Selective Ablation of Human Cancer Cells by Telomerase-Specific

Oncogene (2003) 22, 370–380 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc 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

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