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Journal of Virology, June 1999, p. 5056-5063, Vol. 73, No. 6
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Dynamics of Nontypical Apoptotic Morphological Changes Visualized
by Green Fluorescent Protein in Living Cells with Infectious Pancreatic
Necrosis Virus Infection
Jiann-Ruey
Hong,1,2
Tai-Lang
Lin,1
Jer-Yen
Yang,3
Ya-Li
Hsu,1,* and
Jen-Leih
Wu1,*
Laboratory of Marine Molecular Biology and
Biotechnology, Institute of Zoology, Academia Sinica, Nankang,
Taipei 115,1 Graduate Institute of Life
Sciences, National Defense Medical Center, Taipei
117,2 and Institute of Marine
Biotechnology, National Taiwan Ocean University,
Keelung,3 Taiwan, Republic of China
Received 12 October 1998/Accepted 26 February 1999
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ABSTRACT |
Morphologically, apoptotic cells are characterized by highly
condensed membrane blebbing and formation of apoptotic bodies. Recently, we reported that apoptosis precedes necrosis in a fish cell
line infected with infectious pancreatic necrosis virus
(IPNV). In the present study, we tested the possibility that nontypical apoptosis is a component of IPNV-induced fish cell death. A
variant type of green fluorescent protein (EGFP) was
expressed in a fish cell line such that EGFP served as a protein
marker for visualizing dynamic apoptotic cell
morphological changes and for tracing membrane integrity changes
during IPNV infection. Direct morphological changes were
visualized by fluorescence microscopy by EGFP in living cells infected
with IPNV. The nontypical apoptotic morphological change stage
occurred during the pre-late stage (6 to 7 h postinfection). Nontypical apoptotic features, including highly condensed membrane blebbing, occurred during the middle apoptotic stage. At the pre-late apoptotic stage, membrane vesicles quickly formed, blebbed, and were
finally pinched off from the cell membrane. At the same time, at this
pre-late apoptotic stage, apoptotic cells formed unique small holes in
their membranes that ranged from 0.39 to 0.78 µm according to
examination by scanning electron microscopy and immunoelectron microscopy. Quantitation of the intra- and extracellular release of
EGFP by CHSE-214-EGFP cells after IPNV infection was done by Western
blotting and fluorometry. Membrane integrity was quickly lost during
the late apoptotic stage (after 8 h postinfection), and
morphological change and membrane integrity loss could be prevented and blocked by treatment with apoptosis inhibitors such as
cycloheximide, genistein, and EDTA before IPNV infection. Together, these findings show the apoptotic features at the onset of pathology in
host cells (early and middle apoptotic stages), followed secondarily by
nontypical apoptosis (pre-late apoptotic stage) and then by postapoptotic necrosis (late apoptotic stage), of a fish cell line. Our
results demonstrate that nontypical apoptosis is a component of
IPNV-induced fish cell death.
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INTRODUCTION |
Infectious pancreatic necrosis virus
(IPNV) is the prototype virus of the family Birnaviridae
(8). Birnaviruses also include infectious bursal disease
virus of domestic fowl (28) and drosophila X virus of
Drosophila melanogaster (43). IPNV causes a
lethal disease in both hatchery-reared juvenile salmonids (11,
48) and nonsalmonid fish (5, 11).
There are two major morphologically and biochemically distinct modes of
death in eukaryotic cells: necrosis and apoptosis (9, 18,
51). Necrosis is considered to be a pathological reaction that
occurs in response to perturbations in the cellular environment, such
as complement attack, severe hypoxia, and hyperthermia. These
stimuli increase the permeability of the plasma membrane, resulting in
irreversible swelling of the cells (51). On the other hand,
apoptosis is characterized morphologically by cell shrinkage and
hyperchromatic nuclear fragments and biochemically by chromatin
cleavage into nucleosomal oligomers (51). Apoptosis is
considered to be a physiological process involved in normal tissue turnover which occurs during embryogenesis, aging, and tumor regression (51). However, pathological stimuli, such
as viral infection (14-16, 27, 29, 30), can also
trigger the apoptotic process.
The integrity of the plasma membrane plays an important role in
maintaining Ca2+ homeostasis in cells (22, 33).
An essential role for the lymphocyte plasma membrane in the development
of apoptosis has been proposed (1, 19, 42). It was reported
that protein kinase C is activated during apoptosis induced by gamma
irradiation (32) and glucocorticoids (31). This
activation of protein kinase C may be related to increases in
diacylglycerol, one of the earliest signal-induced breakdown products
of membrane-bound inositol phospholipid.
Green fluorescent protein (GFP) from the jellyfish Aequorea
victoria is a revolutionary report molecule for monitoring gene expression and fusion protein localization in vivo or in situ and in
real time (3, 24, 33, 35, 46). In the present study, we
tested whether nontypical apoptosis is a component of IPNV-induced fish
cell death. A variant type of GFP (EGFP) served as a marker for the
visualization of dynamic apoptotic cell morphological changes and for
tracing membrane integrity changes during IPNV infection. CHSE-214
cells containing the gene for EGFP (CHSE-214-EGFP cells) were
visualized by fluorescence microscopy to detect sequential morphological changes during infection with IPNV. Nontypical apoptotic morphological change occurred in the pre-late stage (6 to 7 h postinfection [p.i.]). At the pre-late stage, apoptotic cells formed unique, small holes in their membranes according to
examination by scanning electron microscopy and immunoelectron
microscopy. Quantitation of the intra- and extracellular release of
EGFP by CHSE-214-EGFP cells after IPNV infection was examined by
Western blotting and fluorometry. The morphological changes and
integrity of membrane loss could be prevented or blocked by treatment
with drugs such as cycloheximide (CHX), genistein, and EDTA. Together, these findings demonstrate that nontypical apoptosis is a component of
IPNV-induced apoptotic cell death in fish. In addition, these findings
regarding typical to nontypical apoptotic morphological changes should
provide important insights into the apoptotic process of virus infection.
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MATERIALS AND METHODS |
Wild-type CHSE-214 cells, CHSE-214-EGFP cells, and viruses.
Chinook salmon embryo (CHSE-214) cells were obtained from the American
Type Culture Collection. Cells were grown at 18°C as monolayers in
plastic tissue culture flasks (Nunc) using Eagle minimum essential
medium (MEM) supplemented with 10% (vol/vol) fetal calf serum (FCS)
and 25 µ g of gentamicin per ml. GFP-producing cells were obtained by
transfection of CHSE-214 cells with a pEGFP-N1 vector (6)
and selection with G418. In these vectors, transcription of insert
sequences is driven by the immediate-early promoter of human
cytomegalovirus. The coding region contains the EGFP gene, which
contains a chromophore mutation which produces fluorescence 35 times
more intense than that of wild-type GFP (6).
The virus isolated, E1-S, a member of the Ab strain of IPNV, was
obtained from Japanese eel in Taiwan (50). E1-S was
propagated in CHSE-214 cell monolayers at a multiplicity of infection
(MOI) of 0.01 particles per cell. Infected cultures were incubated at 18°C until an extensive cytopathic effect was observed. The cells were scraped into a tube with the tissue culture medium and chilled on
ice, and the cells were then sonicated. This virus stock (5 × 107 to 1 × 108 PFU/ml) was dispensed into
1-ml samples and stored at 
70°C. Virus plaque assays were performed
on confluent monolayers of CHSE-214 cells that were infected with the
virus solution for 1 h at room temperature, overlaid with 0.6%
agarose containing 2.5 µg of trypsin per ml, and incubated for 3 days
at 18°C. Cells were then stained with 1% crystal violet in 20%
ethanol (8).
Immunoblotting.
About 105 cells per ml were
seeded on a 60-mm-diameter petri dish and allowed to grow for more than
20 h. The cell monolayers were rinsed twice with
phosphate-buffered saline (PBS), after which they were infected at an
MOI of 1 and incubated for 0, 2, 4, 6, 8, 10, 12, and 24 h p.i.
Uninfected control cells were also incubated for the same periods of
time. At the end of each incubation time, the culture medium was
aspirated. The cells were washed with PBS and then lysed in 0.3 ml of
lysis buffer (10 mM Tris base, 20% glycerol, 10 mM sodium dodecyl
sulfate [SDS], 2% 
-mercaptoethanol, pH 6.8).
Proteins were separated by SDS-polyacrylamide gel electrophoresis
(21), electroblotted, and subjected to immunodetection as
described by Kain et al. (17). Blots were incubated with a
1:7,500 dilution of an immunoglobulin fraction (Clontech) and a 1:1,500
dilution of a peroxidase-labeled goat anti-rabbit conjugate (Amersham).
Chemiluminescence detection was performed in accordance with the
instructions provided with the Western Exposure Chemiluminescent Detection System (Amersham). Chemiluminescent signals were imaged by
exposure of Kodak XAR-5 film (Eastman Kodak, Rochester, N.Y.). Stripping and reprobing of the Western blot (17) and removal of the primary and secondary antibodies from blot were achieved by
incubation in stripping buffer containing 62.5 mM Tris-HCl (pH 6.8),
3.0% (wt/vol) SDS, and 50 mM 1,4-dithiothreitol for 30 min at 55°C
with gentle shaking. The blot was washed three times in PBS containing
0.1% (vol/vol) Tween 20 for 10 min each time and reprobed with
antibodies beginning at the membrane blocking step.
Experiments examining the potency of drugs for preventing morphological
change and blocking membrane integrity loss and those
examining
subsequent EGFP retention during virus infection and
incubation were
carried out as described above, except that extra
CHX (10 µg/ml),
aprotinin (400 µg/ml), leupeptin (400 µg/ml),
genistein (100 µg/ml), tyrphostin (100 µg/ml), and EDTA (2 mM)
were added to
CHSE-214 cells before virus infection and incubation
for 16 h. At
the end of the incubation period, cells were harvested
and samples were
analyzed by Western blotting as previously described
(
17).
Fluorescence microscopy.
A CHSE-214-EGFP monolayer infected
with IPNV (MOI = 1) was examined by light and fluorescence
microscopy using an Olympus IX70 microscope equipped with a BP450-480
pass excitation filter and a BA515 barrier emission filter for
observation of EGFP fluorescence. Photographs were taken with a C-35
AD-4 camera using Kodak Ektachrome 200 film.
DNA preparation and gel electrophoresis.
About
105 cells per ml were seeded on a 60-mm-diameter petri dish
and allowed to grow for more than 20 h. The cell monolayers received virus at an MOI of 1.0 and were incubated for 8 h.
Uninfected control cells were also incubated for 8 h. The two
groups were used for DNA fragmentation studies. At the end of
incubation, the cells were lysed with lysis buffer (10 mM Tris-HCl,
0.25% Triton X-100, 1 mM EDTA, pH 7.4). After treatment with
phenol-chloroform-isoamyl alcohol (25:24:1), the DNA was precipitated
in the presence of 0.3 M sodium acetate and cold absolute ethanol at
70°C for 2 h and then resuspended in 10 mM Tris-HCl (pH
7.4)-1 mM EDTA. Aliquots of 20 µl containing approximately 5 to 10 µg of DNA were then electrophoresed in 1.2% agarose gels for 2 h at 40 V. Gels were stained with ethidium bromide and photographed
under UV transillumination.
Scanning electron microscopy.
Scanning electron microscopy
analysis was carried out by cell seeding on a two-chamber slide.
CHSE-214 cells were infected with virus at an MOI of 1 and incubated
for 0, 4, 8, and 12 h. At the end point, cells were washed twice
with PBS and fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer.
Samples were postfixed with OsO4, dehydrated in ethanol,
critical point dried, and gold sputtered. A Philips 515 scanning
electron microscope was used to examine the specimens.
Immunoelectron microscopy.
CHSE-214-EGFP cells were infected
at an MOI of 1. Infected and uninfected control cells were harvested
8 h after infection. Thin-section electron microscopy and
immunogold labeling were carried out as described by McNulty et al.
(27). The grids were stained with a 1:1,000 dilution of
GFP-specific polyclonal antiserum and a 1:50 dilution of a 15-nm
gold-labeled goat anti-rabbit immunoglobulin G conjugate.
Quantitation of EGFP release by CHSE-214-EGFP cells.
Cellular EGFP and culture medium EGFP protein samples were prepared for
assay in EGFP release experiments. About 105 cells per ml
were seeded on a 60-mm petri dish for more than 20 h. Cell
monolayers were rinsed twice with PBS and then cultured in 3 ml of 10%
FCS-containing MEM. Uninfected cells used as a normal control and cells
that received virus at an MOI of 1 were incubated for 0, 2, 4, 6, 8, 10, 12, and 24 h p.i. At the end of each incubation period, the
culture medium was collected to determine the concentration of retained
EGFP. Cells were washed with PBS and then lysed in 0.3 ml of lysis
buffer (10 mM Tris base, 20% glycerol, 10 mM SDS, 2%
-mercaptoethanol, pH 6.8).
The assay procedure was as follows. First, recombinant GFP purchased
from Clontech was used as the standard. The GFP standard
was diluted
from 1 µg/0.1 ml to 0.001 µg/0.1 ml with 10% FCS-containing
MEM.
Second, 5 µg of lysed cells per sample was diluted with 10%
FCS-containing MEM to a final volume of 100 µl. Third, the
supernatant
was assayed, and 30 µg of supernatant per sample was
diluted with
10% FCS-containing MEM to a final volume of 100 µl.
Protein concentration
was determined by the dye-binding method of
Bradford (
2) using
a commercially available kit (Bio-Rad,
Richmond, Calif.) with
bovine serum as the standard. Fourth, the
fluorescence intensity
of three group samples was counted by a
Fluorolite 1000 (DYNEX).
The EGFP concentrations of the lysed cells and
supernatant were
evaluated by comparing them with that of the GFP
standard by using
a Fluorolite 1000 and dividing by 35 (
6).
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RESULTS |
Visualization of dynamic morphological changes by EGFP.
One of
the most useful aspects of GFP for biological studies is that it can be
monitored in living cells (37). Figure
1 shows the sequence of morphological
changes that occurred in CHSE-214-EGFP cells during virus
infection (MOI = 1). These events were divided into three
stages. First (early stage), detachment of the CHSE-214-EGFP cell
matrix was initiated between 0 and 3 h p.i. Second (middle stage),
the whole cell was rounded up and appeared morphologically more
compact. In this period (3 to 6 h p.i.), the cell volume decreased
to one-third of its original size and the fluorescence intensity was
enhanced. In the third (pre-late) stage, the cells at 6 to 7 h
p.i. quickly underwent severe morphological changes. Membrane vesicles
(MV) were formed from the plasma membrane, and these vesicles
eventually blebbed and finally pinched off from the cell membrane.
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FIG. 1.
Dynamics of the sequential morphological changes
visualized by EGFP in living cells infected with IPNV. Monolayer
cultures of CHSE-214 cells were transfected with pEGFP-N1 by
using Lipofectin and selected with G418. Cells were infected with virus
(MOI of 1), and virus-infected cells were sequentially observed by
fluorescence microscopy from 0 to 7 h p.i. Photographs were taken
with a 40× objective. Scale bar, 3 µm. The arrows indicate the
formation of MV from the apoptotic cell.
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Induction of internucleosomal cleavage by IPNV in CHSE-214-EGFP
cells.
We examined the effect of IPNV infection on host DNA in
CHSE-214-EGFP cells since DNA fragmentation is a well-defined
biochemical marker of apoptosis (40). Virus (MOI = 1)-infected cells were examined for evidence of
internucleosomal fragmentation. Intense internucleosomal
fragmentation of DNA, a pattern highly specific to apoptosis, was
observed in CHSE-214-EGFP cells infected with IPNV (Fig.
2). The IPNV induced DNA fragmentation at
8 h p.i. (Fig. 2, lane 4). The negative control showed no DNA
fragmentation at 0 and 8 h of incubation (Fig. 3, lanes 2 and 3).
Lane 1 contained molecular weight markers that ranged from 500 bp to 1 kb (from MBI Fermantas Inc.).
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FIG. 2.
DNA fragment analysis of CHSE-214-EGFP cells
infected with IPNV E1-S (MOI of 1). DNA was isolated (as described in
Materials and Methods) from uninfected CHSE-214 cells as a negative
control after 0 (lane 2) and 8 (lane 3) h of incubation and from cells
infected for 8 h with an MOI of 1 of E1-S (lane 4),
electrophoresed through 1.2% agarose gels, and visualized by ethidium
bromide staining. Lane 1 contained molecular size markers (1-kb DNA
ladder from USA MBI Fermentas Inc. for sizing of linear fragments
ranging from 500 bp to 1 kb).
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Ultrastructural morphology changes in IPNV-infected
CHSE-214 cells detected by scanning electron microscopy.
Apoptosis
induces characteristic morphological changes in cells, such as
condensation and fragmentation of the nucleus, as well as loss of
cytoplasm (54). To substantiate further that IPNV-infected cells had undergone nontypical apoptotic morphological changes such
that membrane integrity changed, negative control and IPNV-infected CHSE-214 cells were harvested and processed for scanning electron microscopy as shown in Fig. 3. Negative
control cells are shown in Fig. 3A. IPNV-infected CHSE-214
cells at 8 h p.i. displayed detachment, cell rounding,
and blebbing of MV from the plasma membrane at the pre-late
stage of apoptosis (20%; P < 0.05), as shown
in Fig. 3B. Middle-late-stage apoptotic cells (23%; P < 0.05) are shown in Fig. 3C. The cell membrane appears shrunken, and holes are present in the plasma membrane. The hole sizes ranged from 0.39 to 0.78 µ m with about 10 to 20 holes per cell. A
late-stage apoptotic cell (2%; P < 0.05) is shown in
Fig. 3D with the small holes still on the surface of the
late-apoptotic cell.
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FIG. 3.
Scanning electron micrographs of CHSE-214 cells. (A)
Negative control CHSE-214 cell. (B) Pre-late apoptotic CHSE-214 cell.
The formation of MV from the apoptotic cell is indicated by arrows. (C)
Middle-late apoptotic cell. The formation of small holes is indicated
by arrows. (D) Late apoptotic cell. Small holes left on the surfaces of
apoptotic bodies from the IPNV-treated group are indicated by arrows.
Scale bar, 1.5 µm.
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EGFP release is prevented by a protein synthesis inhibitor and a
tyrosine kinase inhibitor.
In EGFP release experiments, EGFP was
used to monitor the integrity of the plasma membrane during apoptosis.
As described above, small holes appeared in middle-late-stage apoptotic
cells (Fig. 3C). We previously proposed that intracellular material might leak out of these small holes to the extracellular region before
secondary necrosis (13). The use of EGFP to monitor the integrity of the plasma membrane of IPNV-infected CHSE-214-EGFP cells
is shown in Fig. 4. The EGFP release
Western blot assay result is shown in Fig. 4A. Fig. 4A, part a, shows
that the amount of GFP decreased, especially between 8 and 16 h
p.i. The internal control, actin protein, is shown in Fig. 4A, part b.
Detection of the EGFP released from the intracellular to the
extracellular region during IPNV infection is shown in Fig. 4A, part c.
The increase of GFP release began between 8 and 16 h p.i., which
is consistent with Fig. 4A, part a. These data indicate that the membrane integrity changed quickly at the middle-late apoptotic stage.
The fluorometric EGFP release assay results are shown in Fig. 4B. The
open squares show that the intracellular amount of EGFP sharply
decreased from 6 to 24 h p.i. but that the largest release of EGFP
occurred between 12 and 24 h p.i. The open diamonds show that the
extracellular amount of EGFP increased between 6 and 24 h p.i.,
which matches the intracellular data described above.
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FIG. 4.
Patterns of EGFP release by CHSE-214-EGFP cells infected
with IPNV. (A, parts a to c) Detection of EGFP release pattern due to
IPNV infection by Western blotting. CHSE-214-EGFP cells were infected
with IPNV (MOI of 1). Samples were electrophoresed on an
SDS-polyacrylamide gel and electroblotted to a nitrocellulose membrane.
The membrane either contained a rabbit polyclonal antiserum directed
against EGFP (part a and c) or was stripped and reprobed with a mouse
IgG monoclonal antibody directed against actin (part b). The
chemiluminescent signal was imaged on Kodak XAR-5 film using a 3-min
(part a), 1.5-min (part b), or 30-min (part c) exposure. (a) Lanes: 1, 0.45 µg of wild-type GFP; 2 to 7, 30 µg of virus-infected CHSE-214
cell lysate at 0, 2, 4, 6, 8, and 16 h p.i., respectively. (b) The
nitrocellulose membrane in part a was stripped and reprobed with
anti-actin monoclonal IgG. (c) A 30-µg sample of supernatant protein
of IPNV-infected CHSE-214-EGFP cells at 0, 2, 4, 6, 8, 10, 12, and
24 h p.i., respectively. (B) Rate of EGFP release by CHSE-214-EGFP
cells infected with IPNV. Cellular and culture medium EGFP samples were
prepared for assay in EGFP release experiments. About 105
cells per ml were seeded on a 60-mm petri dish and incubated for more
than 20 h. Cells that received virus at an MOI of 1 were incubated
for 0, 2, 4, 6, 8, 10, 12, and 24 h p.i. At the end of each
incubation time, the IPNV-infected CHSE-214 cells and culture
medium were collected to determine the concentration of retained EGFP.
Both 5 µg of lysed virus-infected cells per sample and 30 µg of
supernatant medium per sample were counted by a Fluorolite 1000. The
EGFP concentrations of the lysed cells and supernatant were evaluated
by using a Fluorolite 1000 to compare them with standard GFP protein
and dividing by 35 (6).
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EGFP was also used as a protein indicator to directly probe membrane
integrity by immunoelectron microscopy. Normal CHSE-214-EGFP
cells used
as controls are shown in Fig.
5A. Figure
5B shows that
the small vesicle escaped from the membrane hole at the
pre-late
apoptotic cell stage and that the vesicle contains the same
EGFP
labeled by an anti-GFP polyclonal antibody-gold complex.
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FIG. 5.
Immunoelectron micrographs of ultrathin sections of
CHSE-214-EGFP cells that were uninfected or infected with IPNV and
labeled with anti-GFP IgG. (A) Normal CHSE-214-EGFP cell used as a
negative (N) control on which labeled EGFP is present (arrows) and EGFP
formed dimers. (B) CHSE-214-EGFP cell infected with IPNV (MOI of 1) at
8 h p.i. upon which labeled EGFP is present (small arrows).
Nontypical apoptotic morphological changes were observed at this
pre-late apoptotic cell stage such as the formation of MV (large, long
arrow) and, finally, the MV pinching off from the plasma membrane of
the apoptotic cell (large, short arrow).
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Drugs, including the protein synthesis inhibitor CHX, the serine
proteinase inhibitors aprotinin and leupeptin, the tyrosine
kinase
inhibitors genistein and tyrphostin, and the cation chelator
EDTA, were
used before IPNV infection to test the viability of
preventing membrane
integrity change. Some of the drugs, such
as CHX at 10 µg/ml and 2 mM
EDTA, completely prevented EGFP release,
and genistein at 100 µg/ml
partially prevented EGFP release (as
shown in Fig.
6A) to the extracellular region (as shown
in Fig.
6C). The serine proteinase inhibitors aprotinin (400 µg/ml)
and
leupeptin (400 µg/ml) (Fig.
6C, lanes 5 and 6, respectively) and
the tyrosine kinase inhibitor tyrphostin (100 µg/ml) (Fig.
6C,
lane
8) did not prevent EGFP release. The internal control, actin
protein, is shown in Fig.
6B for quantitation of protein loading
per sample.
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FIG. 6.
Western blot assay of the effect of chemical inhibitors
on EGFP release. Protein synthesis inhibitors, serine proteinase
inhibitors, tyrosine kinase inhibitors, and a cation chelator were
added to CHSE-214-EGFP cells before infection with IPNV (MOI of 1).
After infection, the cells were incubated for 16 h. Samples were
electrophoresed on an SDS-12% polyacrylamide gel and electroblotted
to a nitrocellulose membrane. Antigen-specific signals were detected
with either rabbit anti-GFP serum (A and C) or a mouse IgG monoclonal
antibody directed against actin (B). The chemiluminescent signal was
imaged on Kodak XAR-5 film by using a 5-min (A), a 1-min (B), or a
30-min (C) exposure. (A) Lanes: 1, 0.2 µg of recombinant wild-type
GFP as a positive control; 2, normal CHSE-214-EGFP cell lysate; 3, 30 µg of cell lysate protein corresponding to treatment with CHX (100 µg/ml), aprotinin (400 µg/ml), leupeptin (400 µg/ml), genistein
(100 µg/ml), tyrphostin (100 µg/ml), and EDTA (2 mM) and then virus
infected for 16 h, respectively. (B) The nitrocellulose membrane
from panel A was stripped and reprobed with an actin antibody. (C)
Lanes: 1, 100 ng of wild-type GFP; 2, 30 µg of supernatant medium
protein from IPNV-infected cells at 16 h p.i. 4 to 9, 30 µg of
supernatant medium protein corresponding to treatment with CHX (100 µg/ml), aprotinin (400 µg/ml), leupeptin (400 µg/ml), genistein
(100 µg/ml), tyrphostin (100 µg/ml), and EDTA (2 mM) before virus
infection and at 16 h p.i., respectively.
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DISCUSSION |
Here, we provide the first evidence that GFP can be used to
sequentially monitor apoptotic morphological changes in living cells or
probe the change in membrane integrity after IPNV infection. GFP is
stable and species independent and can be monitored noninvasively in
living cells (25, 37). A clone with strong fluorescence intensity and normal morphology, CHSE-214-EGFP, was selected and subcloned as a cell line for experiments (as shown in Fig. 1). The
clones with lower fluorescence intensity did not produce a good image
in sequential morphology studies (4, 10). Working with GFP
raises practical problems. One such problem, common in fluorescence
microscopy of live cells, is that of phototoxicity, which is thought to
be caused mainly by fluorophore-mediated generation of free radicals.
Fortunately, the introduction of mutant GFPs with higher
quantum efficiencies, lower-energy excitation spectra, and better
temperature stability (6, 12, 41) has been advantageous and
has significantly widened the applicability of GFP to the study of
proteins of low abundance. If the cameras and fluorescence microscopes
used become more and more sensitive and efficient, the problems will be
further alleviated.
In our system, cloned cells were expressed with EGFP (32.5 kDa; as
shown in Fig. 4A, lane 2), which is larger in molecular size than
wild-type GFP (27 kDa; as shown in Fig. 4A, lane 1). EGFP may occur by
glycosylation or phosphorylation in CHSE-214 cells and
appears to have a larger molecular size than wild-type GFP. In
addition, GFP is fluorescent either as a monomer or as a dimer. The
ratio of monomeric to dimeric forms depends on the protein
concentration and the environment (47). As described above,
EGFP was also found in both control cells and IPNV-infected cells
either as a monomer or as a dimer (as shown in Fig. 5). We found a
doublet EGFP from the released EGFP, as shown in Fig. 4A (part c, lane
6) and 6C, but whether posttranslational modification of EGFP can be
enhanced by dimerized EGFP could not be ascertained. To substantiate
the significance of our findings, further experiments are required to
evaluate how EGFP produces different expression patterns in our system.
EGFP was used to monitor the dynamic morphological changes in
CHSE-214-EGFP cells infected with IPNV. The results are shown in Fig.
1. We briefly divided this series of events into four stages: (i)
the early apoptotic stage (0 to 3 h p.i.), (ii) the middle
apoptotic stage (3 to 6 h p.i.), (iii) the pre-late apoptotic stage (6 to 7 h p.i.), and (iv) the postapoptotic necrosis stage (after 7 h p.i.). The morphological changes in apoptotic cells observed included cell detachment, rounding up, formation of MV, pinched off MV floating away in the culture medium, and finally, postapoptotic necrosis, as previously observed by Hong et al. (13). We found that these sequential morphological change
events were different from typical apoptotic morphological changes,
such as detachment, rounding up, membrane blebbing, and finally the formation of apoptotic bodies, as described by Wyllie et al.
(52) and Majno and Joris (23). Here, we clearly
defined this process of nontypical apoptotic morphological change by
probing with EGFP after IPNV infection, and we summarize these
findings in Fig. 7.
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FIG. 7.
Diagram illustrating the morphological changes induced
in fish cells by IPNV infection. (A) Normal attached cell. In the early
stage of apoptosis, the cell detaches from the extracellular martix (A
to B, 0 to 3 h p.i.). In the middle stage, the apoptotic cell is
rounded up (A to B, 3 to 6 h p.i.). To enter this pre-late
apoptotic stage (B to C, 6 to 7 h p.i.), there is a rapid process
which follows that includes MV formation and MV pinching off from the
plasma membrane. In the middle stage, the apoptotic cell is left with
small holes in the cell membrane (C to D, 7 to 8 h p.i.). Finally, in
the late apoptotic stage, either membrane-bound apoptotic bodies (D to
E, 8 to 12 h p.i.) are formed or a postapoptotic necrosis process
occurs (D to F, 8 to 12 h p.i.) in which the condensed chromatin
encloses the nuclear membrane.
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To determine the loss of membrane integrity in apoptotic cells, we used
the protein marker EGFP to monitor membrane integrity change after IPNV
infection (as shown in Fig. 4). The direct changes in membrane
integrity identified by immunoelectron microscopy are shown in Fig. 5.
The harmful membrane changes can be prevented by drugs such as the
protein synthesis inhibitor CHX, and the cation chelator EDTA and can
be partially prevented by the tyrosine kinase inhibitor genistein (as
shown in Fig. 6).
The important role of bcl-2 in supporting cell survival has
been well demonstrated, in particular by studies with bcl-2
transgenic (26) or knockout (45) mice. Mcl-1 is a
member of the Bcl-2 family that was identified by differential
screening of cDNA libraries derived from a human myeloid leukemia cell
line induced to undergo differentiation in culture (20). It
was recently shown that transfection of Mcl-1 into Chinese hamster
ovary cells leads to inhibition of apoptosis induced by
c-myc overexpression (36), implying that
mcl-1 is an inhibitor of cell death. In our system, the
study of the apoptotic cell death mechanism during IPNV E1-S (MOI = 1) infection of CHSE-214-EGFP cells showed that IPNV E1-S-induced cell death may be correlated to down-regulation in the Bcl-2 family's Mcl-1 protein expression level (data not shown). We then tested whether
Mcl-1 expression down-regulation can be prevented by apoptosis inhibitors such as protein synthesis inhibitors, a cation chelator, serine proteinase inhibitors, and tyrosine kinase inhibitors. When
CHSE-214-EGFP cells were treated with the protein synthesis inhibitor
CHX at 10 µg/ml, the cation chelator EDTA at 2 µM, and the tyrosine
kinase inhibitor genistein at 100 µg/ml before IPNV infection, viral
protein synthesis was blocked and Mcl-1 was partially down-regulated.
However, the serine proteinase inhibitors aprotinin and leupeptin (each
at 400 µg/ml) and tyrphostin at 100 µg/ml did not have the same
effects (data not shown). These results are consistent with those of
the EGFP release experiments described above. We suggest that the viral
protein might be directly or indirectly correlated to down-regulation
of the Mcl-1 expression level during apoptotic death caused by IPNV
infection. However, to substantiate the significance of our findings,
further experiments are required to evaluate how viral proteins relate
to down-regulation of the Mcl-1 expression level.
IPNV is a highly contagious disease of susceptible hatchery-reared
salmonids (11, 48) and nonsalmonid fish (13, 44). As the name indicates, infection of trout produces marked pancreatic necrosis, but histopathologic changes sometimes also occur in adipose
tissue, in renal hematopoietic tissue, in the gut, and in the liver
(39). We have shown that IPNV can induce nontypical apoptosis with all of its associated characteristics, including DNA
fragmentation, detachment, cell rounding, membrane blebbing, formation
of MV that pinch off from the cell membrane, and finally, postapoptotic
necrosis from middle-stage apoptotic cells. Necrotic cell death may
occur during natural infections, but these features support the
hypothesis that IPNV causes CHSE-214-EGFP cells to undergo apoptosis,
then nontypical apoptosis, and finally, postapoptotic necrosis in vitro.
| |
ACKNOWLEDGMENTS |
We thank R. C. Chen for reviewing the manuscript and
providing comments.
This work was supported by grants awarded to Jen-Leih Wu by the
National Science Council, Republic of China.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory of
Marine Molecular Biology and Biotechnology, Institute of Zoology,
Academia Sinica, Nankang, Taipei 115, Taiwan, Republic of China. Phone: 886-2-27899500. Fax: 886-2-27858059. E-mail:
ZOJLWU{at}ccvax.sinica.edu.tw.
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Abstract
Introduction
