UNCORRECTED PROOF Apoptosis DOI 10.1007/s10495-006-7690-6 Amino acid starvation induced autophagic cell death in PC-12 cells: Evidence for activation of caspase-3 but not calpain-1 1 2 Shankar Sadasivan · Anu Waghray · Stephen F. Larner · William A. Dunn Jr. · Ronald L. Hayes · Kevin K. W. Wang 3 4 5 Published online: xxx 6 C  Springer Science + Business Media, LLC 2006 7 Abstract While the apoptotic and necrotic cell death path- 8 ways have been well studied, there lacks a comprehen- 9 sive understanding of the molecular events involving au- 10 tophagic cell death. We examined the potential roles of 11 the apoptosis-linked caspase-3 and the necrosis/apoptosis- 12 linked calpain-1 after autophagy induction under prolonged 13 amino acid (AA) starvation conditions in PC-12 cells. Au- 14 tophagy induction was observed as early as three hours fol- 15 lowing amino acid withdrawal. Cell death, measured by 16 lactate dehydrogenase (LDH) and -(4,5-dimethylthiazol-2- 17 yl)-2,5-diphenyltetrazolium bromide (MTT) assays occurred 18 within 24 h following starvation and was accompanied 19 by an upregulation in caspase-3 activity but not calpain- 20 1. The cell death that occurred following AA starvation 21 was significantly alleviated by treatment with the autophagy 22 Shankar Sadasivan and Anu Waghray have contributed equally to this work. S. Sadasivan · A. Waghray · S. F. Larner · R. L. Hayes · K. K. W. Wang Center for Traumatic Brain Injury Studies, Department of Neuroscience, McKnight Brain Institute of the University of Florida, Gainesville, Florida R. L. Hayes · K. K. W. Wang () Center for Neuroproteomics and Biomarkers Research, Department of Psychiatry, McKnight Brain Institute of the University of Florida, 100 S. Newell Drive, Box 100256, Gainesville, FL 32610 USA e-mail: kwang@psychiatry.ufl.edu A. Waghray Department of Biochemistry and Molecular Biology, University of Maryland, Rockville, Maryland W. A. Dunn Jr. Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida inhibitor 3-methyl adenine but not with the broad spectrum 23 caspase inhibitors. Thus, this study demonstrates that 3- 24 methyladenine-sensitive autophagic cell death due to AA 25 starvation in PC-12 cells is mechanistically and biochemi- 26 cally similar to, yet distinct from, classic caspase dependent 27 apoptosis. 28 Keywords Autophagy . Autophagic cell death . 29 Apoptosis . Necrosis . Calpain-1 . Caspases . Spectrin 30 breakdown product 31 Introduction 32 Cell death, a highly regulated and well orchestrated pro- 33 cess, is classified into three types: apoptotic (type I), au- 34 tophagic (type II) and necrotic/oncotic (type III) [1, 2]. 35 While mechanisms underlying apoptotic cell death have been 36 well documented [3], autophagic cell death is still poorly 37 understood. Autophagy occurs under stress conditions and 38 aids in replenishing amino acids by degrading intracellular 39 macromolecules [4]. It is characterized by the presence of 40 double membrane vesicles found in the cytoplasm termed 41 “autophagosomes.” Autophagosomes sequester cytoplasmic 42 constituents including organelles and transport them to lyso- 43 somes where they are degraded and the amino acids recycled 44 for rebuilding cellular machinery. Deprivation of nutritional 45 support factors for prolonged periods has been reported to 46 culminate in autophagic cell death [5, 6]. 47 Growth factor or nutrient deprivation mediated autophagic 48 cell death has been documented in neuronal cell culture sys- 49 tems [7, 8]. Autophagic cell death, a conserved integral path- 50 way involved in mammalian cell development [9], is also 51 activated under certain pathological conditions such as the 52 neurodegenerative diseases [10]. A recent study observed 53 Springer