(Received for publication, June 10, 1994; and in revised form, January 4, 1995)
From the
Protein isoprenylation is a post-translational modification
essential for the biological activity of G-proteins. Inhibition of
protein isoprenylation by lovastatin (LOV) induces apoptosis in HL-60
cells, a process of active cell death characterized by the
internucleosomal degradation of genomic DNA. In this article we show
that LOV-induced apoptosis is associated with intracellular
acidification and that activation of the
Na/H
antiporter induces a raise in
pH
which is sufficient to prevent or arrest DNA
digestion. First, LOV induced a decrease in pH
which was dose-dependent and correlated with the extent of
DNA degradation. Flow cytometry analysis revealed that this
acidification was due to the appearance of a subpopulation of cells
whose pH
was 0.9 pH units below control values.
Cell sorting experiments demonstrated that DNA degradation had occurred
only in those cells which had suffered intracellular acidification.
LOV-induced apoptosis could be suppressed by mevalonate
supplementation, inhibition of protein synthesis, and protein kinase C
activation by phorbol myristate acetate. In all three cases,
intracellular acidification was abolished. Inhibition of the
Na
/H
antiporter by
5-N-ethyl-N-isopropyl amiloride induced DNA
degradation in HL-60 cells per se and suppressed the
protective effect of phorbol myristate acetate. LOVinduced
intracellular acidification was not due to a complete inhibition of the
Na
/H
antiporter. In fact, LOV-treated
cells were able to respond to phorbol myristate acetate stimulation of
the Na
/H
antiporter with a marked
increase in pH
. This effect was accompanied by a
rapid arrest of DNA digestion. These observations illustrate the strong
pH dependence of LOV-induced DNA degradation, thus providing a
connection between the activation of the
Na
/H
antiporter and the suppression
of apoptosis.
Apoptosis, or programmed cell death, is an active form of cell death which occurs physiologically during embryonic development as well as in tissue turnover in the adult (for review, see (1, 2, 3) ). Cell death by apoptosis can be initiated by a number of positive signals. During T cell development in the thymus, triggering of the T-cell antigen receptor induces apoptosis in those thymocytes which recognize self antigens(2) . Also, binding of tumor necrosis factor to its receptor triggers apoptosis in several cell types(4, 5) . Alternatively, this form of cell death can occur following the removal of positive signals, like growth factors, cytokines, or hormones, needed for the survival of a particular cell type(2) . In addition, programmed cell death can be induced by a wide range of cytotoxic drugs, many of them used in cancer therapy(6) .
Apoptosis plays a key role in the control of cell populations in the organism. Failure of the cells to die via apoptosis results in abnormally increased cell numbers and oncogenesis(6) . The elucidation of the mechanisms underlying this form of cell death is critical for the understanding of many physiological processes, as well as of the sequence of events leading to oncogenic transformation.
Cells which die via apoptosis usually
suffer similar morphological changes, including nuclear condensation
and internucleosomal DNA digestion by endogenous
endonucleases(1) . Until recently, the activation of a
Ca-dependent endonuclease, DNase I(7) , was
thought to be a key event in the apoptotic process. However, the
implication of a pH-dependent endonuclease, DNase II, in certain forms
of cell death has been recently suggested(8) .
Protein
isoprenylation is a post-translational modification essential for the
membrane localization and biological activity of a number of proteins
(see (9) , for review). The precursor of isoprenoid lipids
involved in protein modification, as well as of cellular sterols, is
mevalonic acid (MVA)()(10) .
Isoprenylated proteins, among which are all the GTP-binding proteins, play crucial roles in the signal transduction pathways from growth factor, cytokine and hormone receptors(11, 12, 13, 14) . Protein isoprenylation is then essential for the control of cell proliferation and differentiation.
We have recently reported that inhibition of isoprenoid biosynthesis by LOV, a competitive inhibitor of the synthesis of MVA, induces apoptosis in HL-60 cells(15) . LOV-induced DNA degradation was specifically prevented by MVA, while other products of the isoprenoid biosynthetic pathway, like cholesterol, dolichol, ubiquinone, and isopentenyl adenosine, failed to protect cells against LOV-induced apoptosis. These findings led us to postulate that LOV-induced apoptosis was due to inhibition of protein isoprenylation.
In the present study we have addressed the mechanism
of LOV action in more detail. For this purpose we have studied the
effects of LOV on the total cell population as well as in individual
cells by using flow cytometry. In addition we have explored the effect
on LOV-induced apoptosis of PMA, CHX, and MVA. The results herein
presented establish a correlation between LOV-induced apoptosis and
intracellular pH under all the conditions studied. Our results suggest
the involvement of DNase II in LOV-induced DNA digestion and provide
evidence for the implication of the Na/H
antiporter in the suppression of apoptosis.
For cytofluorimetric determination of
intracellular pH, HL-60 cells were loaded with BCECF as above and
analyzed on an EPICS-C cytofluorimeter (Coulter
Científica,
Móstoles, Spain), essentially as
described(20, 21) . Excitation of the probe was
performed at 488 nm with a 500 milliwatt ionic argon laser. Emission
fluorescence was filtered through a 525 band pass (green fluorescence)
and a 610 long pass (red fluorescence) filter. Intracellular pH was
estimated from the ratio of the emission fluorescence signals obtained
at 525 and 610 nm. Calibration of the signal was achieved by incubating
BCECF loaded HL-60 cells for 10 min at 37 °C in the presence of
nigericin in high potassium buffer at pH 6.44, 6.83, 7.23, and 7.63.
This gave a linear relationship between pH values and 525/610 nm ratios (r > 0.995). BCECF loaded HL-60 cells were sorted by their
525/610 nm ratio, collected into phosphate-buffered saline, and kept on
ice until they were processed for DNA fragmentation. Continuous
cytofluorimetric measurements of pH variations in HL-60
cells were performed on an EPICS-XL cytofluorimeter (Coulter
Científica,
Móstoles, Spain).
Figure 1:
Effect of LOV on the cell cycle
distribution of HL-60 cells. A, representative examples of the
cell cycle effects of LOV. HL-60 cells were treated with LOV as
indicated, stained with propidium iodide, and their DNA content was
analyzed by fluorescence flow cytometry. The position of the
sub-G peak, integrated by apoptotic cells, is indicated by arrows. The experiment was repeated four times and
representative histograms are presented. B, time course of
LOVinduced cell cycle effects. HL-60 cells were treated with 10
µM LOV. At the indicated time points, cells were analyzed
as in A, and the proportion of cells in each phase of the cell
cycle was quantitated. Results are average values of four experiments
± S.D.
Figure 2:
Suppression of LOV-induced apoptosis by
inhibitors of macromolecular synthesis. After 24 h treatment with the
indicated agents, HL-60 cells were harvested and their fragmented DNA
was obtained as described under ``Experimental Procedures.''
DNA loaded in each lane was from 6 10
cells. 1, 123-bp DNA ladder; 2, untreated cells; 3,
10 µM LOV; 4, 10 µM LOV + 100
ng/ml CHX; 5, 10 µM LOV + 15 ng/ml
actinomycin D (ActD).
Figure 3: Suppression of LOV-induced apoptosis by PMA. A, HL-60 cells were treated with the indicated agents for 24 h. 1, 123-bp DNA ladder; 2, untreated cells; 3, 10 µM LOV; 4, 10 µM LOV + 10 ng/ml PMA; 5, 10 µM LOV + 10 ng/ml PMA + 25 nM staurosporine. B, HL-60 cells were treated with the indicated concentrations of LOV and PMA. 1, 123-bp DNA ladder; 2, control untreated cells; lanes 3-7, cells treated for 24 h with 10 µM LOV in the absence (3) or presence (4-7) of increasing concentrations of PMA. DNA extraction and analysis were as in Fig. 2.
Figure 4:
Effect of EIPA on DNA degradation in HL-60
cells in the presence or absence of LOV and PMA. A,
dose-response of EIPA-induced apoptosis in HL-60 cells. Cells were
treated with the indicated concentrations of EIPA for 24 h. B,
effect of EIPA on PMA suppression of apoptosis. HL-60 cells were either
untreated (2) or treated with 10 µM LOV for 24 h
in the absence (3) or presence (4) of 10 ng/ml PMA
+ 60 µM EIPA. Fragmented DNA from 6 10
cells was loaded in each lane.
Figure 5:
Effect of EIPA on
Na/H
exchange activity and on
pH
in HL-60 cells. A, HL-60 cells were
loaded with BCECF in sodium Hepes buffer as described under
``Experimental Procedures,'' resuspended in the same buffer
containing 3 mg/ml bovine serum albumin and their pH
was continuously monitored fluorimetrically.
Na
/H
exchange activity in the
presence of increasing concentrations of EIPA, was estimated from the
rate of pH
recovery after intracellular
acidification was achieved by addition of 20 mM potassium
acetate to the incubation buffer. Results are expressed as percentage
of the rate of pH
recovery in the absence of EIPA
and are mean ± S.D. values of three experiments. B,
HL-60 cells were treated with increasing concentrations of EIPA for 24
h in the absence (
, control), or presence (
, PMA) of 10
ng/ml PMA. During the final hour of incubation, cells were loaded with
BCECF in Hepes bicarbonate buffer and pH
was
determined. Results are mean ± S.D. values of five
experiments.
In order to test this
hypothesis we measured the pH of HL-60 cells under the
conditions in which we had studied the induction of apoptosis. The
results of these experiments are summarized in Table 1. Under our
experimental conditions, control cells maintained an average pH
of 7.54. Treatment of HL-60 cells with 10 µM LOV for
24 h induced a decrease in pH
of 0.29 pH units. LOV-induced
acidification was completely prevented by the compounds previously
found to prevent LOV-induced apoptosis, namely, MVA, PMA, and
cycloheximide. LOV-induced intracellular acidification was
dose-dependent. Treatment of HL-60 cells with 50 µM LOV
for 24 h caused pH
to drop 0.52 pH units below control
values. These results establish a correlation between pH
and apoptosis under all the conditions studied.
In experiments
performed in isolated nuclei it has been observed that DNase II has an
optimum pH of 5, but it is capable of catalyzing significant
internucleosomal DNA degradation at pH 6.5(8) . We then asked
the question of whether the intracellular acidification induced by LOV
treatment was sufficient to activate this endonuclease. At the
concentrations used, 10 and 50 µM, LOV induced apoptosis
in 20 and 50% of the cells, respectively, as assessed by flow cytometry (Fig. 1A). The possibility remained that only those
cells undergoing apoptosis had suffered intracellular acidification, as
it has been observed in other systems(34) . If this were the
case, fluorimetric pH determinations shown in Table 1would
underestimate the drop in intracellular pH of the apoptotic cells,
since they represent the average pH values of the total
cell population.
To test this possibility we analyzed the
distribution of LOV-treated HL-60 cells according to their pH using flow cytometry (Fig. 6A). We found that
treatment of HL-60 cells with LOV led to the appearance of a
subpopulation of cells with a more acidic pH
than untreated
cells. This subpopulation had a lower 525/610 nm ratio and appeared to
the left of the main population. Calibration of these signals indicated
that the cells in the acidic population maintained an average pH
of 6.64, which is 0.9 pH units below control values, and it is in
the range of the pH reported to activate DNase II(8) .
Meanwhile, the pH
of the cells in the main population did
not change significantly with respect to control values. The proportion
of HL-60 cells in the acidic population was dose-dependent and it
increased from 20% of the cells after treatment with 10 µM LOV for 24 h, to 40-50% of the total cell population with 50
µM LOV (Fig. 6A). It is interesting to
note that the proportion of cells with acidic pH
coincided
with the proportion of apoptotic cells under all conditions studied (Fig. 1A and 6A). Therefore, it was important
to determine whether there was a relationship between intracellular
acidification and apoptosis. If this were true, cells that maintained a
normal pH
should maintain also their DNA integrity, while
the DNA of cells with acidic pH
should have suffered
internucleosomal degradation. To test this hypothesis, HL-60 cells
treated with 50 µM LOV for 24 h were sorted on the flow
cytometer by their pH
, and processed to analyze DNA
fragmentation. Aliquots of the two populations obtained were reanalyzed
by flow cytometry to assess their purity. The therefore considered
``normal pH
'' population contained routinely more
than 95% cells with pH
above 7.05, while the
``acidic'' population contained more than 80% cells with
pH
below 7.05. The DNA analysis of the total population is
shown in Fig. 6B in comparison with the two populations
obtained by cell sorting. As it can be appreciated, HL-60 cells treated
with 50 µM LOV for 24 h showed significant DNA
degradation. When these cells were sorted by their pH
and
reanalyzed, the cells that maintained a normal pH
showed no
detectable DNA degradation, while the cells which had suffered
intracellular acidification showed intense DNA digestion, thus proving
the correlation between LOV-induced intracellular acidification and DNA
degradation.
Figure 6:
Effect of LOV on the intracellular pH and
DNA degradation of HL-60 cells. A, flow cytometry analysis of
LOV-induced intracellular acidification. Cells were incubated for 24 h
with the indicated concentrations of LOV. During the final hour of
incubation, cells were loaded with BCECF and analyzed by flow cytometry
as described under ``Experimental Procedures.'' B,
DNA analysis of HL-60 cells sorted by their intracellular pH. Cells
treated with 50 µM LOV and loaded with BCECF as in A were sorted on the basis of their pH by flow
cytometry and the fragmented DNA of the starting cells, as well as that
of the two cell populations obtained from the sorting was analyzed as
described under ``Experimental Procedures.'' 1,
123-bp DNA ladder; 2, total cell population, 6
10
cells; 3, cells with normal
pH
, 3
10
cells; 4,
cells with acidic pH
, 3
10
cells.
Figure 7:
Effect of LOV on the activity of the
Na/H
antiporter in HL-60 cells. Cells
were incubated for 24 h in the presence or absence of 10 µM LOV. During the final hour of incubation cells were loaded with
BCECF in sodium Hepes buffer and their intracellular pH was
continuously recorded. At the indicated times, sodium propionate (3
mM) or PMA (10 ng/ml) were added, and the changes in
pH
were monitored. The experiments were repeated
at least three times and representative recordings are
presented.
Figure 8:
Flow cytometry analysis of
Na/H
antiporter activity in HL-60
cells. Cells were incubated for 24 h in the presence or absence of 50
µM LOV. During the final hour of incubation cells were
loaded with BCECF in sodium Hepes buffer and their intracellular pH was
continuously monitored by flow cytometry. At the times indicated (arrows), 30 mM sodium propionate was added to the
sample tube and data collection was rapidly restarted. The experiments
were repeated at least three times and essentially the same results
were obtained. The recordings presented correspond to the analysis of 3
10
cells for control cells and 2
10
cells for LOV-treated cells.
Therefore, we studied the protective effects of MVA and PMA when added after the onset of LOV-induced apoptosis (Fig. 9). Twelve hours after the beginning of LOV treatment, a fraction of the cells were collected and their DNA extracted for analysis of DNA fragmentation (Fig. 9, lane 3). To a fraction of the remaining cells, MVA or PMA were added and the treatments were continued for 12 more h (Fig. 9, lanes 4 and 5, respectively). The same was done at 16 h after the beginning of LOV treatment: a fraction of the cells was collected for analysis (Fig. 9, lane 6), while the remaining cells received the addition of either MVA or PMA and the treatments were continued until completion of the 24-h incubation time (Fig. 9, lanes 7 and 8). Untreated cells and cells treated continously for 24 h with 10 µM LOV alone, were used as negative and positive controls, respectively (Fig. 9, lanes 2 and 9).
Figure 9:
Effect of MVA and PMA on LOV-induced DNA
degradation in HL-60 cells after its onset. After 12 and 16 h of
incubation with 10 µM LOV a fraction of the cells were
collected and their fragmented DNA was extracted. To the remaining
cells, either MVA (250 µM) or PMA (10 ng/ml) were added
and the 24-h incubation time was completed. DNA loaded per lane was
from 6 10
cells. 1, 123-bp DNA ladder; 2, control, untreated cells; 3, cells treated with
LOV for 12 h; 4, MVA, or 5, PMA, were added during
the last 12 h of the 24-h treatment with LOV; 6, cells treated
with LOV for 16 h; 7, MVA, or 8, PMA, were added
during the last 8 h of the 24-h treatment with LOV; 9, cells
treated with LOV for 24 h.
We observed that the addition of either MVA
or PMA to HL-60 cells rapidly blocked LOV-induced DNA degradation. As
shown in Fig. 9, cells treated for 24 h with LOV alone showed
extensive DNA laddering. However, when MVA or PMA were included during
the last 12 or 8 h of the incubation the extent of DNA laddering at the
time of analysis (24 h) had not increased with respect to that already
existing at the time of addition (12 or 16 h, respectively).
Suppression of LOV-induced apoptosis by both compounds occurred in less
than 4 h as indicated by the observation that while the cells treated
with LOV alone suffered significant DNA degradation between 12 and 16 h (Fig. 9, lanes 3 and 6), there was no further
increase in cells which received the addition of MVA or PMA at 12 h (Fig. 9, lanes 4 and 5). These observations
suggest that restoring pH may be sufficient to prevent or
arrest LOV-induced apoptosis in HL-60 cells.
Treatment of HL-60 cells with LOV induced apoptosis in a
time- and dose-dependent manner. LOV also induced an accumulation of
the non-apoptotic cells in the G phase of the cell cycle.
The proportion of cells in the S phase of the cell cycle was
significantly reduced by LOV. These results probably reflect the
requirement for isoprenylated proteins for adequate entry in the S
phase(37) .
Inhibitors of RNA and protein synthesis have
been found both to protect cells from apoptosis caused by a variety of
agents (38, 39, 40) and, in other cases, to
induce apoptosis per se(41, 42) . We have
observed that CHX and actinomycin D could induce both kinds of effects
in HL-60 cells depending on the concentration of inhibitor used. This
may indicate that LOV-induced apoptosis needs the transcription of new
genes, while apoptosis induced by high concentrations of CHX or
actinomycin D does not. Alternatively, the protective effect of these
inhibitors could be due, not to the blockade of the apoptotic program,
but to interference with the events that lead to its activation. This
latter possibility is suggested by the observations that CHX was able
to protect cells, not only against apoptosis, but also against other
consequences of the impairment of protein isoprenylation, like the
arrest of non-apoptotic cells in G, or the disorganization
of the actin cytoskeleton(43) . The mechanism(s) by which CHX
can render cells more resistant to LOV action is yet to be explored.
Our results indicate that PMA suppression of LOV-induced apoptosis in HL-60 cells is mediated by PKC activation. LOV-treated cells suffer an impairment of many cellular functions due to the inhibition of G protein isoprenylation(22, 23, 24, 25) . However, in many cases these responses can be still elicited by stimulation with PMA(24, 25) . This indicates that PKC activation bypasses the G protein mediated steps in the corresponding signal transduction pathways. Thus, the protective effect of PMA on apoptosis could be due to the activation of signal transduction pathways at a point distal to that blocked by LOV. Alternatively, PKC activation could directly interfere with the apoptotic process.
Activation of the Na/H
antiport by
PKC and the resulting intracellular alkalinization has been proposed to
be at the basis of the suppression of apoptosis by PMA, interleukin-3,
and granulocyte macrophage colony-stimulating factor in hemopoietic
precursor cells(18) . However, the mechanism by which
intracellular alkalinization led to protection against apoptosis
remained to be determined. Here we have observed that PMA can
effectively protect HL-60 cells against the effects of a partial
inhibition of the Na
/H
antiporter by
EIPA. Both, DNA degradation (results not shown) and intracellular
acidification induced by 20 and 40 µM EIPA could be
prevented by co-incubation with PMA. However, at concentrations of EIPA
which effectively blocked Na
/H
exchange activity, PMA could neither maintain normal
pH
, nor DNA integrity. These results suggest that a
disfunction of the Na
/H
antiporter or
the resulting intracellular acidification could be important events in
the induction of apoptosis.
The recent characterization of a
pH-dependent endonuclease involved in
apoptosis(8, 44) , along with accumulated evidence
suggesting a role for G proteins in the regulation of
pH(31, 32, 33) , prompted us to
study the effect of LOV on the pH
of HL-60 cells. The
results herein reported clearly demonstrate a correlation between
pH
and apoptosis in HL-60 cells. First, there is a
correlation between the concentration of LOV used, the extent of DNA
laddering, and the intensity of intracellular acidification. The drop
in pH
measured in the total population has been shown to be
due to the appearance of a subpopulation of cells whose pH
was 0.9 pH units below control values. Second, when the DNA
content and the pH
of individual cells were considered, it
was obvious that there was a coincidence between the proportion of
cells integrating the sub-G
peak and the proportion of
acidic cells under all the conditions studied. Moreover, when
LOV-treated HL-60 cells were sorted on the basis of their
pH
, only the acidic cells showed DNA fragmentation. The
extent of DNA digestion in the acidic cells could account for the
degradation observed in the total population. The appearance of this
subpopulation is probably due to a higher susceptibility toward the
drug and it could reflect the requirement for certain G protein(s) at a
specific point of the cell cycle.
We have also shown that
suppression of LOV induced apoptosis by three different mechanisms,
namely, MVA supplementation, PKC activation, and inhibition of protein
synthesis, is accompanied in all cases by maintenance of the normal
pH. We have observed that LOV-induced DNA degradation not
only can be prevented, but also stopped, by agents which are able to
induce intracellular alkalinization in the presence of LOV. The strong
pH dependence of LOV-induced apoptosis suggests the involvement of
DNase II in this form of cell death. Interestingly, flow cytometry
analysis of cells treated with 5 µM EIPA revealed that
intracellular acidification occurred uniformly in the whole cell
population. This is probably the reason why EIPA did not induce
apoptosis at this concentration, since, although cells had a decreased
average pH
, there was not a significant proportion of cells
with pH
below 7.05 (less than 10%; results not shown).
Besides the Na/H
antiporter, HL-60
cells possess Na
-dependent and independent
Cl
/CO
H
exchange systems
which can contribute to pH
homeostasis(45) .
However, we have observed that pH
values did not differ
significantly when determined in the presence or absence of
bicarbonate. Moreover, this did not affect the appearance of the two
cell populations after treatment with LOV, nor their proportion
(results not shown).
Once we had established the relationship
between intracellular acidification and DNA degradation, we addressed
the mechanism by which LOV treatment could result in a decrease in
pH. It had been observed previously that inhibitors of
isoprenoid biosynthesis could induce intracellular
acidosis(31, 35) . This effect could be attributed to
a decrease in the activity of the Na
/H
exchanger. However, under our conditions, treatment of HL-60
cells with LOV did not severely interfere with the activity of the
antiporter, as judged by its capacity to respond to exogenously induced
intracellular acidification and PMA stimulation. These results are in
agreement with the observation that osmotic activation of the
Na
/H
antiporter was also not affected
by treatment with LOV(31) .
LOV-induced intracellular
acidification could also be due to the inhibition of G protein mediated
event(s) in the control of pH. Regulation of pH
results from the integration of multiple signals originated both
inside and outside of the cell. Among the external signals are growth
factors and cytokines(46, 47) . Thus, LOV could mimic
the effect of growth factor withdrawal by inhibiting the G
protein-mediated steps in the signal transduction pathways of these
factors. In preliminary experiments aimed at the identification of the
proteins implicated in the induction of apoptosis by LOV, we observed
that treatment of HL-60 cells with pertussis toxin, in conditions under
which at least 95% of the G
-like activity was inhibited,
had no effect on DNA degradation. This suggests that LOV-induced
apoptosis is the consequence of the inhibition of certain proteins and
not of the general impairment of cellular functions. Recently, two G
proteins have been identified, G
and
G
, that activate the Na
/H
antiporter when transfected in COS-1 cells(48) . These G
proteins are ubiquitously expressed and the inhibition of their
function could be implicated in the mechanism of LOV-induced
intracellular acidification.
On the other hand, intracellular
acidification could be a distal event, common to various initially
unrelated apoptotic pathways, and whose function would be to promote
DNA digestion through the activation of the pH-dependent endonuclease.
Two lines of evidence would support this hypothesis. First,
intracellular acidification has been shown to correlate with DNA
degradation during apoptosis induced by widely unrelated stimuli, like
ionomycin(44) , etoposide(34) , a combination of
H ionophore with acidic extracellular pH(8) ,
and now, LOV. Second, intracellular alkalinization by activation of the
Na
/H
exchanger plays an important
role in PMA, interleukin-3, granulocyte macrophage colony-stimulating
factor (18) , and stem cell factor (49) suppression of
apoptosis in interleukin-3 dependent cell lines. It is possible that
activation of the Na
/H
exchanger by
these various agents is actually preventing a drop in pH
that otherwise would directly activate the endonuclease. The
possibility also exists that the circumstance that triggers apoptosis,
more than intracellular acidification per se, is the
impairment of the regulation of the Na
/H
antiporter. It is known that persistent
Na
/H
antiport activity is necessary
for cell cycle progression(50) , and that inhibitors of the
antiport selectively suppress the expression of certain growth
factor-induced cell cycle genes(50) . Thus, a deficiency in the
regulation of the Na
/H
antiport due
to the lack of stimuli during growth factor withdrawal or to the
inhibition of growth factor signaling pathways by LOV could result in
uncoordinated cell cycle regulation that would lead to cell death by
apoptosis.
The hypothesis formulated above are not mutually
excluding. It seems well established now that isoprenylated G proteins
play a crucial role in the regulation of the
Na/H
antiporter and of
pH
. This supports the hypothesis that LOV can induce
intracellular acidification by inhibiting protein isoprenylation. At
the same time, intracellular acidification is emerging as an important
event in the apoptotic process as a phenomenon directly related to
activation of DNA digestion. Our results establish a link between
protein isoprenylation, regulation of pH
, and apoptosis.
Moreover, they provide a biochemical basis for the suppression of
programmed cell death by Na
/H
exchange activation.