Platelet-derived growth factor, insulin-like growth factors, fibroblast growth factors and transforming growth factor beta do not account for the cell growth activity present in bovine milk.
Cation-exchange chromatography effectively concentrates the cell growth activity present in whey and we have used this process as a basis to characterise further the growth factors present in bovine milk. Under neutral conditions, total bioactivity in the growth factor-enriched cation-exchange fraction chromatographed with an apparent molecular mass of 80-100 kDa. In contrast, acid gel-filtration chromatography resolved two peaks of cell growth activity. A peak at 15-25 kDa contained the bulk of growth activity for Balb/c 3T3 fibroblasts while bio-activity for L6 myoblasts and skin fibroblasts eluted with a molecular mass of 6 kDa. A peak of inhibitory activity for Mv1Lu and MDCK cells also eluted at 15-25 kDa. Both IGF-I and IGF-II were purified from fractions that eluted at 6 kDa, although the IGF peptides alone did not account for the total bioactivity recovered. Platelet-derived growth factor (PDGF), identified by radioreceptor assay, eluted at a slightly higher molecular mass than the peak of growth activity for Balb/c 3T3 cells, and an anti-PDGF antibody was without effect on the growth of Balb/c 3T3 cells in response to the whey-derived factors. Further purification of the inhibitory activity for epithelial cells yielded a sequence for transforming growth factor beta (TGF-beta), and all inhibitory activity for Mv1Lu cells was immunoneutralised by an antibody against TGF-beta. In contrast, this antibody decreased the growth of Balb/c 3T3 fibroblasts in the whey-derived extract by only 10%. Finally, a cocktail of recombinant growth factors containing IGF-I, IGF-II, PDGF, TGF-beta and fibroblast growth factor 2 stimulated growth of Balb/c 3T3 cells to a level equivalent to only 51% of that observed in the milk-derived growth factor preparation. We conclude that: (i) cell growth activity recovered from bovine whey is present in acid-labile high molecular weight complexes; (ii) all cell growth inhibitory activity for epithelial cells can be accounted for by TGF-beta; (iii) IGF-I and IGF-II co-elute with the major peak of activity for L6 myoblasts and skin fibroblasts, although the IGF peptides alone do not explain the growth of these cells in the whey-derived extract; and (iv) neither PDGF nor TGF-beta account for the 15-25 kDa peak of Balb/c 3T3 growth activity. These data suggest the presence of additional mitogenic factors in bovine milk.
Belford
D A
DA
Cooperative Research Centre for Tissue Growth and Repair, Women's and Children's Hospital, North Adelaide, Australia.
Rogers
M L
ML
Francis
G L
GL
Payne
C
C
Ballard
F J
FJ
Goddard
C
C
D016475:3T3 Cells; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000032:analysis; D005347:Fibroblasts; D006133:Growth Substances / Q000032:analysis*; D006168:Guinea Pigs; D007334:Insulin-Like Growth Factor I / Q000032:analysis; D007335:Insulin-Like Growth Factor II / Q000032:analysis; D051379:Mice; D008892:Milk / Q000737:chemistry*; D008934:Mitogens / Q000032:analysis*; D018482:Muscle, Skeletal / Q000166:cytology; D010982:Platelet-Derived Growth Factor / Q000032:analysis; D011869:Radioligand Assay; D051381:Rats; D016212:Transforming Growth Factor beta / Q000032:analysis
D006133:Growth Substances; D008934:Mitogens; D010982:Platelet-Derived Growth Factor; D016212:Transforming Growth Factor beta; D016222:Fibroblast Growth Factor 2; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II