Heparin-binding transforming growth factor α-Pseudomonas exotoxin A. A heparan sulfate-modulated recombinant toxin cytotoxic to cancer cells and proliferating smooth muscle cells

E. A. Mesri, R. J. Kreitman, Y. M. Fu, S. E. Epstein, I. Pastan

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

Abstract

TGFα-PE40, a recombinant toxin in which transforming growth factor a (TGFα) 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 TGFa and two different recombinant forms of Pseudomonas exotoxin (PE). One of these is a truncated form of PE devoid of the binding domain (TGFα-PE38); another is a mutant form of full-length toxin containing inactivating mutations in the binding domain and an altered carboxyl terminus (TGFα-PE4EKDEL). The resulting chimeric toxins HB-TGFα-PE38 and HB-TGFα-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 TGFa toxins which were eluted at 0.15 M. Binding studies on A431 cells showed that the HB-TGFα toxins bound to the EGF receptor with an affinity similar to that of the TGFa 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-TGFα-PE4EKDEL was over 10-fold more cytotoxic against proliferating vascular smooth muscle cells (VSMC) than to quiescent VSMC. Moreover, HB-TGFα-PE4EKDEL was 6-fold more potent than TGFα-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. In addition, experiments with heparitinase-treated cells showed that in cells with low numbers of EGF receptors the binding of the HB domain to cell surface heparan sulfate proteoglycans appears to facilitate the internalization of the toxin. We conclude that addition of a HB domain to TGFα-PE38 or TGFα-PE4EKDEL confers the ability to bind to and to be modulated by heparin-like molecules and increases their cytotoxicity to cells expressing low numbers of EGF receptor and proliferating smooth muscle cells.

Original languageEnglish (US)
Pages (from-to)4853-4862
Number of pages10
JournalJournal of Biological Chemistry
Volume268
Issue number7
StatePublished - 1993
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Heparin-binding transforming growth factor α-Pseudomonas exotoxin A. A heparan sulfate-modulated recombinant toxin cytotoxic to cancer cells and proliferating smooth muscle cells'. Together they form a unique fingerprint.

Cite this