Clonal Variation in the Production of Tumor-associated α₂-Macroglobulin in a Malignant Human Melanoma and Association with Growth Stimulation¹

α₂-Macroglobulin (α₂-M) is known as a wide-spectrum proteinase inhibitor and to bind covalently certain growth factors. We have previously characterized tumor-associated α₂-M synthesized and secreted by human tumor cell lines. Of the cell lines studied, the melanoma cell line HMB-2 produced the largest amount of this glycoprotein. Immunofluorescence staining of cultured HMB-2 cells suggested that the cell population is heterogeneous with respect to α₂-M production. We have now isolated clones from the parental HMB-2 cells and characterized eight representative ones in detail. They varied considerably in the quantity of α₂-M secreted, from 4.2 to 46.5% of total protein. No relationship between the production of α₂-M by these clones and their pigmentation or tumorigenicity in nude mice was found. However, statistically there was a strong correlation between the modal chromosome number and population doubling time (r² = 0.88, P < 0.001) and also between the modal chromosome number and α₂-M production (r₂ = 0.73, P < 0.01). The growth rate of the clones correlated with the level of α₂-M in culture medium (r² = 0.69, P < 0.01). Clones with lower α₂-M production had a proportionally shorter population doubling time than the clones with intermediate or high production. Northern hybridization indicated quantitative variation in the α₂-M mRNA expressed by the parental cells and the clones, that was comparable but not identical with the quantity of α₂-M in the respective culture media; both parental cells and clones expressed platelet-derived growth factor A-chain mRNAs with little difference in levels. Serum-free medium from low α₂-M producer clones stimulated normal stationary fibroblasts significantly more than clones producing intermediate or high amounts of α₂-M. α₂-M decreased and anti-α₂-M IgG increased the stimulation. These results suggest that production of tumor-associated α₂-M is related to both autocrine and paracrine growth-stimulating activity of the tumor cells.