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 with a fluoroimmunoassay to determine the levels of ubiquitin-a protein marker of programmed cell death in other systems-during the tissue regression induced by thyroxine (T-4). After a 3-day pretreatment with the 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 levels, demonstrating the same change in vivo. Treatment of cultured tail tips with the protein kinase C inhibitor, H-7, blocks both regression and the rise in ubiquitin seen in tips treated with T-4 alone. Treatment of cultured tips with T-4 and either cycloheximide or actinomycin D inhibits regression compared to T-4 alone; however, the rise in ubiquitin is only blocked by cycloheximide, suggesting that ubiquitin is being made from RNA that was synthesized during the pretreatment period or earlier. These results suggest that ubiquitin will serve as a good molecular marker of tissue regression in the T-4-treated tadpole tail 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.