Genomic instability and telomere fusion of canine osteosarcoma cells.

Canine osteosarcoma (OSA) is known to present with highly variable and chaotic karyotypes, including hypodiploidy, hyperdiploidy, and increased numbers of metacentric chromosomes. The spectrum of genomic instabilities in canine OSA has significantly augmented the difficulty in clearly defining the biological and clinical significance of the observed cytogenetic abnormalities. In this study, eight canine OSA cell lines were used to investigate telomere fusions by fluorescence in situ hybridization (FISH) using a peptide nucleotide acid probe. We characterized each cell line by classical cytogenetic studies and cellular phenotypes including telomere associated factors and then evaluated correlations from this data. All eight canine OSA cell lines displayed increased abnormal metacentric chromosomes and exhibited numerous telomere fusions and interstitial telomeric signals. Also, as evidence of unstable telomeres, colocalization of γ-H2AX and telomere signals in interphase cells was observed. Each cell line was characterized by a combination of data representing cellular doubling time, DNA content, chromosome number, metacentric chromosome frequency, telomere signal level, cellular radiosensitivity, and DNA-PKcs protein expression level. We have also studied primary cultures from 10 spontaneous canine OSAs. Based on the observation of telomere aberrations in those primary cell cultures, we are reasonably certain that our observations in cell lines are not an artifact of prolonged culture. A correlation between telomere fusions and the other characteristics analyzed in our study could not be identified. However, it is important to note that all of the canine OSA samples exhibiting telomere fusion utilized in our study were telomerase positive. Pending further research regarding telomerase negative canine OSA cell lines, our findings may suggest telomere fusions can potentially serve as a novel marker for canine OSA

Characteristics in eight canine OSA cell lines.

*<p>Mean ± SD of chromosome number per cell from more than 150 metaphases.</p>**<p>Mean ± SD of metacentrics per cell from more than 75 metaphases.</p>***<p>SF2: The survival fraction after 2 Gy. Calculated by Graph Pad Prism 5 with linear or linear quadratic regression.</p

Radiation induced survival curves in eight canine OSA cell lines and one human OSA cell line, U2OS.

<p>Experiments were carried out at least three times and error bars indicate the standard error of the means.</p

Telomere abnormalities.

<p>Representative FISH images of the eight canine OSA cell lines’ metaphase chromosomes hybridized with probes against telomeres. Blue represents DNA staining by DAPI and red represents a telomere signal by Cy3. Note the abnormal telomere signals in the magnification box; interstitial telomere signals (A and F), more than one telomere signal in centromere regions (B, D and E), and one or no telomere signal (C) is observed. Note that at the end of chromosomes, there is no telomere signal present (B and E).</p

Sum of telomere abnormalities in ten primary canine OSA cell culture.

*<p>Mean ± SD of chromosome number per cell from more than 100 metaphases.</p>**<p>Sum of four types of telomere abnormalities ± SD per cell from more than 30 cells.</p

Telomere abnormalities distinguished by Rb fusions and interstitial signals in OSA cells.

<p>(A) Four types of telomere abnormalities; ITS¹, one interstitial telomeric sequence, ITS²⁺, more than one interstitial telomeric sequences, Rb¹, Robertsonian translocation with one telomere signal in the centromere region, and Rb²⁺, Robertsonian translocation with more than one telomere signals in the centromere region. Rb⁻ represents Robertsonian translocation with no telomere signal in the centromere region. (B) The number of telomere aberrations per each metaphase cell. Error bars indicate the standard error of the means.</p

Representative images for colocalization of telomere signals and γ-H2AX foci in interphase nuclei of OSA cells.

<p>The D17 cell line is shown in panel A, B and C. D17 shows telomere signals (A) and γ-H2AX (B) and the merged image (C). (D, E and F) represent interphase nuclei of the Grey cell line. Arrows denote colocalizations.</p

Primary canine OSA cell cultures and telomere fusions.

<p>Representative FISH images of the two primary canine OSA cell cultures’ metaphase chromosomes hybridized with probes against telomeres; OSA-1 (A), the sample originated from the limb, OSA-2 (B), the sample originated from the scapula. Note the abnormal telomere signals in the magnification box. Blue represents DNA staining by DAPI and red represents a telomere signal by Cy3. (C) The number of telomere aberrations per each metaphase cell. Error bars indicate the standard error of the means.</p

Telomerase activity in canine OSA cell lines.

<p>TRAP assay confirmed all cell lines expressed enzymatically active TERT. Positive controls were provided by the manufacturer. (−) Non–heated extract, (+) heated extract, IC: internal PCR control.</p