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Trichomonas vaginalis, a urogenital parasite, and its dependence on polyamine backconversion are discussed as is the potential of bis(alkyl) polyamine analogs as antitrichomonad agents. A study has shown that growth of T. vaginalis with DENSpm also brings on polyamine depletion and significant inhibition of growth by a mechanism that involves competition with the transport of spermine and inhibition of SSAT effectively blocking spermidine synthesis. The parasite differs significantly from other eukaryotes in several aspects of its polyamine metabolism. The spermine taken up is backconverted to spermidine through a spermidine:spermine N(super.1)-acetyltransferase (SSAT)/polyamine oxidase-coupled pathway.

Chronic lymphocytic leukaemia (CLL) is a malignant haematological disorder that remains mostly incurable; more than 95% of patients have disease of B-cell origin. Advances with targeted agents such as monoclonal antibodies, antisense therapy, or both these techniques combined with traditional chemotherapy have improved the frequency of remission. The clinical course of CLL is marked by frequent relapse, and there are limited therapeutic options for patients with relapsed or refractory disease. The morphologically mature CLL clone regulates the microenvironment through modulation of the cytokine milieu that aids its growth and survival, and has a role in immune escape. Targeting of the tumour-cell microenvironment has not been investigated as a treatment option for CLL. Immunomodulating agents are a new class of drugs that change expression of various cytokines and that costimulate immune effector cells. [ABSTRACT FROM AUTHOR]

Purpose: To understand the mechanisms behind platinum drug/DENSPM-induced inhibition of cancer cell growth, we compared the effects of oxaliplatin and cisplatin when combined with DENSPM on the induction of SSAT mRNA, activity, polyamines and cell growth in A2780 human ovarian carcinoma cells and their oxaliplatin- and cisplatin-resistant variants A2780/C10B and A2780/CP, respectively., Methods: Parental and Pt-resistant cells were treated with platinum agent alone, DENSPM alone or combination (10 μM each, 20 h). QRT-PCR, radioactive product measurement and HPLC were used for mRNA, activity and polyamine pools, respectively; drug interaction on cell growth was by SRB and isobologram analysis., Results: Both platinum agents induced SSAT mRNA in parental A2780 cells, but not in resistant cells. Platinum drug/DENSPM combinations produced high levels of SSAT activity in parental cells with significant depletion of spermine and spermidine, but not in resistant cells. Co-treatment with platinum agents increased the levels of DENSPM in all cell lines. Oxaliplatin/DENSPM combination was superior to cisplatin/DENSPM in the inhibition of cell growth in parental cells. No synergy was observed in the resistant cells., Conclusions: Increased DENSPM levels following co-treatment with Pt agents enhances the translation and stability of SSAT protein leading to polyamine pool depletion, facilitating more Pt-DNA adduct formation in parental cells. Oxaliplatin/DENSPM combination is superior to cisplatin/DENSPM in cell growth inhibition as DACH-Pt DNA adducts are cytotoxic even at relatively fewer numbers. Reduced platinum uptake in Pt-resistant cells contributes to reduced SSAT mRNA induction and absence of synergy when combined with DENSPM.

The expression of catabolic enzymes spermidine/spermine N(1)-acetyltransferase (SSAT) and spermine oxidase (SMO) increases after ischemic reperfusion injury. We hypothesized that polyamine catabolism is upregulated and that this increase in catabolic response contributes to tissue damage in endotoxin-induced acute kidney injury (AKI). SSAT mRNA expression peaked at threefold 24 h following LPS injection and returned to background levels by 48 h. The activity of SSAT correlated with its mRNA levels. The expression of SMO also increased in the kidney after LPS administration. Serum creatinine levels increased significantly at approximately 15 h, peaking by 24 h, and returned to background levels by 72 h. To test the role of SSAT in endotoxin-induced AKI, we injected wild-type (SSAT-wt) and SSAT-deficient (SSAT-ko) mice with LPS. Compared with SSAT-wt mice, the SSAT-ko mice subjected to endotoxic-AKI had less severe kidney damage as indicated by better preservation of kidney function. The role of polyamine oxidation in the mediation of kidney injury was examined by comparing the severity of renal damage in SSAT-wt mice treated with MDL72527, an inhibitor of both polyamine oxidase and SMO. Animals treated with MDL72527 showed significant protection against endotoxin-induced AKI. We conclude that increased polyamine catabolism through generation of by-products of polyamine oxidation contributes to kidney damage and that modulation of polyamine catabolism may be a viable approach for the treatment of endotoxin-induced AKI.

We have previously showed that platinum drugs up-regulate SSAT and SMO and down-regulate ODC and SAMDC in the polyamine pathway. Several studies including our own established that platinum drugs combined with polyamine analog DENSPM produces synergistic increase in SSAT activity with polyamine depletion. Since polyamine pathway is an important therapeutic target, we investigated whether agents containing both platinum and polyamines have similar effects on the polyamine pathway. Two complexes i) Pt-spermine with two cisplatin molecules linked to a spermine in the center and ii) Pd-spermine with similar structure i, but Pd (II) substituted for Pt (II) were analyzed with respect to their effect on the expression of genes in polyamine pathway, SSAT and SMO protein expression, SSAT activity and polyamine pools. Pt-, Pd-spermine complexes induced significant down-regulation of SMO, arginase 2 and NRF-2, with no change in SSAT, while cisplatin as a single agent or in combination with DENSPM induced significant up-regulation of SSAT and SMO. The SSAT activity was not induced by either Pt- or Pd-spermine in A2780 cells; SMO protein levels were significantly elevated compared to the no-drug control and to a similar extent as cisplatin/DENSPM. The Pd-spm treatment induced a fall in putrescine levels to 33%, spermidine to 62% and spermine to 72% while Pt-spm did not induce such a decline. Comparative cytotoxicity studies in A2780 cells indicated the potency to be cisplatin> Pd-Spm>Pt-Spm. Although both complexes exhibit a lower potency, the degree of resistance itself is much lower for Pt-spermine and Pd-spermine in that order (2.5 and 7.5, respectively) compared to cisplatin ( approximately 12) as tested in cisplatin resistant A2780/CP cells. These studies suggest that Pd (II)-polyamine complexes may constitute a promising group of inorganic compounds for further studies in the development of novel chemotherapy/adjuvant chemotherapy strategies.

Survival has been implicated to play an important role in various pathophysiological processes. However, because of a lack of appropriate animal models, the role and dynamic expression of survivin during pathophysiology are not well defined. We generated a human survivin gene promoter-driven luciferase transgenic mouse model (SPlucTg) so that dynamic survivin gene activity can be monitored during various pathophysiological conditions using in vivo imaging. Our results show that, consistent with survivin positivity in testis, luciferase activity in normal SPlucTg mice was detected in the testis of male mice. Furthermore, similar to the known requirement of transient expression of survivin for pathophysiological responses, we observed a transient luciferase expression in castrated SPlucTg male mice after supplement of androgen. Significantly, it was reported that survivin expression turns on during mouse liver injury and regeneration; a transient and dose-dependent luciferase expression in the mouse liver was observed after administration of carbon tetrachloride into SPlucTg mice. We further demonstrated that luciferase activity closely correlates with endogenous survivin expression. We also demonstrated that only a subset of cells expresses survivin, and its expression overlaps with the expression of several stem cell markers tested. Thus, we have generated a unique animal model for analysis of diverse pathophysiological processes and possible stem cell distribution/activity in vivo.

Folate (vitamin B9) is utilized for synthesis of both S-adenosylmethionine (AdoMet) and deoxythymidine monophosphate (dTMP), which are required for methylation reactions and DNA synthesis, respectively. Folate depletion leads to an imbalance in both AdoMet and nucleotide pools, causing epigenetic and genetic damage capable of initiating tumorigenesis. Polyamine biosynthesis also utilizes AdoMet, but polyamine pools are not reduced under a regimen of folate depletion. We hypothesized that high polyamine biosynthesis, due to the high demand on AdoMet pools, might be a factor in determining sensitivity to folate depletion. We found a significant correlation (P<0.001) between polyamine biosynthesis and the amount of folate required to sustain cell line proliferation. We manipulated polyamine biosynthesis by genetic and pharmacological intervention and mechanistically demonstrated that we could thereby alter AdoMet pools and increase or decrease demand on folate availability needed to sustain cellular proliferation. Furthermore, growing a panel of cell lines with 100 nM folate led to imbalanced nucleotide and AdoMet pools only in cells with endogenously high polyamine biosynthesis. These data demonstrate that polyamine biosynthesis is a critical factor in determining sensitivity to folate depletion and may be particularly important in the prostate, where biosynthesis of polyamines is characteristically high due to its secretory function.

Expression of spermine/spermidine-N1-acetyltransferase (SSAT), the rate-limiting enzyme of polyamine backconversion cascade, increases after ischemia-reperfusion injuries (IRI). We hypothesized that SSAT plays an important role in the mediation of IRI. To test our hypothesis, wild-type (SSAT-wt) and SSAT-deficient (SSAT-ko) mice were subjected to liver or kidney IRI by ligation of hepatic or renal arteries. The liver and kidney content of putrescine (Put), a downstream by-product of SSAT activity, increased in SSAT-wt animals but not in SSAT-ko animals after IRI, indicating that polyamine backconversion is not functional in SSAT-deficient mice. When subjected to hepatic IRI, SSAT-ko mice were significantly protected against liver damage compared with SSAT-wt mice. Similarly, SSAT-ko animals subjected to renal IRI showed significantly greater protection against damage to kidney tubules than SSAT-wt mice. These studies indicate that SSAT-deficient animals are protected against IRI and suggest that SSAT is an important mediator of the tissue damage in IRI.

Recent studies suggest that overexpression of the polyamine-acetylating enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) significantly increases metabolic flux through the polyamine pathway. The concept derives from the observation that SSAT-induced acetylation of polyamines gives rise to a compensatory increase in biosynthesis and presumably to increased flow through the pathway. Despite the strength of this deduction, the existence of heightened polyamine flux has not yet been experimentally demonstrated. Here, we use the artificial polyamine precursor 4-fluoro-ornithine to measure polyamine flux by tracking fluorine unit permeation of polyamine pools in human prostate carcinoma LNCaP cells. Conditional overexpression of SSAT was accompanied by a massive increase in intracellular and extracellular acetylated spermidine and by a 6-20-fold increase in biosynthetic enzyme activities. In the presence of 300 microM 4-fluoro-ornithine, SSAT overexpression led to the sequential appearance of fluorinated putrescine, spermidine, acetylated spermidine, and spermine. As fluorinated polyamines increased, endogenous polyamines decreased, so that the total polyamine pool size remained relatively constant. At 24 h, 56% of the spermine pool in the induced SSAT cells was fluorine-labeled compared with only 12% in uninduced cells. Thus, SSAT induction increased metabolic flux by approximately 5-fold. Flux could be interrupted by inhibition of polyamine biosynthesis but not by inhibition of polyamine oxidation. Overall, the findings are consistent with a paradigm whereby flux is initiated by SSAT acetylation of spermine and particularly spermidine followed by a marked increase in key biosynthetic enzymes. The latter sustains the flux cycle by providing a constant supply of polyamines for subsequent acetylation by SSAT. The broader metabolic implications of this futile metabolic cycling are discussed in detail.

Structural genomics efforts have produced structural information, either directly or by modeling, for thousands of proteins over the past few years. While many of these proteins have known functions, a large percentage of them have not been characterized at the functional level. The structural information has provided valuable functional insights on some of these proteins, through careful structural analyses, serendipity, and structure-guided functional screening. Some of the success stories based on structures solved at the Northeast Structural Genomics Consortium (NESG) are reported here. These include a novel methyl salicylate esterase with important role in plant innate immunity, a novel RNA methyltransferase (H. influenzae yggJ (HI0303)), a novel spermidine/spermine N-acetyltransferase (B. subtilis PaiA), a novel methyltransferase or AdoMet binding protein (A. fulgidus AF&#95;0241), an ATP:cob(I)alamin adenosyltransferase (B. subtilis YvqK), a novel carboxysome pore (E. coli EutN), a proline racemase homolog with a disrupted active site (B. melitensis BME11586), an FMN-dependent enzyme (S. pneumoniae SP&#95;1951), and a 12-stranded beta-barrel with a novel fold (V. parahaemolyticus VPA1032).

While polyamine homoeostasis is clearly important in maintenance of normal cell function, the roles of these cations, as well as the enzymes that regulate their metabolism, in the neoplastic process are not clear. In particular, the polyamine catabolic enzyme SSAT (spermidine/spermine N(1)-acetyltransferase) seems to have different roles in tumorigenesis, depending upon the particular system being analysed. In attempts to clarify the function of SSAT in tumour development, we have utilized the Apc(Min/+) mouse, which carries a mutant allele of the Apc (adenomatous polyposis coli) gene, rendering it susceptible to the formation of multiple adenomas in the small intestine and colon. Using genetically engineered animals (i.e. transgenic and knockout mice), we have shown that SSAT acts as a tumour promoter in the Apc(Min/+) model. Modulation of tumorigenesis is not associated with changes in tissue levels of either spermidine or spermine. These findings, along with those made in other animal models of cancer, have prompted us to propose that metabolic flux through the polyamine biosynthetic and catabolic pathways, and the consequent changes in levels of various metabolites within the cell (i.e. the metabolome), is critical to tumour development. The metabolic flux model represents a novel way of thinking about the role of polyamines in cell physiology and the neoplastic process.

The acetylating enzyme, spermidine/spermine N1-acetyltransferase, participates in polyamine homeostasis by regulating polyamine export and catabolism. Previously, we reported that overexpression of the enzyme in cultured tumor cells and mice activates metabolic flux through the polyamine pathway and depletes the N1-acetyltransferase coenzyme and fatty acid precursor, acetyl-CoA. Here, we investigate this possibility in spermidine/spermine N1-acetyltransferase transgenic mice in which the enzyme is systemically overexpressed and in spermidine/spermine N1-acetyltransferase knock-out mice. Tissues of the former were characterized by increased N1-acetyltransferase activity, a marked elevation in tissue and urinary acetylated polyamines, a compensatory increase in polyamine biosynthetic enzyme activity, and an increase in metabolic flux through the polyamine pathway. These polyamine effects were accompanied by a decrease in white adipose acetyl- and malonyl-CoA pools, a major (20-fold) increase in glucose and palmitate oxidation, and a distinctly lean phenotype. In SSAT-ko mice, the opposite relationship between polyamine and fat metabolism was observed. In the absence of N1-acetylation of polyamines, there was a shift in urinary and tissue polyamines indicative of a decline in metabolic flux. This was accompanied by an increase in white adipose acetyl- and malonyl-CoA pools, a decrease in adipose palmitate and glucose oxidation, and an accumulation of body fat. The latter was further exaggerated under a high fat diet, where knock-out mice gained twice as much weight as wild-type mice. A model is proposed whereby the expression status of spermidine/spermine N1-acetyltransferase alters body fat accumulation by metabolically modulating tissue acetyl- and malonyl-CoA levels, thereby influencing fatty acid biosynthesis and oxidation.

Expression of spermidine/spermine N(1)-acetyltransferase (SSAT) increases in kidneys subjected to ischemia-reperfusion injury (IRI). Increased expression of SSAT in vitro leads to alterations in cellular polyamine content, depletion of cofactors and precursors of polyamine synthesis, and reduced cell proliferation. In our model system, a >28-fold increase in SSAT levels in HEK-293 cells leads to depletion of polyamines and elevation in the enzymatic activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, suggestive of a compensatory reaction to increased polyamine catabolism. Increased expression of SSAT also led to DNA damage and G(2) arrest. The increased DNA damage was primarily due to the depletion of polyamines. Other factors such as increased production of H(2)O(2) due to polyamine oxidase activity may play a secondary role in the induction of DNA lesions. In response to DNA damage the ATM/ATR --> Chk1/2 DNA repair and cell cycle checkpoint pathways were activated, mediating the G(2) arrest in SSAT-expressing cells. In addition, the activation of ERK1 and ERK2, which play integral roles in the G(2)/M transition, is impaired in cells expressing SSAT. These results indicate that the disruption of polyamine homeostasis due to enhanced SSAT activity leads to DNA damage and reduced cell proliferation via activation of DNA repair and cell cycle checkpoint and disruption of Raf --> MEK --> ERK pathways. We propose that in kidneys subjected to IRI, one mechanism through which increased expression of SSAT may cause cellular injury and organ damage is through induction of DNA damage and the disruption of cell cycle.

Purpose: Patients with relapsed or refractory chronic lymphocytic leukemia (CLL) have profound immune defects and limited treatment options. Given the dramatic activity of lenalidomide in other B-cell malignancies and its pleotropic immunomodulatory effects, we conducted a phase II trial of this agent in CLL., Patients and Methods: Patients with relapsed or refractory B-cell CLL (B-CLL) were eligible if they required treatment as per the National Cancer Institute Working Group 1996 guidelines. Lenalidomide was administered orally at 25 mg on days 1 through 21 of a 28-day cycle. Response was assessed after each cycle. Patients were to continue treatment until disease progression, unacceptable toxicity, or complete remission. Rituximab was added to lenalidomide on disease progression., Results: Forty-five patients were enrolled, with a median age of 64 years. Sixty-four percent of the patients had Rai stage III or IV disease, and 51% were refractory to fludarabine. The overall response rate was 47%, with 9% of the patients attaining a complete remission. Fatigue, thrombocytopenia, and neutropenia were the most common adverse effects noted in 83%, 78%, and 78% of the patients, respectively., Conclusion: Lenalidomide is clinically active in patients with relapsed or refractory B-CLL. These findings are encouraging and warrant further investigation of this agent in the treatment of this disorder.

Bacillus subtilis PaiA has been implicated in the negative control of sporulation as well as production of degradative enzymes. PaiA shares recognizable sequence homology with N-acetyltransferases, including those that can acetylate spermidine/spermine substrates. We have determined the crystal structure of PaiA in complex with CoA at 1.9 A resolution and found that PaiA is a member of the N-acetyltransferase superfamily of enzymes. Unexpectedly, we observed the binding of an oxidized CoA dimer in the active site of PaiA, and the structural information suggests the substrates of the enzyme could be linear, positively charged compounds. Our biochemical characterization is also consistent with this possibility, since purified PaiA possesses N1-acetyltransferase activity toward polyamine substrates including spermidine and spermine. Further, conditional overexpression of PaiA in bacteria results in increased acetylation of endogenous spermidine pools. Thus, our structural and biochemical analyses indicate that PaiA is a novel N-acetyltransferase capable of acetylating both spermidine and spermine. In this way, the pai operon may function in regulating intracellular polyamine concentrations and/or binding capabilities. In addition to preventing toxicity due to polyamine excess, this function may also serve to regulate expression of certain bacterial gene products such as those involved in sporulation.

Intracellular polyamine pools are homeostatically maintained by processes involving biosynthesis, catabolism, and transport. Although most polyamine-based anticancer strategies target biosynthesis, we recently showed that activation of polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase-1 (SSAT) suppresses tumor outgrowth in a mouse prostate cancer model. Herein, we examined the effects of differential SSAT expression on intestinal tumorigenesis in the Apc(Min/+) (MIN) mouse. When MIN mice were crossed with SSAT-overproducing transgenic mice, they developed 3- and 6-fold more adenomas in the small intestine and colon, respectively, than normal MIN mice. Despite accumulation of the SSAT product, N(1)-acetylspermidine, spermidine and spermine pools were only slightly decreased due to a huge compensatory increase in polyamine biosynthetic enzyme activities that gave rise to enhanced metabolic flux. When MIN mice were crossed with SSAT knock-out mice, they developed 75% fewer adenomas in the small intestine, suggesting that under basal conditions, SSAT contributes significantly to the MIN phenotype. Despite the loss in catabolic capability, tumor spermidine and spermine pools failed to increase significantly due to a compensatory decrease in biosynthetic enzyme activity giving rise to a reduced metabolic flux. Loss of heterozygosity at the Apc locus was observed in tumors from both SSAT-transgenic and -deficient MIN mice, indicating that loss of heterozygosity remained the predominant oncogenic mechanism. Based on these data, we propose a model in which SSAT expression alters flux through the polyamine pathway giving rise to metabolic events that promote tumorigenesis. The finding that deletion of SSAT reduces tumorigenesis suggests that small-molecule inhibition of the enzyme may represent a nontoxic prevention and/or treatment strategy for gastrointestinal cancers.

Checkpoints that control Myc-mediated proliferation and apoptosis are bypassed during tumorigenesis. Genes encoding polyamine biosynthetic enzymes are overexpressed in B cells from E mu-Myc transgenic mice. Here, we report that disabling one of these Myc targets, Ornithine decarboxylase (Odc), abolishes Myc-induced suppression of the Cdk inhibitors p21(Cip1) and p27(Kip1), thereby impairing Myc's proliferative, but not apoptotic, response. Moreover, lymphoma development was markedly delayed in E mu-Myc;Odc(+/-) transgenic mice and in E mu-Myc mice treated with the Odc inhibitor difluoromethylornithine (DFMO). Strikingly, tumors ultimately arising in E mu-Myc;Odc(+/-) transgenics lacked deletions of Arf, suggesting that targeting Odc forces other routes of transformation. Therefore, Odc is a critical Myc transcription target that regulates checkpoints that guard against tumorigenesis and is an effective target for cancer chemoprevention.

Metabolically stable polyamine derivatives may serve as useful surrogates for the natural polyamines in studies aimed to elucidate the functions of individual polyamines. Here we studied the metabolic stability of alpha-methylspermidine, alpha-methylspermine, and bis-alpha-methylspermine, which all have been reported to fulfill many of the putative physiological functions of the natural polyamines. In vivo studies were performed with the transgenic rats overexpressing spermidine/spermine N(1)-acetyltransferase. alpha-Methylspermidine effectively accumulated in the liver and did not appear to undergo any further metabolism. On the other hand, alpha-methylspermine was readily converted to alpha-methylspermidine and spermidine; similarly, bis-alpha-methylspermine was converted to alpha-methylspermidine to some extent, both conversions being inhibited by the polyamine oxidase inhibitor N(1), N(2)-bis(2,3-butadienyl)-1,4-butanediamine. Furthermore, we used recombinant polyamine oxidase, spermidine/spermine N(1)-acetyltransferase, and the recently discovered spermine oxidase in the kinetic studies. In vitro studies confirmed that methylation did not protect spermine analogs from degradation, whereas the spermidine analog was stable. Both alpha-methylspermidine and bis-alpha-methylspermine overcame the proliferative block of early liver regeneration in transgenic rats and reversed the cytostasis induced by an inhibition of ornithine decarboxylase in cultured fetal fibroblasts.

The enzyme spermidine/spermine N(1)-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 approximately 4 times greater than those of TRAMP/SSAT bigenic mice, and by 36 weeks, they were approximately 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 N(1)-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 approximately 70% reduction in the SSAT cofactor acetyl-CoA and a approximately 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.

A great deal of experimental evidence connects induction of polyamine catabolism via spermidine/spermine N1-acetyltransferase (SSAT) to antiproliferative activity and apoptosis. Following our initial observation from gene expression profiling that platinum drugs induce SSAT, we undertook this present study to characterize platinum drug induction of SSAT and other polyamine catabolic enzymes and to examine how these responses might be enhanced with the well-known inducer of SSAT and clinically relevant polyamine analogue, N1,N11-diethylnorspermine (DENSPM). The results obtained in A2780 ovarian cancer cells by real-time quantitative RT-PCR and Northern blot analysis show that a 2-hour exposure of A2780 cells to platinum drugs induces expression of SSAT, a second SSAT (SSAT-2), spermine oxidase, and polyamine oxidase in a dose-dependent manner. At equitoxic doses, oxaliplatin is more effective than cisplatin in SSAT induction. The most affected enzyme, SSAT, increased 15-fold in mRNA expression and 2-fold in enzyme activity. When combined with DENSPM to further induce SSAT and to enhance conversion of mRNA to activity, oxaliplatin increased SSAT mRNA 50-fold and activity, 210-fold. Polyamine pools declined in rough proportion to levels of SSAT induction. At pharmacologically relevant oxaliplatin exposure times (20 hours) and drug concentrations (5 to 15 micromol/L), these responses were increased even further. Combining low-dose DENSPM with oxaliplatin produced a greater than additive inhibition of cell growth based on the sulforhodamine-B assay. Taken together, the findings confirm potent induction of polyamine catabolic enzymes, such as SSAT by platinum drugs, and demonstrate that these biochemical responses as well as growth inhibition can be potentiated by co-treatment with the polyamine analogue DENSPM. With appropriate in vitro and in vivo optimization, these findings could lead to clinically relevant therapeutic strategies.

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 N(1)-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 approximately 10-fold increase in SSAT mRNA and an increase of approximately 20-fold in enzyme activity. SSAT products N(1)-acetylspermidine, N(1)-acetylspermine, and N(1),N(12)-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 alpha-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.

The gene encoding methylthioadenosine phosphorylase (MTAP), the initial enzyme in the methionine salvage pathway, is deleted in a variety of human tumors and acts as a tumor suppressor gene in cell culture (Christopher, S. A., Diegelman, P., Porter, C. W., and Kruger, W. D. (2002) Cancer Res. 62, 6639-6644). Overexpression of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) is frequently observed in tumors and has been shown to be tumorigenic in vitro and in vivo. In this paper, we demonstrate a novel regulatory pathway in which the methionine salvage pathway products inhibit ODC activity. We show that in Saccharomyces cerevisiae the MEU1 gene encodes MTAP and that Meu1delta cells have an 8-fold increase in ODC activity, resulting in large elevations in polyamine pools. Mutations in putative salvage pathway genes downstream of MTAP also cause elevated ODC activity and elevated polyamines. The addition of the penultimate salvage pathway compound 4-methylthio-2-oxobutanoic acid represses ODC levels in both MTAP-deleted yeast and human tumor cell lines, indicating that 4-methylthio-2-oxobutanoic acid acts as a negative regulator of polyamine biosynthesis. Expression of MTAP in MTAP-deleted MCF-7 breast adenocarcinoma cells results in a significant reduction of ODC activity and reduction in polyamine levels. Taken together, our results show that products of the methionine salvage pathway regulate polyamine biosynthesis and suggest that MTAP deletion may lead to ODC activation in human tumors.

N1,N11-diethylnorspermine (DENSPM) is a polyamine analog that down-regulates polyamine biosynthesis and potently upregulates the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT). In certain cells, such as SKMEL-28 human melanoma cells, induction of SSAT is associated with rapid apoptosis. In this study, we used small interfering RNA (siRNA) to examine the role of SSAT induction in mediating polyamine pool depletion and apoptosis. siRNA duplexes were designed to target three independent sites in the SSAT mRNA coding region (siSSAT). When transfected under nontoxic conditions, two of the duplexes selectively reduced basal SSAT mRNA in HEK-293 cells by >80% and prevented DENSPM-induced SSAT mRNA by 95% in SK-MEL-28 cells. Treatment of SK-MEL-28 cells with 10 muM DENSPM in the presence of 83 nM siSSAT selectively prevented the 1400-fold induction of SSAT activity by approximately 90% and, in turn, prevented analog depletion of spermine (Spm) pools by approximately 35%. siSSAT also prevented DENSPM-induced cytochrome c release and caspase-3 cleavage at 36 h and apoptosis at 48 h as measured by annexin V staining. Overall, the data directly link analog induction of SSAT to Spm pool depletion and to caspase-dependent apoptosis in DENSPM-treated SK-MEL-28 cells. This represents the first use of siRNA technology directed toward a polyamine gene and the first unequivocal demonstration that SSAT induction initiates events leading to polyamine analog-induced apoptosis.

We have previously shown that the clinically relevant polyamine analog N1,N11-diethylnorspermine (DENSPM) causes rapid apoptosis in human melanoma SK-MEL-28 cells via a series of events that include mitochondrial release of cytochrome c and activation of the caspase cascade. Upstream to these events, DENSPM downregulates polyamine biosynthesis and potently upregulates polyamine catabolism at the level of spermidine/spermine N1-acetyltransferase (SSAT). In searching for downstream effectors that either contribute to or abrogate the apoptotic response, we observed that DENSPM treatment of SK-MEL-28 cells for 30 h led to cytosolic release of Smac/Diablo, a mitochondrial protein known to bind and inhibit the function of inhibitor of apoptosis proteins (IAPs). Subsequently, we found that DENSPM markedly lowered survivin and ML-IAP protein (but not XIAP) levels by 18 h via an apparently Smac/Diablo-independent pathway. Proteasome inhibitors fully prevented survivin and ML-IAP protein loss as well as apoptosis, suggesting that the proteasome-mediated degradation of survivin and ML-IAP is causally linked to the cellular outcome. We also observed that structural analogs of DENSPM which differentially induced SSAT and apoptosis lowered survivin and ML-IAP levels in a manner that correlated with enzyme activity. The linkage between IAPs and SSAT was more directly established by the finding that selective prevention of SSAT induction by small interfering RNA prevented survivin and ML-IAP loss as well as apoptosis during DENSPM treatment. Among the melanoma cell lines (SK-MEL-28, MALME-3M, A375 and LOX), survivin degradation correlated temporally with the onset of DENSPM induced apoptosis or growth inhibition. By contrast, ML-IAP degradation occurred only during rapid apoptosis seen in SK-MEL-28 cells. These data suggest a sequence of events whereby DENSPM induction of SSAT leads to loss of IAP proteins and a more fulminate apoptotic response. The findings implicate survivin and ML-IAP as important determinants of polyamine analog drug action in melanoma cells.

In the polyamine back-conversion pathway, spermine and spermidine are first acetylated by spermidine/spermine N(1) -acetyl-transferase (SSAT-1) and then oxidized by polyamine oxidase to produce spermidine and putrescine respectively. Herein we apply homology-search methods to identify novel sequences belonging to a second SSAT, SSAT-2, with a chromosomal location at 17p13.1, which is distinct from SSAT-1 at Xp22. Human SSAT-2 cDNA derived from small-cell lung carcinoma was deduced to encode a 170-amino-acid protein having 46% sequence identity and 64% sequence similarity with SSAT-1. When transiently transfected into HEK-293 cells, SSAT-1 decreased spermidine and spermine pools by approximately 30%, while, at the same time, significantly increasing putrescine, N (1)-acetylspermidine, N (1)-acetylspermine and N (1), N (12)-diacetylspermine pools. By contrast, transfected SSAT-2 had no effect on intracellular polyamine or acetylated polyamine pools. When enzyme activity was assayed on enzyme extracts from transfected cells, both SSAT-1 and SSAT-2 demonstrated much higher acetylating activity than vector-transfected cells. The data suggest that, in intact cells, SSAT-2 may be compartmentalized or it may be inefficient at low intracellular polyamine concentrations. By substituting candidate substrates in the enzyme assay, we determined that SSAT-1 shows the substrate preference norspermidine=spermidine>>spermine> N (1)-acetylspermine>putrescine, whereas SSAT-2 shows the preference norspermidine>spermidine=spermine>> N (1)-acetylspermine=putrescine. Analysis of mRNA levels in cell lines and ESTs (expressed sequence tags) from various tissues by digiNorthern (a web-based tool for virtually displaying expression profiles of query genes based on EST sequences) indicated that SSAT-1 tends to be more widely and highly expressed than SSAT-2. While SSAT-1 mRNA was inducible by polyamine analogues in a variety of cell lines, SSAT-2 was not. The existence of an active, but possibly sequestered, SSAT-2 enzyme suggests that, under certain conditions, it may be recruited into basal or perturbed polyamine metabolism.

The clinically relevant polyamine analogue N(1),N(11)-diethylnorspermine (DENSPM) inhibits cell growth by down-regulating polyamine biosynthesis, up-regulating polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase (SSAT), and depleting intracellular polyamine pools. Among human melanoma cell lines, the analogue causes rapid apoptosis in SK-MEL-28 cells and a sharp G(1) arrest in MALME-3M cells. This study reveals that DENSPM potently activates the mitogen-activated protein kinase (MAPK) pathways in melanoma cells and investigates the role of this response in determining cellular outcomes. Onset of apoptosis was preceded by an intense phosphorylation of the MAPKs, including extracellular signal-regulated kinase 1/2, c-Jun NH(2)-terminal kinase, and p38 in both SK-MEL-28 and MALME-3M cells. A panel of DENSPM analogues differing only in their ability to induce SSAT was used to show that MAPK activation was causally linked to induction of SSAT activity and related oxidative events. The latter was confirmed with the polyamine oxidase inhibitor MDL-75275 and the antioxidant N-acetyl-L-cysteine, which when used in combination with DENSPM, decreased MAPK activation and as previously shown, reduced apoptosis. The MAP/extracellular signal-regulated kinase-1 inhibitor PD 98059 reduced activation of all three kinases but failed to alter apoptosis in DENSPM-treated SK-MEL-28 cells. By contrast, the inhibitor prevented p21(waf1/cip1) induction and enhanced apoptosis in MALME-3M cells as indicated by accelerated caspase-3 activation and positive annexin V staining. The generality of this effect was demonstrated in DENSPM-treated A375 and LOX human melanoma cells. Taken together, the importance of the MAPK pathways in determining the biological response to DENSPM treatment is dependent on the genetic environment of the cell.

Purpose: We tested the polyamine analog SL-11093 (3,8,13,18-tetraaza-10,11-[(E)-1,2-cyclopropyl]eicosane tetrahydrochloride) as an effective chemotherapeutic agent against human prostate cancer grown in nude mice., Methods: NCr-nu mice grafted with DU-145 human prostate tumor cells were treated i.p. with SL-11093 at 50 mg/kg q1dx5 for either three or five cycles separated by intervals of about 10-15 days., Results: In treated animals, tumor growth remained arrested for up to 100 days with minimal animal weight loss. None of the animals died during the treatment and in one experiment two out of six animals showed no palpable tumor. SL-11093 was readily taken up by the tumors, where its levels remained elevated for about 48 h after the end of drug administration. In liver and in kidney, SL-11093 (a (alpha)N,(omega)N-bisethyl derivative) was oxidatively N-deethylated predominantly to its monoethyl and di-deethyl derivatives. In time, the monoethyl derivative was further dealkylated, with a loss of an aminobutyl chain to form an aminomethyl cyclopropyl derivative. In tumor (and in lung), N-dealkylation reactions were less evident., Conclusion: SL-11093 is an effective chemotherapeutic agent against a human prostate tumor xenograft grown in nude mice. The drug accumulation and slow metabolism in tumor compared to other tissues would most likely reduce systemic toxicity of the drug and contribute to a larger therapeutic window for SL-11093 as compared to other cytotoxic polyamine analogs.

Ischemia-reperfusion injury (IRI) is the major cause of acute renal failure in native and allograft kidneys. Identifying the molecules and pathways involved in the pathophysiology of renal IRI will yield valuable new diagnostic and therapeutic information. To identify differentially regulated genes in renal IRI, RNA from rat kidneys subjected to an established renal IRI protocol (bilateral occlusion of renal pedicles for 30 min followed by reperfusion) and time-matched kidneys from sham-operated animals was subjected to suppression subtractive hybridization. The level of spermidine/spermine N(1)-acetyltransferase (SSAT) mRNA, an essential enzyme for the catabolism of polyamines, increased in renal IRI. SSAT expression was found throughout normal kidney tubules, as detected by nephron segment RT-PCR. Northern blots demonstrated that the mRNA levels of SSAT are increased by greater than threefold in the renal cortex and by fivefold in the renal medulla at 12 h and returned to baseline at 48 h after ischemia. The increase in SSAT mRNA was paralleled by an increase in SSAT protein levels as determined by Western blot analysis. The concentration of putrescine in the kidney increased by approximately 4- and approximately 7.5-fold at 12 and 24 h of reperfusion, respectively, consistent with increased functional activity of SSAT. To assess the specificity of SSAT for tubular injury, a model of acute renal failure from Na(+) depletion (without tubular injury) was studied; SSAT mRNA levels remained unchanged in rats subjected to Na(+) depletion. To distinguish SSAT increases from the effects of tubular injury vs. uremic toxins, SSAT was increased in cis-platinum-treated animals before the onset of renal failure. The expression of SSAT mRNA and protein increased by approximately 3.5- and >10-fold, respectively, in renal tubule epithelial cells subjected to ATP depletion and metabolic poisoning (an in vitro model of kidney IRI). Our results suggest that SSAT is likely a new marker of tubular cell injury that distinguishes acute prerenal from intrarenal failure.

In the polyamine back-conversion pathway, spermine and spermidine are first acetylated by spermidine/spermine N1 -acetyltransferase (SSAT) and then oxidized by polyamine oxidase (PAO) to produce spermidine and putrescine respectively. Although PAO was first purified more than two decades ago, the protein has not yet been linked to genomic sequences. In the present study, we apply a BLAST search strategy to identify novel oxidase sequences located on human chromosome 10 and mouse chromosome 7. Homologous mammalian cDNAs derived from human brain and mouse mammary tumour were deduced to encode proteins of approx. 55 kDa having 82% sequence identity. When either cDNA was transiently transfected into HEK-293 cells, intracellular spermine pools decreased by approx. 30%, whereas spermidine increased 2-4-fold. Lysates of human PAO cDNA-transfected HEK-293 cells, but not vector-transfected cells, rapidly oxidized N1-acetylspermine to spermidine. Substrate specificity determinations with the lysate assay revealed a preference ranking of N1-acetylspermine= N1-acetylspermidine> N1,N12-diacetylspermine>>spermine; spermidine was not acted upon. This ranking is identical to that reported for purified PAO and distinctly different from the recently identified spermine oxidase (SMO), which prefers spermine over N1-acetylspermine. Monoethyl- and diethylspermine analogues also served as substrates for PAO, and were internally cleaved adjacent to a secondary amine. We deduce that the present oxidase sequences are those of the FAD-dependent PAO involved in the polyamine back-conversion pathway. In Northern blot analysis, PAO mRNA was much less abundant in HEK-293 cells than SMO or SSAT mRNA, and all three were differentially induced in a similar manner by selected polyamine analogues. The identification of PAO sequences, together with the recently identified SMO sequences, provides new opportunities for understanding the dynamics of polyamine homoeostasis and for interpreting metabolic and cellular responses to clinically-relevant polyamine analogues and inhibitors.

The human methylthioadenosine phosphorylase (MTAP) gene is located on 9p21 and is frequently homozygously deleted, along with p16(cdkN2a/ARF), in a wide variety of human tumors and human tumor-derived cell lines. The function of MTAP is to salvage methylthioadenosine, which is produced as a byproduct of polyamine metabolism. We have reintroduced MTAP into MCF-7 breast adenocarcinoma cells and have examined its effect on the tumorigenic properties of these cells. MTAP expression does not affect the growth rate of cells in standard tissue culture conditions but severely inhibits their ability to form colonies in soft agar or collagen. In addition, MTAP-expressing cells are suppressed for tumor formation when implanted into SCID mice. This suppression of anchorage-independent growth appears to be because of the enzymatic activity of MTAP, as a protein with a missense mutation in the active site does not exhibit this phenotype. MTAP expression causes a significant decrease in intracellular polyamine levels and alters the ratio of putrescine to total polyamines. Consistent with this observation, the polyamine biosynthesis inhibitor alpha-difluoromethylornithine inhibits the ability of MTAP-deficient cells to form colonies in soft agar, whereas addition of the polyamine putrescine stimulates colony formation in MTAP-expressing cells. These results indicate that MTAP has tumor suppressor activity and suggest that its effects may be mediated by altering intracellular polyamine pools.

A well-defined series of 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine analogues was designed and synthesized in order to further ascertain the optimal structural requirements for S-adenosylmethionine decarboxylase inhibition and potentially to augment and perhaps separate their antiproliferative and antitrypanosomal activities. Most structural modifications had a deleterious affect on both the antitrypanosomal and antineoplastic activity of 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine. However, di-O-acetylation of the parent compound produced a potential prodrug that caused markedly pronounced inhibition of trypanosomal and neoplastic cell growth and viability. Moreover, the acetylated derivative of 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine did inhibit HIV-1 growth and infectivity, whereas the parent compound did not.

During polyamine catabolism, spermine and spermidine are first acetylated by spermidine/spermine N(1)-acetyltransferase (SSAT) and subsequently oxidized by polyamine oxidase (PAO) to produce spermidine and putrescine, respectively. In attempting to clone the PAO involved in this back-conversion pathway, we encountered an oxidase that preferentially cleaves spermine in the absence of prior acetylation by SSAT. A BLAST search using maize PAO sequences identified homologous mammalian cDNAs derived from human hepatoma and mouse mammary carcinoma: the encoded proteins differed by 20 amino acids. When either cDNA was transiently transfected into HEK-293 cells, intracellular spermine pools decreased by 75% while spermidine and N (1)-acetylspermidine pools increased, suggesting that spermine was selectively and directly oxidized by the enzyme. Substrate specificity using lysates of oxidase-transfected HEK-293 cells revealed that the newly identified oxidase strongly favoured spermine over N (1)-acetylspermine and that it failed to act on N (1)-acetylspermidine, spermidine or the preferred PAO substrate, N (1), N (12)-diacetylspermine. The PAO inhibitor, MDL-72,527, only partially blocked oxidation of spermine while a previously reported PAO substrate, N (1)-( n -octanesulphonyl)spermine, potently inhibited the reaction. Overall, the data indicate that the enzyme represents a novel mammalian oxidase which, on the basis of substrate specificity, we have designated spermine oxidase in order to distinguish it from the PAO involved in polyamine back-conversion. The identification of an enzyme capable of directly oxidizing spermine to spermidine has important implications for understanding polyamine homoeostasis and for interpreting metabolic and cellular responses to clinically relevant polyamine analogues and inhibitors.

Purpose: SAM486A is a novel inhibitor of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (SAMDC). This study was performed to characterize the toxicity profile and the pharmacological behavior and to determine the maximum tolerated dose (MTD) of SAM486A administered by a 1-h i.v. infusion daily for 5 days every 3 weeks in patients with advanced cancer., Experimental Design: Twenty-three patients received 46 cycles of SAM486A at dose levels ranging from 3.6 to 202.8 mg/m(2)/day. SAM486A plasma concentrations were measured during the first cycle for pharmacokinetic and pharmacodynamic evaluations. Paired tumor biopsy specimens pre- and posttreatment were obtained in 1 patient to assess the impact of SAM486A on intratumoral enzymes and metabolites involved in the polyamine biosynthetic pathway., Results: The dose-limiting toxicity of SAM486A on this schedule was myelosuppression. Nonhematological toxicities, including nausea, vomiting, anorexia, and fatigue, were mild to moderate in severity. The MTD of SAM486A was 102.4 mg/m(2)/day. Pharmacokinetic analyses demonstrated a rapid initial decrease in plasma drug concentrations at the end of infusion, followed by a long terminal elimination phase with a mean (+/- SD) terminal elimination half-life of 65.4 +/- 55.6 h. Dose and area under the concentration-time curve correlated with the appearance of grade 4 neutropenia with correlation coefficients of 0.70 and 0.69, respectively. Analysis of paired tumor biopsy specimens taken before and after SAM486A treatment in 1 patient with metastatic melanoma revealed decreased SAMDC activity, increased ornithine decarboxylase activity, increased levels of putrescine, and depleted levels of decarboxylated S-adenosylmethionine and spermine, all of which are consistent with the proposed mode of action of SAM486A., Conclusions: SAM486A was well tolerated on this schedule of administration with the MTD established at 102.4 mg/m(2)/day. Neutropenia was dose-limiting and correlated with dose and area under the concentration-time curve. Pharmacodynamic assessment of tumoral tissues in 1 study patient demonstrated changes in the levels of polyamines and their biosynthetic enzymes consistent with SAMDC inhibition.

Overexpression of SSAT (polyamine catabolic enzyme) in female mice results in impaired ovarian folliculogenesis and uterine hypoplasia. To identify the molecular basis for this, the gene expression profiles in uterus and ovary and for comparison, liver and kidney, from non-transgenic (NT) and SSAT transgenic (ST) mice were compared. The mRNA abundance for lipoprotein lipase and glyceraldehyde-3-phosphate dehydrogenase was elevated in all four ST (>NT) tissues. The translation initiation factor-3 subunit 5 mRNA, and transcripts related to endogenous murine leukemia provirus (MLV-related) and murine retrovirus-related sequences (MuRRS) were decreased in ST tissues. A novel calmodulin-related mRNA was strongly induced in ST liver and kidney. SSAT overexpression was associated with increased levels of IGF-binding protein-2 (IGFBP-2) in the uterus and ovary, and a reduction in IGFBP-3 mRNA levels in the uterus. Exogenous spermidine and spermine elevated endogenous IGFBP-2 and SSAT mRNA abundance, whereas, putrescine stimulated IGFBP-2 mRNA abundance and transfected IGFBP-2 gene promoter activity in human (Hec-1-A) uterine cells. Sp1 and BTEB1 mRNAs that encode transcription factors for the IGFBP-2 gene also were induced in some ST tissues. The data suggest that SSAT and polyamines are important for the control of molecular pathways underlying reproductive tract tissue growth, phenotype, and function.

The cell cycle regulatory events that interface with polyamine requirements for cell growth have not yet been clearly identified. Here we use specific inhibitors of polyamine biosynthetic enzymes to investigate the effect of polyamine pool depletion on cell cycle regulation. Treatment of MALME-3M cells with either the ornithine decarboxylase inhibitor alpha-difluoromethylornithine or the S-adenosylmethionine decarboxylase inhibitor MDL-73811 lowered specific polyamine pools and slowed cell growth but did not induce cell cycle arrest. By contrast, treatment with the combination of inhibitors halted cell growth and caused a distinct G1 arrest. The latter was associated with marked reduction of all three polyamine pools, a strong increase in p21(WAF1/CIP1/SDI1) (p21), and hypophosphorylation of retinoblastoma protein. All effects were fully prevented by exogenous polyamines. p21 induction preceded p53 stabilization in MALME-3M cells and also occurred in a polyamine-depleted, p53-nonfunctional melanoma cell line, indicating that p21 is induced at least in part through p53-independent mechanisms. Conditional overexpression of p21 in a fibrosarcoma cell line was shown previously to inhibit the expression of multiple proliferation-associated genes and to induce the expression of genes associated with various aspects of cell senescence and organism aging. Polyamine depletion in MALME-3M cells was associated with inhibition of seven of seven tested p21-inhibited genes and with induction of 13 of 14 tested p21-induced genes. p21 expression is also known to induce a senescence-like phenotype, and phenotypic features of senescence were observed in polyamine-depleted MALME-3M cells. Cells increased in size, appeared more granular, and expressed senescence-associated beta-galactosidase. Cells released from the polyamine inhibition lost the ability to form colonies, failed to replicate their DNA, and approximately 25% became bi- or multinucleated. These events parallel the outcome of prolonged p21 induction in fibrosarcoma cells. The results of this study indicate that polyamine pool depletion achieved by specific biosynthetic enzyme inhibitors causes p21-mediated G1 cell cycle arrest followed by p21-mediated changes in gene expression, development of a senescence-like phenotype, and loss of cellular proliferative capacity.

N(1),N(11)-Diethylnorspermine (DENSPM) is a polyamine analogue with clinicalrelevance as an experimental anticancer agent and the ability to elicit a profound apoptotic response in certain cell types. Here, we characterize the polyamine effects and apoptotic signaling events initiated by treatment of SK-MEL-28 human melanoma with 10 microM DENSPM. Maximal induction of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) and polyamine pool depletion were seen by 16 h, whereas early apoptosis was first apparent at 36 h. Intermediate events related to apoptotic signaling were sought between 16 and 36 h. A loss of mitochondrial transmembrane potential (Deltapsi(m)) beginning at 24 h was followed by the release of cytochrome c into the cytosol at 30 h. Loss of mitochondrial integrity was accompanied by caspase-3 activation and poly(ADP-ribose) polymerase digestion from 30 to 36 h. The caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone rendered cells resistant to analogue-induced caspase-3 activation and reduced the apoptotic response in a dose-dependent manner. Because polyamine reduction achieved by inhibitors of polyamine biosynthesis inhibited growth but did not cause apoptosis, we looked for alternative polyamine-related events, focusing on induction of SSAT. Three DENSPM analogues that differentially induced SSAT activity but similarly depleted polyamine pools revealed a close correlation between enzyme induction and cytochrome c release, caspase activation, and apoptosis. Dose-dependent inhibition of polyamine oxidase, an enzyme that oxidizes acetylated polyamines generated by SSAT and releases toxic by-products such as H(2)O(2) and aldehydes, prevented cytochrome c release, caspase activation, and apoptosis. Taken together, the findings indicate that DENSPM-induced apoptosis is at least partially initiated via massive induction of SSAT and related oxidative events and subsequently mediated by the mitochondrial apoptotic signaling pathway as indicated by cytochrome c release and caspase activation.

Amantadine acetylation was demonstrated to occur both in vivo and in vitro using transgenic male mice overexpressing spermidine/spermine N(1)-acetyltransferase (SSAT). We previously reported that neither NAT1 nor NAT2 was responsible for catalyzing acetylation of the primary amine group of amantadine. We hypothesized that the inducible polyamine-catabolizing enzyme, SSAT, was an alternate pathway for acetylating amantadine. Transgenic mice injected s.c. with 3 mg/kg amantadine excreted 4.5 +/- 1% (mean +/- S.E.) of the administered dose as acetylamantadine in 24-h urine samples while, by contrast, nontransgenic control mice failed to excrete any detectable acetylamantadine in their urine. In vitro studies with the cytosolic liver fraction from transgenic mice as the source of SSAT demonstrated spermidine acetylation catalytic activity with an apparent K(m) = 267 +/- 46 microM and V(max) = 0.009 +/- 0.002 nmol/min/mg of protein. Amantadine competitively inhibited spermidine acetylation with an apparent K(i) = 738 +/- 157 microM. Incubation of amantadine, SSAT, and an acetyl CoA-regenerating system produced modest amounts of acetylamantadine. The NAT2 substrate, sulfamethazine, inhibited spermidine acetylation with a calculated K(i) = 3.5 mM, suggesting that SSAT may be an alternate pathway for acetylation of NAT2 substrates. The NAT1 substrate, p-aminobenzoic acid, had no inhibitory effect. These results provide evidence that amantadine can be acetylated by SSAT and may be a specific drug substrate for this enzyme. Further investigation of the role of SSAT as a potential drug-metabolizing pathway is warranted.

Mammals express two isoforms of arginase, designated types I and II. Arginase I is a component of the urea cycle, and inherited defects in arginase I have deleterious consequences in humans. In contrast, the physiologic role of arginase II has not been defined, and no deficiencies in arginase II have been identified in humans. Mice with a disruption in the arginase II gene were created to investigate the role of this enzyme. Homozygous arginase II-deficient mice were viable and apparently indistinguishable from wild-type mice, except for an elevated plasma arginine level which indicates that arginase II plays an important role in arginine homeostasis.

Polyamines are known to be essential for normal cell growth and differentiation. However, despite numerous studies, specific cellular functions of polyamines in general and individual polyamines in particular have remained only tentative, because of a lack of appropriate cell lines in which genes of polyamine-synthesizing enzymes have been disrupted by gene targeting. With the use of homologous recombination technique, we disrupted the gene encoding spermine synthase in mouse embryonic stem cells. The spermine synthase gene is located on X chromosome in mouse and, because the cells used in this study were of XY karyotype, a single targeting event was sufficient to result in null genotype. The targeted cells did not have any measurable spermine synthase activity and were totally devoid of the polyamine spermine. Spermine deficiency led to a substantial increase in spermidine content, but the total polyamine content was nearly unchanged. Despite the lack of spermine, these cells displayed a growth rate that was nearly similar to that of the parental cells and showed no overt morphological changes. However, the spermine-deficient cells were significantly more sensitive to the growth inhibition exerted by 2-difluoromethylornithine, an inhibitor of ornithine decarboxylase. Similarly, methylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, and diethylnorspermine, a polyamine analog, although exerting cytostatic growth inhibition on wild-type cells, were clearly cytotoxic to the spermine-deficient cells. The spermine-deficient cells were also much more sensitive to etoposide-induced DNA damage than their wild-type counterparts.

Acetylation of polyamines by spermidine/spermine N(1)-acetyltransferase (SSAT) has been implicated in their degradation and/or export out of the cell. The relationship of SSAT to polyamine pool dynamics and cell growth is not yet clearly understood. MCF-7 human breast carcinoma cells were transfected with tetracycline-regulated (Tet-off) SSAT human cDNA or murine gene. Doxycycline removal for >2 days caused a approximately 20-fold increase in SSAT RNA and a approximately 10-fold increase in enzyme activity. After 4 days, intracellular putrescine and spermidine pools were markedly lowered, and cell growth was inhibited. Growth inhibition could not be prevented with exogenous polyamines due to a previously unrecognized ability of SSAT to rapidly acetylate influxing polyamines and thereby prevent restoration of the endogenous pools. Instead, cells accumulated high levels of N(1)-acetylspermidine, N(1)-acetylspermine, and N(1), N(12)-diacetylspermine, a metabolite not previously reported in mammalian cells. Doxycycline deprivation before treatment with N(1), N(11)-diethylnorspermine markedly increased analog induction of SSAT mRNA and activity and enhanced growth sensitivity to the analog by approximately 100-fold. Overall, the findings demonstrate that conditional overexpression of SSAT lowers polyamine pools, inhibits cell growth, and markedly enhances growth sensitivity to certain analogs. The enzyme also plays a remarkably efficient role in maintaining polyamine pool homeostasis during challenges with exogenous polyamines.

When Chinese hamster ovary cells were seeded in the presence of the spermine analog N1,N11-diethylnorspermine (DENSPM), cell proliferation ceased; this was clearly apparent by cell counting 2 days after seeding the cells. However, 1 day after seeding there was a slight difference in cell number between control and DENSPM-treated cultures. To investigate the reason for this easily surpassed slight difference, we used a sensitive bromodeoxyuridine/flow cytometry method. Cell cycle kinetics were studied during the first cell cycle after seeding cells in the absence or presence of DENSPM. Our results show that DENSPM treatment did not affect the progression of the cells through G1 or the first G1/S transition that took place after seeding the cells. The first cell cycle effect was a delay in S phase as shown by an increase in the DNA synthesis time. The following G2/M transition was not affected by DENSPM treatment. DENSPM treatment inhibited the transient increases in putrescine, spermidine, and spermine pools that took place within 24 h after seeding. Thus, in conclusion, the first cell cycle phase affected by the inhibition of polyamine biosynthesis caused by DENSPM was the S phase. Prolongation of the other cell cycle phases occurred at later time points, and the G1 phase was affected before the G2/M phase.

Polyamine oxidase functions in the polyamine catabolic pathway, converting N1-acetyl-spermidine and -spermine into putrescine (Put) and spermidine (Spd), respectively, thereby facilitating homeostasis of intracellular polyamine pools. Inhibition of polyamine oxidase in hematopoietic cells by a specific inhibitor, N,N'-bis(2,3-butadienyl)-1,4-butanediamine (MDL-72,527), reduces the levels of Put and Spd and induces the accumulation of N1-acetylated Spd. Although previously thought to be relatively nontoxic, we now report that this inhibitor overrides survival factors to induce cell death of several immortal and malignant murine and human hematopoietic cells, but not of primary myeloid progenitors. Cells treated with MDL-72,527 displayed biochemical changes typical of apoptosis, and cell death was associated with the down-regulation of the antiapoptotic protein Bcl-X(L). However, enforced overexpression of Bcl-X(L), or treatment with the universal caspase inhibitor zVAD-fmk, failed to block MDL-72,527-induced apoptosis in these hematopoietic cells. Despite decreases in Put and Spd pools, MDL-72,527-induced apoptosis was not blocked by cotreatment with exogenous Put or Spd, nor was it influenced by overexpression or inhibition of the polyamine biosynthetic enzyme ornithine decarboxylase. Significantly, MDL-72,527-induced apoptosis was associated with the rapid formation of numerous lysosomally derived vacuoles. Malignant leukemia cells were variably sensitive to the lysosomotropic effects of MDL-72,527, yet pretreatment with the ornithine decarboxylase inhibitor L-alpha-difluoromethylornithine sensitized all of these leukemia cells to the deleterious effects of the inhibitor by stimulating its intracellular accumulation. The lysosomotropic nature of select polyamine analogues may, thus, provide a novel chemotherapeutic strategy to selectively induce apoptosis of malignant hematopoietic cells.

We have recently generated transgenic mice in which polyamine catabolism has been activated by overexpressing the rate-limiting enzyme of polyamine catabolism, spermidine/spermine N1-acetyltransferase (SSAT). These animals have now been tested for their sensitivity to the polyamine analog N1,N11-diethylnorspermine (DENSPM), which is currently undergoing Phase I clinical trial. The analog is known for its ability to potently induce SSAT. Treatment for 4 days with a daily dose (125 mg/kg) of analog caused profound changes in polyamine metabolism in the transgenic animals. Liver SSAT activity was increased by approximately 800-fold while hepatic mRNA increased only 4-fold. Putrescine pools increased while spermidine and spermine pools nearly disappeared, resulting in a compensatory increase in ornithine decarboxylase activity. Similar but less profound changes were also seen in other tissues (spleen, intestine, and skin). This treatment also resulted in a 50% mortality in the transgenic animals, with no apparent histopathological changes in major organs. Nontransgenic animals exhibited no toxicity, and tissue SSAT activity was unchanged or only moderately increased. Polyamine pools were only slightly altered. Greater analog toxicity in transgenic animals may be attributable to higher tissue levels of DENSPM facilitated by SSAT-mediated decreases in spermidine and spermine. To further confirm the enhanced sensitivity of the transgenic animals to the analog, groups of nontransgenic and transgenic animals were subjected to daily injections with DENSPM. On average, transgenic mice died approximately 3 days earlier than their nontransgenic litter-mates. The findings indicate a contributing role for SSAT in whole animal toxicity by SSAT-inducing polyamine analogs.

Although polyamines are well recognized for their critical involvement in cell growth, the cell cycle specificity of this requirement has not yet been characterized with respect to the newly delineated regulatory pathways. We recently reported that polyamine analogues having close structural and functional similarities to the natural polyamines produce a distinct G1 and G2-M cell cycle arrest in MALME-3M human melanoma cells. To determine a molecular basis for this observation, we examined the effects of N1,N11-diethylnorspermine on cell cycle regulatory proteins associated with G1 arrest. The analogue is known to deplete polyamine pools by suppressing biosynthetic enzymes and potently inducing the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase. Treatment of MALME-3M cells with 10 microM N1,N11-diethylnorspermine caused an increase in hypophosphorylated Rb, which correlated temporally with the onset of G1 arrest at 16-24 h. Rb hypophosphorylation was preceded by an increase in wild-type p53 (approximately 100-fold at maximum) and a concomitant increase in the cyclin-dependent kinase inhibitor, p21WAF1/CIP1 (p21; approximately 5-fold at maximum). Another cyclin-dependent kinase inhibitor, p27KIP1, and cyclin D increased slightly, whereas proliferating cell nuclear antigen and p130 remained unchanged. Induction of p21 protein was accompanied by an increase in p21 mRNA, whereas induction of p53 protein was not, suggesting transcriptional activation of the former and posttranscriptional regulation of the latter. SK-MEL-28 human melanoma cells, which contain a mutated p53, failed to induce p53 or p21 and did not arrest in G1. Rather, these cells rapidly underwent programmed cell death within 48 h. Overall, these findings provide the first indication of the cell cycle regulatory pathways by which polyamine antagonists such as analogues might inhibit growth in cells containing wild-type p53 and further suggest a mechanistic basis for differential cellular responses to these agents.