IDENTIFICATION AND INITIAL CHARACTERIZATION OF GROWTH-REGULATORY ACTIVITY IN SERUM-FREE MEDIUM CONDITIONED BY A HUMAN NEUROBLASTOMA CELL LINE (GENE AMPLIFICATION)

The possibility that serum-free medium conditioned by human neuroblastoma cell line BE(2)-C contains growthregulating activity was tested. In limiting serum conditions, culture medium previously exposed to human neuroblastoma BE(2)-C cells stimulated the growth of BE(2)-C and NAP(H) human neuroblastoma cells into colonies on plastic tissue culture surfaces. In addition, BE(2)-C conditioned medium (CM) stimulated DNA synthesis in 7 of 9 human neuroblastoma cell lines (1.4- to 5.8- fold), 1 of 6 non-neuroblastoma human tumor lines (1.3- fold), and 3 of 3 non-human cell lines tested (2.4- to 5.6- fold). The colony forming activity was growth stage-dependent, i.e., found only in medium conditioned by logarithmic phase BE(2)-C cells. Mitogenic activity was, on the other hand, present in medium conditioned by stationary as well as logarithmic phase cultures. Gel filtration chromatography revealed two peaks of mitogenic activity (M(,r) (\u27(TURN)) 12,000 and 9,400 daltons) and two peaks of inhibitory activity (M(,r) (\u27(TURN)) 25,000 and (\u27(TURN)) 5,000 daltons). Both stimulatory and inhibitory activities were stable in 1 M acetic acid (pH 2.2). In addition, the mitogenic activity is heat stable (100(DEGREES)C for up to 10 minutes), partially inactivated by trypsin digestion (100 (mu)g/ml for 2 hours), and completely inactivated by exposure to the disulfide reducing agent, dithiothreitol (0.065 M). Isoelectric focusing of the 12,000 dalton mitogenic fractions revealed a major protein band at pI 6.8 and minor bands at pI 6.2 and 5.4. Comparative studies using a thymidine incorporation bioassay suggest that the mitogenic activity in BE(2)-C conditioned medium is not due to several well characterized growth factors including NGF, EGF, PDGF, FGF, MSA or insulin. Also, receptor binding studies show that the 12,000 dalton mitogenic factor in BE(2)-C CM does not compete with insulin for the insulin receptor. These findings suggest that medium conditioned by human neuroblastoma BE(2)-C cells contains previously unrecognized growth factors that act in an autocrine as well as paracrine manner