The Effector Phase of Physiological Cell Death Relies Exclusively on the Posttranslational Activation of Resident Components Sandra H. Chang,* ,1 Marija Cvetanovic,* Kevin J. Harvey,* ,2 Akira Komoriya,† Beverly Z. Packard,† and David S. Ucker* ,3 *Department of Microbiology and Immunology, University of Illinois College of Medicine, Rm. E803 (M/C 790), 835 South Wolcott, Chicago, Illinois 60612; and †OncoImmunin, Inc., 207A Perry Parkway, Suite 6, Gaithersburg, Maryland 20877 Inhibitors of transcription and translation can pro- tect cells from physiological cell deaths induced by a variety of stimuli. These observations have been taken to suggest that de novo macromolecular synthesis may be an essential component of the cell death process. Paradoxically, the same inhibitors, at higher concen- trations, themselves trigger the death of cells. Previ- ously, we have mapped a conserved and ordered se- quence of events that exerts physiological cell death. Diverse signals converge to activate this lethal path- way, composed of a proteolytic cascade of caspases and subsequent cyclin-dependent kinases. Here we re- port that inhibitors of nuclear gene expression, when they block cell death, act upstream of this lethal pro- cess to prevent its activation. In contrast, when cell death is triggered by high doses of the inhibitors, these same essential molecules are activated, despite the essentially complete blockade of macromolecular synthesis. This inhibitor-induced death response is as- sociated with the release of cytochrome c from mito- chondria and the activation of apical caspase 9 and is blocked by overexpression of Bcl-2. These data dem- onstrate that all essential molecules that exert lethal- ity already are resident within cells and are activated posttranslationally upon stimulation. De novo macro- molecular synthesis pertains idiosyncratically only to upstream, modulatory elements of particular death responses. © 2002 Elsevier Science (USA) Key Words: physiological cell death; programmed cell death; apoptosis; macromolecular synthesis; post- translational modification; caspases; Cdk2; cyclin A. INTRODUCTION Among the earliest observations regarding the pro- cess of physiological cell death on a mechanistic level, the ability of inhibitors of macromolecular synthesis to spare cells from death was striking. Weber [1] first revealed the importance of transcription in the process of developmental cell death with the demonstration that actinomycin D, an inhibitor of RNA synthesis, prevented Xenopus tadpole tail regression in vivo. Tata [2] extended these findings to show that both transcrip- tion and translation were induced actively during tail regression and that either actinomycin D or cyclohex- imide, an inhibitor of protein synthesis, could prevent tail regression. Subsequent work revealed a similar requirement for ongoing macromolecular synthesis in other develop- mental cell deaths, including those occurring during insect metamorphosis [3, 4]. A wide array of studies contributed to the appreciation that macromolecular synthesis plays an essential role in the vast majority of cell deaths that occur physiologically in mammals, in- cluding the deaths of neuronal and lymphoid cells dur- ing developmental repertoire shaping and in the ho- meostatic deaths of lymphoid and other hormonally responsive cells [for examples, see 5–14]. Most dramatically, Martin et al. [8] found that the inhibition of protein synthesis could confer long-term sparing of mature sympathetic neurons that would otherwise die promptly when deprived of nerve growth factor, a requisite survival factor. Significantly, these studies utilized a variety of pharmacologic inhibitors of transcription (5,6-dichloro-1--D-ribofuranosyl benz- imidazole as well as actinomycin D) and translation (anisomycin, emetine, pactamycin, and puromycin in addition to cycloheximide) with distinct modes of ac- tion. A requisite role for macromolecular synthesis of- fered compelling early support for the argument that cell death reflects a genetically determined, cell-auton- omous suicide program, one in which dying cells par- ticipate actively in their own demise. This encouraged the formative view that the new expression of gene products is an obligatory step associated with the func- tion of this endogenous suicide process. Challenge to the importance of macromolecular syn- thesis in cell death came from several sources. The 1 Present address: Department of Environmental Health, Box 357234, University of Washington, Seattle, WA 98195. 2 Present address: Department of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093. 3 To whom correspondence and reprint requests should be ad- dressed. Fax: (312) 996 6415. E-mail: duck@uic.edu. 0014-4827/02 $35.00 15 © 2002 Elsevier Science (USA) All rights reserved. Experimental Cell Research 277, 15–30 (2002) doi:10.1006/excr.2002.5539