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

Background: Antizyme is a critical regulator of cellular polyamine levels d ue to its effect on polyamine transport and its ability to target ornithine decarboxylase for degradation. Antizyme expression is autoregulatory, thro ugh dependence on an unusual +1 translational frameshift mechanism that res ponds to polyamine levels. Results: HEK293 cells were depleted of polyamines by treatment with an orni thine decarboxylase inhibitor, difluoromethylornithine (DFMO), and grown in the presence or absence of exogenous polyamines prior to the analysis of r ibosomal frameshifting levels. Results obtained using an optimized dual luc iferase assay system reveal a 10-fold dynamic range of frameshifting, which correlates positively with polyamine addition. Polyamine addition to cells , which have not been pretreated with DFMO, also resulted in an increase in antizyme frameshifting but to a lesser degree (1.3 to 1.5-fold). In additi on, 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 u tility of a quantifiable cell-based assay for the analysis of polyamines or their analogues on translational frameshifting.