Victoire Ndong
Bio 220 Tuesday lab
Instrutors: P.Gruber & A.Springer
ABSTRACT FOR APOPTOSIS EXPERIMENT
Apoptosis is a programmed cell death which helps regulate the number of cells in a multicellular body and it also helps get rid of cells with damaged DNA in an orderly manner. Apoptosis can be induced by external and internal signals. Those signals will cause cells to aggregate and recruit procaspases which are polypeptide chains that will be activated and initiate the caspase activity. Caspases are members of a family of proteases that are involved in apoptosis. They can be present in the cell as inactive procaspases (polypeptides) that need to be activated by proteolytic processing. There are two types of caspases: Initiator caspases which autoactivate themselves (procaspase-8) and effector caspases like (procaspase 3). The caspase cascade is a series of events that includes the condensation of the chromatin, the fragmentation of the nuclei and the reorientation of the membrane phospholipids. SO in general the role of caspase is to stop DNA replication, disrupt the nucleus membrane and mark the cell for macrophage uptake. The activation of caspase is an essential step during apoptosis. In our experiment, we tried to measure the impact of camptothecin on caspase activity and apoptosis induction by comparing MDCK cells treated with camptothecin to MDCK cells that were not treated. For that we stained MDCK cells with D2-R110 stain which is a substrate for many caspases. When caspases are activated they cleave the two attached aspartic acids causing R110 to fluoresce green. We then compared the digital pictures we took and calculated the percentage of fluorescing cells. Our results showed that 73.4% of the cells treated with camptothecin were showing green fluorescence (thus caspase activity) while only 2.95% of untreated cells were showing fluorescence. From the large difference in number of apoptotic cells between the treated and the control cells, we can say that camptothecin had an important impact on caspase activation thus apoptosis.