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• W1982643460 abstract "Depletion of intracellular polyamine pools invariably inhibits cell growth. Although this is usually accomplished by inhibiting polyamine biosynthesis, we reasoned that this might be more effectively achieved by activation of polyamine catabolism at the level of spermidine/spermine N1-acetyltransferase (SSAT); a strategy first validated in MCF-7 breast carcinoma cells. We now examine the possibility that, due to unique aspects of polyamine homeostasis in the prostate gland, tumor cells derived from it may be particularly sensitive to activated polyamine catabolism. Thus, SSAT was conditionally overexpressed in LNCaP prostate carcinoma cells via a tetracycline-regulatable (Tet-off) system. Tetracycline removal resulted in a rapid ∼10-fold increase in SSAT mRNA and an increase of ∼20-fold in enzyme activity. SSAT products N1-acetylspermidine, N1-acetylspermine, and N1,N12-diacetylspermine accumulated intracellularly and extracellularly. SSAT induction also led to a growth inhibition that was not accompanied by polyamine pool depletion as it was in MCF-7 cells. Rather, intracellular spermidine and spermine pools were maintained at or above control levels by a robust compensatory increase in ornithine decarboxylase and S-adenosylmethionine decarboxylase activities. This, in turn, gave rise to a high rate of metabolic flux through both the biosynthetic and catabolic arms of polyamine metabolism. Treatment with the biosynthesis inhibitor α-difluoromethylornithine during tetracycline removal interrupted flux and prevented growth inhibition. Thus, flux-induced growth inhibition appears to derive from overaccumulation of metabolic products and/or from depletion of metabolic precursors. Metabolic effects that were not excluded as possible contributing factors include high levels of putrescine and acetylated polyamines, a 50% reduction in S-adenosylmethionine, and a 45% decline in the SSAT cofactor acetyl-CoA. Overall, the study demonstrates that activation of polyamine catabolism in LNCaP cells elicits a compensatory increase in polyamine biosynthesis and downstream metabolic events that culminate in growth inhibition. Depletion of intracellular polyamine pools invariably inhibits cell growth. Although this is usually accomplished by inhibiting polyamine biosynthesis, we reasoned that this might be more effectively achieved by activation of polyamine catabolism at the level of spermidine/spermine N1-acetyltransferase (SSAT); a strategy first validated in MCF-7 breast carcinoma cells. We now examine the possibility that, due to unique aspects of polyamine homeostasis in the prostate gland, tumor cells derived from it may be particularly sensitive to activated polyamine catabolism. Thus, SSAT was conditionally overexpressed in LNCaP prostate carcinoma cells via a tetracycline-regulatable (Tet-off) system. Tetracycline removal resulted in a rapid ∼10-fold increase in SSAT mRNA and an increase of ∼20-fold in enzyme activity. SSAT products N1-acetylspermidine, N1-acetylspermine, and N1,N12-diacetylspermine accumulated intracellularly and extracellularly. SSAT induction also led to a growth inhibition that was not accompanied by polyamine pool depletion as it was in MCF-7 cells. Rather, intracellular spermidine and spermine pools were maintained at or above control levels by a robust compensatory increase in ornithine decarboxylase and S-adenosylmethionine decarboxylase activities. This, in turn, gave rise to a high rate of metabolic flux through both the biosynthetic and catabolic arms of polyamine metabolism. Treatment with the biosynthesis inhibitor α-difluoromethylornithine during tetracycline removal interrupted flux and prevented growth inhibition. Thus, flux-induced growth inhibition appears to derive from overaccumulation of metabolic products and/or from depletion of metabolic precursors. Metabolic effects that were not excluded as possible contributing factors include high levels of putrescine and acetylated polyamines, a 50% reduction in S-adenosylmethionine, and a 45% decline in the SSAT cofactor acetyl-CoA. Overall, the study demonstrates that activation of polyamine catabolism in LNCaP cells elicits a compensatory increase in polyamine biosynthesis and downstream metabolic events that culminate in growth inhibition. Cell growth is dependent on a sustained supply of polyamines, which is typically met by the integrated contributions of biosynthesis, catabolism, uptake, and export, each of which is sensitively regulated by effector molecules that, in turn, are controlled by intracellular polyamine pools (1Porter C.W. Regenass U. Bergeron R.J. Dowling R.H. Folsch U.R. Loser C. Falk Symposium on Polyamines in the Gastrointestinal Tract. Kluwer Academic Publishers Group, Dordrecht, Netherlands1992: 301-322Google Scholar). Thus, ornithine decarboxylase (ODC) 1The abbreviations used are: ODC, ornithine decarboxylase; CoA, coenzyme A; AcSpd, N1-acetylspermidine; AcSpm, N1-acetylspermine; dcSAM, decarboxylated S-adenosylmethionine, DiAcSpm, N1,N12-diacetylspermine; DENSPM, N1,N11-diethylnorspermine; DFMO, α-difluoromethylornithine; HPCE, high performance capillary electrophoresis; HPLC, high performance liquid chromatography; MTA, 5′-methylthioadenosine; PAO, polyamine oxidase; Put, putrescine; SAM, S-adenosylmethionine; SAMDC, S-adenosylmethionine decarboxylase; Spd, spermidine; Spm, spermine; SSAT, spermidine/spermine N1-acetyltransferase; Tet, tetracycline; TRAMP, transgenic adenocarcinoma of mouse prostate; tTA, tetracycline-repressible transactivator; dansyl, 5-dimethylaminona-phthalene-1-sulfonyl. and S-adenosylmethionine decarboxylase (SAMDC) control biosynthesis, a polyamine transport system modulates uptake, and spermidine/spermine N1-acetyltransferase (SSAT) regulates polyamine catabolism and export out of the cell. Neoplastic cell growth is associated with elevated polyamine biosynthetic activity, even when the surrounding normal tissue itself is rapidly proliferating, such as the intestinal mucosa (2Porter C.W. Herrera-Ornelas L. Pera P. Petrelli N.F. Mittelman A. Cancer. 1987; 60: 1275-1281Crossref PubMed Scopus (118) Google Scholar, 3Kramer D.L. Nishioka K. Critical Roles of Polyamines in Cancer: Basic Mechanisms and Clinical Approaches. R. G. Landes Co., New York1996: 151-189Google Scholar, 4Thomas T. Thomas T.J. J. Cell Mol. Med. 2003; 7: 113-126Crossref PubMed Scopus (274) Google Scholar). Thus, the rationale for targeting polyamines in antitumor strategies relates to their critical role in supporting neoplastic cell growth and to the overexpression of biosynthetic enzymes in tumor versus normal tissues (3Kramer D.L. Nishioka K. Critical Roles of Polyamines in Cancer: Basic Mechanisms and Clinical Approaches. R. G. Landes Co., New York1996: 151-189Google Scholar, 5Thomas T. Thomas T.J. Cell Mol. Life Sci. 2001; 58: 244-258Crossref PubMed Scopus (771) Google Scholar, 6Seiler N. Curr. Drug Targets. 2003; 4: 537-564Crossref PubMed Scopus (149) Google Scholar). The biology and metabolism of polyamines in the prostate is distinctly different from that of other tissues. In addition to synthesizing these molecules for epithelial cell replacement, the gland produces massive quantities of spermine (Spm) for export into reproductive fluids (7Harrison G.A. Biochem. J. 1931; 25: 1885-1892Crossref PubMed Google Scholar, 8Mann T. The Biochemistry of Semen and of the Male Reproductive Tract. John Wiley, New York1964: 193-200Google Scholar, 9Pegg A.E. Williams-Ashman H.G. Biochem. J. 1968; 108: 533-539Crossref PubMed Scopus (276) Google Scholar, 10Williams-Ashman H.G. Canellakis Z.N. Perspect. Biol. Med. 1979; 22: 421-453Crossref PubMed Scopus (273) Google Scholar). The only major tissue that synthesizes polyamines for export, the prostate, and presumably tumors derived from it, may be dependent on novel and therapeutically exploitable homeostatic mechanisms. For example, we have observed that, in contrast to other cell lines, two of three prostate carcinoma lines failed to regulate polyamine transport in response to polyamine analogues or inhibitors (11Mi 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). Very recently, Rhodes et al. (12Rhodes D.R. Barrette T.R. Rubin M.A. Ghosh D. Chinnaiyan A.M. Cancer Res. 2002; 62: 4427-4433PubMed Google Scholar) performed a meta-analysis of four independent microarray datasets comparing gene expression profiles of benign versus malignant patient prostate samples (12Rhodes D.R. Barrette T.R. Rubin M.A. Ghosh D. Chinnaiyan A.M. Cancer Res. 2002; 62: 4427-4433PubMed Google Scholar). Their study showed that polyamine biosynthesis was the most consistently and significantly affected metabolic, signaling, or apoptotic pathway. More particularly, the study revealed a synchronous network of genes contributing to polyamine biosynthesis was up-regulated while genes detracting from polyamine biosynthesis were down-regulated. In support of these findings, clinical studies by Bettuzzi et al. (13Bettuzzi 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) indicate a significant increase in transcripts of the polyamine biosynthetic enzymes, ODC and SAMDC, in human prostatic cancer relative to benign hyperplasia. In recognition of the unique physiology of the prostate gland, Heston and collaborators (14Heston W.D. Watanabe K.A. Pankiewicz K.W. Covey D.F. Biochem. Pharmacol. 1987; 36: 1849-1852Crossref PubMed Scopus (16) Google Scholar, 15Heston W.D. Cancer Surv. 1991; 11: 217-238PubMed Google Scholar) were among the first to propose that targeting polyamine biosynthesis may be particularly effective against prostate cancer. Most of these efforts have made use of known inhibitors of ODC or SAMDC (16Mamont P.S. Duchesne M.C. Grove J. Bey P. Biochem. Biophys. Res. Commun. 1978; 81: 58-66Crossref PubMed Scopus (371) Google Scholar, 17Regenass U. Mett H. Stanek J. Mueller M. Kramer D. Porter C.W. Cancer Res. 1994; 54: 3210-3217PubMed Google Scholar, 18Kramer D.L. Khomutov R.M. Bukin Y.V. Khomutov A.R. Porter C.W. Biochem. J. 1989; 259: 325-331Crossref PubMed Scopus (54) Google Scholar, 19Danzin C. Marchal P. Casara P. Biochem. Pharmacol. 1990; 40: 1499-1503Crossref PubMed Scopus (48) Google Scholar). Gupta et al. (20Gupta S. Ahmad N. Marengo S.R. MacLennan G.T. Greenberg N.M. Mukhtar H. Cancer Res. 2000; 60: 5125-5133PubMed Google Scholar) showed that the ODC inhibitor, α-difluoromethylornithine (DFMO) effectively suppressed development of prostate cancer in the TRAMP mouse model. As an alternative approach to the use of enzyme inhibition, we propose that disruption of polyamine homeostasis at the level of polyamine catabolism may have unique therapeutic potential against prostate carcinoma. It has been demonstrated, for example, that polyamine analogues such as N1,N11-diethylnorspermine (DENSPM) down-regulate polyamine biosynthesis at the level of ODC and SAMDC and, at the same time, potently (i.e. >200-fold) up-regulate polyamine catabolism at the level of spermidine/spermine N1-acetyltransferase (SSAT) (1Porter C.W. Regenass U. Bergeron R.J. Dowling R.H. Folsch U.R. Loser C. Falk Symposium on Polyamines in the Gastrointestinal Tract. Kluwer Academic Publishers Group, Dordrecht, Netherlands1992: 301-322Google Scholar, 21Bergeron R.J. Feng Y. Weimar W.R. McManis J.S. Dimova H. Porter C. Raisler B. Phanstiel O. J. Med. Chem. 1997; 40: 1475-1494Crossref PubMed Scopus (125) Google Scholar, 22Casero 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, 23Libby P.R. Bergeron R.J. Porter C.W. Biochem. Pharmacol. 1989; 38: 1435-1442Crossref PubMed Scopus (63) Google Scholar, 24Casero Jr., R.A. Ervin S.J. Celano P. Baylin S.B. Bergeron R.J. Cancer Res. 1989; 49: 639-643PubMed Google Scholar, 25Shappell N.W. Miller J.T. Bergeron R.J. Porter C.W. Anticancer Res. 1992; 12: 1083-1089PubMed Google Scholar, 26Pegg A.E. Wechter R. Pakala R. Bergeron R.J. J. Biol. Chem. 1989; 264: 11744-11749Abstract Full Text PDF PubMed Google Scholar, 27Porter C.W. Ganis B. Libby P.R. Bergeron R.J. Cancer Res. 1991; 51: 3715-3720PubMed Google Scholar). Several lines of evidence support the idea that analogue induction of SSAT and hence, activation of polyamine catabolism, is a critical determinant of DENSPM drug action. For example, DENSPM growth inhibition among tumor cell lines correlates with the extent to which SSAT is induced (23Libby P.R. Bergeron R.J. Porter C.W. Biochem. Pharmacol. 1989; 38: 1435-1442Crossref PubMed Scopus (63) Google Scholar, 24Casero Jr., R.A. Ervin S.J. Celano P. Baylin S.B. Bergeron R.J. Cancer Res. 1989; 49: 639-643PubMed Google Scholar, 25Shappell N.W. Miller J.T. Bergeron R.J. Porter C.W. Anticancer Res. 1992; 12: 1083-1089PubMed Google Scholar), and analogues that differentially induce SSAT inhibit cell growth in a correlative manner (22Casero 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, 26Pegg A.E. Wechter R. Pakala R. Bergeron R.J. J. Biol. Chem. 1989; 264: 11744-11749Abstract Full Text PDF PubMed Google Scholar, 27Porter C.W. Ganis B. Libby P.R. Bergeron R.J. Cancer Res. 1991; 51: 3715-3720PubMed Google Scholar). As more direct evidence for this relationship, McCloskey et al. (28McCloskey D.E. Pegg A.E. J. Biol. Chem. 2000; 275: 28708-28714Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) showed that DENSPM-resistant Chinese hamster ovary cells are unable to induce SSAT. Recently, Chen et al. (29Chen Y. Kramer D.L. Li F. Porter C.W. Oncogene. 2003; 22: 4964-4972Crossref PubMed Scopus (39) Google Scholar, 30Chen Y. Kramer D.L. Jell J. Vujcic S. Porter C.W. Mol. Pharmacol. 2003; 64: 1153-1159Crossref PubMed Scopus (33) Google Scholar) reported that small interference RNA interference with DENSPM induction of SSAT prevented polyamine pool depletion while blocking analogue-induced apoptosis in human melanoma cells. The studies cited above relate to SSAT induction in the context of analogue treatment, but they do not address what happens when SSAT is selectively induced in cells. In an earlier report (31Vujcic 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), we showed that conditional overexpression of SSAT leads to polyamine pool depletion and growth inhibition in MCF-7 breast carcinoma cells. On the basis of rationale suggesting that prostate carcinoma may react differently to perturbations in polyamine homeostasis, we investigated the consequences of conditional SSAT overexpression in LNCaP prostate carcinoma cells. Materials—The inhibitor of polyamine oxidase (PAO), N1-methyl-N2-(2,3-butadienyl)butane-1,4-diamine (MDL-72527) was generously provided by Aventis Pharmaceuticals Inc. (Bridgewater, NJ). The ODC inhibitor DFMO was obtained from Ilex, Inc. (San Antonio, TX). Tetracycline (Tet), aminoguanidine, polyamines, and the acetylated polyamines N1-acetylspermidine (AcSpd) and N1-acetylspermine (AcSpm) were purchased from Sigma-Aldrich, whereas N1,N12-diacetylspermine (DiAcSpm) was provided as a gift from Dr. Nikolaus Seiler (Laboratory of Nutritional Oncology, Institut de Recherche Contre les Cancers, Strasbourg, France). SAM was purchased from Sigma-Aldrich, and the SAM metabolites, decarboxylated S-adenosylmethionine (dcSAM) and 5-methylthioadenosine (MTA), were synthesized and kindly provided by Drs. Canio Marasco and Janice Sufrin (Roswell Park Cancer Institute). Radioactive compounds l-[1-14C]ornithine, [acetyl-1-14C] coenzyme A, [α-32P]dCTP were purchased from PerkinElmer Life Sciences, and S-adenosyl-l-[carboxyl-14C]methionine was obtained Amersham Biosciences. Acetyl-coenzyme A (acetyl-CoA) was purchased from Sigma-Aldrich and solubilized as described by Liu et al. (32Liu G. Chen J. Che P. Ma Y. Anal. Chem. 2003; 75: 78-82Crossref PubMed Scopus (25) Google Scholar). Geneticin (G418) and hygromycin B were obtained from Clontech Laboratories, Inc. (Palo Alto, CA) and Invitrogen, respectively. Cell Culture—LNCaP prostate carcinoma cells engineered to constitutively express the tetracycline-repressible transactivator (tTA) (33Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4268) Google Scholar), designated LNGK9 (34Gschwend J.E. Fair W.R. Powell C.T. Prostate. 1997; 33: 166-176Crossref PubMed Scopus (17) Google Scholar), were cultured in RPMI 1640 media supplemented with 2 mm glutamine (Invitrogen), 10% Tet-approved fetal bovine serum (Clontech Laboratories, Inc.), penicillin at 100 units/ml, streptomycin at 100 units/ml (Invitrogen), and 150 μg/ml hygromycin B at 37 °C in the presence of humidified 5% CO2. Aminoguanidine (at 1 mm) was routinely included in the media as an inhibitor of copper-dependent bovine serum amino oxidases to prevent conversion of extracellular polyamines to toxic products. Cells were harvested by trypsinization and counted electronically (Coulter Model ZM, Coulter Electronics, Hialeah, FL). Transfections—LNGK9 cells expressing the tTA were seeded at 2 × 106 cells per 100-mm culture dishes in the absence of hygromycin B and Tet. The following day, fresh media were replaced and cells were cotransfected with the tTA-responsive pTRE-SSAT plasmid (31Vujcic 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 a G418-resistance selection plasmid pcDNA3 (Invitrogen) at a ratio of 20:1 using FuGENE 6 (Roche Applied Science) according to manufacturer's protocol. Stably transfected clones were selected in medium containing 500 μg/ml antibiotic G418, 150 μg/ml hygromycin B, and 1 μg/ml Tet. Healthy G418-resistant clones were selected and tested for SSAT mRNA by Northern blot analysis in the presence or absence of 1 μg/ml Tet. With the Tet-off system, SSAT transcription is induced in the absence of Tet (–Tet) but not in its presence (+Tet). A Tet concentration of 1 μg/ml was found to fully and consistently suppress SSAT gene expression during routine cell culture passage. Clones that expressed low basal level of SSAT mRNA under +Tet conditions and high induced levels of SSAT mRNA under –Tet conditions were selected for further study. Clones were maintained continuously under 1 μg/ml +Tet until experiments were initiated. Northern Blot Analysis—Northern blot analysis was carried out as described by Fogel-Petrovic et al. (35Fogel-Petrovic M. Shappell N.W. Bergeron R.J. Porter C.W. J. Biol. Chem. 1993; 268: 19118-19125Abstract Full Text PDF PubMed Google Scholar) with modifications. Briefly, total RNA was extracted with an RNeasy® Mini kit (Qiagen Inc., Valencia, CA), and its concentration was determined by UV spectrophotometry. RNA samples (5 μg/lane) were separated on 1.5% agarose/formaldehyde gels and transferred to a Duralon-UV membrane (Stratagene, La Jolla, CA). The membrane was cross-linked in a Stratalinker™ 1800, hybridized to [32P]dCTP random-labeled cDNA probes (Stratagene) for detection of SSAT mRNA (36Xiao L. Celano P. Mank A.R. Pegg A.E. Casero Jr., R.A. Biochem. Biophys. Res. Commun. 1991; 179: 407-415Crossref PubMed Scopus (35) Google Scholar), and exposed for autoradiography. A glyceraldehyde-3-phosphate dehydrogenase signal was used as a loading control. Polyamine Enzymes and Polyamine Pools—SSAT, ODC, and SAMDC activities were assayed as described previously (27Porter C.W. Ganis B. Libby P.R. Bergeron R.J. Cancer Res. 1991; 51: 3715-3720PubMed Google Scholar, 37Porter C.W. Cavanaugh Jr., P.F. Stolowich N. Ganis B. Kelly E. Bergeron R.J. Cancer Res. 1985; 45: 2050-2057PubMed Google Scholar). Polyamine enzyme activities were expressed as picomoles of AcSpd generated per minute/mg of protein for SSAT and as nanomoles of CO2/h/mg of protein for ODC and SAMDC. Intracellular polyamines, including acetylated derivatives of spermidine (Spd) and Spm were extracted from cell pellets with 0.6 n perchloric acid, dansylated, measured by reverse phase high-performance liquid chromatography (HPLC) as described by Kramer et al. (38Kramer 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), and expressed as picomoles/106 cells. Extracellular polyamines and acetylated polyamines were extracted from media as described by Kramer et al. (39Kramer D. Stanek J. Diegelman P. Regenass U. Schneider P. Porter C.W. Biochem. Pharmacol. 1995; 50: 1433-1443Crossref PubMed Scopus (14) Google Scholar), containing fetal bovine serum, Tet, and l-glutamine but not G418 or hygromycin B. A total of 50 μl of dansylated sample was injected for HPLC, and data were collected and analyzed as noted above. Extracellular polyamine pools were expressed as nanomoles/equivalent volume (ml)/106 cells. S-Adenosylmethionine and Metabolite Pools—Intracellular SAM and its metabolites, dcSAM and MTA, were extracted from cell pellets with 0.6 n perchloric acid and measured by HPLC according to chromatographic conditions reported by Yarlett and Bacchi (40Yarlett N. Bacchi C.J. Mol. Biochem. Parasitol. 1988; 27: 1-10Crossref PubMed Scopus (45) Google Scholar) with modifications as described by Kramer et al. (38Kramer 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). Briefly, samples (50 μl) were eluted from a C18 column (40 °C) at a flow rate of 0.8 ml/min with a linear gradient starting with solvent A (0.1 m NaH2PO4, 8 mm octane sulfonic acid, 0.05 mm EDTA, 2% acetonitrile) at 80% and solvent B (0.15 mm NaH2PO4, 8 mm octane sulfonic acid, 26% acetonitrile) at 20%. Over the course of 30 min, the gradient increased to 100% solvent B for 10 min. Effluent was monitored with a Waters 2487 dual wavelength UV detector, and data were processed using instrumentation described for polyamine pool analysis and expressed as picomoles/106 cells. Measurement of Acetyl-CoA—High performance capillary electrophoresis (HPCE) separation and quantitation of acetyl-CoA in biological samples followed the method of Liu et al. (32Liu G. Chen J. Che P. Ma Y. Anal. Chem. 2003; 75: 78-82Crossref PubMed Scopus (25) Google Scholar). Cells were lysed and processed by using a solid-phase extraction. Extracts were then analyzed on a Beckman P/ACE MDQ capillary electrophoresis system equipped with a photodiode array detector and an uncoated fused silica CE 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. (32Liu G. Chen J. Che P. Ma Y. Anal. Chem. 2003; 75: 78-82Crossref PubMed Scopus (25) Google Scholar) with modifications. Briefly, the capillary was preconditioned with 1 m NaOH and Milli-Q water for 10 min each at 20 p.s.i. and then equilibrated with 100 mm NaH2PO4 running buffer containing 0.1% β-cyclodextrin (pH 6.0) for 10 min. After each run, the capillary was rinsed with 1 m NaOH, Milli-Q water, and running buffer for 2 min each. The injection was done hydrodynamically at a pressure of 0.5 p.s.i. for 10 s. Injection volume was calculated using CE Expert Lite software from Beckman. Separation voltage was 15 kV at a constant capillary temperature of 15 °C. To establish the standard calibration curves, solutions containing the acetyl-CoA and the internal standard (isobutyryl-CoA, 41 nmol) were prepared at concentrations ranging from 1 to 200 nmol. Standards were processed as described above for cell lysates and resuspended in 10 μl of water. The detector response was (r > 0.99) for all acetyl-CoA species over the above concentration range. Coenzyme As were monitored with a photodiode array detector at the maximum absorbance wavelength (253.5 nm). Data were collected and processed by using Beckman P/ACE 32 Karat software version 4.0. Cellular acetyl-CoA levels were expressed as nanomoles/106 cells. Derivation of Transfected Cells—Cells transfected with the human SSAT cDNA were selected in neomycin and grown as clones in the presence of 1 μg/ml Tet. Of the 100 SSAT/LNGK9 clones screened (data not shown), those most sensitive to Tet regulation were selected according to the differential expression between SSAT mRNA in +Tet (SSAT-off) versus mRNA in –Tet (SSAT-on) (Fig. 1). Based on these criteria, clone 53 was selected for further study, because it displayed low SSAT mRNA in +Tet and an 8-fold increase in total SSAT mRNA (exogenous and endogenous) under –Tet conditions for 48 h. Nearly identical responses were also obtained with several other clones. Transfected human SSAT cDNA (∼1.5 kb) was distinguishable from the smaller endogenous transcript (∼1.3 kb) by differences in polyadenylation, which became apparent during enzyme induction (35Fogel-Petrovic M. Shappell N.W. Bergeron R.J. Porter C.W. J. Biol. Chem. 1993; 268: 19118-19125Abstract Full Text PDF PubMed Google Scholar). Although the exogenous 1.5-kb transcript levels increased with Tet removal, the endogenous 1.3-kb transcript levels remained at basal levels, indicating that the presence or absence of the antibiotic did not affect endogenous gene expression. Tet-regulated expression of SSAT mRNA and activity was characterized from 0 to 144 h in clone 53 (Fig. 2). Following Tet removal, SSAT mRNA increased significantly by 6 h and plateaued by 24 h at levels ranging between 10- and 20-fold greater than the 0-h sample. Induction of mRNA was closely paralleled by increases in SSAT activity, which reached a maximum of ∼20-fold by 24 h (see Figs. 2 and 4).Fig. 2Time-dependent increases in SSAT mRNA and activity in SSAT/LNGK9 clone 53 following Tet removal. Tet was removed for the indicated time, and cells were harvested for total RNA isolation and SSAT enzyme activity. An amount of 5 μg of total RNA was loaded onto each Northern blot lane. Note that following removal of 1 μg/ml Tet, both SSAT mRNA and activity increased rapidly before reaching a plateau between 24 and 48 h. For quantitation, SSAT mRNA bands were scanned fluorometrically, normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal, and expressed as -fold increase (Fold ↑) relative to +Tet at 0 h (lane 1). As expressed by -fold increase, SSAT activity increased in parallel to SSAT mRNA. This blot is representative of findings from three separate experiments. hnRNA, heteronuclear RNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Time-dependent effects of conditional SSAT overexpression on polyamine biosynthetic enzyme activities. Tet was removed from SSAT/LNGK9-clone 53 cells for the indicated time after which cells were harvested for polyamine enzyme activities for SSAT, ODC, and SAMDC. Following Tet removal, both SSAT activity (▪) increased sharply to a maximum of ∼20-fold (A), and ODC activity (▪) increased sharply to ∼10-fold (B) at 48 h before undergoing a steady decline. By contrast, SAMDC activity (•) increased steadily over the course of 144 h to a maximum of ∼18-fold that of basal levels (C). Enzyme activities of SSAT/LNGK9-clone 53 cells grown continuously in the presence of Tet remained relatively unchanged from 0 h (data not shown). Data represents mean values ± S.E., where n is 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Effects of SSAT Overexpression on Cell Growth and Polyamine Metabolism—As shown in Fig. 3, SSAT overexpression caused significant inhibition of cell growth at ∼2 days following Tet removal, which was sustained through the 6-day experiment. Growth inhibition appeared to be cytostatic rather than cytotoxic, because there was no obvious decline in cell number as would be expected with apoptosis and because addition of Tet at 96 h resulted in a rapid resumption of cell growth (Fig. 3). The time-dependent effects of Tet removal on enzyme activities and polyamine pools are shown in Figs. 4 and 5. SSAT increased steadily to 22-fold by 48 h before declining slowly from 48 to 144 h (Figs. 2 and 4). This steady decrease in SSAT activity and mRNA may be due to a homeostatic adjustment of gene expression and/or to a time-dependent selection of cells that express lower levels of SSAT. Consistent with the observed rise in enzyme activity, acetylated polyamines increased under –Tet conditions (Fig. 5). Intracellular AcSpd increased remarkably from undetectable levels (<10 pmol/106 cells) to 10,420 pmol/106 cells by 48 h. Other SSAT products, AcSpm and DiAcSpm, which are rarely seen in cells (31Vujcic 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), accumulated to 390 pmol/106 cells and 2,340 pmol/106 cells, respectively, by 48 h and remained elevated during the course of the 144-h experiment. Putrescine (Put) pools also rose remarkably due presumably to back-conversion of Put from Spd via AcSpd (Fig. 5) and to forward synthesis due to increased ODC activity (described below). Despite the massive accumulation of acetylated polyamines, intracellular levels of Spd and Spm failed to decrease. In fact, the levels of Put, Spd, and Spm increased s" @default.
• W1982643460 created "2016-06-24" @default.
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• W1982643460 date "2004-06-01" @default.
• W1982643460 modified "2023-10-01" @default.
• W1982643460 title "Metabolic and Antiproliferative Consequences of Activated Polyamine Catabolism in LNCaP Prostate Carcinoma Cells" @default.
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