Journal of Histochemistry and Cytochemistry, Vol. 45, 675-684, Copyright © 1997 by The Histochemical Society, Inc.


ARTICLE

Differential Staining of Apoptotic Nuclei in Living Cells: Application to Macronuclear Elimination in Tetrahymena

Solomon S. Mpokea and Jason Wolfea
a Department of Biology, Wesleyan University, Middletown, Connecticut

Correspondence to: Jason Wolfe, Dept. of Biology, Wesleyan University, Middletown, CT 06459.


  Summary
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

Acridine orange (AO) has been used as a vital fluorescent stain to identify apoptotic cells in Drosophila, but little is known about what structures are stained. We explored the specificity of AO staining while studying nuclear apoptosis in Tetrahymena. Using AO alone or together with the vital nuclear stain Hoechst 33342 (HO), we find that lysosomes are generally clustered around the degenerating nucleus and that such nuclei are stained an orange-red color, like lysosomes. Significantly, the combined dyes, more so than with AO alone, distinguish between apoptotic and normal (or necrotic) nuclei by a clear color difference. Moreover, these dyes differentially stain apoptotic and normal nuclei in avian chondrocytes. The differential staining results are nullified in fixed cells or in cytoskeletal preparations treated with RNAse. Similarly, lysosomotrophic agents eliminate the differential staining. Our results are consistent with acidification of the apoptotic nucleus, possibly by fusion with lysosomes. However, even under basic conditions, the macronucleus condenses and is eliminated, suggesting that, if the nucleus is becoming acidified, acidification by itself is not essential for nuclear elimination. The differential staining procedure may provide a useful method for specifically identifying apoptotic cells and separating them for further analysis. (J Histochem Cytochem 45:675-683, 1997)

Key Words: apoptosis, nuclear death, acridine orange, lysosomes, ciliate conjugation, chondrocytes, Hoechst 33342


  Introduction
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Summary
Introduction
Materials and Methods
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Apoptosis, or programmed cell death, plays an important role during development in multicellular organisms (Schwartzman and Cidlowski 1993 ; Gerchenson and Rotello 1992 ; Fesus et al. 1991 ; Oppenheim 1991 ; Wyllie et al. 1980 ) and can contribute significantly to certain disease processes and pathological situations (Ameisen et al. 1995 ; Mignotte et al. 1995 ; Smale et al. 1995 ). It can be recognized by a set of morphological features that include loss of cell volume (Lockshin and Beaulaton 1981 ), blebbing of the plasma membrane, and compaction of chromatin into dense masses that lie at the periphery of the nucleus or, in other cases, condensation of the entire nucleus into a dense ball with the chromatin distributed evenly throughout the nucleus (Wyllie et al. 1980 ).

At the biochemical level, apoptosis is characterized by DNA digestion at the linker regions between nucleosomes to form oligonucleosome-sized fragments that can be separated by agarose gel electrophoresis, yielding a banding pattern that has come to be known as the DNA "ladder" (Compton 1992 ). In tissue sections, DNA fragmentation is often detected by the TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP-biotin Nick End Labeling) technique (Gavrieli et al. 1992 ), in which the transferase enzyme incorporates biotin labeled nucleotides into the free 3'-OH DNA ends of double- or single-strand breaks. The labeled nucleotide can then be detected by a color reaction using avidin-conjugated peroxidase and an appropriate chromogenic substrate (e.g. Wijsman et al. 1993 ), or by fluorescence microscopy using avidin-conjugated fluorochromes (e.g. Gorczyca et al. 1993 ).

Acridine orange (AO) has been used to identify apoptotic cells in Drosophila embryos (White et al. 1994 ; Abrams et al. 1993 ), but few details have been published about what structures in particular are stained. AO has two different staining characteristics. In fixed tissues it is used as a metachromatic dye, differentially staining single-stranded nucleic acids orange and double-stranded nucleic acids green (Darzynkiewicz 1990 ). However, in living cells it serves as a pH indicator, becoming trapped in acidic compartments such as lysosomes and phagosomes, which then fluoresce a brilliant orange-red (Zelenin 1966 ). For its role in identifying apoptotic cells in Drosophila, AO was used as a vital stain. It therefore appeared to us that lysosomes might become highly abundant in apoptotic cells, enabling the latter to be distinguished from non-apoptotic cells.

Conjugating cells of the ciliate protozoan Tetrahymena provide a remarkable model system for the study of nuclear death under conditions in which the cell continues to live. In these binuclear cells, each with a micro- and macronucleus, genetic exchange results in the development of a new macronucleus that replaces the existing, genetically "old" macronucleus (Martindale et al. 1982 ). The latter is eliminated through a regulated developmental process that can be prevented by inhibitors of gene expression (Mpoke and Wolfe 1996 ) or by gene mutation (Kaczanowski 1992 ). Moreover, nuclear death occurs in the context of a precise sequence of nuclear events. The old macronucleus becomes highly condensed, resembling an apoptotic nucleus, just at the time that new micro- and macronuclei begin to be differentiated. We and others have demonstrated that the DNA of the dying nucleus is digested into oligonucleosome-sized pieces, creating a band pattern on gels characteristic of cells dying by apoptosis (Mpoke and Wolfe 1996 ; Davis et al. 1992 ). In addition, the dying macronucleus, alone among five nuclei within the cell, is stained by the TUNEL technique (Mpoke and Wolfe 1996 ). Therefore, selected nuclear apoptosis occurs within a living cell.

We wondered whether lysosomes become abundant or become localized near the degenerating macronucleus during nuclear death in Tetrahymena. This would be consistent with the known role of lysosomes in the autodigestion of degenerating organelles in eukaryotic cells, and might account for AO staining of apoptotic cells in Drosophila. Using AO, we discovered that lysosomes do surround the dying nucleus. More importantly, the dying nucleus itself becomes stained with AO, suggesting that the nucleus becomes an acidic compartment. When AO is used in conjunction with Hoechst 33342 (HO), a vital DNA stain, normal nuclei fluoresce a blue color, whereas apoptotic nuclei stain a bright yellow or brilliant orange, depending on the stage of degeneration. Similar results were obtained with avian chondrocytes. This stain combination, which we call "Vital Apofluor," provides a simple and rapid method for readily distinguishing apoptotic cells from normal cells by a clear color difference. Because it detects apoptosis while the cells are still alive, the procedure may have broad application in apoptosis research.


  Materials and Methods
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Materials and Methods
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Materials and Cell Culture
All chemicals, unless otherwise noted, were purchased from Sigma Chemical (St Louis, MO). The cells used in this study, and their growth, starvation, and conjugation conditions were essentially as previously described (Brown et al. 1993 ; Wolfe et al. 1993 ). Briefly, stock Tetrahymena cells of mating types BIII and BVII (Nanney and Caughey 1953 ) were grown in 25 ml of filtered, sterile 2% proteose peptone (Difco; Detroit, MI) in 250-ml bottles at room temperature and were subcultured weekly. Active cultures were started by inoculating 100 ml of medium in 500-ml screw-cap bottles with 1 ml of stock cells and were grown in a 30C incubator for 2-3 days, reaching a density of about 2-5 x 105 cells/ml.

Before conjugation, cells were washed three times by centrifugation in sterile 10 mM Tris, pH 7.4. The cells were then resuspended in fresh Tris buffer to a cell density of about 2.5 x 105 cells/ml in Erlenmeyer flasks of at least 10 times the volume of the cell suspension and were incubated overnight at 30C. To induce conjugation, equal numbers of complementary mating types were mixed and kept at 30C without shaking. Typically, 80-90% pairing was achieved when conjugation was performed in 50-ml cell suspensions in 500-ml Erlenmeyer flasks.

Cytological analyses
DAPI Staining. The process and staging of nuclear development during conjugation were monitored by staining fixed cells with diamidinophenolindole (DAPI), as follows. Samples were withdrawn at appropriate times, fixed with an equal volume of 20% formalin in 10 mM phosphate buffer, pH 7.0, and kept on ice for 5 min. Excess fixative was washed away by centrifugation and the pellet was resuspended in 1 ml of 10 mM Tris, pH 7.4. Then, 5 µl of a 10 µg/ml stock solution of DAPI in distilled water was added and the cells were washed twice by centrifugation before resuspension in the same buffer containing 25% glycerol and 1% N-propylgallate (NPG) to retard photobleaching (Giloh and Sedat 1982 ). Cells were observed under fluorescence microscopy as described below.

Acridine Orange Staining. Cells at appropriate stages of conjugation were stained with AO as follows. One µl of a 1% stock concentration of AO in distilled water was added to 1 ml of conjugating cells. After mixing briefly, the stained cells were observed immediately with fluorescence microscopy using filters for green fluorescence. In control experiments, conjugating cells were fixed as above before staining with AO. In other experiments, cytoskeletal frameworks were prepared as previously described (Wolfe 1985 ) and either were directly stained with AO or were first treated with RNase (100 µg/ml) (Boehringer; Indianapolis, IN) for 30 min at 37C before staining with AO.

Hoechst Staining. Nuclear DNA was visualized in live cells by staining conjugants with the DNA-specific dye Hoechst 33342 (HO). One ml of conjugating cells was removed and HO was added to a final concentration of 5 µg/ml. Cells were observed immediately with filters for blue fluorescence (the blue channel).

AO/HO Staining. AO/HO is a combination of acridine orange and Hoechst 33342. Used at the concentrations indicated above for the individual stains, AO/HO provides better contrast and allows more distinctive identification of the various classes of nuclei than either stain alone. For AO/HO staining, 1 ml of conjugating cells was doubly stained with 0.001% AO and 5 µg/ml HO. Observations were made with the blue channel.

Ammonium Chloride Treatment
The effect of ammonium chloride on macronuclear condensation was tested by adding various concentrations of ammonium chloride to conjugating cells at 6.5 hr after mixing. At 10 hr, 1-ml samples were withdrawn and fixed with an equal volume of formal phosphate. Fixed samples were stained with DAPI and the numbers of paired cells with condensed macronuclei were scored for each concentration of ammonium chloride. About 200 cells were scored in each case. In some experiments, a solution of ammonium chloride (final concentration 10 mM) was added to conjugating cells at 6.5 hr after mixing opposite mating types, and at 12 hr of conjugation the cells were stained with AO/HO.

Fluorescence Microscopy
All fluorescence observations were made with a Zeiss Axioplan photomicroscope equipped with epifluorescent illumination. DAPI is excited at about 350 nm and emitted light is observed with filters for blue fluorescence. AO-stained cells are usually observed with filters for green fluorescence. The green channel allows excitation of AO molecules at 450-490 nm, and emitted light is filtered through a green filter. In some experiments with AO staining, we made observations with the blue channel. AO/HO-stained cells were also viewed with filters for blue fluorescence. All fluorescent micrographs were taken with an automatic camera loaded with Kodak Gold 200 ASA film.


  Results
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Materials and Methods
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Condensed Macronuclei Are Often Surrounded by Clusters of Lysosomes
When Tetrahymena are stained with AO and observed with green fluorescent microscopy, clusters of lyso-somes and phagosomes (perhaps autophagosomes because the cells have being starving for over 24 hr) fluoresce an intense orange-red. In vegetative cells, lysosomes are randomly distributed throughout the cytoplasm (Figure 1A) and vary in number from cell to cell. In conjugating cells, lysosomes appear to be preferentially localized to the posterior end of the cells (Figure 1B). This is particularly evident at the stage in which the old macronucleus has already condensed. Clusters of lysosomes are often localized close to the condensed macronucleus. Occasionally a red ring of lysosomes is seen tightly encircling the condensed nucleus, suggesting that lysosomes release their digestive contents into the dying nucleus.



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Figure 1. AO staining of Tetrahymena cells to reveal the distribution of lysosomes. Conjugating Tetrahymena cells were stained with AO and observed with fluorescent microscopy using filters for green fluorescence. In single cells (A) with a normal macronucleus (mac), there is a random distribution of lysosomes (lys) throughout the cytoplasm. In conjugating cells (B) the lysosomes are preferentially localized around the degenerating old macronucleus (cm). Bars = 10 µm.

Condensed Macronuclei Stain Red with Acridine Orange
In the course of staining conjugating cells with AO to monitor the distribution of lysosomes, we noted that, unlike other nuclei, condensed macronuclei stain orange-red with AO (Figure 2B). Micronuclei and macronuclei in vegetative cells or early conjugants stain a greenish color, as does the cytoplasm (Figure 2A). These observations indicate that AO stains apoptotic nuclei differently from normal nuclei. The distinctive staining of the condensed macronuclei gives rise to the question: Is this due to lysosomal fusion or is it related to a unique state of nucleic acids?



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Figure 2. AO staining of Tetrahymena cells revealing differential staining of the precondensed and condensed macronuclei AO-treated cells viewed with filters for green (A,B) and blue (C,D) fluorescence. (A) Lysosome-related structures (lys) appear as small red bodies. Micronuclei (arrows), and macronuclei at the precondensed stage (chevron), stain green. (B) Condensed macronuclei stain orange-red (arrowhead) and are often surrounded by lysosomal bodies. In C and D, the high background fluorescence of the cytoplasm with AO staining is eliminated. The precondensed macronucleus stains green; lysosomes stain red and are randomly distributed throughout the cytoplasm (C). In cells with condensed macronuclei (D), the macronuclear anlagen are not visible, micronuclei stain green (arrows), and the condensed macronucleus stains orange-red (arrowhead). Only one member of a conjugant pair is shown in each figure. Bars = 10 µm.

The Distinctive Staining Is Eliminated in Fixed Cells
We sought to distinguish between the two possible staining activities of AO: metachromatic staining of nucleic acids (Darzynkiewicz 1990 ) or accumulation and aggregation of AO in acidic vesicles (Kapuscinski and Darzynkiewicz 1984 ). To test whether AO is staining RNA that might accumulate in the dying nucleus, we prepared cytoskeletal frameworks and compared RNAse-treated samples to untreated controls. In both cases the old macronucleus, as well as the other nuclei, stained green rather than red (not shown). Therefore, a high concentration of RNA in the condensed old macronucleus was ruled out as the basis for the differential AO staining. We next stained fixed whole cells with AO to distinguish them from stained live cells. In fixed cells, the old macronucleus stained green with AO, comparable to the other nuclei (not shown). Because AO does not differentially stain the old macronucleus under conditions in which acidification is disrupted, the data suggest that the mechanism of staining is related to the acidification of the old macronucleus, and accumulation and aggregation of AO within it.

A Double-staining Procedure Identifies Apoptotic Nuclei in Conjugating Cells of Tetrahymena
On the basis of the studies with AO staining alone, there appears to be a close relationship or a potential overlap between the locations of lysosomes and the condensed macronuclei. However, when these specimens were viewed in the green channel, the high autofluorescence from AO staining of the cytoplasm often obscured clear visualization of the relative positions of the various structures in the cell. We utilized another staining procedure that circumvents this problem and allows simultaneous visualization of the various classes of nuclei, as well as the distribution of lysosomes with respect to the apoptotic nucleus in conjugating cells of Tetrahymena. In this double-staining procedure, living cells were stained with AO simultaneously with the vital DNA-binding fluorochrome HO. Observations were then made in a fluorescent microscope with filters for blue fluorescence.

When AO-stained conjugants are observed with filters for blue fluorescence, lysosomes stain red, micronuclei appear greenish, and pre-condensed macronuclei stain green (Figure 2C). In conjugants with condensed macronuclei, the differentiating macronuclei are not visible and the condensed macronucleus stains orange-red (Figure 2D).

Nuclei in living conjugants stained with HO alone and observed with filters for blue fluorescence (Figure 3B) appear comparable to those stained with DAPI in fixed cells (Figure 3D). Figure 3A shows a macronucleus (mac) stained with HO at a stage just before condensation. This stage is identified by the presence of four postzygotic nuclei, two of which have enlarged slightly, an early stage in their differentiation into new macronuclei. In the next stage, which happens only moments later, the macronucleus condenses and (usually) moves to the posterior end of the cell. DNA fragmentation occurs exclusively in the condensed nucleus, as shown by the TUNEL assay (Figure 3C). Micronuclei, macronuclear anlagen, and pre-apoptotic macronuclei are not labeled by TUNEL (Mpoke and Wolfe 1996 ).



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Figure 3. Hoechst 33342 (HO) staining of living cells. (A) A conjugant pair stained with HO at the pre-apoptotic stage. The parental macronucleus has not yet condensed. Each pair has four postzygotic micronuclei (pZN). (B) Hoechst staining of a conjugant pair at the apoptotic stage. The parental macronucleus has become highly condensed (cm), and two of the four micronuclei in each cell have enlarged to become macronuclear anlagen (an). (C) A TUNEL-stained cell showing that only the condensed macronucleus is TUNEL-positive. (D) DAPI-stained conjugant pair, indicating that HO staining is comparable to DAPI staining. Only one member of a conjugant pair is shown in each figure. Bars = 10 µm.

When both vital stains are used together, the color contrast between nuclei and cytoplasm is enhanced and the difference betwen normal nuclei and the degenerating nucleus is more clearly visualized. The cytoplasm is dark, and newly forming macronuclei (macronuclear anlagen) stain a clear blue, whereas the more condensed, differentiating micronuclei stain a somewhat brighter blue (Figure 4). The apoptotic nucleus stains a brilliant orange-red (Figure 4A) or bright yellow (Figure 4B), and lysosomes stain a deep red. Therefore, in a single living cell, three or four different colors are seen simultaneously, each associated with a different structure. The condensed apoptotic macronucleus is clearly distinguished from normal nuclei.



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Figure 4. Different classes of nuclei are distinguishable by double staining with AO/HO. (A) Conjugating cells doubly stained with AO/HO at a late stage of nuclear death show a highly condensed or apoptotic nucleus staining a brilliant orange-red. At a stage just preceding this, the condensed nucleus stains yellow (B). Earlier stages of nuclear death display yellow-green (C) or greenish-blue (D) macronuclei. (E,F) Chicken chondrocytes doubly stained with AO/HO. In normal chondrocytes (E) the nucleus stains blue, and the cytoplasm is rich in orange-red lysosomes and phagosomes. In apoptotic chondrocytes (F) the nucleus stains yellow or orange-red, becoming indistinguishable from the cytoplasm. (G) Ammonium chloride-treated cell stained with AO/HO. Whereas the condensed macronucleus appears orange-red in control cells (e.g., A), here it stains green. (H) Necrotic nuclei stained with AO/HO. Necrosis was induced in Tetrahymena by incubating log-phase cells with 1 mM ZnSO4 for 3 hr, followed by staining with AO/HO. The macronuclei stain blue, indicating that AO/HO does not differentially stain necrotic nuclei. Bars = 10 µm.

The regularity in the temporal sequence of nuclear events during conjugation enabled us to determine that the yellow color is associated with early apoptotic nuclei, whereas the red color is associated with later stages. The transition from yellow to red may reflect increased acidification with time. In any event, this double-staining method not only differentiates between normal and degenerating nuclei, but also distinguishes between early and late stages of degeneration. Occasional precondensed nuclei with a yellow-green (Figure 4C) or greenish-blue fluorescence (Figure 4D) were also seen, suggesting that the very earliest stages of nuclear apoptosis may also be detected by this double stain. Consistent with the initial stage of nuclear death is the observation that the greenish-blue nucleus in Figure 4D is not yet fully condensed. This indicates that the AO/HO staining technique might be used to identify cells or nuclei just as they become apoptotic.

AO/HO Staining Identifies Apoptotic Nuclei in Vertebrate Cells
To investigate whether the AO/HO staining procedure might be applicable to a more typical apoptosis as a means to identify dying cells, we used primary cultures of chicken chondrocytes. These cells can be grown in culture for extended periods of time in the presence of serum. When serum is withdrawn, the cells can remain viable only if seeded at high densities. However, at low densities in the absence of serum, cells begin to die by apoptosis (Bruckner et al. 1989 ).

At 24 hr after induction of apoptosis in chondrocytes by removal of serum from their medium, the still living cells were vitally stained with AO/HO and were observed with the blue channel. Healthy-looking cells have highly noticeable orange-red granules (lysosomes and phagosomes) in their cytoplasm and a blue-staining nucleus (Figure 4E). In contrast, the cytoplasm of apoptotic cells is red-orange, and their nuclei stain yellow. In what is apparently a later stage of apoptosis, the nucleus also stains, becoming hardly distinguishable from the cytoplasm (Figure 4F). These observations show that the apoptotic chondrocytes are readily distinguished with AO/HO, that their nuclei are differentially stained, and that the sequence of color changes in the dying nucleus, from blue through yellow to orange-red, is similar to that observed in Tetrahymena.

Nuclear Acidification May Be the Basis for Differential Staining
To test whether nuclear acidification is required for differential staining by AO/HO, conjugating cells were exposed to two weak bases, ammonium chloride or chloroquine, at 6.5 hr after conjugation. After 12 hr of conjugation the cells were stained with AO/HO. Figure 4G shows that the condensed macronucleus appears green rather than orange-red. The same occurs when cells with already condensed macronuclei (e.g., 14-hr conjugants) are treated with ammonium chloride. Presumably, the weak bases neutralize the acidity, thus reversing the appearance of orange-red nuclei. However, direct demonstration for this will require actual measurement of the nuclear pH.

Interestingly, ammonium chloride does not prevent macronuclear condensation, nor does it prevent nuclear elimination (not shown). Therefore, although acidification might be a general feature of nuclear death, it is not necessary for nuclear death (see Discussion).

AO/HO Differentially Stains Apoptotic but not Necrotic Nuclei
We tested whether AO/HO is specific for apoptosis or whether it also differentially stains necrotic nuclei. Necrotic cell death was induced in Tetrahymena by incubating log phase cells with 1 mM ZnSO4, or 500 nM staurosporine. Under these conditions, most of the cells are dead by 3 hr. [DNA from necrotic Tetrahymena does not show an oligonucleosomal pattern on agarose gels (Mpoke and Wolfe 1996 )]. Stained with AO/HO, the macronuclei of necrotic cells appear blue (Figure 4H). Therefore, AO/HO does not differentially stain necrotic nuclei.


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These studies employ a simple procedure using two vital fluorescent stains, AO and Hoechst 33342, to readily distinguish between apoptotic nuclei and normal nuclei. Because AO/HO differentially stains apoptotic nuclei in living cells, we refer to it as "Vital Apo-fluor." The combined stain provides excellent staining contrast between the nucleus and cytoplasm, and simultaneuosly stains DNA containing structures and lysosomes or lysosome-derived bodies. Although it is too soon to determine the general applicability of the procedure, preliminary data indicate that this simple staining technique may offer additional flexibility to the existing repertoire of methods for identifying and studying apoptotic cells.

The AO/HO staining procedure has several advantages over the TUNEL technique. First, it can be used with living cells, enabling identification and possible selection of apoptotic cells within a population of cells that are in the process of dying. Second, Vital Apofluor is able to distinguish between early and late apoptosis on the basis of the yellow or orange color emitted. Moreover, the Vital Apofluor staining method has the potential to allow detection of subtle color changes associated with the transition to apoptosis even before nuclear condensation has occurred. These color changes might, in turn, be related to the early DNA digestion that has been shown by several studies to occur before chromatin condensation. Finally, Vital Apofluor is specific for apoptotic nuclei and does not differentially stain necrotic nuclei. Several authors have raised concerns about the current difficulties in discrimininating between apoptotic nuclei and necrotic nuclei using the TUNEL assay (Columbano 1995 ; Yasuda et al. 1995 ; D’Herde et al. 1994 ).

On the other hand, the requirement that Vital Apo-fluor be used with living cells precludes its application to studies in which tissues must be fixed and in those when analysis of experimental results must be delayed.

Although the basis of the differential staining with Vital Apofluor is not clear, because the differential is lost in fixed cells a requirement for an active proton pump is suggested. The simplest explanation is that the differential staining is due to acidification of the old macronucleus rather than to staining of single-stranded DNA or RNA. Another possibility is that the AO staining of the old macronucleus is a function of single strand DNA induced by the AO itself. The argument is that a proton pump increases the concentration of AO in the old macronucleus. Then, because the old macronucleus is apoptotic (Mpoke and Wolfe 1996 ; Davis et al. 1992 ), and because apoptotic DNA is susceptible to denaturation by high concentrations of AO (Darzynkiewicz et al. 1992 , Darzynkiewicz et al. 1993 ), the differential staining of the old macronucleus would be due to AO staining of single-stranded DNA. We think this possibility is unlikely because the orange-red coloration of the old macronucleus with Vital Apofluor is virtually instantaneous, occurring within seconds of staining. This is not consistent with a time-dependent denaturation process.

It appears to us that nuclear death is associated with a decrease in the pH of the apoptotic nucleus, conceivably by fusion with lysosomes. Our observations showing clustering and, in some cases, tight encircling of lysosomes around the condensed nucleus is consistent with this view. Moreover, at the ultrastructural level, a vesicle, perhaps derived from lysosomes, envelops the degenerating macronucleus (Weiske-Benner and Eckert 1987 ). This model would also account for the gradual change in the staining color of the apoptotic nucleus: As more lysosomes fuse with the nucleus it becomes more acidic, and the relative emissions from Vital Apofluor shift from blue to green to yellow to orange-red.

Several studies have shown that apoptosis is accompanied by acidification of the entire cell (Gottlieb et al. 1995 ; Li and Eastman 1995 ; Perez-Sala et al. 1995 ). Barry and Eastman 1993 have proposed the existence of an acidic endonuclease in Chinese hamster ovary (CHO) cells that cleaves the DNA in these cells at internucleosomal sites to yield the characteristic apoptotic pattern. This enzyme has identical biochemical features with bovine DNAse II and is considered the CHO homologue of DNAse II. It does not require divalent cations and is not inhibited by Zn2+. Using rat thymocytes induced to undergo apoptosis by {gamma}-ray irradiation, Shiokawa et al. 1994 identified three endonucleases, DNAse {gamma}, {alpha}, and ß. DNAse {gamma} is a cation-dependent, Zn2+-sensitive neutral enzyme, whereas DNAse {alpha} and ß are cation-independent enzymes that require an acidic environment for optimal activity. An acidic endonuclease was also found in various human myelogenous leukemic cell lines exposed to a variety of agents (Yanagisawa-Shiota et al. 1995 ). In Tetrahymena, nuclear elimination is not blocked by Zn2+ or chelation of Ca2+ and Mg2+ (Mpoke and Wolfe 1996 ), indicating that the enzyme involved during nuclear death is likely to be independent of these divalent cations, involving, instead, perhaps acidic enzymes such as DNAse II (Barry and Eastman 1993 ) or DNAse {alpha} and ß (Shiokawa et al. 1994 ).

Lysosomes might therefore play an important role during nuclear death in a number of ways. First, by fusing with the targeted nucleus, lysosomes might release their contents, which may include acidic endonucleases, into the nucleus, thus initiating DNA hydrolysis. Alternatively, the primary role of lysosomes might be to lower the pH of the nucleus, thereby facilitating the activation of resident latent acidic endonucleases which then digest nuclear DNA. Finally, by providing acidic conditions, lysosomes might be playing a role in the degradation of the proteinaceous component of the condensed nucleus. Studies with mouse peritoneal macrophages have indicated that an acidic environment is required for protein degradation because if the pH is raised, protein degradation is inhibited (Ohkuma and Poole 1981 ; Wibo and Poole 1974 ).

Our studies with ammonium chloride showed that when the acidic condition of the old macronucleus is eliminated, the property of differential staining is lost. We wondered whether non-acidic conditions might also cause a delay or inhibition of nuclear death, i.e., to what extent acidification might be a requirement for DNA degradation. Our observations showed that old macronuclei that were not condensed at the time of exposure to ammonium chloride proceed to become condensed. Hence, the first step of nuclear elimination, condensation, is not blocked by ammonium chloride. However, we predicted that the next step, DNA degradation, would be either blocked or delayed, and we expected, as a result, to see an accumulation of cells with condensed nuclei. However, contrary to our expectations, we did not observe such an accumulation. In the presence of ammonium chloride the old macronuclei were eliminated, with kinetics indistinguishable from those of control cells (data not shown). This shows that if the orange-red staining with Vital Apofluor is due to nuclear acidification, then acidification is not necessary for nuclear elimination.

The observation that under basic conditions apoptotic nuclei are eliminated suggests that there may be more than one degradative pathway leading to nuclear death. The pathway that involves lysosomes might be the most efficient because it involves, in addition to acidic endogenous nucleases, a battery of other hydrolytic enzymes. In the presence of ammonium chloride, however, intranuclear pH is raised, which could then allow the activation of non-acidic endonucleases such as DNAse I, and hence the process of DNA digestion and nuclear elimination would still be able to proceed to completion. Others have also discussed the possible existence of separate metabolic pathways in apoptosis (Sun et al. 1994 ) and the utilization of several candidate endonucleases in apoptosis (Yanagisawa-Shiota et al. 1995 ; Shiokawa et al. 1994 ; Barry and Eastman 1993 ; Peitsch et al. 1993 ).

We present here a staining method that is able to detect dying nuclei while the cells are still alive. We suggest that this staining procedure may be useful for the study of apoptosis. In addition to histo- and immunocytology after identification, it may be possible to sort dying cells using flow cytometry. It may also be possible to identify and select early and late apoptotic cells and to perform separate analyses on them. Finally, the technique may prove to be useful in identifying and selecting those cells at the very entry into apoptosis.


  Acknowledgments

Supported by funds from NIH (JW).

We thank members of Dr L. Luken's laboratory for their generous gift of chicken chondrocytes and Drs J. Naegele and W. Firshein for critical reading of this manuscript.

Received for publication May 10, 1996; accepted December 5, 1996.


  Literature Cited
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Summary
Introduction
Materials and Methods
Results
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Literature Cited

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