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Experimental Therapeutics |
Center for Basic Research in Digestive Diseases [H. H., S. F. B., G. J. G.], Molecular Medicine Program [A. B., K. H., R. G. V.], Mayo Clinic, Rochester Minnesota 55905
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-fetoprotein promoter, a gene that is
expressed by many hepatocellular cancers (4)
. Transduction of tumor cells using suicide genes is a common strategy used in cancer gene therapy, in which tumor cells are transduced with genes for enzymes that convert otherwise nontoxic drugs into cytotoxic compounds (5) . A key observation originating from these studies is the so called "bystander effect" (6 , 7) . The bystander effect consists of loss of viability in nontransduced cells after death of transduced neighboring cells. The bystander effect is not well understood but has been observed in vitro and is thought to be even more pronounced in vivo, where the immune system and inflammatory responses enhance this phenomenon (8) . The bystander effect is crucial for effective anticancer therapy because it compensates for the limited efficiency of gene transduction.
A unique gene therapy approach for human cancers is transduction with viral FMGs3 (9) . Several different viruses kill their target cells by causing fusion between infected and noninfected cells via the interaction between the viral envelope with its receptor; the fusion of uninfected cells with infected cells represents a type of bystander effect. The fusogenic portions of membrane glycoproteins have been identified in varicella-zoster virus, measles virus, and human immunodeficiency virus (10, 11, 12, 13) . Transduction of the FMG alone in human cells has been shown to induce cell-cell fusion and formation of lethal multinucleated syncytia (10 , 13) . The ability of viral FMGs to fuse target cells with neighboring cells with a lethal outcome could be exploited as a treatment approach for hepatocellular carcinoma. The cytotoxic activity of the cDNAs of envelope genes from three different classes of viruses including the rhabodoviral VSV-G envelope gene (14) , the combination of the F and H genes from measles virus (13) , and mutated version of the retroviral Gibbon Ape Leukemia virus (GALV) has been evaluated previously (9) .
The mechanism by which the FMG-induced multinucleated syncytia die is unknown. Two mechanisms of cell death are now recognized: (a) necrosis; and (b) apoptosis. Necrosis is characterized by cellular swelling and plasma membrane lysis. On a biochemical level, necrosis results from ATP depletion (15 , 16) . The morphological hallmarks of apoptosis include cell shrinkage, nuclear chromatin condensation, and separation of the cell into membrane-defined fragments (17 , 18) . Apoptosis is mediated by caspase protease activation and requires ATP (16 , 19, 20, 21) . Because necrosis involves release of cellular constituents into the interstitial space and apoptosis does not, necrosis elicits a stronger inflammatory response. Necrosis would, therefore, be expected to enhance the bystander effect because of the resulting inflammatory and immunological response (22) .
Both necrosis and apoptosis involve mitochondrial dysfunction. In necrosis, mitochondrial dysfunction is so severe that oxidative phosphorylation fails, and cellular ATP depletion occurs (16 , 19) . In contrast, in apoptosis the mitochondrial dysfunction is associated with release of intermembrane constituents such as Cyt-c, which activate apoptotic death cascades. In both forms of cell death, mitochondria may lose their membrane potential and release Cyt-c from the intermembrane space. In this context, it may be the magnitude of ATP depletion that determines whether a cell dies by necrosis or apoptosis.
Our overall objective is to develop gene therapy for human hepatocellular carcinoma using FMG. As an initial step toward this goal, we used an in vitro approach to determine whether FMG transduction is cytotoxic for this cell type. We chose GALV-FMG for this study because of its documented efficacy in other cell types (9) . Our specific aims were to answer the following questions: (a) Does GALV-FMG transfection induce multinucleated syncytia in human hepatocellular carcinoma cells? (b) Does the formation of the syncytia result in cell death by a mitochondrial involved pathway? and (c) Does GALV-FMG-induced cell death occur by necrosis or apoptosis? Our results demonstrate that GALV-FMG transduction in a hepatocellular carcinoma cell line causes syncytia formation with mitochondrial dysfunction, ATP depletion, and necrosis. These promising results suggest that this gene therapy approach deserves further study for the treatment of this cancer.
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MATERIALS AND METHODS |
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70%
subconfluent prior to each experiment.
GALV-FMG Transfection.
A cDNA encoding for the fusogenic portion of the envelope glycoprotein
of the GALV was subcloned into the pCR3.1 vector (Invitrogen). Hep3B
cells were transfected with Lipofectamine (Life Technologies, Inc.,
Gaithersburg, MD). Briefly, the cells were transfected by adding 1 ml
of a mixture of 6 µg of Lipofectamine and 1 µg of the DNA plasmid
to each culture dish. The cells were incubated in the above mixture for
5 h at 37°C in a 5% CO2/95% air
incubator. After the incubation, 1 ml of EMEM containing 20% fetal
bovine serum was added to the transfection medium in each culture dish.
Twenty-four h later, the medium was aspirated and replaced with 2 ml of
EMEM containing 10% fetal bovine serum.
Quantitation of Syncytium Formation.
Syncytia formation was quantitated both by phase contrast microscopy
and fluorescence microscopy. Cells were loaded with the nuclear binding
dye DAPI to identify and accurately quantitate nuclei
(24)
. To quantitate cell-cell fusion, individual cells and
nuclei were counted, and a fusion index was calculated as follows: 1-
(number of cells/number of nuclei; Ref. 25
). A minimum of
500 nuclei were counted per datum point.
Determination of Cell Death.
Cell death was quantitated by a propidium iodide exclusion assay
(26)
. Analogous to trypan blue, propidium iodide is
excluded from viable cells but rapidly labels the nuclei of dead cells.
Propidium iodide (5 µM) was added to the culture medium
for 30 min to label dead cells, which were visualized by fluorescence
microscopy using excitation and emission wavelengths of 520 and 605 nm,
respectively. For the detection and quantitation of floating syncytia
in the culture media, culture medium was collected, and the floating
syncytia were collected by a cytospin technique. Two hundred µl of
culture medium were collected and centrifuged using a cytospin
(Cytospin 2; Shandon, Inc., Cheshire, United Kingdom) at 1000
rpm for 10 min. The total number of centrifuged multinucleated syncytia
was counted, and data were expressed as the number of syncytia/200 µl
of medium.
Measurement of LDH Activity.
LDH activity in the culture medium was measured as described previously
(26)
. LDH activity after lysis of the cells with 375
µM digitonin was taken as maximal LDH release
(26)
.
Multiparameter Digitized Fluorescent Microscopy.
Experiments measuring mitochondrial membrane potential, oxidative
stress, and cytosolic free calcium were performed using a
multiparameter digitized fluorescent microscopy system
(23)
. The microscope is an inverted phase/fluorescence
microscope (Axiovert 35M; Carl Zeiss, Inc., Thornwood, NY) equipped
with a thermostat-controlled, heated stage. The software for the system
was the Metafluor quantitative fluorescence software package from
Universal Imaging Corp. (West Chester, PA). Excitation light was
provided by a 100-W mercury vapor lamp or a 75-W xenon lamp and passed
through an interference and neutral density filter wheel assembly
(Eastern Microscope, Research Triangle Park, NC) to select wavelength
and intensity under computer control. A cooled charged coupling device
camera (cooled-CCD camera, model KAF-1400; Photometrics, Tucson, AZ)
collected fluorescent images that were fed to a computer (Data Store
486/50E). A 1316 x 1316 pixel area was digitized with
16-bit resolution using video acquisition for frame averaging,
background subtraction and storage on a hard disk.
Measurement of Mitochondrial Membrane Potential.
The mitochondrial membrane potential was monitored using the membrane
potential-sensitive dye TMRM and digitized fluorescent microscopy
(23)
. TMRM is a fluorescent lipophilic cation that
electrophoretically redistributes across the mitochondrial membrane
according to the mitochondrial membrane potential (27)
.
Cells were loaded with TMRM by incubation in Krebs’-Ringer-HEPES
buffer (115 mM NaCl, 1 mM
KH2PO4, 2 mM
CaCl2, 5 mM KCl, 1.2 mM
MgSO4, and 25 mM sodium-HEPES buffer,
pH 7.4; Ref. 23
) containing 50 nM TMRM for 15
min at 37°C. TMRM fluorescence was observed using a 546-nm excitation
filter, a 580-nm dichroic reflector, and a 590-nm long-pass filter.
Cellular fluorescence was quantitated by multiplying the average
fluorescence intensity in the cells or syncytia by the number of pixels
above background using a threshold of zero.
Detection of Cyt-c Release from Mitochondria.
Release of Cyt-c from cells was detected using both morphological and
cell fractionation approaches. The expression vector pE-GFP-Cyt-c (a
gift of Dr. Anna-Liisa Nieminen, Case Western Reserve University)
encodes a polypeptide that contains GFP fused to the COOH terminus of
Cyt-c (28)
. Hep3B cells (70% subconfluent) were
transiently transfected using Lipofectamine (Life Technologies, Inc.)
as described above. One ml of a mixture containing 10 µg of
Lipofectamine and 1 µg of pE-GFP-Cyt-c DNA plasmid, or 1 ml of a
mixture containing 10 µg of Lipofectamine and 1 µg of pE-GFP-Cyt-c
plus 1 µg of pCR3.1-GALV-FMG, was used for the transfection. Confocal
microscopy of transfected cells was performed using a laser scanning
confocal microscope (LSM 510; Carl Zeiss, Inc., Thornwood, NY). GFP
fluorescence was imaged using excitation and emission wavelengths of
488 and 505 nm, respectively.
Cytosolic extracts from FMG-transfected or untransfected control cells were obtained as described by Leist et al. (29) . At the desired time points, the culture medium was exchanged with premeabilization buffer (210 mM D-mannitol, 70 mM sucrose, 10 mM HEPES, 5 mM succinate, 0.2 mM EGTA, 0.15% BSA, and 80 µg/ml digitonin, pH 7.2). The permeabilization buffer was removed and centrifuged for 10 min at 13,000 x g. Protein from the supernatant was separated on 14% polyacrylamide gels and electroblotted to nitrocellulose. The membrane was blocked with 1% w/v skim milk in 20 mM Tris, 0.5 M NaCl, 0.05% Tween 20 (pH 7.0) for 1 h and then incubated for 1 h with a 1:1000 dilution of mouse anti-Cyt-c (PharMingen, San Diego, CA). After washing, membranes were incubated for 1 h with peroxidase-conjugated goat antimouse IgG (Biosource, Camarillo, CA) and washed again. Bound antibody was visualized using chemiluminescent substrate (ECL; Amersham, Arlington Heights, IL) and exposed to Kodak X-OMAT film.
Measurement of Cellular ATP Level.
Cellular ATP levels were quantitated by the luciferin/luciferase assay
as described previously (30)
. Cells in the culture dished
were rapidly quenched by the addition of 3%
HClO4 at 4°C. The supernatant was neutralized
with 1.0 mol/l KOH, and the precipitate was removed by centrifugation
(7500 x g). ATP was measured following the
directions of the manufacturer of the commercial assay kit.
Bioluminescence was quantitated in a luminometer (TD-20/20; Turner
Designs, Sunnyvale, CA).
Materials and Reagents.
DAPI, propidium iodide, TMRM, fura-2, dihydroethidium,
bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic
acid tetra-(acetoxymethyl)-ester were from Molecular Probes, Inc.
(Eugene, OR). Z-VAD-fmk was obtained from Enzyme Systems Products
(Livermore, CA). Fructose, NaCl,
KH2PO4,
CaCl2, KCl, MgSO4, and
sodium-HEPES were from Sigma Chemical Co. (St. Louis, MO).
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RESULTS |
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20 (Fig. 1A)
. These syncytia increased in size over time, reaching a
maximum of >100 nuclei/syncytium at 72 h after the transfection
(Fig. 1B)
. To quantitate the formation of syncytia, we
calculated the "fusion index" (the percentage of the nuclei in
syncytia versus total nuclei in the microscopic field). The
fusion index was maximal at 72 h after transfection with a value
of 18.2 ± 3.2% (Fig. 1C)
. Syncytia were
never observed in cells transfected with the empty plasmid. Thus,
GALV-FMG transfection in a hepatocellular carcinoma cell line does
result in significant syncytia formation.
|
. The number of propidium iodide-positive syncytia in the
FMG-transfected groups began to increase 3 days after the transfection
and increased to 50% of total syncytia at 5 days (Fig. 2B)
.
In contrast, propidium iodide-positive nuclei were not observed in the
two control groups (untransfected and empty plasmid-transfected cells).
We noted that syncytia frequently detached from the substratum,
resulting in an underestimation of syncytia formation and cell death.
Therefore, we also quantitated the number of detached syncytia in the
culture media. The appearance of floating syncytia also increased 3
days after transfection and reached a maximum at 5 days (Fig. 2C)
. To more fully quantitate cell death, LDH activity in
the culture medium was measured. LDH activity increased in the same
time course as the increase in the number of propidium iodide-positive
syncytia and reached a maximum of 70% at 5 days after the
transfection (Fig. 2D)
. These data demonstrate that
GALV-FMG-induced syncytia result in cell death in the majority of Hep3B
cells. However, the onset of cell death is delayed as compared with
syncytia formation. These data suggest that the act of cell fusion
alone is not lethal but requires additional cellular mechanisms.
|
is a representative photomicrograph of Hep3B cells loaded
with TMRM. The TMRM fluorescence in the syncytia was significantly
decreased as compared with the unfused cells, suggesting mitochondrial
depolarization. Measurements of TMRM fluorescent intensity in syncytia
demonstrated that the mitochondrial membrane potential was 15% of
control cells at all observation periods (Fig. 3B)
. Thus,
syncytia formation appears to result in early mitochondrial
dysfunction.
|
. Cyt-c-GFP
displayed a punctate pattern of fluorescence that was indistinguishable
from that of TMRM in the control cells and single cells in the
transfected cultures. Overlay images of Cyt-c-GFP with TMRM
demonstrated that the transfected protein was in the mitochondria. In
the syncytia induced by cotransfection of Cyt-c-GFP with GALV-FMG,
Cyt-c-GFP displayed a diffuse pattern of fluorescence, indicating a
redistribution of Cyt-c-GFP from mitochondria to the cytoplasm.
Biochemical cell fractionation studies also demonstrated Cyt-c release
into the cytosol, as assessed by immunoblot analysis (see Fig. 5D
). These results suggest that Cyt-c-GFP was released from
mitochondria during multinucleated syncytia formation.
|
, the increase in the number of propidium iodide-positive
syncytia (Fig. 4B)
, or LDH release (Fig. 4C)
induced by FMG transfection. Thus, as assessed by complementary
approaches, loss of syncytial viability occurred by a
caspase-independent mechanism.
|
. GALV-FMG
transfection reduced the total cellular ATP level to 20% of
untransfected cells. High concentrations of fructose have been shown to
prevent ATP depletion in hepatocytes during mitochondrial dysfunction
by enhancing glycolytic generation of ATP (32)
. Therefore,
we determined the effects of fructose on FMG-induced ATP depletion and
cell death. Fructose (20 mM) effectively
attenuated the decrease in cellular ATP level (Fig. 5A)
and reduced the number of propidium iodide-positive
syncytia by 65% at 72 h (Fig. 5B)
. Moreover, fructose
also effectively reduced LDH release into the media (Fig. 5C)
. Fructose effectively attenuated the loss of
mitochondrial membrane potential (Fig. 5D)
and Cyt-c release
from mitochondria into cytosol (Fig. 5E)
. Thus, fructose
protects against the death of multinucleated syncytia by maintaining
cellular ATP concentrations above the lethal threshold. |
DISCUSSION |
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7%,
as assessed by expression of a GFP construct. This significant
bystander effect of GALV-FMG has been observed in other transfected
cells (9)
. Comparative data suggest that the bystander
killing of untransfected cells after GALV-FMG is at least 1 log greater
than the herpes simplex virus thymidine kinase/ganciclovir model
(9)
. Collectively, these data suggest that GALV-FMG would
appear to be a promising construct for gene therapy of hepatocellular
carcinoma cells. Information regarding the mechanisms of cell death in the syncytia is important because cancers are often resistant to many forms of cell death (e.g., overexpression of antiapoptotic proteins, loss of cell cycle checkpoint functions, and others). Several observations suggest that the multinucleated syncytia die by a necrotic process: (a) the syncytia developed lysis of the plasma membrane, a cardinal feature of necrosis, as manifest by uptake of propidium iodide and leakage of LDH into the media; (b) the nuclear morphology was unchanged during loss of viability, an observation consistent with cell death by necrosis but not apoptosis, where chromatin condensation and nuclear fragmentation are morphological hallmarks; (c) caspase inhibitors, which block cell death by apoptosis, did not prevent cell killing after GALV-FMG transduction; and (d) we have shown in other studies that apoptotic DNA cleavage into multiples of oligonucleosomes does not occur in these syncytia (9) . Cell death by necrosis may be advantageous for gene therapy because necrosis elicits an inflammatory response, further enhancing the bystander effect by inflammatory/immune-mediated mechanisms (22) .
Mitochondrial dysfunction with loss of oxidative phosphorylation resulted in a bioenergetic form of syncytial cell death. This conclusion is based on our observation of early and significant mitochondrial depolarization in the syncytia, loss of cellular ATP, and protection against cell death by increasing cellular ATP with the glycolytic substrate fructose. The mitochondrial membrane potential is a critical, if not the predominant mechanism, driving the proton motive force responsible for oxidative phosphorylation. Loss of the mitochondrial membrane potential would therefore be predictive of impairment in oxidative phosphorylation (16 , 20) . In support of this concept, we demonstrated loss of cellular ATP after transfection of GALV-FMG. Moreover, high concentrations of fructose used to generate ATP glycolytically increased ATP to 60% of control values, an observationconsistent with previous studies in anoxic liver (32) . This increase in ATP by fructose appeared to be sufficient to inhibit cell death.
The mitochondrial dysfunction in the syncytia was associated with release of Cyt-c from the intermembrane space of mitochondria into the cytosol, indicating structural alterations in the outer mitochondrial membrane. This structural dysfunction of mitochondria is a feature of cell death either by necrosis or apoptosis (15) . Interestingly, fructose attenuated the release of Cyt-c from mitochondria into the cytosol. This observation suggests that the initial cellular insult after GALV-FMG syncytia formation is failure to synthesize ATP. Loss of cellular ATP then appears to further potentiate mitochondrial dysfunction, causing Cyt-c release. These data suggest that Cyt-c release can be a secondary process in cell death and need not be implicated as primary event. GALV-FMG may cause mitochondrial dysfunction by being mistargeted to mitochondria in the syncytia, causing failure of oxidative phosphorylation. Unfortunately, in the absence of anti-GALV-FMG antiserum, we were unable to test this possibility. Nonetheless, because mammalian cells cannot survive long-term without ATP, cells would not be expected to develop resistance to this form of cell death, further supporting the potential utility of GALV-FMG gene therapy for cancer.
Because hepatocellular carcinomas frequently have mutations in p53 (33, 34, 35) , we used the p53 null Hep3B cells for these studies (36) . GALV-FMG-induced syncytia formation and cell death were efficient in this cell type. These data demonstrate that GALV-FMG cell killing is p53 independent, and therefore, a viable gene therapy approach for cancers, such as hepatocellular carcinoma with p53 mutations.
In conclusion, transduction of GALV-FMG results in efficient formation
of giant syncytia, causing death of hepatocellular carcinoma cells. The
incorporation of adjacent, nontransduced cells into the syncytia is
responsible for a significant bystander effect. It would be anticipated
that because cell death occurs by necrosis and would elicit an
inflammatory response in vivo, the bystander effect would be
accentuated in vivo. Gene therapy with GALV-FMG is,
therefore, a promising approach for the treatment of hepatocellular
carcinoma. Its efficiency in inducing tumor regression in xenografts is
currently been evaluated using plasmid and viral vectors for gene
delivery. Cancer-specific expression constructs using the
-fetoprotein promoter also should be engineered and tested before
its therapeutic promise can be further assessed in humans.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Grant DK41876 from
the NIH, the Mayo Foundation, a grant from the Kanae Foundation for
Life and Socio-Medical Science, and Grant R01 CA85931 from the National
Cancer Institute (to R. V. and A. B.). 
2 To whom requests for reprints should be
addressed, at Mayo Medical School, Clinic, and Foundation, 200 First
Street SW, Rochester, MN 55905. Phone: (507) 284-0686; Fax:
(507) 284-0762; E-mail: gores.gregory{at}mayo.edu
3 The abbreviations used are: FMG, fusogenic
membrane glycoprotein; EMEM, Eagle’s MEM; GALV, Gibbon ape leukemia
virus; DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; LDH,
lactate dehydrogenase; TMRM, tetramethyl rhodamine methyl ester; GFP,
green fluorescent protein; Cyt-c, cytochrome c.
Received 6/26/00. Accepted 9/12/00.
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REFERENCES |
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) or empty plasmid (
) or untransfected cells
(control, 
). The numbers of cells and nuclei were determined, and a
fusion index was calculated as described in "Materials and
Methods." Bars, SD.
