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Experimental Therapeutics |
Research Institute for Diseases of the Chest [K. T., Y. N., K. I., N. H.] and Departments of Cardiovascular Medicine [H. U.] and Ophthalmology [T. S.], Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan, and Pharmaceutical Research Laboratory, Kirin Brewery Company, Ltd., Takasaki 370-1295, Japan [K. S., H. O.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we investigated whether tumor growth could be efficiently suppressed by a soluble form of VEGF receptor (flt-1; fused to Fc portion of human IgG) expressed in a remote organ by adenovirus-mediated gene transfer. Gene transfer using an adenovirus can induce a high-level expression of the transferred gene for a substantial period of time, even with a single application. The soluble receptor should be secreted from infected cells into the blood stream and should reach most, if not all, sites of angiogenesis within the tumor and sequester VEGF from receptors on the target cells, thus achieving an effective suppression of tumor growth. In addition, the soluble receptor may form a heterodimeric complex with a wild-type VEGF receptor and function as a dominant negative receptor (23 , 24) . It has been reported recently that either a direct transfection of a plasmid encoding a soluble VEGF receptor into tumor cells (25) or a regional expression of a soluble receptor near tumor sites (or within the tumor) by adenovirus-mediated gene transfer (26) suppresses tumor growth in vivo. However, in the latter study, systemic delivery of the soluble receptor failed to suppress tumor growth (26) . Theoretically, the soluble receptor should be effective in suppressing tumor growth in a remote area. In the present study, using several cancer cell lines, we investigate whether adenovirus-mediated expression of the soluble receptor can effectively suppress tumor angiogenesis and tumor growth in a remote area, which is an important clinical question.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture.
The following human lung cancer cell lines were used: (a)
NCI-H157, NCI-H460, NCI-H1299, NCI-H322, NCI-H522, and NCI-H358
(generously provided by Dr. A. F. Gazder, University of Texas
Southwestern Medical Center, Dallas, TX); (b) EBC1, PC9,
A549, LK2, and N417 (obtained from the Health Science Research
Resources Bank, Tokyo, Japan); and (c) QG56, QG90, and QG95
(from the National Kyushu Cancer Center, Fukuoka, Japan). The cells
were maintained in RPMI 1640 supplemented with 10% fetal bovine serum
(Life Technologies, Inc., Grand Island, NY) in a humidified incubator
with 5% CO2 at 37°C. Bovine vascular RECs and
COS cells were cultured in DMEM with 10% fetal bovine serum. In
vitro gene transfer into cells was carried out by incubation with
the adenoviral vector in serum-free medium [RPMI 1640 containing
0.05% BSA, 1 µg/ml insulin, 5 µg/ml transferrin, and 25 mmol/liter
HEPES (pH 7.4)] for 2 h at room temperature with gentle
agitation, as described previously (31
, 32)
.
Measurement of VEGF and FGF-2 in Culture Media.
Confluent cancer cells were cultured for 24 h, and then the medium
was collected. After centrifugation, the supernatant was stored at
-80°C until the assay. The VEGF protein in the culture media was
determined using an ELISA kit (Immuno Biological Laboratories,
Tokyo, Japan) according to the manufacturer’s instructions. Each of
the values given here is the mean of triplicate determinations with
respect to standardized cell numbers. FGF-2 was also determined using
an ELISA kit (Amersham International, Buckinghamshire, United
Kingdom).
Measurement of VEGF-ExR Protein in Mouse Serum.
The amount of soluble VEGF-ExR, which was tagged with the Fc portion of
human IgG, in the serum of mice was measured by an ELISA using an
antihuman IgG antibody, as described previously (33)
. Mice
given a single injection of AdVEGF-ExR (5 x 108 pfu) in the femoral muscle were sacrificed at
3, 5, 7, 11, 14, or 21 days after the injection.
[3H]Thymidine Uptake in RECs.
COS cells infected at MOI 10 with either AdLacZ or AdVEGF-ExR or left
uninfected were incubated with serum-free DMEM for 48 h. The
medium was then collected, and the cell debris was removed by
centrifugation (500 x g, 10 min). Confluent
bovine RECs (1 x 104 cells/well)
were incubated with serum-free DMEM for 24 h. The medium was then
exchanged for the supernatant prepared from COS cells. The supernatants
were supplemented with 0.01% BSA and various concentrations of rhVEGF
(0.1–100 ng/ml). Human IgG (1.5 mg/ml) was added into medium prepared
from uninfected COS cells to exclude the specific effect by the IgG Fc
portion tagged to COOH-terminal soluble receptor. The RECs were
incubated for another 18 h and then pulsed with
[3H]thymidine (0.5 mCi/well; Amersham,
Arlington Heights, IL) for 6 h. The insoluble
[3H]thymidine was measured using a
scintillation counter.
Cell Proliferation Assay.
Cell proliferation was monitored spectrophotometrically using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma
Chemical Co., St. Louis, MO), as described previously
(32)
.
Tumor Formation in Nude Mice.
All animals were treated using protocols approved by the animal care
committees of Kyushu University. The experiment was carried out under
both the Guidelines for Animal Experiments of Kyushu University and the
Law (No. 105) and Notification (No. 6) of the Japanese government. Lung
cancer cells (5 x 106) were
injected s.c. into the dorsal skin of nude mice, and tumorigenesis was
monitored for 4 weeks. When tumor formation was seen, tumor volume was
calculated according to the formula
a2 x b, where
a and b are the smallest and largest diameters,
respectively (32)
. Tumor size was measured twice a week
for 1 month. The mice were observed for 120 days to examine the
survival kinetics. Human IgG isolated from normal human serum (DAKO,
Carpinteria, CA) was injected into some mice. VEGF produced by cancer
cells in vivo was detected by immunostaining with polyclonal
anti-VEGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
Student’s t test was used to compare tumor volumes, with
P < 0.05 being considered significant.
In Vivo Angiogenesis Assay.
Growth factor-reduced Matrigel (Becton Dickinson Labware, Bedford, MA)
containing 100 pM rhVEGF (in 200 µl) was injected into
the s.c. space of mice. The mice were then i.m. injected with either
AdVEGF-ExR or AdLacZ at a dose of 5 x 108 pfu. Seven days later, the gel plugs were
resected, fixed in 4% formaldehyde, embedded in paraffin, sectioned at
5 µm, and stained with H&E. Some sections were subjected to
immunostaining with an antibody recognizing factor VIII antigen (DAKO,
Glostrup, Denmark), a biotinylated rabbit antimouse IgG antibody
(Nichirei, Tokyo, Japan), peroxidase-labeled streptavidin, and
diaminobenzidine, as described previously (28)
.
Detection and Quantification of Apoptosis.
Apoptosis among cancer cells in tumor specimens was detected by a DNA
nick end-labeling method (using an in situ Apoptosis
Detection Kit; TAKARA, Tokyo, Japan) according to the manufacturer’s
instructions. The sections were counterstained with hematoxylin.
Apoptotic cells were counted under a light microscope (x200
magnification) in five randomly chosen fields, and the apoptosis index
was calculated as a percentage of all cancer cells in these fields.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Soluble VEGF Receptor Binds to VEGF and Suppresses VEGF-induced
Cellular Response but not Cancer Cell Growth in
Vitro.
We constructed an adenovirus (AdVEGF-ExR) expressing the entire
extracellular domain of the human VEGF receptor (flt-1)
fused to the Fc portion of human IgG. Western blotting analysis showed
that a soluble VEGF receptor of Mr
130,000 was indeed secreted into the culture medium from
AdVEGF-ExR-infected COS cells (data not shown). We confirmed that this
soluble VEGF receptor secreted from the AdVEGF-ExR-infected cells binds
to rhVEGF (data not shown). We examined whether the soluble VEGF
receptor could inhibit the action of VEGF. DNA synthesis in response to
rhVEGF in RECs was measured by [3H]thymidine
incorporation. In the medium prepared from the AdVEGF-ExR-infected COS
cells, VEGF-induced DNA synthesis in RECs was significantly suppressed,
but it was not affected in the medium from AdLacZ-infected cells or
from uninfected COS cells with a considerable amount of human IgG (1.5
mg/ml; Fig. 2
).
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. The results demonstrate that neither AdVEGF-ExR infection nor the
soluble VEGF receptor affects cancer cell growth. The growth of other
cell lines used in this study was also unchanged after AdVEGF-ExR
infection (data not shown).
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. In contrast, when H157 cells infected with AdVEGF-ExR (MOI 10) were
implanted s.c., the tumor stopped growing around day 10 after
inoculation and actually decreased in size thereafter (Fig. 4)
.
Macroscopically, the tumors composed of AdVEGF-ExR-infected H157 cells
looked as red in color as the control tumors until day 7, but the
former turned white in color thereafter, and by day 21, they had become
small and thin. Because AdVEGF-ExR infection did not affect cancer cell
growth in vitro (Fig. 3)
, the inhibitory effect on tumor
formation of AdVEGF-ExR infection was presumably attained via indirect
mechanisms, such as an inhibition of tumor angiogenesis. It should be
noted that whereas the tumor decreased in size quite markedly, it did
not disappear completely. No significant differences in tumor growth or
macroscopic appearance were seen between tumors induced using
uninfected or AdLacZ-infected H157 cells. Some mice were injected with
a large amount of human IgG (10 mg), but no significant differences
were observed regarding tumor growth and appearance (data not shown).
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. The serum level of the soluble receptor peaked on day 7 after the
injection and declined gradually thereafter. However, it was still
detectable on day 21 (n = 4).
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. However, virtually no endothelial cells were found in the
rhVEGF-Matrigel when AdVEGF-ExR (5 x 108 pfu) was injected into the skeletal muscle
(Fig. 6B)
. This inhibitory effect depended on the balance
between the amount of rhVEGF in the gel and the titer of AdVEGF-ExR in
the muscle: the greater the amount of rhVEGF mixed in the gel, the
weaker the inhibition (data not shown). These results demonstrate that
the soluble VEGF receptor produced by AdVEGF-ExR-infected cells can
suppress the action of VEGF in a remote area in vivo.
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, in a mouse injected with AdVEGF-ExR, the tumor began
decreasing in size 10 days after the inoculation and had become only a
white trace by day 21 (but it did not disappear completely). In
contrast, a large reddish tumor was formed in a H157-inoculated mouse
in which either AdLacZ or saline was injected into the muscle. No
inhibitory effect was seen when 10 mg of human IgG were injected
into the muscle once per week for 4 weeks. Representative photographs
(AdLacZ versus AdVEGF-ExR) are shown (Fig. 7G)
. A
similar inhibitory effect was seen in mice inoculated with EBC1 cells
(Fig. 7B)
.
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, and the tumor stroma contained many blood vessels (Fig. 8B
; confirmed by immunostaining with an anti-factor VIII
antibody). In contrast, the tumors in mice given a single injection of
AdVEGF-ExR into the muscle showed extensive necrosis with infiltrated
neutrophils but virtually no living cancer cells (Fig. 8C)
.
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Under the same protocol as the one used in H157 cells, we tested the
growth-inhibitory effects of AdVEGF-ExR using other cell lines (PC9,
QG56, N417, and H460). As shown in Fig. 7, C–E,
partial but
significant inhibitory effects were seen in mice inoculated with PC9,
QG56, and N417 cells. However, no significant suppression of tumor
growth was seen in mice inoculated with H460 cells (Fig. 7F)
, which do not produce VEGF or FGF (Fig. 1)
but secrete
IL-8 at a considerable level (34)
. These data suggest that
a soluble VEGF receptor suppresses the growth of more than one type of
tumor and that it may be effective against many types of tumors.
Apoptosis in Tumors Was Enhanced by Inhibition of Angiogenesis.
To obtain an insight into the mechanisms underlying the inhibitory
effect of the soluble VEGF receptor on tumor growth, we analyzed the
H157 tumors histologically, focusing on the incidence of apoptosis.
First, we analyzed tumors at day 5 after the inoculation, a time at
which tumor cells do not require angiogenesis for their growth
(preangiogenic stage). Although the central area of the tumor was
already necrotic, the majority of cancer cells were alive. No blood
vessels were seen. The nutritional support for the cancer cells was
presumably obtained by simple diffusion from the adjacent host tissues.
Interestingly, TUNEL staining revealed the presence of many apoptotic
cancer cells in the intermediate zone between the central necrotic area
and the peripheral viable area adjacent to the host tissue (Fig. 9A)
. As shown in Fig. 9C
, the cytoplasm of all
cancer cells stained positive for VEGF. In the day 21 tumor
(postangiogenic stage), new vessel formation was observed (compare Fig. 8, A and B
), and only a few cells were apoptotic
(Fig. 9B)
. In contrast, tumors from the AdVEGF-ExR-treated
mice showed extensive necrosis and apoptosis at day 21 after the
inoculation (Fig. 8C)
. To evaluate apoptosis in a
semiquantitative manner, we counted the apoptotic cells by microscopy.
The apoptosis index of the day 5 tumor was 10 times that of the day 21
tumor (Fig. 9D)
. These results suggest that apoptosis may
occur in cancer cells in an ischemic environment, that tumor
angiogenesis rescues cancer cells from apoptosis, and that the soluble
VEGF receptor may suppress tumor angiogenesis, thereby maintaining a
high rate of apoptosis among cancer cells and inhibiting tumor growth.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, in an attempt to inhibit tumor angiogenesis, we
transferred a soluble VEGF receptor (flt-1) either directly
into cancer cells or to a remote organ. Most tumors are thought to
share common angiogenic factors (2)
, thus a successful
antiangiogenic molecule may be effective against a wide range of
tumors. Two major receptors for VEGF, flt-1 and
flk-1 (or KDR), have been identified (36, 37, 38)
.
Because flt-1 has the highest affinity for VEGF (at least
for VEGF165), with a
Kd that, at approximately 10–12
pM, is 7–10 times higher than that of
flk-1 (24
, 37, 38, 39)
, we used a soluble
flt-1 receptor in our study. We confirmed that the soluble
flt-1 produced from the AdVEGF-ExR-infected cells binds to
VEGF (data not shown) and inhibits its action both in vitro
(VEGF-induced DNA synthesis in endothelial cells; Fig. 2
) and in
vivo (VEGF-induced angiogenesis; Fig. 4
). Although we did not
confirm this ourselves, it has been reported that a VEGF receptor
(flt-1) almost identical to ours shows the same high
affinity for VEGF as the wild-type receptor (24
, 40)
. When
we injected AdVEGF-ExR (5 x 108
pfu) into muscle, around 450 ng/ml soluble receptor was detectable in
the circulating blood. This amount would seem to be in excess of the
VEGF present in tumors, as judged by: (a) a rough estimate
of the capacity for VEGF production shown by cancer cells in
vitro (Fig. 1)
; (b) histological examination of the
tumor on day 5 after inoculation (Fig. 9A)
; and
(c) an assumption that the doubling time of cancer cells is
24 h in vivo, although it is 48 h in
vitro. The number of cancer cells present on day 5 after injection
would be 5 x 106 x 24 x 0.1 (8 x 106) because roughly 10% of the cancer cells
seem to have survived in the tumor (Fig. 9A)
. This number of
cells (i.e., 8 x 106)
would secrete 480 pm of VEGF in 24 h. On the basis of this and
their molecular sizes (VEGF, Mr
38,000; the soluble receptor, Mr
130,000), the amount of VEGF-ExR would be over 5000 pm/24 h, which is
about 10 times the amount of VEGF (on a molar basis), leading to the
notion that it would neutralize VEGF completely. In addition to the
sequestering effect of the soluble receptor, both soluble
flt-1 (40)
and flk-1
(23)
can form a heteromeric complex with their wild-type
receptor [soluble flt-1 can also form a complex with
wild-type flk-1 (24)
] and inhibit VEGF
signaling as a dominant negative receptor. If this is true in
animals, then the soluble receptor should achieve inhibition
of VEGF signaling even in the presence of a saturating concentration of
VEGF, provided that the amount of the soluble receptor supplied is high
in comparison with that of the wild-type receptor (41)
.
AdVEGF-ExR-infected cancer cells did not form substantial tumors
in vivo (Fig. 4)
, and not a single mouse died of cancer
during the observation period (4 months). This result is comparable
with that achieved in a study in which cancer cells were stably
transfected with a plasmid encoding soluble flt-1 and then
injected into mice (25)
.
The most important finding in our study was that tumor formation was almost completely suppressed when the soluble receptor was expressed not within the tumor or even close to the tumor, but in a remote organ (by injection of AdVEGF-ExR into skeletal muscle). An inhibitory effect on tumor formation using a similar method has recently been reported using two murine cancer cell lines (42) and systemic i.v. injection of an adenovirus expressing a soluble form of Tie2, an endothelial-specific receptor with tyrosine kinase, which is known to be involved in angiogenesis. Our results and their results (42) demonstrate the effectiveness and usefulness of a strategy involving adenovirus-mediated gene transfer of a soluble receptor for an angiogenic growth factor into a remote organ or the systemic blood, respectively. With a view to future clinical application, the fact that the expression vector does not have to be applied directly into the tumor or even close to the tumor should be of considerable interest in the field. In contrast to our findings, Kong et al. (26) have reported that only regional application of an adenoviral vector expressing a soluble VEGF receptor shows an inhibitory effect and that systemic application does not suppress tumor formation. At present, we do not know the reason for this difference in results between our study and their study. Possible explanations include the different model systems used (our study used human cell lines in nude mice; their study used mouse cell lines in BALB/c mice) or the tagging of the Fc portion of IgG at the COOH-terminal of the receptor (our soluble receptor; no such fusion was used in their study). Provided that a sufficient amount of the soluble receptor was detectable in the blood, we believe that it should inhibit angiogenesis even in a remote area. In their study (26) , the amount of soluble receptor protein that appeared was not measured, and it is not known whether the amount differed between systemic and regional application. Additional studies need to be done because the issue of whether or not the soluble receptor can effectively inhibit cancer located in a remote area is very important clinically.
For our strategy against cancer to be effective, the target
cancer cells need to depend on VEGF for tumor angiogenesis. However,
tumor-associated angiogenesis is known to be promoted by several
cytokines or growth factors (including FGF, VEGF, platelet-derived
endothelial cell growth factor, and IL-8), and many cancer cell lines
produce multiple angiogenic factors. In fact, H157 cells produce FGF-2
(Fig. 1)
and IL-8 at substantial levels (34)
in addition
to VEGF. On the other hand, QC56 and N417 cells do not produce a large
amount of VEGF, at least under normoxic conditions (Fig. 1)
.
Nevertheless, anti-VEGF treatment achieved a significant tumor
suppression in those cancer cell lines (H157, EBC1, QC56, N417, and
PC9) in our study (Fig. 7, A–E)
: it was effective in five
of the six cancer cell lines tested. For QC56 and N417 cells, which
form tumors slowly in vivo, an application of AdVEGF-ExR at
a later stage (e.g., on day 20) instead of on the day of
inoculation (the timing used in the present study) could have achieved
much more powerful effects because the production of the soluble
receptor might already have fallen below the effective level at a time
when it was required to suppress tumor angiogenesis. Our examination of
14 lung cancer cell lines (Fig. 1)
suggests that cancer cells producing
a substantial amount of VEGF in vitro will form tumors
in vivo. Using monoclonal anti-VEGF antibodies, the growth
of a broad spectrum of tumor cells can be inhibited both in
vitro and in vivo (14, 15, 16
, 43
, 44)
. These
findings suggest that VEGF may play a key role in tumor angiogenesis in
a wide variety of cancers. It has not yet been determined whether a
single factor plays a critical role in angiogenesis, even when many
factors are available, or whether multiple factors act in concert to
achieve angiogenesis. Naturally, a combined approach using multiple
soluble receptors for a variety of angiogenic factors would be
interesting and would probably achieve a potent inhibition in a wide
variety of tumors. Indeed, we are currently investigating such an
approach using adenoviruses expressing soluble receptors for either
FGF-2 or platelet-derived growth factor, in addition to AdVEGF-ExR.
Recently, fragments of plasminogen (45) , collagen XVIII (46) , and metalloproteinase 2 (47) , denoted as angiostatin, endostatin, and PEX, respectively, have been shown to inhibit tumor angiogenesis, to induce apoptosis, and to suppress tumor growth. In addition to studies using a recombinant protein of those molecules (48 , 49) , an intratumoral injection of an adenovirus expressing a modified angiostatin to be secretable suppressed angiogenesis, induced apoptosis, and led to a significant arrest of tumor growth (50) . The molecular mechanisms underlying the inhibition of angiogenesis (and the induction of an unexpectedly high level of apoptosis) by those fragmented molecules have yet to be elucidated fully. The enormous amount of recombinant protein required for antitumor effects in vivo (49) may raise the concern that those molecules may not inhibit angiogenesis very efficiently. Nevertheless, a direct comparison between those fragmented molecules and molecules more directly involved in angiogenesis would be interesting. Moreover, an approach using a combination of those molecules should be tried.
In summary, our study shows that VEGF is indeed a critical growth factor for tumor angiogenesis, at least in certain forms of cancer, and our results support the idea that adenovirus-mediated overexpression of a soluble VEGF receptor into a remote organ seems to have potential as a feasible and effective method of cancer gene therapy, although further investigations are required, especially on systemic side effects and its effectiveness against a wide range of cancer cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a Grant-in-Aid for Scientific
Research (to K. T. and H. U.), a Grant-in-Aid for Scientific Research
on Priority Areas from the Ministry of Education, Science and Culture
of Japan (to H. U.), and by grants from Fukuoka Cancer Society (to
K. T. and H. U.) and The Tokyo Biochemical Research Foundation (to
H. U.). 
2 To whom requests for reprints should be
addressed, at Department of Cardiology, Kyushu University School of
Medicine, Fukuoka 812-8582, Japan. Phone: 81-92-642-5361; Fax:
81-92-642-5374; E-mail: ueno{at}cardiol.med.kyushu-u.ac.jp
3 The abbreviations used are: VEGF, vascular
endothelial growth factor; FGF, fibroblast growth factor; REC, retinal
endothelial cell; IL, interleukin; pfu, plaque-forming units; MOI,
multiplicity of infection; rhVEGF, recombinant human
VEGF165 protein; TUNEL, terminal deoxynucleotidyl
transferase-mediated nick end labeling.
Received 5/12/99. Accepted 2/18/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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vß5 may predict the susceptibility to adenovirus-mediated gene transfer in human lung cancer cells. Gene Ther., 5: 361-368, 1998.[Medline]
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P. J. Mahasreshti, J. G. Navarro, M. Kataram, M. H. Wang, D. Carey, G. P. Siegal, M. N. Barnes, D. M. Nettelbeck, R. D. Alvarez, A. Hemminki, et al. Adenovirus-mediated Soluble FLT-1 Gene Therapy for Ovarian Carcinoma Clin. Cancer Res., July 1, 2001; 7(7): 2057 - 2066. [Abstract] [Full Text] [PDF]
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 C. J. Kuo, F. Farnebo, E. Y. Yu, R. Christofferson, R. A. Swearingen, R. Carter, H. A. von Recum, J. Yuan, J. Kamihara, E. Flynn, et al. Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer PNAS, March 22, 2001; (2001) 81615298. [Abstract] [Full Text]
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A. Compagni, P. Wilgenbus, M.-A. Impagnatiello, M. Cotten, and G. Christofori Fibroblast Growth Factors Are Required for Efficient Tumor Angiogenesis Cancer Res., December 1, 2000; 60(24): 7163 - 7169. [Abstract] [Full Text]
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C. J. Kuo, F. Farnebo, E. Y. Yu, R. Christofferson, R. A. Swearingen, R. Carter, H. A. von Recum, J. Yuan, J. Kamihara, E. Flynn, et al. Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer PNAS, April 10, 2001; 98(8): 4605 - 4610. [Abstract] [Full Text] [PDF]
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) or AdLacZ (
) or left uninfected (
). Confluent quiescent
RECs were stimulated with rhVEGF (0.1, 1.0, 10, and 100 ng/ml) in the
above-mentioned medium. Human IgG (1.5 mg/ml) was added to medium
prepared from uninfected COS cells. [3H]Thymidine
incorporation was measured, and the means ± SD
(n = 4) are shown.
) or left uninfected (
