**1. Introduction**

Ricin is a 60–65 kDa glycoprotein toxin derived from the castor bean plant, *Ricinus communis* [1–3]. The toxin, which presumably functions in plant defense, comprises 1–5% of the total dry weight of the bean. The cytotoxicity of ricin is based on its ability to inhibit protein synthesis in all mammalian cell types, including macrophages and epithelial cells [1–3]. Due to its potential to be aerosolized and

deployed as a biological weapon, ricin is classified by the Centers for Disease Control and Prevention (CDC) as a select agen<sup>t</sup> [1–5].

Technically, ricin is a member of the type II family of ribosome-inactivating proteins (RIPs), consisting of a catalytic A subunit (RTA) attached via disulfide bond to a cell-binding B subunit (RTB) [6,7]. RTB is a lectin that binds β-1,4 galactose (Gal) and N-acetylgalactosamine (GalNAc) moieties on glycolipids and glycoproteins on the surface of target cells [8]. Following binding, ricin is internalized via clathrin-dependent endocytosis and then undergoes retrograde transport to the trans Golgi network (TGN) and endoplasmic reticulum (ER) [9]. Recently, it has been shown that fucosylation and the absence of sialylation are vital for the trafficking of ricin to these compartments [9]. Once the toxin reaches the ER, the disulfide link between the A and B subunits is reduced and RTA alone is translocated into the cell cytoplasm [9,10]. RTA inhibits protein synthesis by virtue of its ability to cleave a specific glycosidic bond in the so-called sarcin-ricin loop (SRL) of rRNA in the 60s ribosomal subunit [10–12]. The SRL is critical for the binding of elongation factor 2 to the ribosome, which is necessary for polypeptide synthesis [13,14]. Therefore, depurination of the SRL leads to the cessation of protein synthesis [11,12]. This activity is so potent that it has been noted that a single RTA can inhibit the function of 1500 ribosomes per minute [1]. Ricin is extremely toxic following inhalation [15,16]. Wide-scale damage caused by inhaled ricin leads to acute respiratory distress syndrome (ARDS) which is characterized by a potent proinflammatory response [16–19].

Previously, we reported that the cytokine TNF-α related apoptosis-inducing ligand (TRAIL) modulates the toxicity of ricin as well as the host inflammatory response to this toxin [20]. In particular, we demonstrated that addition of TRAIL enhanced the death of Calu-3 human lung epithelial cells in a caspase-dependent manner and evoked an inflammatory response dominated by IL-6 [20]. Considering that TRAIL is one of a number of potent cell death ligands that accumulate during proinflammatory responses [21–23], we wanted to evaluate the cell death modulatory activities of other cytokines in the context of ricin toxicity. These cytokines include TNF-α and Fas ligand (FasL), both of which, along with TRAIL, are capable of inducing several different programmed cell death pathways [21–23]. In addition, proinflammatory and death-inducing cytokines such as these are abundant components in the bronchoalveolar lavage fluid of animals following ricin inhalation [24–29]. We hypothesize that lung epithelial cells compromised by ricin will be primed to undergo high levels of cell death following contact with death-inducing cytokines. We believe that this heightened cell death response to ricin will be controlled by known programmed cell death pathways. In the current study, we use biochemical approaches to provide a detailed characterization of A549 human lung epithelial cell death responses to ricin administered in combination with TRAIL, TNF-<sup>α</sup>, or FasL. Defining these cell death responses and identifying multiple steps at which they can be inhibited may lead to new therapeutic approaches to ricin toxicity targeted against specific programmed cell death pathways.
