David C. Heimbrook

Epidermal growth factor receptor binding is affected by structural determinants in the toxin domain of transforming growth factor-alpha-Pseudomonas exotoxin fusion proteins.

TGF-alpha-PE40 is a hybrid protein composed of transforming growth factor-alpha (TGF-alpha) fused to a 40,000-dalton segment of Pseudomonas exotoxin A (PE40). This hybrid protein possesses the receptor-binding activity of TGF-alpha and the cell-killing properties of PE40. These properties enable TGF-alpha-PE40 to bind to and kill tumor cells that possess epidermal growth factor (EGF) receptors. Unexpectedly, TGF-alpha-PE40 binds approximately 100-fold less effectively to EGF receptors than does native TGF-alpha (receptor-binding inhibition IC50 = 540 and 5.5 nM, respectively). To understand the factors governing receptor binding, deletions and site-specific substitutions were introduced into the PE40 domain of TGF-alpha-PE40. Removal of the N-terminal 59 or 130 amino acids from the PE40 domain of TGF-alpha-PE40 improved receptor binding (IC50 = 340 and 180 nM, respectively) but decreased cell-killing activity. Substitution of alanines for cysteines at positions 265 and 287 within the PE40 domain dramatically improved receptor binding (IC50 = 37 nM) but also decreased cell-killing activity. Similar substitutions of alanines for cysteines at positions 372 and 379 within the PE40 domain did not significantly affect receptor-binding or cell-killing activities. These studies indicate that the PE40 domain of TGF-alpha-PE40 interferes with EGF receptor binding. The cysteine residues at positions 265 and 287 of PE40 are responsible for a major part of this interference.

Human papillomavirus type 16 E7 protein inhibits DNA binding by the retinoblastoma gene product.

The human papillomavirus E7 gene can transform murine fibroblasts and cooperate with other viral oncogenes in transforming primary cell cultures. One biochemical property associated with the E7 protein is binding to the retinoblastoma tumor suppressor gene product (pRB). Biochemical properties associated with pRB include binding to viral transforming proteins (E1A, large T, and E7), binding to cellular proteins (E2F and Myc), and binding to DNA. The mechanism by which E7 stimulates cell growth is uncertain. However, E7 binding to pRB inhibits binding of cellular proteins to pRB and appears to block the growth-suppressive activity of pRB. We have found that E7 also inhibits binding of pRB to DNA. A 60-kDa version of pRB (pRB60) produced in reticulocyte translation reactions or in bacteria bound quantitatively to DNA-cellulose. Recombinant E7 protein used at a 1:1 or 10:1 molar ratio with pRB60 blocked 50 or greater than 95% of pRB60 DNA-binding activity, respectively. A mutant E7 protein (E7-Ala-24) with reduced pRB60-binding activity exhibited a parallel reduction in its blocking of pRB60 binding to DNA. An E7(20-29) peptide that blocks binding of E7 protein to pRB60 restored the DNA-binding activity of pRB60 in the presence of E7. Peptide E7(2-32) did not block pRB60 binding to DNA, while peptide E7(20-57) and an E7 fragment containing residues 1 to 60 partially blocked DNA binding. E7 species containing residues 3 to 75 were fully effective at blocking pRB60 binding to DNA. These studies indicate that E7 protein specifically blocks pRB60 binding to DNA and suggest that the E7 region responsible for this property lies between residues 32 and 75. The functional significance of these observations is unclear. However, we have found that a point mutation in pRB60 that impairs DNA-binding activity also blocks the ability of pRB60 to inhibit cell growth. This correlation suggests that the DNA-binding activity of retinoblastoma proteins contributes to their biological properties.

A gastrin-releasing peptide antagonist containing a ψ(CH2O) amide bond surrogate

The [Leu26-psi(CH2O)Leu27] derivative of N-Ac-GRP20-27-peptide amide was prepared and evaluated as a gastrin-releasing peptide antagonist. This psi(CH2O) derivative was found to be a more potent inhibitor of [3H-Phe15]GRP15-24NH2 binding and N-Ac-GRP20-27NH2 induced mitogenesis in Swiss 3T3 fibroblasts than the related nitrogen analog [Leu13-psi(CH2NH)Leu14] bombesin. Possible reasons for the improved activity of the (CH2O) insert relative to the (CH2NH) group include increased hydrophobicity and a reduced tendency of the oxygen derivative to form hydrogen bonds.

Biological activity of a transforming growth factor-alpha- Pseudomonas exotoxin fusion protein in vitro and in vivo

SummaryTransforming growth factor-alpha (TGFα)-pseudomonas exotoxin-40 (PE40) is a chimeric protein consisting of an N-terminal TGFα domain fused to a C-terminal 40-kDa segment of the pseudomonas exotoxin A protein. TGFα-PE40 exhibits the receptor binding activity of TGFα and the cell killing activity of PE40. In the current study, we report that a modified TGFα-PE40 derivative significantly prolongs the survival of nude mice bearing tumors derived from cell lines which express the epidermal growth factor receptor (EGFR). In addition, the therapeutic benefit of this protein is mediated by specific binding to the EGF receptor. These results indicate that a therapeutic window exists in vivo for the use of some growth factor-toxin fusion proteins as anticancer agents.

Spontaneously Transformed NRK Cells Lose Their Mitogenic Response to Epidermal Growth Factor

To understand the relationship between growth factor-induced mitogenesis and spontaneous cell transformation, a clonal isolate of epidermal growth factor (EGF)-responsive NRK cells was passed in vitro until morphologically transformed variants arose. Subclones of EGF responsive (Cl-3) and EGF nonresponsive (Cl-10) NRK cells were isolated. Cl-3 cells grew as flat, contact-inhibited monolayers, while Cl-10 cells grew as rounded or spindle-shaped cells that formed dense foci. Cl-10 cells formed colonies in soft agar more efficiently (p less than 0.01) and formed larger tumors in nude mice (p less than 0.05) than Cl-3 cells. Cl-3 cells exhibited a sixfold increase in DNA synthesis in response to 1.0 nM EGF. Cl-10 cells did not increase DNA synthesis on exposure to 100 nM EGF. These different responses to EGF occurred despite similar numbers of receptors and similar receptor.binding affinities for EGF (Cl-3: 7000 receptors, Kd = 0.67 nM; Cl-10: 8000 receptors, Kd = 0.72 nM). No evidence of transforming growth factor-alpha was detected in either of these cell lines using Northern blots, Western blots, or biologic assays. We conclude that NRK cells which undergo spontaneous morphologic transformation and exhibit enhanced anchorage-independent growth lose their mitogenic response to EGF.

Minimal Ligand Analysis of Gastrin‐Releasing Peptide: Receptor Binding and Mitogenesis

Gastrin releasing peptide (GRP) is a peptide hormone containing 27 amino acids which is structurally analogous to the amphibian peptide bombesin. GRP serves a variety of physiological functions and has been implicated in the pathophysiology of small cell lung cancer. Previous work has demonstrated that the modified C terminus of GRP, N-acetyl-GRP-20-27, exerts full agonist activity in a variety of assay systems. However, no systematic comparison of binding of GRP fragments to its receptor and mitogenic potency has been reported. To investigate whether smaller GRP fragments could bind to the GRP receptor without stimulating mitogenesis, we performed binding inhibition and thymidine uptake assays with Swiss 3T3 fibroblasts. These studies were facilitated by the development of a novel tritiated GRP-based radioligand, [3H-Phe15] GRP-15-27, which exhibits enhanced chemical stability compared to iodinated GRP derivatives. We examined a series of C-terminal GRP fragments, from the pentapeptide to the octapeptide, with both N-acetyl and free amine moieties at the N terminus. N-Acetylated derivatives were more potent than their primary amine counterparts in both assays. Deletion of N-terminal residues from GRP-20-27 resulted in significant loss of potency in both assays: the EC50 values of N-acetyl-GRP-21-27 were 10(2)-fold higher than N-acetyl-GRP-20-27, those of N-acetyl-GRP-22-27 were 10(4)-fold higher, and N-acetyl-GRP-23-27 showed minimal activity at concentrations below 100 microM. These results suggest that 1) both His20 and Trp21 play an important role in binding of GRP to the receptor, and 2) for this series of N-terminal deletions, binding to the receptor and mitogenic activity are tightly coupled.