ARTICLE |
Correspondence to: Mark C. Willingham, Dept. of Pathology and Laboratory Medicine, Medical Univ. of South Carolina, 171 Ashley Ave., Charleston, SC 29425.
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Summary |
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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
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Introduction |
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Apoptosis is a term used to describe the terminal morphological and biochemical events seen in programmed cell death (
The gene-directed and/or -regulated events of apoptosis are very different from those seen in necrosis (
A hallmark feature of apoptosis was the observation that nuclear DNA extracted from apoptotic cells was often degraded in an internucleosomal pattern (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 (
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 (
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Materials and Methods |
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Cell Cultures
KB (HeLa markers, cervical carcinoma) (ATCC CCL 17; Rockville, MD), OV2008 (
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 (
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 (
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 (
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.
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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.
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Results |
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Morphological and Kinetic Features of Apoptosis in Cultured Human Carcinoma Cells
Even though many static and dynamic features of apoptosis have been described before (
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.
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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.
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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).
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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.
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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.
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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.
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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 (
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.
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Discussion |
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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.
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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
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Acknowledgments |
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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.
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