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Am J Physiol Cell Physiol 292: C1204-C1215, 2007. First published October 25, 2006; doi:10.1152/ajpcell.00451.2006
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GROWTH, DIFFERENTIATION, AND APOPTOSIS

Spermidine/spermine N1-acetyltransferase overexpression in kidney epithelial cells disrupts polyamine homeostasis, leads to DNA damage, and causes G2 arrest

Kamyar Zahedi,1 John J. Bissler,1 Zhaohui Wang,2 Anuradha Josyula,1 Lu Lu,1 Paula Diegelman,3 Nick Kisiel,3 Carl W. Porter,3 and Manoocher Soleimani2,4

1Division of Nephrology and Hypertension, Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio; 2Division of Nephrology and Hypertension, Department of Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio; 3Roswell Park Cancer Institute, Buffalo, New York; and 4Department of Veterans Affairs Medical Center, Cincinnati, Ohio

Submitted 23 August 2006 ; accepted in final form 18 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Expression of spermidine/spermine N1-acetyltransferase (SSAT) increases in kidneys subjected to ischemia-reperfusion injury (IRI). Increased expression of SSAT in vitro leads to alterations in cellular polyamine content, depletion of cofactors and precursors of polyamine synthesis, and reduced cell proliferation. In our model system, a >28-fold increase in SSAT levels in HEK-293 cells leads to depletion of polyamines and elevation in the enzymatic activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, suggestive of a compensatory reaction to increased polyamine catabolism. Increased expression of SSAT also led to DNA damage and G2 arrest. The increased DNA damage was primarily due to the depletion of polyamines. Other factors such as increased production of H2O2 due to polyamine oxidase activity may play a secondary role in the induction of DNA lesions. In response to DNA damage the ATM/ATR -> Chk1/2 DNA repair and cell cycle checkpoint pathways were activated, mediating the G2 arrest in SSAT-expressing cells. In addition, the activation of ERK1 and ERK2, which play integral roles in the G2/M transition, is impaired in cells expressing SSAT. These results indicate that the disruption of polyamine homeostasis due to enhanced SSAT activity leads to DNA damage and reduced cell proliferation via activation of DNA repair and cell cycle checkpoint and disruption of Raf -> MEK -> ERK pathways. We propose that in kidneys subjected to IRI, one mechanism through which increased expression of SSAT may cause cellular injury and organ damage is through induction of DNA damage and the disruption of cell cycle.

ischemia-reperfusion injury; polyamine depletion; cell proliferation; DNA repair; cell cycle arrest

THE EXPRESSION AND ACTIVITY of spermidine/spermine N1-acetyltransferase (SSAT) is upregulated in kidney, brain, and liver after ischemia-reperfusion injury (IRI) (4, 21). SSAT is the rate-limiting enzyme in polyamine catabolism; it acetylates spermidine (SPD) and spermine (SPM) and leads to their export out of the cell and/or degradation by polyamine oxidase (PAO) (13). Oxidation of acetylated polyamines through the activity of PAO leads to production of potentially toxic metabolites including N-acetyl 3-aminopropanol and H2O2. The depletion of polyamines and accumulation of the by-products of polyamine catabolism also may contribute to tissue damage associated with IRI.

Polyamines (putrescine, SPD, and SPM) are aliphatic cations derived from ornithine (32, 38). Their levels are tightly regulated, and any disturbance in their homeostasis is detrimental to cell growth. These molecules play a fundamental role in the stabilization of DNA structure, regulation of gene expression, protein synthesis, and signal transduction, as well as modulation of cell growth and differentiation (20, 25, 26, 32). Polyamine depletion through inhibition of ornithine decarboxylase (ODC) leads to enhanced expression of p53, p21, and p27 cyclin-dependent kinase inhibitors and G1 arrest (3, 29, 30, 39). In cultured cells, increased SSAT activity leads to depletion of SPD and SPM (53). This disturbance in cellular polyamine homeostasis is associated with an increase in the population of G2 arrested cells and decreased cell growth (42, 53). The cause of decreased cell growth in SSAT-expressing cultures is not clear; however, recent studies indicate that depletion of polyamines, reduction in the cellular content of cofactors required for their biosynthesis, and accumulation of catabolic by-products of polyamine pathway are contributing factors (27, 52).

Regulation of cell proliferation is crucial to recovery from acute renal injuries (36, 37). Cell proliferation is regulated through the interaction of complex signaling pathways that affect the expression and activation of the molecular machinery of cell cycle (35–37, 41). In cells subjected to hypoxia and reoxygenation, DNA replication aberrancies and double-stranded breaks are the major causes of genomic instability (18, 19). The presence of damaged DNA leads to activation of cell cycle checkpoints that slow progression through the cell cycle to allow time for DNA repair (5). The DNA repair process is activated in response to oxidative stress via the activation of ATM and ATR kinases and leads to cell cycle arrest or apoptosis in severely damaged cells (18, 19). Polyamine depletion leads to modification of chromatin and DNA structures, increases DNA susceptibility to damage by various genotoxic agents, and interferes with DNA repair (44–46). The DNA alterations in polyamine-depleted cells may be due to DNA strand breaks as well as inhibition of topoisomerase II function (2). Mammalian cell division also requires the activation of the Raf -> MEK -> ERK pathway for G2/M progression (31, 33, 55). The role of polyamine catabolism in the mediation of DNA damage resulting from hypoxia and reoxygenation is not clear.

The role of SSAT in the pathophysiology of IRI is not understood. Previous studies have suggested that increased SSAT expression in kidney cells leads to reduced cell growth and oxidative stress (53). In current studies, we extend this observation by demonstrating that increased SSAT expression leads to DNA damage, activation of DNA repair pathway, disruption of ERK activation, and G2 arrest. On the basis of these results, we propose that disruption of polyamine metabolism contributes to the development of DNA lesions and cell damage associated with IRI.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Expression vectors and development of stable transfectants. Stable transfectants were developed as previously described (53). To examine the effect of SSAT in more detail, using the Quick Change PCR-based site-directed mutagenesis kit (Stratagene), we generated two constructs that were used to express an inactive SSAT and a stabilized SSAT isoform (12–14). After the sequence alterations were confirmed, HEK-293 cells were transfected with the tetracycline-inducible inactive and stable SSAT expression construct. Stable transfectants were isolated and expanded similarly to the wild-type SSAT.

Cell culture studies. HEK-293 cells were seeded at 1 x 105 cells per 100-mm plate. At 60–70% confluency the cells were growth arrested by replacing the normal growth medium with growth medium containing 1% FBS. Cells were released from arrest by addition of normal growth medium or normal growth medium plus tetracycline (1 µg/ml). Both the adherent and nonadherent cells were harvested and pooled at timed intervals, counted, and processed for flow cytometry, cell extract preparation, comet assay, or analysis of polyamine levels. In the experiments examining the effect of pifithrin-{alpha} (100 µM), SPD (50 µM), and MDL 72527 (50 µM) on cell proliferation, tetracycline-treated and uninduced cells not treated with the aforementioned reagents were used as controls.

Flow cytometric analysis of cell cycle. Cells (2 x 106) were harvested, washed in PBS, fixed in 70% ethanol, and stained with propidium iodide [40 µg/ml propidium iodide (PI), 100 µg/ml RNaseA in calcium- and magnesium-free PBS]. The cells (20,000 independent events) were analyzed for their DNA content with a fluorescence-activated cell sorter.

DNA comet assay. Cells (12,000–15,000) from tetracycline-induced and control cultures were suspended in low-melting-point agarose (Invitrogen; final concentration 0.75%) in PBS and layered on microscope slides. The slides were placed in neutral lysis buffer (1 mM Tris·HCl, pH 7.4, 150 mM NaCl, 4 mM EDTA, and 18 mM N-laurylsarcosine) and then into deionized H2O. Electrophoresis was performed at 200 mA for 10 min in TBE buffer (230 mM Tris, 180 mM boric acid, and 0.2 mM EDTA). After electrophoresis, slides were immersed in a PI solution (2.5 µg/ml; Sigma-Aldrich) and rinsed in deionized H2O, and coverslips were placed on the gels. Slides were analyzed using a Nikon inverted fluorescence microscope with attached charge-coupled device camera. Images were saved and tail moments were determined using CometScore freeware (TriTek, Sumerduck, VA).

Measurement of cellular polyamine levels and activity of enzymes. SSAT activity was assayed radiochemically as described previously (6). Both ODC and S-adenosylmethionine decarboxylase (SAMDC) activities were determined using a CO2 trap assay, as previously described (28), and reported as nanomoles of radiolabeled CO2 per hour per milligram of protein. Polyamine levels were determined as described previously (53, 59). Briefly, intracellular polyamines, including acetylated derivatives of SPD and SPM, were extracted from cell pellets with 0.6 N perchloric acid, dansylated, and measured using reverse-phase high-performance liquid chromatography.

Western blot analysis of cell extracts. Cells were harvested, counted, and washed twice in cold PBS. Extracts were prepared by lysing the cells in lysis buffer (150 mM NaCl, 50 mM TrisHCl, pH 8.0, 2.5 mM MgCl2, 10.3 mM NaF, 1.05 mM Na3VO4, 5 mM sodium pyrophosphate, 1% IGEPAL, 0.8 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 2 µg/ml antipain, 10 µg/ml benzamidine, 1 µg/ml chymostatin, and 1 µg/ml pepstatin) for 20 min at 4°C. The lysates were cleared by centrifugation at 16,000 g for 30 min at 4°C. For Western blot analysis, 20–50 µg of each extract were size-fractionated on a 12% polyacrylamide gel and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% milk proteins and then probed with primary antibodies against p53, p21, Chk1/2, phosphorylated Chk1/2, cyclin B1, and threonine 161-phosphorylated Cdc2, ERK1/2, and phosphorylated ERK1/2. Equal loading of samples was assured by Western blot examination of beta-tubulin levels. After washing, filters were incubated with appropriate secondary antibody conjugated to horseradish peroxidase, and the sites of antigen-antibody interaction on the membranes were visualized using chemiluminescence (ECL kit; Amersham) and captured on light-sensitive imaging film.

Assay for H2O2 production. Production of H2O2 by tetracycline-induced and control cultures was examined using the Amplex red H2O2/peroxidase assay (Invitrogen) following the manufacturer's protocol.

Assay for DNA methylation. Genomic DNA from tetracycline-induced and control cells was extracted. Combined bisulfite restriction analysis (COBRA), a PCR-based method, was used to assess the CpG methylation status of the genomic DNA (9). Briefly, DNA was subjected to bisulfite treatment with the EZ DNA methylation gold kit (Zymo Research) following the manufacturer's protocol. Bisulfite treatment can distinguish nonmethylated from methylated cytosine, since only nonmethylated cytosines will be deaminated. Bisulfite-treated DNA (0.25 ng) was used for PCR reactions with LINE-1 element forward 5'-TTGAGTTGTGGTGGGTTTTATTTAG-3' and reverse 5'-TCATCTCACTAAAAAATACCAAACA-3' primers, using the following amplification protocol: 5 min at 95°C, followed by 35 cycles of amplification (95°C for 30 s, 50°C for 30 s, and 72°C for 30 s). Whereas bisulfite treatment leads to the formation of a Tas1 site in the LINE-1 elements of unmethylated DNA, it leads to the formation of a TaqI site in the LINE-1 elements of methylated DNA. Following amplification, products were digested with TaqI and TasI restriction endonucleases to differentiate methylated and nonmethylated DNA. Digestion of the PCR product of methylated DNA with TaqI restriction endonuclease leads to the formation of two 80-bp fragments, whereas digestion of the PCR product of unmethylated DNA leads to formation of 97- and 63-bp fragments. Restriction digest patterns were compared and quantitated densitometrically in amplicons from SSAT-expressing and control cells. To compare the differences in DNA methylation, the ratios of methylated amplicons to total amplicon content in control and tetracycline-treated cells were compared.

Statistical analyses. Values are expressed as means ± SE. The significance of difference between mean values was examined using ANOVA. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of SSAT overexpression on proliferation of renal epithelial cells. To determine the level of induction of SSAT in HEK cells, we examined the activity of this enzyme in control and tetracycline-induced cells. Our results indicate that the activity of SSAT is increased ~28-fold in tetracycline-induced cells (Table 1). The increase in SSAT expression is accompanied by increases in the activity of ODC and SAMDC, enzymes involved in polyamine anabolism, of ~17–25 and >30-fold, respectively (Table 1). These results indicate the activation of a compensatory response to counter the effect of significant reductions in the cellular content of SPD and SPM by SSAT induction. In addition to significant reductions in SPD and SPM levels, there were dramatic increases in the intracellular contents of the products of SSAT activity, N1-acetylspermine (AC-SPM) and N1-acetylspermidine (AC-SPD) levels, in HEK-293 cells (Table 2). Using direct cell count, we examined the effect of SSAT expression on cell proliferation. Our results indicate that disruption of polyamine homeostasis caused by the induction of SSAT expression leads to decreased cell proliferation without affecting cell viability (Fig. 1A). To determine whether the reduction in cell proliferation was due to increased SSAT activity, HEK-293 cells conditionally overexpressing a stable isoform of SSAT (LH) that is resistant to ubiquitin-mediated degradation or an inactive mutant (Mut) of SSAT were developed. The activity and properties of these proteins have been characterized in previously published studies (13, 14). Our data indicate that although the induction of wild type (Fig. 1A) and stable SSAT (Fig. 1B) reduced cell proliferation, increased expression of inactive SSAT mutant (Fig. 1C) had no effect on cell proliferation.


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  		Table 1. Effect of increased expression of SSAT on the activity of ODC and SAMDC

 

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  		Table 2. Effect of SSAT expression on the polyamine content of HEK-293 cells

 

Figure 1
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  		Fig. 1. Effect of wild-type, stable, and inactive spermidine/spermine N1-acetyltransferase (SSAT) on proliferation of HEK-293 cells. HEK-293 cells capable of conditionally expressing wild type (WT; A) stabilized (LH; B), and inactive (Mut; C) SSAT isoforms were cultured as described in MATERIALS AND METHODS. SSAT expression was induced in semiconfluent monolayers by treatment with tetracycline (1 µg/ml in 70% ethanol). Control cultures were treated with an identical volume of solvent (70% ethanol). Proliferation of SSAT expressing ({circ}) and control (bullet) cultures were examined by direct cell count at timed intervals (24 to 144 h). Similar results were obtained in 3 independent experiments. The data represent mean ± SE from 3 independent experiments. *P ≤ 0.01; **P ≤ 0.005.

 
Effect of SSAT overexpression on cell cycle progression. The cell cycle distribution of HEK-293 cells and HEK-293 cells expressing increased levels of SSAT were examined on days 4 and 6 after addition of tetracycline. Examination of day 4 cultures indicated the presence of an increased population of G2-arrested cells in SSAT-expressing cells (Fig. 2B) but not in control cells (Fig. 2A). Examination of day 6 tetracycline-induced cultures (Fig. 2D) indicates that, in addition to having an increased population of G2-blocked cells, these cultures had a significant population of endoreduplicated cells (third peak in Fig. 2D). Untreated cells did not exhibit any increase in G2 subpopulation, and endoreduplicated cells were completely absent from these cultures (Fig. 2C). These data indicate that increased expression of SSAT in HEK-293 cells leads to G2 arrest and the appearance of endoreduplicated cells.


Figure 2
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  		Fig. 2. Effect of SSAT on cell cycle progression of HEK cells. SSAT expression was induced in semiconfluent monolayers by treatment with tetracycline (1 µg/ml in 70% ethanol). Control cultures were treated with identical volume of solvent (70% ethanol). Flow cytometric analysis of DNA content of the cells was used to examine the cell cycle distribution of control HEK cells on days 4 (A) and 6 (C) and of HEK cells that overexpress SSAT on days 4 (B) and 6 (D). The first (left) peak indicates the number of cells with 2n DNA content (G1 population), the second peak represents the cells with 4n DNA content (G2 population), and the third peak (far right, only present in B and D) represents cells with 8n DNA content (endoreduplicated cells). The increase in the height of the second and third peaks observed in the SSAT overexpressers (B and D) indicates that SSAT overexpression, possibly through polyamine depletion and/or increased expression of toxic metabolites, leads to G2 block and cell cycle arrest. Similar results were obtained in 3 independent experiments.

 
Polyamine supplementation does not alter the effect of SSAT expression on cell proliferation. Decreased proliferation of SSAT-expressing cells may be due to reduced intracellular content of polyamines. We therefore examined the effect of polyamine (50 µM SPD) supplementation on cell growth in normal and SSAT-expressing cells. Examination of polyamine levels in control cultured cells supplemented with SPD compared with levels in cells without the supplement indicates that the SPD content of the former was increased by ~9% in 24-h, 31% in 48-h, 55% in 72-h, and 20% in 96-h control cultures (1,100 to 3,500 pmol/106 cells). Similarly, the SPM content of the control cultures was increased by ~1% in 24-h, 1% in 48-h, 9% in 72-h, and 6% in 96-h control cultures (100 to 1,300 pmol/106 cells). However, SPD supplementation did not lead to increased cellular SPD or SPM content in SSAT-expressing cells and, indeed, led to increased accumulation of AC-SPM in 24-h samples (~37%) and increased putrescine buildup (2- to 19-fold) in samples from all time points. These results are most likely due to prompt acetylation of incoming polyamines by SSAT such that as soon as they enter the cell, they are immediately acetylated and exported or shunted to the PAO pathway. Addition of SPD had no effect on the proliferation of normal cells and did not affect the growth of SSAT-expressing cells (Fig. 3A). Furthermore, comparison of cell cycle profiles of SSAT-expressing cells at 72 h after addition of tetracycline and time-matched control cultures in the absence or presence of SPD revealed that its supplementation does not affect the cell cycle distribution of either the control (Fig. 3B, iii) or SSAT-overexpressing cells (Fig. 3B, iv) compared with control or SSAT-expressing cells in the absence of SPD supplementation (Fig. 3B, i and ii). Other studies have indicated that supplementation with putrescine or SPM also has no effect on cell growth in normal and SSAT-expressing cells (data not shown). These results suggest that polyamine supplementation alone is not sufficient to overcome the adverse effects of SSAT expression.


Figure 3
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  		Fig. 3. Effect of spermidine (SPD) supplementation on proliferation and cell cycle progression of control and SSAT-expressing HEK-293 cells. SSAT expression was induced in HEK-293 cells by treatment with tetracycline (1 µg/ml in 70% ethanol). Control HEK cultures were treated with identical volumes of solvent (70% ethanol). For polyamine supplementation studies, 50 µM SPD was added to cultures receiving the supplement. A: control, control + SPD, tetracycline-induced (Tet), and tetracycline-induced + SPD cultures (Tet+SPD) were harvested at timed intervals. Cell numbers were determined by direct cell count. B: control (i), tetracycline-induced (ii), control + SPD (iii), and tetracycline-induced + SPD cultures (iv) were harvested at 72 h, fixed, and stained with propidium iodide. Cell cycle distribution of each sample was determined by fluorescence-activated cell sorting analysis of cellular DNA content of 20 x 104 cells. Similar results were obtained in 3 independent experiments.

 
SSAT-expressing cells produce increased H2O2 levels. Previous studies in our laboratory (53) indicated that increased expression of SSAT is accompanied by increased expression of PAO. Oxidation of the products of SSAT reaction (acetylated SPD and SPM) by PAO may contribute to cell damage through production of cytotoxic compounds such as H2O2. We therefore compared the production of H2O2 by SSAT-expressing cells with that by control cells. Examination of H2O2 concentrations in culture supernatants indicated that the production of H2O2 by tetracycline-induced SSAT-expressing cells was 2.5- and 1.5-fold higher than control cells at 24 (0.56 ± 0.02 vs. 0.26 ± 0.02 µmol/106 cells) and 48 h (0.31 ± 0.02 vs. 0.17 ± 0.01 µmol/106 cells) after induction of SSAT (Fig. 4). Furthermore, studies qualitatively comparing control and SSAT-expressing cells also revealed the latter to produce higher levels of H2O2 at both 24- and 48-h time points (data not shown).


Figure 4
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  		Fig. 4. Effect of SSAT expression on H2O2 production. The production of H2O2 was examined by measuring the levels of H2O2 in the supernatants of SSAT expressing (Tet) and control cultures normalized for the number of cells at 24- and 48-h time points. Results are means ± SE of 3 samples from 2 independent experiments.

 
We next examined the effect of inhibition of PAO, the activity of which leads to the production of H2O2 and other toxic molecules such as acetylaminopropanol and putrescine, on cell proliferation. The activity of PAO was inhibited with the use of MDL 72527 (50 µM), a proven inhibitor of polyamine and spermine oxidases (SMO) (43). The comparison of cell proliferation in control and SSAT-expressing cells revealed that treatment with MDL 72527 does not affect the SSAT-mediated reduction in cell proliferation (data not shown), suggesting that the inhibition of PAO per se does not reverse the reduction in cell growth. These results suggest that products of PAO-catalyzed reactions are not the primary cause of cellular injury brought about by SSAT overexpression, but rather they may contribute to increased injury in cells that are physiologically compromised due to polyamine depletion.

SSAT overexpression leads to DNA damage and activates the DNA repair response. Polyamines are crucial to the maintenance of DNA structure and integrity (5, 19, 20). We therefore hypothesized that G2 arrest in SSAT-expressing HEK-293 cells might be due to changes in the DNA integrity. To determine the effect of SSAT overexpression on DNA integrity, we subjected control cells and cells expressing high levels of SSAT to neutral comet assay. Comparison of the tail moments (quantitative measure of DNA integrity) indicated that the degree of DNA damage was significantly higher (P < 0.01) in SSAT-expressing cells than in time-matched control cells. Our results indicate that the DNA of cells overexpressing SSAT is damaged (Fig. 5, a and b). In comparison, the DNA of control cells remained intact as evidenced by the absence of a comet tail (Fig. 5, c and d).


Figure 5
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  		Fig. 5. SSAT expression leads to DNA damage. Comet assays were performed on control and SSAT-expressing cells 72 h after induction. Images in a and b represent the comet pattern from SSAT-expressing cells; images in c and d represent control cells. The average tail moments presented below each set of panels are the quantitative measure of DNA damage and are significantly (P < 0.01) higher for SSAT-expressing cells. Similar results were obtained in 3 separate experiments each examining 50 independent comet samples.

 
Cells with damaged DNA initiate DNA repair process and, if the damage is too extensive, may undergo apoptosis. The DNA repair process is initiated via the activation of a cascade of kinases that ultimately affect the cell cycle and apoptosis machinery. HEK-293 cells used in these studies have a defective p53 pathway (48, 49, 58). In support of this observation, our results indicate that the expression of p53 and p21 was unaffected in control and SSAT-overexpressing cells (data not shown). The role of p53 in SSAT-mediated inhibition of HEK-293 cell proliferation was further studied by examining the effect of pifithrin-{alpha}, a p53 inhibitor, on cell proliferation. The addition of pifithrin-{alpha} did not affect the proliferation rate of control or SSAT-overexpressing HEK-293 cells (data not shown).

Alterations in DNA structure and integrity activate the cell cycle checkpoints (5, 18, 19). Since SSAT-overexpressing cells have increased DNA damage (Fig. 5), we examined the effect of elevated SSAT expression on the activation of DNA repair response. DNA damage in response to oxidative stress leads to activation of ATM and ATR kinases. Activated ATM/ATR kinases in turn phosphorylate and activate Chk1 and Chk2 kinases. The activation of Chk1 and Chk2 leads to cell cycle arrest by regulating the activities of Cdc25 phosophatase and Cdc2 kinase (5, 18, 19). Our results demonstrate that SSAT expression led to an increase in the levels of phosphorylated Chk1 and Chk2 (Fig. 6). In comparison, activated Chk1 and Chk2 levels remained very low in the control cultures (Fig. 6). The increase in the active (phosphorylated) form of these kinases was apparent as early as 12 h after induction of SSAT. Our data indicate that the peak activation of these enzymes was observed by day 3 following induction of SSAT and remained elevated for the duration of our studies. These results suggest that ATM/ATR DNA repair pathways are activated as the result of SSAT expression in HEK-293 cells.


Figure 6
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  		Fig. 6. SSAT induction activates the DNA damage checkpoint. Western blot analyses to determine the activation (phosphorylation) of DNA repair checkpoint enzymes Chk1 and Chk2 were performed on cell extracts obtained from control and SSAT-expressing HEK-293 cells at timed intervals (12 to 144 h) after induction. A: increased levels of phosphorylated Chk1 kinase (p-Chk1) indicate that the DNA damage repair response is activated in HEK cells that express SSAT. B: increased levels of phosphorylated Chk2 kinase (p-Chk2) indicate that the DNA damage repair response is activated in HEK cells that express SSAT. Examination of total Chk1 and Chk2 levels indicates that levels of total Chk1 and Chk2 are similar in control and tetracycline-induced cells. Similar results were observed in 3 independent experiments.

 
Determining the effect of SSAT expression and the onset of polyamine anabolic pathway on DNA methylation. The induction of synthesis of polyamines to compensate for the increase in their catabolism leads to increased consumption of S-adenosylmethionine (SAM) and increased production of decarboxylated SAM (dcSAM). The former is a substrate needed for DNA methyltransferases, and the latter is an aminopropyl group donor for polyamine synthesis as well as an inhibitor of DNA methyltransferases. A compensatory increase in polyamine synthesis as evidenced by increased ODC and SAMDC activity leads to depletion of SAM and an increase in dcSAM levels; together, these may contribute to a reduction in DNA methylation and the induction of DNA anomalies (10, 15, 34). To test the above suggestion, we examined the effect of SSAT expression on global genomic DNA methylation using COBRA. Comparing the amplification products of LINE-1 elements from control and SSAT-expressing cells for their sensitivity to digestion with TaqI and TasI indicates that the methylation of LINE-1 elements is not different in SSAT-expressing cells and control cells is not different in SSAT-expressing cells and control cells (data not shown). These results seem to rule out the role of reduced DNA methylation as the primary mechanism of DNA damage in SSAT-overexpressing cells in our system.

MAPK signaling is disrupted in SSAT-expressing cells. To further decipher the signaling pathway involved in SSAT-mediated inhibition of cell proliferation, we decided to examine the activation status of ERK. We chose to examine the ERK activation pathway, since the Raf -> MEK -> ERK pathway has been implicated in the upregulation of expression of cell cycle machinery and promotion of cell division (33, 55). Examination of extracts obtained from control and SSAT-overexpressing HEK cells (Fig. 7) indicates that both populations express equal amounts of ERK1 and ERK2 proteins. Examination of the same extracts for the presence of the active (phosphorylated) form of ERK1 and ERK2 indicates that SSAT overexpression leads to a reduction in ERK activation as early as day 3 postinduction and a drastic reduction in the cellular levels of activated ERK1 and ERK2 by days 6 and 9 post-SSAT induction. These results suggest that the ERK signaling pathway, which plays an important role in regulation of cell proliferation, is disrupted in cells that express SSAT; however, the lateness of disruption in ERK signaling suggests that it is not the primary mechanism involved in the mediation of G2 arrest of SSAT-expressing cells.


Figure 7
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  		Fig. 7. Effect of SSAT induction on ERK signaling. Western blot analyses to determine the activation (phosphorylation) of ERK1/2 were performed on cell extracts obtained from control and SSAT-expressing HEK-293 cells at timed intervals (24 h to 9 days) after induction. Equal loading was assured by examination of beta-tubulin levels. Similar results were observed in 3 independent experiments. pERK, phosphorylated ERK1/2; Kd, kDa.

 
Effect of SSAT expression on cyclin B1 levels and Cdc2 activation. The transition from G2 to M and completion of mitosis depends on the activity of cyclin B-Cdc2 complex (40, 50). Activated as the result of ATM and ATR activation, Chk kinases directly modulate the function of Cdc25 through phosphorylation of its Ser-216 and prevent it from dephosphorylating Cdc2 (1, 8, 56, 57). ERK activity, on the other hand, is involved in the regulation of a number of events that are required for G2/M transition and completion of mitosis (31, 33, 55). These events include, but are not limited to, the regulation of cyclin B-Cdc2 activity via the modulation of Wee1 and Myt1 (11, 57). To determine whether activation of ATM/ATR -> Chk1/2 and disruption of ERK signaling due to expression of SSAT affect cyclin B-Cdc2 signaling, we examined the levels of cyclin B1, Cdc, and activated Cdc2 (Thr-161 phosphorylated) in cell extracts obtained from control and SSAT-expressing HEK-293 cells. Our results indicate that the induction of SSAT reduces the expression of cyclin B1 and interferes with the activation of Cdc2 (Fig. 8).


Figure 8
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  		Fig. 8. SSAT expression reduces the cyclin B1 levels and interferes with Cdc2 activation. Enhanced levels of SSAT in HEK cells interfere with the activation of Cdc2, (p-Cdc2) as determined by the appearance of its Thr-161 phosphorylated form, and expression of cyclin B1. Equal loading was assured by examination of beta-tubulin levels. Similar results were obtained in 3 independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Expression of SSAT increases significantly in organs (e.g., brain, liver, and kidney) subjected to IRI and in cells after ATP depletion and metabolic poisoning (4, 21, 59). We previously demonstrated that increased expression of SSAT can lead to oxidative stress, reduced cell proliferation, and impaired cell adhesion (53). In the current study, we have examined the effect of SSAT on polyamine homeostasis and determined how its increased levels affect cell proliferation. Our results demonstrate that SSAT expression disrupts the polyamine homeostasis in HEK-293 cells, leading to the depletion of SPD and SPM and a concomitant increase in the acetylated forms of these molecules that are either excreted or shunted to the PAO pathway for degradation (Table 2). Furthermore, we observed a compensatory response in SSAT-expressing cells indicating the activation of polyamine anabolic pathways aimed at reestablishment of normal polyamine levels (Table 1). Comet assay data indicate that SSAT-expressing cells show DNA damage (Fig. 5). The patterns of the comet assays for SSAT-expressing cells are similar to those of viable cells with DNA damage and not apoptotic cells (7, 24). Our studies also indicate that increased SSAT expression leads to the activation of ATM/ATR pathway 12 h postinduction (Fig. 6), suggesting that DNA damage is detected by checkpoint machinery early after SSAT induction. In addition to activation of the ATM/ATR DNA repair response, SSAT overexpression disrupts the activation of ERK1 and ERK2 (Fig. 7). The aforementioned signaling cascades are known to affect the cell cycle (1, 8, 31, 33, 55–57) and mediate the onset of G2/M transition arrest in SSAT-expressing cells.

Polyamines play an important role in the regulation of DNA structure, modulation of signal transduction, gene expression, and protein synthesis (20, 25, 26, 32). They are necessary for cell growth, and their depletion reduces cell proliferation. Previous studies indicated that inhibition of polyamine synthesis by {alpha}-difluoromethylornithine (DMFO), an inhibitor of ODC, leads to G1 arrest (3, 29, 30, 39). Cells treated with DMFO exhibit increased expression levels of p53 as well as cyclin-dependent kinase inhibitors p16, p21 and p27 (3, 29, 30, 39). In contrast to DFMO treatment, increased expression of SSAT leads to G2 arrest (42). A number of factors may account for the growth arrest induced by SSAT, including depletion of polyamines, SAM, and acetyl-CoA or increased production of toxic by-products of polyamine catabolism (e.g., H2O2 and aldehydes). Examination of cell lines from different origins indicates that all of the aforementioned factors contribute to reduced proliferative activity of SSAT-overexpressing cells (27, 52). For example, whereas growth retardation of MCF-7 cells conditionally expressing SSAT is thought to be due to polyamine depletion, the reduced proliferative activity of LNCaP prostate carcinoma cells after induction of SSAT appears to be due to depletion of metabolic precursors needed for polyamine synthesis (27, 52).

Our studies indicate that increased SSAT expression leads to DNA damage (Fig. 5). The SSAT-induced DNA damage may be mediated through multiple mechanisms including polyamine depletion and/or increased production of H2O2. Our results demonstrating that the addition of exogenous polyamines had no effect on the proliferative activity of SSAT-expressing HEK-293 cells (Fig. 3) indicate that polyamine supplementation alone does not overcome the effects of increased SSAT activity. This is most likely due to immediate acetylation of incoming polyamines that are then either shunted to the PAO pathway or exported out of the cell. This possibility is supported by our data indicating that polyamine supplementation leads to increased cellular content of polyamines in control but not SSAT-expressing cells. The H2O2 generated during polyamine catabolism results from the activity of PAO and SMO. Shunting of acetylated polyamines to the PAO or SMO pathway leads to increased production of H2O2 and other toxic metabolites that may play a significant role in the mediation of DNA damage and reduction in cell proliferation. Previous studies by our group (51) using catalase suggested that H2O2 does not play a significant role in cell growth and proliferation. We therefore examined the effect of inhibition of PAO on cell proliferation in SSAT-expressing cells. The inhibition of PAO fails to restore the proliferative activity in SSAT-expressing HEK cells (data not shown), confirming published reports (24) and supporting the possibility that the PAO-catalyzed reaction that leads to the production of H2O2 and other toxic metabolites does not play a primary role in the induction of DNA damage and reduction of cell proliferation, at least in vitro. Transcript levels of the polyamine oxidases PAO and SMO increase in kidneys subjected to IRI (Ref. 55; unpublished data). The inhibition of these enzymes leads to reduced tissue damage in cerebral IRI, suggesting that they play a significant role in the induction of tissue damage in ischemic conditions in vivo (16). Coupled to studies that indicate the induction of PAO (49) but not SMO (data not shown) in SSAT-expressing HEK cells, we propose that the dramatic reduction in polyamine levels plays a primary role in the mediation of cell damage. It is very plausible that PAO activation exacerbates the cell damage by increasing the production of H2O2 and other toxic metabolites.

The depletion of SAM levels also may contribute to reduced growth rate in SSAT-expressing cells (27). It is plausible that the upregulation of polyamine synthesis (increased activity of ODC and SAMDC) to compensate for the increase in their catabolism leads to increased consumption of SAM and increased production of dcSAM (17, 22, 23, 27, 51). The former is a substrate needed for DNA methyltransferases, and the latter is an aminopropyl group donor for polyamine synthesis as well as an inhibitor of DNA methyltransferases (17, 22, 23). Therefore, a compensatory increase in polyamine synthesis through depletion of SAM and an increase in dcSAM levels may lead to a reduction in the methylation levels of cellular DNA (17, 22, 23), potentially leading to increased DNA damage, genomic instability, and growth arrest (10, 15, 34). However, our data indicate that increased SSAT expression in HEK cells does not affect the state of DNA methylation (Fig. 7) and that DNA methylation anomalies are not involved in the mediation of DNA damage in our model system.

The depletion of polyamine content and increased H2O2 production correlate closely with the activation of DNA repair checkpoint enzymes. However, since PAO inhibition does not lead to normalization of growth rate, we propose that the induction of PAO and increased production of H2O2 in SSAT-expressing cells are not of primary importance and, in isolation, do not account for the DNA damage and reduction in cell growth in the current model. Our results support the hypothesis that polyamine depletion is the primary factor that affects DNA integrity and cell proliferation in SSAT-overexpressing cells. Previous studies demonstrating that depletion of SPM leads to DNA strand breaks and that polyamine depletion leads to modification of chromatin and DNA structure, increases susceptibility to damage by genotoxic agents, and interferes with DNA repair support our hypothesis that depletion of SPD and SPM is the primary factor involved in the induction of DNA damage and growth arrest in SSAT-expressing cells (2, 45, 47, 54).

SSAT levels increase in tissues (e.g., kidney, brain, and liver) subjected to hypoxia reoxygenation injuries (4, 21, 59). A possible effect of SSAT expression in IRI may be the induction of DNA lesions. In our model, increased SSAT expression leads to DNA damage and cell cycle arrest as evidenced by cell cycle analysis and comet assay results. It is therefore possible that SSAT production partially contributes to the induction of DNA lesions in IRI. The mechanisms and signaling cascades induced by increased DNA damage due to elevated levels of SSAT and polyamine depletion involved in G2/M transition arrest were therefore examined. Our findings indicate that the DNA damage repair response (ATM/ATR -> Chk1/Chk2) is activated in SSAT-expressing cells. The role of ATM/ATR signaling in DNA damage repair subsequent to hypoxia and reoxygenation has been examined (18, 19). The activation of ATM/ATR -> Chk1/Chk2 cascades in response to DNA damage leads to inactivation of Cdc25, disruption of Cdc2 activation, and G2 arrest (1, 8, 56, 57). Our data also indicate that ERK activation is disrupted in cells that express SSAT. The Raf -> MEK -> ERK pathway plays an integral role in the modulation of inhibitory phosphorylation of Cdc2 by Wee1 and Myt1 and in G2/M transition (11, 57). Dysregulation of Cdc2 activity may be an additional mode of induction of G2 arrest in cells that express SSAT. In our studies, we observed a decrease in the cellular content of cyclin B1 and Cdc2 in SSAT-expressing cells. Hence, depletion of polyamines can potentially modulate both the expression of cyclin B1 and the activation of cyclin B1-Cdc2 complex.

Increased SSAT levels seem to affect multiple pathways that regulate cell division. The schematic in GoFig. 9 outlines the potential mechanisms that may be involved in the mediation of the effect of SSAT on cell proliferation (i.e., depletion of cellular contents of precursors of polyamines and polyamines, changes in DNA, and increased levels of toxic metabolites). Our results suggest that SSAT expression enhances the catabolism of SPD and SPM, leading to depletion of polyamine levels, which may lead to changes in chromatin structure and increased DNA instability through a reduced capacity to buffer the repulsion of negatively charged DNA backbone. In addition, in SSAT-expressing cells, the DNA integrity may be compromised through the production of toxic metabolites such as H2O2. However, the latter mechanism is at best of secondary importance in our model system. The DNA damage in SSAT-expressing cells leads to activation of ATM/ATR -> Chk1/Chk2 pathways and G2/M arrest. The mechanism by which SSAT expression downregulates MAPK activation and the importance of disruption of this signaling cascade to early events involved in the modulation of cell proliferation remain to be elucidated. However, based on the role of Raf -> MEK -> ERK in G2/M transition (11, 57), it is not surprising that the disruption of this pathway also adversely affects cell proliferation.


Figure 9
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  		Fig. 9. Schematic of potential pathways affected by SSAT that mediate its antiproliferative activity. In our proposed scheme, increased SSAT expression leads to enhanced catabolism of SPD and SPM(polyamine depletion). Catabolism of polyamines through polyamine oxidase (PAO) activity leads to enhanced production of H2O2. In addition, the onset of a compensatory response aimed at reestablishing polyamine homeostasis may lead to a reduction in S-adenosylmethionine (SAM) levels and a buildup of decarboxylated SAM (dcSAM) and reduced DNA methylation. These alterations may lead to DNA damage, ATM/ATR -> Chk1/2 pathway activation, and G2/M arrest. Polyamine depletion also disrupts the Raf -> MEK -> ERK pathway; based on the role of Raf -> MEK -> ERK in G2/M transition, the disruption of this pathway also may adversely affect cell proliferation. In addition, downregulation of cyclin B1 expression and Cdc2 activity also compromises the proliferative activity of SSAT-expressing cells. Our results suggest that in our system, the primary driving mechanism behind the increased DNA damage and growth reduction is the depletion of cellular polyamines, whereas increased H2O2 production may play a secondary role and DNA methylation plays no role in the induction of DNA damage and reduced cell proliferation. The components of the proposed scheme that contribute to reduced cell proliferation in our model system are shown in boxes with bold outlines.

 
The current studies begin to address how SSAT contributes to the pathophysiology of renal IRI at the molecular level. We propose that in IRI, one of the mechanisms through which increased SSAT expression contributes to tissue damage is disruption of polyamine homeostasis and induction of DNA damage. Furthermore, it is possible that reduction in cell proliferation due to enhanced expression of SSAT can interfere with the tubular repair process and contribute to ongoing renal damage. Other potential effects of increased SSAT levels and disturbances in polyamine homeostasis such as disruption of cell-cell and cell-matrix interactions as well as metabolic anomalies are currently being studied and need to be considered when examining the role of SSAT in IRI.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
These studies were supported by National Institutes of Health Grants CA-22153 and CA-76428 (to C. W. Porter), DK061458 (to J. J. Bissler), and DK-66589 and Department of Veterans Affairs Merit Review Award (to M. Soleimani).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Soleimani, Division of Nephrology and Hypertension, Dept. of Medicine, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, MSB 259G, Cincinnati, OH 45267 (e-mail: manoocher.soleimani{at}uc.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. Abraham RT. Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev 15: 2177–2196, 2001.[Free Full Text]

2. Alm K, Berntsson P, Oredsson SM. Topoisomerase II is nonfunctional in polyamine-depleted cells. J Cell Biochem 75: 46–55, 1999.[CrossRef][Web of Science][Medline]

3. Alm K, Berntsson PS, Kramer DL, Porter CW, Oredsson SM. Treatment of cells with the polyamine analog N,N11-diethylnorspermine retards S phase progression within one cell cycle. Eur J Biochem 267: 4157–4164, 2000.[Web of Science][Medline]

4. Barone S, Okaya T, Rudich S, Petrovic S, Tehrani K, Wang Z, Zahedi K, Casero RA, Lentsch AB, Soleimani M. Distinct and sequential upregulation of genes regulating cell growth and cell cycle progression during hepatic ischemia-reperfusion injury. Am J Physiol Cell Physiol 289: C826–C835, 2005.[Abstract/Free Full Text]

5. Barzilai A, Yamamoto K. DNA damage responses to oxidative stress. DNA Repair (Amst) 3: 1109–1115, 2004.

6. Bernacki RJ, Oberman EJ, Seweryniak KE, Atwood A, Bergeron RJ, Porter CW. Preclinical antitumor efficacy of the polyamine analogue N1,N11-diethylnorspermine administered by multiple injection or continuous infusion. Clin Cancer Res 1: 847–857, 1995.[Abstract]

7. Bowden RD, Buckwalter MR, McBride JF, Johnson DA, Murray BK, O'Neill KL. Tail profile: a more accurate system for analyzing DNA damage using the Comet assay. Mutat Res 537: 1–9, 2003.[Web of Science][Medline]

8. Brown EJ, Baltimore D. Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance. Genes Dev 17: 615–628, 2003.[Abstract/Free Full Text]

9. Chalitchagorn K, Shuangshoti S, Hourpai N, Kongruttanachok N, Tangkijvanich P, Thong-ngam D, Voravud N, Sriuranpong V, Mutirangura A. Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis. Oncogene 23: 8841–8846, 2004.[CrossRef][Web of Science][Medline]

10. Chen RZ, Pettersson U, Beard C, Jackson-Grusby L, Jaenisch R. DNA hypomethylation leads to elevated mutation rates. Nature 395: 89–93, 1998.[CrossRef][Medline]

11. Chen S, Gardner DG. Suppression of WEE1 and stimulation of CDC25A correlates with endothelin-dependent proliferation of rat aortic smooth muscle cells. J Biol Chem 279: 13755–13763, 2004.[Abstract/Free Full Text]

12. Coleman CS, Huang H, Pegg AE. Role of the carboxyl terminal MATEE sequence of spermidine/spermine N1-acetyltransferase in the activity and stabilization by the polyamine analog N1,N12-bis(ethyl)spermine. Biochemistry 34: 13423–13430, 1995.[CrossRef][Medline]

13. Coleman CS, Huang H, Pegg AE. Structure and critical residues at the active site of spermidine/spermine-N1-acetyltransferase. Biochem J 316: 697–701, 1996.

14. Coleman CS, Pegg AE. Proteasomal degradation of spermidine/spermine N1-acetyltransferase requires the carboxyl-terminal glutamic acid residues. J Biol Chem 272: 12164–12169, 1997.[Abstract/Free Full Text]

15. Dodge JE, Okano M, Dick F, Tsujimoto N, Chen T, Wang S, Ueda Y, Dyson N, Li E. Inactivation of Dnmt3b in mouse embryonic fibroblasts results in DNA hypomethylation, chromosomal instability, and spontaneous immortalization. J Biol Chem 280: 17986–17991, 2005.[Abstract/Free Full Text]

16. Dogan A, Rao AM, Hatcher J, Rao VL, Baskaya MK, Dempsey RJ. Effects of MDL 72527, a specific inhibitor of polyamine oxidase, on brain edema, ischemic injury volume, and tissue polyamine levels in rats after temporary middle cerebral artery occlusion. J Neurochem 72: 765–770, 1999.[CrossRef][Web of Science][Medline]

17. Frostesjo L, Holm I, Grahn B, Page AW, Bestor TH, Heby O. Interference with DNA methyltransferase activity and genome methylation during F9 teratocarcinoma stem cell differentiation induced by polyamine depletion. J Biol Chem 272: 4359–4366, 1997.[Abstract/Free Full Text]

18. Hammond EM, Dorie MJ, Giaccia AJ. ATR/ATM targets are phosphorylated by ATR in response to hypoxia and ATM in response to reoxygenation. J Biol Chem 278: 12207–12213, 2003.[Abstract/Free Full Text]

19. Hammond EM, Giaccia AJ. The role of ATM and ATR in the cellular response to hypoxia and re-oxygenation. DNA Repair (Amst) 3: 1117–1122, 2004.

20. Hasan R, Alam MK, Ali R. Polyamine induced Z-conformation of native calf thymus DNA. FEBS Lett 368: 27–30, 1995.[CrossRef][Web of Science][Medline]

21. Hatcher JF, Dempsey RJ, Rao AM. Elevated N1-acetylspermidine levels in gerbil and rat brains after CNS injury. J Neurosci Res 58: 697–705, 1999.[CrossRef][Web of Science][Medline]

22. Heby O. DNA methylation and polyamines in embryonic development and cancer. Int J Dev Biol 39: 737–757, 1995.[Web of Science][Medline]

23. Heby O, Persson L, Smith SS. Polyamines, DNA methylation and cell differentiation. Adv Exp Med Biol 250: 291–299, 1988.[Medline]

24. Husseini GA, O'Neill KL, Pitt WG. The comet assay to determine the mode of cell death for the ultrasonic delivery of doxorubicin to human leukemia (HL-60 Cells) from Pluronic P105 micelles. Technol Cancer Res Treat 4: 707–711, 2005.[Web of Science][Medline]

25. Igarashi K, Kashiwagi K. Polyamines: mysterious modulators of cellular functions. Biochem Biophys Res Commun 271: 559–564, 2000.[CrossRef][Web of Science][Medline]

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

27. Kee K, Foster BA, Merali S, Kramer DL, Hensen ML, Diegelman P, Kisiel N, Vujcic S, Mazurchuk RV, Porter CW. Activated polyamine catabolism depletes acetyl-CoA pools and suppresses prostate tumor growth in TRAMP mice. J Biol Chem 279: 40076–40083, 2004.[Abstract/Free Full Text]

28. Kramer D, Stanek J, Diegelman P, Regenass U, Schneider P, Porter CW. Use of 4-fluoro-L-ornithine to monitor metabolic flux through the polyamine biosynthetic pathway. Biochem Pharmacol 50: 1433–1443, 1995.[CrossRef][Web of Science][Medline]

29. Kramer DL, Chang BD, Chen Y, Diegelman P, Alm K, Black AR, Roninson IB, Porter CW. Polyamine depletion in human melanoma cells leads to G1 arrest associated with induction of p21WAF1/CIP1/SDI1, changes in the expression of p21-regulated genes, and a senescence-like phenotype. Cancer Res 61: 7754–7762, 2001.[Abstract/Free Full Text]

30. Kramer DL, Vujcic S, Diegelman P, Alderfer J, Miller JT, Black JD, Bergeron RJ, Porter CW. Polyamine analogue induction of the p53-p21WAF1/CIP1-Rb pathway and G1 arrest in human melanoma cells. Cancer Res 59: 1278–1286, 1999.[Abstract/Free Full Text]

31. Liu X, Yan S, Zhou T, Terada Y, Erikson RL. The MAP kinase pathway is required for entry into mitosis and cell survival. Oncogene 23: 763–776, 2004.[CrossRef][Web of Science][Medline]

32. Marton LJ, Pegg AE. Polyamines as targets for therapeutic intervention. Annu Rev Pharmacol Toxicol 35: 55–91, 1995.

33. Mirza AM, Gysin S, Malek N, Nakayama K, Roberts JM, McMahon M. Cooperative regulation of the cell division cycle by the protein kinases RAF and AKT. Mol Cell Biol 24: 10868–10881, 2004.[Abstract/Free Full Text]

34. Nichol K, Pearson CE. CpG methylation modifies the genetic stability of cloned repeat sequences. Genome Res 12: 1246–1256, 2002.[Abstract/Free Full Text]

35. Nony PA, Schnellmann RG. Mechanisms of renal cell repair and regeneration after acute renal failure. J Pharmacol Exp Ther 304: 905–912, 2003.[Abstract/Free Full Text]

36. Park SK, Kang MJ, Kim W, Koh GY. Renal tubule regeneration after ischemic injury is coupled to the up-regulation and activation of cyclins and cyclin dependent kinases. Kidney Int 52: 706–714, 1997.[Web of Science][Medline]

37. Park SK, Kim W, Lee CH, Koh GY. Differential changes of CDK activities in glomeruli and tubules during the active DNA synthetic period after ischemic injury. Nephron 86: 306–314, 2000.[CrossRef][Web of Science][Medline]

38. 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]

39. Ray RM, Zimmerman BJ, McCormack SA, Patel TB, Johnson LR. Polyamine depletion arrests cell cycle and induces inhibitors p21Waf1/Cip1, p27Kip1, and p53 in IEC-6 cells. Am J Physiol Cell Physiol 276: C684–C691, 1999.[Abstract/Free Full Text]

40. Riabowol K, Draetta G, Brizuela L, Vandre D, Beach D. The cdc2 kinase is a nuclear protein that is essential for mitosis in mammalian cells. Cell 57: 393–401, 1989.[CrossRef][Web of Science][Medline]

41. Safirstein R, DiMari J, Megyesi J, Price P. Mechanisms of renal repair and survival following acute injury. Semin Nephrol 18: 519–522, 1998.[Web of Science][Medline]

42. Scorcioni F, Corti A, Davalli P, Astancolle S, Bettuzzi S, Marverti G. Manipulation of the expression of regulatory genes of polyamine metabolism results in specific alterations of the cell-cycle progression. Biochem J 354: 217–223, 2001.[CrossRef][Web of Science][Medline]

43. Seiler N, Duranton B, Raul F. The polyamine oxidase inactivator MDL 72527. Prog Drug Res 59: 1–40, 2002.[Medline]

44. Snyder RD. Effects of metal treatment on DNA repair in polyamine-depleted HeLa cells with special reference to nickel. Environ Health Perspect 102, Suppl 3: 51–55, 1994.

45. Snyder RD. Inhibition of X-ray-induced DNA strand break repair in polyamine-depleted HeLa cells. Int J Radiat Biol 55: 773–782, 1989.[Web of Science][Medline]

46. Snyder RD. Polyamine depletion is associated with altered chromatin structure in HeLa cells. Biochem J 260: 697–704, 1989.[Web of Science][Medline]

47. Snyder RD, Lachmann PJ. Hyperthermia, polyamine depletion, and inhibition of X-ray-induced DNA strand break repair. Radiat Res 120: 121–128, 1989.[Web of Science][Medline]

48. Steegenga WT, Shvarts A, Riteco N, Bos JL, Jochemsen AG. Distinct regulation of p53 and p73 activity by adenovirus E1A, E1B, and E4orf6 proteins. Mol Cell Biol 19: 3885–3894, 1999.[Abstract/Free Full Text]

49. Steegenga WT, van Laar T, Riteco N, Mandarino A, Shvarts A, van der Eb AJ, Jochemsen AG. Adenovirus E1A proteins inhibit activation of transcription by p53. Mol Cell Biol 16: 2101–2109, 1996.[Abstract]

50. Th'ng JP, Wright PS, Hamaguchi J, Lee MG, Norbury CJ, Nurse P, Bradbury EM. The FT210 cell line is a mouse G2 phase mutant with a temperature-sensitive CDC2 gene product. Cell 63: 313–324, 1990.[CrossRef][Web of Science][Medline]

51. Tucker JM, Murphy JT, Kisiel N, Diegelman P, Barbour KW, Davis C, Medda M, Alhonen L, Janne J, Kramer DL, Porter CW, Berger FG. Potent modulation of intestinal tumorigenesis in Apcmin/+ mice by the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase. Cancer Res 65: 5390–5398, 2005.[Abstract/Free Full Text]

52. Vujcic S, Halmekyto M, Diegelman P, Gan G, Kramer DL, Janne J, Porter CW. Effects of conditional overexpression of spermidine/spermine N1-acetyltransferase on polyamine pool dynamics, cell growth, and sensitivity to polyamine analogs. J Biol Chem 275: 38319–38328, 2000.[Abstract/Free Full Text]

53. Wang Z, Zahedi K, Barone S, Tehrani K, Rabb H, Matlin K, Casero RA, Soleimani M. Overexpression of SSAT in kidney cells recapitulates various phenotypic aspects of kidney ischemia-reperfusion injury. J Am Soc Nephrol 15: 1844–1852, 2004.[Abstract/Free Full Text]

54. Williams JR, Casero RA, Dillehay LE. The effect of polyamine depletion on the cytotoxic response to PUVA, gamma rays and UVC in V79 cells in vitro. Biochem Biophys Res Commun 201: 1–7, 1994.[CrossRef][Web of Science][Medline]

55. Wright JH, Munar E, Jameson DR, Andreassen PR, Margolis RL, Seger R, Krebs EG. Mitogen-activated protein kinase kinase activity is required for the G2/M transition of the cell cycle in mammalian fibroblasts. Proc Natl Acad Sci USA 96: 11335–11340, 1999.[Abstract/Free Full Text]

56. Xu B, Kim ST, Lim DS, Kastan MB. Two molecularly distinct G2/M checkpoints are induced by ionizing irradiation. Mol Cell Biol 22: 1049–1059, 2002.[Abstract/Free Full Text]

57. Yarden RI, Pardo-Reoyo S, Sgagias M, Cowan KH, Brody LC. BRCA1 regulates the G2/M checkpoint by activating Chk1 kinase upon DNA damage. Nat Genet 30: 285–289, 2002.[CrossRef][Web of Science][Medline]

58. Yu Q, Rose JH, Zhang H, Pommier Y. Antisense inhibition of Chk2/hCds1 expression attenuates DNA damage-induced S and G2 checkpoints and enhances apoptotic activity in HEK-293 cells. FEBS Lett 505: 7–12, 2001.[CrossRef][Web of Science][Medline]

59. Zahedi K, Wang Z, Barone S, Prada AE, Kelly CN, Casero RA, Yokota N, Porter CW, Rabb H, Soleimani M. Expression of SSAT, a novel biomarker of tubular cell damage, increases in kidney ischemia-reperfusion injury. Am J Physiol Renal Physiol 284: F1046–F1055, 2003.[Abstract/Free Full Text]




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