The term “autophagic cell death” describes a form of programmed cell death morphologically distinct from apoptosis and presumed to result from excessive levels of cellular autophagy (6
). In classical apoptosis, or type I programmed cell death, there is early collapse of cytoskeletal elements but preservation of organelles until late in the process. In contrast, in autophagic, or type II, programmed cell death, there is early degradation of organelles but preservation of cytoskeletal elements until late stages. Whereas apoptotic cell death is caspase-dependent and characterized by internucleosomal DNA cleavage, caspase activation and DNA fragmentation occur very late (if at all) in autophagic cell death (Figure ). In contrast with necrosis, both apoptotic and autophagic cell death are characterized by the lack of a tissue inflammatory response.
Figure 3 Ultrastructural examples of apoptotic and autophagic cell death. Electron micrographs of a FasL-treated Jurkat cell undergoing cell death with apoptotic features (A) and of a tamoxifen-treated MCF7 human breast carcinoma cell undergoing cell death with (more ...)
Large numbers of autophagic vacuoles have been observed in dying cells of animals of diverse taxa (reviewed in refs. 6
) (Table ). The consensus view has been that autophagic cell death occurs primarily when the developmental program (e.g., insect metamorphosis) or homeostatic processes in adulthood (e.g., mammary gland postlactational involution, prostate involution following castration) require massive cell elimination. Recently, studies have also described autophagic cell death in diseased mammalian tissues and in tumor cell lines treated with chemotherapeutic agents (Table ). In many of these cases, morphologic features of autophagic and apoptotic cell death or of autophagic and necrotic cell death are observed in the same cell.
Examples of cell death with morphologic features of autophagyA
What is the evidence that autophagy is a death execution mechanism in autophagic cell death? If cell death is truly due to autophagy, then pharmacologic or genetic inhibition of autophagy should prevent the death. Yet, for most of the developmental, disease-associated, and toxic stimulus-induced deaths that are presumed to be autophagic (Table ), the evidence for its role is only correlative. Moreover, in certain cases of autophagic cell death, the available evidence calls into question a causative role of autophagy. For example, in Drosophila
, autophagic cell death but not autophagy observed during salivary gland regression is prevented by mutations in the ecdysone-regulated transcription factors BR-C
). In the slime mold Dictyostelium
, a null mutation in the autophagy gene atg1
blocks vacuolization but not cell death in an in vitro model of autophagic cell death (11
). Thus, in these model systems, autophagy per se is neither sufficient nor required for autophagic cell death. Furthermore, the caspase inhibitor p35 blocks metamorphic cell death in Drosophila
without complete inhibition of autophagy, suggesting that it is caspase-mediated apoptosis, rather than autophagy, that plays a key role in this death process (10
There is, however, some evidence in certain in vitro settings that pharmacologic or genetic inhibition of autophagy can prevent cell death. The pharmacologic inhibitor of autophagy 3-methyladenine (3–MA), a nucleotide derivative that blocks class III PI3K activity (12
), delays or partially inhibits death in starved hepatocytes from carcinogen-treated rats (15
), in anti-estrogen–treated human mammary carcinoma cells (16
), in chloroquine-treated cortical neurons (17
), in nerve growth factor–deprived sympathetic neurons (18
), in serum- and potassium-deprived cerebellar granule cells (19
), in serum-deprived PC12 cells (20
), and in TNF-treated human T lymphoblastic leukemia cells (21
). However, in several of these studies, autophagy occurred in cells thought to die by apoptosis, and it was presumed that autophagy triggered apoptosis, rather than playing a direct role in the death process. Moreover, 3-MA can inhibit kinases other than class III PI3K (18
), some of which may independently affect death signaling, as well as inhibit the permeability transition in mitochondria (22
). Thus, it is not possible to directly implicate autophagy in death execution from these 3-MA inhibitor studies.
Two recent studies provide the first genetic evidence that the autophagy pathway is capable of killing cells (Table ). RNA interference (RNAi) directed against 2 autophagy genes, atg7
and beclin 1
, blocked cell death in mouse L929 cells treated with the caspase inhibitor zVAD (23
). Further, RNAi against autophagy genes atg5
and beclin 1
blocked death of bax–/–
murine embryonic fibroblasts (MEFs) treated with staurosporine or etoposide (24
). Notably, in both of these studies, atg
gene RNAi blocked the death of cells whose apoptotic pathway had been crippled. Although these findings exclude the possibility that autophagy is triggering death through apoptosis induction, they raise the question of whether autophagy is a death mechanism in cells whose apoptotic machinery is intact.
Examples of autophagy gene–dependent cell death, cell survival, and metazoan survival
Interestingly, in etoposide-treated wild-type MEFs (which die by apoptosis), only minimal autophagic activity and no inhibition of death by 3-MA is seen, indicating that autophagy is not involved in the death process unless apoptosis is blocked (24
). These data are consistent with the theory previously proposed by Lockshin and Zakeri that cells preferentially die by apoptosis but will die by any alternative available route, including autophagy, if exposed to harsh enough stimuli (9
). A related possibility is that apoptotic death is faster than autophagic death and, therefore, autophagy is only witnessed playing a role in cell death in apoptotic-deficient cells. This hypothesis is consistent with recent data indicating that growth factor–deprived wild-type cells undergo a rapid apoptotic death, whereas growth factor–deprived bax–/–
cells undergo a slow demise characterized by progressive self-cannibalization (25
Given the uncertain physiologic relevance of autophagy gene–dependent cell death in zVAD-treated cells or in bax–/–, bak–/– cells, it seems premature to conclude that autophagy is a physiologically important cause of cell death. To prove that autophagy is an important cell death pathway in normal cells, it will be necessary to demonstrate cell death resistance phenotypes in apoptotic-competent cells lacking autophagy genes.