Programmed cell death (PCD) is the term used to describe an active form of cell death that occurs during invertebrate and vertebrate development. PCD shares all the attributes of apoptosis, but the term apoptosis is generally restricted to the description of active mammalian cell death, whereas PCD describes all other active cell deaths. PCD pathways have been characterized in several invertebrate systems (1), but none so clearly as that occurring in the nematode worm Caenorhabditis elegans.
A definitive plan of gene expression during cell development and death is seen in C. elegans (2-4). The cell lineage development pattern within this animal has been extensively investigated, which established that exactly 131 cells of the 1090 within the developing nematode die. Some of the genes involved in these deaths are shown in Fig. 1. Of the genes involved in nematode PCD, three have been investigated extensively. Cell death gene 3 (ced-3) and ced-4 are directly responsible for the death of specific cells during development. Loss-of-function mutants of ced-3 or ced-4 result in an animal where cells that normally die during development do not, but instead take up the same differentiated form as their sister cells (2). Ced-3 and Ced-4 gene products act within the cells that die and not as killer signals produced by neighboring or surrounding cells (5). Ced-3 and Ced-4 killing is suppressed by another gene product, Ced-9. Although ced-9 gain-of-function mutants exhibit no cell death, cell death does occur in ced-9 loss-of-function mutants. Moreover, this death occurs not only in the 131 cells that normally die, but also in other cells within the nematode (6). These observations suggest that ced-9 is a general genetic suppressor of cell death within these animals.
Figure 1. The genetic control and pathway of programmed cell death in C. elegans. The ‘killing’ of the cell is carried out by the products of two genes, ced-3 and ced-4, whose activities are regulated by ced-9. Many of the genes that act within this pathway have been conserved throughout the evolution of multicellular organisms, demonstrating the importance of regulated cell death (2, 7, 9)
|
Specification o" |
Programmed ce 1 ceath |
Phagocytosis |
Digestion |
|
|
egl 1 |
ced-3 |
ced-? |
ced 7 |
nuc-1 |
|
ces-1 |
ced-4 |
ced-5 |
ced-3 |
|
|
ces-2 |
 |
ced-6 |
ced-10 |
|
The notion that cell death is essential for the development of the organism is verified by the conservation of death genes throughout multicellular evolution. For example, mammalian homologues of two of the nematode ced genes have been identified. Gene ced-9 is a homologue of the mammalian anti-apoptotic proto-oncogene bcl-2 (7), the B-cell leukemia/lymphoma gene (8) that suppresses apoptotic cell death, and a host of other bcl-2–like genes that define a gene family involved in cell death regulation (9, 10). Transgenic expression of bcl-2 in ced-9 loss-of-function nematode mutants suppresses cell death, demonstrating the conservation of function between these two homologues (11). This conservation of function suggests that genes with functions similar to those of ced-3 and ced-4 should exist in mammalian cells. This hypothesis was borne out by the discovery of a ced-3 mammalian homologue, the interleukin-1b-converting enzyme (ICE) (12). ICE is a thiol proteinase that activates pro-interleukin-1b by proteolytic cleavage to its active form during an inflammatory response (13). Expression of ICE in fibroblasts results in apoptosis, as does expression of Ced-3, demonstrating the conservation of function between these two homologues (12). There are currently 14 members of the Ced-3/ICE gene family in mammals (14-16) (more recently termed caspases for cysteine aspartate proteinases (17)) and two in Drosophila . A mammalian homologue of ced-4 has been identified, and its function is slowly being deciphered (1821). Ced-4 binds both Ced-9 and Ced-3, resulting in a protein complex that somehow acts to regulate PCD (see Apoptosis). Although the exact role of Ced-4 in this complex is unclear, Ced-4 induces death in both mammalian and yeast cells, suggesting some intrinsic death capacity (19, 21).
Other genes within the C. elegans PCD pathway are involved in the phagocytosis of dead cells. Mutant worms that lack one or more of these genes do not phagocytose dead cells; instead, the cells are left within the cellular tissues with no apparent deleterious effects (22), unlike unphagocytosed cells in mammalian tissues. Once again, some of these genes are also conserved in higher organisms, demonstrating the conservation of the PCD pathway as a whole throughout evolution (23).
The fruit fly Drosophila also has conserved PCD pathways. Drosophila embryos exhibit PCD at approximately 7 hours after egg laying (stage 11 embryos), with cell death becoming widespread throughout the embryo at stages 12 and 13. More specific regions of cell death are also seen during dorsal closure (stage 14) and advanced head involution (stage 15) (24). PCD in Drosophila climaxes with prominent cell death throughout the central nervous system as the ventral nerve cord condenses. As in C. elegans, the genes required for PCD in Drosophila are being sought and characterized. One such gene, reaper (rpr), is required for virtually all PCD in the developing embryo. rpr-deficient embryos have many extra cells and fail to hatch, demonstrating the requirement for rpr-regulated cell death during development (25). These embryos are also resistant to X-irradiation-induced death at low doses. That cell death occurs at higher radiation doses suggests that the death pathway is intact but less sensitive in the absence of rpr (26). Two other genes, Grim (27) and Head Involution Defective (hid) (28), are also required for some of the PCD occurring during development. Acting downstream of these genes are the Ced-3-like caspase homologues. Two of these genes have so far been identified, Drosophila cell death proteinase-1 (DCP-1) (29) and Drosophila ICE (DrICE ) (30). Like their mammalian counterparts, these caspase homologues cleave specific protein substrates during PCD. However, unlike the hierarchical arrangement of the caspases in mammalian cells, DrICE appears to be able to autoactivate and carry out PCD in Drosophila cells without requiring DCP-1 activity [Fraser, personal communication].
Other insect models have been successfully used to document genes regulating PCD. Following metamorphosis, the intersegmental muscles of the tobacco hawk moth, Manduca sexta, are no longer required and undergo degeneration, a process triggered by a reduction in the levels of an ecdysone hormone (31-33). This type of PCD involves the activation of several genes, plus the process of attachment of multiple ubiquitin molecules to other proteins, which is probably required for tagging them for protein degradation. Additionally, several genes are downregulated during the commitment of these cells to PCD, and over 30 new polypeptides are translated during muscle degeneration (34).
Examples of PCD are found in all multicellular organisms so far studied, and many of the genes that regulate these deaths have been conserved throughout evolution. This makes the study of invertebrate cell death invaluable in identifying how PCD is regulated in more complex organisms such as mammals.
