ARTICLE |
Correspondence to: Mark C. Willingham, Dept. of Pathology, Wake Forest Univ. School of Medicine, Gray Bldg., Room 2106, Medical Center Blvd., WinstonSalem, NC 27157. E-mail: mwilling@wfubmc.edu
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Summary |
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In an earlier article from this laboratory, the current methods developed to detect apoptosis in cells and tissues were highlighted, along with the challenges in their interpretation. Recent discoveries concerning the underlying biochemical mechanisms of apoptotic effector pathways have made possible further assays that allow a more direct measure of the activation of the apoptotic machinery in cells. This article summarizes some of these newer methods and extends the interpretation of the more classical assays of apoptosis in a defined cell system. We present data in KB and PC3 cell model culture systems induced to undergo apoptosis by the plant toxin ricin. Using a modified in situ nick translation assay (ISNT) with either Bodipy or BUdR labeling, we confirm that most cells showing altered nuclear morphology do not show reactivity with this assay until very late in the apoptotic process. We also show that only a minority of cells label with fluorescent annexin V during apoptosis but that apoptotic cells continue to internalize material from the cell surface through endocytosis after becoming reactive with annexin V. In addition, we describe the utility of a prototype of new assays for caspase substrate cleavage products, the detection of cleaved cytokeratin 18. It is these newer cleavage product assays that perhaps hold the greatest promise for specific detection of apoptosis in cells either in cell culture or in intact tissues. (J Histochem Cytochem 49:821832, 2001)
Key Words: apoptosis, cytochemistry, TUNEL, ISNT, annexin V, caspases, ricin
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Introduction |
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THE REGULATION of apoptosis has gained central importance in many aspects of biology, including studies of embryonic development, the pathogenesis of disease, and the response of cells to therapy (
Fig 1 summarizes a small portion of the apoptosis regulation pathways that have been recently delineated, and it is clear that this new knowledge can be the starting point for the design of more specific assays of apoptosis. Several new assays have recently appeared that depend on newly discovered changes at the molecular level. These changes are relatively more specific for the apoptotic process and offer the possibility of removing some of the controversy surrounding older detection methods. In this article, we describe our recent studies evaluating in situ nick translation (ISNT) methods using Bodipy and BUdR labeling, annexin V binding, and the detection of a caspase-specific cleavage product (cytokeratin 18) in a model system of apoptosis utilizing ricin-induced apoptosis of KB and PC3 cells. This assay system is similar to our previously presented systems in which we compared surface morphological (time-lapse phase-contrast), nuclear morphological (DAPI labeling), and ISNT (biotin-labeled) assays of apoptosis (
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Materials and Methods |
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Cells
KB is a continuous human carcinoma cell line related to HeLa cells. PC3 is a continuous human prostate carcinoma cell line. These cells were obtained from the American Type Culture Collection (Rockville, MD) and were grown in RPMI-1640 medium supplemented with 10% fetal calf serum and penicillinstreptomycin (Sigma; St Louis, MO) at 37C in a 95% air/5% CO2 atmosphere. Cells were subcultured using 2.5% trypsin with 1 mM EDTA.
Chemicals
Annexin V labeled covalently with fluorescein isothiocyanate (FITC) was obtained from Pharmingen (San Diego, CA). Alexa 568annexin V was obtained from BoehringerMannheim Biochemicals (Indianapolis, IN). DAPI (4,6-diamidino-2-phenylindole), ricin toxin, and Dulbecco's PBS were obtained from Sigma. Mouse monoclonal anti-BUdR antibody (clone B44) was obtained from BectonDickinson (San Jose, CA). Mouse monoclonal anti-cytokeratin 18 was obtained from Chemicon (Temecula, CA) and a mouse monoclonal antibody reactive with the caspase-cleavage product of cytokeratin 18 (M30) (
Experimental Design
Apoptosis in KB and PC3 cells was induced using ricin at 1 µg/ml as previously described (
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ISNT Labeling
ISNT was performed using a method similar to that previously presented (
Cells in 35-mm dishes were induced to enter apoptosis using ricin (from 4 to 24 hr, depending on the experiment) as described above, and floating cells were recovered from the dishes, centrifuged, and resuspended in serum-free medium. The floating cells were reattached to 35-mm dishes that had been previously coated with poly-L-lysine (1 mg/ml in PBS for 5 min, washed in PBS) by centrifugation at 700 rpm (200 x g) using a swinging bucket plate rotor (Sorvall RTH-750). Both attached and adherent cells were then fixed in 3.7% formaldehyde in PBS for 10 min at 23C, followed by permeabilization with 0.2% Triton X-100 for 5 min.
The ISNT reaction mix (50 µl) using Bodipy labeling was then added to PBS-washed attached and reattached cells, and spread over the dish by overlaying the cells with a coverslip and incubating for 1 hr at 37C. The mixture consisted of water (37 µl), 10 x Klenow buffer (5 µl), 10 mM dATP, dCTP, and dGTP mix (4.5 µl), 1 mM BodipydUTP (0.5 µl), and 10 U of Klenow enzyme (2 µl). After washing, the cells were postfixed in formaldehyde and mounted under a coverslip in glycerol.
For BUdR labeling, we modified the procedure of
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Annexin V Labeling
For analysis of fluorescent annexin V binding, cells in 35-mm dishes were removed from the 37C incubations, cooled to 4C, and washed in a binding buffer containing 2.5 mM CaCl2, 140 mM NaCl, 20 mM Tris-HCl, pH 7.5. Labeled annexin V was added to cells at 1 µg/ml in binding buffer and incubated at 4C for 1 hr. For initial experiments, the cells were then washed free of unbound annexin V and fixed in 3.7% formaldehyde at room temperature in PBS, then further incubated with formaldehyde in PBS together with 0.1 µg/ml DAPI to label nuclear DNA at 4C overnight. The attached cells were then washed in PBS and mounted under a drop of glycerol under a #1 circular 25-mm diameter coverslip in the dish. The dish was then viewed using a Zeiss Axioplan 2 upright fluorescence microscope equipped with a stage plate designed to hold 35-mm dishes.
Immunocytochemistry/Immunohistochemistry
PC3 cells grown in 35-mm culture dishes were fixed using 80% acetone in water for 10 min. The cells were then incubated in 1% bovine serum albuminPBS saline (BSAPBS) for 10 min, then mouse anti-cytokeratin 18 (10 µg/ml; Chemicon #MAB3234) or mouse anti-caspase cleaved cytokeratin 18 (clone M30; BoehringerMannheim #2140322, 1:20 dilution) in BSAPBS for 30 min at 23C. This was followed by washing in PBS, then by serial incubations in affinity-purified goat anti-mouse IgGrhodamine, followed by rabbit anti-goat IgGrhodamine for signal amplification (25 µg/ml; Jackson ImmunoResearch) in BSAPBS. The dishes were then postfixed in 3.7% formaldehyde in PBS and viewed after mounting under a coverslip in glycerol. For induction of apoptosis, PC3 cells were incubated in 1 µg/ml ricin for 24 hr. To select for apoptotic cells, floating cells were collected, washed in serum-free medium, then reattached by centrifugation onto poly-L-lysine coated dishes before fixation. These cells were also subsequently stained using DAPI to visualize nuclear morphology after the final formaldehyde fixation following antibody labeling.
For immunohistochemistry using MAb M30 on paraffin sections, sections were deparaffinized routinely, treated with Antigen Unmasking Solution (Vector Labs; Burlingame, CA) while heating in a microwave for 10 min, treated with 3% hydrogen peroxide in PBS, then incubated with mouse MAb M30 (1:20 dilution in BSAPBS) for 30 min, followed by sequential incubations with affinity-purified goat anti-mouse IgGhorseradish peroxidase, followed by rabbit anti-goat IgGperoxidase (25 µg/ml; Jackson ImmunoResearch). The peroxidase was detected using diaminobenzidineperoxide in PBS and counterstained with hematoxylin.
Microscopy
For time-lapse microscopy, cells in T-25 flasks were viewed under continuous incubation conditions using a Zeiss Axiovert phase-contrast microscope equipped with a warm stage and an atmospheric controller as previously described (
Fluorescent samples were viewed using a Zeiss Axioplan fluorescence microscope equipped with filters for UV excitation (DAPI), blue light excitation (FITC), and green light excitation (Alexa 568 or rhodamine). The images on individual channels were captured using a SPOT cooled CCD digital camera, or a Dage 300 CCD camera. These images were either retained as grayscale images or recombined as color images with the three channels in overlay using Adobe Photoshop (4.0) software. All of the images shown in the figures represent digital images reproduced at >300 dpi.
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Results |
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The model system for apoptosis employed in our studies involves the induction of cell death in KB cells, a human carcinoma cell line closely related to HeLa cells. We have also used these same assays with PC3 cells, a human prostate carcinoma cell line. Apoptosis was induced in these cells through the use of ricin, the toxic lectin of the castor bean plant. Ricin binds to cells, internalizes through surface clathrin-coated pits into endosomes, and eventually translocates through intracellular membranes into the cytosol, where it irreversibly inactivates ribosomes, inhibiting protein synthesis (reviewed in
ISNT Labeling
ISNT is one method of in situ end-labeling (ISEL) for detecting strand breaks in DNA that occur during apoptosis. Although different enzymes are used in the reaction mix, ISNT and TUNEL assays are closely related. We have chosen to use ISNT rather than TUNEL because, in our experience, ISNT has usually given a stronger signal in cultured cells. Fig 4 shows the use of an ISNT assay in the KBricin system in which the incorporated nucleotide is directly labeled using Bodipy-conjugated dUTP. Bodipy labeling has, in our experience, produced brighter fluorescence signals in comparison to some other fluorescent markers, such as FITC. The left panels in this figure demonstrate ISNT labeling, the center panels demonstrate nuclear morphology using DAPI to label nuclear DNA, and the right panels show the phase-contrast appearance of the cells in each field. When cells undergo apoptosis in this system, cells that enter apoptosis often round up and can be washed from the culture dish. Analysis of these suspended cells provides a high frequency of apoptotic cells, while the cells attached to the plate are often not yet in apoptosis. Fig 4A shows attached cells that have been treated with DNase after fixation, rendering their nuclear DNA sufficiently fragmented to provide a positive control for the ISNT reaction. In Fig 4B, however, the otherwise untreated attached ricin-treated cells fail to show any ISNT signal.
In Fig 4C, some apoptotic cells appear rounded yet have failed to detach from the plate. Such cells show a strong ISNT signal when treated with DNase. However, in the absence of DNase treatment (Fig 4D) most of these cells fail to show an ISNT reaction. This indicates that the ISNT reaction can work in such cells but that most of these apoptotic cells are ISNT-negative at this point. To enrich for cells that have proceeded further into apoptosis, we isolated cells floating in the medium. Figire 4E shows that such cells, when reattached using poly-L-lysine-coated dishes, can be labeled with ISNT when they are treated with DNase after fixation. However, most of these cells are not labeled with ISNT in the absence of DNase treatment (arrowhead in Fig 4F). On the other hand, a few cells in the floating population show labeling with ISNT in the absence of DNase treatment (Fig 4F), and these cells have rounded morphology and segmented nuclear DNA (arrow in Fig 4F). These are the cells that would be labeled with methods such as TUNEL that detect DNA single strand breaks. It is evident from Fig 4F, however, that there are many other cells with the same morphological features (e.g., arrowheads) that fail to label with the ISNT method. Therefore, such methods appear to label only a minority of cells in a heterogeneous population at the early stages of induction of apoptosis, and confirm our prior findings that such labeling in most cells is a late event in the apoptotic process (
Although Bodipy labeling produces a strong signal in the ISNT reaction, the labeled substrate is rather expensive, comparable to the cost of biotin- and digoxigenin-labeled commercial substrates for ISNT and TUNEL reactions. This has often necessitated the use of the smallest possible reaction mix volumes, usually requiring the use of coverslips over cells and often resulting in damage to cells on the culture dish. We attempted to design a system that would be less expensive and allow the use of larger volumes of reaction mix. We employed the use of BUdRtriphosphate as previously described by
Fig 5 demonstrates the results in PC3 cells, both normal and apoptotic, using this BUdRISNT labeling system. Interestingly, when untreated cells were examined with this system, no signal was detected in normal nuclei, as expected (Fig 5A''). However, unexpectedly, a significant signal was detected in organelles with the morphological appearance of mitochondria. We had previously detected a similar pattern using a biotin-labeled ISNT system but had assumed that this background was due to endogenous biotin in mitochondria. However, the present system does not use biotin, which suggests that this ISNT method may be labeling normal mitochondrial DNA. When these cells were treated with DNase after fixation (Fig 5B''), the nuclear DNA labeled strongly but the apparent mitochondrial signal disappeared, perhaps suggesting that the mitochondrial DNA was completely cleaved and not preserved. Apoptotic cells could be found that showed significant mitochondrial signal, but minimal nuclear signal (Fig 5C''), although some apoptotic cells were found that showed peripheral labeling of the nuclear segments (Fig 5D''). Similar to the Bodipy-labeled experiment in Fig 4, however, most apoptotic cells failed to label strongly with this ISNT method, in agreement with our previously published biotin-labeled results (
We also wished to examine the use of this ISNT method using peroxidase labeling. Fig 6 demonstrates an experiment in which cells were labeled with the reaction shown in Fig 3, but subsequently labeled with peroxidase instead of rhodamine. Fixed and permeabilized normal cells showed the same "mitochondrial" pattern using HRP labeling seen with rhodamine, and cells treated with DNase as a positive control showed loss of mitochondrial labeling and gain in nuclear labeling, similar to the results using rhodamine (Fig 5). These results suggest that the BUdRISNT method shown is an inexpensive and accurate ISNT method useful for various labeling techniques.
Annexin V Labeling
Annexin V is a protein that binds in a calcium-dependent manner to exposed phosphatidylserine (PS). When cells undergo apoptosis, PS normally sequestered on the cytoplasmic face of the plasma membrane appears on the exterior of the cell where it can bind to labeled annexin V. Several labeled versions of annexin V are commercially available, and previous reports have described the use of this marker as an assay of apoptosis (
We examined annexin V binding during apoptosis with fluorescently-labeled derivatives using either FITC or Alexa 568 as marker in the KBricin model system. Fig 7 shows KB cells treated with ricin for 6 hr, then labeled with FITCannexin V. Apoptotic cells were easily identified on the basis of their rounded, blebbing morphology and the segmentation of their nuclei detected using DAPI. Some of these cells subsequently incubated with annexin V at 4C showed surface binding of FITC-labeled annexin V, but a significant number failed to show surface labeling (Fig 7A'). We quantitated this event at various times of ricin treatment, as shown in Fig 8. After a lag of 4 hr, cells began to show rounded morphology and altered nuclear shape, and a fraction of these same cells labeled with annexin V. Even at 22 hr after addition of ricin, however, when almost all cells have entered apoptosis, only about one third of the cells labeled with annexin V, even though each of these unlabeled cells showed the other surface and the nuclear morphological features of apotosis.
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One possible explanation for only a minority of apoptotic cells labeling with annexin V at these times was that our assay might have been performed at too early a time, and that cells might show progressive annexin V labeling at later times. To test this idea, we performed a double-label annexin V experiment in which KB cells were labeled with Alexa 568-conjugated annexin V at 6 hr after ricin addition, washed, and allowed to incubate at 37C for an additional 3 hr, and then labeled with FITC-conjugated annexin V. Fig 9 shows results from this experiment, in which some apoptotic cells failed to label at either 6 or 9 hr (Fig 9A, arrowheads), others labeled at both 6 and 9 hr (Fig 9A and Fig 9B, arrow), and still others labeled only at 9 hr (Fig 9B, arrowhead). This might suggest that some apoptotic cells progress in their externalization of PS as they proceed through apoptosis.
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If cells failed to label at 6 hr, it could also be due to the fact that they had not yet begun apoptosis. Therefore, we repeated this experiment using mechanical shaking selection to enrich for floating cells, in which almost all of the harvested cells had entered apoptosis on the basis of their surface morphology (results not shown). When we labeled these cells at 6 hr with Alexaannexin V, washed, then labeled later at 9 hr with FITCannexin V, we saw the results shown in Fig 10. Fig 10A'''' and 10B'''' are composite overlays of the images on individual channels shown in Fig 10A'A''' and 10B'B'''. Some cells showed apoptotic surface and nuclear morphology and did not label at either time with annexin V (results not shown). Other cells labeled at 9 hr but not at 6 hr (Fig 10B, arrow). In addition, some cells labeled at both 6 hr and 9 hr (Fig 10A). Interestingly, cells that had labeled at 6 hr showed internalization of the Alexa-labeled annexin V (Fig 10A''), indicating that apoptotic cells continue to endocytose material from their surfaces even though they have already begun to show other morphological changes typical of apoptosis. In other words, even though the cells have begun the process of dying, they still demonstrate at least one activity typical of living cells. Fig 10 also shows a cell (arrowhead) that demonstrates an interpretive problem with the use of annexin V, in that this cell has proceeded through the final lysis stage of apoptosis, allowing intense labeling of its internal PS directly. Such a result would also be seen with necrotic cells.
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Detection of Caspase-generated Cleavage Products: Cytokeratin 18
Because caspase activity is likely to be the most specific indicator of the apoptotic process, assays of caspase activity through the detection of specific cleavage products in target proteins should represent a promising approach for measuring apoptosis. Such an approach could be applicable to fixed archival tissue blocks. We tested a recently commercially available antibody to the caspase-generated cleavage products of cytokeratin 18 (antibody M30) in PC3 cells, which can be shown to contain cytokeratin 18. This antibody (
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Discussion |
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Apoptosis is a central element in the pathogenesis of many disease processes and in the response to systemic therapies in neoplastic cells. Specific detection of apoptosis in tissue cells is therefore an important cytochemical technique for biomedical studies. The methods developed over the past decade for the detection of apoptosis have often been derived from empirical observations of the properties of dying cells. Recently, however, the molecular and biochemical mechanisms of apoptotic death have been further defined, leading to the prospect of assay methods based on highly specific chemical changes (
In this study, we have used a defined cell culture system, ricin-induced apoptosis in KB and PC3 cells, in which apoptosis can be induced in a highly synchronous and reproducible manner. The characteristics of this process are easily detectable at both the morphological and the biochemical level. We have applied some of the more promising recent cytochemical assays to this system. Because TUNEL and ISNT assays are perhaps the most commonly used cytochemical methods in this field, we have attempted to develop a less expensive alternative ISNT method to allow the use of larger reaction volumes for these assays. Through modification of an existing BUdR labeling method, we tested this method in the ricin-induced apoptosis system and found results similar to those previously reported (
We also examined another commonly used cytochemical assay of apoptosis, the labeling of externalized PS by annexin V. Although this assay detects apoptosis at an earlier stage than ISNT, only a minority of apoptotic cells actually labeled with annexin V before the final lysis of cells at the end of the apoptotic process. This is in agreement with other studies published previously using a different apoptosis system (
Finally, we examined one newly available antibody that detects the specific cleavage fragments of a cytoplasmic target of caspases, cytokeratin 18 (
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Acknowledgments |
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We wish to thank Drs Timothy Kute and Arthur Frankel for helpful discussions. These results include data generated while JMW was the recipient of a summer student scholar research award from the South Carolina Governor's School.
Received for publication November 17, 2000; accepted March 7, 2001.
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