Cell culture analysis of the regulatory frameshift event required for the expression of mammalian antizymes

• Published in: Genes to cells : devoted to molecular & cellular mechanisms Vol. 6; no. 11; pp. 931 - 941
• Main Authors: Howard, Michael T., Shirts, Brian H., Zhou, Jiadong, Carlson, C. Lance, Matsufuji, Senya, Gesteland, Raymond F., Weeks, Reitha S., Atkins, John F.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.11.2001
• Subjects: Animals; antizyme; Base Sequence; Biogenic Polyamines - metabolism; Cell Culture Techniques; Cell Line; difluoromethylornithine; DNA; Eflornithine - pharmacology; Frameshift Mutation; Gene Expression Regulation, Enzymologic - genetics; Humans; Molecular Sequence Data; Proteins - antagonists & inhibitors; Proteins - genetics
• ISSN: 1356-9597; 1365-2443
• DOI: 10.1046/j.1365-2443.2001.00477.x

Background Antizyme is a critical regulator of cellular polyamine levels due to its effect on polyamine transport and its ability to target ornithine decarboxylase for degradation. Antizyme expression is autoregulatory, through dependence on an unusual +1 translational frameshift mechanism that responds to polyamine levels. Results HEK293 cells were depleted of polyamines by treatment with an ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), and grown in the presence or absence of exogenous polyamines prior to the analysis of ribosomal frameshifting levels. Results obtained using an optimized dual luciferase assay system reveal a 10‐fold dynamic range of frameshifting, which correlates positively with polyamine addition. Polyamine addition to cells, which have not been pre‐treated with DFMO, also resulted in an increase in antizyme frameshifting but to a lesser degree (1.3 to 1.5‐fold). In addition, the constructs with the 3′ deletion were more responsive to stimulation by polyamine addition than those with the 5′ deletion. Conclusions The observed regulation of antizyme frameshifting demonstrates the efficiency of a polyamine homeostatic mechanism, and illustrates the utility of a quantifiable cell‐based assay for the analysis of polyamines or their analogues on translational frameshifting.