THYMOMAS AND TELOMERASE
TELOMERASE ACTIVITY IN THYMOMAS AND MAMMARY GLAND
ADENOCARCINOMAS INDUCED BY POLYOMA VIRUS IN AKR MICE*
JAVIER OTERO, NORBERTO
Laboratorio de Patología
Experimental, Departamento de Microbiología, Facultad de Medicina,
Universidad de Buenos Aires
* Partially presented in the Annual Meeting of Sociedad Argentina
de Investigación Clínica, Mar del Plata, November, 1997
Key words: telomerase, Polyoma, TRAP assay, thymoma, mammary
is an enzyme that stabilizes telomere length in transformed cells and
tumors. Its role in tumor development is far from clear. In this
paper, a new experimental model to study telomerase activity during
tumorigenesis is presented. After infection with Polyoma virus, AKR
mice developed thymomas and mammary gland adenocarcinomas. Polyoma
antigens were observed by the peroxidase-antiperoxidase technique on
tissue sections, and by Western blot on tumor extracts. The TRAP assay
was performed to detect telomerase activity. It was not present in
normal mammary gland, but it was positive in mammary gland
adenocarcinomas. A different pattern was seen in thymic tissues:
normal thymus had higher telomerase activity than thymomas. The
incubation of thymoma extracts with normal thymus extracts decreased
telomerase activity in the latter. These results demonstrate two
different patterns of telomerase activity in tumors induced by Polyoma
virus, and suggest the presence of telomerase inhibitory factors in
telomerasa en timomas y adenocarcinomas de mama inducidos por el virus
Polioma en ratones AKR. La telomerasa es una enzima que estabiliza la
longitud de los telómeros en células transformadas y en tumores. Su
función en el desarrollo neoplásico no es clara. En este trabajo se
presenta un nuevo modelo experimental para estudiar la actividad
telomerasa durante la tumorigénesis. Se inocularon ratones AKR con
virus Polioma, los cuales desarrollaron timomas y adenocarcinomas de
mama. Los antígenos de Polioma fueron detectados en cortes
histológicos por peroxidasa-antiperoxidasa, y en extractos tumorales
por Western blot. Para estudiar la actividad telomerasa se empleó el
ensayo TRAP. No se detectó actividad telomerasa en tejido mamario
normal, pero fue positiva en adenocarcinomas de mama. Por el
contrario, el timo normal tuvo mayor actividad telomerasa que los
timomas. La incubación de extractos de timomas con extractos de timo
normal produjo una disminución de la actividad telomerasa en estos
últimos. Estos resultados demuestran dos tipos distintos de actividad
telomerasa en tumores producidos por virus Polioma, y sugieren la
existencia de inhibidores de la telomerasa en los timomas.
Postal address: Dr. Norberto Sanjuan, Departamento de
Microbiología, Facultad de Medicina, Paraguay 2155, 1121 Buenos
Fax: 54-1-508-3705; E-mail: firstname.lastname@example.org
Received: 12-VIII-1998 Accepted: 25-VIII-1998
Telomeres are structures located at the ends of eukaryotic
chromosomes. They are composed of short repeated DNA sequences rich in
G residues on one strand. Humans, mice and other mammals, contain
TTAGGG repeats arranged in tandem, running 5' to 3'. The number of
repeats varies in different species: for example, telomeres of the
laboratory mouse Mus musculus are larger than the human telomeres.
It is acknowledged that telomeres both stabilize chromosome ends and
are involved in the regulation of cell replication timing, chromosome
positioning in the nucleus, and other important functions that control
In every normal somatic cell division, 50-200 nucleotides of telomeric
sequence become lost2. It has been argued that the progressive
shortening of telomeres is a “mitotic clock” that finally conduces
normal cells to replicative senescence3. Immortal cells, however, do
not present any reduction of telomeric DNA tandem repeats. This
strongly indicates that the maintenance of telome-re length is crucial
for the cell to elude senescence and proliferate4. Telomeres are
seemingly directly invol- ved in cellular aging and in the
pathogenesis of can- cer.
Telomerase is an enzyme that either maintains or increases the length
of telomeres. It is a ribonucleoprotein in which the RNA component is
a template for the synthesis of telomeric DNA repeats onto chromosomal
ends1. Telomerase activity is detected in most tumors and stem cells,
but is usually absent in normal tissues5. It can therefore be infered
that telomerase may play some role in tumor production, and is
currently considered a target of anti-tumoral therapy.
Several experimental approaches have been based on this hypothesis.
For example, in vitro transformation of human B lymphocytes by
Epstein-Barr virus has been associated with telomerase activity6,
mouse mammary tumor virus long terminal repeat-driven Wnt-1
proto-oncogene expression in mammary tissue of transgenic mice
produces mammary tumors with high levels of telomerase7, and HPV-16 E6
transduction of human keratinocytes and mammary epithelial cells
Nevertheless, in vitro cell transformation does not correlate
necessarily with the in vivo mechanisms of tumor production. The
construction of transgenic mice or the transduction of cells with
viral oncogenes can provide pertinent information on the relationship
between oncogene expression and telomerase activity, but these systems
are ultimately artificial.
The purpose of this paper is to present a new experimental model, in
which telomerase activity is studied in tumors induced by Polyoma
virus. The use of an infecting oncogenic virus permits highly
reproducible results. Moreover, Polyoma is a natural virus of mice,
and it has the property of infecting and of producing tumors in the
same animal9, 10, thus enabling a step by step study of tumorigenesis.
The PTA strain of Polyoma virus was used in every experiment. This
strain, highly tumorigenic in mice, was obtained as a kind gift from
Dr. Thomas L. Benjamin (Harvard Medical School, Boston, U.S.A.). Virus
stocks were produced in primary cultures of specific-pathogen-free
Balb/c mice kidney epithelial cells (BMK). When cytopathic effect was
completed, cultures were frozen and thawed 3 times and centrifuged at
400 g during 20 minutes. The supernatant was titrated by the
plaque-forming units method (pfu)11. Virus stock was aliquoted, and
maintained at -70°C until used.
Newborn (less than 48 hs of life) AKR mice obtained from the mouse
colony of the Academia Nacional de Medicina, Buenos Aires, were
injected subcutaneously with 105 pfu of virus. Other mice were
injected with the supernatants of uninfected BMK cells, and a third
group was kept without any inoculation to evaluate the eventual
incidence of spontaneous tumors. Each experimental group was composed
of 20 animals, maintained 5 to a box, and fed on pellets ad libitum.
Twice a week mice were observed clinically for the purpose of
Between 60 and 90 days pi, 20/20 mice infected with PTA showed
progressive dyspnea, and were sacrificed with an excess of ether
anesthesia. In all 20 mice, necropsies showed the presence of tumors
in the superior mediastinum. Tumors were large, soft and pink,
occupying most of the thoracic cavity. Two of the mice also had
subcutaneous nodules. Neither mock-infected nor untreated mice
developed any tumors.
Tissues were immediately fixed in Bouin’s fluid, and embedded in
paraffin. Hematoxilin-Eosin, Gomori’s silver impregnation, Masson’s
trichromic, and P.A. Schiff stains were used for histologic tumor
Mediastinal tumors were thymomas, and subcuta-neous neoplasms were
well differentiated intraductal mammary gland adenocarcinomas (Fig.
1). Polyoma structural antigens were detected in all tumors using, on
tissue sections, the peroxidase-antiperoxidase (PAP) technique
counterstained with Hematoxilin. The primary antiserum was a
polyclonal one against Polyoma, prepared in rabbit. Intense positive
labeling was observed in the nuclei of scattered tumor cells. Thymic
tissue of uninfected mice was used as negative control for PAP
technique, and tumors treated with normal rabbit serum as primary
antibody were employed for internal control. Polyoma antigens were not
detected in any control. The presence of Polyoma antigens in tumors
was also comfirmed by SDS-Polyacrylamide Gel Electrophoresis and
Western blot (Fig. 1).
Immediately after the mice were sacrificed, suitable samples of tumor
and of normal tissues were taken and frozen in liquid nitrogen, then
kept at -70 °C until telomerase detection was performed. Telomerase
activity was studied by the TRAP assay, developed by Kim et al.5, and
modified by Blasco et al12. Briefly, tissue were thawed, washed with
Ice-Cold-Wash Buffer (10 mM HEPES-KOH pH 7.5; 1.5 mM Mg2Cl; 10 mM KCl;
1 mM dithiothreitol), then Dounce homogeneized with Ice-Cold-Lysis
Buffer (10 mM Tris-HCl pH 7.5; 1 mM Mg2Cl; 1 mM EGTA; 5 mM
b-mercaptoethanol; 0.1 mM phenyl-methylsulfonyl fluoride; 0.5% w/v
CHAPS; 10% v/v glycerol) and incubated on ice for 30 minutes. The
cellular extract obtained was centrifuged at 16.000 Xg, 30 minutes at
4°C, and supernatants were aliquoted and kept at -70°C. Protein
concentration was determined by the Bradford method (Biorad). An
extension reaction was done using 3 different total protein
concentrations (1 µg, 0.1 µg and 0.01 µg) of each sample to measure
telomerase activity. The final concentration of the components in the
telomerase extention reaction was: 50 mM Tris-Acetate pH 8.5; 1 mM
spermidine; 5 mM b-mercaptoethanol; 3 mM Mg2Cl; 50 mM potassium
acetate; 1 mM EGTA, 0.5 µg of TS oligonucleotide (5'
AATCCGTCGAGCAGAGTT3'); 2 mM each of dTTP, dGTP and dATP. Every
reaction was done in a final volume of 40 µl and incubated 30 minutes
at 30°C. Extracts of testes (where telomerase activity is normally
present in mice) were used for telomerase exten- sion as positive
control. Since telomerase has a RNA component, RNAse and heating at
70°C 10 mi- nutes were employed on the testes extract for internal
control in every experiment. After the extension reac-tion, a PCR
amplification of every sample was do- ne using the TS primer already
described, and the CX primer designed as a telomeric sequence (3'
AATCCCATTCCCATTCCCCATTCCC5'). The reaction was composed of 10 µg of
the extension product in 50 µl of TRAP buffer (20 mM Tris-HCl pH 8.3;
1.5 mM Mg2Cl; 63 mM KCl; 0.005% Tween 20; 1 mM EGTA; 50 µM of all
four dNTPs; 0.1 µg of TS oligonucleotide, 0.1 µg of CX primer; 0.1
mg/ml BSA; 5 µCi of 32P dGTP with a specific activity of 800 Ci/mM; 5
units of Taq polimerase). PCR conditions were 94°C 30 seconds, 50°C
30 seconds, 72°C 90 seconds, 30 cycles. Hot start conditions were
used in order to avoid primer dimerization. 5 µl of every reaction
were analyzed in 12% acrylamide-bisacrylamide- 7 M urea gels, and
later detected by autoradiography.
Results showed no telomerase activity in normal mammary gland tissue,
and a positive reaction in mammary gland adenocarcinomas (Fig. 2). The
intensity of telomerase activity correlated with the protein
con-centration used in every reaction. Protein concentration has been
reported to be critical for telomerase detection and quantitation in
mouse tissues7. Telomerase activity showed a completely different
pattern in the thymic tissue than in mammary gland. Normal thymus had
a strong telomerase activity in the three protein dilutions, while
thymomas showed a marked reduction in telomerase activity as compared
to the normal thymus (Fig. 2). This result was consistently observed,
after repeating 3 times telomerase detection in every single sample.
The fact that mammary gland adenocarcinomas have higher telomerase
activity than normal mammary tissue is in agreement with the idea that
telomerase is involved in the progression of tumor development.
However, the detection of lesser telomerase activity in thymomas than
in normal thymus was unexpected.
This could be explained by different hypotheses. Thymus is an organ
with stem cells and a strong cellular differentiation. Even though
thymomas are tumors, they are composed of well differentiated
epithelial cells, that could eventually have less telomerase activity
than the normal, highly proliferative thymic cells. Another hypothesis
is that thymomas produce an inhibitory factor that eventually
interferes with telomerase activity. To test this possibility,
different concentrations of thymoma extracts were added to normal
thymus extracts, then telomerase reaction was carried out. The
consequence was a decrease in telomerase activity in the normal thymus
extract, observed in 3 independent experiments (Fig. 2). In these 3
experiments, telomerase activity detection was done simultaneously in
normal thymus extracts and on thymoma extracts, using the same
chemicals and experimental conditions. It has been described that
non-especific inhibitors of the PCR step can be present in some mouse
tissues7, 12. In order to check if these extracts can nonspecifically
inhibit the telomerase reaction at the PCR amplification step, thymoma
extracts were added to a control PCR reaction that is known to amplify
Human Immunodeficiency Virus DNA. No inhibition was observed, and
there was no difference between the PCR amplification products with or
without thymoma extract (data non shown). These experiments suggest
that the lesser telomerase activity observed in normal thymus extracts
after being mixed with thymoma extracts occurs in the extension phase
of telomerase reaction.
The results reported here demonstrate that telomerase is present in
tumors produced by Polyoma virus in mice. They also show that
telomerase activity is both qualitatively and quantitatively different
in tumors produced by this DNA-oncogenic virus in 2 different organs
in the same animal, and equally suggest that telomerase-inhibitory
factors could exist in some tumors.
Recently, It has been demonstrated that transfection of telomerase
catalytic subunit into normal (telomerase-negative) human cells
produces elongation of telomeres and active cell division, but no cell
transformation13. This suggests that telomerase expression per se is
not oncogenic. Moreover, an alternative mechanism of telomere
lengthening (ATL) without telomerase activity has been described14.
These results have been observed in cell cultures, albeit not in
tumors. In many reports, telomerase activity has been consistently
detected in human5 and murine15 tumors, supporting the hypothesis
that, somehow, telomerase expression is involved in the development of
cancer. Currently, the pathway used by Polyoma to interact with
telomerase activity is unknown. It is unclear whether this phenomenon
is an early event in tumorigenesis or a late one. The experimental
model described here will hopefully allow a better understanding of
the conditions whereby telomerase contributes to the tumorigenic
Acknowledgements: This work was supported by the University
of Buenos Aires, Argentina. We thank Manuel Gómez Carrillo for
1. Greider CW. Telomere length regulation. Annu Rev Biochem 1996,
2. Harley CB, Futcher AB, Greider CW. Telomeres shorten during aging
of human fibroblasts. Nature 1990; 345: 458-60.
3. Allsopp RC, Vaziri H, Patterson C, Goldstein S, Younglay EV,
Futcher AB, et al. Telomere length predicts replicative capacity of
human fibroblasts. Proc Natl Acad Sci USA 1992; 89: 10114-8.
4. Small MB, Hubbard K, Pardinas JR, Marcus AM, Dhanaraj SN, Sethi KA.
Maintenance of telomeres in SV-40-transformed pre-immortal and
immortal human fibroblasts. J Cell Physiol 1996; 168: 727-36.
5. Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PLC, et al.
Specific association of human telomerase activity with immortal cells
and cancer. Science 1994; 266. 2011-15.
6. Counter CM, Botelho FM, Wang P, Harley CB, Bachetti S.
Stabilization of short telomeres and telomerase acti- vity accompany
immortalization of Epstein-Barr virus-transformed human B lymphocytes.
J Virol 1994; 68: 3410-14.
7. Broccoli D, Goddley LA, Donehower LA, Varmus HE, de Lange T.
Telomerase activation in mouse mammary tumors: lack of detectable
telomere shortening and evidence for regulation of telomerase RNA with
cell proliferation. Moll Cell Biol 1996, 16. 3765-72.
8. Klingelhutz AJ, Foster SA, McDougall JK. Telomerase activation by
the E6 gene product of human papillomavirus type 16. Nature 1996, 380:
9. Freund R, Calderone A, Dawe C, Benjamin TL. Polyoma virus tumor
induction in mice: effects of polymorphisms of VP-1 and Large T
antigen. J Virol 1991; 65: 335-41.
10. Dubensky TW, Freund R, Dawe CJ, Benjamin TL. Polyomavirus
replication in mice: influences of VP-1 type and route of inoculation.
J Virol 1991; 65: 342-49.
11. Turler H, Beard P. Simian Virus 40 and Polyoma virus: growth,
titration, transformation and purification of viral components. In:
Mahy BW (ed). Virology, a practical approach. Oxford: IRL Press, 1985;
12. Blasco MA, Rizen M, Greider CW, Hanahan D. Differential regulation
of telomerase activity and telomerase RNA during multi-stage
tumorigenesis. Nature Genetics 1996, 12: 200-4.
13. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB et
al. Extension of life-span by introduction of telomerase into normal
human cells. Science 1998; 279: 349-52.
14. Bryan TM, Englezou A, Gupta J, Bacchetti S, Reddel RR. Telomere
elongation in immortal human cells without detectable telomerase
activity. EMBO J 1995; 14: 4240-8.
15. Burger AM, Bibby MC, Double JA. Telomerase activity in normal and
malignant tissues: feasibility of telomerase as a target for cancer
chemotherapy. Br J Cancer 1997; 75: 516-22.
Fig. 1.– Histology of thymoma1, mammary gland adenocarcinoma2,
and Western blot to detect Polyoma major capsid protein VP-1 in these
tumors, developed by chemoluminiscence3 A: purified VP-1, B: normal
thymus, C-D: thymomas, E: normal mammary gland, F.G: mammary gland
Fig.2.– Telomerase activity. A. 1: positive control, 2: positive
control treated with RNAse and heat, 3-5: normal mammary gland extract
6-8 mammary gland adenocarcinoma extract. From left to right, lines 3
to 8: 1 µg, 0.1 µg and 0.01 µg of protein in each sample. B. 1-3:
normal thymus extract, 4-6: thymoma extract, 7-9: thymoma extract plus
normal thymus extract. From left to right 1 µg, 0.1 µg and 0.01 µg
of protein in each sample.