Estudio de las nuevas funciones de la telomerasa en la tumorigénesis y desarrollo de nuevas metodologías para la cuantificación de la longitud telomérica: HT Q-FISH y Telomapping
Author
Canela Rodríguez, AndrésAdvisor
Blasco Marhuenda, María AntoniaEntity
UAM. Departamento de Biología Molecular; Centro Nacional de Investigaciones Oncológicas (CNIO)Date
2007-11-30Subjects
Enzimas-Tesis Doctorales; Telómeros-Longitud-Cuantificación-Tesis Doctorales; Biología y Biomedicina / BiologíaNote
Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 30-11-2007Abstract
Telomeres are special structures at the ends of eukaryotic chromosomes that protect
them from degradation and DNA repair activities. Telomerase is a reverse transcriptase that
elongates telomeres in those cells where it is expressed, such as germ cells and stem cell
populations. Telomerase activity levels in adult tissues, however, are not sufficient to prevent
telomere shortening associated to cell division, eventually leading to chromosomal instability
and compromising tissue function. The telomere-driven loss of regenerative capabilities is at
the basis of aging and aging-related diseases. In contrast, cancer cells maintain their
telomeres by up-regulating telomerase and, through this mechanism, expand their proliferative
potential beyond the limits of a normal cell.
The present thesis combines two main objectives, first, the in vivo analysis of the role
of telomerase in tumorigenesis and, second, the development of high-throughput (HT)
telomere length measurement methods for both isolated cells and tissue sections. To
evaluate the role of telomerase in tumorigenesis, we developed a new mouse model with
constitutive expression of the catalytic subunit of telomerase (Tert) targeted to thymocytes and
peripheral T cells (Lck-Tert mice). These Lck-Tert mice showed higher incidences of
spontaneous T-cell lymphoma than the corresponding age-matched wild-type controls,
indicating that constitutive expression of Tert promotes lymphoma. Interestingly, T-cell
lymphomas in Lck-Tert mice were more disseminated than those in wild-type controls,
spreading to nonlymphoid tissues. Importantly, these pro-metastatic role of Tert was
independent of the role of telomerase in telomere length maintenance, since telomere length
distributions were identical in Lck-Tert and wild-type thymocytes. Furthermore, Tert
constitutive expression did not interfere with telomere capping in Lck-Tert primary thymocytes,
although it resulted in greater chromosomal instability upon gamma irradiation in Lck-Tert
primary lymphocytes than in controls, suggesting that Tert may interfere with the cellular
response to DNA damage. Finally, we explored the gene expression changes associated with
constitutive Tert expression. The obtained data confirm the pro-oncogenic potential of Tert as
evidenced by an increase of genes known to boost survival and proliferation at one hand and
the downregulation of genes associated with tumor suppression. Together, these data
establish a novel role for telomerase in facilitating tumorigenesis independent of its known
function in telomere length maintenance.
The second objective of the present thesis, namely the development HT methods to
measure telomere length derives from the fact that, despite the relevance of telomere length
to cancer and age-related diseases, only few studies have succeeded in validating telomere
length as a biomarker in human populations. Similarly, only a few telomere-based anticancer
therapies have been developed to date by screening large compound libraries. These
important drawbacks in telomere research have been due, at least in part, to the lack of fast
and reliable HT quantitative platforms to measure telomere length in large sample sets. During
the present thesis, we have developed an automated HT quantitative telomere method based
on fluorescence in situ hybridization (HT Q-FISH) of interphase nuclei in 96-well format, which
provides the same high quality average telomere length data as conventional Q-FISH analysis
of metaphase spreads on slides. HT Q-FISH has been validated by analysis of human
peripheral blood lymphocytes, revealing that age, gender, geographic, and psychosocial
factors impinge on telomere length. We, therefore, anticipate that the HT Q-FISH method will
be a powerful tool to expand our knowledge on the role of telomere length in human disease
and a powerful prognostic tool in the context of human population and epidemiological studies.
We also envision that the HT Q-FISH technique will provide a useful screening platform for the
development of telomere-based therapeutic strategies. In the clinics, the primary material to
study telomere length would be rather tissue sections from biopsies than cultured cells. For
this reason, we have also developed a method to measure telomere length with high accuracy
in tissue sections. This method, named telomapping. is based on the generation of
topographic FISH maps in paraffin-embedded tissue sections. We have tested this method in
skin sections from mice and found that the longest telomeres are a general feature of adult
stem cell compartments and that telomeres shorten with age in different mouse stem cell
ompartments, suggesting that telomere loss may contribute to stem cell dysfunction with
age. Together, these data confirm that the telomapping platform is suitable to quantify, in a
reliable and rapid manner, telomere length in tissue sections
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