We have recently proposed that intracellular polyamine pools are carefully maintained within a narrow range via a homeostatic complex comprised of three effector systems all of which are rapidly responsive, rate limiting for their specified activity, specific for polyamines and sensitively regulated by intracellular polyamine pools. The effectors include the enzymes ornithine decarboxylase (ODC) and S- adenosylmethionine decarboxylase (SAMDC) which control polyamine biosynthesis; a putative transporter protein which controls uptake; and the acetylating enzyme spermidine/spermine N1 acetyltransferase (SSAT) which has the multifunctional potential to control polyamine back- conversion, binding, export and catabolism. The concept has proven useful in interpreting cellular effects of polyamine analogs and inhibitors and in predicting the effectiveness of their use in anticancer strategies. Herein, we proposed to utilize recently developed probes and biological systems to study unresolved and therapeutically relevant aspects of each effector in an attempt to better understand its function and/or regulation and to better define its relationship to polyamine homeostasis.
The Specific Aims of the program are (1) photoaffinity labelling, and identification of plasma membrane protein(s) involved in polyamine binding and transport; (2) cloning the cDNA(s) for the transporter protein(s) for use in studying the molecular mechanisms underlying regulation of the polyamine transport by polyamines and growth stimuli; (3) to develop a panel of cell line variants which overproduce polyamine biosynthetic enzymes for use in further defining the regulatory interrelationships between the enzymes and other homeostatic effectors; (4) to study the significance of SSAT and SSAT induction in polyamine metabolism polyamine homeostasis and cellular physiology by transfecting and by blocking expression with SSAT mRNA-directed antisense oligonucleotide; (d) to develop 2-fluoroornithine as a HPLC marker for monitoring metabolic flux through the polyamine pools and to use this methodology to studying polyamine homeostasis. It is anticipated that these studies will provide novel information relevant to polyamine pool homeostasis; its validity as a concept; its implications to at least two polyamine-directed agents (with origins in this laboratory) which are now approaching clinical trial and indications by which it might be more effectively exploited and/or targeted in future drug design.
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