Journal of Histochemistry and Cytochemistry, Vol. 45, 923-934, Copyright © 1997 by The Histochemical Society, Inc.


ARTICLE

Major DNA Fragmentation Is a Late Event in Apoptosis

Jae A. Collinsa, Cynthia A. Schandla, Kristy K. Younga, Josef Veselya, and Mark C. Willinghama
a Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina

Correspondence to: Mark C. Willingham, Dept. of Pathology and Laboratory Medicine, Medical Univ. of South Carolina, 171 Ashley Ave., Charleston, SC 29425.


  Summary
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Apoptosis, the terminal morphological and biochemical events of programmed cell death, is characterized by specific changes in cell surface and nuclear morphology. In addition, DNA fragmentation in an internucleosomal pattern is detectable in mass cultures of apoptotic cells. However, DNA fragmentation and nuclear morphological changes may not necessarily be associated events. In this study, we examined OVCAR-3 and KB human carcinoma cells using time-lapse video phase-contrast microscopy to characterize the surface and nuclear morphological features of apoptosis in response to treatment with either taxol or ricin. The surface morphological features of apoptosis were the same in both cell types and with both drugs. Using an in situ nick-translation histochemical assay, these single cells were also examined for DNA strand breaks during apoptosis. Surface morphological changes demonstrated discrete stages of cell rounding, surface blebbing, followed by cessation of movement and the extension of thin surface microspikes, followed much later by surface blistering and cell lysis. Nuclear features examined by DAPI cytochemistry demonstrated apoptotic nuclear condensation very early in this sequence, usually at the time of initial surface blebbing. The nick-translation assay, however, demonstrated DNA strand breaks at a much later time, only after the formation of separated apoptotic bodies or after final cell lysis. This study points out the differences between surface and nuclear morphological changes in apoptosis, and the large temporal separation between nuclear morphological changes and major DNA fragmentation detectable by this in situ technique. This result suggests caution in using in situ nick-translation as a direct correlate of internucleosomal DNA fragmentation in apoptosis. (J Histochem Cytochem 45:923-934, 1997)

Key Words: apoptosis, DNA fragmentation, blebbing, nick-translation, video time-lapse, taxol, ricin, cell death, necrosis


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Apoptosis is a term used to describe the terminal morphological and biochemical events seen in programmed cell death (Wyllie 1992 ). Apoptosis has recently been the subject of great interest because it has been clearly demonstrated to mediate cell death, not only during development but also in neoplasia in response to cancer chemotherapy and radiation (Hickman 1992 ; Eastman 1990 ). Increased interest in apoptosis is also the result of finding several genes, most notably bcl-2, that can regulate the apoptotic process in cells (Hockenbery et al. 1990 ; Tsujimoto and Croce 1985). An understanding of the mechanisms involved in apoptosis will clearly have a major impact on the therapy of cancer and of other pathological conditions that involve active cell death, including autoimmune diseases, neurodegenerative diseases, and viral infections.

The gene-directed and/or -regulated events of apoptosis are very different from those seen in necrosis (Majno and Joris 1995 ). Necrosis is the death of cells as a result of direct injury, usually beginning at the cell surface. Necrotic cells exhibit early lysis of the plasma membrane before any significant alterations in nuclear morphology. Necrotic cells initially swell before lysis, whereas apoptotic cells show cell shrinkage. Necrotic cells eventually exhibit swelling of the nucleus, whereas apoptotic cells exhibit characteristic nuclear morphological changes, including chromatin condensation and hypersegmentation of nuclear chromatin of irregular size. These hypersegmented nuclear structures may then bud from the rapidly blebbing cell surface to form "apoptotic bodies." The surface features of necrosis are also very different from those of apoptosis. Necrotic cells swell and lyse, whereas apoptotic cells show intense cell surface zeiotic blebbing.

A hallmark feature of apoptosis was the observation that nuclear DNA extracted from apoptotic cells was often degraded in an internucleosomal pattern (Compton 1992 ; Arends et al. 1990 ). That is, DNA cleavage during apoptosis occurred at sites between nucleosomes, protein-containing structures that occur in chromatin at ~200-BP intervals. This DNA fragmentation was often analyzed using agarose gel electrophoresis to demonstrate a "ladder" pattern at ~200-BP intervals. Necrosis, on the other hand, is characterized by random DNA fragmentation which forms a "smear" on agarose gels. These differences prompted interest in the specific endonuclease that might be unique to apoptosis, but later work has shown that the morphological features of apoptosis in the cytoplasm can occur in cells that have been enucleated (Jacobson et al. 1994 ; Schultze-Osthoff et al. 1994 ) or in the absence of DNA fragmentation (Falcieri et al. 1993 ; Cohen et al. 1992 ). This suggested that DNA fragmentation might be a secondary consequence, rather than an integral cause, of apoptosis. Other data suggested that the endonuclease involved might be similar to DNAse I, a potential indication that the DNA fragmentation might occur after the release of enzymes from cytoplasmic membrane lysis, an event that would potentially occur only after the final lytic event in the apoptotic sequence (Mannherz et al. 1995 ; Peitsch et al. 1993 ). One could speculate that the ladder pattern of fragmentation in apoptosis might be a consequence of the state of the chromatin at the time of fragmentation rather than of the nature of the endonucleases involved. More recently, data have shown that specific proteases residing in the cytoplasm mediate the terminal events of apoptosis, including those of nuclear morphology (Tewari et al. 1995 ; Lazebnik et al. 1993 ). Even so, the detection of DNA fragmentation and the presence of single strand ends of DNA has continued to be an assay used in many studies to detect apoptotic cells, particularly in intact tissues. This is in spite of the fact that necrosis also produces single-strand DNA ends in cell nuclei. Therefore, the interpretation of these in situ assays of DNA fragmentation [in situ nick-translation (ISNT); terminal transferase (TUNEL)] must be carefully assessed together with morphological features of apoptotic cells.

Even though much work has been performed on the analysis of apoptotic events, little information is available to link the timing of morphological features at the cell surface and in the nucleus to the biochemical degradation of DNA in the same cells. Apoptosis can be initiated by a myriad of different mechanisms in different cell types, and the kinetics of these events vary widely, from only a few minutes to several days depending on the cell system. We wished to analyze the relationship between apoptotic morphological features and DNA fragmentation in different types of cells using different initiating agents. In this study we used taxol, an agent requiring cell cycle events to cause apoptosis (Bhalla et al. 1993 ; Manfredi and Horwitz 1984 ), and compared it with ricin, an agent that can initiate apoptosis independent of cell cycle position (Griffiths et al. 1987 ). Using an in situ nick-translation histochemical assay similar to other previously described assays for detection of apoptotic single-strand ends of DNA (Gold et al. 1993 ; Gorczyca et al. 1993 ; Wijsman et al. 1993 ; Gavrieli et al. 1992 ), we have defined the point in the apoptotic process at which major DNA fragmentation takes place, the point at which most in situ assays of DNA fragmentation would give clear signals.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Cell Cultures
KB (HeLa markers, cervical carcinoma) (ATCC CCL 17; Rockville, MD), OV2008 (Hamilton et al. 1985 ), and OVCAR-3 (ovarian serous carcinoma; ATCC HTB 161) cells are human carcinoma cell lines previously described. The cells were grown in RPMI-1640 medium containing 10% fetal calf serum, penicillin, and streptomycin and were subcultured using trypsin-EDTA. These cell types are adherent to the surface of plastic tissue culture flasks and dishes, but for the nick-translation assays the substratum was also coated by incubation with 1 mg/ml poly-L-lysine (Sigma #P-1399; St Louis, MO) for 5 min at 23C, followed by washing in PBS.

Taxol and Ricin Treatment
Taxol, a cancer chemotherapeutic drug (Paclitaxel) derived from the bark of the Pacific yew tree (Taxus brevifolia) (Calbiochem; La Jolla, CA), causes hyperpolymerization of tubulin and subsequent mitotic arrest, followed by apoptosis (Manfredi and Horwitz 1984 ; Bhalla et al. 1993 ). Taxol's induction of apoptosis is strictly dependent on arrest of the cell cycle in mitosis. Ricin (Sigma) is a highly toxic plant lectin that binds to surface galactose residues, is internalized, and subsequently causes inhibition of protein synthesis through its action on ribosomes. Along with many other protein synthesis-inhibiting toxins, ricin causes apoptosis independent of the cell cycle (Griffiths et al. 1987 ). Taxol (1 µM) or ricin (0.1 µM) was incubated with cells in normal culture medium for the entire period of video recording (ending at 24-72 hr).

Video Time-lapse Microscopy
Tissue culture dishes (35 mm) or T-25 tissue culture flasks, containing cells planted at least 24 hr before drug addition, were incubated with taxol or ricin as indicated above. These culture vessels were placed on the stage of a Zeiss ICM-405 inverted phase-contrast microscope equipped with a warm stage heater/recirculator device that maintained a 5% CO2/95% air atmosphere and a temperature of 37 ± 0.1C (Willingham and Pastan 1985 ). Cells were illuminated with red light and the images obtained with a x40 (NA 0.8) LWD phase 2 oil-immersion objective were captured using a Hitachi KP-M1U CCD camera and recorded using a Panasonic NV-8030 time-lapse video recorder at a fixed rate of 1 frame/ 12 sec (720:1 final time lapse). Secondary recordings from the time-lapse recorder were made using a VHS VCR, and these images were captured using a Macintosh 8500AV computer. These digital images and those scanned from photographic film (below) were then processed using Adobe Photoshop and printed with a Techtronics dye sublimation printer.

Histochemical Methods
Nuclear morphology was selectively identified using histochemical labeling with 0.1 µg/ml DAPI (4',6-diamidine-2'-phenylindole diHCl) (Boehringer; Indianapolis, IN) for 15 min at 37C, a fluorescent dye that selectively labels DNA. In situ nick-translation was performed by a modification of methods previously described (Gold et al. 1993 ; Wijsman et al. 1993 ). Cells were fixed using 3.7% formaldehyde in PBS (15 min, 23C) followed by incubation in 0.1% Triton X-100 to permeabilize nuclei. Positive controls included pre-incubation at this point in 0.1 µg/ml DNAse I (Boehringer) for 1 hr at 23C. Negative controls included deletion of the mouse anti-biotin or deletion of the DNA polymerase described below. The nick-translation reaction (2-3 drops held under a coverslip in each dish) consisted of a solution of 1 ml final volume in H2O containing 100 µl of x10 NT stock buffer, 15 µl dithiothreitol (15 µg/ml stock), 20 µl (100 U) DNA polymerase (endonuclease-free; Boehringer #642-720), 5 µl each (10 µg) of dATP, dCTP, dGTP, and 16 µl of biotin-16-dUTP (Boehringer #1093-070, 50 nmoles). NT buffer x10 included Tris-HCl (60 mg/ml), MgSO4 (25 mg/ml), and crystalline bovine serum albumin (BSA) (0.5 mg/ml). The NT reaction was allowed to proceed for 4 hr at 23C. Immunocytochemical labeling of the incorporated biotin was performed using the following steps: (a) 1 µg/ml mouse monoclonal anti-biotin (Boehringer #1297-597); (b) 25 µg/ml affinity-purified goat anti-mouse IgG conjugated to rhodamine (Jackson ImmunoResearch; West Grove, PA), and (c) 25 µg/ml affinity-purified rabbit anti-goat IgG conjugated to rhodamine (Jackson ImmunoResearch) (for further signal amplification). Each antibody incubation was in a diluent composed of 2 mg/ml crystalline BSA in PBS for 1 hr at 23C. The cells were then postfixed in 3.7% formaldehyde in PBS. This was followed by incubation in 0.1 µg/ml DAPI in PBS for 15 min at 37C. After washing, the cells were mounted under a #1 coverslip in glycerol in the culture dish, and viewed using a Zeiss Axioplan epifluorescence microscope equipped with rhodamine and UV excitation filters and a x40, NA 1.3, phase 3, plan-Neofluar objective. The images of cells were recorded using Kodak Tri-X film developed in Diafine and were digitized using an AGFA-GEVAERT Studioscan IIsi flatbed scanner.

Agarose Gel Electrophoresis
DNA was purified from cultured cells using a DNA extraction kit for high molecular weight DNA (Stratagene #200600; La Jolla, CA). The purified DNA was electrophoresed using a 2% agarose gel with ethidium bromide staining.

Permeability Assay
An assay was devised to examine the intactness of the plasma membrane of cells during apoptotic and necrotic events. The principle of this assay was to identify cells with permeable plasma membranes on the basis of their ability to allow high molecular weight poly-L-lysine to penetrate to the nucleus and bind, in a polyvalent fashion, a subsequently introduced fluorescently labeled immunoglobulin. To demonstrate necrosis, as shown in Figure 1, KB cells were exposed to 0.05% Triton X-100 in water at 23C. Most cells underwent necrotic lysis in less than 2 min. For apoptosis, KB cells were exposed to 10-7 M ricin in normal culture medium for 20 hr at 37C. As a positive control, cells were treated with 80% acetone in water for 5 min to render them completely permeable or, as a negative control, were not exposed to any treatment. After these treatments, the dishes were placed at 4C and incubated with PBS containing 1 mg/ml poly-L-lysine (Sigma) for 5 min, followed by washing in PBS, and then incubated with PBS containing 100 µg/ml affinity-purified goat anti-rabbit IgG conjugated to rhodamine (Jackson ImmunoResearch) for a further 5 min in the absence of any other added protein. After a PBS wash, cells were then fixed in 3.7% formaldehyde in PBS for 5 min at 23C, incubated in DAPI at 10 µg/ml in methanol, and mounted under a #1 coverslip in glycerol.



View larger version (99K):
[in this window]
[in a new window]
 
Figure 1. Morphological features of necrosis in KB cells. KB cells were washed in PBS and water containing 0.1% Triton X-100 was slowly added dropwise to the culture dish. The images were recorded using phase contrast video microscopy. The number in the upper left corner of each panel represents seconds after the first single video frame. The first morphological feature of necrosis is the formation of surface blisters (arrowheads) (at 34 sec), blisters that subsequently break (at 56-58 sec). Bar = 20 µm.

Quantitation of Changes in Nuclear Morphology and Cell Attachment
KB cells were incubated with 10-7 M ricin for 0, 6, 16, or 24 hr. The number of cells attached to the substratum was measured by washing the dishes with fresh medium before trypsinization and cell counting using a Coulter particle counter. The number of cells showing altered nuclear morphology was assessed at those time points by fixing the cells remaining in the dish and staining with DAPI. The number of cells released into the medium was assessed by harvesting culture medium from unwashed cells in parallel experiments and preparing cytospins from these media samples, cytopreps that were then stained with DAPI. These cytopreps were then quantitated by directly counting under the microscope the number of cells with normal or apoptotic nuclear morphology.


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Morphological and Kinetic Features of Apoptosis in Cultured Human Carcinoma Cells
Even though many static and dynamic features of apoptosis have been described before (Wyllie 1992 ; Kung et al. 1990 ), we wished to clarify the timing of apoptotic events and their morphology in the systems used in this study. As a result, we employed video time-lapse microscopy to identify the morphological features of apoptosis. We first examined necrosis for comparison with apoptosis (Figure 1). Necrosis can be easily induced by treatment of cells with low concentrations of detergents in an alkaline medium or in water. In examining two typical human carcinoma cell lines, KB and OVCAR-3, we observed that cell necrosis was typified by initial cell swelling and formation of surface blisters, followed by membrane lysis and dispersal of the cytoplasmic organelles and swollen nuclear contents into the surrounding medium (Figure 1). Depending on the concentration of detergent and temperature, this event could be very rapid (seconds) or relatively slow (minutes). The main feature of this process, however, was its passive nature, and at low magnification it often went unnoticed except for the loss of phase-contrast refractility of the plasma membrane.

Apoptosis, on the other hand, had an entirely different appearance. Apoptosis was induced by treatment either with taxol (1 µM), an agent that leads to hyperpolymerization of tubulin and mitotic arrest, or with ricin (0.1 µM), a potent protein synthesis-inhibiting plant lectin that kills cells independent of cell cycle position. Taxol-induced apoptosis uniformly required mitotic arrest before the onset of apoptosis, and the first evidence of apoptosis in such cells usually was delayed until after 12-18 hr. Ricin-induced apoptosis was independent of the cell cycle and occurred earlier, beginning at ~3-4 hr of treatment. Surprisingly, even though the mechanisms of apoptosis induction are very different, the surface morphological features of apoptosis were very similar with both drugs and were also very similar in both KB and OVCAR-3 cells.

Compared to necrosis, apoptosis was a relatively slow event, requiring time-lapse recording to appreciate its progression. Even so, using time-lapse, the changes in cell shape and structure were extremely dynamic, heralded by a rounding of cells together with the onset of intense cell surface blebbing. Examples of this blebbing activity in static images are shown in Figure 2 and Figure 3, but the time-lapse recordings were much more dramatic and this blebbing sometimes lasted for hours. In contrast to the dynamic nature of blebbing, blister formation (as described below) was extremely slow and irreversible (Figure 3). Blebbing is a normal cell activity observed, e.g., at the onset of cytokinesis during mitosis or as a continuous surface activity in certain cell types, such as Chinese hamster ovary cells. Unlike mitotic cells, however, apoptotic cells failed to stop blebbing in a short time and did not flatten back onto the substratum. This event cannot be distinguished from other physiological causes of surface blebbing if examined only by the surface phase-contrast appearance in static images.



View larger version (63K):
[in this window]
[in a new window]
 
Figure 2. Surface morphological features of OVCAR-3 cells during apoptosis. Rows A and B demonstrate phase-contrast video images of OVCAR-3 cells induced to undergo apoptosis by treatment with ricin (10-7 M). Columns 1-5 represent sequential still video images of each of these fields. (A) A phenomenon commonly seen in OVCAR-3 cultures, in which one cell engulfs another (emperipolesis) (asterisk in A2). Frequently, the engulfed cell undergoes apoptosis but, in this case, the cell on the outside (arrow in A1) undergoes apoptosis. Note the surface blebbing activity in A3 (arrow) and the formation of microspikes (echinoid protrusions) in A4 (arrows). This is followed by the formation of a blister in A5 (arrow). Bar = 12 µm. The central cell in row B (arrow in B1) undergoes apoptosis. Note the formation of blebs in B2 (arrow), the later formation of echinoid protrusions in B3 (arrows), and subsequent blister formation in B4 (arrow). Bar = 17 µm.



View larger version (125K):
[in this window]
[in a new window]
 
Figure 3. Dynamic differences between blebs and blisters. (A-D) Single-frame video images using phase contrast microscopy of OVCAR-3 cells undergoing apoptosis induced by ricin, in which surface blebbing activity has begun. (E-H) Similar images of surface blister formation on the same cells at a later time. The numbers in the upper right of each field represent the time in seconds after the beginning of each sequence. Note that blebbing (arrows) is an extremely rapid and dynamic activity, with dramatic changes in shape and size of blebs, whereas the surface blister in E-H is very static, showing little change in shape or size during a longer period of time. Bar = 15 µm.

Apoptosis proceeded to a subsequent stage after a long interval of surface blebbing. This second stage was characterized by the frequent protrusion of surface microspikes or "echinoid protrusions," a name we have coined because of the similarity of these spikes to the spines of sea urchins (e.g., see Figure 2B3). These are rigid structures that can be seen to protrude actively away from the free cell surface. Therefore, these are very different from "retraction fibers" that form when cells retract away from substratum attachment points. Although similar in formation to the microspikes seen at the margin of lamellar movement in migrating cells, these echinoid protrusions occurred elsewhere on the cell surface, and in some cells, such as OV2008, became very long structures, frequently longer than the diameter of the cell (Figure 4). After the final extension of these echinoid protrusions, the cells showed complete cessation of all active movement. The hallmark of this stage of apoptosis, however, was the static and rigid nature of the entire cell, in which movement of the cell by the flow of medium or the movement of adjacent cells showed that these apoptotic cells were highly inflexible.



View larger version (100K):
[in this window]
[in a new window]
 
Figure 4. Echinoid spikes on the surface of an apoptotic cell. An OV2008 cell is shown at a later stage of apoptosis induced by ricin, in which extensive echinoid spikes (arrows) have protruded from the cell body on the left. Bar = 10 µm.

The next events in apoptosis required a very long time, often 6-12 hr. At this point, cells were still refractile by phase-contrast microscopy, indicating that their plasma membrane was still intact. We examined the intactness of the plasma membrane using the permeability assay described in Materials and Methods. Necrotic cells, such as those shown in Figure 1, showed intense labeling of their nuclei after lysis using this assay. On the other hand, apoptotic cells were impermeable. After examining hundreds of cells at various stages of apoptosis, we found permeability of the cell surface only in cells that had undergone the terminal blistering and lysis events described below. In vivo, this intact, contracted state may well correspond to the time at which apoptotic cells are phagocytosed and destroyed. In culture, however, because no phagocytic cells were present, the apoptotic cells eventually reached a terminal stage not dissimilar from necrosis. After this immobile stage, the cells began to show surface blisters that gradually expanded (e.g., see Figure 3). Eventually these blisters ruptured, releasing the contents of the cells into the surrounding medium or creating a granular "ghost" of the cell with swollen organelles. When the concentration of ricin was held relatively low (0.1 µM), this apoptotic process required 12-24 hr. In other experiments, at very high concentrations of ricin (10 µM), the stages of apoptosis occurred more rapidly and the length of these stages was very short. In fact, in some cells the blebbing was so severe that the cells underwent a lytic event before the blebbing stage had progressed for very long. Such cells then looked very much like necrotic cells. This emphasizes that apoptotic events can have dramatically different time courses depending on the inducing mechanism, and also show remarkable heterogeneity within a single culture. This may explain the difficulty of maintaining synchrony of this process in some cell types and the inability to easily create a DNA ladder pattern in cells showing such heterogeneity. That is, such cell asynchrony would at any one time demonstrate a mixture of apoptotic events together with the terminal necrosis-like events of cell lysis at the end of apoptosis. A comparison of nuclear morphology at these various stages showed that, unlike the complete collection of surface morphological changes, nuclear segmentation occurred at a relatively early stage of the apoptotic process (Figure 5).



View larger version (63K):
[in this window]
[in a new window]
 
Figure 5. Correlation of surface morphological features with nuclear features. KB cells were treated with ricin and then fixed and stained using DAPI. Cells showing intense blebbing (A-D) at the beginning of apoptosis by phase-contrast show either normal nuclear morphology (A',B') or advanced nuclear segmentation (C',D'). A cell showing an apparent echinoid protrusion (E, arrow) also shows advanced nuclear disintegration (E'). These results indicate that nuclear changes in apoptosis are an early event that coincides with the early stages of surface blebbing activity. Bar = 12 µm.

Quantitation of these events was performed, an example of which is shown in Figure 6A. By examining a single video field, the time of initial rounding and blebbing was recorded for all cells in this field after incubation with 0.1 µM ricin. A single cell began rounding and blebbing as early as 3 hr, but the entry of other cells into apoptosis progressed slowly, becoming complete only at 24 hr. The bulk of the cells entered apoptosis within 10 hr. This figure points out the relative asynchrony of the induction process. Figure 6B shows a few examples of the timing of different events in the apoptotic process, including blebbing, echinoid protrusions, cessation of all movements, and final cell lysis. Note that the sequence of events always followed in this order, yet the length of time between individual stages was somewhat variable. This again points out that, at any one time in a culture assayed over a long 24-hr time period, individual cells can be at any stage of this process. This result highlights the difficulty of concluding kinetic information from a mass extraction method, such as agarose gel electrophoresis of DNA.



View larger version (13K):
[in this window]
[in a new window]
 
Figure 6. Quantitation of apoptotic events in OVCAR-3 cells treated with ricin. (A) Quantitation of the time of initial rounding and blebbing of apoptosis in OVCAR-3 cells treated with 0.1 µM ricin as determined from a video phase-contrast recording of a single field (all cells in the field, a total of 15 cells at this magnification, underwent apoptosis during the observation period). Note that in different cells apoptosis begins at a wide range of times (3-24 hr). (B) Analysis of four individual cells in a similar experiment, an analysis that includes determination of the beginning of each of the different events in the apoptotic sequence in the same cell. Note the same absolute order of events in all cells and the long time delay between the last cell movements and the final lysis of the cell membranes.

Figure 7 shows a quantitation of the apoptotic events in KB cells in response to ricin treatment, as measured by examining the number of cells remaining attached to the substratum, the percentage of attached cells showing apoptotic nuclear morphology, and the number of cells with apoptotic nuclear morphology that could be recovered from the culture medium. Figure 7A shows that a majority of cells rounded and detached from the dish at 16 hr of treatment, but the washing procedures used to count the attached cells actually removed many cells at 6 hr of treatment, even though these cells had not floated away into the culture medium (Figure 7C). The nuclear morphology of cells attached to the dish showed a significant number of cells undergoing apoptosis at 6 hr (Figure 7B). By 16 hr, the majority of the cells were disattached, had floated away into the medium (Figure 7C), and almost all of the remaining cells on the dish showed apoptotic nuclear morphology (Figure 7B). By 24 hr, only a few cells remained attached to the dish (Figure 7A) and, of those, fewer had apoptotic nuclear morphology because the bulk of the apoptotic cells had floated away (Figure 7C). At 24 hr, a small fraction of cells with normal nuclear morphology remained but, in other experiments, such cells would undergo apoptosis during the next few hours (results not shown). These results show that apoptosis in this system is massive, and that the apoptotic cells float away from the substratum and can be recovered from the medium, where they show clear evidence of altered nuclear morphology.



View larger version (23K):
[in this window]
[in a new window]
 
Figure 7. Quantitative assessment of cell attachment, nuclear morphology of attached cells, and nuclear morphology and cell number in the culture supernatant during apoptosis. The details of these assays are described in Materials and Methods.

Measurement of DNA Fragmentation
Figure 8 shows an agarose gel electrophoresis analysis at fixed early time points for cells treated with ricin or taxol, demonstrating the presence of the ladder pattern of DNA fragmentation in cultures of KB cells. Both KB and OVCAR-3 cells showed similar apoptotic morphological, kinetic, and DNA analysis features in response to these two very different drugs.



View larger version (56K):
[in this window]
[in a new window]
 
Figure 8. Internucleosomal DNA fragmentation in cells treated with ricin or taxol. KB human carcinoma cells were either untreated (Lane A) or treated with 10-7 M ricin for 9 hr (Lane B) or 1 µM taxol for 48 hr (Lane C). DNA was extracted and electrophoresed in parallel on the same agarose gel with ethidium bromide staining. Lane A shows the lack of DNA fragmentation in untreated cells, but Lanes B and C show that both agents cause a "ladder" of internucleosomal DNA fragments at ~200-BP intervals (arrowheads). Molecular weight standards are shown on the same gel at left.

To analyze the appearance of single-stranded DNA fragments in apoptotic cells in situ, we employed a modified in situ nick-translation system previously described (Gold et al. 1993 ; Wijsman et al. 1993 ). Our first goal was to demonstrate that this assay would faithfully report the presence of DNA fragments without significant background. We performed control experiments in which fixed and permeabilized cells were incubated with the components of the in situ nick-translation assay. In these controls, the cells were incubated either with all of the labeling components for this assay or with selectively deleted protocols to demonstrate the specificity of the labeling method. To generate a sample in which nuclear DNA would contain single-strand DNA ends, we treated one sample with DNAse before histochemical assay. These results (not shown) demonstrated that this assay was both specific and sensitive for the detection of DNA single-strand ends.

We next examined cells that had been treated with either taxol or ricin at different times during which apoptotic events were seen by video time lapse microscopy. These cells were then fixed and incubated with the nick-translation assay, as well as with DAPI to demonstrate the morphology of the nuclear chromatin. Figure 9A and Figure 9B show results from cells treated with taxol. Typical results are shown in Figure 9A-A'', in which cells showed features of apoptotic changes including nuclear segmentation and apoptotic body formation, yet these structures failed to show in situ nick-translation signals of DNA fragmentation. More often, the positive nick-translation signals were found in apoptotic bodies well after they had completely disaggregated from the original cell body, a time corresponding to the lysis stage of apoptosis (Figure 9B). On occasion, cells exhibiting nuclear changes of apoptosis also showed a diffuse cytoplasmic nick-translation signal, as if the DNA fragments were released from the chromatin but were still contained by the intact plasma membrane (Figure 9B). Figure 9C shows at higher magnification the results of cells treated with ricin. A very distinctive feature of the nick-translation signals in apoptotic bodies was the peripheral pattern of labeling around the condensed chromatin core (Figure 9C). Other ricin-induced apoptotic cells showed the result of apoptotic body formation and cell lysis, and occasionally the apoptotic body with its intact plasma membrane exhibited the same diffuse nick-translation signal seen in intact cells (not shown). The overriding pattern found in both cell types in response to both apoptosis inducers, however, was the notable absence of nick-translation signals until very late in the apoptotic process, usually corresponding to the terminal blister stage or after lysis.



View larger version (164K):
[in this window]
[in a new window]
 
Figure 9. In situ nick-translation detection of DNA single-strand ends in apoptotic OVCAR-3 cells. (A-C) Rhodamine channel image representing DNA single-strand ends detected by the in situ nick-translation method. The corresponding DAPI images indicating total DNA are shown in A'-C' and the phase-contrast images of these same cells are shown in A''-C''. Note the absence of nick-translation signals in the apoptotic cells (A) that show extensive chromosomal changes of apoptosis in (A') and shrunken cell morphology in (A'') due to taxol treatment. Bar = 12 µm. (B-B'') An apoptotic body (arrow) is shown that has been derived from segmentation of an apoptotic cell. Note the circular nick-translation signal at the periphery of the chromosomal body, as well as a diffuse nick-translation signal distributed in the remaining cytoplasm of this apoptotic body. Bar = 12 µm. (C) A similar "ring" pattern of nick-translation signal around an apoptotic nuclear fragment (arrow) due to ricin treatment. (A,B, taxol treatment; C ricin treatment.) Bar = 6 µm.


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The goal of this study was, in part, to determine the nature of the morphological changes that occur during apoptosis in cultured human carcinoma cells in response to taxol and ricin. Although the morphological features of apoptosis have been described in many systems, most of those studies involved static images of apoptotic cells. Because we knew that many of the changes in apoptosis were highly dynamic and potentially asynchronous in different cells, it was important to define the timing and nature of apoptotic morphological events in these specific cells and with these specific inducing agents. Furthermore, we wished to determine whether the fragmentation of DNA, as detected by the in situ nick-translation assay, corresponded to an early or late stage of the apoptotic process. This was also intended to provide a basis for comparison with internucleosomal DNA fragmentation gels that detect the generation of internucleosomal DNA fragments often present in only a small percentage of cells in cultured apoptosis systems. Our results are summarized in Figure 10.



View larger version (21K):
[in this window]
[in a new window]
 
Figure 10. Schematic summary of the surface morphological, nuclear shape, and major DNA fragmentation events during apoptosis.

Apoptotic surface morphology was very similar for both taxol and ricin induction and for both cell types examined. This implies that no matter what the induction pathway, once apoptosis begins the cell is likely to follow the same biochemical and morphological pathway. This is consistent with the well-known highly conserved nature of this process. Apoptosis is heralded by cell rounding, presumably through loss of substratum adhesion, followed rapidly by intense surface zeiotic blebbing similar to that seen in mitotic cells during cytokinesis. This was then followed by the protrusion of previously undescribed thin surface echinoid protrusions, along with increasing rigidity of the cell. This was then followed by a very long period of rigidity without cell movement, even though the plasma membrane remained intact. The cells then showed a slow surface blistering process not dissimilar from the plasma membrane damage seen during necrosis. The final event was the physical lysis of the plasma membrane.

When ricin-induced apoptotic nuclear morphological changes were examined in comparison to the surface morphological changes, it was evident that nuclear morphological changes were a very early event in the apoptotic process, corresponding to the time of active surface blebbing. The apoptotic body nuclear remnants remained in some cases within the cell, or in others were protruded with small portions of intact cytoplasm, as described in classical "apoptotic bodies." DNA fragmentation, on the other hand, at least that detected by our in situ nick-translation histochemistry, was a very late event in this process (Figure 10).

These findings, placed in a kinetic context, demonstrate some very important features of this process. First, the entire time span for apoptosis, from the beginning of cell rounding and blebbing to the final lysis of the cell, is a very long process, often taking 12-24 hr. This time is added to any events that are required for the initiation of apoptosis, such as the arrest of cells in mitosis due to taxol treatment (~6-24 hr). Second, the entry into this pathway, even in clonal cell lines, is very asynchronous. Therefore, at any given moment it is possible for different cells in a culture to show any of the stages of these events, from normal cellular morphology all the way through end-stage cell lysis. Third, the changes of nuclear morphology and the major fragmentation of DNA are not necessarily directly temporally related. That these features might be entirely separate is, at first glance, a surprise. However, this raises the question of whether the "ladder" pattern of DNA fragmentation in apoptosis actually correlates with the single-strand ends detected by in situ nick-translation. Indeed, the process of major DNA fragmentation detected at the terminal lytic stage of apoptosis would be no different from that of necrosis except for the fact that the nuclear DNA had been previously condensed during the early stages of apoptotic surface blebbing. This nonspecific DNA fragmentation process, together with the asynchrony of apoptotic entry, may well explain why some cell types and inducing agents show mixed smearing and "laddering" of DNA fragments together, why some rapid apoptotic induction systems show clear early "ladders" with late smearing, or why other slower induction systems show more smearing than "laddering" in DNA gels. That is, the presence of "laddering" implies an apoptotic process, but its absence does not rule it out.

The complexity of this interpretation is highlighted by a recent paper by Negoescu et al. 1996 . Their study utilized a TUNEL procedure to detect strand breaks in glucocorticoid-induced apoptosis in CEM-C7 cells in culture. Their data clearly show that the detectability of DNA fragmentation in apoptotic cells using in situ methods is very dependent on fixation and pretreatment variables. Under conditions similar to those in our experiments, very little DNA fragmentation was detected in most cells with apoptotic nuclear morphology, in agreement with our findings. However, with microwave pretreatment this percentage increased. At the same time, however, the percentage of positive cells with normal nuclear morphology also increased. Although their study defines conditions to optimize specificity of these methods for apoptotic detection, it raises other possibilities concerning the correlation of such assays with agarose gel analysis and with the actual existence of strand breaks during apoptosis in living cells. That is, it is possible that the in situ labeling of apoptotic nuclei can represent both endogenous strand breaks and those that can occur in susceptible, protease-damaged chromatin during the apoptotic process. It is possible that DNA extraction procedures, fixation, permeabilization pretreatments, and other manipulations may create strand breaks in apoptotically sensitive chromatin, breaks that may or may not exist in the living cell. Although we fail to detect strand breaks in our study early in the apoptotic process, either we may have a method of inadequate sensitivity or we may have inadvertently failed to further damage this sensitive DNA during processing. Whatever the explanation, the timing of DNA strand breaks during apoptosis in vivo is still unclear. These results suggest caution in the interpretation of in situ detection of DNA fragmentation and suggest that further study is needed to define the nature and timing of DNA changes that occur during apoptosis.


  Acknowledgments

Supported in part by a grant from the American Cancer Society (CB-144 to MCW).

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


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Arends MJ, Morris RG, Wyllie AH (1990) Apoptosis: the role of the endonuclease. Am J Pathol 136:593-608[Abstract]

Bhalla K, Ibrado AM, Tourlina E, Tang C, Mahoney ME, Huang Y (1993) Taxol induces internucleosomal DNA fragmentation associated with programmed cell death in human myeloid leukemia cells. Leukemia 7:563-568[Medline]

Cohen GM, Sun XM, Snowden RT, Dinsdale D, Skilleter DN (1992) Key morphological features of apoptosis may occur in the absence of internucleosomal DNA fragmentation. Biochem J 286:331-334[Medline]

Compton MM (1992) A biochemical hallmark of apoptosis: internucleosomal degradation of the genome. Cancer Metastasis Rev 11:105-119[Medline]

Eastman A (1990) Activation of programmed cell death by anticancer agents: cisplatin as a model system. Cancer Cells 2:275-280[Medline]

Falcieri E, Martelli AM, Bareggi R, Cataldi A, Cocco L (1993) The protein kinase inhibitor staurosporine induces morphological changes typical of apoptosis in Molt-4 cells without concomitant DNA fragmentation. Biochem Biophys Res Commun 193:19-25[Medline]

Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493-501[Abstract]

Gold R, Schmied G, Rothe G, Zischler H, Breitschopf H, Wekerle H, Lassmann H (1993) Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections. J Histochem Cytochem 41:1023-1030[Abstract/Free Full Text]

Gorczyca W, Gong J, Darzynkiewicz Z (1993) Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res 53:1945-1951[Abstract]

Griffiths GD, Leek MD, Gees DJ (1987) The toxic plant proteins ricin and abrin induce apoptotic changes in mammalian lymphoid tissue and intestine. J Pathol 151:221-229[Medline]

Hamilton TC, Winker MA, Louie KG, Batist G, Behrens BC, Tsuruo T, Grotzinger KR, McKoy WM, Young RC, Ozols RF (1985) Augmentation of adriamycin, melphalan and cisplatin cytotoxicity in drug-resistant and -sensitive human ovarian carcinoma cell lines by buthionine sulfoximine mediated glutathione depletion. Biochem Pharmacol 34:2583-2586[Medline]

Hickman JA (1992) Apoptosis induced by anticancer drugs. Cancer Metastasis Rev 11:121-139[Medline]

Hockenbery D, Nunez G, Milliman C, Schreiber RD, Korsmeyer SJ (1990) Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348:334-346[Medline]

Jacobson MD, Burne JF, Raff MC (1994) Programmed cell death and bcl-2 protection in the absence of a nucleus. EMBO J 13:1899-1910[Abstract]

Kung AL, Zetterberg A, Sherwood AW, Schimke RT (1990) Cytotoxic effects of cell cycle phase specific agents: a result of cell cycle perturbation. Cancer Res 50:7307-7317[Abstract]

Lazebnik YA, Cole S, Cooke CA, Nelson WG, Earnshaw WC (1993) Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis. J Cell Biol 123:7-22[Abstract]

Majno G, Joris I (1995) Review: apoptosis, oncosis and necrosis. An overview of cell death. Am J Pathol 146:3-15[Abstract]

Manfredi JJ, Horwitz SB (1984) Taxol: an antimitotic agent with a new mechanism of action. Pharmacol Ther 25:83-125[Medline]

Mannherz HG, Peitsch MC, Zanotti S, Paddenberg R, Polzar B (1995) A new function for an old enzyme: the role of Dnase I in apoptosis. Curr Top Microbiol Immunol 198:161-174[Medline]

Negoescu A, Lorimier P, Labat-Moleur F, Drouet C, Robert C, Guillermet C, Brambella C, Brambella E (1996) In situ apoptotic cell labeling by the TUNEL method: improvement and evaluation of cell preparations. J Histochem Cytochem 44:959-968[Abstract/Free Full Text]

Peitsch MC, Polzar B, Stephan H, Crompton T, MacDonald HR, Mannherz HG, Tschopp J (1993) Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death). EMBO J 12:371-377[Abstract]

Schultze-Osthoff K, Walczak H, Droge W, Krammer PH (1994) Cell nucleus and DNA fragmentation are not required for apoptosis. J Cell Biol 127:15-20[Abstract]

Tewari M, Quan LT, O'Rourke K, Desnoyers S, Zeng Z, Beidler DR, Poirier GG, Salvesen GS, Dixit VM (1995) Yama/CPP32beta, a mammalian homolog of CED-3 is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell 81:801-809[Medline]

Tsujimoto Y, Croce CM (1986) Analysis of the structure, transcripts and protein products of bcl-2, the gene involved in human follicular lymphoma. Proc Natl Acad Sci USA 83:5214-5218[Abstract]

Wijsman JH, Jonker RR, Keijzer R, van de Velde CJ, Cornelisse CJ, van Dierendonck JH (1993) A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA. J Histochem Cytochem 41:7-12[Abstract/Free Full Text]

Willingham MC, Pastan I (1985) Morphologic methods in the study of endocytosis in cultured cells. In Pastan I, Willingham MC, eds. Endocytosis. New York, Plenum Press, 281-321

Wyllie AH (1992) Apoptosis and the regulation of cell numbers in normal and neoplastic tissues: an overview. Cancer Metastasis Rev 11:95-103[Medline]