Darlene Martineau

Biological and chemical characterization of basic FGF-saporin mitotoxin

Basic fibroblast growth factor (FGF) and saporin-6, a ribosome-inactivating protein, were chemically conjugated and characterized as a cytotoxin to cells expressing the basic FGF receptor. Structural and Western blot analysis of the conjugate showed that it contained saporin and basic FGF in equimolar amounts. The conjugate inhibited protein synthesis in a cell-free system and had potent cytotoxic activity (ID50 = 25pM) for cells expressing the basic FGF receptor. It is equipotent with basic FGF in radioreceptor assays and elutes from heparin Sepharose columns with 2M NaCl. The activity of the mitotoxin can be inhibited by competition with an excess of basic FGF but not nerve growth factor. The possibility that this mitotoxin can be used as an anti-angiogenic factor in paradigms that involve basic FGF is discussed.

Evaluation of antihuman T lymphocyte saporin immunotoxins potentially useful in human transplantation

We have synthesized 3 immunotoxins (ITs) by covalently coupling the saporin-6 hemitoxin (SAP) to OKT11, SOT3, and SOT1a murine monoclonal antibodies that recognize human T lymphocyte CD2, CD3, and CD5 surface antigens, respectively. The resulting ITs, referred to as OKT11-SAP, SOT3-SAP, and SOT1a-SAP, are equally effective in inhibiting eukaryotic protein synthesis in a cell-free system, and all 3 ITs bind to human T lymphocytes in an almost comparable manner. However, these reagents differ markedly in their ability to kill target T lymphocytes as assessed by measuring the inhibition of DNA synthesis and growth of clonable T lymphocytes in response to mitogenic and allogeneic stimuli. Whereas the anti-CD2 IT, OKT11-SAP, shows moderate cytotoxicity against T lymphocytes, the anti-CD3 IT, SOT3-SAP, and the anti-CD5 IT, SOT1a-SAP, are highly effective in eliminating the same target cells. The concentrations inhibiting 50% (IC50) of T lymphocyte DNA synthesis are 60 nM, 4.5 nM, and 1.4 nM for OKT11-SAP, SOT3-SAP, and SOT1a-SAP, respectively. Among 3 tested lysosomotropic amines, i.e., ammonium chloride, chloroquine, and amantadine, the latter only moderately potentiates the cytotoxicity of SOT1a-SAP (IC50 0.36 nM). We show that the conditions under which T lymphocyte killing is accomplished require less than 10 min exposure of T lymphocytes to the ITs, in the absence of adjuvant molecules artificially added to the incubation medium and at physiologic culture pH. These experimental characteristics of unprecedented closeness to a physiologic in-vivo model are likely to reflect the biophysical properties of the SAP moiety of the ITs. We conclude that clinical studies are warranted to define the advantage of using SAP ITs over previously described immunoconjugates.

Basic fibroblast growth factor in cells derived from Dupuytren's contracture: Synthesis, presence, and implications for treatment of the disease☆☆☆

Dupuytrens contracture (DC) is associated with fibroblast and endothelial cell proliferation. We have identified a fibroblast and endothelial cell mitogen, basic fibroblast growth factor (FGF), in cells derived from this tissue and characterized the effects of this growth factor on DC cells. Northern blot analysis of DC cells reveals the presence of basic FGF mRNA species, and the DC cells coexpress multiple forms of basic FGF. Radioreceptor assays establish that the DC cells have high-affinity binding sites for basic FGF and proliferate in response to exogenous recombinant basic FGF. Furthermore, a conjugate between saporin (a ribosome-inactivating protein) and basic FGF, which is cytotoxic to cells possessing the basic FGF receptor, is also cytotoxic to DC cells. The possibility that basic FGF-saporin could be a potential therapeutic agent for prevention of recurrence of the disease after surgery is discussed.

Anti-B16-F10 melanoma activity of a basic fibroblast growth factor-saporin mitotoxin.

Background. The authors attached basic fibroblast growth factor (FGF‐2), a growth factor for numerous tumors and normal cell types, to saporin (SAP), a ribosomeinactivating protein isolated from the plant Saponaria officinalis. The conjugate (FGF‐SAP) then was tested for antitumor activity using B16‐F10 melanoma cells. This rapidly growing murine melanoma cell line has been used classically as a model to screen antitumor agents.

Characterization of a Saporin Mitotoxin Specifically Cytotoxic to Cells Bearing the Granulocyte-Macrophage Colony-Stimulating Factor Receptor

When granulocyte-macrophage colony-stimulating factor (GM-CSF) is chemically conjugated to the ribosome-inactivating protein saporin, the resulting protein conjugate is highly toxic for cells expressing the GM-CSF receptor. Structural and Western blot analyses of the purified conjugate establish that it contains equimolar amounts of the starting materials and is free of any contamination by the non-conjugated components. The resulting bifunctional reagent is specifically cytotoxic to cells expressing the GM-CSF receptor, but is ineffective to cells that do not express the receptor. The cytotoxic activity is inhibited in a dose-dependent manner by GM-CSF, but not by any one of five other peptide growth factors. This is the first report of a mitotoxin for cells that express the GM-CSF receptor and which promises to be a valuable tool to study the expression of the GM-CSF receptor in normal and pathological states.