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Knockdown of ornithine decarboxylase antizyme 1 causes loss of uptake regulation leading to increased N (1), N (11)-bis(ethyl)norspermine (BENSpm) accumulation and toxicity in NCI H157 lung cancer cells

Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Bunting-Blaustein Cancer Research Building 1, 1650 Orleans Street, Baltimore, MD 21231, USA.
Amino Acids (Impact Factor: 3.29). 08/2011; 42(2-3):529-38. DOI: 10.1007/s00726-011-1030-z
Source: PubMed

ABSTRACT

Ornithine decarboxylase antizyme 1 (AZ1) is a major regulatory protein responsible for the regulation and degradation of ornithine decarboxylase (ODC). To better understand the role of AZ1 in polyamine metabolism and in modulating the response to anticancer polyamine analogues, a small interfering RNA strategy was used to create a series of stable clones in human H157 non-small cell lung cancer cells that expressed less than 5-10% of basal AZ1 levels. Antizyme 1 knockdown clones accumulated greater amounts of the polyamine analogue N (1),N (11)-bis(ethyl)norspermine (BENSpm) and were more sensitive to analogue treatment. The possibility of a loss of polyamine uptake regulation in the knockdown clones was confirmed by polyamine uptake analysis. These results are consistent with the hypothesis that AZ1 knockdown leads to dysregulation of polyamine uptake, resulting in increased analogue accumulation and toxicity. Importantly, there appears to be little difference between AZ1 knockdown cells and cells with normal levels of AZ1 with respect to ODC regulation, suggesting that another regulatory protein, potentially AZ2, compensates for the loss of AZ1. The results of these studies are important for the understanding of both the regulation of polyamine homeostasis and in understanding the factors that regulate tumor cell sensitivity to the anti-tumor polyamine analogues.

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    ABSTRACT: Polyamines are essential biogenic poly-cations with important roles in many cellular processes, including translation and DNA replication. High levels of polyamines have been linked to cancer. Increased levels of polyamines, however, have also been shown to promote longevity. Cellular regulation of polyamines involves a variety of complex mechanisms that regulate uptake and excretion as well as synthesis and catabolism of polyamines. A key enzyme in the biosynthesis of polyamines is ornithine decarboxylase (ODC). The activity and stability of this homodimeric protein is controlled by ODC antizyme (OAZ). OAZ binds ODC monomers and thereby targets them for ubiquitin-independent degradation by the proteasome. OAZ is encoded by an interrupted open reading frame. As a consequence, synthesis of the functional protein is slowed and only occurs when a ribosomal frameshift (RFS) event takes place. High polyamine concentrations lead to an increased efficiency of translation of OAZ mRNA. Our studies have revealed that nascent OAZ polypeptide causes a translational arrest of ribosomes when polyamine concentration is low. At higher concentrations, binding of polyamines to nascent OAZ prevents stalling of the ribosomes on OAZ mRNA and thereby promotes synthesis of full-length ODC antizyme. Co-translational sensing of polyamines thereby contributes to a homeostatic regulation of polyamine biosynthesis.
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