Three different human tumor lines in culture, a rhabdomyosarcoma, a bronchogenic carcinoma and a metastatic melanoma, release proteins (transforming growth factors, TGFs) into the medium that confer the transformed phenotype on untransformed fibroblasts. These proteins are acid and heat-stable; produce profound morphologic changes in rat and human fibroblasts; and enable normal anchorage-dependent cells to grow in agar. Removal of the transforming protein results in a reversion of cell phenotype. The major activity interacts with epidermal growth factor (EGF) cell membrane receptors. The peptides from these tumor cells are similar in their action to the sarcoma growth factor (SGF) released by murine sarcoma virus-transformed rodent cells. The most anchorageindependent tumor cells released the most TGFs. EGF-related TGFs were not detectable in fluids from cultures of cells with high numbers of free EGF membrane receptors (normal human fibroblasts and human carcinomas). Mouse sarcoma virus (MSV)-and rat sarcoma virus-transformed cells release a potent growth-stimulating peptide that interacts with epidermal growth factor (EGF) receptors (1, 2). This ability has been utilized to purify sarcoma growth factor (SGF) produced by MSV-transformed cells (3). It has been noted that certain human sarcoma and carcinoma cells (4) and most melanomas (5) lack EGF receptors and, therefore, may produce endogenous factors related to EGF and SGF. To test this possibility, serum-free media were collected from four human tumors and normal human fibroblasts and partially purified. Cells that lack EGF receptors released a potent growth-stimulating activity that enabled normal fibroblasts and epithelial cells to proliferate in soft agar. Supernates from normal human fibroblasts possessed <2% as much activity.A human epidermoid carcinoma cell (A431) with an exceptionally high number of EGF receptors (4, 6, 7) released little growth-stimulating activity when compared to the other tumor cells. That activity did not compete with EGF. Tumor cells that lack EGF receptors and form colonies in soft agar released a greater quantity of the transforming growth factors (TGFs) than did normal or tumor cells that grow poorly in agar.We conclude that certain human tumor cells release potent transforming protein(s) that transform normal indicator cells in a manner similar to SGF. MATERIALS AND METHODSCell Cultures. Cell cultures were maintained at 37°C in 75-cm2 plastic tissue culture flasks (Falcon no. 3024) with Dulbecco's modification of Eagle's medium (DME medium) with 10% calf serum (Colorado Serum). Five human tumor cell cultures were used. The human rhabdomyosarcoma line, A673, and the bronchogenic carcinoma line, 9812, produce progressively growing tumors in immunologically depressed mice and grow readily in soft agar (8). Neither has detectable EGF receptors (4). The human epidermoid carcinoma A431, from a primary vulvar carcinoma in an 85-year-old woman, has an exceptionally high number of EGF receptors (4, 6). The human metastatic m...

Cultured cell lines of human tumour origin as well as cells transformed by various RNA tumour viruses secrete low molecular weight polypeptide transforming growth factors (TGFs). In addition to competing with epidermal growth factor (EGF) for binding to its cellular receptor, TGFs can transform morphologically fibroblast and epithelial cells in culture. In view of accumulating evidence that tyrosine phosphorylation activity is associated with the transforming genes of various tumour viruses, we determined whether phosphotyrosine levels were elevated in these human tumour cells. We show here that TGFs produced by human tumour cells induce phosphorylation of specific tyrosine acceptor sites in the 160,000-molecular weight (160 K) EGF receptor.

Abstract. The ot2-macroglobulin (ot2M) receptor/lowdensity lipoprotein receptor-related protein (LRP) is important for the clearance of proteases, proteaseinhibitor complexes, and various ligands associated with lipid metabolism. While the regulation of receptor function is poorly understood, the addition of high concentrations of the 39-kD receptor-associate d protein (RAP) to cells inhibits the binding and/or uptake of many of these ligands. Previously, we (Kounnas, M. Z., J. Henkin, W. S. Argraves, D. K. Strickland. 1992. J. Biol. Chem. 267:12420-12423) showed that Pseudomohas exotoxin (PE) could bind immobilized LRP. Also, the addition of RAP blocked toxin-mediated cell killing. These findings suggested that PE might use LRP to gain entry into toxin-sensitive cells. Here we report on a strategy to select PE-resistant lines of Chinese hamster ovary cells that express altered amounts of LRP. An important part of this strategy is to screen PE-resistant clones for those that retain sensitivity to both diphtheria toxin and to a fusion protein composed of lethal factor (from anthrax toxin) fused to the adenosine diphosphate-ribosylating domain of PE. Two lines, with obvious changes in their expression of LRP, were characterized in detail. The 14-2-1 line had significant amounts of LRP, but in contrast to wild-type cells, little or no receptor was displayed on the cell surface. Instead, receptor protein was found primarily within cells, much of it apparently in an unprocessed state. The 14-2-1 line showed no uptake of chymotrypsin-aEM and was 10-fold resistant to PE compared with wild-type cells. A second line, 13-5-1, had no detectable LRP mRNA or protein, did not internalize OtEM-chymotrypsin, and exhibited a 100-fold resistance to PE. Resistance to PE appeared to be due to receptor-specific defects, since these mutant lines showed no resistance to a PE chimeric toxin that was internalized via the transferrin receptor. The results of this investigation confirm that LRP mediates the internalization of PE.

Pseudomonas exotoxin (PE) requires proteolytic cleavage to generate a 37-kDa C-terminal fragment that translocates to the cytosol and ADP-ribosylates elongation factor 2. Cleavage within cells is mediated by furin, occurs between arginine 279 and glycine 280, and requires an arginine at both P1 and P4 residues. To study the proteolytic processing of PE-derived chimeric toxins, TGF␣-PE38 (transforming growth factor fused to the domains II and III of PE) and a mutant form, TGF␣-PE38gly279, were each produced in Escherichia coli. When assessed on various epidermal growth factor (EGF) receptor-positive cell lines, TGF␣-PE38 was 100 -500-fold more toxic than TGF␣-PE38gly279. In contrast to PE, where cleavage by furin is only evident at pH 5.5, furin cleaved TGF␣-PE38 over a broad pH range, while TGF␣-PE38gly279 was resistant to cleavage. TGF␣-PE38 was poorly toxic for furin-deficient LoVo cells, unless it was first pretreated in vitro with furin. Furin treatment produced a nicked protein that was 30-fold more toxic than its unnicked counterpart. Using the single chain immunotoxin HB21scFv-PE40 as a substrate, furin-mediated processing of an antibody-based immunotoxin was also evaluated. HB21scFv-PE40, which targets cells expressing the transferrin receptor, was cleaved in a similar fashion to that of TGF␣-PE38 and nicked HB21scFv-PE40 exhibited increased toxicity for LoVo cells. In short-term experiments, the rate of reduction in protein synthesis by furin-nicked immunotoxins was increased compared with unnicked protein, indicating that cleavage by furin can be a rate-limiting step. We conclude that furin-mediated cleavage of PE-derived immunotoxins is important for their cytotoxic activity. Pseudomonas exotoxin (PE)1 is a single chain protein toxin that is toxic for mammalian cells because of its ability to translocate to the cell cytosol and ADP-ribosylate elongation factor 2 (EF2) and inhibit protein synthesis (1). Once synthesized, the toxin folds to form a 3-domain protein composed of an N-terminal domain that mediates receptor binding, a middle domain that mediates translocation and has a prominent arginine-rich loop that allows proteolytic cleavage, and a C-terminal domain that has ADP-ribosylating activity and a endoplasmic reticulum (ER) retention sequence (2-4). In its native form, PE is a proenzyme, and in biochemical experiments, it requires a strong denaturant and reducing agent to reveal its enzyme activity (5). It is, therefore, of considerable interest to learn how toxin molecules unfold and are processed within cells without the use of harsh treatments.The pathway that PE takes to the cytosol begins with its binding to the multi-ligand surface receptor known as the low density lipoprotein receptor-related protein (LRP), also known as the ␣ 2 -macroglobulin receptor (6, 7). Binding leads to endocytosis via coated pits, which brings the toxin to the low pH environment of the endosomal compartment. There, the toxin is cleaved into an N-terminal fragment of 28 kDa and a Cterminal fragment of 37 kDa (8). ...