Am J Physiol Cell Physiol Add DOIs to your references at manuscript stage!
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 293: C705-C711, 2007. First published May 2, 2007; doi:10.1152/ajpcell.00084.2007
0363-6143/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/2/C705    most recent
00084.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Isome, M.
Right arrow Articles by Satriano, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Isome, M.
Right arrow Articles by Satriano, J.

GROWTH, DIFFERENTIATION, AND APOPTOSIS

The antiproliferative effects of agmatine correlate with the rate of cellular proliferation

Masato Isome,1 Mark J. Lortie,1 Yasuko Murakami,2 Eva Parisi,1 Senya Matsufuji,3 and Joseph Satriano1,4

1Division of Nephrology-Hypertension and 4The Stein Institute for Research on Aging, Department of Medicine, University of California San Diego and Veterans Affairs San Diego Healthcare System, La Jolla, California; 2Department of Genetics and Molecular Biology, School of Pharmacy, Musashino University, Nishi-Tokyo, Japan; and 3Department of Biochemistry II, The Jikei University School of Medicine, Tokyo, Japan

Submitted 25 February 2007 ; accepted in final form 29 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Polyamines are small cationic molecules required for cellular proliferation. Agmatine is a biogenic amine unique in its capacity to arrest proliferation in cell lines by depleting intracellular polyamine levels. We previously demonstrated that agmatine enters mammalian cells via the polyamine transport system. As polyamine transport is positively correlated with the rate of cellular proliferation, the current study examines the antiproliferative effects of agmatine on cells with varying proliferative kinetics. Herein, we evaluate agmatine transport, intracellular accumulation, and its effects on antizyme expression and cellular proliferation in nontransformed cell lines and their transformed variants. H-ras- and Src-transformed murine NIH/3T3 cells (Ras/3T3 and Src/3T3, respectively) that were exposed to exogenous agmatine exhibit increased uptake and intracellular accumulation relative to the parental NIH/3T3 cell line. Similar increases were obtained for human primary foreskin fibroblasts relative to a human fibrosarcoma cell line, HT1080. Agmatine increases expression of antizyme, a protein that inhibits polyamine biosynthesis and transport. Ras/3T3 and Src/3T3 cells demonstrated augmented increases in antizyme protein expression relative to NIH/3T3 in response to agmatine. All transformed cell lines were significantly more sensitive to the antiproliferative effects of agmatine than nontransformed lines. These effects were attenuated in the presence of exogenous polyamines or inhibitors of polyamine transport. In conclusion, the antiproliferative effects of agmatine preferentially target transformed cell lines due to the increased agmatine uptake exhibited by cells with short cycling times.

polyamines; antizyme; ornithine decarboxylase; polyamine transport

POLYAMINES (PUTRESCINE, SPERMIDINE, AND SPERMINE) are required components for cellular proliferation (33, 52). Ornithine decarboxylase (ODC), one of the most highly regulated eukaryotic enzymes, is the first and rate-limiting enzyme of polyamine biosynthesis. It is a proto-oncogene that is significantly elevated in animal tumors (43, 44) and whose stepwise increase with the progression from normal colon mucosa to adenocarcinoma suggests a role in multistage carcinogenesis (21, 35, 37). Many of the same factors that induce ODC activity stimulate polyamine transport. As such, rapid polyamine uptake has been noted in many tumor cell lines compared with nontumorigenic cells of the same lineage (4, 15, 28, 31). In vivo, elevated plasma polyamine levels are associated with malignancy and tissue wasting, and tumor cells acquire polyamines released into the circulation (28). Selective inhibition of ODC with inhibitors such as 2-(difluoromethyl)-ornithine results in a compensatory increase in polyamine transport (4). Such upregulation of polyamine transport substitutes for de novo polyamine biosynthesis [for a review, see Ref. 50]. It is for these reasons that inhibition of ODC or the polyamine-converting enzymes alone have often complicated in vivo experimental interpretations as to the importance of polyamines and yielded less than anticipated results in clinical trials (17, 25, 28, 38, 45, 48). The importance of polyamine transport in addition to biosynthesis in affecting intracellular polyamine levels is now well established [for a review, see (49)]. Thus, beneficial intervention must be concerned with polyamine transport as well as biosynthesis when addressing intracellular polyamine availability for growth.

Intracellular polyamine levels are autoregulated via induction of the regulatory protein ODC antizyme (23). Induction of antizyme is via a programmed +1 ribosomal frameshift (23). This novel mechanism of translational induction affords rapid modulation in response to increased intracellular polyamine concentrations. As such, the cell is required to maintain constitutive levels of antizyme mRNA requisite for this response, which underscores the importance of this system in normal cell homeostasis. Four isoforms of antizyme have been described (22). Here, we refer to the most abundant, ubiquitous isoform, ODC antizyme-1. Antizyme binds to ODC, inhibits its activity and accelerates its degradation in a process catalyzed by the 26S proteasome (7, 13, 29). In addition to inhibiting polyamine biosynthesis, antizyme concurrently suppresses polyamine transporter activity (26, 51). This unique two-pronged negative feedback system is effective in limiting intracellular polyamine concentrations.

Arginine decarboxylase (ADC) converts arginine to agmatine. Intracellular concentrations of agmatine vary among organs, with high levels of synthesis and expression in kidney and liver (19, 20, 27). Agmatine inhibits proliferation by suppressing intracellular polyamine levels (1, 9, 42, 53). It induces antizyme expression via a programmed +1 ribosomal frameshift and is the only known endogenous molecule, other than the canonical polyamines, with this capacity (14, 42). Agmatine induces spermidine/spermine acetyltransferase in some cells types, which would promote the back-conversion of higher-order, more highly charged, to lower-order polyamines (53). It may also induce a yet unknown mechanism of suppressing ODC activity independent of antizyme (1). All of these mechanisms reduce intracellular polyamine pools and suppress growth.

We and others have shown that agmatine enters mammalian cells via the polyamine transporter (1, 3, 6, 40). As polyamine transport is positively correlated with proliferation rate, we examined whether agmatine preferentially targets rapidly proliferating, transformed cells (41). In this report, we investigate the effects of agmatine on primary, immortalized, transformed and tumorigenic cell lines and demonstrate that the antiproliferative effects correlate with the rate of proliferation in these cell lines.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. [3H]Agmatine was purchased from American Radiolabeled Chemicals (St. Louis, MO). Polyamine transport inhibitors, MQT1202 (L-Lysine-Spermine) and MQT1483 [L-Lys(e-palmitoyl)-Spermine], were generous gifts from Drs. Reitha S. Weeks, Mark R. Burns, and MediQuest Therapeutics, (Seattle, WA) (5, 54). All other chemicals were purchased from Sigma (St. Louis, MO) unless otherwise noted.

Cells and cell culture. All cell lines were from American Type Culture Collection except Ha-ras (Ras/3T3)- and Src (Src/3T3)-transformed NIH/3T3 cells that were gifts from Dr. Mark Kamps (University of California, San Diego, San Diego, CA) (55), and the breast carcinoma cell lines N2O2 and PC7T were gifts from Dr. Daniel Gold of the Sidney Kimmel Cancer Center (San Diego, CA). Cell lines were maintained in Dulbecco's modified Eagle's medium (Cellgro, Herndon, VA) supplemented with 5% FBS (Atlanta Biologicals, Atlanta, GA), except primary fibroblast cells (CCD-1112Sk) that were maintained in Iscove's modified Eagle's media (Cellgro) supplemented with 5% FBS, unless otherwise noted.

Transport experiments. Monitoring intracellular transport was performed as previously described (39). Briefly, cells were grown in 6-well culture plates for 3 days until near confluence. Twenty-four hours before the uptake experiments, cells were placed in medium with limited (0.1%) FBS. Wells were washed with HEPES buffer (in mM): 25 Na+-free HEPES, 5 KCl, 0.9 CaCl2, 1 MgSO4, 5.6 D-glucose, 137 NaCl. The addition of HEPES buffer containing 10 µM [3H]agmatine at ~200,000 cpm/well started the 30-min uptake period. Three rapid washes with ice-cold PBS and lysis in 3N NaOH terminated the reactions. Bio-Rad Protein Assay (Bio-Rad, Hercules, CA) used a small aliquot of each lysate for protein determination; the remainder of the sample was counted in a beta-scintillation counter to evaluate uptake. Uptake at 4°C, representing nonspecific binding and diffusional transport, was subtracted from the above determinations.

Intracellular agmatine determination. Cells were plated on 10-cm culture dishes. Twenty-four hours before extraction, the cells were placed in serum-free medium, except the primary fibroblast cell line, which required 0.1% FBS. Eight hours after medium change, 100 µM agmatine was added to the experimental samples for a 16-h uptake period at 37°C. Cells were then washed with ice-cold PBS and lysed in 10% TCA in 10 mM HCl. The sample supernatants were transferred to a 10,000 molecular weight spin filter (Millipore) for further purification, and then they were extracted three times with hydrated ethyl ether to remove traces of TCA and lipids. Cell extracts and standards were derivatized for fluorescence detection of primary and secondary amine groups with N-hydroxysuccinimidyl-6-aminoquinoyl carbamate as per kit instructions (AccQ tag; Waters, Franklin, MA). Elution was performed using a Hewlett-Packard 1100 series binary HPLC pump system with a 250-mm 3-µm ODS Hypersil C18 RP column (Hewlett-Packard) maintained at 45°C. Fluorescence was detected in line using a Waters 470 detector linked to the data acquisition system. Elution gradients were based on the AccQ tag kit instructions.

Cell counting. Cells were plated for the days indicated in 10-cm culture dishes or 6-well plates, washed with PBS, and harvested in trypsin/EDTA for quantification in a Coulter Counter (model ZM). In experiments in which cells were preloaded with putrescine, 250 µM putrescine was added to the cells 3 h before the addition of agmatine. Polyamine transport inhibitors MQT1202 (1 µM) and MQT1483 (1 µM) were also added 3 h before agmatine administration.

Western blot analysis. For Western blot analysis, Ras/3T3 cells were collected and lysed [lysis buffer: 1% triton-X 100, 0.5% deoxycholic acid, 1 mM EDTA, 0.1% SDS, 4 mM NaF, Complete protease cocktail (Roche Molecular Biochemicals, Mannheim, Germany), 0.7 µg/ml pepstatin and 1 mM NaVO4 in PBS]. Lysates were resolved on 12% NuPAGE gels in MOPS buffer (Invitrogen, Carlsbad, CA). Gel proteins were transferred to nitrocellulose membranes and immunoblotted with antizyme-1 antibody (24). This antibody cross-reacts weakly with mouse antizyme-2 (data not shown). The secondary antibody was horseradish peroxidase-conjugated (Santa Cruz Biotechnology, Santa Cruz, CA) for autoradiographic detection by ECL Plus (Amersham Pharmacia, Piscataway, NJ), with densitometric analysis by ImageJ Software (National Institutes of Health, Bethesda, MD).

Determination of antizyme activity. The cells were washed with ice-cold PBS and disrupted by three freeze-thaw cycles. Then, 0.5 ml of extract buffer (25 mM Tris·HCl, pH 7.4, 1 mM DTT, and 0.01% Tween 80) was added, and the cell suspension was centrifuged at 18,000 g for 20 min at 4°C. The supernatant contained both active free ODC and inactive ODC bound to antizyme (ODC-antizyme complex). Free ODC activity was assayed by measuring the release of 14CO2 from L-[1-14C] ornithine. The basal reaction mixture contained 0.0625 mCi of L-[1-14C]ornithine, 0.4 mM L-ornithine, 40 mM pyridoxal phosphate, 5 mM DTT, 40 mM Tris·HCl buffer, pH 7.4, 0.01% Tween 80 and enzyme solution in a final volume of 125 ml. Inactive ODC (ODC-antizyme complex) was determined as the increase in ODC activity caused by the addition of an excess amount of recombinant GST fusion antizyme inhibitor (0.1 mg) to the basal reaction mixture described above. Antizyme activity was equated to the amount of ODC-antizyme complex.

Statistical evaluations. Variations between samples within groups were analyzed by ANOVA, with significance determined by Fisher's protected least significant differences post hoc test. StatView software (ver. 4.5; Abacus Concepts, Berkeley, CA) was used for these analyses. All data are means (SD) and represent at least three separate determinations.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Transport and accumulation of agmatine in cell lines. An increased rate of cellular proliferation is associated with increased polyamine uptake. We and others have shown that agmatine import into mammalian cells uses the polyamine transport system (1, 6, 40, 41). Although epithelial cells are primarily chosen for proliferation studies because of their prevalence in cancer, plasticity in epithelial tumor cells toward a fibroblastoid phenotype in progression toward metastasis, that is, an epithelial-mesenchymal transition, is now well established (12). Furthermore, polyamine transport is well characterized in fibroblast cell lines and display a single polyamine transporter in both murine and human (49, 50). Cell lines include the murine fibroblast NIH/3T3 line, NIH/3T3 cells transformed by stable transfection of either H-ras or Src, in Ras/3T3 or Src/3T3 cells, respectively (55), a primary human foreskin fibroblast line (cell designation CCD 1112Sk, from the American Type Culture Collection, Manassas, VA) and the human fibrosarcoma cell line HT1080.

Growth curves of the cell lines over a 4-day period are shown in Fig. 1. After replating, the cells have a lag phase before assuming log phase growth from day 3. The largest differences were observed at day 4. The transformed Ras/3T3, Src/3T3, and HT1080, cell lines grew more rapidly than their nontransformed or primary culture counterparts.


Figure 1
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 1. Cell line growth profiles. Proliferative kinetics of cell lines plated at equal densities over 4 days. Primary, primary foreskin fibroblast cell line.

 
Transport of agmatine significantly increases in transformed Ras/3T3 and Src/3T3 cells relative to the parental murine NIH/3T3 cell line (Fig. 2A). Cellular uptake of agmatine also appreciably increases in human fibrosarcoma cells, HT1080, relative to the primary fibroblast cell line. We observe a significant increase in agmatine import from nontransformed to transformed variants of mammalian cell lines, regardless of whether the cells are of murine or human origin.


Figure 2
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 2. Changes in agmatine uptake rate and accumulation in cell lines. A: uptake of 10 µM [3H]agmatine into the cell lines. B: HPLC determination of intracellular agmatine concentrations in the presence of 100 µM agmatine. There were significant and parallel differences between parental NIH/3T3 cell lines vs. Ras/3T3 and Src/3T3 transformed varients, as with primary fibroblast cells vs. HT1080 fibrosarcoma cells (P < 0.05).

 
HPLC analysis shows an accumulation of intracellular agmatine levels that increase after incubation with 100 µM agmatine for 16 h in transformed cell lines relative to the primary and immortalized lines in a pattern similar to that of agmatine transport (Fig. 2B). The potential of agmatine within the cell depends not only on import, but also on export and metabolism. The equilibrium of these components is represented by the intracellular accumulation of agmatine. Baseline levels of agmatine in cells in culture are very low to undetectable, so we assume the effects observed are due to addition of exogenous agmatine.

Preferential suppression of growth by agmatine in transformed cells. Preferential uptake and accumulation of agmatine into transformed cell lines suggest that these cell lines would be more responsive to the antiproliferative effects of agmatine. We evaluated cell growth, as a function of accrued cell number after 4 days in the presence of agmatine relative to untreated control cells, the latter being set to 100% (Fig. 3). All transformed cell lines demonstrated a significant decrease in cell number relative to their untreated controls by 50 µM agmatine, immortalized NIH/3T3 cells by 250 µM agmatine, and primary fibroblasts were not significantly affected by agmatine within this concentration range. The SV40 transformed the renal proximal tubule cell line, and MCT, displayed a similar sensitivity to agmatine as exhibited here by the transformed variants (42).


Figure 3
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 3. Selective antiproliferative effects of agmatine on cell lines of murine (A), or human origin (B). Cells were plated and allowed to grow for 4 days in the concentrations of agmatine indicated. Cell number was evaluated in a Coulter Counter. Untreated, control cells of all cell lines were placed at 100%. N2O2 and PC7T beast carcinoma cell lines were added to this study to broaden the scope of responses to agmatine.

 
Polyamine supplementation or inhibition of polyamine transport suppresses the effects of agmatine. We preloaded Ras/3T3 cells with the polyamine putrescine and evaluated the effects of agmatine on cell growth, as per Fig. 3. Addition of putrescine to Ras/3T3 cells attenuated the antiproliferative effects of agmatine (Fig. 4A). Competitive inhibition of agmatine uptake by putrescine would also contribute to this effect. Uptake rates of agmatine and putrescine are quite similar in these cells (40). At equimolar concentrations of agmatine and putrescine, that is, at 250 µM, would yield a 50% inhibition of agmatine uptake. The effects of putrescine administration are far greater than can be accounted for by competitive inhibition alone. These data support earlier work that the antiproliferative effects observed with agmatine are due in large part to polyamine depletion (42).


Figure 4
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 4. The effects of agmatine are suppressed by polyamine supplementation or inhibition of polyamine transport. A: Ras/3T3 cells were preincubated with putrescine (Putr; 250 µM) for 3 h before the administration of agmatine. The cells were incubated in the absence or presence of putrescine and/or agmatine, and cell number per well was determined as per Fig. 2B. B: Ras/3T3 cells were incubated with the polyamine transport inhibitor MQT1202 or MQT1483 at a concentration of 1 µM for 3 h before the administration of agmatine and throughout the remainder of the experiment. The cell number per well was determined as above.

 
We next evaluated the significance of the polyamine transport system on the antiproliferative effects of agmatine. Administration of the polyamine transport inhibitors MQT1202 and MQT1483 prevented the inhibitory effects of agmatine on Ras/3T3 cells (Fig. 4B). Thus, polyamine transporters are required for significant import of agmatine into these cells to produce antiproliferative effects.

Effects of agmatine on induction of antizyme. Induction of antizyme occurs in many cell types in response to agmatine administration. Increased uptake of agmatine into the more rapidly proliferating transformed cells should affect antizyme induction, assuming that antizyme function and/or induction is not desensitized by transformation. NIH/3T3, Ras/3T3, and Src/3T3 cells were grown for 4 days in the absence or presence of agmatine (250 µM and 500 µM) and antizyme expression evaluated by Western blot analysis (Fig. 5A). Antizyme expression consistently increased to a greater degree in the transformed cell lines in response to agmatine administration than the parental NIH/3T3 cell line. Putrescine (250 µM), agmatine (1 mM), or a combination of the two-increased antizyme protein expression in Ras/3T3 cells (Fig. 5B). Increases in antizyme in response to agmatine administration were confirmed in antizyme activity assays (not shown).


Figure 5
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 5. Variations in antizyme expression in the presence of agmatine. A: Antizyme protein evaluation by Western blot analysis of NIH/3T3, Src/3T3, and Ras/3T3 cells treated with agmatine for 4 days at the concentrations indicated. The graph represents antizyme protein expressed as a fold increase observed with agmatine over untreated control values (straight line). Ras/3T3 and Src/3T3 cell lines were significantly different from their respective controls, and NIH/3T3 cells at 250 µM and 500 µM agmatine concentrations (P < 0.05). NIH/3T3 cells were different from its untreated controls at 500 µM agmatine (P < 0.05). B: Western blot analysis for antizyme protein expression in Ras/3T3 cells cultured in the absence, control (C), or presence of 250 µM putrescine (Put), 1 mM agmatine (Agm), or a combination of agmatine and putrescine (A+P), for 4 days. Evaluation of beta-actin expression to demonstrate protein loading is shown below the appropriate antizyme bands in both A and B of this figure.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The differential uptake and accumulation of agmatine in transformed relative to nontransformed cell lines correlate with the antiproliferative effects of agmatine. Oncogene transformed murine cells, Ras/3T3 and Src/3T3, and human fibrosarcoma, HT1080, exhibit accelerated growth rates, higher agmatine uptake, and intracellular agmatine concentrations, and they are more sensitive to the antiproliferative effects of agmatine than are their respective immortalized, NIH/3T3, or primary fibroblast counterparts. Two breast carcinoma cell lines, N2O2 and PC7T, also displayed a high sensitivity to agmatine, similar to the fibrosarcoma cell line, HT1080. Akin to the canonical polyamines, agmatine is a cationic molecule with amine groups that are subject to acetylation. This process neutralizes the charge of the molecules and facilitates their cellular export. Unlike the simple amines of polyamines, the guanidino group of agmatine may allow for a different intracellular localization (2) and make it less prone to cellular export.

The kidney, liver, brain, and adrenals maintain high constitutive levels of ADC activity (20, 27). Although ADC was localized to the mitochondrial membrane, agmatine generation was not observed in mitochondrial extracts (8). This is contrary to findings in whole organ preparations (20) and requires further examination. Whether sourced from the gut flora or biosynthesis within organs, agmatine is widely distributed within the plasma (2.8 µM; see Ref. 19) and extracellular fluid and can be selectively concentrated in several organs (19, 36). Concentrations of agmatine in early reports were underestimated due to the lability of the molecule in the derivatization process, a problem that has not been entirely eradicated. More recently, the tissue level of agmatine in the rat kidney was reported to approximate over 400 µM (19). In all of the cell lines evaluated in this report, the growth rates of transformed cells treated with 50 µM agmatine were significantly different from their untreated controls, with a maximal effect at ~500 µM. Rat hepatoma cells, HTC, exhibit a similar profile in response to agmatine with a significance at 50 µM and maximal effects at 500 µM (personal communication, Sebastiano Colombatto, University of Torino, Italy).

Expression of antizyme is primarily controlled at the translational level through ribosomal frameshifting, although there is also a transcriptional mechanism when polyamines are depleted (30). However, there has been no evidence that the polyamine dose response/frameshift kinetics are altered by malignant transformation. The polyamine concentrations required for maximal frameshift efficiency, and the degree of frameshift efficiency was the same in transformed MCT cells as reported for normal rat liver (23, 42). Immunoprecipitation of antizyme from agmatine-treated MCT cell preparations prevented the inhibitory effects on ODC activity (42). We show that agmatine significantly increases antizyme protein levels in two transformed cell lines and also that loading cells with putrescine reduces the effects of agmatine on proliferation. The antiproliferative effects of agmatine thus appear due to polyamine depletion and not due to agmatine functionally replacing or displacing the canonical polyamines (42). Agmatine appears to require cellular import for its antiproliferative effects. It is a multifunctional molecule with both receptor-dependent and -independent functions. As import is required (Fig. 4B), the antiproliferative effects evaluated in this study appear receptor independent. Furthermore, exogenously administered putrescine or agmatine imported into the cytosol induces antizyme expression (Fig. 5B). These studies are in accord with studies demonstrating temporal translocation and compartmentalization of ODC and antizyme (11, 34, 47). Taken together, these data would suggest a mechanism whereby the rapid conversion and utilization of polyamines may result in lower unbound or "free" cytosolic polyamine levels that are insufficient to induce antizyme expression, and/or how the inhibitor may be unable to reach the enzyme, depending upon the physiological status of the cell. However, this hypothesis cannot be directly substantiated by current techniques. We do show that exogenously administered putrescine or agmatine, which imports into the cytosol, is capable of inducing antizyme expression.

Agmatine would be complementary, that is, additive, to the endogenous polyamine pool and thereby effectively lower the threshold levels of polyamines required to bring about the translational induction of antizyme. In addition, induction of antizyme by agmatine could constitute a self-limiting feedback mechanism by inhibiting both polyamine and agmatine uptake (Fig. 6). Taken together, these observations suggest that the polyamine feedback system in these transformed cells is functional, but it may be evaded in proliferating cells. This is not to say that regulation of antizyme expression is not altered in some neoplasms (18, 46) but suggests that such alteration is not as universal in neoplasia as is increased ODC activity. Several studies support the view that induction of antizyme may prove to be a viable method of attenuating neoplastic growth (10, 16, 18, 32).


Figure 6
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 6. Proposed mechanism. Rapidly dividing cells upregulate polyamine transport. Intracellular agmatine accumulation by synthesis and/or transport mediates antizyme induction. Antizyme suppresses intracellular polyamine levels by inhibiting polyamine synthesis and transport and limits further agmatine uptake through the polyamine transporter. Agmatine may also induce spermidine/spermine acetyltransferase activity and reduce ornithine decarboxylase (ODC) activity independent of antizyme (not illustrated). These mechanisms would also reduce intracellular polyamine content. Bars represent negative regulation; ovals in cell membrane symbolize polyamine transporters. ADC. arginine decarboxylase.

 
In summary, the preferential antiproliferative effects of agmatine on several transformed mammalian cell lines occurs from enhanced cellular uptake by polyamine transporters, and subsequent suppression of proliferation. This process exploits the upregulation of polyamine transporters in rapidly cycling cells and may be a useful template for drug development.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants DK-070123, DK-070667, DK-02920, DK-56248, DK-28602, the American Cancer Society, University of California, San Diego, Cancer Center Grant, the Stein Institute for Research on Aging, Center Grant, and funds supplied by the Research Service of the Department of Veterans Affairs.


   ACKNOWLEDGMENTS
 
Eva Parisi is a visiting scholar from the Nephrology Research Laboratory IRBLLEIDA, Hospital Universitari Arnau de Vilanova, Rovira Roure 80. 1a Planta, 25198, Lleida, Spain.

Present address of Dr. M. Isome: Department of Pediatrics, Fukushima Medical University, School of Medicine1, Hikarigaoka Fukushima-City, Fukushima 960-1295, Japan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Satriano, Univ. of California, San Diego, and Veterans Affairs San Diego Healthcare System, Div. of Nephrology-Hypertension, 3350 La Jolla Village Dr., San Diego, CA 92161 (e-mail: jsatriano{at}ucsd.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Babal P, Ruchko M, Campbell CC, Gilmour SP, Mitchell JL, Olson JW, Gillespie MN. Regulation of ornithine decarboxylase activity and polyamine transport by agmatine in rat pulmonary artery endothelial cells. J Pharmacol Exp Ther 296: 372–377, 2001.[Abstract/Free Full Text]

2. Babal P, Ruchko M, Olson JW, Gillespie MN. Interactions between agmatine and polyamine uptake pathways in rat pulmonary artery endothelial cells. Gen Pharmacol 34: 255–261, 2000.[CrossRef][Web of Science][Medline]

3. Blantz RC, Satriano J, Gabbai F, Kelly C. Biological effects of arginine metabolites. Acta Physiol Scand 168: 21–25, 2000.[CrossRef][Web of Science][Medline]

4. Bogle RG, Mann GE, Pearson JD, Morgan DM. Endothelial polyamine uptake: selective stimulation by L-arginine deprivation or polyamine depletion. Am J Physiol Cell Physiol 266: C776–C783, 1994.[Abstract/Free Full Text]

5. Burns MR, Carlson CL, Vanderwerf SM, Ziemer JR, Weeks RS, Cai F, Webb HK, Graminski GF. Amino acid/spermine conjugates: polyamine amides as potent spermidine uptake inhibitors. J Med Chem 44: 3632–3644, 2001.[CrossRef][Web of Science][Medline]

6. Cabella C, Gardini G, Corpillo D, Testore G, Bedino S, Solinas SP, Cravanzola C, Vargiu C, Grillo MA, Colombatto S. Transport and metabolism of agmatine in rat hepatocyte cultures. Eur J Biochem 268: 940–947, 2001.[Web of Science][Medline]

7. Coffino P. Polyamines in spermiogenesis: not now, darling [comment]. Proc Natl Acad Sci USA 97: 4421–4423, 2000.[Free Full Text]

8. Coleman CS, Hu G, Pegg AE. Putrescine biosynthesis in mammalian tissues. Biochem J 379: 849–855, 2004.[CrossRef][Web of Science][Medline]

9. Dudkowska M, Lai J, Gardini G, Stachurska A, Grzelakowska-Sztabert B, Colombatto S, Manteuffel-Cymborowska M. Agmatine modulates the in vivo biosynthesis and interconversion of polyamines and cell proliferation. Biochim Biophys Acta 1619: 159–166, 2003.[Medline]

10. Feith DJ, Shantz LM, Pegg AE. Targeted antizyme expression in the skin of transgenic mice reduces tumor promoter induction of ornithine decarboxylase and decreases sensitivity to chemical carcinogenesis. Cancer Res 61: 6073–6081, 2001.[Abstract/Free Full Text]

11. Gritli-Linde A, Nilsson J, Bohlooly YM, Heby O, Linde A. Nuclear translocation of antizyme and expression of ornithine decarboxylase and antizyme are developmentally regulated. Dev Dyn 220: 259–275, 2001.[CrossRef][Web of Science][Medline]

12. Grunert S, Jechlinger M, Beug H. Diverse cellular and molecular mechanisms contribute to epithelial plasticity and metastasis. Nat Rev Mol Cell Biol 4: 657–665, 2003.[CrossRef][Web of Science][Medline]

13. Hayashi S, Murakami Y, Matsufuji S. Ornithine decarboxylase antizyme: a novel type of regulatory protein. Trends Biochem Sci 21: 27–30, 1996.[CrossRef][Web of Science][Medline]

14. Higashi K, Yoshida K, Nishimura K, Momiyama E, Kashiwagi K, Matsufuji S, Shirahata A, Igarashi K. Structural and functional relationship among diamines in terms of inhibition of cell growth. J Biochem (Tokyo) 136: 533–539, 2004.[Abstract/Free Full Text]

15. Holley JL, Mather A, Wheelhouse RT, Cullis PM, Hartley JA, Bingham JP, Cohen GM. Targeting of tumor cells and DNA by a chlorambucil-spermidine conjugate. Cancer Res 52: 4190–4195, 1992.[Abstract/Free Full Text]

16. Iwata S, Sato Y, Asada M, Takagi M, Tsujimoto A, Inaba T, Yamada T, Sakamoto S, Yata J, Shimogori T, Igarashi K, Mizutani S. Anti-tumor activity of antizyme which targets the ornithine decarboxylase (ODC) required for cell growth and transformation. Oncogene 18: 165–172, 1999.[CrossRef][Web of Science][Medline]

17. Janne J, Alhonen L, Leinonen P. Polyamines: from molecular biology to clinical applications. Ann Med 23: 241–259, 1991.[Web of Science][Medline]

18. Koike C, Chao DT, Zetter BR. Sensitivity to polyamine-induced growth arrest correlates with antizyme induction in prostate carcinoma cells. Cancer Res 59: 6109–6112, 1999.[Abstract/Free Full Text]

19. Lortie MJ, Ishizuka S, Schwartz D, Blantz RC. Bioactive products of arginine in sepsis: tissue and plasma composition after LPS and iNOS blockade. Am J Physiol Cell Physiol 278: C1191–C1199, 2000.[Abstract/Free Full Text]

20. Lortie MJ, Novotny WF, Peterson OW, Vallon V, Malvey K, Mendonca M, Satriano J, Insel P, Thomson SC, Blantz RC. Agmatine, a bioactive metabolite of arginine. Production, degradation, and functional effects in the kidney of the rat. J Clin Invest 97: 413–420, 1996.[Web of Science][Medline]

21. Luk GD, Baylin SB. Ornithine decarboxylase as a biologic marker in familial colonic polyposis. N Engl J Med 311: 80–83, 1984.[Abstract]

22. Mangold U. The antizyme family: polyamines and beyond. IUBMB Life 57: 671–676, 2005.[Web of Science][Medline]

23. Matsufuji S, Matsufuji T, Miyazaki Y, Murakami Y, Atkins JF, Gesteland RF, Hayashi S. Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme. Cell 80: 51–60, 1995.[CrossRef][Web of Science][Medline]

24. Matsufuji S, Miyazaki Y, Kanamoto R, Kameji T, Murakami Y, Baby TG, Fujita K, Ohno T, Hayashi S. Analyses of ornithine decarboxylase antizyme mRNA with a cDNA cloned from rat liver. J Biochem (Tokyo) 108: 365–371, 1990.[Abstract/Free Full Text]

25. McCann PP, Pegg AE. Ornithine decarboxylase as an enzyme target for therapy. Pharmacol Ther 54: 195–215, 1992.[CrossRef][Web of Science][Medline]

26. Mitchell JL, Judd GG, Bareyal-Leyser A, Ling SY. Feedback repression of polyamine transport is mediated by antizyme in mammalian tissue-culture cells. Biochem J 299: 19–22, 1994.[Web of Science][Medline]

27. Morrissey J, McCracken R, Ishidoya S, Klahr S. Partial cloning and characterization of an arginine decarboxylase in the kidney. Kidney Int 47: 1458–1461, 1995.[Web of Science][Medline]

28. Moulinoux JP, Quemener V, Khan NA. Biological significance of circulating polyamines in oncology. Cell Mol Biol 37: 773–783, 1991.[Web of Science][Medline]

29. Murakami Y, Matsufuji S, Hayashi S, Tanahashi N, Tanaka K. Degradation of ornithine decarboxylase by the 26S proteasome. Biochem Biophys Res Commun 267: 1–6, 2000.[CrossRef][Web of Science][Medline]

30. Nilsson J, Koskiniemi S, Persson K, Grahn B, Holm I. Polyamines regulate both transcription and translation of the gene encoding ornithine decarboxylase antizyme in mouse. Eur J Biochem 250: 223–231, 1997.[Web of Science][Medline]

31. Pegg AE. Polyamine metabolism and its importance in neoplastic growth and a target for chemotherapy. Cancer Res 48: 759–774, 1988.[Abstract/Free Full Text]

32. Pegg AE, Feith DJ, Fong LY, Coleman CS, O'Brien TG, Shantz LM. Transgenic mouse models for studies of the role of polyamines in normal, hypertrophic and neoplastic growth. Biochem Soc Trans 31: 356–360, 2003.[CrossRef][Web of Science][Medline]

33. Pegg AE, McCann PP. Polyamine metabolism and function. Am J Physiol Cell Physiol 243: C212–C221, 1982.[Abstract/Free Full Text]

34. Pomidor MM, Ruhl KK, Zheng P, Song Y, Janne OA, Tuan RS, Hickok NJ. Relationship between ornithine decarboxylase and cytoskeletal organization in cultured human keratinocytes: cellular responses to phorbol esters, cytochalasins, and alpha-difluoromethylornithine. Exp Cell Res 221: 426–437, 1995.[CrossRef][Web of Science][Medline]

35. Porter CW, Herrera-Ornelas L, Pera P, Petrelli NF, Mittelman A. Polyamine biosynthetic activity in normal and neoplastic human colorectal tissues. Cancer 60: 1275–1281, 1987.[CrossRef][Web of Science][Medline]

36. Raasch W, Regunathan S, Li G, Reis DJ. Agmatine, the bacterial amine, is widely distributed in mammalian tissues. Life Sci 56: 2319–2330, 1995.[CrossRef][Web of Science][Medline]

37. Radford DM, Nakai H, Eddy RL, Haley LL, Byers MG, Henry WM, Lawrence DD, Porter CW, Shows TB. Two chromosomal locations for human ornithine decarboxylase gene sequences and elevated expression in colorectal neoplasia. Cancer Res 50: 6146–6153, 1990.[Abstract/Free Full Text]

38. Redgate ES, Boggs S, Grudziak A, Deutsch M. Polyamines in brain tumor therapy. J Neurooncol 25: 167–179, 1995.[CrossRef][Medline]

39. Satriano J, Ishizuka S, Archer DC, Blantz RC, Kelly CJ. Regulation of intracellular polyamine biosynthesis and transport by NO and cytokines TNF-alpha and IFN-gamma. Am J Physiol Cell Physiol 276: C892–C899, 1999.[Abstract/Free Full Text]

40. Satriano J, Isome M, Casero RA Jr, Thomson SC, Blantz RC. Polyamine transport system mediates agmatine transport in mammalian cells. Am J Physiol Cell Physiol 281: C329–C334, 2001.[Abstract/Free Full Text]

41. Satriano J, Kelly CJ, Blantz RC. An emerging role for agmatine. Kidney Int 56: 1252–1253, 1999.[CrossRef][Web of Science][Medline]

42. Satriano J, Matsufuji S, Murakami Y, Lortie MJ, Schwartz D, Kelly CJ, Hayashi S, Blantz RC. Agmatine suppresses proliferation by frameshift induction of antizyme and attenuation of cellular polyamine levels. J Biol Chem 273: 15313–15316, 1998.[Abstract/Free Full Text]

43. Scalabrino G, Ferioli ME. Polyamines in mammalian tumors. Part I. Adv Cancer Res 35: 151–268, 1981.[Web of Science][Medline]

44. Scalabrino G, Ferioli ME. Polyamines in mammalian tumors. Part II. Adv Cancer Res 36: 1–102, 1982.[Web of Science][Medline]

45. Schechter PJ, Barlow JLR, Sjoerdsma A. Clinical aspects of inhibition of ornithine decarboxylase with emphasis on therapeutic trials of eflornithine (DFMO) in cancer and protozoan diseases. In: Inhibition of Polyamine Metabolism, edited by McCann PP, Pegg AE, and Sjoerdsma A. Orlando, FL: Academic, 1987, p. 345–367.

46. Schipper RG, Romijn JC, Cuijpers VM, Verhofstad AA. Polyamines and prostatic cancer. Biochem Soc Trans 31: 375–380, 2003.[CrossRef][Web of Science][Medline]

47. Schipper RG, Verhofstad AA. Distribution patterns of ornithine decarboxylase in cells and tissues. Facts, problems, and postulates. J Histochem Cytochem 50: 1143–1160, 2002.[Abstract/Free Full Text]

48. Seiler N. Pharmacological properties of the natural polyamines and their depletion by biosynthesis inhibitors as a therapeutic approach. Prog Drug Res 37: 107–159, 1991.[Medline]

49. Seiler N, Delcros JG, Moulinoux JP. Polyamine transport in mammalian cells. An update. Int J Biochem Cell Biol 28: 843–861, 1996.[CrossRef][Web of Science][Medline]

50. Seiler N, Dezeure F. Polyamine transport in mammalian cells. Int J Biochem 22: 211–218, 1990.[CrossRef][Web of Science][Medline]

51. Suzuki T, He Y, Kashiwagi K, Murakami Y, Hayashi S, Igarashi K. Antizyme protects against abnormal accumulation and toxicity of polyamines in ornithine decarboxylase-overproducing cells. Proc Natl Acad Sci USA 91: 8930–8934, 1994.[Abstract/Free Full Text]

52. Tabor CW, Tabor H. Polyamines. Annu Rev Biochem 53: 749–790, 1984.[CrossRef][Web of Science][Medline]

53. Vargiu C, Cabella C, Belliardo S, Cravanzola C, Grillo MA, Colombatto S. Agmatine modulates polyamine content in hepatocytes by inducing spermidine/spermine acetyltransferase. Eur J Biochem 259: 933–938, 1999.[Web of Science][Medline]

54. Weeks RS, Vanderwerf SM, Carlson CL, Burns MR, O'Day CL, Cai F, Devens BH, Webb HK. Novel lysine-spermine conjugate inhibits polyamine transport and inhibits cell growth when given with DFMO. Exp Cell Res 261: 293–302, 2000.[CrossRef][Web of Science][Medline]

55. White FC, Benehacene A, Scheele JS, Kamps M. VEGF mRNA is stabilized by ras and tyrosine kinase oncogenes, as well as by UV radiation—evidence for divergent stabilization pathways. Growth Factors 14: 199–212, 1997.[Web of Science][Medline]




This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
M. A. Arndt, V. Battaglia, E. Parisi, M. J. Lortie, M. Isome, C. Baskerville, D. P. Pizzo, R. Ientile, S. Colombatto, A. Toninello, et al.
The arginine metabolite agmatine protects mitochondrial function and confers resistance to cellular apoptosis
Am J Physiol Cell Physiol, June 1, 2009; 296(6): C1411 - C1419.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/2/C705    most recent
00084.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Isome, M.
Right arrow Articles by Satriano, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Isome, M.
Right arrow Articles by Satriano, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.