A SAGE Approach to Discovery of Genes Involved in Autophagic Cell Death

Abstract

Programmed cell death (PCD), important in normal animal physiology and disease, can be divided into at least two morphological subtypes, including type I, or apoptosis, and type II, or autophagic cell death. While many molecules involved in apoptosis have been discovered and studied intensively during the past decade, autophagic cell death is not well characterized molecularly. Here we report the first comprehensive identification of molecules associated with autophagic cell death during normal metazoan development in vivo. During Drosophila metamorphosis, the larval salivary glands undergo autophagic cell death regulated by a hormonally induced transcriptional cascade. To identify and analyze the genes expressed, we examined wild-type patterns of gene expression in three predeath stages of Drosophila salivary glands using serial analysis of gene expression (SAGE) [7]. 1244 transcripts, including genes involved in autophagy, defense response, cytoskeleton remodeling, noncaspase proteolysis, and apoptosis, were expressed differentially prior to salivary gland death. Mutant expression analysis indicated that several of these genes were regulated by E93, a gene required for salivary gland cell death. Our analyses strongly support both the emerging notion that there is overlap with respect to the molecules involved in autophagic cell death and apoptosis, and that there are important differences.

Autophagy and signaling: their role in cell survival and cell death

“…79 An analysis of gene expression in Drosophila suggests that the TNFa-like pathway is involved during autophagic cell death in salivary glands. 80 Recently, the interaction of Atg5 with the death domain of FADD has been shown to play a crucial role in interferon-g-induced cell death independently of detectable activation of caspase-8. 81 Elucidation of signaling events downstream of FADD in Atg5-induced cell death would contribute to a better understanding of the control of autophagic cell death.…”

“…Genome-wide scale investigations have shown that, during autophagic cell death in Drosophila, the expression of a large array of genes, including several ATG genes (ATG2, 4-7, and 12), is upregulated. 80,83 Accumulation of ATGs has also been observed in mammalian cells undergoing autophagic cell death, and is dependent on the expression of proteins of the Bcl-2 family (Bcl-2 and Bc1-xL). 68 An accumulation of Beclin 1 has been reported during tamoxifen-induced autophagy.…”

Autophagy and cell death in model organisms

“…56 The overlap between the molecular mechanisms mediating autophagic cell death and apoptosis is also reflected in the results of microarray studies and serial analysis of gene expression during developmental programmed cell death of the Drosophila salivary glands, where genes involved in both apoptosis and autophagy share similar expression profiles. 32,34 Several apoptotic stimuli can promote compensatory autophagic cell death, in particular when the apoptotic pathway is impaired. Suppression of caspase-8 activity induces non-apoptotic cell death in mouse L929 cells.…”

Autophagic programmed cell death in Drosophila

“…Nine of these 11 apgrelated genes are transcribed in dying Drosophila glands, and the seven genes that are most similar to apg2 (CG1241), apg3 (CG6877), apg4 (CG6194), apg5 (CG1643), apg7 (CG5489), apg9 (CG3615), and aut10/cvt18 (CG7986) exhibit increased transcription following the rise in steroid hormone that triggers autophagic programmed cell death of this tissue. 36,64 It is interesting that flies possess genes that are related to the yeast genes involved in both of the autophagy ubiquitin conjugation systems, and that they are also the genes that increase in transcription following the rise in steroid that triggers autophagic cell death. CG1241 encodes a novel protein that is similar to APG2 that functions in the formation and completion of autophagic vacuoles.…”

“…Steroids usually function by activating transcription and, therefore, it is not surprising that transcription regulators are induced in dying salivary glands including the corepressors (Smarter and CG4756), the NFkB regulator cactus, the NFkB family member dif, several other genes encoding DNA-binding proteins (bun, sox14, CG8319), and components of transcription complexes (trap95 and hsf). 36,64 Salivary gland cells exhibit dynamic changes in cell shape and vacuole localization just before autophagic cell destruction, suggesting that cytoplasmic reorganization and proteolysis are important for their death. Consistent with this notion, genes encoding motor proteins including ctp, ck, and dlc90F, and members of the Rho, Rac, and Rab families of small guanosine triphosphates (GTPases) exhibited increase in transcription just before salivary gland autophagic cell death.…”