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Experimental Therapeutics |
Department of Surgery [K. T., K. M. A.], Division of Pulmonary, Allergy and Critical Care Medicine [R. W., V. P. J.], and Department of Pathology and Laboratory Medicine [K. M-K.], University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104
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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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To confer specificity of infection and increase viral spread in the tumor mass, RSAds3 are now being actively developed as cancer therapeutic agents (3, 4, 5, 6) . These viruses have several advantages. First, viral replication amplifies the initial input dose allowing the use of a low dosing regimen. Second, lysis of the cell as part of the virus life cycle induces cell death. Third, the new virus progeny infects neighboring cells and continues to replicate and spread until the tumor is eradicated. Fourth, tumor antigens released from dead cells can enhance antitumor immunity. Fifth, these viruses can be additionally modified either to deliver cytotoxic transgenes (7 , 8) or be enhanced for increased infectivity (9) . Finally, replication is restricted to tumor cells thus avoiding toxicity to normal tissues.
Two commonly used approaches to restrict adenoviral replication to tumor cells are: (a) deleting entire (E1B) or partial regions (E1A) of essential viral genes that normally disarm host-defense mechanisms; and (b) to regulate expression of viral genes (E1A) essential for replication with heterologous promoters that are specifically active in tumor or tissues. For example in the well-characterized Onyx-015 (C1–1042) adenovirus (10) , the E1B 55 kDa gene is deleted so that the virus replicates in p53-deficient tumor cells and not in normal cells, which contain wild-type p53. However, evidence from many studies indicates that the proposed basis for this selective replication is not solely determined by p53 deficiency (4 , 11 , 12) . Nonetheless, its therapeutic potential often in conjunction with chemotherapy has been vigorously evaluated in >240 patients enrolled in Phase I/II/III clinical trials for a variety of carcinomas (13) . Additional modifications of the Onyx-015 virus, including arming it with suicide genes (14 , 15) , deleting the E1B 19 kDa gene (16) , and retargeting its receptor-mediated infection (17) have all provided increased therapeutic benefit. Another strategy used to prevent viral replication in normal cells is to delete the E1A-domain required to bind pRb and inhibit the S phase induction. However, viral replication will proceed in tumor cells, because the pRb/E2F/p16INK4a pathway is often defective. Such E1A mutant adenoviruses have proved to be highly effective in treating many mouse xenograft tumor models (18 , 19) , including metastatic disease models (20) , and have exhibited low toxicities in normal cells (18 , 20) .
In another approach, viral replication has been restricted to tumor cells by regulating the transcription of the E1A gene with a heterologous promoter, which is highly active in given tumor and inactive in normal cells. For example, prostate cancer has been targeted with the adenovirus CN706 in which the E1A gene is under the control of the prostate-specific antigen enhancer element. CN706 has demonstrated a high safety index, selective replication, and a direct oncolytic activity that correlated with prostate-specific antigen level in prostate tumors (21)
. Moreover, CN706 selectivity has been additionally improved by regulating multiple adenoviral genes either with single or different tumor-specific promoters. For example, in CV787 virus (22)
, two different prostate-specific promoters control E1A and E1B genes, respectively. Alternatively, in CV890 adenovirus, a single 
-fetoprotein promoter is used to control the E1A-IRES-E1B cassette to target liver cancer (23)
.
Several other promoters including -fetoprotein (24)
, osteocalcin (25)
, Muc-1 (26)
, L-plastin (27)
, secretory leukocyte protease inhibitor 1 (28)
, and carcinoembryonic antigen 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 tumors because only a limited number of tumors express the targeted tumor markers. However, targeting a rate-limiting gene of a key biochemical pathway that is defective in tumors can increase the range of application with a single RSAd.
E2F-1 is a transcription factor that binds to four E2F-1 binding sites, found in its own promoter, and it positively auto-regulates its own transcription during the G1 to S phase transition (29) . In nonproliferating, normal cells, E2F-1 exists as a pRb/E2F-1 complex, and it is released when pRb is phosphorylated. The unbound form of E2F-1 transactivates several genes of the DNA synthesis pathway to coordinate cell cycle transit through the S phase (30 , 31) . In contrast, in continuously cycling tumor cells, frequent disruptions in the pRB/p16INK4a/cyclin D pathway (32 , 33) keep E2F-1 in a constitutively unbound state. Consequently, E2F-1 is overexpressed in many tumor cells (34 , 35) including lung cancers (36 , 37) . Studies have shown that expression of E2F-1 is increased in tumor cells either because of a deregulated pRb pathway or because of E2F-1 gene amplification (38) , and either mutation or allelic loss of E2F-1 has rarely been detected in cancer cells. Thus it appears that overexpression of E2F-1 generally contributes to tumorigenesis, although experimental evidence indicates that forced overexpression of E2F-1 induces apoptosis in tumor and normal cells (39) .
Taking advantage of this, a replication-defective adenovirus containing the E2F-1 promoter-driven herpes simplex virus-thymidine kinase gene has been used to target E2F-1-expressing cancer cells (40) . Although tumor cell-specific thymidine kinase expression was achieved and treatment with gancyclovir administration reduced tumor growth in a glioma mouse model, poor viral transduction still posed a problem. We have extended this observation to establish direct oncolysis using a RSAd to target E2F-1-expressing tumors without additional transgene delivery.
Here, we report a strategy for the development and preclinical testing of a novel RSAd named AdE2F-1RC. In this virus the E1A gene is placed under the control of the human E2F-1 promoter element to allow us to target a variety of tumors expressing E2F-1, thus exhibiting a deregulated G1-S phase. The AdE2F-1RC virus replicated in and killed a panel of tumor cells to the same extent as the wild-type adenovirus, dl309. More importantly, AdE2F-1RC showed extremely low levels of replication and CPE in nonproliferating normal endothelial, epithelial, and fibroblast cells. In addition, AdE2F-1RC was as potent as the wild-type adenovirus in inhibiting ovarian and lung tumor growth in mouse xenograft models. AdE2F-1RC offers a new treatment platform applicable for any cancer cell of which the hallmark is a deregulated control of cell growth.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cloning of the E2F-1 Promoter.
A 269-bp fragment of the human E2F-1 promoter (GenBank accession no. S74230) was amplified by PCR from human genomic DNA using the forward (5'-GTT-TAA-TTA-ACT-CGA-GGG-TAC-CAT-CCG-GAC-AAA-GCC-TGC-G-3') and reverse (5'-ACT-TAA-TTA-AAA-GCT-TAG-ATC-TCG-AGG-GCT-CGA-TCC-CGC-TCC-GC-3') primers (29)
. PacI and Xho1I (forward primer), and PacI, HindIII, and BglII, sites (reverse primer) were incorporated into the primers to facilitate additional cloning of the E2F-1 promoter. The amplification conditions were an initial step of 94°C for 5 min followed by 35 cycles, with each cycle of 94°C for 30 s and 70°C for 0.5 min, and a final extension step at 72°C for 10 min. The amplified product was subcloned into the pCRII-TOPO plasmid as per the manufacturer’s instructions (Invitrogen, Carlsbad, CA). A positive clone pCRIIE2F-1 was confirmed to contain the E2F-1 promoter by restriction enzyme digestion and DNA nucleotide sequencing. The E2F-1 promoter was digested with XhoI and HindIII from pCRIIE2F-1, and cloned upstream of the luciferase gene into similarly cut pGL2-Basic plasmid (Promega, Madison, WI) to generate pGL2E2F-1.
Analysis of the E2F-1 Promoter Activity.
The activity of the E2F-1 promoter was measured by transiently, cotransfecting 0.2 µg of pSVß-gal plasmid (for transfection-control) with 1.0 µg of either pGL2E2F-1 or pGL2-promoter (positive control), or pGL2-Basic (negative control) into a panel of tumor cells using lipofectin (Life Technologies, Inc. Rockville, MD). Cell extracts were assayed 48 h later for luciferase and ß-galactosidase activities using the Luciferase Assay System and ß-Galactosidase Assay System, respectively, according to the manufacturer’s instructions (Promega, Madison, WI). Luciferase values were normalized for ß-galactosidase activity.
Construction of the AdE2F-1RC Vector.
The pXC1 plasmid has adenovirus 5 sequences from 22–5790 bp containing the E1 gene (Microbix Biosystems Inc., Toronto, Ontario, Canada). A unique PacI site was introduced at nucleotide position 522, essentially as described by Rodriguez et al. (21)
to generate the plasmid pXC1522. A PacI site at nucleotide position 522 was confirmed by restriction digestion with PacI and by DNA nucleotide sequencing. The E2F-1 promoter was cut out of the pCRIIE2F-1 plasmid with PacI and ligated to pXC1522 plasmid (also cut with PacI) to obtain pXC1522E2F-1. The orientation of the E2F-1 promoter was confirmed by DNA nucleotide sequencing.
To construct the AdE2F-1RC virus, homologous recombination was performed between pXC1522E2F-1 plasmid and the right hand side of ClaII digested H5.010CMVGFP adenovirus DNA in 293 cells by standard techniques, including the green/white selection process (42)
. Individual white plaques, visible in 7–10 days, were grown in 293 cells until they exhibited CPE. Low molecular weight DNA isolated from these cells was analyzed for the E2F-1 promoter by restriction digestion and PCR using the primers 7-S (5'-TCG-TGG-CTC-TTT-CGC-GGC-AA-3') and 8-AS (5'-ACC-CAA-GGC-TCT-CTG-CTC-CG-3'), which span the junction between the E2F-1 promoter and the E1A gene as shown in Fig. 1
. After three rounds of plaque purification on 293 cells, one clone was processed for large-scale AdE2F-1RC preparation of and purified by two-step ultra-centrifugation on cesium chloride gradients. Serial dilutions of AdE2F-1RC were plated on 293 cells for plaque assay, and the titer was expressed as pfu/ml. Ad5dl309 (dl309) is a typical wild-type virus but has the 14.7K and RID genes in the E3 region deleted. The E1/E3-deleted AdRSVtk and AdLacZ viruses described elsewhere (41)
are replication defective and contain RSV promoter-driven herpes simplex virus-thymidine kinase gene and cytomegalovirus-driven LacZ cassettes, respectively. All of these viruses were purchased from the Vector Core Facility (Wistar Institute, Philadelphia, PA).
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To measure E1A protein levels, nonproliferating NHLF, HDLF, and WI38 cells were infected with different viruses at MOIs of 2000 to achieve at least 10% infectivity, predetermined with the replication-defective AdLacZ virus. Later (48 h), cell extracts were prepared by scraping the cells into lysis buffer [2% SDS and 0.125 M Tris-Hcl (pH 6.8)] and shearing the DNA with an insulin syringe. Total protein in the nuclear and cell extracts was measured using the BCA protein assay kit (Pierce, Rockford, IL).
Protein (20 µg) was separated on 10% SDS-polyacrylamide gel and electroblotted onto polyvinylidene difluoride (NEN Life Sciences, Boston, MA) membrane, and blocked in 5% milk in TBST (10 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween 20) for 30 min. The membrane was incubated with either monoclonal anti-E2F-1 or polyclonal anti-E1A (clone KH95 and clone13S-5, respectively; Santa Cruz, Inc., Santa Cruz, CA), or monoclonal -actin (Amersham Pharmacia Biotech, Piscataway, NJ) primary antibodies for 1 h and repeatedly washed in TBST. After incubation for 30 min with appropriate secondary horseradish peroxidase-conjugated antibodies and extensive washing with TBST, immunocomplexes on the membrane were detected with the enzyme chemiluminescence reagent substrate according to the manufacturer’s instructions (NEN Life Sciences, Boston, MA).
In Vitro Cell Viability Assay.
Tumor cells were plated at 60–70% confluence in six-well plates and 24 h later, infected with different adenoviral vectors at various MOIs. Normal, HUVEC, and W138 cells were plated in 96-well plates and cultured for another 2 and 4 days, respectively, before being infected. Tumor cells were trypsinized and plated at a density of 3000 cells/100 µl/well in 96-well plates. After 6 (HUVEC) and 9 (W138) days, cell viability was measured by the MTS assay using the Nonradioactive Cell Proliferation Assay kit (Promega, Madison, WI) based on the dehydrogenase enzyme activity of metabolically active cells. The percentage of cell survival was calculated using the formula: % cell survival = A490nm of infected cells/A490nm uninfected cells) x 100%.
In Vitro Cytopathic Assay.
REN, HeLa, A549, and SKOV3 cells were plated at a density of 2–3 x 105 in six-well plates and 24 h later infected with dl309 or AdE2F-1RC at various MOIs. Normal cells (WI38, IMR90, NHDF, NHLF, SAEC, and HMEC) were contact inhibited by growing them for 3–4 days in 10% serum, and infected with dl309 and AdE2F-1RC at MOIs of 1000 (HMEC) and 2000 (WI38, IMR90, NHDF, NHLF, and SAEC). The assay was terminated once dl309-infected cells showed significant CPE (
90–95%). All of the cells were fixed in acetone:methanol (1:1 v/v) for 10 min at room temperature and stained with 1% crystal violet (Sigma, St. Louis, MO) for 1–2 min, the plates dried, and photographed.
Adenoviral Replication Assay.
Contact-inhibited normal cells and log phase tumor cells (plated at 60–70% confluence) were infected with dl309 or AdE2F-1RC at various MOI’s. After infection (48 h), 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 by standard plaque assays on 293 cells. For each cell line, the efficiency of replication of the AdE2F-1RC virus was expressed as VRI, which was calculated using the formula: VRI = number of AdE2F-1RC plaques per 48 h/number of dl309 plaques per 48 h.
Treatment of Established Tumors with AdE2F-1RC.
Female CB17-SCID (Taconic Laboratory, Germantown, NY) mice, aged 6–8 weeks, were acclimatized in a pathogen-free animal facility of the Wistar Institute for at least 1 week before each experiment. All of the animal protocols were in compliance with the Guide for the Care and Use of Laboratory Animals (NIH) and were approved by the Animal Use Committees of the Wistar Institute and the University of Pennsylvania. Four million A549 or 7 x 106 SKOV3 cells in 200 µl of serum-free medium were injected s.c. into both flanks of 21 SCID mice. Tumor sizes were measured regularly, and the volume was calculated using the formula for an ellipsoid, i.e., tumor volume (mm3) = 
/6 x W x L2, where L = length of the tumor and W = width of the tumor. When the tumors had reached approximately 50–100 mm3, mice (n = 7) were randomly divided into 3 treatment groups: (a) medium only; (b) dl309; and (c) AdE2F-1RC. Viruses (109 pfu) in 150 µl of serum-free medium were administered intratumorally for a total of three times every other day and the tumor size monitored. Animals were sacrificed 3 weeks after viral injection and the tumors weighed.
In a second experiment, 3.5 million A549 cells were injected in the flanks of SCID mice and allowed to reach to a size of 30–50 mm3. Flank tumors in four groups of mice (n = 7) were injected directly with a single dose of 109 pfu of dl309, AdE2F-1RC, AdRSVtk, or medium only. Tumor volumes were monitored regularly for 60 days. This experiment was repeated, but the tumors were allowed to grow to an average size of 900 mm3 before they were injected with a single dose of 109 pfu of different viral vectors. Tumor size was monitored periodically for the following 50–55 days.
Statistical Analysis.
The results of the MTS assay and tumor weights are presented as mean ± SE (error bars). Comparisons were made using ANOVA with appropriate pot-hoc testing (Fisher’s PLSD).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Adenoviral DNA was extracted, and recombination was confirmed by restriction enzyme digestion and by PCR amplification of the region between the E2F-1 promoter and the E1A gene using the 7-S and 8-AS primers shown in Fig. 1
. Absence of wild-type virus was confirmed by performing PCR for the E3 gene.
Rationale for Assessing the Selective Replication of the AdE2F-1RC Virus.
We reasoned that comparison of AdE2F-1RC replication between tumor cells overexpressing E2F-1 protein and cells expressing reduced levels of E2F-1 protein would provide a reasonable assessment of the selectivity of this transcriptionally regulated adenovirus. Because, in the clinical setting, the adenovirus would normally be administered to tumor cells in the milieu of normal, nonproliferating cells (i.e., endothelial cells, epithelial cells, and fibroblasts), we reasoned that these cells would be the best controls to assess the selectivity of the AdE2F-1RC virus. Because adenovirus does not replicate in murine cells, assessment of toxicity in animal models is extremely limited. Thus, studying the effects of high doses of the AdE2F-1RC virus on normal human cells provides the best approach for safety studies.
Tumor Cells Express High Levels of E2F-1 Protein.
One consequence of a deregulated pRb/E2F/p16INK4a pathway seen in many tumor cells is the dissociation of E2F-1 protein from pRb/E2F complex with an increased level of "free" E2F-1 protein (32
, 33)
. To verify this, we measured the E2F-1 protein levels in nuclear extracts prepared from tumor and normal cells by immunoblot analysis. E2F-1 protein levels varied widely in lung, mesothelioma, cervical, and ovarian cancer cells as shown in Fig. 2A
. However, in all of the tumor cells, a much higher level of E2F-1 was expressed compared with nonproliferating normal fibroblasts (Fig. 2B)
. In addition, in two tumor cell lines (REN and A549), E2F-1 protein levels in the log phase of cell growth (Fig. 2A
, Lanes 1 and 4, respectively) did not change significantly when these cells were grown to confluence for 3 days before analysis (Fig. 2A
, Lanes 2 and 5, respectively).
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, Lanes 1–3 and 4) nonproliferating normal epithelial and fibroblast cells. A low level was detected in two fibroblast cell lines (Fig. 2B
, Lanes 4 and 5). A positive control (HeLa) is shown in Fig. 2B
, Lane 7, and actin loading control is shown in the bottom panel of Fig. 2B
. It is well known that E2F-1 activity is very tightly regulated during the cell cycle progression from G1 to S phase in normal cells (44)
. This data shows that whereas E2F-1 protein was generally undetectable in a majority of growth-arrested normal cells, E2F-1 protein levels in tumor cells were unaffected by the growth state of the cell.
E2F-1 Promoter Is Activated in Tumor Cells.
To assess the transcriptional activity of the E2F-1 promoter, the human E2F-1 promoter was subcloned upstream of the luciferase gene in the pGL2-Basic plasmid. Using the luciferase reporter assay, the E2F-1 promoter activity was compared with the ubiquitous SV40 promoter in a panel of tumor cells and normalized with the cotransfected, pSVß-gal plasmid. In REN cells, with low levels of E2F-1, the promoter showed 8% of the control SV40 promoter activity. In log phase A549 cells the E2F-1 promoter activity was 23% of control. In H661 cells, with a higher E2F-1 protein expression than A549 cells (Fig. 2A
, Lane 3), the promoter activity was 165% of control. The E2F-1 promoter element demonstrated the capability of directing the transcription of the luciferase gene, and its activity correlated well with E2F-1 protein levels. It was not possible to measure the promoter activity in nonproliferating normal cells, as they could not be easily transfected with the plasmids.
E2F-1 Promoter Has Negligible Activity in Normal Quiescent Cells.
To test the E2F-1 promoter activity in an adenoviral context, we infected nonproliferating normal fibroblasts with dl309 or AdE2F-1RC at MOI of 2000. After infection (48 h), E1A protein was measured by semiquantitative immunoblot analysis, and this is shown in Fig. 2C
. Clearly, dl309 containing the intact E1A promoter expressed 3–4-fold more E1A protein than the AdE2F-1RC virus containing the E2F-1 promoter with a displaced E1A promoter. It is not clear if the low background activity seen in AdE2F-1RC-infected cells is because of a low level of activity from the displaced E1A promoter or is because of the leakiness of the E2F-1 promoter. We would require a construct with no E1A promoter sequences.
Transcriptionally Targeted AdE2F-1RC Has a Potent Antiproliferative Effect in Tumor Cells but not in Normal Cells.
The extent of the antiproliferative effect of AdE2F-1RC was determined by comparing the growth rates of AdE2F-1RC and dl309 infected cells. Normal endothelial (HUVEC) and fibroblast (WI38) cells were infected with AdRSVtk, dl309, or AdE2F-1RC, at MOIs of 10 and 2000, respectively. Cell viability of endothelial cells and fibroblasts was measured 6 and 9 days, respectively, by the MTS assay. This is shown in Fig. 3
. The dl309 virus exhibited a strong antiproliferative effect in both cell lines with 60–80% of the cells dead at the end of the experiment. In contrast, AdE2F-1RC had minimum effect on the growth of cells and appeared to behave in the same manner as the replication defective adenovirus, AdRSVtk.
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. Cell death was a result of viral replicative oncolysis because the replication-defective adenovirus, AdRSVtk, showed minimum effect on tumor cell viability. The cell killing effects of AdE2F-1RC and dl309 viruses on SKOV3, A549, and HeLa cells were comparable. Lowest cell-killing activity was seen in SKOV3 cells with both viruses, suggesting that at the MOIs used the rate of virus production and spread may be a limiting factor. In H661 cells, the antiproliferative effect of AdE2F-1RC was less than that for the dl309, but overall cell viabilities were reduced to 15–38% of uninfected control.
Replication of AdE2F-1RC Is Abrogated in Nonproliferating Normal Cells but not in Tumor Cells.
To assess the replicative ability of AdE2F-1RC, we compared the replication of AdE2F-1RC with that of dl309 in different types of normal and tumor cells. Contact-inhibited normal cells (WI38, IMR90, NHLF, NHDF, HUVEC, HMEC, and SAEC) were infected with AdE2F-1RC and dl309 at MOIs of 10 for HUVEC, 1000 for HMEC, and 2000 for the remainder of the cell lines. Cell lysates were titered on 293 cells 48 h after infection, and the VRI (calculated as a ratio AdE2F-1RC pfu/48 h:dl309 pfu/48 h) calculated is shown in Fig. 4
.
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2 x 106 pfu/ml) than seen with all of the other cell lines (range of 2–9 x 1010 pfu/ml). In contrast, AdE2F-1RC replication was severely attenuated in WI38, IMR90, NHDF, and NHLF cells with low to undetectable levels in the remainder of the cell lines. For example, in WI38, the virus replicated 80–100-fold less than dl309 (Fig. 4
, Lane 1). The somewhat limited extent of viral replication seen in normal cells may be a result of a portion of cells that remain permissive to viral replication, because they may not be growth arrested and may support low E2F-1 activity. We also detected low levels of E1A production in nonproliferating normal fibroblast cells (Fig. 2C)
, although most of these cells do not express E2F-1 protein (Fig. 2B)
.
On the other hand, AdE2F-1RC virus replicated as well as the dl309 in tumor cells (SKOV3, REN, HeLa, and A549) infected at 60–70% confluence with different viral vectors and processed for plaque assay on 293 cells, 48 h later (Fig. 4)
. In A549, AdE2F-1RC replication was reduced 2.5–3.0-fold compared with the dl309 (Fig. 4
, Lane 11) -infected cells.
Transcriptionally Targeted, AdE2F-1RC Causes Extensive CPE in Tumor Cells but not in Normal Cells in Vitro.
To test the selectivity and safety feature of AdE2F-1RC we determined its CPE in several normal cells. We tested the virus on epithelial and fibroblast cells that were contact inhibited, and in a nonproliferative state. Normal cells grown to contact inhibition were infected in parallel with either AdE2F-1RC or dl309, or remained uninfected. Cells were examined regularly for CPE, and when the dl309-infected cells showed almost complete cell death the experiment was halted, and cells were fixed and stained. All of the normal cells were susceptible to dl309 infection and succumbed to its lytic effect within 8–12 days after infection (Fig. 5A)
. In contrast, the morphology of the AdE2F-1RC-infected cells appeared to resemble that of uninfected cells. In fact, remarkable integrity of the monolayer was seen in some cells for up to 12–15 days. The SAEC cell line did not appear as healthy as the uninfected cells, although it did not have any cells that were floating as seen in the dl309-infected wells. Overall, no significant CPE was observed in any of the AdE2F-1RC-infected normal cells (Fig. 5A)
.
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. In REN cells, a dose-dependent CPE was observed (Fig. 5B)
. Moreover, REN cells appeared to be more sensitive, as equivalent killing was observed in A549 and REN cells (by day 8) even though REN cells were infected at a 50-fold lower MOI. In addition, the AdE2F-1RC virus was just as effective in killing tumor cells as the dl309.
Replication-selective Virus AdE2F-1RC Suppresses Growth of Tumors in Vivo.
To evaluate the antitumoral effect of the AdE2F-1RC, subcutaneous tumors were established in flanks of SCID mice using lung (A549) and ovarian cancer (SKVO3) cells. Fourteen (A549) and 17 (SKOV3) days later, macroscopic tumors with a volume of approximately 50–100 mm3 were injected intratumorally with medium only, dl309, or AdE2F-1RCevery other day for a total of three injections. Tumor size was measured with calipers every 3–4 days for 34 days. Tumor growths in dl309- and AdE2F-1RC-treated groups were compared with medium only treated groups. As shown in Fig. 6
, by 31–34 days, there were significant reductions in the tumor size in dl309 and AdE2F-1RC versus medium alone-treated groups for both A549 and SKOV3 tumor models. At the end of the experiment, in dl309- and AdE2F-1RC-treated groups, tumor sizes were significantly reduced by 50% (P < 0.05 for both treatments) and 75–85% (P < 0.01 for both treatments) of control values in A549 (Fig. 6A)
and SKOV3 (Fig. 6B)
tumor models, respectively. Because in vivo efficacy in dl309- and AdE2F-1RC-treated groups were very similar, comparisons between these groups were not made. Although not significant, AdE2F-1RC treatment provided a slightly improved therapeutic benefit compared with dl309 in the SKOV3 tumor model (Fig. 6B)
.
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, right panels). In the dl309-treated group, A549 and SKOV3 tumor weights were reduced significantly by 60% and 85%, respectively (both comparisons, P < 0.001). Similarly, significant reductions in the tumor weights of A549 (55%) and SKOV3 (90%) were obtained in AdE2F-1RC-treated groups (both comparisons, P < 0.001). To confirm intratumoral viral replication, we harvested tumors at an early and at a late time point from both tumor models. Tumor sections were subjected to immunohistochemistry for the detection of adenoviral hexon protein using the antibody described previously (42) . Tumors excised from the dl309- or AdE2F-1RC-treated groups demonstrated a strong presence of hexon protein in the tumor mass. Interestingly, tumors harvested 12 days after viral injection also showed marked hexon staining suggesting viral replication persisting for a prolonged period after injection, in vivo of tumors (data not shown).
Effect of a Single Dose Treatment of AdE2F-1RC on Tumor Growth.
To determine the in vivo efficacy of a low dose of AdE2F-1RC treatment on tumors, at different stages of growth, the experiment was repeated with A549 cells. A single dose (instead of 3 x 109 pfu of viral vectors given previously) of vectors or medium alone was administered intratumorally into very small tumors (30–35 mm3). In another similar experiment the tumors were allowed to grow to a larger size (900 mm3) before being treated with virus. Changes in tumor size of the groups with microscopic tumor treatment and groups with larger tumor treatment are shown in Fig. 7, A and B
, respectively. Microscopic tumors treated with 109 pfu of AdE2F-1RC and dl309 were significantly smaller from the medium alone-treated groups by day 60 (P < 0.05, both comparisons). Injection of a nonreplicating virus AdRSVtk, without administering gancyclovir, had no effect. In addition, compared with the dl309-treated group, a slightly improved efficacy, although not significant, was seen in the AdE2F-1RC-treated group. Surprisingly, animals with a larger tumor burden were also very responsive to treatment with a single dose of vectors. As shown in Fig. 7B
, by day 110, the tumor sizes in both dl309- and AdE2F-1RC-treated groups were significantly smaller than the AdRSVtk (no gancyclovir)-treated group (P < 0.01, both comparisons). The control group was terminated by day 110 as tumors were reaching too large a size.
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DISCUSSION |
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The wild-type adenovirus dl309 replicated in all of the normal cells tested. We reasoned that normal resting cells would be a good model for AdE2F-1RC toxicity tests, because these cells do not express E2F-1 (44
, 45)
and are found in the tumor environment. In contrast to dl309, the replication and CPE of AdE2F-1RC was significantly attenuated in normal cells suggesting that the E2F-1 promoter was not optimally activated. One reason is that the presence of pRb/E2F-1 complex in nonproliferating normal cells renders E2F-1 inactive. Our results agree with studies carried out by Fueyo et al. (18)
who showed that an E1A mutant virus, Ad
24 (unable to bind pRb), did not replicate in growth-arrested CCD32-Luc lung fibroblasts. Nonetheless, we were concerned that E1A expression was not completely abolished in AdE2F-1RC-infected normal fibroblasts (Fig. 2C)
. This may represent infection of a small population of proliferating cells, which had escaped contact inhibition. However, E1A immunostaining in normal AdE2F-1RC-infected NHLF resting cells was not informative (data not shown). Although we formally did not test viral replication in proliferating normal cells, Heise et al. (20)
have demonstrated that their dl922–947 E1A-mutant virus (unable to bind pRb) induced S phase, and replicated in proliferating epithelial and endothelial cells. Howe et al. (19)
also showed that another E1A-mutant virus dl1107 (unable to bind pRb) replicated in proliferating MRC9 fibroblasts. Mechanistically, this may reflect the activity of increased levels of free E2F-1 in S phase cells. The extent to which either a leaky E2F-1 promoter, endogenously activated E1A promoter, or interference from neighboring adenoviral transcriptional elements contributed toward E1A induction in AdE2F-1RC-infected resting normal cells is unclear.
In one respect, AdE2F-1RC and the adenovirus CV890 (23)
have similar construction designs in that both have an intact E1A promoter upstream of their respective heterologous promoters. In CV890, the -fetoprotein promoter drives the E1A-IRES-E1B cassette. When hepatoma and normal HBL100 fibroblasts were infected with CV890, E1A and E1B genes were expressed only in hepatoma cells. In our study, E1B 55kDa and E1B 19kDa proteins were expressed in dl309-infected quiescent normal NHLF fibroblasts and in A549 cells. In comparison, when cells were infected with AdE2F-1RC, both E1B proteins were expressed in A549 cells, and a low level of only E1B 55kDa was expressed in NHLF (data not shown). This may reflect the differential activation of the E1B promoter by E1A protein (46)
. With a view to improving selectivity we are currently examining the effect of removing the E1A-TATA box from AdE2F-1RC.
We found that AdE2F-1RC was as effective as dl309 in killing different tumor cells (Fig. 5B)
with various levels of E2F-1 (Fig. 2A)
. Also, the high selective replication index, seen in tumor cells, correlated reasonably well with deregulated E2F-1 levels in tumor cells. These results suggest that AdE2F-1RC potency in tumor cells correlates with the transactivation of the E2F-1 promoter by E2F-1 protein and it appears not be affected by other genetic lesions, particularly pRb. For example, the virus demonstrated equivalent potency in SKOV3 (pRb+/p53-), A549 (pRb-/p53+), REN (pRb+/p53), and HeLa (pRb-/p53-) cells. The potency effects of AdE2F-1RC may be additionally enhanced by the differential regulation of the E2 promoter in quiescent normal and tumor cells. Although E2F-1 binding sites are present in the promoters of E1A and E2 (Ref. 46
; essential for DNA synthesis) genes, these promoters, because of the free E2F-1, are active only in AdE2F-1RC-infected tumors cells and not in normal quiescent cells. This is because free E2F-1 is recruited to the E2 promoter by the E4-6/7 protein (47)
. Thus, the dual regulation of E1A and E2 in tumor cells most likely improves the viral replication kinetics. This strategy has been used to target colon cancer by using a colon cancer-specific promoter to drive the E2 gene (48)
.
Data from our animal experiments showed that AdE2F-1RC had a significant therapeutic effect in vivo in two xenograft mouse tumor models of ovarian and lung carcinomas after repeated intratumoral viral delivery. The therapeutic effect of AdE2F-1RC was associated with viral replication (we detected viral replication in tumors 10–12 days after virus instillation; data not shown), cell lyses, and viral spread because replication-incompetent virus (AdRSVtk) had minimum effect. In the experiment with A549 cells, a single injection of AdE2F-1RC into small flank tumors significantly inhibited tumor growth. Interestingly, on reinjecting AdE2F-1RC (1 x 109 pfu) into these tumors 60 days after the first treatment, tumor growth continued to regress compared with the control group (data not shown). Why cells that had not responded to the initial treatment and continued to grow for a long time would respond to a second treatment is unclear. Moreover, tumor growth inhibition was maintained over a prolonged time period although not totally eradicated. Our data agrees with that of Harrison et al. (49) , who demonstrated that the growth of A549 tumors could be effectively inhibited by treatment with dl309. However, tumors could never be completely eradicated. Surprisingly, they were able to isolate dl309 virus from tumors 100 days after the first injection. Additional tumor growth retardation was achieved when the E1B 19 kDa gene was deleted, but again tumors did not disappear completely (50) . They attributed tumor persistence to mouse cells growing within the tumors and forming barriers that prevented human adenoviral replication and spread.
The overall therapeutic index for a treatment is determined by the safety and the efficacy of the treatment. A major safety concern for the transition of AdE2F-1RC into the clinic as a therapeutic agent is its replication in normal tissues. A common difficulty in testing replication-selective viruses is that they there are no good laboratory animal models. However, the clinical experience with replicating vectors has indicated them to be generally nontoxic and nonimmunogenic (51 , 52) . Although not directly comparable, Parr et al. (40) developed a replication-incompetent AdE2F-1ß-gal vector in which the ß-galactosidase gene was placed under the control of the E2F-1 promoter. They showed selective expression and low toxicity in normal tissues. After AdE2F-1ß-gal injection into in rat glioma tumors, ß-galactosidase expression was observed only in tumor cells with minimal expression in normal brain tissues.
Other RSAds developed to target the G1-S transition phase (18, 19, 20)
have all used the "partial E1A gene deletion" approach. All of these vectors have demonstrated high therapeutic benefits in preclinical studies, although the effects, including immunological, of E1A mutant proteins in normal cells and any deleterious loss of radio or chemosensitization have not been studied. In this regard our vector expresses wild-type E1A, which has many other beneficial antitumoral activities as shown by its use in clinical trials for breast cancer (53)
. To reduce E1A-mutant protein toxicity in normal tissues, modifications that have been made include transcriptional control with the E2F-1 promoter (ONYX-838; Ref. 54
) and retargeting infectivity by genetically altering the Ad24 virus fiber-knob (55)
. A different modification describes the transcriptional control of a fusion protein, E1A-pRb, which is unable to destabilize the pRb/E2F-1 complex in normal cells with a multicopy p53 response element (56)
.
AdE2F-1RC and other RSAds targeting the G1-S phase pathways may prove to be more effective in treating a broad range of malignancies than vectors with tumor-specific promoters, which have a narrow range of application as they rely on overexpression of a specific tumor or tissue protein (21, 22, 23, 24, 25, 26, 27, 28) . However appealing the notion of an agent that is applicable to a broad range of tumors, caution has to be exercised in assuming that the proposed mechanism of selective replication will hold true in the many different genetic backgrounds in tumors. For example, ONYX-015, which was engineered to target a broad range of tumors with p53 defect has in practice proven to be somewhat controversial in treating every tumor with p53 defect (4 , 5 , 11 , 12) . Nonetheless, ONYX-015 has proven to be beneficial, especially when it is genetically modified to improve viral spread (57, 58, 59) , viral release (16 , 50) , and cell killing (14 , 15) , or is combined with chemotherapeutic agents (4 , 5 , 13) .
The strategy of using RSAds as oncolytic agents has many advantages for cancer therapy. As shown in this study, even if only a small portion of cells is infected (single dose injection), viruses replicate and kill the cells and replicating viruses spread to adjacent cells. Our approach of targeting E2F-1, a critical cell cycle pathway protein, with a tumor-specific promoter controlling the E1A gene allows the targeting of a wide variety of cancers, of which the hallmark is a deregulated cell cycle.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the National Cancer Institute (PO1 66726 and Specialized Programs of Research Excellence Grant P 50-CA-83638), and by the Benjamin Shein Humanity and Mildred Scheel Stitfung (to R. W.) Foundations. 
2 To whom requests for reprints should be addressed, at Room 351 Stemmler Hall, University of Pennsylvania Medical Center, 36th Street and Hamilton Walk, Philadelphia, PA 19104. Fax: (215) 573-2001; E-mail: kunjlata{at}mail.med.upenn.edu
3 The abbreviations used are: RSAd, replication-selective adenovirus; pRb, retinoblastoma protein; NHLF, normal human lung fibroblast; SAEC, small airway epithelial cell; HMEC, human mammary epithelial cell; HUVEC, human umbilical vein endothelial cell; RSVtk, rous sarcoma virus promoter-driven thymidine kinase; TBST, Tris-buffered saline with Tween 20; NHDF, normal human dermal fibroblast-adult; CPE, cytopathic effect; pfu, plaque-forming unit(s); MOI, multiplicity of infection; VRI, virus replication index; FBS, fetal bovine serum; ß-gal, ß-galactosidase.
Received 10/ 2/01. Accepted 4/18/02.
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-the adenovirus 5 packaging signal. Primers, 7-S, and 8-AS used in a PCR assay to confirm the insertion of E2F-1 promoter are shown.
