Coupling of the polyamine and iron metabolism pathways in the regulation of proliferation: Mechanistic links to alterations in key polyamine biosynthetic and catabolic enzymes

Many biological processes result from the coupling of metabolic pathways. Considering this, proliferation depends on adequate iron and polyamines, and although iron-depletion impairs proliferation, the metabolic link between iron and polyamine metabolism has never been thoroughly investigated. This is important to decipher, as many disease states demonstrate co-dysregulation of iron and polyamine metabolism. Herein, for the first time, we demonstrate that cellular iron levels robustly regulate 13 polyamine pathway proteins. Seven of these were regulated in a conserved manner by iron-depletion across different cell-types, with four proteins being down-regulated (i.e., acireductone dioxygenase 1 [ADI1], methionine adenosyltransferase 2α [MAT2α], Antizyme and polyamine oxidase [PAOX]) and three proteins being up-regulated (i.e., S-adenosyl methionine decarboxylase [AMD1], Antizyme inhibitor 1 [AZIN1] and spermidine/spermine-N¹-acetyltransferase 1 [SAT1]). Depletion of iron also markedly decreased polyamine pools (i.e., spermidine and/or spermine, but not putrescine). Accordingly, iron-depletion also decreased S-adenosylmethionine that is essential for spermidine/spermine biosynthesis. Iron-depletion additionally reduced ³H-spermidine uptake in direct agreement with the lowered levels of the polyamine importer, SLC22A16. Regarding mechanism, the “reprogramming” of polyamine metabolism by iron-depletion is consistent with the down-regulation of ADI1 and MAT2α, and the up-regulation of SAT1. Moreover, changes in ADI1 (biosynthetic) and SAT1 (catabolic) partially depended on the iron-regulated changes in c-Myc and/or p53. The ability of iron chelators to inhibit proliferation was rescuable by putrescine and spermidine, and under some conditions by spermine. Collectively, iron and polyamine metabolism are intimately coupled, which has significant ramifications for understanding the integrated role of iron and polyamine metabolism in proliferation.     

Significant induction of spermidine/spermine N1-acetyltransferase without cytotoxicity by the growth-supporting polyamine analogue 1,12-dimethylspermine

The superinduction of the polyamine catabolic enzyme spermidine/spermine N¹-acetyltransferase (SSAT) has been implicated in the cell type-specific cytotoxic activity of some polyamine analogues. We now report that one polyamine analogue, 1, 12-dimethylspermine (DMSpm), produces a large induction of SSAT with no significant effects on growth in the human large cell lung carcinoma line, NCI H157. This cell line has been demonstrated to respond to other analogues with SSAT superinduction and cell death. Treatment of the lung cancer cell line with DMSpm produces a rapid increase in SSAT activity and a near complete depletion of the natural polyamines. Additionally, DMSpm supports cell growth in cells which have been depleted of their natural polyamines by the ornithine decarboxylase inhibitor, 2-difluoromethylornithine. The current results suggest that significant induction of SSAT can occur in the absence of cytotoxicity when the inducing polyamine analogue can support growth and that increased SSAT activity alone is not sufficient for cytotoxicity to occur. © 1995 Wiley-Liss Inc.     

Human lung tumor sensitivity to difluoromethylornithine as related to ornithine decarboxylase messenger RNA levels

The differential response to polyamine depletion has been studied in two types of human lung tumor cells. Small cell lung carcinoma cells die following polyamine depletion by difluoromethylornithine treatment while non-small cell lines demonstrate a typical cytostatic response. We now report that a small cell line, NCI H82, has a lower apparent capacity for polyamine biosynthesis than does a representative non-small cell, NCI H157. In subconfluent cultures, the ornithine decarboxylase activity is 25 times lower in the small cell than the non-small cell and by comparison, the polyamines in the small cell line are markedly reduced. Most significantly, levels of mRNA coding for ornithine decarboxylase are approximately 100-fold lower in the small cell than the non-small cell line, and this difference does not appear to be a result of gene rearrangement. These results suggest that differential sensitivity to polyamine depletion is related to different steady-state levels of ornithine decarboxylase mRNA.     

Comparison of the effects of treatment with the polyamine analogue N1,N8 bis(ethyl)spermidine (BESpd) or difluoromethylornithine (DFMO) on the Topoisomerase II mediated formation of 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA) induced cleavable complex in the human lung carcinoma line NCI H157

The positively charged polyamines putrescine, spermidine, and spermine are thought to be important in the maintenance of chromosomal structure. Polyamine depletion by the ornithine decarboxylase inhibitor, 2-difluoromethyl-ornithine (DFMO) is known to alter the effect of several DNA active agents, presumably resulting from the altered conformation of the polyamine depleted DNA. Here we compare the polyamine depletion effects of DFMO and the spermidine analogue N1,N8 bis(ethyl)spermidine (BESpd) on the formation of Topoisomerase II mediated, 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA) induced cleavable complex formation in human large cell undifferentiated lung carcinoma NCI H157 cells. This human cell line responds in the normal cytostatic manner to DFMO, whereas it responds in an unusual cytotoxic manner to treatment with BESpd. Here we report that neither DFMO nor BESpd alone affects the formation of cleavable complex. However, both compounds significantly enhance the m-AMSA induced formation of cleavable complex, each by approximately 1.6 fold. These results indicate that both DFMO and BESpd lead to a similar depletion of nuclear polyamines. Additionally, although BESpd closely resembles the natural polyamine spermidine, it appears that it cannot substitute for Spd at the level of DNA.     

RepSeq – A database of amino acid repeats present in lower eukaryotic pathogens

Background  

Amino acid repeat-containing proteins have a broad range of functions and their identification is of relevance to many experimental biologists. In human-infective protozoan parasites (such as the Kinetoplastid and Plasmodium species), they are implicated in immune evasion and have been shown to influence virulence and pathogenicity. RepSeq is a new database of amino acid repeat-containing proteins found in lower eukaryotic pathogens. The RepSeq database is accessed via a web-based application which also provides links to related online tools and databases for further analyses.