Knockdown of ornithine decarboxylase antizyme 1 causes loss of uptake regulation leading to increased N1, N11-bis(ethyl)norspermine (BENSpm) accumulation and toxicity in NCI H157 lung cancer cells.

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.

Properties of recombinant human N1-acetylpolyamine oxidase (hPAO): potential role in determining drug sensitivity.

The recent cloning of the mammalian gene coding for N(1)-acetylpolyamine oxidase (PAO) provides the opportunity to directly examine the role of human PAO (hPAO) in polyamine homeostasis as well as its potential role in determining cellular response to antitumor polyamine analogues. To facilitate the study of this enzyme, the production, purification, and characterization of the recombinant hPAO is reported. hPAO oxidizes N(1)-acetylspermidine (K(m)=2.1 microM, K(cat)=15.0 s(-1)) and has very high affinity for N(1)-acetylspermine (K(m)=0.85 microM, K(cat)=31.7 s(-1)). The recombinant hPAO does not efficiently oxidize spermine, thereby demonstrating a significant difference in substrate specificity from the previously described human spermine oxidase PAOh1/SMO. Importantly, hPAO demonstrates the ability to oxidize a subset of antitumor polyamine analogues, suggesting that this oxidase activity could have a significant effect on determining tumor sensitivity to these or similar agents. Transfection of A549 human lung cancer cells with an hPAO-expressing plasmid leads to a profound decrease in sensitivity to those analogues which act as substrates, confirming its potential to alter drug response. One similarity that hPAO shares with human PAOh1/SMO, is that certain oligoamine analogues are potent inhibitors of its oxidase activity. The results of these studies demonstrate how changes in polyamine catabolism may affect drug response.

A perspective of polyamine metabolism.

Polyamines are essential for the growth and function of normal cells. They interact with various macromolecules, both electrostatically and covalently and, as a consequence, have a variety of cellular effects. The complexity of polyamine metabolism and the multitude of compensatory mechanisms that are invoked to maintain polyamine homoeostasis argue that these amines are critical to cell survival. The regulation of polyamine content within cells occurs at several levels, including transcription and translation. In addition, novel features such as the +1 frameshift required for antizyme production and the rapid turnover of several of the enzymes involved in the pathway make the regulation of polyamine metabolism a fascinating subject. The link between polyamine content and human disease is unequivocal, and significant success has been obtained in the treatment of a number of parasitic infections. Targeting the polyamine pathway as a means of treating cancer has met with limited success, although the development of drugs such as DFMO (alpha-difluoromethylornithine), a rationally designed anticancer agent, has revolutionized our understanding of polyamine function in cell growth and provided 'proof of concept' that influencing polyamine metabolism and content within tumour cells will prevent tumour growth. The more recent development of the polyamine analogues has been pivotal in advancing our understanding of the necessity to deplete all three polyamines to induce apoptosis in tumour cells. The current thinking is that the polyamine inhibitors/analogues may also be useful agents in the chemoprevention of cancer and, in this area, we may yet see a revival of DFMO. The future will be in adopting a functional genomics approach to identifying polyamine-regulated genes linked to either carcinogenesis or apoptosis.

Induction of apoptosis in human leukaemic cells by IPENSpm, a novel polyamine analogue and anti-metabolite.

Human promyelogenous leukaemic cells (HL-60) were treated with novel spermine analogue, ( S )- N (1)-(2-methyl-1-butyl)- N (11)-ethyl-4,8-diazaundecane (IPENSpm), and the effects on growth and intracellular polyamine metabolism were measured. IPENSpm was cytotoxic to these cells at concentrations greater than 2.5 microM. It induced apoptosis in a caspase-dependent manner and its toxicity profile was comparable with etoposide, a well-known anti-tumour agent and inducer of apoptosis. IPENSpm decreased intracellular polyamine content as a result of changes in ornithine decarboxylase activity and increases in spermidine/spermine N(1)-acetyltransferase and polyamine export. Analysis showed spermine and spermidine as the major intracellular polyamines, while putrescine and acetyl-polyamines were the main export compounds. IPENSpm used the polyamine transporter system for uptake and its accumulation in cells was prevented by polyamine transport inhibitors. IPENSpm can be classified as a polyamine anti-metabolite and it may be a promising new lead compound in terms of treatment of some human cancers.