(Received for publication, August 3, 1995; and in revised form, September 5, 1995)
From the
Our prior work established that comparable concentrations of N-acetylcysteine (NAC) both block the proliferation of PC12 cells and prevent death of trophic factor-deprived sympathetic neurons and PC12 cells. The present work addresses several aspects of the mechanisms of these actions. NAC increases intracellular levels of glutathione (GSH) by approximately 10-fold in PC12 cells. However, blockade of this increase by treatment with buthionine sulfoximine did not affect either promotion of survival or inhibition of DNA synthesis. Thus, these actions of NAC are independent of its effects on intracellular GSH. NAC's actions in our system do not appear to be dependent on its anti-oxidant/radical scavenger properties, but may be due to its activity as a reductant. Consistent with this, several other reducing agents, the most effective of which was 2,3-dimercaptopropanol, mimicked NAC in blocking DNA synthesis and suppressing death of PC12 cells and sympathetic neurons. Finally, we observed that in striking contrast to nerve growth factor and a number of other trophic agents, the survival-promoting effects of NAC on PC12 cells are blocked by actinomycin D. This suggests that NAC may act by inducing specific gene expression.
Apoptotic cell death is a normal aspect of development as well as a consequence of cellular injury or degeneration(1, 2) . In the nervous system, developmental neuronal apoptotic death appears in part due to competition for limited amounts of target-derived trophic factors(1) . As judged by their palliative effects when administered in experimental models, trophic factors may also be involved in regulation of nerve cell death associated with brain injury or neurodegenerative disease (3, 4, 5) . In an effort to understand the basic mechanisms by which trophic agents prevent death of neuronal and non-neuronal cells and to devise drugs that may be useful for amelioration of maladies characterized by cell death, extensive efforts have been made to identify small molecules that mimic the survival-promoting actions of trophic factors.
Two
model systems that have been used to study regulation of neuronal cell
death by trophic factors and small molecules are cultured neonatal
sympathetic neurons and the PC12 pheochromocytoma cell line. Cultured
neonatal rat sympathetic neurons can be maintained in the presence of
the trophic factor NGF ()and undergo apoptotic death when
the factor is withdrawn(6, 7, 8) . PC12 cells
do not require NGF when grown in serum-containing medium, but rapidly
die by an apoptotic mechanism when deprived of
serum(9, 10, 11, 12) . Under such
serum-free conditions, NGF prevents PC12 cell
death(9, 10) ; conversely, withdrawal of NGF under
serum-free conditions triggers PC12 cell apoptotic death(12) .
Exploitation of the sympathetic neuron and PC12 cell systems has uncovered a variety of small molecules that mimic NGF in preventing death(11, 13, 14) . Recently, we reported that N-acetylcysteine (NAC) effectively maintains the long term survival of sympathetic neurons and PC12 cells in the absence of NGF (14) . NAC also has been found to have survival promoting actions in several other cell systems(15, 16, 17) . Exposure of cells to NAC leads to a large increase in intracellular levels of glutathione (GSH)(18) , and it has been generally assumed that the survival-promoting actions of NAC are due to its direct or indirect (via intracellular GSH) action as an anti-oxidant or free radical scavenging agent.
In our prior work(14) , we made several observations that are relevant to the mechanism by which NAC might prevent cell death caused by withdrawal of trophic support. First, NAC was unexpectedly found to inhibit PC12 cell proliferation with a dose-response curve similar to that for which it prevents cell death. This raised the possibility that capacity of NAC to prevent cell death is due to its anti-proliferative activity and is consistent with the hypothesis that death of PC12 cells and sympathetic neurons (as well as of other cell types) caused by trophic factor withdrawal is due to an aberrant attempt to complete or re-enter the cell cycle(19, 20, 21, 22) . Second, several other anti-oxidants and free radical scavenging agents did not share with NAC the capacity to prevent PC12 cell and sympathetic neuron death or to inhibit DNA synthesis. This suggested a mechanism for NAC other than as an anti-oxidant or free radical scavenger. Third, the D-stereoisomer of NAC, which should not be metabolized, was as effective as L-NAC in preventing apoptosis and blocking proliferation, thereby indicating that a metabolic product of NAC was not responsible for these actions. Last, both L- and D-NAC increased PC12 cell levels of GSH by at least 10-fold. This showed that the effect of NAC on GSH levels was not via direct metabolic conversion as suggested previously, but rather by reduction of extracellular cystine to increase available levels of cysteine, a precursor amino acid of GSH. This observation also raised the possibility that NAC actions, as often suggested, are mediated by GSH.
In the present work, we have continued our investigations of the mechanism of NAC actions. We directly tested whether the induced increase in intracellular GSH is indeed required for the survival and anti-mitotic properties of NAC. We also ascertained whether NAC's actions might be related to its effectiveness as a reducing agent. Last, we determined whether RNA synthesis is required for NAC's survival-promoting activities.
For survival experiments, PC12 cells were washed
extensively with serum-free RPMI 1640 medium as described previously (10) and plated in 0.5 ml of medium in collagen-coated 24-well
plates at a density of 10-15 10
/well. To feed
the cells, and to avoid loss of floating live cells, 0.2 ml of fresh
medium was added every other day.
Primary cultures of dissociated
sympathetic neuronal cultures were obtained from the superior cervical
ganglia of P1-P2 rat pups, dissociated in 0.25% trypsin, and plated in
collagen-coated 24-well dishes at the density of 0.5 ganglion/well
in RPMI 1640 medium supplemented with 10% horse serum and mouse NGF
(100 ng/ml)(24) . A mixture of uridine and fluorodeoxyuridine
(10 µM) was added on the second day to eliminate
non-neuronal cells. After 3-5 days, the cultures were washed
three times with RPMI medium plus 10% horse serum to remove the mouse
NGF. The wells were then cultured in the same medium (0.5 ml/well),
with the indicated additives, in the presence of NGF or anti-NGF rabbit
antibodies at a dilution of 1:200.
For sympathetic neurons, cell counts were carried out by strip counting (26) . Briefly, diametric strips corresponding to 10% of the dish area, were examined under high-power phase-contrast microscopy. Cells with neuron-like morphology that were located on this strip were scored. The same diametric strip was analyzed on each day. Each well was scored individually and the survival expressed relative to the initial cell number in the same well (designated as 100).
Figure 1:
BSO
inhibits the NAC-induced increase in GSH levels (A) but has no
effect on NAC-promoted cell survival (B) or
[H]thymidine incorporation (C). For the
GSH measurement, cells were cultured for 1 or 3 days in serum-free
medium, with either no additive, NGF (100 ng/ml), 60 mM LNAC,
or 60 mM LNAC + 0.2 mM BSO. For the survival
assay, cells were treated for 1 or 3 days with either NGF, 60 mM LNAC, or 60 mM LNAC + 5 mM BSO. Thymidine
incorporation were carried in medium containing full serum, and the
cells were treated for 1 day with either NGF, 60 mM LNAC, or
60 mM LNAC + 5 mM BSO. Results are expressed as
a percentage relative to control, and error bars represent
S.E. (n
3).
Figure 2:
Morphology of naive PC12 cells treated in
serum-free RPMI 1640 for 3 days with the following compounds: no
treatment (A), 100 ng/ml NGF (B), 60 mM LNAC (C); 60 mM NAC + 0.2 mM BSO (D). Magnification: 215.
Our past work (14) showed that the same concentrations of NAC that prevent
PC12 cell apoptosis also inhibit DNA synthesis. To test whether the
latter effect requires elevation of GSH levels, we assessed
incorporation of [H]thymidine in PC12 cell
cultures treated with NAC alone or with NAC plus BSO. Fig. 1C shows that BSO did not affect NAC-induced
inhibition of DNA synthesis. Taken together, these findings indicate
that elevation of intracellular GSH does not account for the mechanism
by which NAC rescues cells from death or inhibits their proliferation.
Figure 3:
Reducing agents maintain survival of PC12
cells and inhibit their [H]thymidine
incorporation. A, survival of PC12 cells after 24 h of
treatment in serum-free medium with either 100 ng/ml NGF, 150
µM DMP, 15 mM thioglycolate, or 1 mM BME. B, relative [
H]thymidine
incorporation after 24 h of treatment with the same compounds as above
in medium supplemented with 3 µM insulin. Results are
expressed as relative to control levels ± S.E. (n = 3).
Figure 4: DMP delays the death of PC12 cells. PC12 cells were cultured in medium without serum and treated with NGF, 150 µM DMP, NGF + DMP, or no additives for the indicated days. Error bars represent S.E.
Our past experiments suggested that withdrawal of trophic
support leads to apoptosis by resulting in uncoordinated cell cycle
progression and showed correlation between the capacity of NAC to block
cell death and to inhibit DNA synthesis. We therefore tested whether
other reducing agents also affect cell division as assessed by
inhibition of [H]thymidine incorporation. PC12
cells were cultured in serum-free medium with insulin, which supports
proliferation and full survival, and treated with the reducing agents
at their respective optimal concentrations. As shown in Fig. 3B, thioglycolate and DMP, which were the most
effective in maintaining cell survival at 24 h of serum deprivation,
also inhibited a substantial fraction of thymidine incorporation at 24
h of treatment. When the potencies of DMP for promoting survival and
for suppressing DNA synthesis at 24 h were examined in further detail,
this revealed a reciprocal relationship with maximal survival occurring
at concentrations yielding maximal inhibition of thymidine
incorporation (Fig. 5). In contrast, BME, which was relatively
ineffective in promoting survival, had relatively little effect on DNA
synthesis (Fig. 3B). These findings indicate that as
for NAC, there is a good correlation between the capacities of several
reducing agents to promote survival and to inhibit the synthesis of
DNA.
Figure 5:
Dose-response curves for DMP-promoted
survival and inhibition of [H]thymidine
incorporation in PC12 cells. The results were obtained after 24 h of
treatment. Cells for survival were cultured in RPMI 1640 without serum,
and cells for [
H]thymidine incorporation were
cultured in RPMI 1640 supplemented with 3 µM insulin.
Results are expressed as relative to untreated cells ± S.E. (n = 3).
, relative number of surviving cells;
, relative [
H]thymidine
incorporation.
We next tested whether DMP, like NAC, can suppress the death of sympathetic neurons brought about by withdrawal of NGF. In this system, the highest concentration of DMP that did not cause morphological changes (i.e. cell detachment from the substrate) in the presence of NGF was 85 µM. As shown in Fig. 6and Fig. 7, at this concentration, DMP significantly delayed neuronal death by up to 5-12 days; we observed the same effect at the concentrations of 50 and 75 µM. However, as in the case of NAC, DMP did not elicit or maintain neurite outgrowth, nor did it interfere with NGF's ability to promote neurite outgrowth.
Figure 6: DMP delays the death of sympathetic neurons after withdrawal of NGF. Primary cultures of rat superior cervical ganglion neurons were deprived of NGF after 3 days in culture and treated with mouse NGF, NGF + 85 µM DMP, DMP, or no treatment. Cell counting was carried out by strip count (26) on the indicated days. Counts were done on triplicate wells and expressed relative to day 0 counts ± S.E.
Figure 7:
DMP
maintains cell survival but not neurite outgrowth. Primary cultures of
rat superior cervical ganglion neurons were deprived of NGF after 6
days in culture and treated with anti-NGF antiserum (A), mouse
NGF (B) 50 µM DMP and anti-NGF (C), 50
µM DMP and NGF for 12 days (D). Magnification:
190.
Figure 8: NAC requires RNA synthesis to promote survival. PC12 cells were washed free of serum, pretreated with 10 µM actinomycin D for 90 min and then treated for 24 h with 100 ng/ml NGF, 60 mM LNAC, or 100 µM chlorophenylthio-cAMP with the continued presence of actinomycin D where indicated. Survival is expressed as relative to control ± S.E.
One well
described property of NAC is to increase intracellular levels of
GSH(18) , and its protective actions have often been attributed
to this effect(30, 43) . We show here that BSO
treatment completely blocks the NAC-induced increase in intracellular
GSH, but has no effect on the capacity of NAC to protect PC12 cells
from apoptotic death or to inhibit their synthesis of DNA. Thus, in
this system, enhanced GSH levels cannot account for NAC's
actions. This conclusion is consistent with several other observations
in the literature. Jones et al.(31) recently reported
that BSO treatment of T cells blocked approximately half of the
increase in GSH levels generated by exposure to NAC, but had no effect
on the capacity of NAC to protect these cells from apoptosis induced by
exposure to anti-CD3 antibodies. NAC has been shown to inhibit viral
replication and in this regard, Mihm et al.(32) showed that although exposure of Molt-4 cells to NAC
or to GSH brought about comparable increases in intracellular GSH
levels, only NAC suppressed cellular production of HIV. NAC also
protects cells from apoptosis induced by Sindbis virus in N18
neuroblastoma cells, and this effect appears to be independent of
increased intracellular GSH. ()
Another possibility is that a metabolic product of NAC mediates its actions in our experiments. However, this appears to be ruled out by the findings that the D-stereoisomer of NAC, which is presumably not metabolized, fully maintains cell survival and inhibits proliferation(14) .
An additional mechanism we considered is that NAC effects are due to its actions as a reducing agent. The efficacy of NAC as a reducing agent in our system is reflected by our observation that D-NAC enhances intracellular GSH levels as well as L-NAC(14) , indicating that it reduces extracellular cystine to cysteine. To assess this notion, we tested several other reducing agents in our paradigm. Both thioglycolate and dimercaptopropanol suppressed apoptotic death and inhibited DNA synthesis. Like NAC, DMP was also found to delay death of NGF-deprived sympathetic neurons and showed close correlation of the dose-response curves for promotion of survival and inhibition of thymidine incorporation. BME, in contrast, was less effective at both preventing death and suppressing synthesis of DNA. These findings are consistent with prior observations regarding the actions of reducing agents and indicate that the efficacy of a given reducing agent may vary with cell type(34, 35, 36, 37) . For instance, BME has been reported to enhance the survival of neurons cultured in serum-free medium (36, 37) and to decrease concanavalin A-induced proliferation of human lymphocytes (34) .
Our observations with reducing agents raise several points. One is that none of the agents we have tested is as effective as L- or D-NAC in maintaining long term survival. The reason for this is unclear, but could include factors such as kinetics of entry and accumulation in cells, reducing potential or additional activities. A second point is that our data provide further correlation between the capacities of agents to inhibit DNA synthesis and to promote neuronal cell survival. Thioglycolate and DMP suppressed both death and thymidine incorporation, while BME was only partially effective in each case. This correlation supports the hypothesis that apoptosis caused by withdrawal of trophic support is due to an inappropriate attempt to re-enter or progress through the cell cycle. Finally, it must be acknowledged that the reducing agents we employed are also anti-oxidants. Thus, we cannot de facto rule out that this property contributes to their activities in our experiments (but see above).
The mechanism by which
NAC might alter transcription to promote survival is presently unclear.
One possibility is via reduction and ligand-independent activation of
receptors for growth factors. We tested this for the Trk NGF receptor
and found that NAC promoted the survival of PC12nnr5 cells despite
their deficiency in Trk expression. ()The respective
contrast between the transcription-dependent and -independent survival
promoting activities of NAC and growth factors appears to further rule
out this mechanism.
An attractive alternative mechanism by which NAC
might affect synthesis is by alteration of the cellular redox state
which in turn might alter the activity of specific transcription
factors. A number of transcription factors including c-Fos,
c-Jun, NF-B, and NFI contain specific cysteine residues
that must be in a reduced state to permit DNA
binding(39, 40, 41) . Incubation of cellular
extracts with BME has been reported to enhance the DNA binding activity
of AP-1 and NF-
B, presumably by reducing oxidized forms of these
factors(42) . In addition, 30 mM NAC has been found to
stimulate AP-1 activity in HeLa cells probably by inducing c-Fos and c-Jun synthesis(43) , while 30 and 50 mM NAC were shown to enhance c-Jun and c-Fos transcripts in rat lens cells(44) . It is of interest that
5 mM NAC, which is without efficacy in our systems, was unable
to stimulate induction of c-Jun and c-Fos in the
latter study. These findings thus serve as examples of a general means
by which reducing agents such as NAC and DMP might influence
transcription of specific genes and thereby promote survival.