N-glycosylation of MDA-7/IL-24 is dispensable for tumor cell-specific apoptosis and "bystander" antitumor activity.

Biochemical and genetic mutation–based analyses confirm that the MDA-7/IL-24 protein can induce transformed cell–specific apoptosis through a mechanism involving endoplasmic reticulum (ER) stress–associated pathways. Covalent modifications by N-linked glycans in the ER contribute to the conformational maturation and biological functions of many proteins. Because MDA-7/IL-24 is a glycosylated protein, we investigated the role of glycosylation in mediating the specific biological and “bystander” antitumor activities of this cytokine. An adenovirus vector expressing a nonsecreted and nonglycosylated version of MDA-7/IL-24 protein was generated via deletion of its signal peptide and point mutations of its three N-glycosylated sites. In this study, we showed that this intracellular nonglycosylated protein was as effective as wild-type MDA-7/IL-24 protein in inducing apoptosis in multiple tumor cell lines. Both constructs ( a ) displayed transformed cell specificity and localization to the ER compartment, ( b ) mediated apoptosis through JAK/STAT-independent and p38 MAPK -dependent pathways, ( c ) induced sustained ER stress as evidenced by expression of ER stress markers (BiP/GRP78, GRP94, XBP-1, and eIF2α), and ( d ) generated proteins that physically interacted with BiP/GRP78. Additionally, an expression construct containing the mda-7 /IL-24 signal peptide linked to the mutated nonglycosylated mda-7 /IL-24 gene retained the ability to induce bystander antitumor activity. These studies reveal that MDA-7/IL-24 glycosylation is not mandatory for inducing cell death or bystander activities in different cancer cells, providing new insights into the mechanism by which MDA-7/IL-24 induces apoptosis and ER stress. (Cancer Res 2006; 66(24): 11869-77)