• View in gallery
    Figure 1—

    Southern blot of TRF smears in normal and neoplastic canine tissues. Lanes 1 through 6 represent normal oral mucosa, and lanes 7 through 10 represent oral squamous cell carcinoma. Numbers on the left side represent the number of kilobases.

  • View in gallery
    Figure 2—

    Box-and-whisker plots illustrating the distribution of mean TRF length for 44 normal and 57 neoplastic canine tissues. Boxes represent the 25th to 75th percentiles, the horizontal bar within each box represents the median value, and the whiskers represent the 95th percentiles. Circles represent outliers in the data set.

  • View in gallery
    Figure 3—

    Box-and-whisker plots illustrating the distribution of mean TRF length for samples of normal lymph nodes (n = 11) and lymphoma-bearing lymph nodes (19) obtained from dogs. See Figure 2 for key.

  • 1.

    Moyzis RK, Buckingham JM, Cram LS, et al. A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the end of human chromosomes. Proc Natl Acad Sci U S A 1988;85:66226626.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Pardue ML, DeBaryshe PG. Telomeres and telomerase: more than the end of the line. Chromosoma 1999;108:7382.

  • 3.

    de Lange T. Protection of mammalian telomeres. Oncogene 2002;41:532540.

  • 4.

    Shay JW, Wright WE. Telomerase therapeutics for cancer: challenges and new directions. Nat Rev Drug Discov 2006;5:577584.

  • 5.

    Counter CM. The roles of telomeres and telomerase in cell life span. Mutat Res 1996;366:4563.

  • 6.

    Allsopp RC, Harley CB. Evidence for a critical telomere length in senescent human fibroblasts. Exp Cell Res 1995;219:130136.

  • 7.

    van Steensel B, Smogorzewska A, deLange T. TRF2 protects human telomeres from end-to-end fusions. Cell 1998;92:401403.

  • 8.

    Hemann MT, Strong MA, Hao LY, et al. The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability. Cell 2001;107:6777.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Harley CB. Telomere loss: mitotic clock or genetic time bomb? Mutat Res 1991;256:271282.

  • 10.

    Flores I, Benetti R, Blasco MA. Telomerase regulation and stem cell behavior. Curr Opin Cell Biol 2006;18:254260.

  • 11.

    Blasco MA. Mice with bad ends: mouse models for the study of telomeres and telomerase in cancer and aging. EMBO J 2005;24:10951103.

  • 12.

    Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 1985;43:405413.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Counter CM, Betelho FM, Wang P, et al. Stabilization of short telomeres and telomerase activity accompany immortalization of Epstein-Barr virus transformed human B lymphocytes. J Virol 1994;68:34103414.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Stewart SA, Hahn WC, O'Connor BF. Telomerase contributes to tumorigenesis by a telomere length-independent mechanism. Proc Natl Acad Sci U S A 2002;99:1260612611.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Mattson MP, Klapper W. Emerging roles for telomerase in neuronal development and apoptosis. J Neurosci Res 2001;63:19.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.

    Yin L, Hubbard AK, Giardina C. NF-κB regulates transcription of the mouse telomerase catalytic subunit. J Biol Chem 2001;275:3667136675.

    • Search Google Scholar
    • Export Citation
  • 17.

    Massard C, Zermati Y, Pauleau AL, et al. hTERT: a novel endogenous inhibitor of the mitochondrial cell death pathway. Oncogene 2006;25:45054514.

  • 18.

    Fu W, Begley JG, Killen MW, et al. Anti-apoptotic role of telomerase in pheochromocytoma cells. J Biol Chem 1999;274:72647271.

  • 19.

    Akiyama M, Hideshima T, Hayashi T, et al. Nuclear factor-κB p65 mediates tumor necrosis factor A-induced nuclear translocation of telomerase reverse transcriptase protein. Cancer Res 2003;63:1821.

    • Search Google Scholar
    • Export Citation
  • 20.

    Kim NW, Piatyszek MA, Prowse KR, et al. Specific associations of human telomerase activity with immortal cells and cancer. Science 1994;266:20112015.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Shay JW, Bacchetti S. A survey of telomerase activity in human cancer. Eur J Cancer 1997;5:787791.

  • 22.

    Stewart SA, Weinberg RA. Telomeres: cancer to human aging. Annu Rev Cell Dev Biol 2006;22:531557.

  • 23.

    Biller BJ, Kitchell BE, Cadile CD. Evaluation of an assay for detecting telomerase activity in neoplastic tissues of dogs. Am J Vet Res 1998;59:15261529.

    • Search Google Scholar
    • Export Citation
  • 24.

    Yazawa M, Okuda M, Setoguchi A, et al. Measurement of telomerase activity in dog tumors. J Vet Med Sci 1999;61:11251129.

  • 25.

    Funakoshi Y, Nakayama H, Uetsuka K, et al. Cellular proliferative and telomerase activity in canine mammary gland tumors. Vet Pathol 2000;37:177183.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Yazawa M, Okuda M, Setoguchi A, et al. Telomere length and telomerase activity in canine mammary gland tumors. Am J Vet Res 2001;62:15391543.

  • 27.

    Argyle DJ, Nasir L. Telomerase: a potential diagnostic and therapeutic tool in canine oncology. Vet Pathol 2003;40:17.

  • 28.

    Kow K, Bailey SM, Williams ES. Telomerase activity in canine osteosarcoma. Vet Comp Oncol 2006;4:184187.

  • 29.

    Renwick MG, Argyle DJ, Long S, et al. Telomerase activity and telomerase reverse transcriptase catalytic subunit expression in canine lymphoma: correlation with Ki67 immunoreactivity. Vet Comp Oncol 2006;4:141150.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Yamaguchi Y, Nozawa K, Savoysky E, et al. Change in telomerase activity of rat organs during growth and aging. Exp Cell Res 1998;242:120127.

  • 31.

    Sedivy JM. Can the ends justify the means? Telomeres and the mechanisms of replicative senescence in mammalian cells. Proc Natl Acad Sci U S A 1998;95:90789081.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32.

    Hipple AK, Colitz CM, Mauldin GN. Telomerase activity and related properties of normal canine lymph node and canine lymphoma. Vet Comp Oncol 2003;1:140151.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33.

    McKevitt TP, Nasir L, Devlin P. Telomere lengths in dogs decrease with increasing donor age. J Nutr 2002;132:1604S1606S.

  • 34.

    Nasir L, Devlin P, McKevitt T, et al. Telomere lengths and telomerase activity in dog tissues: a potential model system to study human telomere and telomerase biology. Neoplasia 2001;3:351359.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35.

    Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature 1990;345:458460.

  • 36.

    Savre-Train I, Gollahon LS, Holt SE. Clonal heterogeneity in telomerase activity and telomere length in tumor-derived cell lines. Proc Soc Exp Biol Med 2000;223:379388.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37.

    Bryan TM, Englezou A, Gupta J, et al. Telomere elongation in immortal human cells without detectable telomerase activity. EMBO J 1995;14:42404248.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38.

    Henson JD, Neumann AA, Yeager TR. Alternative lengthening of telomeres in mammalian cells. Oncogene 2002;21:598610.

  • 39.

    Ulaner GA, Huang HY, Otero J, et al. Absence of telomere maintenance mechanism as a favourable prognostic factor in patients with osteosarcoma. Cancer Res 2003;63:17591763.

    • Search Google Scholar
    • Export Citation
  • 40.

    Perrem K, Colgin LM, Neumann AA. Coexistence of alternative lengthening of telomeres and telomerase in hTERT-transfected GM847 cells. Mol Cell Biol 2001;21:38623875.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41.

    Hultdin M, Gronlund E, Norrback K, et al. Telomere analysis by fluorescence in situ hybridization and flow cytometry. Nucleic Acids Res 1998;26:36513656.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42.

    Reimann N, Rogalla P, Kazmierczak B. Evidence that metacentric and submetacentric chromosomes in canine tumors can result from telomeric fusions. Cytogenet Cell Genet 1994;67:8185.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43.

    Colangelo D, Osella O. Telomerase inhibition and cancer: might platinum based drugs have a future as anti-telomerase pharmacological approach? Curr Med Chem 2005;12:30913102.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44.

    Colangelo D, Ghiglia AL, Viano I, et al. Cis-[Pt(Cl)2(pyridine)(5SO3H-isoquinoline)] complex, a selective inhibitor of telomerase enzyme. Biometals 2003;16:553560.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 45.

    Sun PM, Wei LH, Luo MY, et al. The telomerase activity and expression of hTERT gene can serve as indicators in the anticancer treatment of human ovarian cancer. Eur J Obstet Gynec Reprod Biol 2005;130:249257.

    • Search Google Scholar
    • Export Citation
  • 46.

    Shammas MA, Reis RJS, Li C, et al. Telomerase inhibition and cell growth arrest after telomestatin treatment in multiple myeloma. Clin Cancer Res 2004;10:770776.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 47.

    Tahara H, Shinya K, Seimiya H, et al. G-quadruplex stabilization by telomestatin induces TRF2 protein dissociation from telomeres and anaphase bridge formation accompanied by the loss of the 3′ overhang in cancer cells. Oncogene 2006;25:19551966.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 48.

    Artandi SE, Alson S, Tietze MK, et al. Constitutive telomerase expression promotes mammary carcinomas in mice. Proc Natl Acad Sci U S A 2002;99:81918196.

    • Crossref
    • Search Google Scholar
    • Export Citation

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Telomere length in normal and neoplastic canine tissues

Casey D. CadileDepartment of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Barbara E. KitchellDepartment of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Rebecca G. NewmanDepartment of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Barbara J. BillerDepartment of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Elizabeth R. HetlerDepartment of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802.

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Abstract

Objective—To determine the mean telomere restriction fragment (TRF) length in normal and neoplastic canine tissues.

Sample Population—57 solid-tissue tumor specimens collected from client-owned dogs, 40 samples of normal tissue collected from 12 clinically normal dogs, and blood samples collected from 4 healthy blood donor dogs.

Procedures—Tumor specimens were collected from client-owned dogs during diagnostic or therapeutic procedures at the University of Illinois Veterinary Medical Teaching Hospital, whereas 40 normal tissue samples were collected from 12 control dogs. Telomere restriction fragment length was determined by use of an assay kit. A histologic diagnosis was provided for each tumor by personnel at the Veterinary Diagnostic Laboratory at the University of Illinois.

Results—Mean of the mean TRF length for 44 normal samples was 19.0 kilobases (kb; range, 15.4 to 21.4 kb), and the mean of the mean TRF length for 57 malignant tumors was 19.0 kb (range, 12.9 to 23.5 kb). Although the mean of the mean TRF length for tumors and normal tissues was identical, tumor samples had more variability in TRF length.

Conclusions and Clinical Relevance—Telomerase, which represents the main mechanism by which cancer cells achieve immortality, is an attractive therapeutic target. The ability to measure telomere length is crucial to monitoring the efficacy of telomerase inhibition. In contrast to many other mammalian species, the length of canine telomeres and the rate of telomeric DNA loss are similar to those reported in humans, making dogs a compelling choice for use in the study of human antitelomerase strategies.

Abstract

Objective—To determine the mean telomere restriction fragment (TRF) length in normal and neoplastic canine tissues.

Sample Population—57 solid-tissue tumor specimens collected from client-owned dogs, 40 samples of normal tissue collected from 12 clinically normal dogs, and blood samples collected from 4 healthy blood donor dogs.

Procedures—Tumor specimens were collected from client-owned dogs during diagnostic or therapeutic procedures at the University of Illinois Veterinary Medical Teaching Hospital, whereas 40 normal tissue samples were collected from 12 control dogs. Telomere restriction fragment length was determined by use of an assay kit. A histologic diagnosis was provided for each tumor by personnel at the Veterinary Diagnostic Laboratory at the University of Illinois.

Results—Mean of the mean TRF length for 44 normal samples was 19.0 kilobases (kb; range, 15.4 to 21.4 kb), and the mean of the mean TRF length for 57 malignant tumors was 19.0 kb (range, 12.9 to 23.5 kb). Although the mean of the mean TRF length for tumors and normal tissues was identical, tumor samples had more variability in TRF length.

Conclusions and Clinical Relevance—Telomerase, which represents the main mechanism by which cancer cells achieve immortality, is an attractive therapeutic target. The ability to measure telomere length is crucial to monitoring the efficacy of telomerase inhibition. In contrast to many other mammalian species, the length of canine telomeres and the rate of telomeric DNA loss are similar to those reported in humans, making dogs a compelling choice for use in the study of human antitelomerase strategies.

All eukaryotic chromosomes are capped by numerous kilobases of tandem hexanucleotide repeats (TTAGGG in vertebrates).1 These dynamic and complex terminal regions are called telomeres. In combination with certain proteins, telomeres serve to shield more proximal DNA from degradation and recombination events. Telomeres conceal the ends of chromosomes from repair systems that may recognize chromosomal termini as double-stranded DNA breaks.2

Each 3′ chromosome terminus has an overhang of 150 to 200 nucleotides that consists of single-stranded telomeric DNA, which inserts more proximally into the double-stranded portion of the telomere to form the T loop.3 At birth, human telomeres contain 15 to 20 kb of telomeric DNA, but by middle age, telomeres have decreased in length by approximately half.4 This telomere attrition is attributable to the end-replication problem, which results because DNA polymerase synthesizes DNA in a 5′ to 3′ direction and cells have no means of replacing the short RNA primers that transiently occupy the chromosomal extremity to initiate replication.5 Thus, with each cell division, telomeres of the daughter strands are shorter than those of the parent strands. Human cells lose between 30 and 200 bp of telomeric DNA in each cell division.6 When the telomeres have eroded sufficiently that they no longer provide the essential capping function, the cell is triggered to enter replicative senescence (mortality stage 1).4 This shortage of telomeric DNA leaves the numerous telomeric repeat binding proteins without binding sites, which causes destabilization of T-loop structures.7 When cell-cycle checkpoint mechanisms, such as p53 and pRb, are not operating properly during mortality stage 1, cells continue to divide and consequently lose additional telomeric DNA. This results in erroneous repair, fusion, and recombination events and can lead to mitotic catastrophe, which is referred to as mortality stage 2.4

It has been suggested8 that the shortest telomere in a cell, instead of a critically short mean TRF length of all chromosomes, prompts arrest of the cell cycle.8 This mitotic clock imparts a finite proliferative capacity to a cell.9 Stem and neoplastic cells overcome this by activating telomerase, which restores the health of the telomere and restores replicative capacity to the cell.10 Only 1 in every 106 human epithelial cells and 1 in every 107 human fibroblasts escape the mortality stage to become immortal.4

Telomerase is a ribonucleoprotein enzyme that is the most important factor regulating telomere length in mammalian cells.11 Telomerase is a cellular reverse transcriptase that contains an RNA sequence complementary to the sequence of telomeric DNA. Telomerase consists of 2 components, an RNA template (ie, TERC) and a catalytic protein (ie, TERT). By constructing telomeric repeats and catalyzing their addition to the shortest telomeres, further loss of telomeres is prevented.12 Especially important is the integrity of the T loop, which modulates the accessibility of telomeres to telomerase.11 By offsetting shortening of telomeres and thus extending a cell's proliferative capacity, telomerase confers replicative immortality to the cell.13

In addition, it has been postulated11,14,15 that telomerase enhances survival and proliferation independently of telomere length. Expression of the TERT gene is induced by the transcription factors NFκB and c-myc.15,16 Upregulation of TERT has a role in the survival-promoting qualities of NFκB and c-myc, and TERT specifically appears to be a downstream-effector mechanism that interferes with premitochondrial death signaling.15,17,18 In human cells, NFκB p65 binds with TERT protein during its translocation from the cytoplasm to the nucleus. This movement is modulated by tumor necrosis factor-A.19

The impact of telomerase activation on apoptotic mechanisms is further linked to the survival factor Bcl-2. Overexpression of Bcl-2 protein in human tumor cells results in significant increases in telomerase activity and apoptosis resistance.18 The antiapoptotic effects of telomerase are upstream of the location of the actions of Bcl-2 within the apoptotic cascade.18 Small interfering RNAs can be used to deplete TERT from human cervical and colon cancer cell lines, which results in the facilitation of apoptosis by conventional chemotherapeutic agents.17

In 1994, investigators developed a method (ie, the TRAP assay) for qualitative detection of telomerase activity in human cells, which can detect 1 telomerase-positive cell/10,000 nonexpressing human cells.20 Telomerase is activated in at least 90% of all human tumors.10,11 Normal somatic cells overwhelmingly have negative results when tested for telomerase.21 Exceptions include germ cells and stem cells, which must retain proliferative potential.10 In tumors that arise from stem cells, telomerase may already be active.4 Interestingly, telomerase activation in tumor cells stabilizes existing telomere lengths but does not restore telomeres to the longer lengths evident in adjacent normal cells.22 It has been suggested7 that the ability of telomerase to maintain adequate length of 3′ ends and healthy T loops allows cells to avoid genetic instability and apoptosis.

Our laboratory group evaluated canine tissues for telomerase activity in an effort to validate the TRAP assay for use in the canine species.23 We found that the TRAP assay had sensitivity of 86% and specificity of 95% for detecting malignant canine cells when correlated with a diagnosis established by histologic and cytologic examinations. This provided further evidence to support the use of the TRAP assay as a rapid qualitative screening mechanism for neoplastic cells. Other veterinary groups have reported similar findings, and it is generally accepted that > 90% of all tumors in dogs have telomerase activity.23-29

Biological aspects of telomerase in rodents appear to differ from those of humans and dogs. First, rodent tissues constitutively express telomerase.30 Additionally, cultured rodent cells are able to overcome senescence and become immortal, whereas cultured human cells require a complex series of events to induce transformation and malignancy.31 Finally, mean length of mouse telomeres is 50 kb, whereas mean length of human telomeres is 10 to 20 kb.22 Thus, rodent cells appear to fundamentally differ in telomere biology, compared with that of human cells. Dogs may represent a more appropriate method for study of the human telomerase system.

Several studies26,32-34 have been conducted to evaluate TRF length in canine tissues. In dogs, 47 tumor tissues and 30 normal tissues have been evaluated to determine TRF length. Evaluation of cultured cells, normal tissues, and tumor tissues has revealed a mean telomere length in dogs of 9.7 to 23 kb.

In light of the scant literature currently available on the subject, our objective in the study reported here was to better quantify telomere length in normal and malignant canine tissues. We hypothesized that tumor tissues would have significantly shorter mean TRF length than the TRF length in normal tissues. We also evaluated whether tumor and normal tissues of the same histologic type would have differences in mean TRF length.

Materials and Methods

Animals—Tissue samples were obtained with consent from 57 client-owned dogs that underwent diagnostic procedures at the University of Illinois Veterinary Medical Teaching Hospital between July 1997 and December 2001. Neoplasia in these dogs was confirmed histologically by personnel at the University of Illinois Veterinary Diagnostic Laboratory. In addition, tissue samples were harvested from 12 healthy middle-aged control dogs euthanized at the conclusion of an unrelated orthopedic study. Dogs were euthanized by IV injection of euthanasia solution (a combination of phenobarbital and phenytoin). The control dogs were healthy as judged on the basis of results of physical examination, a CBC, and serum biochemical analysis and gross necropsy findings. Finally, blood samples were obtained from each of 4 healthy blood donor dogs. The protocol for the study was approved by the University of Illinois Laboratory Animal Care Advisory Committee.

Sample collection—Neoplastic tissue samples from the 57 client-owned dogs were collected during incisional or excisional biopsy procedures and consisted of an excess for the amount needed for standard histologic examination. Representative grossly normal tissue samples (n = 40) were collected from the control dogs. Four blood samples were obtained from a peripheral vein of the blood donor dogs. All tissue and PBMC samples were snap-frozen in liquid nitrogen and stored at −80°C until evaluation. All samples were tested by 2 investigators (CDC and RGN) who were not aware of the diagnosis for each tissue sample.

DNA preparation—The DNA was extracted from tissues and quantitated by use of standard methods. Genomic DNA (2.5 μg) was digested with RsaI and HinfI for 12 hours at 37°C.

Southern blot analysis—In accordance with the manufacturer's instructions, experimental DNA fragments were separated overnight on a 0.6% Tris-borate-EDTA agarose gel. Gels also included biotinylated control DNA samples provided in the telomere assay kita and standard predigested L DNA molecular-weight standards. The 2 DNA control samples (2.5 μg each) were HinfI-RsaI digested DNA with a mean TRF length of 11.3 and 3.3 kb, respectively. Gels were stained with ethidium bromide and photographed. Gels were washed with HCl and NaOH, and DNA fragments were transferred onto a positively charged nylon membrane.b Membranes were hybridized for 12 hours at 50°C to a biotinylated probe specific for telomeric repeats included in the kit.a Membranes were washed, blocked, and incubated with streptavidin–horseradish peroxidase complex included in the kit.a Chemiluminescent detection was performed in accordance with the manufacturer's instructions. Nylon membranes were exposed to autoradiography film for 30 seconds. Each of these scanned images was evaluated in a blinded manner, and results were subsequently correlated with the histologic diagnosis.

TRF calculation—Each film was digitally scanned, and image softwarec was used to assess signal intensity. Mean signal data over background signal were generated by use of a classic TRF densitometry equation: L = Σ(ODi × Li)/Σ ODi), where L is mean TRF length, ODi is the signal intensity at position i on the gel, and Li is the TRF length at position i on the gel. This equation translated the densitometric peak measurement into mean TRF length by incorporating the fact that multiple telomere-specific probes hybridize to the longer TRFs, which generates a more intense signal.35

Statistical analysis—A 2-tailed Mann-Whitney U test was used to compare differences in mean and minimum TRF length between groups. Commercially available softwared was used for statistical analysis, and graphing softwaree was used to generate box-and-whisker images.

Results

The telomere assay was performed on 97 canine tissue samples (Table 1). Neoplastic tissue samples included splenic hemangiosarcoma, bronchogenic carcinoma, oral squamous cell carcinoma, lymphoma, and fibrosarcoma. Normal (nonneoplastic) samples included those obtained from the spleen, lungs, oral mucosa, liver, lymph nodes, and PBMCs. We did not stratify results on the basis of breed or age.

Table 1—

The TRF length in normal and neoplastic canine tissues.

Table 1—

Southern blots were made for normal and neoplastic tissues (Figure 1). The 44 normal samples yielded a mean TRF length of 19.0 kb. When PBMCs were excluded, the remaining 40 normal tissue samples yielded a mean for the mean TRF length of 19.1 kb (range, 15.4 to 21.4 kb) and mean of the minimum TRF length was 14.2 kb. For the 57 neoplastic tissues, mean for the mean TRF length was 19.0 kb (range, 12.9 to 23.5 kb) and mean of the minimum TRF length was 13.9 kb. The distribution of mean TRF lengths in neoplastic tissues was compared with the mean TRF lengths for the normal samples (Figure 2).

Figure 1—
Figure 1—

Southern blot of TRF smears in normal and neoplastic canine tissues. Lanes 1 through 6 represent normal oral mucosa, and lanes 7 through 10 represent oral squamous cell carcinoma. Numbers on the left side represent the number of kilobases.

Citation: American Journal of Veterinary Research 68, 12; 10.2460/ajvr.68.12.1386

Figure 2—
Figure 2—

Box-and-whisker plots illustrating the distribution of mean TRF length for 44 normal and 57 neoplastic canine tissues. Boxes represent the 25th to 75th percentiles, the horizontal bar within each box represents the median value, and the whiskers represent the 95th percentiles. Circles represent outliers in the data set.

Citation: American Journal of Veterinary Research 68, 12; 10.2460/ajvr.68.12.1386

The 2-tailed Mann-Whitney U test for nonparametric variables was used to compare results for the 44 normal and 57 neoplastic samples. There was no significant difference in mean (P = 0.88) or minimum (P = 0.65) TRF values. There also was no significant difference in mean TRF length between corresponding normal and neoplastic tissues of the same histologic type (lymph node and lymphoma-bearing lymph node, P = 0.06; spleen and splenic hemangiosarcoma, P = 0.95; lung and bronchiolar carcinoma, P = 0.37; and oral mucosa and oral squamous cell carcinoma, P = 0.29). There was moderate variability of telomere length within various tissues obtained from the same dog. When normal tissue samples from each dog were grouped, the mean ± SD difference between the largest and smallest mean TRF length was 3.6 ± 1.5 kb.

The range of peak signals was wider for neoplastic samples (in which mean TRF length ranged from 12.9 to 23.5 kb), compared with that of normal samples (mean TRF length ranged from 15.4 to 21.4 kb). Of all tissue samples evaluated, the greatest heterogeneity in TRF length was observed in lymph nodes affected by lymphoma (Figure 3). Lymphoma-bearing nodes (n = 19) represented the highest (23.5 kb) and lowest (12.9 kb) recorded mean TRF length as well as the lowest minimum value (8.3 kb).

Figure 3—
Figure 3—

Box-and-whisker plots illustrating the distribution of mean TRF length for samples of normal lymph nodes (n = 11) and lymphoma-bearing lymph nodes (19) obtained from dogs. See Figure 2 for key.

Citation: American Journal of Veterinary Research 68, 12; 10.2460/ajvr.68.12.1386

Discussion

When telomerase becomes active in a tumor cell, telomeres are stabilized at a new shorter length.22 Therefore, telomeres in tumors of humans are generally shorter than those in adjacent normal tissue. We did not find a difference in the mean of the mean TRF length between neoplastic and normal canine tissues. We also found no difference in TRF length between normal and neoplastic tissues for any of the 4 tissue pairs evaluated (oral mucosa and squamous cell carcinoma, lung and bronchogenic carcinoma, spleen and splenic hemangiosarcoma, and lymphoma-bearing and normal lymph node). Ideally, neoplastic and control tissues from the same dog should have been compared to determine effects for age and breed variation in telomere length. However, for practical reasons, we were unable to obtain samples of normal tissues from client-owned tumor-bearing dogs at the time of surgery or biopsy procedures. Because neoplastic samples and corresponding normal tissues were harvested from various dogs, the samples were not matched on the basis of age or breed. Investigators in another study33 evaluated canine PMBCs and detected a significant association between breed and TRF length. In the study reported here, the predominance of mixed-breed dogs and number of distinct tissue categories precluded comparisons of breed- or age-based telomere length.

Of the 12 control dogs, 6 had evaluation of TRF length for tissues obtained from the lungs, oral mucosa, liver, lymph nodes, and spleen. Within each dog, there was moderate variation of TRF length among organs. For the clinically normal dogs, the mean difference between the largest and smallest mean TRF length was 3.6 kb. On the basis of results obtained in telomerase-null mice,8 it has been determined that there are significant differences in TRF length within each animal.

The range of mean TRF signals was much wider in neoplastic samples, compared with the range for normal samples (Figure 2). Of all tissue samples evaluated, the greatest heterogeneity in TRF length was observed in lymph nodes affected by lymphoma (Figure 3). Mean TRF lengths in lymphoma-bearing lymph nodes differed substantially, but not significantly (P = 0.06), from those for the normal lymph nodes. Lymphoma-bearing lymph nodes contained the highest and lowest mean TRF lengths as well as the lowest minimum and highest maximum values. A larger sample size may have increased the ability to detect significant differences, if they existed.

Quantitative in situ fluorescent hybridization has been used on cells derived from telomerase-null mice to determine that within a specific animal and even within each cell, there is significant heterogeneity in telomere length.8 Within clonal populations of tumor cells, there is often pronounced heterogeneity in TRF length.36 Thus, within a single tumor, regulation of telomerase activity can differ substantially among cells.36

Up to 10% of tumors in humans and 35% of immortal cell lines are telomerase-negative and may be examples of ALT, which uses recombination events to replenish lost telomeric DNA.37,38 In contrast to telomerase, the ALT system does not elongate the shortest telomeres preferentially; therefore, one of the hallmark features of ALT is substantial heterogeneity of TRF length within a sample.8,37,39 Samples used in the study reported here were not subjected to telomerase testing, but it is possible that some of the neoplastic samples used ALT. In 2001, investigators transfected an ALT-positive cell line with TERT cDNA and found that telomerase activity and ALT activity can coexist within the same cell.40 In another study,39 27% of osteosarcoma tissue samples from humans had positive results for both telomerase activity and ALT mechanisms of telomere elongation.

Human subtelomeric sequences are between 2.5 and 4 kb,41 but comparable information regarding dog chromosomes is not available. Variable amounts of subtelomeric sequence are included in the TRF length, depending on the specific restriction endonuclease used to digest the genomic DNA. This contributes some variation to the TRF length. Because the length of the subtelomeric region in canine chromosomes is unknown, it is not possible to compare the contributions of these regions between dogs and humans.

Other investigators have determined that the mean TRF lengths in dogs are similar to results obtained by our laboratory group. In 1994, cultured canine cells were used to detect a decrease in telomere length over time.42 In 2001, 27 mammary gland tumors of dogs had a mean for the mean TRF length of 14.5 kb, compared with a value of 18 kb for normal mammary gland tissue.26 In another study,34 mean TRF lengths in 18 soft tissue sarcomas ranged from 7.2 to 23 kb. Mean of the mean TRF length in 63 canine PBMC samples was between 12 and 23 kb. In cultured dermal fibroblasts, TRF length decreased from 20.5 kb at the first passage to 17.5 kb at the 16th passage.34 In 2002, TRF length in 47 PBMC samples from clinically normal dogs ranged from 9.7 to 22.3 kb.33 Finally, mean TRF length in 12 histologically normal lymph nodes ranged from 15 to 23 kb.32

Telomerase activation in cells can be concurrent with immortalization, thereby representing a critical feature of somatic cells that have undergone malignant transformation. It is reasonable to expect that strategies to inhibit telomerase will effectively lead to destabilization and apoptosis of telomerase-positive cells, which represent most neoplasms in humans and dogs. Inhibition strategies, whether by use of antisense oligonucleotides, reverse-transcriptase inhibitors, ribozymes, G-quadruplex binding agents, or RNA interference techniques, may greatly augment traditional anticancer strategies. In 2005, a cisplatin-like compound that reduced telomerase activity in vitro was reported.43,44 The compound promoted shortening of telomeres and apoptosis in human cancer cells by targeting the RNA component of telomerase.43,44 Investigators in another study45 used cisplatin to treat ovarian tumor cells and found that both TERT mRNA concentrations and telomerase activity decreased significantly from pretreatment values.45 The telomerase inhibitor telomestatin, a natural G-quadruplex intercalating agent derived from Streptomyces anulatus, has provided antiproliferative and proapoptotic effects in multiple myeloma cells.46,47 In tumors with long telomeres, slow attrition of telomeres may create a lag period before senescence and apoptosis become apparent. This concern often dampens enthusiasm for telomerase inhibition as a strategy for treatment of patients with cancer. However, intriguing evidence suggests that telomerase activity may play many important roles within cells, such as increasing cellular survival and proliferation, in addition to lengthening telomeres.11,15

Telomerase activity may also promote carcinogenesis in some tissues. Experimental overexpression of TERT in female mice with normal telomeres imparts a high incidence of mammary carcinoma.48

The aforementioned results strengthen the case for antitelomerase treatment strategies, even in cells with relatively long telomeres. The ability to reliably measure telomerase length and telomerase activity in canine cells and tissues supports the value of tumor-bearing dogs as a translational technique for testing inhibition of telomerase as an anticancer therapeutic strategy.

ABBREVIATIONS

kb

Kilobase

TRF

Telomere restriction fragment

TERC

Telomerase RNA component

TERT

Telomerase reverse transcriptase

NFκB

Nuclear factor kappa B

TRAP

Telomeric repeat amplification protocol

PBMC

Peripheral blood mononuclear cell

ALT

Alternative lengthening of telomeres

a.

TeloQuant telomere length assay kit, BD PharMingen, San Diego, Calif.

b.

Hybond N+ nylon membrane, Amersham Biosciences, Piscataway, NJ.

c.

Scion Image for Windows, Scion Corp, Frederick, Md.

d.

GraphPad InStat, GraphPad Software Inc, San Diego, Calif.

e.

SPSS-15 for Windows, SPSS Inc, Chicago, Ill.

References

  • 1.

    Moyzis RK, Buckingham JM, Cram LS, et al. A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the end of human chromosomes. Proc Natl Acad Sci U S A 1988;85:66226626.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Pardue ML, DeBaryshe PG. Telomeres and telomerase: more than the end of the line. Chromosoma 1999;108:7382.

  • 3.

    de Lange T. Protection of mammalian telomeres. Oncogene 2002;41:532540.

  • 4.

    Shay JW, Wright WE. Telomerase therapeutics for cancer: challenges and new directions. Nat Rev Drug Discov 2006;5:577584.

  • 5.

    Counter CM. The roles of telomeres and telomerase in cell life span. Mutat Res 1996;366:4563.

  • 6.

    Allsopp RC, Harley CB. Evidence for a critical telomere length in senescent human fibroblasts. Exp Cell Res 1995;219:130136.

  • 7.

    van Steensel B, Smogorzewska A, deLange T. TRF2 protects human telomeres from end-to-end fusions. Cell 1998;92:401403.

  • 8.

    Hemann MT, Strong MA, Hao LY, et al. The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability. Cell 2001;107:6777.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Harley CB. Telomere loss: mitotic clock or genetic time bomb? Mutat Res 1991;256:271282.

  • 10.

    Flores I, Benetti R, Blasco MA. Telomerase regulation and stem cell behavior. Curr Opin Cell Biol 2006;18:254260.

  • 11.

    Blasco MA. Mice with bad ends: mouse models for the study of telomeres and telomerase in cancer and aging. EMBO J 2005;24:10951103.

  • 12.

    Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 1985;43:405413.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Counter CM, Betelho FM, Wang P, et al. Stabilization of short telomeres and telomerase activity accompany immortalization of Epstein-Barr virus transformed human B lymphocytes. J Virol 1994;68:34103414.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Stewart SA, Hahn WC, O'Connor BF. Telomerase contributes to tumorigenesis by a telomere length-independent mechanism. Proc Natl Acad Sci U S A 2002;99:1260612611.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Mattson MP, Klapper W. Emerging roles for telomerase in neuronal development and apoptosis. J Neurosci Res 2001;63:19.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.

    Yin L, Hubbard AK, Giardina C. NF-κB regulates transcription of the mouse telomerase catalytic subunit. J Biol Chem 2001;275:3667136675.

    • Search Google Scholar
    • Export Citation
  • 17.

    Massard C, Zermati Y, Pauleau AL, et al. hTERT: a novel endogenous inhibitor of the mitochondrial cell death pathway. Oncogene 2006;25:45054514.

  • 18.

    Fu W, Begley JG, Killen MW, et al. Anti-apoptotic role of telomerase in pheochromocytoma cells. J Biol Chem 1999;274:72647271.

  • 19.

    Akiyama M, Hideshima T, Hayashi T, et al. Nuclear factor-κB p65 mediates tumor necrosis factor A-induced nuclear translocation of telomerase reverse transcriptase protein. Cancer Res 2003;63:1821.

    • Search Google Scholar
    • Export Citation
  • 20.

    Kim NW, Piatyszek MA, Prowse KR, et al. Specific associations of human telomerase activity with immortal cells and cancer. Science 1994;266:20112015.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Shay JW, Bacchetti S. A survey of telomerase activity in human cancer. Eur J Cancer 1997;5:787791.

  • 22.

    Stewart SA, Weinberg RA. Telomeres: cancer to human aging. Annu Rev Cell Dev Biol 2006;22:531557.

  • 23.

    Biller BJ, Kitchell BE, Cadile CD. Evaluation of an assay for detecting telomerase activity in neoplastic tissues of dogs. Am J Vet Res 1998;59:15261529.

    • Search Google Scholar
    • Export Citation
  • 24.

    Yazawa M, Okuda M, Setoguchi A, et al. Measurement of telomerase activity in dog tumors. J Vet Med Sci 1999;61:11251129.

  • 25.

    Funakoshi Y, Nakayama H, Uetsuka K, et al. Cellular proliferative and telomerase activity in canine mammary gland tumors. Vet Pathol 2000;37:177183.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Yazawa M, Okuda M, Setoguchi A, et al. Telomere length and telomerase activity in canine mammary gland tumors. Am J Vet Res 2001;62:15391543.

  • 27.

    Argyle DJ, Nasir L. Telomerase: a potential diagnostic and therapeutic tool in canine oncology. Vet Pathol 2003;40:17.

  • 28.

    Kow K, Bailey SM, Williams ES. Telomerase activity in canine osteosarcoma. Vet Comp Oncol 2006;4:184187.

  • 29.

    Renwick MG, Argyle DJ, Long S, et al. Telomerase activity and telomerase reverse transcriptase catalytic subunit expression in canine lymphoma: correlation with Ki67 immunoreactivity. Vet Comp Oncol 2006;4:141150.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Yamaguchi Y, Nozawa K, Savoysky E, et al. Change in telomerase activity of rat organs during growth and aging. Exp Cell Res 1998;242:120127.

  • 31.

    Sedivy JM. Can the ends justify the means? Telomeres and the mechanisms of replicative senescence in mammalian cells. Proc Natl Acad Sci U S A 1998;95:90789081.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32.

    Hipple AK, Colitz CM, Mauldin GN. Telomerase activity and related properties of normal canine lymph node and canine lymphoma. Vet Comp Oncol 2003;1:140151.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33.

    McKevitt TP, Nasir L, Devlin P. Telomere lengths in dogs decrease with increasing donor age. J Nutr 2002;132:1604S1606S.

  • 34.

    Nasir L, Devlin P, McKevitt T, et al. Telomere lengths and telomerase activity in dog tissues: a potential model system to study human telomere and telomerase biology. Neoplasia 2001;3:351359.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35.

    Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature 1990;345:458460.

  • 36.

    Savre-Train I, Gollahon LS, Holt SE. Clonal heterogeneity in telomerase activity and telomere length in tumor-derived cell lines. Proc Soc Exp Biol Med 2000;223:379388.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37.

    Bryan TM, Englezou A, Gupta J, et al. Telomere elongation in immortal human cells without detectable telomerase activity. EMBO J 1995;14:42404248.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38.

    Henson JD, Neumann AA, Yeager TR. Alternative lengthening of telomeres in mammalian cells. Oncogene 2002;21:598610.

  • 39.

    Ulaner GA, Huang HY, Otero J, et al. Absence of telomere maintenance mechanism as a favourable prognostic factor in patients with osteosarcoma. Cancer Res 2003;63:17591763.

    • Search Google Scholar
    • Export Citation
  • 40.

    Perrem K, Colgin LM, Neumann AA. Coexistence of alternative lengthening of telomeres and telomerase in hTERT-transfected GM847 cells. Mol Cell Biol 2001;21:38623875.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41.

    Hultdin M, Gronlund E, Norrback K, et al. Telomere analysis by fluorescence in situ hybridization and flow cytometry. Nucleic Acids Res 1998;26:36513656.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42.

    Reimann N, Rogalla P, Kazmierczak B. Evidence that metacentric and submetacentric chromosomes in canine tumors can result from telomeric fusions. Cytogenet Cell Genet 1994;67:8185.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43.

    Colangelo D, Osella O. Telomerase inhibition and cancer: might platinum based drugs have a future as anti-telomerase pharmacological approach? Curr Med Chem 2005;12:30913102.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44.

    Colangelo D, Ghiglia AL, Viano I, et al. Cis-[Pt(Cl)2(pyridine)(5SO3H-isoquinoline)] complex, a selective inhibitor of telomerase enzyme. Biometals 2003;16:553560.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 45.

    Sun PM, Wei LH, Luo MY, et al. The telomerase activity and expression of hTERT gene can serve as indicators in the anticancer treatment of human ovarian cancer. Eur J Obstet Gynec Reprod Biol 2005;130:249257.

    • Search Google Scholar
    • Export Citation
  • 46.

    Shammas MA, Reis RJS, Li C, et al. Telomerase inhibition and cell growth arrest after telomestatin treatment in multiple myeloma. Clin Cancer Res 2004;10:770776.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 47.

    Tahara H, Shinya K, Seimiya H, et al. G-quadruplex stabilization by telomestatin induces TRF2 protein dissociation from telomeres and anaphase bridge formation accompanied by the loss of the 3′ overhang in cancer cells. Oncogene 2006;25:19551966.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 48.

    Artandi SE, Alson S, Tietze MK, et al. Constitutive telomerase expression promotes mammary carcinomas in mice. Proc Natl Acad Sci U S A 2002;99:81918196.

    • Crossref
    • Search Google Scholar
    • Export Citation

Contributor Notes

Drs. Kitchell and Newman's present address is Center for Comparative Oncology, Small Animal Clinic, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824. Dr. Biller's present address is Robert H. and Mary G. Flint Animal Cancer Center, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80526. Dr. Hetler's present address is Animal Hospital of Streamwood, 904 E Irving Park Rd, Streamwood, IL 60107. Dr. Cadile's present address is Veterinary Medical Specialists, 251 N Amphlett Blvd, San Mateo, CA 94401.

Supported in part by Morris Animal Foundation (grant No. 96CA-14).

Presented in part at the Veterinary Cancer Society Annual Conference, Baton Rouge, La, October 2001.

Address correspondence to Dr. Kitchell.