Telomere dynamics and homeostasis in a transmissible cancer

Abstract

Background: Devil Facial Tumour Disease (DFTD) is a unique clonal cancer that threatens the world's largest carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii) with extinction. This transmissible cancer is passed between individual devils by cell implantation during social interactions. The tumour arose in a Schwann cell of a single devil over 15 years ago and since then has expanded clonally, without showing signs of replicative senescence; in stark contrast to a somatic cell that displays a finite capacity for replication, known as the "Hayflick limit".

Methodology/principal findings: In the present study we investigate the role of telomere length, measured as Telomere Copy Number (TCN), and telomerase and shelterin gene expression, as well as telomerase activity in maintaining hyperproliferation of Devil Facial Tumour (DFT) cells. Our results show that DFT cells have short telomeres. DFTD TCN does not differ between geographic regions or between strains. However, TCN has increased over time. Unlimited cell proliferation is likely to have been achieved through the observed up-regulation of the catalytic subunit of telomerase (TERT) and concomitant activation of telomerase. Up-regulation of the central component of shelterin, the TRF1-intercating nuclear factor 2 (TINF2) provides DFT a mechanism for telomere length homeostasis. The higher expression of both TERT and TINF2 may also protect DFT cells from genomic instability and enhance tumour proliferation.

Conclusions/significance: DFT cells appear to monitor and regulate the length of individual telomeres: i.e. shorter telomeres are elongated by up-regulation of telomerase-related genes; longer telomeres are protected from further elongation by members of the shelterin complex, which may explain the lack of spatial and strain variation in DFT telomere copy number. The observed longitudinal increase in gene expression in DFT tissue samples and telomerase activity in DFT cell lines might indicate a selection for more stable tumours with higher proliferative potential.

Figure 1. The Telomere Restriction Fragment Length analysis of devil samples revealed the presence of restriction enzyme recognition sites intercalated between the telomeric (TTAGGG)n sequences, preventing an accurate estimate of TRF lengths.

Sample names depict the time of collection (10 = 2010). Identical numbers represent different tissue samples collected from the same animal, S depicts spleen and T depicts DFT samples. MWM stands for molecular weight markers. CTRL 1 indicates low molecular weight control DNA (length: 3.2 kbp), CTRL 2 indicates high molecular weight control DNA (length: 10.2 kbp). Control samples were supplied in the Telo TAGGG TL Assay Kit and originated from immortal cell lines.

Figure 2. Tasmanian devil samples were collected from 17 locations across Tasmania.

Tumour progression by 2003 depicted with dashed, by 2005 with dashed-dotted and by 2007 with dotted lines. Dates indicate the progression dates of DFTD. Location abbreviations: Bo = Bothwell, Br = Bronte Park, Bu = Buckland, Fen = Fentonbury, For = Forestier, Fre = Freycinet, Ham = Hamilton, MtW = Mt William, Na = Narawntapu, Ra = Railton, Rav = Ravenswood, Re = Reedy Marsh, S = Sorell, StM = St. Marys, We = Weegena, Wi = Wisedale, WPP = West Pencil Pine.

Figure 3. Relative telomere copy number is lower in DFT samples than in other devil tissues.

Error bars depict standard deviations. Sample names indicate the time of collection (06 = 2006, 07 = 2007, 10 = 2010). Identical numbers represent different tissue samples collected from the same animal. Number of samples used in the analysis: primary tumours collected in 2006: N = 30, 2007: N = 13 and 2010: N = 8; metastasis: N = 4, spleen: N = 5, lung: N = 5.

Figure 4. Relative mean DFT telomere copy number increases with time (2006: N = 30, 2007: N = 13 and 2010: N = 8).

Error bars depict standard deviations. Significant differences in telomere copy numbers (TCN) were observed between years 2006 and 2007 (P = 0.03) and 2006 and 2010 (P = 0.01), but no significant difference in TCN was observed between years 2007 and 2010 (P = 0.6). * indicates significant differences.

Figure 5. TERT and TINF2 genes are up-regulated in DFT samples compared to spleen (P<0.0001).

Number of samples used in the analysis: tumours N = 11 and spleen N = 5. Stippled horizontal lines depict mean relative gene expression, boxplots indicate the range of standard error and bars depict 95% confidence intervals.

Figure 6. TERT and TINF2 gene expression increases with time in DFT samples.

Open bars depict TERT expression, black bars depict TINF2 expression. Number of samples used in the analysis: tumours collected in 2007: N = 5 and 2010: N = 6. * depicts significant differences (P = 0.0173 and P = 0.013, respectively).

Figure 7. Telomerase activity increases over time in DFT cell lines (P = 0.037).

The five cell lines originated from tumour samples collected in 2003, 2006, 2007, 2010 and 2011. Bars represent standard errors between technical repeats (N = 3).