Cancer Research AACR Conference on Molecular Diagnostics - 2008 Tumor Immunology: New Perspectives
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[Cancer Research 63, 7936-7941, November 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Replication of an Adenoviral Vector Controlled by the Human Telomerase Reverse Transcriptase Promoter Causes Tumor-Selective Tumor Lysis

Nicholas A. Lanson, Jr.1, Paul L. Friedlander2, Paul Schwarzenberger1, Jay K. Kolls3 and Guoshun Wang1,4

Gene Therapy Program, Departments of
1 Medicine
4 Genetics,
2 Department of Otolaryngology and Biocommunication, Louisiana State University Health Sciences Center, New Orleans, Louisiana;
3 Department of Pediatrics, Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Telomerase reactivation is a critical step for tumorigenesis, allowing cancer cells to proliferate indefinitely. Taking advantage of this property, we generated an adenovirus vector in which E1 gene expression, and therefore viral replication, is under control of the human telomerase reverse transcriptase (hTERT) promoter. This vector, referred to as Ad5-hTERT-E1, replicated in cancer cells and demonstrated efficient cancer-selective cytolysis in a variety of tumor cell lines, including HT-1080 (fibrosarcoma cells); HeLa (cervical carcinoma cells); A549 (lung carcinoma cells); Hep G2 (hepatocellular carcinoma cells); SCC-4, SCC-25, and SCCLSU-1 (head and neck squamous cell carcinoma cells); T24 (bladder carcinoma); and DU 145 (prostate carcinoma). In contrast, the identical multiplicities of infection of Ad5-hTERT-E1 had no effect on primary cultures of normal human fibroblasts, airway epithelial cells, and bone marrow mesenchymal stem cells. Moreover, a single injection of Ad5-hTERT-E1 into preexisting HT-1080 solid tumors, established s.c. in nu/nu mice, efficiently suppressed tumor growth. Interestingly, this conditionally replicating vector transactivated the replication of an E1-deleted antitumor adenoviral vector, Ad5-RSV-hsvTK, in tumor cells, demonstrating a synergistic antitumor effect in vivo. Combinational injection of a single dose of Ad5-hTERT-E1 and Ad5-RSV-hsvTK vector resulted in significant tumor suppression and regression after ganciclovir treatment. These results suggest that the Ad5-hTERT-E1 vector has potential as a broad-spectrum antitumor agent.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A major problem associated with many current cancer treatments is that they do not offer tumor specificity (1) . Therefore, development of effective alternative approaches with novel tumor-targeting mechanisms is needed. Replication-selective virotherapy holds great promise for the treatment of cancer. Appealing features include tumor-selective targeting, viral self-spreading in cancer cells, and no cross-resistance to current treatments (2, 3, 4) . Replication-selective adenoviruses have attracted considerable interest. ONYX-015 (dl2520) and Ad{Delta}24 are two well-studied replication-selective adenoviruses (5 , 6) . This category of vectors involves directly mutating adenovirus genes, such as E1.5 Such mutations enable viral replication within malignant cells with abnormal p53 or Rb functions. Clinical trials of ONYX-015 demonstrated safety and evidence of antitumoral activity in patients (7, 8, 9) . Because of the unique targeting mechanism, this antitumor agent is largely limited to p53-deficient tumors. Tumor-specific promoters have been used to control E1 gene expression and thus adenoviral replication, including the prostate-specific antigen enhancer/promoter for prostate cancer (10) . Clinical trials demonstrated the safe intraprostatic delivery of this vector and apparent therapeutic effects, as reflected by abolishment of prostate-specific antigen production (11, 12, 13) . Other tumor-selective adenoviral vectors include vectors driven by the {alpha}-fetoprotein gene promoter for hepatocellular carcinoma (14) , the surfactant protein B gene promoter for lung cancers (15) , and the MUC1 promoter for breast carcinoma (16) . Because tissue-specific promoters were used in these vectors, their antitumor spectra were restricted to specific types of cancer.

The selective reactivation of telomerase in tumor cells offers an attractive therapeutic target for developing new broad-spectrum antitumor agents. Such reactivation is required because DNA polymerases cannot completely replicate linear chromosomes de novo (17, 18, 19) . Telomerase in tumor cells compensates for the chromosome shortening and loss of genetic information that normally leads to cell senescence (20, 21, 22) . High telomerase activity is found in nearly all immortal cell lines and in >90% of human tumors (23, 24, 25) . Human telomerase consists of a catalytic protein subunit (hTERT), and a RNA subunit (hTER). The transcriptional regulation of hTERT largely controls telomerase activity (26 , 27) . We therefore hypothesized that the hTERT promoter could be used to restrict adenoviral multiplication to tumors and thus achieve tumor-selective tumor lysis. On the basis of this rationale, we generated a replication-selective adenovirus in which adenovirus E1 gene expression, and thus viral replication and cell lysis, is driven by the hTERT promoter. This vector displayed efficient tumor-selective killing ability in vitro and in vivo. The data suggest that the vector could be developed into a potential therapeutic agent for a wide variety of tumors.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Cell Culture.
HT-1080, HeLa, A549, Hep G2, SCC-4, SCC-25, T24, and DU 145 human cancer cell lines were cultured in the medium recommended by American Type Culture Collection. The adenovirus helper cell line 911 (28) was grown in DMEM-F12 supplemented with 10% FBS, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. SCCLSU-1, a cell line derived in our laboratory from a squamous cell carcinoma of the tongue of an adult male, was grown in DMEM-F12 as above. Primary cultures of human airway epithelial cells and fibroblasts were isolated from donated lung tissues, as described previously (29) . The protocols were approved by the local Institutional Review Board. Human bone marrow mesenchymal stem cells, generously provided by Dr. Darwin J. Prockop (Tulane University Health Sciences Center, New Orleans, LA), were cultured in {alpha}-MEM supplemented with 20% FBS, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (30 , 31) . All cells were cultured in a humidified atmosphere containing 5% CO2 at 37°C.

Construction and Generation of Ad5-TERT-E1.
The 1.7-kb human telomerase promoter was amplified by PCR from DNA isolated from adult human WBCs; its sequence was verified by sequencing of both DNA strands. This telomerase promoter sequence corresponds to the 1720-bp region immediately upstream of the translation start site of hTERT. The 3511-bp fragment of the Ad5 E1 region was amplified by PCR from the plasmid pXC1 (Microbix, Toronto, Canada). The amplicon corresponds to nucleotides 560-4071 of the adenovirus genome and begins at the E1A transcriptional start site but does not include the native E1A promoter. The fidelity of the resultant clone was verified by sequencing. The 1.7-kb hTERT promoter was fused to the 3.5-kb adenovirus E1 region in the AdEasy shuttle vector pShuttle (32) . This shuttle vector was then recombined by homologous recombination with the E1- and E3-deleted pAdEasy-1 adenoviral backbone vector to generate a packagable adenovirus genome. Ad5-hTERT-E1 vector was produced by calcium phosphate precipitation onto A549 cells.

Vector Propagation and Titering.
Ad5-hTERT-E1 was propagated in A549 cells, whereas the E1-, E3-deleted vectors Ad5-RSV-hsvTK, Ad5-CMV-EGFP, and Ad5-ME10 were propagated in the adenovirus packaging cell line 911. Adenoviruses were concentrated and purified by cesium chloride gradient centrifugation, followed by desalting through Sepharose CL-6B columns. Standard plaque assays on 911 cells were used to determine the vector titers (33) .

Crystal Violet Staining Assay to Evaluate Vector-Mediated Tumor Cytolysis in Vitro.
To assay the cytolytic effects of Ad5-hTERT-E1 on tumor and normal cells, we performed a crystal violet assay. Cells grown to 20–40% confluence were infected with 10 MOI of Ad5-hTERT-E1 or control vectors for 30 min at room temperature. Six to 11 days postinfection, the cells were fixed for 20 min at room temperature with 5% glutaraldehyde, washed twice with H2O, stained for 1 h at room temperature in 0.1% crystal violet solution in 200 mM 2-[N-morpholino]ethanesulfonic acid (pH 6.0) and destained by three rinses in H2O.

In Vivo Tumor-Killing Assay.
Three protocols were followed to assess the efficiency of Ad5-hTERT-E1 in killing tumors in vivo. All of the studies were approved by the Louisiana State University Health Sciences Center Institutional and Animal Care Use Committee.

In the first protocol, cultured HT-1080 cells were infected overnight with Ad5-hTERT-E1 at a MOI of 10. The following morning the cells were washed three times with PBS and trypsinized off the plates. We injected 0.5 x 106 cells in 200 µl of EMEM without FBS via the tail vein into five female nu/nu mice (athymic NCR-nu; National Cancer Institute). Control mice received either uninfected HT-1080 cells (four mice) or cells infected with Ad5-ME10 at a MOI of 10 (four mice). Ad5-ME10 is an E1-, E3-deleted adenoviral vector containing a fragment of mouse genomic DNA. Five weeks later the mice were sacrificed, and tumor growth was examined anatomically and microscopically.

In the second protocol, 0.5–2.0 x 106 HT-1080 cells in 100–200 µl of PBS were injected s.c. in the flanks of nude mice. When the tumors were 50–100 mm3 in volume (based on the formula a2 * b * 0.5, where a is the smaller tumor diameter and b is the larger diameter), the tumors were injected one time with 1–3 x 108 PFU of Ad5-RSV-hsvTK (8 mice), UV-light-inactivated Ad5-hTERT-E1 (UV-Ad5-hTERT-E1; 11 mice), or Ad5-hTERT-E1 (13 mice). UV inactivation was done by placing 9.5 x 108 PFU of Ad5-hTERT-E1 in 100 µl of virus storage buffer [150 mM NaCl, 10% glycerol, 20 mM HEPES (pH 7.8)] in an open Petri dish on ice and exposed to UV irradiation for 10 min in a UV Stratalinker 1800 (Stratagene). Cultured HT-1080 cells infected at a MOI of ~1000 with the UV-inactivated Ad5-hTERT-E1 showed no cytopathic effect after 7 days, whereas a MOI of 10 of Ad5-hTERT-E1 produced a full cytopathic effect and eradication of the cells. s.c. tumor volumes were measured three times a week until termination of the experiment.

In the third protocol, s.c. tumors were developed in nude mice as above. When 50–100 mm3 in volume, they were injected one time with either 0.5–1.5 x 108 PFU of Ad5-RSV-hsvTK in 100 µl or PBS (8 mice) or a mixture of 0.5–1.5 x 108 PFU of Ad5-RSV-hsvTK and 1.5–3.0 x 108 PFU of Ad5-hTERT-E1 in 100 µl of PBS (10 mice). In both procedures, 9 days later each mouse received an i.p. injection of 1 ml of PBS containing 1.25 mg of ganciclovir every 12 h for 7 days. Tumor volumes were measured every 3 days.

Statistical Analysis.
Statistical analyses were performed with Student’s t test or the Mann-Whitney unpaired two-group test to determine significance. Pearson’s {chi}2 test was also used to test the hypothesis of no association of columns and rows in tabular data. P < 0.05 was considered significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Construction and Characterization of the Telomerase-Specific, Replication-Selective Ad5-hTERT-E1 Adenoviral Vector.
The 1.7-kb promoter region of the hTERT gene and the 3.6-kb Ad5 E1 region were PCR amplified. The two fragments were fused together by an overlapping PCR approach to form a functional cassette. The cassette was then cloned into the shuttle plasmid of the Adeasy system (32) . A packagable vector genome was generated by homologous recombination in bacteria; the vector’s genomic structure is diagrammed in Fig. 1ACitation . The vector DNA was then used to transfect A549 cells, a non E1-complementing cell line, to produce the Ad5-hTERT-E1 virus. HT-1080 and HeLa cells were transduced with the resultant virus at a MOI of 10. Five days later, apparent cytopathic effects were observed (Fig. 1, C and E)Citation , whereas no such effects were observed in the mock-transfected controls (Fig. 1, B and D)Citation . To confirm that the viral replication was driven by the hTERT promoter and to exclude the possibility of any wild-type contamination or reconstitution, we performed PCR analysis on the collected viruses (clones 2, 5, and 6), using a set of primers chosen from the encapsidation signal and the E1 region, as indicated in Fig. 1FCitation . By prediction, the wild-type virus should yield a 0.48-kb PCR product, whereas the hTERT recombinant virus should give a 2.0-kb product. As seen in Fig. 1GCitation , all three clones propagated in either HT-1080 or HeLa cells only gave the 2.0-kb PCR product, along with the positive control plasmid pTA1-7, which contains the hTERT promoter and E1 gene. However, in a wild-type adenovirus control plasmid (pXC1), a 0.48-kb band was amplified. We concluded that replication of the vector is controlled by the hTERT promoter. Hereafter, we refer to this vector as Ad5-hTERT-E1.



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  		Fig. 1. Genomic structure and characterization of Ad5-hTERT-E1. A, schematic of the Ad5-hTERT-E1 genomic structure. The 1.7-kb human telomerase promoter was fused to the 3.6-kb adenovirus E1 region. To confirm that Ad5-hTERT-E1 is replication-competent and does not require a helper cell line, cultured HT-1080 (B and C) and HeLa cells (D and E) were infected with Ad5-hTERT-E1 (C and E) at a MOI of 10. After 5 days, virus-induced cytopathic effects were observed in the infected (C and E) but not the uninfected cells (B and D). F and G, PCR analysis performed to verify that Ad5-hTERT-E1 contains the human telomerase promoter-E1 construct. The PCR primers were designed such that the forward primer corresponded to the adenovirus encapsidation signal (ES) and the reverse primer to the adenovirus E1 region (F). As predicted, the PCR product of wild-type adenovirus showed a 0.48-kb band, whereas Ad5-hTERT-E1 gave a 2.0-kb product (G). LITR and RITR, left and right inverted terminal repeat; hum. tel., human telomerase; MW stnd., molecular weight standard.

 
The Ad5-hTERT-E1 Vector Has Tumor-Selective Cytolytic Effects.
To test whether Ad5-hTERT-E1, whose replication is dependent on the hTERT promoter, confers tumor-selective killing, we transduced different types of cancer or normal cells with 10 MOI of Ad5-hTERT-E1. Five to 12 days post transduction, crystal violet staining was performed to determine how efficiently and specifically the vector killed cancer cells. As displayed in Fig. 2Citation , Ad5-hTERT-E1 eradicated all of the tumor cells, regardless of whether the tumor cells were freshly isolated from a clinical specimen (SCCLSU-1) or were established tumor cell lines. Tested cells included HT-1080 (fibrosarcoma), HeLa (cervical adenocarcinoma), A549 (lung carcinoma), Hep G2 (hepatocellular carcinoma), SCC-4 and SCC-25 (head and neck squamous cell carcinomas), SCCLSU-1 (primarily isolated head and neck squamous cell carcinoma), T24 (bladder carcinoma), and DU 145 (prostate carcinoma). Strikingly, primary cultures of normal human fibroblasts, airway epithelial cells, and bone marrow stromal stem cells were not affected. Equal MOI of the mock virus control Ad5-ME10 had little or no cytolytic effects on any of the cells. Furthermore, we transduced SCC-4, SCC-25, and SCCLSU-1 with 10 MOI of Ad5-CMV-EGFP. Two days later, flow cytometric analysis demonstrated transduction efficiencies of 1.9, 6.2, and 1.2%, respectively. It has been documented that head and neck squamous cell carcinoma cells, such as SCC-4 and SCC-25, have low levels of expression of the adenovirus receptor (CAR; Ref. 34 ). Of note, Ad5-hTERT-E1 showed full cytolytic effects in these cells, which were not efficiently transduced by adenovirus. We therefore concluded that a low level of Ad5hTERT-E1 entry through CAR appears to be sufficient to kill the target cells via vector replication in the cancer cells. To contrast the tumor-selective killing of Ad5-hTERT-E1 with wild-type adenovirus, we infected normal human fibroblasts and HT-1080 cells with 10 MOI of wild-type Ad5. Four days later, crystal violet staining demonstrated that both types of cells were eradicated (data not shown), which differed from the behavior of Ad5-hTERT-E1 in these cells, further confirming the tumor specificity of this replication-competent vector.



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  		Fig. 2. Analysis of the effect of Ad5-hTERT-E1 on cultured human cancer cells and normal cells. The human cancer cell lines examined were HT-1080 cells from a fibrosarcoma; HeLa cells from a cervical carcinoma; A549 cells from a lung carcinoma; Hep G2 cells from a hepatocellular carcinoma; SCC-4, SCC-25, and SCCLSU-1 cells from squamous cell carcinomas of the tongue; T24 cells from a bladder carcinoma; and DU 145 cells from a metastatic prostate carcinoma. The normal human cells examined were primary cultures of fibroblasts obtained from a lung biopsy of a normal adult, primary cultures of airway epithelial cells obtained from a normal adult, and primary cultures of adult human bone marrow mesenchymal stem cells. Cells were cultured in 6-well dishes until 30–50% confluent and infected at a MOI of 10 with either the replication-incompetent adenovirus Ad5-ME10 or the conditionally replicating adenovirus Ad5-hTERT-E1. Ad5-ME10 is an E1-, E3-deleted adenovirus carrying a fragment of mouse genomic DNA. The cytopathic effect was examined 6–11 days postinfection by crystal violet staining. The conditionally replicating Ad5-hTERT-E1 adenovirus caused cytolysis of all of the cancer cells but not the normal cells. The replication-incompetent Ad5-ME10 had little or no effect on any of the cells.

 
Ad5-hTERT-E1 Suppresses Tumor Growth in Tumor Xenograft Models.
To evaluate whether Ad5-hTERT-E1 suppresses tumor growth in vivo, we first transduced HT-1080 cells with 10 MOI of the vector ex vivo. Sixteen h after transduction, we injected 5 x 105 of the transduced cells i.v. into each nu/nu mouse. As controls, animals received injections of cells that received either no vector or mock virus (Ad5-ME10). Five weeks later, the animals were sacrificed and examined for tumor growth. As summarized in Table 1Citation , tumors were found in all of the control animals, mainly in the lungs and ovaries. However, no tumors were found in any organs of the animals that received Ad5-hTERT-E1-treated cells. Pearson’s {chi}2 test confirmed that there was a significant difference between the experimental group and each control group, respectively (P = 0.0027).


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  		Table 1 Suppression of tumor growth by Ad5-TERT-E1

To assay the efficiency of the conditionally replicative adenovirus Ad5-hTERT-E1 in killing tumor cells, we divided cultured HT-1080 fibrosarcoma cells into three groups. Group A cells were untreated. In group B, cultured HT-1080 cells were infected overnight with Ad5-ME10 at a MOI of 10. Ad5-ME10 is a replication-incompetent mock adenovirus. In group C, cultured HT-1080 cells were infected overnight with Ad5-hTERT-E1 at a MOI of 10. The following morning the cells were trypsinized and counted, and 0.5 x 106 cells were resuspended in 200 µl of EMEM and injected via the tail vein into 5–7-week-old homozygous female nude mice. Five weeks later the mice were euthanized, and tumor growth was examined anatomically and microscopically. All mice receiving untreated HT-1080 cells (group A, four mice) or cells treated with Ad5-ME10 (group B, four mice) developed lung tumors; some also developed ovarian tumors. None of the mice receiving HT-1080 cells preinfected with Ad5-hTERT-E1 (group C, five mice) had any tumors.

 
In a more stringent test to evaluate the tumor-killing ability of Ad5-hTERT-E1, we established s.c. tumors in nu/nu mice by injecting 0.5–2 x 106 HT-1080 cells into their flanks. After 2 weeks, solid tumors with volumes of 50–100 mm3 developed. We then injected a single dose of 1–3 x 108 viable Ad5-hTERT-E1 particles into the tumors, and tumor sizes were measured every 3 days. Similarly, the same dosage of two control vectors, Ad5-RSV-hsvTK (an E1-, E3-deleted vector), and the UV-inactivated Ad5-hTERT-E1 vector, was injected into tumors, respectively. Tumor growth relative to the initial tumor volume is shown in Fig. 3Citation . As noted, Ad5-hTERT-E1 significantly suppressed tumor growth compared with the two control groups. We concluded that a single injection of Ad5-hTERT-E1 was sufficient to suppress tumor growth but not sufficient to eradicate tumors in the tested model system.



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  		Fig. 3. Suppression of growth of preformed s.c. HT-1080 tumors by Ad5-hTERT-E1. HT-1080 tumors were formed by injecting 0.5–2.0 x 106 HT-1080 cells in 100–200 µl of PBS s.c. in the flanks of homozygous female nude mice. After tumors 50–100 mm3 in volume formed, we injected one time into each tumor 1–3 x 108 PFU of Ad5-RSV-hsvTK in 75–200 µl of PBS (8 mice), UV-inactivated Ad5-hTERT-E1 (11 mice), or Ad5-hTERT-E1 (13 mice). The minor (a) and major (b) diameters of the tumors were measured three times a week, and the tumor volumes were estimated based on the formula: a2 * b * 0.5. Ad5-RSV-hsvTK is a replication-incompetent, E1-, E3-deleted adenovirus in which the RSV promoter controls the expression of the hsvTK gene. Ad5-hTERT-E1 is a conditionally replicating adenovirus in which E1 expression is controlled by a 1.7-kb portion of the human telomerase promoter. UV-Ad5-hTERT-E1 is the same as above, but its ability to replicate in and lyse cells has been inactivated by UV irradiation. The fold increase in tumor volume relative to the initial tumor volume at the time of vector infection was obtained. Error bars, SE. The suppression of tumor growth by Ad5-hTERT-E1 is statistically significant from the other two treatment regimes as determined by Student’s t test. Differences with P < 0.1 (*) and P < 0.05 (**) were indicated.

 
Ad5-hTERT-E1 Replicates an E1-Deleted Nonreplicable Adenoviral Vector.
Ad5-hTERT-E1 has full E1 gene functions in tumors. We hypothesized that the E1 gene products provided by Ad5-hTERT-E1 could transactivate the replication of E1-deleted adenoviral vectors. Cultured HT-1080 cells were transduced with Ad5-hTERT-E1, Ad5-CMV-EGFP, or a mixture of both, respectively. Three days later, the cells were subjected to three cycles of freezing-thawing. The supernatants after centrifugation were placed in culture with freshly seeded HT-1080 cells. EGFP transgene expression was examined after 3 days. As shown in Fig. 4Citation , only the coinfection condition produced a second round of EGFP virus transduction (Fig. 4F)Citation , suggesting that the E1-deleted Ad5-CMV-EGFP was replicated with the supplement of E1 functions provided by Ad5-hTERT-E1.



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  		Fig. 4. The conditionally replicating Ad5-hTERT-E1 adenovirus can replicate the E1-deleted replication-incompetent Ad5-CMV-EGFP adenovirus. Cultured HT-1080 cells were transduced with Ad5-hTERT-E1 (A), Ad5-CMV-EGFP (B), or both (C). Ad5-CMV-EGFP is a replication-incompetent adenovirus in which the gene for EGFP is expressed under the control of the CMV promoter. As expected, Ad5-CMV-EGFP gave EGFP expression to HT-1080, as shown in B and C. D–F, to determine whether Ad5-hTERT-E1 could replicate Ad5-CMV-EGFP, we freeze-thawed cells from the cultures shown in A–C three times, centrifuged them at 500xg for 10 min, and filtered the supernatants through 0.45 µm filters. The lysates were then applied to fresh HT-1080 cells. Two days later, the cells were examined for EGFP expression. The conditionally replicating Ad5-hTERT-E1 complemented the E1 gene functions and replicated Ad5-CMV-EGFP (F). The absence of EGFP verified that Ad5-CMV-EGFP alone was replication incompetent (E).

 
Synergistic Antitumor Effects from Coadministration of Ad5-hTERT-E1 and an E1-Deleted Antitumor Adenoviral Vector.
We hypothesized that coinjection of Ad5-hTERT-E1 and an E1-deleted antitumor adenoviral vector would have a synergistic tumor-killing effect. To test this hypothesis, we allowed s.c. HT-1080 tumors to form in nu/nu mice and subsequently treated the tumors with either Ad5-RSV-hsvTK or a mixture of Ad5-hTERT-E1 and Ad5-RSV-hsvTK. Nine days after the viral injections, ganciclovir was administered i.p. twice a day for 1 week. Tumor growth was assessed, and the results are summarized in Table 2Citation . None of the tumors in the mice receiving only with Ad5-RSV-hsvTK demonstrated total tumor regression. During the experimental period, transient tumor suppression was observed (data not shown). In contrast, 4 of 10 tumors into which both Ad5-hTERT-E1 and Ad5-RSV-hsvTK were injected displayed complete tumor regression. To date, no recurrence has been observed (5 months after regression). Pearson’s {chi}2 test confirmed that the difference was significant between the two groups (P = 0.0245).


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  		Table 2 Synergistic antitumor effects of Ad5-hTERT-E1 and Ad5-RSV-hsvTK

We injected 0.5–1.5 x 108 PFU of Ad5-RSV-hsvTK in 100 µl of PBS (group A, 8 mice) or 0.5–1.5 x 108 PFU of Ad5-RSV-hsvTK and 1.5–3 x 108 PFU of Ad5-hTERT-E1 (group B, 10 mice) into preformed 50–200 mm3 HT-1080 s.c. tumors in 5–7-week-old homozygous female nude mice. Beginning 9 days later, all mice received twice daily i.p. injections of 1 ml of PBS containing 1.25 mg ganciclovir for 7 days. The minor (a) and major (b) diameters of the tumors were measured three times a week, and the tumor volumes estimated based on the formula: a2 * b * 0.5.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Replication-selective oncolytic adenoviruses represent a new cancer treatment platform (7 , 35, 36, 37) . The features of tumor-selective targeting, viral self-replication, and intratumoral spreading potentially confer enhanced antitumor potency on this type of vector. Importantly, this new therapeutic platform can complement traditional chemo- and radiotherapies to have synergistic antitumor effects (8 , 38) . In this report we demonstrated a strategy that uses the hTERT gene promoter to control adenoviral replication selectively in human tumors to achieve tumor-specific killing. The Ad5-hTERT-E1 vector displayed high oncolytic activities in various types of tumor cells in vitro and in preexisting tumors in vivo. Furthermore, this vector transactivates the replication of an E1-deleted antitumor adenoviral vector by complementing E1 gene function. A synergistic antitumor effect was noted when the two types of vectors were coadministered. Given the fact that telomerase is reactivated in >90% of tumors, this vector has potential as a wide-spectrum antitumor agent.

Certain types of tumor cells, such as head and neck squamous cell carcinoma cells, are refractory to adenovirus transduction. Interestingly, our replication-selective adenovirus efficiently killed head and neck squamous cell carcinoma cells regardless of the low level of the CAR receptor on the cell membrane. Theoretically, entry of a single viable Ad5-hTERT-E1 virion should be sufficient to achieve full cytolysis in tumor cells because of the conditional replication property of this vector. Therefore, this replication-selective vector may efficiently kill both low- and high-CAR-expressing tumor cells.

In nature, human wild-type adenovirus infection causes mild respiratory symptoms (39) . Various serotypes of wild-type adenoviruses have been administered to cervical cancer patients to treat the cancer (40) . More recently, several clinical trials using genetically engineered replication-competent adenoviruses also confirmed the safety and feasibility of using adenoviruses for cancer therapy (41, 42, 43, 44, 45, 46) . One concern related to this approach is that the virus may target somatic or germinal stem cells. This is especially the case for Ad5-hTERT-E1 because stem cells have been found to have some telomerase activity (47, 48, 49) . Our experimental data demonstrated that Ad5-hTERT-E1 did not affect cultured bone marrow stromal stem cells, suggesting that the level of telomerase activity in stem cells might not be sufficient to support Ad5-hTERT-E1 replication. Furthermore, wild-type adenoviruses have been safely used in patients. It does not appear that our vector has any preferential status greater than wild-type adenoviruses in terms of targeting stem cells. Therefore, we reason that Ad5-hTERT-E1 should be a safe vector to develop for in vivo applications.

Cancer gene therapy with nonreplicating adenoviruses as monotherapy has met with limited success in clinical trials. Because there is little possibility of transducing 100% of the tumor cells, the nontransduced tumor cells may eventually override the therapeutic effects of the vectors. Our experimental data demonstrated that Ad5-hTERT-E1 complements E1 gene functions and replicates an E1-deleted nonreplicable antitumor vector in tumor cells. This finding suggests the potential clinical use of the combination of the two types of vector to achieve maximum antitumor effects. Many nonreplicating antitumor adenoviral vectors have already been developed. The vectors were designed to have a wide variety of tumor-targeting mechanisms, such as immunomodulatory gene therapy (50, 51, 52, 53, 54, 55, 56, 57) , tumor suppressor gene therapy (58, 59, 60, 61, 62, 63) , and chemogene therapy (64, 65, 66) . More relevant to Ad5-hTERT-E1, the hTERT promoter has been used to drive suicide or apoptotic genes, such as Bax, TRAIL, tumor necrosis factor, or bacterial nitroreductase, in the nonreplicable adenovirus system, demonstrating excellent tumor-selective tumor targeting (67, 68, 69, 70, 71, 72) . We believe that the use of Ad5-hTERT-E1 in combination with nonreplicating adenoviral vectors could have synergistic and enhanced tumor-therapeutic effects. While we were revising this manuscript, we noticed that a similar adenovirus system was published by an independent group (73) . In their system, a 255-bp hTERT promoter was used. The authors found that the hTERT adenovirus significantly inhibited tumor growth, confirming the potential therapeutic value of this type of vector.

In conclusion, our findings suggest that the hTERT promoter, when used to control the expression of the adenovirus E1 gene, allows adenovirus replication and cell lysis selectively in tumor cells. Such a vector demonstrated efficient tumor killing in vitro and in vivo. The tumor-selective expression of E1 also facilitates replication of E1-deleted nonreplicable vectors selectively in tumor cells. We therefore predict that combinational treatment with Ad5-TERT-E1 and E1-deleted nonreplicable vectors could result in synergistic antitumor effects greater than any of the vectors alone. Such a protocol may have important applications for future cancer therapies using adenoviral vectors.


   ACKNOWLEDGMENTS
 
We thank Robert Kutner and Cynthia Albores of the Vector Core in the Louisiana State University Health Sciences Center Gene Therapy Program for their help in vector propagation, concentration, and purification. We would also like to thank Christie Delaune for assistance in animal experiments and Donna Bertucci for culturing of cells. Ad5-RSV-hsvTK was a gift from Dr. Steven M. Albelda at the University of Pennsylvania Medical Center (Philadelphia, PA).


   FOOTNOTES
 
Grant support: Supported partially by a research grant (to G. W.) from the Cancer Association of Greater New Orleans, and by the Louisiana Gene Therapy Research Consortium.

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.

Requests for reprints: Guoshun Wang, 642 CSRB, 533 Bolivar Street, Louisiana State University Health Sciences Center, New Orleans, LA 70112. Phone: (504) 568-7908; Fax: (504) 568-8500; E-mail: gwang{at}lsuhsc.edu

5 The abbreviations used are: E1, adenovirus early transcription region 1; hTERT, human telomerase reverse transcriptase; FBS, fetal bovine serum; Ad5, serotype 5 adenovirus; RSV, Rous sarcoma virus; CMV, cytomegalovirus; MOI, multiplicity(ies) of infection; PFU, plaque-forming unit(s); hsvTK, herpes simplex virus thymidine kinase; EGFP, enhanced green fluorescent protein; CAR, Coxsackie and adenovirus receptor. Back

Received 5/ 8/03. Revised 8/17/03. Accepted 9/ 3/03.


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Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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