Search

The field of apoptosis is unusual in several respects. Firstly, its general importance has been widely recognized only in the past few years and its surprising significance is still being evaluated in a number of areas of biology. Secondly, although apoptosis is now accepted as a critical element in the repertoire of potential cellular responses, the picture of the intra-cellular processes involved is probably still incomplete, not just in its details, but also in the basic outline of the processes as a whole. It is therefore a very interesting and active area at present and is likely to progress rapidly in the next two or three years. This review emphasizes recent work on the molecular mechanisms of apoptosis and, in particular, on the intracellular interactions which control this process. This latter area is of crucial importance since dysfunction of the normal control machinery is likely to have serious pathological consequences, probably including oncogenesis, autoimmunity and degenerative disease. The genetic analysis of programmed cell death during the development of the nematode Caenorhabditis elegans has proved very useful in identifying important events in the cell death programme. Recently defined genetic connections between C. elegans cell death and mammalian apoptosis have emphasized the value of this system as a model for cell death in mammalian apoptosis have emphasised the value of this system as a model for cell death in mammalian cells, which, inevitably, is more complex. The signals inducing apoptosis are very varied and the same signals can induce differentiation and proliferation in other situations. However, some pathways appear to be of particular significance in the control of cell death; recent analysis of the apoptosis induced through the cell-surface Fas receptor has been especially important for immunology. Two gene families are dealt with in particular detail because of their likely importance in apoptosis control. These are, first, the genes encoding the interleukin-1β-converting enzyme family of cysteine proteases and, second, those related to the proto-oncogene bcl-2. Both of these families are homologous to cell death genes in C. elegans. In mammalian cells the number of members of both familish which have been identified is growing rapidly and considerable effort is being directed towards establishing the roles played by each member and the ways in which they interact to regulate apoptosis. Other genes with established roles in the regulation of proliferation and differentiation are also important in controlling apoptosis. Several of these are known proto-oncogenes, e.g. c-myc, or tumour suppressors, e.g. p53, an observation which is consistent with the importance of defective apoptosis in the development of cancer. Viral manipulation of the apoptosis of host cells frequently involves interactions with these cellular proteins. Finally, the biochemistry of the closely controlled cellular self-destruction which ensues when the apoptosis programme has been engaged is also very important. The biochemical changes involved in inducing phagocytosis of the apoptotic cell, for example, allow the process to be neatly integrated within the tissues, under physiological conditions. Molecular defects in this area too may have important pathological consequences. [ABSTRACT FROM AUTHOR]

The identification of the most suitable molecular targets for gene and drug therapy is the crucial first step in the development of new disease treatments. The rational identification of such targets depends on a detailed understanding of the pathological changes occuring at the molecular level. We have applied forward genetics approaches to the identification of the critical genes involved in the control of apoptosis in mammalian cells, since defective control of apoptosis underlies many diseases, including cancer and neurodegenerative diseases. We have identified two groups of genes by their effects on cell survival using retroviral cDNA functional expression cloning and retroviral insertional mutagenesis. The identification of these novel genes opens up new areas for apoptosis research and subsequently for the development of new gene and drug therapies.

Vascular smooth muscle cell (VSMC) apoptosis occurs in advanced atherosclerotic plaques where it may contribute to plaque instability. VSMCs express the death receptor Fas but are relatively resistant to Fas-induced apoptosis due in part to the intracellular sequestration of Fas. Although inflammatory cytokines such as interferon (IFN)-gamma present in plaques can prime VSMCs to FasL-induced death, the mechanism of this effect is unclear. We examined Fas expression and FasL-induced apoptosis in human VSMCs in response to IFN-gamma. IFN-gamma induced Fas trafficking to the cell surface within 24 hours, an effect that required Jak2/Stat1 activity. IFN-gamma also stimulated Akt activity, and both Fas trafficking and Stat1 activation were inhibited by blocking PI3K, Akt, or Jak-2. IFN-gamma increased Fas-induced apoptosis in vitro by 46 +/- 8% (mean +/- SEM, P = 0.04), an event that could be abrogated by inhibition of PI3K, Akt, or Jak-2. IFN-gamma also increased Fas-induced apoptosis in vivo 7.5- to 15-fold (P < 0.05) in human arteries transplanted into immunodeficient mice, accompanied by increased Fas and phospho-Ser727-Stat1. We conclude that IFN-gamma primes VSMCs to Fas-induced apoptosis, in part by relocation of Fas to the cell surface, a process that involves PI3K, Akt, and Jak-2/Stat1. IFN-gamma present in plaques may co-operate with FasL to induce VSMC apoptosis in atherosclerosis.

We have discovered that endothelin-1 (ET-1) vasoconstriction is significantly enhanced in aortas of young (8-16-week-old) apolipoprotein E-deficient (ApoE-/-) mice devoid of atherosclerotic lesions (maximum response expressed as a percentage of the mean response to 100 mM KCl (E(MAX)) = 55.7% +/- 19.5% KCl, n = 5) compared to age-matched C57BL/6/J control animals (E(MAX) = 12.6% +/- 2.5% KCl, n = 8), indicating that alterations in the endothelin system may contribute to disease progression, at least in this animal model. There was no difference in the potency of ET-1 to contract aorta from the two groups (C57BL/6/J pD2 = 8.74 +/- 0.30; ApoE-/- pD2 = 8.50 +/- 0.15, P > 0.05). This increased response was specific to ET-1, as it was not observed with phenylephrine or U46619, nor was it due to a non-receptor mediated increase in contractile sensitivity, as there was no change in response to KCl between the two groups. [125I]ET-1 bound with subnanomolar affinity (K(D)) to aorta (K(D) = 0.018 +/- 0.002 nM, n = 4) and, with an order of magnitude lower affinity, to heart (K(D) = 0.47 +/- 0.05, n = 5) of C57BL/6/J mice with binding densities (B(MAX)) of 9.3 +/- 2.4 fmol mg(-1)protein and 100 +/- 14 fmol mg(-1) protein, respectively. Alterations in vascular reactivity to ET-1 could not be explained by increased endothelin receptor density or affinity, as these were not altered in aorta (K(D) = 0.011 +/- 0.003 nM; B(MAX) = 10.1 +/- 3.9 fmol mg(-1), n = 4) and heart (K(D) = 0.43 +/- 0.04 nM; B(MAX) = 115 +/- 26 fmol mg(-1), n == 6) of ApoE-/- animals. The ratio of ET(A) to ET(B) receptors in heart of control and ApoE-/- mice was similar, comprising 89% and 85% ET(A) receptors, respectively. In isolated aorta from ApoE-/- mice on the Western diet, which more closely resembled more advanced stages of the disease in man, the augmented ET-1 vasoconstrictor response was maintained (E(MAX) = 25.2% +/- 6.8% KCl, n = 9); however, it was completely prevented in animals that had received 10 weeks of oral atorvastatin (30 mg kg(-1) day(-1)) (E(MAX) = 4.0% +/- 1.5% KCl, n = 5), a concentration that was chosen because it did not affect plasma cholesterol and triglyceride levels. Therefore, this protective prevention of enhanced ET-1 vasoconstriction in ApoE-/- mice by atorvastatin was independent of its lipid-lowering properties.