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GENOME INSTABILITY AND TRANSFORMATION

Chiara Mondello, Ilaria Chiodi

 

Genome instability is a hallmark of cancer cells. The acquisition of successive genomic alterations through several different mechanisms is the engine of neoplastic transformation. The interest of our laboratory is focused on the stepwise process of transformation and on mechanisms causing genomic instability. Telomeres, the ends of eukaryotic chromosomes, and telomerase, the enzyme deputed to their maintenance, are central elements in our projects.
Human telomeres are formed by tandem repetitions of the TTAGGG hexanucleotide and are essential for chromosome stability. In normal somatic cells, telomeres shorten at each cell division because of the absence of telomerase, and trigger a proliferation block, known as cellular senescence, when they shorten below a threshold length. In contrast, in tumor cells telomerase activity is generally restored, allowing cells to maintain telomeres and to divide indefinitely. Normal cells can acquire the ability to proliferate ad infinitum if telomerase expression is ectopically induced. We have developed a human fibroblast cellular system in which restoration of telomerase activity has been followed not only by immortalization, but also by the acquisition of the neoplastic phenotype. During culture propagation, immortalized cells became more malignant, as shown by a decreased latency in tumor development when injected in immunocompromised mice. By comparing cells at different stages of propagation, we have managed to identify some cellular and molecular alterations specifically associated with different phases of transformation. We are currently interested in exploiting this cellular system to deepen our knowledge on the genomic modifications that accompany cellular transformation. In particular, we are comparing global gene expression in cells with different degrees of malignancy and we are studying the expression of specific genes involved in tumor progression.

 




Figure 1. Telomeres of human chromosomes as detected by fluorescence in situ hybridization with a probe for the TTAGGG hexanucleotide.

 

Telomere length is regulated by complex interactions between telomeric DNA, telomeric proteins and telomerase and is generally maintained within a cell lineage specific range of values. We are interested in investigating elements involved in the maintenance of telomere length homeostasis and in studying the consequences of its loss. To this regard, we are taking advantage of the cellular system we developed, in which we observed a failure in telomere length control; in fact, after transformation, telomere length started increasing, reaching values not yet observed in human cells.
Gene amplification, that is the increase of the copy number of a limited portion of the genome, is a main feature of cancer cells, while it has never been observed in normal cells. Gene amplification plays a role in tumor progression, being one of the mechanisms of oncogene activation and of resistance to chemoterapeutic agents. Increased levels of gene amplification can be harmful to the cells, increasing the probability to change the level of expression of tumor genes. We are interested in identifying genes controlling this process, to this regard we study the ability to amplify in cell lines defective for specific genes. In particular, we are investigating the relevance of genes involved in genome integrity maintenance, such as genes coding for DNA damage response and DNA repair functions.




Figure 2. Amplified genes organized in a ladder-like structure


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