FEEDBACK REGULATION OF POLYAMINE SYNTHESIS AND TRANSPORT

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The polyamines, spermidine and spermine, and their diamine precursor, putrescine, are found in all mammalian cells and are essential for cell growth and viability. Mammalian cells obtain the requisite polyamines by biosynthesis or, if available, by uptake from their environment. In normal cells, intracellular levels of the polyamines are carefully managed, whereas cancer cells and virally transformed cells frequently exhibit abnormal elevations of these compounds. Because of this correlation, there is intense current interest in the development of inhibitors of polyamine synthesis for use as anti-neoplastic, and even anti-carcinogenic, agents. Cancer cells also are noted to incorporate exogenous polyamines more readily than normal cells. Investigators are attempting to exploit this attribute to facilitate the selective incorporation of potentially cytotoxic polyamine analogs into tumor cells. Both these approaches have proven somewhat disappointing so far, in large part because we do not understand the normal control mechanisms utilized by a cell in maintaining polyamine levels. For these reasons, the long-term goal of this project is to elucidate, and potentially to control, the complex of mechanisms whereby mammalian cells regulate the intracellular polyamines levels that are essential for normal physiology. Biochemical and molecular techniques, as well as several hamster and rat cell lines, will be used to investigate three different aspects of polyamine maintenance: a) synthesis of the critical biosynthetic enzyme, ornithine decarboxylase; b) turnover of this enzyme; and c) active polyamine transport. All three processes are sensitively regulated by alterations in polyamine levels. Recently, a variant rat cell line was discovered that is simultaneously deficient in all of these polyamine-feedback responses. This observation suggests that some aspect of an essential spermidine-sensitive reaction is common to these three different cell processes, and this shared intermediate or reaction is deficient in the variant cell line. In the proposed investigation, the mechanism of polyamine feedback that is deficient in the variant cell line will be determined, thereby establishing a common link between the feedback control of ornithine decarboxylase stability, of which something is known, and those of ornithine decarboxylase synthesis and polyamine transport, about which very little is known. This examination of three different control points in the maintenance of cellular polyamine levels will yield a comprehensive understanding of polyamine homeostasis in normal and cancerous mammalian cells, and will guide future efforts to alter abnormal growth by manipulating cellular polyamines.