Genetics of Apoptosis

plates. This is especially important for cell-death research, since many proapoptotic
stimuli, such as H 2 O 2 , UV irradiation, or cytostatic drugs, can be applied in cell
culture.

2.

Cell-culture approaches to isolate genes in apoptosis

Apoptosis is genetically determined. Even if the original stimulus is not encoded by
genes, as in ‘programmed cell death’ (see Introduction), but rather constitutes an
accident, the ensuing signaling pathway is executed by genes and their proteins. As
the process of apoptosis is considered very complex, it is expected that there are still
novel genes to be detected that promise to reveal the inner workings of cells
undergoing apoptosis. Consequently, the isolation of genes is a major effort in
apoptosis research. This review will concentrate on the use of cell-culture techniques
to determine genes involved in apoptosis. It will present some of them and their
relevance for apoptosis induction.
A number of methods, chiefly the two-hybrid assay in yeast (Fields and Song,
1989), allow us to clone genes according to their protein association. As the signaling
pathway for apoptosis is in many cases mediated by protein-protein interactions, such
techniques have turned out to be very powerful (Boldin et al., 1995; Chinnaiyan et
al., 1995; Stanger et al., 1995). However, this overview will focus solely on
mammalian cell culture and its use in apoptosis research.
Apoptosis constitutes a degenerative process that eventually results in the
disappearance of the affected cells. Given this transient nature of apoptosis, three
basic strategies have emerged to isolate genes involved in cell death (Figure 1). The
first is an approach that correlates apoptosis with the transcriptional up- or
downregulation of specific genes. This requires the determination of differentially
regulated genes during the process of apoptosis. The other two approaches are
functional expression assays that allow us to clone genes on the basis of their activity
for apoptosis. The first strategy uses the inactivation of endogenous genes (loss of
function) or the overexpression of inhibitors of apoptosis to select those cells that are
thereby protected against a proapoptotic stimulus. Rather than a selection, the
alternative approach employs multiple transfections in parallel and therefore
constitutes a screen for genes that have the dominant activity to induce apoptosis
upon overexpression. All three strategies will now be described in more detail.

2.1

Correlative strategy

This approach is based on the earlier observation that transcription—or translation-
inhibitors can reduce apoptosis (Lockshin, 1969). This led to the prediction that
apoptosis-inducing proteins do not reside in healthy cells and that only an apoptosis
stimulus causes the synthesis of novel proteins that induce cell death. Intuitively
legitimate as this may be, there are probably more instances where the mediators of

200 GENETICS OF APOPTOSIS