Genetics of Apoptosis

The study of the genetic basis of apoptosis has become a highly complex and fast-moving area of research. Conse quently, this section will only deal with some of the most important aspects of this subject in order to provide a basis for the later discussion on genetic manipulation of the apoptotic pathway in commercially important cell lines.

The genes involved in apoptotic death may be classified into three groups. The first group consists of the modulators of the apoptotic pathway that suppress or induce death. The second group comprises the components of the cell death pathway that mediate the cell death signal. The final component is the group of effector enzymes that is responsible for the death and destruction of the cell. Some examples of these genes are given in Table 1.

Of the modulators of the apoptotic pathway, one group of closely related proteins, the Bcl-2 family, has attracted particular attention. The bcl-2 gene, the first and best characterized member, was identified at the t(14;18)(q32 ~ 1) breakpoint found in human follicular lymphoma (54). It encodes a 24-kDa protein that is located on the outer mitochondrial membrane, the cytosolic face of the nuclear membrane, and endoplasmic reticulum. Numerous studies have demonstrated the ability of this protein to suppress apoptosis in response to a wide variety of inducers. Table 1 lists other members of this family, some of which are functionally similar to Bcl-2, whereas others act as antagonists of Bcl-2 and thereby trigger apoptosis.

Until recently, studies of the molecular basis of cancer were directed at the identification and characterization of genes involved in the regulation of cellular proliferation. However, it is now evident that the failure of cells to undergo apoptosis at the correct time and location is also an important step in malignant transformation. Indeed, studies of bcl-2 in this context have been instrumental in establishing the role of apoptosis in cancer. Mutations that result in the overexpression of bcl-2 lead to the accumulation of cells due to life span extension. These cells then undergo further mutation, most notably involving the c-myc gene, which leads to the formation of high-grade tumors that combine the characteristics of high cellular survival with a high rate of proliferation (61-63). Clearly, these are characteristics that would be desirable in the ideal candidate host for the expression of recombinant proteins.

One of the most common mutations identified in tumors is that of the p53 gene, mentioned earlier. In response to DNA damage, p53 causes cell cycle arrest, allowing the cell to repair damaged DNA, thus ensuring that any potentially carcinogenic mutation is not propagated. However, in some cell types, p53 triggers the induction of apoptosis, again in order to minimize the risk of transformation (48,64).

The well-characterized pattern of development of the nematode Caenorhabtis elegans, which includes the induction of apoptosis in specific cell types, has provided an important insight into the genetic basis of apoptosis. Most notable among the genes identified are ced 9 and ced 3. The former is a bcl-2 homologue that blocks the ability of ced 3 to induce cell death (65). The ced 3gene is a cysteine protease that shares extensive homology with the mammalian protein called interleukin 1b converting enzyme (ICE) (66,67). As the name suggests, this enzyme cleaves the active precursor pro-interleukin 1b to generate the active molecule interleukin 1b, and a number of studies have now implicated it in the induction of apoptosis. However, apoptosis can also be induced in macrophages and thy-mocytes of ICE-negative mice, indicating that ICE is not a universal mediator of apoptosis (68). Indeed, several ICE-related enzymes have been identified in recent years (see Table 2).

The molecular mechanism of apoptosis induced by the interaction of the Fas-ligand with its receptor centers on the activity of ICE-related proteases. Indeed, important progress has now been made in deciphering the earliest events in the signal cascade that transduces the initial death stimulus to the death machinery of the cell. Activation of the Fas receptor by its ligand or agonist antibodies leads to the binding of the adapter protein MORT1/ FADD (Fas-associating protein with death domain) (75,76). This, in turn, binds to the ICE homologue FLICE (FADD-like ICE) or MACH (MORT1-associated ced-3 homologue) (77,78). The targets of this enzyme are still under investigation.

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