Final Activity Report Summary - 3DCAP (Three-dimensional genomic analysis of cancer progression) Cancer is a disease of the cells, caused by abnormal functioning of the machinery that controls the timing and oversees the accuracy of every cell division. Consequently, cancer cells with abnormal genetic content, which in a normal cell would be forced into cell death -also called apoptosis-, manage to survive, thus compromising the normal function of the tissues where they are located. In this process, telomerase, an enzyme whose function is to protect and elongate the ends of cellular chromosomes, play a pivotal role, still not completely understood. Telomerase is not expressed in most somatic cells, and its role seems to be limited to preventing premature death of highly proliferative cells - i.e. stem and transit amplifying cells. However, most cancer cells manage to activate telomerase as a way to achieve immortalization. There is therefore a dual role of telomerase, beneficial in some normal cells and highly deleterious in cancer cells. Our goal in this project was to shed some light on the role of telomerase in normal lung and lung cancer. We wanted to help understanding the mechanism and the timing of telomerase activation in the histological context of the disease. To this end, our goal was first to overcome some important methodological problems that had impeded being able to simultaneously quantify telomerase levels of expression and the length of individual telomeres in intact cells. We have labelled and quantified both, by performing a technically complicated dual immunostaining of telomerase and in situ hybridization of the telomeres. Then, using novel 3D microscopy and image analysis tools, we have been able to measure the length of each telomere in the context of a known level of telomerase expression. Using these unique tools, we have looked at the expression and spatial distribution of telomerase and telomere signals in six lung cancer cells lines and in a normal cell line obtained from normal bronchial epithelia. This way we have confirmed in situ, for the first time, that telomerase positive cancer cells are able to maintain the length of their telomeres as cells cycle, while normal epithelial cells, both telomerase positive and negative are shorten their telomeres as they cycle. Interestingly, we have measured telomerase expression in some normal epithelial cells, in which the rate of telomere shortening is slower than in telomerase negative epithelial cells. These findings seem to indicate the telomerase has a different role in normal and cancer cells: telomerase in cancer cells has a telomere elongation role while telomerase acts in normal cells by protecting, not elongating the chromosomes. This was confirmed by the fact that telomerase activity, measure using biochemical methods, was detected in cancer cells but not in normal cells. Taking advantage of the ability to detect and quantify the length of individual telomeres, we were also able to confirm something that had been postulated, namely, that telomerase does not act in all the telomeres of the cell where it is expressed. Instead, it has preferential activity on the shortest telomeres of the cell. This is consistent with the idea that those are the ones that can become critically short and produce problems during cell division. What remains to complete the project, as it was originally planned, is to use our tools to quantify telomerase activity and telomere length simultaneously in cells in tissue samples, to determine the time of telomerase activation within the histological context of lung cancer progression.