Revival of apoptotic cells that display early-stage dynamic
Tomoki Takashinaa, Manabu Nakayamaa,b,*
aLaboratory of Pharmacogenomics, Graduate School of Pharmaceutical Sciences, Chiba University, 2-6-7, Kazusa-Kamatari,
Kisarazu, Chiba 292-0818, Japan
bDepartment of Human Genome Research, Kazusa DNA Research Institute, 2-6-7, Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan
Received 14 August 2007; accepted 15 August 2007
Available online 24 August 2007
Edited by Barry Halliwell
versible and how cells attain an anti-apoptotic state remain
unknown. Here, we report that apoptotic cells undergoing
early-stage dynamic membrane blebbing revive. We examined
this phenomenon in cell lines that stably express 2DED2DD, a
modified FADD produced by fusing the tandem death effector
domains (DEDs) and tandem death domains (DDs). Induction
of apoptosis caused rapid blebbing. Eight hours later, most cells
shrunk while some detached from the flask. Twenty-four hours
later, when activated caspase 3 decreased, more than half the
cells revived and appeared normal, probably due to the induction
of unidentified anti-apoptotic proteins.
? ? 2007 Federation of European Biochemical Societies. Pub-
lished by Elsevier B.V. All rights reserved.
The critical point at which apoptosis becomes irre-
Keywords: Apoptosis; FADD; Biotechnology;
Membrane blebbing; Anti-apoptotic protein
Whether a cell dies or lives depends on the balance of proa-
poptotic and anti-apoptotic factors within multiple and over-
lapping networks of cellular cascades. Malignant cells are
able to survive because they counteract various apoptotic pro-
teins through various means, including strong induction of
anti-apoptotic proteins, amplification of anti-apoptotic genes,
deletion of apoptotic genes, and depression of apoptotic pro-
teins through epigenesis [1,2].
(FADD) is an adaptor molecule that mediates apoptotic cell
signals by interacting with various cell surface receptors .
Through its C-terminal death domain (DD), FADD can be
recruited by different tumor necrosis factor receptors (TNFRs)
such as Fas/TNFRSF6 receptor, TNFRSF25, and TNFSF10/
TRAIL receptor. The FADD N terminus contains a death
effector domain (DED), which recruits caspase to the death-
inducing signaling complex (DISC) and initiates the apoptotic
caspase cascade . Recruitment of caspase 8 to the receptors
results in oligomerization of the caspase 8 protein, which then
drives its autoactivation through self-cleavage. Finally, acti-
vated caspase 8 activates other downstream caspases.
Previously, we showed that modification of FADD to
2DEDplusE by fusing its tandem DEDs to the E protein of
lambda phage, a head coat protein with self-assembly activity,
greatly increases the apoptosis-inducing activity of FADD in
both adherent NIH3T3 and HEK293 cells . In that study,
we also showed that 2DED2DD protein, which is composed
of tandem DEDs and tandem DDs, displays intermediate
apoptosis-inducing activity. The apoptosis-inducing activity
of 2DED2DD is stronger than that of unmodified FADD
but weaker than that of the 2DEDplusE protein. Five hours
after induction, approximately 40% of cells were affected and
had detached. Interestingly, approximately 24 h after induc-
tion of 2DED2DD expression, the number of adherent cells in-
creased again. It is possible that this increase was due to
reattachment of cells. An alternative but less likely possibility
is that this increase was due to proliferation of surviving cells.
In the present study, we analyzed in detail this phenomenon –
revival of apoptotic cells – because this phenomenon is an
excellent model for analyzing the mechanism underlying the
survival of malignant cells and of normal cells that adopt an
anti-apoptotic state through the balance of pro- and anti-
2. Materials and methods
2.1. Live cell imaging by time-lapse microscopy
Cells were observed using phase contrast microscopy (Olympus
IX71, Tokyo, Japan) with an UplanFL N40·/0.75 Ph objective lens
and a CO2incubator on a heated stage for microscopy (Olympus
MI-IBC-I, Japan). Images were recorded using a CCD camera (Ham-
amatsu ORCA-ER, Japan) and AQUA-C imaging software (Compix
Inc.). Typically, 5 · 104cells were plated onto 35 mm, poly-D D-lysine-
coated glass bottom dishes. Twenty-four hours after subculture, tetra-
cycline was added to the culture medium and the cells were recorded
continuously at 5 min intervals.
2.2. Immunoblot analysis
Cells were plated at a density of 2 · 105cells per 35 mm dish with an
appropriate DMEM medium. Twenty-four hours after subculture, tet-
racycline was added. At the indicated times, both detached and at-
tached cells were pooled, and cell extracts were prepared by washing
the cells with phosphate-buffered saline (PBS) and adding 50 ll of
Chaps Cell Extract Buffer (Cell Signaling Technology, Inc.) or solubi-
lization buffer (25 mM Tris–HCl, pH 6.8; 2% SDS). The protein
Abbreviations: FADD, Fas-associated death domain protein; DISC,
death-inducing signaling complex; cFLIP, cellular FADD-like inter-
leukin-1b converting enzyme-inhibitory protein; PBS, phosphate-
*Corresponding author. Address: Department of Human Genome
Research, Kazusa DNA Research Institute, 2-6-7, Kazusa-Kamatari,
Kisarazu, Chiba 292-0818, Japan. Fax: +81 438 52 3946.
E-mail address: email@example.com (M. Nakayama).
0014-5793/$32.00 ? 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
FEBS Letters 581 (2007) 4479–4484
for his encouragement. This study was supported by Grants from the
Kazusa DNA Research Institute and in part by Grants-in-Aid for Sci-
entific Research from the Ministry of Education, Culture, Sports, Sci-
ence and Technology, the Japanese Government.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.febslet.2007.08.
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T. Takashina, M. Nakayama / FEBS Letters 581 (2007) 4479–4484