title: Heparin-binding transforming growth factor alpha-Pseudomonas exotoxin A. A heparan sulfate-modulated recombinant toxin cytotoxic to cancer cells and proliferating smooth muscle cells.
abstract: TGF alpha-PE40, a recombinant toxin in which transforming growth factor alpha (TGF alpha) is fused to a mutant form of Pseudomonas exotoxin, is selectively cytotoxic to cells bearing epidermal growth factor (EGF) receptors. Heparin binding EGF-like growth factor is a potent mitogen for smooth muscle cells capable of binding to both the EGF receptor and to immobilized heparin (Higashiyama, S., Abraham, J., Miller, J., Fiddes, J., and Klagsbrun, M. (1991) Science 251, 936-938). To study the effect of the heparin-binding domain in a chimeric toxin targeted to the EGF receptor, we fused the DNA sequence corresponding to the putative NH2-terminal heparin-binding (HB) domain of HB-EGF to chimeric toxins composed of TGF alpha and two different recombinant forms of Pseudomonas exotoxin (PE). One of these is a truncated form of PE devoid of the binding domain (TGF alpha-PE38); another is a mutant form of full-length toxin containing inactivating mutations in the binding domain and an altered carboxyl terminus (TGF alpha-PE4EKDEL). The resulting chimeric toxins HB-TGF alpha-PE38 and HB-TGF alpha-PE4EKDEL were expressed in Escherichia coli as inclusion bodies, refolded, and purified by heparin affinity chromatography. Both of the toxins were eluted from heparin at 0.8 M NaCl, in contrast to their respective TGF alpha toxins which were eluted at 0.15 M. Binding studies on A431 cells showed that the HB-TGF alpha toxins bound to the EGF receptor with an affinity similar to that of the TGF alpha toxins. However, cell killing studies on a panel of malignant cell lines showed that cytotoxicity was strongly affected by the presence of the HB domain. Cell lines expressing high numbers of EGF receptors such as A431 and KB were less sensitive to toxins containing the HB domain. Cells with low number of EGF receptors had similar responses to both types of toxins (MCF-7 and LNCaP) or were more sensitive to the toxin with the added HB domain (HEP-G2). HB-TGF alpha-PE4EKDEL was over 10-fold more cytotoxic against proliferating vascular smooth muscle cells (VSMC) than to quiescent VSMC. Moreover, HB-TGF alpha-PE4EKDEL was 6-fold more potent than TGF alpha-PE4EKDEL to proliferating VSMC. Competition studies with EGF and/or heparin showed that heparin blocks the cytotoxicity of HB-TGF toxins and the inhibitory action of heparin is stronger in cells expressing lower number of EGF receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Division of Cancer Diagnosis Biology and Centers, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: E A
authors.initials: EA
authors.lastname: Mesri
authors.forename: R J
authors.initials: RJ
authors.lastname: Kreitman
authors.forename: Y M
authors.initials: YM
authors.lastname: Fu
authors.forename: S E
authors.initials: SE
authors.lastname: Epstein
authors.forename: I
authors.initials: I
authors.lastname: Pastan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002455:Cell Division; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D005098:Exotoxins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D016211:Transforming Growth Factor alpha / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: D005098:Exotoxins; D011993:Recombinant Fusion Proteins; D016211:Transforming Growth Factor alpha; C062005:transforming growth factor type alpha-Pseudomonas exotoxin A; D004815:Epidermal Growth Factor; D006493:Heparin; D004247:DNA; D066246:ErbB Receptors