Figure 4.
Free radical scavengers or BH4 supplementation block MTX-mediated increases in apoptosis sensitivity.A, Jurkat cells were cultured for 48 hr. with MTX and/or the free radical scavenger, NAc, and stimulated with anti-Fas for 5 hr. Results are expressed as % Annexin V+ cells ± S.D. B, as in [A] except cultures were harvested after 48 hrs., processed, and JUN transcript levels determined. Results are expressed as fold induction ± S.D.C, Jurkat cells were treated with MTX for 48 hrs. ROS production was determined by labeling cells with CMH2DCFDA.D, Jurkat cells were treated with MTX for 48 hrs in the presence or absence of NAc. ROS production was determined as in C. Jurkat cells were cultured for 48 with MTX, 0.1 μM, with or without BH4 supplementation, 30 μM. E, % Annexin V+ cells ± S.D. were determined after anti-Fas treatment by flow cytometry. F, ROS levels, expressed as fold increase relative to untreated cells ± S.D., were determined after loading Jurkat cells with CM-H2DCFDA.* P< 0.05 relative to unstimulated cultures. ** P< 0.05 relative to MTX stimulated Jurkat cells.