title: Modifications enhance the apoptosis-inducing activity of FADD.
abstract: The ability to enhance apoptosis-inducing activity in specific cells, despite the presence of cellular antiapoptotic proteins, would allow the removal of target cells from a cell population. Here, we show that modification of Fas-associated protein with death domain (FADD) by fusing the tandem death effector domains (DED) of FADD to the E protein of lambda phage, a head coat protein with self-assembly activity, greatly increases the apoptosis-inducing activity of FADD in both adherent NIH3T3 and HEK293 cells. Induction of apoptosis in cell lines that stably express modified FADD (2DEDplusE) resulted in rapid blebbing, and most cells detached from the flask within 5 h. In contrast, following induction of apoptosis, it took over 24 h for the cells expressing unmodified FADD to exhibit these signs. The cells expressing the modified FADD underwent apoptosis through the typical apoptosis cascade via activation of caspase-3, and apoptosis was inhibited by a caspase inhibitor (i.e., z-VAD-fmk). Theoretically, as our adhesive stable cell lines undergo apoptosis rapidly and in synchrony following mifepristone- or tetracycline-controlled production of a single apoptosis protein without affecting any other cellular pathways, they provide excellent model systems in which to analyze the phenomenon of apoptosis in adhesive cell lines, in particular, blebbing and detachment.
authors.affiliation: Laboratory of Pharmacogenomics, Graduate School of Pharmaceutical Sciences, Chiba University, Kisarazu, Chiba, Japan.
authors.forename: Tomoki
authors.initials: T
authors.lastname: Takashina
authors.forename: Manabu
authors.initials: M
authors.lastname: Nakayama
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001483:Base Sequence; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D017931:DNA Primers; D004789:Enzyme Activation; D053200:Fas-Associated Death Domain Protein / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D051379:Mice; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D053148:Caspase 3