CELL PROLIFERATION GROUP

The role of the polyamines in cell cycle control and programmed cell death

General background

The polyamines - putrescine, spermidine, and spermine - constitute a group of cell components that are important in the regulation of cell proliferation and cell differentiation. There is also evidence suggesting a role for polyamines in programmed cell death. Although their exact functions have not yet been identified, it is clear that the polyamines play important roles in a number of cellular processes such as replication, transcription, and translation. Presumably these roles are exerted by specific interactions that can only be mediated by the cationic polyamines with their characteristic, unique, and flexible charge distributions (see figure below).

The importance of the polyamines in cell function is reflected in a strict regulatory control of their intracellular levels. Adequate cellular polyamine levels are achieved by a careful balance between biosynthesis, degradation, and uptake of the amines. Some of the regulatory mechanisms involved in maintaining a balance in the cellular polyamine pools are truly unique. The polyamine biosynthetic pathway consists of two highly-regulated enzymes, ornithine decarboxylase (1 in the figure below) and S-adenosylmethionine decarboxylase (2 in the figure below), and two constitutively expressed enzymes, spermidine synthase (3 in the figure below) and spermine synthase (4 in the figure below). The biological half-lifes of the two regulatory enzymes ornithine decarboxylase and S-adenosylmethionine decarboxylase (5-60 min) are among the shortest known for mammalian enzymes, allowing the cell to rapidly change the cellular polyamine levels.

The polyamine degradation pathway consists of the highly-regulated enzyme spermidine/spermine N1-acetyl transferase and the constitutively expressed polyamine oxidase. In addition to regulation of polyamine levels by biosynthesis and degradation, cells are equipped with an efficient transport system for utilization of exogenously derived polyamines.

The biosynthesis of polyamines is increased by a great variety of physiological growth stimuli, and polyamine deficiency results in an arrest of cell proliferation, which can be reversed by supplementation with external polyamines. Polyamine deficiency may be achieved by treating cells with specific inhibitors of the polyamine biosynthetic enzymes. In view of the fact that constitutive overproduction of ornithine decarboxylase has been observed in many types of cancer cells, the ornithine decarboxylase gene appears to be of central importance in the regulation of cell growth. When the ornithine decarboxylase gene is transfected into cells and overexpressed, the cells go through malignant transformation.

Most studies concerning the regulation of polyamine biosynthesis and the function of the polyamines have been performed with asynchronous cell systems. Thus, results obtained are mean values depending on the cell cycle phase distributions of the cells and the variations of the parameter studied during the cell cycle. To overcome this technical problem, we have focused on using cell populations that progress synchronously and unperturbed through the cell cycle. In Chinese hamster ovary cells progressing synchronously through the cell cycle, we found that polyamine biosynthesis was activated at the G1 to S and G2 to M transitions. In addition, we found that ornithine decarboxylase and S-adenosylmethionine decarboxylase are regulated at both transcriptional and translational levels during the cell cycle.

Further work is needed, before we clearly understand how polyamine biosynthesis is regulated during the cell cycle. Such work would provide a background frame helping us to understand the role of the polyamines in cell cycle regulation (which is already known to be dependent on oncogenes, tumor suppressor genes, and cyclins with their associated kinases).

The fact that polyamine biosynthesis was activated at the G1 to S transition infers a role for the polyamines in DNA replication. Our earlier results have shown that polyamine depletion affected DNA replication negatively, presumably by reducing the rate of DNA elongation. Recently we presented evidence that polyamine biosynthesis inhibition affects DNA replication in Chinese hamster ovary cells within one cell cycle after seeding them in the presence of polyamine synthesis inhibitors (either ornithine decarboxylase or S-adenosylmethionine decarboxylase inhibitors). The G1 to S transition and the G2 phase progression were affected only after several cell cycles.

Staining of bromodeoxyuridine labelled cells with fluorescently-labelled antibodies against bromodeoxyuridine (which is a thymidine analog) has demonstrated that eukaryotic DNA replication is concentrated to discrete foci in the interphase nucleus. The number and the spatial distribution of replication domains vary in a specific pattern throughout the S phase. Using confocal laser scanning microscopy, we found that neither the number of replication domains nor their spatial distribution was affected in polyamine-depleted cells. This would have been the case if DNA replication initiation was affected. Further work is needed to establish whether the polyamines have a specific role in DNA replication.

DNA replication requires the presence of a number of proteins which have a direct or indirect role for the incorporation of the nucleotides into DNA as well as for the structural integrity of the chromatin. Topoisomerases are required to release various stresses in DNA that might affect the progress of the replication machinery. Using a decatenation assay, we have found that the topoisomerase II activity in cell extracts is the same in control and polyamine-depleted cells. However, when using the single cell gel electrophoresis assay to study the actual efficiency of topoisomerase II in situ, we found that the enzyme was less active in polyamine-depleted cells than in control cells. These results imply that there are conformational restraints on the DNA of polyamine-depleted cells, a notion suggested by other studies as well. It remains, however, an important future task to pinpoint the specific roles of polyamines for chromatin structure.

In most cell types, the biochemical characteristics of programmed cell death include the activation of endogenous calcium and magnesium dependent endonucleases, leading to fragmentation of the chromosomal DNA. Previous results from our and other laboratories indicate that DNA is destabilized in polyamine-depleted cells. This results in a greater vulnerability of DNA to nucleasal attack. Because DNA is known to become degraded during programmed cell death, we have begun to investigate if polyamine degradation plays an active part in the process. As one model system, we are using glucocorticoid-induced programmed cell death in mouse thymus.