Fig. 4. Proposed model for polyamine and polyamine analog activation of SSAT t ra n s c r i p-tion. In the absence of excess polyamines or analogs, Nrf-2 bound to the PRE, but basal transcription remains low. In the presence of inducers, PMF-1 expression increases, binds to Nrf-2, and drives transcriptions of SSAT. However, the possibility exists that yet to be identified transcription cofactors (as indicated by ?) may modify SSAT transcription in response to polyamines and their analogs.

The inducible transcription of human SSAT was found to be under the control of a nine base pair c i s-element 5$-TATGACTAA-3 <t, in a 31-base context starting -1492 base pairs upstream from the transcriptional start site (3 7). This element, referred to as the polyamine responsive element (PRE), was found to be constitutively occupied only in analog-responsive cells by the DNA-binding transcription factor, NF-E2-related transcription factor-2 (Nrf-2). There was no evidence to indicate that treatment with analogs or the natural polyamines altered the binding of Nrf-2 to the PRE. These results suggested the possibility that additional factors were necessary for analog responsiveness.

Therefore, using a yeast two-hybrid strategy an analog-inducible transcription cofac-to r, named polyamine-modulated factor 1 (PMF-1) was identified (3 8). This 20-kDa protein is unable to bind DNA directly because it lacks a functional DNA-binding domain. However, it appears to modulate the transcriptional activation of SSAT via binding to Nrf-2 through a unique leucine zipper/coiled-coil interaction. Interestingly, PMF-1 expression is similar to that of SSAT in that its inducible expression appears to be cell type- and agent type-specific (Fig. 4).

It is also important to note that Nrf-2 and PMF-1 may not be the only factors involved in the transcriptional regulation of SSAT. Nrf-2 is known to have several other binding partners, including the small Maf family transcription cofactors and the Keap1 cytosolic regulatory protein (39-42). Further, PMF-1 has been found to bind to the human homolog of the 7a subunit of the Arab idopsis Cop 9 signalosome (43). Consequently, further study will be required to determine if there are other components contributing to transcriptional regulation of SSAT. Such studies are important because the analog-induced expression of SSAT has been linked to the responsiveness of specific tumor cells and a better understanding as to how SSAT transcription is regulated has the potential of improving drug design. It is also likely that by gaining a better understanding as to how SSAT transcription is controlled, such investigation will allow SSAT to be established as a model gene through which the potential ways that polyamines and their analogs can affect specific gene expression can be investigated.

3.3. Role of SSAT Activity in Cellular Response to Polyamine Analogs

The observations that link SSAT activity and cellular response after exposure to specific polyamine analogs have stimulated interest not only in polyamine catabolism in general, but also into the understanding of how this enzyme's activity can affect cellular survival.

The first indication that highly induced SSAT activity was associated with cytotoxicity was demonstrated in human non-small-cell lung cancer cells (13,14). This pheno-type-specific response to specific antitumor polyamine analogs was subsequently observed in other human tumor cell types (44-50) and confirmed in several in vivo models, demonstrating a significant increase in SSAT activity in response to analog treatment of sensitive tumor types. Importantly, the increased activity observed in vivo was generally specific for the tumor cells (51 -57). Although transient increases in kidney and liver SSAT activities were observed, these enzyme levels rapidly returned to normal after treatment was discontinued. In comparison, the tumor-specific increase in SSAT was not only higher, but persisted considerably longer after cessation of treatment. The tumor selectivity can also be demonstrated for human tumors in situ. When human non-small-cell lung cancer explants from biopsies are exposed, in vitro, to 10 mM BENSpm for 24 h, there is a significant increase in tumor-specific induction of SSAT as determined by specific antibody staining (Fig. 5). These results clearly implicate the activity of SSAT as being associated with the observed tumor-selective cyto-toxicity; however, it was not clear whether the cytotoxicity was the result of increased S SAT enzyme activity, decreased polyamine pools, direct activity by the analog, or some combination of each.

Another indication of a direct link between SSAT and drug response was observed in human lung tumor cells of female origin that possess two active copies of the X-linked SSAT. The NCI H727 carcinoid line expressing both copies were significantly more sensitive to the cytotoxic effects of the analog BESpm than female cells that only expressed a single copy (58). Additional data indicating that increased SSAT expression alone could lead to growth inhibition came when human SSAT was overexpressed in Escherichia coli leading to a near complete depletion of spermidine and decreased bacterial growth (59). However, the clearest evidence indicating a direct role for SSAT activity in the cellular response to the polyamine analogs was observed by Vujcic et al. using a Tet-repressible SSAT expression system in MCF-7 human breast cancer cells (60). Derepression of the expression vector produced a 20-fold increase in SSAT mRNA and a 10-fold increase in enzyme activity. This increased activity was accompanied by a significant decrease in growth rate. When increased SSAT expression was combined with BENSpm treatment, the SS AT-overexpressing cells were significantly more sensitive to the analog than wild-type

Fig. 5. Immunohistochemical staining of a human adenocarcinoma explant after exposure to BENSpm. Adenocarcinoma explants derived from the same aseptic core biopsy were divided into thin (approx 1 mm) sections and incubated in medium containing 10 mM BENSpm (B) or control medium (A) for 24 h. After incubation, tissues were fixed and imbedded in paraffin and processed with a specific anti-SSAT antibody as we have previously reported (54,55). Note the intense staining of only the tumor cells (dashed arrow) in the analog-treated sample (B). No staining was observed in the adjacent normal cells (solid arrow) in the treated sample and no staining was observed in either the tumor (dashed arrow) or normal (solid arrow) in the untreated sample (A).

Fig. 5. Immunohistochemical staining of a human adenocarcinoma explant after exposure to BENSpm. Adenocarcinoma explants derived from the same aseptic core biopsy were divided into thin (approx 1 mm) sections and incubated in medium containing 10 mM BENSpm (B) or control medium (A) for 24 h. After incubation, tissues were fixed and imbedded in paraffin and processed with a specific anti-SSAT antibody as we have previously reported (54,55). Note the intense staining of only the tumor cells (dashed arrow) in the analog-treated sample (B). No staining was observed in the adjacent normal cells (solid arrow) in the treated sample and no staining was observed in either the tumor (dashed arrow) or normal (solid arrow) in the untreated sample (A).

MCF-7 cells or cells containing the expression vector, but not derepressed. Similar results were observed in a normally BENSpm-resistant human small-cell lung cancer line trans-fected with a constitutively expressing SSAT construct (61). However, extreme increases in enzyme protein and activity have been found to be limited to those cells, transfected or not, that are treated with analog, consistent with a major regulatory step of SSAT sup e rin-duction being at the level of protein stabilization.

It is important to note that growth inhibition associated with high SSAT protein expression is dependent on SSAT catalytic activity. McCloskey et al. (62,63) d e m o n-strated that Chinese hamster ovary (CHO) cells expressing even high levels of an SSAT

protein containing a L156F mutation were resistant to the growth inhibitory effects of several polyamine analogs. Additio nally, this mutation resulted in a protein that was not capable of being stabilized by the polyamine analogs.

In addition to growth inhibition, apoptosis associated with increased polyamine catabolism has been observed in several systems. However, the precise mechanisms responsible for the induced apoptosis remain unclear and may be cell type-specific. It is clear that in some cell systems the induction of apoptosis is associated with, and may be dependent on, the induction of SSAT. The best demonstration of this to date are studies reported by Chen et al. using transient transfection of the analog-sensitive human SK-MEL-28 melanoma cells with small interfering RNA targeting SSAT (64). Their results clearly indicate that in this melanoma cell system, the induction of SSAT is necessary for the early downstream events of apoptosis to occur. Unfortunately, because these were transient transfection studies, the longer term effects of SSAT knockdown were not analyzed.

A gene with homology to SSAT has recently been reported and is designated as SSAT2 (65,66). However, because it does not appear that the protein encoded by this gene plays a significant role in polyamine metabolism, it will not be discussed further here (66).

3.4. SSAT Transgenic Models

Cellular transfection models have contributed significantly to our understanding of the importance of SSAT activity in the maintenance of polyamine homeostasis and its potential role in drug response. However, a wealth of information has been derived from a variety of recent transgenic systems (6 7-81). One general feature emerging from these studies that is consistent with in vitro observations is the fact that extremely high SSAT activity is a result of posttranscriptional regulation by polyamine analogs. Without analog exposure, very large increases in SSAT mRNA can occur with only small increases in enzyme protein and activity (67,69, 81) .Another interesting finding in the transgenic animals was that when ODC and SSAT were both overexpressed in the same animals, polyamine catabolism controlled by SSAT overrode polyamine biosynthesis in determining intracellular polyamine concentrations and polyamine metabolites (8 0). The se data are completely consistent with results observed in MCF-7 cells induced to overexpress SSAT, then supplemented with exogenous polyamines (60). Specifically, the added polyamines could not overcome the increased SSAT activity to restore polyamine concentrations to normal levels. Taken together, these results suggest that polyamine catabolism is the major control point of polyamine homeostasis.

It is notable that high systemic overexpression of SSAT results in a mouse that has a similar outward appearance to mice overexpressing ODC (82). In both cases, the mice lose their hair at an early age and have progressive and severe skin wrinkling with age. Because both genotypes result in high putrescine levels in the skin, and because DFMO treatment of the ODC transgenics prevents the skin phenotype, high putrescine levels have been implicated in the ensuing phenotype.

A more recent and targeted SSAT transgenic mouse has been created using the bovine K6 promoter to target SSAT expression to the epidermal keratinocytes in the hair follicle ( 72).Unlike the previous models systemically expressing multiple copies of SSAT, these animals did not lose their hair and, when left untreated, did not exhibit significant changes in SSAT activity or polyamine concentrations in the skin. However, when these mice were challenged with a two-stage tumorigenesis protocol, the transgenic animals were considerably more sensitive to the dimethylbenzanthracine/12-O-tetradodecanoyl-phorbol-13-acetate (DMBA/TPA) treatment than the wild-type mice, resulting in a much greater tumor (98 to 60% after 19 wk of promotion). More importantly, a 31% incidence of malignant carcinomas was observed in the K6-SSAT mice, whereas no malignant tumors were observed in the identically challenged wild-type mice.

The results in the K6-SSAT transgenic mice appear to be in contrast to those observed by Pietilâ et al., in which systemic overexpression of SSAT reduced the number of tumors produced by a similar two-stage protocol. This apparent contradiction could be a result of the different mouse strains used by the investigators, or the result of the specific targeting of the epidermal follicles in the K6-SSAT system, rather than systemic overexpression. More experimentation will be necessary to resolve this apparent contradiction.

In addition to the depletion of polyamines and the production of reactive oxygen species, Kee et al. have recently demonstrated another potential mechanism by which activated SSAT can lead to growth inhibition or cell death in a cell type-specific manner (76,83). Using both in vitro and in vivo prostate models, the high expression of an exogenous SSAT gene leads to a decrease in growth rate that is associated with a significant depletion of acetyl CoA pools. This correlation was demonstrated in both prostate cancer cells and in prostates from TRAMP/SSAT double transgenic mice. In the double transgenic model, the prostate tissue in general is reduced in size, and the tumor progression appears limited based on histopathological scores. The authors propose that the reduction of acetyl CoA pools is owing to an accelerated flux through the polyamine metabolic pathway, resulting from a compensatory increase in polyamine biosynthesis in response to increased SSAT activity. This hypothesis is consistent with their in vitro findings using the LNCaP prostatic cancer cell line in which acetyl CoA depletion by SSAT overexpression could be prevented if the flux through the polyamine metabolic pathway was blocked by inhibiting biosynthesis with DFMO (83). Although it would be preferable to see the results of these studies confirmed using a prostate-t a rgeted SSAT expression mode, these results may have great significance for the development of therapeutic agents targeting SSAT induction in the prostate.

3.5. Expression of SSAT in Response to Stimuli Other Than Polyamines or Polyamine Analogs

Clearly, the transcriptional and posttranscriptional regulation of SSAT by the cell in response to the polyamines and their analogs represent highly regulated processes that are becoming well documented. Although it has long been known that multiple conditions, including toxins, hormones, and various other cellular stresses can lead to increased SSAT expression (19), several previously unidentified inducers have been found to elevate cellular SSAT levels.

Sulindac, a nonsteroidal anti-inflammatory drug is metabolized into two active derivatives, a sulfide that is an effective cyclo-oxygenase (COX) inhibitor and a sul-

fone. Both metabolites are effective in inhibiting colon cancer cell growth. Surprisingly, the sulfone derivative was found to increase the transcription of SSAT through a peroxisome proliferator-activated receptor (PPAR) response element bound by PPARg (84). The results of this study suggest that apoptosis induced by sulindac sulfone is COX-independent, and that PPAR-dependent activation of SSAT leads to polyamine depletion through acetylation and export. Although further study will be necessary to determine the relative effectiveness of the COX-independent induction of apoptosis as compared with the COX-dependent effects, it does open an entirely new avenue for SSAT-associated growth inhibition.

A series of recent reports suggest that specific antitumor agents structurally unrelated to the polyamines are also capable of upregulating the expression of SSAT mRNA (85-87). Using a gene array analysis strategy, Maxwell et al. identified SSAT as being among a select number of genes whose expression was significantly increased in MCF-7 breast cancer cells after exposure to 5-fluorouracil (5-FU). However, because increases in SSAT mRNA have frequently been observed without concurrent increases in activity, and because activity was not measured in this system, the functional significance of the increased mRNA in response to 5-FU alone is not known.

Results from a series of studies in the colon cancer cell line HCT116 suggest that 5-FU can synergize with BENSpm treatment in regard to SSAT expression (87). When 5-FU was used in combination with BENSpm, there were synergistic increases in both SSAT mRNA and apoptosis, as well as decreases in spermidine and spermine concentrations. These results are consistent with observations by Hahm et al. in MCF-7 cells treated with the combination of 5-FU and SSAT-inducing polyamine analogs (8 8). Unfortunately, none of these investigators reported SSAT activity after drug treatment.

In a recent study, Hector et al. examined the combination of oxaliplatin and BENSpm in multiple cell types, including melanoma and ovarian cancer cells. This combination produced a clearly synergistic increase in SSAT mRNA and enzyme activity, and resulted in significantly more growth inhibition than when either drug was used as a single agent (8 6). Most importantly, this study suggests a likely mechanism for the observed synergy. Specifically, it appears that the cytotoxic agent leads to a substantial increase in SSAT mRNA, but without a subsequent elevation of SSAT activity unless the polyamine analog is present to both enhance translation and stabilize the protein. The result is that substantially lower concentrations of each compound can be used to produce much greater growth inhibitory effects, potentially increasing the therapeutic index of the combination.

Taken together, the results from studies demonstrating that compounds other than polyamines or their analogs are capable to significantly regulate SSAT expression suggest that a judicious combination of agents may be used to increase the therapeutic efficacy of both polyamine- and nonpolyamine-targeted agents. Combining compounds targeting polyamine metabolism with traditional chemotherapeutic agents may hold the greatest promise for use in many polyamine pathway-targeting therapies.

Fig. 6. PAO activity. The FAD-dependent PAO oxidizes either N^acetylspermidine (R = H) or N^acetylspermine (R = C3HgNj), producing putrescine or spermidine, N- ac etyl - 3 - ami no-propanal, and H2O2.

3.6. SSAT Expression in Response to Stress

The SSAT gene has been described as a stress response gene and the protein was originally purified from rat liver stimulated with the toxic stressor, carbon tetrachloride (19,89-91). Several more recent reports implicate SSAT as either a marker or mediator of ischemia/reperfusion-associated injury in multiple organ systems (92-98). Although the mechanism of increased SSAT-associated injury has not been elucidated, in vitro results using HEK-293 kidney cells indicate that increased SSAT activity and the downstream oxidation of the acetylated polyamines by PAO lead to the production of toxic H2O2 (9 8). This mechanism is similar to what has been suggested for some cellular responses to the polyamine analogs (99,100). However, it should be emphasized that in this ischemia/reperfusion model, spermine oxidase activity is also highly induced and may play a significant role in the observed events.

4. N1-Acetylpolyamine Oxidase

4.1. PAO Properties, Function, and Expression

The first description of purified rat liver PAO implicated it as a predominantly peroxisomal enzyme capable of oxidizing spermidine and spermine; however, it was much more active in the presence of benzaldehyde (101). The recognition that the N1 -acetylpoly amines were the preferred substrates of PAO led to the belief that polyamine catabolism was a two-step process (102-105), rate-limited by the acetylation of polyamines by SSAT (19). PAO has been excellently reviewed by several authors (17,106-112). However, only recently have mammalian PAOs been cloned and characterized (113-115). Therefore, the focus here will be primarily on the most recent findings relevant to the better understanding of PAO provided by the cloning of the mammalian enzyme.

PAO catalyzes the oxidation of N1-acetylspermine and N1-acetylspermidine to spermidine and putrescine, respectively (Fig. 6). Both reactions occur by cycling the enzyme-bound FAD to the reduced FADH2, and in the presence of molecular oxygen, producing H2O2 and 3-acetoamidopropanal (114).

PAO possesses a terminal peroxisomal localization sequence (-PRL) consistent with the peroxisomal localization of PAO determined by Holtta (101, 114,116), although this targeting sequence does not preclude the existence of a cytosolic protein. It should be noted that both Wu et al. and Vujcic et al. have reported three mammalian PAO s, each of a different amino acid size, including a mouse PAO reported as 504 amino acids, a bovine protein of 452 amino acids, and the human protein of 511 amino acids. Additio nally, 12 splice variants of the human PAO have been identified (115). The existence of multiple splice variants may explain, in part, the various protein sizes initially attributed to PAO isozymes (101,106,117). Another possibility is that some of the previous purifications may have resulted in a mixture of the different polyamine oxidases of similar size. The major human splice variant of spermine oxidase (SMO/PAOhl ) codes for a 551 amino acid protein with both high-domain homology and amino acid identity (39%) with the 511 amino acid PAO. Thus some of the confusion with regard to substrate specificity in the earliest reports of PAO may best be explained as the result of a mixture of two similar-size proteins.

The purified human and mouse PAOs each have high catalytic activity (Kcat = 4.5-31.7 s1) and affinities for the N^acetylated polyamines (Km = 0.85-1.78 mM) , with the highest affinity being reported for N^acetylspermine. The substrate preference for PAO appears to be N^acetylspermine > N^acetylspermidine > N1, N1 2-diacetylspermine >>> spermine (113-115).

4.2. Analogs as Substrates for PAO

Although the mammalian PAOs preferentially oxidize acetylated polyamines, it has long been assumed that they may also oxidize specific polyamine analogs (118,119). Such activity could obviously affect the efficacy of those compounds when used against tumors expressing high PAO activity. However, the determination of which oxidases were active on what compounds awaited the identification and analysis of the individual enzymes. Wu et al. and Vujcic et al. both provide convincing evidence that the symmetrically substituted polyamine analogs typified by BENSpm are effective substrates for PAO (113,114). Additionally, we have demonstrated that recombinant human PAO not only effectively oxidizes the symmetrically substituted analogs, but also efficiently uses the unsymmetrically substituted polyamines as substrates. Consistent with the results of Vujcic et al., the unsymmetrically substituted analogs used in our studies appear to be oxidized at the interior nitrogen, producing the appropriately substituted spermidine analog, rather than the predicted de-ethylation product (113, 115,118). However, in contrast to the observations of Vujcic et al., there was no evidence of PAO activity when BEHSpm was used as a substrate. In fact, none of the analogs possessing aminobutyl terminal moieties were oxidized by the recombinant enzyme (115). The basis for this difference is not clear, although in our studies, purified, recombinant protein was used, whereas Vujcic et al. used lysates from cells transfected with PAO expression vectors (113,115).

Fig. 7. The structure of the polyamine oxidase inhibitor, N1, N4-bis(2,3 - butad i enyl ) -1 ,4 -butanediamine (MDL 72,527).

Fig. 7. The structure of the polyamine oxidase inhibitor, N1, N4-bis(2,3 - butad i enyl ) -1 ,4 -butanediamine (MDL 72,527).

It was recently demonstrated that when human A549 lung adenocarcinoma cells were transfected with PAO, those clones expressing high levels of PAO became wholly resistant to all of the analogs determined to be substrates of PAO (115). These data are entirely consistent with the previous observations of Lawson et al. demonstrating the resistance of CHO cells to CHENSpm, based on high oxidase activity in the CHO cells (119). These data, in combination with other reports (120-122) suggest that the levels of PAO activity within cells may have a profound effect on the sensitivity of various tumors to specific antitumor polyamine analogs (16).

4.3. Inhibitors of PAO

Even before the mammalian enzyme had been cloned and fully characterized, attempts were made to specifically inhibit PAO to understand the significance of its activity. N1, N4-b i s(butadenyl)-1,4-diaminobutane (MDL 72,527; Fig. 7) was the first inhibitor synthesized specifically for this purpose (12 3 ). It was demonstrated that the cotreatment of cells with MDL 72,527 and specific analogs could enhance the activity of those compounds, presumably because MDL 72,527 inhibits their metabolism (124). However, MDL 72,527 has recently been demonstrated to be somewhat less specific in its inhibition than previously thought, as it has been found to be a highly effe ctive inhibitor of the newest member of the polyamine catabolic pathway, SMO/PAOhl (125-128).

Other compounds that are effective inhibitors of human PAO include several antitumor polyamine analogs known as oligoamines, which were synthesized by Frydman and colleagues (10,115,129-132). The mechanism by which these compounds inhibit PAO is unknown; however, it is significant that they also efficie ntly inhibit SMO/PAOhl (12 6).

Although the activity of mammalian PAO has been predicted and studied for several years, only now with its cloning and characterization and with the discovery of the closely related spermine oxidase, will it be possible to fully understand the role of PAO in polyamine homeostasis and drug response. The next few years should be very telling in determining the important roles this enzyme plays in both normal and neoplastic cells.

5. Spermine Oxidase (SMO/PAOh1)

5.1. SMO Regulation, Expression, and Potential Role in Drug Response

SMO/PAOhl is the most recent of the polyamine catabolic enzymes to be discovered. This enzyme catalyzes the oxidation of spermine to spermidine, 3-aminopropanal, and H2O2 (Fig. 8). Our laboratory was the first to clone this oxidase (and named it

Fig. 8. SMO/PAOhl activity. The FAD-dependent SMO/PAOhl oxidize spermine-producing spermidine, 3-aminopropanal, and H2O2

PAOhl as the first human polyamine oxidase to be cloned) based on its homology to the maize plant polyamine oxidase (125,133). Although it had all of the structural domains expected of an acetylpolyamine oxidase, which was the original target of our work, it demonstrated a very high affinity for, and oxidase activity with, spermine as a substrate. Importantly, it was also highly inducible, which set it apart from what was expected for the authentic PAO (125). Vujcic et al. confirmed that this newly cloned enzyme was, in fact, a spermine oxidase, which they named SMO (12 8 ). The human SMO/PAOhl gene is located on chromosome 20p13. Interestingly, this gene codes for multiple splice variants, with the longest open reading frame described thus far for the human gene being 1668 base pairs coding for a 555 amino acid protein with a predicted molecular weight of 61 kD (125,134). The mouse and the human SMO/PAOhl share considerable homology (95% identity) and substrate specificity (128, 135). The mouse gene also codes for multiple splice variants, one of which, mSMOm, is catalyt-ically active and appears to be translocated to the nucleus (136). It is also significant to note that a recently reported nuclear lysine demethylase (LSD1), which is part of a transcriptional corepressor complex, is highly homologous to human spermine oxidase (137,138). Although LSD1 does not appear to use polyamines as substrates, the primary sequence similarity of this FAD-dependent methylase to SMO/PAOh1 strongly suggests that polyamines may interfere with its demethylase reaction, and thereby alter the repressor activity of the demethylase.

In human lung cancer cells, the induction of spermine oxidase in response to various polyamine analogs appears to occur primarily at the level of increased message. This increase appears to result from a combination of modestly increased transcription and a near doubling of mRNA half-life (139). Significant posttranslational regulation of human SMO/PAOh1 has not been described.

Recombinant proteins of both the human and the mouse spermine oxidases have been characterized^! 26,135), and both have very similar properties. The human protein exhibits a Km of 1.6 mMfor spermine and a Fmax of 7.72 mmol/mg protein/min (k = 7.2 s-1) (126). These values are somewhat different than those reported for the mouse homolog, in which the Km and kc were 90-170 mM and 4.5-4.8 s-1, respectively (135,136,140). The basis for the apparent differences in kinetic constants of these very similar homologs is not readily apparent; however, it should be stressed that the methods for preparation of the mouse and human proteins, and the assay systems used to measure the kinetic parameters, were different. The most significant difference was in the preparation of the human protein, where we purified a his-tagged recombinant protein from bacterial inclusion bodies by a denaturation/renaturation protocol (126). In contrast, Mariottini, Federico, and colleagues purified native recombinant protein from periplasmically targeted constructs (135,136,140). Importantly, multiple studies have demonstrated that the affinity and specificity of both mammalian enzymes are suff i-cient to be highly active in situ, and in specific cases, capable of reducing spermine concentrations to below detectable levels (113,125,127,128).

The discovery of this enzyme underscores its potential to alter the response of both normal and tumor cells to the effects of various agents. This is particularly true for the potential effects SMO/PAOhl could have on specific antitumor polyamine analogs. Our original discovery that human non-small-cell lung cancers respond to specific analog treatment with rapid and significant increases in both SMO/PAOhl mRNA and activity, suggested a direct mechanism by which SMO/PAOhl could alter cellular response to these agents (125,127). Specifically, these results suggested that in addition to H2O2 resulting from the SSAT/PAO pathway, H2O2 produced by the direct oxidation of spermine could, in part, lead to the observed apoptotic response of various tumor cells to specific analogs. We originally proposed the production of H2O2 by polyamine catabolism downstream from SSAT activity as a contributing factor to polyamine analog-induced apoptosis; this hypothesis has been corroborated in subsequent studies (99,100). However, these studies were completed prior to the discovery of SMO/PAOhl and used MDL 72,527, which inhibits both PAO and SMO/PAOhl to demonstrate the role of polyamine catabolism in cytotoxicity. Therefore, the origin of the toxic H2O2 is not clear (99,100,113,126,128). An additional issue complicating the interpretation of the origin of H2O2 in analog-induced cells is that most analogs that induce SMO/PAOhl also induce SSAT. To determine the contribution to cellular response by each of these pathways, double knockdown studies using RNA interference targeting either SSAT or SMO/PAOhl , and targeting both SSAT and SMO/PAOhl, are underway in our laboratory. Preliminary results strongly suggest that both pathways contribute to cytotoxicity and that reducing either SSAT or SMO/PAOhl expression results in decreased sensitivity to specific analogs.

It is interesting that unlike PAO, SMO/PAOhl does not effectively oxidize any of the terminally £is-alkylated polyamine analogs examined thus far (115,126). However, ^-monomethylspermine was determined to be a suitable substrate for SMO/PAO hl (128). Additionally, many of the analogs that were found to be substrates for PAO, are effective inhibitors of SMO/PAOhl (115,126). Taken together, these results suggest that analog structure must be critically considered if the goal is to induce cytotoxicity through SMO/PAOhl-produced H2O2.

5.2. Polyamine Catabolism as a Mediator of Cellular Damage and Its Implications in Disease Etiology

The likely link between increased spermine oxidation resulting in the production of H2O2 and its cellular response to specific polyamine analogs is currently under study in several laboratories. How else might normal or dysregulated polyamine catabolism affect cells and organisms? Parchment and Pierce suggested that polyamine oxidation was important for normal embryonic development by producing the H2O2 necessary to kill unwanted cells as the embryo develops (141,142). Although such an activation of polyamine catabolism in development would be beneficial to the organism, the possibility of detrimental activation of polyamine catabolism also exists.

One pathogenic state that leads to the inappropriate expression of spermine oxidase is Helicobacter pylori infection. H. pylori colonizes the mammalian stomach and is associated with gastritis, peptic ulcers, and gastric cancer. One question that persists regarding H. pylori infections is: how do the bacteria escape immune eradication? We have recently determined that infection with H. pylo ri results in a rapid induction of S MO/PAOh1 within the affected macrophages (143). The observed induction of the oxidase produces sufficient H2O2 to lead to macrophage mitochondrial depolarization and apoptotic cell death. These data are entirely consistent with the hypothesis that H. py l o ri-induced SMO/PAOh1 and subsequent macrophage death play a significant role in the bacterium's ability to escape host immune defenses.

In addition to demonstrating a plausible mechanism for allowing persistent H. pylori infection, these results also indicate the possibility that mammalian cells other than macrophages may have their expression of polyamine catabolic enzymes altered by infection. H. pylo ri infection has been associated with gastric cancer, and oxidative stress is directly linked to carcinogenesis from oxidative damage of DNA. The refo re, based on the observations in H. pylori-infected macrophages, we sought to determine if similar effects would result in gastric epithelial cells exposed to H. pylori infection. We hypothesized that if H. pylori infection of gastric epithelial cells (the cells from which gastric cancers originate) results in sufficient spermine oxidase activity and H2O2 production to damage DNA and induce apoptosis, it is possible that this DNA damage will contribute to the genetic mutations necessary for H. py l o ri-induced carcinogenic transformation (Fig. 9). The results of these studies clearly demonstrate that exposure of human gastric epithelial cells to H. pylori results in increased SMO/PAOh1 transcription, oxidase activity, DNA damage (as measured by increased 8-OH deoxy-guanosine production), and apoptosis. Importantly, inhibition of the oxidase activity by MDL 72,527 or knockdown of SMO/PAOh1 with specific small interfering RNAs, prevented the H. pylo«'-induced effects. In vivo results from both human and mouse tissues revealed that H. pylori-infected gastritis tissues demonstrated increased spermine oxidase expression, and when infection was eradicated in humans by antibiotic treatment, there was a concurrent decrease in SMO/PAO h 1 expression. Taken together, these results strongly implicate that H. pylori-induced SMO/PAOh1 activity in gastric epithelial cells plays an integral role in the disease process, and because of attendant oxidative DNA damage, may also be responsible for some of the genetic changes necessary for carcinogenic transformation in the etiology of gastric cancer.

Several questions remain to be answered regarding the potential for infectious agents to upregulate SMO/PAOh1 and potentially produce carcinogenic levels of reactive oxygen species in the form of H2O2. It is not known whether the ability to induce SMO/PAOh1 is limited to H. pylori infection or if other enteric bacterial infections ny. a

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