Paula Diegelman

Identification and characterization of a novel flavin-containing spermine oxidase of mammalian cell origin.

During polyamine catabolism, spermine and spermidine are first acetylated by spermidine/spermine N(1)-acetyltransferase (SSAT) and subsequently oxidized by polyamine oxidase (PAO) to produce spermidine and putrescine, respectively. In attempting to clone the PAO involved in this back-conversion pathway, we encountered an oxidase that preferentially cleaves spermine in the absence of prior acetylation by SSAT. A BLAST search using maize PAO sequences identified homologous mammalian cDNAs derived from human hepatoma and mouse mammary carcinoma: the encoded proteins differed by 20 amino acids. When either cDNA was transiently transfected into HEK-293 cells, intracellular spermine pools decreased by 75% while spermidine and N (1)-acetylspermidine pools increased, suggesting that spermine was selectively and directly oxidized by the enzyme. Substrate specificity using lysates of oxidase-transfected HEK-293 cells revealed that the newly identified oxidase strongly favoured spermine over N (1)-acetylspermine and that it failed to act on N (1)-acetylspermidine, spermidine or the preferred PAO substrate, N (1), N (12)-diacetylspermine. The PAO inhibitor, MDL-72,527, only partially blocked oxidation of spermine while a previously reported PAO substrate, N (1)-( n -octanesulphonyl)spermine, potently inhibited the reaction. Overall, the data indicate that the enzyme represents a novel mammalian oxidase which, on the basis of substrate specificity, we have designated spermine oxidase in order to distinguish it from the PAO involved in polyamine back-conversion. The identification of an enzyme capable of directly oxidizing spermine to spermidine has important implications for understanding polyamine homoeostasis and for interpreting metabolic and cellular responses to clinically relevant polyamine analogues and inhibitors.

Genomic identification and biochemical characterization of the mammalian polyamine oxidase involved in polyamine back-conversion.

In the polyamine back-conversion pathway, spermine and spermidine are first acetylated by spermidine/spermine N1 -acetyltransferase (SSAT) and then oxidized by polyamine oxidase (PAO) to produce spermidine and putrescine respectively. Although PAO was first purified more than two decades ago, the protein has not yet been linked to genomic sequences. In the present study, we apply a BLAST search strategy to identify novel oxidase sequences located on human chromosome 10 and mouse chromosome 7. Homologous mammalian cDNAs derived from human brain and mouse mammary tumour were deduced to encode proteins of approx. 55 kDa having 82% sequence identity. When either cDNA was transiently transfected into HEK-293 cells, intracellular spermine pools decreased by approx. 30%, whereas spermidine increased 2-4-fold. Lysates of human PAO cDNA-transfected HEK-293 cells, but not vector-transfected cells, rapidly oxidized N1-acetylspermine to spermidine. Substrate specificity determinations with the lysate assay revealed a preference ranking of N1-acetylspermine= N1-acetylspermidine> N1,N12-diacetylspermine>>spermine; spermidine was not acted upon. This ranking is identical to that reported for purified PAO and distinctly different from the recently identified spermine oxidase (SMO), which prefers spermine over N1-acetylspermine. Monoethyl- and diethylspermine analogues also served as substrates for PAO, and were internally cleaved adjacent to a secondary amine. We deduce that the present oxidase sequences are those of the FAD-dependent PAO involved in the polyamine back-conversion pathway. In Northern blot analysis, PAO mRNA was much less abundant in HEK-293 cells than SMO or SSAT mRNA, and all three were differentially induced in a similar manner by selected polyamine analogues. The identification of PAO sequences, together with the recently identified SMO sequences, provides new opportunities for understanding the dynamics of polyamine homoeostasis and for interpreting metabolic and cellular responses to clinically-relevant polyamine analogues and inhibitors.

Genetically Altered Expression of Spermidine/Spermine N1-Acetyltransferase Affects Fat Metabolism in Mice via Acetyl-CoA

The acetylating enzyme, spermidine/spermine N1-acetyltransferase, participates in polyamine homeostasis by regulating polyamine export and catabolism. Previously, we reported that overexpression of the enzyme in cultured tumor cells and mice activates metabolic flux through the polyamine pathway and depletes the N1-acetyltransferase coenzyme and fatty acid precursor, acetyl-CoA. Here, we investigate this possibility in spermidine/spermine N1-acetyltransferase transgenic mice in which the enzyme is systemically overexpressed and in spermidine/spermine N1-acetyltransferase knock-out mice. Tissues of the former were characterized by increased N1-acetyltransferase activity, a marked elevation in tissue and urinary acetylated polyamines, a compensatory increase in polyamine biosynthetic enzyme activity, and an increase in metabolic flux through the polyamine pathway. These polyamine effects were accompanied by a decrease in white adipose acetyl- and malonyl-CoA pools, a major (20-fold) increase in glucose and palmitate oxidation, and a distinctly lean phenotype. In SSAT-ko mice, the opposite relationship between polyamine and fat metabolism was observed. In the absence of N1-acetylation of polyamines, there was a shift in urinary and tissue polyamines indicative of a decline in metabolic flux. This was accompanied by an increase in white adipose acetyl- and malonyl-CoA pools, a decrease in adipose palmitate and glucose oxidation, and an accumulation of body fat. The latter was further exaggerated under a high fat diet, where knock-out mice gained twice as much weight as wild-type mice. A model is proposed whereby the expression status of spermidine/spermine N1-acetyltransferase alters body fat accumulation by metabolically modulating tissue acetyl- and malonyl-CoA levels, thereby influencing fatty acid biosynthesis and oxidation.

Potent Modulation of Intestinal Tumorigenesis in Apcmin/+ Mice by the Polyamine Catabolic Enzyme Spermidine/Spermine N1-acetyltransferase

Intracellular polyamine pools are homeostatically maintained by processes involving biosynthesis, catabolism, and transport. Although most polyamine-based anticancer strategies target biosynthesis, we recently showed that activation of polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase-1 (SSAT) suppresses tumor outgrowth in a mouse prostate cancer model. Herein, we examined the effects of differential SSAT expression on intestinal tumorigenesis in the Apc(Min/+) (MIN) mouse. When MIN mice were crossed with SSAT-overproducing transgenic mice, they developed 3- and 6-fold more adenomas in the small intestine and colon, respectively, than normal MIN mice. Despite accumulation of the SSAT product, N(1)-acetylspermidine, spermidine and spermine pools were only slightly decreased due to a huge compensatory increase in polyamine biosynthetic enzyme activities that gave rise to enhanced metabolic flux. When MIN mice were crossed with SSAT knock-out mice, they developed 75% fewer adenomas in the small intestine, suggesting that under basal conditions, SSAT contributes significantly to the MIN phenotype. Despite the loss in catabolic capability, tumor spermidine and spermine pools failed to increase significantly due to a compensatory decrease in biosynthetic enzyme activity giving rise to a reduced metabolic flux. Loss of heterozygosity at the Apc locus was observed in tumors from both SSAT-transgenic and -deficient MIN mice, indicating that loss of heterozygosity remained the predominant oncogenic mechanism. Based on these data, we propose a model in which SSAT expression alters flux through the polyamine pathway giving rise to metabolic events that promote tumorigenesis. The finding that deletion of SSAT reduces tumorigenesis suggests that small-molecule inhibition of the enzyme may represent a nontoxic prevention and/or treatment strategy for gastrointestinal cancers.

Polyamine Acetylation Modulates Polyamine Metabolic Flux, a Prelude to Broader Metabolic Consequences

Recent studies suggest that overexpression of the polyamine-acetylating enzyme spermidine/spermine N1-acetyltransferase (SSAT) significantly increases metabolic flux through the polyamine pathway. The concept derives from the observation that SSAT-induced acetylation of polyamines gives rise to a compensatory increase in biosynthesis and presumably to increased flow through the pathway. Despite the strength of this deduction, the existence of heightened polyamine flux has not yet been experimentally demonstrated. Here, we use the artificial polyamine precursor 4-fluoro-ornithine to measure polyamine flux by tracking fluorine unit permeation of polyamine pools in human prostate carcinoma LNCaP cells. Conditional overexpression of SSAT was accompanied by a massive increase in intracellular and extracellular acetylated spermidine and by a 6-20-fold increase in biosynthetic enzyme activities. In the presence of 300 μm 4-fluoro-ornithine, SSAT overexpression led to the sequential appearance of fluorinated putrescine, spermidine, acetylated spermidine, and spermine. As fluorinated polyamines increased, endogenous polyamines decreased, so that the total polyamine pool size remained relatively constant. At 24 h, 56% of the spermine pool in the induced SSAT cells was fluorine-labeled compared with only 12% in uninduced cells. Thus, SSAT induction increased metabolic flux by ∼5-fold. Flux could be interrupted by inhibition of polyamine biosynthesis but not by inhibition of polyamine oxidation. Overall, the findings are consistent with a paradigm whereby flux is initiated by SSAT acetylation of spermine and particularly spermidine followed by a marked increase in key biosynthetic enzymes. The latter sustains the flux cycle by providing a constant supply of polyamines for subsequent acetylation by SSAT. The broader metabolic implications of this futile metabolic cycling are discussed in detail.

Polyamine biosynthesis impacts cellular folate requirements necessary to maintain S-adenosylmethionine and nucleotide pools

Folate (vitamin B9) is utilized for synthesis of both S‐adenosymiemionine (AdoMet) and deoxythymidine monophosphate (dTMP), which are required for methylation reactions and DNA synthesis, respectively. Folate depletion leads to an imbalance in both AdoMet and nucleotide pools, causing epigenetic and genetic damage capable of initiating tumorigenesis. Polyamine biosynthesis also utilizes AdoMet, but polyamine pools are not reduced under a regimen of folate depletion. We hypothesized that high polyamine biosynthesis, due to the high demand on AdoMet pools, might be a factor in determining sensitivity to folate depletion. We found a significant correlation (P< 0.001) between polyamine biosynthesis and the amount of folate required to sustain cell line proliferation. We manipulated polyamine biosynthesis by genetic and pharmacological intervention and mechanistically demonstrated that we could thereby alter AdoMet pools and increase or decrease demand on folate availability needed to sustain cellular proliferation. Furthermore, growing a panel of cell lines with 100 nM folate led to imbalanced nucleotide and AdoMet pools only in cells with endogenously high polyamine biosynthesis. These data demonstrate that polyamine biosynthesis is a critical factor in determining sensitivity to folate depletion and may be particularly important in the prostate, where biosynthesis of polyamines is characteristically high due to its secretory function.—Bistulfi, G., Diegelman, P., Foster, B. A., Kramer, D. L., Porter, C. W., Smiraglia, D. J. Polyamine biosynthesis impacts cellular folate requirements necessary to maintain S‐adenosylmethionine and nucleotide pools. FASEB J. 23, 2888–2897 (2009). www.fasebj.org

Genomic identification and biochemical characterization of a second spermidine/spermine N1-acetyltransferase.

In the polyamine back-conversion pathway, spermine and spermidine are first acetylated by spermidine/spermine N(1) -acetyl-transferase (SSAT-1) and then oxidized by polyamine oxidase to produce spermidine and putrescine respectively. Herein we apply homology-search methods to identify novel sequences belonging to a second SSAT, SSAT-2, with a chromosomal location at 17p13.1, which is distinct from SSAT-1 at Xp22. Human SSAT-2 cDNA derived from small-cell lung carcinoma was deduced to encode a 170-amino-acid protein having 46% sequence identity and 64% sequence similarity with SSAT-1. When transiently transfected into HEK-293 cells, SSAT-1 decreased spermidine and spermine pools by approximately 30%, while, at the same time, significantly increasing putrescine, N (1)-acetylspermidine, N (1)-acetylspermine and N (1), N (12)-diacetylspermine pools. By contrast, transfected SSAT-2 had no effect on intracellular polyamine or acetylated polyamine pools. When enzyme activity was assayed on enzyme extracts from transfected cells, both SSAT-1 and SSAT-2 demonstrated much higher acetylating activity than vector-transfected cells. The data suggest that, in intact cells, SSAT-2 may be compartmentalized or it may be inefficient at low intracellular polyamine concentrations. By substituting candidate substrates in the enzyme assay, we determined that SSAT-1 shows the substrate preference norspermidine=spermidine>>spermine> N (1)-acetylspermine>putrescine, whereas SSAT-2 shows the preference norspermidine>spermidine=spermine>> N (1)-acetylspermine=putrescine. Analysis of mRNA levels in cell lines and ESTs (expressed sequence tags) from various tissues by digiNorthern (a web-based tool for virtually displaying expression profiles of query genes based on EST sequences) indicated that SSAT-1 tends to be more widely and highly expressed than SSAT-2. While SSAT-1 mRNA was inducible by polyamine analogues in a variety of cell lines, SSAT-2 was not. The existence of an active, but possibly sequestered, SSAT-2 enzyme suggests that, under certain conditions, it may be recruited into basal or perturbed polyamine metabolism.

Use of 4-fluoro-l-ornithine to monitor metabolic flux through the polyamine biosynthetic pathway

The mechanistic effectiveness of various polyamine analogs and enzyme inhibitors is typically determined by their ability to deplete intracellular polyamine pools. In this study, we describe an assay that may prove useful in augmenting this relatively static assessment of drug action. The assay relies upon the substitution of 4-fluoro-L-ornithine (Fl-Orn) for ornithine as a polyamine precursor to provide a means to measure metabolic flux through polyamine pools. At concentrations up to 500 microM, the analog did not inhibit the growth of L1210 murine leukemia cells during incubations of up to 72 hr. Using HPLC, the analog was processed metabolically over time to what was deduced to be 2-fluoroputrescine, 6-fluorospermidine and 6-fluorospermine. The relative proportion of fluorinated polyamine analog to the natural polyamine increased with time and Fl-Orn concentration. The sum of the two was found to be nearly identical to the respective polyamine pool of control cells exposed instead to 500 microM ornithine. This indicates that Fl-Orn was recognized and utilized as a precursor at a rate very similar to that of ornithine itself. Using L1210 cells at different stages of cell growth, it was determined that the metabolic flux through the pools, as indicated by the rate of appearance of individual fluorinated polyamine species, reflected the proliferation status of the cells--non-growing cells failed to incorporate the analog. Likewise, in cell types with varying polyamine pool profiles, such as polyamine enzyme overproducers or those with constitutively different spermidine of spermine ratios, the incorporation of the fluorinated analogs into pools was found to be proportional to the size to the natural polyamine pool. In cells treated with inhibitors of S-adenosylmethionine decarboxylase, Fl-Orn incorporation indicated a total blockade of polyamine synthesis at that enzyme site. Overall, the Fl-Orn assay has demonstrated that polyamine pool profiles generally reflect the rate of flux through the pathway in proliferating cells, suggesting that most intracellular polyamines are freely exchangeable with those undergoing metabolic flux.

Abstract 4257: Pharmacokinetics (PK) and tissue penetration of the novel VEGFR-3/FAK inhibitor, Chloropyramine

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Purpose: Previous reports demonstrate that chloropyramine has anti-tumor efficacy as a single agent, and in combination with cytotoxic agents like doxorubicin through modulation of the FAK-VEGFR-3 pathway. Since limited information is available about the pharmacokinetics of chloropyramine, we investigated the pharmacokinetics and tissue distribution of the drug following a single intraperitoneal (IP) injection. Methods: Female CD-1 mice received a single 50mg/kg dose of chloropyramine; plasma and tissue samples were collected at serial timepoints of 0.5, 1.0, 2.0, 4.0, and 6.0 hours. The tissues included brain, heart, spleen, liver, lung, muscle, and sternum. Each timepoint contained three mice. Chloropyramine concentrations were determined in both plasma and tissues, using a validated LC-MS/MS. Assay was validated with an LLOQ of 0.25ng/ml with the calibration curve ranging from 0.25-100ng/ml (3.42-12.4%CV). Noncompartmental PK analysis was performed using WinNonlin (Pharsight, Version 5.3). Pharmacokinetic parameters including: Maximal concentration (Cmax), terminal elimination half life (T1/2), area under the curve (AUC0-6hr), apparent clearance (Cl/F), apparent volume of distribution (Vd/F), and partition coefficients (Kp). Results: Following IP injection, chloropyramine followed either a mono- or bi-exponential decay in both plasma and tissues. Chloropyramine is cleared from plasma and all tissues within 12hr; thus chloropyramine does not exhibit a great potential for accumulation. Cmax was achieved within 1.0 hour for plasma and all tissues. Chloropyramine distributes well into various tissues such as the spleen, liver, lung, kidney, brain, and heart. The highest level of chloropyramine was achieved in lung tissue with a mean (sd) Cmax concentrations of 41.6 (11.6) μg/ml, respectively. In contrast, the mean Cmax in plasma was approximately 0.5 μg/ml; much less compared to tissues. The mean T1/2 was 0.83 (0.19) hr. The lungs exhibited the highest total tissue penetration of chloropyramine, representing a mean AUC of 51.3 μg-hr/g. The least total exposure was in muscle, with a mean AUC of 8.5 μg-hr/g. The apparent CL/F from plasma was 28.5 L/hr/kg while it was 0.9 L/hr/kg for lung tissue. Conclusions: These results illustrate the wide tissue distribution of chloropyramine, despite of the short plasma half-life. In addition, these results are of great importance because it provides supportive evidence of the tissue levels that will lead to anti-tumor efficacy through inhibition of FAK and VEGFR-3. Thus, evaluation of plasma and tissue concentrations of the drug elucidates the pharmacokinetics of this compound in an effort to identify target tissues into which it has a high degree of penetration to maximize antitumor efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4257. doi:10.1158/1538-7445.AM2011-4257