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Cytometry Part A 57A:70 –74 (2004)
Original Articles
New Method for the Analysis of Cell
Cycle–Specific Apoptosis
Deding Tao, Jianhong Wu, Yongdong Feng, Jichao Qin, Junbo Hu, and Jianping Gong*
The Cancer Research Institute, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology,
Wuhan, Peoples Republic of China
Received 15 May 2003; Revision Received 18 August 2003; Accepted 14 October 2003
Background: In this study, a new method for the analysis
of cell cycle specificity of apoptosis was designed by using
a modified annexin V and propidium iodide (API) method.
Methods: Cells of the human promyelocytic HL-60 line
treated with camptothecin (CPT) or ultraviolet light (UV)
were labeled with fluorescein isothiocyanate– conjugated
annexin V and prefixed with 1% methanol-free formaldehyde on ice, and their DNA was stained stoichiometrically
with propidium iodide in the presence of digitonin. Cellular green and red fluorescences were measured by flow
cytometry.
Results: Cell cycle specificity of apoptosis obtained by
the API method and those analyzed for the presence of
DNA strand breaks by using terminal deoxynucleotidyl
transferase (TdT) assay were similar: CPT- or UV-induced
Apoptosis (programmed cell death) is controlled by a
variety of genes and functional proteins whose transcription and activity are timely induced in response to stress
that triggers this mode of cell death (1–12). Apoptotic
events are highly coordinated with each other, and errors
in execution of apoptosis may lead to severe diseases
(1–16). Therefore, analysis of the incidence of apoptosis
and monitoring of particular events of apoptosis are of
importance not only in basic laboratory studies but also in
the clinical setting.
During the past decade, different methods for analysis of
apoptosis have been developed (1,2,17). The methods using
flow cytometry, by virtue of providing accurate estimates of
apoptotic index in large cell populations and by offering a
possibility of multiparameter analysis, are the most widely
used in detection of apoptosis (18 –22). Because most antitumor drugs and many other agents that induce apoptosis
target cell cycle–specific events, analysis of apoptosis with
respect to the cell cycle position is of particular interest. This
can be accomplished by multiparameter analysis of the flow
cytometric data when cellular DNA content, the parameter
apoptosis preferentially in S- or G1-phase cells, respectively. When the internucleosomal DNA degradation was
prevented by the serine protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone, apoptotic cells could not
be detected by the TdT assay but were identified by the
API method.
Conclusions: The API method, similar to the TdT assay,
accurately detects the cell cycle phase specificity of apoptosis. Also, the API method appears to detect earlier
stages of apoptosis than the TdT assay. © 2004 Wiley-Liss,
Inc.
Key terms: apoptosis; cell cycle; annexin V; terminal
deoxynucleotidyl transferase
that defines the cell cycle position, is measured as one of the
cell attributes (10 –12,14,23–29).
In this study we combined an assay of annexin V binding to identify apoptotic cells with the stoichiometric
staining of cellular DNA using propidium iodide (PI) to
define the cell cycle position. This method (API) was
compared with analysis of DNA strand breaks by using an
exogenous terminal deoxynucleotidyl transferase (TdT; or
terminal dUTP nick end labeling [TUNEL]) assay. The
results showed that the API method is more rapid and has
Contract grant sponsor: China Key Basic Research Program; Contract
grant number: G1998051212; Contract grant sponsor: National Nature
Science Foundation of China; Contract grant numbers: 39670265,
39730270, and 39725027; Contract grant sponsor: Science Foundation of
Ministry of Public Health, Peoples Republic of China; Contract grant
number: 97070239.
*Correspondence to: Jianping Gong, M.D., Ph.D., The Cancer Research
Institute, Tongji Hospital, Tongji Medical College, Huazhong University of
Science and Technology, Wuhan, 430030, Peoples Republic of China.
E-mail: [email protected]
NEW ANALYTIC METHOD FOR CELL CYCLE APOPTOSIS
an advantage over the TUNEL method, which detects
apoptosis in the instances when DNA fragmentation stops
at the 300- to 50-kb section, in not progressing into internucleosomal sections.
MATERIALS AND METHODS
Cell Culture and Induction of Apoptosis
HL-60 cells, purchased from American Type Culture
Collection (Rockville, MD), were maintained in RPMI1640 medium (Gibco, Grand Island, NY) supplemented
with 10% fetal calf serum (Gibco) and 2 mM L-glutamine
(Sigma Chemical Co., St. Louis, MO). The experiments
were performed on cells during their exponential phase
of growth, as described elsewhere (23).
Fresh stock solutions of camptothecin (CPT; Sigma)
were prepared at a drug concentration of 200 ␮g/ml in
dimethyl sulfoxide, and fresh stock solutions of N-tosyl-Lphenylalanine chloromethyl ketone (TPCK; Sigma) were
prepared at a concentration of 50 mM in ethanol. Further
dilutions were made by addition of RPMI-1640 medium.
The cells were incubated at 37°C in the presence of 0.15
␮M CPT or 0.15 ␮M CPT plus 10 ␮M TPCK, respectively,
for up to 4 h. Control cultures were treated with an
equivalent volume of dimethyl sulfoxide and ethanol in
RPMI. In addition, the flasks containing cell cultures during exponential growth were placed directly on the glass
surface of a mini-transilluminator (0.375 W/cm2; Bio-Rad,
Hercules, CA) illuminated with ultraviolet (UV) light for
40 s (150 kJ/m2) and subsequently incubated at 37°C for
4 h. Then the cells were harvested, sedimented (200g, 5
min), resuspended in phosphate buffered saline (PBS;
Sigma), and divided into two portions. One portion was
used for annexin V labeling, and the other was fixed for
the DNA strand break assay and agarose gel electrophoresis. One flask of exponentially growing cells was treated
with 0.15 ␮M CPT, and cell aliquots from that culture
were harvested at 30-min intervals for up to 4 h for the
detection of apoptotic rate.
DNA Gel Electrophoresis
Untreated or drug- or UV-treated cells were collected by
centrifugation and fixed with 75% ethanol pre-cooled to
0 – 4°C by adding ethanol drop-wise under constant agitation. The cells were maintained in ethanol at ⫺20°C for at
least 24 h. The cells were then centrifuged at 200g for 5
min; the ethanol was thoroughly removed; the cell pellets,
in 1.5-ml Eppendorf tubes, were resuspended in 40 ␮l of
0.2 M phosphate citrate buffer, pH 7.8, at room temperature for at least 30 min; after centrifugation at 800g for 5
min, the supernatant was used for agarose gel electrophoresis as described previously (21). The degree of DNA
fragmentation was also assayed by using pulsed filed gel
electrophoresis according to the procedure as described
by Filipski et al (30).
Annexin V and PI Labeling (API)
Common procedure for detection of total apoptosis. Aliquots of freshly collected cells suspended in PBS
were centrifuged (200g, 5 min) and resuspended in bind-
71
ing buffer, pH 7.4, containing 10 mM Hepes (Sigma), 140
mM NaCl, and 2.5 mM CaCl2 to have approximately 106
cells/ml. Five microliters of fluorescein isothiocyanate
(FITC) plus annexin V (Pharmingen, San Diego, CA) and
10 ␮l of PI (Sigma) at a concentration of 50 ␮g/ml in PBS
was then added, and the cells were incubated for 20 min
at room temperature in the dark, as described previously
(31,32).
Procedure for the analysis of cell cycle–specific
apoptosis (API method in brief). Five microliters of
FITC-annexin V was added to 100 ␮l of freshly collected
cells suspended in binding buffer at a density of 106
cells/ml. The cells were placed at room temperature in the
dark for 20 min, rinsed in binding buffer, resuspended in
1 ml of 1% methanol-free formaldehyde in binding buffer
for 30 min on ice, rinsed twice, resuspended in 0.5 ml of
PI solution containing 50 ␮g/ml PI, 0.1% RNase A (Sigma),
500 ␮g/ml digitonin (Sigma), 10 mM PIPES (Sigma), 2 mM
CaCl2 and 0.1 M NaCl, placed at room temperature for 1 h
in the dark, and analyzed by flow cytometry for the cell
cycle specificity of apoptotic cells. Untreated HL-60 cells
served as the negative control.
DNA Strand Break Labeling by TdT
The harvested cells were prefixed in suspension in 1%
methanol-free formaldehyde (Polysciences, Warrington,
PA) in PBS, pH 7.4, on ice for 15 min, as described
previously (20), rinsed and postfixed in 75% cold ethanol,
and placed at ⫺20°C. After fixation, the cells were rinsed
twice with PBS and then resuspended in 50 ␮l of reaction
buffer solution containing 0.2 M potassium cacodylate
(Sigma), 2.5 mM Tris-HCl (pH 6.6), 2.5 mM CoCl2 (Sigma),
0.25 mg/ml bovine serum albumin (Sigma), 5 U of terminal deoxynucleotidyl transferase (Boehringer Mannheim
Biochemical, Indianapolis, IN), and 0.5 nM of biotinylated
dUTP (Boehringer Mannheim). The cells were incubated
in this solution at 37°C for 45 min, rinsed in PBS, and
resuspended in 100 ␮l of a solution containing four times
concentrated saline sodium citrate buffer, 25 ␮g/ml FITCconjugated avidin (Boehringer Mannheim), 0.1% Triton
X-100 (Sigma), and 5% bovine serum albumin, incubated
in this solution for 30 min at room temperature in the
dark, rinsed in PBS containing 0.1% Triton X-100, and
resuspended in 1 ml of PBS containing 5 ␮¿⬎/ml of PI and
0.1% RNase (Sigma). Untreated HL-60 cells served as the
negative control.
Flow Cytometry
The fluorescence of cells stained with FITC-conjugated
avidin and PI in the TdT assay and the fluorescence of cells
stained with FITC-conjugated annexin V and PI in API
assay were measured on a FACSort Flow Cytometer (Becton Dickinson, San Jose, CA). CELLQuest software was
used for acquisition and analysis of data.
RESULTS
Quantitative Analysis of Apoptotic Cells
Table 1 presents the results of apoptotic rates of HL-60
cells measured by common staining with FITC-annexin V
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TAO ET AL.
Table 1
Apoptotic Index (%) Detected by API Method and Standard FITC Plus Annexin V
Staining*
Detecting methods
Apoptotic index (%) after different treatment
UV
CPT
CPT ⫹ TPCK
Standard FITC ⫹ annexin V staining
API method
18.1 ⫾ 1.5
17.3 ⫾ 1.8
35.6 ⫾ 1.7
34.7 ⫾ 1.9
37.0 ⫾ 1.5
35.9 ⫾ 1.8
*API, annexin V plus propidium iodide; CPT, camptothecin; FITC, fluorescein isothiocyanate; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; UV, ultraviolet.
and the API method. The exponentially growing cultures
of HL-60 cell were treated by UV, CPT, and CPT plus
TPCK. The total apoptotic index of each sample was
measured 10 times by using the three approaches described above. The results obtained by the API method
were similar to those obtained by the standard FITCannexin V assay (t-test, P ⬎ 0.05).
Apoptotic indices of the samples treated with UV and
CPT were 10.7 ⫾ 1.6 and 19.5 ⫾ 1.9, respectively, when
estimated by the TdT assay, which is significantly lower
than the API method (17.3 ⫾ 1.8 vs. 34.7 ⫾ 1.9) and
standard annexin V staining (t-test, P ⬍ 0.01).The results
of samples treated with CPT plus TPCK were negative
when measured by the TdT assay (Fig. 1).
Figure 2 shows the relation between CPT-inducing time
and apoptotic index detected by the API method and the
TdT assay. HL-60 cells, which were in the exponential
phase of growth, were incubated at 37°C in the presence
of 0.15 ␮M CPT for different time intervals; every 30 min,
a 10-ml aliquot of cell suspension was removed; and the
apoptotic cells were detected by the API method and TdT
assay. The apoptotic cells were evident after 1.5 h when
detected by the API method, and the apoptotic cells were
not evident until 2.5 h when detected by the TdT assay.
The data indicated the that the API method detects earlier
stages of apoptosis than does the TdT assay.
FIG. 1. Apoptotic indices of HL-60 cells detected by the API method and
the TdT assay. The cells were treated with UV light, 0.15 ␮M CPT, and
0.15 ␮M CPT plus 10 ␮M TPCK.
DNA Agarose Gel Electrophoresis
DNA extracted from the fixed HL-60 cells by phosphate
citrate buffer, as described previously (14), was subjected
to agarose gel electrophoresis. DNA in the gels was visualized under UV light after staining with 5 ␮g/ml of
ethidium bromide (Sigma). As evident in Figure 3, the
DNA extract samples from cultures treated with UV light
and CPT demonstrated a clear ladder pattern on gel electrophoresis, and the ladder intensity with CPT was distinctly higher than that of UV light, whereas no DNA was
detected in the gels from samples representing the untreated (control) cells or the cells treated with CPT plus
TPCK. Analysis of DNA size by pulsed gel electrophoresis
indicated the presence of DNA fragments of at least 50 kb
in extracts of cells treated with CPT combined with TPCK,
similar to that described by Hara et al. (33) (not shown).
Cell Cycle Specificity of Apoptosis
After inducing apoptosis of HL-60 cells by UV, CPT, or
CPT plus TPCK, the cell cycle distribution of apoptotic
and nonapoptotic cells was analyzed with the API method
and the TdT assay, respectively. Figure 4 presents the
results as density contour plots representative of the cells
treated with UV light, CPT, or CPT combined with TPCK.
It is evident that the cells exposed to UV preferentially
underwent apoptosis in G1 phase, those treated with CPT
underwent apoptosis in S phase, and those treated with
FIG. 2. Relation between inducing time and apoptotic index. HL-60
cells were induced with 0.15 ␮M CPT and harvested every 30 min. The
apoptotic cells were identified by the API method and the TdT assay.
NEW ANALYTIC METHOD FOR CELL CYCLE APOPTOSIS
73
result suggested that the sensitivity of the TdT assay is
lower than that of the API method.
After induction of apoptosis by CPT plus TPCK, the
frequency of apoptotic cells detected by the API method
was the same as that induced by CPT only, but the TdT
assay did not detect apoptotic cells.
FIG. 3. DNA gel electrophoresis of HL-60 cells untreated (control; a),
treated for 40 s with UV light (b), treated with 0.15 ␮M CPT (c), or treated
with 0.15 ␮M CPT plus 10 ␮M TPCK (d). All treated cells were incubated
at 37°C for 4 h.
CPT plus TPCK showed no cell cycle phase specificity.
The mean fluorescence intensities of the FITC-positive
(apoptotic) cells in G1 and S phase were 20- and 16.4-fold
higher, respectively, than that of the negative cells when
detected with the API method. In contrast, the mean
fluorescence intensities of FITC-positive cells in G1 and S
phases was only 13- and 11.6-fold higher, respectively,
than that of the negative cells when detected with the TdT
assay. In addition, in the contour plot induced by CPT,
there were no cells in the S phase of the negative control
region when detected with the API method, whereas
there were some residual S-phase cells in the negative
control region when detected by the TdT assay. This
DISCUSSION
Quantitative detection and cell cycle specificity analysis of
apoptosis are very important for the study of the molecular
mechanism of cell death and cell cycle progression. Flow
cytometry offers the advantages of rapid, sensitive, and multiparameter analysis of asynchronous cell populations and
has been used widely for the investigation of the biological
processes associated with cell death (1,2,17–22).
The approach based on DNA strand break labeling in the
assay employing TdT has appeared to be the most specific in
terms of positive identification of apoptotic cells until now
(1,4,22,24). In this way, cellular DNA content of not only
nonapoptotic cells but also apoptotic cells is measured; the
method offers a unique possibility to analyze the cell cycle
position and DNA ploidy of apoptotic cells.
It has been shown that loss of phospholipid asymmetry
leading to exposure of phosphatidylserine on the outside
of the plasma membrane is an early event of apoptosis
(31,32). The principle of standard FITC-annexin V staining
used for the detection of apoptosis is that annexin V can
preferentially conjugate negatively charged phosphatidylserine, and apoptotic cells in different stages have different abilities to resist PI. By this way, we can quantitate
apoptotic cells and distinguish them from necrotic cells.
The standard assay, however, does not provide information of the cell cycle specificity of apoptosis. We observed
that methanol-free formaldehyde can fix cells without
FIG. 4. Results of apoptosis detected by two different methods: API (A–D) and TdT (E–H). HL-60 cells were treated by UV (B, F), CPT (C, G), and CPT
plus TPCK (D, H); untreated cells (A, E) were used as controls.
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TAO ET AL.
dissociation of annexin V, and the optimal concentration
of formaldehyde is 1%. In addition, formaldehyde
crosslinks DNA and, hence, prevents leakage of the fragmented DNA from apoptotic cells. This allows one to
identify the cell cycle position of apoptotic cells. In the
API method, digitonin is used to increase cell permeability
(34) after fixation in formaldehyde. The presence of calcium ions is essential to maintain attachment of annexin V
to phosphatidylserine during fixation and subsequent
staining with PI. We observed that the nonionic detergent
that otherwise is used to permeabilize the plasma membrane causes dissociation of annexin V, which manifests
as decreased fluorescence.
TPCK in this study was used to prevent internucleosomal
DNA fragmentation; in its presence after induction of apoptosis, DNA cleavage stops at 300- to 50-kb sections and does
not progress further (33). Thus, because of paucity of DNA
strand breaks, the TdT assay was unable to identify apoptotic
cells in the TPCK-treated cultures. Therefore, it appears that
TdT assay may fail to detect early apoptotic cells and atypical
apoptotic cells, in which DNA fragmentation did not
progress to internucleosomal DNA sections.
As the present data showed, the API and standard FITCannexin V binding methods produce consistently higher
results than the TUNEL method. One explanation for the
discrepancy may be that the “time window” of the annexin V binding assay is wider, i.e., the cells become first
annexin V positive and sometime later TUNEL positive.
In conclusion, similar to the TdT assay, the API method
accurately detects the cell cycle–phase specificity of apoptosis. However, compared with the TdT assay, it detects
earlier stages of apoptosis and thus has a wider time
window for identification of apoptotic cells. Thus, the
apoptotic index by the API method is higher that the TdT
assay. Because the TdT assay fails to detect apoptosis
when DNA degradation is not progressing to internucleosomal DNA sections, as happens in some cases of “atypical
apoptosis,” the API method is preferred in these instances.
Furthermore, compared with the TdT assay, the API
method has fewer steps, is more rapid, and appears to be
more sensitive. However, the API method also has limitations. Because the key point of API is the conjugation of
annexin V and phosphatidyl serine, the factors that injure
plasma membrane can lead to pseudo-positive results.
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