THE USE OF UBIQUITIN AS A MARKER OF THYROXINE-INDUCED APOPTOSIS IN CULTURED RANA-CATESBEIANA TAIL TIPS

Cultured Rana catesbeiana tadpole tail tips were used in combination w ith a fluoroimmunoassay to determine the levels of ubiquitin-a protein marker of programmed cell death in other systems-during the tissue re gression induced by thyroxine (T-4). After a 3-day pretreatment with t he hormone, tail tips cultured in T-4 showed significant increases in ubiquitin levels by 48 hr. Tail tips taken from tadpoles that had been immersed in T-4 for 6 days showed a parallel increase in ubiquitin le vels, demonstrating the same change in vivo. Treatment of cultured tai l tips with the protein kinase C inhibitor, H-7, blocks both regressio n and the rise in ubiquitin seen in tips treated with T-4 alone. Treat ment of cultured tips with T-4 and either cycloheximide or actinomycin D inhibits regression compared to T-4 alone; however, the rise in ubi quitin is only blocked by cycloheximide, suggesting that ubiquitin is being made from RNA that was synthesized during the pretreatment perio d or earlier. These results suggest that ubiquitin will serve as a goo d molecular marker of tissue regression in the T-4-treated tadpole tai l and that it will be productive to consider tissue regression during amphibian metamorphosis as a specific case of programmed cell death or apoptosis. (C) 1994 Academic Press, Inc.