Fenretinide-induced caspase 3 activity involves increased protein stability in a mechanism distinct from reactive oxygen species elevation

Fenretinide (4-HPR) is a synthetic retinoid that displays a broad range of biological effects and has also demonstrated clinical efficacy as a chemopr eventative agent. One cellular activity of 4-HPR is its ability to induce a poptosis, This effect has been proposed to relate to changes in intracellul ar reactive oxygen species. We show herein that a 1-h treatment of HL-60 ce lls with 4-HPR led to a dose-dependent increase in hydroperoxides, Pretreat ment of cells with the antioxidant vitamin C abolished apoptosis, measured as the appearance of the sub-G(1) peak, in 4-HPR. treated cells. The retino id also elicited a 3.6-fold increase in caspase 3 activity however, this in crease was not affected by vitamin C treatment. Analysis of caspase 3 prote in expression by Western blot analysis revealed that 4-HPR resulted in a si gnificant increase in the appearance of the active p17 subunit without effe cting a concomitant change in p32 pro-caspase 3 le, els. Studies on de novo synthesis and stability of caspase 3 by pulse-chase and immunoprecipitatio n methods show that l-HPR-treated samples had decreased incorporation of ra dioactive amino acid precursors into newly synthesized procaspase 3 but, du ring the chase (for up to 9 h), had more labeled caspase 3 remaining when c ompared with controls, These studies suggest that 4-HPR may effect changes in caspase 3 activity by modulating changes in zgmogen stability by a mecha nism distinct from the retinoid-elicited increase in reactive oxygen specie s.