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Experimental Therapeutics |
Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 [Y-i. Y., M. S., H. H., T. R., T. H., S. T., K. Sh., K. Su.]; Department of Immunology, Medical Institute of Bioregulation, Fukuoka 812-8582 [R. M.]; and Department of Nutrition and Physiological Chemistry, Osaka University Medical School, Osaka 565-0871 [J-i. M.], Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and significantly inhibited the growth not only
of HCC into which the mIL-12 vector had been directly
transferred, but also of the distant HCC. In addition, intratumoral
administration of the mIL-12 vector inhibited spontaneous
lung metastasis and delayed establishment of HCC injected 3 days after
mIL-12 gene therapy. The IL-12 gene therapy
induced more lymphocyte infiltration by NK cells, CD3+
cells, and Mac-1 positive cells into the tumor and reduced the number
of microvessels. Therefore, more terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling-positive tumor cells were
found. These results demonstrate that gene therapy for HCC by
electroporation in vivo using IL-12 is very
efficient and is thus promising for further clinical trial. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Methods of gene transfer for either therapeutic or experimental purposes rely largely on viral vectors. However, serious concerns have been voiced about the use of viral vectors, especially when clinical trials are involved. Retroviral vectors usually suffer from low titers, oncogenic potential, and the requirement of active cell division for integration. Adenoviral vectors can recover in high titers and transfer genes to both dividing and nondividing cells, but host immunogenicity would prevent their repeated use (4) . Electroporation, a rather unfamiliar nonviral gene transfer method in vivo, has been widely used to introduce DNA into various types of cells in vitro (5 , 6) . Recently, gene transfer by electroporation in vivo was found to be effective at introducing DNA into mouse skin (7) , mouse muscle (8) , chick embryos (9) , hearts of chick embryos (10) , rat liver (11) , murine melanoma (12) , and rat glioblastoma (13) . Like other nonviral methods, electroporation has a variety of advantages over viral vectors, because all of the tissues and cells could become targets, in theory. In addition, its handling is easy and quickly completed; no immunogenicity is expected, repeated administration of DNA is possible, no DNA size constraints are imposed, and no specialized process for DNA construction is required. However, electroporation is recognized as having disadvantages in common with other nonviral methods, i.e., gene expression is transient, and gene transfer efficiency is still low in comparison with that of viral vectors. To more closely consider the clinical application of gene therapy for HCC, we used an in vivo electroporation method of gene transfer into HCC. First, we examined the optimal conditions of electric pulses for gene transfer into HCC by luciferase activity to accelerate the efficacy of gene transfer.
IL-12, a bimolecular glycoprotein that consists of a Mr 35,000 and 40,000 subunit, was originally identified as a factor that stimulates natural killer cells (14 , 15) , promotes maturation of CTLs (16) , and induces antiangiogenic effects (17) . It has recently been demonstrated that local or systemic treatment with recombinant IL-12 protein mediates profound antitumor effects in vivo, causing regression of established tumors and their distant metastases (18) . However, systemic administration of IL-12 protein has caused dose-dependent toxicity in mice (19) and in human trials (20) . Alternative approaches, including gene therapy, for the delivery of IL-12 have been pursued. These include injection of genetically engineered fibroblasts for the secretion of IL-12 in an orthotopic melanoma model, direct transfer of IL-12 cDNA via gene gun-mediated transfection of skin tissue overlaying s.c. melanomas, direct intratumoral injection of adenovirus that expresses IL-12 in breast, colon, and prostate cancer models, and intradermal injection of IL-12 cDNA and vaccination using irradiated IL-12-transfected tumor cells in a metastatic colon cancer model (21, 22, 23, 24, 25, 26, 27, 28) . These models suggest that IL-12 gene therapy results not only in tumor regression but also in the suppression of its metastases.
In the present study, we demonstrated some effects of IL-12 gene therapy on the growth of s.c. HCC into which mIL-12 vector was directly transferred and untreated distant HCC using electroporation, and on its spontaneous lung metastasis in a s.c. HCC model in mice.
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MATERIALS AND METHODS |
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Plasmid DNA.
The luciferase gene was isolated from the pGL2 promoter
vector (Promega, Madison, WI) by HindIII and
BamHI digestion. PcLuc and pActLuc
were constructed by cloning the luciferase gene at HindIII
and BamHI sites of pcDNA3. Mouse IL-12
expression plasmid, designated pCAGGS-mIL-12, was
constructed as follows: Both mouse IL-12 p35 and
p40 cDNAs were inserted into the EcoRI site of
pCAGGS expression vector (31)
, resulting
in pCAGGS-p35 and pCAGGS-p40, respectively. The
expression unit for IL-12 p35 included the
cytomegalovirus immediate early enhancer-chicken
ß-actin hybrid promoter. A rabbit ß-globulin poly(A) signal was
excised from pCAGGS-p35 and was inserted downstream of the
IL-12 p40 expression unit of pCAGGS-p40.
Intratumoral DNA Injection and Electroporation.
Mice were anesthetized with pentobarbital sodium. Established s.c. HCC,
the volume of which reached about 0.5 cm3, was
injected with 100 µg of closed circular DNA (pcDNA3Luc,
control pCAGGS, and pCAGGS-mIL-12) at 1.0
µg/µl in saline using an insulin syringe with a 27-gauge needle. A
pair of electrode needles was inserted into the tumor to a depth of 5
mm to encompass the DNA injection sites, and electric pulses were
delivered using an electric pulse generator (CUY-21; BEX Co., Ltd.,
Tokyo, Japan). The shape of the pulse was a square wave,
i.e., the voltage remained constant for the duration of the
pulse. Electrodes consisted of a pair of tungsten needles of 10 mm in
length and 0.4 mm diameter. Pulses, at the rate of one pulse/s, of the
opposite polarity were administered to each DNA injection site.
Luciferase Activity.
Three days after pDNA encoding luciferase was transferred
into HCC by electroporation under various conditions, HCC was resected
under satisfactory diethyl ether anesthesia. Tumors were washed in PBS
solution and placed in a 200-µl Luciferase Cell Culture Lysis agent
(Promega, Madison, WI). The tissue was dissociated until it was
completely dissolved. Twenty µl of each sample were added to 100-µl
substrate and assayed for luciferase activity in a luminometer (LUMAT
LB 9507; EG&G BERTHOLD, Tokyo, Japan). Total protein content for each
sample was determined by the Bradford method using BSA as a standard,
and absorbencies were read at 595 nm on a UV-visible recording
spectrophotometer (Multiscan MS, type 352, version 8; Labsystems,
Helsinki, Finland). Results were normalized to yield luciferase
activity in LU/µg BSA protein.
ELISA of mIL-12 and mIFN-.
Serum samples obtained from the tail vein of mice were assayed for
total mIL-12 (Genzyme, Cambridge, MA) and mIFN- (Biosource
International, Camarillo, CA) using an ELISA kit, according to the
manufacturer’s instructions.
Morphological and Histopathological Analysis of HCC.
In the bilateral s.c. HCC model, control pCAGGS (control
group; n = 5) or pCAGGS-mIL-12
(IL-12 gene therapy group; n = 8)
was transferred only to the right s.c. HCC. For a period of 28 days
after gene therapy, tumor volume was calculated according to the
following formula: V = A x B2
/2
(cm3), where A is the largest diameter
(cm), and B is the smallest diameter (cm). Fourteen days
after gene therapy, another five sections of the bilateral s.c. HCC in
both groups were resected and cut in the middle at the site of the
original tumor inoculation. The tissue was then fixed in 10% buffered
formalin and stained with H&E for histopathological analysis. The ratio
of the viable region of MH134 cells in each tumor at five random areas
was calculated using an image analyzer (MAC SCOP, Nagano, Japan), and
the overall effect of IL-12 gene therapy was evaluated.
For CD 31 immunocytochemistry, paraffin-embedded sections that were 3 µm in thickness of bilateral s.c. HCC in both groups were incubated with 0.1% trypsin in water for 10–15 min at 37°C, then incubated overnight at 4°C with 0.5 µg/ml rat monoclonal antiserum against mouse CD31 (PharMingen, San Diego, CA). Primary antibody was detected with streptavidin-biotin using a Vectastain kit, according to the manufacturer’s instructions (Vector Laboratories, Burlingame, CA). CD31-positive microvessels within or surrounding the HCC were counted. Because microvessels are distributed heterogeneously throughout the tumor, the sections were examined at low magnifications (x40 and x100) to identify the greatest vascular area of the tumor (hot spots), according to the method of Weidner et al. (32 , 33) . Within these areas, a minimum of 10 fields at x400 magnification (total area 1.56 mm2 ) was examined. The average of all of the fields examined within the hot spot area (MVDmean) was then recorded and expressed as counts/mm2 .
TUNEL staining was performed using an Apop Taq in situ apoptosis detection kit (Oncor Inc., Gaithersburg, MD). The sections of bilateral s.c. HCC in both groups were deparaffined and digested in 20 µg/ml proteinase K (Nakarai Tasque, Inc., Kyoto, Japan) for 30 min at room temperature. Then, Apop Taq equilibration buffer, working strength terminal deoxynucleotidyl transferase reaction mixture, working strength stop/wash buffer, and anti-digoxigenin-dUTP were applied sequentially. After staining with diaminobenzidine, the slides were counterstained with hematoxylin, dehydrated, and mounted. An apoptosis index (percentage) was calculated for each of the five samples at five random areas as the proportion of TUNEL-positive MH134 cells with respect to the total number of MH134 cells evaluated multiplied by 100. Only nonnecrotic MH134 cells were quantified to determine the apoptotic index. Approximately 2000 MH134 cells were evaluated for each sample.
Flow Cytometric Analysis of TILs.
The TILs from the mice with bilateral s.c. HCC at the right flank into
which pCAGGS-mIL-12 had been transferred or into which
pCAGGS had been transferred at 14 days after gene therapy
were processed as described earlier (34)
. In brief, the
specimens were minced with scalpels into pieces smaller than 1
mm3 and underwent enzymatic treatment for 2 h. The cell suspensions were centrifuged on differential Ficoll-Hypaque
gradients (Lymphocyte Separation Medium, ICN Biomedical Inc.,
Cleveland, OH; Ref. 35
), and the TILs were
collected from the lower interface, washed with HBSS three times, and
checked for viability and cell counts with the aid of trypan blue dye.
FACS analysis was performed on a FACS Caliber (Becton Dickinson, San
Jose, CA). On the basis of staining with various leukocyte-specific
antibodies and forward- and side-scatter analyses, CD45 positive TILs
could be gated into different groups of infiltrating cell populations.
To block the nonspecific FcR binding of labeled antibodies, 10 µl
of undiluted culture supernatant of 2.4G2 (rat antimouse FcR
monoclonal antibody) was added to the first incubation. All of the
antibodies used in this study were obtained from PharMingen. The
biotin-conjugated anti-CD45 antibody 30F 11.1 (rat IgG2b
) was used,
and streptavidin allophycocyanin was used for biotin labeling.
Phycoerythrin-labeled anti-CD3
antibody 145–2C11 (Armenian hamster
IgG), anti-CD4 antibody H129.19 (rat IgG2a), anti-CD8 antibody 53–6.7
(rat IgG2a), anti-NK1.1 antibody PK136 (mouse IgG2a), and anti-CD
11C antibody HL3 (Armenian hamster IgG
) were used, as were
FITC-labeled anti-CD3 antibody 145–2C11 (Armenian hamster IgG),
anti-B220 antibody (rat IgG2a), and anti-Mac-1 antibody M1/70.15
(rat IgG2b). Propidium iodide staining was used to exclude dead cells.
Assay System for CTL Responses.
In vitro sensitization and cytotoxicity assay were
essentially the same as described previously (36)
. In
brief, 5 x 106 splenic cells from
the mice with s.c. HCC at the right flank into which
pCAGGS-mIL-12 had been transferred or into which
pCAGGS had been transferred at 7 days after gene therapy
were sensitized in vitro to 1 x 105 mitomycin C-treated tumor cells in a 2-ml
volume in 24-well culture plates. Effector cells generated 5 days after
culturing were assayed on the corresponding tumor target cells by
51Cr release assay. The percentage of specific
lysis was calculated as described previously (37)
, and SE
were excluded from the data for the sake of simplicity because they
were consistently <5%. X5563 plasmacytoma derived from C3H strain was
used to confirm the tumor specificity of cytotoxicity.
Distant Site Challenge with Parental Tumor Cells.
Mice with s.c. HCC at the right flank into which
pCAGGS-mIL-12 or pCAGGS had been
transferred were challenged with a tumorigenic dose of MH134 parental
tumor cells (2.5 x 106
cells/mouse) that were implanted into the opposite flank at 3 days
after intratumoral pDNA transfer. Tumor formation was macroscopically
monitored; we defined the day of "distant tumor formation" as the
day upon which the tumor volume reached 0.5 cm3.
Spontaneous Lung Metastasis.
A few mice bearing right s.c. HCC 14 days after gene therapy were
anesthetized with diethyl ether, and the tumor was removed through a
10-mm skin incision and careful dissection. A few mice that eventually
displayed local tumor relapse were excluded from the experiment. After
28 days (14 days after s.c. HCC resection), lungs were removed
bilaterally. Tumor formation was macroscopically monitored. For further
evaluation of spontaneous lung metastasis, the tissue was then fixed in
10% buffered formalin and stained with H&E.
Statistical Analysis.
Statistical evaluations of numerical variables in both groups were
performed using the Mann-Whitney U test, and evaluations of
the qualitative variables were performed using Fisher’s exact
probability test. Differences in tumor growth were statistically
analyzed using the repeated measures ANOVA test. Differences in time to
tumor development challenged at the distant site were statistically
analyzed using the Breslow modification of the Wilcoxon test.
Significance was defined as P < 0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The optimal voltage level of the electric pulses was determined when
other conditions were fixed as follows: eight electric pulses, 50 ms in
duration (Fig. 1
A). The results indicate an increase in mean luciferase
activity with an increase in voltage up to 150 V. HCC that was
subjected to 50 V expressed a mean luciferase activity level of
5.2 ± 3.8 x 103
LU/µg BSA, which increased to 7.0 ± 1.0 x 105 LU/µg BSA in HCC that was subjected to 150
V (P < 0.01; mean ± SE).
However, the mean luciferase activity in HCC that was subjected to 200
V decreased to 1.1 ± 0.25 x 105 LU/µg BSA (P < 0.01).
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B). The results indicate an increase in mean luciferase
activity with an increase in duration up to 50 ms. But when the
duration of the pulse exceeded 50 ms, the mean luciferase level
decreased. A statistical difference was seen between luciferase levels
of HCCs that were subjected to 50 ms (7.0 ± 1.0 x 105 LU/µg BSA) and those subjected to 70 ms
(2.4 ± 0.6 x 105
LU/µg BSA; P = 0.02).
The optimal number of shocks was determined when other conditions were
fixed as follows: 50 ms in duration at 150 V (Fig. 1
C). The
results indicate an increase in mean luciferase activity that occurred
with an increase in the number of shocks up to 10. No statistical
difference was seen between HCCs that were subjected to 8 shocks (7.0
± 1.0 x 105 LU/µg BSA) and those subjected to
10 shocks (8.0 ± 0.8 x 105 LU/µg BSA).
The optimal condition for electroporation gene transfer was shown to occur at 150 V, 50 ms in duration, and 10 shocks.
Time Course of mIL-12 and mIFN-.
The serum mIL-12 level increased gradually after
pCAGGS-mIL-12 transfer into HCC and peaked 5 days after
electroporation (4.4 ± 0.4 ng/ml). Thereafter, serum
mIL-12 levels decreased gradually to approximately 57% of the maximum
value (2.5 ± 0.4 ng/ml) by 1 month after
electroporation (Fig. 2
A). The serum mIFN- level also increased gradually and
peaked 7 days after electroporation (101.6 ± 4.2
pg/ml). This represented a few days of delay, as compared with the
elevation of serum mIL-12 (Fig. 2
B). On the other hand, in
the mice with HCC-transferred pCAGGC, no elevation of mIL-12
was found, and mIFN- was not detectable.
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). After 14 days, the volume of HCC-transferred control pCAGGCrapidly increased to more than 35-fold that of the amount on the
day when the gene was transferred. However, the increase in volume of
HCC-transferred pCAGGS-mIL-12 reduced to about 5-fold that
amount. Suppressed growth was also recognized in the untreated distant
left flank HCC that was inoculated and established at the same time.
This finding was statistically significant when compared with that of
the distant HCC in the control group (P < 0.01; ANOVA; Fig. 3
).
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A, B, and C).
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A, B, and C).
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A, B, and C).
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shows that more tumor-infiltrating NK cells, CD3+
T cells, and Mac-1 positive cells in the
pCAGGS-mIL-12-transferred HCC
(21.2/cm3-tumor,
14.5/cm3-tumor, and
20.3/cm3-tumor) and in the distant HCC at the
opposite site (8.9/cm3-tumor,
7.8/cm3-tumor, and
10.1/cm3-tumor), respectively, were found
when compared with the pCAGGS-transferred HCC
(0.8/cm3-tumor,
1.0/cm3-tumor, and
3.4/cm3-tumor). The increases of NK cells (IL-12:
IL-12 distant: pCAGGS, 26.5: 11.1: 1) and
CD3+ T cells (IL-12: IL-12 distant:
pCAGGS, 14.5: 7.8: 1) were dominant. A
CD3+ T-cell increase was found in both the
CD8+ T-cell population and in the
CD4+ T-cell population.
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. Additional experiments revealed that such augmented cytotoxic
responses are tumor-specific (Table 2)
.
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).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In in vivo electroporation, square pulses should be used,
because they are superior in inducing a higher level of gene expression
than are exponential decay pulses. Unless the tissues or cells are
irreversibly damaged, gene transfection efficiency is roughly
proportional to the amount of heat generated (39)
. In the
present study, optimal conditions for gene transfer into HCC appear to
support this hypothesis, at least in part. Increasing the voltage to
more than 150 V, the time constant to more than 50 ms, or the number of
shocks to more than 10 resulted in a decrease of luciferase activity
transferred into the HCC. In a study of gene transfer into mouse
testis, Muramatsu et al. (40)
also reported
that decreasing the voltage from 100 V to 25 V resulted in a remarkable
reduction of chloramphenicol acetyltransferase activity, whereas
increasing the time constant from 10 to 50 ms at the same 25 V,
chloramphenicol acetyltransferase activity recovered almost to the
level of activity observed at 100 V with 10 ms of exposure. However, in
our study, decreasing the voltage from 150 to 100 or to 50 and
concomitantly increasing the pulse duration or the number of shocks
showed that luciferase activity in HCC could not recover to that found
at 150 V (data not shown). One of the main reasons for this phenomenon
would be that the cell density of the established HCC in
vivo would be higher than that of other normal solid organs such
as the liver. Furthermore, resistance to electric pulses was increased
(HCC, 1.62 ± 0.09 k
; liver, 0.42 ± 0.04 k; n = 8; P < 0.01) in our mouse model. Hence, the voltage necessary
for the poration of cell membranes would be higher than 150 V. We
examined the effects of different electrode voltage, time constants,
and number of shocks, but we fixed other parameters, such as frequency
of pulse (one time/s), DNA concentration (1 µg/µl), volume of
injection fluid (100 µl), and solution type (0.85% NaCl; Ref.
41
, 42
). By optimizing these parameters, the efficiency of
gene transfer might be improved.
IL-12 has been shown to enhance the cytolytic activity of NK cells and
CTLs. IL-12 is also a very strong inducer of IFN- and appears to be
a major determinant in the development of the Th1 immune response
(43)
. Although previous studies (18
, 19) on
IL-12 protein therapy have generally shown that IL-12 is highly
efficacious against a wide range of murine tumors, the present report
is the first to describe the efficiency of IL-12 against this MH134
murine tumor model. When compared with IL-12, another well-known
cytokine, IL-12 protein was found to be more effective and yet less
toxic (44
, 45)
. In similar studies, IL-12 also appeared to
be more potent than IFN- (46)
. However, in the first
clinical trial, IL-12 protein therapy resulted in the death of 2
patients and led to severe toxic effects in 15 others (20
, 47)
. The absence of predosing was suggested to be a cause of
this toxicity (47)
. The present study also tested
IL-12 gene therapy strategy with the capacity to deliver
easily regulated levels of transgenic protein at the site of the tumor.
Previous studies (24)
using an ex vivo gene
transfer approach showed that murine tumor cells transduced by
retroviral vectors containing an IL-12 cDNA were able to
vaccinate mice successfully against tumor challenges. Moreover,
injection of murine fibroblasts that were transfected with an
IL-12 cDNA expression vector at the site of an intradermal
tumor resulted in tumor regression (25)
. By using the
direct transfer of IL-12 cDNA into skin or tumors, local
IL-12 gene therapy resulted in the eradication of
established murine tumors and their metastases, leading to the
generation of tumor-specific immunological memory (23
, 24
, 27
, 28)
.
In the present study, it was shown that administration of
mIL-12 cDNA into HCC mediated by the electroporation method
significantly inhibited the growth of not only mIL-12
directly transferred HCC but also the growth of untreated distant HCC.
In addition, spontaneous lung metastasis was inhibited, and the
establishment of HCC injected 3 days after IL-12 therapy was
inhibited or delayed. These findings support the results of previous
reports. Elevations of IL-12 and IFN- serum levels were observed in
our model. None of the mice subjected to IL-12 gene therapy
died during the experiments. In a recent report, systemic elevation of
IL-12 was shown to be more important than local IL-12 elevation in the
cure of established tumors and spontaneous metastases
(48)
. Thus, the gradual elevation of systemic IL-12 by
means of intratumoral gene transfer by electroporation would be an
ideal model for IL-12 cancer therapy. According to histological
analysis by H&E staining, more lymphocytic infiltration and a smaller
distribution of viable cancer cells were observed not only in the
section of pCAGGS-mIL-12-transferred HCC but also in the
distant HCC, as compared with those of the control group. In this mice
model, the acceleration of the infiltration of lymphocytes into the
distant tumor in the IL-12 gene therapy group was because of
the systemic mIL-12 elevation and the activation of the immune system.
The lymphocytic infiltration of NK cells, CD3+ T
cells (CD8+ T cells and
CD4+ T cells), and Mac-1 positive cells revealed
by FACS analysis of TILs was considered to induce HCC apoptosis. Thus,
more TUNEL-positive cells were found in the
pCAGGS-mIL-12-transferred HCC and the distant HCC than were
found in the control group. Despite the scattered pattern of
TUNEL-positive cells that could not explain the massive area of
necrosis revealed by H&E staining, the antitumor effect of
IL-12 gene therapy for this MH134 mice model was considered
to depend largely or partly on the antiangiogenetic effect revealed by
the analysis of MVDmean. Because of local and
systemic elevation of IL-12 and IFN-, which had antiangiogenetic
effects, fewer microvessels stained by antimouse CD31 antibody were
found in the sections of HCC-transferred pCAGGS-mIL-12 and
distant HCC than were found in the control group. Furthermore, the
suppression of the establishment of s.c. HCC of the delayed injection
on mice with pCAGGS-mIL-12-transferred HCC was recognized.
This result was possibly attributable to the activation of macrophages,
NK cells, and CTLs by systemic IL-12. In addition, the ratio of the
formation of lung metastasis 14 days after resection of the
pCAGGS-mIL-12-transferred HCC was smaller than that of the
pCAGGS-transferred HCC. This phenomenon might be explained
by the fact that the low number of microvessels rendered the MH134
cells more difficult to circulate from the primary HCC-transferred
pCAGGS-mIL-12. In addition, it would be more difficult to
attach and enlarge a tumor in the lung because of the acceleration of
antitumor immunity of macrophages, NK cells, and CTLs with the
elevation of systemic IL-12 and IFN-. But further experimentation is
needed to determine the dominant mechanism of the antitumor effect of
IL-12 gene therapy in this MH134 tumor model using C3H mice:
the activation of the immune system or the antiangiogenic effect.
Because this is the first report of IL-12 gene therapy for HCC and the first use of this MH134 murine tumor model, the comparison of efficiency of the gene transfer and gene therapy against the previously established methods such as viral vectors is impossible. Serious concerns have been voiced about the use of viral vectors, especially when clinical trials are involved. To more closely consider the clinical application of gene therapy for HCC, the in vivo electroporation method of gene transfer has more critical advantages over viral vectors. However, electroporation has certain disadvantages in common with the viral vector method, i.e., gene transfer efficiency is still low, and gene expression is transient. Harrison et al. reported that in vivo electroporation was approximately equivalent to an adenovirus dose of 106 transduction units but was less effective than a 107 dosage evaluated by the percentage of heart tissue of chick embryos expressing green fluorescent protein (GFP) (10) . But the efficiency of gene transfer mediated by electroporation should be accelerated by the examination of the optimal conditions of electric pulses, the type of electrode, and the plasmid concentration. In a recent report, Goto et al. (49) described the efficiency of repeated electro-gene therapy for s.c. tumors using herpes simplex virus thymidine kinase gene, and they reported that low efficiency of gene transfer should be dissolved by repeated use. But additional experiments are required regarding the comparison between electroporation-mediated IL-12 gene therapy and viral vectors against this MH134 tumor model.
In summary, electroporation-mediated IL-12 gene therapy for HCC was directly effective for mIL-12-transferred HCC but was also effective for distant HCC. Furthermore, the present study showed significant inhibition of lung metastasis and delayed establishment of HCC. Therefore, our data suggest that in vivo electroporation-mediated IL-12 gene therapy for HCC may be a promising treatment modality for advanced HCC with intrahepatic metastases in humans.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Department of Surgery and Science, Graduate School of
Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku,
Fukuoka 812-8582, Japan. Phone: 81-92-642-5469; Fax: 81-92-642-5482;
E-mail: yamashi{at}surg2.med.kyushu-u.ac.jp 
2 The abbreviations used are: HCC, hepatocellular
carcinoma; IL-12, interleukin 12; mIL-12, murine interleukin-12; LU,
light units; MVDmean, mean microvessel density; TUNEL,
terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling;
TIL, tumor-infiltrating lymphocyte; FACS, fluorescence-activated cell
sorter.
Received 1/ 3/00. Accepted 11/21/00.
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 M. N. Torrero, W. G. Henk, and S. Li Regression of High-Grade Malignancy in Mice by Bleomycin and Interleukin-12 Electrochemogenetherapy Clin. Cancer Res., January 1, 2006; 12(1): 257 - 263. [Abstract] [Full Text] [PDF]
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 T. Yamaoka, Y. Yonemitsu, K. Komori, H. Baba, T. Matsumoto, T. Onohara, and Y. Maehara Ex vivo electroporation as a potent new strategy for nonviral gene transfer into autologous vein grafts Am J Physiol Heart Circ Physiol, November 1, 2005; 289(5): H1865 - H1872. [Abstract] [Full Text] [PDF]
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 N. Harada, M. Shimada, S. Okano, T. Suehiro, Y. Soejima, Y. Tomita, and Y. Maehara IL-12 Gene Therapy Is an Effective Therapeutic Strategy for Hepatocellular Carcinoma in Immunosuppressed Mice J. Immunol., December 1, 2004; 173(11): 6635 - 6644. [Abstract] [Full Text] [PDF]
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C.-J. Chang, K.-F. Tai, S. Roffler, and L.-H. Hwang The Immunization Site of Cytokine-Secreting Tumor Cell Vaccines Influences the Trafficking of Tumor-Specific T Lymphocytes and Antitumor Efficacy against Regional Tumors J. Immunol., November 15, 2004; 173(10): 6025 - 6032. [Abstract] [Full Text] [PDF]
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Y.-i. Yamashita, M. Shimada, R. Minagawa, E. Tsujita, N. Harimoto, S. Tanaka, K. Shirabe, J.-i. Miyazaki, and Y. Maehara Muscle-targeted interleukin-12 gene therapy of orthotopic hepatocellular carcinoma in mice using in vivo electrosonoporation Mol. Cancer Ther., September 1, 2004; 3(9): 1177 - 1182. [Abstract] [Full Text] [PDF]
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 F Xue, T Takahara, Y Yata, Y Kuwabara, E Shinno, K Nonome, M Minemura, S Takahara, X Li, E Yamato, et al. Hepatocyte growth factor gene therapy accelerates regeneration in cirrhotic mouse livers after hepatectomy Gut, May 1, 2003; 52(5): 694 - 700. [Abstract] [Full Text]
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 J. Jiang, E. Yamato, and J.-i. Miyazaki Sustained Expression of Fc-Fusion Cytokine Following In Vivo Electroporation and Mouse Strain Differences in Expression Levels J. Biochem., April 1, 2003; 133(4): 423 - 427. [Abstract] [Full Text] [PDF]
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 S. Li, X. Zhang, and X. Xia Regression of Tumor Growth and Induction of Long-Term Antitumor Memory by Interleukin 12 Electro-Gene Therapy J Natl Cancer Inst, May 15, 2002; 94(10): 762 - 768. [Abstract] [Full Text] [PDF]
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K. Mikata, H. Uemura, H. Ohuchi, S. Ohta, Y. Nagashima, and Y. Kubota Inhibition of Growth of Human Prostate Cancer Xenograft by Transfection of p53 Gene: Gene Transfer by Electroporation Mol. Cancer Ther., February 1, 2002; 1(4): 247 - 252. [Abstract] [Full Text] [PDF]
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