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Cancer Research 66, 9957-9966, October 15, 2006. doi: 10.1158/0008-5472.CAN-06-1138
© 2006 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Low-Dose Etoposide Enhances Telomerase-Dependent Adenovirus-Mediated Cytosine Deaminase Gene Therapy through Augmentation of Adenoviral Infection and Transgene Expression in a Syngeneic Bladder Tumor Model

Gia-Shing Shieh1,5, Ai-Li Shiau1,2, Yi-Te Yo2, Pey-Ru Lin2, Chao-Ching Chang3, Tzong-Shin Tzai4 and Chao-Liang Wu1,3

1 Institute of Clinical Medicine and Departments of 2 Microbiology and Immunology, 3 Biochemistry and Molecular Biology, and 4 Urology, National Cheng Kung University Medical College, Tainan, Taiwan; and 5 Tainan Hospital, Department of Health, Executive Yuan, Taiwan

Requests for reprints: Chao-Liang Wu, Department of Biochemistry and Molecular Biology, National Cheng Kung University Medical College, 1 Dashiue Road, Tainan 70101, Taiwan. Phone: 886-6-2353535, ext. 5536; Fax: 886-6-2741694; E-mail: wumolbio{at}mail.ncku.edu.tw or Tzong-Shin Tzai, Department of Urology, National Cheng Kung University Medical College, Tainan 70101, Taiwan. Phone: 886-6-2353535, ext. 5253; Fax: 886-6-2383678; E-mail: TTS777{at}mail.ncku.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The human telomerase reverse transcriptase (hTERT) promoter can selectively drive transgene expression in many telomerase-positive human cancer cells. Here we evaluated combination therapy of adenoviral vector Ad-hTERT-CD encoding E. coli cytosine deaminase (CD) driven by the hTERT promoter and low-dose etoposide (0.1 µg/mL) for treating bladder cancer. Ad-hTERT-CD conferred sensitivity to 5-fluorocytosine (5-FC) in bladder cancer cells, which could be enhanced by etoposide treatment, but not in normal cells. Such effect was correlated with up-regulation of hypoxia-inducible factor (HIF)-1{alpha} expression. By contrast, etoposide activated p53 and down-regulated hTERT promoter activity in normal cells. Etoposide also increased adenoviral infection via enhancement of coxsackie-adenovirus receptor expression on bladder cancer and normal cells. Combination index analysis revealed that combined therapy of Ad-hTERT-CD (109 plaque-forming units)/5-FC (200 mg/kg) with etoposide (2 mg/kg) synergistically suppressed tumor growth and prolonged survival in mice bearing syngeneic MBT-2 bladder tumors. This combination therapy regimen induced complete tumor regression and generated antitumor immunity in 75% of tumor-bearing mice. Furthermore, increased infiltrating CD4+ and CD8+ T cells and necrosis within tumors were found in mice receiving combination therapy of Ad-hTERT-CD and etoposide compared with those treated with either treatment alone. Thus, the potential high therapeutic index of the combination therapy may be an appealing therapeutic intervention for bladder cancer. Furthermore, because a majority of human tumors exhibit high telomerase activity, adenovirus-mediated CD gene therapy driven by the hTERT promoter in combination with low-dose etoposide may be applicable to a broad spectrum of cancers. (Cancer Res 2006; 66(20): 9957-66)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Telomerase, a ribonucleoprotein enzyme involved in the synthesis and maintenance of telomeric repeats in the ends of chromosomes, is expressed in immortalized cell lines and in ~90% of human malignancies, but not in most adult somatic tissues (1). The human telomerase reverse transcriptase (hTERT) is the catalytic subunit for human telomerase and its expression is highly associated with telomerase activity (2–4). Telomerase activity and hTERT mRNA expression are detected in most, if not all, cancers including bladder cancer (5–8). By contrast, normal tissues, including those adjacent to cancer, display no or very low levels of telomerase activity. Moreover, the levels of hTERT expression are significantly associated with bladder tumor grade and stage (9). Therefore, hTERT may serve as a good target for gene therapy of bladder cancer and also be useful for targeted transgene expression in human and murine cancers (10–12). The use of hTERT promoter–driven vector system is able to restrict transgene expression to telomerase-positive tumors. Because the vast majority of human bladder cancers express telomerase activity, the hTERT promoter may be exploited for bladder tumor–specific transgene expression in gene therapy of bladder cancer.

Etoposide causes DNA strand breaks and induces apoptosis in a variety of tumor cells. Etoposide up-regulates telomerase activity in human pancreatic cancer cells (13). Furthermore, etoposide at clinically acceptable dosages suppresses humoral and cellular immune responses to adenoviral vectors, thereby enhancing intratumoral transgene expression (14). We hypothesized that low-dose etoposide can increase the infection efficacy of adenoviral vector encoding cytosine deaminase (CD) under the transcriptional control of the hTERT promoter and enhance CD gene expression through up-regulation of the hTERT promoter activity in telomerase-positive bladder cancer cells. Here we show that low-dose etoposide increased the hTERT promoter activity through up-regulation of hypoxia-inducible factor (HIF)-1{alpha} expression and enhanced adenoviral infection via up-regulation of coxsackie-adenovirus receptor (CAR) in bladder cancer cells. Therefore, the combination of etoposide and adenovirus-mediated CD gene therapy driven by the hTERT promoter may have potential applications for the treatment of bladder cancer or other telomerase-positive malignancies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and mice. Normal murine fibroblasts were isolated from adult C3H/HeN mice using standard methods. Seven human cell lines (J82, T24, TSGH-8301, TCC-SUP, and HT-1376 bladder cancer cells as well as WI-38 fetal lung fibroblast and HepG2 hepatocellular carcinoma cells) and four murine cells (MBT-2 bladder transitional cell carcinoma, LL2 Lewis lung carcinoma, NIH 3T3 embryonic fibroblast, and adult fibroblast) were cultured in DMEM supplemented with 10% fetal bovine serum or bovine serum (for NIH 3T3 cells), 2 mmol/L L-glutamine, and 50 µg/mL gentamicin. Male, 6- to 8-week-old C3H/HeN mice were obtained from the Laboratory Animal Center of the National Cheng Kung University. The animals were maintained in specific pathogen-free animal care facility under isothermal conditions with regular photoperiods. The experimental protocol adhered to the rules of the Animal Protection Act of Taiwan and was approved by the Laboratory Animal Care and Use Committee of the National Cheng Kung University.

Analyses of hTERT promoter activity and p53 and HIF-1 transcriptional activities. To quantitatively determine the hTERT promoter activity, cells grown in 24-well plates were cotransfected with 1 µg of pGL3-hTERT (15), a luciferase reporter plasmid driven by the hTERT promoter, and 0.5 µg of pTRELacZ, a ß-galactosidase (ß-gal) reporter vector derived from pTREd2EGFP (Clontech, Palo Alto, CA), with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). In some experiments, at 6 hours posttransfection, cells were treated with various doses of etoposide (Bristol-Myers Squibb, Princeton, NJ). Cell lysates were harvested at 48 hours posttransfection for measuring luciferase activities as previously described (16). Cells were also cotransfected with 1 µg of p53-Luc reporter plasmid and 0.5 µg of pTRELacZ for assessing the transcriptional activity of p53 (16). A DNA fragment containing six tandemly repeated sequences of hypoxia response element (HRE) from the human lactic dehydrogenase A gene (17) just upstream of the cytomegalovirus (CMV) mini promoter derived from pTRE vector (Clontech) was placed upstream of the luciferase reporter gene in a pGL3-Basic vector (Promega, Madison, WI) to generate pHRECMVmini-Luc. Previously, we found that the HIF-1 consensus binding site at –184 relative to the transcription start site of the hTERT promoter was required for induction of the hTERT promoter activity by hypoxia, as determined by deletion analysis and site-directed mutagenesis.6 We therefore used pGL3-184m-hTERT, which carries TTT rather than CAC in the putative HIF-1 binding site located at –184 bp upstream of the transcriptional start site of the hTERT gene, to verify the involvement of HIF-1 in up-regulating hTERT promoter activity by etoposide. The HIF-1 transcriptional activity of etoposide-treated cells was examined using the same protocol as that for assessing p53 transcriptional activity except that pHRECMVmini-Luc or pGL3-184m-hTERT rather than p53-Luc was used. To further confirm the role of HIF-1 on etoposide-induced up-regulation of the hTERT promoter activity, pSUPER vector (ref. 18; kindly provided by Dr. R. Agami, The Netherlands Cancer Institute, Amsterdam, the Netherlands) was used for stable expression of short hairpin RNA (shRNA) targeted to HIF-1{alpha} (19) in cells. MBT-2 cells were cotransfected with pGL3-hTERT and pTRELacZ, as well as 1 µg of pSUPER-HIF-1{alpha} shRNA or control vector (pSUPER), treated with etoposide, and assessed for their luciferase activities.

Adenoviral vectors and infection. The AdEasy system was used to generate Ad-hTERT-CD, an adenoviral vector encoding CD under the control of the hTERT promoter (20). The fragment encompassing the hTERT promoter was excised from pGL3-hTERT by HindIII and KpnI digestion and subcloned into the HindIII/KpnI sites of pShuttle to generate pShuttle-hTERT. The 1.6-kb fragment containing the cDNA of E. coli CD was excised from pCD2 (21) by EcoRI and BamHI digestion, the cohesive ends filled in with Klenow enzyme, and subcloned into pShuttle-hTERT that had been restricted with HindIII and filled in cohesive ends of the linearized fragment with Klenow enzyme, resulting in pShuttle-hTERT-CD. Subsequently, Ad-hTERT-CD was generated as previously described (20). Ad-LacZ encoding ß-gal was used as the control viral vector. Two adenoviral vectors were propagated, purified by CsCl density gradient centrifugation, and quantified by plaque assay. For measuring cell susceptibility to adenovirus infection, various cells were infected with Ad-LacZ at a multiplicity of infection (MOI) of 1 or 10 with or without etoposide (0.1 µg/mL) for 6 hours and then refed with fresh medium without etoposide. The cells were cultured for an additional 42 hours and lysates were assessed for ß-gal activity by a ß-gal reporter gene assay system (Applied Biosystems, Foster City, CA).

In vitro cell viability assay. Cells (2 x 103) grown in 96-well plates overnight were infected with Ad-hTERT-CD in the presence or absence of etoposide for 6 hours. The cells were refed with fresh medium containing 5-fluorocytosine (5-FC; Sigma, St. Louis, MO) with or without etoposide. A colorimetric WST-1 assay (TaKaRa, Shiga, Japan) was used to assess cell viability after 3 days according to the instructions of the manufacturer. Sensitivity to continuous exposure of 5-fluorouracil (5-FU) was assessed for various bladder cancer cells by determining their IC50, which was defined as the concentration at which 50% inhibition of absorbance within 48 hours, with the WST-1 assay.

Immunoblot analysis. MBT-2 cells were cultured with or without etoposide for 6 hours and total cell lysates were harvested for detection of HIF-1{alpha} expression by immunoblot analysis with rabbit polyclonal antibody against HIF-1{alpha} (1:1,000; ab16535, Abcam, Cambridge, United Kingdom). Total lysates from LL2 and HepG2 cells treated with CoCl2 (200 µmol/L) for 6 hours served as the positive control. The blot reprobed with anti-{alpha}-tubulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) served as the internal control.

Flow cytometric analysis of CAR and integrin expressions on bladder cancer cells after etoposide treatment. Cells that had been treated with etoposide (0.1 µg/mL) for 6 hours were incubated on ice with anti-CAR (1:200; RmcB, American Type Culture Collection, Manassas, VA), anti-{alpha}vß3 (1:50; 23C6, PharMingen, San Diego, CA), or anti-{alpha}vß5 (1:200; P1F6, Chemicon, Temecula, CA) monoclonal antibodies for 1 hour followed by incubation for 30 minutes on ice with FITC-conjugated goat anti-mouse antibody. Isotype immunoglobulin G1 (IgG1) was used as a matched control. These samples were analyzed with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA).

Animal studies. MBT-2 cells (2 x 106) were inoculated s.c. into the dorsal flank of C3H/HeN mice at day 0. At day 10, visible and palpable nodules developed at all injection sites ranging from 100 to 300 mm3, with mean tumor volume of 197 ± 13 mm3. Five groups of seven to nine mice were injected i.t. at different tracts and injection sites with 109 plaque-forming units (pfu) of Ad-hTERT-CD or Ad-LacZ in 100 µL of PBS, or with PBS daily at days 11, 12, and 13. One group receiving Ad-hTERT-CD and another group receiving Ad-LacZ were further treated daily with 5-FC (500 mg/kg) i.p. for 14 consecutive days starting on day 12. The remaining three groups received PBS instead. In a separate experiment, four groups of eight tumor-bearing mice were treated with Ad-hTERT-CD (108 or 109 pfu) or PBS daily at days 11, 12, and 13 followed by daily treatment with 5-FC (200 mg/kg) for 7 consecutive days starting on day 12. Additionally, all groups, except one Ad-hTERT-CD (109 pfu) group receiving PBS, were also treated i.p. with etoposide at 2 mg/kg/d, which was defined as a low dose (14), for 8 consecutive days starting on day 11. Tumor volumes were measured as previously described (16). The animals were sacrificed when their primary tumor reached 10% of the body weight.

Histologic and immunohistochemical analyses. To analyze necrosis and cell infiltrates within tumors, tumor-bearing mice treated with different regimens as described above were sacrificed at day 21 and three tumors from each group were excised and fixed in 10% formalin overnight. The tissues were dehydrated and embedded in paraffin and sectioned at 5 µm for H&E or immunohistochemical staining. To detect CD4+ and CD8+ T cells that infiltrated tumors, tumor sections were incubated with rat anti-mouse CD4 (L3T4; GK1.5, PharMingen) or rat anti-mouse CD8a (Ly-2; 53-6.7, PharMingen) antibody and subsequently with biotinylated secondary antibodies and the avidin-biotin-peroxidase complex (DAKO, Carpinteria, CA). Reactions were developed with amino ethyl carbazole (Zymed, South San Francisco, CA) as the chromogenic substrate.

Statistical analysis. The survival analysis was done using the Kaplan-Meier survival curve and the log-rank test. Other statistical differences were assessed with Student's t test. P < 0.05 is regarded statistically significant. Using the CalcuSyn software program (Biosoft, Cambridge, United Kingdom) according to the combination index method of Chou and Talalay (22), significant synergism was determined.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ad-hTERT-CD/5-FC exerted cytotoxic effect in vitro and antitumor effect in vivo against bladder cancer cells. We first assessed the hTERT promoter activity and cell survival following Ad-hTERT-CD/5-FC treatment in various cells using a luciferase reporter assay (Fig. 1A ). MBT-2 cells exhibited much higher hTERT promoter activities compared with mouse fibroblasts and NIH 3T3 cells, indicating that the hTERT promoter was significantly active in murine bladder cancer cells but relatively quiescent in normal fibroblasts. Notably, as NIH 3T3 fibroblasts were derived from spontaneously immortalized mouse embryo culture, they expressed detectable levels of telomerase activity (23). In the presence of 500 µmol/L 5-FC, Ad-hTERT-CD at an MOI of 1 exerted potent cytotoxic effects on MBT-2 cells, whereas cell survival was not affected in NIH 3T3 cells or normal fibroblasts following Ad-hTERT-CD treatment. Regarding human cells, the hTERT promoter activity varied in different human bladder cancer cell lines, whereas it was not detectable in WI-38 fibroblasts. Furthermore, the cytotoxicity of Ad-hTERT-CD/5-FC was largely proportional to the level of hTERT promoter activity. Except for the highest MOIs, Ad-hTERT-CD exerted cytotoxic effects in a dose-dependent manner following treatment with various doses of 5-FC in HT-1376 bladder cancer cells, whereas Ad-hTERT-CD/5-FC did not induce any cytotoxicity in hTERT-negative WI-38 fibroblasts (Fig. 1B). As inherent 5-FU sensitivity significantly contributes to 5-FC response in cells transduced with the CD gene (24), we assessed the sensitivity of bladder cancer cells to 5-FU. The IC50 value for 5-FU was 0.07 µmol/L for HT-1376, 0.35 µmol/L for TCC-SUP, 0.9 µmol/L for J82, 2.12 µmol/L for TSGH-8301, 3.6 µmol/L for T24, and 12.1 µmol/L for MBT-2 cells. Moreover, survival of all the cells tested remained unaffected after treatment with Ad-hTERT-CD or 5-FC alone or with Ad-LacZ plus 5-FC (data not shown). Ad-hTERT-CD/5-FC treatment significantly retarded tumor growth (Fig. 1C) and enhanced survival (Fig. 1D) in MBT-2 tumor–bearing mice compared with Ad-hTERT-CD/PBS, 5-FC, PBS, or Ad-LacZ/5-FC treatment. Collectively, these results show that hTERT promoter effectively drove CD gene expression on 5-FC treatment, which resulted in tumor growth retardation and prolongation of survival of the mice bearing syngeneic MBT-2 bladder tumors.


Figure 1
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  		Figure 1. Ad-hTERT-CD/5-FC exerted cytotoxic effect, which was proportional to the hTERT promoter activity, and antitumor effect on bladder cancer cells. A, various cell lines were assessed for their hTERT promoter activities by a luciferase reporter assay. Cells were infected with Ad-hTERT-CD at an MOI of 1 in the presence of 500 µmol/L 5-FC, and their survival was determined by the WST-1 assay and expressed as the percentage of surviving cells relative to that in the mock-infected cells without 5-FC treatment. B, HT-1376 and WI-38 cells were infected with indicated doses of Ad-hTERT-CD and 5-FC, and their survival was determined. Columns, mean of three (A) or four (B) determinations, which were consistent in two separate experiments; bars, SD. MBT-2 tumor–bearing C3H/HeN mice were treated i.t. with 109 pfu of Ad-hTERT-CD or Ad-LacZ, or with PBS at days 11, 12, and 13, followed by 5-FC (500 mg/kg) or PBS treatment for 14 consecutive days starting on day 12. C, columns, mean tumor volume; bars, SD (P = 0.0002, Ad-hTERT-CD/5-FC versus PBS). D, Kaplan-Meier survival curves (P = 0.0001, Ad-hTERT-CD/5-FC versus PBS).

 
Low-dose etoposide enhanced the hTERT promoter activity through up-regulation of HIF-1{alpha} expression in bladder cancer cells. In MBT-2 cells, etoposide at doses ranging from 0.02 to 0.5 µg/mL enhanced the hTERT promoter activity, with an optimal concentration of 0.1 µg/mL (Fig. 2A ). Because the IC50 of etoposide in MBT-2 cells is >100 µmol/L (25), etoposide at a dose of 0.1 µg/mL (1.7 µmol/L), which was regarded as a low dose, was chosen for subsequent in vitro experiments. Notably, etoposide treatment had no effect on cell survival (Fig. 2A). Etoposide also increased the hTERT promoter activity in human bladder cancer cells displaying various degrees of promoter activity (Fig. 2B). The hTERT promoter activity was enhanced in MBT-2 cells but decreased in NIH 3T3 cells when treated with etoposide (Fig. 2C). These results suggest that following etoposide treatment, the hTERT promoter was significantly active in bladder cancer cells but relatively quiescent in immortalized cells. As etoposide can activate p53 and thereby down-regulate hTERT gene expression (26), we also assessed the effects of p53 status on etoposide-induced up-regulation of the hTERT promoter activity (Fig. 2C). Etoposide increased the hTERT promoter activity ~1.5-fold in MBT-2 cells that harbor mutant p53. By contrast, etoposide decreased the hTERT promoter activity in NIH 3T3 cells, which was associated with enhanced p53 transcriptional activity. Physiologic stress conditions, such as hypoxia, play a role in developing drug resistance in solid tumors. Cancer cells under hypoxic conditions become resistant to multiple drugs, including etoposide. The presence of HIF-1 binding sites on the hTERT promoter prompted us to examine whether HIF-1 was involved in etoposide-induced up-regulation of the hTERT promoter activity in MBT-2 cells. The expression of HIF-1 was significantly up-regulated in etoposide-treated MBT-2 cells, as measured by a hypoxia-responsive luciferase reporter assay (Fig. 3A ) and verified by immunoblot analysis (Fig. 3B). Interestingly, etoposide induced HIF-1 transcriptional activity in MBT-2 but not in NIH 3T3 cells (Fig. 3A). We also used pGL3-184m-hTERT, which carries mutations in the putative HIF-1 binding site of the hTERT gene, to show that mutation of the HIF-1 binding site abolished the activation of the hTERT promoter by etoposide in MBT-2 cells (Fig. 3C). Notably, mutant promoter decreased the basal transcriptional activity by ~30% as compared with the wild-type promoter (Fig. 2A). To further confirm our results, we also determined whether knock-down of HIF-1{alpha} expression by shRNA suppressed etoposide-induced up-regulation of the hTERT promoter activity (Fig. 3D). Whereas control shRNA did not affect the hTERT promoter activity in our reporter assay, HIF-1{alpha} shRNA abrogated etoposide-induced enhancement of the hTERT promoter activity. Taken together, these results suggest that etoposide-induced activation of the hTERT promoter can be mediated via the transcription factor HIF-1 binding to the HRE in the hTERT promoter in bladder cancer cells.


Figure 2
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  		Figure 2. Low-dose etoposide enhanced the hTERT promoter activity in various bladder cancer cells. A, the hTERT promoter activity and survival of MBT-2 cells treated with indicated concentrations of etoposide were assessed by a luciferase reporter assay and the WST-1 assay, respectively. B, etoposide (0.1 µg/mL) increased the hTERT promoter activity in human bladder cancer cells displaying various degrees of promoter activity. C, etoposide (0.1 µg/mL) increased the hTERT promoter activity in MBT-2 cells that harbor mutant p53, but decreased the hTERT promoter activity in NIH 3T3 cells, which was associated with enhanced p53 transcriptional activity.

 

Figure 3
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  		Figure 3. Low-dose etoposide enhanced the hTERT promoter activity through up-regulation of HIF-1{alpha} expression in bladder cancer cells. A, etoposide induced HIF-1 transcriptional activity in MBT-2 but not in NIH 3T3 cells, as measured by a hypoxia-responsive luciferase reporter assay. *, P < 0.05; **, P < 0.01, compared with MBT-2 cells without etoposide treatment. B, detection of HIF-1{alpha} expression in MBT-2 cells treated with or without etoposide by immunoblot analysis. LL2 and HepG2 cells treated with CoCl2 to mimic hypoxic conditions served as the positive control. The expression of {alpha}-tubulin served as the quantitative control. C, mutation of the HIF-1-binding site abolished the activation of the hTERT promoter by etoposide in MBT-2 cells. D, transfection of HIF-1{alpha} shRNA expression vector into MBT-2 cells abrogated etoposide-induced enhancement of the hTERT promoter activity, whereas transfection of the control vector did not have such effect.

 
Low-dose etoposide enhanced adenovirus infection through up-regulation of CAR, but not of {alpha}vß3 or {alpha}vß5 integrins, on bladder cancer cells. Efficient adenovirus infection requires CAR and {alpha}vß integrins for attachment and internalization to enter host cells. To study the effects of etoposide on adenovirus infectivity in bladder cancer cells, expressions of CAR as well as {alpha}vß3 and {alpha}vß5 integrins were detected by flow cytometric analysis. As shown in Fig. 4A , J82 cells expressed moderate levels whereas TCC-SUP cells expressed high levels of CAR. However, both cells expressed little {alpha}vß3 and {alpha}vß5 integrins. Furthermore, etoposide enhanced CAR expression on both cells but had little effects on {alpha}vß3 and {alpha}vß5 integrin expressions. The increase of cell-surface CAR may have led to an increase in transgene expression and subsequent protein production. We also confirmed that the increased CAR level allowed increased adenovirus entry into cells. In J82, TCC-SUP, MBT-2, and NIH 3T3 cells, etoposide treatment enhanced ß-gal activities in Ad-LacZ-infected cells (Fig. 4B), suggesting that enhanced efficiency of adenoviral transgene expression by etoposide treatment may have been attributed, in part, to the up-regulation of CAR expression. Notably, not only bladder cancer cells but also NIH 3T3 fibroblasts became more susceptible to adenoviral infection after etoposide treatment.


Figure 4
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  		Figure 4. Low-dose etoposide enhanced adenovirus infection through up-regulation of CAR on bladder cancer cells. A, J82 and TCC-SUP cells were cultured in the presence or absence of etoposide (0.1 µg/mL) for 6 hours and analyzed for surface expressions of CAR as well as {alpha}vß3 and {alpha}vß5 integrins by flow cytometry. Etoposide-treated (thick line) and untreated cells (dotted line) were incubated with respective monoclonal antibody. Thin line, untreated cells incubated with isotype IgG1 for background staining. For each panel, the top number (+) indicates the mean fluorescence intensity for the etoposide-treated cell population, whereas the bottom number (–) is the untreated population. B, J82, TCC-SUP, MBT-2, and NIH 3T3 cells were infected with Ad-LacZ at an MOI of 1 or 10 with or without etoposide (0.1 µg/mL) for 6 hours. After 42 hours, ß-gal activity in the cell lysate was determined and expressed in relative light units per microgram of protein. Columns, mean of three determinations, which were consistent in two separate experiments; bars, SD.

 
Low-dose etoposide enhanced cytotoxic activity of Ad-hTERT-CD/5-FC in various bladder cancer cells. The effect of low-dose etoposide on the cytotoxicity of Ad-hTERT-CD/5-FC was assessed in human and murine cell lines. Cell survival was decreased as the doses of Ad-hTERT-CD and 5-FC were increased in human TSGH-8301, T24, J82, and TCC-SUP bladder cancer cells (Fig. 5A ). The degree of cytotoxicity seemed to be a function of adenoviral dose and 5-FC concentration. Remarkably, the cytotoxic effects were further enhanced when these cells were concomitantly treated with etoposide. In MBT-2 cells, there was a good dose-response relationship between Ad-hTERT-CD/5-FC doses and cytotoxicity (Fig. 5B). However, Ad-hTERT-CD/5-FC had only negligible effects on cell survival in NIH 3T3 cells regardless of treatment with etoposide (Fig. 5B). Results with higher doses of Ad-hTERT-CD or 5-FC were the exception, displaying a small degree of cytotoxicity in the absence of etoposide. Nevertheless, etoposide treatment suppressed the cytotoxic effect induced by Ad-hTERT-CD/5-FC. Notably, cell survival remained unaffected after treatment with Ad-hTERT-CD, 5-FC, etoposide, or Ad-LacZ/5-FC alone (data not shown). Collectively, Ad-hTERT-CD conferred sensitivity to 5-FC in human and murine bladder cancer cells, which could be enhanced by etoposide, but not in normal cells. These results suggest that low-dose etoposide may improve the therapeutic index of Ad-hTERT-CD/5-FC for killing telomerase-positive bladder cancer cells.


Figure 5
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  		Figure 5. Low-dose etoposide enhanced cytotoxic activity of Ad-hTERT-CD/5-FC in various bladder cancer cells. Human TSGH-8301, T24, J82 and TCC-SUP bladder cancer cells (A) as well as murine MBT-2 and NIH 3T3 cells (B) were infected with Ad-hTERT-CD at indicated MOI in the presence or absence of etoposide (0.1 µg/mL) for 6 hours. The cells were refed with fresh medium supplemented with indicated concentrations of 5-FC and etoposide (0.1 µg/mL). Cell survival was determined after 3 days by the WST-1 assay and expressed as the percentage of surviving cells relative to that in the mock-infected cells without 5-FC treatment. Columns, mean of four determinations, which were consistent in two separate experiments; bars, SD.

 
Ad-hTERT-CD/5-FC in combination with low-dose etoposide synergistically suppressed tumor growth and prolonged survival in mice bearing syngeneic MBT-2 tumors. Although Ad-hTERT-CD alone followed by 5-FC treatment slowed tumor growth in MBT-2 tumor–bearing mice (Fig. 1C), the survival rate was only enhanced from 0% to 30% (Fig. 1D). Based on the in vitro data described above, we sought to enhance the therapeutic effect of Ad-hTERT-CD/5-FC by combination with low-dose etoposide. The severe side effects of 5-FC, including hepatotoxicity and bone marrow depression, are concentration dependent and can be elicited by many other agents, such as immunosuppressive or cytostatic agents (27). To avoid any possible side effects of the combination therapy, 500 mg/kg 5-FC for 14 days, which was considered the standard treatment regimen (28), was replaced by 200 mg/kg 5-FC for 7 days. As shown in Fig. 6A and B , whereas low-dose etoposide had no antitumor effects, Ad-hTERT-CD/5-FC (109 pfu) alone retarded tumor growth (P = 0.0002) and prolonged survival (P = 0.0003) in MBT-2 tumor–bearing mice compared with etoposide treatment alone. Ad-hTERT-CD alone inhibited tumor growth by 22 days, with regrowth of tumors thereafter when 5-FC was no longer administered. Remarkably, Ad-hTERT-CD (109 pfu)/5-FC in combination with etoposide synergistically suppressed tumor growth and enhanced survival in tumor-bearing mice compared with either Ad-hTERT-CD/5-FC (P = 0.0002 for tumor volume and P = 0.0003 for survival) or etoposide (P = 0.0001 for tumor volume and P = 0.0001 for survival) treatment alone. The synergistic effect between etoposide and Ad-hTERT-CD/5-FC treatment groups was verified by combination index values (combination index < 1 represents synergistic activity; combination index = 1 represents additive activity). For the combination therapy, the combination index values from tumor volume data for high and low viral doses were 0.03 and 0.09, whereas those from survival time data were 0.17 and 0.24, respectively. Furthermore, there was no significant difference between mice receiving Ad-hTERT-CD (109 pfu)/5-FC and those receiving Ad-hTERT-CD (108 pfu)/5-FC on tumor volume and survival. Notably, combination of Ad-hTERT-CD/5-FC and etoposide resulted in complete tumor regression in six of eight high-dose Ad-hTERT-CD–treated mice and in four of eight low-dose Ad-hTERT-CD–treated mice without tumor regrowth. Importantly, administration of Ad-hTERT-CD followed by etoposide and 5-FC treatments produced no weight loss or other overt signs of toxicity in mice. In addition, all tumor-free mice that had been rechallenged s.c. with MBT-2 cells (4 x 106) on the opposite flank region at day 70 remained tumor-free after 5 months. Therefore, high-dose and low-dose Ad-hTERT-CD/5-FC in combination with low-dose etoposide induced complete tumor regression in 75% and 50% of tumor-bearing mice and generated antitumor immunity, respectively. As shown in Fig. 5C, H&E staining reveals necrosis within tumors from mice treated with Ad-hTERT-CD/5-FC alone. Furthermore, Ad-hTERT-CD/5-FC plus etoposide induced more tumor necrosis. Immunohistochemical staining shows that CD4+ and CD8+ T cells that infiltrated tumors were significantly increased in the combination treatment group compared with those in the remaining groups. These results suggest that activated immune cells within tumors may have contributed to higher antitumor effects of the combination regimen.


Figure 6
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  		Figure 6. Ad-hTERT-CD/5-FC in combination with low-dose etoposide synergistically suppressed tumor growth and prolonged survival in mice bearing syngeneic MBT-2 tumors. Groups of eight tumor-bearing mice were treated with Ad-hTERT-CD (108 or 109 pfu) i.t. at days 11, 12, and 13 followed by 5-FC (200 mg/kg) treatment for 7 consecutive days starting on day 12, or with etoposide (2 mg/kg) for 8 consecutive days starting on day 11 alone or in combination. A, points, mean tumor volume; bars, SD. P = 0.0001, 0.0001, and 0.0002 for Ad-hTERT-CD (109)/etoposide, Ad-hTERT-CD (108)/etoposide, and Ad-hTERT-CD (109) versus etoposide, respectively; P = 0.0002 and 0.0016 for Ad-hTERT-CD (109)/etoposide and Ad-hTERT-CD (108)/etoposide versus Ad-hTERT-CD (109), respectively. B, Kaplan-Meier survival curves. P = 0.0001, 0.0001, and 0.0003 for Ad-hTERT-CD (109)/etoposide, Ad-hTERT-CD (108)/etoposide, and Ad-hTERT-CD (109) versus etoposide, respectively; P = 0.0003 and 0.0005 for Ad-hTERT-CD (109)/etoposide and Ad-hTERT-CD (108)/etoposide versus Ad-hTERT-CD (109), respectively. C, in a separate experiment, tumors were excised from the treated mice at day 21 and subjected to H&E and immunohistochemical stainings for detection of infiltrating CD4+ and CD8+ T cells that were stained red within tumors.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The hTERT promoter has been used to drive tumor-specific apoptotic transgene expression in human telomerase-positive cells and suppress xenograft tumor growth in nude mice (10–12) because telomerase levels are low in adult murine tissues and primary cultured cells but high in most tumors cells (29–31). In this study, although Ad-hTERT-CD/5-FC retarded tumor growth initially, tumors recurred in all the treated mice when 5-FC was no longer administered. The transduction efficiency of adenoviral vectors in tumors by i.t. injection may be low (32). Moreover, high MOIs of adenoviral vectors encoding suicide gene may be required to achieve effective killing of tumor cells (33). Therefore, inefficient vector delivery to tumor cells and resistance to suicide gene therapy remained to be resolved.

Low-dose etoposide can suppress immune responses to adenoviral vectors and enhance adenovirus-mediated intratumoral transgene expression (14). In this study, we show that low-dose etoposide in conjunction with Ad-hTERT-CD/5-FC significantly enhanced cytotoxic effects against murine bladder cancer cells but failed to induce evident cytotoxicity in NIH 3T3 fibroblasts, even at high MOIs of adenoviral vectors. As NIH 3T3 cells, which were spontaneously immortalized embryonic fibroblasts, expressed detectable levels of telomerase activity (23), they may have expressed CD, albeit to a lesser extent, following high MOIs of Ad-hTERT-CD infection, leading to a small degree of cytotoxic effects. Interestingly, etoposide treatment abolished such effects. Along the same line, our results show that whereas etoposide enhanced the hTERT promoter activity in cancer cells harboring mutant p53, it actually suppressed the promoter activity in NIH 3T3 cells that carry wild-type p53. Moreover, etoposide induced HIF-1 transcriptional activity in MBT-2 but not in NIH 3T3 cells. Our results also show enhanced cytotoxic effects of Ad-hTERT-CD/5-FC by etoposide in a variety of human bladder cancer cells. Possible mechanisms involved in adenoviral transduction seem to be correlated with CAR and integrin expressions on target cells (34–36). It has been shown that adenoviral transduction was enhanced in cisplatin-resistant human laryngeal carcinoma cells, which was associated with increased expressions of {alpha}vß3 integrin and CAR (37). To this end, cisplatin and etoposide enhanced CAR expression in two of five human ovarian cancer cell lines tested, which resulted in adenovirus-mediated transgene expression (38). In this study, we show that adenoviral transduction was significantly enhanced in bladder cancer cells after exposure to etoposide, which was associated with increased expression of CAR. These results suggest that enhancement of CAR expression may be one of the mechanisms contributing to the higher cytotoxic and tumoricidal effects of Ad-hTERT-CD/5-FC plus etoposide on MBT-2 cells in vitro and in vivo. In the clinical settings, low efficiency of gene transfer with adenoviral vectors in cancer gene therapy due to low expression of CAR may be improved by administration of etoposide at a clinically acceptable dosage.

In bladder cancer cell lines, responses to Ad-hTERT-CD/5-FC were variable, which could be attributable to the differences not only in the expression level of CD driven by the hTERT promoter but also in the susceptibility to adenoviral infection and sensitivity to 5-FC. It has been reported that whereas both gene transfer efficiency of adenoviral vector and inherent 5-FU sensitivity significantly contribute to overall 5-FC responses of cells after CD gene transfer, inherent 5-FU sensitivity is a more contributing factor (24). In our in vitro data, cytotoxic effects of Ad-hTERT-CD/5-FC on different cells were largely paralleled with the levels of the hTERT promoter activity. Nevertheless, levels of the hTERT promoter activity were not the only critical factor that determined the cell survival following Ad-hTERT-CD/5-FC treatment. For instance, whereas the hTERT promoter seemed to be less active in HT-1376 cells than in MBT-2 cells (Fig. 1A), they exhibited similar degrees of cytotoxicity induced by Ad-hTERT-CD/5-FC. Based on IC50 values, HT-1376 cells were ~170 times more sensitive to 5-FU than MBT-2 cells, which may have decreased their sensitivity threshold to Ad-hTERT-CD/5-FC. Among different bladder cancer cells used in this study, TSGH-8301 cells, which exhibited the lowest hTERT promoter activity but intermediate 5-FU sensitivity, were the most susceptible bladder cancer cell line to adenoviral infection (39). Nevertheless, TSGH-8301 cells were the least sensitive cell line to Ad-hTERT-CD/5-FC-induced cytotoxicity (Fig. 1A). These data suggest that the CD expression level, which was dependent on the hTERT promoter activity, may be an important factor contributing to the cytotoxicity of Ad-hTERT-CD. The cytotoxic effect of Ad-hTERT-CD plus 5-FC was dose dependent in that the cell survival decreased when the doses of adenoviral vectors and/or 5-FC increased. However, in some cells infected with 10 MOI of Ad-hTERT-CD, such as HT-1376 cells (Fig. 1B), 5-FC produced a dose-response decrease in cell survival at doses up to 50 µmol/L. At higher doses, a plateau was reached, suggesting that 5-FU formation may reach a saturation point due to limitation of the CD enzyme via Ad-hTERT-CD gene transfer. Along this line, HT-1376 cells were the least susceptible cells to adenoviral infection (data not shown) and displayed the lowest IC50 of 5-FU among the bladder cancer cell lines examined here.

In the present study, the diverse effects of etoposide on the cytotoxicity of Ad-hTERT-CD/5-FC between cancer and normal cells may be attributed, in part, to the level of hTERT promoter activity that was modulated by etoposide. Several transcription factors are involved in the regulation of the hTERT promoter activity. c-Myc and SP1 play as activators, whereas Mad1, p53, and zinc-finger factor MZF2 are repressors (40–44). In this study, our data show that low-dose etoposide enhanced the transcriptional activity of p53, and thereby down-regulated the hTERT promoter activity in NIH 3T3 cells that harbor wild-type p53. Up-regulation of the hTERT promoter activity has been shown to correlate with the activation of HIF-1{alpha} transcription factor under hypoxic condition (45, 46). We also show that low-dose etoposide induced HIF-1{alpha} expression in MBT-2 cells, leading to enhanced hTERT promoter activity. The HREs in the hTERT promoter were essential for the up-regulation of the promoter by etoposide because the hTERT promoter was not transactivated by etoposide when the HIF-1 binding site was mutated. Furthermore, we found that the basal level of the hTERT promoter activity was decreased by mutations of the HIF-1 binding site (Fig. 3C), which is in accord with previous work (47). Because the HIF-1 consensus binding site (5'-RCGTG-3') overlaps the E box (CACGTG) known to bind several transcription factors, such as c-Myc, Max, and Mad (48), HIF-1 may compete with these factors for binding to the E box. Taken together, these results suggest that etoposide-induced activation of the hTERT promoter can be mediated via binding of the transcription factor HIF-1 to the HRE in the hTERT promoter in bladder cancer cells.

In conclusion, low-dose etoposide enhances the antitumor effect of Ad-hTERT-CD/5-FC in bladder tumor–bearing mice by increasing adenoviral infection, up-regulating the hTERT promoter activity, and inducing antitumor immune responses. Notably, we evaluated Ad-hTERT-CD and low-dose etoposide as anticancer agents in the syngeneic MBT-2 bladder tumor model, which resembles more closely the situation in human cancer. In addition, as etoposide activates p53, which down-regulates the hTERT promoter activity in normal cells, it does not confer sensitivity to 5-FC. Thus, combination of low-dose etoposide with Ad-hTERT-CD/5-FC provides therapeutic safety and high efficacy in killing telomerase-positive cancer cells while sparing normal cells. The potential high therapeutic index of Ad-hTERT-CD/5-FC system in combination with etoposide may be an appealing therapeutic intervention for bladder cancer. Because intact urothelium can function as an effective barrier, bladder is suitable for intravesical instillation of adenoviral vectors. In line with this notion, intravesical instillation of adenoviral vectors is safe, feasible, and biologically active in patients with bladder cancer (49). As etoposide is widely used in the clinical settings, it serves as a feasible activator to enhance the therapeutic index of hTERT promoter–driven suicide gene therapy. Because a majority of human tumors exhibit high telomerase activity, adenovirus-mediated CD gene therapy driven by the hTERT promoter in combination with low-dose etoposide may be applicable to a broad spectrum of cancers.


   Acknowledgments
 
Grant support: National Science Council grants NSC 89-2318-B-006-013-M51 and 90-2318-B-006-003-M51 (C-L. Wu), Department of Health, the Executive Yuan grant DOH-9011 (G-S. Shieh), and the Foundation of Chen, Jieh-Chen Scholarship, Tainan, Taiwan.

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.

We thank Dr. M. Takakura (Department of Obstetrics and Gynecology, Kanazawa University, Japan), Dr. B. Vogelstein (Johns Hopkins University School of Medicine, Baltimore, MD), and Dr. C.A. Mullen (NIH, Bethesda, MD) for providing pGL3-hTERT-luciferase, plasmids for AdEasy system, and pCD2, respectively.


   Footnotes
 
6 A-L. Shiau, W-S. Wang, G-S. Shieh, and C-L. Wu, unpublished data. Back

Received 3/28/06. Revised 6/29/06. Accepted 8/14/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kim NW, Piatyszek MA, Prowse KR, et al. Specific association of human telomerase activity with immortal cells and cancer. Science 1994;266:2011–5.[Abstract/Free Full Text]
  2. Harrington L, Zhou W, McPhail T, et al. Human telomerase contains evolutionarily conserved catalytic and structural subunits. Genes Dev 1997;11:3109–15.[Abstract/Free Full Text]
  3. Nakamura TM, Morin GB, Chapman KB, et al. Telomerase catalytic subunit homologs from fission yeast and human. Science 1997;277:955–9.[Abstract/Free Full Text]
  4. Meyerson M, Counter CM, Eaton EN, et al. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 1997;90:785–95.[CrossRef][Medline]
  5. Lin Y, Miyamoto H, Fujinami K, et al. Telomerase activity in human bladder cancer. Clin Cancer Res 1996;2:929–32.[Abstract]
  6. Kyo S, Kunimi K, Uchibayashi T, Namiki M, Inoue M. Telomerase activity in human urothelial tumors. Am J Clin Pathol 1997;107:555–60.[Medline]
  7. Ito H, Kyo S, Kanaya T, Takakura M, Inoue M, Namiki M. Expression of human telomerase subunits and correlation with telomerase activity in urothelial cancer. Clin Cancer Res 1998;4:1603–8.[Abstract]
  8. Suzuki T, Suzuki Y, Fujioka T. Expression of the catalytic subunit associated with telomerase gene in human urinary bladder cancer. J Urol 1999;162:2217–20.[CrossRef][Medline]
  9. De Kok JB, Schalken JA, Aalders TW, Ruers TJ, Willems HL, Swinkels DW. Quantitative measurement of telomerase reverse transcriptase (hTERT) mRNA in urothelial cell carcinomas. Int J Cancer 2000;87:217–20.[CrossRef][Medline]
  10. Gu J, Kagawa S, Takakura M, et al. Tumor-specific transgene expression from the human telomerase reverse transcriptase promoter enables targeting of the therapeutic effects of the Bax gene to cancers. Cancer Res 2000;60:5359–64.[Abstract/Free Full Text]
  11. Koga S, Hirohata S, Kondo Y, et al. A novel telomerase-specific gene therapy: gene transfer of caspase-8 utilizing the human telomerase catalytic subunit gene promoter. Hum Gene Ther 2000;11:1397–406.[CrossRef][Medline]
  12. Komata T, Kondo Y, Kanzawa T, et al. Treatment of malignant glioma cells with the transfer of constitutively active caspase-6 using the human telomerase catalytic subunit (human telomerase reverse transcriptase) gene promoter. Cancer Res 2001;61:5796–802.[Abstract/Free Full Text]
  13. Sato N, Mizumoto K, Kusumoto M, et al. Up-regulation of telomerase activity in human pancreatic cancer cells after exposure to etoposide. Br J Cancer 2000;82:1819–26.[CrossRef][Medline]
  14. Bouvet M, Fang B, Ekmekcioglu S, et al. Suppression of the immune response to an adenovirus vector and enhancement of intratumoral transgene expression by low-dose etoposide. Gene Ther 1998;5:189–95.[CrossRef][Medline]
  15. Horikawa I, Cable PL, Afshari C, Barrett JC. Cloning and characterization of the promoter region of human telomerase reverse transcriptase gene. Cancer Res 1999;59:826–30.[Abstract/Free Full Text]
  16. Hsieh JL, Wu CL, Lee CH, Shiau L. Hepatitis B virus X protein sensitizes hepatocellular carcinoma cells to cytolysis induced by E1B-deleted adenovirus through the disruption of p53 function. Clin Cancer Res 2003;9:338–45.[Abstract/Free Full Text]
  17. Kimura H, Weisz A, Ogura T, et al. Identification of hypoxia-inducible factor 1 ancillary sequence and its function in vascular endothelial growth factor gene induction by hypoxia and nitric oxide. J Biol Chem 2001;276:2292–8.[Abstract/Free Full Text]
  18. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296:550–3.[Abstract/Free Full Text]
  19. Ameri K, Lewis CE, Raida M, Sowter H, Hai T, Harris AL. Anoxic induction of ATF-4 through HIF-1-independent pathways of protein stabilization in human cancer cells. Blood 2004;103:1876–82.[Abstract/Free Full Text]
  20. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A 1998;95:2509–14.[Abstract/Free Full Text]
  21. Mullen CA, Kilstrup M, Blaese RM. Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytosine: a negative selection system. Proc Natl Acad Sci U S A 1992;89:33–7.[Abstract/Free Full Text]
  22. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984;22:27–55.[CrossRef][Medline]
  23. Prowse KR, Greider CW. Developmental and tissue-specific regulation of mouse telomerase and telomere length. Proc Natl Acad Sci U S A 1995;92:4818–22.[Abstract/Free Full Text]
  24. Miller CR, Williams CR, Buchsbaum DJ, Gillespie GY. Intratumoral 5-fluorouracil produced by cytosine deaminase/5-fluorocytosine gene therapy is effective for experimental human glioblastomas. Cancer Res 2002;62:773–80.[Abstract/Free Full Text]
  25. Alexander RB, Isaacs JT, Coffey DS. Tumor necrosis factor enhances the in vitro and in vivo efficacy of chemotherapeutic drugs targeted at DNA topoisomerase II in the treatment of murine bladder cancer. J Urol 1987;138:427–9.[Medline]
  26. Chresta CM, Masters JR, Hickman JA. Hypersensitivity of human testicular tumors to etoposide-induced apoptosis is associated with functional p53 and a high Bax:Bcl-2 ratio. Cancer Res 1996;56:1834–41.[Abstract/Free Full Text]
  27. Vermes A, Guchelaar HJ, Dankert J. Flucytosine: a review of its pharmacology, clinical indications, pharmacokinetics, toxicity and drug interactions. J Antimicrob Chemother 2000;46:171–9.[Abstract/Free Full Text]
  28. Huber BE, Austin EA, Good SS, Knick VC, Tibbels S, Richards CA. In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinoma cells genetically modified to express cytosine deaminase. Cancer Res 1993;53:4619–26.[Abstract/Free Full Text]
  29. Chadeneau C, Siegel P, Harley CB, Muller WJ, Bacchetti S. Telomerase activity in normal and malignant murine tissues. Oncogene 1995;11:893–8.[Medline]
  30. Greenberg RA, Allsopp RC, Chin L, Morin GB, DePinho RA. Expression of mouse telomerase reverse transcriptase during development, differentiation and proliferation. Oncogene 1998;16:1723–30.[CrossRef][Medline]
  31. Martin-Rivera L, Herrera E, Albar JP, Blasco MA. Expression of mouse telomerase catalytic subunit in embryos and adult tissues. Proc Natl Acad Sci U S A 1998;95:10471–6.[Abstract/Free Full Text]
  32. Shirakawa T, Gardner TA, Ko SC, et al. Cytotoxicity of adenoviral-mediated cytosine deaminase plus 5-fluorocytosine gene therapy is superior to thymidine kinase plus acyclovir in a human renal cell carcinoma model. J Urol 1999;162:949–54.[CrossRef][Medline]
  33. Freund CT, Tong XW, Block A, et al. Adenovirus-mediated suicide gene therapy for bladder cancer: comparison of the cytomegalovirus- and Rous sarcoma virus-promoter. Anticancer Res 2000;20:2811–6.[Medline]
  34. Wickham TJ, Mathias P, Cheresh DA, Nemerow GR. Integrins {alpha}vß3 and {alpha}vß5 promote adenovirus internalization but not virus attachment. Cell 1993;73:309–19.[CrossRef][Medline]
  35. Bergelson JM, Cunningham JA, Droguett G, et al. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 1997;275:1320–3.[Abstract/Free Full Text]
  36. Li E, Brown SL, Stupack DG, Puente XS, Cheresh DA, Nemerow GR. Integrin {alpha}vß1 is an adenovirus coreceptor. J Virol 2001;75:5405–9.[Abstract/Free Full Text]
  37. Ambriovic-Ristov A, Gabrilovac J, Cimbora-Zovko T, Osmak M. Increased adenoviral transduction efficacy in human laryngeal carcinoma cells resistant to cisplatin is associated with increased expression of integrin {alpha}vß3 and coxsackie adenovirus receptor. Int J Cancer 2004;110:660–7.[Medline]
  38. Hemminki A, Kanerva A, Liu B, et al. Modulation of coxsackie-adenovirus receptor expression for increased adenoviral transgene expression. Cancer Res 2003;63:847–53.[Abstract/Free Full Text]
  39. Hsieh JL, Wu CL, Lai MD, Lee CH, Tsai CS, Shiau AL. Gene therapy for bladder cancer using E1B-55 kD-deleted adenovirus in combination with adenoviral vector encoding plasminogen kringles 1-5. Br J Cancer 2003;88:1492–9.[CrossRef][Medline]
  40. Takakura M, Kyo S, Kanaya T, et al. Cloning of human telomerase catalytic subunit (hTERT) gene promoter and identification of proximal core promoter sequences essential for transcriptional activation in immortalized and cancer cells. Cancer Res 1999;59:551–7.[Abstract/Free Full Text]
  41. Fujimoto K, Kyo S, Takakura M, et al. Identification and characterization of negative regulatory elements of the human telomerase catalytic subunit (hTERT) gene promoter: possible role of MZF-2 in transcriptional repression of hTERT. Nucleic Acids Res 2000;28:2557–62.[Abstract/Free Full Text]
  42. Gunes C, Lichtsteiner S, Vasserot AP, Englert C. Expression of the hTERT gene is regulated at the level of transcriptional initiation and repressed by Mad1. Cancer Res 2000;60:2116–21.[Abstract/Free Full Text]
  43. Kyo S, Takakura M, Taira T, et al. Sp1 cooperates with c-Myc to activate transcription of the human telomerase reverse transcriptase gene (hTERT). Nucleic Acids Res 2000;28:669–77.[Abstract/Free Full Text]
  44. Lin SY, Elledge SJ. Multiple tumor suppressor pathways negatively regulate telomerase. Cell 2003;113:881–9.[CrossRef][Medline]
  45. Yatabe N, Kyo S, Maida Y, et al. HIF-1-mediated activation of telomerase in cervical cancer cells. Oncogene 2004;23:3708–15.[CrossRef][Medline]
  46. Anderson CJ, Hoare SF, Ashcroft M, Bilsland AE, Keith WN. Hypoxic regulation of telomerase gene expression by transcriptional and post-transcriptional mechanisms. Oncogene 2006;25:61–9.[Medline]
  47. Nishi H, Nakada T, Kyo S, Inoue M, Shay JW, Isaka K. Hypoxia-inducible factor 1 mediates up-regulation of telomerase (hTERT). Mol Cell Biol 2004;24:6076–83.[Abstract/Free Full Text]
  48. Wu KJ, Grandori C, Amacker M, et al. Direct activation of TERT transcription by c-MYC. Nat Genet 1999;21:220–4.[CrossRef][Medline]
  49. Kuball J, Wen SF, Leissner J, et al. Successful adenovirus-mediated wild-type p53 gene transfer in patients with bladder cancer by intravesical vector instillation. J Clin Oncol 2002;20:957–65.[Abstract/Free Full Text]



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