Bcl2 and related genes in the regulation of apoptosis

The bcl-2 gene was identified as an oncogene in follicular lymphoma associated with the chromosomal translocation occurring between chromosomes 18 and 14. The bcl-2 gene occurs on chromosome 18q21 (Bakhshi etal., 1985; Cleary and Sklar, 1985; Tsujimoto etal., 1985a,b, 1987). Upon translocation this appears juxtaposed with the immunoglobulin heavy chain gene located on chromosome I4q32. Because the break point in chromosome 18 occurs in the 3'-untranslated region of the bcl-2 gene, this translocation produces a bcl-2/Ig fusion gene. The t(l4;18) translocation occurs in a vast majority of follicular lymphomas and, as a consequence of this translocation, there is an over-expression of bcl-2 RNA and protein, even though the coding regions of the gene are unaltered (Cleary et al., 1986a,b; Graninger et al., 1987; Seto et al.,

1988). This is supposed to be the result of deregulation of the bcl-2/Ig fusion gene. But T-cell lymphomas which do not show this translocation do express bcl-2 protein (Pezzella et al., 1990; Kondo et al., 1992), albeit at a lower level than Bcell lymphomas (Zutter et al., 1991). Therefore, there might be other mechanisms by which bcl-2 expression is deregulated. Two bcl-2 proteins, bcl-2a and bcl-2/?, produced by mRNA splicing have been identified (Seto et al., 1988). The bcl-2 protein is associated with the inner and outer mitochondrial membranes (Hockenbery et al., 1990; Monaghan et al., 1992; Nakai et al., 1993; Nguyen et al., 1993a; Lithgow et al., 1994). However, apoptosis can occur in human mutant cell lines that lack mitochondrial DNA and can be protected from apoptosis by an over-expression of bcl-2 protein. Apoptosis also occurs in anucleated cytoplasts and is prevented by bcl-2 overexpression (Jacobson et al., 1993, 1994). This suggests that the bcl-2 protein may occur at several intracellular sites. It has since been found to be present in the nuclear membrane and the endoplasmic reticulum (Chen-Levy et al., 1989; Alnemri et al., 1992a; Monaghan et al., 1992; Jacobson et al., 1993; Lithgow et al., 1994). The bcl-2 protein suppresses lipid peroxidation and its location may be related to its function as a suppressor of the generation of free radicals (Hockenbery et al., 1993) and in this way it suppresses apoptotic cell death. The opposite view that bcl-2 acts as a prooxidant has been expressed by Steinman (1995). It is known that bcl-2-mediated apoptosis can occur under anaerobic conditions suggesting that reactive oxygen species are not required for apoptosis (Jacobson and Raff, 1995).

The bcl-2 appears to extend cell survival and inhibit apoptosis (Liu et al., 1991; Strasser et al., 1991; Borzillo et al., 1992; Jacobson et al., 1993). This appears to require the full-length bcl-2. The truncated form of the protein does not appear to be able to prolong cell survival (Alnemri et al., 1992b). Expression of bcl-2 extends the life span of memory B-lymphocytes and maintains long-term immune responsiveness (Nunez et al., 1991). This gene is also expressed in cell types with characteristically long life spans. For example, neurones and haemopoietic stem cells and also stem cells of differentiating epithelia of skin and intestine show bcl-2 expression. The gene is also involved in glandular epithelia where hyperplasia and involution are controlled by hormones. In most instances, gene expression is associated with zones of tissues containing cells with long life spans or proliferating cells (Hockenbery et al., 1991). This suggests that its expression exerts a homeostatic effect on cell numbers. In contrast, apoptosis occurring during the involution and remodelling of the mammary gland is regulated by the induction of bax (bar) and bcl-x(S) proteins (Heermeier et al., 1996). The induction of differentiation of leukaemic cells by interleukin-6 and dexamethasone differentially modulates the expression of several bcl-2 family genes (Lotem and Sachs, 1995).

A family of genes related to bcl-2 have now been identified (Table 5). The protein products of these genes show high sequence homology within two high conserved domains called BH1 and BH2 (Yin et al., 1994). The bax protein identified by Oltvai et al. (1993) is a 21 kDa protein possessing a high sequence homology to the bcl-2 protein. This protein forms homodimers and also heterodimers with bcl-2. Over-expression of this protein appears to promote apoptotic cell death and to counteract bcl-2 mediated inhibition of apoptosis. Substitution of an amino acid residue in the BH1 domain can interfere with heterodimer formation between the bax and bcl-2

Apoptosis in tumour growth and metastasis Table 5. The bcl-2 family of apoptosis related proteins

Protein nomenclature

References

Inhibitors of apoptosis (promoters of cell survival)

Cleary et al. (1986a,b) Boise et al. (1993) Gonzalez-Garcia et al. (1995) Un et al. (1993)

Promoters of apoptosis bcl-x(S)

Boise et al. (1993) Yang et al. (1995) Farrow et al. (1995)

bad bak bax

proteins and negates the normal bcl-2 function of inhibition of apoptosis (Yin et al., 1994). It has been postulated that the relative levels of bax and bcl-2 proteins might define the pathway of survival or apoptosis and that the dimerisation between the different components is an essential event in the differential regulation of bcl-2 function. The bax protein has been found to undergo selective dimerisation with other bcl-2 family proteins (Thomas et al, 1995). Bcl-x(L) and Bcl-x(S) (see below) can themselves regulate cell survival and bcl-x(S) can counteract the apoptosis-inhibitory effect of bcl-x(L). However, in vitro, the binding of bcl-x(S) and bcl-x(L) is far weaker than to the bax protein (bar) (Minn et al., 1996). On similar lines, selective dimerisation has been shown to occur between bcl-x(L) and another member of the bcl-2 family, namely the bad protein (Yang et al., 1995). Thus, the bcl-2 family proteins show a wide spectrum of activity ranging from inhibition of cell apoptosis to promotion of the process of cell death. The formation of dimeric complex may provide a mechanism by which cell population homeostasis is finely controlled (see Craig, 1993; Thomas et al., 1995). In other words, the nature of the competing proteins, the strength and the selectivity of their binding can produce a hierarchy of complexes with apoptotic function. These genes also may be differentially regulated, so that the relative levels of the proteins available for dimerisation will vary and thereby control and determine the degree of apoptosis. Among alternative mechanisms suggested is the loss of apoptosis suppressor function of bcl-2 due to enhanced phosphorylation. Agents which affect microtubule dynamics are believed to act in this way (Croce, 1997).

Boise et al (1993) isolated another bc/-2-related gene, bcl-x, which also appears to be involved in the regulation of apoptosis. The bcl-x protein occurs in two forms, bcl-x(L) and bcl-x(S), by the alternative splicing of bcl-x mRNA. A third form of bcl-x protein, the bcl-x0), has been described, which is produced from the unspliced bcl-x mRNA (Gonzalez-Garcia et al, 1994). The bcl-x(L)

protein can inhibit apoptosis and promote cell survival, whereas bcl-x(S) promotes apoptosis and counteracts bcl-2-mediated inhibition of apoptosis. This is compatible with the high bcl-x(L) mRNA levels found predominantly in cells which have a long life span, e.g. in neurones and adult CNS. In contrast, bcl-x(S) mRNA levels found in cell types which show a high turnover (Boise et al., 1993; Gonzalez-Garcia et al., 1995). Microinjection of bcl-x(L) and bcl-x(^) mRNA prevents the apoptosis of primary sympathetic neurones induced by the withdrawal of nerve growth factor (Gonzalez-Garcia et al., 1995). Furthermore, transfection of bcl-x(L) gene into the interleukin-2-dependent CTTL-2 cells promotes their survival even in the absence of the cytokine (Boise et al., 1996). Cells that over-express bcl-x(L) have been found to be resistant to apoptosis following DNA damage inflicted by chemotherapeutic agents (Minn et al., 1995), but no differences in the expression of bcl-2, bcl-x or bar proteins have been encountered between drug-resistant and sensitive cell lines isolated from small cell and non-small cell lung cancers (Reeve et al., 1996). The ced-9 gene of Caenorhabditis elegans is an inhibitor of apoptosis (Hengartner et al., 1992) and it codes for a protein which shows sequence similarities to the bcl-2 protein (Hengartner and Horvitz, 1994). Certain other members of the bcl-2 family have also been found to function antagonistically to bcl-2, such as the bak (Farrow et al., 1995) and bad (Yang et al., 1995). The bad protein bears sequence homology to the BH1 and BH2 domains of bcl-2 protein and dimerise selectively with bcl-2 and bcl-x(L) proteins. It is obviously involved in the regulation of the apoptosis inhibitory function of the bcl-x(L)/bax protein complex. It can displace the bax protein and restore apoptosis (Yang et al., 1995).

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