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[Cancer Research 63, 2506-2512, May 15, 2003]
© 2003 American Association for Cancer Research

Experimental Therapeutics

Identification of the Human IAI.3B Promoter Element and Its Use in the Construction of a Replication-selective Adenovirus for Ovarian Cancer Therapy1

Katsuyuki Hamada2, Shohei Kohno, Mari Iwamoto, Hiroko Yokota, Masato Okada, Masatoshi Tagawa, Susumu Hirose, Kenshi Yamasaki, Yuji Shirakata, Koji Hashimoto and Masaharu Ito

Departments of Obstetrics and Gynecology [K. H., M. Iw., H. Y., M. It.], Neurosurgery [S. K.] and Dermatology [K. Y., Y. S., K. H.], School of Medicine, Ehime University, Ehime 791-0295; Division of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871 [M. O.]; Division of Pathology, Chiba Cancer Center Research Institute, Chiba 260-8717 [M. T.]; and Department of Developmental Genetics, National Institute of Genetics, Shizuoka 411-8540 [S. H.] Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Little is known concerning promoters or gene therapy specific for ovarian cancer. To explore the potential use of IAI.3B isolated from ovarian cancer cells in gene therapy for ovarian cancer, we identified the promoter region of the IAI.3B gene and created a replication-selective adenovirus, AdE3-IAI.3B, driven by the promoter. Transient transfection experiments showed that the DNA segment located between -1816 and -1 bp resulted in preferential expression in ovarian cancer cells with negligible expression in squamous cell carcinoma and normal cells. The promoter activity of IAI.3B was almost the same as that of cytomegalovirus and an order of magnitude higher than those of midkine and cyclooxygenase-2 in ovarian cancer cells. AdE3-IAI.3B replicated as efficiently as the wild-type adenovirus and caused extensive cell killing in a panel of ovarian cancer cells in vitro. In contrast, squamous cell carcinoma and normal cells were not able to support AdE3-IAI.3B replication. In animal studies, AdE3-IAI.3B administered to flank and i.p. xenografts of ovarian cancer cells led to a significant therapeutic effect. These results demonstrate the usefulness of the IAI.3B promoter for generation of ovarian cancer-specific adenoviral vectors and provide a potential for the development of ovarian cancer-specific oncolytic viral therapies.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sufficient encouraging preclinical results have been obtained to justify initiation of clinical Phase I trials of gene therapy for ovarian cancer (1 , 2) . However, results from a recent clinical trial of intracavitary gene transfer indicate that gene delivery is restricted to a few superficial cell layers, and that treatment of larger three-dimensional tumors may still be inadequate (3) . To confer specificity of infection and increase viral spread in the tumor mass, replication-selective adenoviruses are now being actively developed as cancer therapeutic agents (4, 5, 6, 7) .

Several promoters including {alpha}-fetoprotein (8) , osteocalcin (9) , Muc-1 (10) , L-plastin(11) , midkine (12) , and cyclooxgenase-2 (cox-2; 12 ) are being evaluated in other laboratories to restrict viral replication to their cognate tumors. Although all of these vectors are severely replication-attenuated in normal cells and demonstrate high selectivity, their one drawback is that they are available for treatment of only a narrow range of ovarian cancer tumors, because only a limited number of such tumors express the targeted tumor markers. However, targeting a tumor marker broadly expressed in ovarian cancer tumors can increase the range of application of a single replication-selective adenovirus.

The IAI.3B gene was isolated using a polyclonal serum against a high molecular weight fraction derived from the pleural fluid of a patient with ovarian cancer (13) . CA125 is overexpressed in ovarian cancer and widely used for monitoring this type of cancer. IAI.3B and CA125 exhibit very similar patterns of expression in a wide variety of normal and malignant tissues (13) . The deduced peptide sequence of IAI.3B encompasses a B-box/coiled coil motif present in many genes with transformation potential. Although detailed sequence analysis of the IAI.3B gene has been finished, the promoter region of IAI.3B remained to be identified. In the present study, we characterize the promoter element of the IAI.3B gene and use it in a strategy for development of a novel replication-selective adenovirus named AdE3-IAI.3B. In this virus, the E1A gene is placed under the control of the human IAI.3B promoter element to target a variety of ovarian tumors.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Culture Conditions.
The human ovarian teratocarcinoma PA-1 and lung squamous cell carcinoma EBC-1 cell lines were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). The human ovarian clear cell carcinoma RMG-1 cell line was a gift from Dr. Shiro Nozawa, Keio University (Tokyo, Japan). Human ovarian adenocarcinoma OCC1, 420, OVCAR3, 429, and DOV13 cell lines were a gift from Dr. Gordon B. Mills, The University of Texas, M.D. Anderson Cancer Center (Houston, TX). The human ovarian clear cell carcinoma KK cell line, and human ovarian adenocarcinoma KF and MH cell lines were a gift from Dr. Yoshihiro Kikuchi, National Defense Medical College (Tokorozawa, Japan). The normal human keratinocyte K42 and skin fibroblast F27 cell lines were established by Dr. Koji Hashimoto, Ehime University (Ehime, Japan). The human skin squamous cell carcinoma HSC-5 cell line was a gift from Dr. Kazuo Aso, Yamagata University (Yamagata, Japan). Normal human ovarian epithelial NOE1, NOE2, and NOE3 cell lines, and human umbilical vein endothelial HUVEC cell line were established in our laboratory. The human cervical squamous cell carcinoma ME-180 cell line was obtained from American Type Culture Collection (Rockville, MD). Cells were maintained in a humidified 5% CO2/95% air incubator at 37°C. All of the cell lines except K42, NOE1, NOE2, NOE3, and HUVEC NOE3 were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum. K42 was grown in MCDB153 (Nissui Co., Tokyo, Japan) with bovine hypothalamus extract. NOE1, NOE2, NOE3, and HUVEC were grown in MCDB153 with 5% heat-inactivated fetal bovine serum.

Real-Time Quantitative RT-PCR.3
One hundred ng of RNA samples were used in reverse transcription and real-time PCR for RNA expression studies. A reverse transcription and real-time PCR reaction was carried out with the ABI prism 7700 sequence detection system (Applied Biosystems, Foster City, CA) in a total volume of 50 µl that contained TaqMan one-step RT-PCR master mix (Applied Biosystems), 0.3 µM of each forward and reverse primer, and 0.21 µM of TaqMan probe. The forward and reverse primer and TaqMan probe were, respectively, 5'-CCACTTGTTCCATGTGACACAGA-3', 5'-CCGTTTCGTTAACCACTTGTTCTC-3' and 5'-AAGACAAGCCCCCAGACTGGTTCACAAG-3'. The reaction was performed with the following thermal cycling method: 30 min at 48°C for reverse transcription, 5 min at 95°C for AmpliTaq Gold activation, 15 s at 95°C and 60 s at 60°C for 40 cycles. GAPDH was chosen as a housekeeping gene to be tested as an endogenous control.

Cloning of Genomic DNA of the IAI.3B Gene.
A human genomic library (EMBL3 SP6/T7; Clontech Laboratories, Inc., Palo Alto, CA) was screened with the 32P-labeled PCR-amplified human IAI.3B partial DNA fragment between exon 1A and exon 1B. Five positive clones were obtained after the second screening. Genomic DNA encompassing the 5-kb 5'-flanking region, and exon 1B of IAI.3B was directly sequenced by an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The transcription start site of IAI.3B mRNA was determined by a CapFinder method (Clontech).

Assay for IAI.3B Promoter Activity.
Various lengths of DNA fragments upstream from the exon 1B of IAI.3B were PCR-amplified and inserted into the luciferase reporter vector PicaGene Basic, a promoterless and enhancerless vector (Toyo Ink MFG Co., Tokyo, Japan) in sense orientation relative to the luciferase coding sequence between MluI and BglII sites. The sequence of each insert was confirmed by an ABI PRISM 310 Genetic Analyzer. Constructs containing IAI.3B 5'-flanking sequences, which were fused to the Luciferase gene, were transfected into cells in the presence of N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate Liposomal transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN). Dual luciferase assays were performed according to the manufacturer’s protocol (Promega). The 561-bp human midkine and 883-bp human cox-2 promoters were cloned by PCR and subjected to dual luciferase assays.

Construction of the AdE3-IAI.3B Vector.
The pXC1 plasmid has adenovirus 5 sequences from 22 bp to 5790 bp containing the E1 gene (Microbix Biosystems Inc., Toronto, Ontario, Canada). A unique AgeI site was introduced at nucleotide position 552, essentially as described by Rodriguez et al. (14) , to generate the plasmid pXC1-AgeI. The IAI.3B promoter was ligated to pXC1-AgeI plasmid to obtain pXC1-IAI.3B-1816. To construct the AdE3-IAI.3B virus, homologous recombination was performed between pXC1-IAI.3B-1816 plasmid and the right-hand side of pBHGE3 adenovirus DNA containing the E3 region in 293 cells by a standard technique (15) . To construct the wild-type adenovirus AdE3, homologous recombination was performed between pXC1 and pBHGE3 in 293 cells. The replication-defective E1-deleted Ad5CMV-LacZ virus was used to determine transduction efficiencies. The 50% transduction efficiency was assessed by scoring 500 X-Gal-positive cells in each of three replicate dishes and then determining the 50% value for ß-galactosidase-positive blue cells. All of the viruses were purified with double cesium chloride gradients using standard methods, and titered with standard spectrophotometry and plaque assay.

Western Blot Analysis.
Total cell lysates were prepared by lysing cell monolayers in plates with SDS-PAGE sample buffer. Each lane was loaded with 5 µg of cell lysate protein as determined by BCA protein assay (Pierce, Rockford, IL). After electrophoresis at 25 mA for 2.5 h, the proteins in the gels were transferred to Immobilon polyvinylidene difluoride transfer membranes (Millipore Corp., Bedford, MA). Then, the membranes were probed with two primary antibodies, rabbit antiadenovirus-2 E1A polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and human anti-ß-actin monoclonal antibody (Sigma-Aldrich Fine Chemicals, St. Louis, MO), and secondary antibodies, horseradish peroxidase-conjugated sheep antirabbit IgG (Dako, Glostrup, Denmark) and horseradish peroxidase-conjugated sheep antimouse IgG (Amersham, Piscataway, NJ), respectively. The membranes were developed according to the Amersham ECL protocol.

Adenoviral Replication Assay.
Each cell line was infected for 48 h with AdE3-IAI.3B or AdE3 at various MOIs that allow infection of all of the cells. Medium and cells were scraped into 1-ml medium, subjected to three freeze-thaw cycles, and centrifuged to collect the supernatant. Serial dilutions of the supernatant were assayed for live virus particles for 12–14 days by standard plaque assays under semisolid agarose on 293 cells. The efficiency of replication of the AdE3-IAI.3B or AdE3 virus in each cell line was compared with that in 293 cells.

Cell Count Assay.
Cells were plated at a density of 2 x 104 cells/well in 12-well plates. Cells were infected with AdE3, AdE3-IAI.3B, or the Ad5CMV-LacZ viral control. Culture medium alone was used as a mock infection control. After 10 days, cells were harvested and counted to determine the IC50.

Inhibition of s.c. Tumor Growth.
To determine inhibition of s.c. tumor growth, AdE3-IAI.3B was injected into s.c. tumors in female nude (nu/nu) mice (Charles River Laboratories, Tsukuba, Japan). In brief, 5 x 106 PA-1, RMG-1, or ME-180 cells in 100 µl of RPMI 1640 were injected into the right posterior flank of each mouse through an insulin syringe with a 27 1/2-gauge needle. Seven animals were used for each group. After 15 (ME-180), 20 (PA-1), and 25 (RMG-1) days, tumors with a diameter of 5–8 mm were established. Then, 100 µl of AdE3 (1 x 109 PFU), AdE3-IAI.3B (1 x 109 PFU), Ad5CMV-LacZ (1 x 109 PFU), or medium alone were injected intratumorally on days 0, 2, and 4. The tumors were measured every day with calipers in two perpendicular diameters. Tumor volume was calculated by assuming a spherical shape, with the average tumor diameter calculated as the square root of the product of cross-sectional diameters.

Inhibition of i.p. Ovarian Tumor Growth.
To simulate a clinical trial of gene therapy for ovarian cancer, the orthotopic i.p. carcinomatosis model was used, because ovarian cancer remains localized within the peritoneal cavity in a large proportion of patients, ultimately causing local morbidity and lethal complications. Female nude (nu/nu) mice (Charles River) were injected i.p. with 5 x 106 PA-1 cells on day 0. Ten animals were used for each group. On days 4, 5, and 6, mice were injected i.p. with AdE3-IAI.3B (1 x 107 PFU), Ad5CMV-LacZ (1 x 107 PFU), or no virus. The virus was diluted with 100 µl RPMI 1640 in each case. Survival data were plotted on Kaplan-Meier curves, and using the LIFETEST procedure the AdE3-IAI.3B group was compared with the other groups with the log-rank test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ovarian Cancer Cells Express High Levels of IAI.3B mRNA.
The mRNA level of the IAI.3B gene was measured using real-time RT-PCR. We demonstrated the mRNA expressions of the IAI.3B gene relative to that of GAPDH in ovarian cancer cells (PA-1, RMG-1, 420, OCC1, OVCAR3, KK, KF, 429, DOV13, and MH), squamous cell carcinoma cells (ME-180, EBC-1, and HSC-5), normal ovarian epithelial cells (NOE1, NOE2, and NOE3), normal keratinocyte cells (K42), normal fibroblast cells (F27), and normal endothelial cells (HUVEC; Fig. 1Citation ). All of the ovarian cancer cells examined had at least 10–100-fold higher levels of IAI.3B mRNA than other cells (P < 0.05, unpaired t test).



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  		Fig. 1. Relative mRNA levels of IAI.3B in various cell lines as measured by real-time quantitative RT-PCR. The mRNA level of IAI.3B was normalized to that of the endogenous housekeeping gene control GAPDH in ovarian cancer, squamous cell carcinoma, and normal cells. Bars, ±SDs.

 
IAI.3B Promoter Is Activated in Ovarian Cancer Cells.
A 5-kb promoter element of the IAI.3B gene was cloned after screening of a human genomic DNA library. The transcription start site of IAI.3B mRNA was 6-bp upstream of the sequence of exon 1B, which was reported by Brown et al. (16) . Exon 1A, also reported by Brown et al. (16) , was not transcribed in ovarian cancer cells. No TATA box was found. We examined 16 different lengths of 5'-upstream IAI.3B genomic DNA in searching for promoter activity (Fig. 2A)Citation . The transcriptional activities of the IAI.3B promoter were extremely high in ovarian cancer PA-1 cells, when SV40 promoter activity was estimated as 1 (Fig. 2B)Citation . Deletion of the 5'-flanking sequences from -2941 bp to -1816 bp resulted in gradual increase in transcriptional activity, suggesting the presence of a negative regulatory element in the region. A 1816-bp upstream region of IAI.3B (pGV-1816) showed the highest transcriptional activity, at 1.5 times that of control reporter plasmid (pGV-CMV) driven by the CMV enhancer/promoter and 3000 times that of control reporter plasmid (pGV-SV40) driven by the SV40 enhancer/promoter. Additional deletion to -941 bp (pGV-941) resulted in significant decrease in the activity, suggesting the presence of a positive element between -1816 bp and -941 bp. Deletion from -941 bp to -441 bp region revealed gradual increase in transcriptional activity, suggesting the presence of another negative element in the region. These results indicate that distal and proximal promoter elements of IAI.3B reside in the upstream regions between -1816 bp and -941 bp, and between -441 bp and -1 bp, respectively. The region between -1816 bp and -669 bp of the IAI.3B promoter (pGV-1816–669) demonstrated extremely low transcriptional activity, at 7% of that of control reporter plasmid (pGV-SV40). Thus, the sequence 5'-upstream from exon 1A exhibited no significant transcriptional activity.



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  		Fig. 2. Transcriptional activity of IAI.3B promoter. A, a schematic representation of reporter plasmids. The 5'-truncated fragments of the promoter region upstream of the IAI.3B gene were inserted into luciferase (LUC) reporter vector in sense orientation. The arrow indicates the transcription start site. Numbers indicate numbers of bases upstream (-) and downstream (+) of the transcription start site. The name of each reporter construct was assigned according to the 5'-end nucleotide numbers of inserted promoter sequences, upstream of the transcription start site. B, luciferase activities of reporter plasmids of the IAI.3B promoter in ovarian cancer PA-1 cells. The plasmid (pGV-CMV) driven by the CMV enhancer/promoter was used for comparison with the IAI.3B promoter. Luciferase activity in each plasmid was plotted as a ratio to that in the control plasmid (pGV-SV40) driven by the SV40 enhancer/promoter. Bars, ±SDs. C, transcriptional activities of the 1816-bp region of IAI.3B promoter in ovarian cancer cells and other type of cells (squamous cell carcinoma and normal cells). Luciferase activity in each plasmid was plotted as the ratio to that in the control plasmid (pGV-SV40) driven by the SV40 enhancer/promoter. The plasmids driven by the CMV enhancer/promoter, midkine promoter, and cox-2 promoter were used for comparison with the IAI.3B promoter. Bars, ±SDs.

 
To compare the transcriptional activity of the IAI.3B promoter between ovarian cancer cells and various other types of cells, IAI.3B, CMV, midkine, and cox-2 promoter activities were estimated taking SV40 promoter activity to be 1 (Fig. 2C)Citation . IAI.3B promoter activity correlated well with mRNA levels (r = 0.8; P < 0.001; regression analysis). The IAI.3B promoter activity in ovarian cancer cells was 950 times that in squamous cell carcinoma and normal cells (P < 0.001, unpaired t test), 12 times midkine promoter activity in ovarian cancer cells (P < 0.01, paired t test), and 48 times cox-2 promoter activity in ovarian cancer cells (P < 0.05, paired t test). The promoter activities of midkine and cox-2 were not significantly different. Furthermore, the promoter activities of midkine and cox-2 were up-regulated in only 6 of 10 and 5 of 10 ovarian cancer cells used in this study, respectively.

Adenoviral Infection in Ovarian Cancer Cells.
To determine adenovirus E1A protein expression in PA-1, RMG-1, and ME-180 cells after infection with AdE3-IAI.3B and AdE3, Western blot analyses were performed using rabbit antiadenovirus-2 E1A polyclonal antibodies. E1A protein was not detected in PA-1, RMG-1, and ME-180 cells before infection in contrast to 293 cells used as a positive control. AdE3 expressed E1A at significant levels in PA-1, RMG-1, and ME-180 cells. AdE3-IAI.3B expressed E1A at significant levels in PA-1and RMG-1 cells but not in ME-180 cells (Fig. 3A)Citation . Furthermore, AdE3 expressed E1A at significant levels in 8 other ovarian cancer cell lines, 2 other squamous cell carcinoma cell lines, and 6 normal cell lines. AdE3-IAI.3B expressed E1A at significant levels in 8 other ovarian cancer cell lines but not in 2 other squamous cell carcinoma cell lines or 6 normal cell lines (data not shown). Thus, the IAI.3B promoter insertion in AdE3-IAI.3B virus yielded selective E1A expression in IAI.3B-producing cells (ovarian cancer cells) but not in non-IAI.3B-producing cells (squamous cell carcinoma and normal cells).



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  		Fig. 3. Effects of AdE3-IAI.3B infection in vitro. A, expression of E1A by AdE3-IAI.3B. Western blots were probed with antiadenovirus-2 E1A (top) and anti-ß actin antibodies (bottom). Cellular extracts were subjected to SDS-PAGE (Lanes 1–3, PA-1 cells; Lanes 4–6, RMG-1 cells; Lanes 7–9, ME-180 cells; Lane 10, 293 cells). Each cell line was infected for 24 h with AdE3-IAI.3B at 25 MOI (Lanes 3, 6, and 9), AdE3 at 25 MOI (Lanes 2, 5, and 8), or mock infection (Lanes 1, 4, 7, and 10). B, relative PFU levels of AdE3-IAI.3B and AdE3. Cells were infected for 48 h with AdE3-IAI.3B and AdE3 at various MOIs that allow infection of all cells. Plaque assay was tested for virus production for 12–14 days under semisolid agarose on 293 cells. The PFU level in each cell line was plotted as the ratio to that in 293 cells. Bars, ±SDs. C, cytotoxicity of virus and transduction efficiency in ovarian cancer cells and other type of cells. To compare the cytotoxicity of each virus, cells were infected with AdE3-IAI.3B, AdE3, and Ad5CMV-LacZ. Ten days later, cell viability was determined by cell count assay. IC50 (MOI) was calculated. To compare the transduction efficiency of adenovirus, cells were infected with Ad5CMV-LacZ. Bars, ±SDs.

 
To compare the replicative activity of AdE3-IAI.3B between ovarian cancer cells and various other types of cells, relative amounts of live virus particles were estimated taking that in 293 cells to be 1. AdE3 replicated in ovarian cancer, squamous cell carcinoma, and normal cells. On the other hand, AdE3-IAI.3B replicated as efficiently as AdE3 in ovarian cancer cells but not in squamous cell carcinoma or normal cells (Fig. 3B)Citation .

Transcriptionally Targeted AdE3-IAI.3B Has a Potent Antiproliferative Effect in Ovarian Cancer Cells but not in Squamous Cell Carcinoma or Normal Cells.
The extent of the antiproliferative effect of AdE3-IAI.3B was determined by comparing the rate of growth of AdE3-IAI.3B, AdE3, and Ad5CMV-LacZ-infected cells. The IC50 of each adenovirus was determined in adenovirus-infected cells (Fig. 3C)Citation . AdE3-IAI.3B virus exhibited a strong antiproliferative effect in ovarian cancer cells at an IC50 of 0.0006–0.09 MOI with a mean value of 0.03 MOI, which was 700 times lower than those in squamous cell carcinoma and normal cells (P < 0.01, unpaired t test). On the other hand, the IC50 of AdE3 and Ad5CMV-LacZ in ovarian cancer cells did not differ significantly from those in squamous cell carcinoma and normal cells. In ovarian cancer cells, the IC50 of AdE3-IAI.3B did not differ significantly from that of AdE3 but was 1000 times lower than that of Ad5CMV-LacZ (P < 0.001, paired t test). In squamous cell carcinoma and normal cells, the IC50 of AdE3-IAI.3B did not differ significantly from that of Ad5CMV-LacZ but was 4000 times higher than that of AdE3 (P < 0.001, paired t test). Adenoviral infectivity estimated by 50% transduction efficiency did not differ significantly between ovarian cancer cells and other cells (Fig. 3BCitation ; unpaired t test). Thus, AdE3-IAI.3B virus exhibited a strong antiproliferative effect in ovarian cancer cells. In contrast, AdE3-IAI.3B had minimum effect on the growth of squamous cell carcinoma and normal cells, and appeared to behave in the same manner as the replication-defective adenovirus Ad5CMV-LacZ in these cells.

Replication-selective Virus AdE3-IAI.3B Suppresses s.c. Tumor Growth of Ovarian Cancer Cells.
To evaluate the antitumor effect of AdE3-IAI.3B, s.c. tumors were established in flanks of nude mice using ovarian cancer PA-1 and RMG-1 cells, and squamous cell carcinoma ME-180 cells. By 30 days, there were significant reductions in tumor size in the AdE3-IAI.3B and AdE3-treated groups compared with the medium alone, and Ad5CMV-LacZ-treated groups for both the PA-1 and RMG-1 tumor models. AdE3-IAI.3B and AdE3 had completely eradicated all of the PA-1 tumors (P < 0.001, {chi}2 test; Fig. 4ACitation ). In the RMG-1 tumor model, AdE3-IAI.3B and AdE3 significantly reduced tumor sizes by 96–98% compared with medium alone and Ad5CMV-LacZ (P < 0.001, unpaired t test; Fig. 4BCitation ). AdE3-IAI.3B eradicated 1 of 7 RMG-1 tumors, but AdE3 eradicated none of them. In contrast, AdE3-IAI.3B did not significantly reduce the sizes of ME-180 tumors compared with Ad5CMV-LacZ (Fig. 4C)Citation . However, AdE3 significantly reduced the size of these squamous cell carcinoma tumors by 96% compared with Ad5CMV-LacZ (P < 0.001, unpaired t test). Neither AdE3-IAI.3B nor AdE3 eradicated any ME-180 tumors.



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  		Fig. 4. Effects of treatment with AdE3-IAI.3B on s.c. tumor growth in nude mice. Mice were injected s.c. with 5 x 106 cells/mouse. Fifteen (ME-180), 20 (RMG-1), and 25 (PA-1) days later, tumors of 5–8 mm in diameter were obtained. Then 100 µl of medium alone, Ad5CMV-LacZ (1 x 109 PFU), AdE3-IAI.3B (1 x 109 PFU), or AdE3 (1 x 109 PFU) were injected intratumorally on days 0, 2, and 4. Seven mice were used for each treatment group. Bars, ±SDs. A, effect of intratumoral injections of AdE3-IAI.3B on tumor growth of PA-1 cells in nude mice. B, effect of intratumoral injections of AdE3-IAI.3B on tumor growth of RMG-1 cells in nude mice. C, effect of intratumoral injections of AdE3-IAI.3B on tumor growth of ME-180 cells in nude mice.

 
Replication-selective Virus AdE3-IAI.3B Suppresses i.p. Tumor Growth of Ovarian Cancer Cells.
We used a well-established murine model of i.p. carcinomatosis and treated mice with three i.p. doses of AdE3-IAI.3B, the nonreplicative Ad5CMV-LacZ, or no virus on days 4, 5, and 6 (Fig. 5)Citation . In the PA-1 tumor model, the median survival was not reached for AdE3-IAI.3B. For control with no virus and Ad5CMV-LacZ, the median survivals were 56 and 72 days, respectively. The mean survival times for control with no virus, Ad5CMV-LacZ, and AdE3-IAI.3B were 59, 70, and 137 days, respectively. All of the mice in the control group died before day 74. All of the mice treated with AdE3-IAI.3B survived until at least day 85. Survival was significantly better in animals treated with AdE3-IAI.3B (P < 0.001, log-rank test).



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  		Fig. 5. Therapeutic effect of AdE3-IAI.3B in an i.p. model of ovarian cancer. Carcinomatosis was induced with 5 x 106 PA-1 i.p. cells. Four, 5, and 6 days later, AdE3-IAI.3B, Ad5CMV-LacZ (nonreplicative control), or no virus was injected i.p. at doses of 1 x 107 PFU/day.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It has been reported that the IAI.3B gene has exons 1A and 1B (16) . However, we found that exon 1A was not transcribed and that the sequence 5'-upstream from exon 1A exhibited no significant promoter activity. In this study, we for the first time demonstrated the promoter region of the IAI.3B gene and its up-regulation in all 10 of the ovarian cancer cells tested. Campbell et al. (13) reported no significant difference in level of IAI.3BmRNA among seven normal tissues such as heart, brain, and lung in their Northern analyses. However, our quantitative analyses using real-time RT-PCR revealed significant differences in IAI.3B mRNA levels between ovarian cancer cells and other cells tested. The promoter activity of IAI.3B correlated well with mRNA levels in all of these types of cells. The promoter activity of IAI.3B in ovarian cancer cells was extremely high and almost the same as that of CMV. Before this study, ovarian cancer-specific promoter had not been identified, and the midkine and cox-2 promoters were therefore considered best for driving the E1A and E1B genes for oncolytic adenovirus to treat ovarian cancer (12) . However, the promoter activity of IAI.3B was an order of magnitude higher than those of midkine and cox-2 in ovarian cancer cells. Furthermore, the midkine and cox-2 promoters were up-regulated in only half of the ovarian cancer cells used in this study. Altogether, these findings suggest that the IAI.3B promoter is a good candidate for a promoter to drive oncolytic adenovirus for ovarian cancer therapy.

AdE3-IAI.3B has a construction design similar to that of the adenovirus AdE2F-1RC, because both have an intact E1A promoter upstream of their respective heterologous promoters (17) . On infection with the AdE2F-1RC, small cell lung cancer A549 cells expressed E1B55-kDa and E1B 19-kDa proteins, whereas normal cells expressed a low level of only E1B 55-kDa protein (17) . This might reflect the differential activation of the E1B promoter by E1A protein (18) . Although the activity of heterologous promoter composed of E1A and IAI.3B was twice as much as that of the IAI.3B promoter, this heterologous promoter retained the specificity of ovarian cancer cells. On the basis of these results, we used the E1A-IAI.3B composite promoter for construction of a replication-selective adenovirus.

Our study is the first to report a replication-selective adenovirus in which the cell-type specificity of the IAI.3B promoter is used to target ovarian cancer cells. We demonstrated that AdE3-IAI.3B selectively killed ovarian cancer cells expressing high levels of IAI.3B, but did not kill squamous cell carcinoma or normal cells expressing low levels of IAI.3B. Additionally, in mouse flank and i.p. xenograft models, AdE3-IAI.3B exhibited significant therapeutic effects. Replicating incompetent virus (Ad5CMV-LacZ) had minimum effect suggesting that the therapeutic effect of AdE3-IAI.3B was associated with viral replication, cell lysis, and viral spread. At the doses tested, three injections of AdE3-IAI.3B into small flank tumors eradicated all of the PA-1 tumors but only 1 of 7 RMG-1 tumors. This finding may be related to the IAI.3B promoter activities present in both tumor cells. Replication-selective adenoviruses using E2F promoter (17) , AFP promoter (19) , PSA promoter (14) , or without E1B 55-kDa sequence (5) do not completely eradicate tumors, although they maintain tumor growth inhibition over a prolonged time period. In contrast, AdE3-IAI.3B completely eradicated PA-1 tumors. In PA-1 cells, the IAI.3B promoter exhibited potent activity, which was 3000 times that of the SV40 promoter/enhancer and 1.5 times that of the CMV promoter/enhancer, and, thus, induced a potent viral replication of AdE3-IAI.3B. thus, our replication-selective adenovirus driven by the IAI.3B promoter appears to have stronger antiproliferative activity than those reported previously because of its extremely potent promoter activity. In clinical trials, replication-selective adenoviral injections into prostatic tumors, and head and neck tumors induced 45% and 10–20% tumor responses (20 , 21) , respectively. Thus, there is compelling continued development of replication-selective adenoviral vectors. In fact, concomitant treatment of cancer cells with oncolytic adenoviruses and DNA-damaging agents such as certain chemotherapeutic agents can result in supra-additive cell killing (22) . Thus, considering the safety and activity of AdE3-IAI.3B, strong reasons exist for additional laboratory and clinical investigation of AdE3-IAI.3B in conjunction with chemotherapy for the treatment of human ovarian cancer.


   ACKNOWLEDGMENTS
 
We thank Keizo Oka for help in preparing culture medium and Shuzo Sawada for support of this project.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by a grant from the Ministry of Education, Science, Sports and Culture, Japan, and by a Foundation of Industry-University Joint Research Grant from Primmune Corporation, Inc., Osaka, Japan. Back

2 To whom requests for reprints should be addressed, at Department of Obstetrics and Gynecology, School of Medicine, Ehime University, Shitsukawa, Shigenobu, Onsen-gun, Ehime 791-0295 Japan. Phone: 81-899-60-5378; Fax: 81-899-60-5381; E-mail: hamakatu{at}m.ehime-u.ac.jp Back

3 The abbreviations used are: RT-PCR, reverse transcription-PCR; MOI, multiplicity of infection; PFU, plaque forming unit. Back

Received 10/22/02. Accepted 3/19/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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