Implementación sobre Visual DSP++

Normal development requires a balance between cell survival, growth, differentiation and death. Programmed cell death or apoptosis is a physiological process characterised by DNA fragmentation, chromatin condensation and nuclear fragmentation. It occurs naturally within tumour cells and is also the mechanism whereby many anti-cancer treatments exert their effects. If it is blocked, tumour growth potential is unchecked, whereas if it can be induced, prognosis may be improved.

The bcl2 gene, was identified by its proximity to the translocation site 18q21 in t (14; 18) translocations in B cell lymphomas, which juxtaposes it to the immunoglobulin heavy chain locus on 14q32. Transfection of bcl2 into a variety of cell lines results in increased cell survival when the cells are deprived o f growth- factors (Vaux et al. 1988; Nunez et al. 1990; Borzillo et al. 1992). Transfected bcl2 and activated transgenic c-myc synergistically increases tumourigenicity in athymie mice. In vitro, antisense oligonucleotide blockade o f bcl2 expression in leukaemic cell cultures leads to inhibition of growth and decreased cell survival (Reed et al. 1990). Wild-type p53 (Lotem and Sachs, 1993a; Lotem and Sachs, 1993b) induces apoptosis. Deregulated c-myc can enhance apoptosis (Lotem and Sachs, 1993a) but this effect is thought to be overridden if survival is increased or apoptosis inhibited to leave its dominant growth-promoting effect. bcl2 (Lotem and Sachs, 1993a; Ellis et al. 1991; Hockenbery et al. 1990) and mutant p53 (Yonish-Rouach et al. 1991) can suppress apoptosis.

Chapter 1 - Introduction

In baby Fisher rat kidney cells apoptosis induced by adenovirus E l A is mediated by wild-type p53 (Chiou et al. 1994). In these experiments overexpression o f bcl2 with co-expression of a temperature-sensitive p53 mutant (vall35p53) results in no apoptosis at 38®C (non-permissive temperature) or at 32^C (permissive temperature). However the cells do become growth-arrested at the permissive temperature. In bcl2 negative, p53 positive transfectants, cell viability is lost at the lower temperature, suggesting increased survival with increased bcl2 and apoptosis with increased p53. It also suggests separate pathways for growth arrest and programmed cell death. There is some evidence that p53 and bcl2 regulate the same effector pathway, at least in lymphoid cell lines (Marin et al. 1994).

bcl2 belongs to a family of homologous proteins that have differing effects on the apoptosis pathway. These proteins either promote apoptosis, (Bak (Chittenden et al. 1995; Farrow et al. 1995), Bax (Oltvai et al. 1993), Bclxs (Boise et al. 1993)) or promote cell survival (B c Ix l (Boise et al. 1993)). Pathways have recently been discovered that include bcl2-unrelated proteins which promote cell survival such as BAG-1 (Takayama et al. 1995). It is unlikely that the full complement o f apoptosis- regulating proteins have been identified, and the in vitro nature of the studies may not be readily applicable to understanding the normal physiology o f cell death.

Bax protein forms homodimers as well as forming heterodimers with bcl2. The ratio of these two protein products determines the cellular response to death stimuli (Oltvai et al. 1993; Korsmeyer et al. 1993). In p53 negative murine myeloid leukaemia M l cells lines growth arrest and apoptosis occurs on treatment with tumour growth factor Pi (TGFPi) (Selvakumaran et al. 1994b). TGFpi treatment results in no change in Bax expression but does reduce bcl2 expression. p53 transfectants undergo rapid apoptosis with induction of Bax and downregulation of bcl2 expression. In

Chapter 1 - Introduction

cotransfectants with bcl2 and p53, Bax expression increases but bcl2 does not fall in response to TGFPi. The cotransfected cells have delayed but not blocked apoptosis whereas bcl2 transfects treated with TGFpi had no change in Bax status and blocked apoptosis (Selvakumaran et al. 1994a). These observations suggest different pathways exist for apoptosis; both p53-dependent and independent. The p53- dependent pathway appears to coincide with increased Bax and downregulated bcl2, and can be delayed but not prevented by bcl2 transfection. The TGFpi induced apoptosis however can be blocked by bcl2, perhaps due to the lack o f Bax induction. The Bax promoter contains homologous regions to the p53 consensus binding site and in transfection studies with wild-type p53 and a reporter gene, p53 was able to activate the reporter utilising the Bax promoter (Miyashita et al. 1994b; Miyashita and Reed, 1995). This suggests that Bax is transcriptionally activated by wild-type p53. These results suggest that p53 triggers apoptosis by transactivation of Bax protein and alteration of the ratio of Bax:bcl2. Other studies have shown that a bcl2- related protein. Bad, can displace Bax from its heterodimers with bcl2 or BcIxl. It has been suggested that it is the “free” Bax level, not forming heterodimers with BcIxl

(or bcl2), that determines apoptosis/cell survival (Yang et al. 1995). Another interesting discovery is that Bax appears only to be induced by p53 in those cell lines that are actually capable of apoptosis. Cell lines which do not undergo apoptosis become growth-arrested and express normal p53 but this does not induce Bax (Zhan et al. 1994).

The apoptosis story is far from complete. Numerous stimuli trigger cell death; o f which programmed cell death is one mechanism of cell destruction. The effects of these stimuli on cells may occur through pathways involving p53[pj but may by-pass

Chapter 1 - Introduction

this protein altogether. Even the pathways involving p53 protein are poorly understood. There have been no reports on the interrelationships of all these related proteins in vivo.