 ,ul at room temperature for 9 minutes. The slides were
washed 3 times in PBS for 5 minutes each and processed for immunocytochemical detection of biotin either
directly or after dehydration in ethanol (30, 50, 70, 80,
90%, 2 min each). To block unspecific antibody binding,
the slides were incubated in PBS with 0.1% Thimerosal
and 10% FCS for 10 minutes at room temperature. The
Supported by the Bundesminister fOr Jugend, Familie, Frauen und Gesundheit and by the Minister fOr Wissenschaft und Forschung des Landes
Nordrhein-Wesffalen.
Accepted for publication May 22, 1991.
Address reprint requests to Dr. Karin Fehsel, Diabetes Research Institte, Auf'm Hennekamp 65, D-4000 Dusseldorf, FRG.
251
252
Fehsel, Kolb-Bachofen, and Kolb
AJP August 1991, Vol. 139, No. 2
incorporated biotin-dUTP was visualized by the peroxidase reaction (Vecastain kit, Camon, Wiesbaden, Germany) using DAB as enzyme substrate. The slides were
mounted in Eukitt (Kindler GmbH, Freiburg, Germany) for
light microscopy. Stained cells were determined by
counting all cells within adjacent microscopic fields (100
per chamber). In each experiment, tests were done in
duplicate.
Results
Cultures of L929 cells were treated with increasing concentrations of recombinant murine TNFa and assayed for
DNA fragmentation at 16 hours (Figure 1). While nuclei
treated with DNase are homogenously stained, TNFa
obviously attacks only distinct cells. With rising TNFa
concentrations, the amount of staining and the percentage of stained cells increase. At highest TNFa concentration, cell lysis is observed. Control slides with shamtreated cells and processed in parallel revealed only
trace amounts of labelling (Figure 1 A).
Figure 2 shows the sequence of TNFa-induced cell
killing. DNA strand breaks become detectable about 3
hours after incubation with TNF (Figure 2B). In the following 4 hours, the cells do not change morphologically,
whereas DNA fragmentation in the nucleus proceeds
(Figure 2C). Finally, the nucleus membrane disappears
and brown-stained chromatin spreads into the cytoplasm, concomitantly cell lysis becomes apparent (Figure 2D).
A quantitative analysis of cells with DNA strand breaks
is given in Table 1. The parallel analysis of cell viability
showed that a significantly higher percentage of L929
cells had DNA damage than loss of viability. In fact, only
the highest TNFa concentration caused cell death during
the observation period, whereas DNA strand breaks
were seen at all TNFa doses.
Discussion
The method that was described allows for the first time
the analysis of DNA strand breaks at the level of single
Figure 1. L929 cells treated with different TNFa concentrationsfor l6hours. (A) untreated cells, (B) 5.8 ng/ml TNFa, (C) 11.6 ng/ml TNFar,
(D) TNFa untreated cells incubated with DNase I (50 ng/ml; Boebringer) for 9 minutes as described in Materials and Methods, x800.
Figure 2. Characterstic phases of apoptosis in L929 cells induced by TNFa (5.8 ng/ml). (A) untreated cells, (B) cells treatedfor 3 hours, (C)
cells treated for 8 hours, (D) cells treated for 16 hours, x 1730.
Single Cell Analysis of DNA Strand Breaks
253
AJP August 1991, Vol. 139, No. 2
Table 1. Dose Dependency of DNA Strand Breaks and
Cell Death in TNFa-treated L929 Cells
TNFa
Percentage of Cells* with
cells with
loss of
concentration
(ng/ml)
DNA damage dye exclusion
0
2.9
5.8
11.6
<3
14.4 ± 2.3
15.8 ± 1.3
37.7 ± 2.2
<3
4.8 ± 0.2
5.0 ± 0.7
28.8 ± 2.1
P < 0.005
P < 0.001
P < 0.001
Per experiment 100 cells on two separate slides were counted.
Data are mean values of 4-5 experiments + SD.
cells. In situ nick translation has been used previously by
several groups to visualize sites of damage in chromosomes induced by various DNA binding enzymes.1'17
The background staining of normal nuclei by the nick
translation procedure is extremely low"5 as also found
here with L929 cells. This fact allows the detection of
TNFa-induced DNA strand breaks within 3 hours. In situ
nick translation can be started at single and doublestrand breaks, whereas the detection of DNA fragments
by physicochemical methods needs double-strand
breaks, i.e., the method will also detect DNA damage
characterized by extensive single-strand breaks only,
such as induced by DNase treatment on nuclei (Figure
1 D). It is not known whether such conditions occur naturally and what the consequences would be in terms of
subsequent DNA cleavage and cell death. Pharmacologically induced single-strand breaks appear to be cytotoxic via DNA fragmentation and apoptosis.1819 The
method that was described also detects DNA strand
breaks in single cells induced by alkylating agents or
oxygen radicals (Fehsel et al., to be published). Thus
apoptotic as well as non-apoptotic types of DNA damage
are visualized.
During the evaluation of this method, further insight
into the mechanism of TNFa induced DNA strand breaks
could be gained. We found that the response of L929
cells is not homogenous but that with the TNF doses that
were used only a subpopulation of cells reacted with
DNA damage. Furthermore, DNA strand breaks were induced at concentrations of TNFa, which were not toxic.
This indicates that DNA damage during early apoptosis
can be repaired with concomitant escape of cells from
programmed suicide. This view is supported by a previous report that TNFa induces ADP-ribosylation, an enzymatic activity that is associated with DNA repair.20 The
fact that low doses of TNFa desensitize to the lethal effects of TNFo&9 also indicate the presence of defense
mechanism against the apoptotic cell response.
Preliminary results show that the method of in situ
translation also detects DNA strand breaks in tissue sections such as occurring in a large number of thymocytes
(Fehsel and Kolb, manuscript in preparation).
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