Sequential effects of interleukin 2-diphtheria toxin fusion protein on T-cell activation.

Abstract

The interleukin 2-diphtheria toxin-related fusion protein (IL-2-toxin) rapidly inhibits protein synthesis in IL-2 receptor (IL-2R)-bearing phytohemagglutinin-activated T cells but transiently stimulates DNA synthesis. At 7 hr after interaction with IL-2R+ phytohemagglutinin-activated T cells, IL-2-toxin-treated cells bear augmented steady-state levels of c-myc, interferon y, and IL-2R mRNA; these effects are indistinguishable from those produced by recombinant IL-2. Amplification of IL-2 sequences by the polymerase chain reaction reveals an increased level of IL-2 mRNA in cell cultures treated with recombinant IL-2, IL-2-toxin, and cycloheximide. These results suggest that IL-2-toxin can affect de novo IL-2 gene transcription/mRNA stabilization through independent mechanisms exerted by both the IL-2R binding domain and ADP-ribosyltransferase activity of the fusion protein. After 20 hr of culture, IL-2R mRNA was markedly decreased in both IL-2-toxin-and cycloheximide-treated phytohemagglutinin-activated T cells. Although interaction of IL-2-toxin with IL-2R+ T cells initially mimics the stimulatory effects of IL-2 upon c-myc, interferon y, IL-2R, and IL-2 gene expression, the consequences of inhibition of protein synthesis mediated by the ADP-ribosyltransferase activity of the toxin dominate after 7 hr and are indistinguishable from those effects mediated by cycloheximide.A fusion gene encoding the interleukin 2-diphtheria toxin fusion protein (IL-2-toxin) was constructed from a truncated diphtheria toxin gene by replacing DNA sequences coding for the toxin receptor binding domain with sequences coding for amino acids 2-133 of human IL-2 (1). The mature form of IL-2-toxin has a deduced molecular mass of 68 kDa, and the fusion protein retains at least some functional attributes of both its diphtheria toxin and IL-2 components. The fusion protein (i) binds with high affinity to the multimeric IL-2 receptor (IL-2R), (ii) is internalized by IL-2R-mediated endocytosis, and (iii) mediates the ADP-ribosylation of elongation factor 2 in the target-cell cytosol (2).Binding of IL-2 to its high-affinity receptor rapidly activates intracellular processes that lead to gene activation and de novo gene transcription, DNA synthesis, and cell cycle progression (3)(4)(5)(6). Even though IL-2-toxin rapidly inhibits protein synthesis in IL-2R-bearing phytohemagglutinin (PHA)-activated T cells, we now note that this chimeric toxin also transiently stimulates DNA synthesis (e.g., thymidine incorporation).In the present study we demonstrate that binding of the IL-2-toxin to the high-affinity IL-2R initially produces effects in the PHA-activated target cell that are indistinguishable from those mediated by IL-2 itself. Subsequently, the fusion protein inhibits cellular protein synthesis. Additional indirect effects of IL-2-toxin upon T-cell gene activation, produced by the inhibition of protein synthesis, are characterized herein. MATERIALS AND METHODSCell Cultures. Human peripheral blood mononuclear cells were isolated by means...

In vitro and in vivo studies of a VEGF 121 /rGelonin chimeric fusion toxin targeting the neovasculature of solid tumors

“…These findings accord with those of previous studies by Wild et al (32), who showed that conjugates of VEGF and DT were highly toxic to log-phase cells but were not toxic to confluent endothelial cells. The greater sensitivity of dividing endothelial cells to VEGF-toxin constructs may be because of differences in intracellular routing or catabolism of the construct as observed with other targeted therapeutic agents (49).…”

Immunomodulatory effects of RXR rexinoids: modulation of high-affinity IL-2R expression enhances susceptibility to denileukin diftitox

“…Denileukin diftitox has been shown to initiate signal transduction with ligand engagement, even if receptor-mediated internalization of the fusion toxin does not occur. 12 Signaling events mediated by rexinoids and IL-2R-ligand engagement may contribute to the markedly enhanced cytotoxicity observed in this study.…”

“…As shown, low doses of denileukin diftitox alone were associated with increased proliferation, presumably due to cytokinemediated signaling events from binding of denileukin diftitox to intermediate-and low-affinity receptor-expressing cells. 12 To confirm that inhibition of protein synthesis corresponded to increased cytotoxicity, MTT assays were performed. Bexarotene had a greater effect on enhancing cytotoxicity of denileukin diftitox than alitretinoin in both T-and B-leukemia cells, concordant to the degree of protein synthesis inhibition (Figure 4).…”

EGF receptor and p185erbB‐2‐specific single‐chain antibody toxins differ in their cell‐killing activity on tumor cells expressing both receptor proteins

“…Treatment of activated T-cells with a recombinant IL-2-diphtheria-toxin fusion molecule leads to initial stimulation of DNA synthesis and gene transcription through the activity of the IL-2 domain (Walz et al, 1989). Similarly, growth-factortoxin fusion molecules such as TGF-a-PE40 at low doses or (Fan etal., 1993).…”