PARTIAL CHARACTERIZATION OF GLIOMA-DERIVED GROWTH-FACTOR-2 - A NOVEL MITOGENIC ACTIVITY FROM HUMAN CELL-LINE D-54 MG

We have shown that several human malignant glioma cell lines are stimu lated by bacterial lipopolysaccharide (E. coil 0111:B4, 1 mu g/ml) to produce a high molecular weight (> 200 kD) growth activity for BALB 3T 3, clone A31 cells [1,2]. This glioma-derived growth factor (GDGF-2) a cts like a 'competence' factor. Malignant glioma cell line D-54 MG con stitutively produced GDGF-2, which we have partially characterized fro m 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 sensiti ve to > 4 freeze-thaw cycles, alkaline (pH 11, 2 hr) conditions or pre -treatment with proteolytic enzymes. GDGF-2 had a pi of 6.8 determined by preparative isoelectric focusing, bound to DEAF, with elution at 3 5 and 185 mM NaCl and at 43% acetonitrile from a C4 reversed phase col umn. GDGF-2 activity was not neutralized by antibodies to TGF alpha, T GF beta, PDGF, VEGF or TNF alpha indicating that it is not immunochemi cally related to these growth factors. However GDGF-2 co-chromatograph ed 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 l ine produced alpha(2)-macroglobulin (alpha(2)M), which is known to bin d TGF beta; however, immunoprecipitation alpha(2)M did not deplete TGF beta or GDGF-2 activity. Further, neither GDGF-2 or TGF beta can be d issociated into lower molecular weight active components by chromatogr aphy in high salt (2 M NaCl) or 2-ME (0.5 M). GD GF-2 may be a novel a utocrine or paracrine mitogen, stimulating mitotic division or interfe ring with normal cell growth regulation.