©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Intracellular Alkalinization Suppresses Lovastatin-induced Apoptosis in HL-60 Cells through the Inactivation of a pH-dependent Endonuclease (*)

(Received for publication, June 10, 1994; and in revised form, January 4, 1995)

Dolores Pérez-Sala (1)(§) Dolores Collado-Escobar (2) Faustino Mollinedo (1)

From the  (1)Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid and the (2)Instituto López Neyra, Consejo Superior de Investigaciones Científicas, Granada, Spain

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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)(^1)(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.


EXPERIMENTAL PROCEDURES

Materials

Lovastatin was a gift from Merck, Sharp & Dohme. Mevalonic acid lactone, cycloheximide, actinomycin D, PMA, staurosporine, and nigericin were from Sigma. EIPA was from RBI (Natick, MA). BCECF was from Molecular Probes (Eugene, OR). The 123-bp DNA ladder was from Bethesda Research Laboratories (Bethesda, MD).

Cell Culture

Human promyelocytic HL-60 leukemic cells were grown in RPMI 1640 supplemented with 10% (v/v) fetal calf serum (Life Technologies, Inc.), 2 mM glutamine, penicillin (100 units/ml), and gentamycin (24 µg/ml). Lovastatin, was added to the cultures as the sodium salt, obtained from the lactone as described previously(16) .

Cell Cycle Analysis

Approximately 10^6 cells per experimental condition were harvested, washed with phosphate-buffered saline, and resuspended in 250 µl of the same buffer. To this cell suspension, 0.1% Nonidet P-40 and 50 µg/ml propidium iodide, final concentrations, were sequentially added and the content of DNA per cell was estimated by flow cytometry.

DNA Fragmentation

To assess DNA fragmentation, soluble (fragmented) DNA was obtained as described (15) and analyzed on agarose gels.

Intracellular pH Determination

Intracellular pH measurements were performed both fluorimetrically and using flow cytometry. For fluorimetric determination, HL-60 cells (10^7 cells/ml) were loaded with 1 µM BCECF for 30 min at 37 °C with gentle shaking, either in sodium Hepes buffer (25 mM Hepes, pH 7.4, 140 mM NaCl, 5 mM KCl, 0.8 mM MgCl(2), 1.8 mM CaCl(2), 5.5 mM glucose), or in Hepes bicarbonate buffer (15 mM Hepes, pH 7.4, 110 mM NaCl, 20 mM CO(3)HNa, 5 mM KCl, 0.8 mM MgCl(2), 1.8 mM CaCl(2), 5.5 mM glucose), as indicated(17) . BCECF-loaded cells were pelleted by centrifugation at 300 times g for 7 min, resuspended in the same buffer at 10^6 cells/ml, and introduced in a thermostatted cuvette at 37 °C with constant stirring for the continuous recording of the pH-sensitive fluorescence on a Perkin Elmer LS50B luminescence spectrometer. Intracellular pH was estimated from the ratio of the 530-nm fluorescence signals obtained at 500 nm (pH-sensitive) and 450 nm (isosbestic) excitation wavelengths (18) . The signal was calibrated in high potassium Hepes buffer (25 mM Hepes, 145 mM KCl, 0.8 mM MgCl(2), 1.8 mM CaCl(2), 5.5 mM glucose) containing the H/K ionophore nigericin (10 µg/ml) at pH 6.64, 7.4, and 7.57(19) .

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(i) variations in HL-60 cells were performed on an EPICS-XL cytofluorimeter (Coulter Científica, Móstoles, Spain).


RESULTS

Effect of LOV on the Cell Cycle Distribution of HL-60 Cells

In order to assess the extent of LOV-induced apoptosis and to observe whether this process was selective to any phase of the cell cycle, we studied the cell cycle distribution of HL-60 cells treated with LOV (Fig. 1). The appearance of cells with a DNA content less than G(1), characteristic of early apoptotic cells (sub-G(1) peak, Fig. 1A), could be observed between 8 and 12 h after the addition of 10 µM LOV. The cells which did not undergo apoptosis showed an increase in the proportion of cells arrested in G(1), together with a significant decrease in the proportion of cells in the S phase, while the percentage of cells in G(2) plus M was not significantly affected (Fig. 1B). At later stages during LOV treatment there was an almost linear increase in the proportion of apoptotic cells, which amounted to approximately 20% of the total cell population after 24 h. The effect of LOV was dose-dependent. Treatment of HL-60 cells with 50 µM LOV for 24 h induced apoptosis in 40-50% of the cells (Fig. 1A).


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(1) 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.



Macromolecular Synthesis Is Required for LOV-induced Apoptosis

In an attempt to elucidate the mechanisms underlying LOV-induced cell death, we investigated whether the synthesis of macromolecules was necessary for its effect. Fig. 2shows the effect of CHX and actinomycin D on LOV-induced DNA fragmentation. CHX (100 ng/ml) was able to completely prevent DNA degradation induced by 10 µM LOV, while actimomycin D (15 ng/ml) had a partially protective effect. CHX was also able to prevent all the above described effects of LOV on the cell cycle distribution of HL-60 cells, as well as the morphological changes associated with apoptosis (results not shown). However, both CHX and actinomycin D were potent inducers of apoptosis in HL-60 cells when used above 250 and 20 ng/ml, respectively (results not shown).


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 times 10^5 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).



PMA Suppresses LOV-induced Apoptosis in HL-60 Cells

Inhibition of protein isoprenylation by LOV has been shown to impair many G protein-mediated cellular functions(22, 23, 24) . In several cases these functions could be rescued by direct activation of PKC by phorbol esters(24, 25) . Thus, we investigated next whether phorbol esters could provide a protective effect against LOV-induced apoptosis. As shown in Fig. 3, PMA completely prevented LOV-induced DNA fragmentation in HL60 cells. This protective effect was partially reversed by co-incubation with the PKC inhibitor staurosporine (Fig. 3A). PMA is known to induce the differentiation of HL-60 cells into cells with a macrophage-like phenotype(26) . Since the sustained activation of PKC is essential for this process(27) , the ability of several inhibitors of PKC to block PMA-induced differentiation was taken as an index of their effectiveness in our system. The minimum concentration of staurosporine required to inhibit PMA-induced differentiation of HL-60 cells, as assessed by morphological criteria, without affecting cell viability or DNA integrity, was 25 nM. This concentration was found to prevent the protective effect of PMA on LOV-induced apoptosis. However, the use of other inhibitors of PKC was limited by their high cytotoxicity in these cells. Thus, the minimum concentration of H7 required to block differentiation by PMA was found to induce DNA fragmentation in HL-60 cells per se. The suppression of apoptosis by PMA was dose-dependent (Fig. 3B). A concentration of PMA of 1 ng/ml was sufficient to prevent DNA degradation by 10 µM LOV.


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.



Inhibition of the Na/H Antiporter Induces Apoptosis in HL-60 Cells and Suppresses the Protective Effect of PMA

Suppression of apoptosis by phorbol esters has been related to their ability to induce cellular alkalinization(18) . This effect is thought to be mediated by PKC through the phosphorylation of the C-terminal regulatory domain of the Na/H antiporter(28) . This phosphorylation would lead to the activation of the antiporter by increasing its affinity for the intracellular H, thus inducing cytoplasmic alkalinization(29) . To assess whether the activation of the Na/H antiporter was responsible for PMA suppression of LOV-induced apoptosis we studied the effect of the specific inhibitor of the Na/H antiporter, EIPA(30) . We found that EIPA induced DNA degradation in HL-60 cells per se (Fig. 4A). This effect was dose-dependent, being 20 µM the lowest concentration tested which induced significant DNA laddering over the course of a 24-h treatment. At 60 µM, EIPA was also able to overcome the protective effect of PMA on LOV-induced apoptosis (Fig. 4B). We then studied the correlation between the ability of EIPA to inhibit the Na/H antiporter in HL-60 cells and to block PMA prevention of cell death (Fig. 5). A concentration of 10 µM EIPA was sufficient to completely inhibit the Na/H antiporter when assayed in sodium Hepes buffer (results not shown). However, concentrations above 60 µM were required to achieve more than 90% inhibition when the buffer was supplemented with 3 mg/ml bovine serum albumin to mimic the conditions prevailing during cell culture (Fig. 5A). In addition, we explored the effect of EIPA on pH(i) in the presence and absence of PMA in the conditions under which we had studied the induction of apoptosis (Fig. 5B). We observed that treatment of HL-60 cells with EIPA for 24 h induced a dose-dependent decrease in pH(i) which correlated with the ability of EIPA to inhibit Na/H exchange in bovine serum albumin containing medium. Treatment of HL-60 cells with PMA for 24 h did not result in a significant variation of pH(i). However, PMA effectively protected HL-60 cells against EIPA-induced acidification (Fig. 5B). Concentrations of EIPA above 60 µM were necessary to suppress this protective effect. Thus, there is a coincidence between the concentration of EIPA required to block Na/H exchange activity, as shown in Fig. 5A, and that required to suppress the protective effects of PMA (Fig. 4B and 5B). These results suggest that PMA can compensate the effects of a partial inhibition of the antiporter and that near complete inhibition is needed to effectively prevent PMA activation of Na/H exchange.


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 times 10^5 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 (bullet, control), or presence (circle, 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.



LOV-induced Apoptosis Correlates with a Decrease in Intracellular pH

The above data suggested that PMA suppression of LOV-induced apoptosis could be mediated by activation of the Na/H antiporter and subsequent intracellular alkalinization. Moreover, several lines of evidence have indicated the involvement of isoprenylated proteins, presumably G proteins, in the regulation of pH(i)(31, 32, 33) . On the other hand, it has been reported recently that HL-60 cells contain an endonuclease, DNase II, which can be activated in response to intracellular acidification(34) . Thus, LOV-induced inhibition of G proteins isoprenylation could lead to impairment of the regulation of pH(i) and to intracellular acidification, which would then activate the pH-dependent endonuclease.

In order to test this hypothesis we measured the pH(i) 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(i) of 7.54. Treatment of HL-60 cells with 10 µM LOV for 24 h induced a decrease in pH(i) 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(i) to drop 0.52 pH units below control values. These results establish a correlation between pH(i) 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(i) values of the total cell population.

To test this possibility we analyzed the distribution of LOV-treated HL-60 cells according to their pH(i) 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(i) 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(i) 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(i) 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(i) 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(i) should maintain also their DNA integrity, while the DNA of cells with acidic pH(i) 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(i), 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(i)'' population contained routinely more than 95% cells with pH(i) above 7.05, while the ``acidic'' population contained more than 80% cells with pH(i) 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(i) and reanalyzed, the cells that maintained a normal pH(i) 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 times 10^5 cells; 3, cells with normal pH, 3 times 10^5 cells; 4, cells with acidic pH, 3 times 10^5 cells.



LOV-induced Intracellular Acidification Is Not Due to a Complete Inhibition of the Na/H Antiporter

Inhibitors of the enzyme 3-hydroxy-3-methylglutaryl-CoA reductase have been reported to induce intracellular acidification and to diminish the response of the Na/H antiporter to certain stimuli(31, 35) . Therefore, we were interested in studying the activity of the Na/H antiporter in LOV-treated cells. Fig. 7shows representative recordings of the pH(i) changes of control and LOV-treated cells in response to intracellular acidification and to PMA stimulation. These experiments were carried out in HCO(3)-free media, which renders the Na/H antiport the main mechanism for regulation of pH(i)(36) . Na/H exchange activity was estimated from the rate of recovery of pH(i) of HL-60 cells after instantaneous acidification by addition of 3 mM propionate to the incubation medium. After treatment of HL-60 cells with 10 µM LOV for 24 h, a condition under which 20% of the cells had a decreased pH(i), the activity of the Na/H antiporter was not diminished with respect to control cells (Fig. 7, upper panels). The rates of alkalinization after propionate loading averaged 0.09 ± 0.02 pH units/min and 0.15 ± 0.01 pH units/min in control and LOV-treated cells, respectively. LOV-treated cells were also able to respond to PMA stimulation of the Na/H antiport with a marked increase in pH(i) (Fig. 7, lower panels). The extent of intracellular alkalinization in response to PMA was 0.12 ± 0.01 pH units in LOV-treated cells and 0.05 ± 0.01 pH units in control cells. When these experiments were performed with cells treated with 50 µM LOV for 24 h, 40-50% of which had a decreased pH(i), the responses to an acid load and to PMA stimulation were also similar to those of cells treated with 10 µM LOV (results not shown). In order to assess the activity of the Na/H antiporter in the acidic population, we studied the variations in pH(i) of HL-60 cells in response to propionate addition by fluorescence flow cytometry. As it can be appreciated in Fig. 8, HL-60 cells treated with 50 µM LOV for 24 h appeared in two distinct populations of comparable size, the acidic population having a lower 525/610 nm ratio. After acidification with propionate, both populations were able to recover the starting pH(i) with rates similar to that of control cells. These results indicate that treatment with LOV does not severely impair the Na/H antiporter.


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 times 10^5 cells for control cells and 2 times 10^5 cells for LOV-treated cells.



MVA and PMA Can Block LOV-induced DNA Degradation in HL-60 Cells after Its Onset

The results presented show that LOV-treated cells respond to PMA within minutes with a significant activation of Na/H exchange. This activation could be responsible for the recovery and/or maintenance of normal pH(i). If endonuclease activity requires an acidic pH, the raise in pH(i) induced by PMA should be sufficient to stop DNA degradation and/or prevent further activation of the endonuclease. On the other hand, if LOV-induced acidification is due to the inhibition of protein isoprenylation, supplementing the incubation medium of HL-60 cells with MVA should promote the post-translational modification of the unprocessed proteins and allow a rapid recovery of pH(i).

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 times 10^5 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(i) may be sufficient to prevent or arrest LOV-induced apoptosis in HL-60 cells.


DISCUSSION

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(1) 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(1), 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(i), 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(i)(31, 32, 33) , prompted us to study the effect of LOV on the pH(i) of HL-60 cells. The results herein reported clearly demonstrate a correlation between pH(i) 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(i) measured in the total population has been shown to be due to the appearance of a subpopulation of cells whose pH(i) was 0.9 pH units below control values. Second, when the DNA content and the pH(i) of individual cells were considered, it was obvious that there was a coincidence between the proportion of cells integrating the sub-G(1) 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(i), 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(i). 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(i), there was not a significant proportion of cells with pH(i) below 7.05 (less than 10%; results not shown).

Besides the Na/H antiporter, HL-60 cells possess Na-dependent and independent Cl/CO(3)H exchange systems which can contribute to pH(i) homeostasis(45) . However, we have observed that pH(i) 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(i). 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(i). Regulation of pH(i) 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(i)-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, Galpha and Galpha, 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(i) 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(i). 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(i), and apoptosis. Moreover, they provide a biochemical basis for the suppression of programmed cell death by Na/H exchange activation.


FOOTNOTES

*
This work was supported by Grant PM92-0003 from the Dirección General de Investigación Científica y Técnica (DGICYT). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Centro de Investigaciones Biológicas, C.S.I.C., C/Velázquez, 144, 28006 Madrid, Spain. Tel.: 34-1-5611800 (ext. 4302); Fax: 34-1-5627518.

(^1)
The abbreviations used are: MVA, mevalonic acid; G protein, guanine nucleotide-binding protein; LOV, lovastatin; CHX, cycloheximide; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; EIPA, 5-N-ethyl-N-isopropylamiloride; BCECF, 2`,7`-bis(carboxyethyl)-5carboxyfluorescein; H7, 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine; bp, base pair(s).


ACKNOWLEDGEMENTS

We thank Dr. F. J. Cañada and Dr. S. Lamas for helpful comments and discussion and P. Lastres for expert assistance on flow cytometry experiments.


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