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Partial characterization of glioma-derived growth factor 2: A novel mitogenic activity from human cell line D-54 MG

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Summary

We have shown that several human malignant glioma cell lines are stimulated by bacterial lipopolysaccharide (E. coli 0111∶B4, 1 μg/ml) to produce a high molecular weight (> 200 kD) growth activity for BALB 3T3, clone A31 cells [1, 2]. This glioma-derived growth factor (GDGF-2) acts like a ‘competence’ factor. Malignant glioma cell line D-54 MG constitutively produced GDGF-2, which we have partially characterized from serumfree conditioned culture medium. GDGF-2 is resistant to heat (100° C, 5 min), acidic (pH 2, 2 hr) or reducing (0.5 M 2 ME, 30 min) conditions as well as exposure to RNases; however, it is sensitive to > 4 freeze-thaw cycles, alkaline (pH 11, 2 hr) conditions or pre-treatment with proteolytic enzymes. GDGF-2 had a pl of 6.8 determined by preparative isoelectric focusing, bound to DEAE, with elution at 35 and 185 mM NaCl and at 43% acetonitrile from a C4 reversed phase column. GDGF-2 activity was not neutralized by antibodies to TGFα, TGFβ, PDGF, VEGF or TNFα indicating that it is not immunochemically related to these growth factors. However GDGF-2 co-chromatographed on Superose 12 HPLC (250 × 9 mm; 5% isopropanol, 6 mM CHAPS in PBS) with a substance that suppressed growth of mink lung epithelial cells (Mv1Lu), but not BALB 3T3 cells, and could be neutralized by anti-TGFβ antibodies. GDGF-2 activity eluted from heparin columns in 0.6 M NaCl; thus, it is not a heparin binding growth factor. D-54 MG cell line produced alpha2-macroglobulin (α2M), which is known to bind TGFβ; however, immunoprecipitation of α2M did not deplete TGFβ or GDGF-2 activity. Further, neither GDGF-2 or TGFβ can be dissociated into lower molecular weight active components by chromatography in high salt (2 M NaCl) or 2-ME (0.5 M). GDGF-2 may be a novel autocrine or paracrine mitogen, stimulating mitotic division or interfering with normal cell growth regulation.

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  1. Brain Tumor Research Laboratories, Division of Neurological Surgery, University of Alabama at Birmingham, 35294-0006, Birmingham, AL, USA

    Elaine Lyon & G. Yancey Gillespie

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  1. Elaine Lyon
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  2. G. Yancey Gillespie
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Lyon, E., Gillespie, G.Y. Partial characterization of glioma-derived growth factor 2: A novel mitogenic activity from human cell line D-54 MG. J Neuro-Oncol 17, 99–109 (1993). https://doi.org/10.1007/BF01050212

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