Since this family of enzymes is involved in chromatin folding, the scientists wanted to see if HDAC5 played a role in the structure of telomeres, which are located at the ends of chromosomes. "Telomeres are very important since they protect the chromosome from bad events suh as the fusion with another chromosome. They are involved in a number of processes such as cellular ageing," says Denis Mottet. In fact, during each cellular division, telomeres shorten and when they become too short, the cell stops dividing and reaches the state of senescence. Cancer cells, on the other hand, keep telomeres constant, continue to proliferate and do not progress to senescence. "Cancer cells have mechanisms which allow them to maintain the structure and length of their telomeres," explains Denis Mottet.... Which is why they thought HDAC5 might be involved in some way...

Localizing the enzyme using fluorescence

The first objective of the study was to localize the HDAC5 enzyme in cancer cells. "If it was localised in the telomeres, there was a strong chance that it played a role in the structure of these regions," says Denis Mottet. To localise the enzyme, the researchers used different types of cancer cell cultures. "We fixed the cells and injected them with antibodies against HDAC5 that we combined with a fluorescent molecule," explains the researcher. "In parallel, we used a probe that recognizes telomeres, also combined with a fluorescent molecule of a different colour.” Thanks to these immunofluorescence and fluorescence in situ hybridisation (FISH) techniques, Denis Mottet and his team were able to observe both fluorescent colours in cancer cells with long telomeres. Which means that in these cells, HDAC5 is indeed localized at telomeres.
 
"The second part of the research was to inhibit this enzyme using an interference technique which depleted its expression in cancer cells and allowed us to observe the specific consequences on telomeres,” continues Denis Mottet. To do so, the scientists used cancer cells with telomeres of different lengths. The result: "The inhibition of HDAC5 induced telomere shortening only in cancer cells which already had long telomeres," explains Denis Mottet. Cells with shorter telomeres do not seem to be affected by the absence of the enzyme.