ABSTRACT
The vast majority of brain cancers (gliomas) express a receptor (R) for interleukin 13 (IL13). In order to achieve specific targeting of the IL13R in gliomas, we have mutagenized human (h) IL13. The mutation was made to alter IL13 interaction with the shared functional IL13/4 normal tissue receptor, but not with the glioma-associated receptor. We have thus produced hIL13.E13K (glutamic acid at position 13 changed to lysine) and fused it to derivatives of Pseudomonas exotoxin A. The hIL13.E13K-based cytotoxins are less active on normal cells and thus less toxic, and are better antitumor agents compared with the cytotoxins containing nonmutagenized hIL13.
Subject(s)
Antineoplastic Agents/therapeutic use , Brain Neoplasms/drug therapy , Carrier Proteins , Cytotoxins/therapeutic use , Exotoxins/therapeutic use , Glioma/drug therapy , Receptors, Cell Surface/genetics , Receptors, Interleukin/genetics , Brain Neoplasms/genetics , Cell Division/drug effects , Cell Division/genetics , Exotoxins/genetics , Glioma/genetics , Glutamic Acid , Humans , Interleukin-13/genetics , Interleukin-13 Receptor alpha1 Subunit , Lysine , Mutation/genetics , Plasmids , Pseudomonas aeruginosa , Receptors, Interleukin/drug effects , Receptors, Interleukin-13 , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/therapeutic use , Tumor Cells, CulturedABSTRACT
Activation of the transcription factor NF-kappa B by various cellular stimuli involves phosphorylation and subsequent degradation of its inhibitor I kappa B alpha. Both the cytokine tumor necrosis factor alpha (TNF-alpha) and the phosphatase inhibitor calyculin A have been shown to induce rapid phosphorylation and degradation of I kappa B alpha. In the present study, we demonstrate that TNF-alpha and calyculin A stimulate similar although not identical pattern of I kappa B alpha phosphorylation, as demonstrated by phosphopeptide mapping. Interestingly, phosphorylation of I kappa B alpha induced by both inducers involves serine-32 and serine-36 of I kappa B alpha. Furthermore, TNF-alpha- and calyculin A-induced degradation of I kappa B alpha appears to require the same structural domains within I kappa B alpha. In addition to the N-terminal phosphorylation sites and the C-terminal sequences, each of the five ankyrin-like repeats of I kappa B alpha is critically required for the inducible degradation of this NF-kappa B inhibitor. Together, these studies suggest that degradation of I kappa B alpha by both cytokines and phosphatase inhibitors is regulated by site-specific phosphorylation and requires multiple structural domains.