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Partial characterization of glioma-derived growth factor 2: a novel mitogenic activity from human cell line D-54 MG
1Brain Tumor Research Laboratories, University of Alabama at Birmingham 35294-0006.
Abstract:
We have shown that several human malignant glioma cell lines are stimulated by bacterial lipopolysaccharide (E. coli 0111:B4, 1 microgram/ml) to produce a high molecular weight (> 200 kD) growth activity for BALB 3T3, clone A31 cells. 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 serum-free conditioned culture medium. GDGF-2 is resistant to heat (100 degrees 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 TGF alpha, TGF beta, PDGF, VEGF or TNF alpha indicating that it is not immunochemically related to these growth factors. However GDGF-2 co-chromatographed on Superose 12 HPLC (250 x 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-TGF beta 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 alpha 2-macroglobulin (alpha 2M), which is known to bind TGF beta; however, immunoprecipitation of alpha 2M did not deplete TGF beta or GDGF-2 activity. Further, neither GDGF-2 or TGF beta 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.
Insights
Human malignant glioma cells produce a novel growth factor, GDGF-2, which stimulates cell division. This glioma-derived growth factor (GDGF-2) is heat-stable and distinct from known growth factors, suggesting a new role in cell regulation.
Area of Science:
- Oncology
- Cell Biology
- Biochemistry
Background:
- Malignant gliomas are aggressive brain tumors with complex growth regulation.
- Understanding the molecular mechanisms driving glioma proliferation is crucial for developing targeted therapies.
- Several growth factors are implicated in tumor progression, but novel factors may also play a role.
Purpose of the Study:
- To investigate the production and characterization of growth factors secreted by human malignant glioma cell lines.
- To identify and partially characterize a novel growth factor, termed glioma-derived growth factor-2 (GDGF-2), produced by D-54 MG cells.
- To determine the biochemical properties and potential function of GDGF-2 in cell growth regulation.
Main Methods:
- Human malignant glioma cell lines were stimulated with bacterial lipopolysaccharide.
- Growth activity in conditioned media was assessed using BALB 3T3 cells.
- GDGF-2 was partially characterized using techniques including heat stability assays, pH stability, protease sensitivity, isoelectric focusing, and various chromatography methods (DEAE, C4 reversed phase, Superose 12 HPLC, heparin columns).
- Neutralization assays with antibodies against known growth factors (TGF-α, TGF-β, PDGF, VEGF, TNF-α) were performed.
Main Results:
- Human malignant glioma cells stimulated by lipopolysaccharide produced a high molecular weight (> 200 kD) growth activity (GDGF-2) for BALB 3T3 cells.
- GDGF-2 is resistant to heat, acidic conditions, reducing agents, and RNases, but sensitive to alkaline conditions, freeze-thaw cycles, and proteases.
- GDGF-2 has a pI of 6.8, binds to DEAE, and elutes from C4 reversed phase and heparin columns at specific salt concentrations, indicating it is not a heparin-binding growth factor.
- GDGF-2 activity was not neutralized by antibodies to TGF-α, TGF-β, PDGF, VEGF, or TNF-α, suggesting it is immunochemically distinct.
- GDGF-2 co-eluted with a TGF-β-neutralizable growth suppressor on Superose 12 HPLC, but GDGF-2 itself was not TGF-β.
Conclusions:
- Malignant glioma cells constitutively produce GDGF-2, a novel growth factor with unique biochemical properties.
- GDGF-2 acts as a competence factor, potentially mediating autocrine or paracrine signaling in glioma.
- The distinct nature of GDGF-2 from known growth factors highlights its potential as a novel therapeutic target for malignant gliomas.