title: Basic fibroblast growth factor-Pseudomonas exotoxin chimeric proteins; comparison with acidic fibroblast growth factor-Pseudomonas exotoxin.
abstract: We have constructed growth factor-toxin chimeric molecules composed of basic fibroblast growth factor (bFGF) and two different binding mutant forms of Pseudomonas exotoxin termed bFGF-PE40 and bFGF-PE4E KDEL. The chimeric molecules were expressed in Escherichia coli and localized to both inclusion bodies and the spheroplast cytoplasm. The bFGF-toxin fusion protein that was isolated and purified from inclusion bodies was 3-fold more active in inhibiting protein synthesis than that purified from spheroplast cytoplasm. Immunoreactivity of purified bFGF-toxin fusion protein to anti-bFGF antibodies was similar to that of native bFGF, as determined by ELISA analysis. A variety of carcinoma cell lines were sensitive to bFGF-PE40 and bFGF-PE4E KDEL, including H3396 (breast), Hep G2 (hepatocellular), and A431 (epidermoid). The concentration of chimeric toxin that inhibited protein synthesis by 50% (EC50) was 110, 70, and 18 ng/mL for bFGF-PE40 and 15, 1, and 18 ng/mL for bFGF-PE4E KDEL. In comparison with fusion-toxins composed of acidic fibroblast growth factor (aFGF) and either PE40 or PE4EKDEL, bFGF-PE40 and bFGF-PE4E KDEL were similarly cytotoxic on most cell lines tested. Human aortic smooth muscle cells were sensitive to both bFGF and aFGF toxin fusion proteins. However, human aortic endothelial cells were sensitive to the bFGF-toxins but were resistant to both aFGF-toxin forms. Time course studies showed that bFGF-PE40 needed a 4-6-h exposure to target cells for peak inhibition of protein synthesis on both MCF-7 and A431 cells, while aFGF-PE40 was almost fully active within a 2-h incubation.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Bristol-Myers Squibb, Pharmaceutical Research Institute, Molecular Immunology Department, Seattle, Washington 98121.
authors.forename: S L
authors.initials: SL
authors.lastname: Gawlak
authors.forename: I
authors.initials: I
authors.lastname: Pastan
authors.forename: C B
authors.initials: CB
authors.lastname: Siegall
mesh_terms: D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000235:genetics* / Q000633:toxicity*; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004730:Endothelium, Vascular / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005098:Exotoxins / Q000235:genetics* / Q000633:toxicity*; D016220:Fibroblast Growth Factor 1 / Q000235:genetics* / Q000633:toxicity*; D016222:Fibroblast Growth Factor 2 / Q000235:genetics* / Q000633:toxicity*; D015870:Gene Expression / Q000235:genetics; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000187:drug effects; D009369:Neoplasms / Q000188:drug therapy; D010957:Plasmids / Q000235:genetics; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000633:toxicity*; D013997:Time Factors; D014407:Tumor Cells, Cultured / Q000187:drug effects; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D018076:DNA, Complementary; D005098:Exotoxins; D016220:Fibroblast Growth Factor 1; D016222:Fibroblast Growth Factor 2; D006493:Heparin; D017468:Receptors, Fibroblast Growth Factor; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A