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INTRODUCTION
MATERIALS AND METHODS
The continuous growth of tumors depends on the altered regulation
of the cell cycle. A variety of growth factors and their receptors Animals, Cells, and Materials. Four-week-old female nude mice were
mediate the signal transduction pathways that modulate the cell cycle purchased from Harlan-Sprague-Dawley. All human lung cancer cell lines
(1). Among these growth factors are the IGFs1, peptides with molec (human lung adenocarcinomas A549 and SCC5, human lung large cell carci
ular weights of about 7,500.000 that can stimulate cellular prolifera noma NCI H460, and human small cell lung carcinoma NCI H82) were
tion and induce cellular differentiation (2). The IGF-Ir is a het- obtained from Adi F. Gazdar (University of Texas Southwestern Medical
Center, Dallas, TX). All cells were passaged in RPMI 1640 with 8% fetal
erodimer derived from the cleavage of a precursor polypeptide (3), bovine serum. IGF-I and '^I-labeled IGF-1 were purchased from Amersham.
and recently the roles of IGF-I and the IGF-Ir in cancer cells have
Construction of Ad-IGF-Ir/as. The cDNA of the IGF-Ir was made by
been investigated intensively (4). In certain systems, the IGF-Ir seems reverse transcription-PCR of mRNA from NCI H82 (a human small cell lung
to be essential for malignant transformation because fetal fibroblasts cancer cell line). The forward primer used was AGCTG AATTC ATCCC
with a disruption of the IGF-lr gene cannot be transformed by the AAATA AAAGG A, and (he reverse primer used was AGCTG AATTC
SV40 T antigen (5). GGGGA AGAGG TCTCC GAGGC T. The resulting cDNA fragment con
The IGF-Ir may also be important for the maintenance of the tained 321 bp of the IGF-Ir cDNA open reading frame, including the ATG
malignant state. Trojan et til. (6, 7) demonstrated that antisense IGF-I initiation codon. Both ends of the cDNA were engineered to contain EcoRl
could suppress the tumorigenicity of a rat glioblastoma and could restriction sites that were used to clone the fragment in an antisense direction
even cause shrinkage of established tumors. Stable transfection of an into the polylinker site of the pAC shuttle plasmid (a gift of Dr. Robert Gerard.
antisense plasmid expressing the first 300 bp of the IGF-Ir eliminated University of Texas Southwestern Medical Center). pAC contains the CMV
immediate early enhancer and promoter and (he SV40 polyadenylation. The
the tumorigenicity of a variety of tumor cell lines and has been entire insert was sequenced to verify structure, and the resulting pAC-CMV-
reported to induce systemic effects in established, non-gene-modified
IGF-Ir/as and vector plasmid pJM17 (also a gift of Dr. Gerard) were cotrans-
tumors (8, 9). Reduction of IGF-Ir has been shown to induce apoptosis
fected into 293 cells by standard calcium phosphate coprecipitation methods.
in tumors, but it produces only growth arrest in untransformed cells Ad-IGF-Ir/as was generated by homologous recombination ( 16). The resulting
(1). In addition. IGF-receptor knockout mice are viable (although adenovirus was confirmed by sequencing of PCR products and plaque purified
three times. A recombinant adenovirus expressing the luciferase gene under
Received 2/29/96; accepted 4/30/96. the control of CMV promotor was used as a control virus (Ad-luc).
The costs of publication of this article were defrayed in part by the payment of page Cell Growth Assay. Tumor cells were transduced with 10 m.o.i. of Ad-
charges. This article must therefore be hereby marked advertisement in accordance with IGF-Ir/as or Ad-luc for 1 h, and 3 x IO4 cells were plated in 6-well microtiter
18 U.S.C. Section 1734 solely to indicate this fact.
1 Present address: Department of Internal Medicine. Korea Cancer Center Hospital. plates. These were maintained in complete medium for 48 h and then were
215-4. Gongneung-Dong Nowon-Gu. Seoul 139-240. Korea. switched to serum-free medium + 0.1% BSA (fraction V) with or without 10
2 To whom requests for reprints should be addressed, at University of Texas South ng/ml of IGF-I. Cell numbers were counted by hemocytometer after 7 days.
western Medical Center. 5323 Harry HiñesBoulevard. Dallas. TX 75235-8593. Phone: Soft Agar Clonogenicity. Anchorage-independent growth was assessed by
(214) 648-4913: Fax: (214) 648-4950.
' The abbreviations used are: IGF. insulin-like growth factor; IGF-Ir. IGF-I receptor; soft agar clonogenicity assays. Briefly, tumor cells were transduced with
Ad-IGF-Ir/as. adenovirus expressing an antisense IGF-Ir; Ad-luc. adenovirus expressing 20 m.o.i. of Ad-IGF-Ir/as and then were detached and plated in 0.2% agarose
the luciferase gene; m.o.i., multiplicity of infection; CMV, cytomegalovirus. with a 1% underlay (5 X 10' cells/plate). After 1 week, medium with 20 m.o.i.
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ANTITUMOR EFFECTS OF Ad-IGF-lr/as
2.0
2.0
B
1.5-
1.5 -I
LL
aS> 1.0- a
Fig. 1. Scatchard analysis of the IGF-I binding of Ad- u_ 1.0
luc-transduced SCC-5 (A) and Ad-IGF-lr/as-transduced CD
SCC-5 (B). i
0.5- 0.5 -
0.0'
234 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
Ad lue Bound AdIGF Bound
of Ad-IGF-lr/as (Ad-luc as control adenovirus) was added over the soft agar. single bulk transduction, more closely approximating potential clini
The medium overlay was changed after 1 week. Colonies greater than 125 /j.m cal therapeutic situations than previous studies.
were counted after 3 weeks, using a calibrated graticule. In Vitro Growth Characteristics of Ad-IGF-lr/as-transduced
125I-labeled IGF-I Competitive Binding Assay. 125I-labeled IGF-I com
Human Lung Cancer Cell Lines. The ability of Ad-IGF-lr/as to
petitive binding assays were performed as described previously (17). Briefly, block IGF-mediated growth stimulation was tested as described in
human lung cancer cell lines NCI H460 and SCC5 were transduced with "Materials and Methods." In the Ad-luc-transduced group, the addi
10 m.o.i. of Ad-IGF-lr/as or Ad-luc for 1 h. Cells were then incubated for 24 h
in complete medium, and then the medium was changed to serum-free medium
tion of IGF-I resulted in an enhancement ratio of 1.44 ±0.18, and in
the Ad-IGF-lr/as-transduced group, only a 1.15 ±0.08 ratio was
for another 24 h. Cells were detached with a scraper and were washed with
PBS without Ca+2 and Mg+2. Either 1.0 x IO5 NCI H460 cells or 7 X 10" observed (mean ±SE of triplicates). These data suggest that Ad-IGF-
SCC5 cells were distributed into tubes and were incubated with 0.5 ml of Ir/as-transduction can blunt the mitogenic effect of IGF-I.
RPMI + 0.1% BSA containing varying concentrations of IGF-I and a constant Soft Agar Clonogenicity. Previous studies have suggested that the
concentration of 50 pM I25l-labeled IGF-I for 2 h at 4°C.After incubation, cells inhibition of the IGF-Ir has a more profound effect on tumorigenicity
were washed twice with RPMI + 0.1% BSA. Cells were then lysed in 0.5 ml than on in vitro growth on plastic. Therefore, we sought to test the
of lysis buffer (0.1% SDS, 0.01 N NaOH. and 0.1% Triton) to measure cell ability of human lung cancer cells to form colonies in soft agar (Table
bound activity. Radioactivity was counted by gamma counter. The IGF-Ir
1). Three weeks after plating, 448 and 410 colonies were found in
number was calculated by standard Scatchard analysis (18, 19). control and Ad-luc-transduced groups, respectively. The Ad-IGF-lr/as
Therapeutic Studies. To assess the effect of Ad-IGF-lr/as on established
tumors, 3.0 x IO5 NCI H460 cells were injected i.p. into 4-week-old nude mice group showed only 68 colonies (an 84% decrease compared to the
Ad-luc group). This finding suggests that Ad-IGF-lr/as is capable of
irradiated with 300 rad. Three days later, 50 m.o.i. (based on the injected tumor
cell number) of Ad-IGF-lr/as was injected i.p. for 5 consecutive days. Controls suppressing the tumorigenicity of NCI H460 cells.
consisted of animals injected with PBS and Ad-luc on the same schedule. Mice Treatment with Ad-IGF-lr/as Prolongs the Survival of Nude
were euthanized when they developed preterminal symptoms, and the time to Mice with Established Human Lung Cancer Xenografts. To test
this point was assessed as survival. the therapeutic potential of Ad-IGF-lr/as, nude mice bearing estab
lished i.p. H460 cells were treated 3 days after tumor inoculation as
described in "Materials and Methods." Ad-IGF-lr/as treatment re
RESULTS
sulted in a statistically significant prolongation of survival of these
Ad-IGF-lr/as Reduces the Number of IGF-Irs by U5I-labeled mice (median survival of 35 days in the control group, 33 days in
IGF-I Competitive Binding. We were able to confirm the high level Ad-luc-treated group, and 40 days in Ad-IGF-Ir/as-treated group, as
expression of the IGF-Ir RNA in our human lung cancer cell lines by shown in Fig. 2). Thus, there was a 1-week increase in median
RT-PCR using IGF-Ir-specific primers (data not shown). We then survival for animals treated with the antisense virus. No survival
sought to determine whether transduction with the Ad-IGF-lr/as virus benefit was observed with a once per week for 3 weeks dosing
could induce a measurable reduction in the number of surface recep schedule (data not shown), suggesting that perhaps higher infective
tors, as measured by a competitive binding assay. In the NCI H460 doses would achieve greater therapeutic effect.
cells, the observed flmax for control, Ad-luc-, and Ad-IGF-Ir/as-
transduced cells was 5.10 pM, 6.10 pM, and 3.10 pM, respectively (for DISCUSSION
1.0 X 10s cells). The receptor numbers calculated by Scatchard
analysis were 1.53 X 104/cell in the control group, 1.86 X 104/cell in IGF-I seems to be required for the optimal growth of most normal
Ad-luc-transduced group, and 9.3 X 103/cell in the Ad-IGF-Ir/as- cells. Other growth factors vary widely in their necessity in different
transduced group. In SCC5 cells, fimax was 7.63 pM in the control cell types (20). The IGF peptides, binding proteins, and receptors have
group, 8.23 pM in Ad-luc-transduced group, and 3.75 pM in Ad-IGF- very important roles in normal growth and development, including
Ir/as-transduced group (for 7 X IO4 cells; Fig. 1). The calculated
receptor numbers were 3.27 X 104/cell in the control group,
Table 1 Suppression of colony formation in soft agar by transduction of Ad-lGF-lr/as
3.52 X 104/cell in the Ad-luc-treated cells, and 1.60 X 104/cell in the
number"448
H460Control
Group of NCI
Ad-IGF-Ir/as-treated cells. Therefore, a single transduction by Ad-
IGF-lr/as decreased the receptor number by over 50% compared to the Ad-luciferase-transduced 410
control virus-transduced cells. It should be stressed that these cells Ad-IGF-Ir/as-transducedColony 68
were not selected in vitro for transduction but were tested after a " Each number represents the mean of triplicate plates
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ANTITUMOR EFFECTS OF Ad-lGF-Ir/as
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ANTITUMOR EFFECTS OF Ad-IGF-Ir/as
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Antitumor Effects of an Adenovirus Expressing Antisense
Insulin-like Growth Factor I Receptor on Human Lung Cancer
Cell Lines
Choon-Taek Lee, Shan Wu, Dmitry Gabrilovich, et al.
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