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Tumor Biology |
Molecular Oncology Center [P. D. K., Y. H., Ma. K.] and First Department of Biochemistry [L. S-X., Mo. K., F. A., K. N., Y. T., Ma. K.], School of Medicine, Fukuoka University, Fukuoka 814-0180, Japan
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CEA is a highly glycosylated protein of about Mr 180,000 and is widely expressed on the surface of tumor cells of various human tissues as well as on the epithelial cells of normal gastrointestinal tissues and fetal intestine (7) . In normal adult human colon tissues, however, CEA is localized on the luminal surface of the single layer of columnar epithelial cells lining the upper parts of the crypts (8) , so that normal CEA is not directly in contact with blood flow or tissue fluid. On the other hand, in tumor tissues that no longer conform to the single-layer organization by invading through the basement membrane in multicellular arrays, CEA is usually localized at all sides of the cell membranes and is directly facing blood flow or tissue fluid (8) . Hence, tumor CEA can be a useful target molecule for gene therapy using anti-CEA antibody. We have previously prepared an monoclonal antibody clone F11–39, which shows a high affinity to CEA and discriminates CEA in tumor tissues from the other CEA gene family antigens in the normal tissues (9) . A mouse-human chimeric antibody (Ch F11–39) generated from F11–39 has retained the same affinity with that of the parental monoclonal antibody and suppressed tumor growth both in vitro and in vivo when administered with lymphokine-activated killer cells (10 , 11) . Thus, the scFv form of this antibody seems to be applicable to the targeting of retroviral vectors to CEA-expressing tumor cells by expression in the envelope proteins of the virus particles that contain a certain killing or suicidal gene.
The second important issue in this approach is the selection of the gene used for cell killing. NO, one of the smallest biologically active molecules, is a potential toxin for immunological self-defense. The formation of NO from L-arginine in mammalian cells is catalyzed by NOSs including iNOS, endothelial NOS, and brain NOS (12 , 13) . Under physiological conditions, iNOS is absent from mammalian cells, but when cells are activated, it is induced and causes a long-lasting generation of NO, which is associated with a cytotoxic reaction against pathogens and tumor cells (13 , 14) . Previous studies have shown that iNOS transfected into mouse melanoma cells suppressed their tumorigenicity (15) and also lysed bystander murine cells under in vitro and in vivo conditions, which suggested that NO-mediated cell killing does not require transfection of every cell (16) . However, there is no report thus far on the specific-targeted delivery of the iNOS gene against tumor cells.
In the present study, we have generated a bifunctional Moloney murine leukemia virus-based retroviral vector that displays a chimeric envelope protein including an anti-CEA scFv antibody derived from Ch F11–39 and carries the iNOS gene in the genome. The recombinant retrovirus showed a specific delivery of the iNOS gene to human CEA-expressing carcinoma cells and directly killed the infected cells by induction of apoptosis without any additional drugs.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-MEM (Life Technologies, Inc.,
Gaithersburg, MD) supplemented with 10% heat-inactivated fetal bovine
serum, 2 mM glutamine, and penicillin and
streptomycin. The cell cultures were maintained at 37°C in a
humidified atmosphere with 5% CO2.
Plasmid Construction.
The VH and V
cDNA genes were
obtained from the transfectoma Ch F11–39 (10)
by RT-PCR
using oligonucleotide primers corresponding to the 5' and 3' consensus
amino acid sequences of immunoglobulin variable regions, and joined
together using the flexible 14-amino-acid long 212 linker
(18)
by the overlap extension PCR method. The PCR products
thus obtained were cloned into the pT7 blue vector (Novagen, Novagen,
WI). This scFv cDNA was modified at its 5' and 3' ends with
the following PCR primers introducing the unique XhoI and
BstEII sites, respectively (underlined):
5'-TCTCGAGGACATCCAGATGACTCAGTCT-3' and
5'-TGGTGACCTCAGCAGAGACAGTGACCAGA-3'. The modified
scFv cDNA (728 bp, 242 amino acids) was inserted into the
XhoI and BstEII restriction sites of the envelope
expression vector pEnv 20.22 (kindly given by Dr. N. V. Somia,
Salk Institute, La Jolla, CA), which contains a strong
cytomegalovirus immediate-early promoter (19)
, which
yielded the plasmid pscFv-env (Fig. 1)
. This resulted in insertion of the scFv between the sixth and seventh
amino acid of the ecotropic envelope.
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The nucleotide sequences of all of the inserts were confirmed with the ABI Prism 377 DNA sequencing system (PE Applied Biosystem, Foster City, CA).
Reconstruction of Packaging Cells.
To establish a permanent clone of ecotropic packaging cells for
expression of the chimeric envelope displaying anti-CEA scFv antibody,
5 x 105 GP+E-86 cells were
cotransfected with 12 µg of the plasmid pscFv-env and 2 µg of the
hygromycin-B phosphotransferase expression vector pcDNA3.1/Hygro (+)
(Invitrogen, Carlsbad, CA) by the calcium phosphate method. Clones were
selected in the presence of hygromycin B (200 µg/ml; Wako Pure
Chemical, Osaka, Japan) for 10–12 days. The positive clones were
screened by RT-PCR using the primers for anti-CEA scFv (see above) and
by an EPICS flow cytometer (Coulter, Miami, FL) using biotinylated CEA
(20
, 21)
and FITC-conjugated avidin (Vector Laboratories,
Burlingame, CA). The highest positive permanent packaging cell clone
thus obtained was named GPEscFv-env. For control, the GP+E-86 cells
were transfected only with the hygromycin-B phoshphotransferase gene,
selected and designated GPE-hgB.
Production of Viruses.
Retrovirus carrying the iNOS gene was obtained by
transfection of the PA317 packaging cells with pLNC-iNOS using the
calcium phosphate method. After 48 h, the GPEscFv-env or GPE-hgB
packaging cells were infected with the culture supernatant of the
transfected PA317 cells in the presence of Polybrene (4 µg/ml; Sigma
Chemical Co., St. Louis, MO) by spin infection at 2000 rpm for 2 h
at 32°C. After washing, the successfully iNOS-infected
cells were selected for 14 days in the medium containing 2
mM of the specific iNOS inhibitor NMA (Sigma;
Ref. 15
) and 600 µg/ml of G418 (Calbiochem-Novabiochem,
San Diego, CA). For virus production, medium was harvested from
confluent monolayers of the virus-producing cells 18–20 h after a
medium change. The supernatant was filtered through a 0.45-µm pore
filter (Millipore, MA) and used immediately for infection or stored at
-80°C. The clones showing the highest virus titer for the NIH3T3
cells were chosen as virus-producing cells. The resultant recombinant
retrovirus displaying anti-CEA scFv and carrying the iNOS
gene was termed GPEscFv-env/iNOS and the retrovirus only carrying the
iNOS gene as GPE/iNOS. To increase the virus titer, the viral
supernatants were concentrated by centrifugation at 6000 rpm for
18 h at 4°C (5)
.
Immunoblotting.
For detection of envelope proteins, 6 ml of viral supernatants were
centrifuged as described above, and the pellet was dissolved in 100
µl of SDS-PAGE sample buffer. Proteins were then separated by
SDS-PAGE on 8% gel and electroblotted onto nitrocellulose membranes
(Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). After
blocking, the membrane was reacted successively with anti-RLV gp70
antiserum (goat anti-Rouscher leukemia murine envelope gp69/71; Quality
Biotech, Camden, NJ), biotinylated antigoat IgG, and horseradish
peroxidase-conjugated avidin (Vector). Proteins on the membranes were
visualized by using the ECL system and Hyperfilm-ECL (Amersham
Pharmacia Biotech) according to the manufacturer’s instructions.
RT-PCR.
Total RNA was isolated from virus-producing cells or infected cells
using the High Pure RNA Isolation kit (Boehringer Mannheim,
Indianapolis, IN) according to the manufacturer’s instructions.
Isolated RNA (5 µg) was reverse transcribed by using the Superscript
Preamplification System (Life Technologies, Rockville, MD). PCR
amplification was performed using AmpliTaq Gold (Perkin-Elmer, NJ) and
the primers were synthesized from published sequences with sense primer
5'-CTGCAAGAGAACGGAGAACGT-3' and antisense primer
5'-GATCCTCACATACTGTGGACG-3' for iNOS; 5'-TCAGCGCAGGGGCGCCCGGTTCTTT-3'
and 5'-ATCGACAAGACCGGCTTCCATCCGA-3' for neomycin;
5'-GTGGGCCGCTCTAGGCACCAA-3' and 5'-CTCTTTGATGTCACGCACGATTTC-3' for
mouse ß-actin; 5'-TCCTGACCGAGCGTGGCTACAGC-3' and
5'-CTCCTGGAAGGTGGACAGTGAGG-3' for hamster ß-actin;
5'-GGGAATTCGAGCAAGAGATG-3' and 5'-GGGAATTCATAGTCCGCCTAGAA-3' for human
ß-actin; and the above-mentioned oligonucleotides for anti-CEA scFv.
PCR products were electrophoresed on 1.5% agarose gel and stained
with ethidium bromide.
NO Analysis.
The concentration of NO in the culture supernatants of the
virus-producing cells was determined by a microplate assay as described
by Ding et al. (22)
. Briefly, 100 µl of
supernatant cultured in the absence of NMA was mixed with 100 µl of
the Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diamine
dihydrochloride, and 2.5% phosphoric acid; Wako) at room temperature
for 10 min in the dark. The absorbance at 540 nm was monitored by a
microplate reader (Benchmark; Bio-Rad, Hercules, CA). NO concentration
was determined using sodium nitrite as the standard.
Virus-binding Assay.
Target cells were detached from culture flasks by incubation in
trypsin-EDTA solution and were resuspended in cold PBS supplemented
with 2% BSA and 0.1% sodium azide. The cells (1 x 106) were then incubated with the
GPEscFv-env/iNOS or GPE/iNOS retrovirus for 30 min at 4°C for
virus binding. The cells were washed with PBS and were incubated with
goat anti-RLV gp70 antiserum diluted at 1:1000 in PBS. After washed
three times, the cells were incubated with antigoat IgG-FITC conjugate
(DAKO, Glostrup, Denmark) for 30 min at 4°C and were analyzed by the
EPICS.
Virus Infection and Virus Titer Assay.
Target cells were seeded at 1 x 105 cells/well in 6-well plates (Becton
Dickinson, Franklin Lakes, NJ). After 24 h, the target cells were
infected with the GPEscFv-env/iNOS or GPE/iNOS retrovirus in the
presence of Polybrene (4 µg/ml) by spin infection at 2000 rpm for
2 h at 32°C. After washing, the cells were cultured for 12 days
in the presence of 2 mM NMA and 600 µg/ml G418, and the
virus titer was determined after counting G418-resistant colonies and
was calculated by the following formula: G418-R colony-forming
units/ml = number of colonies/virus volume
(ml) x replication factor x fraction of
infected cells plated.
In Vitro Cytotoxicity Assay.
The cytotoxicity of the GPEscFv-env/iNOS or GPE/iNOS retrovirus was
detected by in vitro cytotoxicity assay. Target cells were
infected as described above. The multiplicity of infection used was
adjusted to 0.1. After washing, the cells were cultured for 7 days at
37°C in the absence of NMA. Noninfected control target cells were
incubated for the same days in the plain medium. The remaining cells
were collected in 96-well black plates (Labsystems, Helsinki, Finland),
centrifuged, and incubated with the fluorescent dye H33342 (25 µg/ml;
Calbiochem-Novabiochem) for 1 h at 37°C. Cells were washed and
suspended in 100 µl of PBS, and their fluorescence intensity was
measured at excitation wavelength of 355 nm and emission wavelength of
460 nm by using a Fluoroskan II fluorometer (Labsystems). The results
were expressed as percentage survival, which was obtained by the
formula: (mean test - mean blank/mean control - mean blank) x 100.
Analysis of DNA Fragmentation.
Target cells were infected with the GPEscFv-env/iNOS or GPE/iNOS
retrovirus as described above. After 7-day culture in the absence of
NMA, the low-molecular-mass DNA was extracted from the infected cells
using the Apop Ladder Ex Kit (Takara Biomedicals, Tokyo, Japan)
according to the manufacturer’s instructions. Samples were
electrophoresed together with molecular weight markers on 2% agarose
gel. DNA was visualized by ethidium bromide staining.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Lanes 5 and 6). The GP+E-86 cells
(Lane 2), the PA317 cells (Lane 3), and the
GPE/iNOS retrovirus producing clone (Lane 4) showed no band
for the scFv. The presence of the specific band of iNOS was
also detected in two GPEscFv-env/iNOS clones (Cl-1 and Cl-2; Fig. 2B
, Lanes 5 and 6) as well as in
GPE/iNOS cells (Lane 4) but not in the GP+E-86 and PA317
cells. The presence of neomycin gene in these three packaging cells
(Fig. 2C
, Lanes 4–6) was also confirmed.
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.
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.
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,
Lane 1), whereas the GPEscFv-env/iNOS retrovirus contained
the chimeric envelope protein of about
Mr 97,000 as well as the normal
envelope protein (Lane 2), which confirmed the expected size
of scFv to be about Mr 27,000. These
findings demonstrated that the chimeric envelope protein, as well as
the normal envelope protein, was incorporated into the GPEscFv-env/iNOS
retrovirus.
Specific Binding of Recombinant Retrovirus to CEA-expressing Cells.
The recombinant GPEscFv-env/iNOS retrovirus, which expresses the
anti-CEA scFv chimeric envelope protein, was examined for the binding
activity to CEA-expressing cells by flow cytometry (Fig. 5)
. The GPEscFv-env/iNOS retrovirus showed significant binding activities
to MKN-45, KATO-III, and CHO-CEA cells (Fig. 5
, A-C), all
expressing CEA on their cell surface, but did not do so to
CEA-nonexpressing MKN-74 cells (Fig. 5D)
and parent CHO
cells (Fig. 5E)
. The GPE/iNOS retrovirus, displaying only
normal envelope protein, was unable to bind to CEA-expressing nor to
CEA-nonexpressing cells (Fig. 5
, A-E). These results
indicated that the GPEscFv-env/iNOS retrovirus specifically bound to
CEA-expressing cells.
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. However, only the GPEscFv-env/iNOS retrovirus, which displayed the
anti-CEA scFv chimeric envelope protein, infected CEA-expressing
cells of human and hamster origins (Table 1)
.
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, Lanes 6–8), whereas no band was observed in the
GPEscFv-env/iNOS retrovirus-infected CEA-nonexpressing cells (MKN-74;
Fig. 6
, Lane 5) or in the GPE/iNOS retrovirus-infected cells
(Fig. 6
, Lanes 1–4).
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) as did all of the target cells infected with the GPE/iNOS retrovirus
(data not shown). These findings indicated the specific killing of the
target cells by the GPEscFv-env/iNOS retrovirus carrying the
iNOS gene through the recognition of the cell surface
antigen with the chimeric envelope protein.
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reveal that significant DNA fragmentation occurred in CEA-expressing
cells infected with the GPEscFv-env/iNOS retrovirus whereas neither
CEA-nonexpressing cells infected with the GPEscFv-env/iNOS retrovirus
nor CEA-expressing cells infected with the GPE/iNOS retrovirus showed
DNA fragmentation. The degree of apoptosis was positively correlated
with the amount of NO released into the culture medium from the
infected cells (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Furthermore, when tested on
murine NIH3T3 cells, the infectivity of the GPEscFv-env/iNOS
retroviruses did not decrease as compared with that of the wild-type
GPE/iNOS (Table 1)
. Consequently, the infection titer of the
GPEscFv-env/iNOS retroviruses on CEA-expressing cells was about 10
times higher than that reported by using another anti-CEA scFv gene
(5)
. The main reasons for the high virus titer might be
that our anti-CEA scFv gene was cloned from the Ch F11–39
transfectoma, which produces a chimeric antibody with a very high
affinity to CEA (9
, 10)
, and that the virus was generated
from a helper virus-free ecotropic retroviral packaging cell line
(GP+E-86; 25
), although the titer obtained seems to be
still insufficient for future human gene therapy applications.
Although several gene therapy strategies using the iNOS gene
have been reported for various diseases, only a few reports described
the use of the iNOS gene in cancer gene therapy (15
, 26)
, and there seem to be no publications describing the
specific targeting of the iNOS gene to tumor cells. Our
recombinant GPEscFv-env/iNOS retroviral particles specifically
transfected the iNOS gene into CEA-expressing tumor cells
(Fig. 6)
. However, endogenous iNOS expression in human carcinoma cells
is very diverse (27)
. Some human colon cancer cell lines
(SW480, SW620, and DLD-1) express mRNA for
Ca2+-dependent endothelial NOS as well as for
Ca2+-independent iNOS, whereas a human colon
cancer cell line WiDr does not express iNOS (27)
. The
expression of endogenous iNOS in the human carcinoma and hamster cell
lines used in the present study was undetectable even by RT-PCR (data
not shown), which indicated that the iNOS gene transferred
to these cells could induce and maintain the high production of NO.
Our recombinant GPEscFv-env/iNOS retrovirus also revealed significant cytotoxicity against CEA-expressing cells. To our knowledge, this is the first report of successful killing of CEA-expressing tumor cells by specific targeting of the iNOS gene. The cell death was directly correlated with the amount of NO produced. We have also found that the NO production was augmented with the extension of culture time (data not shown). These findings suggest that NO-mediated cytotoxicity is directly related to the production of NO in culture supernatants. However, the cell survival was not completely inhibited in the present study. The exact reasons for this are unknown, but it may reflect the relatively low efficiency of infection. Higher multiplicities of infection of recombinant retrovirus may lead to a higher number of cells infected and killed. Another possibility is that expression of CEA on the tumor cells is very heterogeneous. It is conceivable that a small number of tumor cells, poorly expressing CEA, could escape from the infection and cytotoxic effect.
It has been reported that the NO released by activated cells can induce
apoptosis through several mechanisms, including break of DNA strands by
NO2 (28)
or by formation of
peroxynitrite (29)
. Induction of DNA fragmentation to
CEA-expressing cells by high levels of NO productions was also
confirmed in the present study (Fig. 8)
.
Our specifically targeted killing approach has several features that make it attractive for clinical gene delivery. First, the recombinant retrovirus obtained specifically bound to CEA-expressing cells. This prevents the uptake of virus by nontarget cells or CEA-nonexpressing cells, which would result in lesser side effects. Second, the targeted cells were directly killed by the biological product of the therapeutic gene, which is very convenient for practical retroviral targeted delivery, because the established suicidal gene therapy systems using, e.g., herpes simplex virus-thymidine kinase (HSV-TK) and cytosine deaminase (CD) need the additional drug treatments (30 , 31) . Furthermore, the "bystander effect" of NO may overcome the limitation of retroviral vector gene transfer efficiency (16) .
Although serum CEA levels vary among the cancer stages, immunohistochemical studies have shown high positivity of CEA in almost all gastrointestinal cancers (32) . Therefore, our strategy may be more useful and effective than other conventional suicidal gene therapy approaches in clinical application. We are in the process of performing additional studies for an in vivo approach of targeted cell killing using a relevant animal model system.
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FOOTNOTES |
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1 Supported in part by Grant-in-Aid for
High-technology Research Center from the Ministry of Education,
Science, Sports and Culture and by a fund from the Sasakawa Health
Science Foundation, Japan. 
2 To whom requests for reprints should be
addressed, at First Department of Biochemistry, School of Medicine,
Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka, 814-0180,
Japan. Phone: 81-92-801-1011, extension 3240; Fax: 81-92-801-3600;
E-mail: kurokima{at}fukuoka-u.ac.jp
3 The abbreviations used are: scFv, single-chain
variable fragment; CEA, carcinoembryonic antigen; NO, nitric oxide;
NOS, NO synthase; iNOS, inducible NOS; NMA,
N-methyl-L-arginine; CHO, Chinese hamster
ovary; RT-PCR, reverse transcription-PCR.
Received 2/21/00. Accepted 10/26/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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