Nerve growth factor expression response to induction agent booster dosing in transfected human embryonic kidney cells

To assess whether nerve growth factor (NGF) expression would respond to booster dosing with the inducing agent ponasterone A, human embryonic kidney cells (HEK-293) were transfected with human NGF cDNA. Cells were cultured for 5 days in media with or without ponasterone A. On day 5, controls received a ponasterone A media replacement, whereas experimental groups received ponasterone A booster media replacement. NGF protein expression bioactivity was assessed using a PC-12 cell bioassay and the concentration of secreted NGF was quantified using NGF enzyme-linked immunosorbent assay. Cells with and without ponasterone A were left for 5 days without changing the medium. On day 5, the supernatants were collected and flash-frozen for enzyme-linked immunosorbent assay. The ponasterone A-positive and -negative booster medium was replaced in the appropriate wells. Supernatants were collected from the wells at 2, 4, and 6 days after the booster dose and removal of original supernatant. The medium was flash-frozen for enzyme-linked immunosorbent assay (1.5 ml), and the remaining 500 mul was transferred to PC-12 cells seeded onto 12-well plates to determine NGF bioactivity. All experiments were performed in quadruplicate. NGF production was measured daily by enzyme-linked immunosorbent assay over a 6-day period after the ponasterone A booster to a maximal release of 1233 +/- 130 pg/ml at day 6 (11 days after original induction). Maximal NGF production per 10(3) cells was 2.5 +/- 0.61 pg at day 6. Bioactivity was determined by percentage differentiation (per 100 cells counted) at 26, 52, and 98 percent for ponasterone A-treated wells on 2, 4, and 6 days after booster dosing (7, 9, and 11 days after induction), respectively. PC-12 cell differentiation was not visualized in the ponasterone A-negative control wells. Human NGF-EcR-293 cells can inducibly secrete bioactive NGF when exposed to the induction agent ponasterone A. Furthermore, repeated bioactive NGF expression peaks beyond that previously demonstrated can be achieved using induction agent booster dosing, indicating the ability to regulate the system over an extended period.