by Shigekazu Nagata Osaka University Medical School, Osaka, Japan,
nagata at genetic.med.osaka-u.ac.jp
Programmed cell death
Apoptosis is a cell death process which occurs during development and aging of animals. It is also induced by cytotoxic lymphocytes (CTL), anti-cancer drugs, c- or UV-irradiation, a group of cytokines called death factors and deprivation of survival factors.
Apoptosis was initially characterized by morphological changes of dying cells. During apoptosis cells shrink, and microvilli on the plasma membrane disappear. The nucleus is also condensed and fragmented. At the final stage of apoptosis the cells themselves are fragmented with all cellular contents inside. One of the biochemical hallmarks of apoptosis is the fragmentation of chromosomal DNA into nucleosome size units (180 bp).
Apoptotic cells can be recognized by staining of the condensed nuclei with fluorescence dyes Hoechst or DAPI. Apoptotic cells expose phosphatidyl-serine to the cell surface, which can be stained with fluoresently labelled annexin V. The fragmented DNA can be detected by TUNEL (terminal deoxynucleotidyltransferase-mediated UTP end labelling) procedure, or by electrophoresis of the isolated DNA on an agarose gel, which yields a ladder of DNA fragments with a unit size of 180 bp.
Cellular & Molecular Regulation
Apoptosis is mediated by a family of proteases called caspases that are activated by processing from its inactive precursor (zymogen). Thirteen members of the human caspase family have been identified. Some of the family members are involved in apoptosis, and these can be divided into two subgroups. The first group consists of caspase 8, caspase 9, and caspase 10, which contain a long prodomain at the N-terminus and function as initiators of the cell death process. The second group contains caspase 3, caspase 6, and caspase 7, which have a short prodomain and work as effectors, cleaving various death substrates that ultimately cause the morphological and biochemical changes seen in apoptotic cells. The other effector molecule in apoptosis is Apaf-1 (apoptotic protease activating factor), which, together with cytochrome C, recruits pro-caspase 9 in an ATP (or dATP)-dependent manner, and stimulates the processing of pro-caspase 9 to the mature enzyme.
The other regulators of apoptosis are the Bcl-2 family members. Eighteen members have been identified for the Bcl-2 family, and divided into three subgroups based on their structure. Members of the first subgroup, represented by Bcl-2 and Bcl-xL have an anti-apoptotic function.Membersof the second subgroup, represented by Bax and Bak [BAK1], as well as members of the third subgroup such as Bid and Bad are pro-apoptotic molecules.
The signal transduction pathway for a death factor (Fas ligand)-induced apoptosis has been well elucidated. Binding of Fas ligand to its receptor results in the formation of a complex (disc, death-inducing signaling complex) consisting of Fas, FADD and pro-caspase 8. Pro-caspase 8 is processed to an active enzyme at the disc. There are two pathways downstream of caspase 8. In some cells, such as thymocytes and fibroblasts, caspase 8 directly activates 3. In type II cells such as hepatocytes, caspase 8 cleaves Bid, a member of the Bcl-2 family. The truncated Bid then translocates to mitochondria and stimulates release of cytochrome c, which activates caspase 9 together with Apaf-1. The activated caspase 9 causes processing of pro-caspase 3 to the mature enzyme. In addition to the death factors, anti-cancer drugs, gamma irradiation or factor-depletion induce apoptotic cell death. Although cytochrome C is released from mitochondria during apoptosis induced by these stimuli, the molecular mechanism that triggers the release of cytochrome C from mitochondria is not known.
Caspase 3 activated downstream of the caspase cascade activates a specific DNase (CAD, caspase-activated DNase). CAD is complexed with its inhibitor, ICAD (inhibitor of CAD), in proliferating cells. When caspase 3 is activated in apoptotic cells, it cleaves ICAD to release CAD. CAD then causes DNA fragmentation in the nuclei. Clinical Relevance Blocking of apoptosis by loss-of-function mutations of apoptosis-inducing molecules such as Fas, Fas ligand and caspases, or overexpression of apoptosis-inhibitory molecule such as Bcl-2, causes cellular hyperplasia. In some cases it leads to tumorigenesis, as evident in B-cell lymphomas, which over-express Bcl-2 due to the translocation of the Bcl-2 gene to the immunoglobulin gene locus. Some multiple myeloma and non-Hodgkin's lymphoma carry loss-offunction mutations in the Fas gene. Somatic mutation in the Fas gene can also be found in patients of autoimmune diseases called Canale-Smith syndrome or autoimmune lymphoproliferative syndrome (ALPS). Exaggeration of apoptosis causes tissue damage. For example, administration of Fas ligand, exposure to c-irradiation, or treatment with a high dose of glucocorticoid kill test animals by causing massive apoptosis in the liver or thymus. Hepatitis, insulitis, graft-versus-host disease, and allergic encephalitis are due to the excessive apoptosis by Fas ligand expressed on CTL. Apoptotic cells are detected in the brain of ischemia or Alzheimer patients, suggesting that apoptosis is at least in part responsible for the disease manifestation in these patients.
A proper dose of anti-cancer drugs or γ-irradiation can kill cancer cells by activating the apoptotic death program in the target cells. Some cancer cells are resistant to these drugs by an unknown mechanism. It is hoped that elucidation of the molecular mechanism of apoptosis leads to development of an efficient cancer therapy.
Table: The apoptosis factory
|Casp 2||ICH1 NEDD2||+||7q35|
|Casp 3||CPP32 Yama apopain||+||4q33|
|Casp 4 TX||ICH-2 ICE-rel-II||+||11q22|
|Casp 7 MCH3 ICE-LAP3 + 10q25||MCH3 ICE-LAP3||+||1025|
|Casp 8||MACH MCH5 FLICE||+||2q33|
|Casp 9||APAF3 MCH6 ICE-LAP6||+||1p36.3 -p36.1|
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