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Targeting the telomere and shelterin complex for cancer therapy: current views and future perspectives.

Bilsland AE, Cairney CJ, Keith WN - J. Cell. Mol. Med. (2011)

Bottom Line: Functioning telomeres are disguised from the DNA damage machinery by DNA remodelling and other activities of the telomere binding complex shelterin.Direct interference with shelterin has been shown to result in cell killing and small molecules directly targeting telomere DNA also have anti-tumour effects partially dependent on shelterin disruption.However, shelterin components have not generally been regarded as therapeutic targets in their own right.

View Article: PubMed Central - PubMed

Affiliation: University of Glasgow, Institute of Cancer Sciences, Beatson Laboratories, Bearsden, Glasgow, UK.

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Cell based screening for inhibitors of telomere uncapping. (A) Infection with an adenoviral vector expressing mutant hTR (Ad-hTR-mut) induces rapid telom-ere uncapping that can be used as a model system to investigate novel bio-markers or signalling events downstream of telomere dysfunction, or as a screening approach for inhibitors of these pathways. (B) Colorectal carcinoma cell line HCT116 was infected with Ad-hTR-mut. One day after infection cells were drugged with 1.25 μM Suramin or ATM/ATR inhibitor CGK733 (Both Merck, Darmstadt, Germany) or 0.078 μM BIBR1532 (Tocris Bioscience, Ellisville, MO, USA). Five days after infection cell viability was assessed by MTT cytotoxicity assay. Viability of treated cells was expressed as fold of virus alone.
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fig02: Cell based screening for inhibitors of telomere uncapping. (A) Infection with an adenoviral vector expressing mutant hTR (Ad-hTR-mut) induces rapid telom-ere uncapping that can be used as a model system to investigate novel bio-markers or signalling events downstream of telomere dysfunction, or as a screening approach for inhibitors of these pathways. (B) Colorectal carcinoma cell line HCT116 was infected with Ad-hTR-mut. One day after infection cells were drugged with 1.25 μM Suramin or ATM/ATR inhibitor CGK733 (Both Merck, Darmstadt, Germany) or 0.078 μM BIBR1532 (Tocris Bioscience, Ellisville, MO, USA). Five days after infection cell viability was assessed by MTT cytotoxicity assay. Viability of treated cells was expressed as fold of virus alone.

Mentions: Cell based assays could provide a second general approach to identify shelterin inhibitors [2]. Importantly, many interactions could be addressed without pre-existing knowledge of the pathway anatomy. We recently reported that promoter screening can identify inhibitors of hTERT expression [50]. Similar assays might be justified for some shelterin related targets such as TRF2, which is overexpressed in several human tumours and may play a role in drug resistance in some settings [6, 51, 52]. Similarly the telomere uncapping phenotype itself could be used in a screening approach to identify potential inhibitors of telomere dysfunction or telomerase itself. In addition, the availability of a good cellular model of telomere uncapping will enable further investigation of the signalling events underlying the telomere uncapping response and may identify new biomarkers of telomere dysfunction, which could aid future drug discovery strategies targeting the telomere (Fig. 2A). We designed a cell based screening assay, which utilizes an adenoviral vector expressing a form of hTR mutated in the template region for reverse transcription by the telomerase catalytic subunit hTERT (Ad-hTR-mut). When incorporated into telomeres this template synthesizes mutant telomere repeats of the sequence TATATATATAA [53], which are predicted to be incapable of binding key components of the shelterin complex, POT1, TRF1 and TRF2. Expression of this construct in cancer cell lines induces rapid telomere uncapping, DNA damage signalling and cyto-toxicity in an hTERT-dependent manner [53–57], thereby circumventing the problem of phenotypic lag which has hampered identification of telomerase inhibitors through screening assays in the past. In the context of this screening assay any inhibitors of telomere uncapping or the downstream signalling events will result in increased cell viability as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay, therefore any compounds inducing non-specific toxic effects will be removed in the initial screening process. With an estimated proportion of 30% of drugs failing in clinical trial due to non-specific toxicity [58] this is a tantalizing prospect from a drug development point of view.


Targeting the telomere and shelterin complex for cancer therapy: current views and future perspectives.

Bilsland AE, Cairney CJ, Keith WN - J. Cell. Mol. Med. (2011)

Cell based screening for inhibitors of telomere uncapping. (A) Infection with an adenoviral vector expressing mutant hTR (Ad-hTR-mut) induces rapid telom-ere uncapping that can be used as a model system to investigate novel bio-markers or signalling events downstream of telomere dysfunction, or as a screening approach for inhibitors of these pathways. (B) Colorectal carcinoma cell line HCT116 was infected with Ad-hTR-mut. One day after infection cells were drugged with 1.25 μM Suramin or ATM/ATR inhibitor CGK733 (Both Merck, Darmstadt, Germany) or 0.078 μM BIBR1532 (Tocris Bioscience, Ellisville, MO, USA). Five days after infection cell viability was assessed by MTT cytotoxicity assay. Viability of treated cells was expressed as fold of virus alone.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3822786&req=5

fig02: Cell based screening for inhibitors of telomere uncapping. (A) Infection with an adenoviral vector expressing mutant hTR (Ad-hTR-mut) induces rapid telom-ere uncapping that can be used as a model system to investigate novel bio-markers or signalling events downstream of telomere dysfunction, or as a screening approach for inhibitors of these pathways. (B) Colorectal carcinoma cell line HCT116 was infected with Ad-hTR-mut. One day after infection cells were drugged with 1.25 μM Suramin or ATM/ATR inhibitor CGK733 (Both Merck, Darmstadt, Germany) or 0.078 μM BIBR1532 (Tocris Bioscience, Ellisville, MO, USA). Five days after infection cell viability was assessed by MTT cytotoxicity assay. Viability of treated cells was expressed as fold of virus alone.
Mentions: Cell based assays could provide a second general approach to identify shelterin inhibitors [2]. Importantly, many interactions could be addressed without pre-existing knowledge of the pathway anatomy. We recently reported that promoter screening can identify inhibitors of hTERT expression [50]. Similar assays might be justified for some shelterin related targets such as TRF2, which is overexpressed in several human tumours and may play a role in drug resistance in some settings [6, 51, 52]. Similarly the telomere uncapping phenotype itself could be used in a screening approach to identify potential inhibitors of telomere dysfunction or telomerase itself. In addition, the availability of a good cellular model of telomere uncapping will enable further investigation of the signalling events underlying the telomere uncapping response and may identify new biomarkers of telomere dysfunction, which could aid future drug discovery strategies targeting the telomere (Fig. 2A). We designed a cell based screening assay, which utilizes an adenoviral vector expressing a form of hTR mutated in the template region for reverse transcription by the telomerase catalytic subunit hTERT (Ad-hTR-mut). When incorporated into telomeres this template synthesizes mutant telomere repeats of the sequence TATATATATAA [53], which are predicted to be incapable of binding key components of the shelterin complex, POT1, TRF1 and TRF2. Expression of this construct in cancer cell lines induces rapid telomere uncapping, DNA damage signalling and cyto-toxicity in an hTERT-dependent manner [53–57], thereby circumventing the problem of phenotypic lag which has hampered identification of telomerase inhibitors through screening assays in the past. In the context of this screening assay any inhibitors of telomere uncapping or the downstream signalling events will result in increased cell viability as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay, therefore any compounds inducing non-specific toxic effects will be removed in the initial screening process. With an estimated proportion of 30% of drugs failing in clinical trial due to non-specific toxicity [58] this is a tantalizing prospect from a drug development point of view.

Bottom Line: Functioning telomeres are disguised from the DNA damage machinery by DNA remodelling and other activities of the telomere binding complex shelterin.Direct interference with shelterin has been shown to result in cell killing and small molecules directly targeting telomere DNA also have anti-tumour effects partially dependent on shelterin disruption.However, shelterin components have not generally been regarded as therapeutic targets in their own right.

View Article: PubMed Central - PubMed

Affiliation: University of Glasgow, Institute of Cancer Sciences, Beatson Laboratories, Bearsden, Glasgow, UK.

Show MeSH
Related in: MedlinePlus