The Cell's Fate, Apoptosis or Senescence, Depends on Dose and Duration of Exposure to DNA Damaging Agents such as Mitomycin C.

摘要

Cytotoxic chemotherapy kills tumor cells by apoptosis often using DNA damaging agents. Typically, the drug doses necessary are toxic to normal cells and, therefore, not well tolerated by patients. Alternatively, it may be possible to render tumor cells senescent ("reproductively dead") by treatment with much lower doses of the same drugs for longer lengths of time.;Mitomycin C (MMC), a DNA damaging agent, was chosen to treat A549 human pulmonary carcinoma cells to define what treatment conditions might cause senescence. Laser scanning cytometry was used to evaluate changes in the cell cycle distribution, rate of DNA synthesis, activation of DNA damage response (DDR), and induction of apoptosis and/or cellular senescence.;The fate, apoptosis or senescence, of A549 cells depended on the concentration and duration of treatment. Pulse exposure of cells to MMC induced the DDR and caused dose-dependent changes in A549's cell cycle distribution, seen as a block in cell transit through S phase. Continuous exposure to MMC also led to induction of the DDR and changes to the cell cycle distributions at concentrations below 1.0 μg/ml whereas higher concentrations triggered apoptosis.;To force A549 cells into senescence, lower MMC concentrations (0.01-0.02 μg/ml) and longer times of exposure (3-6 days) were required. Senescence induced in these cells was characterized by increased β-galactosidase activity, increased expression of cyclin-dependent kinase inhibitors (p21, p27), decreased cell proliferation, and senescent cell specific morphologic changes such as increased nuclear size coupled with a decrease in the staining intensity of DNA.;To determine whether senescence was induced in the entire A549 population and whether the cells completely lost their capacity to replicate DNA, clonogenicity assays were conducted. These assays showed that < 2% of A549 cells treated with 0.01 μg/ml MMC for 6 days were able to proliferate and form colonies, while no cells treated with 0.02 μg/ml were able to develop colonies. Therefore, treatment of A549 cells with a concentration of 0.02 μg/ml MMC for 6 days was, in fact, capable of inducing all cells to undergo senescence.;Thus, drugs used to kill cells by apoptosis at relatively high doses can, at significantly lower doses and increased times of exposure, induce cellular senescence. Therefore, by effectively precluding proliferation of tumor cells it may be possible to achieve results similar to those achieved with more aggressive chemotherapy but with the possibility of fewer side effects.