Selective Ablation of Human Cancer Cells by Telomerase-Specific

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Selective ablation of human cancer cells by telomerase-speciﬁc adenoviral suicide gene therapy vectors expressing bacterial nitroreductase

Alan E Bilsland1, Claire J Anderson1, Aileen J Fletcher-Monaghan1, Fiona McGregor1, TR Jeffry Evans1, Ian Ganly1, Richard J Knox2, Jane A Plumb1 and W Nicol Keith*,1

1Cancer Research UK Department of Medical Oncology, University of Glasgow, Cancer Research UK Beatson Laboratories, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, UK; 2Enact Pharma Plc, Porton Down Science Park, Salisbury SP4 0JQ, UK

Reactivation of telomerase maintains telomere function malignant cells leading to dose-limiting toxicity. Recent and is considered critical to immortalization in most insights into tumour cell biology have provided a wealth human cancer cells. Elevation of telomerase expression in of possibilities for the development of novel mechanism- cancer cells is highly specific: transcription of both RNA based therapeutics (Garrett and Workman, 1999; (hTR) and protein (hTERT) components is strongly Karamouzis et al., 2002; Keith et al., 2002; Scapin, upregulated in cancer cells relative to normal cells. 2002). Therefore, telomerase promoters may be useful in cancer An interesting target for the development of novel gene therapy by selectively expressing suicide genes in anticancer strategies is telomerase, a ribonucleoprotein cancer cells and not normal cells. One example of suicide reverse transcriptase that extends human telomeres by a gene therapy is the bacterial nitroreductase (NTR) gene, terminal transferase activity (White et al., 2001; Keith which bioactivates the prodrug CB1954 into an active et al., 2002; Mergny et al., 2002). Human telomerase cytotoxic alkylating agent. We describe construction of activity is present during embryonic development, but is adenovirus vectors harbouring the bacterial NTR gene repressed in most adult somatic tissues with low levels of under control of the hTR or hTERT promoters. Western activity detected in some tissues with self-renewal blot analysis of NTR expression in normal and cancer capacity. By contrast, telomerase is highly active in the cells infected with adenoviral vectors showed cancer cell- vast majority of human tumours (Kim et al., 1994; Shay specific nitroreductase expression. Infection with adeno- and Bacchetti, 1997; Holt and Shay, 1999). Reactivation viral telomerase–NTR constructs in a panel of seven of telomerase expression in immortal cell populations cancer cell lines resulted in up to 18-fold sensitization to compensates for cell division-associated telomere attri- the prodrug CB1954, an effect that was retained in two tion and is considered to be a critical step in drug-resistant ovarian lines. Importantly, no sensitization immortalization of human cancer cells in vitro and in was observed with either promoter in any of the four vivo (Counter et al., 1992). It is increasingly clear that normal cell strains. Finally, an efficacious effect was telomerase activity is controlled on multiple levels, with observed in cervical and ovarian xenograft models the possibility of many strategies for therapeutic following single intratumoural injection with low doses intervention (White et al., 2001; Keith et al., 2002; of vector, followed by injection with CB1954. Mergny et al., 2002), including inhibitors that lead to Oncogene (2003) 22, 370–380. doi:10.1038/sj.onc.1206168 telomere shortening and apoptosis or senescence in vitro and decreased tumourigenic potential in vivo (Damm Keywords: telomerase; hTERT; hTR; gene therapy; et al., 2001; Hahn et al., 1999; Naasani et al., 1999; nitroreductase; GDEPT; adenovirus Zhang et al., 1999; Pascolo et al., 2002). Transcription of the core RNA (hTR) and reverse transcriptase (hTERT) components constitutes the major level of differential Introduction regulation between normal and cancer cells. Therefore, the differential activities of the hTR and hTERT A major problem with conventional anticancer cyto- promoters may provide a sound basis for the develop- toxic therapies is their low therapeutic index: many ment of transcriptionally directed cytotoxic gene ther- conventional drugs display a lack of selectivity for apy approaches (Plumb et al., 2001). Several recent studies have used hTERT and hTR promoters to drive expression of therapeutic transgenes *Correspondence: WN Keith; Cancer Research UK Department of in tissue culture models of gene therapy (Abdul-Ghani Medical Oncology, University of Glasgow, Cancer Research UK et al., 2000; Gu et al., 2000, 2002; Koga et al., 2000, Beatson Laboratories, Alexander Stone Building, Garscube Estate, 2001; Boyd et al., 2001; Komata et al., 2001; Majumdar Switchback Road, Bearsden, Glasgow G61 1BD, UK; E-mail: [email protected] et al., 2001; Plumb et al., 2001). Additionally, hTERT- Received 19 August 2002; revised 18 October 2002; accepted 22 specific constructs have been administered to xenograft October 2002 models using naked DNA injections, liposomes and Telomerase gene therapy AE BiIsland et al 371 adenovirus vectors, although no study has yet addressed the cytotoxic effects of CB1954 in vitro and in vivo (Zhao in vivo transfer of hTR-specific therapeutic constructs. et al., 1998, 2000; Plumb et al., 2001). A number of therapeutic transgenes are available for In order to compare the efficiency and selectivity of use in cytotoxic gene therapy, and prodrug activating NTR expression between normal and cancer cell lines, systems such as herpes simplexthymidine kinase/ human cervical carcinoma cells (C33a) and human gancyclovir or cytosine deaminase/5-FC have been foetal lung fibroblasts (WI38) were infected for 1 h with widely used in preclinical gene therapy models (Aghi 50 PFU/cell of either Ad-hTR-NTR, Ad-hTERT-NTR et al., 2000). Both systems have been shown to or the reporter virus Ad-CMV-LacZ. NTR expression be efficacious in a variety of models, but their use was analysed by Western blotting (Figure 1a), 48 h in cancer gene therapy may be limited by the require- postinfection. Strong 24 kDa NTR signals were evident ment for division of target cells. Additionally, the in the lanes corresponding to Ad-hTR-NTR- and Ad- multistep enzymatic conversion of these prodrugs hTERT-NTR-infected cervical carcinoma cells, with the may present a kinetic bottleneck limiting their utility. hTR promoter generating a significantly stronger Using plasmid vectors, we previously demonstrated that expression than hTERT. No signal was detected in the hTR- and hTERT-restricted expression of bacterial Ad-hTERT-NTR-infected WI38, but a weak signal was nitroreductase (NTR) in stable cell lines selectively detected in WI38 infected with Ad-hTR-NTR although directs the toxic activation of the weak alkylating agent the differential in expression from the hTR promoter CB1954 to cancer cells. The single enzyme activation of between the two cell lines was extremely large. X-gal CB1954 produces a powerful bifunctional alkylating staining of Ad-CMV-LacZ infected cells 48 h postinfec- agent capable of inducing p53-independent apoptosis in tion indicated that both cell lines were efficiently both dividing and nondividing cells (Anlezark et al., infected by adenovirus at this multiplicity of infection 1992; Cui et al., 1999; Plumb et al., 2001). Thus, the (Figure 1b,c). The proportion of cells infected was NTR/CB1954 system may have some advantages over estimated by counting approximately 500–1000 cells per other suicide gene systems. experiment. For the representative experiment shown, In that study, we showed that the efficiency of the proportions of infected cells were 83%73% (C33a) telomerase-directed gene therapy relies largely on the and 98%71.0% (WI38). Therefore, Ad-hTR-NTR and activity of hTR and hTERT promoters in individual Ad-hTERT-NTR constructs drive efficient expression of cell lines, with cytotoxicity restricted exclusively to NTR specifically in cancer cells, but not in normal cells. highly expressing cells. Here we extend these observa- One concern regarding adenovirus-mediated gene tions with the construction of first-generation adenoviral transfer is that human cellular promoters incorporated (Ad) vectors harbouring hTR-NTR and hTERT-NTR into first-generation adenovirus backbones may lose expression constructs (Ad-hTR-NTR and Ad-hTERT- their tissue specificity because of strong virus-specific NTR). transcriptional regulatory elements (Ring et al., 1996; Shi et al., 1997; Vassaux et al., 1999). In order to confirm that the telomerase promoters retain their predicted cellular transcriptional characteristics in the Results context of the Ad vectors, we defined the initiation sites of NTR transcripts expressed in Ad-hTR-NTR- and Construction and characterization of adenoviral vectors Ad-hTERT-NTR-infected cells. cDNA libraries were The Adeasy system (He et al., 1998) was used to prepared from infected C33a cells and the 50 ends of the construct E1/E3-deleted adenovirus constructs harbour- hTR-NTR and hTERT-NTR transcripts were amplified ing the NTR coding sequence under the transcriptional and sequenced (Figure 2). control of hTR and hTERT promoters. Characteristics The transcriptional start site for the hTR promoter of the telomerase gene therapy adenoviruses Ad-hTR- defined here confirms that transcription of NTR from NTR and Ad-hTERT-NTR are given in Table 1. the Ad-hTR-NTR vector initiates 46 bp upstream of the The ratio of viral particles (VP) at A260 relative to start of the hTR template sequence, at the site that was infectious plaque forming units (PFU) was low for both previously described (Feng et al., 1995). Additionally, vectors (15.6 for Ad-hTR-NTR and 6.5 for Ad-hTERT- we place the hTERT transcriptional start site (TSS3) NTR), indicating that the preparations were of high 64 bp upstream of the hTERT start codon. Several other quality. The 876 bp hTR and 541 bp hTERT promoter transcriptional start points have been defined for fragments incorporated in the vectors have previously hTERT and it is not uncommon for weak, TATA-less been characterized in transfection and cell survival sequences such as the hTERT promoter to show assays and are able to drive sufficient expression of complextranscriptional initiation patterns (Clark et al., NTR to sensitize a range of human tumour cell lines to 1998; Rudge and Johnson, 1999; Dong et al., 2000). The

Table 1 Characteristics of adenoviral vectors Virus name Transgene Promoter Description VP/PFU ratio Ad-hTR-NTR NTR hTR (876 bp) Telomerase gene therapy vector. hTR promoter driving NTR 15.6 Ad-hTERT-NTR NTR hTERT (541 bp) Telomerase gene therapy vector. hTERT promoter driving NTR 6.5 Ad-CMV-LacZ LacZ CMV Infectivity control. CMV promoter driving LacZ Not determined

Oncogene Telomerase gene therapy AE BiIsland et al 372 in vitro, we infected a panel of normal mortal and cancer cells with Ad-hTR-NTR, Ad-hTERT-NTR and exposed cells to CB1954. Preliminary virus titration experiments indicated that high infectious doses had a nonspecific vector-induced cytotoxic effect in some cell lines (data not shown); therefore, throughout this study we have used multiplicities of infection of 10 and 50 PFU/cell at which no nonspecific toxicity was observed. All experiments included equivalent infectious doses of the reporter vector Ad-CMV- LacZ that has been widely used as a control for transduction efficiency in studies of adenovirus- mediated gene transfer. To directly quantify the differential activity of the viral constructs between normal and cancer cells, we performed MTT assays on cells exposed to CB1954 and calculated IC50 values from the cytotoxicity curves in mock-infected cells and in cells infected with 10 and 50 PFU/cell of Ad-hTR-NTR and Ad-hTERT-NTR (Table 2). Based on the IC50 data, values were assigned for the sensitization of virus-infected cells to CB1954 relative to mock-infected cells (Figure 3). Sensitization values are taken to be the fold difference between IC50 for mock- and virus-infected cells of an individual cell strain. Additionally, the Ad-CMV-LacZ vector was included in all experiments in order to estimate the proportion of cells transduced at each infectious dose (Table 2). A clear and efficient promoter and dose-dependent cytotoxic effect was observed in C33a cervical carcinoma, DU145 prostatic carcinoma and A2780 ovarian Figure 1 Expression of NTR in adenovirus-infected cell lines. (a) adenocarcinoma cells on administration of both virus C33a (lanes 2–4) and WI38 cells (lanes 5–7) were mock infected (lanes 4 and 7), or infected for 1 h with 50 PFU/cell either Ad-hTR- and CB1954. C33a cells showed the most pronounced NTR (lanes 2 and 5) or Ad-hTERT-NTR (lanes 3 and 6). Protein, cytotoxic effect with a high dose administra- 48 h post infection, was extracted from infected cells and 20 mg was tion (50 PFU/cell) of Ad-hTR-NTR resulting in an used for Western blot detection of NTR protein expression using 18-fold sensitization to CB1954, while infection with the rabbit anti-NTR antibody R36 and an HRP-conjugated anti- rabbit secondary. For comparison, lane 1 shows the expression of Ad-hTERT-NTR resulted in a fivefold enhancement NTR in C33a cells stably harbouring an hTR-NTR expression of cytotoxicity. In all cell lines that showed sensitisa- plasmid (Plumb et al., 2001). (b) and (c) show C33a (b) and WI38 tion, 50 PFU/cell was more effective than 10 PFU/cell. cells (c) infected with 50 PFU/cell Ad-CMV-LacZ and stained for Thus, adenovirus vectors harbouring the telomerase– expression as described in Materials and methods NTR expression cassettes sensitized C33a cells to the effects of CB1954 in a promoter- and dose- dependent fashion. Similarly, the prostatic carcinoma transcriptional start site defined here is within the cell line DU145 showed strong sensitization to CB1954 transcriptional start region (TSR) 60–112 bp upstream with the hTR-specific construct (fivefold), although of the hTERT translation start codon defined by Wick Ad-hTERT-NTR did not have a powerful effect et al (1999) and lies close to the sites defined by (twofold). Takakura et al (1999) (TSS1) and Horikawa et al (1999) A2780 cells were very poorly transduced with (TSS2). Therefore, our data are consistent with previous adenoviral vectors, reaching only 34% infectivity at reports and, together with the protein expression data the high infectious dose of Ad-CMV-LacZ (Table 2). presented in Figure 1 indicate that the hTR and hTERT Nevertheless, cells infected by Ad-hTR-NTR were promoters retain their native characteristics when the eightfold more sensitive to CB1954 than mock-infected constructs are delivered to human cells in an adenovirus cells. Infection with the hTERT-NTR virus resulted in background. threefold enhancement of cytotoxicity. The low efficacy observed for the hTERT construct in DU145 and A2780 cells presumably reflected both the low efficiency of Selective sensitization of human cancer cells to CB1954 in infection in A2780 (Table 2) and the lower activity of the vitro following infection with telomerase-specific gene therapy viruses hTERT promoter (Plumb et al., 2001). Therefore, Ad- hTR-NTR was the more efficacious vector, while Ad- To determine whether Ad-hTR-NTR and Ad-hTERT- hTERT-NTR resulted only in a modest effect. The data NTR selectively sensitize human cancer cells to CB1954 indicate that it is possible to achieve a cytotoxic effect

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Figure 2 Sequences of the 50 ends of hTR-NTR and hTERT-NTR transcripts in virus-infected cervical carcinoma cells. 50 ends of NTR transcripts expressed in C33a cervical carcinoma cells were amplified from l mg cDNA using the Clontech SMART-RACE kit. The figure shows genomic sequences of (a) hTR and (b) hTERT promoters, in addition to the published transcriptional start sites and the start sites defined from sequence analysis of the 50 ends of NTR transcripts. For the hTR promoter, a single transcriptional start site 46 bp upstream of the template start was established by Feng et al (1995). In contrast, several sites have been reported for the hTERT promoter. The white arrows indicate the transcriptional start region (TSR) identified by Wick et al (1999), while the black (TSS1) and grey (TSS2) arrows represent the start sites defined by Takakura et al (1999) and Horikawa et al (1999), respectively. The start site of hTERT-NTR is indicated by the hatched arrow (TSS3). Nucleotides corresponding to transcriptional starts are underlined, while important structural and regulatory features are indicated by bold lettering. The translational start site of NTR is indicated by bold, enlarged characters when only a minority of cells are transduced, providing several normal human fibroblast cell strains including that the promoter activity is of sufficient strength in WI38, in addition to normal retinal pigmented epithelial infected cells. cells and telomerase negative immortal cells that utilize In contrast, and despite their high permissiveness for an alternative pathway for maintenance of telomere adenovirus infection (98% at 50 PFU), WI38 cells were stability (Gu et al., 2000, 2002; Koga et al., 2000, 2001; not sensitized to CB1954 either by Ad-hTR-NTR or by Komata et al., 2001; Majumdar et al., 2001; Plumb et al., Ad-hTERT-NTR. An important consideration for 2001). In order to extend these findings, we tested telomerase-directed gene therapy must be its specificity normal adult mammary (HMEC), prostate (HPrEC) for a broad range of cancer cells over a broad range of and bronchial (HBrEC) epithelial cell strains. normal cells. The previous studies of telomerase-directed Despite the reasonable permissiveness of both HMEC gene therapy have demonstrated lack of activity in and HBrEC for adenovirus infection (Table 2), none of

Table 2 IC50 values for CB1954 cytotoxicity in cell lines infected with Ad-hTR-NTR and Ad-hTERT-NTR gene therapy viruses hTR hTR hTERT 10 hTERT 10 PFU 50 PFU PFU IC50 50 PFU % Infectivity % Infectivity Mock IC50 IC50 (s.e.) IC50 (s.e.) (s.e.) mM IC50 (s.e.) (s.e.) 10 (s.e.) 50 Cell line (s.e.) mM mM mM mM PFU PFU C33a 176.1 (10.0) 33.4(14.6) 9.8(2.7) 63.1(17.1) 35.9(13.3) 59.2(0.2) 80.3 (2.7) DUI45 39.4 (2.7) 25.9(4.8) 7.1 (2.9) 41.6(5.6) 20.2(1.4) 64.2(10.0) 92.4 (4.8) A2780 28.5(13.9) 10.7(3.7) 3.8(1.4) 17.7(4.4) 10.9(0.6) 16.3(1.8) 33.6 (6.6) A2780-CP70 59.6 (12.1) 25.9 (2.9) 6.0(1.9) 48.3 (9.6) 33.9(3.1) 20.1(5.8) 47.5 (2.9) A2780-ADR 238 (45.1) 54.2(11) 17.6(7) 112.7(30.5) 74.4(15.5) 58.4 (2.7) 71.7(2) UVW 214.4(15.7) 144.6(9.8) 77.7(15.9) 180.8(26.0) 136.2(11.4) 56.9(19.3) 74.5(12.6) 5637 102.9(14.2) 93.5(16.0) 73.4(14.6) 112.6(20.0) 103.2(22.4) 72.3 (11.0) 94.5(1.2) WI38 153.6(48.5) 178.1 (68.0) 164.7(55.2) 148.6 (54.8) 174.1 (74.1) 65.0 (2.6) 97.8(0.1) HMEC 31.7(11.2) 45.4 (20.5) 36.0(11.3) 27.8 (6.3) 29.6(4.5) 34.4(1.1) 66.2 (0.7) HPrEC 266.3 (49.8) 238.7(13.0) 190.5(25.3) 297.3 (32.0) 235.3 (13.5) 8.0 (2.9) 36.8(10.9) HBrEC 188.1 (22.3) 169.2(26.8) 148.9(3.2) 196.1 (27.8) 156.8 (8.0) 24.4(1.1) 68.3 (2.4)

Relative cell densities from optical density measurements at 570 nm to measure the quantity of MTT formazan in individual wells. For an independent experiment, each IC50 value was calculated from the mean value of the 50% y-intercept determined from triplicate plates. IC50 values in the table are the means and s.e., given in brackets, for each virus and multiplicity of infection calculated from three independent experiments

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Figure 3 Mean sensitization to CB1954 and infectivity in adenovirus-infected cancer and normal cell lines. Cell lines were infected with 50 PFU per cell Ad-hTR-NTR or Ad-hTERT-NTR and subjected to CB1954 challenge and MTT assay. IC50 values for individual experiments in each cell line were taken to be the concentration of CB1954 required to reduce cell densities in MTT assays to 50% of those of untreated controls. Sensitization values were the fold difference between the IC50 of the mock-infected cells and those of cells infected with the gene therapy vectors. Black bars correspond to Ad-hTR-NTR-infected cells and grey bars to Ad-hTERT- NTR-infected cells. Data are means and s.e. calculated from three experiments

the normal cell strains tested showed more than 1.4-fold UVW cells most likely reflect similar low promoter reduction in IC50 with either promoter, indicating activities. that the sensitivity of cell lines to CB1954 remained unchanged after administration of the vectors. The normal human prostatic epithelial cell strain HPrEC Telomerase–NTR vectors sensitize drug-resistant ovarian by contrast were relatively refractory to the virus, carcinoma cells to CB1954 reaching only 37% infectivity. However, it is clear from the A2780 data (Table 2) that infection of the A major limitation to current therapies is the develop- total cell population is not a prerequisite for significant ment of drug-resistant phenotypes. The active metabo- enhancement of CB1954-induced cytotoxicity providing lite of CB1954 is a bifunctional alkylating agent, thus it that significant transgene expression can be achieved is important to evaluate the potential of the telomerase in infected cells. Taken together, these data give a strong gene therapy vectors to overcome alkylating agent indication that the hTR and hTERT promoters did resistance. The A2780 ovarian adenocarcinoma deriva- not drive sufficient expression of NTR in normal human tives, A2780–CP70 (selected by cisplatin exposure) and cell lines to result in enhancement of cytotoxicity and A2780–ADR (selected by adriamycin exposure), show suggests that the system has a selective effect for crosstolerance to a range of DNA-damaging agents, cells with high promoter activity. Further evidence of including the purine analogue 6-thioguanine and the the requirement for a high promoter activity in addition alkylating agent N-methyl-N-nitrosourea. Resistance in to efficient infectivity can be seen from the 5637 bladder these cell lines is because of loss of the mismatch repair carcinoma cell line that was not effectively targeted protein hMLHl. (Anthoney et al., 1996; Drummond by either promoter, having sensitization values under 2 et al., 1996; Brown et al., 1997; Strathdee et al., 1999). for all treatments. We previously correlated the To assess whether NTR expression from telomerase- low hTR and hTERT promoter activity of 5637 cells specific vectors is sufficiently high to allow local with a low sensitivity to CB1954 in stable cell activation of CB1954 to overcome the drug-resistant line models harbouring hTR-NTR and hTERT-NTR phenotype in hMLHl negative cells, we analysed the constructs (Plumb et al., 2001). Therefore, the data effects of the vectors in these cell lines (Figure 3). presented here are consistent with high promoter A2780–CP70 cells showed a slightly higher basal activity as a prerequisite for efficient transcriptional tolerance of CB1954 than the parental A2780 cells, with targeting using the telomerase promoters, and we an IC50 of 60 mm for CP70 compared with 29 mm for speculate that the low sensitization values observed in A2780 (Table 2). A2780–ADR had a higher basal

Oncogene Telomerase gene therapy AE BiIsland et al 375 tolerance with an IC50 of 238 mm. Interestingly, however, Distribution of adenovirus vectors in xenograft models neither derivative showed resistance when transduced An important factor for consideration in the evaluation with the gene therapy vectors, exhibiting similar of the efficacy of any virus-mediated gene transfer model cytotoxicity profiles to the parental cells (Figure 3, is the efficiency of virus penetration of the tumour mass. Table 2). Thus, infection with 50 PFU per cell of Ad- In most cases using replication defective vectors, this is hTR-NTR sensitized CP70 cells to CB1954 some 10-fold likely to be the major limiting factor since no reliable and ADR cells 14-fold, while the same infectious dose of systems exist that can transduce all cells of a tumour. To Ad-hTERT NTR resulted in modest sensitizations of address this issue, parallel experiments were performed two-and threefold. Additionally, the cisplatin-resistant to determine the distribution of cells infected and, thus, cell line had a similar permissiveness for Ad-CMV-LacZ infection as the parental cell line, although ADR cells to assess indirectly the potential of the bystander effect in our models. Control C33a and A2780 xenografts were were more permissive, indicating that hTR promoter- injected with infectious doses of Ad-CMV-LacZ (C33a) restricted expression of NTR is sufficiently strong to result in significant cell death in a drug-resistant cell line or Ad-hTR-NTR and Ad-hTERT-NTR (A2780) equivalent to those administered in the tumour reduc- despite transduction of a minority of cells. tion experiments. Tumours were excised 24 h later, corresponding to the time of CB1954 administration in the tumour volume reduction experiments and patterns Ad-hTR-NTR and Ad-hTERT-NTR vectors sensitize of LacZ or NTR expression were examined in frozen human cancer cells to CB1954 in vivo sections derived from four to five separate tumours for each cell type. Representative in vivo expression data are In order to assess the sensitization of model human shown in Figure 4c, d. tumours to Ad-hTR-NTR and Ad-hTERT-NTR vector In keeping with the low dose, single administrations administration followed by CB1954 in vivo, C33a or A2780 xenografts were established in the flanks of of vector employed in these experiments, in most of the xenograft sections analysed for Ad-CMV-LacZ infec- athymic female nude mice. Randomly distributed tion or NTR expression, only a minority of cells showed groups of sixxenograft-bearing animals recieved a transgene expression, suggesting limited xenograft pene- single intratumoural injection of either Ad-hTR-NTR, tration. Excised tumours showed similar heterogeneous Ad-hTERT-NTR or Ad-CMV-LacZ. A single in- patterns of staining whether LacZ or NTR was detected, jection of 80 mg/kg CB1954 was administered by tail with many of the transduced cells restricted mainly to vein injection, 24 h later. Daily calliper measurements the area surrounding the needle track or to the were performed for a further 7 days and tumour peripheral edge of the section. In some cases patchy volumes were estimated from the measurements internal staining was observed, indicating that in some (volume ¼ d 3 Â p/6). LacZ-expressing tumours were harvested for staining on the day of drug injection. instances the vectors had penetrated the tumour mass, The C33a tumours in flanks of control animals with low efficiency, although the proportion of LacZ- or increased in volume at a similar rate, approximately NTR-expressing cells was still considerably lower than doubling in size over 7 days. By contrast, a single the proportion of untransduced cells and staining was injection of telomerase–NTR gene therapy vectors less intense than in peripheral areas. This pattern most coupled to a single CB1954 administration resulted in likely reflects the initial exposure of cells to virus a 40% reduction in relative tumour volume for the supernatant, as it is injected directly into the cell mass hTERT-NTR virus and a 43% reduction for the hTR- and bathing of the cell mass in virus on needle NTR virus. The difference in tumour volumes between withdrawal. It is encouraging that significant therapeutic animals injected with virus only and those injected with effect can be achieved in these models despite low- virus and drug was highly significant at day 7 efficiency tissue penetration. These data point to the activity of a bystander effect mediated by diffusion of (Po0.001). We note with interest that the infectious dose and the number of drug and vector administrations the active lipophilic 4-hydroxylamino derivative of used in this experiment are efficacious, though substan- CB1954 into the cell mass following initial activation tially lower than those utilized in other adenovirus- in a low proportion of virus-tranduced cells with high based reports of telomerase gene therapy (Gu et al., hTR and hTERT promoter activity. 2000; Majumdar et al., 2001). Figure 4b shows the results of an additional experiment in A2780 xenograft- bearing mice. In this experiment, control tumours Discussion increased in size approximately fourfold, while a single intratumour injection with 1 Â l09 PFU, coupled to a A major limitation of current cancer therapeutics is a single intravenous injection of 80 mg/kg CB1954 re- lack of real selectivity for tumour cells in vivo and, sulted in reductions of tumour volume of 54% for the hence, a major aim for the future is the development of hTR-NTR construct and 57% for the hTERT-NTR tumour-specific targeting systems. The hTR and hTERT construct. Again, the difference in tumour volume promoters are interesting candidates for the develop- between virus-only and virus/CB1954 groups was ment of gene therapy systems since they should display significant at day 7 (Po0.001). It should also be noted activity over a range of malignancies. Therefore, in that one animal showed complete tumour regression. order to model delivery of telomerase-specific suicide

Oncogene Telomerase gene therapy AE BiIsland et al 376

Figure 4 Reduction of tumour volume of C33-A and A2780 xenografts after intra tumoural injection of telomerase gene therapy viruses followed by systemic administration of CB1954 (a) and (b)107 C33a or A2780 cells per mouse were injected subcutaneously into the flanks of sixgroups of sixfemale athymic nude mice and allowed to develop for 14 days until tumour diameters were approximately 5 mm. At this time (day 0), four groups of mice were injected intra tumourally with a total of 4 Â l08 PFU for C33a xenografts (panel a) or 1 Â 109 PFU for A2780 (panel b) of Ad-hTR-NTR or Ad-hTERT-NTR per tumour (two groups for each virus). The following day (day 1), three groups (one-drug-only group and 1 each of the virus-injected groups) were intravenously injected with 80 mg/kg CB1954 and the mean tumour volumes of all groups were monitored daily for 7 days. Results given are the mean tumour volumes and standard errors at each time point derived from sixmice per group by the formula volume ¼ d3 Â p/6. A clear reduction in tumour volume over the course of the experiment is evident in the groups injected with both virus and drug, but not in any of the control groups. The differences in tumour volume between virus-only and virus/CB1954 groups were analysed using Students’ t-test and found to be highly significant for both experiments (Po0.001). Filled box: hTR virus with CB1954, open box: hTERT virus with CB1954. Closed triangle: hTR virus alone, open triangle: hTERT virus alone. Closed circle: diluent alone, filled circle: CB1954 alone. (c) and (d) Photomicrographs of vector-infected frozen tissue sections at low magnification (original magnification Â 2.5). C33a xenografts (panel c) were injected with a single dose of 4 Â 108 PFU Ad-CMV-LacZ in parallel with tumour reduction experiments. Tumours were excised and cryosections were stained for LacZ expression and counterstained with eosin, 24 h later. A2780 xenografts (panel d) were infected with Ad-hTR-NTR or Ad-hTERT-NTR at 1 Â 109 PFU. After 24 h, tumours were excised for immunohistochemical analysis of NTR expression and counterstained with haematoxylin. The figure shows a section infected with Ad-hTERT-NTR

gene therapy vectors, we have constructed adenovirus retained a selective effect. It is apparent from this vectors expressing hTR-NTR and hTERT-NTR con- and other studies that although highly efﬁcient cell structs. killing can be directed by both promoters the efﬁciency In the present study, the hTR promoter was stronger of hTERT-promoter directed constructs may be re- than hTERT in all the cancer cell lines assayed, yet it stricted in cancer cells with low promoter activity

Oncogene Telomerase gene therapy AE BiIsland et al 377 (Abdul-Ghani et al., 2000; Gu et al., 2000, 2002; Koga have observed are retained in viral models of gene et al., 2000, 2001; Komata et al., 2001; Plumb et al., transfer and help to validate the general principle 2001). However, the ability of telomerase–NTR vectors underlying a telomerase-directed approach in gene to efficiently sensitize some very poorly transduced therapy. Moreover, the NTR/CB1954 system is cancer cell lines to CB1954 is encouraging. Thus, it is an interesting candidate for development under the unnecessary to infect all telomerase positive cells in a direction of telomerase promoter constructs, since population, providing that the promoter activity is of the safety of pharmacologically relevant doses of sufficient strength to drive high-level expression leading CB1954 in humans has already been demonstrated to a bystander effect in those cells that harbour the in clinical trials (Chung-Faye et al., 2001). In summary, construct. This view is supported by the poor distribu- it is clear that the addition of Ad-hTR-NTR and tion of infection, yet efficient growth retardation in Ad-hTERT-NTR to a potential telomerase-specific xenografts injected with low doses of telomerase–NTR anticancer armoury is an exciting prospect, although vectors. optimal systems for telomerase-directed gene therapy Although the ability to effectively target cytotoxicity will presumably require targeted delivery systems in to cancer cells is paramount in the development of gene addition to a clear understanding of the regulation of therapy strategies, it is highly likely that the develop- hTR and hTERT genes in target tumours (Keith et al., ment of drug-resistant phenotypes such as loss of 2002). mismatch repair function will still represent an important obstacle to effective therapy. In this regard, we note with interest that telomerase-directed NTR expression Materials and methods in hMLHl-deficient drug-resistant derivatives of A2780 Cell lines and viruses results in efficient killing of cells that are crossresistant to both alkylating agents and other common agents that The human cancer cell lines 5637 (bladder carcinoma), DU145 elicit more diverse effects. This raises the intriguing (prostatic carcinoma), C33a (cervical carcinoma) and notion that the use of telomerase-specific molecular UVW (malignant glioma) were obtained from ATCC and therapy to generate high intracellular concentrations of maintained in appropriate media. A2780 (ovarian adenocarcinoma) cells were originally obtained from Dr RF Ozols active alkylating agents can induce sensitivity to cell (FoxChase Cancer Centre, PA, USA). Selection of the drug- death in cancer cells hitherto thought to have a drug- resistant A2780 variants A2780–CP70 and A2780–ADR resistant phenotype. has been described elsewhere (Anthoney et al., 1996). WI38 We conclude from the present data that the sensitizing normal human foetal lung fibroblasts were obtained effect of telomerase-specific adenoviral gene therapy from Coriell Cell Repository (USA) and the normal adult vectors is dependent largely on promoter activity; human mammary epithelial cells (HMEC), prostatic epithelial thus, cell lines with low promoter activity are not cells (HPrEC) and bronchial epithelial cells (HBrEC) were sensitized to CB1954 even in circumstances where obtained from Clonetics (USA) and were subcultured and essentially all of the cells are infected. While a minority maintained according to the instructed culture systems in of tumour cells, such as the 5637 bladder carcinoma each case. The viruses Ad-hTR-NTR and Ad-hTERT-NTR were line and the UVW malignant glioma line, may display generated using the Adeasy system supplied by Qbiogene low hTR and hTERT promoter activity, from previous (Middlesex, UK), according to the manufacturer’s instruc- expression studies it is clear that a number of tumour tions. Ad-hTR-NTR contains a previously characterized types are good candidates for telomerase- 876 bp fragment of the hTR proximal promoter (Zhao et al., directed therapies (Soder et al., 1997, 1998; Sarvesvaran 1998; Plumb et al., 2001) and Ad-hTERT-NTR contains a et al., 1999; Wisman et al., 2000; Downey et al., 541 bp fragment of the hTERT promoter (Plumb et al., 2001). 2001; Hiyama et al., 2001). Importantly, this situation Both vectors contain the Escherichia coli NTR coding is also expected to apply to the majority of normal adult sequence. All ligations into transfer vectors were performed somatic cells in vivo, suggesting that Ad-hTR-NTR and using a rapid ligation kit obtained from Roche Diagnostics Ad-hTERT-NTR/CB1954 will be selectively toxic to Ltd (East Sussex, UK) and electro-cotransformation of adenovirus backbone and transfer vectors were performed cancer cells. Indeed, we have not observed any in a Hybaid (Middlesex, UK) cell shock electroporator. significant enhancement of toxicity following vector Expression cassettes were inserted in a left-to-right orientation transduction in four mortal human cell strains suggest- in the adenovirus backbone and sequence and orientation of ing that telomerase-directed molecular therapeutics may the promoter–NTR constructs in the transfer vectors prove to be a highly effective, yet safe class of anticancer and adenovirus genomic plasmids was confirmed by restriction agent. digest and sequencing. Large-scale amplification was per- A growing body of literature supports the develop- formed by Qbiogene. Since virus-mediated gene transfer ment of telomerase-directed therapeutics as selective, studies commonly include infectivity controls, we obtained yet wide-ranging antitumour agents (Keith et al., 2002). the reporter vector Ad-CMV-LacZ from Qbiogene for use as a A number of very different systems have been proposed positive control allowing an estimate of infection efficiency during cytotoxicity and tumour reduction experiments. Small- to target telomerase expression or activity, but gene scale amplifications on 293 cells were purified using the therapy remains an attractive approach. Taken together, Adenoprep kit (Virapur, USA). Viral titre was checked for the present data confirm that the differential promoter all constructs by plaque assay on 293 cells and by measurement activities of the hTR and hTERT promoters be- of optical density (A260) for Ad-hTR-NTR and Ad-hTERT- tween normal and cancer cells that we and others NTR.

Oncogene Telomerase gene therapy AE BiIsland et al 378 Western blotting MTT assay Purification of the cytoplasmic protein fraction for Western Cells were initially treated in six-well plates with 10 or 50 PFU/ blotting was achieved using the NE-PER differential nuclear cell Ad-hTR-NTR or Ad-hTERT-NTR for 1 h, then trypsi- and cytoplasmic protein extraction kit (Pierce and Warriner nized and plated in 96-well plates for drug treatment and MTT UK Ltd, Chester, UK). Quantification of cytoplasmic protein assay (Plumb et al., 2001). On the day of the drug challenge, extracts was accomplished by BCA/Cu (II)SO4 assay and fourfold serial dilutions of CB1954 were prepared in cell colorimetric changes were quantified using a DynexTechnol- growth medium. For an individual experiment, each data point ogies (West Sussex, UK) MRX II microplate reader. Protein on kill curves was plotted as the mean percentage of the equivalents (20 mg) were electrophoresed in a 12% SDS– untreated control, calculated across triplicate plates for each polyacrylamide gel, then blotted onto nitrocellulose filter and independent drug concentration. Mean IC50 values were blocked overnight at 41C in TBS-T (0.7% Tween 20) contain- calculated from triplicate plates using Softmax2.42 analysis ing 5% nonfat dried milk. The following day, filters were package (Molecular Devices Corporation, CA, USA). Sensi- probed for 2 h with a 1 : 50 dilution of rabbit anti-NTR tization values are the fold difference between the IC50 values primary antibody R36, then with an HRP-conjugated anti- for mock-infected cells and those which were infected by Ad- rabbit secondary. Bound HRP was detected using ECL hTR-NTR or Ad-hTERT-NTR. All experiments were re- Western blotting HRP detection reagents (Amersham Phar- peated at least three times with the exception of HBrEC cells, macia, Buckinghamshire, UK). R36 is a kind gift from Dr which were analysed once in triplicate. Final sensitization Steve Hobbs (CRC Centre for Cancer Therapeutics, Institute values and IC50 values presented are the means and s.e. derived of Cancer Research, Surrey). from all three independent experiments.

In vivo tumour reduction experiments PCR and sequencing C33a and A2780 cells were harvested and resuspended in PBS. Cells (107) were injected subcutaneously into the flanks of Sequence and orientation of NTR expression cassettes in athymic female mice. When mean tumour diameters were at pAdeasy-1 were determined by dideoxy chain termination least 5 mm, the animals were randomized into sixgroups of six sequencing reactions performed using PE Biosystems (Che- animals. At this time (day 0) 4 Â 108 PFU (C33a) or shire, UK) Big Dye Terminator system and reagents according 1 Â l09 PFU (A2780) Ad-hTR-NTR and Ad-hTERT-NTR to the manufacturer’s instructions, using the primers Shuntlf were administered to animals by direct intratumoural injec- (50-ggcgtaaccgagtaagatttgg-30), Shuntlr (50-tgctggatgggctg- tion. The following day, 80 mg/kg CB1954 was administered to tattgc-30) and AdNTseq5a (50-cattccactaaggcatttgatg-30) for 30 all groups except controls. For general estimation of toxicity, end sequencing. Sequence analyses were performed on ABI- animals were weighed daily and tumour volumes were PRISM 377. Generation of cDNA and amplification of estimated from calliper measurements (volume ¼ d3 Â p/6). transcript 50 ends from infected C33a cells was accomplished For statistical analysis, unpaired, two-tailed Student’s t-tests using the Stratagene Europe (Amsterdam, Netherlands) Smart were used. P values of 0.05 were considered significant. All RACE kit. RNA (1 mg) was used in cDNA synthesis reactions o animal experimentation was performed according to United and PCR steps used the primers included in the kit, together Kingdom Home Office regulations and UKCCR guidelines with the gene-specific primer Shuntlr for 50 amplification. were adhered to at all times. At 80 mg/kg CB1954, animals lost Reaction products were subcloned for sequencing in an at most 10% of body weight, but had recovered to weight of Invitrogen (Renfrewshire, UK) TOPO-TAt cloning vector controls by day 6. The combination of CB1954 and virus and were sequenced using the primer set provided in the kit. prolonged the effect of CB1954 alone on body weight loss. However, it should be noted that weight measurement includes the tumour burden. Thus, where a treatment is effective, body LacZ reporter assay weight would be expected to be less than untreated controls. For monolayer experiments, cells were seeded in six-well plates Preparation of NTR-specific rabbit polyclonal antibody and 24 h prior to infection. On the day of infection, cells were mock immunohistochemistry infected or were infected with Ad-CMV-LacZ for 1 h at 371C at either 10 or 50 PFU/cell. Following infection, cells were The NTR-B rabbit polyclonal antibody to NTR, which was incubated in fresh growth medium for a further 48 h, used for all immunohistochemistry, was produced using a corresponding to the day of drug addition. The cell layers peptide consisting of the terminal 16 amino acids of the full- were rinsed and fixed in 0.2% glutaraldehyde, 5 mm EGTA, length NTR protein with an additional cysteine amino acid at 2mm MgCl2 in ice-cold PBS for 20 min. Next, the cells were the NH2 terminus. The peptide NH2–CTPLKSRLPQNITL- rinsed and incubated with 20 mg/ml X-gal, 2.5 mm K3Fe(CN)6, TEV–CO2H was then conjugated to a carrier protein using the s 2.5 mm K4Fe(CN)6 for 24 h in the dark. Proportions of blue Pierce (Chester, UK) Imject Maleimide-activated mcKLH cells were assessed by counting five random fields (500–1000 kit. The peptide was mixed either with complete Freund’s cells). All experiments were repeated at least three times with adjuvant (for the initial injection) or incomplete Freund’s the exception of HBrEC cells, which were analysed only once adjuvant (for subsequent booster injections) at a concentration because of limited replicative potential, and data presented are of 100 mg/ml of peptide-KLH. All animal experimentation the means of three repeats. To determine the tissue distribution was performed to United Kingdom Home Office regulations of Ad-CMV-LacZ in cervical carcinoma xenografts, frozen and UKCCR guidelines were adhered to at all times. sections of xenografts infected with a single administration of For immunohistochemical analysis of NTR expression, 4 Â l08 PFU Ad-CMV-LacZ in parallel with the tumour A2780 ovarian adenocarcinoma xenografts were established reduction experiments were fixed and stained as described and, when tumours reached a minimum diameter of 5 mm, above. Photomicrographs were obtained using Axiovert digital were injected once with 1 Â 109 PFU. Ad-hTR-NTR or video recording equipment (Axiomatic Technology Ltd, Ad-hTERT-NTR. After 24 h, tumours were excised and Nottingham, UK). frozen sections of uninfected or of Ad-hTR-NTR- and

Oncogene Telomerase gene therapy AE BiIsland et al 379 Ad-hTERT-NTR- infected xenografts were prepared and Acknowledgements stained using a Vectastain kit (Vector Laboratories Ltd, This work was supported by the Cancer Research UK, Peterborough, UK) according to the manufacturer’s instruc- the Fifth Framework Program of the European Commission tions. A 1 : 1000 dilution of NTR-B was used for all sections and Glasgow University. We would like to thank Dr Steve analysed. Photomicrographs were obtained using Axiovert Hobbs for the initial gift of NTR antisera. digital video recording equipment (Axiomatic Technology Ltd, Nottingham, UK).

References

Abdul-Ghani R, Ohana P, Matouk I, Ayesh S, Ayesh B, Hiyama E, Hiyama K, Yokoyama T and Shay JW. (2001). Laster M, Bibi O, Giladi H, Molnar-Kimber K, Sughayer Neoplasia, 3, 17–26. MA, de Groot N and Hochberg A. (2000). Mol. Ther., 2, Holt SE and Shay JW. (1999). J. Cell Physiol., 180, 10–18. 539–544. Horikawa I, Cable PL, Afshari C and Barren JC. (1999). Aghi M, Hochberg F and Breakeﬁeld XO. (2000). J. Gene. Cancer Res., 59, 826–830. Med., 2, 148–164. Karamouzis MV, Gorgoulis VG and Papavassiliou AG. Anlezark GM, Melton RG, Sherwood RF, Coles B, Friedlos F (2002). Clin. Cancer Res., 8, 949–961. and KnoxRJ. (1992). Biochem. Pharmacol., 44, 2289–2295. Keith WN, Bilsland A, Evans TRJ and Glasspool RM. Anthoney DA, McIlwrath AJ, Gallagher WM, Edlin AR and (2002). Exp. Rev. Mol Med, http://www.expertreviews.org/ Brown R. (1996). Cancer Res., 56, 1374–1381. 02004507h.htm. Boyd M, Mairs RJ, Cunningham SH, Mairs SC, McCluskey Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, A, Livingstone A, Stevenson K, Brown MM, Wilson L, Ho PL, Coviello GM, Wright WE, Weinrich SL and Shay Carlin S and Wheldon TE. (2001). J. Gene Med., 3, JW. (1994). Science, 266, 2011–2015. 165–172. Koga S, Hirohata S, Kondo Y, Komata T, Takakura M, Brown R, Hirst GL, Gallagher WM, McIlwrath AJ, Margison Inoue M, Kyo S and Kondo S. (2000). Hum. Gene Ther., 11, GP, van der Zee AG and Anthoney DA. (1997). Oncogene, 1397–1406. 15, 45–52. Koga S, Hirohata S, Kondo Y, Komata T, Takakura M, Chung-Faye G, Palmer D, Anderson D, Clark J, Downes M, Inoue M, Kyo S and Kondo S. (2001). Anticancer Res., 21, Baddeley J, Hussain S, Murray PI, Searle P, Seymour L, 1937–1943. Harris PA, Ferry D and Kerr DJ. (2001). Clin. Cancer Res., Komata T, Kondo Y, Kanzawa T, Hirohata S, Koga S, 7, 2662–2668. Sumiyoshi H, Srinivasula SM, Barna BP, Germano IM, Clark MP, Chow CW, Rinaldo JE and Chalkley R. (1998). Takakura M, Inoue M, Alnemri ES, Shay JW, Kyo S and Nucleic Acids Res., 26, 1801–1806. Kondo S. (2001). Cancer Res., 61, 5796–5802. Counter CM, Avilion AA, LeFeuvre CE, Stewart NG, Greider Majumdar AS, Hughes DE, Lichtsteiner SP, Wang Z, CW, Harley CB and Bacchetti S. (1992). EMBO J., 11, Lebkowski JS and Vasserot AP. (2001). Gene Ther., 8, 1921–1929. 568–578. Cui W, Gusterson B and Clark AJ. (1999). Gene Ther., 6, Mergny JL, Riou JF, Mailliet P, Teulade-Fichou MP and 764–770. Gilson E. (2002). Nucleic Acids Res., 30, 839–865. Damm K, Hemmann U, Garin-Chesa P, Hauel N, Kauffmann Naasani I, Seimiya H, Yamori T and Tsuruo T. (1999). Cancer I, Priepke H, Niestroj C, Daiber C, Enenkel B, Guilliard B, Res., 59, 4004–4011. Lauritsch I, Muller E, Pascolo E, Sauter G, Pantic M, Pascolo E, Wenz C, Lingner J, Hauel N, Priepke H, Martens UM, Wenz C, Lingner J, Kraut N, Rettig WJ and Kauffmann I, Garin-Chesa P, Rettig WJ, Damm K Schnapp A. (2001). EMBO J., 20, 6958–6968. and Schnapp A. (2002). J. Biol. Chem., 277, 15566–15572. Dong S, Lester L and Johnson LF. (2000). J. Cell Biochem., Plumb JA, Bilsland A, Kakani R, Zhao J, Glasspool RM, 77, 50–64. KnoxRJ, Evans TR and Keith WN. (2001). Oncogene, 20, Downey MG, Going JJ, Stuart RC and Keith WN. (2001). J. 7797–7803. Oral Pathol. Med., 30, 577–581. Ring CJ, Harris JD, Hurst HC and Lemoine NR. (1996). Gene Drummond JT, Anthoney A, Brown R and Modrich P. (1996). Ther., 3, 1094–1103. J. Biol. Chem., 271, 19645–19648. Rudge TL and Johnson LF. (1999). J. Cell Biochem., 73, Feng J, Funk WD, Wang SS, Weinrich SL, Avilion AA, Chiu 90–96. CP, Adams RR, Chang E, Allsopp RC, Yu J, Le S, West Sarvesvaran J, Going JJ, Milroy R, Kaye SB and Keith WN. MD, Harley CB, Andrews WH, Greider CW and Villepon- (1999). Carcinogenesis, 20, 1649–1651. teau B. (1995). Science, 269, 1236–1241. Scapin G. (2002). Drug Discov. Today, 7, 601–611. Garrett MD and Workman P. (1999). Eur. J. Cancer, 35, Shay JW and Bacchetti S. (1997). Eur. J. Cancer, 33, 2010–2030. 787–791. Gu J, Andreeff M, Roth JA and Fang B. (2002). Gene Ther., 9, Shi Q, Wang Y and Worton R. (1997). Hum. Gene Ther., 8, 30–37. 403–410. Gu J, Kagawa S, Takakura M, Kyo S, Inoue M, Roth JA and Soder AI, Going JJ, Kaye SB and Keith WN. (1998). Fang B. (2000). Cancer Res., 60, 5359–5364. Oncogene, 16, 979–983. Hahn WC, Stewart SA, Brooks MW, York SG, Eaton E, Soder AI, Hoare SF, Muir S, Going JJ, Parkinson EK and Kurachi A, Beijersbergen RL, Knoll JH, Meyerson M and Keith WN. (1997). Oncogene, 14, 1013–1021. Weinberg RA. (1999). Nat. Med., 5, 1164–1170. Strathdee G, MacKean MJ, Illand M and Brown R. (1999). He TC, Zhou S, da Costa LT, Yu J, Kinzler KW and Oncogene, 18, 2335–2341. Vogelstein B. (1998). Proc. Natl. Acad. Sci. USA, 95, Takakura M, Kyo S, Kanaya T, Hirano H, Takeda J, Yutsudo 2509–2514. M and Inoue M. (1999). Cancer Res., 59, 551–557.

Oncogene Telomerase gene therapy AE BiIsland et al 380 VassauxG, Hurst HC and Lemoine NR. (1999). Gene Ther., 6, Zhang X, Mar V, Zhou W, Harrington L and Robinson MO. 1192–1197. (1999). Genes Dev., 13, 2388–2399. White LK, Wright WE and Shay JW. (2001). Trends Zhao JQ, Glasspool RM, Hoare SF, Bilsland A, Szatmari II Biotechnol., 19, 114–120. and Keith WN. (2000). Neoplasia, 2, 531–539. Wick M, Zubov D and Hagen G. (1999). Gene, 232, 97–106. Zhao JQ, Hoare SF, McFarlane R, Muir S, Parkinson Wisman GB, De Jong S, Meersma GJ, Helder MN, Hollema EK, Black DM and Keith WN. (1998). Oncogene, 16, H, de Vries EG, Keith WN and van der Zee AG. (2000). 1345–1350. Hum. Pathol., 31, 1304–1312.

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