ESI-Q-TOF-MS determination of polyamines and related enzyme activity for elucidating cellular polyamine metabolism.

We developed a new procedure for the comprehensive analysis of metabolites and enzymes involved in polyamine metabolism pathways. The procedure utilizes stable isotope-labeled polyamines and directly and precisely determines labeled products from enzymatic reactions by ESI-Q-TOF-MS. The activity of different enzymes could be determined in essentially the same manner by suitably adjusting the reaction conditions for each individual enzyme. We applied the procedure to extracts of regenerating rat liver and analyzed the changes in polyamine-metabolizing enzymes and polyamine contents during recovery from partial hepatectomy. A general outline of polyamine metabolism and information of polyamine dynamics were obtained. This kind of comprehensive information would be valuable in unifying detailed but fragmentary information obtained through conventional analyses focusing on one or a few enzymes and on a limited aspect of polyamine metabolic pathway.

N1-Nonyl-1,4-diaminobutane ameliorates brain infarction size in photochemically induced thrombosis model mice.

Inhibitors for polyamine oxidizing enzymes, spermine oxidase (SMOX) and N1-acetylpolyamine oxidase (PAOX), were designed and evaluated for their effectiveness in a photochemically induced thrombosis (PIT) mouse model. N1-Nonyl-1,4-diaminobutane (C9-4) and N1-tridecyl-1,4-diaminobutane (C13-4) competitively inhibited the activity of PAOX and SMOX in a manner comparable to N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL72527), an irreversible inhibitor of both enzymes. The two compounds were then tested for their effects in the PIT model. Both intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) administration of C9-4 decreased infarct volumes significantly. By contrast, C13-4 reduced the volume of brain infarction by i.c.v. administration, but no reduction was observed after i.p. administration. C9-4 administered by i.p. injection reduced the volume of brain infarction significantly at doses of more than 3 mg/kg, and the dosage of 5 mg/kg or 10 mg/kg demonstrated the most potent effect and were more effective than equivalent doses of the other inhibitors such as MDL72527 and N-benzylhydroxylamine. I.P. injection of 5 mg/kg of C9-4 provided a therapeutic time window of longer than 12 h. This report demonstrates that C9-4 is a potent inhibitor of the polyamine oxidizing enzymes and is useful lead compound for candidate drugs with a long therapeutic time window, to be used in the treatment of ischemic stroke.

Development of irreversible inactivators of spermine oxidase and N1-acetylpolyamine oxidase.

Three functional groups (2-propenyl, 2-propynyl, and 2,3-butadienyl) were introduced onto one of the terminal amino groups of spermidine. Of the six compounds synthesized, N-(3-aminopropyl)-N'-2,3-butadienyl-1,4-butanediamine (N(8)-butadienyl Spd) and N-[3-(2,3-butadienylamino)propyl]-1,4-butanediamine (N(1)-butadienyl Spd) irreversibly inactivated human spermine oxidase (SMO) and N(1)-acetylpolyamine oxidase (APAO). Interestingly, N(8)-butadienyl Spd inactivated SMO far more potently than N,N'-di-2,3-butadienyl-1,4-butanediamine (MDL 72527).

Pentamines as substrate for human spermine oxidase.

Substrate activities of various linear polyamines to human spermine oxidase (hSMO) were investigated. The activities were evaluated by monitoring the amount of H2O2 released from sample polyamines by hSMO. H2O2 was measured by a HPLC method that analyzed fluorescent dimers derived from the oxidation of homovanillic acid in the presence of horseradish peroxidase. Six triamines were tested and were found not to be hSMO substrates. Of sixteen tetramines tested, spermine (Spm) was the most active substrate, followed by homospermine and N-butylated Spm. Pentamines showed a characteristic pattern of substrate activity. Of thirteen pentamines tested, 3343 showed higher substrate activity than Spm, and 4343 showed similar activity to Spm. The activities of the other pentamines were as follows: 3443, 4443, 4344, 3344, 4334, 4444, and 3334 (in decreasing order). Product amines released from these pentamines by hSMO were then analyzed by HPLC. Triamine was the only observed product, and the amount of triamine was nearly equivalent to that of released H2O2. A marked difference in the pH dependency curves between tetramines and pentamines suggested that hSMO favored reactions with a non-protonated secondary nitrogen at the cleavage site. The Km and Vmax values for Spm and 3343 at pH 7.0 and 9.0 were consistent with the higher substrate activity of 3343 compared to Spm, as well as with the concept of a non-protonated secondary nitrogen at the cleavage site being preferred, and 3343 was well degraded at a physiological pH by hSMO.

Increase of N1, N12-diacetylspermine in tissues from colorectal cancer and its liver metastasis.

PURPOSE: N (1),N (12)-Diacetylspermine (DiAcSpm) is a tumor marker featured by increase in the urine of patients with cancers, including early colorectal cancer, but where and how DiAcSpm is made remains unclear. We aimed to clarify whether colorectal cancer tissues produce increased amounts of DiAcSpm, and if they do, to examine whether tissue DiAcSpm level may serve as a criterion of tissue malignancy. METHODS: Tissue samples were obtained from 140 patients (13 low-grade intraepithelial neoplasia, 98 high-grade intraepithelial neoplasia and 29 colorectal cancer) treated for colorectal cancer and intraepithelial neoplasia at Tokyo Metropolitan Komagome Hospital between November 2007 and April 2011. The DiAcSpm level in cancer and adjacent normal tissue extracts was compared, and its relationship with clinical stages of the diseases was analyzed. RESULTS: DiAcSpm levels were higher in colorectal cancer tissue (p < 0.01, n = 12) and its liver metastasis (p < 0.05, n = 5) than in adjacent normal tissues. The tumor/normal ratio of tissue DiAcSpm content was examined for endoscopically obtained tumor and adjacent normal tissues from patients with intraepithelial neoplasia. The ratio was greater than 1.5 in 38 % (5/13) and 78 % (84/108) of low-grade intraepithelial neoplasia and high-grade intraepithelial neoplasia, respectively. CONCLUSIONS: Tissue DiAcSpm levels increase in the tissue of colorectal cancer and also in precancerous lesion, such as high-grade intraepithelial neoplasia. The increase is considered a sign that a tissue is acquiring malignant characteristics. It is likely that the DiAcSpm produced by cancer cells is responsible for the frequent increase in urinary DiAcSpm in early cancer patients.

A mass spectrometric method to determine activities of enzymes involved in polyamine catabolism.

An analytical method for the determination of three polyamines (putrescine, spermidine, and spermine) and five acetylpolyamines [N(1)-acetylspermidine (N(1)AcSpd), N(8)-acetylspermidine (N(8)AcSpd), N(1)-acetylspermine, N(1),N(8)-diacetylspermidine, and N(1),N(12)-diacetylspermine] involved in the polyamine catabolic pathway has been developed using a hybrid tandem mass spectrometer. Heptafluorobutyryl (HFB) derivatives of these compounds and respective internal standards labeled with stable isotopes were analyzed simultaneously by TOF MS, based on peak areas appearing at appropriate m/z values. The isomers, N(1)AcSpd and N(8)AcSpd were determined from their fragment ions, the acetylamidopropyl and acetylamidobutyl groups, respectively, using MS/MS with (13)C(2)-N(1)AcSpd and (13)C(2)-N(8)AcSpd which have the (13)C(2)-acetyl group as an internal standard. The TOF MS method was successfully applied to measure the activity of enzymes involved in polyamine catabolic pathways, namely N(1)-acetylpolyamine oxidase (APAO), spermine oxidase (SMO), and spermidine/spermine N(1)-acetyltransferase (SSAT). The following natural substrates and products labeled with stable isotopes considering the application to biological samples were identified; for APAO, [4,9,12-(15)N(3)]-N(1)-acetylspermine and [1,4,8-(15)N(3)]spermidine ((15)N(3)-Spd), respectively; for SMO, [1,4,8,12-(15)N(4)]spermine and (15)N(3)-Spd, respectively; and for SSAT, (15)N(3)-Spd and [1,4,8-(15)N(3)]-N(1)-acetylspermidine, respectively.

Crystal structures and enzymatic properties of a triamine/agmatine aminopropyltransferase from Thermus thermophilus.

To maintain functional conformations of DNA and RNA in high-temperature environments, an extremely thermophilic bacterium, Thermus thermophilus, employs a unique polyamine biosynthetic pathway and produces more than 16 types of polyamines. In the thermophile genome, only one spermidine synthase homolog (SpeE) was found and it was shown to be a key enzyme in the pathway. The catalytic assay of the purified enzyme revealed that it utilizes triamines (norspermidine and spermidine) and agmatine as acceptors in its aminopropyl transfer reaction; therefore, the enzyme was denoted as a triamine/agmatine aminopropyltransferase (TAAPT). We determined the crystal structures of the enzyme complexed with and without the aminopropyl group donor S-adenosylmethionine. Despite sequence and structural similarity with spermidine synthases from other organisms, a novel C-terminal ß-sheet and differences in the catalytic site were observed. The C-terminal module interacts with the gatekeeping loop and fixes the open conformation of the loop to recognize larger polyamine substrates such as agmatine and spermidine. Additional computational docking studies suggest that the structural differences of the catalytic site also contribute to recognition of the aminopropyl/aminobutyl or guanidium moiety of the substrates of TAAPT. These results explain in part the extraordinarily diverse polyamine spectrum found in T. thermophilus.

Quantification of intermediates of the methionine and polyamine metabolism by liquid chromatography-tandem mass spectrometry in cultured tumor cells and liver biopsies.

By means of liquid chromatography-tandem mass spectrometry we showed recently, that the chromosomal deletion or inactivation of the methylthioadenosine phosphorylase (MTAP) gene led to the accumulation of 5'-deoxy-5'-(methylthio)adenosine (MTA) in cancer cells. Here, we expanded the method to other key intermediates of the methionine and polyamine pathways to further elucidate the molecular consequences of a lack of MTAP activity. Employing multiple-reaction monitoring, limits of detection and lower limits of quantification in the range of 2.5-100 and 5.0-500 nM, respectively, were achieved according to the guidelines of the FDA, thus enabling the direct measurement of the metabolites in biological samples without prior enrichment and derivatization with an analytical repeatability of 1-3%. Relative standards deviations for quadruplicate 80% methanol extractions of metabolites from cultured tumor cells ranged from 1.1 to 25.5%, while the combined methodological and biological variability in metabolite concentrations in 10 liver biopsies was 11.8-51.4%. The method enabled the demonstration of changes in the concentration of intermediates of the methionine and polyamine metabolism other than MTA in hepatocellular carcinoma specimens lacking the enzyme MTAP compared to normal liver tissue.

Identification and determination of urinary acetylpolyamines in cancer patients by electrospray ionization and time-of-flight mass spectrometry.

A method for the quantification of acetylpolyamines, N(1),N(12)-diacetylspermine (DiAcSpm), monoacetylspermidine (AcSpd), and N(1),N(8)-diacetylspermidine (DiAcSpd), identifying each compound simultaneously, was developed with the goal of evaluating these acetylpolyamines as potential biomarkers of cancer. The method consists of prepurification of acetylpolyamines in urine with commercially available cartridges and derivatization with heptafluorobutyric (HFB) anhydride. HFB derivatives of acetylpolyamines were determined simultaneously using (15)N-labeled acetylpolyamines as internal standards by electrospray ionization and time-of-flight mass spectrometry (ESI-TOF MS). After the method was validated, the urinary acetylpolyamines of 38 cancer patients were quantified with this method. A comparison of the concentrations of DiAcSpm with those measured by a colloidal gold aggregation method demonstrated a correlation coefficient of 0.996, showing that the two methods were equally satisfactory. Analysis of the correlation between DiAcSpd or AcSpd and DiAcSpm, performed for the first time, indicated the usefulness of DiAcSpm as a urinary biomarker of cancer. During the course of this work, two simple methods for the preparation of alpha,omega-diacetylpolyamines were developed, and a possibility to separate and determine the concentrations of the two isomers, N(1)-acetylspermidine and N(8)-acetylspermidine in AcSpd, was shown by tandem mass spectrometry (MS/MS).

Spermidine regulates insulin synthesis and cytoplasmic Ca(2+) in mouse beta-TC6 insulinoma cells.

In order to assess the functional role of the polyamines spermidine and spermine in pancreatic beta-cells, we examined the effect of spermidine and spermine synthase inhibitors, trans-4-methylcyclohexylamine (MCHA) and N-(3-aminopropyl)cyclohexylamine (APCHA), on cellular polyamine and insulin contents, insulin secretion, and cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in mouse insulin-secreting Beta-TC6 cells. The cellular spermidine and spermine contents were reduced 90% and 64% by cultivation of cells in the presence of MCHA and APCHA for 3 days, respectively. Addition of spermidine or spermine reversed the polyamine level reduced by MCHA or APCHA, respectively. Insulin secretion was decreased 40~60% in the cells treated with MCHA or APCHA. The reduction by MCHA was reversed to the untreated level by adding spermidine exogenously, while the effect of APCHA was not reversed by treatment with spermine. The cellular insulin content was also reduced by treatment with MCHA but not the expression of insulin 1 and 2 genes, suggesting that spermidine was involved in the translation of insulin mRNAs. The elevation of [Ca(2+)](i), a key event triggering insulin secretion induced by glucose, was reduced in Beta-TC6 cells by MCHA treatment. The spermidine synthase inhibitor also augmented the sustained [Ca(2+)](i) rise induced by carbamylcholine but not by a high concentration of KCl or nicotine. These results suggested that spermidine rather than spermine plays an important role in the regulation of insulin synthesis and the glucose-induced [Ca(2+)](i) rise in Beta-TC6 cells.

The change of antizyme inhibitor expression and its possible role during mammalian cell cycle.

Antizyme inhibitor (AIn), a homolog of ODC, binds to antizyme and inactivates it. We report here that AIn increased at the G1 phase of the cell cycle, preceding the peak of ODC activity in HTC cells in culture. During interphase AIn was present mainly in the cytoplasm and turned over rapidly with the half-life of 10 to 20 min, while antizyme was localized in the nucleus. The level of AIn increased again at the G2/M phase along with ODC, and the rate of turn-over of AIn in mitotic cells decreased with the half-life of approximately 40 min. AIn was colocalized with antizyme at centrosomes during the period from prophase through late anaphase and at the midzone/midbody during telophase. Thereafter, AIn and antizyme were separated and present at different regions on the midbody at late telophase. AIn disappeared at late cytokinesis, whereas antizyme remained at the cytokinesis remnant. Reduction of AIn by RNA interference caused the increase in the number of binucleated cells in HTC cells in culture. These findings suggested that AIn contributed to a rapid increase in ODC at the G1 phase and also played a role in facilitating cells to complete mitosis during the cell cycle.

Antizyme is necessary for conversion of pancreatic tumor cells into glucagon-producing differentiated cells.

Human pancreatic tumor cell lines - AsPC-1, PANC-1, MIA paca2, KP-1 and KP-59 cells - can be induced to differentiate into pancreatic hormone-producing cells by brief trypsin treatment and subsequent culture in a serum-free, chemically defined medium. During culture, AsPC-1 cells formed cell clusters resembling the pancreatic islets, expressed genes associated with the pancreatic development and produced glucagon but not insulin. When PANC-1, MIA paca2, KP-1 and KP-59 cells were treated and cultured the same way, they underwent similar morphological changes and produced insulin and glucagon. We used these systems to identify intracellular regulatory molecules involved in the conversion of pancreatic tumor cells into glucagon-producing cells. We found that the expression of antizyme 1 (AZ1), a negative regulator of ornithine decarboxylase, was increased and its localization was altered from the nucleus to the cytoplasm during AsPC-1 cell differentiation. Transient transfection of AsPC-1 cells with AZ1 siRNA resulted in inhibition of the morphological and functional cell differentiation as well as the specific suppression of AZ1 expression. By contrast, constitutive overexpression of AZ1 in AsPC-1 cells led to the enhancement of glucagon production. We also found that PANC-1 cells reduced the expression of glucagon mRNA when treated with AZ1 siRNA. These results suggested that AZ1 was necessary for the conversion of pancreatic tumor cells into glucagon-producing cells. Glucagon production in AsPC-1 cells was not affected by addition of putrescine, suggesting that the polyamines were not directly involved in the AZ1-mediated conversion of pancreatic tumor cells to differentiated state.

A conceptual model of the polyamine binding site of N1-acetylpolyamine oxidase developed from a study of polyamine derivatives.

We used various polyamine derivatives to study the substrate binding site of N1-acetylpolyamine oxidase (PAO) that was partially purified from rat liver. The substrate activities of acetylpolyamines indicated the presence of two anionic centers corresponding to the 1,3-diaminopropane (1,3-DAP) structure and a hydrophobic region in addition to the cleavage site of the acetamidopropyl group. Based on the results of the inhibitory activities of 1,3-DAP derivatives, we developed a conceptual model of the polyamine binding site of PAO. We used this model to identify a potent competitive inhibitor, N1,N7-dihexyl-1,7-diamino-4-azaheptane, and to develop an affinity column, 1,16-diamino4,13-diazahexadecane-linked Sepharose, which was useful for the purification of PAO.

Chemical derivatization of peptides containing phosphorylated serine/threonine for efficient ionization and quantification in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We describe a useful method for the efficient ionization and relative quantification of peptides containing serine/threonine phosphorylation sites. This method is based on beta-elimination of the phosphate group from serine/threonine phosphorylation sites under alkaline conditions, followed by Michael addition reaction with N-(2-mercaptoethyl)-6-methylnicotinamide (MEMN). As a result of the derivatization reaction, the negatively charged phosphate group is substituted with the nicotinoyl moiety to improve the ionization efficiency of the derivatized peptide. The combination of d(3)-labeled MEMN (d(3)-MEMN) and MEMN (d(0)-MEMN) generates a 3 Da mass difference between d(3)-MEMN-labeled and d(0)-MEMN-labeled peptides, which is a useful signature for the identification of peptides containing serine/threonine phosphorylation sites in the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrum. Moreover, the mass difference is useful for the quantitative analysis of serine/threonine phosphorylation in proteins. In this paper, we describe the synthesis of d(0)/d(3)-labeled MEMN and an application of our approach to model peptides and proteins.

Formation of spermidine or norspermidine from synthetic diacetylpolyamines by acetylpolyamine oxidase in cultured cells.

Prodrugs that can readily release polyamine into cells without the problem of generating cytotoxic compound by serum amine oxidase would be extremely useful for elucidation of polyamine function. As linear polyamines with acetamide groups on both sides are thought to be stable in the presence of serum amine oxidase and produce polyamines by the catalytic reaction of acetylpolyamine oxidase (PAO), a series of diacetyltetraamines, diacetylpentaamines and diacetylhexaamines was prepared as prodrugs and tested for substrate activity against PAO, partially purified from rat liver. Of the compounds, N(1),N(15)-diacetyl-1,15-diamino-4,8,12-triazapentadecane (DA3333) and N(1),N(16)-diacetyl-1,16-diamino-4,8,13-triazahexadecane (DA3343) were found to be stable in culture medium containing newborn bovine serum, and to produce reasonable amounts of norspermidine and spermidine, respectively. DA3333 and DA3343 were then applied to 1-aminooxy-3-aminopropane (AOAP)-treated HTC cells with depleted putrescine and spermidine, and arrested growth. Cell growth recovered with DA3333 and DA3343, but growth rate was reduced in cells with added DA3333 compared with growth rates in cells with added DA3343 and control cells untreated with AOAP. Significant amounts of norspermidine and spermidine were found in cells with added DA3333 and DA3343, respectively. These results show the potential use of diacetylpolyamines in introducing polyamines into cells.

Electrospray ionization and time-of-flight mass spectrometric method for simultaneous determination of spermidine and spermine.

A sensitive method for the determination of polyamines in mammalian cells was described using electrospray ionization and time-of-flight mass spectrometer. This method was 50-fold more sensitive than the previous method using ionspray ionization and quadrupole mass spectrometer. The method employed the partial purification and derivatization of polyamines, but allowed a measurement of multiple samples which contained picomol amounts of polyamines. Time required for data acquisition of one sample was approximately 2 min. The method was successfully applied for the determination of reduced spermidine and spermine contents in cultured cells under the inhibition of aminopropyltransferases. In addition, a new proper internal standard was proposed for the tracer experiment using (15)N-labeled polyamines.

[Regulation of polyamine and cancer].

This review describes my work in the field of polyamine research for the last 35 years. My research started with developing the improved synthesis of decarboxylated S-adenosylmethionine and then moved to the purification of spermidine synthase from rat prostate. I also took considerable efforts to find the synthetic procedure for various polyamines with high yield in order to prepare (15)N-labeled polyamines. On the basis of these methodological work, I searched for the inhibitor of spermidine synthase and found trans-4-methylcyclohexylamine (MCHA), the most effective one at the present time. I also developed a new analytical method for polyamines using stable isotope and ionspray ionization mass spectrometry (IS-MS). Based on these studies I examined the role of polyamines in liver regeneration and found that oral administration of MCHA effectively changed the concentration of polyamines and inhibited the hepatic growth. I also found the close relationship between the concentration ratio of spermidine to spermine and the extent of liver regeneration. These results may shed new light on the control of cell growth by polyamine in vivo.

Inhibition of putrescine aminopropyltransferase influences rat liver regeneration.

A close relationship between rat liver regeneration and the concentration ratio of spermidine to spermine (spd:spm) was demonstrated by the oral administration of trans-4-methylcyclohexylamine (MCHA), a specific inhibitor of putrescine aminopropyltransferase. A decrease in recovery rate of remnant liver with MCHA, as a percentage index of remnant liver weight to body weight, correlated well with a decrease of the spd:spm value, with a correlation coefficient of 0.952 for the remnant livers on day 3 after partial hepatectomy. The decrease in recovery rate could be explained by a prolonged cell cycle based on the data of the proliferating cell nuclear antigen labelling index and mitotic cell index in both livers of day 2 and day 3 after partial hepatectomy. The results presented here will give a new aspect in the field of polyamine regulation to control cell growth in vivo.

N1-aminopropylagmatine, a new polyamine produced as a key intermediate in polyamine biosynthesis of an extreme thermophile, Thermus thermophilus.

In the extreme thermophile Thermus thermophilus, a disruption mutant of a gene homologous to speB (coding for agmatinase = agmatine ureohydrolase) accumulated N1-aminopropylagmatine (N8-amidino-1,8-diamino-4-azaoctane, N8-amidinospermidine), a new compound, whereas all other polyamines produced by the wild-type strain were absent from the cells. Double disruption of speB and speE (polyamine aminopropyltransferase) resulted in the disappearance of N1-aminopropylagmatine and the accumulation of agmatine. These results suggested the following. 1) N1-Aminopropylagmatine is produced from agmatine by the action of an enzyme coded by speE. 2) N1-Aminopropylagmatine is a metabolic intermediate in the biosynthesis of unique polyamines found in the thermophile. 3) N1-Aminopropylagmatine is a substrate of the SpeB homolog. They further suggest a new biosynthetic pathway in T. thermophilus, by which polyamines are formed from agmatine via N1-aminopropylagmatine. To confirm our speculation, we purified the expression product of the speB homolog and confirmed that the enzyme hydrolyzes N1-aminopropylagmatine to spermidine but does not act on agmatine.

Control of spermidine and spermine levels in rat tissues by trans-4-methylcyclohexylamine, a spermidine-synthase inhibitor.

In rat tissues, a decrease in spermidine, accompanied by an increase in spermine was induced by the oral administration (once daily for either 1 week or 1 month) of trans-4-methylcyclohexylamine (4MCHA), a spermidine synthase inhibitor. This is similar to the changes observed in polyamine content when cell growth is arrested. The body-weight gain of the rats tended to decrease with increasing doses of 4MCHA. A decrease in spermidine, combined with a moderate increase in spermine, was observed dose-dependently in all of the tissues tested, with a relatively fast clearance of 4MCHA. Manipulating the polyamine content of tissues, by daily administration of 100 mumol 4MCHA for 1 week, made it possible to estimate the effects of simultaneously added spermidine or spermine on endogenous polyamine contents. The altered polyamine levels, obtained after daily administration for 1 week, were maintained during the extended 1-month period, with growth-dependent alteration. The results show it is possible to produce experimental rats with a higher spermine:spermidine ratio than control rats to investigate the physiological significance of spermidine downregulation and spermine upregulation in vivo.