Events in apoptosis. Acidification is downstream of protease activation and BCL-2 protection.

Cytoplasmic acidification is now recognized as a feature of apoptosis in a variety of systems. However, its relation to other events in the process of apoptosis is not yet characterized. In this work, we examined the effect of BCL-2 overexpression on acidification mediated by cycloheximide treatment or Fas ligation in Jurkat T-lymphoblasts. We find that BCL-2 overexpression attenuates cytoplasmic acidification and apoptosis detected by annexin V labeling. Acidification and phosphatidylserine externalization were found to occur concurrently. We also examined the requirement for protease activation for cytoplasmic acidification to occur and found that inhibition of interleukin-1β converting enzyme/CED-3 family proteases (using carbobenzoxy-Val-Ala-Asp-fluoromethylketone, an inhibitor of these proteases) prevents acidification and apoptosis mediated by Fas ligation. These studies suggest that BCL-2 acts at a point upstream of acidification and that protease activation is also upstream of acidification.

Cytoplasmic acidification is now recognized as a feature of apoptosis in a variety of systems. However, its relation to other events in the process of apoptosis is not yet characterized. In this work, we examined the effect of BCL-2 overexpression on acidification mediated by cycloheximide treatment or Fas ligation in Jurkat Tlymphoblasts. We find that BCL-2 overexpression attenuates cytoplasmic acidification and apoptosis detected by annexin V labeling. Acidification and phosphatidylserine externalization were found to occur concurrently. We also examined the requirement for protease activation for cytoplasmic acidification to occur and found that inhibition of interleukin-1␤ converting enzyme/CED-3 family proteases (using carbobenzoxy-Val-Ala-Asp-fluoromethylketone, an inhibitor of these proteases) prevents acidification and apoptosis mediated by Fas ligation. These studies suggest that BCL-2 acts at a point upstream of acidification and that protease activation is also upstream of acidification.
Acidification has been described as a feature of programmed cell death in a variety of systems, but its relation to other events in the process is unknown. Engagement of the "death machinery" appears to involve activation of a protease or cascade of proteases belonging to the CED-3/interleukin-1␤ converting enzyme (ICE) 1 family (1, 2). Induction of apoptosis by Fas can be prevented by pretreating cells with the tripeptide ICE inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone (ZVAD) (3). BCL-2 protects cells against apoptosis triggered by a variety of stimuli but is only partially protective against Fas-mediated death (4,5). Recently, we have shown that acidification is an early event in apoptosis mediated by Fas and that cytoplasmic alkalinization prevents or delays Fas-mediated apoptosis (6). In order to determine the relationship between BCL-2 and activation of CED-3/ICE family proteases to intracellular acidification, we examined changes in intracellular pH in cells expressing BCL-2 or treated with ZVAD in response to Fas ligation or cycloheximide treatment.

EXPERIMENTAL PROCEDURES
These studies utilized CEM cells stably transfected with pZipneo vector (CEM/Neo) or with vector containing BCL2 (CEM/Bcl-2) as described previously (7,8). CEM cells were induced to undergo apoptosis with 100 ng/ml anti-Fas IgM (clone CH-11, Kamiya Biomedical). Cells were incubated in serum-free RPMI medium for 3 h at 37°C in humidified air, 5% CO 2 . Where noted, cells were pretreated for 60 min with ZVAD (100 M) (Kamiya Biomedical Co.) (3). Intracellular pH was measured by radiometric flow cytometry on a Coulter Elite flow cytometer after loading cells for 30 min with 10 M) carboxy-SNARF-1acetoxymethyl ester (Molecular Probes, Inc., Eugene, OR). Apoptosis was simultaneously assessed by labeling with annexin V-fluorescein isothiocyanate (1 g/ml). Annexin V labeling has been shown to be an early marker of apoptosis (7,9). Simultaneous detection of SNARF-1 fluorescence reflects membrane integrity and intact esterase activity; necrotic cells do not retain SNARF-1.

BCL-2 Potects Against Acidification and Apoptosis-Expres-
sion of BCL-2 has been shown to protect against the induction of apoptosis by cycloheximide (7,8) and to be partially protective against Fas ligation (4,5). We examined the effect of BCL-2 expression upon the induction of acidification and phosphatidylserine externalization in the lymphoid CEM cell line, which undergoes apoptosis in response to Fas ligation. Results of flow cytometric pH analysis are shown in Fig. 1A. Upon treatment with anti-Fas antibody, CEM cells stably transfected with the empty vector (CEM/Neo), and when treated with anti-Fas, CEM cells demonstrate acidification and annexin V labeling. In contrast, CEM cells stably transfected with BCL-2 and treated with anti-Fas exhibit less acidification (that is, fewer cells acidify), although the pH change in the cells that do acidify is similar. The resting pH of untreated CEM/Neo and CEM/Bcl-2 cells is similar. The percentage of acidified cells (defined as cells with a 575/620 nm ratio 2 S.D. below the mean of the control population) in the CEM/Neo cells treated with anti-Fas was 21%, and the percentage of annexin V-labeled cells was 19%. In the CEM/Bcl-2 cells treated with anti-Fas, the percentage of acidified cells was 10%, and the percentage of annexin V-labeled cells was 15%. These results indicate that BCL-2 protects against both acidification and phosphatidylserine externalization. We also found that there was a close correlation between acidified cells and cells that bound annexin V (Fig. 1A, fourth and fifth panels), suggesting that acidification and phosphatidylserine externalization occur concurrently.
Because it is possible that the stably transfected cell lines differ in their levels of expression of Fas antigen, we measured Fas expression on the cell surface by flow cytometry. We found that Fas expression was similar in the two cell lines (Table I).
Because BCL-2 expression has been shown to confer protection against apoptosis mediated by cycloheximide, we examined intracellular pH in CEM cells exposed to 100 g/ml cycloheximide for 3 h (Fig. 1B). As expected, overexpression of BCL-2 protected against acidification and annexin V labeling. The percentage of acidified cells after cycloheximide was 33.8% for CEM/Neo and 7.7% for CEM/Bcl-2. Annexin V labeled 33.0% of the CEM/Neo (versus 7.0% in CEM/Neo control) and 18.4% of the CEM/Bcl-2 cells (versus 17.5% in CEM/Bcl-2 control).
Role of Protease Activation in Cytoplasmic Acidification-If acidification preceded activation of the death protease cascade, then one would expect acidification to occur in response to Fas ligation even in the presence of the ICE family protease inhibitor peptide, ZVAD. If, however, ICE family protease activation is a prerequisite for acidification to occur, then the peptide should inhibit Fas-mediated acidification. Shown in Fig. 2 are represent-ative flow cytometry plots (pH versus forward scatter) showing the effect of ZVAD pretreatment on pH in cells treated with anti-Fas. Pretreatment of cells with ZVAD prevents acidification mediated by Fas ligation. The lack of acidification parallels the absence of annexin V labeling and DNA fragmentation (data not shown). These findings suggest that activation of CED-3/ICE proteases is an event that is upstream of acidification. DISCUSSION These studies suggest that pH homeostasis is tightly linked to the control of apoptosis and that both BCL-2 and CED-3/ICE family proteases affect intracellular pH control. These results are consistent with the recent report by Chinnaiyan et al. (10) that BCL-2 functions upstream of the cysteine proteases Yama (CPP32/apopain) and ICE/LAP3 (Mch3). 2 Activation of the protease cascade is considered to be the point of no return in the process of apoptosis. However, we have previously shown that cytoplasmic alkalinization (e.g. by buffering with imidazole) is protective even if done 60 min after Fas ligation (6), by which time protease activation has occurred (11). Taken together, these findings suggest that the irreversible event may be cytoplasmic acidification. Acidification could serve as a global switch to inac-2 S. J. Martin, manuscript in preparation.  tivate cellular processes that operate at neutral pH and to initiate a previously latent set of enzymes with pH optima below 7.0, such as the acid endonuclease (DNase II) that has been implicated in destruction of the genome (12,13). Quite possibly, other pH-dependent enzymes could become active when the cell enters a new metabolic state upon acidification.