an apoptotic signal are already present in the cell and only have to be activated upon
apoptosis induction (Jacobson et al., 1994; Schulze-Osthoff et al., 1994).Hence, the
determination of genes that are differentially expressed between healthy and apoptotic
cells might be an adequate experiment only for some inducers of cell death. While
the first attempts of this strategy were performed with subtractive cDNA libraries
(Ishida et al., 1992), current experiments involve DNA microarrays to investigate
many genes in parallel (Figure 1a). Several studies have followed this road and come
up with a number of differentially regulated genes. One such experiment was
performed with irradiation as the cell-death inducer (Voehringer et al., 2000), and
another with kainic acid or potassium withdrawal as the proapoptotic stimulus in
neurons (Chiang, L.W. et al., 2001). Hundreds of genes have been found to be
associated with the process of apoptosis, only some of them, such as caspase-3, being
established players in apoptosis. Interestingly, Chiang and colleagues observed
discrete waves of differentially expressed genes, making it possible that earlier genes
influence the expression of the late genes. This suggests that a very complex
transactivation pattern is responsible for apoptosis induction.
Figure 1. Different assays to determine apoptosis-associated genes.
(a) The correlative approach. RNA is isolated from healthy and apoptotic cells and the
differentially expressed genes are compared by subtractive library construction, differential
display, and SAGE or gene microarrays. (b) Selection strategy to isolate apoptosis genes. An
expression library is introduced in a batch format in cells, and a selection is established by the
application of a proapoptotic stimulus. From the cells that survived the induction of cell death,
the transfected DNA is isolated and enriched by several rounds of amplification in bacteria
and selection for survival. (c) A genetic screen to isolate apoptosis genes. Every single cDNA
of an unamplified and normalized library is introduced into a separate population of cells by
an individual transfection. The proapoptotic effect of the genes is then detected by an
appropriate assay for cell death.
CELL-CULTURE SYSTEMS IN APOPTOSIS 201