11.
Cell-culture systems in apoptosis
Stefan Grimm
1.
Introduction: advantages of cell culture to investigate apoptosisCell culture is an invaluable tool to study the behavior of cells, as it allows us to
investigate a great number of them with similar genotype and phenotype. It thereby
circumvents genetic variations between individuals and also the ethical questions of
human and animal experiments. Consequently, the definition of media that support
the growth of cells explanted from tissues was a major achievement in biology (Eagle,
1959). Even though it constitutes a reductionist view of the cells to observe them
without supporting neighboring cells and without the extracellular matrix, many cells
—though usually tumor cells—still reflect their behavior in vivo (Masters, 2000).
Since apoptosis is a rapid process that is usually concluded within a few hours, cell
culture is an adequate instrument to investigate this phenomenon as it allows us to
resolve the sequence of the individual steps. Especially GFP-fusion proteins and
immunofluorescence helped to reveal the dynamic processes that lead to cell death
(Goldstein et al., 2000). Given the ease of studying cells in culture, even genes that
have been isolated by in vivo systems, such as those of Drosophila, are subsequently
analyzed in vitro (McCarthy and Dixit, 1998). This is now aided by a plethora of
different readouts for apoptosis in cell culture, from the demonstration of the DNA
ladder (Wyllie, 1980) or the TUNEL assay that detects the free DNA ends generated
during apoptosis (Gavrieli et al., 1992) to the exposure of surface markers in apoptotic
cells (Koopman et al., 1994). Powerful supplementary methods such as flow
cytometry rely on cell culture and have immensely contributed to the elucidation of
apoptosis (Darzynkiewicz and Traganos, 1998).
An important aspect of cell culture is the easiness with which the cells can be
genetically manipulated. Nowadays, different transfection methods facilitate the
introduction of genetic material into virtually every cell. This permits many genes to
be overexpressed by transfected plasmids. In addition, techniques such as antisense
cDNAs (Albert and Morris, 1994), RNAi (Elbashir et al., 2001), or antisense
oligonucleotides (Ghosh and Iversen, 2000) allow us specifically to inactivate single
mRNAs in order to assess their contribution to cell-death regulation. Moreover,
pharmacologic studies are possible in cell culture, as drugs can be titrated in microtiter