Poster Presentation 25th Lorne Cancer Conference 2013

Loss of ATRX or DAXX facilitates cellular immortalisation via the Alternative Lengthening of Telomeres pathway (#290)

Christine E Napier 1 , Lily I Huschtscha 1 , Kylie Bower 1 , Roger R Reddel 1
  1. Children's Medical Research Institute, Westmead, NSW, Australia

The progressive shortening of telomeres that occurs during normal cell division prevents normal cells undergoing unlimited proliferation. Cancer cells escape this barrier by activating a telomere maintenance mechanism: either the enzyme telomerase, or Alternative Lengthening of Telomeres (ALT). It has previously been shown that many ALT-positive tumours have mutations in a member of the ATRX/DAXX chromatin remodelling complex (1). Therefore, we used shRNA to stably knock down ATRX or DAXX to determine whether the loss of ATRX or DAXX predisposes non-immortal cells to become immortal via the ALT pathway. We transduced four different transformed mortal fibroblast cell strains with an empty vector control, scrambled control or an shRNA vector targeting either ATRX or DAXX. From these experiments, 28 immortal cell lines have been generated. Telomerase was activated in four of the lines, of which three were generated from the parental control cell strains and the fourth line was obtained after transduction with a scrambled control vector. The remaining 24 cultures activated the ALT mechanism. Of the 24 ALT positive immortal cell lines, 16 were obtained after transduction with shATRX or shDAXX and the remaining eight cultures expressed either an empty vector or a scrambled control or were derived from the parental culture. Thus a greater proportion of the immortalization events in shATRX or shDAXX transduced cells was associated with activation of ALT than in control cultures (P=0.02, Fisher’s exact test). The correlation between loss of ATRX or DAXX expression and activation of ALT supports the hypothesis that the ATRX/DAXX complex is a repressor of the ALT mechanism.

1. Heaphy,C.M. et al., Altered telomeres in tumors with ATRX and DAXX mutations. Science 333: 425, 2011.