Tyrosine‐nitration of caspase 3 and cytochrome c does not suppress apoptosis induction in squamous cell carcinoma cells

The influence of tyrosine nitration of cytochrome c and caspase 3 on apoptosis induction was investigated in an established squamous carcinoma cell line, OSC‐4. The intracellular NO and O   − 2levels were increased up to about 110–120% and 140–180% of the control levels, respectively, after the treatment of OSC‐4 cells with 5‐FU (100 μg/ml), PLM (10 μg/ml), CDDP (10 μg/ml), or γ‐rays (20 Gy). The treatment of OSC‐4 cells with ONOO − (1 mM) and the above anticancer agents induced tyrosine nitration of 14, 32 kDa protein among others and nitration of tyrosine residues of cytochrome c and caspase 3 was identified by the Western blotting of immunoprecipitates obtained by antibodies to these proapoptotic proteins. When cytochrome c and procaspase 3 were treated with ONOO − , tyrosine nitration was increased in a ONOO − ‐dose dependent manner. Tyrosine nitration of cleaved (17 kDa) caspase 3, however, was not induced by ONOO − . Procaspase 3 in the cytosol of HeLa cells was activated by the addition of ONOO − ‐treated as well as ONOO − ‐untreated cytochrome c. In addition, cleavage of ICAD and PARP were not suppressed in OSC‐4 cells by pretreatment with ONOO − . Activity of cleaved caspase 3 was not suppressed at low concentrations or by treatment with ONOO − or NO donors, SIN‐1 and SNP. Furthermore, apoptosis of OSC‐4 cells by the anticancer agents was not suppressed by ONOO − . In conclusion, these results suggest that nitration of tyrosine residues of cytochrome c and procaspase 3 is induced by chemoradiotherapy but their nitration does not suppress cancer cell apoptosis. © 2002 Wiley‐Liss, Inc.