166 C
from cells which had been cultured in 4-well chamber of oligonucleosomes was determined as an indicator of
slides and cells washed once in phosphate buffered saline apoptosis. It can be seen that STS induced a 831 increase
PBS. Cells were then fixed for 30–45 min at room in cleavage of the substrate while NMDA produced a
temperature in 4 paraformaldehyde followed by a single modest increase of 225. The difference in the formation
wash with PBS and storage at 48C until staining procedure of nucleosomes when comparing the two agents was much
was performed. An in situ cell death detection kit from less, with STS treatment increasing nucleosome formation
Boehringer Mannheim Indianapolis, IN was used to to 308 of control as compared to 225 for NMDA.
conduct TUNEL staining. Briefly, terminal deoxynu- The finding that LDH leakage occurred with a pro-
cleotidyl transferase is used to catalyze the polymerization apoptotic agent such as STS led us to examine the
of nucleotides to free 39-OH DNA ends. Fluorescein labels timecourse of LDH leakage and caspase activation, and to
incorporated into the nucleotide polymers are detectable by compare it to other inducers of cell death. In Fig. 1A it is
fluorescence microscopy. Cells were permeabilized with shown that over a 20 h time period there is essentially no
0.1 Triton-X 100 for 2 min, washed twice with PBS, and temporal separation of LDH leakage and caspase-3 activa-
incubated with the TUNEL reaction mixture at 378C in the tion in response to STS. Treatment of the cells with 100
dark for 1 h and rinsed with PBS. The chamber walls were mM hydrogen peroxide resulted in LDH leakage of ap-
removed from each slide and one drop of Vectashield proximately 27 of total cellular LDH Fig. 1B. Caspase-
mounting media with DAPI Vector Laboratories, Inc., 3 activity was also elevated but occurred more slowly than
Burlingame, CA was added to each well. A coverslip was LDH leakage. Exposure of the cultures to the nitric oxide
placed on each slide and cells were examined using an oil donor, spermine NONOate, also lead to a similar degree of
immersion lens on a fluorescent microscope at the follow- cell injury as assessed by LDH leakage Fig. 1C. In
ing wavelengths: TUNEL staining, excitation 450 nm, contrast to STS and peroxide treatment, no caspase-3
emission 565 nm; DAPI staining, excitation 360 nm, activity was detected. We have also determined that no
emission 460 nm. oligonucleosomes are generated via NONOate exposure
which also implies a non-apoptotic death with this agent data not shown. These data suggest that caspase-3
3. Results activity is not simply activated non-specifically in response
to injury and that LDH leakage cannot be used to 3.1. Acute cytotoxicity
distinguish necrosis from apoptosis. This finding is con- sistent with Grotton et al. [10] who utilized LDH leakage
Treatment of rat cortical neurons with STS or NMDA in primary mouse cortical neurons as a marker of apop-
for 20 h resulted in a similar 22–24 leakage of total tosis.
cellular LDH Table 1. Immunostaining of similar cul- tures and quantitation of fluorescence suggested that 40–
3.2. Cytotoxicity studied over 48 h 45 of the cells stained positive for neuron specific
enolase results not shown; thus, roughly 50 of the total Considering the indications in the literature that STS
neurons demonstrated LDH leakage during this time induced cell death via apoptotic mechanisms and NMDA
frame. Caspase-3 activity was measured using cleavage of via necrotic mechanisms, it was surprising that the differ-
Ac-DEVD-AFC while endonuclease-dependent formation ence in the magnitude of oligonucleosome formation
Table 1
a
Effect of 20 h treatment with staurosporine STS or NMDA on rat cortical cell cultures on LDH leakage, nucleosome formation, and caspase-3 activity Treatment
500 nM 500 mM
500 nM 500 mM
500 nM 500 mM
STS NMDA
STS NMDA
STS NMDA
Experiment Total
Total Control nucleosome
Control caspase-3 LDH
LDH formation
activity 1
24 20
293 208
697 172
2 19
13 258
209 977
353 3
24 24
373 243
864 221
4 25
28 325
216 980
224 5
28 24
289 249
635 137
Mean 24
22 308
225 831
221 S.E.M.
61 63
620 69
671 637
a
Cells were treated by removing the cell culture media and replacing with a serum-free balanced salt solution containing 500 nM staurosporine, 500 mM NMDA, or vehicle 0.5 DMSO which remained for 20 h. LDH concentration in the media was monitored as a measure of cell membrane integrity,
nucleosome formation was measured in cell lysates by an ELISA as a measure of apoptosis, and caspase-3 activation was measured in cell lysates by the use of the fluorogenic substrate Ac-DEVD-AFC.
C .E. Thomas, D.A. Mayle Brain Research 884 2000 163 –173
167
Fig. 2A, it can be seen that with NMDA, LDH leakage of 48 units that occurs between 20 and 48 h is comparable to
the 57 unit increase in control cells. This contrasts with STS treatment where LDH leakage increases by 135 units
during the 20–48 time period. In a similar manner, oligonucleosomes are not increased at 48 h relative to 20 h
with NMDA treatment; whereas with STS, oligonucleo- some formation was 370 and 880 of control at 20 h
Fig. 1. The effect of staurosporine, hydrogen peroxide or spermine NONOate on LDH leakage and caspase-3 activation in cortical cultures.
Cells were treated by removing the cell culture media and replacing with a serum-free balanced salt solution containing 500 nM staurosporine
STS, 100 mM hydrogen peroxide H O , 1 mM spermine NONOate,
2 2
or vehicle 0.5 DMSO which remained for up to 20 h. LDH concentration in the media was monitored as a measure of cell membrane
integrity and caspase-3 activation was measured in cell lysates by the use of the fluorogenic substrate Ac-DEVD-AFC. A effect of STS treatment
over time; B effect of H O treatment over time; C effect of spermine
2 2
Fig. 2. Comparison of cell damage elicited by NMDA or staurosporine at NONOate treatment over time. Data points represent an average of
20 and 48 h of treatment. Cells were treated by removing the cell culture samples taken from 6 cell culture wells.
media and replacing with a serum-free balanced salt solution containing 500 nM staurosporine, 500 mM NMDA or vehicle 0.5 DMSO which
remained for 20 or 48 h. A LDH concentration in the media was
between STS and NMDA-treated neuronal cultures was not
monitored as a measure of cell membrane integrity; B nucleosome
greater. Also surprising was that the LDH leakage was
formation was measured in cell lysates by an ELISA as a measure of
comparable, especially considering the magnitude of the
apoptosis; C caspase-3 activation was measured in cell lysates by the
difference in caspase activity. Therefore, the effect of 48 h
use of the fluorogenic substrate Ac-DEVD-AFC. Bars represent
exposure to STS or NMDA on the cells was studied. In
mean6standard deviation of three samples.
168 C
and 48 h, respectively Fig. 2B. Caspase-3-like activity death in response to STS, the cells which were sensitive to
was elevated 560 at 20 h in response to STS, but killing by NMDA were eliminated by treatment for 4 days
declined to control level by 48 h Fig. 2C. The decline at with 500 mM NMDA. Table 2 shows the effect of pre-
the later timepoint could be due to loss of measurable treatment of the neurons with NMDA. LDH leakage was
activated protease as a result of cell lysis and release of the completely prevented by inclusion of the NMDA channel
protease into the media. A comparatively modest 82 blocker MK-801 which demonstrates elimination of only
increase in caspase-3 activity was observed upon NMDA NMDA-responsive neurons. As described earlier, there is
treatment after 20 h, this activity was decreased to 41 of little additional LDH leakage in cells exposed to NMDA
control at 48 h. for 20 h as compared to 48 h NMDA treatment Fig. 2A.
Morphological changes observed following STS treat- Furthermore, if cells were pretreated with the NMDA for 4
ment support the progression of apoptosis between 20 and days, washed and exposed again to NMDA, the cells which
48 h. Fig. 3 shows a dramatic increase in the relative survived the initial NMDA pre-treatment were refractory
number of apoptotic bodies which demonstrated positive as indicated by similar levels of LDH leakage as control
TUNEL staining between 20 and 48 h of STS treatment. cells at both 20 and 48 h, suggesting the complete
Co-staining with DAPI showed that control cells retain a depletion of NMDA-sensitive neurons in those cultures
normal nuclear morphology with no evidence of TUNEL results not shown.
staining data not shown. A detailed time course study was performed in cells
which received NMDA pre-treatment and were sub- 3.3. Effect of NMDA pre-treatment on cell death
sequently exposed to STS for 48 h. Staurosporine treat- ment alone yielded the biphasic response with a 163 unit
In an effort to ascertain the nature of the subpopulation increase in LDH leakage, while oligonucleosomes in-
of neurons which undergoes an apparent delayed apoptotic creased from 558 to 1794 of control between 20 and
Fig. 3. TUNEL staining of neurons exposed to staurosporine for 20 or 48 h. Cells were treated by removing the cell culture media and replacing with a serum-free balanced salt solution containing 500 nM staurosporine or vehicle 0.5 DMSO which remained for 20 or 48 h. Cells were then fixed,
permeabilized,and incubated with TUNEL reaction mixture as indicated in the Materials and methods section. Cells were visualized on a fluorescent microscope using a 603 oil-immersion objective. A vehicle-treated control cells after 20 h of treatment; B staurosporine-treated cells after 20 h of
treatment; C vehicle-treated control cells after 48 h of treatment; D staurosporine-treated cells after 48 h of treatment.
C .E. Thomas, D.A. Mayle Brain Research 884 2000 163 –173
169 Table 2
a
Effect of 4-day pre-treatment with 500 mM NMDA on rat cortical cell cultures Pre-treatment condition
LDH leakage U L between day 24 and day 0 Mean6S.D.
None 16.764.7
500 mM NMDA 80.068.9
15 mM MK8011500 mM NMDA 10.060.0
15 mM MK801 11.061.4
a
Cells were pretreated with 500 mM NMDA, 15 mM MK-801 and 500 mM NMDA, 15 mM MK-801 or vehicle which were added in a small volume directly to the culture media 4 days prior to the initiation of treatment. Cells were treated by removing the cell culture media and replacing with a
serum-free balanced salt solution which remained for 20 h. LDH concentration in the media was monitored as a measure of cell membrane integrity.
48 h Fig. 4. NMDA pre-treatment had little effect on the STS-induced LDH leakage or oligonucleosome formation
up to 20 h. Conversely, prior removal of NMDA-sensitive neurons totally abolished the delayed 20–48 h apoptotic
changes as evidenced by oliognucleosome formation Fig. 4B. Caspase-3 activity was elevated 2300 in STS
treated cells as early as 8 h, was maintained for at least 32 h, and declined by 48 h. Depletion of NMDA-sensitive
neurons totally prevented the rise in caspase-3 like activity. Cells treated only with NMDA for 48 h showed little
caspase-3 activity over the 48 h exposure. Analysis of similar treatment conditions for active caspase-3 by West-
ern blot yielded similar results Fig. 5. Staurosporine alone caused notable formation of active caspase-3 17
kDa and decreased levels of pro-caspase-3, but was unable to generate active caspase-3 in cultures which had
been pretreated to remove NMDA-sensitive neurons. These data provide new evidence that NMDA-responsive neurons
are required for activation of pro-caspase-3 by STS.
To further investigate the selective effects of NMDA pre-treatment on STS-induced apoptosis at 20 and 48 h, the
effect of the NMDA antagonist MK-801 was tested in the experiment depicted in Fig. 6. Again, the delayed cell
injury, as determined by LDH leakage and oligonucleo- some formation, was prevented by NMDA pre-treatment.
The effects of NMDA pre-treatment on caspase-3 activa- tion and delayed apoptosis were totally prevented by the
inclusion of MK-801 during the pre-treatment. Thus, MK- 801 protected cells show a pattern comparable to control
cells exposed to STS wherein there is a late phase release of LDH which is accompanied by oligonucleosome forma-
tion. MK-801 treatment by itself had no effect on STS- induced cell killing.
Fig. 4. Timecourse effect of NMDA pre-treatment on staurosporine- induced neuronal cell injury. Cells were pretreated with 500 mM NMDA
4. Discussion
