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Inhibition of Protein Synthesis in Small Cell Lung Cancer Cells Induced by the Diphtheria Toxin-related Fusion Protein DAB₃₈₉ GRP¹

Evans Department of Clinical Research and Department of Medicine, Boston, Massachusetts 02118 [J. C. v., J. A. S., H. Z., J. R. M.], and the National Institutes on Deafness and Other Communication Disorders [J. B.] and Diabetes, Digestive and Kidney Diseases [R. J.], NIH, Bethesda, Maryland 20892

DAB₃₈₉ GRP is composed of the catalytic and transmembrane domains of diphtheria toxin fused to gastrin-releasing peptide (GRP). DAB₃₈₉ GRP is selectively targeted to, and inhibits protein synthesis in, cell lines expressing GRP receptors. Protein synthesis in 5'ET4 cells (BALB/3T3 fibroblasts transfected with the gene encoding the GRP receptor) was inhibited by 50% in the presence of 20 pM DAB₃₈₉ GRP (IC₅₀, 20 pM). DAB₃₈₉ GRP did not inhibit protein synthesis in untransfected BALB/3T3 cells. A second neuropeptide-conjugated toxin, DAB₃₈₉ SP, directed to cells expressing substance P receptors, was not cytotoxic to 5'ET4 cells, nor was DAB₃₈₉ GRP cytotoxic to substance P receptor-bearing cells. DAB₃₈₉ GRP cytotoxic effects were receptor specific and were inhibited either by excess GRP or anti-GRP antibody. Cytotoxicity was mediated by passage through an acidic vesicle, because addition of 10 µM chloroquine to the reaction inhibited cytotoxicity. DAB₃₈₉ GRP and DAB₃₈₉ SP were tested on a number of tumor cell lines. DAB₃₈₉ GRP inhibited protein synthesis in AR42J rat pancreatic acinar cells and HuTu 80 human duodenal adenocarcinoma cells with IC₅₀s of 65 and 200 pM, respectively. DAB₃₈₉ SP had an IC₅₀ of 9.5 pM for the AR42J cells and 12 nM for the HuTu 80 cell line. A number of small cell lung cancer cell (SCLC) lines were tested, and the IC₅₀ for DAB₃₈₉ GRP ranged from 1.1 to 85 nM. Sensitivity to DAB₃₈₉ GRP appeared to be based on receptor number and receptor type (i.e., GRP or neuromedin B preferring). SCLC cells were also sensitive to DAB₃₈₉ SP, with IC₅₀s ranging from 2.4 to 11.5 nM. These results suggest that a potential use exists for diphtheria-based fusion toxins as therapeutic agents for treatment of SCLC and other neuropeptide receptor-bearing cancers.

¹ Supported in part by USPHS Grant CA-60934 from the National Cancer Institute (to J. M.). J. C. v. is a Special Fellow of the Leukemia Society of America.

² To whom requests for reprints should be addressed, at Section of Biomolecular Medicine, Boston University Medical Center Hospital, 88 East Newton Street, Boston, MA 02118.

Received 10/ 4/96. Accepted 12/ 3/96.