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Carcinogenesis Advance Access originally published online on August 3, 2007
Carcinogenesis 2007 28(11):2404-2411; doi:10.1093/carcin/bgm162
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Studies of the mechanism by which increased spermidine/spermine N1-acetyltransferase activity increases susceptibility to skin carcinogenesis

Xiaojing Wang{dagger}, David J. Feith{dagger}, Pat Welsh, Catherine S. Coleman, Christina Lopez1, Patrick M. Woster1, Thomas G. O'Brien2 and Anthony E. Pegg*

Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, PO Box 850, Hershey, PA 17033, USA
1 Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Avenue, Detroit, MI 48202, USA
2 Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA 19096, USA

* To whom correspondence should be addressed. Tel: +1 717 531 8152; Fax: +1 717 531 5157;Email: aep1{at}psu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Previous studies have shown that keratin 6 (K6)–spermidine/spermine N1-acetyltransferase (SSAT) transgenic mice, which modestly over-express SSAT in the skin, are more sensitive to tumor induction by a two-stage tumorigenesis protocol using initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). To evaluate the role of altered levels of polyamines and oxidative stress in this increase, studies were carried out with pharmacologic and genetic manipulation of K6–SSAT mice subjected to DMBA/TPA carcinogenesis. The increased tumor incidence was partially prevented by treatment with 1,4-bis-[N-(buta-2,3-dienyl)amino]butane, an inhibitor of acetylpolyamine oxidase which prevented degradation of the acetylated polyamines. This result suggests that toxic products such as reactive oxygen species and aldehydes liberated by the action of polyamine oxidase on the acetylated polyamines formed by SSAT may enhance tumor development. Breeding of the K6–SSAT mice with K6–antizyme (AZ) mice [which express AZ, a negative regulator of ornithine decarboxylase (ODC)] blocked the development of tumors. In addition, treatment of tumor-bearing K6–SSAT mice with the ODC inhibitor, {alpha}-difluoromethylornithine, resulted in the complete regression of established tumors. In contrast, treatment with N1,N11-bis(ethyl)norspermidine which increased SSAT activity in the tumors did not enhance regression. These results indicate that the tumor progression in K6–SSAT mice is dependent on elevated ODC activity and increased putrescine levels and may be further enhanced by oxidative stress. They support the use of strategies to modulate polyamine levels through the inhibition of ODC activity or polyamine uptake, but not via increased SSAT expression, for cancer chemoprevention in individuals at high risk for skin tumor development.

Abbreviations: APAO, acetylpolyamine oxidase; AZ, antizyme; BE-3-3-3, N1,N11-bis(ethyl)norspermidine; DFMO, {alpha}-difluoromethylornithine; DMBA, 7,12-dimethylbenz[a]anthracene; K6, keratin 6; MDL72527, 1,4-bis-[N-(buta-2,3-dienyl)amino]butane; ODC, ornithine decarboxylase; PCR, polymerase chain reaction; PPAR-{gamma}, peroxisome-proliferator-activated receptor {gamma}; ROS, reactive oxygen species; SSAT, spermidine/spermine N1-acetyltransferase; TPA, 12-O-tetradecanoylphorbol-13-acetate


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
There is considerable evidence that altered polyamine metabolism is associated with neoplastic growth. Polyamine content and synthesis are enhanced during tumor promotion and progression and in cancer cells. Polyamines are downstream targets of several oncogenes and inducers of other oncogenes and are thus intimately involved in neoplastic growth (1–5). Transfection of cells with plasmids that express either ornithine decarboxylase (ODC) or S-adenosylmethionine decarboxylase, which are key enzymes in polyamine biosynthesis, leads to malignant transformation. Transgenic approaches have also demonstrated the importance of polyamines in neoplastic growth. Thus, mice that over-express a stable truncated form of ODC in the skin have an increased incidence of tumors in response to a variety of carcinogens and ultraviolet irradiation (6,7). Mice that express a constitutively active messenger RNA-encoding antizyme (AZ)-1, a protein that reduces ODC and polyamine transport, showed a reduced tumor incidence in response to carcinogens, tumor promoters, ultraviolet radiation and activated oncogenes (7–10). Treatment of rodents with {alpha}-difluoromethylornithine (DFMO), a specific inhibitor of ODC reduces tumor formation in animal models and DFMO is currently being used in several ongoing chemoprevention trials (11).

These studies suggest that elevated levels of polyamines are associated with carcinogenesis. It was therefore quite surprising that when mice expressing low levels of spermidine/spermine N1-acetyltransferase (SSAT) in the skin from the keratin 6 (K6) promoter (K6–SSAT mice) were treated with an initiation/promotion protocol inducing skin papillomas, there was a striking increase in both the number and size of these tumors and in their progression to carcinomas (12). SSAT, which is the product of the Sat1 gene, is a catabolic enzyme that reduces polyamine content. The N1-acetylspermine and N1-acetylspermidine formed by it are good substrates for acetylpolyamine oxidase (APAO), the product of the Paox gene, which ultimately converts them into putrescine. Putrescine and N1-acetylspermidine are also quite readily excreted from the cell (11). Induction of SSAT by polyamine analogs such as N1,N11-bis(ethyl)norspermidine (BE-3-3-3) therefore causes a loss of polyamines and an increase in putrescine (13,14). Widespread transgenic expression of SSAT using its own promoter or a metallothionein promoter led to a substantial reduction in spermidine and/or spermine pools with a large increase in putrescine and in N1-acetylspermidine, which is accentuated by treatment with BE-3-3-3 (15). These mice also showed a wide variety of other defects including hair loss, female infertility, weight loss, central nervous system effects, altered lipid metabolism and a tendency to develop pancreatitis. The pleiotropic effects of these changes complicate the use of these animals for specific studies in carcinogenesis but it was reported that hairless CD2F2 mice with a major increase in the Sat1 gene copy number had a reduced development of papillomas in response to a two-stage skin tumorigenesis protocol (16). Similarly, when these mice were backcrossed to C57BL/6J and were bred with TRAMP mice that develop tumors in the prostate due to an expression of simian virus 40 T antigens from the probasin promoter, the incidence of prostate tumors was reduced (17).

Other studies are also consistent with the concept that activation of the SSAT/APAO catabolic pathway may negatively affect carcinogenesis. Intestinal SSAT is suppressed in response to K-ras activation and increased by non-steroidal anti-inflammatory drugs, which have chemopreventive effects (18,19). Indeed, SSAT inducers have been suggested as possible chemopreventive agents (3,20). However, removal of SSAT via a gene knockout renders ApcMin/+ mice resistant to intestinal tumor development and conversely transgenic over-expression of SSAT enhances intestinal tumorigenesis in these mice (21). Therefore, further investigation of the apparently paradoxical increase in skin tumors was needed. In the experiments described in the present paper, we have studied the effects of AZ, DFMO, BE-3-3-3 and the APAO inhibitor 1,4-bis-[N-(buta-2,3-dienyl)amino]butane (MDL72527) (22,23) on tumor induction in K6–SSAT mice after a two-stage tumorigenesis protocol using initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA).


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Materials
All reagents were purchased from Fisher Scientific (Pittsburgh, PA) or Sigma Chemical Co. (St Louis, MO) unless stated otherwise. Oligodeoxynucleotides were synthesized in the Macromolecular Core Facility, Hershey Medical Center, PA. BE-3-3-3 was kindly provided by Dr R. Casero (Johns Hopkins University, Baltimore, MD). DMBA was from Kodak Laboratory Chemicals (Rochester, NY) and TPA and protease inhibitor cocktail were purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA). [1-14C]Acetyl-coenzyme A was obtained from ICN Biochemicals (Costa Mesa, CA). L-[1-14C]ornithine was purchased from NEN Radiochemicals (Waltham, MA). DFMO was obtained from ILEX Oncology (San Antonio, TX). Antisera to SSAT (24) and AZ (8,9) have been described previously. Monoclonal antibody to proliferating cell nuclear antigen was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

MDL72527 was originally described as an APAO inhibitor in 1985 (23) but is not commercially available, and thus had to be synthesized. The original synthesis is problematic, in that significant side product formation leads to an unacceptably low overall yield. The synthesis of MDL72527 was thus accomplished using an improved synthetic scheme reported here for the first time. Commercially available putrescine was (bis)-N-Boc protected (85.2% yield), and this intermediate was used to alkylate two equivalents of propargyl bromide in the presence of sodium hydride in a 1:5 mixture of dimethylformamide and tetrahydrofuran (59.5% yield) (25). The propargyl groups were then converted to the corresponding allenes (CuBr, formaldehyde and diisopropylamine) (26) to yield the N-Boc-protected precursor (38.7% yield), which was deprotected in the presence of HCl to give the desired target molecule MDL72527 as a white solid in 65.8% yield.

K6–SSAT mice
Although there are two genes termed Sat1 and Sat2 in mammals, only SSAT the product of the Sat1 gene plays a role in polyamine metabolism (27). K6–SSAT mice, which express SSAT were produced on the B6D2F2 background as described previously (12) using a construct that places the SSAT cDNA with 32 bp of 5'- and 3'-untranslated region sequences under the control of the K6 promoter. The mice were backcrossed to both C57BL/6J and to DBA/2J inbred backgrounds (Jackson Laboratories, Bar Harbor, ME) for more than seven generations such that >99% of the genome is derived from the inbred strain. Genotyping was performed by polymerase chain reaction (PCR) amplification of genomic DNA isolated, using a DNeasy tissue kit following the manufacturer's directions (Qiagen, Valencia, CA), from tail clips of 3-week-old offspring. Screening by PCR used a sense primer directed to the K6 promoter region with the sequence 5'-GCAGAAGGAGGGGACAATTATCAC-3' and the anti-sense primer sequence directed to a coding region in the SSAT cDNA was 5'-TGGGCGGATCACGAATTTAGC-3' to amplify a sequence of 400 bp that is only formed when DNA from mice carrying the transgene was used.

K6–AZ mice
There are multiple Oaz genes in mammals (5). AZ, the product derived from the Oaz1 gene was used in the current studies. Mice with constitutive AZ-1 expression from the K6 promoter were produced as described previously (8,10) using a cDNA construct that contains a single nucleotide deletion allowing for translation of functional AZ from the messenger RNA without the need for polyamine-stimulated frameshifting. The mice on the B6D2F2 background were successively backcrossed to C57BL/6J mice for at least five generations so that >98% of their genes are derived from the inbred strain. Transgenic mice were identified by PCR detection of the transgene in genomic DNA isolated from tail clip biopsies using the K6–SSAT primers described above along with the anti-sense primer 5'-GCTGGGAGCTCGATGTAGAG-3' directed to the AZ-coding sequence to amplify an 800 bp sequence only from DNA from mice with the K6–AZ transgene.

Carcinogenesis protocol
The protocol was initiated at 7–9 weeks of age. A region of posterior dorsal skin (~2 x 2 cm) was shaved and at 2–3 days later, the mice were treated with a single dose of DMBA in 200 µl of acetone (400 nmol for C57BL/6J mice and 200 nmol for DBA/2J mice). Tumor promotion was started 1 week later and continued for 12–21 weeks as described with twice weekly application of TPA in 200 µl of acetone (17 nmol for C57BL/6J mice and 6.8 nmol for DBA/2J mice). Tumors >1 mm in diameter were counted weekly. Experimental groups were compared using log-rank test of the survival curves for tumor incidence and two-way analysis of variance for tumor multiplicity (Graphpad Prism, Graphpad Software, San Diego, CA).

Immunohistochemistry
Tumors were removed immediately after killing, fixed overnight in 10% neutral buffered formalin, embedded in paraffin and 5 µm sections were obtained. Immunohistochemistry was performed essentially as described previously for AZ (9) and for SSAT (12). Following deparaffinization and rehydration in graded alcohols, tumor sections were heated in Vector Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA) in a microwave oven (88–95°C, 3 times each for 5 min) before non-specific binding sites were blocked with goat serum. Sections were incubated overnight at 37°C in a humidified chamber with rabbit anti-AZ antibody (9) or rabbit anti-SSAT antibody (12) followed by incubation with biotinylated goat anti-rabbit antibody (1:500 dilution; Vector Laboratories). Slides were then incubated with streptavidin horseradish peroxidase (1:1000 dilution; Dako, Carpinteria, CA). Expression of AZ or SSAT was localized by a final incubation with 3, 3'-diaminobenzidine tetrahydrochloride (Vector Laboratories). Sections were counter stained with hematoxylin and pictures taken on a Nikon TM microscope.

Biochemical analysis
Activating mutations in codon 61 of the c-H-ras gene were detected by PCR amplification of specific alleles using papilloma DNA samples as described previously (10) with normal tail DNA as a negative control.

Tumors were excised and flash frozen in liquid nitrogen for analysis of polyamines and enzyme activity. Polyamines were determined by reverse-phase high-performance liquid chromatography using post-column derivatization with o-phthaldialdehyde and fluorescence detection (28). SSAT activity was measured after homogenization in ice cold 50 mM Tris–HCl pH 7.5, 2.5 mM dithiothreitol and 0.1 mM EDTA containing 1x protease inhibitor cocktail (Calbiochem-Novabiochem Corp.). The extracts were then used to measure the production of [14C]acetylspermidine in an assay mixture containing 3 mM spermidine and 16 µM [14C]acetyl-CoA (50 mCi/mmol) (12). ODC activity was assayed by measuring the conversion of L-[1-14C] ornithine (47.4 mCi/mmol) to 14CO2 as described previously (8).

DFMO, BE-3-3-3 and MDL72527 treatment
Tumor-bearing mice were treated with DFMO by adding 1% (w/v) DFMO to the drinking water for up to 6 weeks. This dose of DFMO had no toxic effects over the experimental period. Tumor-bearing mice were treated with BE-3-3-3 by intra-peritoneal injection (100 mg/kg in saline) after TPA treatments were stopped. The BE-3-3-3 was given twice daily for 5 days/week. Mice were treated with MDL72527 by adding 0.05% (w/v) MDL72527 to the drinking water. In order to avoid toxic effects from the build up of spermine in the blood, which has been reported to occur in mice exposed for long periods to MDL72527 (22,29), the treatment was given for only 5 days a week with 2 days exposure to normal drinking water. Treatment was continued for up to 15 weeks.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Effect of AZ on tumor incidence
K6–SSAT mice on the C57BL/6J background were bred with K6–AZ mice on the same background. The resulting four groups of offspring (wild-type, K6–SSAT, K6–AZ and K6–SSAT/K6–AZ) were treated topically with a single application of 400 nmol DMBA followed by twice weekly administration of 17 nmol of TPA for 21 weeks. Tumor incidence and multiplicity were measured (Figure 1A and B). As reported previously (12), K6–SSAT mice developed more tumors than wild-type and these tumors were significantly larger. There was a 4.3-fold increase in tumors that were >2 mm at the end of the experiment (Table I and Figure 1C). In agreement with previous studies (8,10), the K6–AZ mice developed less tumors than wild-type (Figure 1).


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  		Table I. Size of tumors in wild-type, K6–SSAT, K6–AZ and K6–SSAT/K6–AZ C57BL/6J mice treated with DMBA and promoted with TPA for 22 weeks

 


Figure 1
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  		Fig. 1. Tumor formation in wild-type, K6–SSAT, K6–AZ and K6–SSAT/K6–AZ C57BL/6J mice treated with DMBA and TPA. Panel (A) shows the tumors per mouse seen after 6–22 weeks of promotion with twice weekly treatments of 17 nmol TPA following initiation with 400 nmol of DMBA. Panel (B) shows the percentage of mice with tumors. Results are shown for wild-type (solid squares, n = 24), K6–SSAT (solid circles, n = 15), K6–AZ (open squares, n = 21) and K6–SSAT/K6–AZ (open circles, n = 17). Bars indicate the SEM for each point. The difference in tumor multiplicity between groups was analyzed by two-way analysis of variance. The K6–SSAT group developed more tumors than all other groups (P < 0.01). The K6–AZ and the K6–SSAT/K6–AZ groups both had significantly fewer tumors than the wild-type and K6–SSAT groups (P < 0.001). The difference between the K6–AZ and the K6–SSAT/K6–AZ is not statistically significant (P = 0.66). The tumor incidence in panel (B) was analyzed by log-rank test of the survival curves and the difference between the K6–SSAT/K6–AZ and the K6–SSAT groups was significant (P < 0.001). Panel (C) shows representative mice from each group after 22 weeks of promotion with twice weekly treatments of TPA.

 
The expression of AZ in K6–SSAT/K6–AZ mice greatly reduced the number of tumors to well below wild-type levels. There was a small increase when the K6–SSAT/K6–AZ group was compared with the K6–AZ group but the difference between these groups is not statistically significant. The first detectable tumors appeared at 6 weeks in these mice and, by the end of the experiment, all K6–SSAT mice had tumors with a mean tumor number per mouse of ~8 (Figure 1A). In contrast, in the K6–SSAT/K6–AZ mice, the tumors were not seen until 9 weeks and by the end of the experiment, 43% of the mice had not developed a single tumor and the mean tumor number per mouse was ~1. AZ expression decreased the size of the tumors reducing the mean number of tumors per mouse that exceeded 2 mm by almost 10-fold at 22 weeks (Table I and Figure 1C).

The content of AZ and SSAT in the tumors was investigated by immunohistochemistry (Figure 2). Very low and barely detectable levels of AZ and SSAT were present in the wild-type mice. As expected, these levels were increased in the mice carrying the respective single transgenes. SSAT expression was not altered in the K6–AZ mice but there may be a modest induction of endogenous AZ in the K6–SSAT mice. Importantly, the effect of AZ on tumor development was not related to a reduction of SSAT expression since there was no significant change in SSAT expression in K6–SSAT/K6–AZ mice (Figure 2). Proliferating cell nuclear antigen staining (data not shown) indicated no significant difference in proliferation in any of the four groups.


Figure 2
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  		Fig. 2. Expression of SSAT and AZ in wild-type, K6–SSAT, K6–AZ and K6–SSAT/K6–AZ C57BL/6J mice treated with DMBA and TPA. Mice were treated as in the legend to Figure 1 and sections prepared and stained for AZ or SSAT as indicated under immunohistochemistry in Materials and methods. aThe bar indicated represents 50 µm. bThe bar indicated represents 200 µm.

 
Activating mutations at codon 61 of H-ras, which have previously been shown to occur at a very high frequency in DMBA/TPA-treated papillomas (30), were also detected in our experiments. Using a PCR amplification assay (10), we found that 25/25 tumors from wild-type mice and 31/31 tumors from K6–SSAT mice had such mutations. Therefore, tumors from K6–SSAT mice do not exhibit a reduced frequency of H-ras-activating mutations and increased frequency of K-ras-activating mutations, as observed with K6–ODC mice (31).

Effect of DFMO and BE-3-3-3 on tumor incidence
We next tested the effect of inhibiting ODC activity or inducing SSAT activity by treating tumor-bearing K6–SSAT mice with DFMO or BE-3-3-3, respectively. Tumors were induced by DMBA and TPA in K6–SSAT mice and wild-type littermates as described above. After 16 weeks of promotion with TPA, the mice were treated with DFMO (1% in the drinking water) or normal drinking water for 5 weeks. DFMO caused a rapid regression of the tumors in the K6–SSAT mice so that by 5 weeks they were almost undetectable whereas untreated mice had minimal tumor regression (Figure 3). [Previous studies have shown a similar regression of skin tumors in mice with a transgenic over-expression of ODC alone (32) or with an activated ras oncogene (33) and mice with an activated mitogen-activated protein kinase kinase oncogene treated with DFMO (34).] In contrast, when the mice in each group were treated with 100 mg/kg of BE-3-3-3 (twice daily for 5 days) and this treatment was repeated 3 weeks later, there was no effect on regression in both BE-3-3-3- and saline-treated mice (Table II). This treatment with BE-3-3-3 substantially increased the SSAT activity in the tumors from 193 ± 41 pmol/min/mg protein to 1036 ± 120 pmol/min/mg protein in the K6–SSAT mice.


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  		Table II. Effect of treatment with BE-3-3-3 on tumors in wild-type and K6–SSAT C57BL/6J mice

 


Figure 3
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  		Fig. 3. Effect of DFMO on tumors in K6–SSAT C57BL/6J mice. Mice were treated at 7 weeks of age with 200 nmol DMBA and then with twice weekly treatments of 17 nmol TPA. After 16 weeks of promotion with TPA, seven of the mice were given DFMO (1% in the drinking water) (circles) and the six others received normal drinking water (squares). Bars indicate the SEM for each point.

 
Effect of MDL72527
Tumors were induced by DMBA and TPA as described above in K6–SSAT mice and wild-type littermates on the C57BL/6J background. Owing to the limited supply of the APAO inhibitor MDL72527 and the possibility that prolonged administration of this inhibitor would lead to a toxic build up of spermine in the blood as reported by Seiler et al. (22), this experiment was conducted over a 15 week period. As shown in Figures 1 and 4, this period was sufficient to establish a significant increase in tumors in the K6–SSAT mice compared with wild-type. During the period of promotion, some of the mice were exposed to MDL72527 in the drinking water for 5 days per week. This did not affect water consumption by the mice. The effect on papillomas is shown in Figure 4. Exposure to MDL72527 had no effect at all on the induction of tumors by the two-stage protocol in the wild-type group but significantly reduced the number of tumors and delayed their appearance in the K6–SSAT mice. However, this reduction did not abolish all the increase in this group compared with the wild-type. The exposure to MDL72527 did not alter the size distribution of the tumors in K6–SSAT mice with ~50% being >2 mm diameter in both groups, which is similar to that seen in Table I (data not shown). Treatment with MDL72527 did not affect either ODC or SSAT activity in both wild-type and K6–SSAT mice (data not shown).


Figure 4
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  		Fig. 4. Effect of MDL72527 on tumors in wild-type and K6–SSAT C57BL/6J mice. Mice were treated with 400 nmol DMBA and then with twice weekly treatments of TPA as in the legend to Figure 1. At the commencement of TPA treatment, half of the mice were given MDL72527 (50 mg/100 ml in the drinking water for 5 days/week) and the others received normal drinking water. Panel (A) shows tumors per mouse and panel (B) shows the percentage of the treated mice with tumors. Panel (B) is shown for only 14 weeks since by that time all the mice had at least one tumor. Results are shown for wild-type mice (solid circles), wild-type mice treated with MDL72527 (solid squares), K6–SSAT mice (open circles) and K6–SSAT mice treated with MDL72527 (open squares). Bars indicate the SEM for each point. Each group contained 13–15 mice. The difference between groups was analyzed by analysis of variance. The K6–SSAT group treated with MDL72527 developed fewer tumors than the K6–SSAT group treated with water (P < 0.01). Both groups of SSAT mice developed more tumors than the groups of wild-type mice (P < 0.01). Exposure to MDL72527 had no effect on tumor number or incidence in the wild-type mice.

 
Measurements of polyamines in the tumors harvested at the end of this experiment showed that exposure to the APAO inhibitor had the expected effect in increasing the content of N1-acetylspermidine, reducing the amount of putrescine and reducing the decrease in spermidine and spermine caused by SSAT expression (Figure 5). [The N1-acetylspermine content was also increased but, even in the treated K6–SSAT mice, this polyamine was present at very low levels.] These results provide strong evidence that the previously reported (12) rise in N1-acetylspermidine in the K6–SSAT mice is not responsible for the increased tumor incidence. The extra rise in putrescine due to induction of the SSAT/APAO system may be relevant to the increased tumor burden in the K6–SSAT mice, which is consistent with the studies on AZ and with DFMO described above. However, putrescine content was similar in the wild-type group and the MDL72527-treated K6–SSAT group suggesting that at least part of the increase in tumor development in the latter mice is due to other factors. Excellent candidates for this factor would be the other products of the APAO reaction, which are H2O2 and N-acetyl-3-aminopropanal. The increase in reactive oxygen species (ROS) and a reactive aldehyde could each increase tumor development.


Figure 5
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  		Fig. 5. Effect of MDL72527 on polyamine content in tumors produced in wild-type and K6–SSAT C57BL/6J mice. Mice were treated as described in the legend to Figure 4. Tumors were harvested and the polyamine content measured. Results are shown for tumors from five mice in each group ± standard deviation for the content of putrescine (PUT), spermidine (SPD), spermine (SPM) and N1-acetylspermidine (N1AcSPD). N1-Acetylspermine is not shown because the values are much lower than the other polyamines (0.05 ± 0.05 nmol/mg in wild-type, 0.3 ± 0.2 nmol/mg in wild-type + MDL72527, 0.13 ± 0.08 nmol/mg in K6–SSAT and 0.5 ± 0.07 nmol/mg in K6–SSAT + MDL72527).

 
Effect of SSAT on tumor incidence in DBA/2J mice
In contrast to the results seen in the experiments shown in Figures 1 and 4 and previous publications (12) where K6–SSAT mice on the C57BL/6J background were used, there was only a small increase in tumor incidence and multiplicity when K6–SSAT mice were subjected to DMBA/TPA carcinogenesis on the DBA/2J background (results not shown). On this background, the total number of tumors per mouse was only slightly greater in the K6–SSAT mice than in littermate controls after 21 weeks of promotion with twice weekly treatments of 6.8 nmol TPA following initiation with 200 nmol of DMBA. The K6–SSAT mice did, however, develop twice as many large tumors (>2 mm) (3.92 ± 0.54 per mouse) compared with their littermates (1.83 ± 0.46 per mouse) (P < 0.05). It is likely that the modulation of skin carcinogenesis by SSAT is susceptible to additional genetic modifier loci that differ between these inbred strains.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
Our results confirm the previous study showing that expression of SSAT from the K6 promoter increases susceptibility to carcinogenesis in the skin (12), although this result contrasts with another study where more general SSAT expression due to an increase in the Sat1 gene copy number reduced tumor incidence in the skin (15,16). The reason for this discrepancy is unclear but it should be noted that (i) the study showing a reduction was carried out on a hybrid CD2F2 background and (ii) the general SSAT expression causes a variety of effects mentioned in the introduction that could influence carcinogenesis. However, when backcrossed to C57BL/6J, the same SSAT-over-expressing mice exhibited decreased prostate tumors in TRAMP mice (17) and increased intestinal tumorigenesis in ApcMin/+ mice (21). Loss of the Sat1 gene decreased tumors in the ApcMin/+ model (21). It is therefore apparent that the effects of perturbing polyamine catabolism are system dependent and may involve increased flux resulting from compensatory increases in synthesis as suggested recently (14,21).

The findings reported in this paper showing that AZ expression blocks tumorigenesis in the K6–SSAT mice (Figure 1) and that DFMO treatment (Figure 3) causes their rapid regression is consistent with the concept described by Tucker et al. (21) that increased susceptibility to carcinogenic stimuli in mice over-expressing SSAT is due to a compensatory up-regulation of polyamine biosynthesis to replace those polyamines degraded via the SSAT/APAO pathway. The large increase in putrescine is due to both an increase in synthesis and an increased degradation of the higher polyamines. Both the increase in putrescine and the metabolic consequences of the SSAT/APAO pathway may influence carcinogenesis.

It is clear from the results with AZ expression and with DFMO treatment and the considerations discussed above that continued (and probably elevated) ODC is needed for the SSAT-mediated increase in tumors. However, it is noteworthy that the skin tumors arising in the K6–SSAT mice all had the characteristic mutations at codon 61 of H-ras, which are common in papillomas arising from exposure to polycyclic hydrocarbons (30). This is not the case in K6–ODC mice in which a huge increase in ODC activity leads to many tumors showing K-ras-activating mutations (31). Therefore, increased SSAT expression appears to enhance the later stages of tumor development.

Alterations in the relative ratios of polyamines due to SSAT causing increases in putrescine and N1-acetylspermidine may facilitate skin tumor development and the progression to carcinomas. It is known that polyamines can differentially regulate expression of genes at multiple levels (4) and it has been suggested that the selective transcriptional activation of oncogenes by individual polyamines may be involved in the development of neoplasia (2). Another contributing factor may be the generation of ROS as described below since ROS signaling is known to be involved in aspects of tumor progression including invasion, angiogenesis and metastasis (35). We can clearly rule out the increase in the content of the N1-acetylspermidine metabolite since treatment with the APAO inhibitor, MDL72527 further increased N1-acetylspermidine content but reduced the production of tumors. Prolonged exposure to MDL72527 reduced tumor incidence and multiplicity in the K6–SSAT mice but did not restore it to wild-type levels. Putrescine values were similar in the untreated wild-type and the K6–SSAT + MDL72527 groups but spermidine and spermine were lower in the latter. In order to avoid toxicity, the APAO inhibitor was given for only 5 days a week and the inhibition may not have been complete over the whole time period of exposure.

It is not possible to compare directly the ability of AZ expression and that of MDL72527 treatment in reducing tumors in the K6–SSAT mice since (i) transgenic AZ expression was continuous over the entire period of exposure to DMBA/TPA; (ii) the MDL72527 treatment was only given for 5 days a week for the 15 weeks of promotion and (iii) the overall tumor incidence was lower in all groups in the AZ experiment shown in Figure 1 than in the MDL72527 experiment shown in Figure 4. However, in both experiments, the transgenic expression of SSAT significantly increased tumor development and either AZ or MDL72527 reduced the effects of SSAT expression.

An additional factor in the increased tumor susceptibility of K6–SSAT mice is likely to be the production of ROS and toxic aldehydes by the SSAT/APAO pathway (14). Chemicals and free radicals generating peroxides and hydroperoxides are well known to promote tumors in mouse skin (36–38) and antioxidants block promotion (39). ROS-mediating signaling is widely involved in tumor progression (35). Externally applied H2O2 itself is only weakly active as a promoter (38) but the H2O2 generated internally via APAO may act directly even though APAO has a peroxisomal localizing sequence and is found in peroxisomes where at least part of the released H2O2 is likely to be degraded (14). Additional effects may come from other ROS and the N-acetyl-3-aminopropanal generated by the SSAT/PAO pathway which have been shown to have toxic effects in cells expressing high levels of SSAT (14). The repression of catalase and the increase in ROS has been shown to be important in the progression of skin papillomas to malignant carcinomas (40). There was a much higher rate of formation of carcinomas in the K6–SSAT mice after the two-stage carcinogenesis protocol (12). This was also the case in the present experiments and would be consistent with an effect of the ROS from the APAO/SSAT pathway. Although outside of the scope of the present investigation, measurement of oxidative DNA damage such as the level of 8-oxodeoxyguanosine would be a valuable way in which to investigate the extent to which this pathway contributes to carcinogenesis.

The decreased effect of SSAT over-expression on carcinogenesis in the DBA/2J strain compared with C57BL/6J is not unexpected since many studies have shown that there are genetic modifier loci that can alter the response of mouse skin to two-stage carcinogenesis protocols. These include studies showing that the genetic background modifies the response to ODC over-expression (41), AZ over-expression (10) and to agents generating peroxides and ROS (37,42,43).

SSAT expression is normally very low and the lack of effect of treatment with MDL72527 on carcinogenesis in the control group (Figure 4) suggests that the APAO/SSAT pathway does not contribute to tumor incidence in the basic DMBA/TPA model of carcinogenesis. However, it is very well established that SSAT is greatly increased by a variety of stimuli including hormones, toxins, drugs and cellular stress (13,14) and in these conditions it may be an important contributory factor to carcinogenesis. The induction of SSAT in response to non-steroidal anti-inflammatory agents (44), which occurs via the peroxisomal proliferators response element and receptor PPAR-{gamma} (11,14,45), has been linked to their ability to inhibit carcinogenesis. Some established or experimental antitumor agents such as 5-fluorouracil (46) and BE-3-3-3 (24) also induce SSAT. However, other recent studies have shown that pro-inflammatory cytokines that may promote carcinogenesis such as tumor necrosis factor {alpha} also induce SSAT via nuclear factor-kappa B (47).

In mouse models of colon cancer, ODC is increased via c-myc by mutations inactivating the Apc gene whereas SSAT is reduced by these mutations and also by K-ras via PPAR-{gamma} (11,48). These changes lead to increased polyamine content. However, both increased SSAT and ODC activity have been observed in human colorectal tumors; this increase in SSAT may represent the activation of PPAR-{gamma} expression as a physiological response to reduce polyamines (49). It was therefore suggested that pharmacological activation of PPAR-{gamma} and/or induction of SSAT should be considered as a therapeutic or preventive strategy for treating cancer (49). Our results would favor instead the inactivation of ODC by DFMO and clinical trials with this agent are in progress (11). Other methods of reducing ODC activity such as inducing AZ would also be worthy of consideration. Recent studies have identified some compounds that induce AZ without substituting for the natural polyamines (50). At present, there are no useful inhibitors of SSAT. Such inhibitors would be of great value in studies of the roles of SSAT in normal and neoplastic growth and may provide additional drug candidates.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
National Institutes of Health (CA-018138, GM-026290 and ES-01664).


   Footnotes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgments
 
We wish to thank Suzanne Sass-Kuhn, Kerry Keefer and Chethana Prakashagowda for technical assistance; Dr Diane E. McCloskey for assistance with SSAT assays and Yasser Heakal for assistance with the screening for ras mutations.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received February 26, 2007; revised July 11, 2007; accepted July 12, 2007.


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D. L. Kramer, P. Diegelman, J. Jell, S. Vujcic, S. Merali, and C. W. Porter
Polyamine Acetylation Modulates Polyamine Metabolic Flux, a Prelude to Broader Metabolic Consequences
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