Author/Editor		Druškovič, Mirjam
Title		Povečano izražanje kaspaze-9 sproži proces apoptoze in vivo
Type		monografija
Place		Ljubljana
Publisher		Univerza v Ljubljani, Medicinska fakulteta
Publication year		2003
Volume		str. 36
Language		slo
Abstract		BACKGROUND Apoptosis or programmed cell death is a process by which the cells are actively destroyed. It controls the number and the quality of the cells. Malfunctions of the apoptosis are manifested in malignancies and in neurodegenerative disorders. Caspases are proteases, which have a central role in triggering and executing apoptosis. They are synthesized as inactive procaspases and are activated through proteolytic processing. AIM Caspase-9 is the central activator of apoptosis (e.g. after the tissue ischemia) or it can amplify the apoptosis, when it has been started by other caspases. It was shown that the increased concentration of caspase-9 can induce its activation in vitro. Our goal was to find out whether the increased concentration of caspase-9 triggered the cell death in vivo. HYPOTHESIS The hypotheses were: 1. Overexpression of caspase-9 triggered the cell death. 2. The cells dyed from apoptosis. 3. The apoptosis because of the caspase-9 overexpression was triggered in normal and in malignantly transformed cells. METHODS To observe the process of apoptosis in cultured mammalian cells we used the primary cultures of rat melanotrophs and astrocytes and the human cell line HeLa. The cells were transfected with the DNA encoding the fluorescent fusion protein between the caspase-9 and green fluorescent protein (EGFP). The fluorescent cells were detected by the confocal microscope. We determined the numbers of apoptotic and necrotic cells for fluorescent and non-fluorescent cells by a fluorescent analogue of annexin V and a dye Sytox Green. Then we calculated the percentages of apoptotic cells in relation to all cells on coverslips. For all samples we performed at least 5 independent experiments (n=5) and subjected the results to the statistical analysis by Student's t-test. RESULTS Overexpression of caspase-9 reduced the number of cells in comparison to the cells overexpressing cytochrome c. (Abstract truncated at 2000 characters)
Descriptors		APOPTOSIS CYSTEINE PROTEINASES CELLS, CULTURED MICROSCOPY, CONFOCAL