Partial Purification and Characterization of a Growth Inhibitor Produced By Normal Rat Mammary Epithelial Cells in Vitro

Abstract

Transforming growth factors (TGFs) have been operationally defined as peptides that reversibly induce nonneoplastic cells to express the transformed phenotype as measured by loss of density-dependent inhibition of growth and acquisition of anchorage-independent growth. TGF-beta was originally purified to homogeneity from human platelets and identified as a homodimeric peptide with a molecular weight of 25,000 daltons. TGF-beta is an acid and heat stable peptide growth factor. TGF-beta is a multifunctional growth regulator. The growth inhibitor of the present study was purified from Ham's F12 medium supplemented with BSA, Hepes, EA, HC, Tf, T3, NaSeO3, gentamycin, and Fungizone which was conditioned for 48 hours by normal rat mammary epithelial (RME) cells. The conditioned medium (CM) was assayed under conditions of anchorage-dependent growth. To activate the growth inhibitor, the CM was dialyzed against 1% Acetic Acid. ED50 for the crude twenty-fold concentrate CM was 300 ug protein. A ten-fold purification and ED50 32 ug was achieved by molecular seive chromatography using a Bio-Rad P-60 column. The growth inhibitor eluted in the range of 23,000 to 25,000 daltons. Physical and chemical treatment of the CM revealed the growth inhibitor to be heat stable and protease sensitive. In the present study, partial purification and characterization of a growth inhibitor produced by normal RME cells in vitro suggested that this inhibitor may be TGF-beta or a similar peptide growth inhibitor which may act through an unknown autocrine mechanism.