IL-4 receptors on human medulloblastoma tumours serve as a sensitive target for a circular permuted IL-4-Pseudomonas exotoxin fusion protein

Abstract

Cytotoxins directed to interleukin-4 receptors have shown to mediate relatively selective cytotoxicity against a variety of human cancer cells in vitro and in vivo. In an ongoing Phase I clinical trial, a recombinant protein comprised of circularly permuted IL-4 fused to a mutated form of Pseudomonas exotoxin (the fusion protein termed IL-4(38-37)-PE38KDEL or cpIL4-PE) has shown antitumour activity against malignant glioma. Human medulloblastomas are neuroectodermal tumours that occur in children and have a poor prognosis. The goal of this study was to determine whether human medulloblastoma derived cell lines express interleukin-4 receptor and whether interleukin-4 receptor expression is accompanied by sensitivity to cpIL4-PE. Medulloblastoma cell lines express interleukin-4 receptor at the protein and mRNA levels as determined by binding, indirect immunofluorescence and RT--PCR studies. These cells expressed IL-4Ralpha (also known as IL-4Rbeta) and IL-13Ralpha1 (also known as IL-13Ralpha') chains, however common gamma(c), a component of the interleukin-4 receptor system in immune cells was not detected. Consistent with the expression of IL-4R, cpIL4-PE was found to be highly and specifically cytotoxic to four of five medulloblastoma cell lines. Susceptibility of medulloblastoma cell lines to cpIL4-PE seemed to correlate closely to the functional IL-4 binding sites in general as demonstrated by 125I-IL-4 binding, but did not seem to correlate with mRNA or cell surface immunoreactive receptor protein expression. The sensitivity of medulloblastoma cells to cpIL4-PE could be eliminated by concurrent incubation with IL-4 or IL-13, but not with IL-2. None of these cell lines showed any change in proliferation upon treatment with exogenous IL-4. These studies establish the interleukin-4 receptor as a medulloblastoma-associated target for possible tumour-directed cancer therapy. Further studies are warranted to investigate interleukin-4 receptor expression in primary medulloblastoma tumours and sensitivity to cpIL-4PE in vitro and in vivo.

Figure 1

mRNA transcripts for IL-4R subunits. RT–PCR products were resolved in 2% agarose gel and visualized by EtBR staining. PM-RCC RNA served as positive control for IL-IL-13Rα1 and IL-4Rα chains.

Figure 2

Immunofluorescence analysis of various receptor chains on medulloblastoma cell lines. (A) UW228-2, (B) UW228-3, and (C) D283 medulloblastoma cell lines were stained with either murine IgG1 isotype control or mouse monoclonal antibody to IL-4Rα chain or IL-13Rα1 chains. D283 cell line grew as a suspension culture showing few cells positive in the microscopic field.

Figure 3

Cytotoxicity of cpIL-4-PE against medullobalstoma cell lines. Ten thousand cells were cultured with various concentrations of cpIL4-PE. Cells were then pulsed with 1 μCi [³H]-Leucine and cell-associated radioactivity was measured with a Beta Plate Counter. The results are shown as mean ±s.d. of quadruplicate determinations and the experiment was repeated two times.

Figure 4

Both IL-4 and IL-13, but not IL-2 neutralize the cytotoxic activity of cpIL4-PE: (A) UW-228-2 and (B) UW228-3 cells (10⁴) were cultured with various concentrations of cpIL4-PE. For blocking experiments, cells were pre-incubated with IL-2, IL-4 or IL-13 (2 μg ml⁻¹) for 30 min prior to the addition of cpIL4-PE. Data are shown as mean ±s.d. of quadruplicate determination.

Figure 5

Exogenous IL-4 does not modify the growth of medulloblastoma cells: 2×10⁴ cells of five medulloblastoma cell lines were grown in the absence or presence of 0.1 ng to 1000 ng ml⁻¹ IL-4. The cells were pulsed with 1 μCi of [³H]-thymidine and harvested after 8 h. The incorporated radiolabelled thymidine was measured and shown as per cent of control. Data are shown as mean ±s.d. of quadruplicate determinations.