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• W2134253034 abstract "The enzyme spermidine/spermine N1-acetyltransferase (SSAT) regulates the catabolism and export of intracellular polyamines. We have previously shown that activation of polyamine catabolism by conditional overexpression of SSAT has antiproliferative consequences in LNCaP prostate carcinoma cells. Growth inhibition was causally linked to high metabolic flux arising from a compensatory increase in polyamine biosynthesis. Here we examined the in vivo consequences of SSAT overexpression in a mouse model genetically predisposed to develop prostate cancer. TRAMP (transgenic adenocarcinoma of mouse prostate) female C57BL/6 mice carrying the SV40 early genes (T/t antigens) under an androgen-driven probasin promoter were cross-bred with male C57BL/6 transgenic mice that systemically overexpress SSAT. At 30 weeks of age, the average genitourinary tract weights of TRAMP mice were ∼4 times greater than those of TRAMP/SSAT bigenic mice, and by 36 weeks, they were ∼12 times greater indicating sustained suppression of tumor outgrowth. Tumor progression was also affected as indicated by a reduction in the prostate histopathological scores. By immunohistochemistry, SV40 large T antigen expression in the prostate epithelium was the same in TRAMP and TRAMP/SSAT mice. Consistent with the 18-fold increase in SSAT activity in the TRAMP/SSAT bigenic mice, prostatic N1-acetylspermidine and putrescine pools were remarkably increased relative to TRAMP mice, while spermidine and spermine pools were minimally decreased due to a compensatory 5-7-fold increase in biosynthetic enzymes activities. The latter led to heightened metabolic flux through the polyamine pathway and an associated ∼70% reduction in the SSAT cofactor acetyl-CoA and a ∼40% reduction in the polyamine aminopropyl donor S-adenosylmethionine in TRAMP/SSAT compared with TRAMP prostatic tissue. In addition to elucidating the antiproliferative and metabolic consequences of SSAT overexpression in a prostate cancer model, these findings provide genetic support for the discovery and development of specific small molecule inducers of SSAT as a novel therapeutic strategy targeting prostate cancer. The enzyme spermidine/spermine N1-acetyltransferase (SSAT) regulates the catabolism and export of intracellular polyamines. We have previously shown that activation of polyamine catabolism by conditional overexpression of SSAT has antiproliferative consequences in LNCaP prostate carcinoma cells. Growth inhibition was causally linked to high metabolic flux arising from a compensatory increase in polyamine biosynthesis. Here we examined the in vivo consequences of SSAT overexpression in a mouse model genetically predisposed to develop prostate cancer. TRAMP (transgenic adenocarcinoma of mouse prostate) female C57BL/6 mice carrying the SV40 early genes (T/t antigens) under an androgen-driven probasin promoter were cross-bred with male C57BL/6 transgenic mice that systemically overexpress SSAT. At 30 weeks of age, the average genitourinary tract weights of TRAMP mice were ∼4 times greater than those of TRAMP/SSAT bigenic mice, and by 36 weeks, they were ∼12 times greater indicating sustained suppression of tumor outgrowth. Tumor progression was also affected as indicated by a reduction in the prostate histopathological scores. By immunohistochemistry, SV40 large T antigen expression in the prostate epithelium was the same in TRAMP and TRAMP/SSAT mice. Consistent with the 18-fold increase in SSAT activity in the TRAMP/SSAT bigenic mice, prostatic N1-acetylspermidine and putrescine pools were remarkably increased relative to TRAMP mice, while spermidine and spermine pools were minimally decreased due to a compensatory 5-7-fold increase in biosynthetic enzymes activities. The latter led to heightened metabolic flux through the polyamine pathway and an associated ∼70% reduction in the SSAT cofactor acetyl-CoA and a ∼40% reduction in the polyamine aminopropyl donor S-adenosylmethionine in TRAMP/SSAT compared with TRAMP prostatic tissue. In addition to elucidating the antiproliferative and metabolic consequences of SSAT overexpression in a prostate cancer model, these findings provide genetic support for the discovery and development of specific small molecule inducers of SSAT as a novel therapeutic strategy targeting prostate cancer. Although prostate cancer can be clinically managed in its early phases, the inability to control the more aggressive late stage disease has prompted the search for novel therapies. We became interested in the possibility that strategies targeting polyamine homeostasis may be effective against prostate cancer. The prostate has the highest level of polyamine biosynthesis of any tissue, and it is the only tissue in which polyamines are purposely synthesized for export. More particularly, massive amounts of polyamines are excreted by the prostate into semen. Thus, we reasoned that polyamine homeostasis may be altered in the prostate relative to other tissues and that tumors derived from this gland may exhibit atypical regulatory responses to polyamine analogues and inhibitors (1Mi Z. Kramer D.L. Miller J.T. Bergeron R.J. Bernacki R. Porter C.W. Prostate. 1998; 34: 51-60Crossref PubMed Scopus (28) Google Scholar). An additional rationale for targeting polyamines in prostate cancer derives from a recent meta-analysis of four independent microarray data sets comparing gene expression profiles of benign and malignant patient prostate samples showing that polyamine metabolism was the most systematically affected of all biochemical and signaling pathways (2Rhodes D.R. Barrette T.R. Rubin M.A. Ghosh D. Chinnaiyan A.M. Cancer Res. 2002; 62: 4427-4433PubMed Google Scholar). Genes that supported polyamine biosynthesis were up-regulated, while those that detracted from biosynthesis were down-regulated. The findings agree with earlier clinical studies showing a significant increase in ornithine decarboxylase (ODC) 1The abbreviations used are: ODC, ornithine decarboxylase; AcSpd, N1-acetylspermidine; DFMO, α-difluoromethylornithine; GU, genitourinary; HPCE, high performance capillary electrophoresis; MR, magnetic resonance; Put, putrescine; AdoMet, S-adenosylmethionine; Spd, spermidine; Spm, spermine; SSAT, spermidine/spermine N1-acetyltransferase; Tag, SV40 large T antigen; TRAMP, transgenic adenocarcinoma of mouse prostate; H&E, hematoxylin and eosin; PBS, phosphate-buffered saline; AcSpm, acetylspermine. and S-adenosylmethionine (AdoMet) decarboxylase transcripts in human prostatic cancer relative to benign hyperplasia (3Bettuzzi S. Davalli P. Astancolle S. Carani C. Madeo B. Tampieri A. Corti A. Saverio B. Pierpaola D. Serenella A. Cesare C. Bruno M. Auro T. Arnaldo C. Cancer Res. 2000; 60: 28-34PubMed Google Scholar). Polyamines have been targeted in anticancer strategies for some time (4Thomas T. Thomas T.J. J. Cell. Mol. Med. 2003; 7: 113-126Crossref PubMed Scopus (274) Google Scholar). Various antagonists such as the ODC inhibitor α-difluoromethylornithine (DFMO), AdoMet decarboxylase inhibitor (SAM486), and the polyamine analogue N1,N11-diethylnorspermine have undergone clinical testing as therapeutic and/or preventive agents (5Seiler N. Curr. Drug Targets. 2003; 4: 565-585Crossref PubMed Scopus (108) Google Scholar, 6Seiler N. Curr. Drug Targets. 2003; 4: 537-564Crossref PubMed Scopus (149) Google Scholar). Recognizing the unique physiology of the prostate gland, Heston and collaborators (7Heston W.D. Watanabe K.A. Pankiewicz K.W. Covey D.F. Biochem. Pharmacol. 1987; 36: 1849-1852Crossref PubMed Scopus (16) Google Scholar, 8Heston W.D. Cancer Surv. 1991; 11: 217-238PubMed Google Scholar) have proposed that polyamine inhibitors may be particularly effective against prostate cancer. In support of this concept, Gupta et al. (9Gupta S. Ahmad N. Marengo S.R. MacLennan G.T. Greenberg N.M. Mukhtar H. Cancer Res. 2000; 60: 5125-5133PubMed Google Scholar) have shown that DFMO is effective in depleting polyamine pools and in preventing development of prostate cancer in the transgenic adenocarcinoma of mouse prostate (TRAMP) model (10Greenberg N.M. DeMayo F. Finegold M.J. Medina D. Tilley W.D. Aspinall J.O. Cunha G.R. Donjacour A.A. Matusik R.J. Rosen J.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3439-3443Crossref PubMed Scopus (1083) Google Scholar). Targeting polyamines has traditionally involved interference with or down-regulation of polyamine biosynthesis with small molecule inhibitors or analogues, respectively. As an alternative to blocking biosynthesis, we propose that activation of polyamine catabolism by inducing the rate-limiting enzyme spermidine/spermine N1-acetylspermine transferase (SSAT) may offer distinct advantages. The approach derives from our studies of the polyamine analogue N1,N11-diethylnorspermine that, in addition to down-regulating ODC and AdoMet decarboxylase, very potently up-regulates SSAT in tumor cells and tissues (11Libby P.R. Bergeron R.J. Porter C.W. Biochem. Pharmacol. 1989; 38: 1435-1442Crossref PubMed Scopus (63) Google Scholar, 12Casero Jr., R.A. Ervin S.J. Celano P. Baylin S.B. Bergeron R.J. Cancer Res. 1989; 49: 639-643PubMed Google Scholar, 13Porter C.W. Ganis B. Libby P.R. Bergeron R.J. Cancer Res. 1991; 51: 3715-3720PubMed Google Scholar, 14Shappell N.W. Miller J.T. Bergeron R.J. Porter C.W. Anticancer Res. 1992; 12: 1083-1089PubMed Google Scholar). The latter was shown to occur to a greater degree in human tumor xenografts than in normal host tissues (15Porter C.W. Bernacki R.J. Miller J. Bergeron R.J. Cancer Res. 1993; 53: 581-586PubMed Google Scholar). Correlations between SSAT induction and growth inhibition have been repeatedly suggested by early work in a variety of tumor types (14Shappell N.W. Miller J.T. Bergeron R.J. Porter C.W. Anticancer Res. 1992; 12: 1083-1089PubMed Google Scholar, 16Casero Jr., R.A. Celano P. Ervin S.J. Porter C.W. Bergeron R.J. Libby P.R. Cancer Res. 1989; 49: 3829-3833PubMed Google Scholar, 17McCloskey D.E. Pegg A.E. J. Biol. Chem. 2000; 275: 28708-28714Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Recently that relationship was more precisely defined by the finding that SSAT-targeted small interfering RNA minimizes analogue-mediated enzyme induction and at the same time prevents polyamine pool depletion and apoptosis (18Chen Y. Kramer D.L. Li F. Porter C.W. Oncogene. 2003; 22: 4964-4972Crossref PubMed Scopus (39) Google Scholar, 19Chen Y. Kramer D.L. Jell J. Vujcic S. Porter C.W. Mol. Pharmacol. 2003; 64: 1153-1159Crossref PubMed Scopus (33) Google Scholar). We have previously reported that conditional overexpression of SSAT in MCF-7 breast carcinoma cells leads to polyamine pool depletion and growth inhibition (20Vujcic S. Halmekyto M. Diegelman P. Gan G. Kramer D.L. Janne J. Porter C.W. J. Biol. Chem. 2000; 275: 38319-38328Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). As a prelude to the present study, we showed that conditional enzyme overexpression in LNCaP prostate carcinoma cells causes growth inhibition that differed from that seen in MCF-7 cells in that it was not accompanied by polyamine pool depletion (21Kee K. Vujcic S. Merali S. Diegelman P. Kisiel N. Powell C.T. Kramer D.L. Porter C.W. J. Biol. Chem. 2004; 279: 27050-27058Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Instead cells averted the latter by increasing polyamine biosynthesis at the levels of ODC and AdoMet decarboxylase activities causing heightened metabolic flux through the biosynthetic and catabolic pathways. In a critical experiment, it was shown that interruption of flux by blocking ODC activity prevented growth inhibition (21Kee K. Vujcic S. Merali S. Diegelman P. Kisiel N. Powell C.T. Kramer D.L. Porter C.W. J. Biol. Chem. 2004; 279: 27050-27058Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Additional studies concluded that growth inhibition deriving from overexpression of SSAT was probably attributable to overproduction of pathway products such as acetylated polyamines or to depletion of polyamine precursor metabolites such as AdoMet and/or the SSAT cofactor acetyl-CoA (21Kee K. Vujcic S. Merali S. Diegelman P. Kisiel N. Powell C.T. Kramer D.L. Porter C.W. J. Biol. Chem. 2004; 279: 27050-27058Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Whatever the downstream mechanism, these in vitro data suggest that activation of polyamine catabolism by selective induction of SSAT may constitute an effective antitumor strategy against prostate cancer. The goal of the present study was to further validate the above concept by providing critical in vivo evidence based on a genetic approach. For this purpose, we utilized the TRAMP model that is genetically engineered to develop prostate cancer (10Greenberg N.M. DeMayo F. Finegold M.J. Medina D. Tilley W.D. Aspinall J.O. Cunha G.R. Donjacour A.A. Matusik R.J. Rosen J.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3439-3443Crossref PubMed Scopus (1083) Google Scholar, 22Gingrich J.R. Greenberg N.M. Toxicol. Pathol. 1996; 24: 502-504Crossref PubMed Scopus (94) Google Scholar, 23Gingrich J.R. Barrios R.J. Kattan M.W. Nahm H.S. Finegold M.J. Greenberg N.M. Cancer Res. 1997; 57: 4687-4691PubMed Google Scholar). Cross-breeding these mice with SSAT transgenic mice that systemically overexpress the enzyme (24Pietila M. Alhonen L. Halmekyto M. Kanter P. Janne J. Porter C.W. J. Biol. Chem. 1997; 272: 18746-18751Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) resulted in a profound suppression of prostate tumor outgrowth that may be related to consequences emanating from depletion of acetyl-CoA pools. Materials—The polyamines putrescine (Put), spermidine (Spd), spermine (Spm), and acetylated polyamine N1-acetylspermidine (Ac-Spd) were purchased from Sigma. Acetyl-CoA was also purchased from Sigma and solubilized as described by Liu et al. (25Liu G. Chen J. Che P. Ma Y. Anal. Chem. 2003; 75: 78-82Crossref PubMed Scopus (25) Google Scholar). Breeding and Screening of Transgenic Animals—TRAMP mice (10Greenberg N.M. DeMayo F. Finegold M.J. Medina D. Tilley W.D. Aspinall J.O. Cunha G.R. Donjacour A.A. Matusik R.J. Rosen J.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3439-3443Crossref PubMed Scopus (1083) Google Scholar), heterozygous for the transgene rat probasin-SV40 large T antigen (PBTag) (lineage of founder 8247; Jackson Laboratory, Bar Harbor, ME) were maintained in a pure C57BL/6 background. Mouse tail DNA was isolated using the DNeasy® tissue kit (Qiagen Inc., Valencia, CA). Genotyping of TRAMP animals was performed by PCR according to the Jackson Laboratory protocol. We previously generated mice that systemically overexpressed SSAT under its endogenous murine gene promoter (24Pietila M. Alhonen L. Halmekyto M. Kanter P. Janne J. Porter C.W. J. Biol. Chem. 1997; 272: 18746-18751Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). These SSAT transgenic mice, in the CD2F1 genetic background (24Pietila M. Alhonen L. Halmekyto M. Kanter P. Janne J. Porter C.W. J. Biol. Chem. 1997; 272: 18746-18751Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), were backcrossed for >8 generations into C57BL/6, the same genetic background as the TRAMP mouse. The SSAT transgenic mice are characterized by pronounced hair loss by 3-4 weeks of age (24Pietila M. Alhonen L. Halmekyto M. Kanter P. Janne J. Porter C.W. J. Biol. Chem. 1997; 272: 18746-18751Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), making genotyping unnecessary. Since female SSAT transgenic mice are infertile and male mice have normal reproductive capabilities, the latter were cross-bred with female TRAMP mice to generate the bigenic mice used in this study. Magnetic Resonance (MR) Imaging—Longitudinal analysis of prostate cancer progression in TRAMP mice using MR imaging has been reported by Hsu et al. (26Hsu C.X. Ross B.D. Chrisp C.E. Derrow S.Z. Charles L.G. Pienta K.J. Greenberg N.M. Zeng Z. Sanda M.G. J. Urol. 1998; 160: 1500-1505Crossref PubMed Scopus (45) Google Scholar). More specifically, it was used to assess tumor volume and to track tumor development in TRAMP and TRAMP/SSAT mice. High resolution MR imaging scans were performed using a General Electric CSI 4.7T/33-cm horizontal bore magnet (GE NMR Instruments, Fremont, CA) with upgraded radio frequency and computer systems. MR imaging data were acquired using a custom designed 35-mm radio frequency transceiver coil and a G060 removable gradient coil insert generating a maximum field strength of 950 milliteslas/m. Transaxial, T1-weighted images were acquired through the lower abdomen with a standard spin echo MR imaging sequence. Images were comprised of 20 × 1-mm thick slices with a 3.2× 3.2-cm field of view acquired with a 192 × 192 matrix to provide contiguous image data of the prostate tumor. Acquisition parameters consisted of an echo time/repetition time = 10/724 ms and 4 number of excitations. Pathology—Mouse genitourinary (GU) tracts consisting of bladder, urethra, seminal vesicles, ampullary gland, and the prostate were excised and weighed. The correlation of GU weight as a function of cancer progression in the TRAMP mouse is well documented by Kaplan-Lefko et al. (27Kaplan-Lefko P.J. Chen T.M. Ittmann M.M. Barrios R.J. Ayala G.E. Huss W.J. Maddison L.A. Foster B.A. Greenberg N.M. Prostate. 2003; 55: 219-237Crossref PubMed Scopus (370) Google Scholar). Once GU tracts were grossly examined and documented by fixed angle photography, the dorsal, lateral, ventral, and anterior lobes of the prostate as well as the seminal vesicles were microdissected and placed into multichamber cassettes for fixation in 4% paraformaldehyde for 4 h at 4 °C after which they were paraffin-embedded, sectioned at 5 μm, and stained with hematoxylin and eosin (H&E). H&E slides were reviewed by two experienced morphologists without knowledge of the genotype or age of the mice. Each prostatic lobe (dorsal, lateral, ventral, and anterior) was scored according to the grading system established by Gingrich et al. (28Gingrich J.R. Barrios R.J. Foster B.A. Greenberg N.M. Prostate Cancer Prostatic Dis. 1999; 2: 70-75Crossref PubMed Scopus (199) Google Scholar). The histological scores were then averaged and expressed as mean ± S.E. Immunohistochemistry—Slides containing 5-μm sections were quenched with aqueous 3% hydrogen peroxide for 30 min and rinsed with PBS/T (500 μl/liter Tween 20) to remove endogenous peroxidases. Antigen retrieval involved continuous microwaving of the slides in 10 mm citrate buffer (pH 6.0) for 20 min. Cooled slides were washed for 5 min in PBS/T at room temperature and blocked with 0.03% casein in PBS/T for 30 min prior to the addition of primary antibodies. For anti-SV40 large T antigen staining, monoclonal anti-SV40 large T antigen antibody (catalog number 554149, BD Pharmingen) was used at a 1:400 dilution in a humidity chamber. Following overnight incubation at 4 °C, slides were washed with PBS/T and incubated for 30 min with secondary biotinylated anti-rabbit and anti-mouse immunoglobulins from the LSAB+ kit (DAKO, Carpinteria, CA) diluted according to the manufacturer's protocol. The slides were then washed with PBS/T and complexed with streptavidin (LSAB+ kit, DAKO, prediluted) for 30 min. Immunoreactive anti-SV40 large T antigen was detected by the application of the substrate 3,3′-diaminobenzidine tetrahydrochloride (DAKO) for 5 min. All sections were counterstained with hematoxylin. Analytical Methods—Tissues were snap-frozen in liquid nitrogen, crushed into a fine powder in a mortar or a Bio-Pulverizer (BioSpec Products, Inc., Bartlesville, OK), and then sonicated on ice in Tris/EDTA buffer for polyamine enzyme activities and pool analysis. SSAT activity was assayed as described previously (29Bernacki R.J. Oberman E.J. Seweryniak K.E. Atwood A. Bergeron R.J. Porter C.W. Clin. Cancer Res. 1995; 1: 847-857PubMed Google Scholar) and expressed as pmol of N1-[14C]acetylspermidine generated/min/mg of protein. Decarboxylase activities were determined by a CO2 trap assay and expressed as pmol of CO2 released/h/mg of protein (30Kramer D. Mett H. Evans A. Regenass U. Diegelman P. Porter C.W. J. Biol. Chem. 1995; 270: 2124-2132Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Polyamines and the acetylated derivatives of Spd and Spm were measured by high pressure liquid chromatography following methods reported by Kramer et al. (30Kramer D. Mett H. Evans A. Regenass U. Diegelman P. Porter C.W. J. Biol. Chem. 1995; 270: 2124-2132Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). For Northern blot analysis, frozen tissues were crushed into a fine powder using a mortar and pestle after which total RNA was extracted with guanidine isothiocyanate (31Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar) and purified by CsCl gradient centrifugation (32Ross J. J. Mol. Biol. 1976; 106: 403-420Crossref PubMed Scopus (129) Google Scholar). RNA was loaded onto a gel at 30 μg/lane and subjected to Northern blot analysis following procedures described by Fogel-Petrovic et al. (33Fogel-Petrovic M. Shappell N.W. Bergeron R.J. Porter C.W. J. Biol. Chem. 1993; 268: 19118-19125Abstract Full Text PDF PubMed Google Scholar). Acetyl-CoA Determinations—High performance capillary electrophoresis (HPCE) separation and quantitation of acetyl-CoA in tissue samples as recently described (21Kee K. Vujcic S. Merali S. Diegelman P. Kisiel N. Powell C.T. Kramer D.L. Porter C.W. J. Biol. Chem. 2004; 279: 27050-27058Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) was carried out following the method of Liu et al. (25Liu G. Chen J. Che P. Ma Y. Anal. Chem. 2003; 75: 78-82Crossref PubMed Scopus (25) Google Scholar). Tissues extracts were then analyzed on a Beckman P/ACE MDQ capillary electrophoresis system (Fullerton, CA) equipped with a photodiode array detector and an uncoated fused silica capillary electrophoresis column of 75-μm inner diameter and 60 cm in length with 50 cm from inlet to the detection window (Polymicro Technologies, Phoenix, AZ). Electrophoretic conditions were according to Liu et al. (25Liu G. Chen J. Che P. Ma Y. Anal. Chem. 2003; 75: 78-82Crossref PubMed Scopus (25) Google Scholar) with minor modifications as described previously (21Kee K. Vujcic S. Merali S. Diegelman P. Kisiel N. Powell C.T. Kramer D.L. Porter C.W. J. Biol. Chem. 2004; 279: 27050-27058Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Data were collected and processed by Beckman P/ACE 32 Karat software version 4.0. Acetyl-CoA levels were expressed as nmol/g of tissue. Statistics—Statistical significance (p value) was determined by Student's t test or analysis of variance with Fisher's protected least significant difference test at a 95% confidence level using a StatView computer program (SAS Institute Inc., Cary, NC). The goal of this study was to provide in vivo genetic validation for the concept that activating polyamine catabolism at the level of SSAT will give rise to an antitumor response due to homeostatic perturbations in polyamine metabolism. The effort was catalyzed by our recent reports showing that conditional overexpression of SSAT inhibits in vitro growth of both MCF-7 breast carcinoma cells (20Vujcic S. Halmekyto M. Diegelman P. Gan G. Kramer D.L. Janne J. Porter C.W. J. Biol. Chem. 2000; 275: 38319-38328Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) and LNCaP prostate carcinoma cells (21Kee K. Vujcic S. Merali S. Diegelman P. Kisiel N. Powell C.T. Kramer D.L. Porter C.W. J. Biol. Chem. 2004; 279: 27050-27058Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Consistent with these findings, we now demonstrate that overexpression of SSAT markedly suppresses tumor outgrowth of early and advanced prostatic cancer in TRAMP mice. As will be discussed, this may be due to unusual sensitivity of prostate-derived tumors to polyamine perturbations and/or to novel metabolic disturbances emanating from compensatory responses to activated polyamine catabolism. SSAT-overexpressing transgenic male mice were cross-bred with female TRAMP mice to yield four cohorts of offspring: wild type, SSAT transgenic mice, and TRAMP and TRAMP/SSAT bigenics 15 weeks of age. Prostate and liver tissues were excised from wild-type, SSAT, TRAMP, and TRAMP/SSAT mice to confirm SSAT mRNA expression and enzyme activity. As shown in Fig. 1, prostate gland SSAT mRNA levels were elevated 32- and 35-fold in both SSAT and TRAMP/SSAT cohorts, respectively, relative to wild-type mice. Consistent with SSAT gene overexpression prostatic enzyme activity in both SSAT and TRAMP/SSAT mice was elevated by ∼18-fold. SSAT mRNA in liver of SSAT and TRAMP/SSAT mice was increased 20- and 30-fold over wild-type mice, but unlike the prostate, enzyme activities were only increased 3- and 2-fold, presumably due to tissue-specific differences in translational control (24Pietila M. Alhonen L. Halmekyto M. Kanter P. Janne J. Porter C.W. J. Biol. Chem. 1997; 272: 18746-18751Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The data confirm that overexpression of SSAT occurs in the prostate of transgene-bearing mice. Longitudinal assessment of the prostate by MR imaging was used to monitor tumor appearance and development in representative TRAMP animals. GU tumors were first apparent at ∼20 weeks of age in TRAMP animals. By 30 weeks, all TRAMP mice had visible prostate tumors that, with time, infiltrated the seminal vesicles as is typical in the pure C57BL/6 genetic background (10Greenberg N.M. DeMayo F. Finegold M.J. Medina D. Tilley W.D. Aspinall J.O. Cunha G.R. Donjacour A.A. Matusik R.J. Rosen J.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3439-3443Crossref PubMed Scopus (1083) Google Scholar, 22Gingrich J.R. Greenberg N.M. Toxicol. Pathol. 1996; 24: 502-504Crossref PubMed Scopus (94) Google Scholar, 27Kaplan-Lefko P.J. Chen T.M. Ittmann M.M. Barrios R.J. Ayala G.E. Huss W.J. Maddison L.A. Foster B.A. Greenberg N.M. Prostate. 2003; 55: 219-237Crossref PubMed Scopus (370) Google Scholar, 34Gingrich J.R. Barrios R.J. Morton R.A. Boyce B.F. DeMayo F.J. Finegold M.J. Angelopoulou R. Rosen J.M. Greenberg N.M. Cancer Res. 1996; 56: 4096-4102PubMed Google Scholar). As observed using MR imaging and confirmed at necropsy, some TRAMP mice exhibited predominantly prostatic tumors, while the majority showed significant prostate tumors with seminal vesicle involvement. Both pathologies were reduced in the TRAMP/SSAT mice. On the basis of tumor size in TRAMP mice, the experimental end point was set at week 30. The suppressive effect of SSAT overexpression on tumor outgrowth is apparent in comparisons of dissected GU tracts shown in Fig. 2A. Gross examination of both wild-type and SSAT animals revealed GU tracts that were generally uniform in size and shape. As graphed in Fig. 2B, GU tracts of the SSAT mice were significantly smaller (178 ± 30 mg) than those of the wild-type mice (504 ± 11 mg) despite close similarities in body weight (∼29.7 ± 0.6 versus 28.5 ± 0.5 g, respectively). All of the TRAMP mice displayed visible evidence of prostatic tumors with variable involvement of the seminal vesicles. On the basis of weight, TRAMP GU tracts (1,435 ± 181 mg) were, on average, 4 times larger than those of TRAMP/SSAT mice (356 ± 62 mg). Taken together, the data indicate that SSAT overexpression effectively suppresses tumor outgrowth in the TRAMP model. Since by itself, the 30-week data may reflect a delay in tumor development as opposed to a sustained antitumor effect, we examined tumor size at a later time point. For this, 36 weeks was the longest time possible without encountering tumor excess. Relative to the 30-week data, the average GU tract weight in the TRAMP mice became 200% larger, while that in the TRAMP/SSAT mice at 36 weeks remained statistically unchanged. Thus, suppression of tumor outgrowth became even more exaggerated during the 30-36-week period. Although these findings suggest that the survival time of the TRAMP/SSAT mice would be significantly extended beyond that of the TRAMP mice, such studies were precluded by the strong tendency of the older bigenics to develop skin pathologies. Histopathological analysis from littermates of the TRAMP and SSAT transgenic crosses at 30 weeks demonstrated that the prostate tumors of TRAMP mice were heterogeneous, ranging from high grade prostatic intraepithelial neoplasia (grade 3) to poorly differentiated adenocarcinoma (grade 6), while tumors from TRAMP/SSAT mice were more homogeneous with a moderate range of prostatic intraepithelial neoplasia lesions (grades 2 and 3) and only rare evidence of well differentiated adenocarcinoma (grade 4) (Fig. 3A). Consistent with previous reports (28Gingrich J.R. Barrios R.J. Foster B.A. Greenberg N.M. Prostate Cancer Prostatic Dis. 1999; 2: 70-75Crossref PubMed Scopus (199) Google Scholar), disease in the TRAMP mouse was apparent in the dorsal, lateral, and ventral lobes with substantial seminal vesicle involvement, and it tended to be heterogeneous among mice. When all prostate lobes were averaged (Table I), the mean TRAMP/SSAT mouse grade (4.2) was significantly lower than that of the TRAMP mice (5.0) suggesting interference with disease progression. A similar trend was also seen at 36 weeks. Thus, while the major effect of SSAT overexpression is most obviously manifested as suppression of tumor outgrowth, there is also a delay in tumor progression. Because prostatic disease in the C57BL/6 background infiltrated the seminal vesicles, we derived an index to quantify overall GU disease. Thus, the average histological grades of the four lobes were averaged and then multiplied by the mean GU tract weight to derive a GU disease index for each cohort o" @default.
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• W2134253034 date "2004-09-01" @default.
• W2134253034 modified "2023-09-28" @default.
• W2134253034 title "Activated Polyamine Catabolism Depletes Acetyl-CoA Pools and Suppresses Prostate Tumor Growth in TRAMP Mice" @default.
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