Abstract
The influence of tyrosine nitration of cytochrome c and caspase 3 on apoptosis induction was investigated in an established squamous carcinoma cell line, OSCβ4. The intracellular NO and O levels were increased up to about 110β120% and 140β180% of the control levels, respectively, after the treatment of OSCβ4 cells with 5βFU (100 ΞΌg/ml), PLM (10 ΞΌg/ml), CDDP (10 ΞΌg/ml), or Ξ³βrays (20 Gy). The treatment of OSCβ4 cells with ONOO^β^ (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^β^, tyrosine nitration was increased in a ONOO^β^βdose dependent manner. Tyrosine nitration of cleaved (17 kDa) caspase 3, however, was not induced by ONOO^β^. Procaspase 3 in the cytosol of HeLa cells was activated by the addition of ONOO^β^βtreated as well as ONOO^β^βuntreated cytochrome c. In addition, cleavage of ICAD and PARP were not suppressed in OSCβ4 cells by pretreatment with ONOO^β^. Activity of cleaved caspase 3 was not suppressed at low concentrations or by treatment with ONOO^β^ or NO donors, SINβ1 and SNP. Furthermore, apoptosis of OSCβ4 cells by the anticancer agents was not suppressed by ONOO^β^. 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. Β© 2002 WileyβLiss, Inc.