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The majority of work into the genes involved in the apoptotic pathway has been carried out in the nematode C.elegans. This eukaryote only has over a thousand cells in total, of which one hundred and thirty one die by an intrinsic death program during normal development. Genetic analysis has revealed fourteen genes involved

insult radiation growth factors serum starvation drug treatm ent c o n d e n sa tio n of nuclear chrom atin rise in intercellular calcium

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intact cell and nucleus

cell sh rinkage

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degradation D isruption of m em brane

P h a g o c y to sis of apototic b o d ies

Figure 1.7. The apoptotic process. This can be follow ed through a series o f stages that are unique for apoptosis. After the initial insult, condensation o f the chromatin leads to disruption o f the cellular and nuclear membrane with the subsequent release and phagocytosis o f the apoptotic bodies.

at different stages of this apoptotic pathway. These are termed the ced genes (reviewed by Hengartner and Horvitz, 1994) and isolation of families of highly conserved human homologues has lead to the partial elucidation of the implementation and regulation of the apoptotic pathway in human cells.

1.3.2.1 The bcl-2 family

Bcl-2 was originally cloned from the breakpoint of a t(14;18) translocation present in many human B cell lymphomas (Cleary et al. 1986) which resulted in the overexpression of the bcl-2 protein. It shows high sequence homology with the C. elegans ced-9 gene, which protects cells from programmed cell death (Hengartner and Horvitz, 1994). Bcl-2 protein has been localised to the inner mitochondrial membrane (Hockenbery et al. 1990) although some studies isolate bcl-2 to the endoplasmic recticulum and nuclear membranes (Jacobson et al. 1993). Bcl-2 suppresses cell death induced by a variety of cytotoxic agents including gamma radiation, hypoxia, growth factor withdrawal and chemotherapeutic agents (Sentman et al. 1991; Shimizu et al.

1995; Nunez et al. 1990; Miyashita and Reed, 1993). Failure of bcl-2 to inhibit apoptosis by cytokine deprivation of IL-2 or IL-6 (Nunez et al. 1990)

and the process of negative selection in thymocytes, (Strasser et a l 1991) suggest that multiple independent cellular mechanisms exist for the control of apoptosis.

Recently, it has been suggested that bcl-2 controls programmed cell death by suppressing the production of reactive oxygen species (ROS) (Hockenbery et a l 1993; Kane et a l 1993) and, therefore, functions as an antioxidant. However, the prevention of hypoxia-induced cell death by bcl-2 suggests that this oncogene can exert an anti-cell death function by a mechanism other than regulation of ROS activity (Shimizu et a l 1995).

Further analysis of this oncogene revealed it to be the founding member of a family of highly conserved homologues (reviewed by Rao and White, 1997). Members of this family fall into two functional groups, those that inhibit apoptosis and those that promote it. One such member of this family is the bax protein, which was found to heterodimerise with bcl-2 in vivo (Oltvai, et a l 1993) and accelerate cell death due to cytokine deprivation in an IL-3 dependent cell line. Other bcl-2 family members include bcl-xL and bcl-xS (Boise et a l 1993), bad (Yang et a l 1995), bak (Chittenden et a l

1995), Mcl-l and A l (reviewed by Blandino and Strano, 1997). Three highly conserved genetic regions within the bcl-2 proteins have been identified. These regions, BH l and BH2 (Yin et a l 1994) and BH3 (Boyd et a l 1995) (bcl-2 homology 1, 2 and 3) are critical for dimérisation with partner proteins as mutation of these sites can completely abrogate the protein function. The protein-protein interactions of the bcl-2 family members can control the fate of the cell by promoting apoptosis if the ratio of repressor (e.g. bcl-2) is less than the inducer of apoptosis (e.g. bax), suggesting an antagonistic relationship. Certain complexes form more readily than others and Figure 1.8 outlines some of the combinations of hetero- and homodimers identified and their effect on apoptosis. It has been proposed that apoptosis in some cells is determined by the relative amount of bax homodimers (Korsmeyer, 1995). Therefore, while Bcl-2 and Bcl-xL bind with bax, preventing its homodimerisation and thus suppressing apoptosis, on the other hand bad binds to both bcl-2 and bcl-xL, freeing bax to homodimerise, thus promoting apoptosis.

1.3.2.2 The ICE family

From the work carried out in the C. elegans model, ced-3 was identified as a gene necessary for cell death. The ced-9 gene, whose human homologue is bcl-2, antagonises the function of ced-3 by protecting against cell death (Hengarter and Horvitz, 1994). Similar studies have identified the human homologue of ced-3 to be the cysteine protease interleukin-1 beta-converting enzyme (ICE), a protease important in the inflammatory response (Miura et a l 1993). Numerous cysteine proteases in mammals have been identified, the activity of one or more being increased during apoptosis (reviewed by Kumar, 1995). These include nedd-2/ich-l (Kumar et a l 1994; Wang et

bcIxS

bcl-2

bax ;

LIFE

DEATH

Figure 1.8. D im érisation o f the bcl-2 fam ily and their effect on apoptosis. The protein-protein interactions o f the bcl-2 family can promote or repress apoptosis induced by a variety o f cytotoxic insults. The bcl-2/bcl-2 homcxiimer itself does not affect apoptosis; it needs to be complexed with bax or bad to exert its anti-apoptotic function, bax-bax homodimers are the most potent promoters o f apoptosis and although it com plexes with other promoters o f apoptosis such as bcl-xL, bax shows higher affinity for the homodimer (bold arrow), which is more effective in apoptotic cell death promotion.

al. 1994), TX/Ich-2/ICErel-n (Faucheu et al. 1995; Kamens et al. 1995; Munday etal. 1995), MCH-2 (Nasir et al. 1997) and CPP32/YAMA (Alnemri-Femandes et al. 1995). Overexpression of any of these five proteases in several cell types has been demonstrated to result in apoptosis, and indicate that proteolytic degradation is required for mammalian cell death. Identification of the downstream targets of ICF-like proteins is vital to understanding the exact role these regulators of the apoptotic pathway. There is evidence that ICF family members process themselves, and each other, proteolytically (Rao and White, 1997). Other substrates of these enzymes include poly-ADP ribose (PARP), DNA PK and lamins (Lazebnik et al. 1994, Casciola-Rosenef al. 1996; Lazebnik et al. 1995), suggesting that the ICF proteins regulate the physical processes, such as cellular shrinkage, that occur during apoptosis.

3 .2 .3 p 5 3

In addition to playing a major role in the regulation of the mammalian cell cycle, p53 has been identified as key regulator of the apoptotic response to various cytotoxic agents in mammalian cells. Radiation-induced apoptosis in mouse thymocytes requires functional p53 (Lowe et al. 1993a). Treatment with chemotherapeutic compounds also

requires p53 for execution of apoptosis in mouse embryonic fibroblasts (Lowe et a l 1993b). The human lymphoblast cell lines TK6 and W TKl, which were derived from a single donor and have different p53 status, show differences in both the kinetics and the overall level of radiation-induced apoptosis (Xia et al. 1995). The introduction of wild- type p53 into human cancer cells with deleted p53 enhances apoptosis induced by chemotherapy (Fujiwara et a l 1994) and Hamada et a l (1996) have reported an increase of apoptotic cells in wild-type p53-expressing colorectal tumours compared with mutant p53 tumours.

The exact role of p53 and how it activates the apoptotic pathway is unclear as apoptosis can be induced via p53 independent pathways. Apoptosis is critical for the normal development and p53-knockout mice develop normally into adults (Donehower et a l 1992). However, apoptosis in response to DNA damaging stimuli such as radiation is, in general, p53-dependent. This is consistent with the findings that p53 levels are increased in response to DNA damaging agents (Lu and Lane, 1993). p53 is a well known transcription factor, and this raises the plausible possibility that p53 induces apoptosis via transcription of a 'death gene' in response to radiation. Studies into the effect of p 2 1 ^ ^ ^ 1 ' (which is transcriptionally activated by p53) on apoptosis have shown that increased levels of this protein has no effect on apoptosis (Kobayashi et a l 1996). However, Fan et a l (1994) have demonstrated increased levels of apoptosis in cells deficient in p21 when treated with nitrogen mustard. Treatment with other DNA damaging agents such as gamma irradiation, etopside and campthothecin produced similar effects. Analysis of cell-cycle progression revealed a G l/S block in the p2l4-/+ cells which was absent in p21-/- cells. Therefore, the role of p53 in the apoptotic response to DNA damage may be to transcribe the p21 gene, resulting in a G l arrest. During this arrest the cell presumably attempts to repair the damage, which if it fails, undergoes apoptosis. This implies that the G l arrest and apoptosis functions of p53, while being related, are two separate pathways. This is consistent with the findings that bcl-2 over-expression inhibits p53-triggered apoptosis but not G l arrest (Wang et a l

1995) and that p21 knockout mice demonstrated apoptosis also with no G l arrest (Deng et a l 1995). In predicting radiation response, p53 status has been shown to correlate significantly with survival in some tumour models, and its role in the apoptotic pathway may be partly responsible for this.

The molecular mechanisms involved in p53-dependent apoptosis are under investigation. It has been reported that p53 activates bax while reducing bcl-2 expression in M l myeloid leukaemic cells transfected with a temperature sensitive p53 vector (Selvakumaran et a l 1994), and in ML-1 cells with endogenous p53 (Zhan et a l 1994). Therefore it would appear that p53 regulates apoptosis by regulation of the bcl-2/bax ratio.