Research

Telomeric maintenance is a major limiting factor for continuous cellular growth. In mammalian tumours sufficient telomere length is provided through the action of the reverse transcriptase telomerase or by alternative recombinatorial (ALT) pathways that restore telomeres in the absence of telomerase. Telomere dysfunction provokes chromosome or sister chromatid fusions that induce chromosome aberrations. Chromosomal Instability in Neoplasia (CIN) is a hallmark of human cancers. CIN generates extended intra-tumour heterogeneity that is the main burden for all current and future oncotherapeutic strategies. It is well established that a proportion of structural CIN is related to telomere dysfunction. No detailed information exists on the possible links between numerical chromosomal instability and telomere depletion or de-protection. In human cancer, numerical CIN is operated through massive chromosomal gains or losses, and polyploidization. These lesions produce extended aneuploidy, heteroploidy and loss of heterozygosity (LOH) and drive the genome towards continuous growth. Therefore CIN might be both a leading cause and an epiphenomenon of neoplasia. Our preliminary data in a panel of telomerase positive and ALT continuous human cell lines support a hypothesis of recombinatorial telomere maintenance, operated by chromosomal losses, non-disjunctions and whole genome endoreduplication. This mechanism seems to co-exist with telomerase activity and to be elevated upon silencing of telomerase.

In our current research projects, we combine our high expertise in molecular cytogenetics with state-of-the-art BRFs infrastructure and in vivo and in vitro models of defective telomere metabolism, to study the role of alphoid repeats in the ALT pathway, to attribute a functional role of aneuploidy in continuous growth and to identify key components of CIN. Our studies include a panel of microsatellite unstable (MIN) and CIN human cancer cell lines, fine needle aspiration human biopsies of normal, precancerous and malignant lesions, conditional knock-out mouse models of telomere de-protection, and zebra-fish mutants of telomere metabolism. Introduction of dominant negative mutations or RNA interference is applied in vitro, targeting telomerase activity and the telomere protection proteins of the shelterin complex such as TRF1, TRF2, POT1 and TPP1. The role of the recombination proteins RAD50, RAD51 and MRE11 is also examined. Proteome analysis of inducible CIN phenotypes is performed to identify the role of known, and to reveal unknown factors of this process. A modification of the Q-FISH technique to quantify telomeric length of individual chromosomes and interphase nuclei with great accuracy, sensitivity and repeatability is under development. This protocol is applied, along with the whole arsenal of current cytogenetic methodologies (i.e.: aCGH, multicolor/SKY, multi-band, CO-FISH, Immuno-FISH) to study patterns of chromosomal instability, epigenetic modifications and genomic evolution, related to telomere length or dysfunction.

Gagos Group Research Services include a) Molecular/conventional Karyotyping and evaluation of the rates of CIN in human, mouse, rat and zebrafish continuous and cancer cell lines b) Delineation of possible cell line/cell line contaminations c) Quantification of telomeric length of individual chromosomes and interphase nuclei d) Labelling and evaluation of locus specific FISH probes and e) Identification and enumeration of transgene insertion points.