Recapitulation of Werner syndrome sensitivity to camptothecin by limited knockdown of the WRN helicase/exonuclease
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WRN is a RecQ helicase with an associated exonuclease activity important in DNA metabolism, including DNA replication, repair and recombination. In humans, deficiencies in WRN function cause the segmental progeroid Werner syndrome (WS), in which patients show premature onset of many hallmarks of normal human ageing. At the cellular level, WRN loss results in rapid replicative senescence, chromosomal instability and sensitivity to various DNA damaging agents including the topoisomerase inhibitor, camptothecin (CPT). Here, we investigate the potential of using either transient or stable WRN knockdown as a means of sensitising cells to CPT. We show that targeting WRN mRNA for degradation by either RNAi or hammerhead ribozyme catalysis renders human fibroblasts as sensitive to CPT as fibroblasts derived from WS patients, and furthermore, we find altered cell cycle transit and nucleolar destabilisation in these cells following CPT treatment. Such WS-like phenotypes are observed despite very limited decreases in total WRN protein, suggesting that levels of WRN protein are rate-limiting for the cellular response to camptothecin. These findings have major implications for development of anti-WRN agents that may be useful in sensitising tumour cells to clinically relevant topoisomerase inhibitors.
KeywordsWerner syndrome WRN RecQ Camptothecin Topoisomerase RNAi Ribozyme Aging Cancer
We thank Mrs Christine Borer for technical support to LSC and MAB. This work was funded by the BBSRC grants [107/EGH16152 and 107/ERA16270] to RGAF, JLEB, KJ-B and JL, BBSRC grants [BB/E000924/1] and [43/ERA16310] and ESRC programme grant [ES/G037086/1] (under the cross-council New Dynamics of Ageing initiative) to LSC, and NIH grant AG024399 to JC.
Conflicts of interest
The authors state no conflicts of interest.
- Agrelo R, Cheng WH, Setien F, Ropero S, Espada J, Fraga MF, Herranz M, Paz MF, Sanchez-Cespedes M, Artiga MJ, Guerrero D, Castells A, von Kobbe C, Bohr VA et al (2006) Epigenetic inactivation of the premature aging Werner syndrome gene in human cancer. Proc Natl Acad Sci USA 103(23):8822–8827PubMedCrossRefGoogle Scholar
- Citti L, Eckstein F, Capecchi B, Mariani L, Nevischi S, Poggi A, Rainaldi G (1999) Transient transfection of a synthetic hammerhead ribozyme targeted against human MGMT gene to cells in culture potentiates the genotoxicity of the alkylation damage induced by mitozolomide. Antisense Nucleic Acid Drug Dev 9(2):125–133PubMedCrossRefGoogle Scholar
- Clingen PH, Lowe JE, Green MHL (2000) Measurement of DNA damage and repair capacity as a function of age using the Comet assay. In: Barnett YA, Barnett CR (eds) Methods in molecular medicine, vol 38: ageing methods and protocols. Human Press, Totowa, NJ, pp 143–157Google Scholar
- Machwe A, Xiao L, Lloyd RG, Bolt E, Orren DK (2007) Replication fork regression in vitro by the Werner syndrome protein (WRN): Holliday junction formation, the effect of leading arm structure and a potential role for WRN exonuclease activity. Nucleic Acids Res 35(17):5729–5747PubMedCrossRefGoogle Scholar
- Sidorova JM, Li N, Folch A, Monnat RJ, Jr (2008) The RecQ helicase WRN is required for normal replication fork progression after DNA damage or replication fork arrest. Cell Cycle 7(6):796–807Google Scholar