An AU-rich sequence present within the 3' untranslated region has been shown to mark some short-lived mRNAs for rapid degradation. We demonstrate by label transfer and gel shift experiments that a 32-kDa polypeptide, present in nuclear extracts, specifically interacts with the AU-rich domains present within the 3' untranslated regions of human granulocyte-macrophage colony-stimulating factor, c-fos, and c-myc mRNAs and a similar domain downstream of the poly(A) addition site of the adenovirus IVa2 mRNA. Competition experiments and partial protease analysis indicated that the same polypeptide interacts with all four RNAs. A single AUUUA sequence in a U-rich context was sufficient to signal binding of the 32-kDa polypeptide. Insertion of three copies of this minimal recognition site led to markedly reduced accumulation of j-globin RNA, while the same insert carrying a series of U-to-G changes had little effect on RNA levels. Steady-state levels of ,-globin-specific nuclear RNA, including incompletely processed RNA, and cytoplasmic mRNA were reduced. Cytoplasmic mRNA containing the AU-rich recognition sites for the 32-kDa polypeptide exhibited a half-life shorter than that of mRNA with a mutated insert. We suggest that binding of the 32-kDa polypeptide may be involved in the regulation of mRNA half-life. mRNA turnover plays a major role in the regulation of mRNA steady-state levels in animal cells (reviewed in references 2 and 33). The stabilities of different mRNAs vary widely. While ,B-globin mRNA has a relatively long half-life (more than 17 h; 43), other mRNAs are degraded at relatively high rates. Beta-interferon mRNA (31), c-fos and c-myc mRNAs (15), and the granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA (36) exhibit halflives of about 30 min. The rates of turnover of some mRNAs can change in response to a variety of physiological signals. For example, intracellular iron levels control the stability of the transferrin receptor mRNA (5, 25), and histone mRNA stability is regulated by the cell cycle (35).Specific sequence determinants that influence the degradation pathway have been identified in several cases, and in some of these systems ongoing translation is required for destabilization of mRNA (reviewed in reference 9). The autoregulated tubulin mRNA instability requires sequences in the coding region of the gene and is dependent on protein synthesis (12,49). For histones and transferrin receptor mRNAs, different 3'-end stem-loop structures determine their stability (5,14,28). The presence of free histones and ongoing translation are essential for histone mRNA degradation (30, 38). The iron-dependent regulation of transferrin receptor mRNA stability involves a protein factor (21,29).Recent studies have shown that AU-rich sequences within the 3' untranslated regions of some short-lived mRNAs play a role in their rapid degradation. Shaw and Kamen (36) demonstrated that removal of the AU-rich domain increased the half-life of the GM-CSF mRNA, while 3-globin mRNA was destabilized by insertion...

Summary Epidermal growth factor (EGF) receptor-overexpression is characteristic of many human tumours of epithelial origin and has been correlated with unfavourable patient prognosis. Its involvement in the malignant process, its elevated expression in tumours and its accessibility on the tumour cell surface make the EGF receptor a potential target for directed tumour therapy. We have previously characterized a recombinant antibody -Pseudomonas exotoxin A fusion protein, scFv(225)-ETA, which displayes antitumoral activity towards EGF receptor-overexpressing tumour cells but is less potent in tumour cell killing than TGF-a-ETA, a recombinant toxin using the natural EGF receptor ligand transforming growth factor a (TGF-a) as a targeting domain. Here, we describe the construction and functional characterization in vitro of a novel single-chain antibody-toxin, scFv(14E1)-ETA, based on the independently isolated EGF receptor-specific monoclonal antibody 1 4E1. ScFv(14E1 )-ETA binds to an EGF receptor epitope that is very similar or identical to that of scFv(225)-ETA with nine times higher affinity than the latter and displays more than tenfold higher cytotoxic activity on EGF receptor-overexpressing tumour cells. ScFv(14E1)-ETA cell killing activity was very similar to that of TGF-a-ETA on receptor-overexpressing cells but, in contrast to the latter, scFv(1 4E1 )-ETA was much more selective and did not display significant cytotoxic activity on cells expressing moderate EGF receptor levels.Keywords: single chain Fv; epidermal growth factor receptor; exotoxin A; directed tumour therapy The erbB/EGF receptor-related gene family encodes growth factor receptors with intrinsic tyrosine kinase activity. Four members of this family have been identified: ErbBlEGF receptor, ErbB-2, ErbB-3 and ErbB-4 (Peles and Yarden, 1993). Members of this family have been implicated in the development of a variety of human malignancies. EGF receptor gene amplification and overexpression have been observed in a high percentage of primary human carcinomas of epithelial origin, including glioblastoma and cancer of the lung, breast, head and neck and bladder, and correlates with an unfavourable prognosis for the patients (Gullick, 1991). Increased receptor expression in tumour cells is often accompanied by increased production of TGF-a (Derynck et al, 1987; Van de Vijver et al, 1991), which leads to receptor activation by an autocrine pathway and contributes to malignant transformation. Because of its accessibility on the cell surface, its overexpression in several types of cancer and its involvement as a marker for an unfavourable prognosis, the EGF receptor is under intensive scrutiny as a therapeutic target for novel anti-tumour reagents.Various strategies have been used to target the EGF receptor for tumour therapy. Monoclonal antibodies directed towards the extracellular domain of the EGF receptor have proven effective in the inhibition of tumour cell growth. The EGF receptor-specific monoclonal antibody (MAb) 225 competes with EGF for bind...

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