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Analysis of Apoptosis in FUCCI HeLa Cells

INTRODUCTION MATERIALS AND METHODS


Later stages of apoptotic cell death are characterized Cell Culture and Irradiation
by the fragmentation of cellular DNA (1). For example, HeLa.S-FUCCI cells (6) were grown in DMEM with 10%
HeLa cells exposed to a lethal dose of UVC irradiation (30 fetal bovine serum and penicillin/streptomycin. UVC lamp of
J/m2) undergo apoptosis and exhibit DNA fragmentation 254 nm (Phillips) was used to irradiate cells at 30 J/m2. Immu-
(2). DNA fragmentation, which results in reduced DNA noblots were performed using an anti-GFP (FL) antibody
content, can be assessed using several methods, the most (Santa-Cruz sc-8334) and antiactin antibody (Hybridoma
common being cell analysis by flow cytometry. The stand- bank JLA-20).
ard measure for the level of apoptosis in response to a
tested treatment is the fraction of cells with sub-G1 DNA Time-Lapse Microscopy
content, indicating that the DNA of these cells underwent Cells were grown on a Zeiss LMS 710 microscope
fragmentation and DNA loss (3). However, sub-G1 DNA equipped with an incubator and X40 Plan-Neofluar lens (NA
content is reached only after a significant amount of cellu- 0.6). mAG was excited by a 488-nm laser line (Argon laser),
lar DNA is lost. We predicted that in cells that start under- and fluorescence signals were collected at 493–578 nm. mKO2
going apoptosis at late stages of the cell cycle, in which was excited by a 561-nm laser line (DPSS 561-10) and fluores-
DNA content is higher, it would take longer for cellular cence signals were collected at 578–696 nm.
DNA content to decrease to the sub-G1 DNA level. In
addition, cells in which apoptosis was triggered in G2/M
Flow Cytometry and TUNEL Assay
would pass through stages of having G2/M, S, and then G1
Cells were UV irradiated, harvested after the indicated
levels of DNA content. Thus, the population of cells identi- times, fixed in 75% ethanol, and stored at 220C. The fixed
fied by flow cytometry as containing a G1 level of DNA cells were rehydrated in PBS on ice for 30 min, and stained
may include apoptotic cells in which DNA fragmentation is using Apo-BrdU-Red in situ DNA fragmentation assay kit
incomplete. Ignoring this population of cells may lead to (BioVision), using Hoechst 33342 instead of 7-AAD. Cells
significant underestimation of the fraction of cells under- were analyzed in a BD LSRII (Becton Dickinson) flow cytome-
going apoptosis at certain time points. In addition, the ter. mAG and BrdU-Red were excited by a 488-nm laser line,
assumption that all cells in the G1 fraction are living may and fluorescence signals were collected at 530 nm (530/30; 505
lead to misinterpretation of the relationship between apo- LP) for mAG and at 575 nm (575/25)(550LP) for BrdU-Red.
ptosis and cell cycling. Indeed, this phenomenon has been Hoechst 33342 was excited by a 355 laser line, and fluores-
demonstrated in HL60 cells (4,5). cence signals were collected at (450/50). mKO2 fluorescence
Here, we used HeLa cells stably expressing fluorescent was below detection level in these experiments.
ubiquitination-based cell cycle indicator (FUCCI) fusion pro-
teins as cell cycle sensors to perform a systematic quantitative RESULTS AND DISCUSSION
analysis of the kinetics of DNA depletion in cells at different To test the possibility that some apoptotic cells may be
stages of cell cycle progression and to specifically follow cells mistakenly identified by flow cytometry analysis as live cells,
that started undergoing apoptosis in S-G2/M phases (6). We we exposed HeLa cells to a lethal dose of UV irradiation and
demonstrate that the longer time required for DNA fragmen- used flow cytometry to analyze their DNA content at different
tation in these cells may lead to misinterpretation of flow cyto- time points after irradiation. To distinguish between cells in
metry analysis results. different phases of the cell cycle, we used HeLa cells stably

Received 8 February 2011; Revision Received 20 February 2011; Published online 11 March 2011 in Wiley Online Library
Accepted 22 February 2011 (wileyonlinelibrary.com)
*Correspondence to: Sharon Eden, Department of Developmental DOI: 10.1002/cyto.a.21051
Biology and Cancer Research, IMRIC, The Hebrew University- © 2011 International Society for Advancement of Cytometry
Hadassah Medical School, Jerusalem 91120, Israel.
E-mail: sharoned@ekmd.huji.ac.il

Cytometry Part A • 79A: 243 246, 2011


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expressing FUCCI fusion proteins as cell cycle sensors (6). modified the fixation conditions to maintain cell integrity (see
These cells express Cdt1-Kusabira Orange fusion protein, methods). Using this new approach, we observed TUNEL-
which confers red fluorescence in G1 phase, and Geminin- positive cells with sub-G1 DNA content 4 h after UV exposure
Azami Green fusion protein, resulting in green fluorescence in as expected (Fig. 1C). The peaks of TUNEL-positive cells cor-
S, G2, and M phases of the cell cycle. FUCCI cells in cytokin- responded to populations of cells with different DNA content:
esis do not express any fluorescent protein. It has been the largest peak of TUNEL-positive cells had sub-G1 level
reported that Cdt1 is degraded rapidly after UV-induced DNA DNA content, whereas another peak had approximately S-level
damage (7,8). However, the protein domain required for this of DNA content, similar to the major peak of cells with green
degradation was localized to amino acids 1–53 of Cdt1, and fluorescence at this time point. By 16 h, all peaks of cells with
the Cdt1-Kusabira Orange fusion protein contains only amino either green or red fluorescence also stained positive for
acids 30–120 of Cdt1. We verified that the Cdt1-Kusabira TUNEL despite the fact that the DNA content of most of the
Orange fusion protein is not degraded in HeLa cells after green cells remained higher than sub-G1. This observation fur-
exposure to UV irradiation by immunoblot with anti-GFP ther supports the idea that DNA content alone is not an accu-
antibody, which recognizes the Kusabira Orange protein (Fig. rate indicator of cell cycle phase or cell survival in cells
1A). In addition, we followed cell death using time-lapse mi- exposed to apoptosis-inducing agents. It also confirms that
croscopy and observed red fluorescence several hours after UV not all apoptotic cells appear in the sub-G1 fraction.
irradiation in both living and dead cells (Fig. 1B). These data This experiment also allowed us to compare the
confirm that the HeLa FUCCI cells should allow us to investi- kinetics of DNA degradation in cells that began apoptosis
gate cell death in S-G2/M phase HeLa cells following UV irra- during S/G2/M phases to cells that became apoptotic in G1.
diation. Time-lapse microscopy also showed that both red and Throughout the experiment, the green cells exhibited faster
green fluorescent cells exhibited apoptotic morphology after kinetics of apoptosis, as indicated by TUNEL staining, than
UV exposure and remained fluorescent several hours after they cells that started undergoing apoptosis in G1 (red cells).
began to exhibit apoptotic morphology (Fig. 1B). Four hours after UV exposure, only 12.7% of the cells with
We used the FUCCI cells to differentiate between cells that red fluorescence were TUNEL positive, whereas 27.5% of
began to undergo apoptosis in S/G2/M and those that began apo- the green cells were TUNEL positive. This discrepancy was
ptosis in G1. We were particularly interested in the S/G2/M observed for the duration of the analysis, further indicating
population, because these cells have higher DNA content and that cells with higher DNA content start undergoing apo-
may take longer to deplete their DNA to sub-G1 DNA levels. We ptosis earlier than cells in G1 phase.
exposed FUCCI-HeLa cells to UV irradiation and assessed their Despite the more rapid kinetics, the green cells took lon-
DNA content every 2 h using flow cytometry analysis, identifying ger to reach sub-G1 DNA content. Sixteen hours after UV irra-
the S/G2/M population by gating for green fluorescence. The diation, only 55.3% of total cells had sub-G1 DNA content, but
nongreen population consists primarily of red-fluorescent cells 86.1% of all cells were TUNEL positive, demonstrating that
in G1 phase, although it also includes a very small fraction of using sub-G1 content as a marker for apoptosis results in a sig-
nonfluorescent cells in cytokinesis. To simplify description of our nificant underestimate. Comparing the proportion of red and
results, we will refer to the nongreen cell population as red cells. green cells with TUNEL staining revealed that this discrepancy
As expected, at the time of irradiation, green cells were mainly is due primarily to the longer time course of DNA loss in cells,
gated to the predicted DNA content of S and G2/M phases, and which begin apoptosis in S2/G/M. Sixteen hours after irradia-
red cells were primarily gated to G1 DNA content (Fig. 1C). By tion, 76.9% of red cells stained positive for TUNEL, and a sim-
16 h postirradiation, 83.9% of the red cells possessed sub-G1 ilar percentage (83.9%) of red cells presented sub-G1 DNA
DNA content, but only 11.6% of the green cells had reached sub- content. At the same time point, 92.4% of the green cells
G1 DNA content. These results suggest that cells that started to stained positive for TUNEL, but only 11.6% exhibited sub-G1
undergo apoptosis while in G2/M required a significantly longer DNA content. In addition, DNA content deficit cannot be used
time to deplete their DNA to a sub-G1 level. The percentage of as a sole marker of apoptotic cells. Mechanical cell degradation
green cells with G1 DNA content increased from 2 to 16 h after and lysis of mitotic cells or micronuclei may appear as apopto-
UV exposure, indicating that cells that became apoptotic in G2/ tic cells. In addition to DNA deficit, other parameter should be
M went through a period where their DNA content was equiva- used to positively identify apoptotic cells (9). We conclude that
lent to G1-level before it deteriorated to sub-G1 levels. These measuring the fraction of cells with sub-G1 DNA content is
results indicate that some of the cells with G1-levels of DNA con- useful for determining whether certain treatments induce apo-
tent have started undergoing apoptosis. ptosis but is not an accurate quantitative measure of apoptosis
To confirm that DNA fragmentation is already underway and can be misleading regarding the relationship between apo-
in the population of green cells with G1-equivalent DNA con- ptosis and cell cycle progression.
tent, we performed flow cytometry analysis of cells stained for Combining flow cytometry analysis with TUNEL analysis
fragmented DNA using a TUNEL assay adapted for flow cyto- did not resolve this issue because TUNEL staining labels only
metry analysis. The harsh fixing conditions required for cells in late stages of apoptosis, albeit earlier than DNA dete-
TUNEL staining resulted in fragile cells, and a very significant rioration following fragmentation. Our results also indicate
reduction in the fraction of cells with sub-G1 DNA content, that DNA fragmentation in cells with higher DNA content
regardless of their DNA content at the time of irradiation. We occurs with faster kinetics than in cells with G1-level DNA con-

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Figure 1. Analysis of DNA content in UV-exposed FUCCI HeLa cells. (A) Cdt1-Kusabira Orange is stable after UV irradiation. FUCCI HeLa
cells were exposed to 30 J/m2 UVC irradiation, lysed at the indicated time points, and the cell lysates were subjected to immunoblot analy-
sis using anti-GFP antibody and anti-beta-actin antibody as a loading control. Cdt1-Kusabira Orange fusion protein was detected at 34Kd,
and no degradation was observed 2 h after UV irradiation. (B) FUCCI HeLa cells maintain fluorescence after UV irradiation. FUCCI HeLa
cells were exposed to 30 J/m2 UVC irradiation and visualized by time-lapse microscopy. Frames of the indicated time points are shown.
Cells with both red and green fluorescence maintain their color after they exhibit apoptotic morphology. (C) Analysis of DNA content in
UV-exposed FUCCI HeLa cells. FUCCI HeLa cells were exposed to 30 J/m2 UVC irradiation. The cells were labeled for TUNEL staining, and
the DNA was stained with Hoechst 33342. Flow cytometry analysis of HeLa cells after exposure to UV irradiation at the indicated time
points after irradiation is shown. Green, cell with green fluorescence; red, cells with red fluorescence; blue, TUNEL positive cells.

tent. Although the apoptotic process can be followed using phase, the cell density and the fraction of dividing cells should
other markers, the different kinetics exhibited by cell popula- be taken into account when analyzing cell death by flow cyto-
tions in different cell cycle phases will skew the results regard- metry. This is of particular importance when studying apopto-
less of the method used to identify apoptotic cells. We propose sis in the context of cell cycle progression. Similar conclusions
that the most accurate method of measuring the level of apo- were presented in a recent work, demonstrating that combin-
ptosis is to follow the population of cells in each cell cycle ing cytometry analysis with the FUCCI probe may contribute
phase separately as demonstrated here. In addition, because to development of an assay that integrates the complexity of
the kinetics of DNA fragmentation is dependent on cell cycle cell cycle regulation into drug discovery screens (10).

Cytometry Part A  79A: 243 246, 2011 245


COMMUNICATION TO THE EDITOR

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