Kinetics of tetracycline action and dose response analysis of tetracycline-regulated luciferase expression.

(A) HL-60 cells expressing luciferase under the control of tTA were grown in the absence of tetracycline. At time 0, cells were separated in several batches and grown in the presence of various doses of tetracycline: 1 ng/mL (♦), 10 ng/mL (■), 20 ng/mL (▪), 50 ng/mL (○), and 100 ng/mL (●). At indicated times, aliquots containing the same amounts of cells were withdrawn and assayed for luciferase activity. No change in growth behavior or cell morphology occurred for any concentration of antibiotic. (B) Cells maintained in the presence of doses of tetracycline that repress luciferase expression were washed twice with phosphate-buffered saline and recultured in the absence of tetracycline. At indicated times, aliquots containing the same amount of cells were withdrawn and assayed for luciferase activity. A substantial increase in luciferase activity could be detected only after several passages when cells had been treated with 100 ng/mL tetracycline (●). Results are given in relative luciferase activity (RLU). Average values of 2 independent experiments are given.